Preparation method of fermented yeast polypeptide composition with whitening effect

Abstract

The invention discloses a preparation method of a fermented yeast polypeptide composition with a whitening effect, and relates to the technical field of biological fermentation technologies, bioactive polypeptides and cosmetic raw materials. The method comprises the following steps: by taking schizosaccharomyces pombe as a raw material, carrying out liquid fermentation with a nitrogen-enhanced culture medium, carrying out high-pressure homogenization wall breaking, separating protein and carrying out directional enzymolysis with compound protease, so as to obtain an active product mainly comprising micromolecule polypeptide. The molecular weight of the obtained polypeptide is mainly smaller than 1 kDa, and the polypeptide has good biocompatibility and stability. In-vitro cell experiments, zebra fish model and enzymology experiment results show that the yeast polypeptide composition can significantly inhibit tyrosinase activity and reduce melanogenesis, has anti-oxidation and anti-inflammatory effects, is high in safety, and is suitable for being used as a whitening functional component in cosmetics, skin care products or external preparations.

Description

Technical Field

[0001] This invention relates to the fields of bio-fermentation technology, bioactive peptides and cosmetic raw materials, and in particular to a method for preparing a fermented yeast peptide composition with whitening effects. Background Technology

[0002] With increasing public awareness of skin tone evenness and skin health, skin-whitening cosmetics have become an important part of functional skincare products. Commonly used skin-whitening active ingredients include vitamin C and its derivatives, arbutin, niacinamide, and tranexamic acid. However, these ingredients generally suffer from problems in practical applications, such as insufficient stability, strong irritation, controversies surrounding long-term safety, or limited efficacy.

[0003] In recent years, bioactive peptides derived from microorganisms have gradually become a hot topic in cosmetic raw material research and development due to their small molecular weight, good biocompatibility, low toxicity, and multi-target regulatory capabilities. Yeast, as a relatively safe microorganism, has had its metabolites reportedly used in the skincare field. However, current technologies mostly focus on yeast fermentation broth or cell lysates, which contain complex components, have unclear activity mechanisms, and lack targeted preparation and efficacy verification of peptide components.

[0004] Especially in the field of skin whitening, there is a lack of yeast-derived polypeptide products that are obtained through systematic fermentation control, cell wall disruption extraction, and enzymatic hydrolysis processes, with small molecule active polypeptides as the main component, and whose effects of inhibiting melanin production, anti-inflammation, and anti-oxidation have been confirmed by in vitro and in vivo multi-model experiments. Therefore, it is necessary to develop a yeast polypeptide skin whitening raw material that is highly safe, has clear efficacy, and is suitable for industrialization. Summary of the Invention

[0005] Based on the problems raised in the background art mentioned above, the present invention proposes a method for preparing a fermented yeast polypeptide composition with whitening effect.

[0006] The technical solution is as follows: A method for preparing a fermented yeast polypeptide composition with whitening effects includes the following steps: (1) Yeast fermentation culture steps: Prepare 100 parts of fermentation culture medium by weight of the following raw materials: 8-15 parts yeast extract; 15-30 parts peptone; 15-30 parts glucose; 3-8 parts ammonium sulfate; 1-4 parts of potassium dihydrogen phosphate; Magnesium sulfate heptahydrate, 0.2–1.0 parts; Organic acid regulator 0.3–1.0 parts; Deionized water balance; Adjust the pH of the culture medium to 5.5–6.0, sterilize it, inoculate with Saccharomyces cerevisiae, and carry out liquid fermentation culture at 28–32℃ and 180–240 rpm. Collect the cells when the OD600 of the bacterial solution reaches 10–18. (2) Bacterial cell collection and washing steps: Centrifuge the fermentation broth at 3000-6000×g for 3-10 min, collect the cells, and wash them 1-3 times with buffer solution; (3) High-pressure homogenization and cell wall disruption steps: The washed bacterial cells were resuspended in buffer solution and subjected to high-pressure homogenization at 1000–1300 bar for 3–5 cycles, with the temperature controlled at 2–8°C, to obtain crude bacterial extract. (4) Protein component separation steps: Centrifuge the crude bacterial extract at 8000-12000×g for 5-20 min, and take the supernatant as the protein component solution. (5) Enzymatic hydrolysis for preparing polypeptides: The protein component solution was adjusted to a protein mass fraction of 3-8% and a pH of 7.0-7.5. Enzymatic hydrolysis was carried out at 45-60°C by adding protease at a protein mass of 0.5-2.0% for 3-8 hours. (6) Enzyme inactivation and polypeptide acquisition steps: The enzymatic hydrolysate is heated to 85–100°C and held for 10–30 minutes to inactivate the enzyme. Then, the supernatant is collected by centrifugation and filtered for sterilization to obtain a fermented yeast polypeptide composition with whitening effects.

[0007] The organic acid regulator is selected from one or more of malic acid, citric acid, or tartaric acid.

[0008] The inoculation amount of the yeast *Schizosacchariformis* was 0.5–3.0% of the culture medium mass.

[0009] The buffer solution is selected from one or a combination of Na2HPO4, KH2PO4, NaCl, and KCl.

[0010] The single homogenization time for the high-pressure homogenization is 10–60 s.

[0011] The protease is a modified neutral protease, and its preparation method is as follows: By weight, mix 40-60 parts of neutral protease with 220-380 parts of 0.15-0.35M KH₂PO₄-K₂HPO₄ (pH 8.0) buffer, add 3-8 parts of glycerol as a stabilizer, and add 1-3M CaCl₂ stock solution to a final concentration of 2mM. Separately, take 2-5 parts of Angelica dahurica root extract and prepare a 5%-10% (w / v) stock solution with cosmetic-grade propylene glycol, controlling the final concentration of propylene glycol in the system to 5%-10%. Slowly add the stock solution dropwise to the enzyme solution (1-3 mL / min, stirring continuously), then add 0.5-0.9 parts of sodium bicarbonate, and react at 32-36℃ and 100-200 rpm under light protection and N₂ for 1-3 hours. The reaction solution is then directly loaded onto a Sephadex G-25 gel column with 0.02-0.05M potassium phosphate buffer (pH 8.0). Isocratic elution was performed, and the main enzyme peak was collected. A 5-10 kDa molecular weight cutoff ultrafiltration membrane was used to concentrate the enzyme under low-speed stirring at ≤25℃ and 0.1-0.3 MPa transmembrane pressure. The concentrate was then replaced with 0.02-0.05 M potassium phosphate (pH 8.0) buffer by ultrafiltration, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography with a linear gradient of 0.1→0.5 M NaCl. The highly active fraction was collected to obtain the cosmetic-grade modified neutral protease.

[0012] The pH of the ethanol is 7.0-7.6.

[0013] The concentration of the NaOH solution is 0.05-0.2 mol / L.

[0014] The enzymatic hydrolysis process is carried out by stirring at a speed of 100–500 rpm.

[0015] The average molecular weight of the fermented yeast polypeptide composition prepared is less than 1 kDa.

[0016] Mechanism of reaction of modified neutral protease: The amino group in the neutral protease molecule undergoes a ring-opening addition reaction with the epoxy group on the root extract of *Angelica dahurica*, achieving chemical bonding through a covalent bond. This reaction preserves the core catalytically active structure of the neutral protease while optimizing enzyme stability through structural modification with *Angelica dahurica* root extract. Under optimized enzymatic hydrolysis conditions (such as controlling the degree of hydrolysis), the modified protease exhibits improved stability in a yeast proteolysis system, contributing to the yield of polypeptide products with a more concentrated molecular weight distribution. Effects of modified neutral protease: The amino-epoxy ring-opening addition reaction allows for precise control of the degree of enzyme modification, avoiding excessive loss of enzyme activity. The modified neutral protease exhibits enhanced stability under enzymatic hydrolysis conditions, enabling it to maintain its catalytic activity and improve peptide conversion. Covalent modification ensures a strong bond between the enzyme and the modifying agent, preventing detachment during the reaction and guaranteeing product homogeneity.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. Obtain yeast active products mainly composed of small molecule peptides, with high safety; 2. It synergistically inhibits melanin production across multiple targets, with a clear mechanism of action; 3. The preparation process is controllable, green and environmentally friendly, and suitable for industrial scale-up; 4. It can be widely used in cosmetics and skin care products. Attached Figure Description

[0018] Figure 1 To detect the molecular weight distribution of peptides in fermenting yeast (Tricine-SDS-PAGE experiment). Figure 2 For yeast polypeptide cytotoxicity testing; Figure 3 Detection of yeast peptide toxicity in a zebrafish developmental model; Figure 4 The graph shows the detection of anti-inflammatory activity - NO. Figure 5 An attempt to detect anti-inflammatory activity and reactive oxygen species; Figure 6 Image of zebrafish test for whitening activity; 1XPTU-propylthiouracil; Glutathione; Nonapeptide-1; Mixed-yeast polypeptide composition; Glycyrrhizin; Peptidone-Peptidone.

[0019] Figure 7 This is a statistical graph showing the inhibition rate of yeast polypeptides on melanin synthesis in B16 cells.

[0020] Figure 8 This is a statistical graph showing the inhibition of tyrosinase activity by yeast polypeptides. Detailed Implementation

[0021] The features of the present invention are further illustrated below through embodiments, but the scope of protection of this patent is not limited to the embodiments. Example

[0022] A method for preparing a fermented yeast polypeptide composition with whitening effects includes the following steps: (1) Yeast fermentation culture steps: Prepare 100g of fermentation culture medium by weight, with the following amounts of raw materials: 8g yeast extract, 15g peptone, 15g glucose, 3g ammonium sulfate, 1g potassium dihydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.3g organic acid regulator (malic acid, purity ≥99.0%), and 47.5g deionized water (to bring the total to 100g). Add the above raw materials to a 500mL Erlenmeyer flask in sequence, stir to dissolve, and then adjust the pH of the culture medium to 5.5 with 0.1mol / L hydrochloric acid; then place the Erlenmeyer flask in an autoclave (model LS-50HD) and sterilize at 121℃ for 20min, and cool to room temperature for later use. Inoculate 0.5% of the culture medium mass with *Schizosaccharomyces cerevisiae* (strain number ATCC 24843), with an inoculation amount of 0.5g. Place the inoculated culture medium in a constant temperature shaker (model THZ-300), set the temperature to 28℃ and the rotation speed to 180rpm, and carry out liquid fermentation culture. During the fermentation process, take samples every 2 hours to detect the OD600 value of the bacterial solution. When the OD600 reaches 10, stop the fermentation and collect the fermentation broth.

[0023] (2) Cell collection and washing steps: Transfer the collected fermentation broth to a high-speed refrigerated centrifuge (model TGL-20M), set the centrifugation speed to 3000×g and the temperature to 4℃, and centrifuge for 3min; after centrifugation, discard the supernatant and collect the bottom cells. Use Na2HPO4-KH2PO4 buffer (concentration 0.05mol / L, pH 7.0) as the washing solution, add 50mL of washing solution to resuspend the cells, centrifuge again (3000×g, 4℃, 3min), repeat the washing once, and finally collect the washed cells.

[0024] (3) High-pressure homogenization cell disruption step: The washed bacterial cells were resuspended in 60 mL of Na2HPO4-KH2PO4 buffer (0.05 mol / L, pH 7.0) to prepare a bacterial suspension; the bacterial suspension was transferred to a high-pressure homogenizer (model AH-BASIC), the homogenization pressure was set to 1000 bar, the temperature was 2℃ (temperature controlled by a low-temperature circulation system), the homogenization time was 10 s per cycle, and the homogenization was repeated 3 times; after homogenization, the crude bacterial extract was obtained and placed in an ice bath for later use.

[0025] (4) Protein component separation steps: Transfer the crude bacterial extract into a high-speed refrigerated centrifuge, set the centrifugation speed to 8000×g and the temperature to 4℃, and centrifuge for 5min; after centrifugation, carefully aspirate the supernatant, which is the protein component solution, and the protein concentration is 2.8mg / mL.

[0026] 5. (5) Enzymatic hydrolysis preparation of peptides: Take 50 mL of protein fraction solution, add an appropriate amount of deionized water to adjust the protein mass fraction to 3%, and adjust the pH to 7.0 with 0.1 mol / L NaOH solution; transfer the solution to a constant temperature water bath stirrer (model HH-S), set the temperature to 45℃ and the stirring speed to 100 rpm, and stir at a constant temperature for 10 min. Add modified neutral protease (addition amount 0.042 g) at 0.5% of the protein mass, start the enzymatic hydrolysis reaction, and continue stirring for 3 h. During this period, monitor the pH every 30 min to ensure that the pH is stable at 7.0±0.1.

[0027] The modified neutral protease is prepared as follows: by weight, the protease is a modified neutral protease, and its preparation method is as follows: By weight, 40 parts of neutral protease and 220 parts of 0.15M KH2PO4-K2HPO4 (pH 8.0) buffer were mixed, and 3 parts of glycerol were added as a stabilizer. 1M CaCl2 stock solution was added by volume to bring the final concentration of the system to 2mM. Separately, 2 parts of Angelica dahurica root extract were prepared into a 5% (w / v) stock solution using cosmetic-grade propylene glycol. The final concentration of propylene glycol in the system was controlled at 5%. The stock solution was slowly added dropwise to the enzyme solution (1mL / min, with stirring), and then 0.5 parts of sodium bicarbonate were added. The reaction was carried out at 32℃ and 100rpm under light protection and N2 protection for 1 hour. The reaction solution was directly loaded onto a Sephadex G-25 gel column and eluted isocratically with 0.02M potassium phosphate buffer (pH 8.0). The main enzyme peak was collected, and an ultrafiltration membrane with a 5kDa molecular weight cutoff was used at ≤25℃ and 0.1MPa. The solution was concentrated by low-speed stirring under transmembrane pressure; the concentrate was replaced with 0.02M potassium phosphate (pH 8.0) buffer by ultrafiltration, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography with a linear gradient of 0.1M NaCl. The highly active component was collected to obtain cosmetic-grade modified neutral protease, which was then sealed and refrigerated for later use.

[0028] (6) Enzyme inactivation and peptide acquisition steps: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysate is transferred to a constant temperature water bath, heated to 85°C, and kept at a constant temperature for 10 min to inactivate the enzyme; then the enzyme-inactivated solution is transferred to a high-speed refrigerated centrifuge and centrifuged (8000×g, 4°C, 5 min), and the supernatant is collected; the supernatant is filtered through a 0.22μm polyethersulfone filter membrane (model SLGP033RB) to remove bacteria, and a clear fermented yeast peptide composition is obtained.

[0029] According to gel permeation chromatography (GPC), the average molecular weight of the composition is 850 Da (less than 1 kDa), which meets the requirements for whitening peptides. Example

[0030] A method for preparing a fermented yeast polypeptide composition with whitening effects includes the following steps: (1) Yeast fermentation culture steps: Prepare 100g of fermentation culture medium by weight, with the following amounts of raw materials: 12.75g yeast extract, 25.75g peptone, 25.75g glucose, 6.25g ammonium sulfate, 3g potassium dihydrogen phosphate, 0.75g magnesium sulfate heptahydrate, 0.8g organic acid regulator (tartaric acid and citric acid mixed in a 1:1 mass ratio, with a purity ≥99.0%), and 35.95g deionized water (to bring the total to 100g). Add the above raw materials to a 500mL Erlenmeyer flask in sequence, stir to dissolve, and then adjust the pH of the culture medium to 5.8 with 0.1mol / L hydrochloric acid; then place the Erlenmeyer flask in an autoclave and sterilize at 121℃ for 20min, and cool to room temperature for later use. Inoculate 2.25% of the culture medium mass with *Schizosaccharomyces cerevisiae* (strain number ATCC 24843), with an inoculation amount of 2.25g. Place the inoculated culture medium in a constant temperature shaker, set the temperature to 30.5℃ and the rotation speed to 220rpm, and carry out liquid fermentation culture. During the fermentation process, take samples every 2 hours to detect the OD600 value of the bacterial solution. When the OD600 reaches 15.5, stop the fermentation and collect the fermentation broth.

[0031] (2) Cell collection and washing steps: Transfer the collected fermentation broth to a high-speed refrigerated centrifuge, set the centrifugation speed to 5000×g and the temperature to 4℃, and centrifuge for 8 min; after centrifugation, discard the supernatant and collect the bottom cells. Use Na2HPO4-KCl buffer (concentration 0.1mol / L, pH 7.4) as the washing solution, add 70mL of washing solution to resuspend the cells, centrifuge again (5000×g, 4℃, 8 min), repeat the washing 3 times, and finally collect the washed cells.

[0032] (3) High-pressure homogenization cell disruption step: The washed bacterial cells were resuspended in 80 mL of Na2HPO4-KCl buffer (0.1 mol / L, pH 7.4) to prepare a bacterial suspension; the bacterial suspension was transferred to a high-pressure homogenizer, and the homogenization pressure was set to 1200 bar and the temperature to 6 °C (controlled by a low-temperature circulation system). The homogenization time was 45 s for each homogenization, and the homogenization was repeated 5 times; after homogenization, the crude bacterial extract was obtained and placed in an ice bath for later use.

[0033] (4) Protein component separation steps: Transfer the crude bacterial extract into a high-speed refrigerated centrifuge, set the centrifugation speed to 10500×g and the temperature to 4℃, and centrifuge for 15min; after centrifugation, carefully aspirate the supernatant, which is the protein component solution, and the protein concentration is 3.2mg / mL.

[0034] (5) Enzymatic hydrolysis preparation of peptides: Take 50 mL of protein fraction solution, add an appropriate amount of deionized water to adjust the protein mass fraction to 6.5%, and adjust the pH to 7.4 with 0.1 mol / L NaOH solution; transfer the solution to a constant temperature water bath stirrer, set the temperature to 55℃ and the stirring speed to 350 rpm, and stir at a constant temperature for 10 min. Add modified neutral protease (0.102 g) at 1.5% of the protein mass to start the enzymatic hydrolysis reaction, and continue stirring for 6.5 h. During this period, monitor the pH every 30 min to ensure that the pH is stable at 7.4 ± 0.1.

[0035] The modified neutral protease is prepared as follows: by weight, the protease is a modified neutral protease, and its preparation method is as follows: By weight, 50 parts of neutral protease and 280 parts of 0.25M KH2PO4-K2HPO4 (pH 8.0) buffer were mixed, and 6 parts of glycerol were added as a stabilizer. 2M CaCl2 stock solution was added to a final concentration of 2mM. Separately, 4 parts of Angelica dahurica root extract were prepared into a 7% (w / v) stock solution using cosmetic-grade propylene glycol. The final concentration of propylene glycol in the system was controlled at 7%. The stock solution was slowly added dropwise to the enzyme solution (2mL / min, with stirring), followed by the addition of 0.6 parts of sodium bicarbonate. The reaction was carried out at 34℃ and 150rpm under light protection and N2 protection for 2 hours. The reaction solution was directly loaded onto a Sephadex G-25 gel column and eluted isocratically with 0.04M potassium phosphate buffer (pH 8.0). The main enzyme peak was collected, and an 80kDa molecular weight cutoff ultrafiltration membrane was used for filtration at ≤25℃ and 0.2MPa. The solution was concentrated by low-speed stirring under transmembrane pressure; the concentrate was replaced with 0.04M potassium phosphate (pH 8.0) buffer by ultrafiltration, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography with a linear gradient of 0.3M NaCl to collect the highly active component, which yielded the cosmetic-grade modified neutral protease.

[0036] (6) Enzyme inactivation and peptide acquisition steps: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysate is transferred to a constant temperature water bath, heated to 95°C, and kept at a constant temperature for 23 min to inactivate the enzyme; then the enzyme-inactivated solution is transferred to a high-speed refrigerated centrifuge and centrifuged (10500×g, 4°C, 12 min), and the supernatant is collected; the supernatant is filtered through a 0.22μm polyethersulfone filter membrane to remove bacteria, and a clear fermented yeast peptide composition is obtained, named whitening peptide 2.

[0037] According to gel permeation chromatography (GPC), the average molecular weight of the composition is 720 Da (less than 1 kDa), which meets the requirements for whitening peptides. Example

[0038] A method for preparing a fermented yeast polypeptide composition with whitening effects includes the following steps: (1) Yeast fermentation culture steps: Prepare 100g of fermentation culture medium by weight, with the following amounts of raw materials: 15g yeast extract, 30g peptone, 30g glucose, 8g ammonium sulfate, 4g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 1.0g organic acid regulator (tartaric acid, purity ≥99.0%), and 31g deionized water (to bring the total to 100g). Add the above raw materials to a 500mL Erlenmeyer flask in sequence, stir to dissolve, and then adjust the pH of the culture medium to 6.0 with 0.1mol / L hydrochloric acid; then place the Erlenmeyer flask in an autoclave and sterilize at 121℃ for 20min, and cool to room temperature for later use. Inoculate 3.0% of the culture medium mass with *Schizosaccharomyces cerevisiae* (strain number ATCC24843), with an inoculation amount of 3.0g. Place the inoculated culture medium in a constant temperature shaker, set the temperature to 32℃ and the rotation speed to 240rpm, and carry out liquid fermentation culture. During the fermentation process, take samples every 2 hours to detect the OD600 value of the bacterial solution. When the OD600 reaches 18, stop the fermentation and collect the fermentation broth.

[0039] (2) Cell collection and washing steps: Transfer the collected fermentation broth to a high-speed refrigerated centrifuge, set the centrifugation speed to 6000×g and the temperature to 4℃, and centrifuge for 10 min; after centrifugation, discard the supernatant and collect the bottom cells. Use Na2HPO4-KH2PO4-KCl buffer (concentration 0.1mol / L, pH 7.6) as the washing solution, add 80mL of washing solution to resuspend the cells, centrifuge again (6000×g, 4℃, 10 min), repeat the washing 3 times, and finally collect the washed cells.

[0040] (3) High-pressure homogenization cell disruption step: The washed bacterial cells were resuspended in 90 mL of Na2HPO4-KH2PO4-KCl buffer (0.1 mol / L, pH 7.6) to prepare a bacterial suspension; the bacterial suspension was transferred to a high-pressure homogenizer, and the homogenization pressure was set to 1300 bar and the temperature to 8 °C (controlled by a low-temperature circulation system). The homogenization time was 60 s for each homogenization, and the homogenization was repeated 5 times; after homogenization, the crude bacterial extract was obtained and placed in an ice bath for later use.

[0041] (4) Protein component separation steps: Transfer the crude bacterial extract into a high-speed refrigerated centrifuge, set the centrifugation speed to 12000×g and the temperature to 4℃, and centrifuge for 20min; after centrifugation, carefully aspirate the supernatant, which is the protein component solution, and the protein concentration is 3.5mg / mL.

[0042] (5) Enzymatic hydrolysis preparation of peptides: Take 50 mL of protein fraction solution, add an appropriate amount of deionized water to adjust the protein mass fraction to 8%, and adjust the pH to 7.5 with 0.1 mol / L NaOH solution; transfer the solution to a constant temperature water bath stirrer, set the temperature to 60℃ and the stirring speed to 500 rpm, and stir at a constant temperature for 10 min. Add modified neutral protease (0.14 g) at 2.0% of the protein mass to start the enzymatic hydrolysis reaction, and continue stirring for 8 h. During this period, monitor the pH every 30 min to ensure that the pH is stable at 7.5 ± 0.1.

[0043] The protease is a modified neutral protease, and its preparation method is as follows: By weight, 60 parts of neutral protease and 380 parts of 0.35M KH2PO4-K2HPO4 (pH 8.0) buffer were mixed, and 8 parts of glycerol were added as a stabilizer. 3M CaCl2 stock solution was added by volume to bring the final concentration of the system to 2mM. Separately, 5 parts of Angelica dahurica root extract were prepared into a 10% (w / v) stock solution using cosmetic-grade propylene glycol. The final concentration of propylene glycol in the system was controlled at 10%. The stock solution was slowly added dropwise to the enzyme solution (3mL / min, with stirring), and then 0.9 parts of sodium bicarbonate were added. The reaction was carried out at 36℃ and 200rpm under light protection and N2 protection for 3 hours. The reaction solution was directly loaded onto a Sephadex G-25 gel column and eluted isocratically with 0.05M potassium phosphate buffer (pH 8.0). The main enzyme peak was collected, and an ultrafiltration membrane with a 10kDa molecular weight cutoff was used at ≤25℃ and 0.3MPa. The solution was concentrated by low-speed stirring under transmembrane pressure; the concentrate was replaced with 0.05M potassium phosphate (pH 8.0) buffer by ultrafiltration, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography with a linear gradient of 0.5M NaCl to collect the highly active component, which yielded the cosmetic-grade modified neutral protease.

[0044] (6) Enzyme inactivation and peptide acquisition steps: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysate is transferred to a constant temperature water bath, heated to 100℃, and kept at a constant temperature for 30 min to inactivate the enzyme; then the enzyme-inactivated solution is transferred to a high-speed refrigerated centrifuge and centrifuged (12000×g, 4℃, 15 min), and the supernatant is collected; the supernatant is filtered through a 0.22μm polyethersulfone filter membrane to remove bacteria, and a clear fermented yeast peptide composition is obtained, named whitening peptide 3.

[0045] According to gel permeation chromatography (GPC), the average molecular weight of the composition is 680 Da (less than 1 kDa), which meets the requirements for whitening peptides.

[0046] Comparative Example 1 Competitor 1.

[0047] Comparative Example 2 Competitor 2.

[0048] Detection of the efficacy of fermented yeast peptides: 1) Yeast peptide cytotoxicity assay. Cell viability was assessed using CCK-8 assay. Logarithmically growing HepG2 cells were seeded at 2000–5000 cells / well in 96-well plates (100 μL / well) and cultured overnight at 37°C with 5% CO2. 50 μL of culture medium was aspirated, and 50 μL of complete culture medium containing 2× peptide was added. The medium was filtered to remove bacteria (0.22 μm), resulting in final concentrations of 12.5, 25, 50, 100, 200, and 400 μg / mL. A blank control (without peptide) was also performed. Cells were cultured for another 24 or 48 hours, with 3–6 replicates per concentration. 10 μL of CCK-8 reagent was added to each well, gently mixed, and incubated at 37°C in the dark for 1–4 hours. The OD value at 450 nm was measured using a microplate reader. Cell viability was calculated. The results showed that yeast peptides, whitening peptide 2, and whitening peptide 3 had no significant cytotoxicity at concentrations ranging from 12.5 to 200 ug / ml, while whitening peptide 3 exhibited significant toxicity at extremely high concentrations (400 ug / ml). Compared with the tested samples, competing products 1 and 2, commonly used whitening ingredients in the skincare market, both showed high cytotoxicity.

[0049] 2) Zebrafish developmental model for detecting yeast peptide toxicity. Fertilized eggs were obtained from natural spawning and cultured in E3 embryo culture medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.2). 4–6 hours post-fertilization (hpf), normally developing and undamaged blastocysts were selected for the experiment. Based on CCK8 results, the yeast peptide was diluted to 400 μg / mL using E3 medium. The solution was filtered to sterilize (0.22 μm). 20 embryos + 2 mL of peptide solution were added to each well of a 24-well plate. The plates were incubated at 28.5°C in the dark, with the solution replaced every 24 hours (to prevent degradation / contamination). Observations and records were kept until 120 hpf. Photographs were taken using a stereomicroscope. Results showed that the yeast peptide and whitening peptide 2 had no significant toxic effects; however, whitening peptide 3 showed extremely strong toxicity, and all eggs failed to develop.

[0050] 3) Yeast peptide anti-inflammatory activity assay (NO method). RAW264.7 macrophages were cultured at 2–5 × 10⁻⁵ ppm. 4Cells were seeded in 96-well plates (100 μL complete medium / well) and incubated overnight at 37°C with 5% CO2 (to ensure adherence). The old medium was discarded, and each well was supplemented with 90 μL fresh medium + 10 μL of the sample solution to be tested, to a final concentration of 50 μg / mL. After 30 minutes, LPS (final concentration 1 μg / mL) was added. A blank control was provided: no cells + medium; a negative control was provided: cells + LPS (no peptides). Incubation continued for 18–24 hours, and the supernatant was collected to measure NO. 50 μL of the cell culture supernatant was transferred to a new 96-well plate. 50 μL of Griess reagent (equal volumes of solution A: 1% sulfonamide; solution B: 0.1% NED) was added, and the reaction was carried out at room temperature in the dark for 10–15 minutes. The OD value was measured at 540 nm using a microplate reader. The OD was converted to NO concentration (μM) based on the NaNO2 standard curve (0–100 μM), and the NO inhibition rate (%) was calculated.

[0051] The results showed successful model establishment; whitening peptide 3 had no significant inhibitory effect on LPS-induced NO production, while whitening peptide 2 and yeast peptide both significantly reduced LPS-induced NO production; competitor 2 also showed a strong reducing effect. Competitor 1 was cytotoxic, leading to cell death.

[0052] 4) Detection of anti-inflammatory activity of yeast peptides (reactive oxygen species assay). RAW264.7 cells were cultured at 2–5 × 10⁻⁶ cells / year. 4 Cells were seeded in 96-well black-walled transparent plates (or ordinary 96-well plates) and cultured overnight at 37°C with 5% CO2 (to ensure adherence). The old medium was discarded, and 90 μL of fresh medium + 10 μL of peptide solution was added to each well, resulting in a final concentration of 50 μg / mL. After 30 minutes, LPS (final concentration 1 μg / mL) was added. A blank control (cell-free + medium) and a negative control (cells + LPS, peptide-free) were provided. Cells were cultured at 37°C for 4–6 hours (ROS peaks earlier than NO). The supernatant was discarded, and 100 μL of serum-free medium containing 10 μM DCFH-DA (2',7'-dichlorofluorescein diacetate) was added to each well. The cells were incubated at 37°C in the dark for 30–60 minutes. The cells were gently washed 1–2 times with PBS to remove probes that had not entered the cells. 100 μL of PBS was added to each well, and the cells were detected using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. Read the fluorescence intensity of each well and calculate the ROS content.

[0053] The results showed that the model was successfully established. Yeast peptides reduced the effect of LPS-induced reactive oxygen species production, while whitening peptides 2 and 3 had no reducing effect. Competitors 1 and 2 also did not show a reducing effect, and competitor 2 actually increased the effect of LPS-induced reactive oxygen species production.

[0054] 5) Effect of yeast peptides on melanin production in zebrafish. Fertilized eggs were obtained through natural mating and cultured in E3 medium at 28.5°C. At 24 hours post-fertilization (hpf), normally developing and malformed embryos were selected for the experiment. Embryos were transferred to 24-well plates, 10–15 embryos per well + 2 mL E3 medium. Test sample solutions were prepared at a concentration of 10 mg / mL. 1.5–20 μl of the test sample was added to each solution. The solution was filtered to remove bacteria (0.22 μm). The embryos were cultured at 28.5°C in the dark until 72–96 hpf (the period of massive melanin deposition). Melanin patches in the head were observed using a stereomicroscope. Embryos were fixed and photographed under the same lighting and magnification. Image analysis was performed using ImageJ software, selecting the average grayscale value of the head.

[0055] 6) Inhibition of tyrosinase activity by yeast peptides. Yeast peptide solutions were desalted, lyophilized, and prepared into sample solutions with concentrations of 5 mg / ml, 10 mg / ml, 15 mg / ml, and 20 mg / ml using 0.1 M phosphate buffer (pH 6.8). Arbutin at the same concentration was prepared as a positive control, and the buffer solution served as a negative control. In a 96-well plate, 50 μL of yeast peptide or arbutin solution and 100 μL of L-DOPA solution (1 mM) were added to each well. For the negative control, 150 μL of buffer solution was added. After incubation at 37°C for 5 min, 50 μL of tyrosinase solution was quickly added to each well, mixed, and placed in a microplate reader. No enzyme was added to the blank control to subtract spontaneous oxidation. Absorbance was read every 30 seconds at a wavelength of 475 nm, and the reaction curve was recorded after 5 min. Analyze the data, calculate the initial reaction rate for each group, and plot the concentration-inhibition rate curve using the formula: Inhibition rate (%) = [1 − (Sample rate / Negative control rate)] × 100%. Then, fit the curve to determine the IC50. 50 .

[0056] 7) Inhibitory effect of yeast peptides on melanin synthesis in B16 cells. B16 cell culture medium: RPMI 1640 (Pronosai CM-0029); cell plating: DMEM high-glucose medium. On day 1, cells were plated at a density of 2 × 10⁵ cells / well in 6-well plates and incubated overnight for 24 h. On day 2, the yeast peptide concentrations were added at 10 ppm, 1 ppm, and 0.1 ppm. On day 3, the corresponding concentrations of yeast peptide-containing medium were prepared according to the above protocol, and the medium was changed. On day 4 (48 h of drug treatment), the original medium was discarded, the cells were washed three times with PBS, digested with 500 μL of trypsin, neutralized with serum-containing medium at a 1:2 ratio, and the cell suspension was transferred to 1.5 mL centrifuge tubes. After centrifugation at 5000 rpm for 3 min, the supernatant was discarded, 1 mL of PBS was added, and the mixture was thoroughly mixed. The cell suspension was then added to a cell counting chamber for cell counting and recording. Melanin dissolution: After centrifugation and discarding the PBS supernatant, add 550 μL of 1M NaOH solution to each tube. Vortex thoroughly and heat in a 90℃ metal bath for 40 min, vortexing every 20 min. Centrifuge at 14,000×g for 10 min. Transfer the supernatant to a new 1.5 mL centrifuge tube, vortex to mix, and add 100 μL to each of five wells (96-well plate). Measure the absorbance at 405 nm. Use 1M NaOH solution as a blank control. Relative melanin content (%) = A sample / A blank × 100%.

[0057] Figure 1 Results: The molecular weight of yeast polypeptides was less than 1 kDa.

[0058] Figure 2 Results: The yeast polypeptide composition, whitening peptide 2, and whitening peptide 3 showed no toxicity at concentrations ranging from 12.5 to 200 ug / ml. Whitening peptide 3 exhibited toxicity at extremely high concentrations (400 ug / ml). Compared with the test samples, competing products 1 and 2 both showed high cytotoxicity.

[0059] Figure 3 Results: The ultra-high concentration (400 ug / ml) of yeast polypeptide composition and whitening peptide 2 showed no obvious toxic effects; however, whitening peptide 3 showed extremely strong toxic effects, and all fish eggs failed to develop.

[0060] Figure 4 Results: LPS-induced macrophage model was successfully established; both the yeast polypeptide composition and whitening peptide 2 significantly reduced LPS-induced NO production; whitening peptide 3 did not significantly inhibit LPS-induced NO production.

[0061] Figure 5Results: LPS-induced macrophage model was successfully established; yeast polypeptide composition, whitening peptide 2 and whitening peptide 3 did not significantly reduce LPS-induced reactive oxygen species production; competitor products 1 and 2 also did not show a reducing effect, and competitor product 2 actually increased the effect of LPS.

[0062] Figure 6 Results: Obvious melanin patches were visible on the heads of zebrafish in the control group (Control), while the 1×PTU group (positive control) showed a significant reduction in melanin patches due to inhibition of melanin synthesis. Among the experimental groups, the glutathione treatment group showed a significant decrease in the number of melanin patches, with the effect increasing with concentration, but cell viability also decreased. Yeast polypeptide composition and nonapeptide-1 significantly inhibited melanin formation in the zebrafish head in a dose-dependent trend, with no significant toxic effects on zebrafish. Glycyrrhizin and peptidylcholine induced zebrafish embryonic death, but no valid data were available.

[0063] Figure 7 Results: The yeast polypeptide composition exhibited a concentration-dependent inhibitory effect on melanin production in B16 cells. The relative melanin content in B16 cells decreased sequentially with increasing concentration of the yeast polypeptide composition.

[0064] Figure 8 Results: Both the yeast polypeptide composition and arbutin showed significant inhibitory effects on tyrosinase within a certain concentration range, and the inhibition rate was positively correlated with the concentration. The IC50 of the yeast polypeptide composition was calculated to be 11.3 mg / mL, significantly stronger than that of the positive control arbutin (IC50 = 16.81 mg / mL), indicating that the yeast polypeptide composition can significantly inhibit tyrosinase activity.

[0065] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a fermented yeast polypeptide composition with whitening effects, characterized in that, Includes the following steps: (1) Yeast fermentation culture steps: Prepare 100 parts of fermentation culture medium by weight of the following raw materials: 8-15 parts yeast extract; 15-30 parts peptone; 15-30 parts glucose; 3-8 parts ammonium sulfate; 1-4 parts of potassium dihydrogen phosphate; Magnesium sulfate heptahydrate, 0.2–1.0 parts; Organic acid regulator 0.3–1.0 parts; Deionized water balance; Adjust the pH of the culture medium to 5.5–6.0, sterilize it, inoculate with Saccharomyces cerevisiae, and carry out liquid fermentation culture at 28–32℃ and 180–240 rpm. Collect the cells when the OD600 of the bacterial solution reaches 10–18. (2) Bacterial cell collection and washing steps: Centrifuge the fermentation broth at 3000-6000×g for 3-10 min, collect the cells, and wash them 1-3 times with buffer solution; (3) High-pressure homogenization and cell wall disruption steps: The washed bacterial cells were resuspended in buffer solution and subjected to high-pressure homogenization at 1000–1300 bar for 3–5 cycles, with the temperature controlled at 2–8°C, to obtain crude bacterial extract. (4) Protein component separation steps: Centrifuge the crude bacterial extract at 8000-12000×g for 5-20 min, and take the supernatant as the protein component solution. (5) Enzymatic hydrolysis for preparing polypeptides: The protein component solution was adjusted to a protein mass fraction of 3-8% and a pH of 7.0-7.

5. Enzymatic hydrolysis was carried out at 45-60°C by adding protease at a protein mass of 0.5-2.0% for 3-8 hours. (6) Enzyme inactivation and polypeptide acquisition steps: The enzymatic hydrolysate is heated to 85–100°C and held for 10–30 minutes to inactivate the enzyme. Then, the supernatant is collected by centrifugation and filtered for sterilization to obtain a fermented yeast polypeptide composition with whitening effect.

2. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The organic acid regulator is selected from one or more of malic acid, citric acid, or tartaric acid.

3. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The inoculation amount of the yeast *Schizosacchariformis* is 0.5–3.0% of the culture medium mass.

4. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The buffer solution is selected from one or a combination of Na2HPO4, KH2PO4, NaCl, and KCl.

5. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The single homogenization time for the high-pressure homogenization is 10–60 s.

6. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The protease is a modified neutral protease, and its preparation method is as follows: By weight, mix 40-60 parts of neutral protease with 220-380 parts of 0.15-0.35M KH₂PO₄-K₂HPO₄ (pH 8.0) buffer, add 3-8 parts of glycerol as a stabilizer, and add 1-3M CaCl₂ stock solution to a final concentration of 2mM. Separately, take 2-5 parts of Angelica dahurica root extract and prepare a 5%-10% (w / v) stock solution with cosmetic-grade propylene glycol, controlling the final concentration of propylene glycol in the system to 5%-10%. Slowly add the stock solution dropwise to the enzyme solution (1-3 mL / min, stirring continuously), then add 0.5-0.9 parts of sodium bicarbonate, and react at 32-36℃ and 100-200 rpm under light protection and N₂ for 1-3 hours. The reaction solution is then directly loaded onto a Sephadex G-25 gel column with 0.02-0.05M potassium phosphate buffer (pH 8.0). Isocratic elution was performed, and the main enzyme peak was collected. A 5-10 kDa molecular weight cutoff ultrafiltration membrane was used to concentrate the enzyme under low-speed stirring at ≤25℃ and 0.1-0.3 MPa transmembrane pressure. The concentrate was then replaced with 0.02-0.05 M potassium phosphate (pH 8.0) buffer by ultrafiltration, and then subjected to DEAE-Sepharose Fast Flow ion exchange chromatography with a linear gradient of 0.1→0.5 M NaCl. The highly active fraction was collected to obtain the cosmetic-grade modified neutral protease.

7. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 6, characterized in that: The pH of the ethanol is 7.0-7.

6.

8. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 6, characterized in that: The concentration of the NaOH solution is 0.05-0.2 mol / L.

9. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The enzymatic hydrolysis process is carried out by stirring, with a stirring speed of 100-500 rpm.

10. The method for preparing a fermented yeast polypeptide composition with whitening effect according to claim 1, characterized in that: The average molecular weight of the fermented yeast polypeptide composition prepared is less than 1 kDa.

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