A cherry blossom fermentation product, its preparation method, composition and application

Abstract

The present invention provides a cherry blossom fermentation product, a preparation method thereof, a composition and an application, belonging to the technical field of cosmetics. It includes: S1. Freeze the cherry blossoms in liquid nitrogen, thaw them, and break their cell walls to obtain cherry blossom powder; S2. Add the cherry blossom powder to an aqueous solution of a deep eutectic solvent for extraction, filter to obtain solid residue 1; add sodium phosphate to the filtrate for precipitation to obtain solid residue 2, and dry the filtrate to prepare an extract; S3. Culture Streptococcus thermophilus to obtain a seed solution of the strain; S4. Inoculate and ferment the seed solution of the strain, centrifuge to collect the bacterial sludge, and freeze-dry the supernatant to prepare a Streptococcus thermophilus cherry blossom ferment; S5. Enzymatically hydrolyze the bacterial sludge, break its cell walls under high pressure, add ethanol to the cell wall-breaking solution for precipitation to obtain cell wall polysaccharide, and dry the filtrate to prepare a lysate; S6. Mix the extract, the Streptococcus thermophilus cherry blossom ferment, the cell wall polysaccharide and the lysate to prepare a product, which has the effects of moisturizing and repairing, anti-glycation, antioxidant, brightening the skin, anti-aging, anti-wrinkle and anti-damage.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a cherry blossom fermentation product, a preparation method thereof, a composition and an application. Background Art

[0002] Skin care is a related process of cleaning, maintaining, protecting and skin barrier maintenance for different skin conditions at different times.

[0003] In the infant period, there are significant differences in the microscopic structure between the skin of infants and that of adults. The density of the skin grid-like structure formed by the stratum corneum of infants is higher than that of adults. The thickness of the stratum corneum of infants is 30% thinner than that of adults, the epidermis is 20 - 30% thinner than that of adults, and the total thickness of the dermis is also lower than that of adults. Therefore, the skin of infants has characteristics such as worse thermoregulation function than adults, faster trans-epidermal water loss, being more vulnerable to external stimuli, imperfect immune system, being prone to sunburn and rash. Therefore, maintaining the skin barrier, moisturizing and repairing in the infant period are more necessary and important skin care methods for them.

[0004] Generally, when the skin of infants is damaged due to being stimulated, sunburned, having a rash, etc., inflammatory factors will be produced. Blocking the stimulus source and controlling the production of inflammatory factors are also important repair means. Therefore, the condition of skin barrier damage and its repair ability can be observed by monitoring inflammatory factors. In addition to the integrity of the skin barrier, the skin hydration ability also directly determines the skin moisturizing ability. Usually, hydration can increase the water content of the stratum corneum from the normal 10% to more than 50%. Aquaporin 3 (AQP3) is a substance on the cell membrane, responsible for the transport of substances such as water, glycerol and urea, belonging to a kind of transport protein factor, and is mainly expressed in keratinocytes and skin fibroblasts. AQP3 not only participates in skin hydration and barrier function, but also plays an important role in skin injury, repair and healing, and is an important guarantee for maintaining the normal form and function of the skin. By regulating AQP3, the skin hydration and barrier function can also be improved, thereby enhancing the skin moisturizing and repair ability.

[0005] For adults, skin care will have to face the problem of skin aging. However, skin aging is a normal physiological process. Generally, under multiple factors such as natural environment, living habits and personal stress, the aging speed of skin varies greatly among people. This difference sometimes leads to huge differences in the degree of skin aging between two different individuals of the same age base or even between different parts of the same body. Glycated collagen is a feedback of dermal matrix glycation and functional decline. It is accompanied by the degradation of related matrix and looks yellowish. The skin will also lose its white texture. Generally, glycated collagen in adult skin can be observed at the age of 20. With age and skin aging, AGEs in the skin increase. The proportion of glycation can exceed 30%-50% at the age of 80. Hydroxyl free radical (·OH) is an oxidant with stronger activity than active oxygen. Hydroxyl free radical can inactivate amino acids and proteins and oxidatively decompose them. At the same time, the aging process of skin cells is also accompanied by various skin problems such as cell damage, free radical invasion, inflammatory response, excessive skin pigmentation, membrane peroxidation, wrinkles, age spots, freckles, rash spots, darkening of color, uneven color tone, etc.

[0006] Cherry blossoms are a plant of the genus Prunus, subgenus Prunus, in the Rosaceae family. They are cultivated all over the world and are rich in vitamins A, B, and E. They have the effects of relieving cough, relieving asthma, clearing the lungs, moistening the intestines, sobering up, inhibiting malignant tumors, beautifying and nourishing the skin, strengthening mucous membranes, and promoting sugar metabolism. Cherry blossoms are also used and reported in cosmetics. CN 112206191 provides a cherry blossom extract, an extraction method, and an application thereof. CN1950390A provides a genkwain and a sakurain derivative, their cosmetic and therapeutic uses, and a preparation method thereof.

[0007] Infants and young children need to pay attention to skin moisturizing and skin barrier maintenance and repair, while the skin of adults also needs targeted anti-oxidation, anti-glycation, anti-aging and repair. At present, a variety of skin treatment methods are used to delay, minimize or even eliminate problems related to excessive skin aging, including skin resurfacing, laser correction, photorejuvenation, etc. Usually, those effective treatments require more time, physical strength and financial resources. However, applying professional cosmetics such as patches and masks, and oral vitamins can also effectively solve the corresponding problems in a targeted manner, and can save more time, physical strength and financial resources accordingly. However, the use of probiotics thermophilic streptococci, thermophilic bacteria lysates, and cherry blossoms to produce a fermentation product composition with moisturizing and repairing, anti-glycation, antioxidant and anti-aging, and effectively solve the problems related to excessive skin aging has not been reported. Summary of the invention

[0008] The object of the present invention is to provide a cherry blossom fermentation product, its preparation method, composition and application, which contain rich active ingredients such as vitamin A, vitamin B, vitamin E, cherry blossom antioxidant elements (cherry blossom flavonoids and flavonoid glycosides), amino acids and polypeptides, and have the functions of moisturizing and repairing, anti-glycation, antioxidant, skin brightening and anti-aging, cell growth ability (anti-wrinkle), and anti-cell damage ability, especially the abilities of moisturizing, repairing, anti-glycation and anti-aging.

[0009] The technical solution of the present invention is realized as follows:

[0010] The present invention provides a preparation method of a cherry blossom fermentation product, comprising the following steps:

[0011] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them with liquid nitrogen, thaw them, dry them, break the cell walls by pulverization, and inactivate the enzymes by ultrasonic treatment to obtain cherry blossom powder;

[0012] S2. Deep eutectic solvent extraction: Add the cherry blossom powder obtained in step S1 to an aqueous solution of deep eutectic solvent, heat and stir for extraction, filter, and keep the solid residue 1 for drying; Add sodium phosphate to the filtrate to precipitate, keep the solid residue 2 for drying, and dry the filtrate to obtain an extract;

[0013] S3. Bacteria expansion: Resuscitate Streptococcus thermophilus, add it to a seed medium, sterilize it, and culture it to obtain a bacterial seed solution;

[0014] S4. Fermentation: Inoculate the bacterial seed solution in step S3 into a fermentation substrate for fermentation, centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain a Streptococcus thermophilus cherry blossom ferment;

[0015] S5. Preparation of cell wall polysaccharide and lysate: Dilute the bacterial sludge obtained in step S4 with PBS solution, add snailase and lysozyme, carry out enzymatic hydrolysis, inactivate the enzymes, centrifuge, discard the precipitate, take the upper supernatant, break the cell walls under high pressure, add ethanol to precipitate the broken cell wall solution, wash the solid, and dry it to obtain cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry it to obtain a lysate;

[0016] S6. Preparation of cherry blossom fermentation product: Mix the extract obtained in step S2, the Streptococcus thermophilus cherry blossom ferment obtained in step S4, the cell wall polysaccharide and lysate obtained in step S5 evenly to obtain a cherry blossom fermentation product.

[0017] As a further improvement of the present invention, the conditions for enzyme inactivation by ultrasonic treatment in step S1 are to treat at a frequency of 35 - 45 kHz for 25 - 35 min.

[0018] As a further improvement of the present invention, the water content in the deep eutectic solvent aqueous solution in step S2 is 50-70 wt%, and the rest is the deep eutectic solvent, which is prepared by mixing choline chloride and glucose in a molar ratio of 1:2. The solid-liquid ratio of the cherry blossom powder and the deep eutectic solvent aqueous solution is 1:5-10 g / mL. The temperature of the heating and stirring extraction is 40-50 °C, and the time is 1-3 h.

[0019] As a further improvement of the present invention, the formula of the seed culture medium in step S3 is as follows: 5-15 g of skimmed milk powder, 3-7 g of yeast powder, 0.02-0.04 wt% of MgSO4, 0.05-0.15% of MnSO4, 0.05-0.15 wt% of ZnSO4, 0.2-0.7 wt% of glycerophosphate disodium, 0.02-0.07 wt% of ascorbic acid, and the balance is water up to 1 L. The culture conditions are 35-40 °C, 100-200 r / min, and the culture time is 20-24 h. The Streptococcus thermophilus is Streptococcus thermophilus CICC6223, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

[0020] As a further improvement of the present invention, the inoculation volume ratio of the bacterial seed liquid in step S4 is 1-2:100. The fermentation substrate is adding 1-3 wt% of solid residue 1 and 0.5-1 wt% of solid residue 2 to the seed liquid culture medium. The fermentation conditions are stirring at 150-200 rpm, adjusting the pH value to 7.0±1.0 with PBS, culturing at 35-40 °C for 20-24 h, then raising the temperature to 40-45 °C for fermentation culture for 5-7 h, and the ventilation volume is 80-120 m 3 / h.

[0021] As a further improvement of the present invention, the conditions for high-pressure cell disruption in step S5 are circulating cell disruption 2-4 times at 2-6 °C and 60-100 MPa in a high-pressure cell disruptor. The dilution factor is 7-10 times. The pH of the PBS solution is 7.2-7.5. The mass ratio of the cell sludge, snailase, and lysozyme is 100:1-2:0.5-1. The temperature of the enzymatic hydrolysis is 45-50 °C, and the time is 1-2 h.

[0022] As a further improvement of the present invention, the mass ratio of the extract, Streptococcus thermophilus cherry blossom ferment, cell wall polysaccharide, and lysate in step S6 is 10-12:25-35:3-5:5-7.

[0023] The present invention further protects a skin care composition, which is characterized in that it is composed of the above-mentioned cherry blossom fermentation product and a transdermal absorption promoter in a mass ratio of 100:1-2. The transdermal absorption promoter includes eucalyptus oil and a transdermal compound, with a mass ratio of 3-5:2. The structural formula of the transdermal compound is shown in Formula I:

[0024]

[0025] The present invention further protects the application of the above-mentioned cherry blossom fermentation product and the above-mentioned composition in cosmetics.

[0026] The present invention has the following beneficial effects:

[0027] The transdermal absorption promoting effect of menthol, on the one hand, changes the microstructure of the stratum corneum, forms continuous hydrogen bonds with the head groups of lipid molecules, destroys the hydrogen bond network between lipids, makes the stratum corneum structure disordered and loose, and thus reduces the skin barrier effect; on the other hand, it has a strong affinity with cholesterol in the lipid molecular layer, increases the fluidity of the lipid bilayer of the stratum corneum, and improves the drug penetration rate. However, pure menthol has strong volatility and is easy to dissipate when added to skin care products, reducing its activity.

[0028] Azone can increase the structural disorder by extracting lipids and changing the keratin conformation, reduce the skin barrier function, and has an inhibitory effect on the active epidermal cell Ca 2+ pump, and promotes drug penetration through the skin by affecting the Ca 2+ balance inside and outside the membrane and increasing membrane fluidity. However, direct use of azone has greater irritation to the skin and sometimes causes skin problems such as red rashes and itching.

[0029] In the present invention, after triethanolamine is chlorinated through acyl chloride and then coupled with caprolactam, a structure similar to azone is obtained, but the irritation to the skin is greatly reduced and it is safer to use. At the same time, it is coupled with 2 molecules of menthol, which not only increases the molecular weight of menthol, greatly increases its boiling point, makes it not easy to volatilize, and thus improves the stability of the prepared product. The prepared transdermal compound also has the high-efficiency transdermal absorption promoting effect of menthol and azone, and the effect is better. The preparation method is simple, the conditions are mild, and the synthesis efficiency is high.

[0030] In the present invention, the cherry blossoms are treated by inactivating enzymes, which can keep a high concentration of flavonoid components from being enzymatically hydrolyzed, preserve freshness and maintain a high content of sakuranetin. By extracting the active components with a deep eutectic solvent, the extraction rate is high, the conditions are mild, and the activity of the components will not be damaged. In the obtained extract, the content of flavonoid glycoside components of cherry blossoms is higher, the solubility is improved, it has better stability, and has better soothing ability.

[0031] After extraction, the phosphorylcholine obtained by reaction with phosphoric acid is mixed with the extracted cherry solid residue for fermentation, which not only provides the raw material of the probiotic Streptococcus thermophilus, promotes its large-scale proliferation and produces rich bacterial mud, but also produces rich vitamins and amino acids in the product, as well as cherry antioxidants mainly composed of flavonoids and their glycosides. The obtained Streptococcus thermophilus cherry fermentation product contains a variety of natural moisturizers, skin brightening agents and anti-aging agents. The combination of these active substances can provide synergistic beneficial effects.

[0032] The bacterial mud is hydrolyzed by snail enzyme and lysozyme, and the resulting thermophilic Streptococcus lysate is rich in active amino acids, vitamins, short-chain fatty acids, antimicrobial peptides, antioxidant peptides and other components, which can better resist glycation, antioxidant, skin brightening and anti-aging effects, while the bacterial wall polysaccharides have excellent moisturizing and antioxidant effects.

[0033] The cherry blossom fermentation product prepared by the present invention contains rich active ingredients such as vitamin A, vitamin B, vitamin E, cherry blossom antioxidants (cherry blossom flavonoids and flavonoid glycosides), amino acids and polypeptides, and has moisturizing and repairing, anti-glycation, anti-oxidation, skin brightening and anti-aging, cell growth ability (anti-wrinkle), anti-cell damage ability, especially moisturizing, repairing, anti-glycation, and anti-aging abilities.

[0034] Among them, sakura element compounds have multiple phenolic hydroxyl groups, which can scavenge free radicals, chelate metal ions, and hinder the intermediate reaction of oil oxidation, thereby exerting antioxidant properties and improving the oxidative stability of the emulsion. Flavonoid phenolic components can also bind to proteins through various non-covalent interactions such as hydrogen bonds and hydrophobic interactions, and can synergize with proteins to improve their emulsification properties. In addition, it actually increases the immunity of the skin by enhancing the hydration capacity and water retention of the skin, increasing the skin's tightness and elasticity. It can promote cell regeneration, strengthen skin metabolism and have a unique exfoliating effect, prevent skin aging, brighten, anti-aging and anti-inflammatory, repair, etc., effectively remove deep dirt in the pores, tighten the skin, and prevent fine lines and nasolabial folds.

[0035] Vitamins have specific metal chelating ability and can inhibit the formation of AGEs by blocking ROS and free transition metal ions.

[0036] Amino acids and peptides are mostly effective electron donors and have metal ion chelating activity. They can form complexes with metals and limit the reactivity of metals. They can also react with the carbonyl groups of proteins (cross-linking the amino groups with amino acids, or clearing modified proteins on macrophages through RAGE) to remove hydroxyl radicals, thereby reducing the level of free radicals. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Streptococcus thermophilus is Streptococcus thermophilus of CICC6223, purchased from

[0039] snailase

[0040] lysozyme

[0041] Synthesis of the transdermal compound in Preparation Example 1

[0042] Synthesis route:

[0043]

[0044] Preparation method:

[0045] S1. Add 0.1 mol of triethanolamine to 200 mL of dichloromethane, dropwise add 50 mL of a dichloromethane solution containing 0.31 mol of thionyl chloride, stir and react at room temperature for 1 h, remove the solvent and unreacted raw materials under reduced pressure to obtain Intermediate 1; ESI-MS calculated value: C6H 13 Cl3N(M+H) + 205.53, measured value: 205.5, NMR result: 1 H NMR(300 MHz, CDCl3) δ 3.48(t, 6H), 2.62(t, 6H).

[0046] S2. Add 0.11 mol of Intermediate 1, 0.1 mol of caprolactam and 0.5 mol of triethylamine to 300 mL of chloroform, heat to 60 °C, stir and react for 3 h, remove the solvent under reduced pressure, and recrystallize with ethanol to obtain Intermediate 2; ESI-MS calculated value: C 12 H 23 Cl2N2O(M+H) + 282.22, measured value: 282.2, NMR result: 1 HNMR(300 MHz, CDCl3) δ 3.50(t, 4H), 3.30(t, 2H), 3.2(t, 2H), 2.62 - 2.65(m, 6H), 2.17(t, 2H), 1.55 - 1.57(m, 4H), 1.29(m, 2H).

[0047] S3. Add 0.1 mol of intermediate 2, 0.21 mol of menthol, and 0.7 mol of triethylamine to 400 mL of acetonitrile. Heat the mixture to 70 °C and stir for 4 h. Remove the solvent under reduced pressure and recrystallize with acetone / ethanol to obtain the product with a total yield of 82.9%. ESI-MS calculated value: C 32 H 61 N2O3(M+H)+521.83, measured value: 521.8. NMR result: 1 H NMR(300 MHz, CDCl3) δ 3.45(t, 4H), 3.28(t, 2H), 3.22(t, 2H), 2.78(m, 2H), 2.62(t, 2H), 2.52(t, 4H), 2.17(t, 2H), 1.77 - 1.80(m, 6H), 1.57 - 1.62(m, 12H), 1.32 - 1.37(m, 6H), 1.04 - 1.07(m, 18H).

[0048] Example 1

[0049] This example provides a method for preparing a cherry blossom fermentation product, which includes the following steps:

[0050] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them in liquid nitrogen, thaw them, dry them, crush them into powder, and perform ultrasonic treatment at a frequency of 35 kHz for 25 min to inactivate the enzymes, obtaining cherry blossom powder;

[0051] S2. Deep eutectic solvent extraction: Add 10 g of the cherry blossom powder prepared in step S1 to 50 mL of an aqueous deep eutectic solvent solution. Heat the mixture to 40 °C and stir for 1 h. Filter to obtain solid residue 1 for drying and retention; Add 3 g of sodium phosphate to the filtrate and precipitate for 1 h to obtain solid residue 2 for drying and retention. Dry the filtrate to obtain the extract;

[0052] The water content in the aqueous deep eutectic solvent solution is 50 wt%, and the rest is the deep eutectic solvent, which is prepared by mixing choline chloride and glucose in a molar ratio of 1:2;

[0053] S3. Bacteria expansion: Resuscitate Streptococcus thermophilus, add it to the seed medium, sterilize it, and culture it at 35 °C and 100 r / min for 20 h to obtain a bacterial seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0054] The formula of the seed medium is as follows: 5 g of skim milk powder, 3 g of yeast powder, 0.02 wt% of MgSO4, 0.05% of MnSO4, 0.05 wt% of ZnSO4, 0.2 wt% of glycerophosphate disodium, 0.02 wt% of ascorbic acid, and the balance is water up to 1 L;

[0055] S4. Fermentation: Inoculate the strain seed liquid in step S3 into the fermentation substrate for fermentation. The inoculation volume ratio of the strain seed liquid is 1:100. Centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain the Streptococcus thermophilus cherry blossom ferment;

[0056] The fermentation substrate is prepared by adding 1 wt% of solid residue 1 and 0.5 wt% of solid residue 2 to the seed liquid medium;

[0057] The fermentation conditions are as follows: under stirring at 150 rpm, adjust the pH value to 7.0 ± 1.0 with PBS, culture at 35 °C for 20 h, then raise the temperature to 40 °C for fermentation culture for 5 h, and the ventilation volume is 80 m 3 / h;

[0058] S5. Preparation of cell wall polysaccharide and lytic products: Dilute 100 g of the bacterial sludge obtained in step S4 with PBS solution at pH = 7.2 by 7 times, add 1 g of snail enzyme and 0.5 g of lysozyme, enzymolyze at 45 °C for 1 h, inactivate the enzyme, centrifuge, discard the precipitate, take the upper supernatant, and circulate and break the wall 2 times in a high-pressure cell disruptor at 2 °C and 60 MPa. Add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, and dry to obtain the cell wall polysaccharide. Recover ethanol from the filtrate and freeze-dry to obtain the lytic products;

[0059] S6. Preparation of cherry blossom fermentation product: Stir and mix 10 g of the extract obtained in step S2, 25 g of the Streptococcus thermophilus cherry blossom ferment obtained in step S4, 3 g of the cell wall polysaccharide obtained in step S5, and 5 g of the lytic products for 10 min to obtain the cherry blossom fermentation product.

[0060] Example 2

[0061] This example provides a method for preparing a cherry blossom fermentation product, including the following steps:

[0062] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them with liquid nitrogen, thaw, dry, break and pulverize them, and perform ultrasonic treatment at a frequency of 45 kHz for 35 min to inactivate the enzyme to obtain cherry blossom powder;

[0063] S2. Deep eutectic solvent extraction: Add 10 g of the cherry blossom powder obtained in step S1 to 100 mL of an aqueous solution of deep eutectic solvent, heat to 50 °C, stir and extract for 3 h, filter, and obtain solid residue 1 for drying and retention; Add 3 g of sodium phosphate to the filtrate, precipitate for 1 h, obtain solid residue 2 for drying and retention, and dry the filtrate to obtain the extract;

[0064] The water content in the aqueous solution of the deep eutectic solvent is 70 wt%, and the rest is the deep eutectic solvent, which is prepared by mixing choline chloride and glucose in a molar ratio of 1:2;

[0065] S3. Bacteria expansion: Resuscitate Streptococcus thermophilus, add it to the seed medium, sterilize, culture at 40 °C and 200 r / min for 24 h to obtain a bacterial seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0066] The formula of the seed medium is as follows: 15 g of skim milk powder, 7 g of yeast powder, 0.04 wt% of MgSO4, 0.15% of MnSO4, 0.15 wt% of ZnSO4, 0.7 wt% of glycerol phosphate disodium, 0.07 wt% of ascorbic acid, and the balance is water up to 1 L;

[0067] S4. Fermentation: Inoculate the bacterial seed solution in step S3 into the fermentation substrate for fermentation. The inoculation volume ratio of the bacterial seed solution is 2:100. Centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain the Streptococcus thermophilus cherry blossom ferment;

[0068] The fermentation substrate is the addition of 3 wt% of solid residue 1 and 1 wt% of solid residue 2 to the seed liquid medium;

[0069] The fermentation conditions are as follows: Stir at 200 rpm, adjust the pH value to 7.0 ± 1.0 with PBS, culture at 40 °C for 24 h, raise the temperature to 45 °C for fermentation culture for 7 h, and the ventilation volume is 120 m 3 / h;

[0070] S5. Preparation of cell wall polysaccharide and lysate: Dilute 100 g of the bacterial sludge obtained in step S4 with PBS solution at pH = 7.3 by 10 times, add 2 g of snail enzyme and 1 g of lysozyme, enzymolyze at 50 °C for 2 h, inactivate the enzyme, centrifuge, discard the precipitate, take the upper supernatant, and circulate and break the wall 4 times in a high-pressure cell disruptor at 6 °C and 100 MPa. Add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, and dry to obtain the cell wall polysaccharide. Recover ethanol from the filtrate and freeze-dry to obtain the lysate;

[0071] S6. Preparation of cherry blossom fermentation product: Stir and mix 12 g of the extract obtained in step S2, 35 g of the Streptococcus thermophilus cherry blossom ferment obtained in step S4, 5 g of the cell wall polysaccharide obtained in step S5, and 7 g of the lysate for 10 min to obtain the cherry blossom fermentation product.

[0072] Example 3

[0073] This example provides a method for preparing a cherry blossom fermentation product, including the following steps:

[0074] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them in liquid nitrogen, thaw, dry, break and pulverize them, and perform ultrasonic treatment at a frequency of 40 kHz for 30 min to inactivate the enzyme to obtain cherry blossom powder;

[0075] S2. Deep eutectic solvent extraction: Add 10 g of the cherry blossom powder prepared in step S1 to 70 mL of an aqueous deep eutectic solvent solution, heat to 45 °C, stir and extract for 2 h, filter, and keep the solid residue 1 for drying; add 3 g of sodium phosphate to the filtrate, precipitate for 1 h, keep the solid residue 2 for drying, and dry the filtrate to obtain the extract;

[0076] The water content in the aqueous deep eutectic solvent solution is 60 wt%, and the rest is the deep eutectic solvent, which is prepared by mixing choline chloride and glucose in a molar ratio of 1:2;

[0077] S3. Bacteria expansion: Resuscitate Streptococcus thermophilus, add it to the seed medium, sterilize, culture at 37 °C and 150 r / min for 22 h to obtain a bacterial seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0078] The formula of the seed medium is as follows: 10 g of skim milk powder, 5 g of yeast powder, 0.03 wt% of MgSO4, 0.1% of MnSO4, 0.1 wt% of ZnSO4, 0.5 wt% of glycerophosphate disodium, 0.05 wt% of ascorbic acid, and the balance is water to 1 L;

[0079] S4. Fermentation: Inoculate the bacterial seed solution in step S3 into the fermentation substrate for fermentation. The inoculation volume ratio of the bacterial seed solution is 1.5:100. Centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain the Streptococcus thermophilus cherry blossom ferment;

[0080] The fermentation substrate is prepared by adding 2 wt% of solid residue 1 and 0.7 wt% of solid residue 2 to the seed liquid medium;

[0081] The fermentation conditions are as follows: under stirring at 170 rpm, adjust the pH value to 7.0 ± 1.0 with PBS, culture at 37 °C for 22 h, raise the temperature to 42 °C and ferment for 6 h, and the ventilation volume is 100 m 3 / h;

[0082] S5. Preparation of cell wall polysaccharide and lysate: Dilute 100 g of the bacterial sludge prepared in step S4 with a PBS solution of pH = 7.3 by 8 times, add 1.5 g of snail enzyme and 0.7 g of lysozyme, enzymolyze at 47 °C for 1.5 h, inactivate the enzyme, centrifuge, discard the precipitate, take the upper supernatant, and cycle and break the wall 3 times in a high-pressure cell disruptor at 4 °C and 80 MPa. Add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, dry to obtain the cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry to obtain the lysate;

[0083] S6. Preparation of cherry blossom fermentation product: Stir and mix 11 g of the extract prepared in step S2, 30 g of the Streptococcus thermophilus cherry blossom ferment prepared in step S4, 4 g of the cell wall polysaccharide prepared in step S5, and 6 g of the lysate for 10 min to obtain the cherry blossom fermentation product.

[0084] Comparative Example 1

[0085] Compared with Example 3, the difference lies in that ultrasonic treatment was not performed in step S1.

[0086] Specifically as follows:

[0087] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them in liquid nitrogen, thaw them, dry them, break the cell walls and pulverize them to obtain cherry blossom powder.

[0088] Comparative Example 2

[0089] Compared with Example 3, the difference lies in that step S2 was not carried out.

[0090] Specifically as follows:

[0091] S1. Enzyme inactivation: Wash the cherry blossoms, freeze them in liquid nitrogen, thaw them, dry them, break the cell walls and pulverize them, and perform ultrasonic treatment at a frequency of 40 kHz for 30 min to inactivate the enzymes to obtain cherry blossom powder;

[0092] S2. Bacteria expansion: Resuscitate Streptococcus thermophilus, add it to the seed medium, sterilize it, culture it at 37 °C and 150 r / min for 22 h to obtain a bacterial strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0093] The formula of the seed medium is as follows: 10 g of skim milk powder, 5 g of yeast powder, 0.03 wt% of MgSO4, 0.1% of MnSO4, 0.1 wt% of ZnSO4, 0.5 wt% of disodium glycerophosphate, 0.05 wt% of ascorbic acid, and the balance is water to 1 L;

[0094] S3. Fermentation: Inoculate the bacterial strain seed solution in step S2 into the fermentation substrate for fermentation. The inoculation volume ratio of the bacterial strain seed solution is 1.5:100. Centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain the Streptococcus thermophilus cherry blossom ferment;

[0095] The fermentation substrate is the seed solution medium added with 2.7 wt% of the cherry blossom powder in step S1;

[0096] The fermentation conditions are as follows: Stir at 170 rpm, adjust the pH value to 7.0 ± 1.0 with PBS, culture at 37 °C for 22 h, raise the temperature to 42 °C and ferment for 6 h, and the ventilation volume is 100 m 3 / h;

[0097] S4. Preparation of cell wall polysaccharide and cell lysate: Dilute 100 g of the cell sludge obtained in step S3 with PBS solution at pH = 7.3 by 8 times, add 1.5 g of snailase and 0.7 g of lysozyme, enzymolyze at 47 °C for 1.5 h, inactivate the enzyme, centrifuge, discard the precipitate, take the supernatant, circulate and break the wall 3 times in a high-pressure cell disruptor at 4 °C and 80 MPa. Add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, and dry to obtain cell wall polysaccharide. Recover ethanol from the filtrate and freeze-dry to obtain cell lysate;

[0098] S5. Preparation of cherry blossom fermentation product: Stir and mix 11 g of the extract obtained in step S2, 30 g of Streptococcus thermophilus cherry blossom ferment obtained in step S3, 4 g of cell wall polysaccharide obtained in step S4, and 6 g of cell lysate for 10 min to obtain cherry blossom fermentation product.

[0099] Comparative Example 3

[0100] Compared with Example 3, the difference is that solid residue 1 is not added in step S4.

[0101] Specifically as follows:

[0102] S4. Fermentation: Inoculate the strain seed liquid in step S3 into the fermentation substrate for fermentation. The inoculation volume ratio of the strain seed liquid is 1.5:100. Centrifuge the fermentation broth to collect cell sludge, and freeze-dry the supernatant to obtain Streptococcus thermophilus cherry blossom ferment;

[0103] The fermentation substrate is adding 2.7 wt% of solid residue 2 to the seed liquid medium;

[0104] The fermentation conditions are under stirring at 170 rpm, adjusting the pH value to 7.0 ± 1.0 with PBS, culturing at 37 °C for 22 h, raising the temperature to 42 °C for fermentation culture for 6 h, and the ventilation volume is 100 m 3 / h.

[0105] Comparative Example 4

[0106] Compared with Example 3, the difference is that solid residue 2 is not added in step S4.

[0107] Specifically as follows:

[0108] S4. Fermentation: Inoculate the strain seed liquid in step S3 into the fermentation substrate for fermentation. The inoculation volume ratio of the strain seed liquid is 1.5:100. Centrifuge the fermentation broth to collect cell sludge, and freeze-dry the supernatant to obtain Streptococcus thermophilus cherry blossom ferment;

[0109] The fermentation substrate is adding 2.7 wt% of solid residue 1 to the seed liquid medium;

[0110] The fermentation conditions are as follows: under stirring at 170 rpm, the pH value is adjusted to 7.0 ± 1.0 with PBS, cultured at 37 °C for 22 h, then the temperature is raised to 42 °C for fermentation culture for 6 h, and the ventilation volume is 100 m 3 / h.

[0111] Comparative Example 5

[0112] Compared with Example 3, the difference lies in that no snail enzyme was added in step S5.

[0113] Specifically as follows:

[0114] S5. Preparation of cell wall polysaccharide and lysate: Dilute 100 g of the cell paste obtained in step S4 with PBS solution at pH = 7.3 by 8 times, add 2.2 g of snail enzyme, enzymolyze at 47 °C for 1.5 h, inactivate the enzyme, centrifuge, discard the precipitate, take the upper supernatant, circulate and break the wall 3 times in a high-pressure cell disruptor at 4 °C and 80 MPa, add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, dry to obtain cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry to obtain the lysate.

[0115] Comparative Example 6

[0116] Compared with Example 3, the difference lies in that no lysozyme was added in step S5.

[0117] Specifically as follows:

[0118] S5. Preparation of cell wall polysaccharide and lysate: Dilute 100 g of the cell paste obtained in step S4 with PBS solution at pH = 7.3 by 8 times, add 2.2 g of snail enzyme, enzymolyze at 47 °C for 1.5 h, inactivate the enzyme, centrifuge, discard the precipitate, take the upper supernatant, circulate and break the wall 3 times in a high-pressure cell disruptor at 4 °C and 80 MPa, add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, dry to obtain cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry to obtain the lysate.

[0119] Comparative Example 7

[0120] Compared with Example 3, the difference lies in that no enzymolysis was carried out in step S5.

[0121] Specifically as follows:

[0122] S5. Preparation of cell wall polysaccharide and lysate: Dilute 100 g of the cell paste obtained in step S4 with PBS solution at pH = 7.3 by 8 times, centrifuge, discard the precipitate, take the upper supernatant, circulate and break the wall 3 times in a high-pressure cell disruptor at 4 °C and 80 MPa, add ethanol to the disrupted solution until the ethanol content in the system is 70 wt%, precipitate for 0.5 h, wash the solid, dry to obtain cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry to obtain the lysate.

[0123] Test Example 1

[0124] The sakura fermentation products prepared in Examples 1-3 and Comparative Examples 1-7 of the present invention were subjected to content component tests, and the results are shown in Table 1.

[0125] Determination method:

[0126] (1) Polypeptide test:

[0127] A standard aqueous solution of casein phosphopeptide with a concentration of 12 mg / mL was prepared with casein phosphopeptide standard. 0 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL were taken respectively and made up to 10 mL with water; then 6.0 mL of the standard solution was taken respectively, 4.0 mL of biuret reagent was added, mixed evenly, allowed to stand for 10 minutes, centrifuged at 2000 r / min for 10 minutes, and the supernatant was taken to measure the OD value at 540 nm (using the first tube as the blank control). With the peptide concentration as the abscissa and the OD value as the ordinate, a standard curve was made. The sample was tested under the above conditions, and the polypeptide content was calculated through the standard curve.

[0128] (2) Total amino acid test:

[0129] It was determined according to the method of "QB / T 2409-1998 Determination of Amino Acid Content in Cosmetics".

[0130] (3) Polysaccharide test:

[0131] A standard glucose solution with a concentration of 0.1001 mg / mL was prepared with glucose standard. 0.1 mL, 0.4 mL, 0.8 mL, 1.6 mL, and 2.0 mL were taken respectively and made up to 2 mL with pure water; then 1 mL of phenol solution (concentration 5%) and 5 mL of concentrated sulfuric acid were added respectively, reacted for 15 min, cooled in cold water for 30 min for color development; the absorbance was measured with an ultraviolet spectrophotometer under the condition of 490 nm. The sample was tested under the above conditions, and the polysaccharide content was calculated through the standard curve.

[0132] (4) Content of sakura elements (flavonoids and their glycosides) (rutin method):

[0133] Prepare a rutin standard solution with a concentration of 0.416 mg / mL using rutin standard (the solvent is 30% ethanol). Respectively take 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and make up to 5 mL with 30% ethanol; then respectively add 0.3 mL of 10% aqueous Al(NO3)3 solution, 0.3 mL of 5% NaNO2 solution, 3 mL of NaOH solution (1 mol / L), and 1.4 mL of 30% ethanol; after reacting for 15 min, measure the absorbance with a UV spectrophotometer under the condition of 510 nm, establish a standard curve, test the sample under the above conditions, and determine its content through the standard curve.

[0134] (5) Glutathione peroxidase activity:

[0135] Add 0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.20 mL of 1.0 mol / L GSH to 1.60 mL of 0.61 mol / L trichloroacetic acid (TCA), and dilute with double-distilled water to prepare 2 mL of GSH standard solutions with concentrations of 0, 10, 20, 40, 60, 80, 100 μmol / L respectively. Then successively add 2.5 mL of 0.32 mol / L Na2HPO4, 120 μL of 4 mol / L NaOH solution, and 0.5 mL of 1.0 mol / L 2-nitrobenzoic acid (DTNB) solution to each tube. Use the blank tube to calibrate the zero point, measure the absorbance value at a wavelength of 423 nm, and make a GSH standard curve. Glutathione peroxidase activity:

[0136] The sample reacts at 37 °C and pH 6.5. Referring to the standard curve method, successively add 1 mL of 1.0 mol / L GSH, 2.5 mL of 0.32 mol / L Na2HPO4, 120 μL of 4 mol / L NaOH, and 0.5 mL of 1.0 mol / L DTNB. The sample is measured for absorbance value using the same method, and the content is calculated through the standard curve. Taking a 1 μmol decrease in GSH concentration as 1 enzyme activity unit (U).

[0137] Table 1

[0138]

[0139] As can be seen from the above table, the cherry blossom fermentation products prepared in Examples 1-3 of the present invention contain rich active components.

[0140] Test Example 2 Anti-glycation ability test

[0141] When sugar meets protein, under specific conditions, it will turn brown, which is an important factor leading to dull and aging skin. In this experiment, the inhibition rate of the glycation reaction is quantified through the glycation reaction experiment.

[0142] In a container containing 5 mL of 2.0 mol / L amino acid aqueous solution, 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of 1.0 mol / L glucose solution were added respectively. The mixture was heated at 95 °C for 3 hours, made up to 10 mL with double-distilled water, and the absorbance at 420 nm was measured to prepare a standard curve.

[0143] The samples of cherry blossom fermentation products prepared in Examples 1-3 and Comparative Examples 1-7 were measured respectively. During the measurement, 1 mL of the sample was taken, 1 mL of 1.0 mol / L glucose solution was added, and the absorbance was measured according to the above method (made up to 10 mL). Using double-distilled water as the blank control, the glucose content was calculated through the standard curve, and the inhibition rate of the saccharification reaction was calculated according to the following formula:

[0144]

[0145] The results are shown in Table 2.

[0146] Table 2

[0147] Group Glycation Inhibition Rate (%) Example 1 76.9 Example 2 77.2 Example 3 78.1 Comparative Example 1 72.4 Comparative Example 2 66.7 Comparative Example 3 68.9 Comparative Example 4 73.4 Comparative Example 5 70.3 Comparative Example 6 68.5 Comparative Example 7 64.2

[0148] As can be seen from the above table, the cherry blossom fermentation products prepared in Examples 1-3 can all significantly inhibit the saccharification reaction.

[0149] Test Example 3 Promote the Expression of Aquaporin

[0150] Logarithmic-phase HSF cells were collected, made into a cell suspension with a density of 1×105 cells / mL in 1640 complete medium, inoculated into a 6-well culture plate, 1 mL of the cell suspension was added to each well, and then 1.5 mL of complete medium was added. It was incubated in an incubator at 37 °C and 5% CO2 for 24 h. The old medium was removed, and then 1 mL of medium containing 500 μmol / L H2O2 and 1.5 mL of medium containing 1 wt% of the cherry blossom fermentation products prepared in Examples 1-3 or Comparative Examples 1-7 were added to each well, and 0.1% Vc ethyl ether was used as the positive control group. After adding the cells, they were cultured in a 37 °C carbon dioxide incubator for 48 hours. The cells were scraped off from the 6-well culture plate with a cell scraper and transferred into a centrifuge tube together with the medium. The cells and the medium were ultrasonically broken: ultrasonic for 8 min; ultrasonic for 8 s each time, with an interval of 5 s. Then, it was centrifuged at 1000×g for 10 min, and the supernatant was removed to obtain the sample to be measured. The content of AQP-3 was detected using a related kit from Nanjing Jiancheng Bioengineering Institute. The results are shown in Table 3.

[0151] Table 3

[0152] Group AQP-3 Content (ng / mL) Serum Group 0.0808 Vc Ethyl Ether 0.1591 Example 1 0.6122 Example 2 0.6108 Example 3 0.6145 Comparative Example 1 0.5967 Comparative Example 2 0.5674 Comparative Example 3 0.5788 Comparative Example 4 0.6034 Comparative Example 5 0.5842 Comparative Example 6 0.5709 Comparative Example 7 0.5310

[0153] As can be seen from the above table, compared with the serum group, the cherry blossom fermentation product samples prepared in Examples 1-3 have a better effect on promoting the expression of aquaporin.

[0154] Test Example 4 Ability to control inflammatory factor levels

[0155] Collect RAW264.7 cells in the logarithmic phase and prepare a cell suspension with a density of 1×10 4 cells / mL with DMEM complete medium. Inoculate the cell suspension into a 96-well culture plate, add 100 μL of the cell suspension to each well, and then add 100 μL of the complete medium. The wells around the plate are filled and sealed with sterile phosphate buffer solution (pH 6.8), and incubated in an incubator at 37 °C and 5% CO2 for 24 hours. Remove the old medium, and then add 10 μL of 40 μg / mL lipopolysaccharide to each well, and supplement the volume to 200 μL with the medium containing 1 wt% of the cherry blossom fermentation product prepared in Examples 1-3 or Comparative Examples 1-7. Set the serum control group to add only the complete medium, the induction group to add lipopolysaccharide and the complete medium, and the positive control group to add 10 μL of 12.5 μg / mL dexamethasone, 10 μL of 40 μg / mL lipopolysaccharide, and 180 μL of the complete medium. After adding the cells, culture them in a carbon dioxide incubator at 37 °C for 24 hours. After the culture is completed, aspirate the cell supernatant and transfer it into 1.5 mL centrifuge tubes, which are the samples to be tested. Use the corresponding ELISA kit to detect the content of inflammatory factors in the samples. At the same time, use 60 ppm dexamethasone to replace the cherry blossom fermentation product in the experimental group as the positive control group; use the cells without ultraviolet irradiation and without adding the cherry blossom fermentation product as the blank control. The rest are the same as in Example 1, and the supernatant is taken to measure the inflammatory factors. The inflammatory factors include IL-6, and the detection is carried out using the kit. The experimental results are shown in Table 4.

[0156] Table 4

[0157] Group IL-6 Content (ng / mL) Blank Control Group 238.18 Dexamethasone 124.97 Example 1 41.04 Example 2 40.67 Example 3 40.12 Comparative Example 1 52.61 Comparative Example 2 61.86 Comparative Example 3 58.67 Comparative Example 4 45.48 Comparative Example 5 48.64 Comparative Example 6 49.23 Comparative Example 7 41.06

[0158] As can be seen from Table 4, compared with the induction group, the cherry blossom fermentation product samples prepared in Examples 1-3 of the present invention have a better effect on the content of IL-6. After culturing RAW264.7 cells with the cherry blossom fermentation product of the present invention, the inflammatory factor IL-6 is significantly reduced, and it is better than the positive control group, indicating that the cherry blossom fermentation product of the present invention has a strong anti-inflammatory and repair ability on RAW264.7 cells.

[0159] Test Example 5 NO clearance rate experiment

[0160] Collect RAW264.7 cells in the logarithmic phase and culture them in DMEM (containing double antibodies) culture medium with 10% fetal bovine serum at 37 °C and 5% CO2 with saturated humidity until the logarithmic growth phase. Digest the cells and adjust the concentration to 1×10 4Cells were inoculated at a density of 1×10 cells / mL, and 100 μL of the cell suspension was added to each well of a 96-well plate. The outermost wells were filled with 100 μL of PBS to maintain a humid environment, and the cells were cultured for 24 h until they adhered to the plate. After 24 h, the culture medium was aspirated. In the first column (control group), 100 μL of the culture medium containing 30 ng / mL lipopolysaccharide (LPS) solution was added. In the subsequent columns (experimental groups), additives (including 1 wt% of the cherry blossom fermentation products prepared in Examples 1-3 or Comparative Examples 1-7 and 30 ng / mL lipopolysaccharide) were added, and the cells were cultured for an additional 72 h. Then, the culture medium in each well was aspirated and transferred to different centrifuge tubes, and centrifuged at 1000 rmp for 5 min. According to the operating instructions of the nitric oxide assay kit from Beyotime, the nitric oxide content was measured at a wavelength of 540 nm, and the average nitric oxide content of each group was calculated.

[0161] The results are shown in Table 5.

[0162] Table 5

[0163] Group NO Content (μmol / L) Serum Group 2.04 Induction Group 2.67 Dexamethasone 1.79 Example 1 0.45 Example 2 0.46 Example 3 0.42 Comparative Example 1 0.69 Comparative Example 2 0.94 Comparative Example 3 0.83 Comparative Example 4 0.52 Comparative Example 5 0.89 Comparative Example 6 0.93 Comparative Example 7 1.21

[0164] As can be seen from the above table, after culturing RAW264.7 cells with the cherry blossom fermentation products prepared in Examples 1-3 of the present invention, the NO content decreased significantly, and the down-regulation rates were significantly better than those of the dexamethasone (positive group).

[0165] Test Example 6 Ability to promote cell growth

[0166] HACAT cells in the logarithmic growth phase were collected and prepared into a cell suspension with a density of 1×10 4 cells / mL in a 1640 complete medium. The cell suspension was inoculated into a 96-well culture plate, with 100 μL added to each well. The wells around the plate were filled and sealed with sterile phosphate buffer solution (pH 6.8). The plate was incubated in an incubator at 37 °C and 5% CO2. After the cells adhered to the plate, drugs were added. Culture media containing 1 wt% of the cherry blossom fermentation products prepared in Examples 1-3 or Comparative Examples 1-7 were added, 100 μL per well, for a total of 200 μL. A serum control group was set up, adding cells and complete culture medium, 200 μL per well. A blank control group was set up, adding only complete culture medium, 200 μL per well. Each group had 6 replicate wells as parallel wells. After incubating in an incubator at 37 °C and 5% CO2 for 24 h, the old culture medium in the 96-well culture plate was discarded, and 200 μL of phosphate buffer solution (pH 6.8) was added for washing twice. Then, 200 μL of complete medium and 20 μL of MTT solution were added to each well, and the plate was incubated in a CO2 incubator for 3 h. The culture medium in the wells was carefully aspirated, and 100 μL of dimethyl sulfoxide was added to each well. The 96-well plate was placed on a shaker and shaken for 10 minutes to fully dissolve the crystals. Then, the absorbance of each well was measured at 490 nm using an enzyme-linked immunosorbent assay reader. Based on the blank control group and the serum control group, the cell survival rate of each group was calculated.

[0167] The results are shown in Table 6.

[0168] Table 6

[0169] Group Cell Viability Rate (%) Serum Group 100 Example 1 156.2 Example 2 155.9 Example 3 157.4 Comparative Example 1 132.5 Comparative Example 2 116.9 Comparative Example 3 120.5 Comparative Example 4 149.6 Comparative Example 5 124.9 Comparative Example 6 122.1 Comparative Example 7 113.6

[0170] The above results show that the cherry blossom fermentation products prepared in Examples 1-3 of the present invention can effectively promote cell activity, and the cell viability is significantly improved.

[0171] Scavenging rate of hydroxyl radicals in Test Example 7

[0172] Prepare a 1 wt% cherry blossom fermentation product prepared in Examples 1-3 or Comparative Examples 1-7 as a sample solution. Take 2 mL of 1.00×10 -2 mol / L salicylic acid-ethanol solution, 2 mL of 9.00×10 -3 mol / L FeSO4 solution and the sample solution and add them to a test tube, then add 2 mL of 8.8×10 -2 mol / L H2O2 solution, shake well and place in a water bath at 37°C for 30 min, and measure the absorbance A i at 510 nm; measure the absorbance A j with 2 mL of pure water instead of the salicylic acid solution; measure the absorbance A0 with 2 mL of pure water instead of the sample solution. Calculate the scavenging rate (%) of ·OH according to the formula. Use vitamin C as the control group. The results are shown in Table 7.

[0173] Scavenging rate of ·OH (%) = [1 - (A i - A j ) / A0] × 100%

[0174] Table 7

[0175] Group ·OH Scavenging Rate (%) Control Group 36.7 Example 1 81.2 Example 2 81.9 Example 3 82.5 Comparative Example 1 75.6 Comparative Example 2 68.8 Comparative Example 3 72.2 Comparative Example 4 79.6 Comparative Example 5 75.4 Comparative Example 6 73.6 Comparative Example 7 67.3

[0176] As can be seen from the above table, the cherry blossom fermentation products prepared in Examples 1-3 of the present invention have good antioxidant properties.

[0177] Example 4

[0178] A skin care composition is composed of the cherry blossom fermentation product prepared in Example 1 and a transdermal absorption promoter in a mass ratio of 100:1. The transdermal absorption promoter is a mixture of eucalyptus oil and the transdermal compound prepared in Preparation Example 1, and the mass ratio is 3:2.

[0179] Example 5

[0180] A skin care composition is composed of the cherry blossom fermentation product prepared in Example 2 and a transdermal absorption promoter in a mass ratio of 100:2. The transdermal absorption promoter is a mixture of eucalyptus oil and the transdermal compound prepared in Preparation Example 1, and the mass ratio is 5:2.

[0181] Example 6

[0182] A skin care composition is composed of the cherry blossom fermentation product prepared in Example 3 and a transdermal absorption promoter in a mass ratio of 100:1.5. The transdermal absorption promoter is a mixture of eucalyptus oil and the transdermal compound prepared in Preparation Example 1, and the mass ratio is 4:2.

[0183] Example 7

[0184] Compared with Example 6, the difference is that the transdermal absorption promoter is a single eucalyptus oil.

[0185] Example 8

[0186] Compared with Example 6, the difference is that the transdermal absorption promoter is a single transdermal compound prepared in Preparation Example 1.

[0187] Comparative Example 8

[0188] Compared with Example 6, the difference is that no transdermal absorption promoter is added.

[0189] Test Example 8

[0190] Eligible subjects were selected and divided into 6 groups, namely Example 4 - 8 and Comparative Example 8 groups, with 10 people in each group. The subjects used the skin care compositions prepared in Example 4 - 8 and Comparative Example 8 as test products according to the usage requirements. The test site was the face. Skin data was collected at the corresponding test sites of the subjects, and the changes in skin data before and after using the samples in the test area were analyzed.

[0191] Change rate (%) = (Last data - Initial data) / Initial data × 100%

[0192] The specific test process is as follows:

[0193] Test conditions: On the test day, the subjects cleaned their faces with the designated cleansing sample and sat quietly in a laboratory at a temperature of (25 ± 2) °C and a humidity of 50% ± 5% RH for 30 minutes.

[0194] Test method: The Corneometer was used to measure the water content of the skin stratum corneum (the higher the water content, the higher the value), and the Cutometer was used to measure the elastic R2 value (the higher the elasticity, the higher the value) and the firmness F4 value (the higher the firmness, the lower the value).

[0195] Before the experiment started, the initial skin data of the subjects was measured. After the initial data collection was completed, the samples were distributed to the subjects for full-face application once a day for 1 week. After 1 week, the final skin data of the subjects was measured under the same test conditions.

[0196] The results are shown in Table 8.

[0197] Table 8

[0198] Group Change Rate of Skin Stratum Corneum Water Content (%) Change Rate of Skin Elasticity R2 Value (%) Change Rate of Skin Firmness F4 Value (%) Example 4 55.6 24.3 -33.1 Example 5 54.4 24.0 -33.6 Example 6 56.2 25.1 -34.9. Example 7 45.6 21.0 -26.7 Example 8 43.2 20.4 -25.5 Comparative Example 1 39.4 17.5 -20.3

[0199] As can be seen from the above table, the skin care compositions prepared in Examples 1-3 of the present invention have good effects of increasing skin moisture content, improving skin elasticity, and improving skin firmness.

[0200] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a cherry blossom fermentation product, characterized in that, It includes the following steps: S1. Enzyme inactivation: Wash the cherry blossoms, freeze them with liquid nitrogen, thaw, dry, break the cell walls by pulverization, and inactivate the enzymes by ultrasonic treatment to obtain cherry blossom powder; S2. Deep eutectic solvent extraction: Add the cherry blossom powder obtained in step S1 to an aqueous solution of the deep eutectic solvent, heat and stir for extraction, filter, and keep the obtained solid residue 1 for drying; Add sodium phosphate to the filtrate to precipitate, keep the obtained solid residue 2 for drying, and dry the filtrate to obtain the extract; The water content in the aqueous solution of the deep eutectic solvent is 50 - 70 wt%, and the rest is the deep eutectic solvent, which is prepared by mixing choline chloride and glucose in a molar ratio of 1:2; S3. Bacteria expansion: Resuscitate Streptococcus thermophilus CICC6223, add it to the seed medium, sterilize, and culture to obtain the bacterial strain seed liquid; S4. Fermentation: Inoculate the bacterial strain seed liquid in step S3 into the fermentation substrate for fermentation, centrifuge the fermentation broth to collect the bacterial sludge, and freeze-dry the supernatant to obtain the Streptococcus thermophilus cherry blossom ferment; The fermentation substrate is the seed liquid medium added with 1 - 3 wt% of solid residue 1 and 0.5 - 1 wt% of solid residue 2; S5. Preparation of cell wall polysaccharide and lysate: Dilute the bacterial sludge obtained in step S4 with PBS solution, add snailase and lysozyme, carry out enzymatic hydrolysis, inactivate the enzymes, centrifuge, discard the precipitate, take the upper clear liquid, break the cell walls under high pressure, add ethanol to precipitate the broken cell wall liquid, wash the solid, and dry to obtain the cell wall polysaccharide, recover ethanol from the filtrate, and freeze-dry to obtain the lysate; S6. Preparation of cherry blossom fermentation product: Mix the extract obtained in step S2, the Streptococcus thermophilus cherry blossom ferment obtained in step S4, the cell wall polysaccharide and the lysate obtained in step S5 evenly to obtain the cherry blossom fermentation product.

2. The preparation method according to claim 1, wherein The conditions for enzyme inactivation by ultrasonic treatment in step S1 are to treat at a frequency of 35 - 45 kHz for 25 - 35 min.

3. The preparation method according to claim 1, wherein The solid-liquid ratio of the cherry blossom powder and the aqueous solution of the deep eutectic solvent in step S2 is 1:5 - 10 g / mL, the temperature for heating and stirring extraction is 40 - 50 °C, and the time is 1 - 3 h.

4. The preparation method according to claim 1, wherein, The formula of the seed culture medium described in step S3 is as follows: 5 - 15 g of skim milk powder, 3 - 7 g of yeast powder, 0.02 - 0.04 wt% of MgSO4, 0.05 - 0.15% of MnSO4, 0.05 - 0.15 wt% of ZnSO4, 0.2 - 0.7 wt% of disodium glycerophosphate, 0.02 - 0.07 wt% of ascorbic acid, and the balance is water up to 1 L; the conditions for the culture are 35 - 40 °C, 100 - 200 r / min, and the culture is carried out for 20 - 24 h. The bacterial content of the bacterial strain seed liquid is 10 8 -10 9 cfu / mL.

5. The preparation method according to claim 1, characterized in that, The inoculation volume ratio of the bacterial strain seed solution described in step S4 is 1-2:

100. The fermentation conditions are as follows: under stirring at 150-200 rpm, the pH value is adjusted to 7.0±1.0 with PBS, cultured at 35-40°C for 20-24 h, then the temperature is raised to 40-45°C for fermentation culture for 5-7 h, and the ventilation volume is 80-120 m 3 / h.

6. The preparation method according to claim 1, wherein The conditions for high-pressure cell wall breaking in step S5 are to cycle and break the cell walls 2 - 4 times at 2 - 6 °C and 60 - 100 MPa in a high-pressure cell wall breaker, the dilution multiple is 7 - 10 times, the pH of the PBS solution is 7.2 - 7.5, the mass ratio of the bacterial sludge, snailase, and lysozyme is 100:1 - 2:0.5 - 1, the temperature for enzymatic hydrolysis is 45 - 50 °C, and the time is 1 - 2 h.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the extract, the Streptococcus thermophilus cherry blossom ferment, the cell wall polysaccharide, and the lysate in step S6 is 10 - 12:25 - 35:3 - 5:5 - 7.

8. A cherry blossom fermentation product prepared by the preparation method according to any one of claims 1 - 7.

9. A skin care composition, characterized in that, Composed of the cherry blossom fermentation product described in claim 8 and a transdermal absorption promoter in a mass ratio of 100:1 - 2, the transdermal absorption promoter includes eucalyptus oil and a transdermal compound, with a mass ratio of 3 - 5:2, and the structural formula of the transdermal compound is shown as Formula I: Formula I.

10. Use of the cherry blossom fermentation product according to claim 8 in cosmetics.

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