Extraction method of transdermally absorbable tremella fuciformis extract

Abstract

The invention relates to an extraction method of a transdermally absorbable tremella extract, and belongs to the technical field of fungus extraction. The tremella extract is obtained through warm water soaking, hydrogen peroxide pretreatment, two-stage fermentation, two times of extraction, composite enzymolysis and final ultrafiltration, the molecular weight of the tremella extract is gradually reduced through layer-by-layer treatment, and the tremella extract can be effectively applied to the fields of cosmetics and the like and has a good transdermal absorption effect.

Description

Technical Field

[0001] The invention belongs to the technical field of fungus extraction and relates to a method for extracting a tremella extract that can be absorbed through the skin. Background Art

[0002] Tremella extract is a natural ingredient extracted from Tremella fuciformis. It has a variety of skin care benefits because it contains active ingredients such as polysaccharides, proteins, amino acids, trace elements, etc., especially in moisturizing, anti-oxidation, and promoting skin repair. With the increasing demand of consumers for natural ingredients and safe skin care products, the market potential of Tremella fuciformis extract is rapidly expanding and has become one of the important raw materials in the skin care industry.

[0003] The main component of Tremella fuciformis polysaccharide is Tremella fuciformis polysaccharide. Since Tremella fuciformis polysaccharide itself is a complex polysaccharide composed of multiple monosaccharide units connected by glycosidic bonds, Tremella fuciformis extract usually exists in the form of macromolecules. However, traditional extraction methods such as hot water extraction and alcohol precipitation extraction mostly rely on physical and chemical means, and the obtained Tremella fuciformis extract often retains its macromolecular form more. This Tremella fuciformis extract with a macromolecular structure cannot penetrate into the deep layers of the skin well to exert its biological activity. It is also easy to form a thicker film on the skin when used, affecting the skin's breathing and comfort. Summary of the invention

[0004] The object of the present invention is to provide a method for extracting a tremella extract that can be absorbed through the skin. The method comprises the following steps: warm water soaking, hydrogen peroxide pretreatment, two-stage fermentation, two extractions, composite enzymolysis, and finally ultrafiltration to obtain the tremella extract. After layer-by-layer treatment, the molecular weight of the tremella extract is gradually reduced, and the transdermal absorption capacity of the tremella extract is improved.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for extracting a transdermally absorbable tremella extract, comprising the following steps:

[0007] (1) soaking dried Tremella fuciformis in 35° C. warm water for 8-12 hours to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0008] (2) After preparing the culture medium, sterilize it at 121°C for 10-20 min and set aside;

[0009] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0010] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:5-7, extracting in an 80° C. water bath for 1.5-2.5 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0011] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0012] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0013] Furthermore, the pretreatment in step (1) refers to soaking the Tremella fuciformis particles in a 0.5-1.5% hydrogen peroxide solution, stirring at a speed of 20-60 rpm for 20-40 min at room temperature, rinsing with clean water after stirring, and drying to obtain pretreated Tremella fuciformis particles.

[0014] Furthermore, the raw materials of the culture medium in step (2) include 10-20 g / L of pretreated tremella particles, 1-3 g / L of green tea extract, 4-6 g / L of glucose, 2.5-3.5 g / L of xylose, 2.8-3.2 g / L of yeast powder, 1.5-2.5 g / L of peptone, 0.3-0.7 g / L of potassium dihydrogen phosphate, 0.2-0.4 g / L of magnesium sulfate, 0.15-0.25 g / L of calcium nitrate and the remainder of deionized water; the initial pH value of the culture medium is 5.2-5.8.

[0015] Furthermore, in step (3), the bacterial agent A is composed of Aspergillus oryzae and Rhizopus in a mass ratio of 1:1, and the effective bacterial content of Aspergillus oryzae is 1.2-1.6×10 7 CFU / g, the effective bacterial content of the Rhizopus is 0.9-1.1×10 7 CFU / g, the mass ratio of Aspergillus oryzae to the culture medium is 0.01-0.03:1; the fermentation tank parameters in the first stage of fermentation are: fermentation temperature is 28-32°C, fermentation speed is 100-140rmp, fermentation time is 44-52h, and oxygen ventilation during fermentation is 1-2vvm.

[0016] Furthermore, in step (3), the bacterial agent B is lactic acid bacteria, and the effective bacterial content of the lactic acid bacteria is 1.5-2.5×10 7 CFU / g, the mass ratio of the lactic acid bacteria to the culture medium is 0.02-0.04:1; the parameters of the fermentation tank in the second stage of fermentation are: fermentation temperature is 26-30°C, fermentation speed is 80-120rmp, fermentation time is 22-26h, and oxygen ventilation during fermentation is 1-2vvm.

[0017] Furthermore, the centrifugation speed and time in step (3) are 2000-4000 rpm and 10-20 min, respectively.

[0018] Furthermore, the filtration in step (4) and step (5) uses a 0.45 μm filter membrane; the speed and time of the centrifugation in step (5) are 6000-10000 rpm and 10-20 min, respectively.

[0019] Furthermore, in step (6), the ratio of the complex enzyme to the extract is 0.03-0.05:1 g / ml, and the complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 0.6-0.74:0.4-0.5:1-1.2; the parameters of the enzymatic hydrolysis are: enzymatic hydrolysis temperature of 47-53°C, enzymatic hydrolysis time of 3-4h, and enzymatic hydrolysis pH of 5.3-5.7; and the ultrafiltration uses an ultrafiltration membrane with a molecular weight of 9-11 kDa.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention obtains the Tremella fuciformis extract through warm water soaking, hydrogen peroxide pretreatment, two-stage fermentation, two-stage extraction, composite enzymatic hydrolysis, and finally ultrafiltration. After layer-by-layer treatment, the molecular weight of the Tremella fuciformis extract is gradually reduced, and the transdermal absorption capacity of the Tremella fuciformis extract is improved.

[0022] (2) The Tremella extract of the present invention is rich in natural polysaccharides. The Tremella is effectively softened by warm water immersion and hydrogen peroxide pretreatment. The introduction of hydrogen peroxide can partially degrade the polymer chains of polysaccharides, reduce their molecular weight, and improve the efficiency of subsequent fermentation. At the same time, green tea extract is introduced into the culture medium to protect the Tremella extract from being oxidized and destroyed during the extraction process. The Tremella is treated by a two-stage fermentation process. Amylase and cellulase are produced during the fermentation process of Aspergillus oryzae, which are helpful for the release of active ingredients such as polysaccharides in the Tremella extract. Rhizopus contains a variety of enzymes that can synergistically enhance the degradation of Tremella with Aspergillus oryzae. The decomposition ability of Tremella fuciformis is improved, the fermentation efficiency and the extraction rate of Tremella fuciformis extract are improved, and lactic acid bacteria are subsequently introduced to enhance the polysaccharide production ability, thereby improving the skin affinity of Tremella fuciformis extract; the Tremella fuciformis extract is extracted through two hot water extractions, and the cellulose, hemicellulose, pectin and other impurities in the Tremella fuciformis are degraded by a composite enzyme, thereby further improving the degradation efficiency of Tremella fuciformis and releasing the effective ingredients in the Tremella fuciformis. Ultrafiltration is then used to remove impurities with larger molecular weight, and different treatments are coordinated to further grade the Tremella fuciformis and optimize the extraction ability of active ingredients such as Tremella fuciformis polysaccharides, thereby gradually reducing the molecular weight of the Tremella fuciformis extract and improving the transdermal absorption capacity of the Tremella fuciformis extract. DETAILED DESCRIPTION

[0023] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0024] The green tea extracts in all embodiments and comparative examples of the present invention are purchased directly from the market, and are purchased from Gansu Yishengxiang Biotechnology Co., Ltd.; the strain collection number of Aspergillus oryzae is CICC 40214; the Rhizopus is purchased directly from the market, and is purchased from Shanghai Huzheng Biotechnology Co., Ltd., with the product number GIM3.230; the lactic acid bacteria are purchased directly from the market, and are purchased from Shandong Xinxiong Biotechnology Co., Ltd., and the product name is Lactobacillus plantarum; the cellulases are purchased directly from the market, and are purchased from Jiangxi Huayuyuan Biotechnology Co., Ltd.; the hemicellulases are purchased directly from the market, and are purchased from Jiangxi Huayuyuan Biotechnology Co., Ltd.; the pectinases are purchased directly from the market, and are purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0025] Example 1

[0026] A method for extracting a transdermally absorbable tremella extract. The method for extracting a transdermally absorbable tremella extract of this embodiment comprises the following steps:

[0027] (1) soaking dried Tremella fuciformis in 35° C. warm water for 8 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0028] (2) After preparing the culture medium, sterilize it at 121°C for 10 min and set aside;

[0029] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0030] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:5, extracting in an 80° C. water bath for 1.5 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0031] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0032] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0033] The pretreatment in step (1) of this embodiment refers to soaking the Tremella fuciformis particles in a 0.5% hydrogen peroxide solution, stirring at 20 rpm for 20 min at room temperature, rinsing with clean water after stirring, and drying to obtain pretreated Tremella fuciformis particles.

[0034] The raw materials of the culture medium in step (2) of this embodiment include 10 g / L of pretreated Tremella fuciformis particles, 1 g / L of green tea extract, 4 g / L of glucose, 2.5 g / L of xylose, 2.8 g / L of yeast powder, 1.5 g / L of peptone, 0.3 g / L of potassium dihydrogen phosphate, 0.2 g / L of magnesium sulfate, 0.15 g / L of calcium nitrate and the remainder of deionized water; the initial pH value of the culture medium is 5.2.

[0035] The bacterial agent A in step (3) of this embodiment is composed of Aspergillus oryzae and Rhizopus in a mass ratio of 1:1, and the effective bacterial content of Aspergillus oryzae is 1.2×10 7 CFU / g, the effective bacterial content of Rhizopus is 0.9×10 7 CFU / g, the mass ratio of Aspergillus to culture medium was 0.01:1; the fermentation tank parameters in the first stage of fermentation were: fermentation temperature was 28°C, fermentation speed was 100rmp, fermentation time was 44h, and oxygen ventilation during fermentation was 1vvm.

[0036] The bacterial agent B in step (3) of this embodiment is lactic acid bacteria, and the effective bacterial content of lactic acid bacteria is 10 7 CFU / g, the mass ratio of lactic acid bacteria to culture medium was 0.02:1; the parameters of the fermentation tank in the second stage of fermentation were: fermentation temperature was 26°C, fermentation speed was 80rmp, fermentation time was 22h, and oxygen ventilation during fermentation was 1vvm.

[0037] The centrifugation speed and time in step (3) of this embodiment are 2000 rpm and 10 min respectively.

[0038] In this embodiment, the filtration in steps (4) and (5) uses a 0.45 μm filter membrane; the speed and time of centrifugation in step (5) are 6000 rpm and 10 min, respectively.

[0039] The ratio of the complex enzyme to the extract in step (6) of this embodiment is 0.03:1 g / ml, and the complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 0.6:0.4:1; the parameters of enzymatic hydrolysis are: enzymatic hydrolysis temperature is 47°C, enzymatic hydrolysis time is 3h, and enzymatic hydrolysis pH is 5.3; the ultrafiltration membrane used is a 9 kDa molecular weight ultrafiltration membrane.

[0040] Example 2

[0041] A method for extracting a transdermally absorbable tremella extract. The method for extracting a transdermally absorbable tremella extract of this embodiment comprises the following steps:

[0042] (1) soaking dried Tremella fuciformis in 35° C. warm water for 12 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0043] (2) After preparing the culture medium, sterilize it at 121°C for 20 min and set aside;

[0044] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0045] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:7, extracting in an 80° C. water bath for 2.5 h, and filtering to remove solid residue to obtain a filtrate after the extraction is completed;

[0046] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0047] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0048] The pretreatment in step (1) of this embodiment refers to soaking the Tremella fuciformis particles in a 1.5% hydrogen peroxide solution, stirring at 60 rpm for 40 min at room temperature, rinsing with clean water after stirring, and drying to obtain pretreated Tremella fuciformis particles.

[0049] The raw materials of the culture medium in step (2) of this embodiment include 20 g / L of pretreated Tremella fuciformis particles, 3 g / L of green tea extract, 6 g / L of glucose, 3.5 g / L of xylose, 3.2 g / L of yeast powder, 2.5 g / L of peptone, 0.7 g / L of potassium dihydrogen phosphate, 0.4 g / L of magnesium sulfate, 0.25 g / L of calcium nitrate and the remainder of deionized water; the initial pH value of the culture medium is 5.8.

[0050] The bacterial agent A in step (3) of this embodiment is composed of Aspergillus oryzae and Rhizopus in a mass ratio of 1:1, and the effective bacterial content of Aspergillus oryzae is 1.6×10 7 CFU / g, effective bacterial content of Rhizopus 1.1×10 7 CFU / g, the mass ratio of Aspergillus to culture medium was 0.03:1; the fermentation tank parameters in the first stage of fermentation were: fermentation temperature was 32°C, fermentation speed was 140rmp, fermentation time was 52h, and oxygen ventilation during fermentation was 2vvm.

[0051] The bacterial agent B in step (3) of this embodiment is lactic acid bacteria, and the effective bacterial content of lactic acid bacteria is 2.5×10 7 CFU / g, the mass ratio of lactic acid bacteria to culture medium was 0.04:1; the parameters of the fermentation tank in the second stage of fermentation were: fermentation temperature was 30°C, fermentation speed was 120rmp, fermentation time was 26h, and oxygen ventilation during fermentation was 2vvm.

[0052] The centrifugation speed and time in step (3) of this embodiment are 4000 rpm and 20 min respectively.

[0053] In this embodiment, the filtration in steps (4) and (5) uses a 0.45 μm filter membrane; the speed and time of centrifugation in step (5) are 10,000 rpm and 20 min, respectively.

[0054] In step (6) of this embodiment, the ratio of the complex enzyme to the extract is 0.05:1 g / ml, and the complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 0.74:0.5:1.2; the parameters of enzymatic hydrolysis are: enzymatic hydrolysis temperature is 53°C, enzymatic hydrolysis time is 4h, and enzymatic hydrolysis pH is 5.7; the ultrafiltration membrane used is a molecular weight of 11 kDa.

[0055] Example 3

[0056] A method for extracting a transdermally absorbable tremella extract. The method for extracting a transdermally absorbable tremella extract of this embodiment comprises the following steps:

[0057] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0058] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0059] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0060] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0061] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0062] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0063] The pretreatment in step (1) of this embodiment refers to soaking the Tremella fuciformis particles in a 1% hydrogen peroxide solution, stirring at 40 rpm for 30 min at room temperature, rinsing with clean water after stirring, and drying to obtain pretreated Tremella fuciformis particles.

[0064] The raw materials of the culture medium in step (2) of this embodiment include 15 g / L of pretreated Tremella fuciformis particles, 2 g / L of green tea extract, 5 g / L of glucose, 3 g / L of xylose, 3 g / L of yeast powder, 2 g / L of peptone, 0.5 g / L of potassium dihydrogen phosphate, 0.3 / L of magnesium sulfate, 0.2 g / L of calcium nitrate and the remainder of deionized water; the initial pH value of the culture medium is 5.5.

[0065] The bacterial agent A in step (3) of this embodiment is composed of Aspergillus oryzae and Rhizopus in a mass ratio of 1:1, and the effective bacterial content of Aspergillus oryzae is 1.4×10 7 CFU / g, effective bacterial content of Rhizopus 1×10 7 CFU / g, the mass ratio of Aspergillus to culture medium was 0.02:1; the fermentation tank parameters in the first stage of fermentation were: fermentation temperature was 28-32°C, fermentation speed was 120rmp, fermentation time was 48h, and oxygen ventilation during fermentation was 1.5vvm.

[0066] The bacterial agent B in step (3) of this embodiment is lactic acid bacteria, and the effective bacterial content of lactic acid bacteria is 2×10 7 CFU / g, the mass ratio of lactic acid bacteria to culture medium was 0.03:1; the parameters of the fermentation tank in the second stage of fermentation were: fermentation temperature was 28°C, fermentation speed was 100rmp, fermentation time was 24h, and oxygen ventilation during fermentation was 1.5vvm.

[0067] The centrifugal speed and time in step (3) of this embodiment are 3000 rpm and 15 min respectively.

[0068] In this embodiment, the filtration in steps (4) and (5) uses a 0.45 μm filter membrane; the speed and time of centrifugation in step (5) are 8000 rpm and 15 min, respectively.

[0069] The ratio of the complex enzyme to the extract in step (6) of this embodiment is 0.04:1 g / ml, and the complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 0.67:0.45:1.1; the parameters of enzymatic hydrolysis are: enzymatic hydrolysis temperature is 50°C, enzymatic hydrolysis time is 3.5h, and enzymatic hydrolysis pH is 5.5; the ultrafiltration membrane used is a molecular weight of 10 kDa.

[0070] Comparative Example 1

[0071] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0072] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles;

[0073] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0074] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0075] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0076] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0077] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0078] Comparative Example 2

[0079] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0080] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0081] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0082] (3) inoculating the bacterial agent A into the culture medium in a fermentation tank for fermentation. The fermentation tank parameters are as follows: fermentation temperature of 30° C., fermentation speed of 120 rpm, fermentation time of 48 h, oxygen ventilation of 1.5 vvm during fermentation, centrifugation, and separation to obtain the fermentation liquid;

[0083] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0084] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0085] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0086] Comparative Example 3

[0087] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0088] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0089] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0090] (3) inoculating the bacterial agent B into the culture medium in a fermentation tank for fermentation. The parameters of the fermentation tank are as follows: fermentation temperature of 28° C., fermentation speed of 100 rpm, fermentation time of 24 h, oxygen ventilation of 1.5 vvm during fermentation, centrifugation, and separation to obtain a fermentation liquid;

[0091] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0092] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0093] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0094] Comparative Example 4

[0095] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0096] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0097] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0098] (3) inoculating bacterial agent A and bacterial agent B into the culture medium in a mass ratio of 1:1 in a fermenter for fermentation. The parameters of the fermenter are as follows: fermentation temperature of 28° C., fermentation speed of 100 rpm, fermentation time of 72 h, oxygen ventilation of 1.5 vvm during fermentation, centrifugation, and separation to obtain a fermentation liquid;

[0099] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0100] (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract;

[0101] (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0102] Comparative Example 5

[0103] On the basis of Example 3, Aspergillus oryzae in the microbial agent A was removed and replaced with an equal weight of Rhizopus, and other conditions were consistent with Example 3.

[0104] Comparative Example 6

[0105] On the basis of Example 3, the Rhizopus in the bacterial agent A was removed and replaced with an equal weight of Aspergillus oryzae, and other conditions were consistent with Example 3.

[0106] Comparative Example 7

[0107] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0108] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0109] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0110] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0111] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0112] (5) adding a complex enzyme to the filtrate, performing enzymatic hydrolysis and passing the filtrate through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0113] Comparative Example 8

[0114] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0115] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0116] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0117] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0118] (4) adding a complex enzyme to the fermentation broth, performing enzymatic hydrolysis and filtering the broth through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

[0119] Comparative Example 9

[0120] On the basis of Example 3, keeping other conditions the same, the extraction method of the transdermal absorbable Tremella fuciformis extract was changed to:

[0121] (1) soaking dried Tremella fuciformis in 35° C. warm water for 10 h to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles;

[0122] (2) After preparing the culture medium, sterilize it at 121°C for 15 min and set aside;

[0123] (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid;

[0124] (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:6, extracting in an 80° C. water bath for 2 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate;

[0125] (5) Repeat step (4) to extract the obtained filtrate, centrifuge and filter after the extraction, to obtain a transdermal absorbable Tremella fuciformis extract.

[0126] Comparative Example 10

[0127] On the basis of Example 3, the cellulase in the complex enzyme was removed and replaced with an equal weight of hemicellulase. Other conditions were the same as those in Example 3.

[0128] Comparative Example 11

[0129] On the basis of Example 3, the hemicellulase in the complex enzyme was removed and replaced with an equal weight of pectinase, and the other conditions were the same as those in Example 3.

[0130] Comparative Example 12

[0131] On the basis of Example 3, the pectinase in the complex enzyme was removed and replaced with an equal weight of cellulase, and the other conditions were the same as those in Example 3.

[0132] 1. Patch test

[0133] Fifteen volunteers with healthy skin were selected, half of them were male and half were female, and their ages were between 20 and 45. All volunteers had no history of skin disease, no known allergy to the test materials, and had not used any drugs or cosmetics that might affect skin reactions within at least 2 weeks before the test.

[0134] The Tremella extract obtained in Examples 1-3 was used as a sample, and 0.02±0.005 of a 5% concentration of the sample was added dropwise, and the sample was attached to the flexion side of the forearm of the subject with a low-allergenic tape in a spot tester. The spot tester was removed after 24 hours, and the skin reactions were observed 0.5 hours, 24 hours, and 48 hours after removal. The results were recorded according to the skin reaction grading standard in the "Technical Specification for Safety of Cosmetics" (2015 edition), where the scoring is divided into 0-4 levels, 0 being a negative reaction, and the test results are shown in Table 1.

[0135] Table 1

[0136]

[0137] As can be seen from Table 1, the Tremella extract obtained by the method of the present invention is mild to the skin and has no adverse reactions when used.

[0138] 2. Transdermal absorption test

[0139] The dorsal skin of healthy male Wistar rats was used as the permeability barrier and the test was conducted in an in vitro Franz diffusion cell. The parameters of the diffusion cell were: effective diffusion area of ​​about 3.25 cm 2 , the volume of the receiving tank was about 8 mL, the magnetic stirring speed was 600 rpm, and the temperature of the receiving liquid was 37.0 ± 0.1 °C.

[0140] The Tremella extracts prepared in Example 3 and Comparative Examples 1-12 were used as samples. 1 mL of the samples with a concentration of 5% were respectively taken and evenly coated on the back skin of rats. The skin was fixed between the receiving pool and the supply pool (the inner layer of the skin faced the receiving pool). A 20% ethanol physiological saline solution was prepared as the receiving solution. One side of the skin was completely in contact with the receiving solution, and it was ensured that there were no bubbles between the skin and the receiving solution. 1 mL of samples were taken at 1, 2, 4, 6, 8, and 24 hours (at the same time, an equal amount of isothermal receiving solution was supplemented). The concentration of the Tremella extract in the receiving solution filtered through a 0.22 μm filter membrane was determined by HPLC, and the cumulative penetration of the Tremella extract at different times was calculated.

[0141] The calculation formula of the cumulative transdermal amount of Tremella fuciformis extract per unit area is:

[0142]

[0143] In the above formula, Q is the cumulative transdermal amount, S is the effective diffusion area, V is the volume of saline in the receiving pool, and C i is the concentration of Tremella fuciformis extract in the receiving solution from the first sampling to the last sampling, C n is the concentration of Tremella fuciformis extract in the receiving solution during the sampling, m 0 is the theoretical sample weight, m ​​is the actual sample weight; calculate the proportion of the cumulative permeation amount of the Tremella fuciformis extract after 12 hours in the initial Tremella fuciformis extract content to obtain the 12-hour permeability (%), and record it as shown in the following Table 2.

[0144] Table 2

[0145]

[0146]

[0147] As shown in Table 2, the Tremella extract obtained by the present invention is easier to penetrate the skin than the Tremella extract obtained by Comparative Examples 1-12, and has a better transdermal absorption effect. However, Comparative Examples 1-12 arbitrarily changed a certain step or the composition of the reagents in the extraction method of the present invention, and could not achieve the effect of the present invention of degrading Tremella step by step through layer-by-layer treatment, thereby reducing the molecular weight of the Tremella extract, so the transdermal absorption capacity was weakened.

[0148] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any indirect modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for extracting a transdermally absorbable Tremella fuciformis extract, characterized in that: The method for extracting the transdermally absorbable Tremella fuciformis extract comprises the following steps: (1) soaking dried Tremella fuciformis in 35° C. warm water for 8-12 hours to obtain expanded Tremella fuciformis, crushing the Tremella fuciformis and passing it through a 10-mesh sieve to obtain Tremella fuciformis particles, and pre-treating the Tremella fuciformis particles to obtain pre-treated Tremella fuciformis particles; (2) After preparing the culture medium, sterilize it at 121°C for 10-20 min and set aside; (3) Inoculating bacterial agent A into a culture medium in a fermentation tank to carry out a first stage of fermentation, then inoculating bacterial agent B into the culture medium to carry out a second stage of fermentation, and centrifuging to separate and obtain a fermentation liquid; (4) mixing the fermentation broth and 80° C. hot water in a volume ratio of 1:5-7, extracting in an 80° C. water bath for 1.5-2.5 h. After the extraction is completed, filtering to remove solid residues to obtain a filtrate; (5) Repeat step (4) to extract the obtained filtrate, centrifuge after the extraction, filter, and obtain an extract; (6) adding a complex enzyme to the extract, performing enzymatic hydrolysis and filtering the extract through an ultrafiltration membrane to obtain a transdermal absorbable Tremella fuciformis extract.

2. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: The pretreatment in step (1) refers to soaking the Tremella fuciformis particles in a 0.5-1.5% hydrogen peroxide solution, stirring at a speed of 20-60 rpm for 20-40 min at room temperature, rinsing with clean water after stirring, and drying to obtain pretreated Tremella fuciformis particles.

3. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: The raw materials of the culture medium in step (2) include 10-20 g / L of pretreated tremella particles, 1-3 g / L of green tea extract, 4-6 g / L of glucose, 2.5-3.5 g / L of xylose, 2.8-3.2 g / L of yeast powder, 1.5-2.5 g / L of peptone, 0.3-0.7 g / L of potassium dihydrogen phosphate, 0.2-0.4 g / L of magnesium sulfate, 0.15-0.25 g / L of calcium nitrate and the balance of deionized water; the initial pH value of the culture medium is 5.2-5.

8.

4. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: Step (3) The bacterial agent A is composed of Aspergillus oryzae and Rhizopus in a mass ratio of 1:1, and the effective bacterial content of Aspergillus oryzae is 1.2-1.6×10 7 CFU / g, the effective bacterial content of the Rhizopus is 0.9-1.1×10 7 CFU / g, the mass ratio of Aspergillus oryzae to the culture medium is 0.01-0.03:1; the fermentation tank parameters in the first stage of fermentation are: fermentation temperature is 28-32°C, fermentation speed is 100-140rmp, fermentation time is 44-52h, and oxygen ventilation during fermentation is 1-2vvm.

5. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: In step (3), the bacterial agent B is lactic acid bacteria, and the effective bacterial content of the lactic acid bacteria is 1.5-2.5×10 7 CFU / g, the mass ratio of the lactic acid bacteria to the culture medium is 0.02-0.04:1; the parameters of the fermentation tank in the second stage of fermentation are: fermentation temperature is 26-30°C, fermentation speed is 80-120rmp, fermentation time is 22-26h, and oxygen ventilation during fermentation is 1-2vvm.

6. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: The centrifugal speed and time in step (3) are 2000-4000 rpm and 10-20 min respectively.

7. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: The filtration in step (4) and step (5) uses a 0.45 μm filter membrane; the speed and time of the centrifugation in step (5) are 6000-10000 rpm and 10-20 min, respectively.

8. The method for extracting a transdermally absorbable Tremella fuciformis extract according to claim 1, wherein: In step (6), the ratio of the complex enzyme to the extract is 0.03-0.05:1 g / ml, and the complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 0.6-0.74:0.4-0.5:1-1.2; the parameters of the enzymatic hydrolysis are: enzymatic hydrolysis temperature of 47-53°C, enzymatic hydrolysis time of 3-4h, and enzymatic hydrolysis pH of 5.3-5.7; and the ultrafiltration uses an ultrafiltration membrane with a molecular weight of 9-11 kDa.

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