A fermentation product composition of a filamentous yeast-like fungus with multiple efficacies and its use

Abstract

The application provides a kind of filamentous yeast-like fungus fermentation product composition with multiple efficacy and its application, by screening different filamentous yeast-like fungus fermentation product and ring hexapeptide, while limiting the relative content of both, finally a kind of filamentous yeast-like fungus fermentation composition with superior permeability, higher and more full efficacy is prepared.The obtained fermentation composition can realize more significant improvement of the ability of resisting skin photoaging, repairing photo-damage, reducing cell apoptosis or anti-aging and wrinkle removal compared with original fermentation liquor and original cyclic peptide, significantly reduces the generation of free radicals, inhibits the synthesis of matrix metalloproteinase, and also significantly improves the content of collagen, has the efficacy of soothing, relieving oxidative stress, inhibiting inflammatory factors or inflammatory mediators, and has stronger application potential in the field of cosmetic skin care and medical anti-aging.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation and cosmetic application technology. Specifically, it relates to a filamentous yeast-like fungal fermentation product composition with multiple functions and its application. Background Technology

[0002] Human skin is exposed to ultraviolet (UV) radiation from the sun every day. Sunlight's UV radiation is mainly classified into three types based on its wavelength: long-wave ultraviolet (UVA, wavelength 320–400 nm), medium-wave ultraviolet (UVB, wavelength 290–320 nm), and short-wave ultraviolet (UVC, wavelength 200–290 nm). UVC, with wavelengths shorter than 290 nm, is blocked by the ozone layer; UVA has extremely strong penetrating power and can affect the dermis and even subcutaneous tissue, leading to oxidative stress and tissue inflammation; UVB can directly interact with DNA to produce dipyrimidine photoproducts, causing DNA damage during DNA replication. Acute UV radiation can cause sunburn, abnormal pigmentation, and immunosuppression. Long-term exposure to UV radiation leads to premature skin aging, also known as photoaging. Studies have shown that photoaging is mainly related to reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals, and hydrogen peroxide. Normal physiological metabolism and environmental influences such as UV radiation and environmental pollution can all lead to the production of ROS. Excessive levels of reactive oxygen species (ROS) generate oxidative stress, which causes oxidative damage primarily affecting cellular components such as cell membranes, DNA, lipids, and proteins. This damage can occur by inducing the synthesis of matrix metalloproteinases (MMPs), which degrade collagen and elastin in the ECM and inhibit their synthesis. Furthermore, ROS can promote inflammatory responses by activating nuclear factor-κB (NF-κB), thereby influencing the expression of pro-inflammatory cytokines such as interleukin-1 (IL-1), epidermal growth factor (EGF), and tumor necrosis factor-α (TNF-α). Oxidative stress further leads to reduced keratinocyte vitality and slower cell turnover in the epidermis, weakening the skin barrier and causing dryness and flaking. In the dermis, the number of fibroblasts gradually decreases, collagen synthesis slows, and collagen breakdown accelerates. Therefore, photoaged skin manifests as roughness, sagging, deep wrinkles, telangiectasia with redness, and pigmentation. Based on these findings, preventing skin photo-oxidation or alleviating UV damage to delay photoaging has been a major concern. Antioxidants are a crucial way to combat skin aging. Due to various safety and efficiency issues, most traditional sunscreens and whitening agents can absorb some ultraviolet rays, but they cannot fully meet the needs of cosmetic applications such as improving photoaging of the skin.

[0003] At present, some natural fermentation products contain components such as vitamins, amino acids, polypeptides, minerals, polysaccharides, flavonoids, polyphenols, etc. have certain antioxidant effect, higher safety, more prominent function and good biocompatibility, etc. gradually become the focus of people's development and research. For example, the Chinese invention patent with the authorization announcement number CN101724006A discloses a preparation method of a filamentous yeast-like fungus fermentation product. A pure natural culture medium prepared by filamentous yeast-like fungus and tricholoma matsutake, white willow bark, ganoderma lucidum, white truffle or rice is used for multiple fermentation to improve cell safety, improve skin care effect of fermentation product, save cost, suitable for production and manufacturing, mainly applied to moisturizing, oil control, skin color lightening and other cosmetic and skin care products, but does not involve the use of anti-photoaging and other effects.

[0004] One of the classic anti-aging ingredients, polypeptides, are widely used in cosmetics, beauty and skin care fields, but there are certain application pain points. On the one hand, its penetration is low, and the amount actually entering the skin to play a role is limited. On the other hand, most polypeptides are inactivated by protease degradation in blood and skin tissue, losing efficacy. Therefore, the bioavailability of polypeptide raw materials is very low, and it cannot maximize its efficacy. In recent years, scientists have confirmed that cyclic peptides (or cyclic peptides) have long in vivo half-life, stable structure and strong penetration, etc. Therefore, the use of external cyclic peptide products can play a certain care and defense, and slow down the aging effect. The development of cyclic peptides in the field of beauty and skin care also has certain innovation and practicality. Chinese invention patent CN116162135A discloses a cyclic hexapeptide and its application. Efficacy research found that it has more outstanding efficacy than linear peptides in skin anti-wrinkle and firming. It is mainly applied to cosmetics and medical beauty firming and anti-wrinkle fields, but does not involve the application of anti-photoaging, antioxidant and soothing repair effects.

[0005] Therefore, it is urgent to find a filamentous yeast-like fungus fermentation product composition with multiple effects. By screening fermentation products and cyclic hexapeptides, a composition is compounded to significantly improve anti-skin photoaging, repair photo-damage or anti-aging and wrinkle removal, while also significantly improving collagen content, inhibiting MMP-1, soothing and repairing, relieving oxidative stress, inhibiting inflammatory factors or inflammatory mediators. SUMMARY

[0006] To solve the problems in the prior art, the application provides a filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof. By screening different filamentous yeast-like fungus fermentation products and cyclic hexapeptides, limiting the relative content of the two, a group of filamentous yeast-like fungus fermentation compositions with superior permeability, higher and more comprehensive effects are finally obtained, which have the effects of significantly reducing the generation of free radicals, inhibiting the synthesis of matrix metalloproteinase, protecting against ultraviolet damage, reducing cell apoptosis, preventing photoaging and the like, and have stronger application potential in the fields of beauty care, skin care and medical anti-aging.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following scheme:

[0008] In one aspect, the application provides a composition comprising a filamentous yeast-like fungus fermentation product and a cyclic hexapeptide.

[0009] In some embodiments, the filamentous yeast-like fungus fermentation product contains rich amino acids, polypeptides and the like, which can directly supply skin cells with nutrients, and preferably in a certain proportion, can further promote the proliferation ability of keratinocytes and fibroblasts, activate cells, and provide a basis for realizing the efficacy.

[0010] Further, the composition comprises 0.05% to 90% of the filamentous yeast-like fungus fermentation product, 0.05% to 5% of the cyclic hexapeptide, and the balance of the base component, by mass percentage, and the sum of the mass percentages of the above components is 100%.

[0011] Further, the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

[0012] In some embodiments, the cyclic hexapeptide with high purity is prepared by using an optimal preparation method, and the composition obtained by compounding the cyclic hexapeptide with the filamentous yeast-like fungus fermentation product has significant synergistic effect and better beauty care effect.

[0013] Further, the preparation method of the filamentous yeast-like fungus fermentation product comprises the following steps:

[0014] (1) culturing the filamentous yeast-like fungus to obtain an inoculum;

[0015] (2) inoculating the inoculum into a liquid natural product culture medium for primary fermentation;

[0016] (3) Collecting the supernatant produced by centrifugation at the end of each fermentation, and mixing the supernatant to obtain the fermentation product;

[0017] The natural material includes any one of rice, white willow bark, Ganoderma lucidum, white truffle, Tricholoma matsutake or white birch fungus; when the natural material is rice, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus rice fermentation product; when the natural material is white willow bark, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus white willow bark fermentation product; when the natural material is Ganoderma lucidum, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus Ganoderma lucidum fermentation product; when the natural material is white truffle, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus white truffle fermentation product; when the natural material is Tricholoma matsutake, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus Tricholoma matsutake fermentation product; and when the natural material is white birch fungus, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus white birch fungus fermentation product.

[0018] In some embodiments, the filamentous yeast-like fungus fermentation product is prepared by using a 4-fold fermentation technique, which has the highest safety and the best skin care efficacy compared to fermentation products of other multiple fermentation times. The fermentation product has a higher protein content level and better skin care efficacy, and the fermentation product filtrate has a higher cell safety concentration range.

[0019] Preferably, the composition includes 0.05% to 10% of the filamentous yeast-like fungus rice fermentation product, or 0.05% to 15% of the filamentous yeast-like fungus white willow bark fermentation product, or 0.05% to 7.5% of the filamentous yeast-like fungus Ganoderma lucidum fermentation product, or 0.05% to 10% of the filamentous yeast-like fungus white truffle fermentation product, or 0.05% to 10% of the filamentous yeast-like fungus Tricholoma matsutake fermentation product, or 0.05% to 15% of the filamentous yeast-like fungus white birch fungus fermentation product, by mass percentage.

[0020] In some embodiments, the safe concentration of the fermentation product and the cyclic hexapeptide in the composition is screened respectively, and the experimental results show that when the rice fermentation product is 0.05% to 10%, the white willow bark fermentation product is 0.05% to 15%, the ganoderma lucidum fermentation product is 0.05% to 7.5%, the white truffle fermentation product is 0.05% to 10%, the tricholoma matsutake fermentation product is 0.05% to 10%, the white birch fungus fermentation product is 0.05% to 15%, or the cyclic hexapeptide is 0.05% to 5%, the cell survival rate can reach more than 70%, and meanwhile, the high-concentration peptide is compounded on the basis of the original fermentation liquid, without changing the safe tolerance of the cell, and has a strong application value. Compared with the commercially available yeast fermentation product, the cell tolerance of the filamentous yeast-like fungus fermentation product composition of the present application is higher, and the biocompatibility is better. Preferably, the combination 2 (white willow bark fermentation liquid + cyclic hexapeptide) and the combination 6 (white birch fungus fermentation liquid + cyclic hexapeptide) have higher concentrations under the premise of safety, indicating that they have low biological toxicity, high safety, and can be used at a higher concentration. Meanwhile, at a higher concentration, the effect of the composition is more significant.

[0021] Preferably, in the composition, the filamentous yeast-like fungus rice fermentation product accounts for 2.5% by mass percentage, or the filamentous yeast-like fungus white willow bark fermentation product accounts for 2.5% by mass percentage, or the filamentous yeast-like fungus ganoderma lucidum fermentation product accounts for 2.5% by mass percentage, or the filamentous yeast-like fungus white truffle fermentation product accounts for 2.5% by mass percentage, or the filamentous yeast-like fungus tricholoma matsutake fermentation product accounts for 2.5% by mass percentage, or the filamentous yeast-like fungus white birch fungus fermentation product accounts for 2.5% by mass percentage; and the cyclic hexapeptide accounts for 2% by mass percentage.

[0022] In another aspect, the present application provides a use of a composition for preparing an agent for resisting skin photoaging or repairing photo-damage or improving penetration capacity, wherein the composition comprises any one of a filamentous yeast-like fungus rice fermentation product, a filamentous yeast-like fungus white willow bark fermentation product, a filamentous yeast-like fungus ganoderma lucidum fermentation product, a filamentous yeast-like fungus white truffle fermentation product, a filamentous yeast-like fungus tricholoma matsutake fermentation product, a filamentous yeast-like fungus white birch fungus fermentation product, and a cyclic hexapeptide.

[0023] In some embodiments, under the optimal ratio (2.5% of the fermentation product and 2% of the cyclic hexapeptide), the filamentous yeast-like fungus fermentation product can achieve the effect of promoting the transdermal absorption of the cyclic hexapeptide, and when the white willow bark fermentation product + cyclic hexapeptide combination is preferred, the penetration effect is optimal.

[0024] Further, the composition comprises a filamentous yeast-like fungus rice fermentation product and a cyclic hexapeptide, and the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

[0025] In some embodiments, the ability of the filamentous yeast-like fungus fermentation product composition to repair light damage is tested, and the results show that the filamentous yeast-like fungus fermentation product composition has significant light damage repair ability when the concentration of the filamentous yeast-like fungus fermentation product is 0.1% to 10% and the concentration of the cyclic hexapeptide is 0.5 to 3%, slightly promotes cell activity, and has the strongest light damage repair ability when the fermentation product is 2.5% and the cyclic hexapeptide is 2%. Meanwhile, when the preferred combination 1 (rice fermentation liquid + cyclic hexapeptide) is used, the best anti-skin photoaging, light damage repair ability, and cell activity promotion effect are achieved, and the application value is extremely high.

[0026] In another aspect, the present application provides a use of a composition for preparing a preparation for improving soothing and repairing ability, inhibiting inflammatory factors or inflammatory mediators, the composition comprising any one of a filamentous yeast-like fungus rice fermentation product, a filamentous yeast-like fungus white willow bark fermentation product, a filamentous yeast-like fungus ganoderma lucidum fermentation product, a filamentous yeast-like fungus white truffle fermentation product, a filamentous yeast-like fungus tricholoma matsutake fermentation product, a filamentous yeast-like fungus white birch fungus fermentation product, and a cyclic hexapeptide.

[0027] Further, the composition comprises the filamentous yeast-like fungus white willow bark fermentation product and the cyclic hexapeptide, and the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

[0028] In some embodiments, under the optimal ratio (fermentation product 2.5% and cyclic hexapeptide 2%), the filamentous yeast-like fungus fermentation product composition has significant effects of inhibiting the expression of pro-inflammatory cytokines TNF-α and inflammatory factors IL-1α and IL-8, and soothing skin damage, and the synergistic effect of the compounded composition is significant, and when the composition is the preferred combination 2 (white willow bark fermentation liquid + cyclic hexapeptide), the best effects of inhibiting the expression of inflammatory factors and soothing ultraviolet light damage of the skin are achieved.

[0029] In another aspect, the present application provides a use of the composition as in any one of the above technical solutions for preparing a preparation for anti-aging and wrinkle removal, increasing collagen content, or inhibiting MMP-1.

[0030] In some embodiments, under the optimal ratio (fermentation product 2.5% and cyclic hexapeptide 2%), the filamentous yeast-like fungus fermentation product composition has significant effects of promoting the expression of Collagen I, Collagen III, and Collagen IV and inhibiting the expression of MMP-1, and the synergistic effect of the compounded composition is significant, and when the composition is the preferred combination 2 (white willow bark fermentation liquid + cyclic hexapeptide), the best anti-wrinkle effect is achieved.

[0031] In still another aspect, the present application provides a use of the composition according to any one of the above technical solutions for preparing a preparation for relieving oxidative stress and improving antioxidant capacity.

[0032] In some embodiments, at the optimal ratio (fermentation product 2.5%, cyclic hexapeptide 2%), both the complexed filamentous yeast-like fungus fermentation product and the cyclic hexapeptide can significantly improve the antioxidant capacity of the composition, achieving a synergistic effect; and when the preferred combination 2 (white willow bark fermentation broth + cyclic hexapeptide) is used, the antioxidant capacity is the strongest.

[0033] The present application has the following beneficial effects:

[0034] 1. The series of filamentous yeast-like fungus fermentation product compositions provided by the present application have multiple effects, are convenient to ferment and synthesize, have high biocompatibility and are safe and non-irritating to the human body after being optimized at the optimal ratio, and can be applied in the field of cosmetics.

[0035] 2. The series of filamentous yeast-like fungus fermentation products provided by the present application can regulate the skin epidermis layer as a skin conditioner, promote the penetration and absorption of the original cyclic hexapeptide, achieve a synergistic effect, and make the cyclic hexapeptide have a stronger effect.

[0036] 3. The series of filamentous yeast-like fungus fermentation products provided by the present application contain rich amino acids and polypeptides, which can directly provide nutrients for skin cells; after being optimized at the optimal ratio, the composition obtained by compounding the filamentous yeast-like fungus fermentation product and the cyclic hexapeptide has a significant repair capacity for the photodamage of keratinocytes and fibroblasts, significantly enhances the proliferation capacity of the keratinocytes and fibroblasts, activates the cells, and provides a basis for the realization of the efficacy.

[0037] 4. The series of filamentous yeast-like fungus fermentation product compositions provided by the present application can significantly inhibit the expression of ROS / inflammatory factors or inflammatory mediators IL-1, IL-8 and TNF-α, achieve the effect of relieving and alleviating oxidative stress, and significantly promote the expression of key genes related to the barrier function, thereby helping to relieve the problems of skin dryness and desquamation caused by weakened barrier function.

[0038] 5. The series of filamentous yeast-like fungus fermentation product compositions provided by the present application can obviously promote the expression of collagen I, collagen III and collagen IV genes under a photoaging model after being optimized at the optimal ratio, significantly improve the effect of the fermentation composition on improving the content of collagen, provide a theoretical basis for the production of collagen by the skin, and can also significantly inhibit the production of MMP-1, thereby slowing down the degradation of collagen. The two-pathway mechanism of promoting production and inhibiting degradation balances and supplements collagen, thereby being capable of playing a better anti-aging and wrinkle-removing effect on the skin. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Column chart of Collagen I gene expression amount in each group.

[0040] Figure 2 Column chart of Collagen III gene expression amount in each group.

[0041] Figure 3 Column chart of Collagen IV gene expression amount in each group.

[0042] Figure 4 Column chart of MMP-1 gene expression amount in each group.

[0043] Figure 5 Column chart of IL-1α content expression.

[0044] Figure 6 Column chart of IL-8 content expression.

[0045] Figure 7 Column chart of TNF-α content expression. DETAILED DESCRIPTION

[0046] The application will be further described in conjunction with the following examples. It should be noted that the following examples are intended to facilitate the understanding of the application, and do not limit the application in any way.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0048] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0049] In the present application, the definition of "anti-photoaging" is "resisting damage caused by ultraviolet light irradiation, having the effect of repairing cell photo-damage, significantly improving cell survival rate, having significant antioxidant capacity, significantly reducing ROS content, and also having significant anti-wrinkle effect, significantly promoting collagen gene expression amount, inhibiting MMP-1 expression, and also having significant soothing ability and significantly inhibiting expression of inflammatory factors and pro-inflammatory factors".

[0050] Example 1 Preparation of filamentous yeast-like fungus fermentation product and cyclic hexapeptide

[0051] In this example, the optimal method for preparing the filamentous yeast-like fungus fermentation product and the cyclic hexapeptide is selected, and the specific preparation process is as follows:

[0052] I. Preparation of fermentation product of filamentous yeast-like fungi: including preparation of natural product culture medium, supplementary nutrient solution and inoculum, and subsequent preparation of fermentation (the filamentous yeast-like fungi Galactomyces Citri-Aurantii is purchased from China Microbial Strain Network, the purchase website is https: / / www.biobw.org / China-strain / bio-113662.html; the origins of the six natural products are respectively tomorrow Xingyou organic congee rice, Shandong white willow bark, Changbai Mountain Ganoderma lucidum, frozen Yunnan white truffle fungus, Yunnan Shangri-La Tricholoma matsutake and Jilin Changbai Mountain white birch).

[0053] 1) Preparation of culture medium: use Tricholoma matsutake, white willow bark, Ganoderma lucidum, rice, white truffle and white birch as natural product configuration liquid natural product culture medium, the process is as follows: weigh 100g of natural product, powder the natural product, mix with pure water, the volume ratio of natural product powder to pure water is 1:20, high temperature sterilization treatment, form a semi-solid paste and stratify after standing (upper layer of suspended clear liquid and lower layer of turbid precipitate), seal and store in cold storage (4℃) for standby. Respectively get Tricholoma matsutake culture medium, white willow bark culture medium, Ganoderma lucidum culture medium, rice culture medium, white truffle culture medium and white birch culture medium. Preparation of supplementary nutrient solution: preparation of 1% sucrose sterile nutrient solution (sucrose: pure water = 1:100) as supplementary nutrient solution for scale-up fermentation.

[0054] 2) Preparation of inoculum: filamentous yeast-like fungi Galactomyces Citri-Aurantii purchased from China Microbial Strain Network is cultured in YM medium small 0.5L liquid, fermented at 26℃ for 3 days, stirred and shaken once a day, the OD value of measured absorbance turbidity is >1.50 as inoculum. 600

[0055] 3) Fermentation product of filamentous yeast-like fungi through first fermentation: take the inoculum to inoculate into the fermentation tank, carry out liquid fermentation of natural product culture medium (the volume ratio of inoculum to culture medium is 1:1000, the OD value is 1.50), ferment for 72h, the fermentation temperature is 26±3℃, after the fermentation is completed, high-speed centrifugation (5000rpm) is carried out, the supernatant of first fermentation is separated, stored in cold storage for standby, and the precipitate is high temperature and high pressure sterilized for standby.

[0056] 4) Second fermentation: take all the precipitate layer separated by first fermentation, carry out second fermentation with 1:1000 of solid-liquid ratio (precipitate layer: 1% sucrose sterile nutrient solution), the volume ratio of inoculum to culture medium is 1:1000, the OD value is 1.50, ferment for 72h, the fermentation temperature is 26±3℃, after the fermentation is completed, high-speed centrifugation (5000rpm) is carried out, the supernatant of second fermentation is separated, stored in cold storage for standby, and the precipitate is high temperature and high pressure sterilized for standby.

[0057] ​5) Third fermentation: take all the second fermentation of the separation of the precipitate layer, precipitate due to the consumption of less than the last time, also with 1:1000 of the ratio of liquid (precipitate layer: 1% sucrose sterile nutrient solution) for the third fermentation (inoculum and medium volume ratio of 1:1000, OD value of 1.50), fermentation 72h, fermentation temperature: 26±3℃, after the end of fermentation high speed centrifugation (5000rpm), separation of the third fermentation supernatant, stored in the refrigerator for standby, precipitate autoclaving standby.

[0058] 6) Fourth fermentation: take all the third fermentation of the separation of the precipitate layer, precipitate further reduced, also with 1:1000 of the ratio of liquid (precipitate layer: 1% sucrose sterile nutrient solution) for the fourth fermentation (inoculum and medium volume ratio of 1:1000, OD value of 1.50), high fermentation 72h, fermentation temperature: 26±3℃, after the end of fermentation high speed centrifugation (5000rpm), separation of the fourth fermentation supernatant, stored in the refrigerator for standby.

[0059] 7) The 4 fermentation collected supernatant mixture to obtain the filamentous yeast-like fungus fermentation product, add 0.5% 1,2-hexanediol and 0.4% p-hydroxyacetophenone as preservative to obtain the filamentous yeast-like fungus fermentation product filtrate, finally respectively obtain the following six kinds of filamentous yeast-like fungus fermentation product:

[0060] A, when the natural material is rice, obtain the filamentous yeast-like fungus rice fermentation product (i.e. filamentous yeast-like fungus rice fermentation liquid);

[0061] B, when the natural material is white willow bark, obtain the filamentous yeast-like fungus white willow bark fermentation product (i.e. filamentous yeast-like fungus white willow bark fermentation liquid);

[0062] C, when the natural material is ganoderma, obtain the filamentous yeast-like fungus ganoderma fermentation product (i.e. filamentous yeast-like fungus ganoderma fermentation liquid);

[0063] D, when the natural material is white truffle, obtain the filamentous yeast-like fungus white truffle fermentation product (i.e. filamentous yeast-like fungus white truffle fermentation liquid);

[0064] E, when the natural material is truffle, obtain the filamentous yeast-like fungus truffle fermentation product (i.e. filamentous yeast-like fungus truffle fermentation liquid);

[0065] F, when the natural material is white birch fungus, obtain the filamentous yeast-like fungus white birch fungus fermentation product (i.e. filamentous yeast-like fungus truffle fermentation liquid).

[0066] II. Preparation of cyclohexapeptide:

[0067] 1) The specific preparation process is as follows: sequentially using amino acid raw materials Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH for linear peptide connection, then cutting to obtain the fully protected linear peptide H-Gln(Trt)-Gly-Pro-Gln(Trt)-Gly-Pro-OH, then cyclizing the linear peptide H-Gln(Trt)-Gly-Pro-Gln(Trt)-Gly-Pro-OH to obtain the ring-closed product Cyclo(Gln(Trt)-Gly-Pro-Gln(Trt)-Gly-Pro), and finally cutting again to obtain the crude peptide Cyclo(Gln-Gly-Pro-Gln-Gly-Pro). After cutting to obtain the crude product, the finished product Cyclo(Gln-Gly-Pro-Gln-Gly-Pro) is obtained by purification and freeze-drying, and the purity is 99.2%.

[0068] 2) The cyclic hexapeptide and its derivatives or salts prepared above can exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids comprising them above can have L-, D- configurations, or be racemic independently of each other. It is thus possible to obtain isomeric mixtures as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the isomers or isomeric mixtures present. The preferred structure of the peptide used in this embodiment is a pure isomer, i.e., an enantiomer or a diastereomer.

[0069] A filamentous yeast-like fungus fermentation product is prepared in this embodiment, wherein the fermentation product filtrate of the filamentous yeast-like fungus after multiple fermentations has higher cell safety and better skin care efficacy; at the same time, a small molecule cyclic hexapeptide with anti-wrinkle efficacy is also prepared in this embodiment. By screening different combinations of filamentous yeast-like fungus fermentation products and small molecule cyclic hexapeptides, while limiting the relative content of both, a filamentous yeast-like fungus fermentation composition with superior penetration, higher and more comprehensive efficacy is finally prepared. The obtained fermentation composition can achieve more significant improvements in the abilities to resist skin photoaging, repair photo-damage, reduce cell apoptosis or anti-aging and wrinkle removal, significantly reduce the production of free radicals, inhibit the synthesis of matrix metalloproteinases, and also significantly increase the collagen content, has the efficacy of soothing and repairing, relieving oxidative stress, inhibiting inflammatory factors or inflammatory mediators, and has stronger application potential in the fields of cosmetic skin care and medical anti-aging.

[0070] Example 2 Cell safety detection of filamentous yeast-like fungus fermentation product composition and screening of composition concentration

[0071] To obtain the applicable range of the filamentous yeast-like fungus fermentation product composition in Example 1, the applicable range was screened by cell safety test on the composition in this example, and the specific experimental principle and method were as follows:

[0072] Test principle and method: MTT method is also called MTT colorimetric method, which is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet formazan and deposit in cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value can be determined by enzyme-linked immunoassay instrument, which can indirectly reflect the number of living cells. Within a certain range of cell number, the amount of MTT crystal formation is proportional to the cell number. It is mainly used in laboratory to evaluate cytotoxicity and promote cell proliferation ability, and the experimental method is carried out according to the "MTT method for determining cell activity operation standard". Cell safety evaluation: when the cell survival rate measured by MTT method is less than 70%, it is toxic, and the higher the value, the higher the safety.

[0073] The filamentous yeast-like fungus fermentation product plant substrates were respectively: rice, white willow bark, ganoderma lucidum, white truffle, tricholoma matsutake and white birch fungus, which were divided into samples 1-6, sample 7 was a commercially available yeast fermentation product, and the fermentation compositions were respectively sample 1-7 control composition 1-7. During the test, first select the corresponding high concentration to start the test, and then reduce the concentration in turn, to obtain the highest concentration of sample cell activity greater than 70%, the higher the highest concentration, the lower the biological toxicity; first screen the single fermentation product and the single cyclic peptide respectively, screen the safe highest concentration, and then compound them, and the specific information and concentration are shown in Table 1:

[0074] Table 1 sample information table

[0075] Sample group Test sample group Screening safe concentration Combination 1 Rice fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 2 White willow bark fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 3 Ganoderma lucidum fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 4 Truffle fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 5 Matsutake fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 6 White birch fungus fermentation broth + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5% Combination 7 Commercially available yeast fermentation product + cyclohexapeptide Fermentation broth from 0.05% to 20%; cyclopeptide from 0.05 to 5%

[0076] In this example, the highest concentration of the sample with a cell survival rate of about 70% was counted, and the MTT determination results in the test sample showed that the highest concentration of the cyclic hexapeptide was 5%, the highest concentrations of fermentation liquid samples 1-7 were 10%, 15%, 7.5%, 10%, 10%, 15% and 5% respectively, and the corresponding composition results after compounding were shown in Table 2:

[0077] Table 2 composition safety result table

[0078]

[0079]

[0080] From Table 2, on the one hand, compared with the commercially available yeast fermentation product, the filamentous yeast-like fungus fermentation product composition of the present application has higher cell tolerance and better biocompatibility; on the other hand, on the basis of the original fermentation broth, high-concentration peptides are compounded, the safety tolerance to cells is not changed, and it has extremely strong application value; on the other hand, compared with other four groups of filamentous yeast-like fungus fermentation product compositions, combination 2 (white willow bark fermentation broth + cyclic hexapeptide) and combination 6 (white birch fungus fermentation broth + cyclic hexapeptide) can be used at a higher concentration on the premise of safety, indicating that they have low biological toxicity, high safety, and can be used at a higher concentration. At the same time, the effect of the composition is more significant at a higher concentration.

[0081] It can be seen that the filamentous yeast-like fungus fermentation product composition in the present embodiment has low biological toxicity, high safety, and more significant use effect, and at the same time, the preferred combination 2 (white willow bark fermentation broth + cyclic hexapeptide) and combination 6 (white birch fungus fermentation broth + cyclic hexapeptide).

[0082] Example 3 Test of light damage repair ability of filamentous yeast-like fungus fermentation product composition

[0083] In order to test the light damage repair ability of the six filamentous yeast-like fungus fermentation product compositions in Example 1, comparative experiments were carried out on the compositions within a certain proportion range in the present embodiment, and the specific experimental process was as follows:

[0084] 1) Cell inoculation: fibroblasts were inoculated into a 96-well plate at a cell inoculation amount of 3.5×10 3 cells / well, and incubated in an incubator (37℃, 5% CO2) overnight;

[0085] 2) Drug administration: when the plating rate of the cells in the 96-well plate reached 20% to 30%, drug administration was carried out in groups, and the drug administration amount per well was 200μL. Each group had 3 replicate wells. The culture was carried out in an incubator (37℃, 5% CO2) for 24h, 48h and 72h respectively. The groups that needed to be incubated and cultured for 48h and 72h were changed every day;

[0086] 3) UVA irradiation: according to the test scheme, the groups that needed to be irradiated were irradiated with UVA, and the irradiation dose was 45J / cm 2 . After the irradiation was completed, it was placed in an incubator (37℃, 5% CO2) for continuous culture for 24h;

[0087] 4) MTT detection: the supernatant was discarded, MTT working solution (0.5mg / mL) was added, and it was incubated at 37℃ in the dark for 4h. After the incubation was completed, the supernatant was discarded, 150μL DMSO was added to each well, and the OD value was read at 490nm. At the same time, after 48h and 72h, the above MTT detection operation was carried out;

[0088] 5) Cell relative viability calculation: According to the formula, cell relative viability (%) = (sample well OD - zero well OD) / (solvent control well OD - zero well OD) * 100%.

[0089] There are several ways:

[0090] 1. Blank group: not subjected to ultraviolet irradiation;

[0091] 2. Negative control group: subjected to ultraviolet irradiation;

[0092] 3. Positive control group: adding drug TGF-β1 (100 ng / mL; Peprotech, transforming growth factor), subjected to ultraviolet irradiation;

[0093] 4. Cyclohexapeptide group: 2% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0094] 5. Rice fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0095] 6. White willow bark fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0096] 7. Ganoderma lucidum fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0097] 8. Truffle fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0098] 9. Tricholoma matsutake fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0099] 10. White Birch fungus fermentation broth group: 2.5% cyclohexapeptide alone was added for experiment, subjected to ultraviolet irradiation;

[0100] 11. Combination 1 (rice fermentation broth + cyclohexapeptide): different proportions of 2.5% + 0.5%, 2.5% + 1, 2.5% + 2%, and 2.5% + 3% were selected for experiment, subjected to ultraviolet irradiation;

[0101] 6. Combination 2 (white willow bark fermentation broth + cyclohexapeptide): different proportions of 2.5% + 0.5%, 2.5% + 1, 2.5% + 2%, and 2.5% + 3% were selected for experiment, subjected to ultraviolet irradiation;

[0102] 7. Combination 3 (Ganoderma lucidum fermentation broth + cyclohexapeptide): different proportions of 2.5% + 0.5%, 2.5% + 1, 2.5% + 2%, and 2.5% + 3% were selected for experiment, subjected to ultraviolet irradiation;

[0103] 8. Combination 4 (Truffle fermentation broth + cyclohexapeptide): 2.5% + 0.5%; 2.5% + 1; 2.5% + 2%; 2.5% + 3% of different proportions were selected for experiments, and were subjected to UV irradiation.

[0104] 9. Combination 5 (Truffle fermentation broth + cyclohexapeptide): 2.5% + 0.5%; 2.5% + 1; 2.5% + 2%; 2.5% + 3% of different proportions were selected for experiments, and were subjected to UV irradiation.

[0105] 10. Combination 6 (White Birch Truffle fermentation broth + cyclohexapeptide): 2.5% + 0.5%; 2.5% + 1; 2.5% + 2%; 2.5% + 3% of different proportions were selected for experiments, and were subjected to UV irradiation.

[0106] 11. Combination 7 (Commercially available yeast fermentation product + cyclohexapeptide): 2.5% + 0.5%; 2.5% + 1; 2.5% + 2%; 2.5% + 3% of different proportions were selected for experiments, and were subjected to UV irradiation.

[0107] The specific results are shown in Table 3:

[0108] Table 3 Test results of combinations 1-7 for photodamage prevention / repaired cells

[0109]

[0110]

[0111]

[0112] Note: p < 0.05 indicates a significant difference.

[0113] From Table 3, 1) the cell survival rate of the positive control group is significantly higher than that of the negative control group, which shows that the positive control is effective this time; 2) compared with the negative control group, after ultraviolet irradiation, combinations 1-6 have strong repair ability for the photodamage of keratinocytes and fibroblasts; in subsequent experiments, further experiments were carried out on the concentration of filamentous yeast-like fungus fermentation product in the range of 0.1% to 10%, and it can be known from this experiment that the cell survival rate of the filamentous yeast-like fungus fermentation product composition in the range of 0.1% to 10% and the concentration of cyclic hexapeptide in the range of 0.5 to 3% is more than 90%, and the photodamage repair ability is significantly enhanced; and when the fermentation product in the composition is 2.5% and the cyclic hexapeptide is 2%, the photodamage repair ability reaches the strongest; 2) compared with the negative control group, although combination 7 can improve the photodamage repair ability of cells, but compared with combinations 1-6, the effect is not obvious, combinations 1-6 can significantly improve the photodamage repair ability of keratinocytes and fibroblasts, and compared with combination 7, p<0.05, the difference is significant, which shows that the filamentous yeast-like fungus fermentation product composition of the application has strong photodamage repair ability, and the photodamage repair ability is extremely significant compared with the commercially available yeast fermentation product composition; 3) compared with the single cyclic hexapeptide group and the single fermentation product in combinations 1-6, the keratinocyte survival rate and fibroblast survival rate of the filamentous yeast-like fungus fermentation product composition in combinations 1-6 are significantly improved, which shows that the single cyclic hexapeptide and the single fermentation product do not have significant photodamage repair ability, and only after the combination of the two, the photodamage repair ability of the composition will be significantly improved; 4) among the 6 compositions, combination 1 has the highest keratinocyte and fibroblast survival rate under the best ratio concentration, which shows that combination 1 (rice fermentation liquid + cyclic hexapeptide) has the best photodamage repair ability; 5) compared with the blank group, the keratinocyte survival rate and fibroblast survival rate of combinations 1-6 are obviously improved, which shows that the active ingredients contained in the single fermentation liquid have a certain effect, but not to the stage of promoting growth, and the cyclic hexapeptide also does not have a significant growth-promoting effect, but the keratinocyte survival rate and fibroblast survival rate after the combination are obviously higher than those of the blank group, which shows that the composition after the combination has a promoting effect on cell survival rate and promotes cell activity.

[0114] Therefore, in this embodiment, when the concentration of the filamentous yeast-like fungus fermentation product is 0.1% to 10% and the concentration of the cyclic hexapeptide is 0.5 to 3% in the filamentous yeast-like fungus fermentation product composition, the photodamage repair ability is significantly enhanced, and the cell activity is slightly promoted, and when the fermentation product is 2.5% and the cyclic hexapeptide is 2%, the photodamage repair ability reaches the strongest; at the same time, when the preferred combination 1 (rice fermentation liquid + cyclic hexapeptide) is used, the best photodamage repair ability and the promoting effect on cell activity are achieved, which has extremely strong application value.

[0115] Test of permeability of the fermentative product composition of filamentous yeast-like fungi of Example 4

[0116] To test the permeability of the six filamentous yeast-like fungi fermentative product compositions of Example 1, comparative experiments were carried out in this example on different compositions at the optimal ratio, and the specific experimental principle is as follows: based on Franz cell diffusion tank analysis of polypeptides containing free amino acids in vitro transdermal absorption, pigskin as the research model, using quantitative detection method to detect the target content in the receiving liquid to calculate the cumulative permeation, penetration rate, diffusion percentage, the transdermal absorption of the sample to be tested was quantified, and the in vitro transdermal absorption of polypeptides containing free amino acids was analyzed. And compare the effect of the composition on the permeation behavior of polypeptides.

[0117] The experimental method is as follows: this test is based on pigskin system, and the concentration of sample in the receiving liquid at different time points is determined by high performance liquid chromatography method to evaluate the skin permeation behavior of the sample. The determination sample is the selected optimal concentration, different plant-based 2.5% fermentation substrate + 2% cyclohexapeptide composition 1-7 and cyclohexapeptide alone. Test system: pigskin; materials and equipment: TK-12D type drug transdermal absorption Franz diffusion cell, constant temperature magnetic stirrer (IKA).

[0118] ①The cumulative permeation Q calculation formula is as follows: Q = [Cn x V + ∑Ci x V0] / S (i = 1, 2, 3, …, n-1), note: Q: cumulative permeation; S: effective diffusion area; V: receiving liquid volume in receiving chamber; V0: volume of each sampling; Ci: drug concentration in receiving liquid from the first sampling to the last sampling; Cn: sample concentration measured at the nth sampling point;

[0119] ②Diffusion percentage P = Pt / Po*100%, note: Pt is the sample content in the receiving pool, and Po is the theoretical content of the sample in the release pool.

[0120] Note: p < 0.05 indicates significant difference.

[0121] The experimental scheme is shown in Table 4:

[0122] Table 4 Test scheme of in vitro permeability

[0123]

[0124] The experimental results are shown in Table 5:

[0125] Table 5 Test results of transdermal permeation rate of sample at different times

[0126]

[0127]

[0128] From Table 5, it can be seen that 1) the single fermentation liquid group has no penetration ability, and at the same time, compared with the single cyclohexapeptide group, the diffusion percentage of combinations 1-6 is significantly improved after 24 h, p<0.05, there is a significant difference, and when combination 2 (white willow bark fermentation liquid + cyclohexapeptide) is selected, the diffusion percentage reaches the highest, at this time the penetration effect is best, which shows that the complex filamentous yeast-like fungus fermentation product can promote the transdermal absorption of cyclohexapeptide, and when the white willow bark fermentation product is preferred, the promotion effect is best; 2) after 24 h, the diffusion percentage of combination 7 has no significant difference with the cyclohexapeptide group, p>0.05, which shows that the ordinary yeast fermentation product filtrate cannot improve the transdermal penetration rate of cyclopeptide.

[0129] Therefore, it can be seen that in this embodiment, at the best ratio, the complex filamentous yeast-like fungus fermentation product can promote the transdermal absorption of cyclohexapeptide, and when the white willow bark fermentation product + cyclohexapeptide combination is preferred, the promotion effect is most obvious, at this time the penetration effect is best.

[0130] Example 5: Test of antioxidant capacity of filamentous yeast-like fungus fermentation product composition

[0131] When the ROS content exceeds the body's ability to remove it, the balance between oxidation and antioxidant is broken, causing oxidative stress. As the outermost tissue of the human body, the skin is directly exposed to the environment and is more likely to cause oxidative stress and oxidative damage. Oxidative stress can cause skin aging and the occurrence of skin diseases. To test the antioxidant capacity of the six filamentous yeast-like fungus fermentation product compositions in Example 1, this embodiment uses an in vitro free radical scavenging experiment to verify the differences in antioxidant capacity of the relevant sample groups. The specific experimental process is as follows:

[0132] Antioxidant capacity test: Take the above optimal concentration of fermentation broth as the test solution. Take 1.0 mL of 50 mmol / L Tris-HCl buffer solution with pH 8.2 in a 10 mL EP tube, add 0.2 mL of test solution to it, mix well, and incubate at 25°C for 10 min, then add 10 mmol / L preheated at 25°C. 0.1 mL of o-phenyl triol solution, the total volume is 6 mL, shake quickly, and record the increase value (AS) of absorbance at 320 nm wavelength within 1 min by ultraviolet-visible spectrophotometer. Calculate the increase value of absorbance per minute in the linear range. Take another reagent as above, replace the sample solution with an equal volume of water, and determine the increase value of absorbance at 320 nm wavelength within 1 min. According to the formula of superoxide anion radical scavenging rate (%) = (Ao-AS) / Ao x 100%, calculate the superoxide anion radical scavenging rate, and draw the inhibition curve of different concentrations of test solution. According to the drawing curve, estimate the concentration of test solution when the superoxide anion radical scavenging rate reaches 50% (the optimal concentration in the said cyclic hexapeptide, sample and composition: cyclic hexapeptide is 2%, sample is 2.5% fermentation product, and composition is 2.5% fermentation product + 2% cyclic hexapeptide).

[0133] The experimental results are shown in Table 6:

[0134] Table 6 Comparison of antioxidant capacity of samples

[0135]

[0136]

[0137] Note: p<0.05 indicates significant difference.

[0138] From Table 6, it can be seen that 1) compared with the single cyclic hexapeptide group and the single sample group (single fermentation product), the concentration of the filamentous yeast sample composition when the superoxide anion radical scavenging rate reaches 50% is significantly reduced, p<0.05, there is a significant difference, which shows that the fermentation product composition is significantly better than the single fermentation product and the single cyclic hexapeptide group in terms of radical scavenging ability, indicating that after compounding, the antioxidant effect is synergistically enhanced, and the antioxidant capacity of the composition is significantly improved; and when the combination 2 (white willow bark fermentation broth + cyclic hexapeptide) is selected, the antioxidant capacity is the strongest; 2) compared with sample 7, the concentration of combination 7 when the superoxide anion radical scavenging rate reaches 50% is increased, which shows that the combination of ordinary yeast fermentation product and cyclic hexapeptide cannot improve the antioxidant capacity of the composition, and does not have synergistic effect; at the same time, compared with combinations 1-6, the antioxidant capacity is significantly decreased.

[0139] Therefore, in this embodiment, at the optimal ratio, the combination of the filamentous yeast-like fungus fermentation product and the cyclic hexapeptide can significantly improve the antioxidant capacity of the composition, achieving a synergistic effect; and when the preferred combination 2 (willow bark fermentation broth + cyclic hexapeptide) is used, the antioxidant capacity is the strongest.

[0140] Example 6 Test of in vitro anti-wrinkle efficacy of filamentous yeast-like fungus fermentation product composition

[0141] Collagen is mainly produced by fibroblasts present in the dermis layer of the skin, and is an important element for supporting the skin. Collagen accounts for about 80% of the dermis layer of the skin, making the skin full and plump, promoting the increase of Collagen I content, and achieving the effect of resisting wrinkle formation. Collagen I is the main collagen in the dermis, and Collagen III is also a main component of the extracellular matrix of the dermis. Collagen I and Collagen III are assembled into collagen fibers, which play an important role in the toughness of the skin. Collagen IV is the main component of the hemidesmosome basement membrane band complex and the key protein at the junction of the epidermis. MMP-1 belongs to the collagenase in matrix metalloproteinase, and its main hydrolysis substrate is fibrous collagen, i.e. Collagen I and Collagen III. After the content of MMP-1 decreases, the degree of collagen degradation decreases, thereby achieving a certain anti-wrinkle effect. In this experiment, fibroblasts were used as a research model, and a UVA irradiation was used to establish an in vitro photoaging model. In this embodiment, the combination 2 with the highest penetration performance in Example 4 was taken as an example (the composition was the optimal ratio: 2.5% fermentation product + 2% cyclic hexapeptide). The anti-photoaging and anti-wrinkle efficacy of the test substance was analyzed by detecting the changes of the enzyme matrix metalloproteinase MMP-1 and the collagen content Collagen I, Collagen III and Collagen IV. At the same time, in this embodiment, it has been confirmed through comparative experiments that among the combinations 1-6, the combination 2 has the best in vitro anti-photoaging and anti-wrinkle efficacy. Therefore, in this embodiment, the combination 2 with the best effect was selected for subsequent comparative experiments.

[0142] The cells used in this test were fibroblasts, which were obtained through a commercial channel. The main reagents were DMEM culture medium (Gibco), PBS (Solabio), MTT (Sigma), DMSO (Sigma), RNAiso Plus (Takara), reverse transcription kit (PrimeScript RT reagent Kit) (Takara), SYBR Premix Ex Taq (Takara), and so on. TM RT reagent Kit) (Takara), SYBR Premix Ex Taq TMII Fluorochrome (Takara), sterile ddH2O (Takara) and TGF-β1 (Peprotech), wherein Transforming growth factor beta (TGF-β) is a class of multifunctional cytokines that can be produced by a variety of tissue cells. The TGF-β signaling pathway is a pathway composed of a variety of members of multifunctional cytokines, corresponding receptors, and intracellular signal transduction molecules, which can affect the occurrence and development of diseases, regulate gene transcription, control cell cycle, affect cell proliferation, differentiation, adhesion, metastasis and apoptosis, and is a classic cell growth factor, which is usually used as a positive control to regulate cell proliferation. Main equipment CO2 incubator (Thermo, 150I), super clean bench (Sunan Antai, SW-CJ-1F), constant temperature box (Testor), ordinary PCR instrument (Bio-Rad), fluorescent quantitative PCR instrument (Bio-Rad, CFX-96), inverted microscope (Olympus, CKX41), UVA irradiation instrument (Philips).

[0143] Experimental process: the surface of the sample group was added with the corresponding concentration of sample working solution, the sample was uniformly distributed on the model surface, and was incubated in a CO2 incubator (37℃, 5% CO2) for 24h. After incubation, the residual test substance on the model surface was washed with sterile PBS solution, and the residual liquid in and outside the model was wiped off with a sterile cotton swab. The specific experimental design is shown in Table 7:

[0144] Table 7, experimental design

[0145]

[0146] Note: the blank control is normal fibroblasts, normal culture: fibroblasts are inoculated into a 96-well plate at a cell inoculation amount of 3.5×10 3 cells / well, incubated in an incubator (37℃, 5% CO2) overnight, and treated with the same UVA ultraviolet irradiation, but without the addition of the remaining substances; the positive control is normal fibroblasts, normal culture: fibroblasts are inoculated into a 96-well plate at a cell inoculation amount of 3.5×10 3 cells / well, incubated in an incubator (37℃, 5% CO2) overnight, and treated with the same UVA ultraviolet irradiation, but without the addition of the remaining substances; the positive control is normal fibroblasts, normal culture: fibroblasts are inoculated into a 96-well plate at a cell inoculation amount of 3.5×10 3 cells / well, incubated in an incubator (37℃, 5% CO2) overnight, and treated with the same UVA ultraviolet irradiation, but without the addition of the remaining substances; the positive control is normal fibroblasts, normal culture: fibroblasts are inoculated into a 96-well plate at a cell inoculation amount of 3.5×10

[0147] The experimental results are shown in Table 8, wherein Figures 1-4 Figure 1 ​The Collagen I gene expression column chart of each group, Figure 2 The Collagen III gene expression column chart of each group; Figure 3 The Collagen IV gene expression column chart of each group; Figure 4 The MMP-1 gene expression column chart of each group; in the chart, the vertical coordinate represents the amplification multiple relative to the blank control.

[0148] From Figure 1 It can be known from the above that 1) the Collagen I gene expression of the PC group is higher than that of the NC group, and the positive control is effective this time; 2) after being irradiated with UVA for 72 h, the Collagen I gene expression of the combination 2 reaches the highest, reaches 2.5 times close to the blank control, and the Collagen I gene expression of the combination 2 is significantly increased compared with the negative control group and the positive control group, which shows that the combination 2 not only has a significant anti-photoaging and anti-wrinkle effect, but also has a significantly higher effect than the transforming growth factor TGF-β1, and the Collagen I gene expression of the combination 2 is also significantly increased compared with the blank control, which shows that the combination 2 also has a significant effect of promoting the expression of the Collagen I gene; 3) compared with the cyclic peptide, the sample 2, the sample 7 and the combination 7, the Collagen I gene expression of the combination 2 is also significantly increased, which shows that the anti-photoaging and anti-wrinkle effects of the combination 2 are significantly better than those of the single fermentation product and the cyclic hexapeptide group, and when the filamentous yeast-like fungal fermentation product and the cyclic hexapeptide are compounded at the best ratio, a significant synergistic effect is produced, while the combination 7 has no significant synergistic effect.

[0149] From Figure 2 It can be known from the above that 1) the Collagen I gene expression of the PC group is higher than that of the NC group, and the positive control is effective this time; 2) after being irradiated with UVA for 72 h, the Collagen I gene expression of the combination 2 reaches the highest, reaches 2.5 times close to the blank control, and the Collagen I gene expression of the combination 2 is significantly increased compared with the negative control group and the positive control group, which shows that the combination 2 not only has a significant anti-photoaging and anti-wrinkle effect, but also has a significantly higher effect than the transforming growth factor TGF-β1, and the Collagen I gene expression of the combination 2 is also significantly increased compared with the blank control, which shows that the combination 2 also has a significant effect of promoting the expression of the Collagen I gene; 3) compared with the cyclic peptide, the sample 2, the sample 7 and the combination 7, the Collagen I gene expression of the combination 2 is also significantly increased, which shows that the anti-photoaging and anti-wrinkle effects of the combination 2 are significantly better than those of the single fermentation product and the cyclic hexapeptide group, and when the filamentous yeast-like fungal fermentation product and the cyclic hexapeptide are compounded at the best ratio, a significant synergistic effect is produced, while the combination 7 has no significant synergistic effect.

[0150] From Figure 3The results show that: 1) the expression level of Collagen III gene in group PC was higher than that in group NC, indicating that the positive control was effective; 2) after 72 hours of UVA irradiation, the expression level of Collagen IV gene in combination 2 reached its highest level, 2.7 times that of the blank control. Compared with the negative control and the positive control group, the expression level of Collagen IV gene in combination 2 was significantly increased. This indicates that combination 2 not only has significant anti-photoaging and anti-wrinkle effects, but also has effects significantly higher than those of transforming growth factor TGF-β1. Compared with the blank control, the expression level of Collagen IV gene in combination 2 was also significantly increased, indicating that combination 2 also has a significant effect on promoting the expression of Collagen IV gene; 3) compared with cyclic peptide, sample 2, sample 7 and combination 7, the expression level of Collagen IV gene in combination 2 was also significantly increased. This indicates that the anti-photoaging and anti-wrinkle effects of combination 2 are significantly better than those of the fermentation product and cyclic hexapeptide group alone. When combined with filamentous yeast-like fungal fermentation product and cyclic hexapeptide in the optimal ratio, a significant synergistic effect is produced, while combination 7 has no significant synergistic effect.

[0151] from Figure 4 The results show that: 1) Compared to the NC group, the MMP-1 expression level in the PC group was significantly reduced, indicating that the positive control was effective; 2) After 72 hours of UVA irradiation, the MMP-1 expression level of combination 2 was significantly reduced compared to both the negative and positive control groups, indicating that combination 2 not only has significant anti-photoaging and anti-wrinkle effects, but also has significantly higher effects than transforming growth factor TGF-β1. At the same time, compared to the blank control, the MMP-1 expression level of combination 2 was also significantly reduced, indicating that combination 2 significantly inhibits the expression of MMP-1 and has significant anti-photoaging and anti-wrinkle effects; 3) Compared with cyclic peptide, sample 2, sample 7, and combination 7, the MMP-1 expression level of combination 2 was also significantly reduced, indicating that the anti-photoaging and anti-wrinkle effects of combination 2 are significantly better than those of the fermentation product and cyclic hexapeptide alone. Moreover, when combined with filamentous yeast-like fungal fermentation product and cyclic hexapeptide in the optimal ratio, a significant synergistic effect is produced, while combination 7 has no significant synergistic effect.

[0152] Therefore, in this embodiment, under the optimal ratio, the filamentous yeast-like fungal fermentation product composition has the effect of significantly promoting the expression of Collagen I, Collagen III and Collagen IV and inhibiting the expression of MMP-1. Moreover, the synergistic effect of the compounded composition is significant. Furthermore, when the composition preferably consists of combination 2 (white willow bark fermentation broth + cyclic hexapeptide), it has the best anti-photoaging and anti-wrinkle effects.

[0153] Example 7: Test of the soothing ability of filamentous yeast-like fungal fermentation product composition

[0154] After the skin is exposed to ultraviolet radiation, a clinically visible redness phenomenon occurs. Taking keratinocytes as the research object, an in vitro damage model is established by UVB irradiation, and the soothing effect of the test compound is analyzed by detecting the changes in the content of inflammatory factors. TNF-α, an inflammatory cytokine, activates the κ-light chain enhancement (NF-KB) signaling pathway of B cells, and can also promote the production of various inflammatory factors, interleukin-1α (IL-1α), interleukin-8 (IL-8), and thus trigger skin inflammation. The increase in IL-1α and IL-8 produced by white blood cells can exacerbate the destruction of the epidermal barrier, and the activation of inflammatory factors such as tumor necrosis factor TNF-α can cause a fever.

[0155] In this embodiment, the soothing effect of the single fermentation broth, the cyclohexapeptide group and the different compositions after ultraviolet light UVB irradiation is quantitatively analyzed and evaluated by detecting the content of pro-inflammatory factors (IL-1α, IL-8, TNF-α) after ultraviolet light irradiation. The specific culture and detection process is as follows: select HaCaT cells in the logarithmic growth phase, perform routine treatment, adjust the cell concentration of the cell suspension to 2×10 4 Inoculate cells / cone in a 48-well plate and place in a 37℃, 5% CO2 incubator for 24h; after UVA (30J / cm 2 ) stimulation, administer according to the grouping, and then culture for 24h to obtain the supernatant. Use the corresponding ELISA kit (Abeam) to detect the amount of each inflammatory factor / mediator produced.

[0156] Specifically, it is divided into the following groups (the remaining conditions are the same; the cyclohexapeptide, fermentation broth and composition are all at the best concentration: cyclohexapeptide is 2%, fermentation broth is 2.5%, and composition is 2.5% fermentation broth + 2% cyclohexapeptide; at the same time, in this embodiment, it has been confirmed through comparative experiments that combination 2 has the best soothing ability in combinations 1-6, so combination 2 with the best effect is selected for subsequent comparative experiments).

[0157] 1. Blank control: keratinocytes without ultraviolet irradiation;

[0158] 2. Negative control (NC control): keratinocytes without adding any ingredients, and subjected to ultraviolet irradiation;

[0159] 3. Fermentation broth 2: keratinocytes only added with fermentation broth 2 (white willow bark fermentation broth) and subjected to ultraviolet irradiation;

[0160] 4. Fermentation broth 7: keratinocytes only added with fermentation broth 7 (ordinary commercially available yeast fermentation broth) and subjected to ultraviolet irradiation;

[0161] 5. Cyclic hexapeptide: Keratinocytes, with only cyclic hexapeptide added, after ultraviolet irradiation;

[0162] 6. Combination 2: Keratinocytes, with the addition of Combination 2 (white willow bark fermentation liquid + cyclic hexapeptide), and subjected to ultraviolet irradiation;

[0163] 7. Combination 7: Keratinocytes, with the addition of Combination 7 (commonly available yeast fermentation broth + cyclic hexapeptide), and subjected to ultraviolet irradiation;

[0164] Experimental results are as follows Figures 5-7 As shown, where Figure 5 The bar chart shows the expression of IL-1α levels. Figure 6 A bar chart showing IL-8 content expression. Figure 7 The bar chart shows the expression of TNF-α content, with the vertical axis representing the average concentration (pg / mL).

[0165] from Figure 5 The results show that: 1) After 24 hours of UVB irradiation, the IL-1α content of combination 2 was significantly reduced to about 5 pg / mL compared with the NC control group. At the same time, there was no significant difference between combination 2 and the blank control (p>0.05). This indicates that combination 2 has a significant effect in reducing inflammatory factors or inflammatory mediators in UV irradiation, thereby achieving the effect of soothing skin damage. 2) Compared with fermentation broth 2, fermentation broth 7, cyclic hexapeptide and combination 7, the IL-1α content of combination 2 was also significantly reduced. This indicates that the inhibitory effect of combination 2 on inflammatory factors and the effect of soothing skin damage are significantly better than those of fermentation broth alone, cyclic hexapeptide group and ordinary commercially available yeast fermentation broth group. Moreover, when combined with filamentous yeast-like fungal fermentation products and cyclic hexapeptide in the optimal ratio, a significant synergistic effect is produced, while combination 7 has no significant synergistic effect.

[0166] from Figure 6 The results show that: 1) After 24 hours of UVB irradiation, the IL-8 content of combination 2 was significantly reduced to about 20 pg / mL compared with the NC control group. At the same time, there was no significant difference between combination 2 and the blank control (p>0.05). This indicates that combination 2 has a significant effect in reducing inflammatory factors or inflammatory mediators in UV irradiation, thereby achieving the effect of soothing skin damage. 2) Compared with fermentation broth 2, fermentation broth 7, cyclic hexapeptide and combination 7, the IL-8 content of combination 2 was also significantly reduced. This indicates that the inhibitory effect of combination 2 on inflammatory factors and the effect of soothing skin damage are significantly better than those of fermentation broth alone, cyclic hexapeptide group and ordinary commercially available yeast fermentation broth group. Moreover, when combined with filamentous yeast-like fungal fermentation products and cyclic hexapeptide in the optimal ratio, a significant synergistic effect is produced, while combination 7 has no significant synergistic effect.

[0167] from Figure 7It can be known that 1) after irradiation with UVB for 24h, the TNF-α content of combination 2 is significantly reduced to about 4 pg / mL compared with the NC control group, and the TNF-α content of combination 2 is also significantly reduced compared with the blank control, which indicates that combination 2 has a significant effect of inhibiting pro-inflammatory cytokine TNF-α, thereby achieving the effect of soothing skin damage; 2) compared with fermentation broth 2, fermentation broth 7, cyclohexapeptide and combination 7, the TNF-α content of combination 2 is also significantly reduced, which indicates that the effect of combination 2 in inhibiting pro-inflammatory cytokines and soothing skin damage is significantly better than that of single fermentation broth, cyclohexapeptide group and ordinary commercial yeast fermentation broth group, and when the fermentation product of the filamentous yeast-like fungus and the cyclohexapeptide are compounded at the best ratio, a significant synergistic effect is produced, while combination 7 has no significant synergistic effect.

[0168] It can be known that in the present embodiment, at the best composition ratio, the filamentous yeast-like fungus fermentation product composition has a significant effect of inhibiting the expression of pro-inflammatory cytokine TNF-α and inflammatory factors IL-1α and IL-8, and soothing skin damage, and the synergistic effect of the compounded composition is significant, and when the composition is preferred combination 2 (fermentation broth of white willow bark + cyclohexapeptide), the best effect of inhibiting the expression of inflammatory factors and soothing skin damage by ultraviolet light is achieved.

[0169] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. The use of a composition in the preparation of formulations with improved permeability, antioxidant capacity, anti-photoaging properties, and anti-wrinkle effects, characterized in that, The composition comprises, by weight percentage, 2.5% filamentous yeast-like fungus fermentation product of white willow bark and 2% cyclic hexapeptide, with the balance being the basic component, and the sum of the weight percentages of the above components is 100%; the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

2. A composition, characterized in that, The product comprises, by mass percentage, 0.05–15% fermentation product of filamentous yeast-like white willow bark fungus and 0.05–5% cyclic hexapeptide, with the remainder being the basic component, and the sum of the mass percentages of the above components is 100%; the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

3. The composition according to claim 2, characterized in that, The preparation method of the fermentation product of the filamentous yeast-like fungus *Galactomyces Citri-Aurantii* is as follows: multiple fermentation is carried out using the filamentous yeast-like fungus *Galactomyces Citri-Aurantii*, that is, the precipitate obtained by centrifugation after the first fermentation is sterilized and used as a component of the culture medium for the next fermentation. The multiple fermentation is carried out in 4 stages, including the following steps: (1) Culture filamentous yeast-like fungi to obtain inoculum; (2) The inoculum was inoculated into a liquid natural culture medium for the first fermentation; (3) Collect the supernatant produced by centrifugation at the end of each fermentation, mix them and use them as the fermentation product; The natural substance in the culture medium is white willow bark.

4. The use of a composition in the preparation of an formulation for enhancing the efficacy of soothing skin damage caused by ultraviolet light, characterized in that, The composition comprises, by weight percentage, 2.5% filamentous yeast-like fungus fermentation product of white willow bark and 2% cyclic hexapeptide, with the balance being the basic component, and the sum of the weight percentages of the above components is 100%; the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

5. Use of the composition according to any one of claims 2 to 3 for preparing formulations that enhance antioxidant capacity, anti-aging and wrinkle reduction, and increase collagen content.

6. Use of a composition in the preparation of an agent that enhances the ability to repair photodamage, characterized in that, The composition comprises, by weight percentage, 0.1-10% rice fermentation product of filamentous yeast-like fungi and 0.5-3% cyclic hexapeptide, with the balance being the basic component, and the sum of the weight percentages of the above components is 100%; the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

7. A composition, characterized in that, The product comprises, by mass percentage, 0.05-10% rice fermentation product of filamentous yeast-like fungi and 0.05-5% cyclic hexapeptide, with the remainder being the basic component, and the sum of the mass percentages of the above components is 100%; the sequence of the cyclic hexapeptide is Cyclo(Gln-Gly-Pro-Gln-Gly-Pro).

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