Eurotium cristatum fermentation mycoplasm for promoting collagen generation and extract, preparation method and application of eurotium cristatum fermentation mycoplasm

Abstract

The invention provides a eurotium cristatum fermentation mycoplasm for promoting collagen generation, an extract thereof, a preparation method and an application of the eurotium cristatum fermentation mycoplasm. Eurotium cristatum is adopted to ferment phalaenopsis amabilis, the obtained fermentation mycoplasm and the extract of the fermentation mycoplasm have the effect of promoting skin fibroblasts to express type I collagen, the eurotium cristatum and phalaenopsis amabilis have a synergistic effect in a fermentation system, and the active ingredients and functions of the two products are greatly improved compared with those of traditional products; the compound is expected to be further developed into a cosmetic functional raw material, can be used for preparing an anti-skin-aging composition, and is simple in preparation method and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a fermentation product of a gold flower fungus for promoting collagen production, an extract thereof, a preparation method and application thereof. BACKGROUND

[0002] The secondary metabolites of orchid plants have been intensively studied, and the active ingredients are polysaccharides, phenols, alkaloids, organic sulfides, terpenes, etc., which have good skin care effects. In order to extract more active ingredients from butterfly orchids, the extraction process of butterfly orchids has been optimized in the market, such as hot reflux extraction, supercritical carbon dioxide extraction, etc. However, due to the fact that plant chemical substances mainly exist in the cell sap, and the plant cell wall contains a large amount of cellulose, pectin, lignin, etc., which hinders the extraction of active chemical substances in plants, causing resource waste. In patent CN 117017851 A, butterfly orchids are fermented by bifidobacterium, which produces more effective substances. However, bifidobacterium is an anaerobic bacterium, mainly existing in the digestive tract of humans and animals, and is a probiotic, but is not a dominant bacterium in terms of degrading cellulose. Therefore, in order to improve the development and utilization of butterfly orchid resources in the field of cosmetics, it is necessary to find a more suitable treatment method to improve the extraction rate and bioavailability of skin care functional ingredients of butterfly orchids, and to promote the good application of butterfly orchids in skin anti-aging. SUMMARY

[0003] The present application aims to overcome the above-mentioned deficiencies in the prior art, and provides a fermentation product of a gold flower fungus for promoting collagen production, an extract thereof, a preparation method and application thereof. The butterfly orchid fermentation product is mild, has little skin irritation, and can well improve the skin anti-wrinkle effect. Compared with liquid fermentation, solid-state fermentation can better improve the activity of the butterfly orchid fermentation product of the gold flower fungus, thereby better promoting the expression of skin collagen.

[0004] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: The first aspect of the present application is to provide a preparation method of a fermentation product of a gold flower fungus for promoting collagen production, comprising the following steps: S1, drying fresh-cut flowers of butterfly orchids at low temperature, steam sterilizing, and reserving; S2, inoculating a gold flower fungus activation liquid, the bacterial concentration of the gold flower fungus activation liquid being 1.0x10 5 ~1.0x10 7 cfu / mL; S3, culturing the inoculated butterfly orchid flowers at 28±2℃ and 65~85% RH humidity, to obtain the fermentation product of the gold flower fungus.

[0005] Further, in step S1, the moisture content of the dried fresh-cut flowers is controlled to be 5%~15%.

[0006] Further, in step S1, the steam sterilization is performed at a temperature of 100-125℃ for 10-30 minutes.

[0007] Further, in step S2, the inoculation amount is 0.8-1.2% of the activated bacterial solution based on the weight of the fresh-cut flowers.

[0008] The second aspect of the present application is to provide a fermented mycelium of the golden flower, which is prepared by the above method.

[0009] The third aspect of the present application is to provide an extract of the fermented mycelium of the golden flower, which is obtained by extracting the above-mentioned fermented mycelium in a solvent, wherein the solvent includes a polar solution or a non-polar solution.

[0010] Further, the polar solution includes water, ethanol or a eutectic solvent.

[0011] Further, the non-polar solution includes squalane, vegetable oil or supercritical carbon dioxide.

[0012] The fourth aspect of the present application is to provide an anti-aging composition, which includes the above-mentioned fermented mycelium of the golden flower or the above-mentioned extract.

[0013] The fifth aspect of the present application is to provide the use of the above-mentioned anti-aging composition in the preparation of daily-use cosmetics.

[0014] The sixth aspect of the present application is to provide a lip essence, which includes the above-mentioned fermented mycelium of the golden flower or the above-mentioned extract.

[0015] Compared with the prior art, the present application has the following advantages: The present application uses the golden flower to ferment the Phalaenopsis, and the fermented mycelium and the extract of the fermented mycelium have the effect of promoting the expression of collagen type I by skin fibroblasts. The golden flower and the Phalaenopsis have a synergistic effect in the fermentation system, and the active ingredients and functions of the two products are greatly improved compared with traditional products. The two products are expected to be further developed as functional raw materials for cosmetics, and can be used to prepare anti-aging compositions. Moreover, the preparation method is simple and suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Picture of the fermented mycelium prepared by solid-state fermentation of the golden flower on the Phalaenopsis prepared in Example 1; Figure 2 Picture of the fermented mycelium prepared by solid-state fermentation of the golden flower on the Phalaenopsis prepared in Example 2; Figure 3 Picture of the fermented mycelium prepared in Comparative Example 1; Figure 4 Mass spectrum of uvaol in the oil of Phalaenopsis for Example 2; Figure 5 Raman spectrum of transdermal test for Example 2 and Comparative Example 4; Figure 6 Human efficacy test chart for Example 3, wherein 3A is the improvement effect of lip line, 3B is the improvement effect of lip plumpness, and 3C is the improvement chart of lip contour. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present application clearer, the specific embodiments of the present application are further described in detail below with reference to the specific embodiments and the accompanying drawings. If the specific techniques or conditions are not specified in the embodiments, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained on the market.

[0018] The present application provides a preparation process of Phalaenopsis fermented by Chrysosporium sp. After the fermentation is completed by using the process, Chrysosporium sp. on the Phalaenopsis grows luxuriantly and can well ferment and convert the Phalaenopsis. The preparation process comprises the following steps: 1) Fresh-cut Phalaenopsis is dried at low temperature, and the water content is kept at 5% to 15% for standby 2) The Phalaenopsis is steamed, the steam temperature is 100°C to 125°C, and the time is about 10 to 30 min 3) 1% w / w of Chrysosporium sp. activation liquid is inoculated, the strain concentration of the activation liquid is 1.0×10 5 cfu / ml to 1.0×10 7 cfu / ml 4) The inoculated Phalaenopsis is cultured at 28±2°C and 65% to 85% RH for 5 days, and the pile is turned over once a day, and then dried for standby.

[0019] The present applicant found in the research that after the solid-state fermentation of Phalaenopsis, the ability of the Phalaenopsis extract to promote the expression of skin collagen can be increased whether polar solution extraction or non-polar solution extraction is used.

[0020] In some specific embodiments, the polar solution includes but is not limited to aqueous solution, ethanol solution, eutectic solvent, etc.

[0021] In some specific embodiments, the non-polar solution includes but is not limited to squalane, vegetable oil, supercritical carbon dioxide, etc.

[0022] In some specific embodiments, the extraction process can be direct immersion extraction, or one or more of stirring, ultrasonic wave, microwave, etc. can be used for auxiliary extraction.

[0023] The present applicant also found in the research that the fermented fungus of the present application or the extract thereof can promote the expression of collagen type 1 by skin fibroblasts, indicating that it has the effect of resisting skin aging.

[0024] The present application also provides an application of the Phalaenopsis flower fermentation product in cosmetics. The present application provides a cosmetic product comprising the Phalaenopsis flower fermentation product described in the above technical solution. In some specific implementation manners, the cosmetic product can be a lip essence oil, for example, comprising: the above-mentioned Phalaenopsis flower fermentation product oil solution and base oil.

[0025] In the present application, a typical formula of the essence oil comprises: 0.1-5% (w / w) of the above-mentioned Phalaenopsis flower fermentation product solution; 5-30% (w / w) of low-viscosity base oil; 5-80% (w / w) of medium-high-viscosity base oil; In some specific embodiments, the low-viscosity base oil includes but is not limited to ethylhexyl palmitate, coco-caprylate / caprate, caprylic / capric triglyceride, octyldodecanol, etc., including one or more thereof.

[0026] In some specific embodiments, the medium-high-viscosity base oil includes but is not limited to diisostearyl malate, VP / eicosene copolymer, dipolyglycerin dipolylinoleate, etc., including one or more thereof.

[0027] In some specific embodiments, the formula of the essence oil is: 1% (w / w) of the Phalaenopsis flower fermentation product oil solution; 24.75% (w / w) of coco-caprylate / caprate; 74.25% (w / w) of diisostearyl malate.

[0028] The present application has achieved some positive effects in the process of research and development or use, and indeed has great advantages compared with the prior art. The following content is described in combination with the data, charts, etc. of the test process.

[0029] The following examples of the number of strains of golden flower fungus (Eurotium cristatum): CICC 2099, China Industrial Microbial Culture Collection Center. 1,2-hexanediol and p-hydroxyacetophenone from Symrise Fragrance & Flavor (Nantong) Co., Ltd. Squalane from Empresa Figueirense de Pesca Lda. Phalaenopsis Sogo Yukidian is selected. The yeast is selected as Candida blank, and the strain number is CICC 33217, China Industrial Microbial Culture Collection Center.

[0030] Example 1 This example provides a golden flower fungus fermented fungus and an extract of the golden flower fungus fermented fungus, and the preparation method is as follows: 1) The fresh cut flowers of Phalaenopsis were dried at low temperature, and the water content was kept at 5% to 15%. 2) The Phalaenopsis flowers were steamed at 100°C for 20 minutes, and then cooled to room temperature.

[0031] 3) The golden flower fungus was inoculated on PDA flat plate medium and cultured at 28±2°C for 72 hours. The spores on the flat plate were scraped and collected, and the spore concentration was adjusted to about 2.0×10 5 Cfu / ml suspension to obtain the golden flower fungus activation liquid. 1% (w / w) of the golden flower fungus activation liquid was inoculated on the steamed Phalaenopsis. The inoculated Phalaenopsis was cultured at 28±2°C and 80±5% RH for 5 days, and the pile was turned over once a day. After fermentation, it was dried for standby, and the golden flower fungus fermented fungus was obtained.

[0032] 4) The golden flower fungus fermented fungus was crushed through a 40-mesh sieve, and purified water was added at a ratio of 1:10 (w / w) of the Phalaenopsis flowers. Stir for 2 hours, filter, and obtain the water-soluble fermentation product of Phalaenopsis. Add 0.6% (w / w) of p-hydroxyacetophenone and 0.6% (w / w) of 1,2-hexanediol for preservation.

[0033] Example 2 This example provides a golden flower fungus fermented fungus and an extract of the golden flower fungus fermented fungus, and the preparation method is as follows 1) The fresh cut flowers of Phalaenopsis were dried at low temperature, and the water content was kept at 5% to 15%. 2) The Phalaenopsis flowers were steamed at 121°C for 10 minutes, and then cooled to room temperature.

[0034] 3) The golden flower is inoculated on PDA flat plate culture medium and placed in a 28±2℃ environment for 72h of culture. Spores on the flat plate are scraped and collected. The spore concentration is adjusted to about 2.0×10^7 cfu / ml suspension to obtain a golden flower activation solution. 1% (w / w) of the golden flower activation solution is inoculated on the steamed butterfly orchid. The inoculated butterfly orchid is placed in a 28±2℃, 70±5% RH humidity environment for 5 days of culture, with the pile turned over once a day. After the fermentation is completed, the butterfly orchid is dried for standby use, and the golden flower fermentation fungus is obtained.

[0035] 4) The golden flower fermentation fungus is crushed through a 40-mesh sieve. Squalane is added at a ratio of 1:10 (w / w) of the butterfly orchid, and the mixture is extracted for 24h. Filtration is performed to obtain a butterfly orchid fermentation product oil solution.

[0036] Comparative Example 1 The difference from Example 1 is that the butterfly orchid is not steamed, but is subjected to ultraviolet sterilization at 100W for 30min. The temperature and humidity control during the solid-state fermentation process is 33±2℃ and a humidity of 35±5% RH.

[0037] Comparative Example 2 The difference from Example 1 is that the golden flower is subjected to liquid-state fermentation, and the specific steps are as follows. 1) The fresh cut butterfly orchid is dried at low temperature, with the water content maintained at 5%-15%. 2) The butterfly orchid is crushed through a 40-mesh sieve. Purified water is added at a ratio of 1:10, and the mixture is uniformly mixed and sterilized at 121℃ for 20min to obtain a butterfly orchid liquid-state fermentation medium.

[0038] 3) The golden flower is inoculated on PDA flat plate culture medium and placed in a 28±2℃ environment for 72h of culture. Spores on the flat plate are scraped and collected. The spore concentration is adjusted to about 2.0×10 5 cfu / ml suspension to obtain a golden flower activation solution. 1% (w / w) of the golden flower activation solution is inoculated in the butterfly orchid liquid-state fermentation medium. The inoculated butterfly orchid is placed in a 28±2℃ environment for 3 days of shaking culture. After the fermentation is completed, the mixture is filtered to obtain a butterfly orchid liquid-state fermentation product aqueous solution. 0.6% (w / w) of p-hydroxyacetophenone and 0.6% (w / w) of 1,2-hexanediol are added for preservation.

[0039] Comparative Example 3 This comparative example provides a butterfly orchid water extract, and the specific operation is as follows. 1) The fresh cut butterfly orchid is dried at low temperature, with the water content maintained at 5%-15%. 2) The Phalaenopsis is crushed through a 40-mesh sieve, and purified water is added at a ratio of 1:10 (w / w) of Phalaenopsis flowers to water, stirred for 2 h, filtered, to obtain a Phalaenopsis water extract, and 0.6% (w / w) of p-hydroxyacetophenone and 0.6% (w / w) of 1,2-hexanediol preservatives are added.

[0040] Comparative Example 4 This comparative example provides a Phalaenopsis oil extract, and the specific operation is as follows 1) The fresh-cut flowers of Phalaenopsis are dried at low temperature, and the water content is maintained at 5% to 15%, 2) The Phalaenopsis is crushed through a 40-mesh sieve, and squalane is added at a ratio of 1:10 (w / w) of Phalaenopsis flowers to squalane, and is extracted for 24 h, filtered, to obtain a Phalaenopsis oil extract.

[0041] Comparative Example 5 The difference from Example 1 is that yeast CICC 33217 is used for fermentation.

[0042] Figures 1-3 The photos after the end of solid-state fermentation of Phalaenopsis in Examples 1-2 and Comparative Example 1 can be seen from the figure, and it can be seen that under the conditions of Examples 1-2, the golden flower fungus grows more luxuriantly, proving that the conditions of Examples 1-2 are more conducive to the growth of the golden flower fungus, and the fermentation of Phalaenopsis is more sufficient.

[0043] In order to better illustrate the efficacy of the golden flower fungus fermentation fungus and the extract of the golden flower fungus fermentation fungus provided by the present application, the inventors have carried out the following research: 1, Collagen type I content determination: 1.1) Cell culture: Freeze-preserved human fibroblasts (HSF) were inoculated into a conventional culture medium (DMEM high-sugar medium) at an appropriate density for testing.

[0044] 1.2) Cell activity detection: HSF cells were plated in a 96-well plate, incubated at 37°C, 5% CO2 for 24 h, the culture medium was aspirated, and the basic culture medium containing 0.125%, 0.25%, 0.5%, 1%, 2% concentration test samples was added, and then incubated at 37°C, 5% CO2 for 24 h, and then the OD value was detected by MTT method, and the effect of the test sample on the activity of HSF cells was obtained by calculation and analysis. 490

[0045] 1.3) Type I collagen content determination: ​Take HSF cells to spread 96-well plates, 37℃, 5% CO2 incubation 24h, replace with 1% concentration test sample (all within the safe concentration of cells) containing basic medium, 37℃, 5% CO2 incubation 24h again, collect cell supernatant, centrifugal. Absorb the supernatant, use ELISA kit to determine the content of type I collagen in cell culture supernatant, and obtain the effect of test sample on the content of type I collagen expressed by fibroblast. The detection results of examples 1-2 and comparative examples 1-4 promoting the content of type I collagen expressed by skin fibroblast are shown in table 1.

[0046] Table 1.

[0047] From table 1, it can be seen that the effect of the extract of pholiota nameko fermented material of examples 1 and 2 on promoting the expression of type I collagen by skin fibroblast is higher than that of the direct extraction of phalaenopsis amabilis flower of comparative examples 3 and 4, which confirms that pholiota nameko fermentation can improve the skin care activity of phalaenopsis amabilis. At the same time, the effect of examples 1 and 2 is higher than that of comparative examples 1 and 2, which proves that the fermentation method has a great influence on the activity of phalaenopsis amabilis. The effect of example 1 is higher than that of comparative example 5, and the fermentation effect of pholiota nameko is better than that of candida blankiata, which proves that pholiota nameko fermentation has better advantages in improving the activity of phalaenopsis amabilis. In summary, the product of phalaenopsis amabilis fermented by pholiota nameko in the solid state fermentation method provided by the present application has better effect on promoting the expression of type I collagen by skin fibroblast.

[0048] 2, Content detection of triterpenoid substances A large number of studies have proved that pholiota nameko fermentation of plants can improve the yield of water-soluble active ingredients of plants, such as soluble polysaccharides, small molecule proteins and peptides. However, there are few reports on the improvement of oil-soluble active ingredients of plants by pholiota nameko fermentation. It is known from the published literature that terpenoids are important physiological active substances in orchid plants, and ursolic acid is a typical pentacyclic triterpenoid compound existing in plants. Therefore, the content of ursolic acid in the phalaenopsis amabilis oil extract obtained from examples 2 and comparative example 4 is detected, and the dissolution of terpenoids is compared.

[0049] Detection method LC-MS.

[0050] Equipment: Agilent 6546 LC / Q-TOF Analysis conditions: C18 analysis column (5μm, 250.0mm×4.6mm), mobile phase is a mixture of methanol and 5% acetic acid (volume ratio 4:6), the detection wavelength of ultraviolet detector is 252nm. The standard product is HPLC grade ursolic acid. The results are shown in table 2. Figure 4 The mass spectrum of ursolic acid target peak in phalaenopsis amabilis oil (E+ and E- scanning).

[0051] Table 2.

[0052] From Table 2, it can be seen that after fermentation by Auricularia auricula, the dissolution rate of triterpenoids is higher, which proves that Auricularia auricula solid-state fermentation is helpful for the dissolution of the soluble active ingredients of Phalaenopsis oil.

[0053] 3. Transdermal permeability test results Equipment: LabRAM Soleil ultra-high resolution microconfocal Raman spectrometer The laser power used in the experiment was 72 μW, and the integration time of a single point was 0.5 s. Depth Raman imaging was performed in the X-Z direction, and the spectrum data were collected by point-by-point scanning. The longitudinal step interval for collecting spectrum data was 10 μm, the scanning area was 20 μm × 120 μm, and the entire spectrum measurement process was completed within 3 min.

[0054] The front end of the human arm was selected for human Raman testing: a test skin area of 1 × 1 cm 2 was selected, and tests were performed at time points of 0 h, 0.5 h, 1 h, 2 h, and 4 h. The number of test subjects was 1, and the test area was 3 pieces of test skin area of 1 × 1 cm 2 . Five points in each test area were selected for parallel testing three times. The overall relative permeability after using the sample of Example 2 and Comparative Example 4 was calculated. The results are shown in Table 3.

[0055] Table 3.

[0056] From Tables 3 and Figure 4 , it can be directly seen that compared with Comparative Example 4, the relative permeability of Example 2 is significantly higher and the penetration depth is deeper, which proves that after Auricularia auricula solid-state fermentation, the active ingredients of Phalaenopsis are more easily absorbed by the skin.

[0057] Example 3 This example provides a ratio of a lip essence oil The Phalaenopsis fermentation product oil solution obtained in Example 2 was added to the base oil at 1% (w / w), and the base oil was a mixture of cocoyl octanoate / cocoyl decanoate and diisostearyl malate at a ratio of 1:3.

[0058] Human lip efficacy test 32 healthy male or female subjects aged 18-60 years old with rough skin and obvious lip lines were recruited to use the test sample continuously for 14 days and 28 days, and the changes before and after using the test sample were observed by instrument testing.

[0059] Test instrument: The human skin rapid optical imaging system PRIMOS-CR tests the lip volume and lip lines; The facial image analyzer VISIA-CR tests the lip contour.

[0060] The results are shown in Table 4 and Figure 5 It can be observed from the table that after 14 days of continuous use of the test sample, the lip lines, lip plumpness and lip contour of the subjects were significantly improved compared with the baseline values. After 28 days of continuous use of the test sample, the lip lines, lip plumpness and lip contour of the subjects were further improved. It is proved that the essence oil containing the product of the gold flower fungus fermented phalaenopsis has good lip anti-aging effect.

[0061] Table 4.

[0062] The above not involved, applicable to the prior art.

[0063] The present application is not limited to the specific methods or specific compositions described herein and can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims and equivalents thereof.

Claims

1. A method for preparing fermented mycelium of *Aureobasidium aureum* that promotes collagen production, characterized in that, Includes the following steps: S1. Dry the fresh-cut flowers of Phalaenopsis orchids at low temperature and sterilize them with steam, then set aside. S2. Inoculate with *Aureobasidium aureum* activation solution. The bacterial concentration of the *Aureobasidium aureum* activation solution is 1.0 × 10⁻⁶. 5 ~1.0×10 7 cfu / mL; S3. Inoculate the Phalaenopsis orchids and culture them at 28±2℃ and 65~85%RH humidity. The fermentation inoculum of Aureobasidium aureum is thus obtained.

2. The preparation method according to claim 1, characterized in that, In step S1, the moisture content of the dried cut flowers is controlled at 5%~15%.

3. The preparation method according to claim 2, characterized in that, In step S1, the conditions for steam sterilization are: steam temperature of 100℃~125℃, and time of about 10~30 minutes.

4. The preparation method according to claim 3, characterized in that, In step S2, the inoculation amount is 0.8-1.2% activated bacterial solution based on the weight of the cut flowers.

5. A fermentation mycelium for promoting collagen production, characterized in that, It is prepared by the preparation method described in any one of claims 1-4.

6. An extract of the fermented mycelium of *Aureobasidium aureum*, characterized in that, The *Aureobasidium aureum* fermentation mycelium as described in claim 5 is extracted in a solvent, wherein the solvent includes a polar solution or a non-polar solution.

7. The extract according to claim 6, characterized in that, The polar solution includes water, ethanol, or a eutectic solvent; the non-polar solution includes squalane, vegetable oil, or supercritical carbon dioxide.

8. An anti-skin aging composition, characterized in that, It includes the fermentation mycelium of *Aureobasidium* as described in claim 5, or the extract as described in any one of claims 6-8.

9. The use of the anti-skin aging composition as described in claim 8 in the preparation of daily cosmetic products.

10. A lip essence oil, characterized in that, It includes the fermentation mycelium of *Aureobasidium* as described in claim 5, or the extract as described in any one of claims 6-8.

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