Skin-repairing active ingredients based on dual-fermentation and peony flower extract

Abstract

This invention relates to the field of cosmetic technology, disclosing a skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract, comprising: active functional components and an aqueous carrier; the dual-fermentation lysate is prepared by stepwise directional symbiotic fermentation, gradient ultrasonic lysis-low temperature inactivation, and ultrafiltration purification of peony seed meal complex hydrolysate-peony petal polysaccharide complex as fermentation substrate by *Saccharomyces cerevisiae* and *Bifidobacterium bifidum*; the peony-derived active nanoliposomes are prepared by encapsulating peony flower flavonoid aglycones with yeast β-glucan isolated from the dual-fermentation lysate as hydrophilic wall material and α-linolenic acid monoglyceride from peony seed oil obtained by directional glycerolysis as the core lipid phase. This invention significantly improves the bioavailability, batch stability, and multiple skin care effects of the active substance, including skin barrier repair, anti-oxidation and anti-aging, photoaging damage repair, and anti-inflammatory stabilization.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract. Background Technology

[0002] Peony is a traditional Chinese plant used for both medicinal and ornamental purposes. It is widely cultivated and has abundant resources in my country. Peony seed oil, made from peony seeds, a byproduct of peony cultivation, is rich in unsaturated fatty acids, and peony flower extract contains abundant flavonoid active substances. Both have excellent antioxidant and skin repair potential. Bifida ferment lysate and Saccharomyces cerevisiae fermentation products are recognized as core repair ingredients in the cosmetics industry. The synergy of these two can build a multi-dimensional skin repair system, and related research and applications have been gradually carried out. However, existing technologies for the compound development of peony-derived active ingredients and dual-fermentation synergistic raw materials are limited to simple physical compounding in the finished product preparation stage. This approach fails to address the inherent defects of peony-derived fat-soluble active ingredients, such as easy oxidation and rancidity, poor compatibility with water-based systems, and low transdermal absorption efficiency. Furthermore, it cannot address the issues of strain competition, metabolic interference, and low yield of target active ingredients during fermentation caused by differences in the growth and metabolic characteristics of the two strains. Moreover, it cannot achieve deep molecular-level fusion and synergistic efficacy between peony-derived active ingredients and dual-fermentation products. The main technical problem this invention aims to solve is that existing technologies cannot construct an integrated technical system that simultaneously achieves high-value utilization of peony resources, precise control of dual-fermentation, stable and efficient delivery of active ingredients, and synergistic enhancement of multiple repair effects. This results in low bioavailability, limited efficacy, and poor storage stability of the obtained compound active ingredients, making it difficult to meet the development needs of high-end functional cosmetics. Summary of the Invention

[0003] The purpose of this invention is to provide a skin-repairing active substance based on dual-fermentation and peony flower extract to solve the above-mentioned technical problems. To achieve the above objectives, the present invention provides the following solution: Skin-repairing active ingredients based on dual-fermentation synergistic fermentation and peony flower extract include: active functional components and aqueous carrier; The active functional component is composed of a dual-fermentation lysate and peony-derived active nanoliposomes in a mass ratio of (3-12):1. The dual-fermentation lysate is prepared by stepwise directional symbiotic fermentation, gradient ultrasonic lysis-low temperature inactivation, and ultrafiltration purification of peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex as fermentation substrate by *Saccharomyces cerevisiae* and *Bifidobacterium bifidum*. The peony-derived active nanoliposomes were prepared by encapsulating peony flower flavonoid aglycones with yeast β-glucan isolated from the lysate of the double yeast directional fermentation as the hydrophilic wall material and α-linolenic acid monoglyceride of peony seed oil obtained by directional glycerolysis as the core lipid phase. The average particle size of the peony-derived active nanoliposomes is 50-150 nm, and the encapsulation efficiency of active ingredients is ≥85%. Preferably, the preparation method of the peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex is as follows: Using peony seed meal, a byproduct of cold pressing of peony seed oil, as raw material, after degreasing and crushing, a compound enzyme system is added for stepwise enzymatic hydrolysis. After enzyme inactivation and centrifugation, the supernatant is collected, and then peony petal polysaccharide is added to prepare a fermentation substrate with a solid content of 10-18%. The compound enzyme system consists of cellulase, neutral protease, and pectinase in a mass ratio of 3:2:1. The stepwise enzymatic hydrolysis temperature is 45-55℃, and the total enzymatic hydrolysis time is 4-6 hours. Preferably, the specific process of the stepwise directional symbiotic fermentation is as follows: (1) Pre-fermentation stage: The activated seed liquid of *Saccharomyces cerevisiae* is inoculated into the fermentation substrate at an inoculation rate of 2-4%, and pre-fermented for 12-18 hours at 28-30℃ and an aeration rate of 1-1.2 vvm to degrade the macromolecular substances of the substrate and enrich the active enzyme system. (2) Main fermentation stage: Activated Bifidobacterium bifidum seed culture is inoculated into the pre-fermentation broth at an inoculation rate of 3-5%. The pH of the system is adjusted to 5.8-6.5. The main fermentation is carried out at 36-38℃ with a microaerobic aeration rate of 0.3-0.5 vvm for 36-48 hours to complete the directional symbiotic fermentation. The viable counts of both the *Diplostomum tumefaciens* seed culture and the *Bifidobacterium bifidum* seed culture are ≥1×10⁻⁶. 8 CFU / mL. Preferably, after ultrafiltration purification, the lysate from the dual-fermentation directed fermentation contains ≥30% by mass of peony-derived modified small molecule active peptides with a molecular weight of less than 1000 Da, and also contains ≥15% by mass of yeast β-glucan, amylase, protease, β-glucosidase, as well as free amino acids and B vitamins. The peony-derived modified small molecule active peptide is a small molecule collagen-promoting peptide with terminal grafted α-linolenic acid generated from the enzymatic hydrolysate of peony seed meal during the fermentation process by yeast metabolism. Preferably, the preparation method of the α-linolenic acid monoglyceride from peony seed oil is as follows: using cold-pressed peony seed oil as raw material, immobilized lipase is added, and a directional glycerolysis reaction is carried out in a solvent-free system at 35-40°C and a vacuum of 0.08-0.1 MPa. After the reaction is completed, the product is separated and purified. The cold-pressed peony seed oil contains ≥90% total unsaturated fatty acids, of which α-linolenic acid accounts for 38-42% by mass; the α-linolenic acid monoglyceride in the peony seed oil has a mass percentage of ≥60%, an acid value of ≤0.5mg / g, and a peroxide value of ≤3mmol / kg. Preferably, the method for preparing the peony-derived active nanoliposomes is as follows: (1) Preparation of lipid phase: Peony seed oil α-linolenic acid monoglyceride, peony flower flavonoid aglycone and hydrogenated lecithin are mixed in a mass ratio of 5:1:2, dissolved in anhydrous ethanol, stirred in a water bath at 35-40℃ until completely dissolved, and the organic solvent is removed by rotary evaporation to form a uniform lipid film. (2) Aqueous phase preparation: Yeast β-glucan was isolated and purified from the lysate of the two yeasts in a directional fermentation and prepared into an aqueous solution with a mass fraction of 1-3%. Propylene glycol was added as a co-emulsifier. (3) Liposome preparation: The aqueous phase was injected into the lipid membrane and hydrated in a water bath at 35-40℃ for 30 min. After high-speed shearing, it was homogenized by high-pressure homogenizer for 3-5 cycles and sterilized by 0.22μm filter membrane to obtain peony-derived active nanoliposomes. Preferably, the total mass percentage of the active functional component in the composition is 3-28%. Preferably, the aqueous carrier includes at least one of a humectant, a chelating agent, a pH adjuster, and a preservative. Preferably, the skin-repairing active ingredient is prepared by the following steps: (1) Preparation of fermentation substrate: Prepare peony seed meal complex enzymatic hydrolysate, mix it with peony petal polysaccharide, prepare fermentation substrate, sterilize and set aside; (2) Directed fermentation of dual yeasts: Fermentation broth was prepared according to the stepwise directional symbiotic fermentation process. After fermentation, the broth was subjected to low-temperature high-pressure inactivation and gradient ultrasonic lysis. The supernatant was centrifuged, purified by ultrafiltration membrane fractionation, and freeze-dried to obtain the freeze-dried powder of the lysate of the directional fermentation of dual yeasts. At the same time, yeast β-glucan was separated and purified. (3) Preparation of active substances derived from peony: Peony seed oil was prepared by cold pressing, and α-linolenic acid monoglyceride of peony seed oil was obtained by directional glycerolysis reaction; Peony flower flavonoid aglycones were prepared by ultrasonic extraction of ethanol-enzymatic hydrolysis of glycosidic bonds. (4) Preparation of nanoliposomes: Using yeast β-glucan as the wall material, peony seed oil α-linolenic acid monoglyceride and peony flower flavonoid aglycone were encapsulated to prepare peony-derived active nanoliposomes; (5) Preparation of the final product of the composition: The freeze-dried powder of the lysate of the dual-fermentation direction fermentation, the active nanoliposomes of peony source and the aqueous carrier acceptable to cosmetics are mixed in proportion, and the mixture is homogenized, vacuum degassed and sterilized by filtration to obtain the skin repair active substance. On the other hand, the present invention also protects the application of the above-mentioned skin repair active substance based on dual-fermentation and peony flower extract in the preparation of cosmetics with skin barrier repair, photoaging damage repair, antioxidant and anti-aging, soothing redness, and anti-inflammatory and stabilizing effects. The cosmetic product is available in any one of the following forms: serum, lotion, face cream, eye cream, or facial mask. The skin repair nanodelivery composition is added to the cosmetic product at a mass ratio of 0.8-30%. Compared with the prior art, the beneficial effects of the present invention are as follows: This invention abandons conventional synthetic fermentation media and uses peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex as the fermentation substrate for dual-fermentation, achieving a dual breakthrough in principle: First, the plant proteins and cellulose in peony seed meal, a byproduct of cold pressing of peony seed oil, are transformed into small-molecule plant peptides and oligosaccharides through complex enzymatic hydrolysis, which can be directly used as nitrogen and carbon sources for yeast growth, completely replacing components such as peptone and glucose in conventional synthetic media. This achieves high-value closed-loop utilization of peony processing byproducts and solves the problem of wasting resources from the entire peony plant. Second, during the metabolic proliferation of the two strains, peony-derived α-linolenic acid and plant active peptides can be covalently grafted to the ends of small-molecule collagen-promoting peptides generated by yeast metabolism through microbial transpeptidation and esterification modification, forming peony-derived modified functional active peptides. This achieves molecular-level covalent fusion of dual-fermentation metabolites and peony active substances from the fermentation source, rather than the physical mixing of existing technologies. This invention, based on the differences in growth and metabolic characteristics between *Saccharomyces cerevisiae* (strictly aerobic) and *Bifidobacterium bifidum* (microaerobic), designs a stepwise, directional symbiotic fermentation process, fundamentally eliminating growth competition and metabolic inhibition between strains: In the pre-fermentation stage, aerobic environment control prioritizes the rapid proliferation of *Saccharomyces cerevisiae*. Simultaneously, its secreted amylase, protease, and other complex enzyme systems further degrade large polysaccharides and proteins in the fermentation substrate into easily absorbed monosaccharides and polypeptides, providing suitable nutrient substrates for the subsequent growth of *Bifidobacterium bifidum*. Furthermore, the aerobic respiration of *Saccharomyces cerevisiae* consumes free oxygen in the system, creating a suitable microaerobic growth environment for *Bifidobacterium bifidum*. In the main fermentation stage, microaerobic aeration and precise pH control provide optimal growth conditions for *Bifidobacterium bifidum*. At this stage, the nutritional and environmental requirements of the two strains are perfectly matched, eliminating oxygen and nutrient competition and achieving symbiotic and synergistic metabolism. This invention utilizes yeast β-glucan produced by dual-fermentation as the hydrophilic wall material and α-linolenic acid monoglyceride prepared by directed glycerolysis of peony seed oil as the core lipid phase to construct nanoliposomes encapsulating peony flower flavonoid aglycones. From a formulation perspective, this achieves stable encapsulation and efficient delivery of active ingredients: Firstly, the wall material, yeast β-glucan produced by fermentation, is homologous to dual-fermentation lysate and has natural compatibility with aqueous carriers in cosmetics. Simultaneously, β-glucan can specifically bind to the dectin-1 receptor on keratinocytes, exhibiting active targeted repair rather than being an inert carrier, thus achieving a unity of carrier function and active ingredient efficacy; Secondly, the core lipid phase uses peony-derived α-linolenic acid. Compared to the original triglyceride structure of peony seed oil, monoglycerides have stronger emulsifying and stratum corneum compatibility, and can efficiently fuse with the lipid bilayer of the skin barrier. At the same time, the unsaturated double bonds they contain can form intermolecular hydrogen bonds with the phenolic hydroxyl groups of flavonoid aglycones, stabilizing the active structure of flavonoids. Combined with the core-shell closed structure of liposomes, they can prevent the oxidative degradation of unsaturated fatty acids and flavonoids by oxygen and light, thus solving the defect of easy oxidation and rancidity of peony-derived active ingredients in principle. Thirdly, the liposome particle size is precisely controlled to 50~150nm through high-pressure homogenization process. This particle size range can be delivered across the barrier through the intercellular spaces of the stratum corneum, avoiding the problem that large molecule oils cannot penetrate the stratum corneum. The integrated technical system of this invention, through the synergistic combination of dual-fermentation directed fermentation lysate and peony-derived active nanoliposomes, achieves multi-target synergistic efficacy across the entire physiological pathway of skin repair, achieving a synergistic effect of 1+1>2 in principle: the complex active enzyme system in the dual-fermentation lysate can gently degrade old and dead keratinocytes in the stratum corneum, opening transdermal channels for active ingredients; simultaneously, the peony-derived modified small molecule active peptides with a molecular weight below 1000 Da can penetrate into the dermis, stimulating fibroblasts to synthesize type I and type III collagen, inhibiting the degradation of collagen by metalloproteinases, and achieving anti-aging and firming effects in the dermis. The peony-dermal active nanoliposomes, delivered transdermally, deliver α-linolenic acid to replenish intercellular lipids in the skin barrier, repair damaged lipid bilayers, and enhance skin barrier resistance. Flavonoid aglycones achieve antioxidant and anti-photoaging effects by scavenging free radicals and upregulating the Nrf2 antioxidant pathway, while inhibiting the NF-κB inflammatory pathway and reducing the release of pro-inflammatory factors, thus achieving anti-inflammatory and soothing effects. The liposome wall material, yeast β-glucan, activates the repair pathway of keratinocytes and upregulates the expression of tight junction proteins Occludin and Claudin-1, further strengthening skin barrier function. These active ingredients act on multiple key physiological pathways, including epidermal barrier repair, dermal anti-aging, antioxidant protection, and anti-inflammatory stabilization, forming a synergistic repair system across the entire chain. Compared to single active ingredients, the antioxidant, barrier repair, and anti-photoaging effects are significantly enhanced. Detailed Implementation

[0004] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1 Materials and Instruments 1. Strains Information Saccharomycopsis fibuligera: purchased from China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC12221; stored on slant at 4℃, subcultured every 3 months. Bifidobacterium bifidum: purchased from China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC1.2287, type strain; stored on anaerobic slant at 4℃, subcultured once a month. 2. Reagents and Raw Materials Peony raw materials: Peony seed meal (residual oil rate ≤0.5%, crude protein 28.6%, crude fiber 32.1%), dried petals of Fengdan peony during its peak blooming period (moisture content ≤8%), and peony seed oil cold-pressed at 45℃ (total unsaturated fatty acids 91.2%, α-linolenic acid 40.2%, acid value 0.28mg / g, peroxide value 1.8mmol / kg, conforming to GB2716-2018). Enzyme preparations: Cellulase (food grade, 100,000 U / g, Shandong Longket), Neutral protease (food grade, 100,000 U / g, Shandong Longket), Pectinase (food grade, 100,000 U / g, Shandong Longket), β-glucosidase (food grade, 100,000 U / g, Shanghai Yuanye), Immobilized lipase Novozym435 (10,000 U / g, Novozymes, commercially available); Microbial culture medium reagents (all biochemical grade, Beijing Aoboxing): peptone, yeast extract, glucose, potassium dihydrogen phosphate, magnesium sulfate, diammonium hydrogen citrate, sodium acetate, Tween-80, L-cysteine ​​hydrochloride, agar powder; Cosmetic raw materials (all cosmetic grade, commercially available compliant products): hydrogenated lecithin (Germany Lipoid S75), propylene glycol (USA Dow), glycerin (Malaysia Procter & Gamble), sodium hyaluronate (Bloomage Biotechnology, molecular weight 1.2 million Da), disodium EDTA, citric acid, sodium citrate, phenoxyethanol. Other reagents: Anhydrous ethanol, n-hexane, methanol, etc. were all analytical grade (Sinopharm Group), and the experimental water was ultrapure water (resistivity 18.2 MΩ·cm). The formulation for this embodiment is shown in Table 1. Table 1 Active functional components Lyophilized Powder of Directed Fermentation Lysate 133.33 13.33% Active functional components Peony-derived active nanoliposomes 16.67 1.67% Aqueous carrier glycerin 80 8.00% Aqueous carrier Propylene glycol 30 3.00% Aqueous carrier Sodium hyaluronate 2 0.20% Aqueous carrier Disodium EDTA 0.5 0.05% Aqueous carrier Citric acid 1 0.10% Aqueous carrier Sodium citrate 0.8 0.08% Aqueous carrier Phenoxyethanol 5 0.50% Aqueous carrier Ultrapure water 730.7 73.07% total - 1000 100.00% 3. Instruments and Equipment 50L Fully Automatic Fermentation Tank: Shanghai Baoxing BIOTECH-50JS (with online pH, DO, temperature, aeration rate, and stirring speed control); High-pressure homogenizer: Shanghai Donghua SRH60-70; Rotary evaporator: German IKARV10autopro; High-speed refrigerated centrifuge: Hunan Xiangyi H2500R-2; Ultrasonic cell disruptor: Ningbo Xinzhi JY92-IIN; Malvern laser particle size analyzer: Malvern Zetasizer Nano ZS90 (UK); UV-Vis spectrophotometer: Shimadzu UV-2600 (Japan); High-performance liquid chromatograph: Agilent 1260 Infinity II; Franz diffusion cell: Shanghai Kaixin TK-6H; Vacuum freeze dryer: Beijing Boyikang FD-1A-80; Anaerobic incubator: Shanghai Xinmiao YQX-II; Constant temperature and humidity incubator: Shanghai Yiheng LRH-250. Preparation process Step 1: Seed liquid preparation (1) Preparation of Seed Culture of *Saccharomyces cerevisiae* Slant activation: Under aseptic conditions, pick two loops of CGMCC12221 slant bacterial growth and inoculate them into YPD slant medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar, pH natural) and incubate at 29℃ for 48 h to complete activation; Primary seed culture: Pick a single colony from the activated slant and inoculate it into a 500mL shake flask (100mL liquid volume) of YPD liquid medium. Incubate at 29℃ and 200r / min for 24h. Secondary seed culture: Inoculate 5% of the primary seed culture into 5L shake flasks (1L total volume) of YPD liquid medium and incubate at 29℃ and 200 rpm for 18 hours; Quality control standard: viable count ≥1.2 × 10⁻⁶ using the plate spread method. 8 CFU / mL, refrigerate at 4℃ for use, and store for no more than 12 hours. (2) Preparation of Bifidobacterium bifidum seed culture Slant activation: Under aseptic conditions, pick two loops of CGMCC1.2287 anaerobic slant bacterial growth and inoculate them into MRS modified slant medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 2 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1 mL / L Tween-80, 0.5 g / L L-cysteine ​​hydrochloride, 20 g / L agar, pH 6.2), and anaerobic incubate at 37℃ for 48 h to complete activation; Primary seed culture: Pick a single colony from the activated slant and inoculate it into a 500mL anaerobic bottle (containing 200mL of liquid) of MRS modified liquid medium, and incubate at 37℃ for anaerobic incubation for 24h. Secondary seed culture: Inoculate the primary seed culture at a rate of 5% into a 5L anaerobic bottle (containing 2L of medium) of MRS modified liquid culture medium and incubate anaerobically at 37℃ for 18 hours; Quality control standard: viable count ≥1.2×10⁻⁶ using the anaerobic plate spread method. 8 CFU / mL, store in an anaerobic environment for later use, and do not store for more than 12 hours. Step 2: Preparation of fermentation substrate (peony seed meal complex enzymatic hydrolysate - peony petal polysaccharide complex, reproducible 1:1) (1) Preparation of peony petal polysaccharides Weigh 1 kg of peony petal powder, add 10 L of ultrapure water, with a material-to-liquid ratio of 1:10, and extract by stirring in a 95℃ water bath for 2 hours. Extract twice, combine the extracts, and centrifuge at 4000 r / min for 10 min to collect the supernatant. The supernatant was concentrated by rotary evaporation to 1 / 5 of its original volume. Anhydrous ethanol was added to the concentrate until the final volume fraction of ethanol was 80%. The mixture was allowed to stand at 4°C for 12 hours to precipitate, and then centrifuged at 4000 r / min for 10 minutes to collect the precipitate. The precipitate was washed twice each with anhydrous ethanol and acetone, redissolved in ultrapure water, deproteinized five times using the Sevag method, dialyzed for 48 hours using a dialysis bag (molecular weight cutoff 3500 Da), and freeze-dried to obtain peony petal polysaccharide; quality control standard: polysaccharide content ≥85% as determined by phenol-sulfuric acid method. (2) Stepwise compound enzymatic hydrolysis of peony seed meal Peony seed meal pretreatment: The deoiled peony seed meal was crushed and passed through an 80-mesh sieve. 2.5 kg of meal powder was weighed and added to 25 L of ultrapure water at a material-to-liquid ratio of 1:10. The mixture was stirred evenly and the pH was adjusted to 4.8 with 1 mol / L citric acid. Step 1 Enzymatic hydrolysis: Add 6.25g of cellulase and 2.08g of pectinase (total addition is 0.33% of the meal mass), stir at 50℃ and 100r / min for 2h to degrade cellulose and pectin; Step 2: Enzymatic hydrolysis: Adjust the pH of the system to 6.0, add 4.17g of neutral protease (0.17% of the weight of the meal flour), stir at 50℃ for 3 hours, and the total enzymatic hydrolysis time is 5 hours. Enzyme inactivation centrifugation: Immediately after enzymatic hydrolysis, heat to 100℃ and incubate for 10 min to inactivate the enzyme. Cool to room temperature, centrifuge at 4000 r / min for 15 min, collect the supernatant, and discard the precipitate. (3) Final preparation and sterilization of fermentation substrate Add the prepared peony petal polysaccharide to the supernatant, stir until completely dissolved, add ultrapure water to adjust the solid content of the system to 15%, and obtain the fermentation substrate; Pump 30L of fermentation substrate into a 50L fermenter and autoclave at 121℃ and 0.1MPa for 20 minutes. After sterilization, gradually cool down to 29℃ for later use. Step 3: Stepwise directional symbiotic fermentation and post-treatment (1) Stepwise directional symbiotic fermentation Pre-fermentation stage: Under aseptic conditions, the secondary seed culture of *Saccharomyces cerevisiae* was inoculated into the fermenter at a 3% inoculation rate (900 mL). The parameters were set as follows: temperature 29℃, stirring speed 200 r / min, aeration rate 1.1 vvm (33 L / min), pH at rest, and aerobic pre-fermentation for 15 h. The quality control standards for pre-fermentation were: amylase activity ≥120 U / mL, protease activity ≥80 U / mL, and reducing sugar content ≤5 g / L. After passing the test, the fermentation broth was allowed to proceed to the main fermentation stage. Gradient transition: After the pre-fermentation is completed, the tank temperature gradient is increased to 37℃ within 1 hour, the aeration rate gradient is reduced to 0.4vvm, the stirring speed is reduced to 100r / min, and the pH of the system is adjusted to 6.2 with 1mol / L sodium hydroxide to avoid sudden environmental changes that may cause the strain to become inactive. Main fermentation stage: Under aseptic conditions, inoculate with secondary seed culture of Bifidobacterium bifidum at an inoculation volume of 4% (1200 mL) and maintain the following parameters: temperature 37℃, stirring speed 100 r / min, aeration rate 0.4 vvm (12 L / min), pH 6.2, and microaerobic main fermentation for 42 h; Fermentation termination: After the main fermentation is completed, the fermentation liquid is placed in a tank. Quality control standards: polysaccharide content ≥8g / L and small molecule peptide content ≥15g / L. (2) Post-treatment of fermentation broth (modify the lysis-inactivation logic to ensure full release of active substances) Gradient ultrasonic lysis: The fermentation broth was transferred to an ultrasonic cell disruptor and ultrasonically lysed under ice bath conditions. The parameters were: power 200W, ultrasonic 3s, interval 5s, total duration 30min. The system temperature was controlled to be ≤10℃ throughout the process to fully disrupt the cells and release intracellular active substances. Low-temperature inactivation: The lysed fermentation broth is transferred to an inactivation tank and kept at 60°C for 20 minutes to completely inactivate the strain. Then it is cooled to room temperature to avoid degradation of active substances due to high temperature. Centrifugation to remove impurities: Centrifuge at 8000 r / min and 4℃ for 20 min, then collect the supernatant and discard the bacterial precipitate; Ultrafiltration purification: The supernatant was purified using a PES ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The operating pressure was 0.3 MPa and the temperature was 25 °C. The solution was dialyzed with ultrapure water three times and the permeate was collected. This permeate was the purified lysate for the directional fermentation of yeast. At the same time, the retentate with a molecular weight cutoff of 100,000 Da was collected for the separation of yeast β-glucan. Freeze-drying: The lysate was pre-frozen at -40℃ for 8 hours and then freeze-dried under a vacuum of ≤10Pa for 48 hours to obtain freeze-dried powder of lysate from the directional fermentation of dual yeasts; quality control standards: the proportion of small molecule active peptides with a molecular weight of less than 1000Da is ≥30%, the yeast β-glucan content is ≥8%, and it is sealed and stored at -20℃ for later use. Isolation and purification of yeast β-glucan: Add 3 volumes of anhydrous ethanol to 100,000 Da cutoff buffer, let stand at 4℃ for 12 h to precipitate, centrifuge to collect the precipitate, remove protein by Sevag method, purify by DEAE-cellulose column chromatography, and freeze dry to obtain pure yeast β-glucan; quality control standard: glucan content ≥90%, used for nanoliposome preparation. Step 4: Preparation of Peony-derived Active Substances (1) Preparation of α-linolenic acid monoglyceride from peony seed oil Raw material pretreatment: Cold-pressed peony seed oil and glycerin were added to a solvent-free reaction vessel at a molar ratio of 1:4, stirred evenly, and the air in the system was purged with nitrogen. Targeted glycerol hydrolysis: Add immobilized lipase Novozym435 (8% of the weight of peony seed oil), set parameters: temperature 38℃, vacuum degree 0.09MPa, stirring speed 150r / min, react for 8h, and purge with trace amounts of nitrogen to remove water throughout the process; Separation and purification: After the reaction, the immobilized enzyme was removed by filtration, and the α-linolenic acid monoglyceride was separated and purified by secondary molecular distillation (evaporation temperature 160℃, vacuum degree 0.1Pa); quality control standards: α-linolenic acid monoglyceride content ≥60%, acid value ≤0.5mg / g, peroxide value ≤3mmol / kg. (2) Preparation of peony flower flavonoid aglycones Flavonoid crude extraction: Add 1 kg of peony petal powder to 10 L of 70% ethanol, and extract by ultrasonic extraction at 50℃ for 40 min (power 300W). Extract twice, combine the extracts, centrifuge to collect the supernatant, and concentrate by rotary evaporation until there is no ethanol odor. Glycosidic bond enzymatic hydrolysis: Adjust the pH of the concentrate to 5.0, add β-glucosidase (0.3% of the concentrate volume), enzymatically hydrolyze at 50℃ for 4 hours, and inactivate the enzyme at 100℃ for 10 minutes; Purification and drying: The enzymatic hydrolysate was adsorbed onto AB-8 macroporous resin, eluted with 70% ethanol, and the eluent was concentrated and freeze-dried to obtain peony flower flavonoid aglycones; quality control standard: total flavonoid content ≥45%. Step 5: Preparation of Peony-derived Active Nanoliposomes Lipid phase preparation: Weigh 50g of α-linolenic acid monoglyceride, 10g of peony flavonoid aglycone and 20g of hydrogenated lecithin, add 500mL of anhydrous ethanol, stir in a 38℃ water bath until completely dissolved to form a homogeneous lipid phase solution. Thin film preparation: The lipid phase solution was transferred into a rotary evaporator at 38°C, vacuum degree 0.09MPa, and rotation speed 80r / min to evaporate and remove ethanol, forming a uniform and pore-free lipid film on the inner wall of the bottle. Vacuum drying was continued for 30min to remove residual ethanol. Aqueous phase preparation: Weigh 20g of the above-mentioned yeast β-glucan pure product, add 1L of ultrapure water to prepare a 2% aqueous solution, add 20g of propylene glycol, stir evenly, and keep warm in a 38℃ water bath for later use. Hydration film formation: The aqueous phase at 38℃ was rapidly injected into a liposome-shaped flask, and hydration was carried out by stirring at 38℃ and 150r / min for 30min to form a liposome proemulsion. Homogenization and refinement: colostrum was sheared at 10000r / min for 5min, homogenized at 60MPa for 4 cycles, and the temperature was controlled at ≤40℃ throughout the process; Sterilization and packaging: Sterilization filtration through a 0.22μm filter membrane to obtain peony-derived active nanoliposomes; quality control standards: average particle size 50-150nm, PDI≤0.2, active ingredient encapsulation rate≥85%, refrigerated at 4℃ for later use. Step 6: Preparation of final product (can be reproduced 1:1, in compliance with cosmetic production standards) Preparation of aqueous carrier: Add 730.70g of ultrapure water to the mixing tank, and add 0.50g of disodium EDTA, 1.00g of citric acid, 0.80g of sodium citrate, 80.00g of glycerol, and 30.00g of propylene glycol in sequence while stirring at 80r / min, and stir until completely dissolved; add 2.00g of sodium hyaluronate, and stir at 150r / min for 30min until completely dissolved and without lumps; Addition of active ingredients: Cool the system to 30℃, add 133.33g of freeze-dried powder of lysozyme from dual-fermentation directional fermentation and 16.67g of peony-derived active nanoliposomes in sequence while stirring at 100r / min, and stir for 30min until uniformly dispersed; Preservative and volume adjustment: Add 5.00g of phenoxyethanol, stir for 15min, and add ultrapure water to a total mass of 1000g; Homogenization and degassing: Homogenize twice at 30 MPa, then degas under vacuum at -0.09 MPa for 10 min; Sterile filling: Sterile filtration with a 0.22μm filter membrane, aseptic filling, to obtain the final skin repair active substance product. Example 2 The only difference between this embodiment and Embodiment 1 is the core proportions and process boundary parameters. The total mass percentage of the active functional components is 3%, of which the mass ratio of the lyophilized powder of the dual-fermentation lysate to the active nanoliposomes of peony source is 3:1, that is, 22.5g of the lyophilized powder of the dual-fermentation lysate and 7.5g of the active nanoliposomes of peony source; the components and amounts of the aqueous phase carrier are completely consistent with those in Example 1, and ultrapure water is added to make up to 1000g. Process parameter adjustment Fermentation substrate: Peony seed meal compound enzymatic hydrolysate was prepared with a solids content of 10% by mass. Pre-fermentation stage: 2% inoculum of *Saccharomyces cerevisiae*, 28℃, aeration rate of 1.0 vvm, pre-fermentation for 12 h; Main fermentation stage: Bifidobacterium bifidum inoculum 3%, system pH 5.8, 37℃, aeration rate 0.3 vvm, main fermentation 36h; Preparation of nanoliposomes: High-pressure homogenization cycled 3 times, the final liposome average particle size was 145.8 nm, PDI≤0.2, and the active ingredient encapsulation efficiency was 86.2%. The steps for seed liquid preparation, enzymatic hydrolysis, post-fermentation treatment, preparation of peony-derived active ingredients, and final product formulation are all completely consistent with those in Example 1. Example 3 The only difference between this embodiment and Example 1 is the core ratio and process boundary parameters; the other raw materials, strains, operating procedures, and quality control standards are completely identical. The core formula was adjusted (total batch 1000g). The total mass ratio of active functional components was 28%, of which the mass ratio of lyophilized dual-fermentation lysate to peony-derived active nanoliposomes was 12:1, i.e., 258.46g of lyophilized dual-fermentation lysate and 21.54g of peony-derived active nanoliposomes. The aqueous phase carrier components and dosage were completely consistent with those in Example 1, and ultrapure water was added to make up to 1000g. Key process parameter adjustment Fermentation substrate: Peony seed meal compound enzymatic hydrolysate was prepared with a solids content of 18% by mass. Pre-fermentation stage: 4% of *Saccharomyces cerevisiae* was inoculated at 30℃ with an aeration rate of 1.2 vvm for 18 hours. Main fermentation stage: 5% inoculum of Bifidobacterium bifidum, system pH 6.5, 37℃, aeration rate 0.5 vvm, main fermentation for 48 h; Preparation of nanoliposomes: High-pressure homogenization cycle 5 times, the final average particle size of liposomes was 52.7 nm, PDI≤0.2, and the encapsulation efficiency of active ingredients was 89.5%. The remaining operations—seed liquid preparation, enzymatic hydrolysis, post-fermentation treatment, preparation of peony-derived active ingredients, and final product formulation—are completely consistent with those in Example 1. Comparative Example 1 Formula composition (total batch 1000g): 13.33% conventional double fermentation product, 1.5% cold-pressed peony seed oil, 0.167% peony flower flavonoid aglycone, the aqueous phase carrier components and dosage are the same as in Example 1, and ultrapure water is added to 1000g. Preparation of conventional dual-fermentation product: Using conventional synthetic culture medium (20 g / L glucose, 15 g / L peptone, 10 g / L yeast extract), *Bifidobacterium bifidum* and *Diplostomum tumefaciens* were simultaneously inoculated, fermented at 37°C for 48 h, and the supernatant was freeze-dried after inactivation and centrifugation. Preparation method: All raw materials are stirred and mixed evenly at room temperature to obtain the final composition. There is no stepwise fermentation or nanoliposome preparation step. Comparative Example 2 The only difference between this comparative example and Example 1 is that the stepwise directional symbiotic fermentation process is eliminated, and a dual-strain synchronous mixed fermentation is adopted. All other raw materials, substrates, and formulation steps are completely identical. Fermentation operation: The seed culture of *Saccharomyces cerevisiae* (3% inoculum) and the seed culture of *Bifidobacterium bifidum* (4% inoculum) were simultaneously inoculated into the peony-derived fermentation substrate and fermented simultaneously for 57 h at 37℃, aeration rate of 0.8 vvm and pH 6.2. The remaining operations—post-fermentation treatment, nanoliposome preparation, and final product formulation—are completely consistent with those in Example 1. Comparative Example 3 The only difference between this comparative example and Example 1 is that the fermentation substrate used is a conventional synthetic culture medium, rather than the peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex. All other processes, ratios, and preparation steps are completely identical. Fermentation substrate: conventional synthetic culture medium (20 g / L glucose, 15 g / L peptone, 10 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate), sterilized at 121℃ for 20 min for later use; The remaining operations—stepwise fermentation process, post-fermentation treatment, preparation of nanoliposomes, and formulation of the final product—are completely consistent with those in Example 1. Comparative Example 4 The only difference between this comparative example and Example 1 is that the preparation step of peony-derived active nanoliposomes is omitted, and an equal amount of active material is directly added exogenously. All other fermentation processes and raw material ratios are completely identical. Formula adjustment: Replace the peony-derived active nanoliposomes in Example 1 with an equal mass of peony seed oil α-linolenic acid monoglyceride, peony flower flavonoid aglycone, and yeast β-glucan mixture (in the same proportion as the liposome feed ratio). Preparation method: The preparation steps of the lysate from the dual-fermentation directional fermentation are completely consistent with those in Example 1. All active raw materials and aqueous carriers are directly mixed at room temperature by stirring, without liposome preparation or high-pressure homogenization steps. The performance test results and analysis are shown in Table 2. Table 2 Example 1 3.26 92.7 4.12 96.3 42.58 92.4 2.87 Example 2 4.58 87.2 4.85 95.1 38.62 88.7 2.53 Example 3 3.41 91.5 4.37 95.8 40.19 90.6 2.69 Comparative Example 1 28.74 42.6 38.92 52.7 8.73 56.2 1.12 Comparative Example 2 12.63 68.3 5.21 82.4 21.35 71.5 1.76 Comparative Example 3 8.72 75.1 4.68 93.2 28.47 78.3 1.98 Comparative Example 4 10.25 70.8 27.64 61.5 12.46 75.9 1.84 Comparative Example 5 15.86 59.4 - - - 68.7 1.35 Results Analysis Comparative Example 2 differs from Example 1 only in the single variable of fermentation process (Comparative Example 2 involves simultaneous mixed fermentation of two strains, while Example 1 involves stepwise directional symbiotic fermentation). Experimental data show that Example 1 scavenges EC with DPPH. 50 Compared to control group 2, the expression level decreased by 74.2%, while the expression level of Occludin protein increased by 63.1%. The reasonable reason is that simultaneous fermentation cannot simultaneously achieve the optimal growth environment for both aerobic *Bifidobacterium tumefaciens* and microaerobic *Bifidobacterium bifidum*. Competition for oxygen and nutrients exists between strains, resulting in a significantly lower yield of target active peptides and polysaccharides compared to the stepwise fermentation process, ultimately manifesting as a decrease in activity. The only difference between Comparative Example 3 and Example 1 in verifying the effect of the peony-derived fermentation substrate was the fermentation substrate (Comparative Example 3 used a conventional synthetic culture medium, while Example 1 used a peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide substrate). Experimental data showed that Example 1 scavenged EC with DPPH. 50 Compared to control 3, the concentration of active ingredients in the skin decreased by 62.6%, while the amount of active ingredients retained in the skin increased by 49.6%. During the fermentation process, the peony-derived substrate was metabolized by yeast to generate small molecule active peptides modified with peony-derived ingredients, achieving molecular-level fusion of the dual-fermentation product and peony active ingredients. Compared to fermentation products from conventional culture media, it additionally possesses the antioxidant and repairing effects of peony active ingredients, while the small molecule peptides have better transdermal permeability. The only difference between Comparative Example 4 and Example 1 in terms of the efficacy verification of the nanoliposome delivery system was the formulation process (Comparative Example 4 involved direct addition of the active ingredient without nanoliposome encapsulation; Example 1 involved encapsulation with nanoliposomes using a homologous wall material). Experimental data showed that Example 1 exhibited a 77.9% lower rate of change in peroxide value over 6 months compared to Comparative Example 4, a 56.6% higher retention rate of the active ingredient, and a 132.2% higher amount of active ingredient retained in the skin compared to Comparative Example 4. The core-shell structure of the nanoliposomes effectively isolates the active ingredient from oxygen, significantly mitigating the oxidative rancidity of unsaturated fatty acids. Simultaneously, the 50-150 nm particle size adapts to the permeation channels in the intercellular spaces of the stratum corneum, significantly increasing transdermal retention compared to free lipid-soluble active ingredients. This validates the improved stability and bioavailability of the active ingredient by the nanodelivery system of this invention. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract, characterized in that, include: Active functional components and aqueous carrier; The active functional component is composed of a dual-fermentation lysate and peony-derived active nanoliposomes in a mass ratio of (3-12):

1. The dual-fermentation lysate is prepared by stepwise directional symbiotic fermentation, gradient ultrasonic lysis-low temperature inactivation, and ultrafiltration purification of peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex as fermentation substrate by *Saccharomyces cerevisiae* and *Bifidobacterium bifidum*. The peony-derived active nanoliposomes were prepared by encapsulating peony flower flavonoid aglycones with yeast β-glucan isolated from the lysate of the double yeast directional fermentation as the hydrophilic wall material and α-linolenic acid monoglyceride of peony seed oil obtained by directional glycerolysis as the core lipid phase. The average particle size of the peony-derived active nanoliposomes is 50-150 nm.

2. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The preparation method of the peony seed meal complex enzymatic hydrolysate-peony petal polysaccharide complex is as follows: Using peony seed meal, a byproduct of cold pressing of peony seed oil, as raw material, after degreasing and crushing, a compound enzyme system is added for stepwise enzymatic hydrolysis. After enzyme inactivation and centrifugation, the supernatant is collected, and then peony petal polysaccharide is added to prepare a fermentation substrate with a solid content of 10-18%. The compound enzyme system consists of cellulase, neutral protease, and pectinase in a mass ratio of 3:2:

1. The stepwise enzymatic hydrolysis temperature is 45-55℃, and the total enzymatic hydrolysis time is 4-6 hours.

3. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The specific process of the stepwise directional symbiotic fermentation is as follows: (1) Pre-fermentation stage: The activated seed liquid of *Saccharomyces cerevisiae* is inoculated into the fermentation substrate at an inoculation rate of 2-4%, and pre-fermented for 12-18 hours at 28-30℃ and an aeration rate of 1-1.2 vvm to degrade the macromolecular substances of the substrate and enrich the active enzyme system. (2) Main fermentation stage: Activated Bifidobacterium bifidum seed culture is inoculated into the pre-fermentation broth at an inoculation rate of 3-5%. The pH of the system is adjusted to 5.8-6.

5. The main fermentation is carried out at 36-38℃ with a microaerobic aeration rate of 0.3-0.5 vvm for 36-48 hours to complete the directional symbiotic fermentation. The viable counts of both the *Diplostomum tumefaciens* seed culture and the *Bifidobacterium bifidum* seed culture are ≥1×10⁻⁶. 8 CFU / mL.

4. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, After ultrafiltration purification, the mass percentage of peony-derived modified small molecule active peptides with a molecular weight of less than 1000 Da in the lysate from the dual-fermentation directional fermentation is ≥30%. The peony-derived modified small molecule active peptide is a small molecule collagen-promoting peptide with terminal grafted α-linolenic acid generated from the enzymatic hydrolysate of peony seed meal during the fermentation process by yeast metabolism.

5. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The preparation method of the α-linolenic acid monoglyceride of peony seed oil is as follows: using cold-pressed peony seed oil as raw material, immobilized lipase is added, and a directional glycerolysis reaction is carried out in a solvent-free system at 35-40℃ and a vacuum degree of 0.08-0.1MPa. After the reaction is completed, the product is separated and purified. The cold-pressed peony seed oil contains ≥90% total unsaturated fatty acids, of which α-linolenic acid accounts for 38-42% by mass; the α-linolenic acid monoglyceride in the peony seed oil has a mass percentage of ≥60%, an acid value of ≤0.5mg / g, and a peroxide value of ≤3mmol / kg.

6. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The preparation method of the peony-derived active nanoliposomes is as follows: (1) Preparation of lipid phase: Peony seed oil α-linolenic acid monoglyceride, peony flower flavonoid aglycone and hydrogenated lecithin are mixed in a mass ratio of 5:1:2, dissolved in anhydrous ethanol, stirred in a water bath at 35-40℃ until completely dissolved, and the organic solvent is removed by rotary evaporation to form a uniform lipid film. (2) Aqueous phase preparation: Yeast β-glucan was isolated and purified from the lysate of the two yeasts in a directional fermentation and prepared into an aqueous solution with a mass fraction of 1-3%. Propylene glycol was added as a co-emulsifier. (3) Liposome preparation: The aqueous phase was injected into the lipid membrane and hydrated in a water bath at 35-40℃ for 30 min. After high-speed shearing, it was homogenized by high-pressure homogenizer for 3-5 cycles and sterilized by 0.22μm filter membrane to obtain peony-derived active nanoliposomes.

7. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The active functional component accounts for 3-28% of the total mass of the composition.

8. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to claim 1, characterized in that, The aqueous carrier includes at least one of a humectant, a chelating agent, a pH adjuster, and a preservative.

9. The skin-repairing active substance based on dual-fermentation synergistic fermentation and peony flower extract according to any one of claims 1-8, characterized in that, The skin-repairing active ingredient is prepared through the following steps: (1) Preparation of fermentation substrate: Prepare peony seed meal complex enzymatic hydrolysate, mix it with peony petal polysaccharide, prepare fermentation substrate, sterilize and set aside; (2) Directed fermentation of dual yeasts: Fermentation broth was prepared according to the stepwise directional symbiotic fermentation process. After fermentation, gradient ultrasonic lysis and low-temperature high-pressure inactivation were performed. The supernatant was centrifuged, purified by ultrafiltration membrane fractionation, and freeze-dried to obtain freeze-dried powder of lysate from the directional fermentation of dual yeasts. At the same time, yeast β-glucan was separated and purified. (3) Preparation of active substances derived from peony: Peony seed oil was prepared by cold pressing, and α-linolenic acid monoglyceride of peony seed oil was obtained by directional glycerolysis reaction; Peony flower flavonoid aglycones were prepared by ultrasonic extraction of ethanol-enzymatic hydrolysis of glycosidic bonds. (4) Preparation of nanoliposomes: Using yeast β-glucan as the wall material, peony seed oil α-linolenic acid monoglyceride and peony flower flavonoid aglycone were encapsulated to prepare peony-derived active nanoliposomes; (5) Preparation of the final product of the composition: The freeze-dried powder of the lysate of the dual-fermentation direction fermentation, the active nanoliposomes of peony source and the aqueous carrier acceptable to cosmetics are mixed in proportion, and the mixture is homogenized, vacuum degassed and sterilized by filtration to obtain the skin repair active substance.

10. The application of the skin-repairing active substance based on dual-fermentation and peony flower extract as described in any one of claims 1-8 in the preparation of cosmetics with skin barrier repair, photoaging damage repair, and antioxidant and anti-aging effects; The cosmetic product can be any one of the following: serum, lotion, face cream, eye cream, or facial mask. The skin-repairing active ingredient accounts for 3-30% of the total mass of the cosmetic product.