A method for preparing mulberry leaf extract through multiple fermentation, and the mulberry leaf extract and its applications.

Abstract

This invention relates to a method for preparing mulberry leaf extract through multiple fermentation, and the mulberry leaf extract and its applications, belonging to the field of cosmetic raw material extraction technology. This method, through a complex enzymatic pretreatment, three-stage specific fermentation, and ultrafiltration purification process, produces a mulberry leaf extract that is non-irritating to the skin, exhibits good safety, high bioavailability, and excellent skin penetration, demonstrating significant effects in enhancing the skin barrier, moisturizing, and reducing wrinkles.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material extraction technology, and in particular to a method for preparing mulberry leaf extract through multiple fermentation, the mulberry leaf extract and its applications. Background Technology

[0002] Mulberry leaf extract, rich in flavonoids, polysaccharides, polyphenols, and other active ingredients, possesses antioxidant and anti-aging effects, making it valuable in the cosmetics industry. Conventional extraction methods, including water extraction, alcohol extraction, acid-base extraction, and ultrasonic or microwave-assisted extraction, generally suffer from low extraction efficiency, easy destruction of active ingredients, high impurity content, and solvent residue. In contrast, bio-fermentation can gently release and enhance active substances while reducing impurities and solvent residue, thus becoming a new direction for the green and efficient preparation of mulberry leaf extract. For example, CN119157790A discloses a mulberry leaf extract, its preparation method, and its application in cosmetics. This method uses *Candida utilis*, *Lactobacillus plantarum*, and *Bacillus subtilis* as a complex fermentation strain. The mulberry leaf extract prepared by this method has high flavonoid, chlorogenic acid, and peptide content, and exhibits good inhibitory effects on tyrosinase and DPPH free radical scavenging. However, mixed fermentation suffers from metabolic interference issues. The glycolysis of *Candida utilis* and the protease secretion of *Bacillus subtilis* compete for substrates, resulting in low levels of small-molecule peptides and other active ingredients, limited bioavailability, and poor barrier repair effects. CN119286943A discloses a method for fermenting mulberry leaves with a compound probiotic, along with the fermented mulberry leaf products and their applications. This method uses *Staphylococcus xylose* and *Lactobacillus plantarum* at a volume ratio of 1–3:1–3 to ferment mulberry leaves. The fermentation products inhibit tyrosinase, and the fermented mulberry leaf products can be used to prepare whitening cosmetics. However, this method is a single-stage fermentation, resulting in incomplete degradation and lack of enzymatic pretreatment. This leads to insufficient cell wall disruption of mulberry leaves, low total flavonoid extraction rate, and a single type of metabolite.

[0003] Based on the problems of existing technologies, such as interference with microbial metabolism in mixed fermentation and incomplete degradation, insufficient conversion, poor extract quality, and low bioavailability in single-stage fermentation, this invention proposes a method for preparing mulberry leaf extract through multiple fermentation. By combining compound enzymatic hydrolysis with three-stage stepwise fermentation, the above-mentioned defects are specifically addressed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing mulberry leaf extract through multiple fermentation, as well as the mulberry leaf extract and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing mulberry leaf extract through multiple fermentation, comprising the following steps:

[0007] S1. Pretreatment with compound enzymatic hydrolysis: Weigh mulberry leaf powder and deionized water at a mass ratio of 1:100, mix and stir evenly to form a culture medium, add a compound enzyme of cellulase and pectinase, the inoculation amount of the compound enzyme is 1-2 wt% of the culture medium, the mass ratio of cellulase to pectinase is (1-3):2, and enzymatic hydrolysis is carried out at 48-52℃ and pH 4.8 for 4.5-5.5 hours. After enzymatic hydrolysis, the hydrolysate is sterilized and cooled.

[0008] S2, First stage fermentation: Transfer the sterilized enzymatic hydrolysate to a fermenter, inoculate with brewer's yeast culture, and ferment for 28-32 hours under anaerobic conditions at 26-30℃ and pH 6.0-6.4.

[0009] S3, Second stage fermentation: Inoculate with Lactobacillus acidophilus-Staphylococcus epidermidis compound bacterial solution and ferment for 38-42 hours at 28-32℃, pH 5.0-5.4, under static micro-aerobic environment;

[0010] S4. Third stage fermentation: Inoculate with Bacillus licheniformis culture and ferment for 48-52 hours at 33-37℃, pH 6.8-7.2, and with aeration.

[0011] S5. Purification: The fermentation broth is extracted by ultrasonication, centrifugation, ultrafiltration, vacuum concentration and freeze drying to obtain mulberry leaf extract.

[0012] In this invention, the pretreatment step of compound enzymatic hydrolysis before fermentation effectively breaks down the cell walls of mulberry leaves, allowing for the effective release of polysaccharides and proteins, providing a substrate basis for the subsequent three-stage stepwise fermentation. This invention employs a three-stage stepwise fermentation metabolic regulation strategy, achieving the targeted transformation and enhancement of mulberry leaf active ingredients through the orderly collaboration of the microbial community. In the first stage, *Saccharomyces cerevisiae* degrades polysaccharides, providing favorable conditions for subsequent fermentation; in the second stage, a compound microbial community creates an acidic environment, promoting component transformation; in the third stage, *Bacillus licheniformis* converts proteins into small molecule peptides, free amino acids, and other small molecule components. The entire process, through the orderly metabolism of "polysaccharide degradation → acid hydrolysis → protein hydrolysis," forms a three-dimensional synergistic system of "small molecule sugars - free flavonoids - active peptides / free amino acids." This solves the problems of incomplete component degradation and insufficient transformation caused by single enzymatic hydrolysis or single-strain fermentation in existing technologies, while avoiding the problems of metabolic interference from mixed fermentation strains, poor extract efficacy, and low bioavailability. Ultimately, it achieves the synergistic effects of enhancing the skin barrier, long-lasting moisturizing, and dynamic anti-wrinkle.

[0013] In a preferred embodiment of the preparation method described in this invention, in step S2, the concentration of the brewing yeast culture is 1.0 × 10⁻⁶. 8 -2.0×10 8 CFU / mL, inoculum volume is 2-3 wt% of the culture medium.

[0014] More preferably, in step S3, the mass ratio of Lactobacillus acidophilus to Staphylococcus epidermidis is 1:2, and the total concentration of the compound bacterial solution is 2.0 × 10⁻⁶. 8 -3.0×10 8 CFU / mL, with an inoculum volume of 3-5 wt% of the culture medium.

[0015] More preferably, in step S3, the micro-oxygen environment is controlled by nitrogen replacement to maintain dissolved oxygen at 0.5-1.0 mg / L.

[0016] More preferably, in step S4, the concentration of Bacillus licheniformis bacterial solution is 0.8 × 10⁻⁶. 8 -1.2×10 8 The inoculum volume is 2-4 wt% of the culture medium, and the aeration rate is 1.5 L / min.

[0017] More preferably, in step S5, the ultrasonic extraction power is 180-220W, and the treatment is carried out at 45-55℃ for 25-35 minutes; the ultrafiltration uses a 10kDa ultrafiltration membrane.

[0018] Secondly, the present invention provides a mulberry leaf extract prepared by the above method.

[0019] Thirdly, the present invention provides the application of the above-mentioned mulberry leaf extract in the preparation of cosmetics that enhance the skin barrier function.

[0020] Fourthly, the present invention provides the application of the above-mentioned mulberry leaf extract in the preparation of moisturizing cosmetics.

[0021] Fifthly, the present invention provides the application of the above-mentioned mulberry leaf extract in the preparation of anti-wrinkle cosmetics.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. Through the synergistic effect of compound enzymatic hydrolysis pretreatment and three-stage stepwise fermentation, the active ingredients of mulberry leaves are fully converted and synergistically enhanced. The entire process forms a three-dimensional synergistic system of "small molecule sugars-free flavonoids-active peptides / free amino acids" through orderly metabolism of "polysaccharide degradation → acid hydrolysis conversion → protein hydrolysis". This not only solves the problems of incomplete degradation and insufficient conversion of components caused by single enzymatic hydrolysis or single-strain fermentation in existing technologies, but also avoids the problems of bacterial metabolism interference, poor extract effect and low bioavailability in mixed fermentation. Ultimately, it achieves the synergistic effects of enhancing the skin barrier, long-lasting moisturizing and dynamic anti-wrinkle.

[0024] 2. The mulberry leaf extract prepared by this invention has been verified by safety testing to be non-irritating to the skin, highly compatible with cells, and suitable for all skin types. It can be widely used in various cosmetics that enhance the skin barrier, moisturize and reduce wrinkles. Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0026] The microbial sources of this invention are cellulase (enzyme activity of 50,000 U / g) and pectinase (enzyme activity of 50,000 U / g), both purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0027] Saccharomyces cerevisiae: accession number CCTCC NO:M 2021941, deposited at the China Center for Type Culture Collection; Lactobacillus acidophilus: accession number CICC22150, deposited at the China Industrial Microbiological Culture Collection Center; Staphylococcus epidermidis: accession number GDMCC No.60607, deposited at the Guangdong Provincial Center for Microbiological Culture Collection; Bacillus licheniformis: accession number CGMCC NO.3336, deposited at the China General Microbiological Culture Collection Center.

[0028] Example 1: Preparation of mulberry leaf extract C1

[0029] S1. Pretreatment with compound enzymatic hydrolysis: Take 100g of mulberry leaf powder, add 1000g of deionized water, mix and stir evenly to prepare the culture medium. Add a compound enzyme of cellulase and pectinase, with an inoculation amount of 1.5wt% of the culture medium and a mass ratio of cellulase to pectinase of 2:2. Enzymatic hydrolysis is carried out at 50℃ and pH 4.8 for 5 hours. After enzymatic hydrolysis, the hydrolysate is autoclaved at 115℃ for 15 minutes and then cooled to 30℃.

[0030] S2. First stage fermentation: Transfer the sterilized enzymatic hydrolysate to the fermenter, and inoculate with Saccharomyces cerevisiae at 2.5 wt% of the culture medium, with a bacterial concentration of 1.5 × 10⁻⁶. 8 CFU / mL, fermented for 30 hours at 28℃, pH 6.2, under anaerobic conditions.

[0031] S3. Second stage fermentation: Inoculate the culture medium with a 4 wt% Lactobacillus acidophilus-Staphylococcus epidermidis compound bacterial solution, with a mass ratio of Lactobacillus acidophilus to Staphylococcus epidermidis of 1:2, and a total concentration of the compound bacterial solution of 2.5 × 10⁻⁶. 8 Fermentation was carried out at CFU / mL for 40 hours at 30℃, pH 5.2, under static microaerobic conditions (dissolved oxygen was controlled at 0.8 mg / L by nitrogen replacement).

[0032] S4. Third stage fermentation: Inoculate with Bacillus licheniformis culture at 3 wt% of the culture medium, with a culture concentration of 1.0 × 10⁻⁶. 8 Fermentation was carried out at CFU / mL at 35℃, pH 7.0, and an aeration rate of 1.5 L / min for 50 hours.

[0033] S5. Purification: The fermentation broth was extracted by ultrasonic extraction at a power of 200W and treated at 50℃ for 30 minutes. After centrifugation, the supernatant was filtered through a 10kDa ultrafiltration membrane. The permeate was collected, concentrated under vacuum, and freeze-dried to obtain mulberry leaf extract C1.

[0034] Example 2: Preparation of mulberry leaf extract C2

[0035] S1. Pretreatment with compound enzymatic hydrolysis: Take 100g of mulberry leaf powder, add 1000g of deionized water, mix and stir evenly to prepare the culture medium. Add a compound enzyme of cellulase and pectinase, with an inoculation amount of 1wt% of the culture medium and a mass ratio of cellulase to pectinase of 1:2. Enzymatic hydrolysis is carried out at 48℃ and pH 4.8 for 4.5 hours. After enzymatic hydrolysis, the hydrolysate is autoclaved at 115℃ for 15 minutes and then cooled to 30℃.

[0036] S2. First stage fermentation: Transfer the sterilized enzymatic hydrolysate to a fermenter, and inoculate with *Saccharomyces cerevisiae* broth at 2 wt% of the culture medium, with a broth concentration of 1.0 × 10⁻⁶. 8 CFU / mL, fermented for 28 hours at 26℃, pH 6.0, under anaerobic conditions.

[0037] S3. Second stage fermentation: Inoculate the culture medium with a 3 wt% Lactobacillus acidophilus-Staphylococcus epidermidis compound bacterial solution, with a mass ratio of Lactobacillus acidophilus to Staphylococcus epidermidis of 1:2, and a total concentration of the compound bacterial solution of 2.0 × 10⁻⁶. 8 CFU / mL, fermented for 38 hours at 28℃, pH 5.0, under static microaerobic conditions (dissolved oxygen controlled at 0.5 mg / L by nitrogen replacement).

[0038] S4. Third stage fermentation: Inoculate with Bacillus licheniformis culture at 2 wt% of the culture medium, with a culture concentration of 0.8 × 10⁻⁶. 8 Fermentation was carried out at CFU / mL at 33℃, pH 6.8, and an aeration rate of 1.5 L / min for 48 hours.

[0039] S5. Purification: The fermentation broth was extracted by ultrasonic extraction at a power of 180W at 45℃ for 25 minutes. After centrifugation, the supernatant was filtered through a 10kDa ultrafiltration membrane. The permeate was collected, concentrated under vacuum, and freeze-dried to obtain mulberry leaf extract C2.

[0040] Example 3: Preparation of mulberry leaf extract C3

[0041] S1. Pretreatment with compound enzymatic hydrolysis: Take 100g of mulberry leaf powder, add 1000g of deionized water, mix and stir evenly to form a culture medium. Add a compound enzyme of cellulase and pectinase, with an inoculation amount of 2wt% of the culture medium and a mass ratio of cellulase to pectinase of 3:2. Enzymatic hydrolysis is carried out at 52℃ and pH 4.8 for 5.5 hours. After enzymatic hydrolysis, the hydrolysate is autoclaved at 115℃ for 15 minutes and cooled to 30℃.

[0042] S2. First stage fermentation: Transfer the sterilized enzymatic hydrolysate to a fermenter, and inoculate with *Saccharomyces cerevisiae* broth at 3 wt% of the culture medium, with a broth concentration of 2.0 × 10⁻⁶. 8 CFU / mL, fermented for 32 hours at 30℃, pH 6.4, under anaerobic conditions.

[0043] S3. Second stage fermentation: Inoculate the culture medium with a 5 wt% Lactobacillus acidophilus-Staphylococcus epidermidis compound solution, where the mass ratio of Lactobacillus acidophilus to Staphylococcus epidermidis is 1:2, and the total concentration of the compound solution is 3.0 × 10⁻⁶. 8 Fermentation was carried out at CFU / mL for 42 hours at 32℃, pH 5.4, under static microaerobic conditions (dissolved oxygen was controlled at 1.0 mg / L by nitrogen replacement).

[0044] S4. Third stage fermentation: Inoculate with Bacillus licheniformis culture at 4 wt% of the culture medium, with a culture concentration of 1.2 × 10⁻⁶. 8 Fermentation was carried out at CFU / mL at 37℃, pH 7.2, and an aeration rate of 1.5 L / min for 52 hours.

[0045] S5. Purification: The fermentation broth was extracted by ultrasonic extraction at a power of 220W at 55℃ for 35 minutes. After centrifugation, the supernatant was filtered through a 10kDa ultrafiltration membrane. The permeate was collected, concentrated under vacuum, and freeze-dried to obtain mulberry leaf extract C3.

[0046] Comparative Example 1

[0047] Compared with Example 1, the difference is that the S1 complex enzymatic hydrolysis pretreatment step is omitted and replaced by mechanical crushing (homogenization at 8000 r / min for 30 minutes), while the rest is the same as Example 1.

[0048] Comparative Example 2

[0049] Compared with Example 1, the difference is that the S1 compound enzymatic hydrolysis pretreatment step omits pectinase and replaces it with an equivalent amount of cellulase; the rest is the same as in Example 1.

[0050] Comparative Example 3

[0051] Compared with Example 1, the difference is that the S1 complex enzymatic hydrolysis pretreatment step omits cellulase and replaces it with an equivalent amount of pectinase; the rest is the same as in Example 1.

[0052] Comparative Example 4

[0053] Compared with Example 1, the difference is that the second and third stages of fermentation are omitted, and only the first stage of fermentation is carried out. The bacterial solutions in the second and third stages are replaced with the bacterial solutions in the first stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0054] Comparative Example 5

[0055] Compared with Example 1, the difference is that the first and third stages of fermentation are omitted, and only the second stage of fermentation is carried out. The bacterial solutions in the first and third stages are replaced with the bacterial solutions in the second stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0056] Comparative Example 6

[0057] Compared with Example 1, the difference is that the first and second stages of fermentation are omitted, and only the third stage of fermentation is carried out. The bacterial solutions of the first and second stages are replaced with the bacterial solutions of the third stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0058] Comparative Example 7

[0059] Compared with Example 1, the difference is that the first stage of fermentation is omitted, and only the second and third stages of fermentation are carried out. The bacterial solution in the first stage is replaced with the bacterial solution in the second stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0060] Comparative Example 8

[0061] Compared with Example 1, the difference is that the second stage of fermentation is omitted, and only the first and third stages of fermentation are carried out. The bacterial solution in the second stage is replaced with the bacterial solution in the third stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0062] Comparative Example 9

[0063] Compared with Example 1, the difference is that the third stage of fermentation is omitted, and only the first and second stages of fermentation are carried out. The bacterial solution in the third stage is replaced by the bacterial solution in the first stage with an equivalent inoculation amount. The rest is the same as in Example 1.

[0064] Comparative Example 10

[0065] Compared with Example 1, the difference is that the strains for the first, second and third stages of fermentation are inoculated simultaneously (with an equivalent inoculation amount to Example 1) for synchronous fermentation, while the rest is the same as Example 1.

[0066] Effect testing methods

[0067] Skin irritation test: A human patch test was used. Mulberry leaf extracts prepared in Examples 1-3 (each prepared into a 10 wt% solution with deionized water) were applied to the back skin of volunteers using patch applicators soaked in the above solutions. The patches were changed every 24 hours and observed continuously for 72 hours. Whether irritation reactions such as erythema, edema, and itching occurred were recorded. The skin irritation was scored according to the skin irritation scoring criteria in the "Cosmetic Safety Technical Specifications (2015 Edition)". The results are shown in Table 1.

[0068] Cytotoxicity assay: Using the MTT assay with human skin fibroblasts as a model, different concentrations of mulberry leaf extracts from Examples 1-3 (prepared with deionized water to concentrations of 0.1 wt%, 0.5 wt%, 1.0 wt%, 5.0 wt%, and 10.0 wt%, respectively) were co-cultured with cells for 24 hours. After culturing, MTT solution was added, and the cells were cultured for another 4 hours. The supernatant was discarded, and dimethyl sulfoxide was added to dissolve and crystallize the cells. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated. The results are shown in Table 2.

[0069] TEWL test: Using a transdermal water loss meter, under the same environmental conditions (temperature 25±1℃, relative humidity 40±5%), 39 healthy volunteers were randomly divided into 13 groups of 3 people each. Mulberry leaf extracts prepared in Examples 1-3 and Comparative Examples 1-10 (prepared with deionized water to a concentration of 5wt%) were applied to the skin surface of each group. TEWL values ​​were measured before application and 24 hours after application. The average value for each group was taken, and the TEWL reduction rate was calculated. The results are shown in Table 3.

[0070] Stratum corneum moisture content test: Using a skin moisture meter, the skin stratum corneum moisture content was measured before and after applying the extract under the same environmental conditions (temperature 25±1℃, relative humidity 40±5%). (The mulberry leaf extracts prepared in Examples 1-3 and Comparative Examples 1-10 were each prepared into 5wt% solutions using deionized water). Thirty-nine healthy volunteers were randomly divided into 13 groups of 3 people each. The skin stratum corneum moisture content was measured before application and at 3 and 6 hours after application. The average value of each group was taken, and the increase rate of stratum corneum moisture content was calculated. The results are shown in Table 4.

[0071] Wrinkle depth test: A skin wrinkle tester was used to measure specific wrinkle areas on the volunteers' faces. Thirty-nine healthy volunteers were randomly divided into 13 groups of 3 people each. Wrinkle depth was measured before and 4 weeks after applying the extract (the mulberry leaf extracts prepared in Examples 1-3 and Comparative Examples 1-10 were respectively prepared into 5wt% solutions with deionized water). The average value of each group was taken, and the wrinkle depth reduction rate was calculated. The results are shown in Table 5.

[0072] Skin permeability test: A Franz diffusion cell was used. Pig skin (simulating human skin) was fixed between the supply and receiving chambers of the diffusion cell. Mulberry leaf extracts from Examples 1-3 and Comparative Examples 1-10 (prepared with deionized water to a concentration of 5 wt%) were added to the supply chamber. Phosphate-buffered saline (PBS, pH 7.4) was added to the receiving chamber. The experiment was conducted at 32°C and a stirring speed of 300 r / min. Samples were taken from the receiving chamber after 24 hours, and the content of active ingredients (flavonoids) in the samples was determined by HPLC to calculate the skin permeability. After the Franz diffusion cell experiment, the pig skin was removed, processed, and the content of active ingredients in the skin tissue was measured. The bioavailability was calculated by comparing the content with the initial content of active ingredients in the supply chamber. The results are shown in Table 6.

[0073] Test Results

[0074] Table 1 Results of skin irritation test

[0075]

[0076]

[0077] Table 2. Cytotoxicity test results (cell viability)

[0078]

[0079] This invention removes some tannins through glycolysis by brewer's yeast, and the acid metabolism of the compound bacteria further neutralizes irritating substances, reducing irritation. Skin irritation tests in Examples 1-3 showed no erythema, edema, itching, or other irritating reactions. In cytotoxicity tests, even at a high concentration of 10 wt%, the cell survival rate of the examples remained at 81.4%-83.5%. This is attributed to the three-stage fermentation process, which degrades macromolecules (such as crude protein and crude fiber) in mulberry leaves into small peptides, free amino acids, and small sugars, components with good skin compatibility. Simultaneously, the organic acids produced in the second stage are consistent with the weakly acidic environment of healthy skin, avoiding the risk of strong irritation. This demonstrates that multiple fermentation not only enhances efficacy but also significantly improves the safety of raw materials.

[0080] Table 3 TEWL Test Results

[0081]

[0082]

[0083] The TEWL reduction rate of Examples 1-3 was significantly higher than that of Comparative Examples 1-10, especially significantly better than the unenzymatically hydrolyzed control group (Comparative Example 1). Comparative Example 1, due to only mechanical disruption and ineffective release of polysaccharides and proteins, had a TEWL reduction rate of only 20.4%. The single-enzyme hydrolysis groups (Comparative Examples 2-3) were still inferior to Examples 1-3 due to incomplete enzymatic hydrolysis. This indicates that compound enzymatic hydrolysis is a key prerequisite for enhanced efficacy, providing a substrate basis for the subsequent three-stage fermentation. Compound enzymatic hydrolysis can break down the cell walls of mulberry leaves, synergistically enhancing the skin barrier in conjunction with the small-molecule sugars, organic acids, and free flavonoids produced during the three-stage fermentation. Single-stage fermentation (Comparative Examples 4-6), dual-stage fermentation (Comparative Examples 7-9), or mixed simultaneous fermentation groups (Comparative Example 10) lacked a complete metabolic chain, failing to form a synergistic system, resulting in decreased skin barrier function.

[0084] Table 4 Results of the test on the moisture content of the stratum corneum

[0085]

[0086]

[0087] The moisturizing effects of Examples 1-3 were significantly better than those of Comparative Examples 1-10. Comparative Example 1, lacking an enzymatic hydrolysis step, had insufficient release of active ingredients, resulting in a 4.7% increase in stratum corneum moisture content over 6 hours. The increase rates of the single-enzyme hydrolysis groups (Comparative Examples 2-3) were also significantly lower than those of Examples 1-3, indicating that the component transformation in the single-enzyme hydrolysis groups was insufficient. Compound enzymatic hydrolysis can promote the release of moisturizing components and enhance the moisturizing effect of the extract. Example 3 achieved a 47.3% increase in stratum corneum moisture content over 6 hours, thanks to the rapid hydration of small-molecule sugars and the long-lasting water-locking effect of free amino acids and small-molecule peptides. However, single-stage fermentation (Comparative Examples 4-6), two-stage fermentation (Comparative Examples 7-9), or mixed simultaneous fermentation groups (Comparative Example 10) lacked a complete metabolic chain, failing to form a synergistic system and exhibiting poor moisturizing durability.

[0088] Table 5. Results of Anti-wrinkle Function Test

[0089]

[0090]

[0091] The wrinkle depth reduction rate (31.9%-44.5%) of Examples 1-3 after 4 weeks was significantly higher than that of Comparative Examples 1-10, demonstrating that the synergistic effect of compound enzymatic hydrolysis and staged fermentation can significantly enhance the anti-wrinkle effect. In the third stage, the protease secreted by Bacillus licheniformis degrades mulberry leaf protein into small molecule peptides, which can penetrate the stratum corneum and stimulate fibroblasts to synthesize collagen; the free flavonoids produced in the second stage reduce collagen degradation through antioxidant effects. The anti-wrinkle effect of Comparative Examples 1-10 was significantly reduced due to defects in the preparation process, indicating that stepwise fermentation can achieve the orderly metabolism of effective ingredients, thereby improving the anti-wrinkle effect.

[0092] Table 6 Results of Bioavailability and Skin Penetration Tests

[0093]

[0094]

[0095] The 24-hour skin penetration rate (48.7%-56.8%) and bioavailability (47.8%-55.9%) of Examples 1-3 were significantly higher than those of Comparative Examples 1-10. The main reason is the regulation of the molecular weight of the components by the compound enzymatic hydrolysis and multi-stage fermentation process. The compound enzymatic hydrolysis (cellulase and pectinase) breaks down the cell walls of mulberry leaves, thereby increasing the release rate of active ingredients. The three-stage fermentation degrades large polysaccharides into small sugars, hydrolyzes flavonoids into free flavonoids, and converts proteins into small peptides and free amino acids. These components can penetrate efficiently through the gaps in the stratum corneum.

[0096] Comparative Examples 1-10 showed reduced permeability due to defects in the preparation process. The single-enzyme hydrolysis group (Comparative Examples 2-3) could only degrade some cellulose or pectin, and the remaining large molecules hindered penetration. The mixed simultaneous fermentation group (Comparative Example 10) produced some polymers due to interference from microbial metabolism, which blocked the stratum corneum channels, resulting in a skin permeability of only 23.5% after 24 hours. This indicates that the synergistic effect of compound enzymatic hydrolysis and staged multi-stage fermentation processes can achieve the degradation and efficient penetration of small molecule sugars, free flavonoids, small molecule peptides, free amino acids, and other components, ultimately achieving the synergistic effects of strengthening the skin barrier, long-lasting moisturizing, and dynamic anti-wrinkle.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention 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 the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing mulberry leaf extract through multiple fermentation, characterized in that, Includes the following steps: S1. Pretreatment with compound enzymatic hydrolysis: Weigh mulberry leaf powder and deionized water at a mass ratio of 1:100, mix and stir evenly to form a culture medium, add a compound enzyme of cellulase and pectinase, the inoculation amount of the compound enzyme is 1-2 wt% of the culture medium, the mass ratio of cellulase to pectinase is (1-3):2, and enzymatic hydrolysis is carried out at 48-52℃ and pH 4.8 for 4.5-5.5 hours. After enzymatic hydrolysis, the hydrolysate is sterilized and cooled. S2, First stage fermentation: Transfer the sterilized enzymatic hydrolysate to a fermenter, inoculate with brewer's yeast culture, and ferment for 28-32 hours under anaerobic conditions at 26-30℃ and pH 6.0-6.

4. S3, Second stage fermentation: Inoculate with Lactobacillus acidophilus-Staphylococcus epidermidis compound bacterial solution and ferment for 38-42 hours at 28-32℃, pH 5.0-5.4, under static micro-aerobic environment; S4. Third stage fermentation: Inoculate with Bacillus licheniformis culture and ferment for 48-52 hours at 33-37℃, pH 6.8-7.2, and with aeration. S5. Purification: The fermentation broth is extracted by ultrasonication, centrifugation, ultrafiltration, vacuum concentration and freeze drying to obtain mulberry leaf extract.

2. The method according to claim 1, characterized in that, In step S2, the concentration of the brewer's yeast culture is 1.0 × 10⁻⁶. 8 -2.0×10 8 CFU / mL, inoculum volume is 2-3 wt% of the culture medium.

3. The method according to claim 1, characterized in that, In step S3, the mass ratio of Lactobacillus acidophilus to Staphylococcus epidermidis is 1:2, and the total concentration of the compound bacterial solution is 2.0 × 10⁻⁶. 8 -3.0×10 8 CFU / mL, with an inoculum volume of 3-5 wt% of the culture medium.

4. The method according to claim 1, characterized in that, In step S3, the micro-oxygen environment is achieved by controlling dissolved oxygen at 0.5-1.0 mg / L through nitrogen replacement.

5. The method according to claim 1, characterized in that, In step S4, the concentration of Bacillus licheniformis bacterial solution is 0.8 × 10⁻⁶. 8 -1.2×10 8 The inoculum volume is 2-4 wt% of the culture medium, and the aeration rate is 1.5 L / min.

6. The method according to claim 1, characterized in that, In step S5, the ultrasonic extraction power is 180-220W, and the treatment is carried out at 45-55℃ for 25-35 minutes; the ultrafiltration uses a 10kDa ultrafiltration membrane.

7. A mulberry leaf extract prepared by the method according to any one of claims 1-6.

8. The use of the mulberry leaf extract according to claim 7 in the preparation of cosmetics that enhance the skin barrier function.

9. The use of the mulberry leaf extract according to claim 7 in the preparation of moisturizing cosmetics.

10. The use of the mulberry leaf extract according to claim 7 in the preparation of anti-wrinkle cosmetics.

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