An ultra-low molecular weight tremella polysaccharide extract with moisturizing, soothing and anti-aging effects, and a preparation method and application thereof

Abstract

The application belongs to the technical field of cosmetic raw materials, and specifically discloses an ultra-low molecular weight tremella polysaccharide extract with moisturizing, soothing and anti-aging effects, and a preparation method and application thereof. The application adopts a fermentation extraction mode, uses Candida utilis as a fermentation strain, and adopts a fermentation process to extract tremella to obtain an ultra-low molecular weight tremella polysaccharide extract with a molecular weight of less than 3000 Da. The tremella extract contains a large amount of active substances, and the content of tremella polysaccharide is more than 17.3 mg / ml. In addition, the low molecular weight tremella polysaccharide extract obtained by fermentation extraction contains a large amount of active substances, and the final product can promote the expression of water channel protein AQP3 gene, and has a long-acting moisturizing effect. In addition, the low molecular weight tremella polysaccharide extract also has good soothing and anti-aging effects, and is suitable for application in cosmetics.

Description

Technical Field

[0001] This application relates to the field of cosmetic raw material technology, and in particular to an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing and anti-aging effects, its preparation method and application. Background Technology

[0002] Tremella fuciformis Berk, also known as white fungus, is a delicious edible fungus and a globally recognized health food. In my country, Tremella is mainly produced in Sichuan, Guizhou, Hubei, Shaanxi, Fujian, and Zhejiang provinces. my country has relatively abundant Tremella resources, and research on it is increasingly deepening not only in culinary applications but also in the pharmaceutical and cosmetic fields. The fruiting body of Tremella contains various nutrients such as protein, carbohydrates, vitamins, and amino acids, and can be used for medicinal and tonic purposes. One of the main active ingredients of the fruiting body is Tremella polysaccharide. Tremella polysaccharide is mainly a heteropolysaccharide with α-(1-3)-D-mannose as its main chain, and it has a wide range of biological activities, including immunomodulation, anti-tumor, antioxidant, hypoglycemic and lipid-lowering, anticoagulant and thrombotic, anti-ulcer, protein synthesis promotion, antiviral, nerve cell growth promotion, and memory improvement.

[0003] Conventional methods for extracting polysaccharides from Tremella fuciformis include hot water extraction and acid extraction. However, the polysaccharides extracted in these methods are mostly macromolecular structures with molecular weights exceeding 1 million. Furthermore, the extracts of Tremella fuciformis polysaccharides have high viscosity, making them difficult to filter. Consequently, they suffer from low extraction rates and are not easily absorbed or exert their skin-care activities. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging effects, as well as its preparation method and application. The ultra-low molecular weight Tremella fuciformis polysaccharide extract obtained using the preparation method of this application can enhance the moisturizing and anti-aging effects of cosmetics.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, this application provides a method for preparing an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging effects, comprising the following steps:

[0007] S1. Inoculate *Candida utilis* into *Tremella fuciformis* fermentation medium and ferment to obtain crude fermentation broth;

[0008] S2. The crude fermentation liquid is sterilized and filtered to obtain an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects.

[0009] This application employs a fermentation extraction method, using *Candida utilis* as the fermentation strain, and utilizes a fermentation process to extract *Tremella fuciformis* polysaccharides, yielding ultra-low molecular weight *Tremella fuciformis* polysaccharides with a molecular weight below 3000 Da. The obtained *Tremella fuciformis* polysaccharide extract contains a large amount of active substances, with the *Tremella fuciformis* polysaccharide content exceeding 17.3 mg / ml. The molecular weight of the *Tremella fuciformis* polysaccharide extract obtained in this application is significantly lower than that obtained by hot water extraction and acid extraction methods. This preparation method solves the problems of high molecular weight, high viscosity, difficulty in filtration, low extraction rate, and poor absorption and skin-care activity of *Tremella fuciformis* polysaccharides obtained by hot water extraction and acid extraction methods.

[0010] In a preferred embodiment of the method for preparing the ultra-low molecular weight Tremella fuciformis polysaccharide extract described in this application, the Tremella fuciformis fermentation culture medium comprises the following components in weight percentage:

[0011] 0.5–1.8% white fungus, 1–4% carbon source, 0.3–1.2% nitrogen source, 0.35–0.8% inorganic salts, with the remainder being water.

[0012] In some specific embodiments, the tremella is tremella powder.

[0013] As a preferred embodiment of the preparation method of the ultra-low molecular weight Tremella polysaccharide extract described in this application, the Tremella includes at least one of Tongjiang Tremella, Gutian Tremella, Guizhou Tremella, and Yunnan Tremella, preferably Tongjiang Tremella; the carbon source is at least one of fructose syrup, dextrin, soybean starch, potato starch, and molasses; the nitrogen source is at least one of soybean peptone, yeast extract, cottonseed meal, and trypsin; and the inorganic salt is sulfate and / or phosphate.

[0014] In some specific embodiments, the inorganic salt is magnesium sulfate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0015] Preferably, the tremella fermentation culture medium comprises the following components by weight percentage:

[0016] 1.2% Tongjiang Tremella powder, 2% fructose syrup, 0.8% soybean peptone, 0.3% magnesium sulfate, 0.15% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate and the balance water.

[0017] In the technical solution of this application, the origin of the fruiting body of Tremella fuciformis and the fermentation strain have a great influence on the molecular weight and content of Tremella fuciformis polysaccharide extract. The Tremella fuciformis polysaccharide extract obtained by fermentation with Candida utilis has a higher content of Tremella fuciformis polysaccharide and a lower molecular weight of polysaccharide, which is conducive to skin absorption. The above effect cannot be obtained by using other strains for fermentation. The type of fruiting body of Tremella fuciformis also has a great influence on the molecular weight and content of Tremella fuciformis polysaccharide extract. This application prefers Tremella fuciformis from Tongjiang for fermentation.

[0018] The types of carbon and nitrogen sources affect the growth of *Candida utilis*, ultimately significantly impacting the molecular weight and content of the polysaccharides in the product. Using the aforementioned carbon and nitrogen sources, *Candida utilis* grows better in *Tremella fuciformis* culture medium, yielding lower molecular weights and higher polysaccharide content. Experiments have shown that if the *Tremella fuciformis* fermentation medium lacks inorganic salts, or if the inorganic salts are limited to either phosphate or magnesium sulfate, the growth of *Candida utilis* is inhibited, resulting in fewer active substances produced in the final product, thus leading to poorer moisturizing, anti-aging, and soothing effects.

[0019] The tremella fermentation culture medium of this application contains the above-mentioned inorganic salts, and the growth of Candida utilis is not inhibited. The final tremella polysaccharide extract has a small molecular weight and a high content of polysaccharides.

[0020] In a preferred embodiment of the preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract described in this application, the *Candida utilis* is a *Candida utilis* seed culture, and the inoculation amount of the *Candida utilis* seed culture is 2-5%; the cell concentration of the *Candida utilis* seed culture is 10. 7 ~10 9 CFU / mL. Preferably, the cell concentration of the *Candida utilis* seed culture is 10. 8 CFU / mL.

[0021] In some specific embodiments, the Candida utilis strain is Candida utilis GDMCC2.148, which was purchased from the Guangdong Provincial Center for Microbial Culture Collection.

[0022] Using the *Candida utilis* strain with the above-mentioned accession number as the fermentation strain, a combined fermentation process was used to ferment and extract *Tremella fuciformis*, resulting in an ultra-low molecular weight *Tremella fuciformis* polysaccharide fermentation extract with a molecular weight below 3000 Da. This extract contains a large number of active substances, with the content of *Tremella fuciformis* polysaccharide exceeding 17.3 mg / ml, demonstrating a high fermentation extraction efficiency.

[0023] In a preferred embodiment of the preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract described in this application, in step S1, the fermentation temperature is 28-33°C, the fermentation time is 40-60 h, and the fermentation speed is 100-200 rpm.

[0024] In a preferred embodiment of the preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract described in this application, in step S2, the filtration is microfiltration or filtration through a diatomaceous earth filter, the sterilization temperature is 80-100°C, and the sterilization time is 15-30 min.

[0025] During fermentation, the shaking speed of the fermentation flask and the fermentation time have a significant impact on the molecular weight and content of the final product, *Tremella fuciformis* polysaccharide. The shaking speed affects the dissolved oxygen content of the fermentation medium; a higher shaking speed results in a higher dissolved oxygen content, which can promote the production of *Candida utilis* metabolites to some extent. However, if the dissolved oxygen content exceeds a certain level, the growth and metabolism of *Candida utilis* will be inhibited, leading to a higher molecular weight and lower polysaccharide content in the final product. The preferred fermentation speed in this application is 100–200 rpm. The fermentation time also affects the final product. Specifically, if the fermentation time is too short, the fermentation will end once the growth and metabolites of Candida utilis reach a certain amount, resulting in a larger molecular weight and lower content of Tremella fuciformis polysaccharides in the final product. If the fermentation time is too long, there will be too many Candida utilis, which will consume the nutrients in the Tremella fuciformis fermentation broth to maintain their own growth, resulting in a lower content of Tremella fuciformis polysaccharides in the final product. Therefore, the fermentation time of this application is preferably 40-60 hours, with 50 hours being the optimal time. This results in a smaller molecular weight of Tremella fuciformis polysaccharides and an increased content of Tremella fuciformis polysaccharides.

[0026] As a preferred embodiment of the preparation method of the ultra-low molecular weight Tremella polysaccharide extract described in this application, in step S1, the preparation method of the Candida utilis seed liquid is as follows: pick Candida utilis colonies into liquid culture medium, and culture them at 28-33℃ for 36-60h to obtain the Candida utilis seed liquid.

[0027] Secondly, this application provides an ultra-low molecular weight Tremella fuciformis polysaccharide extract obtained by the above preparation method.

[0028] Thirdly, this application provides the application of the above-mentioned ultra-low molecular weight Tremella fuciformis polysaccharide extract in the preparation of cosmetics.

[0029] As a preferred embodiment of the application described in this application, the amount of the ultra-low molecular weight Tremella fuciformis polysaccharide extract added to the cosmetic is 0.1-20% by mass percentage.

[0030] The ultra-low molecular weight Tremella fuciformis polysaccharide extract obtained through fermentation in this application contains a large number of active substances. The final product can promote the expression of the aquaporin AQP3 gene, resulting in a long-lasting moisturizing effect. Furthermore, the ultra-low molecular weight Tremella fuciformis polysaccharide extract also has excellent soothing and anti-aging effects, making it suitable for use in cosmetics.

[0031] The Tremella fuciformis fermentation culture medium using the above-mentioned components and their contents can improve the moisturizing, anti-aging and soothing effects of cosmetics.

[0032] Compared with the prior art, this application has the following beneficial effects:

[0033] This application provides an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging effects, its preparation method, and its applications. This application employs a fermentation extraction method, using *Candida utilis* as the fermentation strain, and uses a fermentation process to extract Tremella fuciformis polysaccharides, obtaining an ultra-low molecular weight Tremella fuciformis polysaccharide extract with a molecular weight below 3000 Da. The extract contains a large number of active substances, with the Tremella fuciformis polysaccharide content exceeding 17.3 mg / ml. Furthermore, due to the high content of active substances, the low molecular weight Tremella fuciformis polysaccharide extract obtained by fermentation extraction of this invention can promote the expression of the aquaporin AQP3 gene, resulting in a long-lasting moisturizing effect. In addition, the low molecular weight Tremella fuciformis polysaccharide extract also has excellent soothing and anti-aging effects, making it suitable for application in cosmetics. Detailed Implementation

[0034] To better illustrate the purpose, technical solution, and advantages of this application, the following will provide further explanation of this application in conjunction with specific embodiments.

[0035] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0036] Unless otherwise specified, all percentages mentioned in this application are percentages by mass.

[0037] In the following examples and comparative examples, the sources of the main raw materials are as follows:

[0038] The Candida utilis used in this application is Candida utilis GDMCC2.148, which was purchased from the Guangdong Provincial Center for Microbial Culture Collection.

[0039] The Saccharomyces cerevisiae used in this application is Saccharomyces cerevisiae BNCC336054, and the Lactobacillus plantarum is Lactobacillus plantarum BNCC336421. Both strains were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0040] The *Candida utilis* seed culture of this application is prepared by the following method (other seed culture preparation methods can also be used): Colonies of the above-mentioned *Candida utilis* are placed in malt extract culture medium and cultured at 28–33°C for 36–60 h to obtain the *Candida utilis* seed culture. The cell concentration of the *Candida utilis* seed culture is 10-1. 7 ~10 9 CFU / mL.

[0041] The *Saccharomyces cerevisiae* seed culture of this application was prepared by the following method (other seed culture preparation methods can also be used): *Saccharomyces cerevisiae* colonies were placed in malt extract culture medium and cultured at 28-33℃ for 36-60 hours to obtain the *Saccharomyces cerevisiae* seed culture. The cell concentration of the *Saccharomyces cerevisiae* seed culture was 10-1. 7 ~10 9 CFU / mL.

[0042] The *Lactobacillus plantarum* seed culture in this application was prepared using the following method (other seed culture preparation methods may also be used): *Lactobacillus plantarum* colonies were placed in MRS medium and cultured at 33–40°C for 36–60 h to obtain the *Lactobacillus plantarum* seed culture. The cell concentration of the *Lactobacillus plantarum* seed culture was 10-1. 7 ~10 9 CFU / mL.

[0043] The preparation method of malt extract culture medium is as follows: add 4 times the amount of water to dry malt powder, incubate at 60℃ for 3 hours for saccharification, and check with iodine solution until complete saccharification is obtained to obtain malt extract sugar. Dilute the malt extract sugar with water to a sugar content of 9 baling degrees, adjust the pH to 6.3, and sterilize at 121℃ for 15 minutes for later use.

[0044] The MRS medium is prepared as follows: 10 g / L protein powder, 5 g / L beef meal, 4 g / L yeast powder, 2 g / L glucose, 1 mL / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and the remaining water are added and mixed evenly. The mixture is then sterilized at 121℃ for 15 min before use.

[0045] The preparation method of the tremella powder used in this application is as follows: place the dried tremella fruiting body in a grinder, grind it, sieve the powder, and take the lower layer of powder for later use.

[0046] Example 1

[0047] This embodiment provides a method for preparing an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging effects, comprising the following steps:

[0048] S1. The concentration of the bacterial cells prepared above is 10. 8 CFU / mL Candida utilis seed culture was inoculated into Tremella fuciformis fermentation medium at a 3% inoculation rate and fermented at 30℃ and 150 rpm for 50 h to prepare crude fermentation broth.

[0049] S2. The obtained crude fermentation liquid is kept at 90℃ for 20 minutes. After cooling, the crude fermentation liquid is filtered through a microfiltration membrane. The resulting filtrate is an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects.

[0050] The preparation method of the tremella fermentation culture medium is as follows:

[0051] Mix 1.2% Tongjiang Tremella powder, 2% fructose syrup, 0.8% soybean peptone, 0.3% magnesium sulfate, 0.15% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate and the remaining water evenly, and sterilize at 121℃ for 15 minutes for later use.

[0052] Examples 2-11

[0053] The preparation methods of ultra-low molecular weight Tremella polysaccharide extracts in Examples 2-11 are basically the same as those in Example 1, except for some differences in the Tremella fermentation culture medium and step S1. The remaining steps and parameters are the same as those in Example 1. The differences between the preparation methods of Examples 2-11 and Example 1 are shown in Table 1 below. The parts in the table that are the same as the parameters in Example 1 are indicated by —.

[0054] Table 1

[0055]

[0056]

[0057] Example 12

[0058] S1. Set the bacterial cell concentration to 10. 7 CFU / mL Candida utilis seed culture was inoculated into Tremella fuciformis fermentation medium at a 5% inoculation rate and fermented at 33℃ and 180 rpm for 55 h to prepare crude fermentation broth.

[0059] S2. Keep the crude fermentation liquid at 100℃ for 15 minutes. After cooling, filter the crude fermentation liquid with a diatomaceous earth filter. The resulting filtrate is an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects.

[0060] The preparation method of the tremella fermentation culture medium is as follows:

[0061] Mix 0.5% Tongjiang white fungus powder, 4% potato starch, 0.3% cottonseed powder, 0.4% magnesium sulfate, 0.1% sodium dihydrogen phosphate, 0.3% disodium hydrogen phosphate and the remaining water evenly, and sterilize at 121℃ for 15 minutes for later use.

[0062] Example 13

[0063] S1. Set the bacterial cell concentration to 10. 9 CFU / mL Candida utilis seed culture was inoculated into Tremella fuciformis fermentation medium at a 2% inoculation rate and fermented at 28℃ and 120 rpm for 45 h to prepare crude fermentation broth.

[0064] S2. Keep the crude fermentation liquid at 80℃ for 30 minutes. After cooling, filter the crude fermentation liquid through a microfiltration membrane. The resulting filtrate is an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects.

[0065] The preparation method of the tremella fermentation culture medium is as follows:

[0066] Mix 1.8% Tongjiang Tremella powder, 1% fructose syrup, 1.2% yeast extract, 0.2% magnesium sulfate, 0.2% sodium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and the remaining water evenly, and sterilize at 121℃ for 15 minutes for later use.

[0067] Comparative Example 1

[0068] The Candida utilis strain used in the fermentation in Example 1 was replaced with Saccharomyces cerevisiae (the concentration of the Saccharomyces cerevisiae seed culture was 10). 8 (CFU / mL), the remaining steps and parameters are the same as in Example 1.

[0069] Comparative Example 2

[0070] The Candida utilis strain used in the fermentation in Example 1 was replaced with Lactobacillus plantarum (the cell concentration of the Lactobacillus plantarum seed culture was 10). 8 (CFU / mL), the remaining steps and parameters are the same as in Example 1.

[0071] Comparative Example 3

[0072] The 2% fructose syrup in Example 1 was replaced with 2% glucose, and the remaining steps and parameters were the same as in Example 1.

[0073] Comparative Example 4

[0074] The 0.8% soybean peptone in Example 1 was replaced with 0.8% deionized water, and the remaining steps and parameters were the same as in Example 1.

[0075] Comparative Example 5

[0076] In Example 1, 0.3% magnesium sulfate, 0.15% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate were replaced with 0.55% deionized water, while the remaining steps and parameters were the same as in Example 1.

[0077] Comparative Example 6

[0078] The 0.3% magnesium sulfate, 0.15% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate in Example 1 were replaced with 0.55% magnesium sulfate, and the remaining steps and parameters were the same as in Example 1.

[0079] Comparative Example 7

[0080] The 0.3% magnesium sulfate, 0.15% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate in Example 1 were replaced with 0.55% potassium dihydrogen phosphate, and the remaining steps and parameters were the same as in Example 1.

[0081] Comparative Example 8

[0082] The fermentation shaking speed of 150 rpm in Example 1 was changed to 250 rpm, and the remaining steps and parameters were the same as in Example 1.

[0083] Comparative Example 9

[0084] The fermentation time of 50h in Example 1 was changed to 30h, and the remaining steps and parameters were the same as in Example 1.

[0085] Experimental Example 1: Determination of Polysaccharide Content and Molecular Weight of Tremella Polysaccharide Extract

[0086] The method for determining the molecular weight of Tremella fuciformis polysaccharide is referenced in the determination method described in the study on quality control of Tremella fuciformis polysaccharide based on high performance gel permeation chromatography, [J] Chinese Traditional and Herbal Drugs, 2011, 42(09): 1732-1735.

[0087] The test method for the content of polysaccharides in Tremella fuciformis is as follows: The content of polysaccharides in Tremella fuciformis is determined by the phenol-sulfuric acid method in the Chinese Pharmacopoeia.

[0088] Test samples: Tremella polysaccharide extracts prepared in Examples 1-13 and Comparative Examples 1-9.

[0089] The test results are shown in Table 2 below:

[0090] Table 2

[0091]

[0092]

[0093] Test results show that, compared with the comparative examples, the Tremella fuciformis polysaccharide extract prepared in this application has a lower molecular weight and a higher content of Tremella fuciformis polysaccharides. Comparing Examples 1 and 5-7, and Comparative Examples 1 and 2, it is evident that the origin of the Tremella fuciformis fruiting body and the fermentation strain have a significant impact on the molecular weight and polysaccharide content of the Tremella fuciformis polysaccharide extract. The Tremella fuciformis polysaccharide extract obtained by fermentation with *Candida utilis* in this application has a higher content of Tremella fuciformis polysaccharides and a lower molecular weight, which is beneficial for skin absorption. Using other strains for fermentation cannot achieve the above effects. The type of Tremella fuciformis fruiting body also has a significant impact on the molecular weight and polysaccharide content of the Tremella fuciformis polysaccharide extract. This application preferably uses *Tremella fuciformis* from Tongjiang for fermentation.

[0094] Comparing Examples 1-4 and Comparative Example 3, it can be seen that the types of carbon and nitrogen sources have a great influence on the molecular weight and content of polysaccharides in the final product. In this application, the carbon source is selected from at least one of fructose syrup, dextrin, soybean starch, potato starch, and molasses, and the nitrogen source is selected from at least one of soybean peptone, yeast extract, cottonseed flour, and trypsin. The resulting Tremella fuciformis polysaccharide extract has a low molecular weight and a high content.

[0095] Comparing Example 1 and Comparative Examples 5-7, it can be seen that if inorganic salts are lacking in the fermentation medium of Tremella fuciformis, or if the inorganic salts are only one of phosphate or magnesium sulfate, the growth of Candida utilis will be inhibited to a certain extent, and the final product will have a larger molecular weight and lower content of Tremella fuciformis polysaccharide. Therefore, in the Tremella fuciformis fermentation medium of this application, the inorganic salts should be selected from sulfate and phosphate.

[0096] Comparative examples 1, 8-11, and 8 and 9 show that the shaking speed of the fermentation flask and the fermentation time have a significant impact on the molecular weight and content of the polysaccharides in the final product. The shaking speed affects the dissolved oxygen content of the Tremella fuciformis fermentation medium. A higher shaking speed results in higher dissolved oxygen content, which can promote the production of Candida utilis metabolites to some extent. However, if the dissolved oxygen content exceeds a certain level, the growth and metabolism of Candida utilis will be inhibited, leading to a higher molecular weight and lower polysaccharide content in the final product. If the fermentation time is too short, the growth and metabolites of Candida utilis will reach a certain level, and fermentation will end, resulting in a higher molecular weight and lower content of polysaccharides in the final product. If the fermentation time is too long, the number of Candida utilis will be excessive. To maintain their growth, Candida utilis will consume nutrients in the Tremella fuciformis fermentation broth, resulting in a lower polysaccharide content in the final product. The preferred fermentation time in this application is 40-60 hours, with 50 hours being optimal.

[0097] Test Example 2: Aquaporin 3 (AQP3) Gene Expression Test

[0098] Test Principle: Aquaporin (AQP), also known as water-porin protein, is a protein located on the cell membrane (intrinsic membrane protein). It forms "pores" in the cell membrane, controlling the entry and exit of water, acting like a "water pump" for the cell. Aquaporin (AQP3) is expressed in the epidermis and basal layer, serving as a natural moisturizing factor. It maintains the hydration levels of the intercellular matrix and intracellular spaces, and may play a key role in skin hydration and moisturization. This study uses in vitro experiments to detect the effect of samples on AQP3 gene expression in keratinocytes, assessing whether the samples have a moisturizing effect.

[0099] Test samples: Tremella polysaccharide extracts prepared in Examples 1-13 and Comparative Examples 1-9 were diluted with cell culture medium to a mass concentration of 2%.

[0100] Test Procedure: HaCaT human keratinocytes were seeded into 6-well plates (200,000 cells / well) and cultured at 37°C for 24 hours. After culture, the original culture medium was discarded. DMEM medium containing 10% serum was added to the negative control group, and the test sample was added to each well of the sample group. Cultures were then incubated for 24 hours. The supernatant was discarded after culture. The expression level of AQP3 mRNA in HaCaT human keratinocytes was detected using qRT-PCR. The relative expression level was calculated based on the mRNA expression level in the negative control group, and the average value was taken. The test results are shown in Table 3 below.

[0101] Table 3

[0102]

[0103]

[0104] Test Example 3: Long-lasting moisturizing and anti-aging efficacy test

[0105] Test samples: emulsions and blank emulsions prepared from the tremella polysaccharide extracts obtained in Examples 1-13 and Comparative Examples 1-9.

[0106] The formula for the emulsion is shown in Table 4 below:

[0107] Table 4

[0108]

[0109] For the blank emulsion, replace 3% Tremella fuciformis polysaccharide extract in the above formula with 3% deionized water, and keep the rest consistent with the above formula table.

[0110] The preparation methods for emulsions and blank emulsions containing Tremella fuciformis polysaccharide extract are as follows: Xanthan gum in component A is dispersed with glycerol and 1,3-butanediol, and then the remaining materials are added sequentially. The mixture is stirred and heated to 80°C, and kept warm for later use. All materials in component B are mixed evenly, and the mixture is heated to 80°C. Component B is then added to component A and homogenized for 5 minutes. The mixture is cooled to 40°C, and all materials in component C are added and mixed evenly to obtain an emulsion or blank emulsion containing Tremella fuciformis polysaccharide extract.

[0111] Test Procedure: 230 healthy volunteers aged 45-60 years with significant facial wrinkles, a facial skin stratum corneum moisture content below 20 C.U. were selected. Ten volunteers used one lotion each, for a total of 23 groups. The facial skin stratum corneum moisture content was measured before and 28 days after sample use. The rate of change in skin stratum corneum moisture content after sample use was calculated, and the magnitude of the change was used to determine the moisturizing effect of the lotion.

[0112] Meanwhile, before and 28 days after using the sample, photos of the volunteers' facial skin were taken. The proportion of wrinkles on the volunteers' facial skin was calculated using software. The rate of change in the proportion of wrinkles on the facial skin after using the sample was calculated, and the magnitude of the rate of change was used to judge the anti-aging effect of the lotion.

[0113] Change rate of skin stratum corneum moisture content / % = (Skin stratum corneum moisture content after 28 days of sample use - Skin stratum corneum moisture content before sample use) / Skin stratum corneum moisture content before sample use × 100%

[0114] Testing instrument: CM825 skin moisture meter, Courage & Khazaka, Germany.

[0115] Change rate of facial skin wrinkles percentage / % = (Percentage of facial skin wrinkles after 28 days of sample use - Percentage of facial skin wrinkles before sample use) / Percentage of facial skin wrinkles before sample use × 100%.

[0116] Testing instruments and software: VISIA-CR facial imaging system, Canfield Scientific, USA; Image-Pro Plus skin analysis software, Media Cybernetics, USA.

[0117] The test results are shown in Table 5 below.

[0118] Table 5

[0119]

[0120]

[0121] Test results show that, from both cellular and human efficacy perspectives, the Tremella fuciformis polysaccharide extract of this application exhibits excellent moisturizing and anti-aging effects. In contrast, the blank emulsion showed poorer moisturizing and anti-aging effects. Comparing the results of Example 1 and Comparative Examples 1 and 2, it is evident that the Tremella fuciformis polysaccharide extract obtained by fermenting Tremella fuciformis with Candida utilis not only activates the expression of the aquaporin 3 gene at the cellular level but also provides long-lasting moisturizing and anti-aging effects when applied to the human body. Fermentation with different strains resulted in poorer moisturizing and anti-aging effects for the Tremella fuciformis polysaccharide extract.

[0122] Comparative Examples 1-4 and Comparative Example 3 show that the types of carbon and nitrogen sources affect the types of active substances in the final Tremella polysaccharide extract, thereby affecting the moisturizing and anti-aging effects of the final product. In this application, the carbon source of the Tremella fermentation culture medium is preferably at least one of fructose syrup, dextrin, soybean starch, potato starch, and molasses, and the nitrogen source is preferably at least one of soybean peptone, yeast extract, cottonseed powder, and tryptone.

[0123] Comparing Example 1 and Comparative Examples 5-7, it can be seen that if the tremella fermentation culture medium lacks inorganic salts, or if the inorganic salts are only one of phosphate or magnesium sulfate, the growth of Candida utilis will be inhibited to a certain extent, resulting in less active substances produced in the final product, which in turn leads to poor moisturizing and anti-aging effects of the product. Therefore, in the tremella fermentation culture medium of this application, the inorganic salts should be selected from sulfate and phosphate. Comparing Examples 1, 8-11, and 8 and 9, it is evident that the shaking speed of the fermentation flask and the fermentation time significantly affect the final content of active substances during fermentation. The shaking speed affects the dissolved oxygen content in the Tremella fuciformis fermentation medium; a higher shaking speed results in higher dissolved oxygen content, which can promote the production of Candida utilis metabolites to some extent. However, if the dissolved oxygen content exceeds a certain level, the growth and metabolism of Candida utilis will be inhibited, leading to poor moisturizing and anti-aging effects in the final product. If the growth and metabolites of Candida utilis reach a certain level, fermentation will end, resulting in a higher sugar molecular weight and lower sugar content in the final product. Conversely, if the fermentation time is too long, the number of Candida utilis will be excessive. To maintain its growth, Candida utilis will consume nutrients in the Tremella fuciformis fermentation broth, resulting in poor moisturizing and anti-aging effects in the final product. The preferred fermentation time in this application is 40-60 hours, with 50 hours being optimal.

[0124] Test Example 4: Soothing Performance Test

[0125] Test sample: Same as test example 3.

[0126] Test Procedure: 230 healthy volunteers with no injury on the inner arm were selected and divided into 23 groups of 10 each. A 1.5×1.5cm skin area on the inner arm of each volunteer was randomly selected for patch application. 0.08mL of a 10% (w / w) mixed solution (mass ratio SDS:capsaicin:lactic acid = 5:3.5:1.5) was titrated into each patch. The patches were removed after 24 hours, and the number of erythema spots in the test area was measured 24 hours after patch removal. The erythema was then measured at (2.0±0.1) mg / cm². 2 Apply the appropriate amount of sample to the test sample, and after 2 hours, detect the number of erythema spots on the test skin. Calculate the rate of change in the number of erythema spots before and after sample application to determine the soothing effect of the test sample.

[0127] Change rate of skin erythema number (%) = (Number of skin erythema after sample application - Number of skin erythema before sample application) / Number of skin erythema before sample application × 100%

[0128] Testing instrument: MX18 skin pigmentation analyzer, Courage & Khazaka, Germany.

[0129] The test results are shown in Table 6 below:

[0130] Table 6

[0131]

[0132]

[0133] Test results show that the Tremella fuciformis polysaccharide extracts of this application all have excellent soothing effects. The blank emulsion, however, has a poor soothing effect. Comparing the results of Example 1, Comparative Examples 1 and 2, it can be seen that the Tremella fuciformis polysaccharide extract obtained by fermenting Tremella fuciformis with Candida utilis not only activates the expression of the aquaporin 3 gene at the cellular level, but also has a soothing effect when applied to the human body. When the fermentation was carried out with different strains (Saccharomyces cerevisiae or Lactobacillus plantarum), the soothing effect of the Tremella fuciformis polysaccharide extract was poor.

[0134] Comparative Examples 1-4 and Comparative Example 3 show that the types of carbon and nitrogen sources affect the types of active substances in the final Tremella polysaccharide extract, thereby affecting the soothing effect of the final product. In this application, the carbon source of the Tremella fermentation culture medium is preferably at least one of fructose syrup, dextrin, soybean starch, potato starch, and molasses, and the nitrogen source is preferably at least one of soybean peptone, yeast extract, cottonseed powder, and tryptone, so as to better improve the soothing effect of the cosmetic.

[0135] Comparing Example 1 and Comparative Examples 5-7, it can be seen that if the tremella fermentation culture medium lacks inorganic salts, or if the inorganic salts are only one of phosphate or magnesium sulfate, the growth of Candida utilis will be inhibited to a certain extent, resulting in less active substances produced in the final product, which in turn leads to a poor soothing effect of the product. Therefore, in the tremella fermentation culture medium of this application, the inorganic salts should be selected from sulfate and phosphate.

[0136] Comparative Examples 1, 8-11, and 8 and 9 show that the shaking speed of the fermentation flask and the fermentation time have a significant impact on the final content of active substances during fermentation. The shaking speed affects the dissolved oxygen content of the Tremella fuciformis fermentation medium during fermentation. The higher the shaking speed, the higher the dissolved oxygen content, which can promote the production of Candida utilis metabolites to a certain extent. However, if the dissolved oxygen content exceeds a certain level, the growth and metabolism of Candida utilis will be inhibited, resulting in a poor soothing effect of the final product. If the growth and metabolites of Candida utilis reach a certain level, fermentation will end, leading to a higher sugar molecular weight and lower sugar content in the final product. If the fermentation time is too long, the number of Candida utilis will be too large. To maintain its own growth, Candida utilis will consume the nutrients in the Tremella fuciformis fermentation broth, resulting in a poor soothing effect of the final product. The preferred fermentation time in this application is 40-60 hours, with 50 hours being the optimal time.

[0137] This application employs a fermentation extraction method, using *Candida utilis* as the fermentation strain, to ferment and extract *Tremella fuciformis*. The resulting fermented extract contains a large amount of active substances, with *Tremella fuciformis* polysaccharide content exceeding 17.3 mg / ml. This yields ultra-low molecular weight *Tremella fuciformis* polysaccharide with a molecular weight below 3000 Da, significantly lower than that obtained by hot water extraction or acid extraction. Furthermore, due to the high content of active substances, the fermented extract of *Tremella fuciformis* from this invention can promote the expression of the aquaporin AQP3 gene, resulting in a long-lasting moisturizing effect. In addition, the *Tremella fuciformis* polysaccharide extract also possesses excellent soothing and anti-aging effects, making it suitable for application in cosmetics.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing an ultra-low molecular weight Tremella fuciformis polysaccharide extract with moisturizing, soothing, and anti-aging effects, characterized in that, Includes the following steps: S1. Inoculate *Candida utilis* into *Tremella fuciformis* fermentation medium and ferment to obtain crude fermentation broth; S2. The crude fermentation liquid is sterilized and filtered to obtain an ultra-low molecular weight Tremella polysaccharide extract with moisturizing, soothing and anti-aging effects. The tremella fermentation culture medium comprises the following components by weight percentage: 0.5-1.8% Tremella fuciformis, 1-4% carbon source, 0.3-1.2% nitrogen source, 0.35-0.8% inorganic salts, with the remainder being water; The tremella includes at least one of Tongjiang tremella, Gutian tremella, Guizhou tremella, and Yunnan tremella; the carbon source is at least one of fructose syrup, dextrin, soybean starch, potato starch, and molasses; the nitrogen source is at least one of soybean peptone, yeast extract, cottonseed meal, and tryptone; and the inorganic salts are sulfates and phosphates. In step S1, the fermentation temperature is 28~33℃, the fermentation time is 40~60h, and the fermentation speed is 100~200rpm. The molecular weight of the ultra-low molecular weight Tremella polysaccharide extract is less than 3000 Da.

2. The preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract as described in claim 1, characterized in that, The *Candida utilis* strain is a *Candida utilis* seed culture, and the inoculation amount of the *Candida utilis* seed culture is 2-5%; the cell concentration of the *Candida utilis* seed culture is 10. 7 ~10 9 CFU / mL.

3. The preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract as described in claim 1, characterized in that, In step S2, the filtration is microfiltration or filtration through a diatomaceous earth filter, the sterilization temperature is 80~100℃, and the sterilization time is 15~30min.

4. The preparation method of the ultra-low molecular weight Tremella fuciformis polysaccharide extract as described in claim 2, characterized in that, In step S1, the method for preparing the Candida utilis seed liquid is as follows: pick up Candida utilis colonies and put them into liquid culture medium, and culture them at 28~33℃ for 36~60h to obtain the Candida utilis seed liquid.

5. The ultra-low molecular weight Tremella fuciformis polysaccharide extract obtained by the preparation method according to any one of claims 1 to 4.

6. The application of the ultra-low molecular weight Tremella fuciformis polysaccharide extract as described in claim 5 in the preparation of cosmetics.

7. The application as described in claim 6, characterized in that, The amount of the ultra-low molecular weight Tremella polysaccharide extract added to cosmetics is 0.1-20% by mass percentage.

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