Composition and fermentation product thereof and application in preparing cosmetics

Abstract

The present invention relates to the technical field of cosmetics, and in particular to a composition and a fermentation product thereof and an application thereof in the preparation of cosmetics. The present invention prepares a composition from honeysuckle, frankincense, myrrh, angelica, red peony root and bacterial strains, and studies the effects of fermenting the Chinese medicine composition by different combined fermentation methods. The results show that the fermentation of plant lactobacillus and yeast significantly promotes the release of skin care efficacy substances in the Chinese medicine composition, significantly improves its efficacy in skin care, and the resulting composite fermentation liquid has the effects of inhibiting oil, inflammation, inhibiting Propionibacterium acnes and anti-oxidation.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a composition and a fermentation product thereof and applications in preparing cosmetics. Background Art

[0002] my country is rich in Chinese medicine resources. Chinese medicine is rich in nutrients such as amino acids and trace elements. It also contains effective active ingredients such as polysaccharides, brass, saponins, volatile oils, organic acids and alkaloids. The traditional method of using Chinese medicine is generally to take it after simple decoction, and the remaining medicine residue is discarded, which not only causes waste but also fails to fully extract the effective ingredients of Chinese medicine, resulting in the inability to fully exert its medicinal value. There are three main methods for extracting effective ingredients of Chinese medicine. One is solvent extraction, which mainly uses organic solvents as extractants; one is steam distillation, which uses steam above 100°C to achieve the purpose of extraction; the second is sublimation. After the solid Chinese medicine is directly heated, its effective ingredients are gasified and condensed and collected. However, the operating steps of these two extraction methods are relatively cumbersome, and they have virtually increased many process steps and costs. Therefore, it is necessary to find an efficient Chinese medicine extraction and effective method. Chinese medicine fermentation is another important method for processing Chinese medicine. It uses enzymes and organic acids produced during the growth and metabolism of probiotics to process Chinese medicine. Microorganisms produce a variety of extracellular enzymes during the metabolic process, such as cellulase, protease, pectinase, etc., which can decompose the cell walls of Chinese herbal plant cells, cause cell rupture, and dissolve a large amount of biologically active ingredients. They can also decompose many large-molecule effective active substances that cannot be absorbed and utilized by the human body into small-molecule active substances. The probiotics-transformed Chinese medicine has a mild effect on the extraction of active ingredients of Chinese medicine, which can retain the natural active ingredients of Chinese medicine to the greatest extent, and also produce new effective active ingredients, thereby increasing the extraction rate of effective ingredients of Chinese medicine and improving the efficacy of Chinese medicine. At the same time, microorganisms can also decompose certain toxic components in Chinese medicine, thereby reducing its toxic side effects and making it safer to use.

[0003] There are many kinds of microorganisms, and the enzyme systems they produce are different. The substances that microorganisms metabolize and utilize are also different. Not all strains are suitable for fermentation, and not all Chinese medicines are suitable for fermentation. The efficacy of most Chinese medicines does not change significantly after fermentation, while the efficacy of a few Chinese medicines decreases significantly after fermentation. Therefore, it requires a lot of experimental research and demonstration to select appropriate fermentation strains according to different Chinese medicines to enhance the efficacy of Chinese medicines after fermentation, increase the extraction rate of effective ingredients, reduce toxic and side effects, produce new active ingredients, and maximize the utilization rate of drugs. At the same time, since the effective active ingredients contained in a single Chinese medicine are not enough to meet the skin's needs for various nutrients, and the use of multiple Chinese medicines in combination also requires higher requirements for fermentation strains, how to make the compounded Chinese medicines work together to exert better efficacy has broad application prospects and development value. For example, honeysuckle, frankincense, myrrh, angelica, red peony root, etc. have a long tradition of use as traditional medicinal materials and foods. There are few studies on the fermentation of one of the ingredients alone or in combination with other ingredients. At the same time, the skin care effects of the fermented products of these Chinese medicines have not been effectively developed, and it is unclear which specific probiotics or which specific Chinese medicine combinations can achieve better skin beautifying effects. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a composition and a fermentation product thereof and application thereof in the preparation of cosmetics.

[0005] The composition provided by the invention comprises honeysuckle, frankincense, myrrh, angelica, red peony root and bacterial strains; wherein the bacterial strains comprise saccharomyces cerevisiae and lactobacillus, wherein the lactobacillus can produce cellulase.

[0006] The composition provided by the present invention is composed of two parts: a traditional Chinese medicine and a bacterial strain, wherein each part can be mixed or exist independently. Each part can exist in the form of a liquid or a solid, for example, a solution, an extract or a powder, which is not limited by the present invention.

[0007] Early experiments show that honeysuckle, frankincense, myrrh, angelica and red peony are used for compatibility, and each medicinal taste cooperates with each other, coordinates with each other, and plays a good oil-suppressing, anti-acne, anti-oxidation and / or anti-inflammatory effect together. Each component is indispensable, and under a specific ratio, a more significant effect can be exerted. In the composition of the present invention, the mass ratio of honeysuckle, frankincense, myrrh, angelica and red peony is (1-5): (0.5-2): (0.5-2): (1-3): (1-3). In some embodiments, the mass ratio of honeysuckle, frankincense, myrrh, angelica and red peony is 5:1:1:3:3, or 1:2:2:3:3, or 4:2:1:2:2, or 5:1:2:2:3, or 0.5:3:2:2:3. Among them, when the mass ratio of honeysuckle, frankincense, myrrh, angelica and red peony root is 5:1:1:3:3 or 1:2:2:3:3, the obtained fermentation product has the best efficacy, which is significantly better than the effects obtained at other ratios.

[0008] In the strains described in the present invention, the two strains can exist as a composite strain or can exist independently of each other. They can be bacterial powder, bacterial suspension or frozen strains, which are not limited in the present invention. In the present invention, the brewer's yeast is BNCC337309; in the present invention, the lactobacillus is Lactobacillus plantarum 5b4m2.

[0009] Furthermore, the present invention also provides a fermentation product, which is obtained by fermenting the composition as described above.

[0010] The preparation method of the fermentation product of the present invention comprises the steps of extracting the components of traditional Chinese medicine to obtain an extract, and then fermenting the extract with bacteria.

[0011] Among them, honeysuckle, frankincense, myrrh, angelica, and red peony can be extracted separately and then fermented with lactobacillus and yeast respectively, and then merged to obtain fermentation products; at least two of honeysuckle, frankincense, myrrh, angelica, and red peony can also be combined and extracted, and then fermented with lactobacillus and yeast respectively; or honeysuckle, frankincense, myrrh, angelica, and red peony are combined and extracted and then fermented with lactobacillus and yeast respectively. Or honeysuckle, frankincense, myrrh, angelica, and red peony can be extracted separately in the Chinese medicine components, and then co-fermented with lactobacillus and yeast, or at least two of honeysuckle, frankincense, myrrh, angelica, and red peony are combined and extracted, and then co-fermented with lactobacillus and yeast, or honeysuckle, frankincense, myrrh, angelica, and red peony are combined and extracted and then co-fermented with lactobacillus and yeast. The specific fermentation order is not limited in the present invention. As preferably, the preparation of fermentation products includes the following three methods:

[0012] In some embodiments, the method for preparing the fermentation product comprises:

[0013] After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid;

[0014] The activated seed liquid of the brewer's yeast is mixed with the liquid to be fermented, and ripened at 32-37° C. for 3-5 hours under a closed condition to obtain a first product;

[0015] The activated seed liquid of the lactobacillus is mixed with the liquid to be fermented, and ripened at 32-37° C. for 3-5 hours under closed conditions to obtain a second product;

[0016] The first product and the second product are mixed, and matured at 32-37° C. for 3-5 hours under closed conditions to obtain a fermentation product.

[0017] In this embodiment, the preparation of the activated seed solution of Saccharomyces cerevisiae includes inoculating Saccharomyces cerevisiae into a YPD medium for activation until the bacterial content is 10 7 ~10 8 The preparation of the lactobacillus seed solution comprises inoculating lactobacillus into MRS medium for activation until the bacterial content is 10 9 ~10 10 cfu / ml. The volume ratio of the activated Lactobacillus seed liquid to the liquid to be fermented is 1:(1-10); the volume ratio of the activated Saccharomyces cerevisiae seed liquid to the liquid to be fermented is 1:(1-10). The first product and the second product are mixed in equal volumes.

[0018] In some other embodiments, the method for preparing the fermentation product comprises:

[0019] After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid;

[0020] After adding carbon source A and nitrogen source A to the fermented liquid, inoculating lactobacillus, and fermenting at 35-37° C. for 24-32 hours to obtain product A;

[0021] After adding carbon source B and nitrogen source B to the fermented liquid, inoculating brewer's yeast, and fermenting at 25-28° C. for 18-24 hours to obtain product B;

[0022] The product A and the product B are mixed to obtain a fermentation product.

[0023] In this embodiment, the inoculation amount of lactobacillus is 5%, the inoculation amount of yeast is 8%, and product A and product B are mixed in equal volumes.

[0024] In some other embodiments, the method for preparing the fermentation product comprises:

[0025] After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid;

[0026] After adding carbon source C and nitrogen source C to the fermented liquid, inoculate lactobacillus, ferment at 35-37°C for 24-32 hours, inactivate, add carbon source D and nitrogen source D, inoculate saccharomyces cerevisiae, ferment at 25-28°C for 18-24 hours, and obtain a fermentation product.

[0027] In this embodiment, the inoculation amount of Lactobacillus is 5%, and the inoculation amount of yeast is 8%.

[0028] In the present invention, honeysuckle, frankincense, myrrh, angelica, and red peony root are ground into a particle size of 100 to 200 meshes as Chinese medicine powder. 100 mL of water is used to prepare a fermentation liquid for every 7.8 to 13 g of Chinese medicine powder. The preparation method includes extraction, reflux extraction, decoction or distillation. In the present invention, after the Chinese medicine is ultrafinely ground, it is dissolved in water to prepare a fermentation liquid. Citric acid is used to adjust the pH value, and after adjusting the pH, a pasteurization step is also included. The pasteurization conditions include: 65°C constant temperature water bath for 30 minutes.

[0029] In the present invention, the nitrogen sources A, B, C, and D are independently selected from ammonium sulfate, yeast extract, and ammonium chloride; the carbon sources A, B, C, and D are independently selected from glucose, fructose, or maltose. The amount of the carbon source added is 5wt% to 20wt%, and the amount of the nitrogen source added is 5wt% to 20wt%. The amounts of the nitrogen source and the carbon source added in each step may be the same or different.

[0030] After the fermentation product is obtained by the method as described above, the method further includes the steps of centrifugation, filtration and sterilization, and separation to obtain a supernatant. Furthermore, the supernatant is also diluted and / or freeze-dried.

[0031] In the present invention, the main thing used in the fermentation process is the rich enzyme metabolites formed by the strain after self-fermentation, and the Chinese medicine composition is subjected to ripening enzymolysis. During the enzymolysis process, the appropriate temperature and the closed environment can be ensured. During the enzymolysis process, live bacteria are not required to work, and the growth and reproduction of bacteria are not required. Early experiments have shown that the preparation of fermentation products by metabolizing the active ingredients of Chinese medicine with live bacteria can also obtain certain oil control, anti-acne, anti-oxidation or anti-inflammatory effects. However, the effect is not as good as the process of ripening enzymolysis only. In addition, during the combined fermentation, it is necessary to select suitable fermentation conditions and a long fermentation time to ensure the normal growth and reproduction metabolism of the strain, and the conditions need to be strictly controlled. The method of sealed ripening with seed liquid in the present invention can not only simplify the process, but also improve the physiological activity of the product.

[0032] The study found that two agings are more conducive to the production of physiological activity of the fermentation product, and that the same aging time has significant differences in efficacy. By adjusting the aging time and aging conditions, the present invention obtains a fermentation product with better physiological activity.

[0033] Furthermore, the present invention also provides the use of the composition, fermentation product, and fermentation product obtained by the preparation method as described above in the preparation of cosmetics.

[0034] The cosmetics prepared by the composition and fermentation product of the present invention have the effects of oil inhibition, anti-acne, anti-oxidation and / or anti-inflammatory. In the present invention, the anti-acne includes inhibiting Propionibacterium acnes. The anti-oxidation includes anti-ROS and scavenging DPPH. The anti-inflammatory includes inhibiting the level of inflammatory factors. The inflammatory factors are interleukins. Specifically, IL-1β and IL-8.

[0035] The present invention also provides an oil-suppressing, anti-acne, anti-oxidation and / or anti-inflammatory cosmetic, which comprises the above-mentioned composition, fermentation product, and fermentation product obtained by the preparation method.

[0036] The cosmetics described in the present invention refer to chemical industrial products or fine chemical products that are spread on any part of the human body surface, such as skin, hair, nails, lips and teeth, by smearing, spraying or other similar methods, to achieve the purpose of cleaning, maintenance, beautification, modification and change of appearance, or correction of human body odor and keeping in good condition. They include facial cleansers, bath gels, shampoos, lotions, perfumes, skin cleansing water, makeup removers, essences, stock solutions, honey, milk, hair care milk, essence milk, face cream, foundation cream, shampoo cream, concealer cream, hair treatment cream, essence cream, pre-makeup cream, scented powder, talcum powder, loose powder, cleansing powder, honey powder, pressed powder, blush powder, lipstick, hair wax or makeup remover oil, moisturizing oil, hair oil, essence oil, facial mask, eye mask, lip mask or hand mask.

[0037] The present invention also provides a method for inhibiting oil, resisting acne, resisting oxidation and / or resisting inflammation, which comprises administering the cosmetic of the present invention. The administration method includes but is not limited to smearing, spraying and applying.

[0038] The invention prepares a composition with honeysuckle, frankincense, myrrh, angelica, red peony root and bacterial strains, and studies the effects of fermenting the traditional Chinese medicine composition by different combined fermentation methods. The results show that the fermentation of plant lactobacillus and yeast significantly promotes the release of skin care functional substances in the traditional Chinese medicine composition, significantly improves its skin care efficacy, and the obtained composite fermentation liquid has the effects of inhibiting oil, inhibiting inflammation, inhibiting Propionibacterium acnes and anti-oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The relative secretion rate of oil in each group is shown;

[0040] Figure 2 The inhibition rate of each group on Propionibacterium acnes is shown;

[0041] Figure 3 Shows the ROS levels in each group;

[0042] Figure 4 Shows the DPPH inhibition rate of each group;

[0043] Figure 5 The relative inhibition rate of IL-1β in each group is shown;

[0044] Figure 6 The relative inhibition rate of IL-8 in each group is shown. DETAILED DESCRIPTION

[0045] The present invention provides a composition and a fermentation product thereof and an application thereof in the preparation of cosmetics. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] In the present invention, the raw materials of the Chinese medicine composition include honeysuckle, frankincense, myrrh, angelica, and red peony root. The raw materials can be synergistically combined, and the protein, starch, trace elements, etc. contained therein can also provide raw materials for the fermentation of microorganisms. By using the plant lactobacillus of this laboratory and the purchased brewing yeast to jointly ferment the Chinese medicine composition, the various types of effective substances and various active enzymes produced by microorganisms during the growth process are used to promote the increase in the content of different effective ingredients of the Chinese medicine composition, thereby improving its overall skin care effect. Among them, plant lactobacillus and brewing yeast can be obtained by purchase. Lactobacillus plantarum 5b4m2 was deposited in the China Center for Type Culture Collection on June 14, 2022, with a deposit number of CCTCC No: M2022885, and the deposit address is Wuhan University, Wuhan, China. Other lactobacilli, such as Lactococcus lactis cb18m1, Lactobacillus acidophilus 5B5Y5, Lactobacillus plantarum cb41m2, and Lactobacillus salivarius cb13m2, all come from the Guangdong National Nanotechnology Innovation Institute of the Guangdong-Hong Kong-Macao Greater Bay Area.

[0047] Saccharomyces cerevisiae can be obtained through commercial channels. The strain number of Saccharomyces cerevisiae is BNCC337309, which was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0048] The method for preparing the composite fermentation filtrate of the traditional Chinese medicine composition provided by the invention comprises the following steps: using honeysuckle, frankincense, myrrh, angelica and red peony root as the traditional Chinese medicine composition to prepare a fermentation liquid.

[0049] The preparation of the Chinese medicine fermentation liquid comprises: the Chinese medicine composition has a ratio of honeysuckle: frankincense: myrrh: angelica; red peony root = 1-5:0.5-2:0.5-2:1-3:1-3, the Chinese medicine composition is crushed and passed through a 100-200 mesh sieve, deionized water is used to prepare the Chinese medicine fermentation liquid, 5-15 parts of the Chinese medicine composition are added to 100 parts of deionized water by weight, the pH of the fermentation liquid is adjusted to 5.0-6.0 with citric acid, the fermentation liquid is pasteurized in a 65°C constant temperature water bath for 30 minutes, the fermentation strain is selected from plant lactobacillus 5b4m2 and saccharomyces cerevisiae, and the optimized fermentation steps are as follows:

[0050] 1) Lactobacillus primary culture: 5% of Lactobacillus plantarum seed solution was inoculated into MRS medium for fermentation and cultured at 25°C-37°C for 24-32h to obtain a Lactobacillus primary culture with a bacterial content of 10 9 ~10 10 cfu / ml.

[0051] 2) Primary culture of Saccharomyces cerevisiae: Prepare the seed solution of Saccharomyces cerevisiae, inoculate the seed solution into YPD medium at an inoculum rate of 8% for fermentation and culture at 28°C for 18-24h to obtain the primary culture of Saccharomyces cerevisiae with a bacterial content of 10 7 ~10 8 cfu / ml.

[0052] 3) Ripening: After fermentation is completed, a ripening treatment is performed, wherein the primary culture of Lactobacillus and the Chinese medicine to be fermented liquid are mixed in a ratio of 1:1-10 and ripened in a closed state at 32°C-37°C for 3-5h to obtain a first product, and the primary culture of Saccharomyces cerevisiae and the Chinese medicine to be fermented liquid are ripened in a closed state at 32°C-37°C for 3-5h to obtain a second product, and the first fermentation product and the second fermentation product are mixed in equal volumes and then continued to be ripened in a closed state at 32°C-37°C for 3-5h.

[0053] 4) Filtration: The supernatant of the composite fermentation is obtained by centrifugation and filtration for sterilization.

[0054] The composite fermentation filtrate can be used in the preparation of external skin preparations as an active ingredient for antibacterial, anti-oil, anti-oxidation, anti-inflammatory, etc., and its dosage is 1%-10%. It can be added to daily chemical products such as cleansing cream, lotion, cream, essence, toner (such as softener, toner), and facial mask.

[0055] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0056] Example 1

[0057] 1. Screening of lactic acid bacteria

[0058] Use cellulose culture medium screening plate to screen strains with high cellulase activity. Then use an inoculation needle to activate the isolated strains and then inoculate them on the cellulose screening medium plate. Incubate them at 30℃ for 2 days. Stain them with 0.1% Congo red staining solution and then decolorize them with 1mol / L NaCl solution for 5 minutes. If the strain produces cellulase, a clear transparent circle will appear around the colony.

[0059] Table 1: Cellulase production results

[0060] Strain number Strain name Cellulase 5B5Y5 Lactobacillus acidophilus + cb41m2 Lactobacillus plantarum + cb13m2 Lactobacillus salivarius + 5b4m2 Lactobacillus plantarum + cb18m1 Lactococcus lactis -

[0061] The strains were screened for cellulase production, and the results are shown in Table 1, showing that Lactobacillus acidophilus 5B5Y5, Lactobacillus plantarum cb41m2, Lactobacillus salivarius cb13m2 and Lactobacillus plantarum 5b4m2 can all produce cellulase.

[0062] 2. Fermentation Steps

[0063] 1. Activate lactic acid bacteria and yeast strains and prepare seed solution: In a sterile environment, use an inoculation loop to select strains from the glycerol cryopreservation tube of Lactobacillus plantarum, streak inoculate on MRS solid slant culture, observe the colony morphology on the plate, pick a single bacterium on the plate and inoculate it into MRS liquid culture medium, shake flask culture to prepare seed solution, and culture at 37°C for 24 h; In a sterile environment, use an inoculation loop to select strains from the glycerol cryopreservation tube of Saccharomyces cerevisiae, streak inoculate on YPD solid slant culture, observe the colony morphology on the plate, pick a single bacterium on the plate and inoculate it into YPD liquid culture medium, shake flask culture to prepare seed solution, and culture at 28°C for 24 h. (YPD medium: glucose 2%, tryptone 2%, yeast powder 1%, initial pH 6.5. MRS medium: tryptone 1%, K2HPO4 0.2%, glucose 2%, sodium acetate 0.5%, triammine citrate 0.2%, beef extract 1%, yeast powder 0.5%, MgSO4·7H2O 0.04%, Tween-80 0.1mL, MnSO4·H2O 0.005%, initial pH 6.4.)

[0064] 2. Preparation of the fermented liquid: The ratio of the Chinese medicine composition is honeysuckle: frankincense: myrrh: angelica; red peony root = (1-5): (0.5-2): (0.5-2): (1-3): (1-3), which are crushed and passed through a 100-200 mesh sieve, dissolved in deionized water to prepare the Chinese medicine fermented liquid, the pH of the fermented liquid is adjusted to 5.0-6.0 with citric acid, and the fermented liquid is pasteurized in a constant temperature water bath at 65°C for 30 min.

[0065] 3. Fermentation:

[0066] Fermentation method 1: After fermentation, the plant lactobacillus seed liquid was inoculated into MRS medium at a 5% inoculation rate for fermentation and culture at 35-37°C for 24-32h to obtain the primary culture of lactobacillus with a bacterial content of 10 9 ~10 10 cfu / ml. At the same time, the brewer's yeast seed liquid was inoculated into the YPD medium at an inoculation rate of 8% for fermentation and cultured at 25-28°C for 18-24 hours to obtain the brewer's yeast primary culture with a bacterial content of 10 7 ~10 8 cfu / ml, the primary culture of Lactobacillus and the primary culture of Saccharomyces cerevisiae are respectively mixed with the fermented liquid in a ratio of 1: (1-10) and matured at 32°C-37°C for 3-5h in a closed state to obtain the first product and the second product respectively. Finally, the first product and the second product are mixed in equal volumes and further matured at 32°C-37°C for 3-5h in a closed state.

[0067] Fermentation method 2: fermentation of Chinese medicine separately, inoculate the plant lactobacillus seed liquid into the fermented liquid (5wt%-20wt% glucose and 5wt%-20wt% yeast extract are added to the fermented liquid) at an inoculation rate of 5%, and culture at 35-37°C for 24-32h. Inoculate the saccharomyces cerevisiae seed liquid into the fermented liquid (5wt%-20wt% glucose and 5wt%-20wt% ammonium sulfate are added to the fermented liquid) at an inoculation rate of 8%, and culture at 25-28°C for 18-24h. Mix the fermentation products of the two separate fermentations in equal volumes.

[0068] Fermentation method 3: step-by-step fermentation, inoculate plant lactobacillus seed liquid with 5% inoculation amount into the fermented liquid (nitrogen source and carbon source, 5wt%-20wt% glucose, 5wt%-20wt% yeast extract are added to the fermented liquid), cultivate at 35-37°C for 24-32h, and heat inactivate after fermentation. Inoculate saccharomyces cerevisiae seed liquid with 8% inoculation amount again, and add 5wt%-20wt% glucose and 5wt%-20wt% ammonium sulfate added to the fermented liquid, and cultivate at 25-28°C for 18-24h.

[0069] 4. The supernatant of the composite fermentation is obtained by centrifugation and filtration sterilization.

[0070] 3. Screening of Chinese medicine components

[0071] According to the formulation in Table 2, fermentation was carried out according to the above method, using fermentation method 1, and then the oil inhibition and antioxidant level of the obtained product were tested.

[0072] Table 2

[0073]

[0074] The results showed that the combination of honeysuckle: frankincense: myrrh: angelica: red peony root = 3g:0.6g:0.6g:1.8g:1.8g had the strongest antioxidant capacity and the effect of inhibiting oil secretion.

[0075] Embodiment 2:

[0076] The ratio of the Chinese medicine composition in the fermented liquid is honeysuckle: frankincense: myrrh: angelica; red peony root = 3g: 0.6g: 0.6g: 1.8g: 1.8g) / 100ml.

[0077] The fermentation method 1 in Example 1 was used for fermentation, specifically: the Lactobacillus plantarum seed liquid was inoculated into 100 ml of MRS medium at an inoculum size of 5% for fermentation and cultured at 37°C for 24 h to obtain a primary culture of Lactobacillus with a bacterial content of 2×10 9 cfu / ml, 50 ml of the initial culture of Lactobacillus was mixed with 50 ml of the fermented liquid and then matured for 5 hours to obtain the first product. At the same time, the saccharomyces cerevisiae seed liquid was inoculated into the YPD medium at an inoculum rate of 8% for fermentation and cultured at 28°C for 18 hours to obtain the initial culture of saccharomyces cerevisiae with a bacterial content of 1.0×10 8 cfu / ml, 50ml of the initial culture of Saccharomyces cerevisiae was mixed with 50ml of the fermentation liquid and matured at 37℃ for 5h in a sealed state to obtain the second product. Finally, the first product and the second product were mixed in equal volumes in a sealed state at 37℃ for a second maturation for 3h, and the supernatant of the composite fermentation was obtained by centrifugation, filtration and sterilization, i.e., the composite fermentation liquid.

[0078] Embodiment 3:

[0079] The ratio of the Chinese medicine composition in the fermented liquid is honeysuckle: frankincense: myrrh: angelica; red peony root = 3g: 0.6g: 0.6g: 1.8g: 1.8g) / 100ml.

[0080] Fermentation was carried out by fermentation method 2, specifically, inoculating 100 ml of the fermented liquid with Lactobacillus plantarum seed liquid at an inoculation rate of 5%, adding nitrogen source and carbon source, and culturing at 37° C. for 24 h. Inoculating 100 ml of the fermented liquid with Saccharomyces cerevisiae seed liquid at an inoculation rate of 8%, adding nitrogen source and carbon source, and culturing at 28° C. for 18 h, taking 50 ml of the fermentation products of the two fermentations in equal volumes, mixing, centrifuging, filtering and sterilizing to obtain the supernatant of the composite fermentation.

[0081] Embodiment 4:

[0082] The ratio of the Chinese medicine composition in the fermented liquid is honeysuckle: frankincense: myrrh: angelica; red peony root = 3g: 0.6g: 0.6g: 1.8g: 1.8g) / 100ml.

[0083] Fermentation was carried out using fermentation method 3, specifically, the following steps: inoculating plant lactobacillus seed liquid into 100 ml of a fermented liquid (with nitrogen source and carbon source added) at an inoculation rate of 5%, culturing at 37° C. for 24 h, performing pasteurization and heat inactivation after the fermentation was completed, inoculating saccharomyces cerevisiae seed liquid again at an inoculation rate of 8%, and adding nitrogen source and carbon source at the same time, culturing at 28° C. for 18 h, and obtaining a composite fermentation supernatant by centrifugation and filtration sterilization after the fermentation was completed.

[0084] Example 5-17

[0085] As shown in Table 3, the experiment was carried out according to the same steps as in Example 2 to obtain various composite fermentation broths.

[0086] Table 3:

[0087]

[0088] Comparative Example 1:

[0089] The fermentation method 2 in Example 1 was used for treatment, except that Lactobacillus plantarum was added: Lactobacillus plantarum seed solution was inoculated into 100 ml of the fermented liquid at an inoculum amount of 5% (the mass ratio of honeysuckle: frankincense: myrrh: angelica: red peony root was 3g:0.6g:0.6g:1.8g:1.8g), and nitrogen source and carbon source were added, and cultured at 37°C for 24h.

[0090] Comparative Example 2:

[0091] The fermentation method 2 in Example 1 was used for treatment, except that only Saccharomyces cerevisiae was added: the Saccharomyces cerevisiae seed solution was inoculated into 100 ml of the fermented liquid at an inoculation rate of 8% (the mass ratio of honeysuckle: frankincense: myrrh: angelica: red peony root was 3g:0.6g:0.6g:1.8g:1.8g), and a nitrogen source and a carbon source were added, and the culture was carried out at 28°C for 18h.

[0092] Comparative Example 3:

[0093] No bacteria were added for fermentation, and only the Chinese medicine composition (the mass ratio of honeysuckle: frankincense: myrrh: angelica: red peony root was 3g:0.6g:0.6g:1.8g:1.8g) was dissolved in 100ml of deionized water.

[0094] Experimental testing

[0095] Cell efficacy evaluation

[0096] 1. Oil inhibition cell test

[0097] Methods: SZ95 cells were cultured at 3×10 4 Cells were inoculated at a density of 100 μg / well and cultured for 24 h before washing once with PBS.

[0098] Negative control: add fresh DMEM medium;

[0099] Model group: fresh culture medium containing 0.125% oleic acid was added;

[0100] Sample well: add fresh culture medium containing 0.125% oleic acid, and add the fermentation supernatant of each example or comparative example to a volume fraction of 10%. ;

[0101] After 24 hours of culture, discard the supernatant and prepare the oil red staining solution and staining process:

[0102] (1) Prepare the oil red working solution: Dissolve 1g of oil red in 200ml of isopropanol, mix well and stir to dissolve, prepare the storage solution, and store at 4 degrees in the dark. Prepare the working solution with a ratio of 3:2 of oil red storage solution to ultrapure water, let it stand for a while, and filter it with filter paper.

[0103] (2) The adipocytes were washed once with PBS, fixed with 4% formaldehyde for 5 min, discarded, washed once with PBS, and then treated with 60% isopropanol for 5 min and discarded.

[0104] (3) Staining: Add 120 μl of Oil Red working solution to each well and stain in the dark for 1 hour.

[0105] (4) Washing solution: After staining, use a vacuum pump to remove the dye solution, add 200ul of 60% isopropanol to each well, treat for 3 minutes, and discard. Add 200ul of 100% isopropanol to each well, protect from light, and incubate on a shaker for 5 minutes to fully wash away the oil red bound to fat.

[0106] (5) Transfer 150 μl of sample to a 96-well plate and measure the absorbance at OD500 nm using a microplate reader.

[0107] Table 4:

[0108] Group Relative oil secretion rate Control group 0 Model Group 100％ Example 2 18.32％ Example 3 35.34％ Example 4 44.19％ Example 5 72.32％ Example 6 81.34％ Example 7 61.18％ Example 8 23.87％ Example 9 15.91％ Example 10 19.15％ Embodiment 11 36.97％ Example 12 43.21％ Embodiment 13 27.29％ Embodiment 14 31.23％ Embodiment 15 57.92％ Example 16 19.31％ Embodiment 17 49.91％ Comparative Example 1 49.39％ Comparative Example 2 54.11％ Comparative Example 3 68.60％

[0109] From Table 2 and Figure 1It can be seen that the composite fermentation liquid in this scheme has a good inhibitory effect on oil secretion, with an inhibition rate of more than 72.71%, and the Chinese medicine compositions have a synergistic effect. When the ratio is not within the scope of this scheme, such as Example 11, the oil secretion inhibition rate is significantly reduced. Among them, compared with the model group, the relative oil secretion rates of the embodiments and comparative examples to which active substances are added are significantly reduced. Embodiments 2, 3, and 4 are composite fermentation liquids prepared by different fermentation methods. The results show that the relative oil secretion rate of fermentation method 1 is the lowest. Embodiments 2, 5, 6, and 7 are composite fermentation liquids using different lactic acid bacteria. The results show that the relative oil secretion rate of the composite fermentation liquid using plant lactobacillus 5b4m2 is the lowest. Embodiments 2, 12, 13, 14, and 15 are different post-ripening times, showing that with the extension of the post-ripening time, the relative oil secretion first decreases and then increases. The post-ripening time is preferably 3-5h, and the relative oil secretion rate is 37.29% to 18.32%, that is, the oil secretion rate is inhibited by 62.71% to 81.68%.

[0110] 2. Experiment on inhibiting Propionibacterium acnes

[0111] Prepare Propionibacterium acnes in the logarithmic growth phase, the number of which is about 1×10 8 cfu / ml~1×10 9 cfu / ml, the pathogen cultured to the logarithmic growth phase was inoculated into the blank medium at a 10% inoculation rate. The Propionibacterium acnes cultured to the logarithmic growth phase was taken and diluted with blank BHI medium to obtain a culture medium containing 10 cfu / ml of P.ances. 5 CFU / ml of bacterial suspension.

[0112] Preparation of antibacterial system: Prepare antibacterial systems with different sample concentrations for group testing. Negative control: 1000ul bacterial suspension + 400ul PBS / blank culture medium (the same solvent as the test sample); 10% concentration composite fermentation broth: 1000ul Propionibacterium acnes suspension + 140ul fermentation supernatant prepared in each embodiment or comparative example + 260ul PBS / blank culture medium. The prepared antibacterial systems of various concentrations were placed in a 37°C incubator for 48 hours, and then the bacterial content of each system was detected. The antibacterial rate of the raw material was calculated according to the antibacterial effect test method (suspension quantitative method) of antibacterial daily chemical products in QB / T 2738-2012 "Evaluation method for antibacterial and antibacterial effects of daily chemical products". (Note: If the antibacterial rate is ≥50% to 90%, the product has an antibacterial effect; if the antibacterial rate is ≥90%, the product has a strong antibacterial effect.)

[0113] Table 5:

[0114] Group Antibacterial rate (%) Negative control 0％ Example 2 100％ Example 3 99.30％ Example 4 97％ Example 5 86.70％ Example 6 74.00％ Example 7 91.30％ Example 8 99％ Example 9 99％ Example 10 100％ Embodiment 11 100％ Example 12 93％ Embodiment 13 97.30％ Embodiment 14 99.90％ Embodiment 15 91.40％ Example 16 99.21％ Embodiment 17 100％ Comparative Example 1 85.10％ Comparative Example 2 94.50％ Comparative Example 3 95％

[0115] From the above table and Figure 2It can be seen that the composite fermentation liquid and the traditional Chinese medicine mixed liquid prepared in this scheme both have a strong inhibition rate on Propionibacterium acnes.

[0116] 3. ROS scavenging assay of HaCaT keratinocytes

[0117] Cells: Primary human keratinocytes HaCaT

[0118] Materials: DCFH-DA, 3% hydrogen peroxide (H2O2) solution.

[0119] Stimulation conditions: Hydrogen peroxide (H2O2) induced an increase in the ROS content in keratinocytes. The concentration of hydrogen peroxide used was 120 μM and the treatment time was 1 hr.

[0120] Experimental method: 1×10 4 / well (200ul) (the degree of fusion reaches 45%-60% 24h after inoculation) is inoculated into a 96-well plate. After the inoculation is completed, it is placed in a CO2 incubator for incubation for 24h±2h. The culture medium in each well of the well plate is discarded, and the experimental group is added with the prepared DMEM culture medium containing 5vol% of the fermentation supernatant of each embodiment and comparative example, and normal culture medium is added to the blank control well and the model well. After the addition of the sample, the well plate is placed in a CO2 incubator for 24h±2h. After the incubation is completed, the liquid is not discarded. Except for the blank control, the remaining wells need to be induced with H2O2. The induction time of H2O2 is 1hr, and the induction dose is 120μM. Discard the culture medium in the wells, wash three times with PBS, add 100uL DCFH-DA working solution (sampling according to the instructions of the Biyuntian Reactive Oxygen Detection Kit) to each well, incubate in a CO2 incubator for 30min, and after the incubation, wash the cells in each well three times with serum-free DMEM. After the operation, place the 96-well plate on the detection platform of the fluorescent microplate reader, set the incident light wavelength to 525nm, the excitation light wavelength to 488nm, and read the readings.

[0121] Table 6:

[0122]

[0123]

[0124] From the above table and Figure 3It can be seen that the active oxygen free radical ROS scavenging effect of Example 2, Example 9 and Example 10 is the best. At the same time, when the composite fermentation liquid is prepared by different fermentation methods, fermentation method 1 has a better active oxygen free radical ROS scavenging effect than fermentation method 2 and fermentation method 3, and the free radical scavenging ability of its composite fermentation liquid is better than that of the mixed liquid of the Chinese medicine composition. At the same time, the selection of which strain will affect the release of the antioxidant active ingredients of the Chinese medicine composition. Among them, the active oxygen free radical ROS scavenging effect of the combined fermentation of Lactobacillus plantarum 5b4m2 and Saccharomyces cerevisiae is the best, and when the post-ripening time is 3-5h, it has better ROS free radical scavenging ability and strong antioxidant capacity.

[0125] 4. DPPH clearance rate

[0126] Preparation of DPPH test solution: Dissolve 4 mg of DPPH in about 10 mL of anhydrous ethanol, shake thoroughly to mix evenly, and the working concentration is 0.1 mM.

[0127] 100ul blank MRS and 100ul anhydrous ethanol were added to well A0, 100ul anhydrous ethanol solution and 100μL 0.2mmol / L DPPH anhydrous ethanol solution were added to well A1, 100ul of 5vol% supernatant of the composite fermentation broth prepared in each embodiment or comparative example (5% fermentation broth + 95%) was taken to well A2 of the multi-well plate, and then 100μL 0.2mmol / L DPPH anhydrous ethanol solution was added to well A2, mixed, incubated at 37°C in a dark environment for 30min, and the absorbance was measured at 517nm by a microplate reader.

[0128] Calculation method of free radical scavenging rate: (A1-A2) / (A1-A0)×100%

[0129] (A0 is the OD value after adding blank MRS plus anhydrous ethanol, A1 is the OD value after adding anhydrous ethanol for 0 hours, and A2 is the OD value after adding the composite fermentation supernatant and incubating for 30 minutes)

[0130] Table 7:

[0131]

[0132]

[0133] From the above table and Figure 4 It can be seen that Examples 2, 3, 9 and 10 have better DPPH free radical scavenging effect and better antioxidant effect than the single Chinese medicine mixture and other Examples.

[0134] 5. Anti-inflammatory test

[0135] Induce THP-1 into inflammatory cells: centrifuge the cultured Propionibacterium acnes bacterial suspension (the OD490 absorbance of the bacterial suspension is 1.3-1.5) at 1000 rpm for 5 min, discard the supernatant, resuspend the bacteria with PBS, centrifuge and discard the supernatant, repeat 3 times, remove impurities outside the bacteria, and resuspend the bacteria with 1640 culture medium of the original volume of the bacterial suspension to prepare Propionibacterium acnes working solution; culture THP-1 cells until they are in good condition, collect THP-1 cells, count and adjust the cell density to 10 using the Propionibacterium acnes working solution 6 / mL, plated into 48-well plates, and induced for 12-24h.

[0136] Addition of active substances: Add 5% of the compound fermentation broth and culture in a carbon dioxide incubator for 24 hours;

[0137] Model group: complete culture medium + cells + Propionibacterium acnes working solution; Experimental group: complete culture medium + cells + Propionibacterium acnes working solution + composite fermentation solution;

[0138] Collect cell culture supernatant: Use a sterile tube to collect the cell culture supernatant, and centrifuge for 10 minutes before collecting the supernatant;

[0139] ELISA kit detection

[0140] (1) Addition of standard samples: set up standard sample wells and sample wells, add 50 μL of standard samples of different concentrations to each standard sample well, and add 50 μL of sample diluent to well 0;

[0141] (2) Sample addition: Set up blank wells (blank control wells without adding sample and enzyme-labeled reagent, and the rest of the steps are the same) and test sample wells (fermentation broth supernatant prepared in each embodiment or comparative example). Add 50 μL of sample to the test sample well on the enzyme-labeled plate, add to the bottom of the well, try not to touch the well wall, and gently shake to mix.

[0142] (3) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank well.

[0143] (4) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 min.

[0144] (5) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0145] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 60 seconds and then discard. Repeat this process 5 times and pat dry.

[0146] (7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 15 min.

[0147] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0148] (9) Determination: Use the blank well as the zero setting and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be performed within 15 minutes after adding the stop solution.

[0149] Table 8:

[0150]

[0151] According to the above table and Figures 5-6 It is shown that, based on the comprehensive anti-inflammatory factors IL-1β and IL-8, Examples 2, 9, 10, 16, and 17 have better anti-inflammatory effects compared with other examples and the comparative example.

[0152] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fermentation product for preparing cosmetics, characterized in that: Prepared by fermenting the composition; The composition comprises honeysuckle, frankincense, myrrh, angelica, red peony root and bacterial strains; wherein the bacterial strains are composed of saccharomyces cerevisiae and lactobacillus, wherein the lactobacillus can produce cellulase; The mass ratio of honeysuckle, frankincense, myrrh, angelica and red peony root is (1-5): (0.5-2): (0.5-2): (1-3): (1-3); The preparation method of the fermentation product comprises the steps of extracting the components of the traditional Chinese medicine to obtain an extract, and then fermenting the extract with a strain of bacteria.

2. The fermentation product according to claim 1, characterized in that The strain number of the brewer's yeast is BNCC337309; the lactobacillus is Lactobacillus plantarum 5b4m2.

3. The method for preparing the fermentation product according to claim 1 or 2, characterized in that: include: After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid; The activated seed liquid of the brewer's yeast is mixed with the liquid to be fermented, and the mixture is matured at 32-37° C. for 3-5 hours under a closed condition to obtain a first product; The activated seed liquid of the lactobacillus is mixed with the liquid to be fermented, and ripened at 32-37° C. for 3-5 hours under closed conditions to obtain a second product; The first product and the second product are mixed, and matured at 32-37° C. for 3-5 hours under closed conditions to obtain a fermentation product.

4. The method for preparing the fermentation product according to claim 1 or 2, characterized in that: include: After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid; After adding carbon source A and nitrogen source A to the fermented liquid, inoculating lactobacillus, and fermenting at 35-37° C. for 24-32 hours to obtain product A; After adding carbon source B and nitrogen source B to the fermented liquid, inoculating brewer's yeast, and fermenting at 25-28° C. for 18-24 h to obtain product B; Mixing product A and product B to obtain a fermentation product; The nitrogen sources A and B are independently selected from ammonium sulfate, yeast extract, and ammonium chloride; The carbon sources A and B are independently selected from glucose, fructose or maltose.

5. The method for preparing the fermentation product according to claim 1 or 2, characterized in that: include: After crushing honeysuckle, frankincense, myrrh, angelica, and red peony root, dissolving them in water, adjusting the pH value to 5.0-6.0, and sterilizing to obtain the fermentation liquid; After adding carbon source C and nitrogen source C to the fermented liquid, inoculating lactobacillus, fermenting at 35-37° C. for 24-32 hours, adding carbon source D and nitrogen source D after inactivation, and then inoculating saccharomyces cerevisiae, fermenting at 25-28° C. for 18-24 hours to obtain a fermentation product; The nitrogen sources C and D are independently selected from ammonium sulfate, yeast extract, and ammonium chloride; The carbon sources C and D are independently selected from glucose, fructose or maltose.

6. Use of the fermentation product according to claim 1 or 2, or the fermentation product obtained by the preparation method according to any one of claims 3 to 5 in the preparation of cosmetics; The cosmetic has the effects of oil inhibition, anti-acne, anti-oxidation and / or anti-inflammatory.

7. An oil-suppressing, anti-acne, anti-oxidation and / or anti-inflammatory cosmetic, comprising the fermentation product according to claim 1 or 2, or a fermentation product obtained by the preparation method according to any one of claims 3 to 5.

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