Selaginella tamariscina fermentation extracting solution, preparation method, application and external preparation for skin

Abstract

The embodiment of the invention discloses a selaginella tamariscina fermentation extracting solution, a preparation method, application and a skin external preparation. The preparation method of the selaginella tamariscina fermentation extracting solution can improve the content of polysaccharide in a selaginella tamariscina extract, and the preparation method comprises the following steps: inoculating a fermentation substrate with a fermentation strain, and performing fermentation treatment to obtain a selaginella tamariscina fermentation solution; wherein the fermentation substrate comprises selaginella tamariscina and water; the fermentation strain comprises first saccharomyces cerevisiae; the first saccharomyces cerevisiae is saccharomyces cerevisiae with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.28462, and is preserved in the China General Microbiological Culture Collection Center; and sterilizing and filtering the selaginella tamariscina fermentation liquor to obtain the selaginella tamariscina fermentation extract.

Description

Technical Field

[0001] The invention relates to the technical field of Selaginella extraction, and in particular to a Selaginella fermentation extract, a preparation method, an application and a skin external preparation. Background Art

[0002] Polysaccharides are widely present in organisms and are one of the basic substances for maintaining the normal operation of life activities. Plant polysaccharides, also known as plant polysaccharides, are generally derived from plants. Plant polysaccharides have a variety of biological activities, so they have broad application prospects at present.

[0003] Selaginella is a perennial herb commonly used as a traditional Chinese medicine. A large number of flavonoids, phenylpropanoids, acetylphenols, steroids, glycosides and other compounds have been isolated and identified from Selaginella. At present, the research on the extraction of Selaginella mainly adopts the water extraction method. For example, in the Chinese patent document with the publication number CN101461829A, a method for extracting Selaginella and its main component, sedum biflavonoids, is disclosed, and it is proved that the Selaginella extract has the application prospect of protecting the liver.

[0004] However, the water extraction method has a defect, that is, the polysaccharide content in the obtained Selaginella extract is limited, which restricts the application of Selaginella extract in terms of polysaccharide activity.

[0005] Therefore, how to increase the polysaccharide content in Selaginella extract is a problem to be solved. Summary of the invention

[0006] An object of an embodiment of the present invention is to solve at least one of the above problems and provide corresponding beneficial effects.

[0007] Another object of the embodiment of the present invention is to provide a method for preparing a Selaginella fermentation extract, which can increase the polysaccharide content in the Selaginella extract, thereby obtaining a Selaginella fermentation extract rich in polysaccharides.

[0008] Another object of the embodiment of the present invention is to provide a Selaginella fermentation extract rich in polysaccharides.

[0009] Another purpose of the embodiment of the present invention is to provide an application of Selaginella fermentation extract.

[0010] Another object of an embodiment of the present invention is to provide a skin preparation based on the Selaginella fermented extract.

[0011] The embodiments of the present invention mainly adopt the following technical solutions to achieve the above objectives.

[0012] In a first aspect, the present invention provides a method for preparing a Selaginella fermentation extract, comprising:

[0013] The fermentation strain is inoculated into the fermentation substrate, and subjected to fermentation treatment to obtain a Selaginella fermentation liquid; wherein the fermentation substrate includes Selaginella and water; the fermentation strain includes a first brewer's yeast, which is classified and named as Saccharomyces cerevisiae, and is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC), with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit date of September 15, 2023, a deposit registration number of CGMCC No. 28462, and a deposit certificate showing that it is in a living state;

[0014] The Selaginella fermentation liquid is sterilized and filtered to obtain the Selaginella fermentation extract.

[0015] In a second aspect, an embodiment of the present application provides a Selaginella fermented extract prepared according to the method for preparing the Selaginella fermented extract described in the first aspect.

[0016] In a third aspect, an embodiment of the present application provides a skin topical preparation comprising the Selaginella fermented extract described in the second aspect.

[0017] In a fourth aspect, an embodiment of the present application provides a use of the Selaginella fermented extract described in the second aspect in the preparation of a skin topical preparation.

[0018] In a fifth aspect, an embodiment of the present application provides a use of the Selaginella fermentation extract described in the second aspect as an antioxidant.

[0019] The embodiments of the present application include at least the following beneficial effects:

[0020] In some embodiments, the preparation method provided in the embodiments of the present application uses a fermentation strain containing the first brewer's yeast to extract Selaginella, which is beneficial to increasing the sugar content in the extract, so a Selaginella fermentation extract with a higher polysaccharide content than the Selaginella water extract can be obtained.

[0021] In some embodiments, in the preparation method of the present application, the fermentation strain contains the first brewing yeast, which is beneficial to improving the stability of the Selaginella fermentation extract.

[0022] In some embodiments, the Selaginella fermentation extract prepared according to the preparation method of the embodiments of the present application is rich in polysaccharides and can achieve higher antioxidant properties.

[0023] In some embodiments, the skin topical preparation of the present invention has excellent antioxidant capacity due to the presence of Selaginella fermentation extract.

[0024] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram showing the appearance of the Selaginella fermented extract provided in Example 1 of the present application during a stability test;

[0026] Figure 2 A schematic diagram showing the appearance of the Selaginella fermented extract provided in Example 2 of the present application during a stability test.

[0027] Microbial strain description

[0028] First Saccharomyces cerevisiae:

[0029] It is classified and named Saccharomyces cerevisiae and deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC). The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 15, 2023. The deposit registration number is CGMCC No. 28462. The deposit certificate shows that it is in a living state. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] The terms "include", "comprise", "contain" and any variations thereof in the embodiments of the present application are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to the process, method, product or device.

[0032] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0033] In this document, unless otherwise specified, the term "%" means "percentage"; the term "°C" means: degrees Celsius; the term "μm" means: micrometer; the term "nm" means: nanometer.

[0034] Herein, unless otherwise specified, the term "%" refers to the total weight of the composition of the present application.

[0035] In this article, unless otherwise specified, all defined ranges are meant to include each specific range within a given range and a combination of sub-ranges between given ranges. For example, a range of 1 to 5 specifically includes 1, 2, 3, 4 and 5, and also includes sub-ranges such as 2 to 5, 3 to 5, 2 to 3, 2 to 4, 1 to 4, etc.

[0036] Unless otherwise indicated, the term "mL" refers to milliliters.

[0037] Unless otherwise indicated, the term "min" refers to minutes and the term "h" refers to hours.

[0038] In a first aspect, the present invention provides a method for preparing a Selaginella fermentation extract, comprising the following steps S100 to S200:

[0039] Step S100, inoculating the fermentation strain into the fermentation substrate, and subjecting the fermentation treatment to obtain the Selaginella fermentation liquid; wherein the fermentation substrate comprises Selaginella and water; the fermentation strain comprises a first brewer's yeast; the first brewer's yeast is selected from the brewer's yeast strain with a deposit number of CGMCC No. 28462, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration; the deposit date is September 15, 2023;

[0040] Specifically, a fermentation substrate and a fermentation strain may be obtained first, and then the fermentation strain may be inoculated into the fermentation substrate, and then the fermentation treatment may be performed to obtain a Selaginella fermentation liquid.

[0041] In some embodiments, the Selaginella in the fermentation substrate is the whole herb of Selaginella; the whole herb of Selaginella is the dried whole herb of Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata (HooK. et Grev.) Maxim. of the Selaginaceae family.

[0042] Step S200, sterilizing and filtering the Selaginella fermentation liquid to obtain the Selaginella fermentation extract.

[0043] The preparation method provided in the embodiment of the present application uses a fermentation strain containing the first brewer's yeast to extract Selaginella, which is beneficial to increasing the sugar content in the extract, so a Selaginella fermentation extract having a higher polysaccharide content than Selaginella water extract can be obtained.

[0044] In addition, since the fermentation strain contains the first brewing yeast, it is also beneficial to improve the stability of the Selaginella fermented extract. For example, compared with the Selaginella fermented extract prepared by the fermentation strain composed of only the second brewing yeast, the Selaginella fermented extract prepared in the embodiment of the present application has better stability.

[0045] It is further understood that when the Selaginella fermented extract with a high polysaccharide content prepared by the preparation method according to the embodiment of the present application is applied to a skin topical preparation, a higher antioxidant property can be achieved.

[0046] In some embodiments, step S100 includes steps S110 to S130:

[0047] S110, obtaining a Selaginella fermentation substrate according to the fermentation substrate.

[0048] S120, obtaining fermentation strain seed liquid according to the fermentation strain.

[0049] S130, adding 5-10% of the fermentation bacteria seed liquid into the Selaginella fermentation substrate according to the volume ratio, and fermenting for 19 hours to 5 days at a rotation speed of 180-200 rpm and a temperature of 20-45° C. to obtain the Selaginella fermentation liquid.

[0050] Specifically, in step S130, the volume ratio refers to the ratio of the volume of the fermentation bacteria seed liquid to the volume of the Selaginella fermentation substrate, that is, the volume percentage of the fermentation bacteria seed liquid to the volume percentage of the Selaginella fermentation substrate.

[0051] In some embodiments, the Selaginella in the fermentation substrate is the whole herb of Selaginella, and step S110 includes steps S111-S112:

[0052] Step S111, washing and drying the whole herb of Selaginella, and then crushing it into powder to obtain a powder, and mixing the powder with water to obtain a Selaginella powder liquid;

[0053] Step S112, sterilizing the Selaginella powder liquid at a temperature of 110-121° C. for 30 minutes and then cooling it to room temperature to obtain a Selaginella fermentation matrix, wherein the mass percentage of Selaginella in the Selaginella fermentation matrix is ​​1-5%.

[0054] In some embodiments, when the fermentation strain is a first brewing yeast, S120 includes:

[0055] The first brewer's yeast was cultured to a live bacterial concentration of 0.8-2×10 8 CFU / g, and obtain the fermentation bacteria seed liquid.

[0056] Specifically, the first brewer's yeast can be inoculated from the slant into a triangular flask, and then cultured for a first period of time and then expanded until the live bacterial concentration of the strain is 2×10 8 CFU / g, and obtain the first bacterial seed solution. In some embodiments, the first time is 12 to 60 hours, and optionally 48 hours.

[0057] In some embodiments, the fermentation strain further comprises a second Saccharomyces cerevisiae;

[0058] The second brewer's yeast is classified as Saccharomyces cerevisiae, with a deposit number of CGMCC No. 17452, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of March 27, 2019;

[0059] The second brewer's yeast has been recorded and published in a Chinese patent document with an application date of 202211638075.3, a publication number of CN116115542A, a publication date of May 16, 2023, and an invention name of "A fermented emulsion, a skin topical preparation containing the same, and a preparation method and application thereof".

[0060] When the fermentation strains include the first brewing yeast and the second brewing yeast, step S120 includes S1201-1203:

[0061] Step S1201: Cultivate the first brewer's yeast until the live bacterial concentration of the strain is 0.8 to 2×10 8 CFU / g, and obtain the first strain seed liquid.

[0062] Specifically, the first brewer's yeast can be inoculated into a triangular flask from a slant surface, and then cultured statically for a first period of time and then expanded until the live bacterial concentration of the strain is 0.8 to 2×10 8 CFU / g, and obtain the first strain seed liquid.

[0063] In some embodiments, the first time is 12 to 60 hours, optionally 48 hours.

[0064] Step S1202: Cultivate the first brewer's yeast until the live bacterial concentration of the strain is 0.8 to 2×10 8 CFU / g, to obtain the first strain seed solution;

[0065] The second brewer's yeast was inoculated from the slant into the triangular flask, and after being cultured for a second period of time, the culture was expanded until the live bacterial concentration of the strain was 0.8-2×10 8 CFU / g, and obtain the second bacterial species seed liquid.

[0066] In some embodiments, the second time is 12 to 60 hours, optionally 48 hours.

[0067] Step S1203, mixing the first bacterial seed liquid and the second bacterial seed liquid at a volume ratio of 0.5 to 3:1 to obtain a fermentation bacterial seed liquid. Optionally, the volume ratio is 1 to 3:1, and optionally, the volume ratio is 1 to 2:1.

[0068] In some embodiments, step S200, filtering the Selaginella fermentation liquid to obtain the Selaginella fermentation extract, comprises S210-S230:

[0069] Step S210: sterilize the Selaginella fermentation broth at a temperature of 100-110°C for 30 minutes, and then centrifuge at a rotation speed of 4000-5000 rpm for 10 minutes to 5 hours to separate the liquid phase portion from the solid phase portion of the Selaginella fermentation broth to obtain a liquid phase portion; microfiltrate the liquid phase portion to remove the bacteria in the liquid phase portion to obtain a first filtrate; in some embodiments, the centrifugation time is 30 minutes.

[0070] Microfiltration refers to microfiltration (MF). Microfiltration is a widely used membrane filtration separation technology. Microfiltration is used to separate particles of 0.02 to 10 μm, and the pressure required for the process is in the range of 0.07 to 0.2 MPa. Microfiltration can be used to intercept particles, bacteria, pollutants, etc. from gas and liquid phases.

[0071] Step S220: Filter the first filtrate in a plate-and-frame filter with a filter cloth mesh number of 60 to 100 meshes and a pressure not exceeding 4 MPa to obtain a second filtrate; in some embodiments, the filter cloth mesh number is 80 meshes.

[0072] Step S230: Select a microporous filter membrane with a pore size of 0-0.8 μm to filter the second filtrate to obtain a Selaginella fermentation extract.

[0073] In a second aspect, the embodiments of the present application provide a Selaginella fermentation extract.

[0074] The Selaginella fermented extract is prepared according to the preparation method of the first embodiment.

[0075] In a third aspect, the embodiments of the present application provide a skin preparation, comprising the Selaginella fermented extract described in the embodiments of the second aspect.

[0076] In some embodiments, the skin preparation is a topical skin preparation.

[0077] In addition, in certain embodiments, the present application provides a use of a Selaginella fermented extract in the preparation of a skin topical preparation.

[0078] In certain embodiments, the present application also provides a use of a Selaginella fermentation extract as an antioxidant.

[0079] <Isolation of the First Saccharomyces cerevisiae (Deposit Number: CGMCC No. 28462)>

[0080] In some embodiments, the first Saccharomyces cerevisiae can be isolated and obtained according to the following method, which specifically comprises the following steps:

[0081] (1) taking an appropriate amount of sample from sauerkraut and placing it in a sterile test tube; adding sterile physiological saline, and fully shaking and mixing to release the microorganisms therein, thereby obtaining a mixed solution;

[0082] (2) using sterile physiological saline to perform gradient dilution (10 times, 100 times, 1000 times) on the mixed solution to reduce the density of microorganisms and obtain a diluted liquid;

[0083] (3) Take a small amount of the diluted liquid with a sterile pipette and drop it onto one end of the YPD agar plate;

[0084] (4) After dropping, use a sterile inoculating loop to streak the diluted liquid on the plate from one end to the other to form gradually sparse single colonies. Select colonies that have the characteristics of Saccharomyces cerevisiae (usually round, smooth, and cream-colored);

[0085] (5) Place the streaked plate in a constant temperature incubator, usually at 30°C for 48 hours, and observe the single colonies formed on the plate;

[0086] (6) Use an inoculation loop to pick up the target colony for further purification, transfer the selected single colony to a new YPD plate, repeat the above steps to ensure purification, observe the cell morphology under a microscope, and further identify it through molecular biological methods (such as PCR) to obtain the first brewer's yeast (deposit number CGMCC No. 28462).

[0087] <Preparation Example of Selaginella Extract>

[0088] Example 1

[0089] The Selaginella extract prepared in Example 1 is a Selaginella fermentation extract, and the preparation method comprises the following steps:

[0090] Step S101: taking Selaginella, wherein the Selaginella is the whole plant of Selaginella, washing and drying the whole plant of Selaginella, and then crushing it into powder to obtain a powder, mixing the powder with water to obtain a Selaginella powder liquid; sterilizing the Selaginella powder liquid at a temperature of 110° C. for 30 minutes and then cooling it to room temperature to obtain a Selaginella fermentation matrix, wherein the mass percentage (mass fraction) of Selaginella in the Selaginella fermentation matrix is ​​5%;

[0091] Step S102: Take the first brewer's yeast as the fermentation strain, inoculate the first brewer's yeast from the slant into the triangular flask, and culture it statically for 48 hours to expand the culture until the live bacterial concentration of the strain is 2×10 8 CFU / g, and the fermentation strain seed liquid was obtained;

[0092] Step S103: adding 10% of the fermentation bacteria seed liquid into the fermentation matrix according to the volume ratio, and fermenting for 68-72 hours at a rotation speed of 200 rpm and a temperature of 45° C. to obtain Selaginella fermentation liquid;

[0093] Step S104: sterilizing the Selaginella fermentation liquid at 110° C. for 30 min, and then centrifuging at 5000 rpm for 30 min to separate the liquid phase from the solid phase of the Selaginella fermentation liquid to obtain a liquid phase; removing the bacterial cells in the liquid phase by microfiltration to obtain a first filtrate;

[0094] Step S105: filtering the first filtrate in a plate-and-frame filter with a filter cloth having a mesh number of 80 and a pressure not exceeding 4 MPa to obtain a second filtrate;

[0095] Step S106: Select a microporous filter membrane with a pore size of 0.4 μm to perform a second filtration on the second filtrate to obtain a Selaginella fermentation extract.

[0096] Example 2

[0097] The Selaginella extract prepared in Example 2 is a Selaginella fermented extract, and the preparation method is basically the same as that in Example 1, the main difference being that the fermentation strains are different. The fermentation strains in Example 2 include a first brewer's yeast and a second brewer's yeast.

[0098] Specifically, the preparation method of Example 2 comprises the following steps:

[0099] Step S201: taking Selaginella, wherein the Selaginella is the whole plant of Selaginella, washing and drying the whole plant of Selaginella, and then crushing it into powder to obtain a powder, mixing the powder with water to obtain a Selaginella powder liquid; sterilizing the Selaginella powder liquid at a temperature of 110° C. for 30 minutes and then cooling it to room temperature to obtain a Selaginella fermentation matrix, wherein the mass percentage (mass fraction) of Selaginella in the Selaginella fermentation matrix is ​​5%;

[0100] Step S202: Inoculate the first brewer's yeast from the slant into a triangular flask and culture it statically for 48 hours to expand the culture until the live bacterial concentration of the strain reaches 2×10 8 CFU / g, and the first strain seed solution was obtained; the second brewer's yeast was inoculated into the triangular flask from the slant, and static culture was cultured for 48 hours to expand the culture until the live bacterial concentration of the strain was 2×10 8CFU / g, obtaining a second bacterial seed solution; mixing the first bacterial seed solution and the second bacterial seed solution in a volume ratio of 1:1 to obtain a fermentation bacterial seed solution;

[0101] Step S203: adding 10% of the fermentation bacteria seed liquid into the fermentation matrix according to the volume ratio, and fermenting for 68-72 hours at a rotation speed of 200 rpm and a temperature of 45° C. to obtain Selaginella fermentation liquid;

[0102] Step S204: sterilizing the Selaginella fermentation liquid at 110° C. for 30 min, and then centrifuging at 5000 rpm for 30 min to separate the liquid phase from the solid phase of the Selaginella fermentation liquid to obtain a liquid phase; removing the bacterial cells in the liquid phase by microfiltration to obtain a first filtrate;

[0103] Step S205: filtering the first filtrate in a plate-and-frame filter with a filter cloth having a mesh number of 80 and a pressure not exceeding 4 MPa to obtain a second filtrate;

[0104] Step S206: Select a microporous filter membrane with a pore size of 0-0.8 μm to perform a second filtration on the second filtrate to obtain a Selaginella fermentation extract.

[0105] Example 3

[0106] The Selaginella extract prepared in Example 3 is a Selaginella fermented extract, and the preparation method is basically the same as that in Example 2, except that the volume ratio of the first strain seed liquid and the second strain seed liquid in the fermented strain seed liquid is different, that is:

[0107] The first bacterial seed liquid and the second bacterial seed liquid were mixed in a volume ratio of 0.5:1 to obtain a fermentation bacterial seed liquid.

[0108] Example 4

[0109] The Selaginella extract prepared in Example 4 is a Selaginella fermented extract, and the preparation method is basically the same as that in Example 2, except that the volume ratio of the first strain seed liquid and the second strain seed liquid in the fermented strain seed liquid is different, that is:

[0110] The first bacterial seed liquid and the second bacterial seed liquid are mixed in a volume ratio of 2:1 to obtain a fermentation bacterial seed liquid.

[0111] Example 5

[0112] The Selaginella extract prepared in Example 5 is a Selaginella fermented extract, and the preparation method is basically the same as that in Example 2, except that the volume ratio of the first strain seed liquid and the second strain seed liquid in the fermented strain seed liquid is different, that is:

[0113] The first bacterial species seed liquid and the second bacterial species seed liquid were mixed in a volume ratio of 3:1 to obtain a fermentation bacterial species seed liquid.

[0114] Comparative Example 1

[0115] The Selaginella extract prepared in Example 2 is a Selaginella fermented extract, and the preparation method is basically the same as that in Example 1, except that the fermentation strain in step S102 is different, and the fermentation strain is a second Saccharomyces cerevisiae. Accordingly, step S102 of comparative example 1 is specifically as follows:

[0116] Take the second brewer's yeast as the fermentation strain, inoculate the second brewer's yeast from the slant into the triangular flask, and culture it statically for 48 hours to expand the culture until the live bacterial concentration of the strain is 2×10 8 CFU / g, and obtain the fermentation bacteria seed liquid.

[0117] Comparative Example 2

[0118] The Selaginella extract prepared in Comparative Example 1 is a Selaginella water extract, and the preparation method comprises the following steps:

[0119] Take Selaginella, the Selaginella is the whole plant of Selaginella, wash and dry the whole plant of Selaginella, and then grind it into powder to obtain a powder; mix the powder with water to obtain a Selaginella powder liquid, wherein the mass fraction of Selaginella in the Selaginella powder liquid is 5%;

[0120] The Selaginella powder liquid was extracted twice in a water bath at 85°C, each extraction lasting 3 hours, and the two supernatants were combined and concentrated to half of the original volume to obtain the Selaginella water extract.

[0121] The Selaginella used in the above embodiments and comparative examples was purchased from Anhui Boyao Qiancao Chinese Medicine Co., Ltd.; origin: Linyi, Shandong; batch number: YC2211020.

[0122] In the above embodiments and comparative examples, the first brewer's yeast and the second brewer's yeast were selected from Zhejiang Chenhai Life Sciences Co., Ltd.

[0123] <Experimental Example 1, Polysaccharide Content Detection>

[0124] The Selaginella extracts of Examples 1 to 5 and Comparative Examples 1-2 were taken as test liquid samples, respectively, to measure the polysaccharide content. Experimental Example 1 The polysaccharide content in the Selaginella extract was determined by phenol-sulfuric acid spectrophotometry, and the specific method was as follows:

[0125] (1) Weigh 0.01 g (accurate to 0.0001 g) of glucose standard and dissolve it in distilled water. After it is fully dissolved, add dilute to a 10 mL volumetric flask to obtain a 1 mg / mL anhydrous glucose standard stock solution.

[0126] (2) Accurately pipette 2, 1, 0.5, 0.25, and 0.125 mL of the anhydrous glucose standard stock solution into 10 mL volumetric flasks, respectively, and dilute to volume with distilled water to obtain 0.2, 0.1, 0.05, 0.025, and 0.0125 mg / mL glucose standard solutions.

[0127] (3) Add 1.0 mL of glucose standard solution of different concentrations, 0.5 mL of 6% phenol, and 3.0 mL of concentrated sulfuric acid, shake well, and place in a boiling water bath for 20 min, then cool to room temperature. Pipette 200 μL of the above reaction solution into a 96-well plate and measure the absorbance at 490 nm. Draw a standard curve with the glucose standard concentration as the horizontal axis and the absorbance as the vertical axis.

[0128] (4) Accurately measure 1.0 mL of the test liquid sample into a 10 mL colorimetric tube, then repeat step (3), measure the absorbance at a wavelength of 490 nm, and calculate the total sugar content W in the test liquid sample according to the standard curve equation 1.

[0129] Formula 1: W = [(c × v) / m] × F × 0.9 × 100%; where,

[0130] W—total sugar content in the sample, in %;

[0131] c—the sugar content in the sample solution to be tested is obtained from the standard curve, in mg / mL;

[0132] v—the volume of the sample solution taken during colorimetric determination, in mL;

[0133] m—sample mass, in g;

[0134] F—dilution factor;

[0135] 0.9—Correction factor for converting glucose to polysaccharides.

[0136] Results and analysis of Experimental Example 1

[0137] The results are shown in Table 1.

[0138] Table 1

[0139]

[0140]

[0141] According to Table 1, the polysaccharide content of the Selaginella extracts of Examples 1 to 5 of the present application is significantly higher than that of the comparative example, indicating that the polysaccharide content of the Selaginella extracts can be increased according to the preparation method provided in the examples of the present application.

[0142] <Experiment 2, DPPH free radical scavenging rate detection>

[0143] The DPPH method is often used to determine the in vitro antioxidant capacity of extracts.

[0144] Therefore, in Experimental Example 2, the Selaginella extracts of Examples 1 to 5 and Comparative Examples 1-2 were taken as Selaginella test solutions, respectively, and the DPPH free radical scavenging rate of the Selaginella extracts was detected by the DPPH method to determine the antioxidant capacity.

[0145] The detection method of DPPH clearance rate is as follows:

[0146] (1) Prepare 2 mmol / L DPPH solution (stock solution): Accurately weigh 3.9 mg DPPH (accurate to 0.0001 g), add 5 mL anhydrous ethanol, mix well, and freeze in a dark place (prepare before use).

[0147] (2) Prepare 12.5 μg / mL vitamin C solution: Accurately weigh 2.0 mg of vitamin C (accurate to 0.0001 g), add 80 mL of distilled water, mix well and set aside.

[0148] (3) Determination of DPPH activity

[0149] Before use, take the above 2mmol / L DPPH solution and dilute it with anhydrous ethanol. The OD value is required to be 0.7±0.05 after subtracting the absorbance value of the blank control from the absorbance value at 515nm. Take the DPPH dilution solution of the corresponding concentration as the DPPH test solution for subsequent experiments.

[0150] Referring to Table 2, a sample group A1 was set on a 96-well plate. The sample to be tested and the DPPH test solution were added to the wells of the 96-well plate corresponding to the sample group A1 in sequence. The reaction was carried out at room temperature in the dark for 30 minutes, and the light absorption value at 515 nm was measured. Each well was repeated 3 times. The sample to be tested of the sample group A1 was selected from the aforementioned Selaginella test solution.

[0151] Referring to Table 2, a positive control group A2 was set up on a 96-well plate. In the small wells of the 96-well plate corresponding to the positive control group A2, the sample to be tested and the absolute ethanol solution were added in sequence, reacted at room temperature in the dark for 30 minutes, and the light absorption value at 515 nm was measured; each well was repeated 3 times. The sample to be tested in the positive control group A2 was selected from the vitamin C solution.

[0152] Referring to Table 2, a blank control group A0 was set on a 96-well plate. DPPH test solution and anhydrous ethanol solution were added to the small wells of the 96-well plate corresponding to the blank control group A0 in sequence. The reaction was carried out at room temperature in the dark for 30 minutes, and the light absorption value at 515 nm was measured; each well was repeated 3 times.

[0153] Table 2. Sample addition table for DPPH free radical scavenging experiment

[0154] Sample to be tested (μL) DPPH test solution (μL) Anhydrous ethanol solution (μL) Sample Group A1 100 100 0 Positive control group A2 100 0 100 Blank control group A0 0 100 100

[0155] (4) Calculation of DPPH radical scavenging rate of Selaginella test solution

[0156] The clearance rate was calculated according to the following formula 2.

[0157] Formula 2: Clearance rate = {1-[(A1-A2) / A0]}×100%;

[0158] Where:

[0159] A1——average absorbance of sample group;

[0160] A2——average absorbance of the positive control group wells;

[0161] A0——average absorbance of blank control group.

[0162] Results and analysis of Experimental Example 2

[0163] The results are shown in Table 3.

[0164] Table 3

[0165] Clearance rate (%) Example 1 52.51±2.01 Example 2 87.08±0.05 Example 3 60.14±4.18 Example 4 67.13±0.83 Example 5 67.00±2.14 Comparative Example 1 28.42±1.99 Comparative Example 2 29.57±1.72

[0166] According to Table 3, the comparative examples all did not exceed 30%, while the clearance rates of Examples 1 to 5 were all above 50%, far exceeding the comparative examples. Therefore, the Selaginella fermented extract prepared by the preparation method of the present application example has excellent antioxidant capacity.

[0167] <Experimental Example 3, Stability Test>

[0168] The Selaginella extracts of Examples 1 to 5 and Comparative Examples 1-2 were taken as test samples, respectively, and the stability was tested under the conditions of high temperature, circulation, light, low temperature, and room temperature.

[0169] Stability testing methods include:

[0170] (1) High temperature test: The test samples are placed at 60°C for 15 days, and samples are taken at specified times to test the appearance and physical and chemical indicators.

[0171] (2) Room Temperature (RT) test: Place the test sample at 25±2℃, 75%RH±5%RH for 1 month, take samples at the specified time, and test the appearance and physical and chemical indicators. RH stands for Relative Humidity, which refers to humidity.

[0172] (3) Low temperature test: Place the test samples at -20℃ for 1 month, take samples at specified times, and test the appearance and physical and chemical indicators.

[0173] (4) Cyclic experimental test: The test samples were placed in a cyclic stability test chamber (50°C-4°C) for 1 month, and samples were taken at specified times to test the appearance and physical and chemical indicators of the fermentation filtrate.

[0174] (5) Lighting test: Place the test sample in a room with a critical light intensity of 4500Lx, a total light intensity of no less than 1.2×106lux·hr, and a near-ultraviolet light energy of no less than 200W·hr / m 2 Place the samples in a UV light box at room temperature for 1 month, and then test their appearance and physical and chemical indicators.

[0175] Stability test results and analysis

[0176] The results are shown in Table 4 and Figure 1-2 shown.

[0177] Figure 1 A schematic diagram of the appearance of the test sample provided in Example 1 after stability testing under different investigation conditions is shown. Figure 1 The five sample containers in the figure correspond, from left to right, to the appearance of the test samples for high temperature experiment test, normal temperature experiment test, low temperature experiment test, cycle experiment test, and illumination experiment test.

[0178] Figure 2 A schematic diagram of the appearance of the test sample provided in Example 2 after stability testing under different investigation conditions is shown. Figure 2 The five sample containers in the figure correspond, from left to right, to the appearance of the test samples for high temperature experiment test, normal temperature experiment test, low temperature experiment test, cycle experiment test, and illumination experiment test.

[0179] Table 4 shows the stability results of each test sample.

[0180] Table 4

[0181]

[0182]

[0183] From the above results, it can be seen that the test samples corresponding to Examples 2 and 4 were basically unchanged after being tested under the conditions of high temperature, cycle, light, low temperature, and room temperature. The test sample corresponding to Example 1 only slightly changed color in the high temperature test, the test sample corresponding to Example 3 only slightly changed color in the high temperature and light tests, and the test sample corresponding to Example 5 only slightly changed color in the high temperature test, and there was no change under other conditions.

[0184] Different from Examples 1-5, the test sample corresponding to Comparative Example 1 changes significantly after being tested under the conditions of high temperature, cycle, light, low temperature, and room temperature, indicating that the sample is unstable.

[0185] Therefore, the Selaginella extract prepared according to the preparation method of the embodiment of the present application has better stability.

[0186] <Experimental Example 4, Patch Test>

[0187] The Selaginella extracts of Examples 1 to 5 and Comparative Examples 1-2 were taken as test samples, respectively, and patch test was carried out.

[0188] The test method is as follows:

[0189] Take 0.2mL to 0.025mL of the liquid test sample, drop it on the filter paper, and then place the filter paper in the spot tester. On the spot tester, the test sample control spot tester hole is used as a blank control group, and the control spot tester hole is added with a solvent equal to the test sample, and the solvent is distilled water (in some embodiments, olive oil can also be used);

[0190] Select 30 suitable volunteers as subjects, and the subjects are randomly selected in the age range of 18-60 years old. Determine the test area on the back of the subject's arm, and use non-irritating tape to fix the patch tester on the back of the subject's arm. The test cycle lasts for 24 hours. After 24 hours, remove the patch tester, let it stand for 30 minutes, wait for the indentation to disappear, and observe the skin's reaction. If the test result is negative, it is necessary to observe again 24 hours and 48 hours after the patch test.

[0191] Results and analysis of Experimental Example 4

[0192] The patch test results are shown in Table 5 below.

[0193] Table 5

[0194]

[0195]

[0196] Note:

[0197] Negative reaction: no adverse results;

[0198] Suspected reaction: only slight erythema;

[0199] Weak positive reaction (erythema reaction): erythema, infiltration, edema, and papules;

[0200] Strong positive reaction (herpes reaction): erythema, infiltration, edema, papules, herpes; the reaction may extend beyond the tested area;

[0201] Very strong positive reaction (confluent herpes reaction): obvious erythema, severe infiltration, edema, confluent herpes; the reaction exceeds the test area.

[0202] From the above results, it can be seen that the Selaginella extracts provided in Examples 1 to 5 of the present application will not produce adverse reactions after being used on human skin.

[0203] Application Example 1

[0204] An essence liquid includes the Selaginella fermented extract prepared in Example 2 of the present invention. The specific formula thereof is shown in Table 5, and the preparation method can be prepared according to the conventional preparation method of the essence liquid.

[0205] Table 6

[0206]

[0207]

[0208] Since the essence contains the Selaginella fermented extract provided in Example 2, it also has the advantages brought by the Selaginella fermented extract.

[0209] Application Example 2

[0210] A cream comprises the Selaginella fermented extract prepared in Example 2 of the present invention. The specific formula thereof is shown in Table 7, and the preparation method can be prepared according to the conventional preparation method of cream.

[0211] Table 7

[0212] Element Mass fraction (%) Butanediol 6 Phenoxyethanol 0.8 Hexanediol 0.8 Xanthan gum 0.1 Hydroxyethyl Acrylate / Sodium Acryloyldimethyl Taurate Copolymer 0.8 EDTA-2Na 0.02 Isononyl Isononanoate 3 Pentaerythritol tetraethyl acetate 5 Six sticky silicone oil 2 Octyl silicone oil 3 C16 / C18 fatty alcohol / cetostearyl alcohol 2.5 Polyoxyethylene (21) stearyl alcohol ether 0.8 Glyceryl Stearate Citrate / PEG-100 Stearate / Behenyl Alcohol 2 Example 3 Selaginella fermented extract provided 5 Deionized water margin

[0213] Since the above cream contains the Selaginella fermented extract provided in Example 2, it also has the advantages brought by the Selaginella fermented extract.

[0214] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Selaginella fermentation extract, characterized in that: include: The fermentation strain is inoculated into the fermentation substrate, and subjected to fermentation treatment to obtain Selaginella fermentation liquid; wherein the fermentation substrate includes Selaginella and water; the fermentation strain includes a first brewer's yeast; the first brewer's yeast is: a brewer's yeast with a preservation number of CGMCC No. 28462, which is preserved in the General Microbiology Center of China Microorganism Culture Collection Administration; The Selaginella fermentation liquid is sterilized and filtered to obtain the Selaginella fermentation extract.

2. The method for preparing the Selaginella fermented extract according to claim 1, characterized in that: The method of inoculating the fermentation bacteria into the fermentation substrate and subjecting the fermentation process to obtain the Selaginella fermentation liquid comprises: Obtaining Selaginella fermentation substrate according to fermentation substrate; Obtaining fermentation strain seed liquid according to the fermentation strain; 5-10% of the fermentation bacteria seed liquid is added to the Selaginella fermentation substrate according to the volume ratio, and fermented for 19 hours to 5 days at a rotation speed of 180-200 rpm and a temperature of 20-45° C. to obtain the Selaginella fermentation liquid.

3. The method for preparing the Selaginella fermented extract according to claim 1, characterized in that: The method of obtaining the Selaginella fermentation substrate according to the fermentation substrate comprises: The Selaginella is washed and dried, and then crushed into powder to obtain a powdery substance, and the powdery substance is mixed with water to obtain a Selaginella powder liquid; The Selaginella powder liquid is sterilized at a temperature of 110-121° C. and then cooled to room temperature to obtain a Selaginella fermentation matrix, wherein the mass percentage of Selaginella in the Selaginella fermentation matrix is ​​1-5%.

4. The method for preparing the Selaginella fermented extract according to claim 1, characterized in that: The fermentation strain is a first brewing yeast, and obtaining the fermentation strain seed liquid according to the fermentation strain comprises: The first brewer's yeast was cultured to a viable bacterial concentration of 2 × 10 8 CFU / g, and obtain the fermentation bacteria seed liquid.

5. The method for preparing the Selaginella fermented extract according to claim 2, characterized in that: The fermentation strains further include a second Saccharomyces cerevisiae, wherein the second Saccharomyces cerevisiae is a Saccharomyces cerevisiae with a deposit number of CGMCC No. 17452 and is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee; The step of obtaining the fermentation strain seed liquid according to the fermentation strain comprises: The first brewer's yeast was cultured until the live bacterial concentration of the strain was 2×10 8 CFU / g, to obtain the first strain seed solution; The second brewer's yeast was cultured until the live bacterial concentration of the strain was 2×10 8 CFU / g, and the second bacterial species seed solution was obtained; The first bacterial seed liquid and the second bacterial seed liquid are mixed in a ratio of 0.5 to 3:1 to obtain a fermentation bacterial seed liquid.

6. The method for preparing the Selaginella fermented extract according to claim 1, characterized in that: The step of filtering the Selaginella fermentation liquid to obtain the Selaginella fermentation extract comprises: The Selaginella fermented liquid is sterilized at a temperature of 100-110° C. for 30 minutes, and then centrifuged at a speed of 4000-5000 rpm for 30 minutes to separate the liquid phase part and the solid phase part of the Selaginella fermented liquid to obtain a liquid phase part; microfiltration is performed to remove the bacterial cells in the liquid phase part to obtain a first filtrate; Filtering the first filtrate through a plate-and-frame filter with a filter cloth having a mesh number of 80 and a pressure not exceeding 4 MPa to obtain a second filtrate; A microporous filter membrane with a pore size of 0 to 0.8 μm is selected to filter the second filtrate to obtain a Selaginella fermentation extract.

7. A method for preparing a Selaginella fermentation extract, characterized in that: Take Selaginella, wash and dry it, and then crush it into powder to obtain a powder, mix the powder with water to obtain a Selaginella powder liquid, wherein the Selaginella is the whole herb of Selaginella; sterilize the Selaginella powder liquid at a temperature of 110° C. for 30 minutes and then cool it to room temperature to obtain a Selaginella fermentation matrix, wherein the mass percentage of Selaginella in the Selaginella fermentation matrix is ​​5%; The first brewer's yeast was inoculated from the slant into a triangular flask and cultured for 48 h to expand the culture until the live bacterial concentration of the strain reached 2×10 8 CFU / g, and the first strain seed solution was obtained; the second brewer's yeast was inoculated into the triangular flask from the slant, and static culture was cultured for 48 hours to expand the culture until the live bacterial concentration of the strain was 2×10 8 CFU / g, obtaining a second strain seed liquid; mixing the first strain seed liquid and the second strain seed liquid in a ratio of 0.5 to 3:1 to obtain a fermentation strain seed liquid; wherein the first brewer's yeast is a brewer's yeast with a deposit number of CGMCC No.28462, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee; and the second brewer's yeast is a brewer's yeast with a deposit number of CGMCC No.17452, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee; Inoculate 10% of the fermentation bacteria seed liquid into the fermentation substrate according to the volume ratio, and ferment for 19 hours to 5 days at a rotation speed of 200 rpm and a temperature of 45° C. to obtain a Selaginella fermentation liquid; sterilizing the Selaginella fermentation liquid at a temperature of 110° C. for 30 minutes, and then centrifuging at a speed of 5000 rpm for 30 minutes to separate the liquid phase portion and the solid phase portion of the Selaginella fermentation liquid to obtain a liquid phase portion; removing the bacterial cells in the liquid phase portion by microfiltration to obtain a first filtrate; Filtering the first filtrate through a plate-and-frame filter with a filter cloth having a mesh number of 80 and a pressure not exceeding 4 MPa to obtain a second filtrate; A microporous filter membrane with a pore size of 0 to 0.8 μm is selected to perform a second filtration on the second filtrate to obtain a Selaginella fermentation extract.

8. The Selaginella fermented extract prepared according to the method for preparing the Selaginella fermented extract according to any one of claims 1 to 7.

9. A skin external preparation, characterized in that: The invention comprises the Selaginella fermented extract as claimed in claim 8.

10. Use of the Selaginella fermented extract according to claim 8 in the preparation of external skin preparations.

11. Use of the Selaginella fermented extract according to claim 8 as an antioxidant.

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