A kind of forsythia fruit fermentation product and its preparation method and application

Abstract

The present disclosure provides a method for preparing a fermented forsythia fruit, comprising: a fermentation substrate preparation step: after mixing forsythia fruit powder and water in proportion, sterilizing and cooling to obtain a fermentation substrate; a fermentation step: inoculating plant lactobacillus into the fermentation substrate, fermenting, then sterilizing and separating to obtain a supernatant to obtain a forsythia fruit fermented liquid, or further drying the supernatant to obtain a forsythia fruit fermented dry powder. The above-mentioned preparation method uses plant lactobacillus to ferment forsythia fruit, retains all the effective components and their activity of the plant, and avoids the loss of active components caused by other extraction methods; the forsythia fruit fermented pulp has a pH of 4.3-4.5, which is suitable for human skin. The forsythia fruit fermented product is rich in functional substances such as polyphenols, polysaccharides, and lignin, and has strong anti-aging and anti-inflammatory functions for the skin when used in cosmetics, and also has good human safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and in particular relates to a forsythia fruit fermentation product and a preparation method and application thereof. Background Art

[0002] Forsythia suspensa (Thunb.) Vahl is a deciduous shrub belonging to the genus Forsythia of the family Oleaceae. Forsythia suspensa has many chemical components, including phenylethanol glycosides, lignans, phenolic acids, flavonoids, terpenes and volatile oils, C6-C2 natural alcohols and their glycosides, among which phenylethanol glycosides and lignans are the main chemical components of Forsythia suspensa.

[0003] Forsythia fruit is rich in nutrients, and its main functions are as follows: 1) Antioxidant: Forsythia contains phenylethanol glycosides and flavonoids, which have the function of scavenging free radicals in the body; 2) Anti-inflammatory: It can reduce the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum of mice with carrageenan-induced inflammation model, indicating that it has a certain anti-inflammatory effect; 3) Antiviral: Forsythia water extract can inhibit the proliferation of viruses in the body; 4) Antipyretic and analgesic effects; 5) Hepatoprotective effects; 6) Immunomodulatory effects.

[0004] The application fields of Forsythia suspensa are mainly concentrated in the fields of medicine, health food and cosmetics. Forsythia suspensa products are mainly primary processed such as tea and essential oil, and deep processing is less, resulting in a waste of resources. The primary products of Forsythia suspensa mainly include Forsythia suspensa tea, Forsythia suspensa essential oil, preservatives, etc., and the fermented products mainly include fermented beverages, tea, etc. At present, the method of fermenting Forsythia suspensa has been applied to the field of health food, but there is relatively little research progress in the field of cosmetics, and no research has been found on the use of lactic acid bacteria to ferment Forsythia suspensa in the field of cosmetics. In the prior art, the fermentation cycle of Forsythia suspensa fermented products is long, and the fermentation steps are cumbersome, resulting in high energy consumption during the fermentation process and high product costs. Summary of the invention

[0005] A brief overview of the disclosure is given below in order to provide a basic understanding of certain aspects of the disclosure. It should be understood that this overview is not an exhaustive overview of the disclosure. It is not intended to identify key or important parts of the disclosure, nor is it intended to limit the scope of the disclosure. Its purpose is simply to give certain concepts in a simplified form as a prelude to a more detailed description discussed later.

[0006] In order to solve the above technical problems, the technical solution provided by the present disclosure is:

[0007] In a first aspect, the present disclosure provides a method for preparing a fermented product of Forsythia suspensa fruit, comprising:

[0008] The fermentation substrate preparation step is: forsythia fruit powder and water are mixed in proportion, sterilized, and cooled to obtain the fermentation substrate;

[0009] Fermentation step: inoculating plant lactobacillus into the fermentation substrate, fermenting, sterilizing, separating and taking the supernatant to obtain Forsythia suspensa fermentation liquid, or further drying the supernatant to obtain Forsythia suspensa fermentation dry powder.

[0010] Furthermore, in the above-mentioned method for preparing the fermented forsythia fruit, in the fermentation substrate preparation step, the dosage ratio of the forsythia fruit powder and water is 1g:10ml-1g:40ml (such as 1g:15ml, 1g:20ml, 1g:25ml, 1g:30ml, 1g:35ml, etc.).

[0011] Furthermore, the Lactobacillus plantarum is Lactobacillus plantarum subsp. plantarum.

[0012] Furthermore, in the above-mentioned method for preparing the fermented product of Forsythia suspensa, the method for preparing the bacterial solution used for inoculation includes: inoculating Lactobacillus plantarum in a suitable culture medium for activation, purification, and expansion culture, and the OD value of the bacterial solution is 0.5-1.0; the volume ratio of the bacterial solution to the fermentation substrate is 1:8-20 (for example, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, etc.).

[0013] Furthermore, in the method for preparing the fermented forsythia fruit, the forsythia fruit powder is obtained by drying and crushing the forsythia fruit and then passing it through a 50-mesh sieve.

[0014] Furthermore, in the above-mentioned preparation method of Forsythia fruit fermentation product, in the fermentation step, the fermentation treatment temperature is 37-45°C (such as 38°C, 40°C, 42°C, 44°C, etc.), and the time is 6-36h (such as 8h, 10h, 15h, 20h, 25h, 30h, 34h, etc.), preferably 6-16h; preferably, the fermentation treatment is carried out in a shaking table with a rotation speed of 150r / min-180r / min (such as 155r / min, 160r / min, 165r / min, 170r / min, 175r / min).

[0015] Furthermore, in the above-mentioned preparation method of Forsythia suspensa fermented product, the separation treatment adopts a centrifugation method; more preferably, the centrifugal speed is 4500r / min-5000r / min (such as 4600r / min, 4700r / min, 4800r / min, 4900r / min, etc.), and the centrifugation time is 20min-40min (such as 25min, 30min, 35min, etc.).

[0016] Furthermore, in the method for preparing the fermented product of Forsythia suspensa, the drying treatment of the supernatant can be performed by freeze drying or spray drying.

[0017] In a second aspect, the present disclosure provides a Forsythia fruit fermented product prepared by the above method, including Forsythia fruit fermented liquid, Forsythia fruit fermented dry powder, etc.

[0018] In a third aspect, the present disclosure provides an application of the forsythia fruit fermentation product in cosmetics.

[0019] The beneficial effects of the present disclosure include but are not limited to:

[0020] 1. The present invention uses lactic acid bacteria, especially Lactobacillus plantarum, to ferment Forsythia suspensa fruit, thereby retaining all the effective components and their activity of the plant and avoiding the loss of active components caused by other extraction methods;

[0021] 2. The fermentation method adopted in the present disclosure does not add any organic reagents during the extraction of the effective components of Forsythia suspensa fruit, the fermentation temperature and the fermentation pH are moderate, the structure of the plant active components is not destroyed, and the natural activity of the plant is maintained;

[0022] 3. The fermented puree of Forsythia suspensa fruit provided by the present disclosure has a pH value of 4.3-4.5, is suitable for human skin, has high safety to the human body, and can be directly used as a cosmetic;

[0023] 4. The fermented product of Forsythia suspensa provided by the present invention is rich in functional substances such as polyphenols, polysaccharides, lignin, etc., which can be used in cosmetics to have strong anti-aging and anti-inflammatory functions on the skin, and also have good human safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the anti-inflammatory efficacy test results of the forsythia fruit fermented liquid prepared in Example 1;

[0025] Figure 2 Schematic diagram of the test results of the antioxidant efficacy of the fermented liquid of Forsythia suspensa obtained in Example 1, wherein A is a DPPH free radical scavenging rate curve, B is an OH free radical scavenging rate curve, and C is a schematic diagram of the total antioxidant capacity test results;

[0026] Figure 3This is a schematic diagram of the results of reactive oxygen species (ROS) detection in the antioxidant efficacy test of the forsythia fruit fermented liquid prepared in Example 1;

[0027] Figure 4 This is a photo of a chicken embryo chorioallantoic membrane test on the Fructus Forsythiae fermented liquid prepared in Example 1;

[0028] Figure 5 The hemolysis curve of the hemolysis experiment of red blood cells on the fermented liquid of Forsythia suspensa obtained in Example 1;

[0029] Figure 6 The figure is a schematic diagram of the sensory evaluation results of the subjects obtained by the questionnaire survey on the use of the forsythia fruit fermented liquid prepared in Example 1 as a skin care product. DETAILED DESCRIPTION

[0030] The technical solution of the present disclosure will be described below in conjunction with exemplary embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] The forsythia fruit powder in the following examples is obtained by using forsythia fruit purchased from the market, drying and crushing, and then sieving (through a 50-mesh sieve) to remove larger particles.

[0034] The lactic acid bacteria in the following examples are Lactobacillus plantarum purchased from China Industrial Microbiological Culture Collection Center with the strain number CICC20261; the culture medium used is MRS culture medium; the culture temperature for activation, purification and expansion of lactic acid bacteria is 30-37°C.

[0035] Example 1 Lactobacillus plantarum forsythia fruit fermentation experiment

[0036] The specific preparation steps of the forsythia fruit fermentation product of this embodiment are as follows:

[0037] (1) Activation of bacterial strains: Pick colonies from the preserved slant and place them in liquid culture medium, then place them in a shaker to activate the bacterial strains and maintain higher vitality.

[0038] (2) Strain purification: The activated strain is diluted and plated in a gradient manner to obtain single colonies.

[0039] (3) Strain expansion: The strain to be used is inoculated into the corresponding liquid culture medium and cultured in a shaker at an appropriate temperature until its OD value is 0.5-1.0, when the strain is in the logarithmic phase, which is the appropriate inoculation concentration.

[0040] (4) Preparation of fermentation substrate: Forsythia suspensa fruit powder and water are mixed in a ratio of 1 g:20 ml to 1 g:40 ml to prepare Forsythia suspensa fruit powder culture medium, i.e., fermentation substrate;

[0041] (5) Inoculation and shaking: The bacterial solution obtained in step (3) was inoculated into the Forsythia suspensa fruit powder culture medium, with the volume ratio of the bacterial solution to the fermentation substrate being 1:5-1:20, and fermented in a shaking incubator at a rotation speed of 180 r / min, a fermentation temperature of 45° C., and a fermentation time of 6-16 h. For details, see Table 1.

[0042] (5) Sterilization: The fermented lactic acid bacteria culture solution of Forsythia suspensa is sterilized by high pressure to inactivate the bacteria.

[0043] (6) Centrifugation: The fermentation liquid was centrifuged at 4800 r / min for 30 min, and the supernatant was retained to obtain a series of Forsythia suspensa fermentation liquids. See Table 1 below for details. It can be seen from the table that the optimal preparation conditions are a material-liquid ratio (ratio of Forsythia suspensa powder to water in the fermentation substrate, g / mL) of 1:20, an inoculum amount (ratio of bacterial liquid volume to fermentation substrate volume, mL / mL) of 1:10, a fermentation time of 15 h, and a fermentation temperature of 45°C. The Forsythia suspensa fermentation liquid prepared under these conditions was subjected to subsequent performance testing.

[0044] Table 1 Multiple groups of fermentation process conditions and product performance in Example 1

[0045]

[0046] The physical and chemical properties of the forsythia fruit fermented liquid prepared under the above optimal conditions were analyzed. Its appearance is a viscous liquid with a color ranging from light pink to brownish red; pH value is 4.5, viscosity is 180cP, soluble solid content is 4.5%, total colony count is less than 50CFU / ml, and no pathogenic bacteria were detected. According to the cosmetic hygiene standard GB7916-87, the total number of bacteria in cosmetics is not higher than 1000CFU / ml, so the forsythia fruit fermented liquid meets the quality requirements of cosmetics.

[0047] The forsythia fruit fermentation liquid was prepared under the above optimal conditions for active ingredient analysis. As shown in Table 2, compared with the water extract (the water extract is the sample control of the fermentation liquid, the preparation steps are the same as the fermentation liquid, and lactic acid bacteria are not added, and the water extract below is also the same), the fermentation liquid contains higher contents of total phenols, total sugars, total proteins, lignin, etc.

[0048] Table 2 Determination of active substance content

[0049]

[0050] Example 2 Performance evaluation experiment of the forsythia fruit fermented liquid obtained in Example 1

[0051] 1. Analysis of anti-inflammatory effects

[0052] Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of inflammatory HaCat cytokines TNF-α, IL-8, IL-1β, KLK-7, and IL-6. For specific methods, refer to the instructions of the Solebol kit.

[0053] 1. Treatment of cell culture supernatant: Collect cell culture supernatant from 6-well plate, collect the supernatant with a sterile tube, centrifuge for about 20 minutes (2000-3000r / min), and carefully collect the supernatant for later use. If precipitation is formed during storage, centrifuge again.

[0054] 2. Dilution of standard: Set 10 standard wells on the enzyme-labeled plate, add 100 μL of standard to the first and second wells respectively, then add 50 μL of standard diluent to the first and second wells respectively, mix well; then take 100 μL from the first and second wells respectively and add to the third and fourth wells respectively, then add 50 μL of standard diluent to the third and fourth wells respectively, mix well; then take 50 μL from the third and fourth wells respectively and discard them, then take 50 μL from each well and Add to the fifth and sixth wells, then add 50μL of standard diluent to the fifth and sixth wells respectively and mix; after mixing, take 50μL from the fifth and sixth wells respectively and add to the seventh and eighth wells respectively, then add 50μL of standard diluent to the seventh and eighth wells respectively, after mixing, take 50μL from the seventh and eighth wells respectively and add to the ninth and tenth wells, then add 50μL of standard diluent to the ninth and tenth wells respectively, after mixing, take 50μL from the ninth and tenth wells respectively and discard.

[0055] 3. Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0056] 4. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0057] 5. Liquid preparation: Dilute the concentrated detergent with distilled water and set aside.

[0058] 6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0059] 7. Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0060] 8. Incubation: Same operation as 3.

[0061] 9. Washing: Same operation as 5.

[0062] 10. Color development: Add a certain amount of color developer as required by the instructions, gently shake to mix, and develop the color at 37°C in the dark for 15 minutes.

[0063] 11. Termination: Add 50 μL of stop solution to each well to terminate the reaction (the color changes at this time).

[0064] 12. Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0065] 13. Calculation: Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0066] Results Figure 1 , indicating that compared with water extract, Forsythia suspensa fruit fermented liquid has stronger anti-inflammatory ability.

[0067] 2. Analysis of Antioxidant Efficacy

[0068] 1.DPPH free radical scavenging experiment

[0069] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0070] Dilute the fermented liquid of Forsythia suspensa fruit with water to prepare samples of different volume concentrations. See Table 3 for sample addition requirements. Add solvent to all test tubes (T, T0, C, C0). Use water for water-soluble samples and 95% ethanol for oil-soluble samples. Make up to 3mL and mix well. Add 1mL of DPPH ethanol solution to the sample tube (T) and DPPH tube (C). Replace the sample background (T0) and solvent background (C0) with 95% ethanol. Shake gently and let stand at room temperature for 5 minutes. Transfer each reaction solution into a 1cm cuvette and measure the absorbance at 517nm.

[0071] Table 3 Sample addition requirements

[0072] T-sample tube <![CDATA[T0 - Sample background]]> C-DPPH tube <![CDATA[C0 - Solvent Background]]> Sample solution (mL) 1 1 - - Water or 95% ethanol solvent (mL) 2 2 3 3 DPPH ethanol solution (mL) 1 - 1 - 95% ethanol (mL) - 1 - 1 Parallel times 3 / Sample 1 / sample 3 / Experimental 1 / Experiment

[0073] The DPPH free radical scavenging rate was calculated by the following formula:

[0074]

[0075] In the formula: T is the absorbance of the sample tube, that is, the absorbance of the solution after the sample reacts with DPPH; T0 is the background absorbance of the sample; C is the average of the absorbance of the DPPH tube three times, that is, the absorbance of the DPPH solution when no sample is added; C0 is the background absorbance of the solvent.

[0076] DPPH free radical scavenging rate curve see Figure 2 A in.

[0077] 2. Hydroxyl radical scavenging experiment

[0078] Take 0.5mL 0.75mmol / L o-phenanthroline anhydrous ethanol and dissolve it in a test tube. Add 1mL 0.15mol / L phosphate buffer solution (PBS, pH=7.4) and 0.5mL distilled water in turn. After fully mixing, add 0.5mL 0.75mmol / L ferrous sulfate solution (FeSO4). After mixing, add 0.5mL 0.01% hydrogen peroxide (H2O2). After 60min in a 37℃ water bath, measure the absorbance at 536nm. The data obtained is the absorbance A of the damaged tube. 损伤 ; For the undamaged tube, replace the 0.5mL 0.01% hydrogen peroxide in the damaged tube with 0.5mL distilled water. The operation method is the same as that of the damaged tube. The absorbance value A of the undamaged tube can be measured. 未损伤 ; The sample tube is replaced with the sample (diluted with water to make different volume concentrations of forsythia fruit fermentation liquid) instead of the distilled water in the damaged tube. The operation method is the same as that of the damaged tube. The absorbance A in the sample tube can be measured 样品 ; Calculate the OH radical scavenging rate of the sample according to the following formula:

[0079] Clearance rate I (%) = 100% * (A 样品 -A 损伤 ) / (A 未损伤 -A 损伤 )

[0080] OH radical scavenging rate curve see Figure 2 B in.

[0081] 3. Total Antioxidant Capacity Determination

[0082] Standard curve drawing: dilute the standard with distilled water. Dilute the 10mM Trolox standard solution to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM.

[0083] Sample determination: Add 200 μl of ABTS working solution to each well of the 96-well plate, add 10 μl of distilled water or PBS or other appropriate solution to the blank control well; add 10 μl of Trolox standard solution of various concentrations to the standard curve detection well; add 10 μl of various samples (i.e., water extract and Forsythia suspensa fermentation liquid stock solution) to the sample detection well. Mix gently. After incubation for 2-6 minutes, measure the absorbance value A734 at 734nm.

[0084] Total clearance Figure 2 C in.

[0085] 4. Reactive oxygen species detection (ROS)

[0086] Experimental method: Take HaCat cells that grow well in the logarithmic phase, inoculate each well of a six-well plate with 2 ml of HaCat cell suspension diluted with complete DMEM culture medium, and set up a blank control group, a model group, and a sample group. The number of cells in each well is controlled to be 300,000. After culturing in a 37°C, 5% CO2 incubator for 12 hours, discard the culture medium and replace it with serum-free DMEM. After culturing in a 37°C, 5% CO2 incubator for 12 hours, discard the culture medium and add a small amount of PBS (pH = 7.4) to just cover the cells. Use UVB to stimulate the cells, and the blank group is not irradiated. Aspirate and discard PBS, add samples of different concentrations to incubate the cells for 24 hours, add 1 mL of diluted reactive oxygen fluorescent probe (DCFH-DA) (serum-free DMEM: DCFH-DA = 1:1000), and incubate in a 37°C cell incubator for 20 minutes, inverting every 3-5 minutes. Wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells. Discard the cell culture medium, add 1 ml PBS, and take pictures. Figure 3 .

[0087] See also Figure 2 , Figure 3 , indicating that compared with water extract, Forsythia suspensa fruit fermented liquid has stronger antioxidant capacity.

[0088] 3. Safety and efficacy analysis

[0089] 1. Chicken embryo chorioallantoic membrane test (HET-CAM)

[0090] The chicken embryo chorioallantoic membrane test (HET-CAM) is a classic in vitro assessment method for eye irritation, which is suitable for the evaluation of cosmetic products or raw materials. The chorioallantoic membrane (CAM) is a respiratory membrane located around the chicken embryo. This test uses the characteristics of the intact, clear and transparent CAM vascular membrane system in the middle of incubation. A certain amount of the test substance is directly exposed to the CAM. After a period of action, the changes in the toxic effect indicators of the chorioallantoic membrane (such as bleeding, coagulation and vascular melting) are observed, and a score is obtained by combining them to evaluate the eye irritation of the test substance. These indicators reflect the changes in the morphological structure, color and permeability of blood vessels and vascular networks, as well as phenomena such as chorioallantoic membrane protein denaturation and the degree of damage.

[0091] For tests conducted using the endpoint evaluation method, the endpoint score (ES) should be calculated. Based on the degree of bleeding, coagulation, and vascular melting observed in the chicken embryo, six parallel tests are performed on each sample, and the vascular reaction scores are added together. The vascular reaction score with the highest total score is used as the final score, and the highest score in the group is used as the reaction ES value. Based on the ES value, the eye irritation of the test substance is classified according to Table 4.

[0092] Table 4 Eye irritation prediction model of HET-CAM endpoint evaluation method

[0093]

[0094] The test substances included: negative control (0.9% sodium chloride), positive control (0.1 mol / L sodium hydroxide) and forsythia fruit fermentation liquid (6 parallels). Figure 4 , the numbers 1, 2, 3, 4, 5, and 6 in the figure are parallel samples. It can be seen from the figure that there is no bleeding phenomenon before and after the sample is used, and according to the score, it can be known that the fermented liquid of Forsythia fruit has no eye irritation.

[0095] The CAM of the negative control (0.9% sodium chloride), the positive control (0.1 mol / L sodium hydroxide) and the fermented broth of Forsythia suspensa before and after addition were observed and scored morphologically, and the results were obtained as shown in Table 5, where numbers 1, 2, 3, 4, 5, and 6 are parallel samples, respectively.

[0096] Table 5 Experimental record of chicken embryo chorioallantoic membrane of forsythia fruit fermented liquid

[0097]

[0098] 2. Red blood cell hemolysis test

[0099] The red blood cell test (RBC) is one of the alternative methods to the rabbit eye irritation test (Draize test). Red blood cells are considered to be the best biological source for studying biofilm effects, with strong operability and good homogeneity. The basic principle of the RBC test is to evaluate the irritation of chemicals to eye tissues by detecting the amount of hemoglobin leakage and the degree of protein denaturation in red blood cells. The RBC test is also widely used internationally in the study of eye irritation of chemicals such as cosmetic products and raw materials.

[0100] The specific steps of this experiment are as follows:

[0101] 1. Pretreatment of RBC

[0102] (1) Blood collection and transportation

[0103] Take fresh rabbit blood from the slaughterhouse and put it in a polyethylene plastic container. Add the anticoagulant citrate buffer at a ratio of 1:9 and mix well. Immediately keep the mixed blood sample warm in an incubator at 21-22°C. Transport it to the laboratory within 30 minutes. If the blood sample is not contaminated, the time can be extended to 1 hour.

[0104] (2) Isolation of RBCs

[0105] 1) Aliquoting and dilution: collect fresh blood (treated with citrate anticoagulant) and dilute it with PBS solution (blood: PBS = 4:10 volume ratio);

[0106] 2) Centrifugation to remove impurities: Centrifuge the above dilution at 1500×g for 10 minutes at room temperature, carefully aspirate the supernatant and the light yellow white blood cell layer after centrifugation, then add PBS and repeat the above washing and centrifugation steps 2-3 times (turn on the microplate reader for preheating for 15 minutes during the second centrifugation).

[0107] 3) Preparation of RBC suspension: After the last centrifugation, dilute the precipitated cells with PBS to a concentration of about 2% red blood cell suspension (about 2 mL precipitate + 98 mL PBS), and shake gently to mix evenly.

[0108] 4) RBC concentration calibration: Take 0.5mL of the above cell suspension in a 10mL EP tube, add distilled water to dilute to 5mL, mix and react for 1 minute, and measure at 541nm with an enzyme marker (PBS is a blank control, and the absorbance is adjusted with red blood cell suspension). The ideal absorbance value should be 0.5 (±5%). Seal the treated RBC suspension and store it at 4℃.

[0109] 2. Determination of hemolysis curve

[0110] (1) The fermented liquid of Forsythia suspensa was diluted with water to prepare sample solutions with volume concentrations of 20%, 40%, 60%, 80%, and 100%, and then each concentration gradient sample solution was added to the RBC suspension at a ratio of 3:1 (750 μL sample + 250 μL RBC) and mixed.

[0111] (2) Incubate the test substance RBC mixture on a shaker at room temperature for 60 min.

[0112] (3) Place each EP tube in a centrifuge and centrifuge at 10,000 rpm / min for 1 minute to terminate the incubation.

[0113] (4) Take the supernatant and measure its absorbance at 540 nm. Perform three replicates for each concentration and take the average value.

[0114] (5) Simultaneously measure the control group:

[0115] Negative control (zero hemolysis): 750 μL PBS + 250 μL RBC, the hemolysis rate of the negative control was set to 0%;

[0116] Positive control: 750 μL 0.1% SDS water + 250 μL RBC;

[0117] Complete hemolysis control: 750 μL water + 250 μL RBC. The hemolysis rate of the complete hemolysis control is set to 100%.

[0118] 3. Data processing

[0119] The absorbance OD value and concentration gradient curve measured at a wavelength of 560nm for each group were taken as the linear region for regression line, and the difference between the OD values ​​of the negative control and the positive control at 560nm was substituted into the regression line equation to obtain the half-hemolysis rate H50 (expressed in terms of concentration in the test system).

[0120] The calculation formula is as follows:

[0121]

[0122] The hemolysis curve of the red blood cell hemolysis experiment is shown in Figure 5 ,Depend on Figure 5 It can be seen that the hemolysis rate of Forsythia suspensa fermented liquid is lower than that of the water extract.

[0123] 4. Consumer Questionnaire Survey

[0124] The forsythia fruit fermented liquid obtained in Example 1 was used as a skin care product, and its efficacy was explored through a consumer use questionnaire according to the "Technical Specification for Safety of Cosmetics" (2015 edition).

[0125] 1. Subject selection

[0126] According to the Declaration of Helsinki, the selection of volunteers follows the medical and ethical standards for human testing, and all volunteers must be tested voluntarily and sign an informed consent form before the test. Before the volunteer signs the informed consent form, the tester needs to inform the volunteer of the purpose of the test, possible benefits, potential risks and problems, and related rights and obligations.

[0127] Inclusion criteria: (1) Volunteers aged 18 to 60 years old, both men and women, except pregnant or lactating women, or women who are planning to become pregnant within six months. (2) The test site has not received skin treatment, cosmetic treatment or other tests that may affect the results. (3) Those who have not used hormone drugs or immunosuppressants in the past month. (4) Those whose test site has not participated in other clinical trials now or in the past three months. (5) Volunteer to participate and sign the informed consent form. (6) Able to complete the specified content according to the test protocol requirements.

[0128] Exclusion criteria: Those who meet any of the following criteria will be excluded: (1) those with severe systemic diseases, immunodeficiency or autoimmune diseases; (2) those who have used histamine in the past week or immunosuppressants in the past month; (3) those who have used any anti-inflammatory drugs in the test area in the past two months; (4) patients with insulin-dependent diabetes; (5) patients who are receiving treatment for respiratory diseases; (6) people with allergic constitution, allergic dermatitis, etc., and those with skin diseases or a history of diseases; (7) people who are receiving dermatological treatment; (8) those with large birthmarks, scratches, white spots, pigmented moles, keloids, etc. in the test area that affect the experiment.

[0129] Trial suspension criteria: (1) If a serious safety issue occurs during the trial, the clinical trial should be suspended immediately. (2) If a major error is found in the clinical trial protocol, making it difficult to evaluate the efficacy; or a well-designed protocol has serious deviations during implementation, making it difficult to evaluate the efficacy. The trial sponsor requests suspension or the administrative department requests suspension.

[0130] 2. Experimental Design

[0131] Sample usage: After cleansing at night, take an appropriate amount of this product (undiluted forsythia fruit fermented liquid) and apply it evenly on the face. Wait for 10-15 minutes and then wash the face with water.

[0132] Number of evaluators: 20 (valid data for 20 people).

[0133] Follow-up time: Use once every other day, wash off after 10-15 minutes and follow up as normal skin care. Follow-up at 0, 2 weeks, and 4 weeks respectively.

[0134] Follow-up evaluation form: questionnaire survey.

[0135] Adverse reactions: No adverse reactions.

[0136] Note: Instruct the tester to report immediately if any allergy or discomfort occurs, stop the test, and wash the test area with plenty of water.

[0137] from Figure 6 According to the results of the sensory questionnaire in Table 6, after using the samples for 2 and 4 weeks, the subjects said that the samples had sustained improvements in overall improvement, hydration and nourishment, even skin tone, lightening of fine lines, firming skin, improvement of sensitivity, and acne removal. Among them, the number of people who increased in firming skin, improvement of sensitivity, and acne removal was relatively large. From the perspective of effectiveness, after using the samples for 2 weeks, the subjects evaluated that the products had an improvement efficiency of 50% to 100%. After continuing to use them for 4 weeks, the effectiveness was improved to varying degrees.

[0138] Table 6 Sensory evaluation table of subjects

[0139] Overall Improvement Hydrating and nourishing Evens out skin tone Reduce fine lines Firming skin Improve sensitivity Acne 4 weeks 80％ 95％ 80％ 85％ 85％ 85％ 95％ 2 weeks 50％ 65％ 60％ 70％ 55％ 55％ 60％

[0140] Table 7 is a rating table for whether the subjects experienced erythema, flushing, papules, itching, tightness, scaling, stinging, acne breakouts, etc., where very much = 3 points, a lot = 2 points, a little = 1 point, and not at all = 0 points. According to the results of the questionnaire, the subjects did not experience erythema, flushing, papules, itching, tightness, scaling, stinging, acne breakouts, etc. during the 2nd and 4th week of using the sample.

[0141] Table 7 Scoring table for improvement of erythema, flushing, etc.

[0142]

[0143]

[0144] Comparative Example

[0145] Referring to Table 8, this comparative example uses different lactic acid bacteria to ferment Forsythia suspensa fruit powder, and the bacteria are purchased from China Industrial Microbiological Culture Collection Management Center, and the names of the bacteria are respectively: CICC20241-Lactobacillus paracasei, CICC20261-Lactobacillus kefiri, CICC20243-Lactobacillus helveticus, and the fermentation process is the same as the optimal fermentation conditions in Example 1, and the bacteria are screened by detecting the active substance content in the fermented liquid. As shown in Table 8, Lactobacillus plantarum subsp. Plantarum is superior in lignans, total sugar, polyphenols, and total protein content compared to the other three bacterial fermentation products.

[0146] Table 8 Content of active substances in different lactic acid bacteria fermentation

[0147]

[0148]

[0149] Finally, it should be noted that, in the present disclosure, if any, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0150] Although the present disclosure has been disclosed above through the description of the specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed by the present disclosure.

Claims

1. A method for preparing a fermented product of Forsythia suspensa fruit, characterized in that: include: The fermentation substrate preparation step is: forsythia fruit powder and water are mixed in proportion, sterilized, and cooled to obtain a fermentation substrate; the forsythia fruit powder and water are used in an amount ratio of 1g:10ml-1g:40ml; The method comprises the following steps: inoculating Lactobacillus plantarum into the fermentation substrate, performing fermentation treatment, sterilizing, separating and treating the supernatant to obtain a Forsythia fruit fermentation liquid, or further drying the supernatant to obtain a Forsythia fruit fermentation dry powder; the Lactobacillus plantarum is Lactobacillus plantarum subsp. plantarum, and the strain number is CICC20261; the preparation method of the bacterial liquid used for inoculation comprises the following steps: inoculating Lactobacillus plantarum into a suitable culture medium for activation, purification and expansion culture, wherein the OD value of the bacterial liquid is 0.5-1.0; and the volume ratio of the bacterial liquid to the fermentation substrate is 1:10-20.

2. The preparation method according to claim 1, characterized in that In the method for preparing the fermented forsythia fruit, the forsythia fruit powder is obtained by drying and crushing the forsythia fruit and then passing it through a 50-mesh sieve.

3. The preparation method according to claim 1 or 2, characterized in that: In the fermentation step, the fermentation treatment temperature is 37-45° C. and the time is 6-36 hours.

4. The preparation method according to claim 3, characterized in that: In the fermentation step, the fermentation time is 6-16 hours.

5. The preparation method according to any one of claims 1 to 2 and 4, characterized in that: The fermentation process is carried out in a shaking incubator at a rotation speed of 150 r / min-180 r / min.

6. The preparation method according to any one of claims 1 to 2 and 4, characterized in that: The separation process adopts a centrifugal method.

7. The preparation method according to claim 6, characterized in that: The centrifugal speed is 4500r / min-5000r / min, and the centrifugal time is 20min-40min.

8. A fermented product of Forsythia suspensa fruit prepared by the method according to any one of claims 1 to 7.

9. Use of the forsythia fruit fermentation product as claimed in claim 8 in the preparation of cosmetics.

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