Jasmine double bacteria ferment, and preparation method and application thereof

Abstract

The application discloses a double-bacterium fermentation product of calendula, and a preparation method and application thereof, and relates to the technical field of plant fermentation. The preparation method of the double-bacterium fermentation product of calendula is as follows: first fermentation is carried out by using lactobacillus plantarum, second fermentation is carried out by using saccharomyces cerevisiae, and the fermentation liquor of calendula after the two times of fermentation is subjected to sterilization and filtration treatment, so that the fermentation liquor of calendula is obtained. The fermentation liquor of calendula is subjected to combined fermentation by using lactobacillus plantarum and saccharomyces cerevisiae, so that the content of polyphenols and flavones in the double-bacterium fermentation product of calendula is significantly improved. Meanwhile, the double-bacterium fermentation product of calendula has the effects of moisturizing, antioxidation, soothing and oil control, and has a wide application prospect in the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant fermentation, in particular to a calendula double-bacterial fermentation product and a preparation method and application thereof. BACKGROUND

[0002] Calendula officinalis is a herbaceous plant of the Asteraceae family, originally from southern Europe, and now widely cultivated around the world. Its height is 30-60 cm, the whole plant is covered with white hair, the leaves are alternate, the capitula are solitary on the top of the stem, the flowers are mainly golden yellow or orange yellow, and the flowering period lasts for 4-9 months. It has strong adaptability, is cold-resistant, and prefers sunny and loose slightly acidic soil. The main active substances are saponins, which have anti-inflammatory and hypolipidemic effects, can significantly reduce serum cholesterol and triglycerides in test animals, flavonoids and carotenoids (quercetin, lutein, etc.), which are strong antioxidants with outstanding free radical scavenging ability, and triterpenoids and organic acids (such as salicylic acid), which have synergistic antibacterial and wound healing promoting effects.

[0003] Lactobacillus plantarum is a gram-positive lactic acid bacteria that widely exists in nature, especially in fermented foods such as vegetables, meat products, dairy products, and wine. It is also an important probiotic in the human gastrointestinal tract and plays an important role in maintaining human health. Modern research has found that Lactobacillus plantarum not only plays an important role in fermented foods, but also has many applications in the cosmetics industry, such as the fermented broth of Lactobacillus plantarum has the effects of regulating skin flora balance and enhancing skin barrier.

[0004] Saccharomyces cerevisiae is a fungus that widely exists in nature, especially in fermented foods such as wine. Modern research has found that yeast not only plays an important role in fermented foods, but also has many applications in the cosmetics industry, such as the fermentation product of yeast has the effects of regulating skin flora balance and enhancing skin barrier.

[0005] The existing technology has the following limitations:

[0006] Calendula contains sugars, proteins, organic acids, polyphenols, flavonoids, saponins and other substances, rich in potassium, sodium and other elements. Simple water extraction is difficult to release the active ingredients in Calendula, and the extraction effect is poor. Chinese patent CN119185158A discloses a preparation method of Calendula double bacteria fermentation, which includes the steps of using yeast and Bifidobacterium adolescentis to ferment Calendula successively, but the strain combination is not optimized; CN113662894A discloses a method of using the fermentation enzyme liquid of Lentinula edodes to pretreat Calendula, and then selecting Lactobacillus plantarum and Saccharomyces cerevisiae to ferment it to obtain an enzyme hydrolysis fermentation, which has the effects of antioxidant, anti-allergic and anti-inflammatory, and the process is complex. There is no report on the use of Lactobacillus plantarum and Saccharomyces cerevisiae to obtain Calendula double bacteria fermentation without the use of enzyme hydrolysis step, and the active substance content in the fermentation broth is significantly improved and has skin care effect. SUMMARY

[0007] To solve the problems of redundant enzyme hydrolysis step and inefficient fermentation process in the prior art, the purpose of the present application is to provide a preparation method and application of Lactobacillus plantarum and Saccharomyces cerevisiae fermented Calendula. Through the combined fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae, the active ingredients can be efficiently released without enzyme hydrolysis, the content of polyphenols and flavonoids is significantly improved, and the fermentation broth has skin care effects such as skin moisturizing, antioxidant, soothing and oil control. The present application is as follows:

[0008] In the first aspect of the present application, a preparation method of Calendula double bacteria fermentation is provided, which comprises the following steps:

[0009] S1. Using Lactobacillus plantarum to ferment the culture medium containing Calendula for the first time to obtain fermentation broth A;

[0010] S2. Using Saccharomyces cerevisiae to ferment the fermentation broth A for the second time to obtain fermentation broth B;

[0011] S3. Sterilizing and filtering the fermentation broth B successively to obtain Calendula double bacteria fermentation.

[0012] Further, the Lactobacillus plantarum is selected from at least one of Lactobacillus plantarum with preservation number CGMCC. 32813 or Lactobacillus plantarum with number HC1775 purchased from Weikaihaisheng (Shandong) Biological Engineering Co., Ltd.; and the Saccharomyces cerevisiae is selected from at least one of Saccharomyces cerevisiae with preservation number GDMCC2.139 or Saccharomyces cerevisiae with batch number 20240422 purchased from Shandong Hezhongguangyuan Biological Technology Co., Ltd.

[0013] Further, the inoculation amount of Lactobacillus plantarum in step S1 is 1% to 4%, and the inoculation amount of Saccharomyces cerevisiae in step S2 is 1% to 4%.

[0014] Optionally, the inoculation amount of Lactobacillus plantarum in step S1 is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or any value between them; and the inoculation amount of Saccharomyces cerevisiae in step S2 is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or any value between them.

[0015] Further, the fermentation condition in step S1 is 35-40℃, anaerobic culture, and the time is 36-48h; and the fermentation condition in step S2 is 25-35℃, rotation speed 150-250rpm, and the fermentation time is 36-48h.

[0016] Further, the culture medium containing Calendula contains Calendula powder, glucose and water.

[0017] Further, the Lactobacillus plantarum is Lactobacillus plantarum CGMCC. 32813; and the Saccharomyces cerevisiae is Saccharomyces cerevisiae GDMCC2. 139.

[0018] Further, the sterilization and filtration in step S3 specifically include the following operations:

[0019] The filtrate is filtered by 100-200 mesh filter cloth, and then subjected to homogenization and wall breaking at 1400-1500 MPa (≤30℃, 2-4 cycles), and then centrifuged at 12000-15000 rpm in a tubular centrifuge to obtain a centrifugal liquid, which is sterilized at high temperature to obtain the Calendula double-bacterial fermentation liquor.

[0020] In the second aspect of the present application, the Calendula double-bacterial fermentation liquor obtained by the preparation method is provided.

[0021] In the third aspect of the present application, a composition is provided, which comprises the Calendula double-bacterial fermentation liquor and an additive acceptable in the cosmetic field.

[0022] Further, the mass fraction of the Calendula double-bacterial fermentation liquor in the composition is ≤50%, preferably ≤40%, and more preferably ≤30%.

[0023] Further, the additive comprises at least one of a preservative, an emulsifier, a humectant, a thickening agent, a pH regulator, a surfactant, a stabilizer, an antioxidant and a moisturizing agent.

[0024] Further, the additive comprises at least one of phenoxyethanol, parabens, vitamin E (tocopherol), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), polysorbate (Tween series), glyceryl stearate, carbomer, xanthan gum, glycerol, citric acid, sodium hydroxide, sodium lauryl sulfate (SLS), cocamidopropyl betaine, ethylenediaminetetraacetic acid (EDTA), ethylhexyl palmitate.

[0025] It should be noted that the present application does not strictly limit the composition and mass fraction of the additive acceptable in the field of cosmetics, and all the one or more additives having the functions of preservative, emulsifier, moisturizer, thickening agent, pH regulator, surfactant, stabilizer, antioxidant, nourishing agent harmless to human body selected according to the dosage form of the product or the mass fraction of the additive adjusted according to the actual need are within the protection scope of the present application.

[0026] Further, the composition further comprises other ingredients having the functions of antioxidant and / or anti-inflammatory and / or moisturizing and / or oil control, preferably, the other ingredients having the functions of antioxidant and / or anti-inflammatory and / or moisturizing and / or oil control comprise at least one of pueraria extract, Lotus corniculatus seed extract, nicotinamide, peony extract, peony root bark extract, peony seed oil, ceramide, citrus fruit extract, jasminum mesnyi flower extract, 4-tert-butylcyclohexanol, chrysanthemum extract, Rosa gallica flower extract, rose flower water, and plant fermented oil.

[0027] Further, the dosage form of the composition comprises at least one of water, emulsion, essence, cream, ointment, gel, mask, and powder.

[0028] In one specific embodiment of the present application, the composition comprises the Calendula bifida dual bacterial ferment and the Pueraria montana var. lobata extract, preferably, the mass concentration of the Calendula bifida dual bacterial ferment in the composition is 0.5% to 4%, and the mass concentration of the Pueraria montana var. lobata extract is 1.5% to 4%.

[0029] In a fourth aspect of the present application, the preparation method or the Calendula bifida dual bacterial ferment or the composition is applied in any of the following aspects:

[0030] A1. Application in the preparation of antioxidant products;

[0031] A2. Application in the preparation of anti-inflammatory products;

[0032] A3. Application in the preparation of 5α-reductase activity inhibiting products;

[0033] A4. Application in the preparation of moisturizing products;

[0034] A5. Use in the preparation of oil control products;

[0035] A6. Use in the preparation of soothing products;

[0036] A7. Use in the preparation of anti-aging products.

[0037] Further, the product at least includes cosmetics. Preferably, the dosage form of the cosmetic includes at least one of water, emulsion, essence, cream, paste, gel, mask, and powder.

[0038] Further, the product can achieve the effect of anti-aging by scavenging free radicals. Further, the product can also achieve the effect of soothing by inhibiting the release of inflammatory factors, preferably, the inflammatory factors include at least one of TNF-a, IL-6, and NO. Further, the product can achieve the effect of oil control by inhibiting the activity of 5a-reductase.

[0039] The beneficial effects of the present application include but are not limited to:

[0040] The present application optimizes the strain and fermentation conditions of Calendula officinalis fermentation, and first uses Lactobacillus plantarum fermentation and then uses Saccharomyces cerevisiae fermentation. The fermentation method described in the present application has simple raw materials, only Calendula officinalis powder and glucose are needed for fermentation; the fermentation steps are simple, no additional enzyme hydrolysis is needed, and the preparation period is short, which lays a foundation for large-scale industrial production.

[0041] By combined fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae, the content of polyphenols and flavonoids in Calendula officinalis double-strain fermentation product is significantly improved, and the double-strain fermentation process significantly improves the active substances compared with single-strain fermentation. At the same time, the Calendula officinalis double-strain fermentation product has the effects of moisturizing, antioxidant, soothing, and oil control, and has a broad application prospect in the field of cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0042] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0043] Figure 1 is a flow chart for preparing Calendula officinalis double-strain fermentation product in the embodiments of the present application. DETAILED DESCRIPTION

[0044] The present application will be described in detail below in conjunction with embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are purchased through commercial channels.

[0045] 1. Materials and reagents:

[0046] Calendula officinalis: produced in Yunnan, China;

[0047] Lactobacillus plantarum 1: with the preservation number of CGMCC.32813, recorded in the patent CN119351288A;

[0048] Saccharomyces cerevisiae 1: purchased from Guangdong Microbial Culture Collection Center, with the preservation number of GDMCC2.139;

[0049] Lactobacillus plantarum A: purchased from Weikaihaisi (Shandong) Biological Engineering Co., Ltd., with the name of Lactobacillus plantarum, strain number HC1775, specification 10,000 billion cfu / g, batch number 20230201;

[0050] Saccharomyces cerevisiae B: purchased from Shandong Hezhong Kangyuan Biological Technology Co., Ltd., with the name of Saccharomyces cerevisiae, specification 200 billion cfu / g, batch number 20240422;

[0051] Pectinase (Shandong Longkete Enzyme Preparation Co., Ltd.);

[0052] MRS broth medium (Shanghai Bomei Microbial Technology Co., Ltd.), proteose peptone (Angel Yeast Co., Ltd.), yeast extract powder (Angel Yeast Co., Ltd.), glucose monohydrate (Shandong Xiwang Sugar Industry Co., Ltd.).

[0053] 2. Medium formula:

[0054] (1) MRS liquid medium:

[0055] MRS broth medium 49.3 g, add water to 1 L.

[0056] (2) YPD liquid medium:

[0057] Yeast extract powder 10 g, proteose peptone 20 g, glucose monohydrate 22 g, add water to 1 L.

[0058] (3) Calendula officinalis fermentation medium:

[0059] Calendula officinalis powder 100.0 g, glucose monohydrate 22.0 g, added to 1.0 liter of purified water, sterilized in a sterilized pot at 105℃ for 20 min to obtain the fermentation medium.

[0060] Instrument and equipment:

[0061] 100L automatic fermentation tank of Shanghai Bailong Biological Technology Co., Ltd.; 722 type ultraviolet-visible spectrophotometer; constant temperature shaker.

[0062] 3. Culture method

[0063] (1) Culture conditions

[0064] 1) Lactobacillus plantarum 1: Lactobacillus plantarum 1 was inoculated into MRS liquid medium and incubated anaerobically at 37°C for 24 h.

[0065] 2) Saccharomyces cerevisiae 1: Saccharomyces cerevisiae 1 was inoculated into YPD liquid medium and incubated at 28°C, 200 rpm for 24 h.

[0066] 3) Lactobacillus plantarum A: Lactobacillus plantarum A was inoculated into MRS liquid medium and incubated anaerobically at 37°C for 24 h.

[0067] 4) Saccharomyces cerevisiae B: Saccharomyces cerevisiae B was inoculated into YPD liquid medium and incubated at 28°C, 200 rpm for 24 h.

[0068] Example 1: Saccharomyces cerevisiae - Lactobacillus plantarum sequential fermentation

[0069] Pre-treatment: The Calendula officinalis dry flowers were ground into powder and sieved through a 100-mesh sieve. 7 kg of the Calendula officinalis powder and 1.54 kg of monohydrated glucose were added to a 100-L fermentation tank, and the volume was made up to 70 L with deionized water, and sterilized at 105-110°C for 20-30 min.

[0070] Fermentation method: Saccharomyces cerevisiae 1 was inoculated into the Calendula officinalis fermentation medium (inoculation amount 2%), and incubated at 28°C for 48 h; then Lactobacillus plantarum 1 was inoculated (inoculation amount 2%), and incubated at 37°C for 48 h.

[0071] Post-treatment method: The product was filtered through a 100-mesh filter cloth, and then homogenously broken at 1400-1500 MPa (≤30°C, 2-4 cycles), and then centrifuged at 14000 rpm in a tubular centrifuge. The centrifugate was sterilized at 115°C for 20 min to obtain the Calendula officinalis double-strain fermentation product.

[0072] Example 2: Lactobacillus plantarum - Saccharomyces cerevisiae sequential fermentation

[0073] Pre-treatment: The same as the pre-treatment method of Example 1.

[0074] Fermentation method: Lactobacillus plantarum 1 was inoculated into the Calendula officinalis fermentation medium (inoculation amount 2%), and incubated at 37°C for 48 h; then Saccharomyces cerevisiae 1 was inoculated (inoculation amount 2%), and incubated at 28°C for 48 h.

[0075] Post-treatment method: The same as the post-treatment method of Example 1.

[0076] Example 3: Lactobacillus plantarum - Saccharomyces cerevisiae sequential fermentation after enzymatic pre-treatment

[0077] Pre-treatment: Dry Calendula flowers were ground into powder and sieved through 100 mesh. The Calendula fermentation medium was added with pectinase (3000 U / L) and enzymolysis at 50°C for 2 hours. The Calendula fermentation medium was obtained by inactivating at 95°C for 30 minutes.

[0078] Fermentation method and post-treatment method: same as the fermentation and post-treatment method of Example 2.

[0079] Comparative Example 1: Preparation of Calendula water extract

[0080] Comparative Example 2: Single-strain fermentation of Lactobacillus plantarum 1

[0081] Comparative Example 3: Single-strain fermentation of Saccharomyces cerevisiae 1

[0082] Example 4: Sequential fermentation of Lactobacillus plantarum A-Saccharomyces cerevisiae 1

[0083] Example 5: Sequential fermentation of Lactobacillus plantarum 1-Saccharomyces cerevisiae B

[0084] Example 6: Sequential fermentation of Lactobacillus plantarum A-Saccharomyces cerevisiae B

[0085] Example 7: Sequential fermentation of Lactobacillus plantarum A-Saccharomyces cerevisiae B

[0086] Test Example 1: Active ingredient content test

[0087] (1) Test method:

[0088] 1) Determination of polyphenol content (Folin-Ciocalten method)

[0089] Test principle:

[0090] Folin phenol method is to determine the content of polyphenols with Folin phenol as reagent. Folin phenol is a phenolic compound that can react with polyphenols to produce a dark brown product, the absorbance of which is proportional to the content of polyphenols. Therefore, by measuring the absorbance of the dark brown product, the content of polyphenols can be calculated.

[0091] Reagents and materials: 0.2 mol / L Folin phenol reagent, 15% sodium bicarbonate solution, gallic acid standard solution, sample solution.

[0092] Test steps:

[0093] Prepare the standard curve: accurately pipette 100 μg / L of gallic acid standard solution 0, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, respectively, then add 5.0 mL of 0.2 mol / L Folin phenol reagent, shake well and stand for 5 min, then add 2.0 mL of 15% Na2CO3 solution, and place at room temperature in the dark for 1 h. Take 0 sample as the blank to correct the transmittance to 100%, and measure the absorbance at 765 nm. Draw the standard curve with the standard solution concentration (μg / mL) as the abscissa and the absorbance as the ordinate.

[0094] Sample solution determination: pipette 1 mL of the sample solution into a test tube, repeat the above steps, and measure the absorbance at 765 nm. According to the regression equation established by the standard curve, calculate the content.

[0095] 2) Determination of flavonoid content (spectrophotometric method)

[0096] Test principle:

[0097] The adjacent carbonyl and hydroxyl groups in flavonoids can complex with metals to produce color. The total flavonoid content is determined by the depth of color (i.e. the size of absorbance A value).

[0098] Reagents and materials: 5% sodium nitrite solution, 10% aluminum nitrate solution, 4% sodium hydroxide solution, rutin standard solution, sample solution.

[0099] Test steps:

[0100] Pipette the diluted rutin standard solution with gradient concentration into 50 mL volumetric flasks, add 60% ethanol solution to 12 mL, respectively, add 5% sodium nitrite solution 2 mL, shake well, and stand for 6 min, add 10% aluminum nitrate solution 2 mL, shake well, and stand for 6 min, add 4% sodium hydroxide solution 20 mL, shake well, and stand for 5 min, add 60% ethanol solution to the mark, shake well, and stand for 15 min, then measure the sample absorbance at 508 nm wavelength. Draw the standard curve with absorbance as abscissa and rutin concentration as ordinate.

[0101] Take 0.5 mL of the sample to be tested, place it in a 50 mL volumetric flask, and add the chromogenic reagent according to the method above. Measure the absorbance at 508 nm. Calculate the content according to the regression equation established by the standard curve.

[0102] (2) Test results:

[0103] The test results are shown in Table 1.

[0104] Table 1. Increase rate of active substance of Calendula double bacteria fermentation compared to water extraction (Comparative Example 1)

[0105]

[0106] The universal advantage of the fermentation process: Examples 1-3, Comparative Examples 2-3, compared to the water extraction method of Comparative Example 1, the polyphenol extraction rate was increased by 68.1%-237.5%, and the flavone extraction rate was increased by 77.2%-196.1%, confirming that microbial fermentation can significantly release active ingredients of Calendula.

[0107] Synergistic effect of double bacteria: Under the premise of using the same strain, the double bacteria fermentation process of Examples 1-3 significantly improved the extraction rates of polyphenols and flavones compared to single bacteria fermentation (Comparative Examples 2-3).

[0108] The key of fermentation sequence: The polyphenol extraction rate of Example 2 (first Lactobacillus plantarum and then Saccharomyces cerevisiae) was higher than that of Example 1 (first Saccharomyces cerevisiae and then Lactobacillus plantarum) (237.5% vs 68.1%), and the flavone extraction rate was also higher (196.1% vs 77.2%), overcoming the technical bias of low fermentation efficiency of Saccharomyces cerevisiae in the prior art.

[0109] The enzyme step is not necessary: The active substance extraction rate of Example 2 is better than that of Example 3, confirming that the enzyme pretreatment step in the double bacteria fermentation process of Calendula in the present application cannot achieve the effect of promoting strain fermentation and improving the yield of target products (polyphenols, flavones).

[0110] At the same time, by using other strains to replace the strains in Example 2, the flavone and polyphenol contents of Examples 4-6 also significantly increased compared to Example 1, and were better than Example 3.

[0111] Preferred scheme description: Based on the highest active substance extraction rate (polyphenol 237.5%, flavone 196.1%) achieved by Example 2, the subsequent efficacy verification was carried out using the double bacteria fermentation liquid obtained by this process.

[0112] Test Example 2 Efficacy Test

[0113] (1) Test method

[0114] 1) Moisturizing test

[0115] Test principle: The ability of the sample to slow down the evaporation of moisture (weight loss) under a specific humidity environment is evaluated by weighing, reflecting the ability of the sample to actively grab and lock the environmental moisture.

[0116] Reagents and materials: Artificial leather (6cm*6cm), deionized water.

[0117] Positive control: 5% glycerol aqueous solution.

[0118] Test steps:

[0119] Cleaning: Clean the 6cm*6cm artificial leather with cleaning agent, and clean it with an ultrasonic cleaning instrument for 10min.

[0120] Pre-treatment: Adjust the temperature and humidity of the constant temperature and humidity chamber to 21℃ and 80RH%, respectively, and place the artificial leather in the environment for 24h.

[0121] Formal test: Weigh the artificial leather after 24h of equilibrium as a carrier, and record the mass as M0. Uniformly apply 1ml of deionized water on the artificial leather, and this 1ml of deionized water is the basic moisture; uniformly apply 0.2g of sample on the surface of the artificial leather, and weigh it, and record the mass as M 样 , each sample was measured in triplicate, and after weighing, the artificial leather was placed in a constant temperature and humidity chamber, and was placed in the constant temperature and humidity chamber for 1, 2, and 4h, respectively, and was weighed and recorded, and the moisture retention rate at this time was calculated.

[0122] Data analysis:

[0123] The moisture retention rate was calculated according to the following formula:

[0124] ;

[0125] In the formula:

[0126] M t - the mass of the artificial leather at the corresponding time point;

[0127] M0- the initial mass of the artificial leather;

[0128] M 样 - the mass of the artificial leather after adding the sample.

[0129] 2) Antioxidant test (DPPH clearance rate)

[0130] Test principle: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable long-life free radical, its ethanol solution is dark purple, and has strong absorption at 517 mm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to the pairing of single electron. The fading degree of DPPH ethanol solution is linearly related to the number of electrons it receives, so the ability of the test sample to scavenge free radicals, i.e. the strength of antioxidant activity, can be evaluated.

[0131] Reagents and materials:

[0132] 2×104mol / L DPPH working solution, 99% ethanol solution, 0.04 mg / ml vitamin C solution.

[0133] Test steps:

[0134] 100 μL of the test sample, 100 μL of DPPH were added in the 96-well plate, 3 replicates were set for each reaction, and the reaction was shaken at room temperature for 5 min. At the same time, blank control and self-control groups were set; 100 μL of DPPH working solution + 100 μL of pure water solution was the blank control group; 100 μL of sample solution + 100 μL of pure water solution was the self-control group. The absorbance value was measured at 517 nm wavelength using an enzyme-labeled instrument.

[0135] Data analysis:

[0136] The DPPH scavenging rate was calculated according to the following formula:

[0137] ;

[0138] In the formula:

[0139] A t - The sample tube absorbance value, i.e. the absorbance value of the solution after the sample and DPPH reaction;

[0140] A r - The absorbance value of the self-control group;

[0141] A0- The average absorbance value of the blank control group.

[0142] 3) Soothing (anti-inflammatory) test

[0143] Test principle: LPS binds to the surface antigen recognition receptor of raw 264.7 cells, induces macrophages to secrete inflammatory factors such as interleukin-1β (IL-1β), IL-6, TNF-α, NO, etc., and realizes its cytotoxicity, antiviral, immunomodulation and apoptosis and other biological functions. LPS-induced raw 264.7 model is a classic cell model for studying inflammatory factors, and the difference in the content of inflammatory factors secreted by RAW264.7 after negative control and sample administration is analyzed to evaluate the soothing effect of the sample to be tested. This test is based on the LPS-induced raw 264.7 inflammatory cell model, and the soothing effect of the sample to be tested is evaluated by detecting the changes in TNF-a and IL-6 expression.

[0144] Materials:

[0145] Main reagents: DMEM culture medium (gibco), fetal bovine serum, thiazolyl blue (MTT), LPS, dexamethasone sodium phosphate, IL-6 Elisa kit (Quanzhou Ruixin Biological Technology Co., Ltd.), TNF-a. Elisa kit (Quanzhou Ruixin Biological Technology Co., Ltd.) and the like.

[0146] Main equipment: cell incubator, constant temperature incubator, enzyme label instrument.

[0147] Test method:

[0148] A. Cytotoxicity test

[0149] Raw 264.7 cells were cultured in vitro at an appropriate density in a 96-well plate, and the sample was prepared into 8 different gradient concentrations with serum-free culture medium and added to the cell well, 100 μL / well, 6 replicates per group. At the same time, set up a blank control group (BC) of culture medium, incubate at 37°C, 5% CO2 for 20-28h after adding sample, and detect cell toxicity by MTT method.

[0150] B. Cell soothing test

[0151] According to the results of the cytotoxicity test, select 3 concentrations as the detection concentration of the efficacy test. Cultivate Raw 264.7 cells in vitro at an appropriate density, discard the culture medium in the 96-well plate, add LPS solution and sample solution of different concentrations to each well, and set up positive control group (PC), negative control group (NC) and blank control group (BC) at the same time. The positive control group is dexamethasone sodium phosphate working solution, the negative control group is culture medium, and the blank control group is test culture medium without LPS. Each group has 6 replicates. Incubate at 37°C, 5% CO2 for 20-28h after adding sample, collect the liquid in the 96-well plate, and perform IL-6 and TNF-α tests according to the ELISA kit instructions.

[0152] 4) Oil control efficacy test

[0153] Test principle: 5α-reductase (5α-Reductase, 5αR) is a membrane protein located in the target cell microsomes, which is a key enzyme for the metabolism of androgen testosterone (Testosterone, T). It can reduce the 4, 5 unsaturated double bond of testosterone under the condition of providing electrons by reduced coenzyme II (NADPH), and convert it into more active dihydrotestosterone (Dihydrotestosterone, DHT). Dihydrotestosterone, as a more potent androgen than testosterone, can accelerate the proliferation of human sebaceous gland cells and promote excessive secretion of sebum, which is easy to cause oily skin and other symptoms. Therefore, by inhibiting the activity of 5α-reductase to reduce sebum secretion, it can be used to evaluate the oil control effect of cosmetics.

[0154] Reagents and materials:

[0155] 5α-reductase Elisa detection kit (Wuhan Jilide Biological Technology Co., Ltd.), ultrapure water, incubator, enzyme label instrument (CALT / 2017R004).

[0156] Test steps:

[0157] Take out the required board from the aluminum foil bag after equilibration at room temperature for 20 min, and put the remaining board back into the 4℃ with a self-sealing bag.

[0158] Set standard hole (0 U / mL, 5 U / mL, 10 U / mL, 20 U / mL, 40 U / mL, 80 U / mL) and sample hole, and add different concentrations of standard 50 μL to each standard hole;

[0159] Add 10 μL of the sample to be tested to the sample hole, and then add 40 μL of sample diluent; the blank hole is not added.

[0160] Except for the blank hole, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard hole and the sample hole, cover the reaction hole with a sealing film, and incubate in a 37℃ water bath or incubator for 60 min.

[0161] Discard the liquid, dry it on the blotting paper, add enough washing solution to each well, stand for 1 min, shake off the washing solution, dry it on the blotting paper, and repeat the plate washing for 5 times.

[0162] Add 50 μL of substrate A and B to each well, and incubate at 37℃ in the dark for 15 min.

[0163] Add 50 μL of termination solution to each well, and measure the OD value of each well at 450 nm wavelength within 15 min.

[0164] Data analysis:

[0165] The standard curve of 5-a reductase content was drawn according to the measured absorbance value, and the concentration of 5-a reductase in the sample was calculated by the standard curve. (The sample has been diluted 5 times during operation, and the final result needs to be multiplied by 5).

[0166] (2) Test results

[0167] 1) Moisturizing test results

[0168] Table 2. Moisturizing efficacy test results of samples

[0169]

[0170] The moisturizing test results are shown in Table 2, and the test data show that the moisturizing rate of the Calendula double bacterial fermentation liquid sample after 1 hour and 2 hours is 83.02% ± 1.30%, 57.17% ± 1.67% respectively, which has moisturizing efficacy under the test conditions.

[0171] 2) Antioxidant test results (DPPH clearance rate)

[0172] The average absorbance value and average clearance rate of the sample and positive control are shown in Table 3.

[0173] Table 3. DPPH clearance rate test results of samples

[0174]

[0175] The test data show that the DPPH clearance rate of the Calendula double bacterial fermentation liquid sample at 15%, 8%, 1.5%, 1% mass concentration is 92.34 ± 1.13%, 57.89% ± 2.16%, 46.55% ± 0.09%, 39.51% ± 0.54% respectively, and the Calendula double bacterial fermentation has antioxidant effect under the test conditions.

[0176] 3) Soothing test results

[0177] Cytotoxicity experiment

[0178] Compared with the blank control group (BC), the sample at a mass concentration of 10.00% and below has no toxicity to macrophage RAW264.7, and the subsequent efficacy detection concentration is set to 8.00%, 1.50%, 1.00%.

[0179] Cell soothing experiment

[0180] IL-6 inflammatory factor test

[0181] The IL-6 inhibition rates of the Calendula double bacterial fermentation liquid samples with mass concentrations of 8.00%, 1.50%, and 1.00% were 36.10% (P<0.05), 36.79% (P<0.05), and 37.03% (P<0.05), respectively, compared with the negative control group (NC).

[0182] TNF-α inflammatory factor test

[0183] The TNF-α inhibition rates of the Calendula double bacterial fermentation liquid samples with mass concentrations of 8.00%, 1.50%, and 1.00% were 72.23% (P<0.05), 32.79% (P<0.05), and 25.86% (P<0.05), respectively, compared with the negative control group (NC).

[0184] In summary, the Calendula double bacterial fermentation liquid samples at concentrations of 8.00%, 1.50%, and 1.00% can exert soothing effects by inhibiting the expression of inflammatory factors IL-6 and TNF-α in macrophages.

[0185] 4) Test results of oil control efficacy (5α-reductase inhibition rate)

[0186] The average absorbance values and average clearance rates of the samples and the positive control are shown in Table 4.

[0187] Table 4. Test results of 5α-reductase inhibition rate of samples

[0188]

[0189] The test data showed that the inhibition rate of 5α-reductase activity measured for the Calendula double bacterial fermentation liquid sample with a mass concentration of 1.5% was 61.49%. That is, the sample "Calendula double bacterial fermentation product" can exert oil control efficacy by inhibiting 5α-reductase activity under the test conditions.

[0190] Example 7 Verification of oil control efficacy of Calendula double bacterial fermentation product composition

[0191] Composition: Calendula double bacterial fermentation liquid 0.5%-4% synergized with Kudzu root extract (purchased from Beijing Dongfangmiansen Biotechnology Co., Ltd., product number ACC-Pure Geqingyan) 1.5%-4%.

[0192] Test purpose: By testing the 4h and 1-week secretion amounts of human sebum, it is verified that the composition composed of Calendula double bacterial fermentation liquid and Kudzu root extract has synergistic oil control efficacy.

[0193] Test basis: T / CAB 0152-2022. Cosmetic anti-wrinkle, firming, moisturizing, oil control, repair, nourishing, soothing seven efficacy test methods [S]. Beijing: China Association for Promotion of Industry-University-Research Cooperation, 2022.

[0194] Test instrument: Sebumeter SM 815 (Courage & Khazaka, Germany)

[0195] Test procedure:

[0196] 1. Selection of subjects

[0197] Inclusion criteria:

[0198] 1) Healthy subjects aged 18-29 years, both male and female;

[0199] 2) Positive lactic acid stinging test, total score ≥3 points;

[0200] 3) Forehead sebum amount exceeding 120 μg / cm2 within 8 hours;

[0201] 4) Self-perceived dry and rough skin;

[0202] 5) Ability to accept skin examination and pretreatment of the test area;

[0203] 6) Ability to understand the test procedure, willingness to participate in the test and signing of the written informed consent form.

[0204] Exclusion criteria:

[0205] 1) Pregnant or lactating women, or those with recent plans for pregnancy;

[0206] 2) Highly sensitive constitution, allergic diseases, history of cosmetic allergy;

[0207] 3) Suffering from skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, etc., or suffering from other chronic systemic diseases;

[0208] 4) Presence of skin phenomena such as birthmarks, pigmentation, inflammation, scars, pigmented nevi, hypertrichosis, etc. on the test site;

[0209] 5) Oral or external use of corticosteroids or other anti-inflammatory drugs within the past 1 month;

[0210] 6) Use of cosmetics with similar efficacy or other products with similar efficacy within the past 2 weeks;

[0211] 7) Participation in a cosmetic human clinical trial within the past 1 month;

[0212] 8) Other clinical assessments deemed unsuitable for participation in the test.

[0213] 108 subjects who met the above inclusion criteria and exclusion criteria were selected, and finally 108 subjects completed the test, divided into nine groups, 12 people in each group, with an average age of 24 years old.

[0214] 2. Test design

[0215] In the single and continuous use for 1 week efficacy test, the researchers randomly selected the left side of the subject's face as the experimental group and the right side as the control group. The experimental group used water solvent with the corresponding mass percentage of efficacy substances. The control group used water solvent. The group information is shown in Table 5.

[0216] Table 5. Combination of Calendula double bacterial ferment and wild kudzu root extract composition

[0217]

[0218] 3. Usage steps

[0219] 1) Before using the product (BL)

[0220] Face cleaning, constant temperature and humidity environment waiting for 30 min, forehead skin oil content measurement (Sebumeter SM 815 & MPA 4), product usage explanation, test product distribution and weighing, on-site product usage by subjects, constant temperature and humidity environment waiting.

[0221] 2) 4h after using the product

[0222] Forehead skin oil content measurement (Sebumeter SM 815 & MPA 4), constant temperature and humidity environment waiting.

[0223] 3) 1 week after using the product

[0224] Information registration (verify identity information), product weighing and understanding of subject usage, face cleaning, constant temperature and humidity environment waiting for 30 min, discomfort symptom tracking inquiry (on-site), forehead skin oil content measurement (Sebumeter SM 815 & MPA 4).

[0225] 4. Statistical methods

[0226] Excel software was used to perform descriptive statistics on each measurement, including mean, standard deviation, median, minimum and maximum, etc.

[0227] The SPSS analysis software was used to compare the measured values at different time points with the base values, and Shapiro-Wilk Test was used for significance test of normal distribution of data difference. If p (two-sided) > 0.05, it was normally distributed, and paired sample t test was performed, and the significance difference level a was 0.05. If p (two-sided) < 0.05, it was non-normally distributed, and Wilcoxon signed rank test was performed, and the significance difference level a was 0.05.

[0228] The experimental group and the control group (between groups) at different time points were compared, and Shapiro-Wilk Test was used for significance test of normal distribution of data difference (control group difference-experimental group difference). If p (two-sided) > 0.05, it was normally distributed, and paired sample t test was performed, and the significance difference level a was 0.05. If p (two-sided) < 0.05, it was non-normally distributed, and Wilcoxon signed rank test was performed, and the significance difference level a was 0.05. The calculation formula is as follows:

[0229] The change amount after using the product (the difference of the average value) = the average value after use-the average value before use;

[0230] The change rate after using the product = (the average value after use-the average value before use) / the data before use.

[0231] 5. Test results

[0232] Table 6 Calming oil test results of Calendula double bacteria fermented product-wild kudzu root extract composition

[0233]

[0234] The test results are shown in Table 6, and the test data shows that 4h after using the test efficacy substance, the forehead skin oil content of the experimental group shows an upward trend, the forehead skin oil content of the control group shows an upward trend, and the change trend of the skin oil content of the experimental group is better than that of the control group; the change trend of the skin oil content of experimental group 1 is better than that of experimental group 2 and experimental group 3. The change trend of the skin oil content of experimental group 4 is better than that of experimental group 5 and experimental group 6. The change trend of the skin oil content of experimental group 7 is better than that of experimental group 8 and experimental group 9. The change trend of the skin oil content of experimental group 7 is better than that of experimental group 1 and experimental group 4, but the amplitude of the change trend is reduced.

[0235] After using the test product for 1 week, the forehead skin oil content of the experimental group 1 was significantly reduced by 19.54% (p<0.001), and the forehead skin oil content of the control group had no obvious change, and the change trend of the skin oil content of the experimental group was better than that of the control group. The skin oil content of the experimental group 1 was significantly reduced by 19.54% (p<0.001) compared with the experimental group 2 and the experimental group 3, which was significantly better than the experimental group 2: significantly reduced by 11.10% (p<0.001) and the experimental group 3: significantly reduced by 12.29% (p<0.001).

[0236] After using the test product for 1 week, the forehead skin oil content of the experimental group 4 was significantly reduced by 36.78% (p<0.001), and the forehead skin oil content of the control group had no obvious change, and the change trend of the skin oil content of the experimental group was better than that of the control group. The skin oil content of the experimental group 4 was significantly reduced by 36.78% (p<0.001) compared with the experimental group 5 and the experimental group 6, which was significantly better than the experimental group 5: significantly reduced by 23.18% (p<0.001) and the experimental group 6: significantly reduced by 20.98% (p<0.001).

[0237] After using the test product for 1 week, the forehead skin oil content of the experimental group 7 was significantly reduced by 39.79% (p<0.001), and the forehead skin oil content of the control group had no obvious change, and the change trend of the skin oil content of the experimental group was better than that of the control group. The skin oil content of the experimental group 7 was significantly reduced by 39.79% (p<0.001) compared with the experimental group 8 and the experimental group 9, which was significantly better than the experimental group 8: significantly reduced by 28.95% (p<0.001) and the experimental group 9: significantly reduced by 25.75% (p<0.001).

[0238] Test results: the efficacy composition composed of 0.5%~4% Calendula double-strain fermentation liquor and 1.5%~4% wild kudzu root extract has a synergistic oil control effect.

[0239] The above only describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a calendula ferment by two strains, characterized in that, The method includes the following steps: S1. The culture medium containing calendula was fermented for the first time using Lactobacillus plantarum to obtain fermentation broth A, wherein Lactobacillus plantarum was selected from Lactobacillus plantarum with preservation number CGMCC No. 32813, the inoculum amount of Lactobacillus plantarum was 1%~4%, the fermentation conditions were 35~40℃, anaerobic culture, and the fermentation time was 36~48h; S2. The fermentation broth A is fermented a second time using Saccharomyces cerevisiae to obtain fermentation broth B, wherein the Saccharomyces cerevisiae is selected from Saccharomyces cerevisiae with preservation number GDMCC No. 2.139, the inoculum amount of Saccharomyces cerevisiae is 1%~4%, the fermentation conditions are a temperature of 25~35℃, a rotation speed of 150~250rpm, and a fermentation time of 36~48h; S3. The fermentation broth B is sterilized and filtered sequentially to obtain the calendula dual-strain fermentation product.

2. The preparation method according to claim 1, characterized in that, The culture medium containing calendula includes calendula powder, glucose, and water.

3. Calendula spp. fermentation product obtained by the preparation method according to any one of claims 1-2.

4. A composition, characterized in that, The composition comprises the calendula ferment as described in claim 3 and additives acceptable in the cosmetics field.

5. A composition, characterized in that, The composition comprises 0.5% to 4% by mass of the calendula double-strain fermentation product as described in claim 3 and 1.5% to 4% by mass of kudzu root extract, wherein the kudzu root extract is purchased from Beijing Dongfang Miaosen Biotechnology Co., Ltd., with the product number ACC-Pure Ge Qingyan.

6. The use of the calendula ferment as described in claim 3 or the composition as described in claim 4 or 5 in any of the following aspects: A1. Applications in the preparation of antioxidant products; A2. Applications in the preparation of anti-inflammatory products; A3. Applications in the preparation of moisturizing products; A4. Applications in the preparation of oil-controlling products; A5. Applications in the preparation of soothing products; A6. Applications in the preparation of anti-aging products.

7. The application according to claim 6, characterized in that, The products include at least cosmetics.

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