CN117017844A

CN117017844A - Filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof

Info

Publication number: CN117017844A
Application number: CN202311161749.XA
Authority: CN
Inventors: 李小静; 黄虎; 朱慧林
Original assignee: Shanghai Zhongyi Daily Chemical Co ltd
Current assignee: Shanghai Zhongyi Daily Chemical Co ltd
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2023-11-10

Abstract

The invention provides a filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof, and the filamentous yeast-like fungus fermentation composition with superior permeability and higher full effects is finally prepared by screening and compounding different filamentous yeast-like fungus fermentation products and cyclohexapeptide and limiting the relative content of the filamentous yeast-like fungus fermentation products and the cyclohexapeptide. Compared with the original fermentation liquid and the original cyclic peptide, the obtained fermentation composition can remarkably improve the capabilities of resisting skin photoaging, repairing photodamage, reducing apoptosis or resisting aging and removing wrinkles, remarkably reduce the generation of free radicals, inhibit the synthesis of matrix metalloproteinase, remarkably improve the content of collagen, and has the effects of relieving and repairing, relieving oxidative stress and inhibiting inflammatory factors or inflammatory mediators, and has stronger application potential in the fields of beauty and skin care and medical and anti-aging.

Description

Filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof

Technical Field

The invention belongs to the technical field of biological fermentation and cosmetic application, and particularly relates to a filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof.

Background

Human skin is exposed to solar Ultraviolet (UV) radiation daily. Ultraviolet rays in sunlight mainly include 3 types according to wavelength division thereof, long-wave ultraviolet rays (UVA, wavelength 320 to 400 nm), medium-wave ultraviolet rays (UVB, wavelength 290 to 320 nm) and short-wave ultraviolet rays (UVC, wavelength 200 to 290 nm). UVC having a wavelength shorter than 290nm is blocked by the ozone layer; UVA has extremely strong penetrating power, and can influence the dermis of the skin and even subcutaneous tissue areas, so that oxidative stress and tissue inflammation are caused; UVB can interact directly with DNA to produce bipyrimidine photoproducts, resulting in DNA damage during DNA replication. Acute ultraviolet radiation can lead to skin sunburn, abnormal pigmentation, and immunosuppression, and prolonged exposure to Ultraviolet (UV) radiation can lead to premature aging of the skin, i.e., photoaging of the skin. Research has shown that photoaging occurs primarily in association with Reactive Oxygen Species (ROS), including superoxide anions, hydroxyl radicals, hydrogen peroxide, and the like. The generation of ROS is caused by normal physiological metabolism of the human body and environmental influences such as ultraviolet irradiation, environmental pollution, etc. Excessive ROS levels produce oxidative stress that causes oxidative damage mainly including damage to cellular components such as cell membranes, DNA, lipids, or proteins, such as by inducing synthesis of matrix protein metalloenzymes (MMPs), which degrade collagen and elastin in ECM, and inhibit collagen and elastin synthesis. In addition, ROS can promote the development of inflammatory responses by activating nuclear factor- κB (NF- κB), thereby affecting the expression of proinflammatory cytokines such as interleukin-1 (IL-1), epidermal Growth Factor (EGF), and tumor necrosis factor- α (TNF- α). Oxidative stress further results in decreased keratinocyte viability, slower turnover, decreased barrier function, dryness, desquamation, etc. of the skin, such as in the epidermis; the number of fibroblasts in the dermis layer gradually decreases, collagen synthesis slows down, and decomposition accelerates. Photoaged skin thus appears as rough, loose, deep wrinkles, telangiectasia reddening, and darkened pigmentation, etc. Based on the above, how to prevent skin photooxidation or alleviate ultraviolet damage of skin and delay skin photoaging has been greatly paid attention to. Antioxidant is a vital route to combat skin aging. Most conventional sunscreens and whitening agents and the like can absorb part of the ultraviolet rays due to various safety and efficiency problems, but they do not fully satisfy the need for cosmetic applications such as improving skin photoaging.

At present, some natural fermented products contain components such as vitamins, amino acids, polypeptides, minerals, polysaccharides, flavones, polyphenols and the like, have certain antioxidation efficacy, and the natural fermented products have the advantages of higher safety, more outstanding functionality, good biocompatibility and the like, so that the natural fermented products gradually become hot spots for people to develop and research. For example, the Chinese patent publication No. CN101724006A discloses a preparation method of a fermentation product of filamentous yeast-like fungi, which adopts a pure natural culture medium prepared from the filamentous yeast-like fungi and pine mushroom, white willow bark, ganoderma lucidum, white truffle or rice to carry out multiple fermentation, improves the cell safety, improves the skin care effect of the fermentation product, saves the cost, is suitable for production and manufacture, is mainly applied to the beauty and skin care products such as moisturizing, oil control, skin color brightening and the like, but does not relate to the use of the effects of resisting photoaging and the like.

The polypeptide which is one of the anti-aging classical components is widely applied to the fields of cosmetics and beauty and skin care, however, certain application pain points exist, on one hand, the permeability is low, the amount of the polypeptide which actually enters the skin to play a role is limited, on the other hand, most of the polypeptide can be degraded and deactivated by protease in blood and skin tissues to lose efficacy, so that the bioavailability of the polypeptide raw material is very low, and the efficacy cannot be exerted to the greatest extent. In recent years, scientists prove that the cyclic peptide (or cyclic peptide) has the characteristics of long in vivo half-life, relatively stable structure, strong permeability and the like, so that the external cyclic peptide-containing product can play a certain role in nursing, defending and slowing down aging, and the development of the cyclic peptide has a certain innovation and practicability in the field of beauty and skin care. The Chinese patent No. 116162135A discloses a cyclohexapeptide and application thereof, and efficacy researches on the cyclohexapeptide show that the cyclohexapeptide has more excellent efficacy than linear peptide in the aspects of skin wrinkle resistance and tightening, is mainly applied to the fields of cosmetics, medical tightening wrinkle resistance and the like, but does not relate to the application of the efficacy of photoaging resistance, oxidation resistance, relaxation repair and the like.

Therefore, it is highly desirable to find a filamentous yeast-like fungus fermentation product composition with multiple effects, and to compound a composition by screening fermentation products and cyclohexapeptide, so as to achieve the effects of remarkably improving skin photoaging resistance, photodamage repair or aging resistance and wrinkle removal, remarkably improving collagen content, and inhibiting MMP-1, relieving repair, relieving oxidative stress, and inhibiting inflammatory factors or inflammatory mediators.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a filamentous yeast-like fungus fermentation product composition with multiple effects and application thereof. The composition has the advantages of remarkably reducing the generation of free radicals, inhibiting the synthesis of matrix metalloproteinase, protecting ultraviolet injury, reducing apoptosis, preventing photoaging and the like, and has stronger application potential in the fields of beauty and skin care and medical and aesthetic aging resistance.

In order to achieve the above purpose, the invention is realized by adopting the following scheme:

In one aspect, the invention provides a composition comprising a filamentous yeast-like fungal fermentation product and a cyclohexapeptide.

In some embodiments, the filamentous yeast-like fungus ferment contains abundant amino acids, polypeptides and the like, can directly supply skin cell nutrients, and can further cooperate with cyclic peptides to promote proliferation capacity of keratinocytes and fibroblasts and activate cells after the preferred proportion, thereby providing a basis for realizing efficacy.

Further, the composition comprises, by mass, 0.05% -90% of a filamentous yeast-like fungus fermentation product, 0.05% -5% of a cyclohexapeptide, and the balance being a base component, wherein the sum of the mass percentages of the above components is 100%.

Further, the sequence of the cyclohexapeptide is Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

In some embodiments, the high-purity cyclohexapeptide prepared by using the optimal preparation method and the composition obtained by compounding the cyclohexapeptide with the filamentous yeast-like fungus fermentation product have remarkable synergistic capacity and better effects of beautifying and protecting skin.

Further, the preparation method of the filamentous yeast-like fungus fermentation product comprises the following steps: performing multiple fermentation with filamentous yeast-like fungus Galactomyces Citri-Aurantii, namely sterilizing the precipitate obtained by centrifugation after the previous fermentation is used for the culture medium component of the next fermentation, wherein the number of times of the multiple fermentation is 4, and the method comprises the following steps:

(1) Culturing a filamentous yeast-like fungus to obtain an inoculation liquid;

(2) Inoculating the inoculating liquid into a liquid natural substance culture medium for primary fermentation;

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

the natural substances in the natural substance culture medium comprise any one of rice, white willow bark, ganoderma lucidum, white truffle, matsutake or betulin; when the natural product is rice, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus rice fermentation product; when the natural product 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 product is ganoderma lucidum, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus ganoderma lucidum fermentation product; when the natural product is white truffle, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus white truffle fermentation product; when the natural product is tricholoma matsutake, the filamentous yeast-like fungus fermentation product is a filamentous yeast-like fungus tricholoma matsutake fermentation product; when the natural product is Fuscoporia obliqua, the fermentation product of the filamentous yeast-like fungus is Fuscoporia obliqua fermentation product of the filamentous yeast-like fungus.

In some embodiments, the filamentous yeast-like fungal fermentation product is prepared using a 4-fold fermentation technique, which is the most safe and skin care effective compared to other times of multiple fermentations. The fermentation product has higher protein content level and better skin care effect, and the filtrate of the fermentation product has higher cell safety concentration range.

Preferably, the composition comprises, by mass, 0.05% -10% of a rice fermentation product of a filamentous yeast-like fungus, or 0.05% -15% of a white willow bark fermentation product of a filamentous yeast-like fungus, or 0.05% -7.5% of a ganoderma fermentation product of a filamentous yeast-like fungus, or 0.05% -10% of a white truffle fermentation product of a filamentous yeast-like fungus, or 0.05% -10% of a pine mushroom fermentation product of a filamentous yeast-like fungus, or 0.05% -15% of a white fudge fermentation product of a filamentous yeast-like fungus.

In some embodiments, the safe concentrations of the fermentation product and the cyclohexapeptide in the composition are respectively screened, and the experimental result shows that the cell survival rate can reach more than 70% in the range of 0.05% -10% of rice fermentation product, 0.05% -15% of white willow bark fermentation product, 0.05% -7.5% of ganoderma lucidum fermentation product, 0.05% -10% of white truffle fermentation product, 0.05% -15% of betulin fermentation product or 0.05% -5% of cyclohexapeptide, meanwhile, the safe tolerance to cells is not changed on the basis of the original fermentation liquid, and the filamentous yeast-like fungus fermentation product composition has extremely high cell tolerance and better biocompatibility compared with the commercial yeast fermentation product. Preferably, combination 2 (white willow bark broth + cyclohexapeptide) and combination 6 (betulin broth + cyclohexapeptide) can be used at a higher concentration on the basis of safety, indicating that they have low biotoxicity and high safety, can be used at a higher concentration, and at the same time, the more remarkable the effect produced by the composition at the higher concentration.

Preferably, the composition comprises, by mass, 2.5% of rice fermentation product of filamentous yeast-like fungi, or 2.5% of white willow bark fermentation product of filamentous yeast-like fungi, or 2.5% of ganoderma fermentation product of filamentous yeast-like fungi, or 2.5% of white truffle fermentation product of filamentous yeast-like fungi, or 2.5% of tricholoma matsutake fermentation product of filamentous yeast-like fungi, or 2.5% of fumaron fermentation product of filamentous yeast-like fungi; also included was a cyclic hexapeptide of 2%.

In another aspect, the present invention provides a use of a 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 matsutake fermentation product, a filamentous yeast-like fungus betulina fermentation product, and a cyclohexapeptide for preparing a preparation for preventing skin photoaging or repairing photodamage or improving permeability.

In some embodiments, at the optimal ratio (2.5% fermentation product and 2% cyclohexapeptide), the compounded filamentous yeast-like fungal fermentation product achieves an effect of promoting transdermal absorption of cyclohexapeptide, and the osmotic effect is optimal when white willow bark fermentation product + cyclohexapeptide combination is preferred.

Further, the composition comprises a filamentous yeast-like fungal rice fermentation product and a cyclohexapeptide having the sequence Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

In some embodiments, the photodamage repair capability of the filamentous yeast-like fungus fermentation product composition is tested, and experimental results show that the photodamage repair capability of the filamentous yeast-like fungus fermentation product composition is remarkable when the concentration of the filamentous yeast-like fungus fermentation product is 0.1% -10% and the concentration of the cyclohexapeptide is 0.5% -3%, and meanwhile, the photodamage repair capability of the filamentous yeast-like fungus fermentation product composition slightly promotes the cell activity, and the photodamage repair capability of the filamentous yeast-like fungus fermentation product composition reaches the strongest when the concentration of the fermentation product is 2.5% and the concentration of the cyclohexapeptide is 2%; meanwhile, when the combination 1 (rice fermentation liquor and cyclohexapeptide) is preferred, the effects of resisting skin photoaging, repairing photodamage and promoting cell activity are achieved, and the application value is extremely high.

In yet another aspect, the present invention provides a use of a composition comprising a cyclohexapeptide and any one of a filamentous yeast-like fungi rice fermentation product, a filamentous yeast-like fungi white willow bark fermentation product, a filamentous yeast-like fungi ganoderma lucidum fermentation product, a filamentous yeast-like fungi white truffle fermentation product, a filamentous yeast-like fungi matsutake fermentation product, a filamentous yeast-like fungi betulina fermentation product, for preparing a formulation that enhances the ability to repair, inhibits inflammatory factors or inflammatory mediators.

Further, the composition comprises a fermentation product of the filamentous yeast-like fungus white willow bark and a cyclohexapeptide having the sequence Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

In some embodiments, the filamentous yeast-like fungus fermentation product composition has the efficacy of significantly inhibiting the expression of pro-inflammatory cytokines TNF- α and inflammatory factors IL-1α and IL-8, and soothing skin lesions at an optimal ratio (2.5% fermentation product and 2% cyclohexapeptide), and the compounded composition has the efficacy of significantly synergistically enhancing the efficacy of optimally inhibiting inflammatory factor expression and soothing skin uv light lesions when the composition is preferably combined with 2 (white willow bark fermentation broth + cyclohexapeptide).

In a further aspect, the invention provides the use of a composition according to any one of the above technical schemes for the preparation of an anti-ageing, wrinkle-removing, collagen content-increasing or MMP-1-inhibiting formulation.

In some embodiments, at the optimal ratio (2.5% fermentation product and 2% cyclohexapeptide), the filamentous yeast-like fungus fermentation product composition has the efficacy of significantly promoting the expression of Collagen I, collagen III and Collagen IV and inhibiting the expression of MMP-1, and the compounded composition has significant synergistic effect and has optimal anti-wrinkle efficacy when the composition is preferably combined with 2 (white willow bark fermentation broth+cyclohexapeptide).

In a further aspect, the present invention provides the use of a composition according to any one of the above technical solutions for the preparation of a formulation for alleviating oxidative stress and improving antioxidant capacity.

In some embodiments, under the optimal ratio (2.5% of fermentation product and 2% of cyclohexapeptide), the antioxidant capacity of the composition can be obviously improved by compounding the filamentous yeast-like fungus fermentation product and the cyclohexapeptide, and the synergistic effect is achieved; and when the combination of 2 (white willow bark fermentation broth + cyclohexapeptide) is preferred, the oxidation resistance is the strongest.

The beneficial effects of the invention are as follows:

1. the series of filamentous yeast-like fungus fermentation product compositions with multiple effects provided by the invention are convenient to ferment and synthesize, have high biocompatibility and are safe and non-irritating to human bodies after optimal proportion is optimized, and can be applied to the field of cosmetics.

2. The series of filamentous yeast-like fungus fermentation products provided by the invention are used as skin conditioning agents for conditioning skin epidermis layers, promote the osmotic absorption of original cyclohexapeptide, play a role in synergy, and enable the fermentation products to exert a stronger effect.

3. The series of filamentous yeast-like fungus fermentation products provided by the invention contain rich amino acids, polypeptides and the like, can directly supply skin cell nutrients, and after optimal proportion is optimized, the composition obtained by synergistic cyclohexapeptide compounding has obvious repairing capability on photodamage of keratinocytes and fibroblasts, and simultaneously obviously enhances the proliferation capability of the cells, activates the cells, and provides a foundation for realizing the efficacy of the composition.

4. The series of filamentous yeast-like fungus fermentation product compositions with multiple effects provided by the invention can obviously inhibit the expression of ROS/inflammatory factors or inflammatory mediators IL-1, IL-8 and TNF-alpha after optimizing the optimal proportion, realize the effects of relieving and alleviating oxidative stress, obviously promote the expression of key genes related to a barrier, and help to alleviate the problems of skin dryness, desquamation and the like caused by the weakening of the barrier function.

5. The series of filamentous yeast-like fungus fermentation product compositions with multiple effects provided by the invention can obviously promote the expression of collagen I, collagen III and collagen IV genes under a photoaging model after optimizing the optimal proportion, obviously improve the effect of the fermentation composition on improving the content of collagen, provide theoretical basis for collagen production of skin, and also can obviously inhibit MMP-1 production, so that the degradation of collagen can be slowed down. The mechanism of promoting the generation and inhibiting the degradation of the double channels balances and supplements the collagen so as to have better anti-aging and wrinkle-removing effects on the skin.

Drawings

FIG. 1 is a bar graph showing the expression level of the Collagen I gene of each group.

FIG. 2 is a bar graph showing the expression level of the Collagen III gene in each group.

FIG. 3 is a bar graph showing the expression level of the Collagen IV gene in each group.

FIG. 4 is a histogram showing the MMP-1 gene expression level of each group.

FIG. 5 is a histogram of IL-1. Alpha. Content expression.

FIG. 6 is a bar graph showing IL-8 content expression.

FIG. 7 is a bar graph of TNF- α content expression.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto.

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

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the invention, the definition of ' anti-photoaging ' is ' resisting damage caused under ultraviolet irradiation ', has the effects of repairing photodamage of cells, remarkably improving the survival rate of the cells, remarkably resisting oxidation capability, remarkably reducing ROS content, simultaneously remarkably reducing wrinkle resistance, remarkably promoting the expression quantity of collagen genes, inhibiting the expression of MMP-1, remarkably relieving capability and remarkably inhibiting the expression of inflammatory factors and pro-inflammatory factors '.

EXAMPLE 1 preparation of a fermentation product of a filamentous Yeast-like fungus and a Cyclohexapeptide

In this example, the best method was chosen for the preparation of filamentous yeast-like fungal fermentation products and cyclohexapeptides, the specific preparation process being as follows:

1. preparation of a filamentous yeast-like fungal fermentation product: comprises the steps of preparing a natural culture medium, supplementing nutrient solution and inoculating solution, and preparing fermentation (the filamentous yeast-like fungi Galactomyces Citri-Aurantii is purchased from Chinese microorganism strain net, the purchase website is https:// www.biobw.org/China-strain/bio-113662.Html; and the six natural products are respectively Mingri organic porridge rice, shandong white willow bark, changbaishan ganoderma lucidum, frozen Yunnan white truffle, yunnan Shanglira pine mushroom and Jilin Changbaishan birch).

1) Preparation of the culture medium: adopts pine mushroom, white willow bark, ganoderma lucidum, rice, white truffle and betulin as natural substances to prepare liquid natural substance culture medium, and the flow is as follows: weighing 100g of natural materials, powdering the natural materials, mixing the natural materials with pure water, wherein the volume ratio of the natural material powder to the pure water is 1:20, sterilizing at high temperature to form semi-solid paste, standing, layering (upper layer suspension clear liquid and lower layer turbid precipitate), and sealing and storing in a cold storage (4 ℃) for standby. Obtaining pine mushroom culture medium, white willow bark culture medium, ganoderma lucidum culture medium, rice culture medium, white truffle culture medium and white birch mushroom culture medium respectively. Preparing a supplementary nutrient solution: a 1% sucrose sterile nutrient solution (sucrose: pure water=1:100) was prepared as a supplemental nutrient solution for the extended fermentation.

2) Preparing an inoculation liquid: culturing filamentous yeast-like fungus Galactomyces Citri-Aurantii purchased from Chinese microorganism strain net in YM medium small 0.5L liquid state, fermenting at 26deg.C for 3 days, stirring every day, shaking for 1 time, and measuring OD of light absorption turbidity ₆₀₀ Is that>1.50 as inoculum.

3) The filamentous yeast-like fungal fermentation product undergoes a first fermentation: inoculating the inoculating liquid into a fermentation tank, and performing natural culture medium liquid fermentation (the volume ratio of the inoculating liquid to the culture medium is 1:1000, and the OD value is 1.50) for 72h at the fermentation temperature: and (3) carrying out high-speed centrifugation (5000 rpm) after fermentation at 26+/-3 ℃, separating the supernatant fluid of the first fermentation, storing in a cold storage for standby, and sterilizing the precipitate at high temperature and high pressure for standby.

4) And (3) secondary fermentation: taking all the precipitate layers separated by the first fermentation, and mixing with 1:1000 (sediment layer: 1% sucrose sterile nutrient solution), and performing secondary fermentation (volume ratio of inoculating solution to culture medium is 1:1000, OD value is 1.50) for 72h, fermentation temperature: and (3) carrying out high-speed centrifugation (5000 rpm) after fermentation at 26+/-3 ℃, separating a supernatant obtained by the second fermentation, storing in a cold storage for standby, and sterilizing a precipitate at high temperature and high pressure for standby.

5) And (3) fermenting for the third time: taking all the sediment layers separated by the second fermentation, the sediment is reduced compared with the last time due to consumption, and the sediment layer is also taken as a sediment layer with the following formula 1: the third fermentation (the volume ratio of the inoculation liquid to the culture medium is 1:1000, and the OD value is 1.50) is carried out for 72 hours by the feed liquid ratio of 1000 (sediment layer: 1% sucrose sterile nutrient solution), and the fermentation temperature is as follows: and (3) carrying out high-speed centrifugation (5000 rpm) after fermentation at 26+/-3 ℃, separating supernatant fluid of the third fermentation, storing in a cold storage for standby, and sterilizing precipitate at high temperature and high pressure for standby.

6) Fourth fermentation: taking all the third fermentation separated sediment layers, further reducing sediment, and also taking 1: the fermentation is carried out for the fourth time (the volume ratio of the inoculating solution to the culture medium is 1:1000, the OD value is 1.50) according to the feed liquid ratio of 1000 (sediment layer: 1% sucrose sterile nutrient solution), the fermentation is carried out for 72 hours, and the fermentation temperature is: and (3) after the fermentation is finished, carrying out high-speed centrifugation (5000 rpm), separating a supernatant obtained by the fourth fermentation, and storing the supernatant in a cold storage for standby.

7) Mixing the supernatant collected after the 4 times of fermentation to obtain a filamentous yeast-like fungus fermentation product, adding 0.5% of 1, 2-hexanediol and 0.4% of p-hydroxyacetophenone as preservatives to obtain a filtrate of the filamentous yeast-like fungus fermentation product, and finally obtaining the following 6 filamentous yeast-like fungus fermentation products respectively:

A. when the natural product is rice, obtaining a filamentous yeast-like fungus rice fermentation product (i.e., a filamentous yeast-like fungus rice fermentation broth);

B. when the natural product is white willow bark, obtaining a fermentation product of the white willow bark of the filamentous yeast-like fungus (namely a fermentation liquid of the white willow bark of the filamentous yeast-like fungus);

C. when the natural product is ganoderma lucidum, obtaining a filamentous yeast-like fungus ganoderma lucidum fermentation product (namely, a filamentous yeast-like fungus ganoderma lucidum fermentation liquid);

D. When the natural substance is white truffle, obtaining a fermentation product of the white truffle of the filamentous yeast-like fungi (namely a fermentation liquid of the white truffle of the filamentous yeast-like fungi);

E. when the natural product is tricholoma matsutake, obtaining a fermentation product of the tricholoma matsutake (namely tricholoma matsutake fermentation liquid) of the filamentous yeast-like fungus;

F. when the natural product is Fuscoporia obliqua, the fermentation product of Fuscoporia obliqua (i.e. fermentation broth of Fuscoporia obliqua) is obtained.

2. Preparation of the cyclohexapeptide:

1) The preparation process comprises the following steps: the method comprises the steps of sequentially adopting amino acid raw materials Fmoc-Pro-OH, fmoc-Gly-OH, fmoc-Gln (Trt) -OH, fmoc-Pro-OH, fmoc-Gly-OH and Fmoc-Gln (Trt) -OH to carry out linear peptide connection, then cutting to obtain full-protection linear peptide H-Gln (Trt) -Gly-Pro-Gln (Trt) -Gly-Pro-OH, cyclizing the linear peptide H-Gln (Trt) -Gly-Pro-Gln (Trt) -Gly-Pro-OH to obtain a closed-loop product Cyclo (Gln (Trt) -Gly-Pro-Gln (Trt) -Gly-Pro), and finally cutting again to obtain crude peptide Cyclo (Gln-Gly-Pro). After the crude product is obtained by cutting, the purified and freeze-dried product Cyclo (Gln-Gly-Pro-Gln-Gly-Pro) is obtained, and the purity is 99.2 percent.

2) The cyclohexapeptide prepared as described above and its derivatives or salts thereof may exist as stereoisomers or a mixture of stereoisomers; for example, the above amino acids containing them may have the configuration L-, D-, or be racemic independently of each other. Thus, it is possible to obtain isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the isomer or isomeric mixture present. The preferred structure of the peptides used in this example are pure isomers, i.e., enantiomers or diastereomers.

The filamentous yeast-like fungi fermentation product is prepared in the embodiment, wherein the filtrate of the fermentation product of the filamentous yeast-like fungi subjected to multiple fermentation has higher cell safety and better skin care effect; meanwhile, a small molecule cyclohexapeptide with anti-wrinkle effect is also prepared in the embodiment. The filamentous yeast-like fungus fermentation composition with superior permeability and higher full efficacy is finally prepared by screening and compounding different filamentous yeast-like fungus fermentation products and small molecule cyclohexapeptide and limiting the relative content of the filamentous yeast-like fungus fermentation products and the small molecule cyclohexapeptide. Compared with the original fermentation liquid and the original cyclic peptide, the obtained fermentation composition can remarkably improve the capabilities of resisting skin photoaging, repairing photodamage, reducing apoptosis or resisting aging and removing wrinkles, remarkably reduce the generation of free radicals, inhibit the synthesis of matrix metalloproteinase, remarkably improve the content of collagen, and has the effects of relieving and repairing, relieving oxidative stress and inhibiting inflammatory factors or inflammatory mediators, and has stronger application potential in the fields of beauty and skin care and medical and anti-aging.

EXAMPLE 2 detection of cell safety and screening of composition concentration of fermentation product of filamentous Yeast-like fungi

To obtain the applicable ratio ranges of the filamentous yeast-like fungus fermentation product composition of example 1, the applicable ratio ranges are selected by performing cell safety detection on the composition in this example, and the specific experimental principles and methods are as follows:

test principle and method: the MTT method is also called MTT colorimetric method, and is a method for detecting the survival and growth of cells. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystal formazan and deposit the blue-violet crystal formazan in cells, and dead cells have no function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value can be measured by an enzyme-linked immunosorbent assay (ELISA) to indirectly reflect the number of living cells. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers. The method is mainly used for evaluating cytotoxicity and promoting cell proliferation capacity in a laboratory, and the experimental method is implemented by referring to MTT method cell activity determination operation standard. Cell safety assessment: when the MTT method is used for measuring the cell survival rate to be less than 70%, the MTT method is toxic, and the higher the value, the higher the safety degree is.

The plant substrates of the fermentation products of the filamentous yeast-like fungi are respectively as follows: the rice, white willow bark, lucid ganoderma, white truffle, tricholoma matsutake and betulina are respectively divided by samples 1-6, sample 7 is a fermentation product of certain commercial saccharomycetes, and the fermentation compositions are respectively compared with compositions 1-7 according to samples 1-7. In the testing process, firstly, selecting a corresponding high concentration to start the test, and sequentially reducing the concentration to obtain the highest concentration of data with the cell activity of the sample being more than 70%, wherein the higher the highest concentration is, the lower the biotoxicity is; screening a single fermentation product and a single cyclic peptide respectively, and compounding the single fermentation product and the single cyclic peptide after screening to obtain the safe highest concentration, wherein the specific information and the concentration are shown in the following table 1:

Table 1 sample information table

Sample group	Test sample group	Screening for safe concentrations
			Combination 1	Rice fermentation liquor + cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
Combination 2	White willow bark fermentation liquor and cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
			Combination 3	Ganoderma lucidum fermentation liquor and cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
Combination 4	Truffle fermentation liquor and cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
			Combination 5	Tricholoma matsutake fermentation broth + cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
Combination 6	Inonotus obliquus fermentation broth + cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent
			Combination 7	Commercial yeast fermentation product +cyclohexapeptide	Fermentation liquor is from 0.05% to 20%; the cyclic peptide accounts for 0.05 to 5 percent

In this example, the statistical cell viability reached about 70% of the highest concentration of the sample, and the test sample showed that the highest concentration of the cyclohexapeptide was 5% and the highest concentrations of the fermentation broth samples 1-7 were 10%, 15%, 7.5%, 10%, 15%, 5%, respectively, and the results of the corresponding compositions after compounding are shown in table 2 below:

table 2 composition safety results table

As can be seen from table 2, on the one hand, the filamentous yeast-like fungus fermentation product composition of the present invention has higher cell tolerance and better biocompatibility than the commercially available yeast fermentation products; on the other hand, the high-concentration peptide is compounded on the basis of the original fermentation liquid, so that the safety tolerance to cells is not changed, and the method has extremely high application value; in yet another aspect, combination 2 (white willow bark broth + cyclohexapeptide) and combination 6 (betulin broth + cyclohexapeptide) can be used at higher concentrations than other 4 groups of filamentous yeast-like fungus fermentation product compositions based on safety, indicating that they are less biotoxic, safer, can be used at higher concentrations, and at higher concentrations the more pronounced the effect produced by the composition.

From these results, the filamentous yeast-like fungus fermentation product composition of this example is low in biotoxicity, high in safety and more remarkable in use effect, and preferably, combination 2 (white willow bark broth+cyclohexapeptide) and combination 6 (betulin broth+cyclohexapeptide) are used.

EXAMPLE 3 testing of photodamage repair Capacity of filamentous Yeast-like fungus fermentation product compositions

To test the photodamage repair capability of 6 filamentous yeast-like fungus fermentation product compositions of example 1, comparative experiments were performed on the compositions in a range of ratios in this example, and the specific experimental procedure was as follows:

1) Cell inoculation: fibroblast cell at 3.5X10 ³ Cell inoculum size of each well was inoculated into 96-well plates, incubator (37 ℃, 5% CO) ₂ ) Incubating overnight;

2) Administration: when the plating rate of cells in the 96-well plate reaches 20% -30%, grouping drug administration is carried out, the drug administration amount of each well is 200 mu L, and 3 compound wells are arranged in each group. Incubator (37 ℃, 5% CO) ₂ ) Respectively culturing for 24 hours, 48 hours and 72 hours. The groups needing to be incubated for 48 hours and 72 hours are subjected to liquid exchange treatment every day;

3) UVA irradiation: according to the test scheme, UVA irradiation is carried out on the groups needing to be irradiated, and the irradiation dose is 45J/cm ² . After the irradiation, the mixture was placed in an incubator (37 ℃ C., 5% CO) ₂ ) Continuously culturing for 24 hours;

4) MTT detection: the supernatant was discarded, MTT work (0.5 mg/mL) was added, incubated at 37℃for 4h in the absence of light, after the incubation was completed, 150. Mu.L of DMSO was added to each well, and the OD was read at 490 nm. After 48h and 72h simultaneously, carrying out the MTT detection operation;

5) Cell relative viability calculation: according to the formula, the relative cell viability (%) = (sample well OD-zeroed well OD)/(solvent control well OD-zeroed well OD) ×100%.

There are several ways:

1. blank group: ultraviolet irradiation is not carried out;

2. negative control group: irradiating with ultraviolet;

3. positive control group: adding TGF-beta 1 (100 ng/mL; peprotech, transforming growth factor) and irradiating with ultraviolet;

4. cyclic hexapeptide group: independently adding 2% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

5. rice fermentation broth group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

6. white willow bark fermentation broth group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

7. ganoderma lucidum fermentation liquor group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

8. truffle fermentation liquor group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

9. Matsutake fermentation broth group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

10. fuscoporia obliqua fermentation broth group: independently adding 2.5% of cyclohexapeptide, performing experiment, and irradiating with ultraviolet;

11. combination 1 (rice broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

6. combination 2 (white willow bark broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

7. combination 3 (ganoderma lucidum broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

8. combination 4 (truffle broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

9. combination 5 (tricholoma matsutake broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

10. combination 6 (Chaba broth + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

11. Combination 7 (commercial yeast fermentation product + cyclohexapeptide): 2.5% +0.5% are selected respectively; 2.5% +1;2.5% +2%; experiments were carried out at different ratios of 2.5% +3%, irradiated with ultraviolet light;

the specific results are shown in Table 3:

TABLE 3 compositions 1-7 photodamage defense/repair cell test results

Note that: p <0.05 indicates that there is a significant difference.

As can be seen from table 3, 1) the positive control group has significantly higher cell survival rate than the negative control group, indicating that the positive control is effective; 2) Compared with a negative control group, after ultraviolet irradiation, the combination 1-6 has stronger repairing capability on photodamage of keratinocytes and fibroblasts; in the subsequent experiments, further experiments are carried out on the concentration of the fermentation product of the filamentous yeast-like fungi within the range of 0.1-10%, and the combination of the experiments shows that in the fermentation product composition of the filamentous yeast-like fungi, the cell survival rate of the fermentation product of the filamentous yeast-like fungi within the range of 0.1-10% and the concentration of the cyclohexapeptide within the range of 0.5-3% is more than 90%, so that the photodamage repairing capability is obviously enhanced; and when the fermentation product in the composition is 2.5% and the cyclohexapeptide is 2%, the photodamage repairing capability is strongest; 2) Compared with a negative control group, the composition 7 can improve the photodamage repair capability of cells, but compared with compositions 1 to 6, the composition 1 to 6 can obviously improve the photodamage repair capability of keratinocytes and fibroblasts, and compared with composition 7, p is less than 0.05, the difference is obvious, so that the filamentous yeast-like fungus fermentation product composition has strong photodamage repair capability, and compared with the yeast fermentation product composition sold in the market, the photodamage repair capability is extremely obvious; 3) Compared with the single cyclohexapeptide group and the single fermentation products in the compositions 1-6, the keratinocyte survival rate and the fibroblast survival rate of the filamentous yeast-like fungus fermentation product composition in the compositions 1-6 are both obviously improved, which indicates that the single cyclohexapeptide and the single fermentation product have no obvious photodamage restoration capability, and only after the combination of the single cyclohexapeptide and the single fermentation product, the photodamage restoration capability of the composition is obviously improved; 4) Of the 6 groups of compositions, combination 1 had the highest keratinocyte and fibroblast viability at the best proportioned concentrations, indicating that combination 1 (rice broth + cyclohexapeptide) had the best photodamage repair capacity; 5) Compared with the blank group, the keratinocyte survival rate and the fibroblast survival rate of the compositions 1-6 are obviously improved, which indicates that the active ingredients contained in the single fermentation liquid have a certain effect, but do not reach the growth promoting stage, and the cyclohexapeptide does not promote obvious growth effect, but the keratinocyte survival rate and the fibroblast survival rate of the two compositions after being compounded are obviously higher than those of the blank group, which indicates that the compounded composition has a promotion effect on the cell survival rate and promotes the cell activity.

From this, in the present example, the filamentous yeast-like fungus fermentation product composition has a remarkable photodamage repair ability when the concentration of the filamentous yeast-like fungus fermentation product is 0.1% to 10% and the concentration of the cyclohexapeptide is 0.5% to 3%, while slightly promoting the cell activity, and the photodamage repair ability is strongest when the fermentation product is 2.5% and the cyclohexapeptide is 2%; meanwhile, when the combination 1 (rice fermentation liquor and cyclohexapeptide) is preferred, the effects of achieving the optimal photodamage repair capability and promoting the cell activity are achieved, and the application value is extremely high.

EXAMPLE 4 testing of the penetration Capacity of filamentous Yeast-like fungus fermentation product compositions

To test the penetration capacity of the 6 filamentous yeast-like fungus fermentation product compositions of example 1, a comparative experiment was performed on the different compositions, preferably in the optimal ratio, in this example, with the following experimental principles: based on Franzcell diffusion cell analysis of the transdermal absorption of the polypeptide containing free amino groups in vitro, pig skin is taken as a research model, the content of target substances in receiving liquid is detected by adopting a quantitative detection method to calculate the cumulative permeation quantity, permeation rate and diffusion percentage, the transdermal absorption quantification of a sample to be detected is carried out, and the in vitro transdermal absorption of the polypeptide containing free amino groups is analyzed. And comparing the effect of the composition on the osmotic behavior of the polypeptide.

The experimental method is as follows: the test is based on a pigskin system, the concentration of the sample in the receiving liquid at different time points is measured by a high performance liquid phase method, and the skin permeation behavior of the sample is evaluated. Samples were assayed for selection of optimal concentrations, 2.5% fermentation substrate+2% cyclohexapeptide compositions 1-7 and cyclohexapeptide alone for different plant bases. Test system: pigskin; materials and equipment: TK-12D drug transdermal absorption Franz diffusion cell, isothermal magnetic stirrer (IKA).

(1) The cumulative permeation quantity Q is calculated as follows: q= [ cn×v + Σci×v0]/S (i=1, 2, 3, …, n-1), note: q: cumulative permeation quantity; s: an effective diffusion area; v: a receiving volume of liquid in the receiving chamber; v0: the volume of each sample; ci: the drug concentration in the liquid is received from the 1 st time to the last sampling; cn: sample concentration measured at the nth sampling point;

(2) diffusion percentage p=pt/Po 100%, note: pt is the sample content in the receiving cell and Po is the theoretical content of the sample in the releasing cell.

Note that: p <0.05 indicates significant differences.

The experimental protocol is shown in table 4:

table 4 in vitro penetration capacity test protocol

The experimental results are shown in table 5:

table 5 results of the transdermal penetration test of samples at various times

As can be seen from table 5, 1) the single broth group has no penetration ability, and the diffusion percentage of the combination 1 to 6 is significantly improved after 24 hours compared with the single-loop hexapeptide group, p <0.05, there is a significant difference, and when combination 2 (white willow bark fermentation broth+cyclohexapeptide) is selected, the diffusion percentage reaches the highest, at this time, the penetration effect is optimal, which indicates that the compound filamentous yeast-like fungus fermentation product can achieve the effect of promoting the percutaneous absorption of cyclohexapeptide, and the promotion effect is optimal when the white willow bark fermentation product is preferred; 2) After 24h, the diffusion percentage of combination 7 was not significantly different from that of the cyclohexapeptide group, p >0.05, indicating that the common yeast fermentation product filtrate could not enhance the transdermal permeability of the cyclic peptides.

Therefore, in this example, the effect of promoting the transdermal absorption of the cyclohexapeptide can be achieved by compounding the fermentation product of the filamentous yeast-like fungus at the optimal ratio, and the promoting effect is most remarkable when the combination of the fermentation product of white willow bark and the cyclohexapeptide is preferable, and the permeation effect is optimal.

Example 5 test of antioxidant Capacity of filamentous Yeast-like fungus fermentation product composition

The ROS content exceeds the capability of human body clearance, the balance of oxidization and antioxidation is broken, oxidative stress is caused, the skin is taken as the outermost tissue of the human body, and is directly exposed to the environment, so that oxidative damage caused by the oxidative stress is more easily caused, and skin aging and skin diseases are caused by the oxidative stress. To test the antioxidant capacity of the 6 filamentous yeast-like fungus fermentation product compositions of example 1, this example used an in vitro radical scavenging test to verify the differences in antioxidant capacity of the relevant sample groups, the procedure was as follows:

antioxidant Capacity test: taking the fermentation liquor with the optimal concentration as a sample solution. 1.0mL of 50mmol/LpH 8.2.2 Tris-HCl buffer solution was taken in a 10mL EP tube, 0.2mL of the sample solution was added thereto, the mixture was uniformly mixed and incubated at 25℃for 10 minutes, then 0.1mL of 10mmol/L pyrogallol solution preheated at 25℃was added, the total volume was 6mL, and after rapid shaking, the increase in Absorbance (AS) at 320nm for 1 minute was recorded with an ultraviolet-visible spectrophotometer. The increase in absorbance per minute in the linear range was calculated. The reagent was replaced with the same volume of water as above, and the absorbance increase (Ao) at 320nm was measured for 1 min. The superoxide anion radical clearance is calculated according to the formula of superoxide anion radical clearance (%) = (Ao-AS)/ao×100%, inhibition curves of test solution with different concentrations are drawn, and the concentration of the test solution when the superoxide anion radical clearance reaches 50% is estimated according to the drawn curves (the optimum concentration of the cyclohexapeptide, the sample and the composition is 2% of the cyclohexapeptide, 2.5% of the fermentation product is obtained by the sample, and the composition is 2.5% of the fermentation product+2% of the cyclohexapeptide).

The experimental results are shown in table 6:

table 6 results of comparison of antioxidant capacity of samples

Note that: p <0.05 indicates significant differences.

As can be seen from table 6, 1) compared with the single-ring hexapeptide group and the single-sample group (single fermentation product), the concentration of the filamentous yeast-like fungus fermentation product composition is obviously reduced when the scavenging rate of superoxide anion free radicals reaches 50%, p is less than 0.05, and the significant difference exists, which indicates that the fermentation product composition is significantly better than the single-ring hexapeptide group and the single-ring hexapeptide group in scavenging capability, and the synergistic antioxidation effect is achieved after the combination is illustrated, and the antioxidation capability of the composition is significantly improved; and when combination 2 (white willow bark fermentation broth + cyclohexapeptide) is selected, the antioxidant capacity is strongest; 2) Compared with sample 7, when the scavenging rate of superoxide anion free radical reaches 50%, the concentration is increased instead, which indicates that the combination of the common saccharomycete fermentation product and the cyclohexapeptide can not improve the antioxidation capability of the composition and does not have synergistic capability; meanwhile, compared with the combination 1-6, the antioxidant capacity of the composition is obviously reduced.

Therefore, in the embodiment, under the optimal proportion, the compound filamentous yeast-like fungus fermentation product and the cyclohexapeptide can obviously improve the oxidation resistance of the composition and achieve the synergistic effect; and when the combination of 2 (white willow bark fermentation broth + cyclohexapeptide) is preferred, the oxidation resistance is the strongest.

Example 6 test of in vitro anti-wrinkle efficacy of filamentous Yeast-like fungus fermentation product compositions

Collagen is mainly produced by fibroblasts existing in the dermis layer of the skin, is an important element for supporting the skin, and accounts for about 80% of the dermis layer of the skin, so that the skin is full and full, the increase of the content of Collagen I is promoted, and the effect of resisting the generation of wrinkles can be achieved. Wherein, collagen I is the main Collagen in dermis, collagen III is also a main component of the extracellular matrix of dermis, and Collagen fibers are assembled together with Collagen I to play an important role in the toughness of skin. Collagen type IV (Collagen IV) is the main component of the semi-desmosome basement membrane band complex, the key protein at the junction of the dermis, MMP-1 belongs to collagenase in matrix metalloproteinases, and the main hydrolysis substrates are fibrous collagens, namely, collagen I and Collagen III. After the MMP-1 content is reduced, the degradation degree of collagen is reduced, so that a certain anti-wrinkle effect is achieved. In the experiment, fibroblasts are taken as a research model, UVA irradiation is adopted to establish an in vitro photoaging model, the highest combination 2 of permeability in the example 4 is taken as an example (the composition is the optimal proportion: 2.5 percent of fermentation products and 2 percent of cyclohexapeptide), and the photoaging and wrinkle resisting effects of an analyte are analyzed by detecting the change of ECM degraded enzyme matrix metalloproteinase MMP-1 and Collagen contents Collagen I, collagen III and Collagen IV; meanwhile, in this example, it has been confirmed through comparison experiments that in the compositions 1 to 6, the combination 2 has the optimal anti-photoaging in vitro anti-wrinkle effect, so in this example, the combination 2 having the optimal effect was selected for the subsequent comparison experiments.

The cells used in this test were fibroblasts and were obtained by commercial means. The main reagent comprises: DMEM broth (Gibco), PBS (Soxhausto), MTT (Sigma), DMSO (Sigma), RNAiso Plus (Takara), reverse transcription kit (PrimeScript) ^TM RT reagent Kit)(Takara)、SYBR Premix Ex Taq ^TM II fluorescent dye (Takara), sterile ddH ₂ O (Takara) and TGF-beta 1 (Peprotech), wherein transforming growth factor (Transforming growth factor beta, TGF-beta) is a multifunctional cytokine that can be produced by a variety of tissue cells. The TGF-beta signal channel is a multifunctional cytokine composed of a plurality of members, and a channel composed of the multifunctional cytokine and corresponding receptor and intracellular signal transduction molecules, can influence the occurrence and development of diseases, regulate the transcription of genes, control the cell cycle, influence the proliferation, differentiation, adhesion, transfer and apoptosis of cells, and is a classical cell growth factor which is commonly used as a cation marker to regulate the proliferation of cells. Main plant CO ₂ Incubator (Thermo, 150I), ultra clean bench (Sujingtai, SW-CJ-1F), incubator (Teste), general PCR instrument (Bori), fluorescent quantitative PCR instrument (BioRad, CFX-96), inverted microscope (Olympus, CKX 41), UVA irradiator (Philips).

The experimental process comprises the following steps: adding sample working solution with corresponding concentration on the surface of the sample group, uniformly distributing the samples on the surface of the model, and placing the samples on CO ₂ Incubator (37 ℃,5% CO) ₂ ) And incubated for 24h. After the incubation, the sample remained on the surface of the model was washed with sterile PBS solution, and the inner and outer residual liquids of the model were wiped off with sterile cotton swabs. The specific experimental design is shown in table 7:

TABLE 7 design of experiments

Note that: blank control was normal fibroblasts, normal culture: fibroblast cell at 3.5X10 ³ Cell inoculum size of each well was inoculated into 96-well plates, incubator (37 ℃,5% CO) ₂ ) Is not treated by UVA ultraviolet irradiation; negative control was normal fibroblasts, normal culture: fibroblast cell at 3.5X10 ³ Cell inoculum size of each well was inoculated into 96-well plates, incubator (37 ℃,5% CO) ₂ ) Is subjected to the same UVA ultraviolet irradiation treatment, but no other substances are added; positive control was normal fibroblasts, normal culture: fibroblast cell at 3.5X10 ³ Cell inoculum size of each well was inoculated into 96-well plates, incubator (37 ℃,5% CO) ₂ ) Is incubated overnight, and is treated by the same UVA ultraviolet irradiation, and TGF-beta 1 is added; p is p<0.05 indicates significant differences.

The experimental results are shown in figures 1-4, wherein figure 1 is a colragen I gene expression level bar chart of each group, and figure 2 is a colragen III gene expression level bar chart of each group; FIG. 3 is a bar graph of the expression level of the Collagen IV gene for each group; FIG. 4 is a histogram of MMP-1 gene expression levels for each group; in the figure, the ordinate indicates the amplification factor with respect to the blank.

As can be seen from FIG. 1, 1) the expression level of the Collagen I gene in the PC group is higher than that in the NC group, and the positive control is effective; 2) After irradiation with UVA for 72 hours, the expression level of the Collagen i gene of the combination 2 reaches the highest, which is 2.5 times that of the blank control, and compared with the negative control group and the positive control group, the expression level of the Collagen i gene of the combination 2 is obviously increased, which indicates that the combination 2 has obvious effects of resisting photoaging and wrinkling, and the effect is obviously higher than the effect of transforming growth factor TGF-beta 1, and simultaneously compared with the blank control, the expression level of the Collagen i gene of the combination 2 is also obviously increased, which indicates that the combination 2 also has the effect of obviously promoting the expression of the Collagen i gene; 3) Compared with the cyclic peptides, sample 2, sample 7 and combination 7, the expression level of the Collagen I gene of combination 2 is also significantly increased, which indicates that the anti-photoaging and anti-wrinkle effects of combination 2 are significantly better than the independent fermentation products and the cyclic hexapeptide group, and that when the filamentous yeast-like fungus fermentation products and the cyclic hexapeptide are compounded in the optimal ratio, a significant synergistic effect is produced, while the composition 7 has no significant synergistic effect.

As can be seen from FIG. 2, 1) the PC group Collagen III gene expression level was higher than that of NC group, and this positive control was effective; 2) After 72 hours of irradiation with UVA, the expression level of the Collagen iii gene of combination 2 was significantly increased compared to the negative and positive control groups, which indicates that combination 2 has not only significant anti-photoaging and anti-wrinkle effects, but also significantly higher effects than the transforming growth factor TGF- β1, while the expression level of the Collagen iii gene of combination 2 was also significantly increased compared to the blank control, which indicates that combination 2 also has significant effect of promoting the expression of the Collagen iii gene; 3) The increased Collagen iii gene expression of combination 2 compared to cyclopeptides, sample 2, sample 7 and combination 7 was also significant, with significant differences, p <0.05, demonstrating that both the anti-photoaging and anti-wrinkle effects of combination 2 were significantly better than the individual fermentation products and the cyclopeptide group, and that significant synergy was produced when the filamentous yeast-like fungus fermentation products and the cyclopeptide were compounded at the optimal ratio, whereas composition 7 did not.

As can be seen from FIG. 3, 1) the PC group Collagen III gene expression level was higher than that of NC group, and this positive control was effective; 2) After 72 hours of irradiation with UVA, the expression level of the Collagen IV gene of the combination 2 reaches the highest and reaches 2.7 times of that of a blank control, and compared with a negative control group and a positive control group, the expression level of the Collagen IV gene of the combination 2 is obviously increased, which indicates that the combination 2 has obvious effects of resisting photoaging and wrinkling, and the effect is obviously higher than that of transforming growth factor TGF-beta 1, and simultaneously compared with the blank control, the expression level of the Collagen IV gene of the combination 2 is also obviously increased, which indicates that the combination 2 also has the effect of obviously promoting the expression of the Collagen IV gene; 3) The increased expression of the Collagen iv gene in combination 2 compared to cyclopeptides, sample 2, sample 7 and combination 7 also showed that both the anti-photoaging and anti-wrinkle effects of combination 2 were significantly better than the individual fermentation products and the cyclopeptide group, and that when the filamentous yeast-like fungus fermentation products and the cyclopeptide were formulated at the optimal ratio, a significant synergy was produced, whereas composition 7 did not.

As can be seen from fig. 4, 1) the expression level of MMP-1 in the PC group was significantly reduced compared with that in the NC group, and this positive control was effective; 2) After 72 hours of irradiation with UVA, the MMP-1 expression level of combination 2 is significantly reduced compared with both the negative control group and the positive control group, which indicates that combination 2 has not only significant anti-photoaging and anti-wrinkle effects, but also effects significantly higher than those of transforming growth factor TGF- β1, and that the MMP-1 expression level of combination 2 is also significantly reduced compared with the blank control, which indicates that combination 2 significantly inhibits the expression of MMP-1, and has significant anti-photoaging and anti-wrinkle effects; 3) The significantly reduced MMP-1 expression of combination 2 compared to cyclopeptides, sample 2, sample 7 and combination 7, demonstrated that both the anti-photoaging and anti-wrinkle effects of combination 2 were significantly better than the individual fermentation products and the cyclopeptide group, and that significant synergy was produced when the filamentous yeast-like fungal fermentation products and the cyclopeptide were compounded at the optimal ratio, whereas no significant synergy was produced for composition 7.

It can be seen that, in this example, the filamentous yeast-like fungus fermentation product composition has the effects of significantly promoting the expression of Collagen I, collagen III and Collagen IV and inhibiting the expression of MMP-1 in the optimal ratio, and the compounded composition has significant synergistic effect, and has optimal photoaging and wrinkle resistance when the composition is preferably combined with 2 (white willow bark fermentation broth+cyclohexapeptide).

EXAMPLE 7 test of the ability of a filamentous Yeast-like fungus fermentation product composition to relieve

After the skin is irradiated by ultraviolet, a clinically visible redness phenomenon can occur, keratinocytes are taken as a research object, an in-vitro damage model is built by adopting UVB irradiation, and the relief efficacy of the compound to be tested is analyzed by detecting the change of the inflammatory factor content. TNF-alpha pro-inflammatory cytokines cause activation of the kappa-light chain enhanced (NF-KB) signaling pathway of B cells, and can also promote the production of various inflammatory factors, interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8), by the cells, thereby causing skin inflammation. Increased IL-1 alpha and IL-8 production by leukocytes exacerbates the disruption of the epidermal barrier, increased epidermal water loss, and the activation of inflammatory factors such as tumor necrosis factor TNF-alpha, which can cause a fever.

In this example, individual fermentation broths, groups of cyclohexapeptides and different compositions were quantitatively evaluated in UV UVB radiation from the detection of the content index of pro-inflammatory factors (IL-1. Alpha., IL-8, TNF-alpha.) after UV radiation by examining the skin model after the action of each group of active ingredientsThe relaxing effect after shooting is realized by the following specific culture and detection processes: selecting HaCaT cells in logarithmic growth phase, performing conventional treatment, and regulating cell concentration of cell suspension to 2×10 ⁴ Inoculating the cells/wells into 48-well plate, placing at 37deg.C, 5% CO ₂ Culturing in an incubator for 24 hours; UVA (30J/cm) ² ) After cells were stimulated, the cells were dosed in groups and the supernatant was collected by incubation for 24 hours, and the respective inflammatory factor/mediator production was measured using the corresponding ELISA kit (Abcam).

Specifically, the compositions were divided into the following groups (the other conditions not illustrated were the same; the optimum concentrations of the cyclohexapeptide, the fermentation broth and the composition were all 2%, the fermentation broth was 2.5%, the composition was 2.5% and the fermentation broth+2% cyclohexapeptide; meanwhile, in this example, it was confirmed by comparative experiments that in compositions 1 to 6, combination 2 had the optimum soothing ability, and therefore in this example, combination 2 having the optimum effect was selected for the subsequent comparative experiments):

1. Blank control: keratinocytes, not irradiated with ultraviolet light;

2. negative control (NC control): keratinocytes, without any added components, are irradiated by ultraviolet rays;

3. fermentation broth 2: keratinocytes were irradiated with ultraviolet light by adding only fermentation broth 2 (white willow bark fermentation broth);

4. fermentation broth 7: keratinocytes were irradiated with ultraviolet light by adding only fermentation broth 7 (a common commercially available yeast fermentation broth);

5. cyclic hexapeptide: keratinocytes, to which only cyclohexapeptide is added, are irradiated with ultraviolet light;

6. combination 2: keratinocytes, added with combination 2 (white willow bark fermentation broth + cyclohexapeptide), and irradiated with ultraviolet rays;

7. combination 7: keratinocytes, added with combination 7 (common commercial yeast fermentation broth + cyclohexapeptide), were irradiated with ultraviolet light;

the experimental results are shown in FIGS. 5 to 7, wherein FIG. 5 is an IL-1α content expression histogram, FIG. 6 is an IL-8 content expression histogram, FIG. 7 is a TNF- α content expression histogram, and the ordinate is the average concentration of the content (pg/mL).

As can be seen from fig. 5, 1) after 24 hours of irradiation with UVB, the IL-1 α content of combination 2 is significantly reduced to about 5pg/mL compared to NC control, while combination 2 has no significant difference from the blank, p >0.05, which indicates that combination 2 has significant effect of reducing inflammatory factors or inflammatory mediators in uv irradiation, thereby achieving the effect of relieving skin injury; 2) The IL-1 alpha content of combination 2 was also significantly reduced compared to fermentation broth 2, fermentation broth 7, cyclohexapeptide and combination 7, demonstrating that both the inflammatory factor inhibition and skin lesion relief efficacy of combination 2 was significantly better than that of fermentation broth alone, the cyclohexapeptide group and the common commercial yeast fermentation broth group, and that significant synergy was produced when the filamentous yeast-like fungus fermentation product and cyclohexapeptide were compounded at the optimal ratio, whereas composition 7 did not.

As can be seen from fig. 6, 1) after 24 hours of irradiation with UVB, the IL-8 content of combination 2 is significantly reduced to about 20pg/mL compared to NC control, while combination 2 has no significant difference from the blank, p >0.05, which indicates that combination 2 has significant effect of reducing inflammatory factors or inflammatory mediators in uv irradiation, thereby achieving the effect of relieving skin injury; 2) The IL-8 content of combination 2 was also significantly reduced compared to fermentation broth 2, fermentation broth 7, cyclohexapeptide and combination 7, demonstrating that both the inflammatory factor inhibition and skin lesion relief efficacy of combination 2 was significantly better than that of fermentation broth alone, the cyclohexapeptide group and the common commercial yeast fermentation broth group, and that significant synergy was produced when the filamentous yeast-like fungus fermentation product and cyclohexapeptide were compounded at the optimal ratio, whereas composition 7 did not.

As can be seen from fig. 7, 1) after 24 hours of irradiation with UVB, the TNF- α content of combination 2 was significantly reduced to about 4pg/mL compared to NC control, and the TNF- α content of combination 2 was also significantly reduced compared to blank control, which indicates that combination 2 has the effect of significantly inhibiting pro-inflammatory cytokine TNF- α, thereby achieving the effect of relieving skin injury; 2) The TNF- α content of combination 2 was also significantly reduced compared to fermentation broth 2, fermentation broth 7, cyclohexapeptide and combination 7, demonstrating that both the pro-inflammatory cytokine inhibition and skin lesion relief efficacy of combination 2 was significantly better than that of the individual fermentation broths, cyclohexapeptide group and the common commercial yeast fermentation broth group, and that significant synergy was produced when the filamentous yeast-like fungus fermentation product and cyclohexapeptide were compounded at the optimal ratio, whereas composition 7 did not.

It is understood that, in the present embodiment, the filamentous yeast-like fungus fermentation product composition has the effects of significantly inhibiting the expression of proinflammatory cytokines TNF- α and inflammatory factors IL-1α and IL-8, and relieving skin injury, and the compounded composition has significant synergistic effect, and has the effects of optimally inhibiting the expression of inflammatory factors and relieving skin ultraviolet injury when the composition is preferably combined with 2 (white willow bark fermentation broth+cyclohexapeptide).

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A composition comprising a filamentous yeast-like fungal fermentation product and a cyclohexapeptide.

2. The composition according to claim 1, wherein the composition comprises, in mass percent, 0.05 to 90% of a filamentous yeast-like fungus fermentation product, 0.05 to 5% of a cyclohexapeptide, and the balance being a base component, the sum of the mass percentages being 100%.

3. The composition of claim 2, wherein the cyclohexapeptide has the sequence Cyclo-Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

4. A composition according to claim 3, wherein the filamentous yeast-like fungus fermentation product is prepared by a process comprising: performing multiple fermentation with filamentous yeast-like fungus Galactomyces Citri-Aurantii, namely sterilizing the precipitate obtained by centrifugation after the previous fermentation is used for the culture medium component of the next fermentation, wherein the number of times of the multiple fermentation is 4, and the method comprises the following steps:

(1) Culturing a filamentous yeast-like fungus to obtain an inoculation liquid;

5. Use of a composition for preparing a formulation for combating skin photoaging or repairing photodamage or improving penetration capacity, characterized in that the composition comprises any one of a filamentous yeast-like fungi rice fermentation product, a filamentous yeast-like fungi white willow bark fermentation product, a filamentous yeast-like fungi ganoderma lucidum fermentation product, a filamentous yeast-like fungi white truffle fermentation product, a filamentous yeast-like fungi matsutake fermentation product, a filamentous yeast-like fungi betulin fermentation product, and a cyclohexapeptide.

6. The use according to claim 7, wherein the composition comprises a filamentous yeast like fungal rice fermentation product and a cyclohexapeptide having the sequence Cyclo-Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

7. Use of a composition for preparing a formulation for enhancing the ability to repair, inhibiting inflammatory factors or inflammatory mediators, the composition comprising a rice fermentation product of filamentous yeast-like fungi, a white willow bark fermentation product of filamentous yeast-like fungi, a ganoderma fermentation product of filamentous yeast-like fungi, a white truffle fermentation product of filamentous yeast-like fungi, a matsutake fermentation product of filamentous yeast-like fungi, a fumagule fermentation product of filamentous yeast-like fungi, and a cyclohexapeptide.

8. The use according to claim 9, wherein the composition comprises a fermentation product of the filamentous yeast like fungi salix alba bark and a cyclohexapeptide having the sequence Cyclo (Gln-Gly-Pro-Gln-Gly-Pro).

9. Use of a composition according to any one of claims 1 to 4 for the preparation of an anti-ageing, wrinkle-removing, collagen content increasing or MMP-1 inhibiting formulation.

10. Use of a composition according to any one of claims 1 to 4 for the preparation of a formulation for alleviating oxidative stress and improving antioxidant capacity.