Small molecule rejuvenating fermentation composition and its application

Abstract

The present invention provides a small molecule rejuvenating fermentation composition and its application, belonging to the field of cosmetic technology. The fermentation composition of the present invention includes, by weight: 95-99 parts of composite plant fermentation concentrate and 1-5 parts of protective agent; the composite plant fermentation concentrate uses polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, and ginseng root extract as composite plant raw materials, and obtains secondary metabolites through composite lactobacillus fermentation; the composite lactobacillus is composed of at least 2 of plant lactobacillus, fermented mucus lactobacillus, casei lactobacillus, and acidophilus. The fermentation composition of the present invention is obtained by fermentation using a variety of plant extracts, and the multi-link anti-aging effect is significant. It comprehensively improves skin wrinkles by scavenging free radicals, promoting type I collagen, elastin regeneration, anti-glycation and other ways, and has good anti-wrinkle and firming effects, good anti-inflammatory and soothing effects, better safety, and broad application prospects in the fields of beauty or daily chemicals.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a small molecule skin-rejuvenating fermentation composition and application thereof. Background Art

[0002] Skin aging is a complex physiological process, a necessary stage in the process of biological life activities, and a gradual, genetically influenced, irreversible decline of the complex morphological structure and physiological function of the whole body as the organism matures and grows older. Skin aging refers to the process of skin external morphology, structure, and function decline under the influence of exogenous or endogenous factors. From the perspective of internal factors, the natural aging of cells gradually slows down the metabolism rate and prolongs the cell renewal cycle. Among the external factors, ultraviolet rays are one of the key factors leading to skin aging. Long-term exposure to ultraviolet rays will destroy the collagen and elastic fibers in the skin, thereby accelerating the formation of wrinkles. Environmental pollution, smoking, and bad eating habits can also cause damage to skin health. The endogenous and exogenous aging of the skin are interrelated and affect each other. The clinical manifestations of skin aging are dry, rough, thin, reduced elasticity, loss of luster, sagging, atrophy, wrinkles, pigmentation, capillary dilation, etc., as well as the disorder of skin physiological structure and functional degradation. Therefore, how to alleviate the aging process of the skin has become a hot topic in the field of anti-aging research.

[0003] Cosmetic compositions are playing an increasingly important role in combating skin aging. Patent CN114306112B provides an anti-aging composition and a preparation method thereof, wherein the anti-aging composition comprises the following components: dipotassium glycyrrhizinate sodium hyaluronate, sucrose stearate, vitamin E, fucoidan, polygonatum extract, saffron extract, glycerin, 1,3-butylene glycol and deionized water, which can achieve good anti-aging, moisturizing and anti-inflammatory effects and improve anti-aging effects. Patent CN107802540B discloses an anti-aging composition, which comprises a polypeptide component and an anti-aging plant extract, which can play a synergistic role and achieve better anti-aging effects.

[0004] However, the existing anti-aging compositions cannot delay aging from multiple pathways comprehensively, and need to be combined with substances from other pathways to achieve better anti-wrinkle and firming effects. Therefore, it is necessary to develop a comprehensive anti-wrinkle and firming composition with a more comprehensive mechanism and more significant effect, which can achieve a faster, more continuous and effective improvement effect on wrinkles and skin elasticity, and solve the anti-aging problem. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a small molecule rejuvenating fermentation composition and its application. The fermentation composition provided by the present invention includes, by weight: 95-99 parts of composite plant fermentation concentrate and 1-5 parts of protective agent; the composite plant fermentation concentrate uses polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, and ginseng root extract as composite plant raw materials, and is fermented by composite lactobacillus to obtain secondary metabolites; the composite lactobacillus is composed of plant lactobacillus, fermented mucus lactobacillus, casei lactobacillus and acidophilus. The fermentation composition of the present invention is obtained by fermentation using a variety of plant extracts, and has a significant multi-link anti-aging effect. It improves skin wrinkles comprehensively by scavenging free radicals, promoting type I collagen, elastin regeneration, anti-glycation and other pathways, has good anti-wrinkle and firming effects, has good anti-inflammatory and soothing effects, is safe, and has broad application prospects in the fields of beauty or daily chemicals.

[0006] the term:

[0007] In the present invention, the term "lactobacillus fermentation lysate" refers to the product of inactivation and filtration after lactobacillus fermentation. The lactobacillus fermentation lysate is rich in lactic acid, peptidoglycan, teichoic acid, protein, phospholipids, sterols, fatty acids, peptides and amino acids, nucleotides, extracellular polysaccharides, and multiple vitamins, and can be used for skin conditioning, anti-aging, antibacterial, anti-inflammatory, moisturizing, etc.

[0008] In the present invention, the term "half clearance rate (EC50)" means that the lower the sample concentration that achieves 50% clearance of DPPH free radicals, the stronger the free radical scavenging ability of the substance.

[0009] In the present invention, the term "ascorbic acid equivalent (AEAC)" refers to how much mg ascorbic acid (VC) the antioxidant capacity of 100 g of a sample is equivalent to.

[0010] In the present invention, the term "zebrafish embryo" refers to a zebrafish embryo at a developmental stage that relies on yolk for energy.

[0011] In the present invention, the term "10% lethal concentration (LC10)" means: the concentration of the test substance that causes 10% of the tested zebrafish to die. Zebrafish whose hearts do not beat within one minute are considered dead.

[0012] In the present invention, the term "no heartbeat" means "the zebrafish heart does not beat within one minute.

[0013] The technical solution of the present invention includes:

[0014] In a first aspect, the present invention provides a fermentation composition, which comprises, by weight: 95-99 parts of composite plant fermentation concentrate and 1-5 parts of a protective agent.

[0015] Specifically, the composite plant fermentation concentrate uses polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, and ginseng root extract as composite plant raw materials, and is fermented with composite lactobacillus to obtain secondary metabolites.

[0016] More specifically, the composite lactobacillus is composed of at least two of Lactobacillus plantarum, Lactobacillus mucus fermentation, Lactobacillus casei, and Lactobacillus acidophilus.

[0017] The plant lactobacillus is plant lactobacillus 14917;

[0018] The fermented mucus lactobacillus is fermented mucus lactobacillus CICC 25124

[0019] The Lactobacillus casei is human Lactobacillus casei NCU011061;

[0020] The Lactobacillus acidophilus is Lactobacillus acidophilus NCFM.

[0021] Preferably, the composite lactobacillus is composed of Lactobacillus plantarum, Lactobacillus mucus fermentation, Lactobacillus casei and Lactobacillus acidophilus.

[0022] Specifically, the composite plant fermentation concentrate is prepared from the following raw materials in weight percentage: 4%-11% polygonatum root extract, 2%-9% tuckahoe sclerotium extract, 1%-6% avocado fruit extract, 1%-6% ginseng root extract, 2%-8% composite lactobacillus and the remainder water.

[0023] Preferably, the composite plant fermentation concentrate is prepared from the following raw materials in weight percentage: 5%-10% polygonatum root extract, 3%-8% tuckahoe sclerotium extract, 1%-5% avocado fruit extract, 1%-5% ginseng root extract, 3%-7% composite lactobacillus and the remainder water.

[0024] Preferably, the water comprises distilled water and / or deionized water.

[0025] More preferably, the water is deionized water.

[0026] Preferably, the protective agent includes, but is not limited to, one or more of 1,2-hexanediol, 1,2-pentanediol, caprylyl glycol, and ethylhexylglycerol.

[0027] More preferably, the protective agent is 1,2-hexanediol.

[0028] In a second aspect, the present invention provides a method for preparing the above-mentioned fermentation composition, the preparation method comprising the following steps:

[0029] After mixing polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, ginseng root extract and water, adding the cultured composite lactobacillus, filtering after fermentation, adding a protective agent, and stirring evenly, a fermentation composition is obtained.

[0030] Specifically, the fermentation condition is 35-39°C fermentation.

[0031] Preferably, the fermentation condition is 37°C.

[0032] Specifically, the fermentation time is 5-10 days.

[0033] Preferably, the fermentation time is 7 days.

[0034] Specifically, the filtration is carried out using a ceramic membrane.

[0035] Preferably, the filtration is carried out using a 0.1 μm plate and frame filter.

[0036] In a third aspect, the present invention provides the use of the above-mentioned fermentation composition in the preparation of cosmetics with anti-glycation, antioxidant, anti-wrinkle, firming and / or soothing effects.

[0037] In a fourth aspect, the present invention provides a cosmetic comprising the above-mentioned fermentation composition.

[0038] Specifically, the cosmetic has anti-glycation, anti-oxidation, anti-wrinkle, firming and / or soothing effects.

[0039] Preferably, the cosmetics can promote the regeneration of type I collagen and elastin.

[0040] Specifically, the cosmetics include but are not limited to: facial cleanser, facial cleanser, toner, lotion, facial cream, essence, facial mask, foundation, concealer, sunscreen, sunscreen spray, shampoo, conditioner, shower gel, body lotion, microemulsion, nanoemulsion, lotion, ointment, foam, patch, makeup powder, makeup cotton, eye essence, eye mask, eye shadow, eye gel or eye cream.

[0041] Specifically, the cosmetic also includes cosmetically acceptable additives.

[0042] Preferably, the additives include, but are not limited to: one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

[0043] The beneficial effects of the present invention are:

[0044] The fermentation composition of the present invention is prepared by fermenting a variety of plant extracts, has a significant multi-link anti-aging effect, comprehensively improves skin wrinkles by scavenging free radicals, promoting type I collagen, elastin regeneration, anti-glycation and other pathways, has good anti-wrinkle and firming effects, has good anti-inflammatory and soothing effects, is relatively safe, and has broad application prospects in the fields of beauty and cosmetics or daily chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a bar graph of anti-glycation experiment; in the figure, A is the anti-glycation effect of the fermentation composition with a concentration of 2.5%; B is the anti-glycation effect of the fermentation composition with a concentration of 5%.

[0046] Figure 2 This is the scavenging effect diagram of DPPH free radicals.

[0047] Figure 3 This is the scavenging effect diagram of ABTS free radicals.

[0048] Figure 4 This is a bar graph of the relative expression of type I collagen gene in zebrafish; * in the figure represents a significant difference (p < 0.05).

[0049] Figure 5 This is a bar graph of the relative expression of zebrafish elastin gene; * in the figure represents a significant difference (p < 0.05).

[0050] Figure 6 This is a representative diagram of the soothing efficacy test results; A in the figure represents the model control group; B represents the positive control group; and C represents the sample treatment group.

[0051] Figure 7 This is a bar graph of the soothing efficacy test results; * in the graph represents a significant difference (p < 0.05). DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0053] The materials used in the present invention are shown in Table 1:

[0054] Table 1 Materials used in the present invention

[0055]

[0056] Example 1 Preparation of fermentation composition

[0057] 1. Preparation of compound lactobacillus culture solution

[0058] The composite lactobacillus culture solution contains, by weight percentage, 8% of polygonatum sibiricum extract, 6% of tuckahoe sclerotium extract, 3% of avocado fruit extract, 3% of ginseng root extract and the balance of deionized water.

[0059] The above-mentioned composite lactobacillus culture solution was added into a triangular flask, mixed evenly, a cotton plug was added to the bottle mouth, and the bottle mouth was wrapped with newspaper, and sterilized at 115° C. for 20 min in a portable high-pressure sterilizer, and then taken out for use.

[0060] 2. Expansion of bacterial strains

[0061] Under sterile conditions, use an inoculation loop to pick a loop of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, and Lactobacillus acidophilus, transfer them into different sterilized test tubes, draw a curve, and culture them in horizontal test tubes at 32°C for 7 days. After the colonies grow, transfer them again under the same operating conditions, and the culture conditions are the same as above. The strains are used in the experiment after 3 generations of activation process.

[0062] 3. Preparation of composite plant fermentation concentrate

[0063] The selected culture temperature is 37±2°C, sterile water is added to the activated inclined test tube to wash out the spores, the spore suspension is aspirated with a sterile pipette, and a 5% inoculum amount (the weight ratio of Lactobacillus plantarum 14917, Lactobacillus fermentative CICC 25124, Lactobacillus casei NCU011061 and Lactobacillus acidophilus NCFM is 1:1:1:1) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0064] 4. Preparation of fermentation composition

[0065] 97 parts of the composite plant fermentation concentrate were taken, 3 parts of 1,2-hexanediol were added, and the mixture was stirred evenly to obtain a fermentation composition.

[0066] Example 2 Preparation of fermentation composition

[0067] 1. Preparation of compound lactobacillus culture solution

[0068] The composite lactobacillus culture solution contains, by weight percentage, 4% of polygonatum sibiricum extract, 9% of tuckahoe sclerotium extract, 1% of avocado fruit extract, 1% of ginseng root extract and the balance of deionized water.

[0069] The above-mentioned composite lactobacillus culture solution was added into a triangular flask, mixed evenly, a cotton plug was added to the bottle mouth, and the bottle mouth was wrapped with newspaper, and sterilized at 115° C. for 20 min in a portable high-pressure sterilizer, and then taken out for use.

[0070] 2. Expansion of bacterial strains

[0071] Under sterile conditions, use an inoculation loop to pick a loop of Lactobacillus plantarum and Lactobacillus fermentans respectively, transfer them into different sterilized test tubes, draw a curve, and culture them in horizontal test tubes at 32°C for 7 days. After the colonies grow, transfer them again under the same operating conditions, and the culture conditions are the same as above. The strains are used in the experiment after 3 generations of activation process.

[0072] 3. Preparation of composite plant fermentation concentrate

[0073] The selected culture temperature is 37±2°C, sterile water is added to the activated slant test tube to wash out the spores, the spore suspension is sucked with a sterile pipette, and an inoculum amount of 8% (the weight ratio of Lactobacillus plantarum 14917 and Lactobacillus fermentative mucus CICC 25124 is 1:1) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120 r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1 μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0074] 4. Preparation of fermentation composition

[0075] 95 parts of the composite plant fermentation concentrate were taken, 5 parts of 1,2-hexanediol were added, and the mixture was stirred evenly to obtain a fermentation composition.

[0076] Example 3 Preparation of fermentation composition

[0077] 1. Preparation of compound lactobacillus culture solution

[0078] The composite lactobacillus culture solution contains, by weight percentage, 11% of polygonatum sibiricum extract, 2% of tuckahoe sclerotium extract, 6% of avocado fruit extract, 6% of ginseng root extract and the balance of deionized water.

[0079] The above-mentioned composite lactobacillus culture solution was added into a triangular flask, mixed evenly, a cotton plug was added to the bottle mouth, and the bottle mouth was wrapped with newspaper, and sterilized at 115° C. for 20 min in a portable high-pressure sterilizer, and then taken out for use.

[0080] 2. Expansion of bacterial strains

[0081] Under sterile conditions, use an inoculation loop to pick a loop of Lactobacillus casei and Lactobacillus acidophilus respectively, transfer them into different sterilized test tubes, draw a curve, and culture them in horizontal test tubes at 32℃ for 7 days. After the colonies grow, transfer them again under the same operating conditions. The culture conditions are the same as above. The strains are used in the experiment after 3 generations of activation process.

[0082] 3. Preparation of composite plant fermentation concentrate

[0083] The selected culture temperature is 37±2°C, sterile water is added to the activated slant test tube, spores are washed off, the spore suspension is aspirated with a sterile pipette, and a 2% inoculation amount (the weight ratio of human Lactobacillus casei NCU011061 and Lactobacillus acidophilus NCFM is 1:1) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120 r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1 μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0084] 4. Preparation of fermentation composition

[0085] Take 99 parts of the composite plant fermentation concentrate, add 1 part of 1,2-hexanediol, stir evenly, and obtain a fermentation composition.

[0086] Comparative Example 1 Preparation of Fermentation Composition

[0087] Comparative Example 1 was prepared with reference to Example 1, and the only difference between Comparative Example 1 and Example 1 was that the composition of the composite lactobacillus culture solution was different.

[0088] The composition of the composite lactobacillus culture solution of Comparative Example 1 is as follows: 8% of polygonatum root extract, 6% of tuckahoe sclerotium extract, 3% of avocado fruit extract and the balance of deionized water.

[0089] Comparative Example 2 Preparation of Fermentation Composition

[0090] Comparative Example 2 was prepared with reference to Example 1. The only difference between Comparative Example 2 and Example 1 was that the composition of the composite lactobacillus culture solution was different.

[0091] The composition of the composite lactobacillus culture solution of Comparative Example 2 is: 8% polygonatum sibiricum extract, 6% tuckahoe sclerotium extract, 3% ginseng root extract and the balance deionized water.

[0092] Comparative Example 3 Preparation of Fermentation Composition

[0093] Comparative Example 3 was prepared with reference to Example 1. The only difference between Comparative Example 3 and Example 1 was that the composition of the composite lactobacillus culture solution was different.

[0094] The composition of the composite lactobacillus culture solution of Comparative Example 3: 8% polygonatum root extract, 3% ginseng root extract and the balance deionized water.

[0095] Comparative Example 4 Preparation of Fermentation Composition

[0096] Comparative Example 4 was set up with reference to Example 1. The only difference between Comparative Example 5 and Example 1 is that the composition of the composite lactobacillus culture solution is different.

[0097] The composition of the composite lactobacillus culture solution of Comparative Example 4: 8% of polygonatum root extract and the balance of deionized water.

[0098] Comparative Example 5 Preparation of Fermentation Composition

[0099] Comparative Example 5 is set with reference to Example 1. The difference between Comparative Example 5 and Example 1 is only that: "Step 3, preparation of composite plant fermentation concentrate" is different. The specific process is as follows:

[0100] The selected culture temperature is 37±2°C, sterile water is added to the activated inclined test tube to wash out the spores, the spore suspension is aspirated with a sterile pipette, and a 10% inoculum amount (the weight ratio of Lactobacillus plantarum 14917, Lactobacillus fermentative CICC 25124, Lactobacillus casei NCU011061 and Lactobacillus acidophilus NCFM is 1:1:1:1) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0101] Comparative Example 6 Preparation of Fermentation Composition

[0102] Comparative Example 6 is set with reference to Example 2. The only difference between Comparative Example 6 and Example 2 is that "Step 3, preparation of composite plant fermentation concentrate" is different. The specific process is as follows:

[0103] The selected culture temperature is 37±2°C, sterile water is added to the activated slant test tube, spores are washed off, the spore suspension is sucked with a sterile pipette, and an 8% inoculum (Lactobacillus plantarum 14917) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120 r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1 μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0104] Comparative Example 7 Preparation of Fermentation Composition

[0105] Comparative Example 7 is set with reference to Example 2. The only difference between Comparative Example 7 and Example 2 is that "Step 3, preparation of composite plant fermentation concentrate" is different. The specific process is as follows:

[0106] The selected culture temperature is 37±2°C, sterile water is added to the activated slant test tube to wash out the spores, the spore suspension is sucked with a sterile pipette, and an inoculum amount of 8% (the weight ratio of Lactobacillus plantarum NDC75017 and Lactobacillus fermentative mucus CICC25124 is 1:1) is transferred into the composite lactobacillus culture solution prepared in step 1, and the culture is shaken at a speed of 120r / min and a culture temperature of 37±2°C for 7 days. Filter with a 0.1μm plate and frame filter to obtain a composite plant fermentation concentrate.

[0107] Experimental Example 1 Anti-glycation experiment

[0108] Since the key to the formation of AGEs by glycation reaction is the condensation of the carbonyl group of reducing sugar and the free amino group of protein, we can use glucose and bovine serum albumin to react with each other in vitro to generate AGES and then add anti-glycation products to evaluate its anti-glycation effect. Most AGES have fluorescent properties, so the fluorescence intensity can be measured by an enzyme marker to reflect the content of AGEs. By measuring the changes in AGES content after sample administration, it is tested whether the sample has the effect of inhibiting AGES synthesis. In this way, it is determined whether the sample has anti-glycation effect. The specific experimental process is as follows:

[0109] 1) Modeling: The fermentation composition prepared in Examples 1-3 and Comparative Examples 1-7 (at a concentration of 2.5% and 5%), bovine serum albumin (PBS, 8.00 mg / mL) and glucose (PBS, 0.2 mol / L) mixed solution were added to a 96-well plate (test system: 320 μL / well); the model control group was not added with the fermentation composition, but with an equal volume of deionized water. Incubate at 60°C for about 40 hours.

[0110] 2) Detect fluorescence intensity: After incubation, use a microplate reader to measure the fluorescence intensity at 370nm excitation wavelength and 440nm emission wavelength. Calculate the anti-glycation effect of the sample based on the fluorescence intensity. The calculation formula is as follows:

[0111]

[0112] Where S represents the fluorescence intensity.

[0113] The results of the test are as follows Figure 1 shown.

[0114] Experimental Example 2 Antioxidant Experiment

[0115] 1. DPPH removal experiment

[0116] Antioxidant activity can be characterized by evaluating the inhibition rate of the test sample on DPPH free radicals. DPPH analysis is a simple method for screening free radical scavengers and evaluating antioxidant activity. The ethanol solution of DPPH appears purple, with a maximum absorption wavelength of 517nm. When a free radical scavenger is added to the DPPH solution, its lone pair of electrons is paired, and the absorption disappears or weakens, causing the solution to become lighter in color, appearing yellow or light yellow, and the absorbance at 517nm becomes smaller. The degree of change is linearly related to the free radical scavenging intensity, so it can be quantitatively analyzed by spectrophotometry. This method can be expressed by the scavenging rate. The larger the scavenging rate, the stronger the scavenging ability of the substance.

[0117] DPPH determination process: Prepare a series of test sample solutions with multiple concentrations, and the test samples are divided into the fermentation composition prepared in Example 1. Add the test samples of various concentrations and VC of various concentrations (as a positive control) to a 96-well plate. Add 300 μL of DPPH working solution to each well and mix well. Measure the absorbance at 517 nm at 30 min, and calculate the DPPH free radical scavenging rate according to the following formula.

[0118]

[0119] Where: A is the absorbance of the reaction between the sample and DPPH; A1 is the absorbance of the control when anhydrous ethanol replaces DPPH; A0 is the blank absorbance when 70% ethanol replaces the sample.

[0120] The results of DPPH scavenging experiment are as follows Figure 2 shown.

[0121] The half catalytic elimination efficiency (EC50) and ascorbic acid equivalent (AEAC) were calculated based on the DPPH free radical scavenging results.

[0122]

[0123] The AEAC determination results of the DPPH removal experiment are shown in Table 2:

[0124] Table 2 EC50 and AEAC results of DPPH scavenging experiment

[0125]

[0126] When the addition amount of the fermentation composition prepared in Example 1 is 0.032%, the effect of scavenging half of the DPPH free radicals can be achieved. The antioxidant capacity of each 100g of the fermentation composition is equivalent to 190.51g of Vc, indicating that the fermentation composition of the present invention has significant antioxidant capacity.

[0127] 2. ABTS removal experiment

[0128] ABTS reacts with potassium persulfate to generate green ABTS·+ free radicals. The free radical has a maximum absorption at 752nm. Therefore, by detecting the absorbance at 752nm, its concentration can be determined. After a substance is added to the ABTS·+ free radical working solution, if the absorbance at 752nm decreases, it means that the substance has free radical scavenging activity and is an antioxidant.

[0129] ABTS determination process: Prepare a series of test sample solutions with multiple concentrations, and the test samples are divided into the fermentation composition prepared in Example 1. 300 μL of anhydrous ethanol (as a blank control), test samples of various concentrations, and VC of various concentrations (as a positive control) are mixed with 300 μL of ABTS working solution and ABTS working solution, respectively, and react for 30 minutes. Move to a 96-well plate, measure the absorbance at 752 nm, and calculate the ABTS free radical scavenging rate according to the following formula.

[0130]

[0131] In the formula: A control is the absorbance of the ABTS working solution replaced by anhydrous ethanol;

[0132] The results of ABTS clearance assay are shown in Figure 3 shown.

[0133] The half catalytic elimination efficiency (EC50) and ascorbic acid equivalent (AEAC) were calculated based on the DPPH free radical scavenging results.

[0134]

[0135] The EC50 and AEAC results of the ABTS clearance experiment are shown in Table 3:

[0136] Table 3 EC50 and AEAC results of ABTS clearance experiment

[0137]

[0138] When the addition amount of the fermentation composition prepared in Example 1 is 0.012%, the effect of scavenging half of the ABTS free radicals can be achieved. The antioxidant capacity of each 100g of the fermentation composition is equivalent to 232.67g Vc, indicating that the fermentation composition of the present invention has significant antioxidant capacity.

[0139] Experimental Example 3 col1a1a, col1a1b and col1a2 Expression level determination

[0140] Collagen is the most abundant extracellular matrix protein in the human body, and type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans and shows a high degree of conservation with humans. Their innate expression decreases significantly at 6 days after fertilization and becomes very low between 10 and 12 days after fertilization. col1a1a , col1a1b and col1a2 ) expression, compare the relative expression of type I collagen gene in zebrafish between the treatment group and the blank control group, and calculate the promotion rate of type I collagen gene expression to evaluate the anti-wrinkle and firming effects of raw materials or products. The specific experimental methods are as follows:

[0141] 1. Experimental Preparation

[0142] 1) Test organisms:

[0143] Wild-type AB strain zebrafish ( Danio Rerio ) Spawning test. The broodstock should have good reproductive capacity - the best age is 6-12 months. Cross-crossing should be used as much as possible to maintain genetic diversity. A new batch of broodstock should be replaced after 5 generations of pure strain broodstock. The broodstock should not have obvious visible signs of infection and disease, and should not have been treated with drugs within 2 months. Before the breeding and spawning test, the broodstock should be domesticated for more than 14 days after being introduced into the laboratory.

[0144] Breeding requirements: The water temperature should be controlled at 26-28.5℃, and the indoor temperature should be controlled at 20-25℃. The breeding density should be controlled at 1-2 fish per liter of water, and the light should be fixed at 12-16 hours per day, and a good filtration system should be maintained. Feed at least 2 times a day, including at least 1 time of brine shrimp (Artemia salina) larvae, with a feeding interval of more than 3 hours to avoid overfeeding that affects water quality and cleanliness.

[0145] Spawning requirements: If you use a spawning tank to collect fish eggs, put male and female fish in a ratio of 2:1 into the spawning tank 1-2 hours before turning off the lights on the day before spawning. To avoid genetic deviation, mix the fish eggs collected from at least 3 spawning tanks and select them for use. If you use a breeding fish tank to collect fish eggs, put the egg collection box into the breeding fish tank where the fish eggs are to be collected before turning off the lights on the day before spawning or before turning on the lights on the day of spawning. To prevent the eggs from being eaten by adult fish, cover the egg collection box with an inert net. Mating, spawning and fertilization are completed within about 30 minutes after turning on the lights, and the egg collection box can be removed from the fish tank at that time. After the fish eggs are removed from the egg collection box, clean the fish embryos with fish embryo culture medium. Select healthy zebrafish embryos and culture them at a density of no more than 1 fish embryo in 200μL fish embryo culture medium at 28℃±1℃ until 6 days after fertilization.

[0146] 2) Preparation of fish embryo culture medium: Weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolve them in 10 L of water. The pH value is 6.5-8.5. All chemicals are of analytical grade.

[0147] 3) Preparation of acetyl hexapeptide-8 solution: Use fish embryo culture medium to prepare 0.8g / L acetyl hexapeptide-8 solution, and prepare it fresh before use.

[0148] 4) Preparation of test solution: The fermented composition prepared in Example 1 was prepared into a test solution with a concentration of 2% using fish embryo culture medium.

[0149] 2. Experimental Grouping

[0150] The test set up a blank control group, a positive control group and a sample treatment group, and the settings of each group are as follows:

[0151] 1) Blank control group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate, with 3 wells, each containing 12 zebrafish and 2.5 mL of fish embryo culture medium.

[0152] 2) Positive control group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate, with 3 wells, each well containing 12 zebrafish and 2.5 mL of acetyl hexapeptide-8 working solution.

[0153] 3) Sample treatment group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate with 3 wells, each containing 12 zebrafish and 2.5 mL of the test solution.

[0154] The above groups were placed in a 28°C±1°C incubator for 24h±1h. Twelve zebrafish from each well were collected into a 1.5mL tube, the solution was removed, 0.5mL RNAlater solution was added, and the tube was frozen for storage.

[0155] 3. RNA Extraction

[0156] Remove RNAlater solution and rinse with PBS solution three times. Place in ice bath, remove PBS solution, and add 500μL TRIzol reagent. Homogenize embryos with a pellet pestle and place in ice bath for 10 minutes. Add 100μL chloroform, vortex for 1 minute, and place in ice bath for 5 minutes. Centrifuge samples for 20 minutes, transfer the upper layer solution to a 1.5mL centrifuge tube, add 250μL isopropanol, vortex for 1 minute, place in ice bath for 10 minutes, and centrifuge samples for 20 minutes. Remove all supernatant, add 500μL ethanol, vortex for 1 minute, centrifuge samples for 5 minutes, and place in ice bath for 10 minutes. Repeat this step 3 times. Remove ethanol, centrifuge samples for 5 minutes, and air-dry samples in a fume hood with the lid open for 10 minutes. Add 10μL DNase / RNase-free ultrapure distilled water and heat at 55℃ for 15 minutes.

[0157] 4. cDNA Synthesis

[0158] The RNA sample was diluted to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. The RNA was synthesized into cDNA using the PrimeScript RT Kit with gDNA Eraser (Takara; Cat no. RR047A) and stored frozen.

[0159] 5. Perform real-time RT-PCR

[0160] Prepare primer mix and real-time PCR master mix for each primer. Dilute cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9µL PCR master mix and 1µL diluted cDNA sample to each well of 96-well PCR plate. Seal the 96-well PCR plate with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification. Real-time PCR kit: SYBR Premix ExTaq (Takara; Cat no. RR420A).

[0161] 6. Perform real-time RT-PCR

[0162] Collect real-time PCR data. Ct is used as the amplification result. β-actin The gene amplification amount was used as the housekeeping gene, and the relative expression of each gene was calculated as the test result.

[0163] The results of the test are as follows Figure 4As shown, the results show that the fermentation composition of Example 1 at a 2% formula addition concentration has a promotion rate of 89% (p=0.0088), 108% (p=0.013) and 106% (p-0.0066) on the expression of zebrafish collala, collalb and col1a2 genes, respectively. This shows that the fermentation composition provided by the present invention can significantly promote the expression of zebrafish type I collagen gene, can promote the regeneration of type I collagen, and has anti-wrinkle and firming effects.

[0164] Experimental Example 4 Elna Expression level determination

[0165] Elastin in human skin is a protein that allows many tissues in the body to maintain their original shape after stretching and contracting, and allows the skin to return to its original shape after being squeezed. As we age, the elastin fibers in the skin will gradually break and lose, causing the skin to wrinkle and lose elasticity. Compared with human skin, zebrafish skin is highly similar to human skin in structure, except that the epidermis is differentiated epidermal cells and mucus layer instead of stratum corneum. The elastin gene is highly conserved, and the elastin in the human body is produced by Eln Gene synthesis, the corresponding gene of this gene in zebrafish is Elna, and the test sample is used to test whether it promotes Elna The expression of genes can be used to determine whether the sample can promote the regeneration of elastin, help the skin resist wrinkles, and achieve skin firming. The specific experimental methods are as follows:

[0166] 1. Experimental Preparation

[0167] The experimental preparation is the same as that of Experimental Example 3.

[0168] 2. Experimental Grouping

[0169] The test set up a blank control group, a positive control group and a sample treatment group, and the settings of each group are as follows:

[0170] 1) Blank control group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate, with 3 wells, each containing 12 zebrafish and 2.5 mL of fish embryo culture medium.

[0171] 2) Positive control group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate, with 3 wells, each well containing 12 zebrafish and 2.5 mL of acetyl hexapeptide-8 working solution.

[0172] 3) Sample treatment group setting: 36 zebrafish were randomly selected and evenly distributed into a 24-well plate with 3 wells, each containing 12 zebrafish and 2.5 mL of the test solution.

[0173] The above groups were placed in a 28°C±1°C incubator for 24h±1h. Twelve zebrafish from each well were collected into a 1.5mL tube, the solution was removed, 0.5mL RNAlater solution was added, and the tube was frozen for storage.

[0174] 3. RNA extraction and cDNA synthesis

[0175] RNA extraction and cDNA synthesis were the same as in Experimental Example 3

[0176] 4. Perform real-time RT-PCR

[0177] Prepare primer mix and real-time PCR master mix for each primer. Dilute cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9µL PCR master mix and 1µL diluted cDNA sample to each well of 96-well PCR plate. Seal the 96-well PCR plate with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification. Real-time PCR kit: SYBR Premix ExTaq (Takara; Cat no. RR420A).

[0178] 5. Perform real-time RT-PCR

[0179] Collect real-time PCR data. Ct is used as the amplification result. β-actin The gene amplification amount was used as the housekeeping gene, and the relative expression of each gene was calculated as the test result.

[0180] The results of the test are as follows Figure 5 As shown in Figure 2, the sample at a 2% formula addition concentration promoted the expression of zebrafish Elna gene by 171% (p=0.00096). This shows that the fermentation composition provided by the present invention can promote Elna Gene expression can promote the regeneration of elastin, and has anti-wrinkle and firming effects.

[0181] Experimental Example 5: Soothing Effect Verification

[0182] Soothing means helping to improve conditions such as skin irritation. When the skin is stimulated by physical, chemical or pathogenic invasion, it will produce an immune response, causing discomfort such as redness and swelling, and showing signs of sensitive skin. At this time, immune white blood cells (including neutrophils and macrophages) gather in the skin to engulf and remove harmful substances such as infections, foreign bodies or damaged cells in the skin. Inhibiting the aggregation of white blood cells at the stimulated site can soothe the skin irritation state. The neutrophils of zebrafish embryos (the main immune white blood cells) and human neutrophils are highly similar in morphology, biochemistry and physiological functions. Therefore, the test results obtained by establishing a stimulated zebrafish model are well derivable for human skin efficacy. Neutrophils of zebrafish embryos can be visualized in vivo or observed by Sudan black staining through transgenic strains of fluorescently labeled neutrophils. This experiment used a copper sulfate-induced model in which the neuromast cells in the lateral line region of zebrafish embryos were stimulated to cause neutrophil aggregation. The changes in the number of neutrophils in the lateral line region of fish embryos in the test group and the model control group were compared, and the neutrophil aggregation inhibition rate was calculated to evaluate whether the raw materials, formulas or products have soothing stimulation to achieve a soothing effect.

[0183] 1. Experimental Preparation

[0184] 1) Test organisms: The test organisms are the same as those in Example 3.

[0185] 2) Preparation of fish embryo culture medium: Weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolve them in 10 L of water. The pH value is 6.5-8.5. All chemicals are of analytical grade.

[0186] 3) Preparation of copper sulfate solution: Prepare 1mM (160mg / L) anhydrous copper sulfate stock solution with water and store at room temperature. The working solution concentration is 10µM (1.6mg / L), diluted with fish embryo culture medium, and prepared fresh before use.

[0187] 4) Preparation of indomethacin solution: Prepare 1mM (3.6g / L) indomethacin stock solution with dimethyl sulfoxide and keep refrigerated. The working solution concentration is 10µM (3.6mg / L) indomethacin, diluted with fish embryo culture medium and prepared fresh before use.

[0188] 5) Preparation of paraformaldehyde: Prepare a solution containing 4% paraformaldehyde and 0.8% sodium hydroxide, pH 7.2-7.4, in phosphate buffered saline (PBS) and refrigerate.

[0189] 6) Preparation of Sudan black staining solution: Sudan black storage solution: Prepare 0.4% Sudan black storage solution with 70% alcohol. Refrigerate. Phenol solution: Dissolve 8g phenol in 15mL ethanol solution, dissolve 0.3g disodium hydrogen phosphate dodecahydrate in 50mL water, then mix the two solutions to make a phenol solution, store at room temperature, valid for half a year. Sudan black staining solution: Mix 900µL 70% alcohol solution, 100µL Sudan black storage solution and 1µL phenol solution. Prepare fresh before use.

[0190] 7) Preparation of PBST: Prepare 0.04% Triton X-100 in PBS solution and store at room temperature.

[0191] 8) Preparation of bleaching solution: Prepare a solution containing 3% hydrogen peroxide and 1% potassium hydroxide with water. Prepare freshly before use.

[0192] 9) Preparation of clearing solution 1: Prepare a solution containing 20% ​​glycerol and 0.25% potassium hydroxide with water and store at room temperature.

[0193] 10) Preparation of test solution: The fermented composition prepared in Example 1 was prepared into a test solution with a concentration of 10% using fish embryo culture medium.

[0194] 2. Experimental Grouping

[0195] The test requires setting up a blank control group, a model control group, a positive control group and a sample treatment group. The settings of each group are as follows:

[0196] 1) Blank control group setting: Randomly select 24 fish embryos and place them in a 3 cm culture dish, and add 5 mL of fish embryo culture medium.

[0197] 2) Model control group setting: 24 fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 10 µM anhydrous copper sulfate was added.

[0198] 3) Positive control group setting: Randomly select 24 fish embryos and place them in a 3 cm culture dish. Add 5 mL of fish embryo culture medium containing 10 µM anhydrous copper sulfate and 10 µM indomethacin.

[0199] 4) Sample treatment group setting: Randomly select 24 fish embryos and place them in a 3 cm culture dish. Add 5 mL of fish embryo culture medium containing 10 µM anhydrous copper sulfate and the test substance.

[0200] The above groups were placed in a constant temperature box at 28°C ± 1°C and cultured for 40-45 minutes.

[0201] 3. Fish embryo fixation and staining

[0202] After fixing each test group of fish embryos in paraformaldehyde for at least 1 hour, treat the fish embryos with PBST 3 times, 5 minutes each time, and then treat with 50% ethanol for 3 minutes. After the fish embryos were stained with Sudan black staining solution at room temperature for 1 hour, they were immersed in 70% ethanol 4 times, 5 minutes each time, and then treated with PBST 2 times, 5 minutes each time. Treat the fish embryos with bleach solution for 10 minutes. If treated in a test tube, keep the lid open. Then treat with 70% ethanol solution for 5 minutes, PBST for 1 minute, clear solution 1 for 15 minutes, clear solution 2 for 10 minutes, and then PBST for 3 minutes. Lay the fish embryos on their side. Then put them under a stereo microscope to take pictures of the tail of the fish embryos. The dark spots are stained neutrophils.

[0203] 4. Data and result calculation

[0204] Count the number of neutrophils in the lateral line region of the tail three quarters of the way from the anus of each fish embryo. Calculate the neutrophil aggregation inhibition rate:

[0205]

[0206] Wherein: S is the average number of neutrophils in fish embryos of the sample treatment group, in units of granules per tail (granules / tail); M is the average number of neutrophils in fish embryos of the model control group, in units of granules per tail (granules / tail); B is the average number of neutrophils in fish embryos of the blank control group, in units of granules per tail (granules / tail).

[0207] The results of the test are as follows Figure 6-Figure 7 As shown, the dark dots in the figure are stained neutrophils. The results showed that the soothing effect of the model control group was not significant (0% neutrophil aggregation inhibition rate); 10µM indomethacin had a significant soothing effect, with a 30% neutrophil aggregation inhibition rate); the fermented composition of Example 1 of the present invention had a significant soothing effect, with a 42% (p=0.00000000062) neutrophil aggregation inhibition rate.

[0208] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A fermentation composition, characterized in that The fermentation composition comprises, by weight: 95-99 parts of composite plant fermentation concentrate and 1-5 parts of protective agent; The composite plant fermentation concentrate uses polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, and ginseng root extract as composite plant raw materials, and is fermented with composite lactobacillus to obtain secondary metabolites; The composite lactobacillus is composed of at least two of Lactobacillus plantarum, Lactobacillus fermented mucus, and Lactobacillus acidophilus; the Lactobacillus plantarum is Lactobacillus plantarum 14917, the Lactobacillus fermented mucus is Lactobacillus fermented mucus CICC25124, and the Lactobacillus acidophilus is Lactobacillus acidophilus NCFM; The composite plant fermentation concentrate is prepared from the following raw materials in percentage by weight: 4%-11% polygonatum root extract, 2%-9% tuckahoe sclerotium extract, 1%-6% avocado fruit extract, 1%-6% ginseng root extract, 2%-8% composite lactobacillus and the balance water.

2. The fermentation composition according to claim 1, characterized in that The composite plant fermentation concentrate is prepared from the following raw materials in percentage by weight: 5%-10% polygonatum root extract, 3%-8% tuckahoe sclerotium extract, 1%-5% avocado fruit extract, 1%-5% ginseng root extract, 3%-7% composite lactobacillus and the remainder water.

3. The fermentation composition according to claim 1, characterized in that The preparation method of the fermentation composition comprises: mixing polygonatum root extract, tuckahoe sclerotium extract, avocado fruit extract, ginseng root extract and water, adding the cultured composite lactobacillus, filtering after fermentation, adding a protective agent, and stirring evenly to obtain the fermentation composition.

4. The fermentation composition according to claim 1, characterized in that The protective agent includes one or more of 1,2-hexanediol, 1,2-pentanediol, caprylyl glycol, and ethylhexylglycerol.

5. Use of the fermented composition according to any one of claims 1 to 4 in the preparation of cosmetics having anti-glycation, antioxidant, anti-wrinkle, firming and / or soothing effects.

6. A cosmetic, characterized in that: The cosmetic comprises the fermented composition according to any one of claims 1 to 4.

7. The cosmetic according to claim 6, characterized in that: The cosmetic has anti-glycation, anti-oxidation, anti-wrinkle, firming and / or soothing effects.

8. The cosmetic according to claim 6, characterized in that: The cosmetics include: cleansing milk, toner, lotion, cream, essence, facial mask, foundation, concealer, sunscreen, sunscreen spray, shampoo, conditioner, shower gel, foam, patch, makeup powder, cotton pad, eye essence, eye mask, eye shadow, eye gel or eye cream.

9. The cosmetic according to claim 6, characterized in that: The cosmetic also includes cosmetically acceptable additives.

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