A torulopsis pullulans fermentation product filtrate, and a preparation method and application thereof

Abstract

The application relates to the field of cosmetics, and relates to a Saccharomycopsis fibuligera fermentation product filtrate, a preparation method thereof and application, which comprises the following steps: crushing peony leaves to obtain peony leaf powder, mixing the peony leaf powder with ethanol, ultrasonic extraction, concentration, adding protein peptone, yeast paste and glucose to prepare a Saccharomycopsis fibuligera fermentation culture medium, inoculating the Saccharomycopsis fibuligera to ferment, filtering and centrifuging the fermentation liquid to obtain supernatant, sterilizing the supernatant, and then adding a preservative to obtain the Saccharomycopsis fibuligera fermentation product filtrate. The Saccharomycopsis fibuligera fermentation product filtrate prepared by the method has excellent anti-aging, whitening, moisturizing and skin repairing effects, and has a good application prospect in the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a filtrate of yeast fermentation product with a membrane-forming structure, its preparation method, and its application. Background Technology

[0002] The peony is a treasure trove; its roots, bark, leaves, flowers, and seed oil are all listed in the "Catalogue of Used Cosmetic Raw Materials" (2015 edition). Compared to other parts of the peony, the leaves have advantages such as high yield and multiple harvests per year. Peony leaves contain high levels of protein, carbohydrates, fats, and vitamins, with a protein content as high as 15%, making them highly nutritious and valuable. They also contain reducing sugars, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, benzoyloxypaeoniflorin, gallic acid, and other polyphenols, as well as flavonoids, flavonoids, tannins, chlorogenic acid, and other active functional ingredients. Among the active ingredients, the reducing sugar content is as high as 12%, the total phenol content is 8.5%, the total flavonoid content is 1.6%, and the paeoniflorin content is 1.93%, with the total phenol content even higher than that of the bark.

[0003] Existing technologies often use organic solvents such as ethanol and acetone to extract active ingredients from peonies. This not only causes environmental pollution but also has high extraction costs. The extracts may contain residual organic solvents, and the purification process after extraction is cumbersome and the experimental operation is somewhat dangerous, making it unsuitable for use in cosmetics and food ingredients.

[0004] To address this issue, existing technologies include methods that enhance the efficacy of peony leaves through microbial fermentation. For example, Chinese patent CN114209624A discloses a peony fermentation liquid and its preparation method and application. This method uses Lactobacillus plantarum and Lactobacillus bulgaricus to ferment the peony, reducing the loss of active ingredients in the peony. The resulting peony fermentation liquid has good antioxidant, whitening, and skin-nourishing effects.

[0005] Saccharomycopsis fibuligera, also known as Saccharomycopsis fibuligera, contains natural components such as free amino acids, minerals, and organic acids essential for healthy skin through its extracellular fermentation product filtrate. It has multiple cosmetic effects, including anti-inflammatory, anti-UV, moisturizing, skin repair, and antioxidant properties. Long-term use can maintain skin stability, improve skin texture, and make the skin more delicate. It has been included in the "List of Used Cosmetic Raw Material Names" (2015 edition) and is also a major ingredient in famous cosmetics such as SK-II Facial Treatment Essence.

[0006] In existing technologies, *Saccharomyces cerevisiae* primarily utilizes glucose as a substrate for fermentation, but this approach has limitations in terms of antioxidant and whitening effects. Fermentation methods combining peony leaves and *Saccharomyces cerevisiae* are rarely reported. Summary of the Invention

[0007] The present application aims to provide a Saccharomycopsis fibuligera fermentation product filtrate and a preparation method and application thereof, which improves the anti-aging, whitening, moisturizing, skin repair and other effects of the fermentation filtrate by combining the peony leaf fermentation liquor with Saccharomycopsis fibuligera.

[0008] To solve the above technical problems, the specific scheme adopted by the present application is as follows: a preparation method of a Saccharomycopsis fibuligera fermentation product filtrate, wherein seed liquid of Saccharomycopsis fibuligera is added into a culture medium prepared from peony leaf filtrate, and then fermentation and filtration are performed to obtain the Saccharomycopsis fibuligera fermentation product filtrate.

[0009] As a further optimization of the above technical solution, the method comprises the following steps:

[0010] S1: crushing peony leaves to obtain peony leaf powder;

[0011] S2: mixing the peony leaf powder with an organic solvent, ultrasonic extraction, and filtration to obtain peony leaf filtrate;

[0012] S3: adding peptone, yeast extract and glucose to the peony leaf filtrate after concentration, and sterilizing to obtain a Saccharomycopsis fibuligera fermentation culture medium;

[0013] S4: adding seed liquid of Saccharomycopsis fibuligera to the Saccharomycopsis fibuligera fermentation culture medium for primary fermentation, and then ultrasonic treatment; and then secondary fermentation to obtain an initial fermentation liquor;

[0014] S5: filtering and centrifuging the initial fermentation liquor, high-temperature instant sterilization of the supernatant after centrifugation, cooling to room temperature, and adding a preservative to obtain the Saccharomycopsis fibuligera fermentation product filtrate.

[0015] As a further optimization of the above technical solution, the preparation method of the peony leaf filtrate in step S2 is as follows: uniformly mixing the peony leaf powder with 70% ethanol at a mass-volume ratio of 1:10, ultrasonic extraction, and filtration to obtain filtrate and residue, retaining the filtrate, uniformly mixing the residue with 50% ethanol at a mass-volume ratio of 1:8, second ultrasonic extraction, and filtration to obtain filtrate and residue, retaining the filtrate, uniformly mixing the residue with 20% ethanol at a mass-volume ratio of 1:8, third ultrasonic extraction, and filtration to obtain filtrate and residue, and combining the filtrates obtained in the three times to obtain the peony leaf filtrate.

[0016] As a further optimization of the above technical solution, in step S3, the peony leaf filtrate is first concentrated to half of the original volume at 60-70 DEG C, and then peptone, yeast extract and glucose are added.

[0017] As a further optimization of the above technical solution: in step S4, the preparation method of the Saccharomycopsis fibuligera seed liquid is as follows: a Saccharomycopsis fibuligera glycerol freezing tube is taken out, and a sterile operation is performed on a super-clean bench to streak on a solid culture medium plate; the plate is placed in a 28-32 DEG C incubator and cultured for 3 days; after the colonies grow, a single colony is picked and inoculated into a liquid culture medium, and placed in a 30 DEG C, 180 rpm / min shaker for 48 h to obtain the Saccharomycopsis fibuligera seed liquid.

[0018] As a further optimization of the above technical solution: in step S4, the volume ratio of the Saccharomycopsis fibuligera seed liquid added to the Saccharomycopsis fibuligera fermentation medium is 3%-10%.

[0019] As a further optimization of the above technical solution: in step S4, the ultrasonic treatment power is 50-70 W, 0.5-0.7 W / ml, the frequency is 30-50 kHz, the temperature is 28-32 DEG C, and the ultrasonic treatment time is 50-70 min.

[0020] As a further optimization of the above technical solution: in step S4, the conditions of the first fermentation and the second fermentation are both as follows: the temperature is 28-32 DEG C, the rotation speed is 180 rpm / min, and the fermentation time is 20-30 h.

[0021] The Saccharomycopsis fibuligera fermentation product filtrate is prepared by using the preparation method of the Saccharomycopsis fibuligera fermentation product filtrate.

[0022] The application of the Saccharomycopsis fibuligera fermentation product filtrate in cosmetics.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The Saccharomycopsis fibuligera fermentation product filtrate provided by the application has good anti-skin aging effect, whitening effect, and moisturizing effect, and has good skin repair effect; the content of total flavonoids, total polyphenols, and various active ingredients in the Saccharomycopsis fibuligera fermentation product filtrate is significantly improved.

[0025] The Saccharomycopsis fibuligera fermentation product filtrate provided by the application not only has a conversion effect on the active ingredients in the peony leaf, but also affects the extracellular metabolic products of the Saccharomycopsis fibuligera. During the fermentation process, due to the interaction between the peony leaf and the Saccharomycopsis fibuligera, the moisturizing, whitening, and antioxidant effects of the Saccharomycopsis fibuligera fermentation filtrate are greatly improved. During the Saccharomycopsis fibuligera fermentation process, the antioxidant activity and tyrosinase enzyme inhibition activity of the peony leaf are not simply added together, but during the fermentation process, the low-activity ingredients in the peony leaf are converted into high-activity ingredients, and the active ingredients in the peony leaf affect the extracellular metabolism of the Saccharomycopsis fibuligera, thereby improving the antioxidant activity and whitening activity of the Saccharomycopsis fibuligera extracellular fermentation filtrate.

[0026] This invention uses peony leaf filtrate as a substrate for fermentation with *Saccharomyces cerevisiae*, which can significantly improve the antioxidant and whitening effects of the fermentation product filtrate. During fermentation, *Saccharomyces cerevisiae* secretes various extracellular enzymes that enzymatically break down the cell walls of peony leaves, promoting the dissolution of active ingredients. Furthermore, a large amount of flavonoids, polyphenols, acids, and sugars in peony leaves are bound together and fixed in the cell walls and intercellular matrix as insoluble bound phenols. Through fermentation with *Saccharomyces cerevisiae*, glycosidic bonds and lipokinase bonds are enzymatically broken down, converting the bound phenols into soluble free phenols, thus increasing the content of flavonoids and polyphenols, thereby enhancing antioxidant activity and tyrosinase inhibition rate.

[0027] The fermentation product filtrate of *Saccharomyces cerevisiae* provided by this invention employs a low-frequency, low-power ultrasound-assisted fermentation method. This method promotes the growth of *Saccharomyces cerevisiae*. Ultrasound exerts a certain degree of damage on the yeast, but this damage is reversible. After damage, the cell membrane permeability increases, leading to increased dissolution of intracellular enzymes and substances. These intracellular enzymes act on various components of the peony leaf, increasing the concentration of nutrients in the yeast's environment. Furthermore, *Saccharomyces cerevisiae* grows rapidly in the peony leaf suspension, forming dendritic pseudohyphae. Ultrasound disperses these pseudohyphae, separating them into individual yeast cells, increasing the surface area for exchange with the external environment, facilitating nutrient absorption, and promoting yeast growth. On the other hand, the rapid growth of *Saccharomyces cerevisiae* accelerates the conversion of active ingredients in the peony leaf. These two factors interact, jointly enhancing the antioxidant and whitening effects of the *Saccharomyces cerevisiae* peony leaf fermentation filtrate. Attached Figure Description

[0028] Figure 1 Flowchart for the preparation of filtrate from yeast fermentation products with encapsulated membranes;

[0029] Figure 2 This is a graph showing the change in the number of capsule-forming yeasts over time during fermentation.

[0030] Figure 3 Morphological image of yeast with clasped capsules before ultrasound;

[0031] Figure 4 Morphological image of cladocyst-bound yeast after ultrasound;

[0032] Figure 5 To demonstrate the anti-cellular aging activity of yeast fermentation product filtrate with encapsulated membranes;

[0033] Figure 6 A comparison of TIC (Total Intake) before and after fermentation of the capsule-coated yeast;

[0034] Figure 7 A graph showing the changes in the composition of peony leaves before and after fermentation with sac-covered yeast. DETAILED DESCRIPTION

[0035] The application is further described below in connection with specific embodiments. It is understood that these embodiments are merely for illustrative purposes and do not limit the scope of the application. Furthermore, it is understood that those skilled in the art can make various modifications or alterations to the application after reading the content of the present application, and these equivalent forms also fall within the scope of the appended claims.

[0036] In the following examples, the Saccharomycopsis fibuligera is purchased from the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 21624, and the address of No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0037] The activated solid culture medium of Saccharomycopsis fibuligera is: 20 g of tryptone, 20 g of glucose, 10 g of yeast powder, and 20 g of agar powder are added to 1 L of water, and then placed in a high-pressure steam sterilization pot for sterilization at 121°C for 15 min, and then cooled for standby use.

[0038] The seed liquid liquid culture medium of Saccharomycopsis fibuligera is: 20 g of tryptone, 20 g of glucose, and 10 g of yeast powder are added to 1 L of water, and then placed in a high-pressure steam sterilization pot for sterilization at 121°C for 15 min, and then cooled for standby use.

[0039] The inoculation amount of Saccharomycopsis fibuligera is expressed by percentage, which means that the amount of Saccharomycopsis fibuligera accounts for the volume percentage of the Saccharomycopsis fibuligera fermentation medium.

[0040] The activation method of the Saccharomycopsis fibuligera strain is as follows: the Saccharomycopsis fibuligera glycerol freezing tube is taken out from the -80°C refrigerator, and then streaked on the activated solid culture medium plate of Saccharomycopsis fibuligera in a sterile operation in a clean bench, and then placed in a 30°C incubator for inverted culture for 3 days to grow colonies.

[0041] The preparation method of the Saccharomycopsis fibuligera seed liquid is as follows: a single colony is picked up by a sterile inoculation loop, inoculated into a 250 ml flask containing 100 ml of Saccharomycopsis fibuligera seed liquid liquid culture medium, and then placed in a 30°C, 180 rpm / min shaker for culture for 48 h to obtain the seed liquid.

[0042] A preparation method of a Saccharomycopsis fibuligera fermentation product filtrate is provided. The Saccharomycopsis fibuligera seed liquid is added to a culture medium prepared from a peony leaf filtrate, and then fermented and filtered to obtain the Saccharomycopsis fibuligera fermentation product filtrate. The specific steps include the following steps:

[0043] S1: grinding peony leaves to obtain peony leaf powder; peony leaves are the leaves of Paeonia ostii collected in April to June, which are washed under running water for 3-4 times and then dried at 50°C until constant weight, and then immediately used or stored in a refrigerator at 4°C; when used, the peony leaves are ground into peony leaf powder with a mesh size of 60 or more;

[0044] S2: mixing the peony leaf powder with an organic solvent to obtain a peony leaf filtrate by filtration; the organic solvent is 70% ethanol, specifically, the peony leaf powder (dry weight) is mixed with 70% ethanol at a ratio of 1:10 (g:ml) to obtain a mixture, which is extracted by ultrasonic wave, and then filtered to obtain a filtrate and a residue; the residue (wet weight) is mixed with 50% ethanol at a ratio of 1:8 (g:ml) to obtain a mixture, which is extracted by ultrasonic wave again under the same conditions to obtain a filtrate and a residue; the residue (wet weight) is mixed with 20% ethanol at a ratio of 1:8 (g:ml) to obtain a mixture, which is extracted by ultrasonic wave again under the same conditions to obtain a filtrate and a residue; and the filtrates obtained in the three times of extraction are combined;

[0045] wherein the ultrasonic wave extraction conditions are as follows: ultrasonic wave power 250-350 W, 2.5-3.5 W / ml, frequency 20-40 kHz, temperature 45-55°C, stirring speed 80-120 rpm / min, and time 50-70 min;

[0046] S3: adding proteose peptone, yeast extract and glucose to the peony leaf filtrate, and sterilizing to obtain a fermenting medium for Saccharomycopsis fibuligera; first, the filtrate is concentrated to half of the original volume at 60-70°C, and then 1-2 g of proteose peptone, 1-2 g of yeast extract and 1-2 g of glucose are added to each 100 ml of the concentrated solution; the mixture is sterilized in a high-pressure steam sterilization pot at 121°C for 15 min, and then cooled to obtain the fermenting medium for Saccharomycopsis fibuligera;

[0047] S4: adding a seed solution of Saccharomycopsis fibuligera to the fermenting medium for Saccharomycopsis fibuligera, and performing primary fermentation at a temperature of 28-32°C and a stirring speed of 180 rpm / min for 20-30 h, and then performing ultrasonic wave treatment; and then performing secondary fermentation at a temperature of 28-32°C and a stirring speed of 180 rpm / min for 20-30 h; the ultrasonic wave treatment conditions are as follows: ultrasonic wave power 50-70 W, 0.5-0.7 W / ml, frequency 30-50 kHz, ultrasonic wave temperature 28-32°C, and ultrasonic wave time 50-70 min;

[0048] S5: filtering and centrifuging the initial fermentation liquid of Saccharomycopsis fibuligera, and then sterilizing the supernatant obtained after centrifugation at high temperature, cooling to room temperature, and adding a preservative to obtain a Saccharomycopsis fibuligera fermentation product filtrate;

[0049] High temperature instant sterilization conditions are temperature 130-150℃, time 5-15 seconds;

[0050] The preservative is p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the initial fermentation liquor of the Saccharomycopsis fibuligera is 0.5%-1%, and the mass percentage of the 1,2-hexanediol in the initial fermentation liquor of the Saccharomycopsis fibuligera is 0.5%-1%.

[0051] Example 1

[0052] Step 1, pick fresh leaves of the 4-month peony variety Fengdan, wash under running water for 3-4 times, and then dry in a vacuum drying box at 50℃ until the weight is constant. Use a laboratory small crusher to crush the peony leaves to pass through 60 mesh to obtain peony leaf powder.

[0053] Step 2, mix the peony leaf powder with 70% ethanol according to the mass volume ratio (g:ml) 1:10, and under the conditions of ultrasonic power 250W, 2.5W / ml, frequency 20kHz, temperature 55℃, and stirring speed 120rpm / min, ultrasonic extraction for 70min. Filter the filtrate and residue, and reserve the filtrate. Add 50% ethanol to the residue, mix according to the mass (wet weight) volume ratio (g:ml) 1:8, and under the same conditions, extract for the second time. Filter the filtrate and residue, and reserve the filtrate. Add 20% ethanol to the residue, mix according to the mass (wet weight) volume ratio (g:ml) 1:8, and under the same conditions, extract for the third time. Filter the filtrate and residue, and combine the filtrates of the three times;

[0054] Step 3, concentrate the obtained filtrate at 60℃ to half of the original volume, and add 1g of proteose peptone, 1g of yeast paste, and 1g of glucose to every 100ml of the concentrated solution. Put it into a high-pressure steam sterilization pot and sterilize at 121℃ for 15min, and then cool to obtain the Saccharomycopsis fibuligera fermentation medium;

[0055] Step 4, add 3% of the Saccharomycopsis fibuligera seed solution by volume to the Saccharomycopsis fibuligera fermentation medium, and under the conditions of temperature 32℃ and stirring speed 180rpm / min, ferment for 20h. Then under the conditions of ultrasonic power 50W, 0.5W / ml, frequency 30kHz, and ultrasonic temperature 32℃, ultrasonic for 50min;

[0056] Step 5, then continue to ferment under the conditions of temperature 32℃ and stirring speed 180rpm / min for 20h to obtain the initial fermentation liquor of the Saccharomycopsis fibuligera;

[0057] Step 6, after the initial fermentation broth of Saccharomycopsis fibuligera is filtered, centrifuged at 5000 r / min for 10 min, the precipitate is discarded, and the supernatant is cooled to room temperature after sterilization at 130℃ for 15 seconds. Then, 0.5% of p-hydroxyacetophenone and 1% of 1,2-hexanediol are added to the initial fermentation broth of Saccharomycopsis fibuligera, and the fermentation product filtrate of Saccharomycopsis fibuligera is obtained.

[0058] Example 2

[0059] Step 1, fresh leaves of peony variety Fengdan in April are picked, washed under running water for 3-4 times, and then dried in a vacuum drying box at 50℃ until the weight is constant. The peony leaves are crushed to pass through an 80-mesh screen using a laboratory small crusher, and peony leaf powder is prepared.

[0060] Step 2, the peony leaf powder is uniformly mixed with 70% ethanol according to a mass-volume ratio (g:ml) of 1:10. Ultrasonic extraction is performed at an ultrasonic power of 300W, 3.0W / ml, a frequency of 30kHz, a temperature of 50℃, a stirring speed of 100rpm / min, and for 60min. Filtrate and residue are obtained by suction filtration, and the filtrate is retained. The residue is mixed with 50% ethanol according to a mass (wet weight):volume ratio (g:ml) of 1:8, and second extraction is performed under the same conditions. Filtrate and residue are obtained by suction filtration, and the filtrate is retained. The residue is mixed with 20% ethanol according to a mass (wet weight):volume ratio (g:ml) of 1:8, and third extraction is performed under the same conditions. Filtrate and residue are obtained by suction filtration, and the filtrate of the three extractions is combined.

[0061] Step 3, the obtained filtrate is concentrated at 65℃ to half of the original volume. Then, 1.5g of proteose peptone, 1.5g of yeast extract, and 1.5g of glucose are added to every 100ml of the concentrated solution. The solution is sterilized in a high-pressure steam sterilization pot at 121℃ for 15min, and then cooled to prepare the fermentation medium for Saccharomycopsis fibuligera;

[0062] Step 4, 6% of the seed solution of Saccharomycopsis fibuligera is added to the fermentation medium for Saccharomycopsis fibuligera. Fermentation is performed at a temperature of 30℃ and a stirring speed of 180rpm / min for 25h. Then, ultrasonic extraction is performed at an ultrasonic power of 60W, 0.6W / ml, a frequency of 40kHz, and an ultrasonic temperature of 30℃ for 60min.

[0063] Step 5, then, fermentation is continued at a temperature of 30℃ and a stirring speed of 180rpm / min for 25h, and the initial fermentation broth of Saccharomycopsis fibuligera is prepared.

[0064] Step 6, the initial fermentation broth of Saccharomycopsis fibuligera peony leaf was filtered, centrifuged at 5000 r / min for 10 min, the precipitate was discarded, and the supernatant was cooled to room temperature after sterilization at 140℃ for 10 seconds. 0.7% of 1,2-hexanediol and 0.7% of 4-hydroxyacetophenone were added to the initial fermentation broth of Saccharomycopsis fibuligera, and the Saccharomycopsis fibuligera fermentation product filtrate was obtained.

[0065] Example 3

[0066] Step 1, pick fresh leaves of peony variety Fengdan in April, rinse 3-4 times under running water, dry to constant weight in a vacuum drying oven at 50℃, and then crush the peony leaves to pass through a 100 mesh screen using a laboratory small crusher to obtain peony leaf powder.

[0067] Step 2, mix the peony leaf powder with 70% ethanol according to a mass to volume ratio (g:ml) of 1:10, ultrasonic extract for 50 min under the conditions of ultrasonic power 350 W, 3.5 W / ml, frequency 40 kHz, temperature 45℃, stirring speed 80 rpm / min, filter to obtain filtrate and residue, and retain the filtrate; add 50% ethanol to the residue, mix according to a mass (wet weight) to volume ratio (g:ml) of 1:8, ultrasonic extract for 50 min under the same conditions, filter to obtain filtrate and residue, and retain the filtrate; add 20% ethanol to the residue, mix according to a mass (wet weight) to volume ratio (g:ml) of 1:8, ultrasonic extract for 50 min under the same conditions, filter to obtain filtrate and residue, and combine the filtrates of the three times;

[0068] Step 3, concentrate the obtained filtrate to half of the original volume at 70℃, add 2 g of proteose peptone, 2 g of yeast paste, and 2 g of glucose to every 100 ml of the concentrated solution, and then sterilize in a high-pressure steam sterilization pot at 121℃ for 15 min, and cool to obtain a Saccharomycopsis fibuligera fermentation medium;

[0069] Step 4, add 10% of the Saccharomycopsis fibuligera seed solution by volume to the Saccharomycopsis fibuligera fermentation medium, and then ultrasonically treat at an ultrasonic power of 70 W, 0.7 W / ml, a frequency of 50 kHz, and an ultrasonic temperature of 28℃ for 70 min under the conditions of a temperature of 28℃ and a stirring speed of 180 rpm / min;

[0070] Step 5, then continue to ferment at a temperature of 28℃ and a stirring speed of 180 rpm / min for 30 h to obtain an initial fermentation broth of Saccharomycopsis fibuligera;

[0071] Step 6, after the initial fermentation broth of Saccharomycopsis fibuligera was filtered, centrifuged at 5000 r / min for 10 min, the precipitate was discarded, and the supernatant was cooled to room temperature after sterilization at 150℃ for 5 seconds. 1% p-hydroxyacetophenone and 0.5% 1,2-hexanediol were added to the initial fermentation broth of Saccharomycopsis fibuligera, and the Saccharomycopsis fibuligera fermentation product filtrate was obtained.

[0072] Comparative Example 1

[0073] The steps of this comparative example are basically the same as those of Example 2, except that:

[0074] Steps 4-5 are missing, and the obtained is the peony leaf extract without Saccharomycopsis fibuligera fermentation;

[0075] Comparative Example 2

[0076] The steps of this comparative example are basically the same as those of Example 2, except that:

[0077] Steps 1-2 are missing;

[0078] Step 3, 3g of proteose peptone, 3g of yeast paste, and 3g of glucose were added to 100ml of water, and the mixture was sterilized in a high-pressure steam sterilization pot at 121℃ for 15min and then cooled to prepare the Saccharomycopsis fibuligera fermentation medium;

[0079] Steps 4-6 are the same as those of Example 2, and the obtained is the Saccharomycopsis fibuligera fermentation product filtrate without the addition of peony leaves;

[0080] Comparative Example 3

[0081] The steps of this comparative example are basically the same as those of Comparative Example 2, except that:

[0082] Step 6, after the Saccharomycopsis fibuligera fermentation broth was filtered, it was centrifuged at 5000 r / min for 10 min, and the precipitate was discarded. The supernatant was cooled to room temperature after sterilization at 140℃ for 10 seconds. An equal volume of the solution obtained in Comparative Example 1 was added, and the mixture was concentrated to half the original volume at 50℃ to obtain the peony leaf extract and the Saccharomycopsis fibuligera fermentation product filtrate without the addition of peony leaves at the same concentration;

[0083] Comparative Example 4

[0084] The steps of this comparative example are basically the same as those of Example 2, except that:

[0085] Step 4, 6% of the Saccharomycopsis fibuligera seed solution was added to the Saccharomycopsis fibuligera fermentation medium, and the mixture was fermented at 30℃ and 180 rpm / min for 25h to obtain the Saccharomycopsis fibuligera fermentation product filtrate without the use of low-frequency ultrasonic fermentation process;

[0086] Effect Example

[0087] Microbial count

[0088] The number of cladocysteine-covered yeasts was calculated using the hemocytometer method. The fermentation broth was thoroughly shaken, 1 ml of the fermentation broth was taken and diluted with sterile physiological saline, an appropriate amount of glass beads were added and shaken for 5 minutes, and then placed in a hemocytometer for counting under a microscope.

[0089] DPPH free radical experiment

[0090] (1) Take 100ul of the diluted test solution, add 100ul of ethanol, mix, and then mix with 1300ul of DPPH solution with a concentration of 50ug / ml. This is tube A.

[0091] (2) Take 100 μL of the test solution diluted in the same proportion and mix it with 1400 μL of anhydrous ethanol to form Ax0 tube;

[0092] (3) Take 200ul of anhydrous ethanol and mix it with 1300ul of DPPH solution with a concentration of 50ug / ml. This is tube A0.

[0093] (4) After reacting in the dark for 30 minutes, the samples were taken at A, A0, and A0, respectively. x0 Add 250 μL to a 96-well plate and measure the absorbance at 517 nm using a microplate reader. Perform three replicates for each sample and take the average of the results.

[0094]

[0095] In the formula: A0 is the absorbance of the methanol blank (buffer solution replaces the sample); A is the absorbance of the sample; A x0 This represents the absorbance of the sample control.

[0096] ABTS free radical experiment

[0097] (1) Take 900 μL of deionized water and mix it with 600 μL of ABTS solution of appropriate concentration until the final absorbance is between 0.7 and 0.9. This is tube A0.

[0098] (2) Take 300 μL of the diluted test solution, add 600 μL of deionized water, mix, and then mix with 600 μL of ABTS solution of the same concentration as tube A0 to form tube A.

[0099] (3) Take 100 μL of the test solution diluted in the same proportion and mix it with 1400 μL of deionized water to form the Ax0 tube;

[0100] (4) After reacting in the dark for 30 minutes, the samples were taken at A, A0, and A0, respectively. x0 Add 250 μL to a 96-well plate and measure the absorbance at 734 nm using a microplate reader. Perform three replicates for each sample and take the average of the results.

[0101]

[0102] A0 is the absorbance of deionized water blank (buffer instead of sample); A is the absorbance of sample; A x0 is the absorbance of sample control.

[0103] Hydroxyl radical experiment

[0104] (1) Take 100ul of the diluted sample, add 750ul of salicylic acid solution with a concentration of 4×10 -3 mol / L, add 750ul of ferrous sulfate heptahydrate solution with a concentration of 4×10 -3 mol / L, add 750ul of hydrogen peroxide solution with a concentration of 5×10 -3 mol / L, and finally add 150ul of deionized water, mix well, and make A x tube.

[0105] (2) Take 750ul of salicylic acid solution with a concentration of 4×10 -3 mol / L, add 750ul of ferrous sulfate heptahydrate solution with a concentration of 4×10 -3 mol / L, add 750ul of hydrogen peroxide solution with a concentration of 5×10 -3 mol / L, and finally add 250ul of deionized water, mix well, and make A0 tube.

[0106] (3) Take 100ul of the diluted sample, add 750ul of salicylic acid solution with a concentration of 4×10 -3 mol / L, add 750ul of ferrous sulfate heptahydrate solution with a concentration of 4×10 -3 mol / L, and finally add 900ul of deionized water, mix well, and make A x0 tube.

[0107] (4) After 30min of light-free reaction, take 250ul from A x , A0, A x0 tube and add to 96-well plate, measure absorbance at 510nm wavelength on enzyme marker, do 3 parallel for each sample, take the average value of the results.

[0108]

[0109] A0 is the blank group (pure water instead of sample);

[0110] A x is the sample group;

[0111] A x0 is the sample control group (pure water instead of H2O2 solution).

[0112] Tyrosinase inhibition experiment

[0113] ((1) Add 750 uL of phosphate buffer with pH 6.8, 400 uL of 0.05% L-tyrosine, and finally 400 uL of tyrosinase solution with enzyme activity of 200 U / mL to A tube in sequence;

[0114] 2) Add 1000 uL of phosphate buffer with pH 6.8, 400 uL of 0.05% L-tyrosine to A0 tube in sequence;

[0115] (3) Add 350 uL of phosphate buffer with pH 6.8, 400 uL of 0.05% L-tyrosine, 400 uL of sample solution diluted appropriately, and finally 400 uL of tyrosinase solution with enzyme activity of 200 U / mL to C tube in sequence;

[0116] (4) Add 600 uL of phosphate buffer with pH 6.8, 400 uL of 0.05% L-tyrosine, and 400 uL of sample solution diluted appropriately to C0 tube in sequence.

[0117]

[0118] In the formula, A is the absorbance of the mixture without sample extract and enzyme; A0 is the absorbance of the mixture without sample extract and enzyme; C is the absorbance of the mixture with sample extract and enzyme; and C0 is the absorbance of the mixture with sample extract and without enzyme.

[0119] Determination of polyphenol content

[0120] (1) Preparation of standard curve

[0121] Accurately take 0.4 ml of gallic acid control solution with different concentrations into a 10 mL volumetric flask, add 1.5 mL of Folin phenol reagent, mix well, add 2.0 mL of 10% sodium carbonate solution, and react at 25°C in the dark for 30 min. Add distilled water to constant volume, and measure the absorbance of the standard solution with different concentrations at 770 nm. Take the solution concentration (C) as the abscissa and the absorbance (A) as the ordinate to draw the standard curve. The gallic acid has a good linear relationship with the absorbance in the range of 2.018-10.09 ug / mL.

[0122] (2) Determination of polyphenol content of fermentation product

[0123] Dilute the fermentation broth by an appropriate ratio, take 0.4 mL, and add Folin reagent, 10% sodium carbonate solution, and distilled water in sequence according to the above standard curve determination method to measure the absorbance. According to the standard curve, the polyphenol content is calculated.

[0124] Determination of flavone content

[0125] (1) Standard curve drawing

[0126] Precisely pipette 2.5 ml of rutin reference solution of different concentrations into a 25 mL volumetric flask, add 0.5 ml of 5% NaNO2 in turn, shake well, and let stand for 5 min, then add 0.5 ml of 10% Al(NO3)3, shake well, and let stand for 6 min, then add 17.5 ml of 1 mol / L NaOH solution, shake well, dilute to 25 ml with 50% ethanol, shake well, and let stand for 15 min. The solution is brown red. Measure the absorbance of rutin standard solution of different concentrations at 510 nm. Draw the standard curve with absorbance A as the abscissa and concentration as the ordinate.

[0127] (2) Determination of the content of flavonoids in fermentation product

[0128] Dilute the fermentation broth according to the appropriate proportion, take 2.5 ml, and add NaNO2, Al(NO3)3, NaOH solution and 50% ethanol in turn according to the above standard curve determination method to measure the absorbance. According to the standard curve, calculate the content of flavonoids.

[0129] Active ingredient analysis of peony leaf

[0130] The detection of active ingredients of peony leaf was analyzed and identified by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.

[0131] (1) Ultra-high performance liquid chromatography conditions: Agilente eclipse plus C18 chromatographic column (2.1 mm□100 mm, 1.8 μm); mobile phase is 0.01% formic acid aqueous solution (A)-0.01% formic acid acetonitrile (B), gradient elution (0-3 min, 5% B; 3-63 min, 5%-95% B; 63-68 min, 95% B; 68-69 min, 95%-5% B; 69 min-73 min, 5% B); flow rate 0.3 mL / min; column temperature 30℃; injection volume 3 μL.

[0132] (2) Mass spectrometry conditions: compound scanning range m / z 50 Da-1500 Da; ionization temperature (TEM): 500℃; atomizing gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi; curtain gas (CUR): 30 psi; de-clustering voltage (DP): -80 V; collision energy (CE): -7 eV; spray voltage (ISVF) in negative ion mode is -4500 V. Information correlation acquisition is adopted; the parameter settings of sub-ion scanning mode are as follows: molecular weight scanning range 50-1000 m / z, collision energy (CE): (35±15) eV; other main parameters are the same as TOF-MS scanning mode, and CDS is used for automatic calibration of molecular weight accuracy.

[0133] (3) Identification of active components in the leaf of Paeonia suffruticosa Andr. fermented by Starmerella bombicola

[0134] First, the sample to be tested was subjected to LC-MS detection using the Sciexos analysis software of SCIEX Corporation, and the peak components of the standard were identified by comparing the XIC graph. The compound peaks identified above were integrated, and the integral results were corrected, and the relative content of the compound was calculated by peak area.

[0135] Analysis of active components in extracellular products of Starmerella bombicola

[0136] The active components of Paeonia suffruticosa leaves were analyzed and identified by GC-MS.

[0137] (1) Chromatographic conditions: The chromatographic column was an HP-5MS (30 m x 0.25 mm x 0.25 μm) flexible quartz capillary column, the column temperature was 80 (retained for 3 min), the temperature was increased to 150℃ at 5℃ / min and maintained for 10 min, the temperature was increased to 280℃ at 10℃ / min and maintained for 10 min, the carrier gas was high-purity He gas (99.999%), the pre-column pressure was 7.62 psi, the carrier gas flow rate was 1.0 mL / min, the injection volume was 1 uL, the split ratio was 10:1, and the solvent delay time was 3 min.

[0138] (2) The ion source was an EI source, the ion source temperature was 230℃, the quadrupole rod temperature was 150℃, the electron energy was 70 eV, the interface temperature was 280℃, and the mass scan range was 20-800 amu.

[0139] (3) Identification and quantification of active components in extracellular products of Starmerella bombicola

[0140] First, the sample to be tested was subjected to GC-MS detection, and the peak components of the standard were identified by comparing the TIC graph. This includes integrating the compound peaks identified above based on retention time and quantitative ions, and correcting the integral results, and calculating the content of the compound according to the peak area.

[0141] Determination of anti-cell aging activity of Starmerella bombicola fermentation product filtrate

[0142] (1) Cell culture

[0143] Mouse embryonic fibroblasts (NIH3T3) were cultured in 10% FBS (fetal bovine serum) and 1% Penicillin-Streptomycin in DMEM complete medium, and placed in a 37℃ carbon dioxide incubator containing 5% carbon dioxide.

[0144] (2) Establishment of mouse embryonic fibroblast aging model

[0145] The mouse embryonic fibroblast cells were treated with different concentrations of hydrogen peroxide, and the effect of hydrogen peroxide on the viability of mouse embryonic fibroblast cells was detected by WST (water-soluble tetrazolium) cytotoxicity detection kit, and the cells were treated with sublethal concentration of hydrogen peroxide to establish a cell aging model.

[0146] (3) Anti-aging activity determination

[0147] The mouse fibroblasts were cultured to 80-90% cell adhesion. The adherent cells were prepared into a cell suspension of 1 x 10 5 / mL, and then added to a 96-well plate after gentle shaking, and incubated for 24 h. After the culture ended, the samples in each group were added to the 96-well plate for 1 h of pretreatment, and then sublethal concentration of hydrogen peroxide was added to the 96-well plate for 5 h of treatment, and the cell viability of mouse fibroblasts was analyzed by WST kit.

[0148] Skin texture improvement experiment

[0149] (1) Experimental instruments

[0150] Skin moisture loss tester: Germany CK Tewameter™ 300 MDD4, which evaluates the skin barrier function by measuring the trans-epidermal water loss rate.

[0151] Skin moisture tester: Germany CK Corneometer CM825 MDD4, which is used to test the water content of the skin of the subjects to determine the moisturizing effect of the cosmetic.

[0152] Skin elasticity tester: Germany dual MPA580, which is used to test the skin elasticity and aging degree.

[0153] Multifunctional skin tester: Germany CK MPA10, which is used to test skin melanin, hemoglobin, pH, oil content, skin color, skin gloss, skin friction, etc.

[0154] (2) Experimental subjects: 50 healthy female volunteers aged 18-24 years old, with an average age of 20.5 years old, without serious systemic diseases, without active allergic diseases, without serious history of allergy to cosmetics, and without participating in other clinical tests. The volunteers who met the standards were randomly divided into a blank control group (blank control), a Saccharomyces boulardii filtrate group (Example 2), a peony leaf extract group (Comparative Example 1), a Saccharomyces boulardii extracellular product group (Comparative Example 2), and a mixed group (Comparative Example 3).

[0155] (3) Test method: control the environmental conditions at room temperature at 22℃, humidity 40%-50%, 2-3 days before testing, the test site does not use any cosmetics, the tester enters the test environment quietly 30 min in advance, randomly selects the inner side of the forearm of the left and right hands as the test area, before using the sample, the volunteer wipes the test site with a dry face tissue, marks a 3cm x 3cm test area on the inner side of the arm, the volunteer uses the sample at a dosage of 2ml / cm2, the blank control uses the same amount of distilled water instead, once in the morning, noon and evening, measures the moisture content, elasticity and other data of each test area after 20 days. Place the probe of the detector vertically on the skin, after 3-5 seconds, read the value when the data is stable, measure the moisture, elasticity and other values of the marked area, repeat the measurement 3 times at each test point, and take the average value.

[0156] Skin damage repair experiment

[0157] (1) Experimental materials

[0158] 60 SPF level female healthy nude mice, weighing 10-13g.

[0159] (2) Measurement method

[0160] When measuring, the test probe of different detectors is pressed vertically on the surface of the mouse back skin, the top of the probe is pressed back a distance, a prompt sound is heard and the result is displayed on the host. Each 1 site is measured 3 times, and the average value is taken.

[0161] (3) Preparation of model

[0162] Use the ultraviolet phototherapy instrument, place the nude mice under the ultraviolet phototherapy instrument lamp, irradiate at a distance of 40cm, irradiate for 20min per day, pause irradiation until the symptoms subside, irradiate for 15 days, and reach a cumulative irradiation dose of UVB of 0.15mJ / cm 2 .

[0163] (4) Experimental method

[0164] Divide the 60 female nude mice into 6 groups, namely the blank group (blank control), the model group (model control), Example 2 (filter liquor of Saccharomycopsis fibuligera fermentation product), Comparative Example 1 (peony leaf extract), Comparative Example 2 (Saccharomycopsis fibuligera fermentation liquor), and Comparative Example 3 (mixture of Saccharomycopsis fibuligera fermentation liquor and peony leaf extract). The model group, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 samples are each applied to the back skin of the nude mice at a dosage of 2ml / cm2 per day, and the applied nude mice are irradiated under uVB; the model group is massaged every day and irradiated under uVB; the blank group is massaged every day without uVB irradiation. After 15 days, the back skin of the nude mice is detected using a skin tester to observe the effect of silibinin on the back skin of the nude mice.

[0165] Results analysis

[0166] <Whitening antioxidant activity>

[0167] Table 1 Whitening antioxidant activity of Saccharomycopsis fibuligera fermentation product filtrate of different examples

[0168]

[0169] Note: Different letters in the upper right corner of the same column indicate significant differences (P <0.05)

[0170] DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate reflect the strength of in vitro antioxidant capacity, the smaller the radical scavenging rate value in IC50 / %, the stronger the radical scavenging capacity, from the data of examples 1-3, it can be seen that the effect of Saccharomycopsis fibuligera fermentation product filtrate example 2 prepared by the method described in the application is the best, and the DPPH radical scavenging rate, OH radical scavenging rate and ABTS radical scavenging rate are all the smallest. The tyrosinase inhibition rate reflects the size of the whitening effect, the smaller the tyrosinase inhibition rate in IC50 / %, the stronger the whitening ability, according to the method described, the effect of Saccharomycopsis fibuligera peony leaf fermentation stock solution example 2 is the best. But within the implementation range of the method, the radical scavenging rate (example 2 hydroxyl radical scavenging rate is significantly lower than examples 1 and 3) and tyrosinase inhibition rate of examples 1-3 do not show significant differences (P>0.05). Polyphenols and flavonoids are the main active ingredients in peony leaves, and the higher the content of polyphenols and flavonoids, the better the efficacy activity is often indicated, the polyphenol content of example 2 is the highest, and the flavonoid content of example 1 is the highest, but there is no significant difference (P>0.05).

[0171] Comparing Example 2 and Comparative Example 1, Comparative Example 1 lacks the Saccharomycopsis fibulata fermentation steps of steps 4-5 in Example 2, the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate and tyrosinase inhibition rate IC50 / % values in Example 2 are all less than those of Comparative Example 1, and there are significant differences (P<0.05), the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate of the Saccharomycopsis fibulata fermentation product filtrate are increased by 2 times, 1.6 times and 1.5 times respectively, and the tyrosinase inhibition rate is increased by 1.7 times. It shows that the antioxidant efficacy and whitening efficacy of the Saccharomycopsis fibulata fermentation product filtrate can be significantly improved by Saccharomycopsis fibulata fermentation. The polyphenol content is often positively correlated with the antioxidant activity of the compound, and Example 2 is significantly higher than Comparative Example 1, and there are significant differences (P<0.05). During the fermentation process, Saccharomycopsis fibulata secretes a variety of extracellular enzymes, which hydrolyze the cell wall of the leaves of Paeonia suffruticosa, promote the dissolution of active ingredients, and a large amount of flavonoids, polyphenols and acids, sugars and other substances in the leaves of Paeonia suffruticosa are combined in the form of insoluble combined phenols in the cell wall and intercellular substance of the leaves of Paeonia suffruticosa. Through Saccharomycopsis fibulata fermentation, glycosidic bond and lipid bond are hydrolyzed, and combined phenols are converted into soluble free phenols, so that the content of flavonoids and polyphenols is increased, thereby causing the improvement of antioxidant activity and tyrosinase inhibition rate.

[0172] Comparing Example 2 and Comparative Example 2, the medium of Comparative Example 2 does not add the leaves of Paeonia suffruticosa, and the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate and tyrosinase inhibition rate IC50 / % values in Example 2 are all significantly less than (P<0.05) those of Comparative Example 2, and the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate are increased by 8.5 times, 2.9 times and 3.1 times respectively, and the tyrosinase inhibition rate is increased by 2.6 times. It shows that adding the leaves of Paeonia suffruticosa extract during the Saccharomycopsis fibulata fermentation process can significantly improve the antioxidant efficacy and whitening efficacy of the Saccharomycopsis fibulata fermentation product filtrate.

[0173] Compared with Comparative Example 3, Comparative Example 3, after concentration, contains the same amount of peony leaf extract and extracellular fermentation filtrate of *Saccharomyces cerevisiae* as in Example 2. In Example 2, the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate, and tyrosinase inhibition rate were all lower than those in Comparative Example 3, while the flavonoid and polyphenol contents were higher. Among them, the DPPH radical scavenging rate, OH radical scavenging rate, and flavonoid content showed significant differences (P<0.05). The DPPH radical scavenging rate, OH radical scavenging rate, and ABTS radical scavenging rate increased by 1.4 times, 1.3 times, and 1.3 times, respectively, and the tyrosinase inhibition rate increased by 1.3 times. This indicates that the addition of peony leaves during the fermentation of *Saccharomyces cerevisiae* does not result in a simple additive effect of antioxidant and tyrosinase inhibitory activities. It is possible that during fermentation, low-activity components in peony leaves are converted into high-activity components, or that the active components in peony leaves affect the extracellular metabolism of *Saccharomyces cerevisiae*, or that both effects are present, jointly enhancing the antioxidant and whitening activities of the extracellular fermentation filtrate of *Saccharomyces cerevisiae*.

[0174] Compared with Comparative Example 4, Example 2 lacked the ultrasonic-promoted fermentation step of capsule-forming yeast in Example 2 (see Example 2). Figure 1 In Example 2, the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate and tyrosinase inhibition rate were all lower than those in Comparative Example 4, but only the OH radical scavenging rate showed a significant difference (P<0.05).

[0175] When ultrasound was applied during fermentation, compared with the control group, the DPPH radical scavenging rate, OH radical scavenging rate, ABTS radical scavenging rate, and tyrosinase inhibition rate (IC50 / %) were all reduced. Specifically, the IC50 / % of DPPH radical scavenging rate decreased by 24.48%, the IC50 / % of OH radical scavenging rate decreased by 19.38%, the IC50 / % of ABTS radical scavenging rate decreased by 20.51%, and the IC50 / % of tyrosinase inhibition rate decreased by 24.42%.

[0176] Within the scope of this method, low-frequency, low-intensity ultrasound (ultrasound power 50-70W, 0.5-0.7W / ml, frequency 30-50kHz, ultrasound temperature 28-32℃, ultrasound time 50-70min) can significantly promote the growth of *Saccharomyces cerevisiae* (see [link to relevant documentation]). Figure 2 Ultrasound has a certain damaging effect on *Saccharomyces cerevisiae*, but this damage is reversible. After damage, the cell membrane permeability of *Saccharomyces cerevisiae* increases, and the dissolution of various intracellular enzymes and substances also increases. Intracellular enzymes act on various components of the peony leaf, increasing the concentration of nutrients in the *Saccharomyces cerevisiae* environment. Furthermore, *Saccharomyces cerevisiae* grows rapidly in peony leaf suspension, forming dendritic pseudohyphae (see...).Figure 3 ), ultrasound has a dispersing effect, which separates the pseudohyphae into single yeast cells (see Figure 4 ), increases the exchange area with external substances, and is more conducive to obtaining nutritional ingredients, thereby promoting the growth of Torula sp. On the other hand, the rapid growth of Torula sp. accelerates the conversion of active ingredients in the leaves of Paeonia suffruticosa, and the two interact to improve the antioxidant and whitening effects of the Torula sp. leaf fermentation stock solution. The changes in flavonoid and polyphenol contents were opposite to the changes in free radical scavenging rate and tyrosinase inhibition rate, and Example 2 was higher than Comparative Example 4, but no significant difference was observed (P>0.05).

[0177] <Anti-cell aging activity>

[0178] As shown in Figure 5 , the cell viability of the hydrogen peroxide model group was significantly lower than that of the blank group (P<0.05), indicating that the model was successfully created. All test samples could inhibit the reduction of cell viability induced by hydrogen peroxide to varying degrees, and the cell viability of the Torula sp. fermentation product filtrate group (filtrate group) was the highest (93.4%), which was very close to the blank group and had no significant difference (P>0.05) with the Paeonia suffruticosa leaf extract (extract group) and the mixed group of Torula sp. fermentation broth and Paeonia suffruticosa leaf extract (mixed group), but was significantly higher than the model group and the yeast group (P<0.05), indicating that the addition of Paeonia suffruticosa leaves during the fermentation of Torula sp. could significantly improve the anti-aging activity of the fermentation product.

[0179] <Improvement effect on skin quality>

[0180] Table 2 Improvement effect of Torula sp. fermentation product filtrate on skin quality

[0181] Group Skin moisture (a.u.) Amount of skin moisture loss (g m -2 ·h -1 )]]> Skin elasticity Color Blank control 43.64 a ±13.82]]> 17.65 a ±0.32]]> 64.74 a ±8.74 19.38 a ±4.26]]> Example 2 55.72 b ±8.45]]> 15.26 b ±0.24 80.04 c ±9.58]]> 16.37 a ±6.73 Comparative Example 1 48.36 ab ±7.34]]> 17.71 a ±0.19]]> 73.92 b ±6.28]]> 18.29 a ±5.14 Comparative Example 2 50.07 b ±9.28]]> 16.93 ab ±031]] 66.56 a ±8.11]]> 19.03 a ±5.76 <!-- 12 -->]]> Comparative Example 3 52.91 b ±7.53]]> 15.38 b ±0.42 74.37 b ±7.39]]> 17.82 a ±4.52]]>

[0182] Note: Different letters in the upper right corner of the same column indicate significant differences (P<0.05)

[0183] The skin moisture content of the Torula sp. fermentation product filtrate group (Example 2) was the highest among the four sample groups, and the three sample groups of Example 2, Torula sp. extracellular product group (Comparative Example 2), and mixed group (Comparative Example 3) were significantly higher than the blank control group (P<0.05), but there was no significant difference between the sample groups (P>0.05), indicating that the moisturizing effect of the Torula sp. product filtrate was obvious, and the addition of Paeonia suffruticosa leaves during the fermentation of Torula sp. could significantly improve the moisturizing efficacy of its extracellular product.

[0184] The skin moisture loss amount and the skin pigment index mainly reflect the integrity of the skin. The skin moisture loss amount of Example 2 is the lowest, and Example 2 and Comparative Example 3 are significantly lower than the blank control group and Comparative Example 1 (P<0.05), indicating that the skin repair effect of the filtrate of the Saccharomycopsis fibuligera fermentation product is obvious. The skin pigment of Example 2 is the lowest, indicating that the filtrate of the Saccharomycopsis fibuligera product can improve the ability of the skin to maintain integrity, but there is no significant difference between each experimental group and the blank group (P>0.05).

[0185] The skin elasticity mainly reflects the aging degree of the skin. Example 2, Comparative Example 1 and Comparative Example 3 are significantly higher than the blank control group and Comparative Example 2 (P<0.05), indicating that the filtrate of the Saccharomycopsis fibuligera product and the peony leaf extract can obviously improve the aging of the skin. The skin elasticity of Example 2 is significantly higher than Comparative Example 1 and Comparative Example 3 (P<0.05), indicating that after adding peony leaves in the fermentation process of Saccharomycopsis fibuligera, the skin elasticity effect is not only higher than that of Saccharomycopsis fibuligera fermentation liquid and peony leaf extract, but also higher than that of the sum of the two.

[0186] <Repairing effect on skin>

[0187] Table 3 Repairing effect of Saccharomycopsis fibuligera fermentation product filtrate on skin

[0188] Group Skin moisture (a.u.) Melanin Hematin PH value Blank control 57.34 a ±7.06]]> 157.28 a ±8.3]]> 427.36 a ±10.2]]> 6.5 a ±0.10]]> Model control 35.77 b ±6.82]]> 186.77 b ±8.7]]> 533.92 b ±9.4]]> 7.4 b ±0.12]]> Example 2 67.28 c ±5.73]]> 136.37 c ±7.1]]> 431.69 a ±10.5]]> 6.03 c ±0.21]]> Comparative Example 1 47.09 d ±4.38]]> 151.29 ad ±5.4]]> 504.28 d ±9.6]]> 6.50 a ±0.15]]> Comparative Example 2 41.62 bd ±8.91]]> 155.91 a ±8.4]]> 483.75 e ±11.7]]> 6.71 a ±0.14]]> Comparative Example 3 57.28 a ±6.74]]> 146.73 d ±6.7]]> 460.63 f ±8.8 6.34 ac ±0.15]]>

[0189] Note: Different letters in the upper right corner of the same column indicate significant difference (P<0.05)

[0190] The skin moisture content of the three sample groups of the Saccharomycopsis fibuligera fermentation product filtrate (Example 2), the peony leaf extract (Comparative Example 1) and the mixture of the Saccharomycopsis fibuligera extracellular product and the peony leaf extract (Comparative Example 3) is significantly higher than that of the model control group (P<0.05), indicating that the Saccharomycopsis fibuligera fermentation product filtrate, the peony leaf extract and the Saccharomycopsis fibuligera extracellular product and the peony leaf extract can all alleviate the decrease of skin moisture content caused by ultraviolet radiation. The skin moisture content of Comparative Example 3 is basically the same as that of the blank control group, and the skin moisture content of Example 2 where the Saccharomycopsis fibuligera fermentation product filtrate is located is even higher than that of the blank control group, showing good skin repair and moisturizing effect.

[0191] The skin melanin and hemoglobin index mainly reflects the integrity of the skin and the skin pigmentation. After the mice were excessively irradiated by ultraviolet rays, the skin melanin and hemoglobin pigmentation was obvious. The four experimental groups can significantly reduce (P<0.05) the pigmentation of skin melanin and hemoglobin. The effect of Example 2 is the most obvious. The hemoglobin is restored to the level comparable to the blank control, and the melanin is even lower than the blank control. It is shown that the filtrate of Saccharomycopsis fibuligera fermentation product (Example 2) has a significant effect of maintaining the integrity of the skin and reducing pigmentation. After the mice skin was excessively irradiated by ultraviolet rays, the PH value was significantly increased. The four experimental groups can significantly improve (P<0.05) the skin PH value. The effect of the filtrate of Saccharomycopsis fibuligera fermentation product (Example 2) is the most obvious. It is shown that the filtrate of Saccharomycopsis fibuligera fermentation product can significantly improve the increase of PH value of the mouse skin caused by excessive ultraviolet irradiation. The filtrate of Saccharomycopsis fibuligera fermentation product can improve the moisture and pH value of the skin, reduce the pigmentation of the skin, thereby reducing the damage of ultraviolet irradiation to the skin, and has an important role in improving the quality of the skin and delaying skin aging.

[0192] <Changes in the content of main active ingredients in peony leaves before and after fermentation>

[0193] Table 4 Changes in the content of main active ingredients in peony leaves before and after fermentation

[0194]

[0195] Note: The peak area is used to represent the amount of content, and the scientific notation is used. The same row with different letters in the upper right corner indicates a significant difference (P<0.05)

[0196] From Figure 7As can be seen, about 5000 compounds were obtained by LC-MS analysis of the suspension of peony leaf fermented by P. toruloides, of which 343 compounds were significantly increased or newly appeared (P<0.05), 587 compounds were significantly decreased or reduced to undetectable (P<0.05), and other components had no obvious change. Through standard comparison, 17 components derived from peony leaf can be identified (see Table 4), and according to the peak area change of each compound before and after fermentation in Table 4, 7 compounds such as gallic acid, paeonol protolignan B, sophoricoside, pentagalloyl glucose, benzoylpaeoniflorin, benzoyloxypaeoniflorin and rutin were significantly reduced (P<0.05) or reduced to undetectable (such as pentagalloyl glucose). 6 compounds such as paeoniflorin, galloyloxypaeoniflorin, galloylpaeoniflorin, 2,5-dihydroxy-4-methoxyacetophenone, quercetin and benzoic acid were significantly increased (P<0.05). Benzoic acid, paeoniflorin, galloyloxypaeoniflorin and galloylpaeoniflorin may be converted from gallic acid, pentagalloyl glucose, benzoylpaeoniflorin and benzoyloxypaeoniflorin, 2,5-dihydroxy-4-methoxyacetophenone may be derived from the bioconversion and hydroxylation of paeonol protolignan B and sophoricoside, and quercetin may be derived from the bioconversion of rutin. There was no obvious change in 6 compounds such as p-hydroxybenzoic acid, gallic acid methyl ester, oxypaeoniflorin, paeonol new glycoside, paeoniflorin C and paeonol.

[0197] Effect of peony leaf on extracellular metabolites of P. toruloides

[0198] Table 5 Effect of peony leaf on extracellular metabolites of P. toruloides

[0199]

[0200] Note: The same row with different letters in the upper right corner indicates significant difference (P<0.05)

[0201] Through standard comparison, 23 components derived from the metabolites of P. toruloides can be identified (see Table 5), of which 8 components were significantly increased after fermentation, including 5 amino acids such as valine, alanine, glycine, proline and threonine, 2 vitamins such as VB2 and VB5, and 1 acid component lactate, and other components had no obvious change. The increase of the content of 5 amino acids may be derived from the decomposition of protein macromolecules in peony leaf or the increase of amino acid synthesis in the corresponding metabolic pathway of P. toruloides, and the increase of VB2, VB5 and lactate may be derived from the change of extracellular metabolites of P. toruloides. At present, since vitamin antioxidants contain antioxidant components, they can scavenge free radicals and promote skin metabolism, so they are added to cosmetics to smooth wrinkles and improve skin texture.

[0202] In summary, the filtrate of the fermentation product of P. toruloides provided by the present application has good anti-skin aging, moisturizing, skin repair and other effects.

[0203] The aging of skin and the increase of free radical content, ultraviolet irradiation, skin damage, etc. are closely related. Experiments prove that the filtrate of the fermentation product of Pachysolen tannophilus has significant antioxidant effect.

[0204] The OH radical scavenging rate IC50 / % of the filtrate of the fermentation product of Pachysolen tannophilus is 13.73, the DPPH radical scavenging rate IC50 / % is 1.45, and the ABTS radical scavenging rate IC50 / % is 0.31. Compared with the original extracellular fermentation liquid of Pachysolen tannophilus, the DPPH radical scavenging rate, OH radical scavenging rate and ABTS radical scavenging rate of the filtrate of the fermentation product of Pachysolen tannophilus obtained in the application are increased by 8.5 times, 2.9 times and 3.1 times, respectively. Compared with the extract of peony leaves, the DPPH radical scavenging rate, OH radical scavenging rate and ABTS radical scavenging rate are increased by 2 times, 1.6 times and 1.5 times, respectively. Compared with the mixed sample of the extract of peony leaves and the extracellular fermentation product of Pachysolen tannophilus at the same concentration, the DPPH radical scavenging rate, OH radical scavenging rate and ABTS radical scavenging rate are increased by 1.4 times, 1.3 times and 1.3 times, respectively (see Table 1). The filtrate of the fermentation product of Pachysolen tannophilus can significantly inhibit the decrease of cell viability induced by hydrogen peroxide, and the cell viability (93.4%) is very close to the normal cell viability level, indicating that the filtrate of the fermentation product of Pachysolen tannophilus has significant anti-aging activity (see Table 1). Figure 5 The filtrate of the fermentation product of Pachysolen tannophilus can significantly improve skin elasticity. Compared with before use (blank control), the skin elasticity is increased by 23.63% after using the filtrate of the fermentation product of Pachysolen tannophilus obtained in the application. Compared with the original extracellular fermentation liquid of Pachysolen tannophilus, the skin elasticity is increased by 20.25%. Compared with the extract of peony leaves, the skin elasticity is increased by 8.28%. Compared with the mixed sample of the extract of peony leaves and the extracellular fermentation product of Pachysolen tannophilus at the same concentration, the skin elasticity is increased by 7.62% (see Table 2).

[0205] The filtrate of the fermentation product of Pachysolen tannophilus provided in the application has good whitening effect.

[0206] Experiments prove that the tyrosinase inhibition rate IC50 / % of the filtrate of the fermentation product of Pachysolen tannophilus is 1.64. Compared with the original extracellular fermentation liquid of Pachysolen tannophilus, the tyrosinase inhibition rate of the filtrate of the fermentation product of Pachysolen tannophilus obtained in the application is increased by 2.6 times. Compared with the extract of peony leaves, the tyrosinase inhibition rate is increased by 1.7 times. Compared with the mixed sample of the extract of peony leaves and the extracellular fermentation product of Pachysolen tannophilus at the same concentration, the tyrosinase inhibition rate is increased by 1.3 times (see Table 1).

[0207] The filtrate of the fermentation product of Pachysolen tannophilus provided in the application has good moisturizing effect.

[0208] The skin moisture of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is 55.72 au, which is increased by 27.68% compared with that before use (blank control), and the skin moisture of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is increased by 11.28% compared with that of the original extracellular fermentation liquid of Saccharomycopsis, and the skin moisture of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is increased by 15.22% compared with that of the peony leaf extract, and the skin moisture of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is increased by 5.31% compared with that of the mixed sample of the peony leaf extract and the extracellular fermentation product of Saccharomycopsis (see Table 2).

[0209] The filtrate of the fermentation product of Saccharomycopsis provided by the application has good skin repair effect

[0210] The skin moisture loss of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is 15.26 g·m -2 ·h -1 , which is decreased by 13.85% compared with that before use (blank control), and the skin moisture loss of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is decreased by 7.56% compared with that of the original extracellular fermentation liquid of Saccharomycopsis, and the skin moisture loss of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is decreased by 13.83% compared with that of the peony leaf extract, and the skin moisture loss of the mouse after using the filtrate of the fermentation product of Saccharomycopsis is even lower than that of the mixed sample of the peony leaf extract and the extracellular fermentation product of Saccharomycopsis, and in addition, the skin pigment content is also reduced to different degrees (see Table 2). In terms of repairing skin damage caused by excessive ultraviolet radiation, the filtrate of the fermentation product of Saccharomycopsis can significantly alleviate the decrease of skin moisture content, pigmentation and increase of PH value caused by ultraviolet radiation damage. After using the filtrate of the fermentation product of Saccharomycopsis, the skin moisture content is even higher than that of the blank control, the hemoglobin and melanin are significantly lower than those of the blank control, and the use effect is significantly higher than that of the original extracellular fermentation liquid of Saccharomycopsis and the peony leaf extract alone, and even significantly higher than that of the mixed sample of the peony leaf extract and the extracellular fermentation product of Saccharomycopsis (see Table 3).

[0211] The content of total flavonoids and total polyphenols in the filtrate of the fermentation product of Saccharomycopsis provided by the application is significantly increased

[0212] The content of total flavonoids and total polyphenols in the filtrate of the fermentation product of Saccharomycopsis provided by the application is significantly increased

[0213] The content of total flavonoids and total polyphenols in the filtrate of the fermentation product of Saccharomycopsis provided by the application is significantly increased The content of total flavonoids and total polyphenols in the filtrate of the fermentation product of Saccharomycopsis provided by the application is significantly increased.

[0214] Compared with before fermentation, the contents of many components increased or decreased, and even new active components appeared (see Figure 6 , Figure 7 ), among which 587 components were significantly reduced (P<0.05, and Fc<1 / 2), and 343 components were significantly increased (P<0.05, and Fc>2) (see Figure 7 ), and through standard substance comparison, 17 components were identified from the leaves of Paeonia suffruticosa (see Table 4), among which 7 compounds, including gallic acid, paeonol glycoside B, sophoraflavone, pentagalloyl glucose, benzoylpaeoniflorin, benzoyloxypaeoniflorin, and rutin, were significantly reduced (P<0.05), or reduced to undetectable (such as pentagalloyl glucose). Six compounds, including paeoniflorin, galloyloxypaeoniflorin, galloylpaeoniflorin, 2,5-dihydroxy-4-methoxyacetophenone, quercetin, and benzoic acid, were significantly increased (P<0.05). Benzoic acid, paeoniflorin, galloyloxypaeoniflorin, and galloylpaeoniflorin may be converted from gallic acid, pentagalloyl glucose, benzoylpaeoniflorin, and benzoyloxypaeoniflorin, 2,5-dihydroxy-4-methoxyacetophenone may be bioconverted and hydroxylated from paeonol glycoside B and sophoraflavone, and quercetin may be bioconverted from rutin. The increase of active components such as paeoniflorin and galloyloxypaeoniflorin is the main factor for the increase of the multiple effects of the filtrate of the fermentation product of Saccharomycopsis fibuligera, and these active components have many skin-related physiological and biochemical activities, such as inhibiting the secretion of inflammatory cytokines, resisting oxidative stress, and regulating the expression of aging-related genes, so that the filtrate of the fermentation product of Saccharomycopsis fibuligera provided by the present application has more extensive potential application value.

[0215] The above only describes the preferred embodiments of the present application and should not be used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method of preparing a filter liquor of a Saccharomyces boulardii fermentation product, characterized in that: The Saccharomycopsis fibuligera seed liquid is added into the culture medium prepared from the peony leaf filtrate, and then fermentation and filtration are performed to obtain the Saccharomycopsis fibuligera fermentation product filtrate. Specifically, the method comprises the following steps: S1: crushing peony leaves to obtain peony leaf powder; S2: mixing the peony leaf powder with an organic solvent, performing ultrasonic extraction, and performing suction filtration to obtain a peony leaf filtrate; S3: adding protein peptone, yeast extract, and glucose to the peony leaf filtrate after concentration, and performing sterilization treatment to obtain a Saccharomycopsis fibuligera fermentation culture medium; S4: adding a Saccharomycopsis fibuligera seed liquid to the Saccharomycopsis fibuligera fermentation culture medium for primary fermentation, and then performing ultrasonic treatment; and then performing secondary fermentation to obtain an initial fermentation liquid; S5: filtering and centrifuging the initial fermentation liquid, performing high-temperature instantaneous sterilization on the supernatant after centrifugation, cooling to room temperature, and adding a preservative to obtain the Saccharomycopsis fibuligera fermentation product filtrate. In step S2, the preparation method of the peony leaf filtrate is as follows: the peony leaf powder is uniformly mixed with 70% ethanol at a mass-volume ratio of 1:10, ultrasonic extraction is performed, suction filtration is performed to obtain filtrate and residue, the filtrate is reserved, the residue is uniformly mixed with 50% ethanol at a mass-volume ratio of 1:8, second ultrasonic extraction is performed, suction filtration is performed to obtain filtrate and residue, the filtrate is reserved, the residue is uniformly mixed with 20% ethanol at a mass-volume ratio of 1:8, third ultrasonic extraction is performed, suction filtration is performed to obtain filtrate and residue, and the filtrates obtained in the three times of extraction are combined to obtain the peony leaf filtrate. The Saccharomycopsis fibuligera is purchased from the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 21624, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology.

2. The method for preparing the filtrate of yeast fermentation product with a membrane-coated capsule according to claim 1, characterized in that: In step S3, the peony leaf filtrate is first concentrated to half of the original volume at 60-70°C, and then protein peptone, yeast extract, and glucose are added.

3. The method for preparing the filtrate of yeast fermentation product with a capsule-like membrane according to claim 1, characterized in that: In step S4, the preparation method of the Saccharomycopsis fibuligera seed liquid is as follows: a Saccharomycopsis fibuligera glycerol freezing tube is taken out, and a sterile operation is performed on a clean bench to streak a solid culture medium plate, which is placed in a 28-32°C incubator for inverted culture for 3 days, and after the colonies grow, a single colony is picked and inoculated into a liquid culture medium, and placed in a 30°C, 180 rpm / min shaker for culture for 48 h to obtain the Saccharomycopsis fibuligera seed liquid.

4. The method for preparing the filtrate of yeast fermentation product with a capsule-like membrane according to claim 1, characterized in that: In step S4, the volume ratio of the Saccharomycopsis fibuligera seed liquid added to the Saccharomycopsis fibuligera fermentation culture medium is 3%-10%.

5. The method for preparing the filtrate of yeast fermentation product with a membrane-covered capsule according to claim 1, characterized in that: In step S4, the ultrasonic treatment has a power of 50-70 W, 0.5-0.7 W / ml, a frequency of 30-50 kHz, and a temperature of 28-32°C, and the ultrasonic treatment time is 50-70 min.

6. The method for preparing the filtrate of yeast fermentation product with a capsule-like membrane according to claim 1, characterized in that: In step S4, the conditions of the primary fermentation and the secondary fermentation are both as follows: a temperature of 28-32°C, a rotation speed of 180 rpm / min, and a fermentation time of 20-30 h.

7. The Saccharomycopsis fibuligera fermentation product filtrate prepared by the preparation method of the Saccharomycopsis fibuligera fermentation product filtrate in any one of claims 1-6.

8. The use of the Saccharomycopsis fibuligera fermentation product filtrate in claim 7 in the preparation of cosmetics.

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