Preparation method of centella asiatica fermentation product with repairing and anti-inflammatory effects as well as product and application of centella asiatica fermentation product

Abstract

The invention relates to a preparation method of a centella asiatica fermentation product with repairing and anti-inflammatory effects as well as a product and application of the centella asiatica fermentation product. The preparation method comprises the following steps: mixing a centella asiatica raw material with compound enzyme and water, performing enzymolysis, performing enzyme deactivation and filtration on enzymatic hydrolysate to obtain an extracting solution, inoculating a compound strain into the extracting solution, and performing fermentation, filtration and sterilization to obtain the centella asiatica fermentation product with repairing and anti-inflammatory effects. A centella asiatica fermentation product is obtained; wherein the compound enzyme comprises a combination of cellulase, pectinase, xylanase and beta-glucosidase; the compound strain comprises bifidobacterium and lactobacillus plantarum. The centella asiatica fermentation product is prepared in a manner of combining biological enzymolysis with microbial fermentation, various unique pharmacological components such as asiaticoside in centella asiatica can be effectively extracted and fully utilized, the efficacy of the finally prepared centella asiatica fermentation product is linearly improved, the centella asiatica fermentation product permeates the bottom of the skin layer by layer, and the health care effect of the centella asiatica is improved. The moisturizing and soothing effects can be realized; the skin can be compacted and refined.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to a method for preparing Centella asiatica ferment products with repairing and anti-inflammatory effects, as well as the products and applications thereof. Background Technology

[0002] Centella asiatica (L.) Urban, also known as iron lamp grass, money grass, copper coin grass, horse hoof grass, and thunder root, is a perennial herbaceous plant belonging to the genus Centella in the family Apiaceae. Its stems are creeping, slender, and root at the nodes; leaves are membranous to herbaceous, round, kidney-shaped, or horseshoe-shaped; umbels bear 3-4 flowers with ovate petals, purplish-red or creamy-white; fruits are laterally flattened, with a hairy or smooth surface; flowering and fruiting period is from April to October.

[0003] Centella asiatica has the effects of clearing heat and dampness, promoting blood circulation and stopping bleeding, and detoxifying and reducing swelling. It is mainly used to treat fever, cough and asthma, sore throat, enteritis, dysentery, etc. It has a tender texture and good palatability, and can be eaten raw, used as a herbal tea, or eaten as a vegetable. Centella asiatica has beautiful leaves, is evergreen all year round, and is resistant to trampling. It has a good effect on preventing soil erosion and is an excellent lawn plant. Its extract has a significant promoting effect on the healing of wounds and ulcers, so it is also often used in the cosmetics industry.

[0004] Centella asiatica contains asiaticoside and hydroxyasiatic acid, which can stimulate collagen synthesis, making it suitable for sensitive and damaged skin. Its polysaccharides can enhance the skin's water-locking ability and strengthen the barrier function of the stratum corneum. It also possesses multiple skin-improving effects, including anti-inflammatory, antioxidant, and anti-aging properties. Microbial fermentation refers to the process of using functional Chinese herbal plants, where various enzymes produced by microbial metabolism break down the cell walls, allowing for the full extraction of active ingredients within the cells. These active ingredients are then transformed into highly active small molecules by the microorganisms. Compared to traditional plant extraction techniques, this method can more efficiently extract and fully utilize the effective components from plants. Therefore, providing a method for efficiently extracting and utilizing the active ingredients in Centella asiatica through fermentation to prepare related products has significant application value. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing Centella asiatica ferment products with repair and anti-inflammatory effects, as well as the products and applications thereof.

[0006] To achieve this objective, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a Centella asiatica ferment product with repairing and anti-inflammatory effects, the preparation method comprising the following steps:

[0008] Centella asiatica raw material is mixed with compound enzyme and water for enzymatic hydrolysis. The hydrolysate is then inactivated and filtered to obtain an extract. The compound bacterial strain is inoculated into the extract for fermentation, filtered, and sterilized to obtain Centella asiatica fermentation product.

[0009] The complex enzyme comprises a combination of cellulase, pectinase, xylanase, and β-glucosidase; the complex bacterial strain comprises Bifidobacterium and Lactobacillus plantarum.

[0010] This invention uses a combination of enzymatic hydrolysis and microbial fermentation to prepare Centella asiatica fermentation products. This method can effectively extract and fully utilize various unique pharmacological components in Centella asiatica, such as asiaticoside. The resulting Centella asiatica fermentation products have significantly enhanced efficacy, penetrating deep into the skin to moisturize, soothe, firm, and refine the skin.

[0011] Furthermore, the four enzymes used—cellulase, pectinase, xylanase, and β-glucosidase—work synergistically to enhance the extraction efficiency of relevant active ingredients from Centella asiatica, thereby increasing the content of active ingredients in the final product. Through subsequent microbial fermentation, the active ingredients are further transformed into smaller molecules that are more easily absorbed, effectively improving skin condition.

[0012] Preferably, the mass ratio of cellulase, pectinase, xylanase and β-glucosidase is 1:(0.5-3):(0.1-1):(0.1-2);

[0013] The "0.5-3" can be, for example, 0.5, 1, 1.5, 2, 2.5, 3, etc.

[0014] The "0.1-1" can be, for example, 0.1, 0.2, 0.4, 0.6, 0.8, 1, etc.;

[0015] The "0.1-2" can be, for example, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 1.8, 2, etc.

[0016] Based on the synergistic relationship among the four enzymes mentioned above, their extraction effect is better when the four enzymes are used in the specific dosage ratio range mentioned above.

[0017] Preferably, the ratio of viable counts of Bifidobacterium to Lactobacillus plantarum is 1:10-10:1, for example, it can be 1:10, 1:9, 1:8, 1:5, 1:4, 1:2, 1:1, 1:5, 1:10, etc.

[0018] Preferably, the total viable bacterial concentration of the composite strain is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g, for example, could be 1×10 8CFU / mL (CFU / g), 2×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 8×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0019] Preferably, the enzymatic hydrolysis process is as follows: after drying and pulverizing the Centella asiatica raw material, it is first mixed with water and subjected to ultrasonic extraction. The extract is collected, and a compound enzyme is added for enzymatic hydrolysis.

[0020] Preferably, the ratio of the Centella asiatica raw material to water is 1:10-1:20, g / mL, for example, it can be 1:10, 1:12, 1:15, 1:18, 1:19, 1:20, etc.

[0021] Preferably, the amount of the compound enzyme added is 0.4%-1% of the water mass, for example, it can be 0.4%, 0.5%, 0.6%, 0.8%, 0.9%, 1%, etc.

[0022] Preferably, the power of the ultrasonic extraction is 200-500 W, such as 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, etc.; the frequency is 20-40 kHz, such as 20 kHz, 22 kHz, 25 kHz, 28 kHz, 30 kHz, 32 kHz, 35 kHz, 38 kHz, 40 kHz, etc.; and the time is 20-30 min, such as 20 min, 22 min, 25 min, 26 min, 28 min, 30 min, etc.

[0023] Preferably, the enzymatic hydrolysis temperature is 40-60℃, such as 40℃, 45℃, 50℃, 55℃, 58℃, 60℃, etc.; and the time is 1-3 h, such as 1 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, etc.

[0024] Preferably, the enzymatic hydrolysis process also includes ultrasonic treatment.

[0025] Preferably, the ultrasonic treatment is performed once every 20-30 minutes (e.g., 20 min, 22 min, 25 min, 26 min, 28 min, 30 min, etc.), and each treatment lasts 5-10 minutes, such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0026] Preferably, the power of the ultrasonic treatment is 100-300 W, such as 100 W, 250 W, 200 W, 250 W, 280 W, 300 W, etc.; and the frequency is 20-40 KHz, such as 20 KHz, 25 KHz, 30 KHz, 35 KHz, 38 KHz, 40 KHz, etc.

[0027] Preferably, the enzyme inactivation temperature is 70-90℃, for example, 70℃, 72℃, 75℃, 78℃, 80℃, 85℃, 90℃, etc.; the time is 8-15 min, for example, 8 min, 10 min, 12 min, 13 min, 14 min, 15 min, etc.

[0028] Preferably, the inoculation amount of the compound strain is 1%-5% of the mass of the extract, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0029] Preferably, the fermentation temperature is 30-35℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.; and the time is 24-48 h, such as 24 h, 26 h, 28 h, 30 h, 35 h, 38 h, 40 h, 42 h, 45 h, 48 h, etc.

[0030] Preferably, the fermentation process also includes ultrasonic treatment.

[0031] Preferably, the ultrasonic treatment is performed once every 6-12 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.), for 5-10 minutes each time, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0032] Preferably, the power of the ultrasonic treatment is 100-300 W, such as 100 W, 250 W, 200 W, 250 W, 280 W, 300 W, etc.; and the frequency is 20-40 KHz, such as 20 KHz, 25 KHz, 30 KHz, 35 KHz, 38 KHz, 40 KHz, etc.

[0033] Preferably, the sterilization temperature is 80-90℃, such as 80℃, 82℃, 85℃, 88℃, 90℃, etc.; and the time is 10-15 min, such as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0034] In a second aspect, the present invention provides a Centella asiatica fermentation product prepared according to the preparation method described in the first aspect.

[0035] Thirdly, the present invention provides an application of the Centella asiatica fermentation product according to the second aspect in the preparation of cosmetics.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention employs a combination of enzymatic hydrolysis and microbial fermentation to prepare Centella asiatica fermentation products. This method effectively extracts and fully utilizes various unique pharmacological components from Centella asiatica, such as asiaticoside. The resulting Centella asiatica fermentation product exhibits significantly enhanced efficacy, penetrating deep into the skin to moisturize, soothe, and firm the skin. Furthermore, the addition of four enzymes—cellulase, pectinase, xylanase, and β-glucosidase—synergistically enhances the extraction efficiency of relevant active ingredients from Centella asiatica, thereby increasing the content of active ingredients in the final product. Subsequent microbial fermentation further transforms these active ingredients into more easily absorbed smaller molecules, effectively improving skin condition. Detailed Implementation

[0038] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0039] The enzyme information mentioned below is as follows: cellulase, pectinase, xylanase and β-glucosidase are products purchased from Yuanye Biotechnology with model numbers S10041, S10007, S23566 and S10047, respectively.

[0040] The following strain information is involved: Bifidobacteria: Bifidobacterium longum ATCC15697, Bifidobacterium thermophilum ATCC25525, Bifidobacterium adolescentis ATCC15703; Lactobacillus plantarum: CICC25125, CICC20272, CICC20717.

[0041] Example 1

[0042] This embodiment provides a Centella asiatica fermentation product, and the specific steps are as follows:

[0043] (1) Wash, dry and crush the Centella asiatica leaves and mix them with deionized water at a ratio of 1:15 (g / mL). Sonicate at 30 KHz and 350 W for 25 min.

[0044] (2) Adjust the pH of the treatment solution to 5, add a compound enzyme (composed of cellulase, pectinase, xylanase and β-glucosidase in a mass ratio of 1:1:0.5:0.8), the amount of compound enzyme added is 0.6% of the mass of deionized water, enzymatic hydrolysis at 55℃ for 2 h, during the enzymatic hydrolysis process, use ultrasound to treat for 8 min every 25 min (frequency 30 KHz, power 200 W), and then heat the enzymatic hydrolysate to 80℃ and keep it for 10 min to inactivate the enzyme.

[0045] (3) Filter with activated carbon to obtain the extract, and then add the compound strain (Bifidobacterium longum ATCC15697 and Lactobacillus plantarum CICC25125 with a viable count ratio of 1:1, and a total viable count concentration of 1×10⁻⁶) to the extract. 8 CFU / mL was inoculated into the extract for fermentation. The mixture was then allowed to stand at 32°C under microaerobic (5% DO) conditions for 36 h. During the fermentation process, the mixture was treated with ultrasound for 8 min every 9 h (frequency 30 kHz, power 200 W).

[0046] (4) After fermentation, heat the fermentation liquid to 80°C and keep it for 14 minutes to inactivate the strain.

[0047] (5) The bacteria and residue were removed by filter bag filtration, the filtrate was collected, and further filtered using a 0.22 μm microfiltration membrane. The filtrate was then concentrated, sterilized, and freeze-dried to obtain the Centella asiatica fermentation product.

[0048] Example 2

[0049] This embodiment provides a Centella asiatica fermentation product, and the specific steps are as follows:

[0050] (1) Wash, dry and crush the Centella asiatica leaves and mix them with deionized water at a ratio of 1:20 (g / mL). Sonicate at 40 KHz and 500 W for 20 min.

[0051] (2) Adjust the pH of the treatment solution to 6, add a compound enzyme (composed of cellulase, pectinase, xylanase and β-glucosidase in a mass ratio of 1:2.5:1:2), the amount of compound enzyme added is 0.4% of the mass of deionized water, enzymatic hydrolysis at 58℃ for 1.5 h, during the enzymatic hydrolysis process, use ultrasound to treat for 6 min every 30 min (frequency 40 KHz, power 100 W), and then heat the enzymatic hydrolysate to 90℃ and keep it for 8 min to inactivate the enzyme.

[0052] (3) Filter with activated carbon to obtain the extract, and then add the compound strain (Bifidobacterium thermophilum ATCC25525 and Lactobacillus plantarum CICC20272 with a viable count ratio of 3:1, and a total viable count concentration of 1×10⁻⁶). 8 CFU / mL was inoculated into the extract for fermentation. The mixture was then allowed to stand at 30°C under microaerobic (5% DO) conditions for 48 h. During the fermentation process, the mixture was treated with ultrasound for 6 min every 12 h (frequency 40 kHz, power 100 W).

[0053] (4) After fermentation, heat the fermentation liquid to 90°C and keep it for 10 min to inactivate the strain.

[0054] (5) The bacteria and residue were removed by filter bag filtration, the filtrate was collected, and further filtered using a 0.22 μm microfiltration membrane. The filtrate was then concentrated, sterilized, and freeze-dried to obtain the Centella asiatica fermentation product.

[0055] Example 3

[0056] This embodiment provides a Centella asiatica fermentation product, and the specific steps are as follows:

[0057] (1) Wash, dry and crush the Centella asiatica leaves and mix them with deionized water at a ratio of 1:10 (g / mL). Sonicate at 20 kHz and 200 W for 30 min.

[0058] (2) Adjust the pH of the treatment solution to 4, add a compound enzyme (composed of cellulase, pectinase, xylanase and β-glucosidase in a mass ratio of 1:0.5:0.2:1), the amount of compound enzyme added is 1% of the mass of deionized water, enzymatic hydrolysis at 48℃ for 2.5 h, during the enzymatic hydrolysis process, use ultrasound to treat for 10 min every 20 min (frequency 20 KHz, power 300 W), and then heat the enzymatic hydrolysate to 75℃ and keep it for 12 min to inactivate the enzyme.

[0059] (3) Filter with activated carbon to obtain the extract, and then add the compound strain (Bifidobacterium adolescentis ATCC15703 and Lactobacillus plantarum CICC20717 with a viable count ratio of 1:5 and a total viable count of 1×10⁻⁶) to the extract. 8 CFU / mL was inoculated into the extract for fermentation. The mixture was then allowed to stand at 35°C under microaerobic (5% DO) conditions for 24 h. During the fermentation process, the mixture was treated with ultrasound for 10 min every 6 h (frequency 20 kHz, power 300 W).

[0060] (4) After fermentation, heat the fermentation liquid to 85°C and keep it for 12 minutes to inactivate the strain.

[0061] (5) The bacteria and residue were removed by filter bag filtration, the filtrate was collected, and further filtered using a 0.22 μm microfiltration membrane. The filtrate was then concentrated, sterilized, and freeze-dried to obtain the Centella asiatica fermentation product.

[0062] Comparative Example 1

[0063] This comparative example provides a Centella asiatica fermentation product, using Centella asiatica leaves from Example 1. The only difference between this example and Example 1 is step (2). The difference is that the complex enzyme does not contain cellulase, and its reduced amount is proportionally allocated to pectinase, xylanase and β-glucosidase. The remaining steps and related parameters are the same as in Example 1.

[0064] Comparative Example 2

[0065] This comparative example provides a Centella asiatica fermentation product, using Centella asiatica leaves from Example 1. The only difference between this example and Example 1 is step (2). The difference is that the compound enzyme does not contain pectinase, and its reduced amount is proportionally allocated to cellulase, xylanase and β-glucosidase. The remaining steps and related parameters are the same as in Example 1.

[0066] Comparative Example 3

[0067] This comparative example provides a Centella asiatica fermentation product, using Centella asiatica leaves from Example 1. The only difference between this example and Example 1 is step (2). The difference is that the complex enzyme does not contain xylanase, and its reduced amount is proportionally allocated to cellulase, pectinase and β-glucosidase. The remaining steps and related parameters are the same as in Example 1.

[0068] Comparative Example 4

[0069] This comparative example provides a Centella asiatica fermentation product, using Centella asiatica leaves from Example 1. The only difference between this example and Example 1 is step (2). The difference is that the complex enzyme does not contain β-glucosidase, and its reduced amount is proportionally allocated to cellulase, pectinase, and xylanase. The remaining steps and related parameters are the same as in Example 1.

[0070] Comparative Example 5

[0071] This comparative example provides a Centella asiatica fermentation product, which uses the enzyme-inactivated treatment liquid after step (2) of Example 1. The only difference between this example and Example 1 is that the compound strain is replaced with an equal number of viable bacteria and an equal amount of Bifidobacterium longum ATCC15697 for inoculation and fermentation. All other steps and related parameters are the same as in Example 1.

[0072] Comparative Example 6

[0073] This comparative example provides a Centella asiatica fermentation product, which uses the enzyme-inactivated treatment liquid from step (2) of Example 1. The only difference between this example and Example 1 is that the composite strain is replaced with an equal number of viable bacteria and an equal amount of Lactobacillus plantarum CICC25125 for inoculation and fermentation. All other steps and related parameters are the same as in Example 1.

[0074] Comparative Example 7

[0075] This comparative example provides a Centella asiatica extract, which uses the enzyme-inactivated treatment solution after step (2) of Example 1. The only difference between this example and Example 1 is that no bacterial fermentation treatment is performed. The enzyme-inactivated treatment solution after step (2) is filtered through activated carbon to obtain the extract, which is then directly processed into the Centella asiatica extract in step (5). All other steps and related parameters are the same as in Example 1.

[0076] Test Example 1

[0077] Moisturizing efficacy test

[0078] (1) Test principle: Based on the different forces that different moisturizers exert on water molecules, their ability to absorb and retain water also varies slightly. The moisturizing rate of a moisturizer can be characterized by testing it in the laboratory (weighing method).

[0079] (2) Instruments and materials:

[0080] Instrument: Constant temperature and humidity chamber;

[0081] Test materials: Centella asiatica fermentation product / Centella asiatica extract (test substance), water (blank control), 10% glycerol solution (standard reference).

[0082] (3) Test environment: temperature 20±1℃, humidity 45%±2RH.

[0083] (4) Test method: Weigh the sample and place it in a constant temperature and humidity chamber. Weigh the sample at 2, 8 and 20 h, record the data and calculate the moisture retention rate. Select a reagent with known moisturizing ability as a standard reference.

[0084] (5) Calculation of test results: According to the experimental design, the values ​​of Mt (mass of test substance), Yt (mass of blank control), and Ht (mass of standard reference) for each time period were measured respectively. The relative moisturizing rate of each group of test substances in each time period was calculated according to the following moisturizing rate calculation formula.

[0085] Relative moisturizing rate % = [P] 样 -P 空 ] / [P 标 -P 空 ×100%;

[0086] In the formula, the moisturizing rate P 样=(Mt / M0)×100%; Moisturizing rate P 空 =(Yt / Y0)×100%; Moisturizing rate P 标 =(Ht / H0)×100%.

[0087] The experimental results are shown in Table 1.

[0088] Table 1

[0089]

[0090] According to the data in the table,

[0091] (1) As can be seen from Examples 1 to 3, the Centella asiatica fermentation product provided by the present invention has excellent moisturizing effect.

[0092] (2) According to the comparison between Example 1 and Comparative Examples 1-4, the present invention uses a complex enzyme including four specific enzymes, namely cellulase, pectinase, xylanase and β-glucosidase, to extract the active ingredients from Centella asiatica. The four enzymes can work synergistically to effectively improve the efficiency of related enzyme hydrolysis. The final product has a high content of active ingredients and has excellent moisturizing effect.

[0093] (2) According to the comparison between Example 1 and Comparative Examples 5-7, the preparation of Centella asiatica fermentation products by combining biological enzymatic hydrolysis and microbial fermentation in this invention can effectively extract and fully utilize the multiple pharmacological components in Centella asiatica. After fermentation treatment by microbial strains, the active ingredients in Centella asiatica can be further converted into easily absorbed small molecules. The final product prepared has a better moisturizing effect. Furthermore, it was found that the combination of Bifidobacterium and Lactobacillus plantarum used in this invention can synergistically enhance the effect during fermentation.

[0094] Test Example 2

[0095] Anti-inflammatory efficacy test

[0096] Experimental methods: Interleukin-1α (IL1-α) and interleukin-6 (IL-6) are cellular inflammatory factors. The two inflammatory factors were detected by ELISA to evaluate the anti-inflammatory effect of the test samples.

[0097] Experimental grouping and treatment: Blank group: HaCat cells without any treatment were cultured in 12-well plates; Model group: HaCat cells were stimulated with 1 μg / mL LPS and cultured in 12-well plates for 24 h; Sample group: After stimulating HaCat cells with 1 μg / mL LPS for 24 h, 100 μL of the above-mentioned test sample dilution solution (the sample lyophilized powder was diluted with deionized water to a concentration of 1%) was added to each well of the 12-well plate; Each experimental group was cultured in a 37°C, 5% CO2 incubator for 72 h, with 3 replicates per group. Cell culture supernatant was collected, and the expression levels of IL-1α and IL-6 were detected by an ELISA kit. The results were averaged. The levels detected in the blank group were used as the reference (i.e., 100%). The relative levels of IL-1α and IL-6 in each experimental group are shown in Table 2.

[0098] Table 2

[0099]

[0100] According to the data in the table, the anti-inflammatory effect of the Centella asiatica fermentation products prepared in Examples 1-3 is better than that in Comparative Examples 1-4. This indicates that the absence of any one of the four enzymes (cellulase, pectinase, xylanase, and β-glucosidase) in the complex enzyme used for enzymatic extraction will affect the skin improvement effect of the final extract. In addition, the anti-inflammatory effect of Example 1 is higher than that in Comparative Examples 5-7, indicating that the Centella asiatica extract after microbial fermentation is more effective. Furthermore, the combined use of Bifidobacterium and Lactobacillus plantarum also has a certain synergistic effect in improving the anti-inflammatory effect of the final product.

[0101] Test Example 3

[0102] Soothing test

[0103] Test method: Keratinocytes were used, and the relative expression level of capsaicin receptor (TRPV1) protein was detected by immunofluorescence (IF) to evaluate the soothing efficacy of glycosyl isomers.

[0104] According to 4.5×10 4 Cells were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2, 95% RH). When the cell deposition rate in the 24-well plates reached 40%-60%, the following treatments were administered: Blank control group: no capsaicin stimulation; Model control group: capsaicin stimulation; Sample group: 2% of a diluted solution of Centella asiatica fermentation products / Centella asiatica extracts prepared in Examples 1-3 and Comparative Examples 1-7 was added, followed by capsaicin stimulation.

[0105] After drug administration, the 24-well plate was incubated in an incubator for 24 h. After incubation, the supernatant was discarded, the plate was washed three times with PBS, the PBS was discarded, and 200 μL of goat serum was added to each well. The plate was blocked at 25°C for 60 min. The goat serum blocking solution was discarded, and the diluted primary antibody Anti-VR1 antibody (200 μL / well) (1:150 dilution with goat serum) was added to each well. The plate was incubated overnight at 4°C. The primary antibody was discarded, and the plate was washed three times with PBS for 5 min each time. The residual PBS on the plate was blotted dry with absorbent paper. The diluted fluorescent secondary antibody Goat pAb to Rb IgG (Alexa Fluor® 488) (200 μL / well) (1:500 dilution with PBS) was added to each well. The plate was incubated at room temperature in the dark for 1 h. The secondary antibody was discarded, and the plate was washed three times with PBS for 5 min each time. The residual PBS on the plate was blotted dry with absorbent paper. The Hochest 33342 (200 μL / well, PBS) was added to each well. (1:500 dilution), incubate at 25℃ for 5 min; discard Hochest 33342, wash 3 times with PBS, 5 min each time. Blot dry the residual PBS liquid on the slide with absorbent paper, pick up the slide with a needle, add a drop of anti-quenching agent to the slide, and place the slide upside down on the slide, then mount the slide (use a cut yellow pipette tip to draw up the anti-quenching agent); detect the relative expression inhibition rate of capsaicin receptor (TRPV1) of each group of samples under a fluorescence microscope, and calculate the results shown in the table below.

[0106] Table 3

[0107]

[0108] According to the data in the table, the Centella asiatica fermentation product obtained by the preparation process provided by the present invention can significantly downregulate the expression level of TRPV1 in keratinocytes and has a good soothing effect. Among them, the soothing effect of Example 1 is better than that of Comparative Examples 1-7, indicating that the extraction and preparation process conditions of Centella asiatica fermentation product used in the present invention have different degrees of influence on the soothing effect of the final extracted product.

[0109] Test Example 4

[0110] Security test

[0111] Fifteen participants were selected according to the inclusion criteria and randomly divided into three groups (two males and three females in each group) to undergo the trial. An appropriate amount of Centella asiatica fermentation product sample prepared in Examples 1-3 (mixed with pure water to adjust the extract concentration to 5% before testing) was placed in the drug compartment of the plaster test tape. The plaster tester containing the sample was applied to the flexor surface of the subject's forearm skin using medical tape for 24 hours. Thirty minutes after removing the plaster tester, and after the indentation disappeared, the skin reaction was observed. For those with negative results, the skin reaction was observed again at 24 hours and 48 hours after the trial, and the results were recorded. The grading criteria for the degree of reaction are shown in the table below.

[0112]

[0113] The results showed that the products prepared in Examples 1-3 all exhibited negative reactions at 24 h and 48 h after the test, with no irritation or erythema. Therefore, the Centella asiatica fermentation product prepared by this invention is non-irritating to the skin and shows no positive reaction, proving that the product is highly safe, has low irritation, and is relatively mild.

[0114] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of the product of this invention, and the addition of auxiliary components, as well as the selection of specific methods, all fall within the protection scope and disclosure scope of this invention.

[0115] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0116] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing a fermenting product of Centella asiatica having a repairing and anti-inflammatory effect, characterized by, The preparation method comprises the following steps: The enzyme solution is subjected to enzyme inactivation, filtration to obtain an extract, the complex strain is inoculated into the extract to perform fermentation, filtration, sterilization, and thus a snowbell fern fermentation product is obtained. The complex enzyme comprises a combination of cellulase, pectinase, xylanase and beta-glucosidase; and the complex strain comprises Bifidobacterium and Lactobacillus plantarum.

2. The production method according to claim 1, characterized by, The mass ratio of the cellulase, pectinase, xylanase and beta-glucosidase is 1:(0.5-3):(0.1-1):(0.1-2).

3. The production method according to claim 1, characterized by, The ratio of the viable bacterial count of the Bifidobacterium to the viable bacterial count of the Lactobacillus plantarum is 1:10-10:

1. Preferably, the total viable bacterial concentration of the complex strain is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

4. The method of claim 1, wherein, The enzyme inactivation process specifically comprises: after the snowbell fern raw material is dried and crushed, the snowbell fern raw material is mixed with water to perform ultrasonic extraction, the extract is collected, the complex enzyme is added, and enzyme inactivation is performed. Preferably, the ratio of the snowbell fern raw material to water is 1:10-1:20, g / mL. Preferably, the addition amount of the complex enzyme is 0.4%-1% of the mass of the water.

5. The preparation method according to claim 4, characterized in that, The ultrasonic extraction is performed at a power of 200-500 W, a frequency of 20-40 KHz, and for a time of 20-30 min.

6. The production method according to claim 1 or 4, characterized by, The enzyme inactivation is performed at a temperature of 40-60℃ for a time of 1-3 h. Preferably, ultrasonic treatment is further performed during the enzyme inactivation process. Preferably, the ultrasonic treatment is performed once every 20-30 min for 5-10 min. Preferably, the ultrasonic treatment is performed at a power of 100-300 W and a frequency of 20-40 KHz.

7. The preparation method according to claim 1, characterized in that, The inoculation amount of the complex strain is 1%-5% of the mass of the extract. Preferably, the fermentation is performed at a temperature of 30-35℃ for a time of 24-48 h.

8. The method of claim 1, wherein, The sterilization is performed at a temperature of 80-90℃ for a time of 10-15 min. 9.A snowbell fern fermentation product prepared by the preparation method according to any one of claims 1-8. 10.Use of the snowbell fern fermentation product according to claim 9 in the preparation of a cosmetic.

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