Preparation method of tremella gum

Abstract

The invention belongs to the technical field of daily chemical products, and particularly relates to a preparation method of tremella gum, which comprises the following steps: (1) crushing and sieving dried tremella sporocarp to obtain tremella powder; (2) mixing the tremella powder with an extraction solvent, and performing ultrasonic treatment to obtain a mixture; and (3) adding inorganic alkali into the mixture, stirring and mixing, slowly dropwise adding epoxypropane while stirring, carrying out heat preservation reaction, adding an acid agent to adjust the pH value to be 5-8, and drying to obtain the tremella gum. Wherein the extraction solvent comprises betaine, polyethylene glycol and lactic acid in a molar ratio of 1: (2-4): (0.4-0.8). According to the present invention, the prepared white fungus glue has high viscosity and good light transmission under the normal temperature condition and the low concentration, can be used as the thickening agent in the daily chemical product, further has good antioxidant and whitening effects, and widens the application range of the white fungus glue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of daily chemical products, and particularly relates to a preparation method of tremella gum. BACKGROUND

[0002] Tremella, also known as white fungus, is a kind of edible fungus with medicinal and edible properties, rich in polysaccharides, amino acids, proteins and various minerals, and has the functions of antioxidant, anti-aging, anti-tumor, blood pressure reduction and immune enhancement. These functional properties promote the extraction of tremella polysaccharides from tremella, which has been widely used in medical treatment, health care, food processing, skin care and beauty, and animal husbandry.

[0003] The tremella polysaccharides contained in the fruiting body of tremella include intracellular polysaccharides, extracellular polysaccharides and cell wall polysaccharides. Due to the distribution of tremella polysaccharides in different cell structures of the fruiting body of tremella, the traditional process is often used in the processing of tremella, which often leads to low extraction efficiency of effective components or damage to other active components. For example, using high-concentration ethanol solution for processing will cause denaturation of proteins in tremella, thereby reducing the quality of the finished product.

[0004] In view of the above, it is urgent to develop a preparation method of tremella gum which can fully extract effective components from tremella and reduce the loss of raw materials to meet the high demand of the market for tremella gum. SUMMARY

[0005] In view of the above, a kind of tremella gum and its preparation method are proposed to solve the above problems.

[0006] In the first aspect, the present application provides a preparation method of tremella gum, comprising the following steps:

[0007] (1) crushing the dried tremella fruiting body, sieving, and obtaining tremella powder;

[0008] (2) mixing the tremella powder and the extraction solvent, and then performing ultrasonic treatment to obtain a mixture;

[0009] (3) adding an inorganic base to the mixture and stirring, slowly adding propylene oxide while stirring, and then performing heat preservation reaction, adding an acid agent to adjust the pH to 5-8, and drying to obtain the tremella gum;

[0010] The extraction solvent comprises betaine, polyethylene glycol and lactic acid in a molar ratio of 1:(2-4):(0.4-0.8).

[0011] The present application can fully destroy the cell structure of tremella fruiting body by adding betaine, polyethylene glycol and lactic acid in the extraction solvent, so as to fully release the active ingredients from the cell structure. Specifically, betaine acts as a hydrogen bond acceptor, polyethylene glycol and lactic acid act as hydrogen bond donors, and the three together form a hydrogen bond network of the eutectic solvent, which can effectively destroy the hydrogen bond or van der Waals force of the cell structure of tremella fruiting body, so that the active ingredients in the tremella fruiting body are released. Moreover, by selecting polyethylene glycol and lactic acid as hydrogen bond donors, the polarity of the eutectic solvent and the pH value of the eutectic solvent can be adjusted to help the extraction solvent better penetrate into the cell structure of tremella fruiting body, so that the active ingredients in the tremella fruiting body are better dissolved.

[0012] Further, in step (2), the water content of the extraction solvent is 15wt%-20wt%.

[0013] The water content of the extraction solvent in the present application is 15wt%-20wt%, and the reasonable water content of the extraction solvent can improve the viscosity and flowability of the extraction solvent, thereby realizing the purpose of dissolving the active ingredients in the tremella fruiting body from the cell structure of the tremella fruiting body.

[0014] Further, in step (2), the solid-liquid ratio of the tremella powder and the extraction solvent is 1g:5-10mL.

[0015] The solid-liquid ratio of the tremella powder and the extraction solvent in the present application is 1g:5-10mL, and the reasonable solid-liquid ratio can not only ensure that the tremella powder and the extraction solvent are in sufficient contact to promote the dissolution of the active ingredients in the tremella fruiting body into the extraction solvent, but also can avoid the inconsistent dissolution of intracellular polysaccharide, extracellular polysaccharide and cell wall polysaccharide, which is converted into reducing sugar in the extraction solvent, resulting in the decrease of the viscosity and use effect of the finished product.

[0016] Further, the ultrasonic treatment is ultrasonic treatment at a temperature of 40-80℃ and an ultrasonic frequency of 100-300W for 13-17min.

[0017] Further, the ultrasonic treatment is ultrasonic treatment at a temperature of 40-50℃ and an ultrasonic frequency of 100-180W for 10-12min, and then ultrasonic treatment at a temperature of 70-80℃ and an ultrasonic frequency of 200-300W for 3-5min.

[0018] In the present application, by controlling the ultrasonic temperature, ultrasonic power and ultrasonic time, the extraction solvent can better penetrate into the cell structure of tremella fruiting body, fully destroy the cell structure of tremella fruiting body, and further improve the extraction efficiency of the active ingredients in tremella fruiting body; and the denaturation of proteins in tremella fruiting body can be avoided, so that the cell structure of tremella fruiting body is not blocked, thereby improving the dissolution efficiency of the active ingredients in tremella fruiting body.

[0019] Further, the amount of propylene oxide is 30%-50% based on the mass of the tremella powder.

[0020] Further, the amount of the inorganic base is 10%-25% based on the mass of the tremella powder.

[0021] Further, in step (3), the inorganic base is any one of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and potassium bicarbonate.

[0022] Further, the acid agent is any one of hydrochloric acid, citric acid, glacial acetic acid, tannic acid, and trichloroacetic acid.

[0023] Further, in step (3), the stirring speed is 200-400 r / min, the temperature is 45-55 DEG C, and the time is 0.25-0.5 h; when the propylene oxide is added dropwise, the stirring speed is 200-300 r / min, the temperature is 50-70 DEG C, and the time is 1-2 h; and when the reaction is kept, the stirring speed is 50-150 r / min, the temperature is 50-70 DEG C, and the time is 2-4 h.

[0024] In the present application, the pretreated tremella powder is treated with propylene oxide, so that the viscosity and transparency of the finished product can be further improved.

[0025] Further, the polyethylene glycol is polyethylene glycol 400.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] In the present application, the tremella fruiting bodies are first treated with a eutectic solvent under ultrasonic treatment, and then propylene oxide is added dropwise under alkaline conditions for stirring reaction, so that the prepared tremella gum has high viscosity and good light transmission under normal temperature conditions and at a low concentration, and can be used as a thickening agent in daily chemical products. More importantly, the prepared tremella gum also has good antioxidant and whitening effects, which widens the application range of the tremella gum.

[0028] In the present application, betaine, polyethylene glycol, and lactic acid are added to the extraction solvent, and by controlling the water content, the solid-liquid ratio, the ultrasonic process, and other parameters in the extraction solvent, the cell structure of the tremella fruiting bodies can be fully destroyed, so that the effective components are fully released from the cell structure. More importantly, the betaine, polyethylene glycol, lactic acid, and tremella gum in the present application interact with each other, so that the moisture retention performance of the finished product can be further improved. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Ion chromatogram of the tremella gum in Example 3 of the present application

[0030] Figure 2 Gel permeation chromatogram of tremella gum in embodiment 3 of the present application

[0031] Figure 3 Schematic diagram of depilation part of experimental object in skin phototoxicity test of the present application

[0032] Figure 4 Results of multiple skin irritation test of tremella gum in embodiment 3 of the present application

[0033] Figure 5 Results of acute eye irritation test of tremella gum in embodiment 3 of the present application

[0034] Figure 6 Results of skin allergy test of tremella gum in embodiment 3 of the present application

[0035] Figure 7 Results of skin phototoxicity test of tremella gum in embodiment 3 of the present application DETAILED DESCRIPTION

[0036] The present application provides a kind of tremella gum and its preparation method and application, to make the purpose, technical scheme and effect of the present application more clear, definite, the following further detailed description of the present application.It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0037] The test method used in the examples is a conventional method unless otherwise specified;The materials, reagents, etc., can be obtained from commercial channels unless otherwise specified.

[0038] Example 1

[0039] A preparation method of a kind of tremella gum, comprising the following steps:

[0040] (1) the dried tremella fruiting body is crushed, and is passed through 100 mesh screen to obtain tremella powder;

[0041] (2) the tremella powder and extraction solvent are mixed according to the ratio of 1g:5mL, first under the condition of temperature 40 DEG C, ultrasonic frequency 100W ultrasonic 12min, then under the condition of temperature 70 DEG C, ultrasonic frequency 200W ultrasonic 5min, to obtain a mixture;

[0042] (3) sodium hydroxide is added to the mixture and stirred, and propylene oxide is slowly added while stirring, and the reaction is kept warm, hydrochloric acid is added to adjust the pH to 5, dried, and the finished product is prepared;

[0043] In step (2), the preparation of the extraction solvent is as follows: betaine, polyethylene glycol 400 and lactic acid are mixed in a molar ratio of 1:2:0.4, deionized water is added and mixed to prepare an extraction solvent with a water content of 15wt%.

[0044] In step (3), the amount of sodium hydroxide is 10% and the amount of propylene oxide is 30% according to the mass of the tremella powder; the stirring speed is 200 r / min, the temperature is 45℃, and the time is 0.5 h; the stirring speed is 200 r / min, the temperature is 50℃, and the time is 2 h when the propylene oxide is added dropwise; the stirring speed is 50 r / min, the temperature is 50℃, and the time is 4 h for the heat preservation reaction.

[0045] Example 2

[0046] A method for preparing a tremella gum includes the following steps:

[0047] (1) crushing the dried tremella fruiting body, passing through a 100-mesh sieve, and obtaining tremella powder;

[0048] (2) mixing the tremella powder and an extraction solvent according to a solid-liquid ratio of 1 g:10 mL, first ultrasonicating at a temperature of 50℃ and an ultrasonic frequency of 180 W for 10 min, then ultrasonicating at a temperature of 80℃ and an ultrasonic frequency of 300 W for 3 min, and obtaining a mixture;

[0049] (3) adding sodium hydroxide to the mixture and stirring, slowly adding propylene oxide while stirring, heat preservation, adding hydrochloric acid to adjust the pH to 8, drying, and obtaining a finished product;

[0050] In step (2), the extraction solvent is prepared by mixing betaine, polyethylene glycol 400, and lactic acid according to a molar ratio of 1:4:0.8, adding deionized water, and mixing uniformly to obtain an extraction solvent with a water content of 20 wt%;

[0051] In step (3), the amount of sodium hydroxide is 25% and the amount of propylene oxide is 50% according to the mass of the tremella powder; the stirring speed is 400 r / min, the temperature is 55℃, and the time is 0.25 h; the stirring speed is 300 r / min, the temperature is 70℃, and the time is 1 h when the propylene oxide is added dropwise; the stirring speed is 150 r / min, the temperature is 70℃, and the time is 2 h for the heat preservation reaction.

[0052] Example 3

[0053] A method for preparing a tremella gum includes the following steps:

[0054] (1) crushing the dried tremella fruiting body, passing through a 100-mesh sieve, and obtaining tremella powder;

[0055] (2) mixing the tremella powder and the extraction solvent according to a solid-liquid ratio of 1 g:8 mL, first under the condition of a temperature of 45℃ and an ultrasonic frequency of 150 W for 11 min, and then under the condition of a temperature of 75℃ and an ultrasonic frequency of 250 W for 4 min to obtain a mixture;

[0056] (3) adding sodium hydroxide into the mixture and stirring and mixing, slowly adding propylene oxide while stirring, incubating for reaction, adding hydrochloric acid to adjust the pH to 7, drying to obtain a finished product;

[0057] In step (2), the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid according to a molar ratio of 1:3:0.5, adding deionized water and mixing to obtain an extraction solvent with a water content of 18wt%;

[0058] In step (3), according to the mass of the tremella powder, the amount of sodium hydroxide is 15%, and the amount of propylene oxide is 40%; the stirring speed for mixing is 300 r / min, the temperature is 50℃, and the time is 0.4 h; the stirring speed for adding propylene oxide is 250 r / min, the temperature is 60℃, and the time is 1.5 h; the stirring speed for incubation is 100 r / min, the temperature is 60℃, and the time is 3 h.

[0059] Comparative Example 1

[0060] Compared with Example 3, the only difference is that the amount of raw materials in the extraction solvent is different. In Comparative Example 1, the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid according to a molar ratio of 1:0.5:3, adding deionized water and mixing to obtain an extraction solvent with a water content of 18wt%, and the remaining steps and parameters are the same as those in Example 3.

[0061] Comparative Example 2

[0062] Compared with Example 3, the only difference is that the amount of raw materials in the extraction solvent is different. In Comparative Example 2, the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid according to a molar ratio of 2:3:0.5, adding deionized water and mixing to obtain an extraction solvent with a water content of 18wt%, and the remaining steps and parameters are the same as those in Example 3.

[0063] Comparative Example 3

[0064] Compared with Example 3, the only difference is that the type of raw material in the extraction solvent is different, i.e. lactic acid is replaced by citric acid, and the remaining steps and parameters are the same as those in Example 3.

[0065] Comparative Example 4

[0066] The difference between the example 3 and the example 4 is that the raw material of the extraction solvent is different, i.e. polyethylene glycol 600 is used instead of polyethylene glycol 400, and the other steps and parameters are the same as the example 3.

[0067] Comparative example 5

[0068] The difference between the example 3 and the example 5 is that the ratio of the tremella powder and the extraction solvent is different, i.e. the tremella powder and the extraction solvent are mixed according to the ratio of 1g:3mL, and the other steps and parameters are the same as the example 3.

[0069] Comparative example 6

[0070] The difference between the example 3 and the example 6 is that the ratio of the tremella powder and the extraction solvent is different, i.e. the tremella powder and the extraction solvent are mixed according to the ratio of 1g:12mL, and the other steps and parameters are the same as the example 3.

[0071] Comparative example 7

[0072] The difference between the example 3 and the example 7 is that the ultrasonic process parameters are different, i.e. the tremella powder and the extraction solvent are mixed according to the ratio of 1g:8mL, and the mixture is ultrasonically treated at the temperature of 45℃ and the ultrasonic frequency of 150W for 15min, and the other steps and parameters are the same as the example 3.

[0073] Comparative example 8

[0074] The difference between the example 3 and the example 8 is that the ultrasonic process parameters are different, i.e. the tremella powder and the extraction solvent are mixed according to the ratio of 1g:8mL, and the mixture is ultrasonically treated at the temperature of 75℃ and the ultrasonic frequency of 250W for 15min, and the other steps and parameters are the same as the example 3.

[0075] Comparative example 9

[0076] The difference between the example 3 and the example 9 is that the ultrasonic process parameters are different, i.e. the mixture is ultrasonically treated at the temperature of 45℃ and the ultrasonic frequency of 150W for 4min, and then ultrasonically treated at the temperature of 75℃ and the ultrasonic frequency of 250W for 11min, and the other steps and parameters are the same as the example 3.

[0077] Test example 1

[0078] The viscosity, the light transmittance, the moisturizing property and the antioxidant property of the tremella gum prepared in the examples 1-3 and the comparative examples 1-9 are detected.

[0079] The testing method of the viscosity and the light transmittance is that the prepared product is added with deionized water to prepare a 1% mass concentration of the to-be-tested solution under the condition of 25℃, the viscosity of the to-be-tested solution is detected by using a NDJ-1 rotary viscometer, and the light transmittance is detected at 400nm by using an ultraviolet spectrophotometer.

[0080] Moisture retention performance is reflected by hygroscopicity and moisture retention rate data, and the test method is as follows:

[0081] A saturated Na2CO3 solution was prepared and placed in a desiccator to form a test environment with a relative humidity of 43%;

[0082] Hygroscopicity determination: 0.3g of tremella gum products prepared in Examples 1-3 and Comparative Examples 1-9 was accurately weighed into a weighing bottle that had been dried to a constant weight, and then placed in a desiccator with a humidity of 43%. The weight of the sample was measured after 5h, and the in vitro hygroscopicity was calculated according to the following formula:

[0083] Hygroscopicity (%) = (M2-M1) / M1x100%

[0084] In the formula, M1 is the mass before being placed in the desiccator, and M2 is the mass when being taken out of the desiccator.

[0085] Moisture retention rate determination: Deionized water was used to prepare 1wt% sample solutions of the tremella gum products prepared in Examples 1-3 and Comparative Examples 1-9, which were respectively placed in weighing bottles that had been dried to a constant weight, and then placed in a desiccator with a humidity of 43%. The weight of the sample was measured after 12h, and the in vitro moisture retention rate was calculated according to the following formula:

[0086] Moisture retention rate (%) = M2 / M1x100%

[0087] In the formula, M1 is the mass before being placed in the desiccator, and M2 is the mass when being taken out of the desiccator.

[0088] Antioxidant performance is reflected by hydroxyl radical scavenging rate data, and the test method is as follows:

[0089] Deionized water was used to prepare 1wt% sample solutions of the tremella gum products prepared in Examples 1-3 and Comparative Examples 1-9;

[0090] Anhydrous ethanol was used to prepare a 2mmol / L salicylic acid-ethanol solution;

[0091] FeSO4·6H2O was prepared into a 6mmol / L ferrous sulfate solution using deionized water;

[0092] H2O2 was diluted into a 6mmol / L H2O2 solution using deionized water;

[0093] Sample group: 1mL of the sample solution, 1mL of the ferrous sulfate solution, 1mL of the salicylic acid-ethanol solution, and 1mL of the H2O2 solution were mixed uniformly, and then placed in a water bath at 37℃ in the dark for 30min. The absorbance at 510nm was measured, three parallel tests were set, the average value was calculated, and recorded as A1;

[0094] Control group: 1 mL of the sample solution to be tested, 1 mL of ferrous sulfate solution, 1 mL of salicylic acid-ethanol solution, 1 mL of H2O are mixed uniformly, and the absorbance at 510 nm is measured under the condition of 37 DEG C water bath for 30 min in the dark, three parallel tests are set, the average value is calculated, and is recorded as A2;

[0095] Blank group: 1 mL of deionized water, 1 mL of ferrous sulfate solution, 1 mL of salicylic acid-ethanol solution, 1 mL of H2O2 solution are mixed uniformly, and the absorbance at 510 nm is measured under the condition of 37 DEG C water bath for 30 min in the dark, three parallel tests are set, the average value is calculated, and is recorded as A0;

[0096] The clearance rate calculation formula is as follows:

[0097] The clearance rate (%) = [1-(A1-A2) / A0] * 100%

[0098] The viscosity, light transmittance, moisturizing property and antioxidant property test results are shown in Table 1.

[0099] Table 1

[0100] Group Viscosity / mPa.s Transmittance / % Moisture absorption / % Moisture retention / % Removal rate (%) Example 1 11394 85.6 15.2 94.3 92.4 Example 2 11432 84.2 14.8 95.1 93.6 Example 3 11527 88.5 16.3 95.7 95.1 Comparative Example 1 10271 54.3 7.5 80.2 79.5 Comparative Example 2 10539 43.8 7.1 77.3 81.3 Comparative Example 3 11247 82.2 13.6 89.2 90.2 Comparative Example 4 11308 82.8 12.8 92.4 91.3 Comparative Example 5 11283 79.6 12.5 90.1 90.8 Comparative Example 6 11214 76.1 11.1 87.6 88.4 Comparative Example 7 11225 70.5 11.8 86.2 86.7 Comparative Example 8 10952 63.7 8.6 82.9 84.6 Comparative Example 9 10914 67.4 10.2 84.4 85.1

[0101] The experimental results show that the tremella gum prepared in the application has high viscosity, good light transmittance and can be used as a thickening agent in daily chemical products; and has good moisturizing property, can supplement water for the skin, help the skin to quickly absorb, maintain a certain water content in the cuticle layer, delay water loss, increase the water permeability between the epidermis and the dermis to restore the luster and elasticity of the skin; and has good antioxidant property.

[0102] Compared with Example 3, adjusting the raw material ratio of the extraction solvent in Comparative Example 1-2 and adjusting the type of raw material in Comparative Example 3-4 leads to the performance of the finished product being reduced. This may be because the hydrogen bond network of the eutectic solvent formed by betaine, polyethylene glycol and lactic acid can effectively destroy the hydrogen bond or van der Waals force of the cell structure of the tremella fruiting body, so that the active ingredients in the tremella fruiting body are released; and the polyethylene glycol and lactic acid selected as the hydrogen bond donor in the application can also enhance the polarity of the eutectic solvent and adjust the pH value of the eutectic solvent, which helps the extraction solvent to better penetrate into the cell structure of the tremella fruiting body, so that the active ingredients in the tremella fruiting body are better dissolved.

[0103] Compared with Example 3, adjusting the ratio of tremella powder and extraction solvent in Comparative Example 5-6 leads to the performance of the finished product prepared to be reduced. This may be because the reasonable ratio of tremella powder and extraction solvent not only ensures that the active ingredients in the tremella fruiting body are fully contacted and dissolved into the extraction solvent, but also avoids the inconsistent dissolution of intracellular polysaccharide, extracellular polysaccharide and cell wall polysaccharide, which is converted into reducing sugar in the extraction solvent, resulting in the viscosity of the finished product being reduced.

[0104] Compared with Example 3, adjusting the process parameters of ultrasonic treatment in Comparative Examples 7-9 leads to the performance of the finished product prepared to be reduced. This may be because the reasonable ultrasonic treatment process parameters not only enable the extraction solvent to better penetrate into the cell structure of the tremella fruiting body, fully destroy the cell structure of the tremella fruiting body, and thus improve the extraction efficiency of the active ingredients in the tremella fruiting body; but also avoid the denaturation of proteins in the tremella fruiting body, which changes the cell structure of the tremella fruiting body, thereby reducing the dissolution efficiency of the active ingredients in the tremella fruiting body.

[0105] Test Example 2

[0106] The tremella gum finished products prepared in Examples 1-3 are diluted with deionized water to different concentrations, and the transdermal absorption of the finished products is tested according to GB / T 27818-2011 "Chemical skin absorption in vitro test method".

[0107] The tyrosinase inhibition test is carried out according to T / SHRH 015-2018 Cosmetics-Tyrosinase Activity Inhibition Test Method, 3 parallel experiments are carried out for each finished product, the average value ± standard deviation is calculated, and the test results are shown in Table 2.

[0108] Table 2

[0109]

[0110] In the skin melanin biosynthesis, tyrosinase is a key enzyme that acts on dopa to form dopaquinone, which spontaneously undergoes a series of reactions to finally form melanin. The whitening effect of the finished product is evaluated by detecting the activity of tyrosinase.

[0111] The experimental results show that the tremella gum prepared by the present application has obvious inhibitory effect on the activity of tyrosinase.

[0112] Test Example 3

[0113] The transdermal performance of the tremella gum finished products prepared in Examples 1-3 is detected according to GB / T 27818-2011 "Chemical skin absorption in vitro test method" and using PYJ-12B drug transdermal diffusion tester, 3 parallel experiments are carried out for each finished product, the average value is calculated, and the test results are shown in Table 3.

[0114] Test process:

[0115] 1. Preparation of test sample

[0116] Deionized water is used as a solvent to adjust the pH value to 5-6;

[0117] The finished products of examples 1-3 are respectively prepared into test samples with a polysaccharide content of 6.5 mg / g and a concentration of 1 wt% by using a solvent;

[0118] 2, Device preparation

[0119] A matched magnetic stirrer is placed in an 8 mL receiving pool, and a pigskin sample is fixed on a Franz transdermal cell with the dermis layer facing the receiving pool containing PBS, the stratum corneum is exposed in the drug delivery chamber, the pigskin is pulled tight with a forceps, and is fixed with an iron clamp. Air is exhausted to make the bottom surface of the pigskin in close contact with the receiving liquid;

[0120] 3, Skin sampling

[0121] According to the sampling amount of 0.125 g / cm 2 , 0.275 g of the test sample is accurately weighed on the fixed pigskin in a constant temperature water bath at 32℃±1℃, a stirrer is placed in the receiving pool, and is continuously stirred at 300 r / min, and the transdermal test is carried out for 24 h under light-proof conditions.

[0122] 4, Sampling and sample preparation

[0123] Subcutaneous receiving liquid sampling: 1 mL of the subcutaneous receiving liquid at the corresponding time point (1 h, 1.5 h, 2 h, 4 h, 8 h, 24 h) is taken for detection, and 1 mL of PBS with a pH value of 7.2 is added to the receiving pool of each detection group to continue the transdermal test at the subsequent time point.

[0124] Table 3

[0125] Name Example 1 Example 2 Example 3 24h polysaccharide accumulation (ug / cm 2 )]]> 15.03 14.85 15.27

[0126] The experimental results show that the tremella gum prepared by the present application has good transdermal effect, can penetrate the skin barrier, reach the bottom layer of the skin, and play the effects of antioxidant and whitening.

[0127] Test example 4

[0128] 1, The tremella gum of example 3 is prepared into a sample solution with a concentration of 10 ppm by using deionized water, is uniformly vortexed and oscillated, is filtered by using a 0.22 μm water filter membrane, and is prepared for use, mineral matter is determined by using an ICS-6000 ion chromatograph, and the results are shown in Figure 1 .

[0129] 2, The tremella gum sample of example 3 is accurately weighed, is prepared into a sample solution with a concentration of 5 mg / mL by using deionized water, is uniformly vortexed and oscillated, is filtered by using a 0.22 μm water filter membrane, is transferred to a 1.8 mL sample injection vial, and is prepared for use, and the molecular weight is determined by using a gel permeation chromatograph, and the results are shown inFigure 2 .

[0130] 3. Weigh 10 mg of the standard sample and the finished product of Example 3 precisely with a tin capsule or an aluminum capsule. After sealing them tightly, put each sample into the auto-sampler of a Flashsmart organic elemental analyzer to start the detection, and determine the nitrogen element content. The nitrogen content in the tremella glue of Example 3 of the present invention is 0.7 wt%.

[0131] See Figure 1 , indicating that the preparation method of the tremella glue of the present invention retains the mineral active ingredients in tremella, enabling it to achieve the purpose of skin repair and anti-aging by enhancing the skin barrier function and promoting epidermal cell differentiation.

[0132] See Figure 2 , the weight-average molecular weight of the tremella glue prepared by the present invention is about 1.3 million. The tremella glue not only contains polysaccharide structures with large molecular weights, but also polysaccharide structure homologues, and there are also many substances with different molecular weights. Therefore, the molecular weight distribution of the tremella glue prepared by the present invention is the broadest, which may be because the preparation method of the present invention fully retains all the nutritional components of the tremella glue.

[0133] Test Example 5

[0134] The tremella glue prepared in Example 3 was subjected to multiple skin irritation tests, acute eye irritation tests, skin allergy tests, and skin phototoxicity tests in accordance with the "Technical Specifications for Cosmetic Safety" (2015 Edition), Chapter 6 - Toxicological Test Methods. The test results are shown in Figure 4-7 .

[0135] Multiple skin irritation tests:

[0136] 1. Test animals: 4 ordinary-grade New Zealand rabbits (female, 1912.35 - 2010.54 g), provided by the Guangdong Provincial Medical Experimental Animal Center (Sanshui Base), production license number: SCXK (Guangdong) 2023 - 0035; quality certificate number: 44411600017507. Adapt in the experimental animal house for at least 7 days before the test.

[0137] 2. Feeding environment: Ordinary environment, temperature 16°C - 26°C, relative humidity 30% - 70%, use license number: SYXK (Guangdong) 2022 - 0194.

[0138] 3. Feed source: Guangdong Provincial Medical Experimental Animal Center, production license number: Guangdong Feed Certificate (2024) 05073, production date: 2025 - 06 - 04; feed certificate number: NO.2506060043 - 1.

[0139] 4. Test method: 24 h before the test, the test animals were shaved on both sides of the dorsal spine, without damaging the epidermis, with an area of 3 cm x 3 cm on the left and right. 0.5 mL of the test substance (Example 3 finished product diluted with deionized water to a concentration of 1 mg / mL) was applied to the left side of the shaved skin with an area of 2.5 cm x 2.5 cm, and the other side of the skin served as a control. Apply once a day for 14 consecutive days. From the second day, shave before each application, remove residual test substance with warm water, and observe the local reaction of the skin and score 1 h later.

[0140] Acute eye irritation test:

[0141] 1. Test animals: 3 ordinary New Zealand rabbits (female, 1728.49-1750.95 g) were provided by Guangdong Medical Laboratory Animal Center (Sanshui Base), production license number: SCXK (Yue) 2023-0035; quality certificate number: 44411600017507. The test animals were acclimated in the experimental animal room for at least 7 days before the test.

[0142] 2. Rearing environment: ordinary environment, temperature 16℃-26℃, relative humidity 30%-70%, use license number: SYXK (Yue) 2022-0194.

[0143] 3. Source of feed: Guangdong Medical Laboratory Animal Center, production license number: Yue Shijian (2024) 05073, production date: 2025-05-06; feed certificate number: NO.2506060043-1.

[0144] 4. Test method: 0.1 mL of the test substance (Example 3 finished product diluted with deionized water to a concentration of 0.1 mg / mL) was dropped into the conjunctival sac of one side of the rabbit, and was passively closed for 1 s without washing. The other side was not treated as a control. The eyes of the animals were examined at 1 h, 24 h, 48 h, 72 h, and 4 d and 7 d after the test substance was dropped. If no irritation reaction occurs within 72 h, the test can be terminated. If corneal involvement or other eye irritation is found, and the animal does not recover within 7 days, the observation time is extended to determine the reversibility or irreversibility of the damage, which generally does not exceed 21 days, and observation reports at 7 d, 14 d and 21 d are provided.

[0145] Skin allergy test:

[0146] 1. Test animals: 30 Hartley guinea pigs (male) were provided by Guangzhou Tianzhu Biological Technology Co., Ltd., production license number: SCXK (Yue) 2024-0071; quality certificate number: 44838300000858. The test animals were acclimated in the experimental animal room for at least 7 days before the test.

[0147] 2. Feeding environment: ordinary environment, temperature 18-29℃; relative humidity 30%-70%, use license number: SYXK(Yue)2022-0194.

[0148] 3. Feed source: Guangdong Medical Experimental Animal Center, production license number: Guangdong Feed License (2024) 05073, production date: 2025-05-16; feed certificate number: NO.2506060043-2.

[0149] 4. Positive: 2,4-dinitrochlorobenzene (brand: Xia reagent; batch number: 20230614); after pre-dissolution with acetone, it is prepared with olive oil; the induction concentration is 0.5%, and the challenge concentration is 0.2%.

[0150] 5. Test method:

[0151] 5.1 24h before the test, the guinea pigs were shaved on the left side of the back, with a range of about 4cm 2 -6cm 2 ;

[0152] 5.2 Induction phase: 0.2mL of the test substance was applied to the left side of the back of the test guinea pigs in the test area of the 2cm×2cm depilation area, then covered with two layers of gauze and one layer of plastic film, and then fixed with non-irritating adhesive tape. After 6h, the test substance was removed with warm water. On the 7th day and the 14th day, the same method was repeated once. The negative control group was not induced.

[0153] 56.3 Challenge phase: 14 days after the last induction, 0.2mL of the test substance (Example 3 finished product diluted with deionized water to a concentration of 1mg / mL) was applied to the right side of the back of the test guinea pigs in the test area of the 2cm×2cm depilation area (shaved 24h before contact), then covered with two layers of gauze and one layer of plastic film, and then fixed with non-irritating adhesive tape. After 6h, the test substance was removed with warm water.

[0154] 5.4 Animal observation: 24h and 48h after the end of the challenge contact, the skin reaction was observed and scored.

[0155] Skin phototoxicity test:

[0156] 1. Test animals: 6 ordinary Hartley guinea pigs (half male and half female). Provided by Guangdong Medical Experimental Animal Center, production license number: SCXK(Yue)2023-0035; quality certificate number: NO.SCXK(Yue)2023-00352025003963. The animals were acclimated in the experimental animal room for at least 7 days before the test.

[0157] 2. Feeding environment: ordinary environment, temperature 18-29℃, relative humidity 30%-70%, use license number: SYXK(Yue)2022-0194.

[0158] 3. Feed source: Guangdong Medical Experimental Animal Center, production license number: Yue Shijian (2024) 05073, production date: 2025-07-29; feed certificate number: NO.2508080046.

[0159] 4. Instrument and equipment: skin phototoxicity test instrument (manufacturer: Tianjin Development Zone He Pu Trade Co., Ltd.; model: HOPE-MED8130B).

[0160] 5. Positive: 8-methoxy psoralen (brand: Aladdin; batch number: 52317054; concentration: 0.05%), prepared with 95% ethanol.

[0161] 6. Test method:

[0162] 6.1 Test animal preparation: 24h before the test, the back of the test animal was shaved on both sides of the spine without damaging the epidermis, and the test site skin was intact. Four depilation areas were prepared, each with an area of about 2cmx2cm, as shown in Figure 3 .

[0163] 6.2 Dyeing operation: fix the animal, take 0.2mL of the test substance and apply it to the animal depilation areas 1 and 2, 30min later, cover the left side (depilation areas 1 and 3) with aluminum foil, fix it with tape, and irradiate the right side with UVA, the actual irradiation dose of the sample group is 10.0J / cm 2 , the UVB irradiation dose does not exceed 0.1J / cm 2 .

[0164] 6.3 After the irradiation, observe the skin reaction and score at 1h, 24h, 48h and 72h.

[0165] See Figure 4- Figure 7 , the test results show that the finished product prepared by the present application meets the relevant provisions of "Cosmetic Safety Technology Specification" (2015 edition), and will not cause harm to human health under normal use conditions.

[0166] The above description is a detailed description of the preferred and feasible embodiments of the present application, but the embodiments are not intended to limit the scope of the patent application of the present application, and any equivalent changes or modifications made under the technical spirit of the present application should be included in the patent scope covered by the present application.

Claims

1. A method for preparing tremella gelatin, characterized in that, The preparation method of the tremella gel includes the following steps: (1) The dried fruiting body of Tremella fuciformis is crushed and sieved to obtain Tremella fuciformis powder; (2) The mixture of tremella powder and extraction solvent is subjected to ultrasonic treatment to obtain a mixture; (3) Add an inorganic base to the mixture and stir to mix. While stirring, slowly add propylene oxide, keep the reaction at a constant temperature, add an acid to adjust the pH to 5-8, and dry to obtain the tremella gel. The extraction solvent comprises betaine, polyethylene glycol, and lactic acid in a molar ratio of 1:(2-4):(0.4-0.8).

2. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (2), the water content of the extraction solvent is 15wt%-20wt%.

3. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (2), the ratio of the tremella powder to the extraction solvent is 1g:5-10mL.

4. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (2), the ultrasonic treatment is performed for 13-17 minutes at a temperature of 40-80℃ and an ultrasonic frequency of 100-300W.

5. The method for preparing Tremella fuciformis gel as described in claim 4, characterized in that, The ultrasonic treatment involves first ultrasonicating for 10-12 minutes at a temperature of 40-50℃ and an ultrasonic frequency of 100-180W, and then ultrasonicating for 3-5 minutes at a temperature of 70-80℃ and an ultrasonic frequency of 200-300W.

6. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (3), the amount of propylene oxide used is 30%-50% based on the mass of the tremella powder.

7. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (3), the amount of inorganic alkali used is 10% to 25% based on the mass of the tremella powder.

8. The method for preparing Tremella fuciformis gel as described in claim 7, characterized in that, The inorganic base is any one of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and potassium bicarbonate.

9. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, In step (3), the stirring speed is 200-400 r / min, the temperature is 45-55℃, and the time is 0.25-0.5 h. When adding propylene oxide, the stirring speed is 200-300 r / min, the temperature is 50-70℃, and the time is 1-2 h. The stirring speed for the heat preservation reaction is 50-150 r / min, the temperature is 50-70℃, and the time is 2-4 h.

10. The method for preparing Tremella fuciformis gel as described in claim 1, characterized in that, The polyethylene glycol is polyethylene glycol 400.

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