Bifidobacterium fermented cosmetic functional raw material based on placenta protein and preparation method of bifidobacterium fermented cosmetic functional raw material

Abstract

The invention discloses a placenta protein-based bifidobacterium fermented cosmetic functional raw material and a preparation method thereof, and relates to the technical field of cosmetic preparation. According to the method, by optimizing extraction and purification processes of placenta protein, radix ophiopogonis and lucid ganoderma, the content of effective components in an extracting solution is greatly increased; by screening high-activity bifidobacterium strains and compounding the high-activity bifidobacterium strains with lactic acid bacteria, the conversion of effective components in the raw materials is promoted by utilizing the synergistic effect of the strains, so that the yield of short-chain fatty acids and the antioxidant activity are greatly improved; by optimizing parameters such as fermentation temperature, pH value and time, the conversion efficiency of effective components is maximized, and the contents of placenta protein hydrolysate, radix ophiopogonis steroid saponin and ganoderma triterpenoids in fermentation products are greatly increased; moreover, by adding auxiliary components, the efficacy and the stability are improved, the 24-hour moisture retention rate reaches 90% or above, the product quality meets the cosmetic hygienic standard, and the safety is high.

Description

Technical Field

[0001] This invention relates to the field of cosmetic preparation technology, and in particular to a placental protein-based Bifidobacterium fermentation cosmetic functional ingredient and its preparation method. Background Technology

[0002] With the development of the cosmetics industry, consumers are increasingly demanding higher standards for the safety, functionality, and bioactivity of cosmetic ingredients. Placental protein, rich in growth factors, proteins, and trace elements, possesses cell regeneration, anti-inflammatory, and antioxidant properties, making it a commonly used active ingredient in cosmetics. Steroidal saponins and polysaccharides in Ophiopogon japonicus can moisturize and provide antioxidant effects; polysaccharides and triterpenoids in Ganoderma lucidum can enhance skin immunity and delay aging. All three have excellent potential for cosmetic applications. Short-chain fatty acids and vitamins produced during Bifidobacterium fermentation can regulate the skin's microecology and enhance its barrier function; its fermentation lysate also possesses anti-wrinkle and repairing effects.

[0003] Currently, placental proteins are mostly extracted and used alone. The pretreatment of Ophiopogon japonicus and Ganoderma lucidum often employs traditional pulverization and extraction methods, resulting in low dissolution rates and insufficient purity of active ingredients. During Bifidobacterium fermentation, the selection of single strains and the lack of system optimization of fermentation parameters lead to low conversion efficiency of active ingredients and poor synergistic effects in the fermentation products. Furthermore, existing compound raw materials lack targeted quality control systems, making it difficult to guarantee product stability and safety. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides the following technical solution.

[0005] The first aspect of this invention provides a method for preparing a placental protein-based Bifidobacterium fermentation cosmetic functional ingredient, comprising:

[0006] Bifidobacterium strains and lactic acid bacteria strains were mixed at a ratio of 3:1 and inoculated into a fermentation medium containing the compound raw materials. The mixture was fermented at 35-37℃ and pH 5.8-6.2 for 24-28 hours to obtain the fermentation broth.

[0007] Adding excipients to the fermentation broth, followed by purification and freeze-drying, yields powdered cosmetic functional ingredients.

[0008] The volume percentage of the compound raw materials added to the fermentation culture medium is as follows: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution.

[0009] The Ophiopogon japonicus extract was prepared according to the following method:

[0010] Prepare Ophiopogon japonicus ultrafine powder with a particle size of 10-20 μm;

[0011] Deionized water was added to Ophiopogon japonicus ultrafine powder, and a second reflux extraction was performed to prepare Ophiopogon japonicus aqueous extract;

[0012] Within 2-3 minutes, add anhydrous ethanol to the Ophiopogon japonicus aqueous extract to make the volume fraction of anhydrous ethanol reach 60-70%. After stirring evenly, let stand and centrifuge to dissolve the precipitate and obtain the crude extract of Ophiopogon japonicus.

[0013] The crude extract of Ophiopogon japonicus was purified and concentrated to obtain an extract solution with a solid content of 10-15%.

[0014] The Ganoderma lucidum extract solution was prepared according to the following method:

[0015] Ganoderma lucidum seed powder was subjected to cell wall breaking treatment to obtain Ganoderma lucidum cell wall breaking liquid;

[0016] Deionized water was added to the Ganoderma lucidum cell wall breaking solution, and the solution was placed in an ultrasonic-microwave synergistic extractor to prepare the extract.

[0017] Centrifuge the extract, ultrafilter the supernatant, and collect the permeate.

[0018] The extract was concentrated under reduced pressure to obtain Ganoderma lucidum extract.

[0019] Preferably, the purified placental protein solution is prepared according to the following method:

[0020] Take a fresh placenta, wash it to remove blood and fascia, and cut it into 1-2cm pieces. 3 Add 3-5 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat them for 20-30 minutes at a power of 200-300W and a temperature of 35-40℃.

[0021] Add a complex protease to the ultrasonically treated mixture at a concentration of 0.5-1.0% of the placental mass, adjust the pH to 6.5-7.0, and hydrolyze at 40-45℃ for 2-3 hours to obtain the hydrolysate; the activity of the complex protease is ≥5000U / g.

[0022] Centrifuge the enzymatic hydrolysate at 5000-8000 r / min for 15-20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa. Collect the permeate.

[0023] The permeate was purified by ion exchange chromatography with 0.1-0.2 mol / L NaCl solution as the eluent and a flow rate of 1-2 mL / min. The main peak eluent was collected. The permeate was then further purified by gel filtration chromatography with phosphate buffer at pH 7.0 as the eluent and a flow rate of 0.8-1.2 mL / min. The target component was collected.

[0024] The purified target component was placed in a vacuum concentrator and concentrated to a solid content of 15-20% under conditions of 45-50℃ and a vacuum of -0.08 to -0.09 MPa to obtain purified placental protein solution.

[0025] Preferably, the Ophiopogon japonicus extract is prepared according to the following method:

[0026] Take dried Ophiopogon japonicus and pulverize it using an ultrafine pulverizer, controlling the particle size to be 10-20μm, to obtain Ophiopogon japonicus ultrafine powder;

[0027] Add 8-10 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 80-85℃ for 2-3 hours, filter, and collect the first extract; add 6-8 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain the Ophiopogon japonicus aqueous extract.

[0028] Slowly add anhydrous ethanol to the aqueous extract of Ophiopogon japonicus to make the ethanol volume fraction reach 60-70%. After stirring evenly, let it stand for 12-16 hours. Centrifuge at 5000-6000 r / min for 15 minutes, take the precipitate, dissolve it with deionized water to obtain crude extract of Ophiopogon japonicus.

[0029] The crude extract of Ophiopogon japonicus was purified by adsorption with macroporous resin at a flow rate of 1-1.5 mL / min. After loading, the column was rinsed with 3-5 column volumes of deionized water and then eluted with 70-80% ethanol solution at a flow rate of 1-1.5 mL / min. The eluent was collected.

[0030] The eluent was concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09 MPa until the solid content was 10-15%, thus obtaining the Ophiopogon japonicus extract.

[0031] Preferably, the Ganoderma lucidum extract solution is prepared according to the following method:

[0032] Take dried Ganoderma lucidum fruiting bodies, crush them and pass them through an 80-100 mesh sieve. Add 3-5 times the volume of deionized water and soak for 2-3 hours. Then, use a low-temperature cell wall breaker to break the cell wall for 30-40 minutes at a temperature of 5-10℃ and a speed of 10000-12000r / min to obtain Ganoderma lucidum cell wall break liquid.

[0033] Add 6-8 times the volume of deionized water to the Ganoderma lucidum cell wall breaking solution, place it in an ultrasonic-microwave synergistic extraction instrument, and extract for 1-1.5 hours under ultrasonic power of 150-200W, microwave power of 300-400W, and temperature of 60-65℃ to obtain the extract.

[0034] Centrifuge the extract at 6000-7000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa, and collect the permeate.

[0035] The permeate was concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09 MPa until the solid content was 10-15%, thus obtaining Ganoderma lucidum extract.

[0036] Preferably, the Bifidobacterium strain is screened according to the following method:

[0037] Purified placental protein solution, Ophiopogon japonicus extract and Ganoderma lucidum extract were added to MRS medium at volumes of 5%, 3%, and 3%, respectively, to obtain screening medium.

[0038] Each candidate Bifidobacterium strain was inoculated into the screening medium and cultured to obtain the first culture medium;

[0039] The placental protein hydrolysis rate, the retention rate of Ophiopogon japonicus steroidal saponins, and the retention rate of Ganoderma lucidum triterpenoids in the first culture medium were detected. The candidate Bifidobacterium strains corresponding to the first culture medium that meet the first preset conditions were taken as the final Bifidobacterium strains.

[0040] Preferably, the ratio of Bifidobacterium strains to Lactobacillus strains is determined according to the following method:

[0041] Purified placental protein solution, Ophiopogon japonicus extract and Ganoderma lucidum extract were added to MRS medium at volumes of 5%, 3%, and 3% respectively to obtain screening medium.

[0042] Bifidobacterium strains and lactic acid bacteria strains were mixed in different proportions and inoculated into screening medium to obtain a second culture medium.

[0043] The content of short-chain fatty acids and the DPPH free radical scavenging rate in the second culture medium were detected, and the compounding ratio corresponding to the second culture medium that meets the second preset conditions was taken as the final compounding ratio.

[0044] Preferably, the volume percentage of the compound raw material added to the fermentation culture medium is determined according to the following method:

[0045] Using the amounts of purified placental protein solution, Ophiopogon japonicus extract, and Ganoderma lucidum extract as independent variables, a Box-Behnken response surface methodology was employed, with the amounts of purified placental protein solution, Ophiopogon japonicus extract, and Ganoderma lucidum extract set at 8-12%, 3-7%, and 3-7%, respectively. A multi-factor experiment was conducted, using the DPPH free radical scavenging rate and moisturizing performance of the fermentation broth as evaluation indicators.

[0046] Regression analysis was performed on the experimental data, and a quadratic regression model was established. The solution yielded the following results: when the DPPH free radical scavenging rate of the fermentation broth was ≥88% and the moisturizing performance was ≥90%, the volume percentage of the compound raw materials added to the fermentation culture medium was: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution.

[0047] Preferably, the fermentation conditions are determined according to the following method:

[0048] Fermentation was carried out at various temperatures, and the number of microorganisms, the content of effective components, and the DPPH free radical scavenging rate in the fermentation broth obtained at each temperature were detected. The temperature corresponding to the highest number of microorganisms, the highest content of effective components, and the DPPH free radical scavenging rate ≥82% was taken as the final fermentation temperature. The effective components included: placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, and Ganoderma lucidum triterpenoids.

[0049] Fermentation was carried out at a determined fermentation temperature and at various pH values. The pH change curves, enzyme activity, and conversion efficiency of effective components of the fermentation broth were detected at each pH value. The pH value corresponding to the highest enzyme activity and the best conversion efficiency of effective components was taken as the final fermentation pH value.

[0050] Fermentation was carried out at a determined fermentation temperature and pH value; the number of microorganisms, the content of active ingredients, the content of short-chain fatty acids, and the DPPH free radical scavenging rate in the fermentation broth were detected; the time corresponding to when the number of microorganisms ≥10 CFU / mL, the content of active ingredients reaches its peak, the total content of short-chain fatty acids ≥18 mmol / L, and the DPPH free radical scavenging rate ≥85% was taken as the final fermentation time.

[0051] Preferably, the addition of excipients to the fermentation broth, followed by purification and freeze-drying, yields powdered cosmetic functional ingredients, including:

[0052] Add vitamin C at a volume percentage of 0.2-0.3% and vitamin E at a volume percentage of 0.1-0.2% to the fermentation broth, and stir well.

[0053] Add sodium hyaluronate at a volume percentage of 0.3-0.5% and glycerin at a volume percentage of 2-3%, and stir for 15-20 minutes.

[0054] Add xanthan gum and sodium carboxymethyl cellulose at a volume percentage of 0.1-0.2% and stir at 300-500 r / min for 30 min to obtain a mixture.

[0055] Centrifuge the mixture at 6000-8000 r / min for 20-30 min and collect the supernatant;

[0056] The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities;

[0057] The filtered liquid was placed in a freeze dryer and freeze-dried for 24-30 hours at a temperature of -50 to -45°C and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials.

[0058] The second aspect of the present invention provides a placental protein-based Bifidobacterium fermentation cosmetic functional ingredient, which is prepared using the preparation method described in the first aspect.

[0059] The beneficial effects of this invention include:

[0060] 1. This invention optimizes the extraction and purification process of placental proteins by employing ultrasound-assisted enzymatic hydrolysis combined with ultrafiltration and chromatography purification techniques, reducing the destruction of placental protein activity, increasing the extraction rate to over 85%, and achieving a purity of over 90%; Ophiopogon japonicus is purified using ultrafine pulverization combined with water extraction, alcohol precipitation, and macroporous resin purification, increasing the dissolution rate of effective components (steroidal saponins and polysaccharides) by 30-40%; Ganoderma lucidum is extracted using low-temperature cell disruption combined with ultrasound-microwave synergistic extraction, increasing the extraction rates of triterpenoids and polysaccharides by 25-35% and 20-30%, respectively.

[0061] 2. By screening highly active Bifidobacterium strains and combining them with lactic acid bacteria, the synergistic effect between strains can promote the conversion of effective components in raw materials, increase the yield of short-chain fatty acids by 40-50%, and increase the antioxidant activity (DPPH free radical scavenging rate) to over 85%.

[0062] 3. Fermentation parameters such as fermentation temperature, pH value, and time were optimized for the compound system to maximize the conversion efficiency of effective components. The contents of placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, and Ganoderma lucidum triterpenoids in the fermentation products reached more than 5.0%, more than 1.0%, and more than 1.0%, respectively.

[0063] 4. The optimal ratio of compound raw materials is determined through response surface methodology, and auxiliary ingredients are added to enhance efficacy and stability. The 24-hour moisturizing rate reaches over 90%, and the product quality meets cosmetic hygiene standards, ensuring high safety. Detailed Implementation

[0064] To better understand the above technical solution, the following will provide a detailed explanation of the technical solution in conjunction with specific implementation methods.

[0065] The purpose of this invention is to overcome the shortcomings of the prior art and provide a functional raw material for Bifidobacterium fermentation compound cosmetics based on placental protein, Ophiopogon japonicus, and Ganoderma lucidum, and its preparation method. By optimizing the raw material processing technology, fermentation conditions, and component compounding ratio, the extraction rate, conversion efficiency, and synergistic effects of the effective components in the raw material are improved, ensuring product stability and safety.

[0066] This invention provides a method for preparing a placental protein-based Bifidobacterium fermentation cosmetic functional ingredient, comprising:

[0067] Bifidobacterium strains and lactic acid bacteria strains were mixed at a ratio of 3:1 and inoculated into a fermentation medium containing the compound raw materials. The mixture was fermented at 35-37℃ and pH 5.8-6.2 for 24-28 hours to obtain the fermentation broth.

[0068] Adding excipients to the fermentation broth, followed by purification and freeze-drying, yields powdered cosmetic functional ingredients.

[0069] The volume percentage of the compound raw materials added to the fermentation culture medium is as follows: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution.

[0070] In one embodiment of the present invention, the following steps may be taken.

[0071] I. Raw material pretreatment

[0072] 1. Placental protein extraction and purification

[0073] (1) Take a fresh placenta, clean it to remove blood and fascia, and cut it into 1-2cm pieces. 3 Add 3-5 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat them for 20-30 minutes at a power of 200-300W and a temperature of 35-40℃.

[0074] (2) Add a complex protease (protease activity ≥5000U / g) to the ultrasonically treated mixture. The amount of complex protease added is 0.5-1.0% of the placental mass. Adjust the pH to 6.5-7.0 and enzymatically hydrolyze for 2-3 hours at a temperature of 40-45℃ to obtain the enzymatic hydrolysate.

[0075] (3) Centrifuge the enzyme hydrolysate at 5000-8000 r / min for 15-20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa. Collect the permeate.

[0076] (4) The permeate was purified by ion exchange chromatography (using a DEAE-cellulose chromatography column), with the eluent being 0.1-0.2 mol / L NaCl solution and the flow rate being 1-2 mL / min. The main peak eluent was collected. The permeate was then further purified by gel filtration chromatography (using a Sephadex G-75 chromatography column), with the eluent being pH 7.0 phosphate buffer and the flow rate being 0.8-1.2 mL / min. The target component was collected.

[0077] (5) The purified target component is placed in a vacuum concentrator and concentrated at a temperature of 45-50℃ and a vacuum of -0.08 to -0.09MPa until the solid content is 15-20% to obtain purified placental protein solution.

[0078] 2. Preparation of Ophiopogon japonicus extract

[0079] (1) Take dried Ophiopogon japonicus, pulverize it using an ultra-micro pulverizer, and control the pulverized particle size to be 10-20μm to obtain Ophiopogon japonicus ultra-micro powder;

[0080] (2) Add 8-10 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 80-85℃ for 2-3 hours, filter, and collect the first extract; add 6-8 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain Ophiopogon japonicus aqueous extract.

[0081] (3) Slowly add anhydrous ethanol to the water extract of Ophiopogon japonicus to make the volume fraction of ethanol reach 60-70%. After stirring evenly, let it stand for 12-16 hours. Centrifuge at 5000-6000 r / min for 15 minutes, take the precipitate, dissolve it with a small amount of deionized water to obtain crude extract of Ophiopogon japonicus.

[0082] (4) The crude extract of Ophiopogon japonicus was purified by adsorption with macroporous resin (AB-8 type macroporous resin). The loading flow rate was 1-1.5 mL / min. After loading, the column was rinsed with 3-5 column volumes of deionized water and then eluted with 70-80% ethanol solution at a flow rate of 1-1.5 mL / min. The eluent was collected.

[0083] (5) The eluent is concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09MPa until the solid content is 10-15% to obtain the Ophiopogon japonicus extract.

[0084] 3. Preparation of Ganoderma lucidum extract

[0085] (1) Take dried Ganoderma lucidum fruiting body, crush it and pass it through an 80-100 mesh sieve, add 3-5 times the volume of deionized water, soak for 2-3 hours, and then use a low temperature cell wall breaker to break the cell wall for 30-40 minutes at a temperature of 5-10℃ and a speed of 10000-12000r / min to obtain Ganoderma lucidum cell wall break liquid.

[0086] (2) Add 6-8 times the volume of deionized water to the Ganoderma lucidum cell wall breaking liquid, place it in an ultrasonic-microwave synergistic extractor, and extract for 1-1.5 hours under ultrasonic power of 150-200W, microwave power of 300-400W, and temperature of 60-65℃ to obtain the extract.

[0087] (3) Centrifuge the extract at 6000-7000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa to collect the permeate;

[0088] (4) The permeate is concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09MPa until the solid content is 10-15% to obtain Ganoderma lucidum extract.

[0089] II. Bifidobacterium screening and compounding

[0090] 1. Screening of Bifidobacterium strains

[0091] (1) Bifidobacterium strains were identified by morphological observation (Gram positive, non-spore-forming, Y-shaped or V-shaped arrangement), biochemical identification (able to ferment glucose and lactose to produce acid, but not ferment sucrose) and 16S rRNA gene sequence analysis.

[0092] (2) Each Bifidobacterium strain was inoculated into MRS medium containing purified placental protein solution (5%), Ophiopogon japonicus extract solution (3%), and Ganoderma lucidum extract solution (3%), and cultured at 37°C under anaerobic conditions (5% CO2 volume fraction) for 24 h.

[0093] (3) The hydrolysis rate of placental protein in the fermentation broth was detected (the content of free amino acids was determined by Kjeldahl method and the hydrolysis rate was calculated), the retention rate of Ophiopogon japonicus steroidal saponins was determined by high performance liquid chromatography with Ophiopogon japonicus saponin D as a reference standard), and the retention rate of Ganoderma lucidum triterpenoids was determined by high performance liquid chromatography with Ganoderma lucidum acid A as a reference standard. Bifidobacterium strains with placental protein hydrolysis rate ≥30%, Ophiopogon japonicus steroidal saponin retention rate ≥85%, and Ganoderma lucidum triterpenoids retention rate ≥85% were screened out and recorded as strain B.

[0094] 2. Strains in combination

[0095] (1) Select lactic acid bacteria (Lactobacillus plantarum, strain L) and strain B for compounding, and set the inoculation ratio of strain B to strain L to be 1:1, 2:1, 3:1, and 4:1.

[0096] (2) Strains with different compound ratios were inoculated into MRS medium containing the above compound raw materials and cultured at 37°C under anaerobic conditions for 24 h. The content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and antioxidant activity (DPPH free radical scavenging rate) in the fermentation broth were detected by gas chromatography.

[0097] (3) Screening was conducted to select compound ratios with a total short-chain fatty acid content ≥15mmol / L and a DPPH free radical scavenging rate ≥80%, and the optimal compound ratio was determined to be strain B: strain L = 3:1.

[0098] III. Fermentation Process Optimization

[0099] 1. Fermentation temperature optimization

[0100] (1) The compound strain (strain B: strain L = 3:1) was inoculated into the fermentation medium (containing purified placental protein liquid, Ophiopogon japonicus extract liquid, Ganoderma lucidum extract liquid, initial solid content 12%) at an inoculation amount of 5%, and cultured for 24h under anaerobic conditions at temperatures of 30℃, 32℃, 35℃, 37℃, 40℃, and 42℃ respectively.

[0101] (2) The number of microorganisms in the fermentation broth at each temperature was detected (using plate counting method, the number of Bifidobacteria ≥10 CFU / mL), the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids) and the DPPH free radical scavenging rate were detected.

[0102] (3) The optimal fermentation temperature is determined to be 35-37℃, at which point the number of microorganisms meets the standard, the content of effective ingredients is the highest, and the DPPH free radical scavenging rate is ≥82%.

[0103] 2. Fermentation pH optimization

[0104] (1) Under the optimal fermentation temperature (35-37℃), add phosphate buffer (0.1mol / L, pH 5.5, 6.0, 6.5, 7.0, 7.5) to the fermentation medium to adjust the initial pH value, inoculate the compound strain (inoculation amount 5%), and anaerobic culture for 24h.

[0105] (2) Detect the pH change curve, enzyme activity (protease activity, cellulase activity) and effective component conversion efficiency of the fermentation broth at each pH value;

[0106] (3) The optimal initial pH value is determined to be 6.0-6.5. During the fermentation process, the pH value is maintained at 5.8-6.2. At this time, the enzyme activity is the highest (protease activity ≥120U / mL, cellulase activity ≥80U / mL), and the conversion efficiency of effective components is the best.

[0107] 3. Fermentation time optimization

[0108] (1) Under the optimal fermentation temperature (35-37℃) and initial pH (6.0-6.5), the compound strain (inoculation amount 5%) was inoculated and cultured for 12h, 16h, 20h, 24h, 28h and 32h respectively;

[0109] (2) Samples were taken every 4 hours to detect the number of microorganisms, the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids), the content of short-chain fatty acids and the DPPH free radical scavenging rate in the fermentation broth.

[0110] (3) Plot the fermentation curve and determine the optimal fermentation time as 24-28h, at which time the number of microorganisms is ≥10CFU / mL, the content of effective ingredients reaches the peak, the total content of short-chain fatty acids is ≥18mmol / L, and the DPPH free radical scavenging rate is ≥85%.

[0111] IV. Ingredient Combination and Post-processing

[0112] 1. Determining the optimal compound ratio

[0113] (1) Using the amount of purified placental protein solution (A), Ophiopogon japonicus extract (B), and Ganoderma lucidum extract (C) as independent variables, a Box-Behnken response surface design was adopted, with the addition amount of A set at 8-12%, B at 3-7%, and C at 3-7%.

[0114] (2) Using the antioxidant activity (DPPH free radical scavenging rate, Y) and moisturizing performance (24h moisturizing rate, Y) of the fermented compound raw materials as evaluation indicators, a multi-factor experiment was conducted.

[0115] (3) The experimental data were subjected to regression analysis using Design-Expert software. A quadratic regression model was established and the optimal compound ratio was obtained as follows: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution. At this time, Y≥88% and Y≥90%.

[0116] 2. Addition of auxiliary ingredients

[0117] (1) Add natural antioxidants to the fermented compound raw materials: vitamin C (0.2-0.3%) and vitamin E (0.1-0.2%), and stir well;

[0118] (2) Add moisturizing agents: sodium hyaluronate (0.3-0.5%) and glycerin (2-3%), and continue stirring for 15-20 minutes;

[0119] (3) Add stabilizers: xanthan gum (0.1-0.2%) and sodium carboxymethyl cellulose (0.1-0.2%), stir for 30 min at 300-500 r / min to obtain a mixture;

[0120] 3. Post-processing

[0121] (1) Centrifuge the mixture at 6000-8000 r / min for 20-30 min and take the supernatant;

[0122] (2) The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities;

[0123] (3) The filtered liquid was placed in a freeze dryer and freeze-dried for 24-30 hours at a temperature of -50 to -45℃ and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials.

[0124] V. Quality Control

[0125] 1. Appearance: Powder, light yellow to light brown in color, odorless;

[0126] 2. pH value: Take a 1% aqueous solution of the raw material and measure it with a pH meter. The pH value is 6.0-7.0.

[0127] 3. Content of active ingredients: The content of placental protein hydrolysate (calculated as glycine) was determined by high performance liquid chromatography (HPLC) and was ≥5.0%, ≥1.0% of Ophiopogon steroidal saponins (calculated as Ophiopogon saponin D) and ≥1.0% of Ganoderma triterpenoids (calculated as Ganoderma acid A).

[0128] 4. Microbiological indicators: Total colony count ≤100 CFU / g, mold and yeast ≤10 CFU / g, and no pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) should be detected.

[0129] 5. Hazardous substances: heavy metals (lead ≤10mg / kg, mercury ≤1mg / kg, arsenic ≤2mg / kg), pesticide residues (hexachlorocyclohexane and DDT ≤0.1mg / kg). Specific Implementation Example 1

[0131] The method for preparing Bifidobacterium fermented cosmetic functional ingredients based on placental protein provided in this embodiment may include the following steps:

[0132] S1. Raw material pretreatment

[0133] S1.1 Placental protein extraction and purification

[0134] (1) Take a fresh bovine placenta, wash it to remove blood and fascia, and cut it into 1cm pieces. 3 Add 3 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat for 20 minutes at a power of 200W and a temperature of 35℃.

[0135] (2) Add a complex protease (protease activity 5000 U / g) to the ultrasonically treated mixture. The amount of complex protease added is 0.5% of the bovine placenta mass. Adjust the pH to 6.5 and enzymatically hydrolyze at 40℃ for 2 hours to obtain the enzymatic hydrolysate.

[0136] (3) Centrifuge the enzyme hydrolysate at 5000 r / min for 15 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. Collect the permeate.

[0137] (4) The permeate was purified by DEAE-cellulose chromatography column with 0.1 mol / L NaCl solution as the eluent and a flow rate of 1 mL / min. The main peak eluent was collected. The permeate was then further purified by Sephadex G-75 chromatography column with phosphate buffer at pH 7.0 as the eluent and a flow rate of 0.8 mL / min. The target component was collected.

[0138] (5) The purified target component was placed in a vacuum concentrator and concentrated at a temperature of 45°C and a vacuum of -0.08MPa until the solid content was 15% to obtain purified placental protein solution.

[0139] S1.2 Preparation of Ophiopogon japonicus extract

[0140] (1) Take dried Ophiopogon japonicus, pulverize it using an ultra-micro pulverizer, and control the pulverized particle size to 10μm to obtain Ophiopogon japonicus ultra-micro powder;

[0141] (2) Add 8 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 80℃ for 2 hours, filter, and collect the first extract; add 6 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain Ophiopogon japonicus aqueous extract.

[0142] (3) Slowly add anhydrous ethanol to the water extract of Ophiopogon japonicus to make the volume fraction of ethanol reach 60%. After stirring evenly, let it stand for 12 hours. Centrifuge at 5000 r / min for 15 minutes, take the precipitate, dissolve it with a small amount of deionized water to obtain the crude extract of Ophiopogon japonicus.

[0143] (4) The crude extract of Ophiopogon japonicus was purified by adsorption using AB-8 macroporous resin at a flow rate of 1 mL / min. After the sample was loaded, the resin was rinsed with 3 column volumes of deionized water and the water was discarded. Then, the eluent was eluted with 5 column volumes of 60% ethanol solution at a flow rate of 1 mL / min.

[0144] (5) The eluent was concentrated under reduced pressure at 50°C and -0.08 MPa until the solid content was 15% to obtain Ophiopogon japonicus extract.

[0145] S1.3 Preparation of Ganoderma lucidum extract

[0146] (1) Take dried Ganoderma lucidum and place it in a low-temperature high-speed blender. Blend the Ganoderma lucidum for 30 minutes at a speed of 10,000 r / min to obtain Ganoderma lucidum powder.

[0147] (2) Add 6 times the volume of deionized water to the Ganoderma lucidum cell wall breakage powder, place it in an ultrasonic-microwave synergistic extractor, and extract for 1 hour under ultrasonic power of 150W, microwave power of 300W and temperature of 60℃ to obtain Ganoderma lucidum extract;

[0148] (3) Centrifuge the Ganoderma lucidum extract at 6000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa to collect the permeate;

[0149] (4) The permeate was concentrated under reduced pressure at 50°C and -0.08 MPa until the solid content was 10% to obtain Ganoderma lucidum extract.

[0150] S2, Bifidobacterium screening and compounding

[0151] S2.1, Strain Screening

[0152] (1) Bifidobacterium strains were identified by morphological observation (Gram positive, non-spore-forming, Y-shaped or V-shaped arrangement), biochemical identification (able to ferment glucose and lactose to produce acid, but not ferment sucrose) and 16S rRNA gene sequence analysis.

[0153] (2) Each Bifidobacterium strain was inoculated into MRS medium containing purified placental protein solution (5%), Ophiopogon japonicus extract solution (3%), and Ganoderma lucidum extract solution (3%), and cultured at 37°C under anaerobic conditions (5% CO2 volume fraction) for 24 h.

[0154] (3) The hydrolysis rate of placental protein in the fermentation broth was detected (the content of free amino acids was determined by Kjeldahl method and the hydrolysis rate was calculated), the retention rate of Ophiopogon steroidal saponins was determined by high performance liquid chromatography with Ophiopogon saponin D as a reference standard, and the retention rate of Ganoderma triterpenoids was determined by high performance liquid chromatography with Ganoderma acid A as a reference standard. Bifidobacterium strains with placental protein hydrolysis rate ≥30%, Ophiopogon steroidal saponin retention rate ≥85%, and Ganoderma triterpenoid retention rate ≥85% were screened out and recorded as strain B;

[0155] S2.2, strain compound

[0156] (1) Select lactic acid bacteria (Lactobacillus plantarum, strain L) and strain B for compounding, and set the inoculation ratio of strain B to strain L to be 2:1.

[0157] (2) Strains with different compound ratios were inoculated into MRS medium containing the above compound raw materials and cultured at 37°C under anaerobic conditions for 24 h. The content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and antioxidant activity (DPPH free radical scavenging rate) in the fermentation broth were detected by gas chromatography.

[0158] S3, Fermentation Process Optimization

[0159] (1) S3.1, Fermentation temperature optimization

[0160] (2) The compound strain (strain B: strain L = 3:1) was inoculated into the fermentation medium (containing purified placental protein liquid, Ophiopogon japonicus extract liquid, Ganoderma lucidum extract liquid, initial solid content 12%) at an inoculation amount of 5% and cultured at 32-35℃ under anaerobic conditions for 24h.

[0161] (3) The number of microorganisms in the fermentation broth at each temperature was detected (using plate counting method, the number of Bifidobacteria ≥10 CFU / mL), the content of effective ingredients (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids) and the DPPH free radical scavenging rate were detected.

[0162] S3.2, Fermentation pH Optimization

[0163] (1) At the fermentation temperature (32-35℃), add phosphate buffer (0.1mol / L, pH 6.5-7.0) to the fermentation medium to adjust the initial pH value, inoculate the compound strain (inoculation amount 5%), and anaerobic culture for 24h.

[0164] (2) Detect the pH change curve, enzyme activity (protease activity, cellulase activity) and effective component conversion efficiency of the fermentation broth at each pH value;

[0165] S3.3, Fermentation Time Optimization

[0166] (1) Under the conditions of fermentation temperature (32-35℃) and initial pH (6.5-7.0), the compound strain (inoculation amount 5%) was inoculated and cultured for 20h respectively;

[0167] (2) Samples were taken every 4 hours to detect the number of microorganisms, the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids), the content of short-chain fatty acids and the DPPH free radical scavenging rate in the fermentation broth.

[0168] S4. Ingredient formulation and post-processing

[0169] S4.1 Determination of the optimal compound ratio

[0170] (1) Placental protein solution (A), Ophiopogon japonicus extract (B), Ganoderma lucidum extract (C), with the addition amount of A set at 12%, B at 3%, and C at 3%;

[0171] (2) Using the antioxidant activity (DPPH free radical scavenging rate, Y) and moisturizing performance (24h moisturizing rate, Y) of the fermented compound raw materials as evaluation indicators, a multi-factor experiment was conducted.

[0172] S4.2, Addition of auxiliary ingredients

[0173] (1) Add natural antioxidants to the fermented compound raw materials: vitamin C (0.2%) and vitamin E (0.1%), and stir well;

[0174] (2) Add moisturizers: sodium hyaluronate (0.3%) and glycerin (2%), and continue stirring for 15 minutes;

[0175] (3) Add stabilizers: xanthan gum (0.1%) and sodium carboxymethyl cellulose (0.1%), stir at 300 r / min for 30 min to obtain a mixture;

[0176] S4.3 Post-processing

[0177] (1) Centrifuge the mixture at 6000 r / min for 20 min and take the supernatant;

[0178] (2) The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities;

[0179] (3) The filtered liquid was placed in a freeze dryer and freeze-dried for 24 hours at a temperature of -50℃ and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials. Specific Implementation Example 2

[0181] The method for preparing Bifidobacterium fermented cosmetic functional ingredients based on placental protein provided in this embodiment may include the following steps:

[0182] S1. Raw material pretreatment

[0183] S1.1 Bovine placental protein extraction and purification

[0184] (6) Take a fresh placenta, clean it to remove blood and fascia, and cut it into 1cm pieces. 3 Add 5 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat them for 30 minutes at a power of 200W and a temperature of 35℃.

[0185] (7) Add a complex protease (protease activity 5000 U / g) to the ultrasonically treated mixture. The amount of complex protease added is 1.0% of the bovine placenta mass. Adjust the pH to 7.0 and enzymatically hydrolyze for 3 hours at 40°C to obtain the enzymatic hydrolysate.

[0186] (8) Centrifuge the enzyme hydrolysate at 5000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. Collect the permeate.

[0187] (9) The permeate was purified by DEAE-cellulose chromatography column with 0.2 mol / L NaCl solution as the eluent and a flow rate of 2 mL / min. The main peak eluent was collected. The permeate was then further purified by Sephadex G-75 chromatography column with phosphate buffer at pH 7.0 as the eluent and a flow rate of 1.2 mL / min. The target component was collected.

[0188] (10) The purified target component was placed in a vacuum concentrator and concentrated at a temperature of 45°C and a vacuum of -0.08MPa until the solid content was 20% to obtain purified placental protein solution.

[0189] S1.2 Preparation of Ophiopogon japonicus extract

[0190] (6) Take dried Ophiopogon japonicus, pulverize it using an ultra-micro pulverizer, and control the pulverized particle size to 20μm to obtain Ophiopogon japonicus ultra-micro powder;

[0191] (7) Add 8 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 85℃ for 3 hours, filter, and collect the first extract; add 8 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain Ophiopogon japonicus aqueous extract.

[0192] (8) Slowly add anhydrous ethanol to the water extract of Ophiopogon japonicus to make the volume fraction of ethanol reach 70%. After stirring evenly, let it stand for 12 hours. Centrifuge at 6000 r / min for 15 minutes, take the precipitate, dissolve it with a small amount of deionized water to obtain the crude extract of Ophiopogon japonicus.

[0193] (9) The crude extract of Ophiopogon japonicus was purified by adsorption using AB-8 macroporous resin at a flow rate of 1.5 mL / min. After the sample was loaded, the resin was rinsed with 5 column volumes of deionized water and the water was discarded. Then, 5 column volumes of 80% ethanol solution were used for elution at a flow rate of 1.5 mL / min, and the eluent was collected.

[0194] (10) The eluent was concentrated under reduced pressure at a temperature of 55℃ and a vacuum degree of -0.09MPa until the solid content was 20% to obtain the Ophiopogon japonicus extract.

[0195] S1.3 Preparation of Ganoderma lucidum extract

[0196] (1) Take dried Ganoderma lucidum and place it in a low-temperature high-speed blender. Blend the Ganoderma lucidum for 40 minutes at a speed of 10,000 r / min to obtain Ganoderma lucidum powder.

[0197] (2) Add 8 times the volume of deionized water to the Ganoderma lucidum cell wall breakage powder, place it in an ultrasonic-microwave synergistic extractor, and extract for 1 hour under ultrasonic power of 200W, microwave power of 400W and temperature of 65℃ to obtain Ganoderma lucidum extract;

[0198] (3) Centrifuge the Ganoderma lucidum extract at 7000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to collect the permeate;

[0199] (4) The permeate was concentrated under reduced pressure at a temperature of 55°C and a vacuum of -0.09MPa until the solid content was 15% to obtain Ganoderma lucidum extract.

[0200] S2, Bifidobacterium screening and compounding

[0201] S2.1, Strain Screening

[0202] (1) Bifidobacterium strains were identified by morphological observation (Gram positive, non-spore-forming, Y-shaped or V-shaped arrangement), biochemical identification (able to ferment glucose and lactose to produce acid, but not ferment sucrose) and 16S rRNA gene sequence analysis.

[0203] (2) Each Bifidobacterium strain was inoculated into MRS medium containing purified placental protein solution (5%), Ophiopogon japonicus extract solution (3%), and Ganoderma lucidum extract solution (3%), and cultured at 37°C under anaerobic conditions (5% CO2 volume fraction) for 24 h.

[0204] (3) The hydrolysis rate of placental protein in the fermentation broth was detected (the content of free amino acids was determined by Kjeldahl method and the hydrolysis rate was calculated), the retention rate of Ophiopogon steroidal saponins was determined by high performance liquid chromatography with Ophiopogon saponin D as a reference standard, and the retention rate of Ganoderma triterpenoids was determined by high performance liquid chromatography with Ganoderma acid A as a reference standard. Bifidobacterium strains with placental protein hydrolysis rate ≥30%, Ophiopogon steroidal saponin retention rate ≥85%, and Ganoderma triterpenoid retention rate ≥85% were screened out and recorded as strain B;

[0205] S2.2, strain compound

[0206] (1) Select lactic acid bacteria (Lactobacillus plantarum, strain L) and strain B for compounding, and set the inoculation ratio of strain B to strain L to be 1:1.

[0207] (2) The compounded strain was inoculated into MRS medium containing the above compound raw materials and cultured at 37°C under anaerobic conditions for 24 h. The content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and antioxidant activity (DPPH free radical scavenging rate) in the fermentation broth were detected by gas chromatography.

[0208] (3) Screening out compound ratios with a total short-chain fatty acid content ≥15mmol / L and a DPPH free radical scavenging rate ≥80%;

[0209] S3, Fermentation Process Optimization

[0210] S3.1 Fermentation temperature optimization

[0211] (1) The compound strain (strain B: strain L = 1:1) was inoculated into the fermentation medium (containing purified placental protein liquid, Ophiopogon japonicus extract liquid, Ganoderma lucidum extract liquid, and initial solid content of 12%) at an inoculation amount of 5% and cultured at 30-35℃ under anaerobic conditions for 24h.

[0212] (2) The number of microorganisms in the fermentation broth at each temperature was detected (using plate counting method, the number of Bifidobacteria ≥10 CFU / mL), the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids) and the DPPH free radical scavenging rate were detected.

[0213] S3.2, Fermentation pH Optimization

[0214] (1) Under the conditions of fermentation temperature (30-35℃), add phosphate buffer (0.1mol / L, pH5.5) to the fermentation medium to adjust the initial pH value, inoculate the compound strain (inoculation amount 5%), and anaerobic culture for 24h;

[0215] (2) The initial pH value is 5.5-6.0, and the pH value is maintained at 4.5-5.5 during fermentation;

[0216] S3.3, Fermentation Time Optimization

[0217] (1) Under the conditions of optimal fermentation temperature (30-35℃) and initial pH (5.5-6.0), inoculate the compound strain (inoculation amount 5%) and culture for 12h;

[0218] (2) Samples were taken every 4 hours to detect the number of microorganisms, the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids), the content of short-chain fatty acids and the DPPH free radical scavenging rate in the fermentation broth.

[0219] S4. Ingredient formulation and post-processing

[0220] S4.1, A is added at 12%, B at 3%, and C at 7%.

[0221] S4.2, Addition of auxiliary ingredients

[0222] (1) Add natural antioxidants to the fermented compound raw materials: vitamin C (0.3%) and vitamin E (0.2%), and stir well;

[0223] (2) Add moisturizing agents: sodium hyaluronate (0.5%) and glycerin (3%), and continue stirring for 20 minutes;

[0224] (3) Add stabilizers: xanthan gum (0.2%) and sodium carboxymethyl cellulose (0.2%), stir at 500 r / min for 30 min to obtain a mixture;

[0225] S4.3 Post-processing

[0226] (1) Centrifuge the mixture at 6000-8000 r / min for 30 min and take the supernatant;

[0227] (2) The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities;

[0228] (3) The filtered liquid was placed in a freeze dryer and freeze-dried for 30 hours at a temperature of -50℃ and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials. Specific Implementation Example 3

[0230] The method for preparing Bifidobacterium fermented cosmetic functional ingredients based on placental protein provided in this embodiment may include the following steps:

[0231] S1. Raw material pretreatment

[0232] S1.1 Placental protein extraction and purification

[0233] (11) Take a fresh bovine placenta, wash it to remove blood and fascia, and cut it into 1cm pieces. 3 Add 5 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat them for 30 minutes at a power of 200W and a temperature of 35℃.

[0234] (12) Add a complex protease (protease activity 5000 U / g) to the ultrasonically treated mixture. The amount of complex protease added is 1.0% of the bovine placenta mass. Adjust the pH to 7.0 and enzymatically hydrolyze for 3 hours at 40°C to obtain the enzymatic hydrolysate.

[0235] (13) Centrifuge the enzyme hydrolysate at 5000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. Collect the permeate.

[0236] (14) The permeate was purified by DEAE-cellulose chromatography column with 0.2 mol / L NaCl solution as the eluent and a flow rate of 2 mL / min. The main peak eluent was collected. The permeate was then further purified by Sephadex G-75 chromatography column with phosphate buffer at pH 7.0 as the eluent and a flow rate of 1.2 mL / min. The target component was collected.

[0237] (15) The purified target component was placed in a vacuum concentrator and concentrated at a temperature of 45°C and a vacuum of -0.08MPa until the solid content was 20% to obtain purified placental protein solution.

[0238] S1.2 Preparation of Ophiopogon japonicus extract

[0239] (11) Take dried Ophiopogon japonicus, pulverize it using an ultra-micro pulverizer, and control the pulverized particle size to 20 μm to obtain Ophiopogon japonicus ultra-micro powder;

[0240] (12) Add 8 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 85℃ for 3 hours, filter, and collect the first extract; add 8 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain Ophiopogon japonicus aqueous extract.

[0241] (13) Slowly add anhydrous ethanol to the water extract of Ophiopogon japonicus to make the volume fraction of ethanol reach 70%. After stirring evenly, let it stand for 12 hours. Centrifuge at 6000 r / min for 15 minutes, take the precipitate, dissolve it with a small amount of deionized water to obtain the crude extract of Ophiopogon japonicus.

[0242] (14) The crude extract of Ophiopogon japonicus was purified by adsorption using AB-8 macroporous resin at a flow rate of 1.5 mL / min. After the sample was loaded, the resin was rinsed with 5 column volumes of deionized water and the water was discarded. Then, 5 column volumes of 80% ethanol solution were used for elution at a flow rate of 1.5 mL / min, and the eluent was collected.

[0243] (15) The eluent was concentrated under reduced pressure at 55°C and -0.09 MPa until the solid content was 20% to obtain Ophiopogon japonicus extract.

[0244] S1.3 Preparation of Ganoderma lucidum extract

[0245] (1) Take dried Ganoderma lucidum and place it in a low-temperature high-speed blender. Blend the Ganoderma lucidum for 40 minutes at a speed of 10,000 r / min to obtain Ganoderma lucidum powder.

[0246] (2) Add 8 times the volume of deionized water to the Ganoderma lucidum cell wall breakage powder, place it in an ultrasonic-microwave synergistic extractor, and extract for 1 hour under ultrasonic power of 200W, microwave power of 400W and temperature of 65℃ to obtain Ganoderma lucidum extract;

[0247] (3) Centrifuge the Ganoderma lucidum extract at 7000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to collect the permeate;

[0248] (4) The permeate was concentrated under reduced pressure at a temperature of 55°C and a vacuum of -0.09MPa until the solid content was 15% to obtain Ganoderma lucidum extract.

[0249] S2, Bifidobacterium screening and compounding

[0250] S2.1, Strain Screening

[0251] (1) Bifidobacterium strains were identified by morphological observation (Gram positive, non-spore-forming, Y-shaped or V-shaped arrangement), biochemical identification (able to ferment glucose and lactose to produce acid, but not ferment sucrose) and 16S rRNA gene sequence analysis.

[0252] (2) Each Bifidobacterium strain was inoculated into MRS medium containing purified placental protein solution (5%), Ophiopogon japonicus extract solution (3%), and Ganoderma lucidum extract solution (3%), and cultured at 37°C under anaerobic conditions (5% CO2 volume fraction) for 24 h.

[0253] (3) The hydrolysis rate of placental protein in the fermentation broth was detected (the content of free amino acids was determined by Kjeldahl method and the hydrolysis rate was calculated), the retention rate of Ophiopogon steroidal saponins was determined by high performance liquid chromatography with Ophiopogon saponin D as a reference standard, and the retention rate of Ganoderma triterpenoids was determined by high performance liquid chromatography with Ganoderma acid A as a reference standard. Bifidobacterium strains with placental protein hydrolysis rate ≥30%, Ophiopogon steroidal saponin retention rate ≥85%, and Ganoderma triterpenoid retention rate ≥85% were screened out and recorded as strain B;

[0254] S2.2, strain compound

[0255] (3) Select lactic acid bacteria (Lactobacillus plantarum, strain L) and strain B for compounding, and set the inoculation ratio of strain B to strain L to be 3:1.

[0256] (4) The compounded strain was inoculated into MRS medium containing the above compound raw materials and cultured at 37°C under anaerobic conditions for 24 h. The content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and antioxidant activity (DPPH free radical scavenging rate) in the fermentation broth were detected by gas chromatography.

[0257] (4) Screen out the compound ratios with a total short-chain fatty acid content ≥15mmol / L and a DPPH free radical scavenging rate ≥80%;

[0258] S3, Fermentation Process Optimization

[0259] S3.1 Fermentation temperature optimization

[0260] (1) The compound strain (strain B: strain L = 3:1) was inoculated into the fermentation medium (containing purified placental protein liquid, Ophiopogon japonicus extract liquid, Ganoderma lucidum extract liquid, and initial solid content of 12%) at an inoculation amount of 5% and cultured at 35-37℃ under anaerobic conditions for 24h.

[0261] (2) The number of microorganisms in the fermentation broth at each temperature was detected (using plate counting method, the number of Bifidobacteria ≥10 CFU / mL), the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids) and the DPPH free radical scavenging rate were detected.

[0262] S3.2, Fermentation pH Optimization

[0263] (1) Under the conditions of fermentation temperature (35-37℃), add phosphate buffer (0.1mol / L, pH6.0) to the fermentation medium to adjust the initial pH value, inoculate the compound strain (inoculation amount 5%), and anaerobic culture for 24h;

[0264] (2) The initial pH value is 6.0-6.5, and the pH value is maintained at 5.8-6.2 during fermentation;

[0265] S3.3, Fermentation Time Optimization

[0266] (1) Under the conditions of fermentation temperature (35-37℃) and initial pH (6.0-6.5), inoculate the compound strain (inoculation amount 5%) and culture for 32h;

[0267] (2) Samples were taken every 4 hours to detect the number of microorganisms, the content of effective components (placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, Ganoderma lucidum triterpenoids), the content of short-chain fatty acids and the DPPH free radical scavenging rate in the fermentation broth.

[0268] S4. Ingredient formulation and post-processing

[0269] S4.1, A is added at 10%, B at 5%, and C at 5%.

[0270] S4.2, Addition of auxiliary ingredients

[0271] (1) Add natural antioxidants to the fermented compound raw materials: vitamin C (0.3%) and vitamin E (0.2%), and stir well;

[0272] (2) Add moisturizing agents: sodium hyaluronate (0.5%) and glycerin (3%), and continue stirring for 20 minutes;

[0273] (3) Add stabilizers: xanthan gum (0.2%) and sodium carboxymethyl cellulose (0.2%), stir at 500 r / min for 30 min to obtain a mixture;

[0274] S4.3 Post-processing

[0275] (1) Centrifuge the mixture at 6000-8000 r / min for 30 min and take the supernatant;

[0276] (2) The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities;

[0277] (3) The filtered liquid was placed in a freeze dryer and freeze-dried for 30 hours at a temperature of -50℃ and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials.

[0278] Test results:

[0279] 1. DPPH free radical scavenging rate detection

[0280] (1) Detection method: The sample solution to be tested was mixed with DPPH ethanol solution (concentration of 0.2 mmol / L) at a volume ratio of 1:1 and reacted at room temperature in the dark for 30 min. The absorbance value was measured at a wavelength of 517 nm. Anhydrous ethanol was used as a blank control instead of the sample solution. The DPPH free radical scavenging rate was calculated according to the formula: DPPH free radical scavenging rate (%) = (1 - A sample / A blank) × 100%, where A sample is the absorbance value of the sample solution and DPPH ethanol solution after mixing, and A blank is the absorbance value of the blank control.

[0281] (2) The test results are shown in the table below:

[0282] Specific Implementation DPPH maximum clearance rate Specific Implementation Example 1 60％ Specific Implementation Example 2 76.5％ Specific Implementation Example 3 >80％

[0283] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compounding ratio, the maximum DPPH scavenging rate of the sample in Example 3 was significantly higher than that in Examples 1 and 2. This indicates that suitable experimental conditions during fermentation have a significant effect on improving the antioxidant efficacy of the product.

[0284] 2. Short-chain fatty acid content test

[0285] (1) Test method: Gas chromatography. Specific procedure: Take the fermentation broth sample, add an appropriate amount of internal standard (e.g., n-heptanoic acid), extract with an organic solvent (e.g., n-hexane), shake well, centrifuge, and take the upper organic phase for gas chromatography analysis. Gas chromatography conditions: Use a polar capillary column, high-purity nitrogen as the carrier gas, set the flow rate according to the column specifications, set the injection port temperature to 200-250℃, set the detector temperature to 250-300℃, and use a programmed temperature ramp. The initial temperature is set to 80-100℃, held for a certain time, and then increased to 200-220℃ at a certain rate, held until the analysis is complete. By comparing the gas chromatogram with that of the standard sample, qualitative analysis is performed based on retention time, and quantitative analysis is performed using the peak area ratio of the internal standard method. The content of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the fermentation broth is calculated.

[0286] (2) The test results are shown in the table below:

[0287] Specific Implementation Short-chain fatty acid content Specific Implementation Example 1 6.8 mmol / L Specific Implementation Example 2 12.5 mmol / L Specific Implementation Example 3 ≥15mmol / L

[0288] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compounding ratio, the short-chain fatty acid content of the sample in Example 3 is significantly higher than that in Examples 1 and 2. This indicates that Example 3 has a higher degree of fermentation.

[0289] 3. Evaluation of moisturizing performance

[0290] (1) Evaluation method:

[0291] The weighing method was used. The specific procedure was as follows: Take a dry sponge block of a certain mass (m), immerse it completely in the sample solution to be tested, soak it for a certain period of time, remove it, gently absorb excess solution from the surface of the sponge block with filter paper, and then weigh it immediately, recording the mass (m). The moisture retention rate (%) was calculated using the formula: Moisture retention rate (%) = (mm) / m × 100%.

[0292] Where m is the mass of the dry sponge block and m is the mass of the sponge block after absorbing liquid. This formula can accurately evaluate the moisturizing performance of functional ingredients in compound cosmetics, providing an important basis for product quality assessment.

[0293] (3) The evaluation results are shown in the table below:

[0294]

[0295]

[0296] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compounding ratio, the long-lasting moisturizing effect of the sample in Example 3 is significantly higher than that in Examples 1 and 2. This indicates that the polysaccharides and other products in Example 3 have a higher degree of molecular size during fermentation, resulting in better skin absorption.

[0297] 4. Active ingredient content test

[0298] (1) Placental protein hydrolysate test

[0299] Test Method: High-performance liquid chromatography (HPLC) was used for determination. The specific procedure is as follows: First, standard solutions of placental protein hydrolysates at different concentrations were prepared. Under selected HPLC conditions (using a suitable C18 reversed-phase column, an acetonitrile-water mixture as the mobile phase, adjusting the ratio according to actual conditions, controlling the flow rate at 0.8-1.2 mL / min, and selecting an appropriate detection wavelength based on the characteristics of the placental protein hydrolysates, generally within the range of 200-250 nm), the samples were injected for analysis, and a standard curve was plotted. Then, an appropriate amount of fermentation broth sample was taken, and after appropriate treatment (such as centrifugation to remove impurities), it was injected under the same chromatographic conditions. The content of the corresponding placental protein hydrolysates was determined from the peak area on the standard curve. This method can accurately determine the content of placental protein hydrolysates in the fermentation broth, thereby assessing the hydrolysis of placental protein during fermentation.

[0300] The test results are shown in the table below:

[0301] Specific Implementation Placental protein hydrolysate content Specific Implementation Example 1 1.2％ Specific Implementation Example 2 3.7％ Specific Implementation Example 3 5.6％

[0302] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compounding ratio, the content of placental protein hydrolysate in the sample of Example 3 is significantly higher than that in Examples 1 and 2. This indicates that Example 3 has a higher degree of fermentation, and the protein hydrolysis effect increases with the increase of the degree of fermentation.

[0303] (2) Test of Ophiopogon japonicus steroidal saponin content

[0304] Test method:

[0305] High-performance liquid chromatography (HPLC) was used for determination. First, standards for ophiopogonin steroidal saponins were prepared, and a series of standard solutions of different concentrations were prepared. These standard solutions were sequentially injected into the HPLC system under pre-set chromatographic conditions (e.g., using a suitable C18 reversed-phase column, an acetonitrile-water mobile phase, adjusting the mobile phase ratio according to the properties of ophiopogonin steroidal saponins to achieve better separation, a flow rate typically set at 0.8-1.2 mL / min, and a detection wavelength selected at the wavelength of maximum absorption of ophiopogonin steroidal saponins, generally in the range of 200-220 nm). The peak areas of each standard solution were recorded, and a standard curve was plotted with the concentration of the standard solution on the x-axis and the peak area on the y-axis. Next, an appropriate amount of fermentation broth sample was taken, pretreated by centrifugation to remove solid impurities, and then injected for analysis under the same chromatographic conditions. The corresponding ophiopogonin steroidal saponin content was determined from the standard curve based on the obtained sample peak area. This method can accurately determine the content of ophiopogonin steroidal saponins in the fermentation broth, thereby assessing the retention of ophiopogonin steroidal saponins during fermentation.

[0306] The test results are shown in the table below:

[0307] Specific Implementation Ophiopogon japonicus steroidal saponin content Specific Implementation Example 1 0.24％ Specific Implementation Example 2 0.53％ Specific Implementation Example 3 1.86％

[0308] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compounding ratio, the content of Ophiopogon japonicus steroidal saponins in Example 3 is significantly higher than that in Examples 1 and 2. This indicates that suitable temperature, pH value, and fermentation time play an important role in the retention and accumulation of Ophiopogon japonicus steroidal saponins during fermentation. Simultaneously, the selection and ratio of the compounded bacterial strains also positively impacted the content of Ophiopogon japonicus steroidal saponins, resulting in a higher level of Ophiopogon japonicus steroidal saponins in Example 3, providing strong assurance for improving the quality of functional raw materials in compound cosmetics.

[0309] (3) Test of Ganoderma lucidum triterpenoid content

[0310] Test method:

[0311] High-performance liquid chromatography (HPLC) was used for determination. First, a precise amount of Ganoderma lucidum triterpenoid standards were weighed, dissolved in an organic solvent such as methanol, and diluted to a specific volume to prepare standard solutions of different concentrations. These standard solutions were sequentially injected into the HPLC system and analyzed under suitable chromatographic conditions (generally a C18 reversed-phase column, a methanol-water mixture as the mobile phase, with the ratio adjusted according to the properties of Ganoderma lucidum triterpenoids, a flow rate controlled at 0.8-1.2 mL / min, and a detection wavelength selected at the characteristic absorption wavelength of Ganoderma lucidum triterpenoids, typically in the range of 240-260 nm). The peak areas of each standard solution were recorded, and a standard curve was plotted with the standard solution concentration on the x-axis and the peak area on the y-axis. Then, an appropriate amount of fermentation broth sample was taken, pretreated by centrifugation to remove solid impurities, and analyzed under the same chromatographic conditions. The corresponding Ganoderma lucidum triterpenoid content was determined from the standard curve based on the sample peak area. This method can accurately determine the content of Ganoderma lucidum triterpenoids in the fermentation broth, thereby assessing the retention of Ganoderma lucidum triterpenoids during fermentation.

[0312] The test results are shown in the table below:

[0313] Example Ganoderma triterpenoid content Specific Implementation Example 1 0.12％ Specific Implementation Example 2 0.45％ Specific Implementation Example 3 1.62％

[0314] The test results above show that, with continuous optimization of the fermentation process and adjustment of the compound ratio, the content of Ganoderma lucidum triterpenes in Example 3 is significantly higher than that in Examples 1 and 2. This indicates that suitable temperature, pH value, and fermentation time play an important role in the retention and accumulation of Ganoderma lucidum triterpenes during fermentation. Simultaneously, the selection and ratio of the compound strains also positively impacted the content of Ganoderma lucidum triterpenes, resulting in a higher level of content in Example 3, providing strong assurance for improving the quality of functional raw materials in compound cosmetics.

[0315] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention is also intended to include these modifications and modifications.

Claims

1. A method for preparing a placental protein-based Bifidobacterium fermented cosmetic functional ingredient, characterized in that, include: Bifidobacterium strains and lactic acid bacteria strains were mixed at a ratio of 3:1 and inoculated into a fermentation medium containing the compound raw materials. The mixture was fermented at 35-37℃ and pH 5.8-6.2 for 24-28 hours to obtain the fermentation broth. Adding excipients to the fermentation broth, followed by purification and freeze-drying, yields powdered cosmetic functional ingredients. The volume percentage of the compound raw materials added to the fermentation culture medium is as follows: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution. The Ophiopogon japonicus extract was prepared according to the following method: Prepare Ophiopogon japonicus ultrafine powder with a particle size of 10-20 μm; Deionized water was added to Ophiopogon japonicus ultrafine powder, and a second reflux extraction was performed to prepare Ophiopogon japonicus aqueous extract; Within 2-3 minutes, add anhydrous ethanol to the Ophiopogon japonicus aqueous extract to make the volume fraction of anhydrous ethanol reach 60-70%. After stirring evenly, let stand and centrifuge to dissolve the precipitate and obtain the crude extract of Ophiopogon japonicus. The crude extract of Ophiopogon japonicus was purified and concentrated to obtain an extract solution with a solid content of 10-15%. The Ganoderma lucidum extract solution was prepared according to the following method: Ganoderma lucidum seed powder was subjected to cell wall breaking treatment to obtain Ganoderma lucidum cell wall breaking liquid; Deionized water was added to the Ganoderma lucidum cell wall breaking solution, and the solution was placed in an ultrasonic-microwave synergistic extractor to prepare the extract. Centrifuge the extract, ultrafilter the supernatant, and collect the permeate. The extract was concentrated under reduced pressure to obtain Ganoderma lucidum extract.

2. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The purified placental protein solution was prepared according to the following method: Take a fresh placenta, wash it to remove blood and fascia, and cut it into 1-2cm pieces. 3 Add 3-5 times the volume of physiological saline to the small pieces, place them in an ultrasonic extractor, and ultrasonically treat them for 20-30 minutes at a power of 200-300W and a temperature of 35-40℃. Add a complex protease to the ultrasonically treated mixture at a concentration of 0.5-1.0% of the placental mass, adjust the pH to 6.5-7.0, and hydrolyze at 40-45℃ for 2-3 hours to obtain the hydrolysate; the activity of the complex protease is ≥5000U / g. Centrifuge the enzymatic hydrolysate at 5000-8000 r / min for 15-20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa. Collect the permeate. The permeate was purified by ion exchange chromatography with 0.1-0.2 mol / L NaCl solution as the eluent and a flow rate of 1-2 mL / min. The main peak eluent was collected. The permeate was then further purified by gel filtration chromatography with phosphate buffer at pH 7.0 as the eluent and a flow rate of 0.8-1.2 mL / min. The target component was collected. The purified target component was placed in a vacuum concentrator and concentrated to a solid content of 15-20% under conditions of 45-50℃ and a vacuum of -0.08 to -0.09 MPa to obtain purified placental protein solution.

3. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The Ophiopogon japonicus extract was prepared according to the following method: Take dried Ophiopogon japonicus and pulverize it using an ultrafine pulverizer, controlling the particle size to be 10-20μm, to obtain Ophiopogon japonicus ultrafine powder; Add 8-10 times the volume of deionized water to the Ophiopogon japonicus ultrafine powder, reflux extract at 80-85℃ for 2-3 hours, filter, and collect the first extract; add 6-8 times the volume of deionized water to the residue, repeat the reflux extraction once, and collect the second extract; combine the two extracts to obtain the Ophiopogon japonicus aqueous extract. Slowly add anhydrous ethanol to the aqueous extract of Ophiopogon japonicus to make the ethanol volume fraction reach 60-70%. After stirring evenly, let it stand for 12-16 hours. Centrifuge at 5000-6000 r / min for 15 minutes, take the precipitate, dissolve it with deionized water to obtain crude extract of Ophiopogon japonicus. The crude extract of Ophiopogon japonicus was purified by adsorption with macroporous resin at a flow rate of 1-1.5 mL / min. After loading, the column was rinsed with 3-5 column volumes of deionized water and then eluted with 70-80% ethanol solution at a flow rate of 1-1.5 mL / min. The eluent was collected. The eluent was concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09 MPa until the solid content was 10-15%, thus obtaining the Ophiopogon japonicus extract.

4. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The Ganoderma lucidum extract solution was prepared according to the following method: Take dried Ganoderma lucidum fruiting bodies, crush them and pass them through an 80-100 mesh sieve. Add 3-5 times the volume of deionized water and soak for 2-3 hours. Then, use a low-temperature cell wall breaker to break the cell wall for 30-40 minutes at a temperature of 5-10℃ and a speed of 10000-12000r / min to obtain Ganoderma lucidum cell wall break liquid. Add 6-8 times the volume of deionized water to the Ganoderma lucidum cell wall breaking solution, place it in an ultrasonic-microwave synergistic extraction instrument, and extract for 1-1.5 hours under ultrasonic power of 150-200W, microwave power of 300-400W, and temperature of 60-65℃ to obtain the extract. Centrifuge the extract at 6000-7000 r / min for 20 min, take the supernatant, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa, and collect the permeate. The permeate was concentrated under reduced pressure at a temperature of 50-55℃ and a vacuum degree of -0.08 to -0.09 MPa until the solid content was 10-15%, thus obtaining Ganoderma lucidum extract.

5. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The Bifidobacterium strains were screened according to the following method: Purified placental protein solution, Ophiopogon japonicus extract and Ganoderma lucidum extract were added to MRS medium at volumes of 5%, 3%, and 3%, respectively, to obtain screening medium. Each candidate Bifidobacterium strain was inoculated into the screening medium and cultured to obtain the first culture medium; The placental protein hydrolysis rate, the retention rate of Ophiopogon japonicus steroidal saponins, and the retention rate of Ganoderma lucidum triterpenoids in the first culture medium were detected. The candidate Bifidobacterium strains corresponding to the first culture medium that meet the first preset conditions were taken as the final Bifidobacterium strains.

6. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The ratio of Bifidobacterium strains to Lactobacillus strains was determined as follows: Purified placental protein solution, Ophiopogon japonicus extract and Ganoderma lucidum extract were added to MRS medium at volumes of 5%, 3%, and 3% respectively to obtain screening medium. Bifidobacterium strains and lactic acid bacteria strains were mixed in different proportions and inoculated into screening medium to obtain a second culture medium. The content of short-chain fatty acids and the DPPH free radical scavenging rate in the second culture medium were detected, and the compounding ratio corresponding to the second culture medium that meets the second preset conditions was taken as the final compounding ratio.

7. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The volume percentage of the compound raw material added to the fermentation medium was determined according to the following method: Using the amounts of purified placental protein solution, Ophiopogon japonicus extract, and Ganoderma lucidum extract as independent variables, a Box-Behnken response surface methodology was employed, with the amounts of purified placental protein solution, Ophiopogon japonicus extract, and Ganoderma lucidum extract set at 8-12%, 3-7%, and 3-7%, respectively. A multi-factor experiment was conducted, using the DPPH free radical scavenging rate and moisturizing performance of the fermentation broth as evaluation indicators. Regression analysis was performed on the experimental data, and a quadratic regression model was established. The solution yielded the following results: when the DPPH free radical scavenging rate of the fermentation broth was ≥88% and the moisturizing performance was ≥90%, the volume percentage of the compound raw materials added to the fermentation culture medium was: 10% purified placental protein solution, 5% Ophiopogon japonicus extract solution, and 5% Ganoderma lucidum extract solution.

8. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, Fermentation conditions were determined as follows: Fermentation was carried out at various temperatures, and the number of microorganisms, the content of effective components, and the DPPH free radical scavenging rate in the fermentation broth obtained at each temperature were detected. The temperature corresponding to the highest number of microorganisms, the highest content of effective components, and the DPPH free radical scavenging rate ≥82% was taken as the final fermentation temperature. The effective components included: placental protein hydrolysate, Ophiopogon japonicus steroidal saponins, and Ganoderma lucidum triterpenoids. Fermentation was carried out at a determined fermentation temperature and at various pH values. The pH change curves, enzyme activity, and conversion efficiency of effective components of the fermentation broth were detected at each pH value. The pH value corresponding to the highest enzyme activity and the best conversion efficiency of effective components was taken as the final fermentation pH value. Fermentation was carried out at a determined fermentation temperature and pH value; the number of microorganisms, the content of active ingredients, the content of short-chain fatty acids, and the DPPH free radical scavenging rate in the fermentation broth were detected; the time corresponding to when the number of microorganisms ≥10 CFU / mL, the content of active ingredients reaches its peak, the total content of short-chain fatty acids ≥18 mmol / L, and the DPPH free radical scavenging rate ≥85% was taken as the final fermentation time.

9. The method for preparing Bifidobacterium fermented cosmetic functional raw materials based on placental protein as described in claim 1, characterized in that, The addition of excipients to the fermentation broth, followed by purification and freeze-drying, yields powdered cosmetic functional ingredients, including: Add vitamin C at a volume percentage of 0.2-0.3% and vitamin E at a volume percentage of 0.1-0.2% to the fermentation broth, and stir well. Add sodium hyaluronate at a volume percentage of 0.3-0.5% and glycerin at a volume percentage of 2-3%, and stir for 15-20 minutes. Add xanthan gum and sodium carboxymethyl cellulose at a volume percentage of 0.1-0.2% and stir at 300-500 r / min for 30 min to obtain a mixture. Centrifuge the mixture at 6000-8000 r / min for 20-30 min and collect the supernatant; The supernatant was filtered using a 0.22μm microporous membrane to remove microorganisms and impurities; The filtered liquid was placed in a freeze dryer and freeze-dried for 24-30 hours at a temperature of -50 to -45°C and a vacuum degree of ≤10Pa to obtain powdered compound cosmetic functional raw materials.

10. A placental protein-based Bifidobacterium fermented cosmetic functional ingredient, characterized in that, It is prepared using the preparation method described in any one of claims 1-9.

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