CN115074393A

CN115074393A - Pichia pastoris fermentation lysate filtrate, preparation method and application

Info

Publication number: CN115074393A
Application number: CN202210933234.6A
Authority: CN
Inventors: 李佳佳; 纪白慧; 钱松; 曹凯杰; 陈聚印; 石叶飞
Original assignee: Jiangsu Chuangjian Medical Technology Co ltd
Current assignee: Jiangsu Chuangjian Medical Technology Co ltd
Priority date: 2022-08-04
Filing date: 2022-08-04
Publication date: 2022-09-20
Anticipated expiration: 2042-08-04
Also published as: CN115074393B; WO2024027798A1

Abstract

The invention relates to a Pichia pastoris fermentation lysate filtrate, a preparation method and application, and belongs to the technical field of cosmetic raw materials; the invention provides a fermentation lysate filtrate prepared by fermenting pichia pastoris for expressing recombinant collagen; the fermentation lysate filtrate comprises total protein, recombinant collagen, polysaccharide, amino acid and multiple vitamins; the invention realizes the preparation of the cosmetic raw material by expressing the recombinant human collagen through the pichia pastoris fermentation thalli for the first time, reduces the waste treatment pressure brought by the yeast fermentation thalli and changes waste into valuable; the preparation method is a whole-course online and automatic preparation process, and the preparation process is quick and efficient; meanwhile, the whole preparation process flow can effectively control the breeding of harmful bacteria; the lysate filtrate of the invention has the effects of relieving skin, enhancing skin micro-ecological barrier, controlling oil, preventing sun and the like, and expands the application range of the lysate filtrate in preparing cosmetics.

Description

Pichia pastoris fermentation lysate filtrate, preparation method and application

Technical Field

The invention relates to a Pichia pastoris fermentation lysate filtrate, a preparation method and application, and belongs to the technical field of cosmetic raw materials.

Background

Collagen (collagen) plays an important role in cell migration, cell metabolism, signal pathway response, maintenance, regulation of normal physiological functions of cells, tissues and organs, and repair of damage. As an important natural biological protein, the protein has unique functional characteristics of good biocompatibility, bioactivity, degradability and the like, and has wide application prospects in various fields of chemical industry, medicines, foods, medical cosmetology, cosmetics and the like. The recombinant human collagen produced by modern genetic engineering technology has stable property, no disease hidden danger and immunogenicity of animal-derived collagen, and industrial production is not limited by raw material sources, and is now a hot spot of the field and capital attention of the national key development support. At present, the largest large-scale production line for fermenting and producing collagen by domestic recombinant collagen production enterprises reaches the 20 ton level, and the production scale and the number of the recombinant collagen production enterprises can be continuously enlarged and increased in the future.

The recombinant collagen is expressed in pichia pastoris by secretion expression, and can generate a large amount of fermentation thalli (20-30% of the volume of the fermentation thalli in a general fermentation tank) which are used as wastes to be treated. Wastes (wastewater, thalli and the like) generated in the yeast fermentation industry (such as beer production and bio-pharmaceuticals) are required to be subjected to harmless treatment and can be discharged after meeting relevant standards, and because the quantity of the wastes is large, huge pressure is brought to the treatment capacity of environmental protection facilities of enterprises. At present, waste fermentation thalli are gradually utilized, such as preparation of single cell protein (protein content is reduced), fermentation into alcohol (cellulose generated during fermentation is utilized, the significance of thalli per se is not great), development into organic fertilizer for agricultural production (certain treatment is required, a larger field is also required), production of other biomass energy, preparation of animal feed, preparation of yeast extract, yeast extract and the like, extraction of glucan, preparation of yeast extract and the like. The yeast fermentation thalli has various utilization modes, but generally speaking, the processed products have limited additional value, small market space or more replaceable products, and have certain requirements on production equipment, production places and environment, so that most production enterprises prefer to discharge thalli after harmless treatment.

The use of microbial lysates and extracts as cosmetic raw materials has become a hot spot in the related field. In addition to the above uses, yeast cells can be used as a raw material for cosmetics, which is a treatment method with a high added value. The yeast can be directly used as raw material, and can also be prepared into various forms of cosmetic raw materials such as yeast derivatives.

The skin micro-ecology is an ecosystem which is composed of various microorganisms such as bacteria, fungi, viruses, mites and arthropods, tissues on the surface of the skin, various secretions of cells, micro-environments and the like. In recent years, with the progress of research, skin micro-ecology has also been the focus of attention in the cosmetics world. In cosmetic applications, micro-ecological skin care is also the focus of product development for many international brands companies. International brands such as ashira, lancome, eleya, Baojie and the like develop and promote related products successively, such as 'shenxian water' of SK-II under Baojie flag, wherein the component of the core raw material Pitera is galactose yeast-like bacteria fermentation product filtrate, and the barrier function of skin can be improved by promoting the expression of tight junction protein and increasing transmembrane resistance. The core raw materials used by the star product 'Xiaozui bottle' of the ashira and the 'Xiaohei bottle' of the lancome are yeast fermentation product lysate of bifidus, the components of the lysate are that although yeast is contained in the name, the lysate is not saccharomyces but is derived from bifidobacterium, and metabolites, cytoplasm components, cell wall components and a plurality of organelle fragments are obtained by culturing and cracking thallus cells. In addition, each major international mainstream raw material company focuses on micro-ecological skin care in a dispute, and various post-biotic raw materials such as Relipidium of basf are successively pushed out, wherein the main component is hydrolyzed yeast protein, so that the skin colony balance is restored; disemann's OXY 229PF contains yeast lysate as a major component, which reduces Corynebacterium klebsiella, and thus relieves facial redness.

The skin microecology, the physical barrier of the skin and the skin immunity jointly form a first defense system of an organism, resist the stimulation of various external physical and chemical factors and maintain the normal functions of the organism. Therefore, the integrity of the skin barrier is of great importance to healthy skin, and the development of safer and more effective skin barrier repair materials is also a problem to be solved in the current development of skin care products. The existing cosmetic barrier repair raw materials are mainly used for repairing physical barriers of stratum corneum layers, and cosmetic raw materials with skin barrier repair effects developed by taking improvement of skin micro-ecological barriers and enhancement of immune barriers as starting points are lacked.

In the development of cosmetic raw materials having skin barrier repair effects, pichia pastoris-derived raw materials are deficient; moreover, the efficacy of the existing yeast cosmetics mostly focuses on the aspects of moisture retention, anti-aging, sun protection, whitening and the like, and raw materials for enhancing skin micro-ecological barriers, immune barriers and controlling sebum secretion as basic efficacy mechanisms are also deficient.

In addition, the application of collagen in medicine, beauty and cosmetics is generally accepted, but the pichia pastoris fermentation thalli for expressing recombinant collagen is used for preparing cosmetic raw materials, particularly fermentation lysate filtrate, has no precedent, and related research and development are blank; the preparation mode and the efficacy research of related products are not disclosed in the published information; whether cosmetic raw materials (especially fermentation lysate filtrate) prepared from pichia pastoris expressing recombinant human collagen is feasible, better and more effective is not reported; the recombinant collagen production enterprises also have no corresponding product release or patent and research literature publication.

In addition, when yeast is used for preparing extract or lysate filtrate or other cosmetic raw materials, the wall breaking and cracking of yeast are firstly completed, the cell wall of pichia pastoris is thicker (70nm-200nm) and has compact structure, high mechanical strength, long treatment time and low efficiency in a conventional treatment mode, and possible primers and exogenous substances (enzymes) are also provided. For the production of fermentation cells for industrial scale production: the period of the fermentation thalli generation is short, the amount of the fermentation thalli is large, harmful thalli are easy to breed in a longer treatment time, and pyrogen substances such as endotoxin and the like are generated; the breaking of the thalli during the scale production needs to be simple, stable and efficient, and the introduction of exogenous substances is avoided (meanwhile, the cost is also favorably controlled). In addition, the crushed pichia pastoris lysate is mixed with insoluble and soluble components, and an effective treatment mode is also needed for effective separation.

Disclosure of Invention

The invention aims to overcome some technical problems in the prior art, mainly fills the blank in the aspects of products, preparation methods, product effects and application of the products, namely 'preparation of fermentation lysate filtrate by pichia pastoris for expressing recombinant collagen', and solves the technical problems of long treatment time, low efficiency, possibility of introducing exogenous substances and the like in the preparation method of the lysate filtrate in the prior art.

Therefore, the invention provides a Pichia pastoris fermentation lysate filtrate, a preparation method and application.

In order to achieve the above technical objects, the present invention provides a method for preparing a fermentation lysate filtrate, wherein the fermentation lysate filtrate is prepared from cells obtained by fermentation of pichia pastoris.

Further, the pichia pastoris is pichia pastoris for expressing recombinant collagen.

Further, the pichia pastoris for expressing recombinant collagen is selected from one or more of the strains with the preservation number of CGMCC NO.7189, CGMCC NO.14057, CGMCC NO.17147, CGMCC NO.17150, CGMCC NO.17148, CGMCC NO.17149, CGMCC NO.20626, CGMCC NO.20627, CGMCC NO.21891 or CGMCC NO. 21892.

Wherein the thallus is obtained by performing solid-liquid separation on a fermentation product.

The preparation method of the fermentation lysate filtrate comprises thallus dilution, high-pressure homogeneous lysis, centrifugation and filtration.

Further, the thallus is diluted to be thallus suspension with pure water to the mass concentration of 5-30% (W/W); preferably, the cell dilution is a cell suspension diluted with pure water to a mass concentration of 10% (W/W).

The high-pressure homogeneous cracking is to crush the thallus suspension for 1-3 times under the pressure of more than 100MPar to obtain a lysate; preferably, the high pressure homogeneous lysis is to crush the bacterial suspension at a pressure of 1200 MPar.

The centrifugation is to centrifuge the lysate at 10000-20000 g to obtain supernatant.

The filtration is to filter the supernatant with 0.22 μm pore size, and obtain fermentation lysate filtrate.

The invention also provides the fermentation lysate filtrate prepared by the preparation method, wherein the fermentation lysate filtrate comprises total protein, recombinant collagen, polysaccharide, amino acid and a plurality of vitamins.

According to the embodiment of the present invention, the total protein content is 0.1-2.5%, preferably 1% (10mg/mL) by mass volume fraction in the fermentation lysate filtrate; the content of the recombinant collagen is 1-15 mug/mL, generally more than 10 mug/mL, preferably 10-15 mug/mL; the polysaccharide content is about 0.1-1.5mg/mL, preferably 1 mg/mL; the total amount of amino acids is about 0.2% to 1%, preferably 0.6% to 0.82%.

The fermentation lysate filtrate has the effects of resisting allergy, relieving, resisting inflammation, preventing sunburn, regulating skin micro-ecology, controlling oil and repairing skin barriers.

The invention also provides a composition, the composition includes the fermentation lysate filtrate.

Further, the content of the fermentation lysate filtrate in the composition is 1 to 10 wt%, preferably 5 wt%.

The invention also provides application of the fermentation lysate filtrate and the composition in anti-allergy cosmetics, soothing cosmetics, anti-inflammation cosmetics, sun protection cosmetics, skin micro-ecology regulation cosmetics, oil control cosmetics and skin barrier repair cosmetics.

The invention also provides the use of said fermentation lysate filtrate, said composition in the cosmetic field. The cosmetics comprise anti-allergy, soothing, anti-inflammatory, sunscreen, skin micro-ecology regulating, oil controlling and skin barrier repairing products.

Wherein the repairing of the skin barrier comprises enhancing the skin micro-ecological barrier, increasing the skin immune barrier.

Furthermore, the anti-allergy, soothing and anti-inflammatory mainly comprise the functions of improving and relieving stratum corneum irritation symptoms and improving epidermal tissue damage; reducing the level of inflammatory mediator PGE2 secretion, and alleviating inflammatory reactions of the skin, such as reducing vascular permeability in the skin, thereby alleviating the occurrence of redness in the skin; has the effect of relieving skin irritation through a mechanism of inhibiting the expression of TRPV1 protein.

The sun protection is expressed as the ability to absorb ultraviolet light.

The expression of regulating the skin microecology is that the microecology is regulated by improving the expression of antibacterial peptide and a mode recognition receptor, and the skin barrier is improved, so that the repairing effect is achieved; under the action of the Pichia pastoris fermentation lysate filtrate, the contents of proteins HBD1, HBD2, LL-37 and TLR2 are obviously increased.

The oil control is shown to inhibit skin lipid droplet synthesis.

The invention also provides a cosmetic, wherein the fermentation lysate filtrate is contained in the cosmetic, and the cosmetic comprises but is not limited to skin care essence, facial mask and cream.

Further, the content of the fermentation lysate filtrate in the cosmetic is 1 to 10% by weight, preferably 5% by weight.

According to an embodiment of the present invention, the present invention further provides a skin care essence, wherein the skin care essence comprises 1-10 wt%, preferably 5 wt% of fermentation lysate filtrate, and further comprises deionized water, glycerol, butylene glycol, glyceryl polyether-26, EDTA-2Na, sodium hyaluronate, allantoin, betaine, xanthan gum, and 1, 2-pentanediol.

The invention also provides a facial mask, which comprises 1-10 wt%, preferably 5 wt% of fermentation lysate filtrate, deionized water, glycerol, EDTA-2Na, sodium hyaluronate, xanthan gum, carbomer, a pH regulator, 1, 2-pentanediol and 1, 2-hexanediol.

The invention also provides a skin-care cream which comprises 1-10 wt%, preferably 5 wt% of fermentation lysate filtrate, glycerol, disodium EDTA, xanthan gum, acryloyldimethyl ammonium taurate/VP copolymer, p-hydroxyacetophenone, 1, 2-hexanediol, cetearyl glucoside/cetearyl alcohol, caprylic capric triglyceride, hydrogenated polyisobutene, polydimethylsiloxane and deionized water.

The invention has the beneficial effects that:

(1) the invention realizes the preparation of the cosmetic raw material by expressing the recombinant human collagen pichia pastoris fermentation thallus for the first time, increases an effective and high-added-value utilization way of the recombinant human collagen fermentation thallus, reduces the waste treatment pressure brought by the yeast fermentation thallus and changes waste into valuable.

(2) The pichia pastoris fermentation thalli for expressing recombinant human collagen in the invention is used for preparing lysate filtrate, contains a certain amount of collagen, and is not added additionally.

(3) The preparation method of the fermentation lysate filtrate is a whole-course online and automatic preparation process, physical crushing and cracking are carried out on pichia pastoris fermentation thalli by high-pressure homogenization, solid-liquid separation is carried out after cracking is carried out by using a continuous flow centrifuge, the lysate filtrate is obtained after filtration, the preparation process is rapid and efficient, large-scale preparation can be carried out, and no external component is introduced; meanwhile, the whole preparation process flow can realize full-automatic operation, the materials are transmitted by a full-automatic online pipeline, sterile filtration and filling are finished in a clean area, and the breeding of harmful bacteria can be effectively controlled.

(4) The lysate filtrate prepared by the method has no safety problem when being applied to cosmetics, and has the following effects except the effects of traditional yeast-derived cosmetic raw materials: the secretion of the antibacterial peptide is promoted, the micro-ecological balance is adjusted, and the effect of the skin micro-ecological barrier is enhanced; the skin immunity barrier is improved and the skin is relieved by inhibiting the expression of inflammatory factors; oil control and sun protection effects; these effects, which have not been seen before for the starting material "Pichia pastoris fermentation lysate filtrates", open up the scope of their use for the preparation of cosmetics.

(5) The prepared pichia pastoris fermentation lysate filtrate has double barrier repair effects on a skin micro-ecological barrier and an immune barrier, and the mechanism of the effect of the pichia pastoris fermentation lysate filtrate applied to cosmetics is explained to a certain extent.

(6) The prepared pichia pastoris fermentation lysate filtrate has the effects of reducing the secretion of cell lipid by experiments besides the effects, and further expands the application range of the pichia pastoris fermentation lysate filtrate in preparing cosmetics (oil control type cosmetics).

Drawings

FIG. 1 is a flow diagram of a method of preparing a fermentation lysate filtrate according to the present invention.

FIG. 2 shows the gram-staining results of post-high-pressure homogenized Pichia pastoris lysates obtained under different parameters in the examples, wherein A1 is the lysate obtained after 3 times lysis under 100MPar, B1 is the lysate obtained after 2 times lysis under 120MPar, C1 is the lysate obtained after 2 times lysis under 150MPar, and D1 is the lysate obtained after 2 times lysis under 200 MPar.

FIG. 3 is an SDS-PAGE (A, loading 10. mu.L) and WB (B) of the lysate filtrates prepared in example 1, wherein in FIG. 3B, lane 1 is the lysate filtrate, loading 10. mu.L, lane 2 is a blank control, lane 3 is recombinant type III human collagen, loading 2.5. mu.g, lane 4 is recombinant type III human collagen, and loading 1. mu.g.

FIG. 4 is a standard curve fitted during calculation of the concentration of recombinant collagen in the sample in the examples.

FIG. 5 is a standard curve and a curve equation fitted during calculation of total protein content in samples in the examples.

FIG. 6 is a standard curve and a curve equation fitted in the process of calculating the polysaccharide content in the sample in the examples.

FIG. 7 is a 3D-based epidermal skin model of an embodiment

After cultivation, H was performed&E, a tissue morphology graph under a microscope after staining, wherein the graph A is a blank control, B is a negative control, namely an SLS treatment group, C is a positive control, namely SLS and 0.01% dexamethasone are added at the same time, and D is an experimental group, namely SLS and 2% filtrate are added at the same time.

FIG. 8 is a histogram of the results of the PGE2 content measurements in the examples.

FIG. 9 shows the results of immunofluorescence assay of capsaicin receptor (TRPV1) in examples.

Fig. 10 is a histogram of the relative IOD values of TRPV1 protein in the examples.

FIG. 11 shows the UV light region 200-400nm wavelength UV rate of the fermentation lysate filtrate and the dilution solution with different multiples obtained by the present invention.

FIG. 12 is a 3D-based epidermal skin model

The result of staining with HBD1 protein.

FIG. 13 is a diagram of a 3D-based epidermal skin model

Histogram of Integrated Optical Density (IOD) values of HBD1 protein.

FIG. 14 is a 3D-based epidermal skin model

The result of staining with HBD2 protein.

FIG. 15 is a 3D-based epidermal skin model

Histogram of Integrated Optical Density (IOD) values of HBD2 protein.

FIG. 16 is a diagram of a 3D-based epidermal skin model

Staining results for TLR2 protein.

FIG. 17 is a 3D-based epidermal skin model

Histogram of TLR2 protein Integrated Optical Density (IOD) values.

FIG. 18 is a 3D-based epidermal skin model

The result of staining with LL-37 protein.

FIG. 19 is a 3D-based epidermal skin model

Histogram of Integrated Optical Density (IOD) values of LL-37 protein.

FIG. 20 shows the results of fat drop synthetic oil red O staining; the scale size in the figure is 50 μm.

Fig. 21 is a histogram of integrated optical density of lipid droplets (IOD) values.

FIG. 22 is a graph showing the effect of the volunteers using the essence containing Pichia pastoris fermentation lysate filtrate.

FIG. 23 is a graph showing the effect of facial mask pack applied to volunteers with the lysate filtrate containing Pichia pastoris.

FIG. 24 is a graph showing the effect of cream on volunteers using a fermentation lysate filtrate containing Pichia pastoris.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following embodiments of the present invention are described in detail, but the following embodiments do not limit the scope of the present invention.

In the embodiments of the present invention, what is not described in detail is accomplished by using a conventional experimental method, and what is not described in the embodiments is understood and easily accomplished by those skilled in the art according to the product specification or the basic knowledge in the art, and thus, will not be described in detail.

The raw material sources related in the invention are as follows:

the fermentation lysate preparation raw material strains are all from the applicant unit of the invention, are all subjected to corresponding strain preservation, are all proprietary pichia pastoris engineering bacteria for expressing recombinant collagen, and are all preserved in the common microorganism center of the China Committee for culture preservation management of microorganisms; comprises strains with the preservation number of CGMCC NO.7189 in the public number CN103102407B, the preservation number of CGMCC NO.14057 in the public number CN107090458A, the preservation number of CGMCC NO.17147 in the public number CN111004319A, the preservation number of CGMCC NO.17150 in the public number CN110964099A, the preservation number of CGMCC NO.17148 in the public number CN110606896B, the preservation number of CGMCC NO.17149 in the public number CN110747198B, the preservation number of CGMCC NO.20626 in the public number CN113185604B, the preservation number of CGMCC NO.20627, the preservation number of CGMCC NO.21891 in the public number CN114106150A and the preservation number of CGMCC NO. 21892. The recombinant human collagen expressed by the engineering bacteria has a 6 XHis-Tag label at the amino terminal (N terminal) or the carboxyl terminal (C terminal), which is beneficial to detection.

In addition to the above list, it is within the scope of the present invention to add collagen to other strains producing collagen or to common yeast lysate filtrates to achieve the technical effects of the present invention.

The expression and production of the recombinant human collagen by the pichia pastoris engineering bacteria are secretory expression: the protein is translated and enters the endoplasmic reticulum, after signal peptide is cut off, the protein is processed by Golgi apparatus and secreted to the outside of cell, the translation, processing and secretion are the last three stages of recombinant human collagen expression and production, and when the cell expresses a large amount of protein, the three stages are carried out simultaneously, so that although most of the recombinant human collagen is secreted to the outside of the cell, the pichia pastoris cell still has collagen remained. The recombinant human collagen is produced by large-scale fermentation, and 20-30% of the volume of the recombinant human collagen in a fermentation tank is fermentation thalli. After batch fermentation is finished, bacteria and bacteria liquid of supernatant of fermentation liquor are separated (a ceramic membrane or a continuous flow centrifuge is mostly used), bacteria slurry with high bacteria concentration is generated, the main components are water and wet bacteria, the wet bacteria concentration can reach about 50% (W/W), and the bacteria slurry can be directly used as a raw material for preparing solution filtrate.

The wet cell concentration in the present invention is the ratio of (aqueous non-dry) cell weight/suspended cell liquid weight, and the calculation method and results are given in the specific case.

Example 1: preparation of lysate filtrates

The yeast cell wall is thicker, the structure is more complex than that of the bacterial cell wall, the property is stable, the integrity of the structure and the components of the yeast cell wall can be still maintained after the protoplast dies or dies, the function of a biological membrane system of the dead protoplast is lost, enzymes in lysosomes enter cytoplasm to largely decompose substances in cytoplasm and nucleus, various small molecular substances escape after passing through the cell wall, but macromolecular substances (such as nucleic acid, protein and the like) can only pass through the cell wall after being degraded into the small molecular substances, and the integrity of the cell wall can still be maintained after being slightly influenced. This is also a big difference between yeast cells and bacteria and animal cells during cell disruption, i.e., only complete cell wall lysis can be achieved during cell disruption.

The invention selects a high-pressure homogenization method to effectively break the cell wall of the yeast, which is a rapid and efficient physical breaking mode without primer exogenous substances. After the disruption, the lysate is subjected to separation of insoluble matter (cell wall-insoluble glucan, insoluble protein, etc.) and soluble matter in the lysate using a high-speed continuous flow centrifuge. The lysate filtrate was prepared by taking the fermentation strain of the engineering bacterium with the preservation number of CGMCC NO.7189 in publication No. CN103102407B as an example.

(1) Taking fermented bacterial pulp, centrifuging at 12000rpm for 10min, weighing, and determining wet bacterial concentration of wet bacterial/bacterial pulp supernatant.

(2) The bacterial cell slurry was diluted with purified water to obtain a bacterial cell suspension having a wet bacterial cell concentration of 5% to 30% (W/W), and in this example, a wet bacterial cell concentration of 10% (W/W) is preferred.

(3) The bacterial suspension is input into a high-pressure homogenizer (AH30-100 plus, Anthony nanotechnology (Suzhou) Co., Ltd.), a cold water system is started for refrigeration, the crushing temperature is controlled to be 4-10 ℃, the pressure of 100MPar is used for crushing for 1-3 times, and then the lysate is obtained, in the embodiment, the pressure of 120MPar is preferred, and the lysate is obtained after 2 times of crushing.

(4) Feeding the lysate into high-speed continuous flow centrifuge (GQ105B, Jiayi (Shanghai) mechanical equipment Co., Ltd.), starting cold water system for refrigeration (controlling temperature at 4-10 deg.C), centrifuging at 10000g-20000g centrifugal force to obtain supernatant, preferably 10000g centrifugal force in this embodiment.

(5) In a clean workshop, the supernatant was introduced into a plate and frame filter system (CW300-10, Chuangwei filtration plant equipment Co., Hainin), and the supernatant was filtered through a 0.45 μm PP filter and a 0.22 μm PES filter in this order to obtain a lysate filtrate.

(6) Adding a small amount of antiseptic, diluting according to the requirement, and packaging to obtain the final product.

In this example, the preparation effect of the obtained fermentation lysate filtrate and the intermediate product in the preparation process were examined, and the examination specifically included:

firstly, detecting the thallus cracking effect in the step (3):

the cracking effect of the pichia pastoris can be detected in a microscopic examination mode, a gram staining method (a gram staining kit is purchased from Beijing Solebao science and technology Co., Ltd.) can be used for microscopic examination, and the principle is similar to that of gram staining: after the crystal violet is primarily dyed and the iodine solution is mordant, a water-insoluble crystal violet and iodine compound is formed in cell walls, cytoplasm is dyed to be purple, the cell walls of yeast are thicker and denser than those of gram-positive bacteria, meshes are reduced due to water loss during ethanol decoloration, and the ethanol decoloration treatment does not contain lipoid, so that gaps can not appear during the ethanol treatment, the crystal violet and iodine compound can be firmly remained in the walls, and the cell walls are still purple under the condition of complete cell walls. If the cell wall of yeast is broken and treated by ethanol, the crystal violet and iodine complex is still colorless after being dissolved and decolored, and is red after being counterdyed by red dyes such as safranin and the like. After gram staining, the cells with unbroken cell walls are purple, the cells with broken cell walls (including the types of cell morphology that remain relatively intact) and the material released from the cells are stained red, and the whole of the high pressure homogenous disruption product forms a uniform red background in the field of view.

The experimental procedure is briefly as follows:

before disruption, yeast cells were counted using a hemocytometer and the total was C.

After cell disruption, taking the disruption solution, smearing, and gram staining: the same general gram staining is required, the coating is uniform and not thick, and the ethanol decolorization time is controlled. And (4) performing phase contrast microscope or oil-lens observation, counting the number of purple cells (n1) and red cells (n2) in each visual field under each observation, and replacing different positions on the glass slide to obtain a plurality of different visual fields for a plurality of times. Calculating the ratio a of the cells which are not subjected to wall breaking after wall breaking to be n1/(n1+ n2), and taking an average value;

and (3) similarly taking a crushed solution after the cells are crushed, centrifuging at a high speed, removing a supernatant, resuspending and precipitating by using pure water with the same volume as diluted bacterial plasma, staining gram or only staining crystal violet, and further facilitating observation, counting by using a blood counting plate (preferably using a phase contrast microscope), and counting the total number of the yeast cells which still have a complete cell shape after being crushed, wherein the total number is counted as C'.

Calculating the wall breaking rate, and performing statistical analysis:

d (100%): wall breaking rate; c: number of cells before disruption; c': total number of cells after disruption; a: the ratio of cells not broken after wall breaking.

When the yeast is broken, the most fundamental requirement is that the cell wall is broken, and the gram staining method is an extremely visual and effective detection method, clear and definite and high in resolution. The fermentation strain with preservation number of CGMCC NO.7189 in the publication number CN103102407B is obtained by taking the wall breaking rate of the fermentation strain of the engineering strain with preservation number of CGMCC NO.7189 under different breaking conditions as an example: 100MPar (3 times): the wall breaking rate is 95.9%; 120MPar (2 times): the wall breaking rate is 97.3%; 150MPar (2 times) with wall breaking rate of 99.4%; 200MPar (2 times): the wall breaking rate is 99.5%, and the breaking and cracking effects are shown in figure 2. The 120Mpar disruption for 2 times can completely meet the cracking requirement of pichia pastoris.

Second, the detection of recombinant human collagen, total protein, polysaccharide, amino acid, vitamin in the lysate filtrate obtained by the method of this example

The recombinant human collagen expressed by the engineering bacteria adopted in the embodiment has a 6 XHis-Tag label at the amino terminal (N terminal) or the carboxyl terminal (C terminal), which is beneficial to detecting the collagen in the filtrate, and Western Blot can be used for visual qualitative detection and Elisa can be used for quantitative detection. Taking the engineering bacterium with preservation number of CGMCC NO.7189 in the publication number CN103102407B as an example, lysate filtrate prepared by fermenting the bacterium with 10% wet bacterium concentration is adopted for corresponding detection.

SDS-PAGE electrophoresis and WB detection results:

the lysate filtrate was prepared from Pichia pastoris (purchased from Saimer Feishell technology (China)) as a blank control, which was not transferred to express the collagen gene, and subjected to SDS-PAGE electrophoresis and Western Blot detection, resulting in the following results:

FIG. 3A is a SDS-PAGE electrophoresis of lysate filtrates prepared at 10% wet cell concentration, with different sized proteins distributed from top to bottom; FIG. 3B shows Western Blot results of lysate filtrates prepared at 10% wet cell concentration (Anti-His, antibody purchased from Biotechnology (Shanghai) Ltd., mouse Anti-His monoclonal antibody, product No. D199987), and from FIG. 3B (ECL chemiluminescence chromogenic, full-automatic chemiluminescence image analysis System Tanon 5200 synthesizes protein molecular weight standards into images), it can be seen that 6 × His tag can detect bands against 6 × His tag, the size of the band of interest is consistent with the molecular weight in electrophoresis of purified lyophilized recombinant type III human collagen expressed in patent publication No. CN103102407B, indicating that the lysate filtrates contain recombinant type III human collagen, while the blank control (lysate filtrate prepared by cleavage of Pichia pastoris strain not transformed to express collagen gene) has no band.

Elisa detection of 5D collagen content:

taking lysate filtrate prepared by 10% wet thallus concentration to carry out Elisa detection, simultaneously taking purified recombinant III type human collagen freeze-dried sponge to carry out Elisa standard curve formulation, taking blank Pichia pastoris yeast which is not transferred to express collagen gene to prepare lysate filtrate as a blank control, using an indirect Elisa method, and briefly describing the following experimental steps:

(1) the lysate filtrate (10, 100 times) was diluted with PBS gradient and added to the microplate, and the lyophilized sample of recombinant type III human collagen purified expressed in patent publication CN103102407B was dissolved and diluted with PBS gradient, and also added to the microplate, and left to stand at 4 ℃ overnight.

(2) The enzyme-labeled plate was discarded, and a 5% skimmed milk powder solution (obtained from Biotechnology (Shanghai) Co., Ltd., mass/volume ratio, e.g., 5g skimmed milk powder dissolved in 100mL PBS) diluted with PBS was subjected to sealing treatment at 37 ℃ for 2 hours.

(3) Discarding the liquid in the elisa plate, washing with PBS twice, and then adding 5% skim milk powder solution to dilute the primary antibody (Anti-His, antibody purchased from bio-engineering (shanghai) gmbh, mouse Anti-His monoclonal antibody, cat # D199987) solution at a dilution ratio of 1: 5000, and standing for 1 hour at 37 ℃.

(4) Discarding the liquid in the elisa plate, washing with PBS twice, adding 5% skimmed milk powder solution to dilute HRP-labeled secondary antibody (purchased from bio-engineering (shanghai) gmbh, secondary antibody matched with mouse anti-His monoclonal antibody, product number D199987) solution, with a dilution ratio of 1: 2000, standing at 37 ℃ for 1 hour.

(5) Discarding the liquid in the ELISA plate, washing with PBS twice, adding TMB color development solution (TMB color development P0209-100mL, Bi Yun Tian Lian), adding stop solution (P0215-100mL, Bi Yun Tian Lian) after obvious blue appears, and loading in ELISA reader (Varioskan) ^TM LUX multifunctional microplate reader-VL 0L0TD0, Saimer Feishell science and technology (China) Co., Ltd.) read the absorbance value at 450 nm.

(6) And fitting the standard curve by GraphPad Prism 5, performing data processing, and calculating the concentration of the recombinant collagen in the sample. The fitted standard curve is shown in FIG. 4, with the abscissa being the logarithm of the collagen concentration and the ordinate being the absorbance value at 450 nm. The curve fitting equation is:

Y＝0.0007221+(1.372-0.0007221)/(1+10^((-0.1103-X)*1.341)))

wherein Y is the protein concentration and X is the absorbance at 450nm of the sample detected.

The results of Elisa test (450nm absorbance values) of lysate filtrates at different concentrations are as follows:

TABLE 1 measurement of absorbance values at 450nm for different concentrations of lysate filtrates Elisa

Dilution factor	/	10	100
				Lysate filtrate absorbance values	0.2379	0.4120	0.1375
Absorbance value of blank control	0.0025	0.0036	0.0037

From analysis on curve fitting, the better detection range is OD450 is in the range of about 0.25 to about 1.25. Among the experimental groups that can be effectively calculated is ddH ₂ O is the OD450 value of Elisa experiment after 10-fold dilution of centrifugal supernatant of the crushing liquid. The lysate filtrate had a concentration of 11.44. mu.g/mL of recombinant type III human collagen. In general, the recombinant collagen content of lysate filtrate prepared by 10% wet cell concentration is about 10-15 μ g/mL, and the recombinant human collagen content also increases with the increase of the wet cell concentration. And OD450 of lysate filtrate prepared by the blank pichia pastoris which is not transferred to express the collagen gene is basically out of the lower limit of the effective detection range, and the collagen can not be detected.

c. Detection of Total protein content in lysate filtrates

The experimental method refers to the fifth method in the protein content determination method of pharmacopoeia of the people's republic of China (2020 edition): the detection of the total protein content was carried out by the Coomassie Brilliant blue method (Bradford method). The lysate filtrate is prepared by using the fermentation thalli of CGMCC NO.7189 engineering bacteria in the patent with the publication number of CN103102407B and the concentration of 0.2-20% wet thalli, the Bradford protein concentration determination reagent is purchased from Biyunnan biology (P0006), and the experimental steps are briefly described as follows:

(1) protein standards (5mg/mL BSA) were diluted with purified water to 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mg/mL.

(2) mu.L of protein standards of different concentrations were added to the protein standard wells of the 96-well plate, while 5. mu.L of lysate filtrate samples were added to the sample wells of the 96-well plate.

(3) Add 250. mu. L G250 staining solution to each well.

(4) Using a microplate reader (Varioskan) ^TM LUX multifunctional microplate reader-VL 0L0TD0, Saimer Feishell science and technology (China) Co., Ltd.) to determine the absorbance value at 595 nm.

(5) The data were processed to calculate the protein concentration in the sample from the standard curve and the sample volume used. The standard curve and the curve equation are shown in fig. 5. Total protein content (mg/mL) results in lysate filtrates prepared from 0.2% to 20% wet cell concentrations are shown in Table 2:

TABLE 2.0.2% -20% Wet cell concentration Total protein content (mg/mL) in lysate filtrates prepared

Slope (R) of the standard curve in the Bradford method ² )>0.98, with the lysate filtrate preparation of wet bacterial concentration increases, the lysate filtrate total protein content increases, 0.192 ~ 26.9mg/mL, when the wet bacterial concentration is above 10%, the lysate filtrate total protein content is greater than 10mg/mL (II) ((II))>1%) up to 25mg/mL (2.5%) or more, and the total protein content in the filtrate produced by multiple times of lysis with a bacterial concentration of 10% is generally 10-15 mg/mL. Other methods such as BCA method, biuret method and Kjeldahl method in protein content determination method of pharmacopoeia of the people's republic of China (2020 edition) are also selected according to actual conditions for detecting the total protein content.

d. Detection of polysaccharide content in lysate filtrates

The technical information, 2011(10) page 127-128, the lysate filtrate is prepared by the method in the patent with the publication No. CN103102407B of 10-20% of the wet cell concentration of the fermentation cell of the CGMCC NO.7189 engineering bacteria, and the polysaccharide content is detected.

The experimental procedure is briefly as follows:

(1) preparation of mannose Standard solution (0.1 mg/mL): accurately weighing 500mg of mannose dried to constant weight at 105 ℃, placing the mannose in a 500mL volumetric flask, adding water to dissolve the mannose and diluting the mannose until scales are uniformly shaken to obtain a stock solution of 1mg/mL for later use. And (5) transferring 10mL of the upper solution by using a10 mL pipette, and diluting to 100mL to obtain 0.1mg/mL of mannose standard solution.

(2) Preparation of 85% phenol solution: heating phenol in a water bath to 45 ℃, taking 85mL of phenol solution, dissolving in 15mL of water, and obtaining 85% phenol solution after complete dissolution. And putting the mixture into a refrigerator with 4 degrees, and keeping the mixture away from light. Can be used for a long time.

Preparation of 5% phenol solution: the 85% phenol solution was taken out and dissolved by heating in a water bath at 45 ℃ to obtain 1mL of a solution, which was then dissolved in 16mL of pure water to obtain a 5% phenol solution. Stored in dark place, and is prepared for use first.

(3) Preparing a standard sample and drawing a standard curve: a mannose standard solution (0.1mg/mL) was accurately aspirated, and prepared as shown in Table 3. Adding 0.5mL of phenol solution and 2.5mL of concentrated sulfuric acid (rapid, precise addition), mixing for 20 s, standing in 80 deg.C water bath for 30min, cooling, and measuring absorbance at 490 nm. And (3) drawing a standard curve by taking a blank reference experiment, the absorbance value as a vertical coordinate and the mannose concentration as a horizontal coordinate, and establishing a standard curve regression equation.

TABLE 3 preparation and testing of Standard samples

(4) And detecting the sample to be detected by the method.

(5) And (3) calculating the content of polysaccharide:

and (5) substituting the absorbance of the measured sample into a standard curve equation. The corresponding solubility of the polysaccharide is obtained and divided by the solubility of the polysaccharide in the filtrate of the sample lysate to obtain the content of the polysaccharide.

W: the content of the sample polysaccharide; w1: calculating the solubility of the obtained polysaccharide under a standard curve regression equation; w2: solubility of polysaccharides in sample lysate filtrate.

The standard curve and the curve equation are shown in fig. 6:

the polysaccharide content (mg/mL) in the lysate filtrate obtained by the preparation was as follows:

when the wet cell concentration (W/W) in the preparation of the lysate filtrate was 10%, the polysaccharide content reached 1.15 mg/mL; when the wet thallus concentration is 15%, the polysaccharide content reaches 1.45 mg/mL; when the wet bacterial concentration is 20%, the polysaccharide content reaches 1.59 mg/mL. The results of the assay were combined at other concentrations, indicating that the polysaccharide content of the lysate filtrate was 1-1.6 mg/mL.

e. Detection of amino acid and vitamin content in lysate filtrate

The lysate filtrate was prepared at 10% wet cell concentration of the fermentation cells of the engineering bacteria CGMCC NO.7189 as published under No. CN 103102407B. The method comprises the steps of detecting amino acid in lysate filtrate by using a second method for detecting nicotinic acid and nicotinamide in GB 5009.89-2016 food safety national standard food, a first method for detecting vitamin B2 in GB 5009.85-2016 food safety national standard food, a second method for detecting amino acid in GB 5009.124-2016 food safety national standard food, a first method for detecting choline in GB 5413.20-2013 food safety national standard infant food and dairy food, a second method for detecting inositol in GB 5009.270-2016 food safety national standard food, a first method for detecting vitamin B6 in GB 5009.154-2016 food safety national standard food, a first method for detecting pantothenic acid in GB 5009.210-2016 food safety national standard food, a first method for detecting folic acid in GB 737373829 food safety national standard food, and a second method for detecting biotin in GB 4-2016 food safety national standard food, And (4) entrusting Shanghai Tianxiang quality technology service Limited company to complete detection of the content of the vitamin and issuing a detection report.

The results are shown in Table 4.

TABLE 4 detection results of amino acids and microorganisms in lysate filtrates

The results show that the lysis filtrate prepared with 10% wet cell concentration contains various vitamin B group and amino acids, the total amount of amino acids can reach about 0.82%, and the lysis filtrate can be used as raw material to provide abundant active substance components for cosmetics.

Thirdly, safety detection of the lysate filtrate obtained

a. Detection of residual DNA

The DNA in the lysate filtrate was mainly the residue of Pichia pastoris genomic DNA, and the DNA in the lysate filtrate prepared at a bacterial concentration of 10% was detected by PCR-fluorescent probe method, which was submitted to Shenke Biotechnology Inc. of Huzhou for completion and reported detection. The basic process is briefly described as follows, and related reagents in the experimental process are produced by Shenke biotechnology, Inc. in Huzhou province or are commercially available:

(1) after the lysate-containing filtrate sample is diluted, the lysate-containing filtrate sample is purified by a magnetic bead method;

(2) specific primers and probes (Pichia pastoris residual DNA detection kit (PCR-fluorescent probe method) from SK030205P100, applied to Shenzhou, Shenke, Biotechnology Co., Ltd.) for in vitro amplification of specific fragments of DNA in the lysate filtrate sample (quantitative PCR apparatus FQD-96A, LineGene 9600Plus quantitative PCR system);

(3) in the PCR amplification process, the specific probe is released from the template DNA, and then a fluorescence signal can be detected by using a fluorescence quantitative PCR instrument, and the data of the fluorescence signal is analyzed by detection software of the PCR instrument.

The detection results are as follows:

TABLE 5 lysate residual DNA test results

In the table, the number of the first and second,

and (3) detection of the mean value: average value of the detection values. CV: CV (coefficient of variance), the ratio of standard deviation to mean, the values used to characterize the coefficient of variation in statistics, both CVs have the same meaning. Adding a scalar quantity: refers to the amount of standard added. And (4) adding standard detection mean value: adding the average value of the detection values of the standard substance and the reference substance.

As a result, the DNA content in the lysate filtrate prepared by the present invention was determined to be 31.4. mu.g/mL, which is lower than the residual amount of DNA required in biopharmaceuticals, and is in a safe range.

b. Detection of heavy metals, methanol content, etc

According to the related detection methods of the first methods of chapter 1.6 and chapter 2.22 of the fourth chapter of the technical Specification for safety of cosmetics 2015, the contents of heavy metals and methanol in lysate filtrate are detected, and the detection report is issued by the Suzhou customs integration technology center.

The results are shown in table 6 below:

TABLE 6 lysate filtrate heavy metal, methanol detection results

Item	Result Unit	Technical requirements	The result of the detection	Method for detecting concentration	Single item judgment
						Lead (II)	mg/kg	≤10	＜0.03	0.03	Conform to
Arsenic (As)	mg/kg	≤2	＜0.001	0.001	Conform to
						Mercury	mg/kg	≤1	＜0.001	0.001	Conform to
Cadmium (Cd)	mg/kg	≤5	＜0.001	0.001	Conform to
						Methanol	mg/kg	≤2000	＜25	25	Meet with

The results show that the contents of heavy metal and methanol in the lysate filtrate prepared by the method meet the safety requirements, and the lysate filtrate is a cosmetic raw material, so that the actual dilution effect can be generated in the preparation and use of an actual product, and the safety problem is avoided.

c. Detection of microbial limits

According to the microbial limit detection method in the cosmetic safety technical specification 2015 edition, the lysate filtrate is subjected to microbial limit detection, the detection is finished by the Suzhou customs integrated technology center and a detection report is issued, the results all meet the safety requirements, the detection results are shown in the following table 7, and the results show that the microbial results in the lysate filtrate prepared by the method all meet the requirements.

TABLE 7 microbial detection results of lysate filtrates

d. Toxicology testing

According to the toxicology test method in the cosmetic safety technical Specification 2015 edition, the lysate filtrate is subjected to toxicology safety evaluation, and the Ningbo customs technical center is entrusted to complete detection and provide a detection report.

The statistics of the detection results are shown in the following table 8, the experimental methods listed in the table are all conventional methods specified in the technical Specification for cosmetic safety, and the experiments are all completed in the Ningbo Customs technical center. As a result, the toxicological detection results of the lysate filtrate prepared by the method all meet the requirements, and the safety is guaranteed.

TABLE 8 results of toxicological safety test of lysate filtrates

(1) Acute oral toxicity test

Processing a test sample: 5.0806g of sample (namely lysate filtrate prepared by the invention) is weighed and placed in a beaker, a small amount of pure water is taken, the sample is uniformly mixed and transferred into a 20mL volumetric flask, the beaker is washed by a small amount of pure water for multiple times and transferred into the volumetric flask, the pure water is added to a constant volume to a scale mark, the sample is transferred to a sample tube mark for standby after being fully shaken up, and the sample is ready to use. The intragastric volume is 2.0mL/100 g.BW.

Experimental animals: the weight of 10 SPF grade ICR mice (Shanghai Jiesi laboratory animals Co., Ltd.) with the age of 6 weeks, female and male halves (female animal is not pregnant and has not fared), is controlled between 18.5g and 21.4g (the weight of the same sex organism does not exceed the average value +/-20%)

Animals before test are adapted to a barrier environment animal room (barrier environment, 23.0-23.8 ℃, relative humidity of 40.8-59.7%) for 8 days (Co60 irradiates mouse materials, Jiangsu province cooperative medical and biological engineering Limited liability company; first-grade RO ultrafiltration water (sodium hypochlorite is added, free chlorine content in the water is controlled to be 2-3ppm for sterilization) is directly supplied to the animals for free drinking through a drinking nozzle), and the animals are fasted overnight before contamination.

After the test is started, the intragastric administration dose is 5080.6mg/kg by a one-time limiting method. After the infection, fasting is continued for 3 hours, and symptoms of the poisoning or behavioral changes are observed daily and weighed once a week. Observing and recording the poisoning and death conditions of the animals in the infection process and the observation period, wherein the observation period is 14 days, and after the observation period is finished, the surviving animals are sacrificed and roughly dissected.

And (3) detection results:

the experimental animals do not have any toxic symptoms and toxic death within 14 days of the infection; the body weight of the male and female animals is not abnormal. After the experimental observation is finished, the gross anatomy examination of the tested animal has no abnormal change. LD50 > 5080.6mg/kg, detailed results are as follows:

TABLE 9 acute oral toxicity test results

It can be seen that under the conditions of this test, the acute oral LD50 of the test samples to ICR mice is > 5000 mg/kg. The sample was classified as practically nontoxic according to acute oral toxicity.

(2) Acute transdermal toxicity test

Treating a test sample: test with raw sample lysate filtrate.

Experimental animals: 10 SPF-grade SD rats (Shanghai Jiesi laboratory animals Co., Ltd.) with the body weight of 201.2 g-218.2 g are female and male half (female is not pregnant and has not fared).

Animals before test are adapted to a barrier environment animal room (the barrier environment is 22.0-22.9 ℃, and the relative humidity is 45.8-55.5%) for 7 days (Co60 irradiates mouse materials, Jiangsu province cooperative medical and biological engineering Limited liability company; first-grade RO ultrafiltration water (sodium hypochlorite is added, and the content of free chlorine in the water is controlled at 2-3ppm for sterilization) and is directly and freely drunk by the animals through a drinking nozzle, and the animals before contamination are not fast and freely drink water.

After the test is started, a one-time limit method is adopted, and the contamination dose is 2500 mg/kg. Animals were housed individually during the trial.The weight range of the contamination precursor is 201.2 g-218.2 g, and the calculated 10 percent of the body surface area is 31.3cm ² ～33.1cm ² . 24 hours before the test, the affected area on the back of the animal was shaved and the skin preparation area was 5cm by 7 cm. Weighing 0.5g of test sample required by each animal, uniformly applying to skin preparation area, covering with a thin film, fixing with non-irritating adhesive plaster to prevent animal licking, sealing and contacting for 24 hr, removing the fixture and the covering, and washing residual test sample on skin with water. The animals were observed and recorded for intoxication and death during the course of the infection and observation period, weighed once a week for an observation period of 14 days, and after the observation period was complete, the surviving animals were sacrificed and gross dissection was performed.

And (3) test results: the experimental animals do not have any toxic symptoms and toxic death within 14 days of the infection; the body weight of the male and female animals is not abnormal. After the experimental observation is finished, the gross anatomy examination of the tested animal has no abnormal change. LD50 is more than 2500 mg/kg. The results are given in Table 10 below.

TABLE 10 results of acute transdermal toxicity test

It can be seen that under the conditions of this test, the test sample had an acute transdermal LD50 > 2180mg/kg for SD rats, which is of a minor toxicity grade according to the acute skin toxicity classification.

(3) Acute skin irritation test

Treating a test sample: test with raw sample lysate filtrate.

Experimental animals: 4 common-grade New Zealand rabbits (family farm of Vietnam street, Jiashan county); male and female halves (female animals selected for non-pregnant and non-farrowing); the weight is controlled between 2.27kg and 2.98 kg.

Before the experiment, the animals at least adapt to 3d in the environment of an experimental animal room, and are raised in a single cage, wherein the raising environment temperature is as follows: 22.8-23.6 ℃, relative humidity: 52.9 to 61.4 percent of 4; raising a feed experimental rabbit compound feed (maintenance type, oriental feed limited liability company in tung county); the first-level RO ultrafiltration water (sodium hypochlorite is added, the content of free chlorine in the water is controlled at 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking nozzle.

The experimental method comprises the following steps: the two sides of the spine of the experimental animal are cut off in 24 hours before the experiment, and the hair removing range is about 3cm multiplied by 3cm respectively. 0.5mL of the test sample was applied directly to the dehaired skin on one side, the area of application was 2.5cm by 2.5cm, then covered with two layers of gauze (2.5cm by 2.5cm) and a layer of cellophane paper, and then fixed with a non-irritating adhesive tape and bandage. The other side of the skin served as a control. After 4h of the blocking test, the residual test sample is removed with warm water. And observing the skin reaction of the smearing part at 1h, 24h, 48h and 72h after the test sample is removed, grading the skin reaction, and grading the stimulation intensity according to the highest integral mean value of each observation time point of 24h, 48h and 72 h.

And (3) test results: during the test period, no abnormal symptoms appear in the experimental animals, and the mean value of the highest skin irritation integral at each observation time point (24h, 48h and 72h) is 0.00. Under the test conditions, the acute skin irritation of the test sample to the rabbit is as follows: has no irritation.

TABLE 11 results of acute skin irritation test

(4) Multiple skin irritation test

Treating a test sample: test with raw sample lysate filtrate.

Experimental animals: 4 common-grade New Zealand rabbits (family farm of Vietnam street, Jiashan county); male and female halves (female animals selected for non-pregnant and non-farrowing); the weight is controlled between 2.34kg and 2.92 kg.

Before the experiment, the animals at least adapt to 3d in the environment of an experimental animal room, and are raised in a single cage, wherein the raising environment temperature is as follows: 22.0-23.9 ℃, relative humidity: 40.5% -58.6%; raising a feed experimental rabbit compound feed (maintenance type, oriental feed limited liability company in tung county); the first-level RO ultrafiltration water (sodium hypochlorite is added, the content of free chlorine in the water is controlled at 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking nozzle.

The experimental method comprises the following steps: the two sides of the spine of the experimental animal are cut off in 24 hours before the experiment, and the hair removing range is 3cm multiplied by 3cm respectively. The smearing area is 2.5cm multiplied by 2.5 cm. 0.5mL of the test sample is smeared on one side of the unhaired skin, the other side of the unhaired skin is used as a control, and the test sample is smeared for 1 time every day and is smeared for 14 days continuously. From the next day, the hair was cut before each application and the remaining test sample was removed with warm water. The results were observed after one hour.

And (3) test results: during the test period, no abnormal symptoms appeared in the experimental animals, and the average integral of each animal in 14 days is 0.00. See table 12 below for details. Under the test conditions, the tested sample has no irritation to the rabbit skin irritation for many times.

TABLE 12 results of multiple skin irritation tests

(5) Acute eye irritation test

Treating a test sample: test with raw sample lysate filtrate.

Experimental animals: 3 common-grade New Zealand rabbits (family farm of Vietnam street, Jiashan county); male and female halves (female animals selected for non-pregnant and non-farrowing); the weight is controlled between 2.40kg and 2.61 kg.

Before the experiment, the animals at least adapt to 3d in the environment of an experimental animal room, and are raised in a single cage, wherein the raising environment temperature is as follows: 23.0-24.1 ℃, relative humidity: 49.8% -60.7%; feeding: the experimental rabbit compound feed (maintenance type, oriental feed limited liability company in tung county); the first-level RO ultrafiltration water (sodium hypochlorite is added, the content of free chlorine in the water is controlled at 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking nozzle.

The test method comprises the following steps: both eyes of the test animals were examined 24h before the start of the test (including examination using a 2% sodium fluorescein solution). Animals with eye irritation symptoms, corneal defects, and conjunctival damage were not used for the test. The lower eyelid of one eye of the rabbit is slightly pulled open, 0.1mL of the test sample is dripped into the conjunctival sac, and the upper eyelid and the lower eyelid are passively closed for 1s to prevent the test sample from being lost. The other eye was not treated for self-control. Eyes are not washed within 24 hours after the test sample is dripped. The eyes of the animals were examined 1h, 24h, 48h, 72h and 4d and 7d after instillation of the test samples. If no stimulus response occurs for 72 hours, the test is terminated.

And (3) test results: during the test period, the experimental animals have no abnormal symptoms, and the integral mean values of the individual animals are all 0.00. See table 13 below for details. Under the test conditions, the acute eye irritation of the test sample to the rabbit is as follows: has no irritation.

TABLE 13 acute eye irritation test results

Individual animal score mean means the average score for each animal in terms of cornea, iris, conjunctival congestion and conjunctival edema for three different observation times (24h, 48h and 72h) (i.e. the sum of the 24h, 48h and 72h scores for each animal divided by the observation time point number 3).

(6) Skin phototoxicity test

Processing a test sample: the lysate filtrate of the original sample was used for testing. Positive control: 8-Methoxypsoralen (batch: L630V 07; Beijing Bailingwei science and technology Co., Ltd.), solvent: absolute ethanol, concentration: 0.05 percent; the dosage is as follows: 2.4000 mg.

Experimental animals: 6 common-grade guinea pig test groups and 6 positive control groups (Yuyao Simen town Jianfei laboratory rabbit farm); male and female (female animal selected for non-pregnant and non-farrowing); the weight is controlled between 261.2g and 272.4 g.

Animals were acclimated for at least 3d in the laboratory environment prior to testing. Raising environment temperature: 23.3-24.0 ℃; relative humidity: 49.5% -56.7%; feeding: guinea pig compound feed (maintenance, biotechnology, army, su); and (3) filtering the primary RO water (adding sodium hypochlorite, and controlling the content of free chlorine in the water to be 2-3ppm for sterilization). The water can be directly drunk by animals through the drinking mouth.

The test method comprises the following steps: irradiation dose: 10000mJ/cm ² Average light intensity：15.1mW/cm ²

The formula for the calculation of the exposure time is:

note: 1mW/cm ² ＝1mJ/cm ² /sec

Inputting the irradiation dose and the average light intensity in a UVA-TOXI phototoxicity instrument, and obtaining the irradiation time of 00:10:64 (namely the irradiation time is 664s) according to a calculation formula;

the test steps are as follows: animals were acclimated for at least 3 days in the laboratory environment prior to the trial. Approximately 24 hours before the test, the skin on two sides of the spine of the animal is unhaired, and the skin on the test part needs to be intact without damaging the abnormality. 4 depilatory regions were prepared, each depilatory region having a depilatory area of approximately 2cm by 2 cm. Animals were fixed and 0.2mL of test sample was applied to the depilated

areas

1 and 2 of the animals as shown in chapter 7 skin phototoxicity test table 1 of technical specifications for cosmetic safety (2015 edition). After 30min, the left side (depilated areas 1 and 3) was covered with aluminum foil, taped and the right side was irradiated with UVA. After finishing, the skin reaction is observed for 1h, 24h, 48h and 72h respectively, and the skin reaction score of each animal is judged according to the skin phototoxicity test table 2 of chapter 7 of cosmetic safety technical Specification (2015 edition). The positive control group was prepared in the same manner.

And (3) test results: during the test period, no abnormal symptom appears in the experimental animals, the animal score at each observation time point is 0, and the tested sample is classified according to the skin irritation response, so that the skin phototoxicity is not seen. The phototoxicity test results of the positive control on the skin of the guinea pigs are shown in Table 14, and the phototoxicity test results of the test samples on the skin of the guinea pigs are shown in Table 15. Under the test conditions, the test sample has the following phototoxicity test result on the skin of the guinea pig: no phototoxicity to the skin was observed.

TABLE 14 phototoxicity test results of positive control on guinea pig skin

Note: in the table head, 1,2, 3 and 4 are test areas shown in fig. 1 of chapter 7 of technical Specification for cosmetic safety (2015) and skin phototoxicity test.

TABLE 15 phototoxicity test results of test samples on guinea pig skin

Note: the tables 1,2, 3 and 4 are the test areas shown in fig. 1 of chapter 7 of technical Specification for cosmetic safety (2015) and skin phototoxicity test.

(7) Skin allergy test

Processing a test sample: the lysate filtrate of the original sample was used for testing. Positive control: alpha-hexyl cinnamaldehyde (batch No.: LV30V 28; Beijing Bailingwei technologies, Inc.); solvent: absolute ethanol (analytical grade, lot # 20210119, national group chemical Co., Ltd.), acetone (analytical grade, lot # 20170918, national group chemical Co., Ltd.), and Millipore pure water.

Induction concentration: 60 percent, taking 3.6mL of alpha-hexyl cinnamaldehyde and 2.4mL of 80 percent ethanol solution to be uniformly mixed to prepare the compound; excitation concentration: 30 percent, and 3.0mL of alpha-hexyl cinnamaldehyde is uniformly mixed with 7.0mL of 80 percent acetone solution to prepare the compound; the dosage is as follows: 6.6 mL.

Experimental animals: common grade Netherlands guinea pigs (Simen town Yuyao city build flying laboratory rabbit farm); the test group and the positive control group are respectively 20, and the negative control group is 10; male and female (female is not pregnant and has not born); the weight is controlled between 257.8g and 273.4 g.

Animals were acclimated for at least 3 days in the laboratory environment prior to the trial. Raising environment temperature: 22.5-24.1 ℃; relative humidity: 43.4% -61.9%; feed: guinea pig compound feed (maintenance type, department of biotechnology, ammu, Suzhou), first-level RO ultrafiltration water (sodium hypochlorite was added to control the free chlorine content in the water to 2-3ppm for sterilization). The water can be directly drunk by animals through a drinking mouth.

The test method comprises the following steps: approximately 24 hours before the test, the guinea pig was depilated on the left side of the back to a range of approximately 6cm ² 。

And (3) induction contact: 0.2mL of the test sample was applied to the dehaired skin of 2cm × 2cm of test animals, covered with two layers of gauze and one layer of cellophane, and sealed and fixed with a non-irritating adhesive plaster for 6 h. The same procedure is repeated for 7d and 14 d. The positive control group was treated with a 60% α -hexylcinnamaldehyde solution in the same manner as the test group except that the test sample was not administered to the negative control group.

And (3) exciting contact: at 14d after the last induction, 0.2mL of the test sample was applied to the right 2cm x 2cm depilated area (24h before contact) of the back of the test and negative control guinea pigs, which was then covered with two layers of gauze and a layer of cellophane and fixed with a non-irritating adhesive plaster for 6 h. The positive control group was treated with 30% α -hexylcinnamaldehyde solution in the same manner. Skin reactions were observed 24h and 48h after the end of challenge exposure, scored and judged for sensitization intensity.

And (3) test results: during the test period, no abnormal symptom appears in the experimental animals, and the sensitization rate at each observation point is 0%. See tables 16 and 17. Under the test conditions, the result of the test of the skin allergy of the tested sample to the guinea pig is as follows: skin allergy is not seen.

TABLE 16 summary of body weights of the animals tested

Group of	Animal number (only)	Initial body weight (g)	Weight termination (g)
				Negative control	10	262.2±3.1	316.3±5.1
Test sample set	20	265.6±4.7	327.5±12.0
				Positive control	20	286.6±5.0	368.9±5.8

TABLE 17 results of skin allergy test on guinea pigs for test samples or positive controls (BT method)

Note: when the skin reaction integral is 0, 1,2, 3 … …, the skin reaction intensity column indicates the ratio of the number of animals that reacted to the number of test animals; the table as such refers to the invention prepared lysate filtrate.

(8) Bacterial reverse mutation assay

Treating a test sample: sterile pure water is used as a solvent to prepare the concentration of each dosage group.

Negative control: sterile pure water, dimethyl sulfoxide (DMSO, colorless clear liquid)

Positive control:

(1) without S9 (purchase, molthox, usa):

dixon (Dexon): batch number: g1063485; the manufacturer: dr. enrnstorfer GmBH, germany; solvent: sterile pure water; concentration: 500 mu g/mL; the dosage is as follows: 0.1 mL/dish;

sodium Azide (SA): batch number: 20120615, respectively; the manufacturer: shanghai Epimei chemical Co., Ltd; solvent: sterile pure water; concentration: 15 mu g/mL; the dosage is as follows: 0.1 mL/dish.

(2) Adding S9:

2-aminofluorene (2-AF): batch number: c11449586; the manufacturer: shanghai Michelin Biochemical technology, Inc.; solvent: dimethyl sulfoxide (DMSO); concentration: 100 mu g/mL; the dosage is as follows: 0.1 mL/dish;

1, 8-dihydroxyanthraquinone (1, 8-DHQ): batch number: c10429233; the manufacturer: shanghai Michelin Biochemical technology, Inc.; solvent: DMSO; concentration: 500 mu g/mL; the dosage is as follows: 0.1 mL/dish;

cyclophosphamide (CP): batch number: k1812016; the manufacturer: aladdin reagent (Shanghai) Inc.; solvent: sterile pure water; concentration: 2000. mu.g/mL; the dosage is as follows: 0.1 mL/dish.

Experimental strains: salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) were purchased from MOLTOX, USA, and the tested strains were identified to meet the standard requirements.

The testing steps are as follows: four dose groups of 12.5. mu.L/dish, 25.0. mu.L/dish, 50.0. mu.L/dish and 100.0. mu.L/dish were selected, along with a blank control, a solvent control (negative control), a positive mutagen control and a sterile control group. The bacterial strain is subjected to enrichment culture to obtain enrichment liquid, and then the enrichment liquid is subjected to plate doping. In the experiment, 2.0mL of top layer culture medium containing 0.5mmol/L histidine and 0.5mmol/L biotin solution is subpackaged in test tubes, heat preservation is carried out in a water bath at 45 ℃, then 0.1mL of enrichment medium, 0.1mL of tested sample and 0.5mL of phosphate buffer solution or 0.5mL of S9 mixed solution (when metabolic activation is needed) are sequentially added into each tube, the mixture is fully and uniformly mixed, the mixture is rapidly poured onto the bottom layer culture medium, and the plate is rotated to be uniformly distributed. Horizontally placing for condensation and solidification, turning the plate, and inversely placing in an incubator at 37 ℃ for incubation for 48 h. Positive mutagen control, solvent control, blank control and sterility control were also run in this manner. The number of colonies transformed per dish was counted.

The number of colonies per dish for each dose group of test, blank control (spontaneous regression), solvent control, and positive mutagen control were recorded and averaged and standard deviation was calculated.

And (3) test results: results of the standard plate incorporation tests are shown in Table 18. The solvent control (negative control) results show that each group grows in a sterile colony without adding the test strain salmonella typhimurium and can be used for tests; the numbers of revertant colonies of the blank control and the positive mutagen control groups are within an acceptable range, and the test is effective;

under the test condition, the number of the reverted colonies detected by each dosage group of the test object by a plate doping method with or without a metabolic activation system does not exceed more than 2 times of that of a solvent control group, and the dosage response relation is not presented.

TABLE 18 Salmonella typhimurium Return mutation test results (individual/dish, x. + -.s)

(9) In vivo mammalian cell micronucleus assay

Treating a test sample: the sample lysate filtrate was formulated into each dose group solution using pure water as a solvent.

Negative control: pure water

Positive control: cyclophosphamide (batch: F1812016; brand: Allantin; solvent: pure water; concentration: 3mg/mL)

Experimental animals: 30 SPF grade ICR mice (Shanghai Jie Si Jie laboratory animals Co., Ltd.) with female and male halves (female animal is not pregnant and has not born), weight control is 25.0 g-27.4 g, and the same sex weight of the same sex does not exceed plus or minus 20% of the average value.

Before the test, animals are directly and freely drunk by the animals through a drinking nozzle in a quarantine environment animal room (the quarantine environment is 22.8-23.5 ℃, and the relative humidity is 51.6-57.8 percent) (Co60 irradiates mouse materials, Jiangsu province cooperative medical and biological engineering Limited liability company; first-level RO ultrafiltration water (sodium hypochlorite is added, and the content of free chlorine in the water is controlled to be 2-3ppm for sterilization) for 4 days, and the animals are not fasted and freely drink water before the contamination.

The test method comprises the following steps: dose setting: a test substance group, a negative control group (pure water) and a positive control group (60mg/kg cyclophosphamide) were set. Based on the preliminary test results, the final concentrations of the test substance groups were set to 1000mg/kg, 2000mg/kg, and 5000 mg/kg.

The administration mode comprises the following steps: performing oral gavage; administration volume: 20 mL/kg. BW.

The test steps are as follows: the animal is infected by oral gavage for 30h, i.e. the interval between two times of infection is 24h, and the material is taken 6h after the second time of infection. The animals were sacrificed by cervical dislocation and the femurs were removed. The muscles were stripped off and the blood stain was wiped off. Both ends of the femur were cut to expose the medullary cavity. Sucking 0.1mL calf serum by using a syringe, washing a conventional smear of a washing liquid for a marrow cavity, and drying in the air or drying by hot air. The dried smear was fixed in methanol for 10 min. Staining with Jiemsa application liquid for 15min, washing with clear water, and air drying. The positive and negative control groups were operated in the same manner as the test group. Selecting the area with uniform and complete cell distribution and proper coloring. The number of PCEs (pleochromophilic erythrocytes) containing micronuclei was counted under an oil lens. PCE is grayish blue and NCE (mature red blood cells) is pink. Each animal was counted for the number of micronuclei contained in 2000 PCEs. Micronucleus cell rate (MN ‰) refers to the number of PCEs containing micronuclei, expressed in thousandths. There are 2 or more micro-cores present in a1 PCE, still counting by 1. The micronucleus rates between groups are compared using the poisson distribution u test or other suitable significance test method.

And (3) test results: the test result shows that compared with the negative control group, the number of the polyblast picostaining cells containing micronucleus in the bone marrow of the mice in the positive control group is obviously increased (P <0.05), and compared with the negative control group, the micronucleus rate of the polyblast picostaining cells in each test object group has no statistical significance (P > 0.05).

Under the test condition, the test result of the micronucleus test of the mammalian erythrocytes in the tested object is negative, and the sample is a non-mutagenic agent. The filtrate has good toxicological safety according to a plurality of detection results of acute toxicity detection (no toxicity in acute oral toxicity test detection, micro toxicity in acute transdermal toxicity test detection), skin irritation/corrosivity, skin sensitization, skin phototoxicity, genetic toxicity and the like.

TABLE 19 statistical results of micronucleus rates for micronucleus test

Note: compared with the negative control group, P is less than 0.05. Micronucleus rate and PCE/NCE were counted in mice as mean. + -. standard deviation.

e. Patch test on human skin

According to the method for testing human body safety in the cosmetic safety specification 2015 edition, the lysate filtrate is subjected to a human body skin patch test, and an ingel detection technology service (Shanghai) limited company is entrusted to complete and provide a detection report.

Totally 33 subjects are selected for testing, and the test results show that 32 subjects have no skin reaction after 0.5h, 24h and 48h after the test substance spot tester is removed, the scoring grades are all 0; the grade of 1 test subject is 124 h after the test subject spot tester is removed, and the specific experimental results are shown in the following table 20.

TABLE 20 lysate filtrate skin occlusion Patch test results

TABLE 21 skin closed patch test skin response grading Standard

According to the regulations in the cosmetic technical assessment points, the number of people with grade 1 adverse skin reactions in 30 subjects is not more than 5 (5 are not included), and the specific grading standards are shown in the table 21. The results show that the test results meet the specified safety requirements, and therefore, the lysate filtrate prepared by the method is considered to be safe for human body application.

In conclusion, the pichia pastoris fermentation lysate filtrate prepared by the method is subjected to heavy metal content, methanol content, microbial limit, safe toxicology and human skin patch test detection according to a method in the technical specification of cosmetic safety, and residual DNA detection is carried out at the same time, so that the safety of the pichia pastoris fermentation lysate filtrate meets the requirement of being applied to cosmetics.

Example 2: efficacy testing of fermentation lysate filtrates

In this example, using the lysate filtrate prepared in example 1 as an example, a number of efficacy evaluations were performed.

First, detection of soothing efficacy

The epidermis is used as a tissue for separating an organism from the external environment, besides being used as a physical barrier, keratinocytes can receive external 'danger signals' to stimulate and convert and transmit to immune cells in the skin for early warning, so that an immune barrier of the skin is formed, and the skin immune barrier function regulation disorder can cause skin inflammatory reaction to cause skin problems and diseases. Some cosmetics or ultraviolet rays with strong skin contact irritation can cause clinical acute injury to skin barrier, so that the skin is dry and red spot is generated. The anionic surfactant Sodium Lauryl Sulfate (SLS) has amphiphilic (hydrophilic and lipophilic) characteristics and can damage the skin barrier, especially the lipid components in the barrier and the cell membrane, after contacting the skin at a relatively high concentration. Thus, this experiment uses an in vitro recombinant epidermal model

For the research object, SLS stimulation is adopted to cause a damage model, the lysate filtrate obtained in example 1 is processed, the related inflammatory factors and the tissue morphology change are used as indexes, the soothing effect of the lysate filtrate prepared by the invention is evaluated by detecting the changes of the tissue morphology, the content of inflammatory mediators PGE2 and the content of capsaicin receptor TRPV-1, and Shaanxi Boxi general detection science and technology Limited company is entrusted to detect and provide a detection report.

The experimental protocol is shown in table 22 below.

TABLE 22 lysate filtrate soothing efficacy detection grouping and detection method

(1) The model was transferred to a 6-well plate (0.9 mL of model culture solution was added in advance) according to the test protocol of table 22 above, and the test group number was marked on the 6-well plate.

(2) NC and PC group surface adding 25 u L concentration of 0.2% SLS solution, sample group surface adding 12.5 u L concentration of 0.4% SLS solution and 12.5 u L corresponding concentration of sample working solution (i.e. the lysate filtrate prepared by the invention), sample evenly distributed on the model surface, CO placed ₂ Incubator (37 ℃, 5% CO) ₂ ) And (4) performing medium incubation for 24 h.

(3) After the incubation is finished, the test object remained on the surface of the model is washed by sterile PBS solution, and the residual liquid inside and outside the model is lightly wiped off by a sterile cotton swab.

(4) Inflammatory mediator (PGE2) test: after the administration and incubation for 24h, 3D epidermal skin model culture solution is collected in an EP tube, and after the collection, samples for ELISA detection are placed in a refrigerator at-80 ℃ for freezing storage, and detection analysis is carried out according to the operation instruction of a PGE2 ELISA kit.

(5) And (3) testing the tissue morphology: the model for tissue morphology detection (i.e., epidermal model treated according to table 22) was fixed with 4% paraformaldehyde for 24H, and the model was cut out, examined for H & E staining, photographed under a microscope, and photographed and analyzed.

(6) Capsaicin receptor (TRPV1) test: the model for detection (i.e., the epidermis model treated according to table 22) was fixed with 4% paraformaldehyde for 24 hours, and after fixation, the model was cut out from the ring, subjected to immunofluorescence detection of capsaicin receptor (TRPV1), observed by taking a photograph under a microscope, and photographed and analyzed.

And (4) result statistical analysis: plotting by using GraphPad Prism, and expressing the result as Mean + -SD; the comparison among the groups adopts t-test statistical analysis; statistical analysis was two-tailed. P <0.05 was considered to have a significant difference, and P <0.01 was considered to have a very significant difference.

Analyzing the detection result of the tissue morphology:

the tissue morphology test result is shown in fig. 7, fig. 7 is a tissue morphology diagram under a microscope after H & E staining is performed on the epidermis model after the filtrate culture is finished, wherein fig. 7A is a blank control, 7B is a negative control, namely an SLS treatment group, 7C is a positive control, namely SLS and 0.01% dexamethasone are simultaneously added, and 7D is an experimental group, namely SLS and 2% filtrate are simultaneously added.

The tissue morphology is the microscopic physiological structural analysis of the tissue after HE staining, the change condition of the skin barrier under different treatment conditions can be seen through the model histological morphology, when the skin barrier is loosened and the thickness of the living cell layer is reduced after the injury is caused by SLS stimulation, and whether the injury has the effect of improving the injury is judged through the morphological change after the treatment by adding active substances. The tissue morphology detection result shows that compared with a blank group, the number of negative group model cells after SLS stimulation is reduced, the arrangement is disordered, the stratum corneum is loose, and the vacuole phenomenon appears, which indicates that the stimulation condition of the test is effective. Compared with a negative group, the loose and vacuole conditions of the viable cuticle of the positive control group model are obviously improved, and the positive control detection is proved to be effective. Compared with the negative group, the degree of stratum corneum looseness and vacuole are obviously improved after the 2% pichia pastoris fermentation lysate filtrate treatment, which shows that the 2% filtrate under in vitro conditions is beneficial to relieving the stimulation reaction caused by SLS chemical stimulation and improving the epidermal tissue damage caused by stimulation.

Analysis of PGE2 test results:

the results of the PGE2 test are shown in table 23, and the histogram of the results of the PGE2 content test is shown in fig. 8.

TABLE 23 summary of the results of the PGE2 assay in the lysate filtrates

Remarking: when the statistical analysis is carried out by using the t-test method, compared with the BC group, the significance is represented by #, the P-value <0.05 is represented by #, and the P-value <0.01 is represented by # #; significance was expressed as P-value <0.05 and P-value <0.01 compared to the NC group.

The experimental result shows that compared with the BC group, the secretion level of PGE2 in the NC group is remarkably increased, which indicates that the SLS stimulation can remarkably improve the secretion amount of PGE2, and the SLS stimulation condition is effective in the test. Compared with the NC group, the PGE2 secretion level of the PC group is extremely obviously reduced, which indicates that the positive control detection is effective. The levels of PGE2 secretion were very significantly reduced for the 2% pichia pastoris fermentation lysate filtrate compared to the NC group.

Prostaglandins play a very important role in the inflammatory response, the best known of which is PGE 2. PGE2 is also the PG currently known to be most synthesized and most widely distributed in mammals. The primary source of PG in skin is keratinocytes, the PG predominantly synthesized by keratinocytes including PGE2, PGF2a, and PGD 2.

The inflammatory response of the skin is often accompanied by one to several important clinical features: erythema, swelling, fever or increased local skin temperature, pain, itching, and may be accompanied in part by dandruff. The four manifestations of the inflammatory reaction on the skin (red, swelling, heat, pain) are closely related to PGE 2: PGE2 has vasodilatory and vasodilatory effects, manifested as flushing skin; under the synergistic effect of histamine and kinin, the vascular permeability is increased, so that local red and swelling tissues can be caused, local fever can be caused, PGE2 entering blood can be combined with a receptor of hypothalamus to cause the change of body temperature center, and then general fever is caused; in addition, PGE2 may stimulate nerve endings to cause pain. During the course of trauma, PGE2 is involved as an inflammatory mediator in the development and progression of the inflammatory response through multiple pathways. PGE2 is a potent vasodilator, slowing blood flow and promoting leukocyte adhesion and adhesion. In addition, PGE2, as a pro-inflammatory mediator, may also mediate the production of other inflammatory mediators and inflammatory factors such as IL-6. Experiments show that the 2% pichia pastoris fermentation lysate filtrate can obviously reduce the secretion level of PGE2, and the fermentation lysate filtrate can relieve skin inflammatory reaction by mediating the secretion of inflammatory mediators PGE2, such as reducing the vascular permeability in the skin, thereby relieving the generation of the skin redness phenomenon.

Analysis of TRPV1 test results:

the result of the TTRPV1 immunofluorescence assay shows that compared with the BC group, the fluorescence intensity of the NC group model TRPV1 is remarkably increased, which indicates that the stimulation condition of the test is effective. Compared with the NC group, the fluorescence intensity of the TRPV1 of the PC group model is obviously reduced, which indicates that the positive control detection is effective. Compared with the NC group, the fluorescence intensity of the pichia pastoris fermentation lysate filtrate treatment group model TRPV1 with a sample concentration of 2% was significantly reduced. The TRPV1 immunofluorescence detection results are shown in FIG. 9, the TRPV1 data results are shown in Table 24, and the TRPV1 protein relative IOD value histogram is shown in FIG. 10.

TABLE 24 TRPV1 data results summary Table

The experimental data show that compared with the BC group, the TRPV1 protein content of the NC group is obviously increased, which indicates that the stimulation condition of the test is effective. Compared with the NC group, the TRPV1 protein content of the PC group is obviously reduced, which indicates that the positive control detection is effective. Compared with the NC group, the TRPV1 protein content after the action of 2% Pichia pastoris fermentation lysate filtrate is obviously reduced. The filtrate can play a role in relieving inflammation through a mechanism of inhibiting the expression of the TRPV1 protein.

Transient receptor potential vanilloid type 1 (TRPV1), also known as capsaicin receptor, is an important nociceptor expressed on peripheral primary afferent neurons and is involved in the formation of acute inflammatory pain sensitivity. Is Ca capable of efficiently mediating ²⁺ The cation channel that flows in is widely distributed in cells such as neurons, immune cells, various organ epithelial cells, and keratinocytes. It was found that TRPV1 channel can be activated by a variety of stimuli, such as various physical and chemical stimuli, including high temperature (temperature 43 deg.C or higher), low pH (acidic environment), voltage, osmolarity, endogenous and exogenous vanilloids, such as Capsicum annuumPlain, etc. When TRPV1 is activated, calcium ion channel is opened, calcium ion flows in, and calcium ion concentration in cytoplasm is increased, which causes neuron and its fiber to release neuropeptides such as substance P, neurokinin A, calcitonin gene related peptide, vasoactive intestinal peptide and excitatory amino acids such as glutamic acid and aspartic acid. On the skin, TRPV1 is expressed in cutaneous sensory nerves, keratinocytes and mast cells, and activated TRP channels mediate not only temperature sensing and regulation but also take part in the onset of itching pain and inflammation of cutaneous nerve origin, and thus TRP channels have become important molecular targets for the treatment of inflammatory skin diseases and itching. Therefore, the test selects TRPV1 as a target molecule, and the relieving effect of the filtrate on inflammation is evaluated by measuring the change of the expression level of the TRPV1 under the action of chemical stimulation on an epidermal model.

Second, detection of ultraviolet absorption effect

The fermentation lysate filtrate obtained by the invention and the dilution liquid with different times (pure water dilution) are subjected to ultraviolet region (200- ^TM LUX multifunction microplate reader-VL 0L0TD0, seimer feishell science and technology (china), and the absorption rate was calculated as the transmittance, and the result is shown in fig. 11.

The ultraviolet light is generally divided into UVC region (200-280nm), UVB region (280-320nm) and UVA region (320-400 nm). 98 percent of ultraviolet rays reaching the earth surface are UVA, have the strongest penetrability and can directly reach the dermis layer, and are the main cause of skin tanning; in addition, only less than 2% of UVB is the main cause of sunburn and erythema, while UVC is absorbed by the ozone layer generally, but short-wave ultraviolet rays have the greatest harm to human bodies, and long-term irradiation can cause skin cancer. The test results show that the fermentation lysate filtrate of the present invention has an average absorbance in the UVC region of approximately 100.00%, an average absorbance in the UVB region of > 95%, and an average absorbance in the UVA region of > 80%.

With the increase of dilution times, the ultraviolet absorption capacity of the diluted lysate filtrate is also reduced, the absorption rate of the filtrate with the concentration of 50% in the UVC region is not obviously reduced, but the absorption rate of the filtrate in the UVB region and the UVA region is obviously reduced, but is more than 40%; a lysate filtrate at a concentration of 25% with an average absorbance in the UVC region of > 90%, an average absorbance in the UVB region decreasing with increasing uv wavelength from 89% to 65%, and an average absorbance in the UVA region of > 27%; 10-fold dilution of the lysate filtrate, with an average absorbance of > 90% in the UVC region, an increase in the average absorbance of UVB in the uv wavelength from 87% to 34%, and an average absorbance of > 20% in the UVA region; the effect on ultraviolet absorption gradually decreased with increasing dilution factor, but the average absorption rate in UVC region was > 60% and the average absorption rate in UVB was maintained at about 20% even when diluted to 1%. The most likely 5% dilution to be used in the future can reach 74% -99% average absorption in the UVC region, a decrease in UVB from 67% to 23% with increasing uv wavelength, and > 15% average absorption in the UVA region.

The result shows that the fermentation lysate filtrate can absorb most of medium-short wave ultraviolet rays and effectively resist the damage of the ultraviolet rays to the skin. The sunscreen effect of the lysate filtrate is similar to the effect of common UVB absorbers such as aminobenzoate and derivatives thereof, salicylate and derivatives thereof, cinnamate and camphor derivatives, and the diluted lysate filtrate also has good sunscreen and skin care effects on ultraviolet rays in a certain wavelength range.

Third, regulating microecological result analysis

In the embodiment, the skin model is based on the 3D epidermis

And evaluating the effect of the sample to be tested on the protein level expression quantity of the skin antibacterial peptide and the TOLL-like receptor by detecting the changes of the proteins of HBD1, HBD2, LL-37 and TLR 2. The test entrusts Shaanxi Boxi general detection technology company to detect and issue a detection report.

(1) The test method comprises the following steps:

the model was transferred to 6-well plates (0.9 mL of the corresponding grouping of EpiGrowth medium was added in advance) according to the test protocol of table 25 below, and the 6-well plates were marked with test group numbers.

TABLE 25 lysate filtrate conditioning microecological detection method

Adding working solution with corresponding concentration on the surface of the sample group into a 6-well plate, and placing the plate in CO ₂ Incubator (37 deg.C, 5% CO) ₂ ) And (5) performing medium incubation for 24 h. After the incubation is finished, the test object remained on the surface of the model is washed by a wash bottle filled with sterile PBS solution, and the residual liquid inside and outside the model is wiped off by a sterile cotton swab. The model for immunohistochemical detection was used for immunohistochemical testing. Results statistics were plotted using GraphPad Prism and the results were expressed as Mean ± SD. The comparison between groups was performed by statistical analysis of t-test. Statistical analysis was two-tailed. P is<0.05 considered to have a significant difference, P<0.01 is considered to have a very significant difference.

(2) The result of the detection

analysis of HBD1 results

The staining results of HBD1 protein in each group are shown in FIG. 12, the brown part indicated by red arrow in the picture is HBD1 protein, and the darker brown color indicates the higher content of HBD1 protein. The summary of HBD1 versus IOD values is shown in Table 26, and the histogram of HBD1 Integrated Optical Density (IOD) values is shown in FIG. 13.

TABLE 26 HBD1 summary of relative IOD values

Group of	Relative IOD mean value	SD	P-Value
				BC	1.00	0.10	/
PC(1,25(OH) ₂ VD ₃ )	6.26	0.38	0.000##
				Pichia pastoris fermentation lysate filtrate (0.5%)	3.79	0.36	0.000**
Pichia pastoris fermentation lysate filtrate (1.0%)	5.34	0.16	0.000**

As a result, compared with the BC group, the content of the HBD1 protein in the PC (1, 25-dihydroxy vitamin D3) group is obviously increased, which indicates that the positive control is effective in the test. Compared with the BC group, the HBD1 protein content of the samples, namely-0.5% of the filtrate of the Pichia pastoris fermentation lysate and-1.0% of the filtrate of the Pichia pastoris fermentation lysate, is obviously increased.

Analysis of HBD2 results

The staining results of HBD2 protein in each group are shown in FIG. 14, the brown part indicated by red arrow in the picture is HBD2 protein, and the darker brown color indicates the higher content of HBD2 protein. A summary of HBD2 versus IOD values is shown in table 27, and a histogram of HBD1 Integrated Optical Density (IOD) values is shown in fig. 15.

TABLE 27 HBD2 summary of relative IOD values

Group of	Relative IOD average	SD	P-Value
				BC	1.00	0.10	/
PC(1,25(OH) ₂ VD ₃ )	8.76	0.24	0.000##
				Pichia pastoris fermentation lysate filtrate (0.5%)	4.21	0.20	0.000**
Pichia pastoris fermentation lysate filtrate (1.0%)	6.79	0.23	0.000**

As a result, compared with the BC group, the content of the HBD2 protein in the PC (1, 25-dihydroxy vitamin D3) group is obviously increased, which indicates that the positive control is effective in the test. Compared with the BC group, the HBD2 protein content of the samples of-0.5% of the Pichia pastoris fermentation lysate filtrate and-1.0% of the Pichia pastoris fermentation lysate filtrate is obviously increased.

TLR2 result analysis

The staining results of the TLR2 protein of each group are shown in figure 16, wherein the brown part indicated by a red arrow in the picture is TLR2 protein, and the darker brown color indicates that the content of the TLR2 protein is higher. A summary of TLR2 versus IOD values is shown in table 28, and a histogram of TLR2 Integrated Optical Density (IOD) values is shown in fig. 17.

TABLE 28 TLR2 summary Table of relative IOD values

Group of	Relative IOD mean value	SD	P-Value
				BC	1.00	0.10	/
PC(1,25(OH) ₂ VD ₃ )	1.75	0.24	0.007##
				Pichia pastoris fermentation lysate filtrate (0.5%)	1.38	0.04	0.004**
Pichia pastoris fermentation lysate filtrate (1.0%)	1.73	0.20	0.005**

The result shows that compared with the BC group, the content of the TLR2 protein of the PC (1, 25-dihydroxy vitamin D3) group is obviously increased, which indicates that the positive control detection is effective. Compared with the BC group, the TLR2 protein content can be obviously increased after the action of the Pichia pastoris fermentation lysate filtrate with the concentration of 0.5% and 1.0%.

LL-37 result analysis

The staining results for LL-37 proteins in each group are shown in FIG. 18, where the red arrows indicate a tan portion of LL-37 protein, and a darker brown color indicates a higher level of LL-37 protein. A summary of LL-37 versus IOD values is shown in Table 29, and a histogram of LL-37 Integrated Optical Density (IOD) values is shown in FIG. 19.

TABLE 29 LL-37 relative IOD value summary Table

Group of	Relative IOD mean value	SD	P-Value
				BC	1.00	0.13	/
PC(1,25(OH) ₂ VD ₃ )	1.58	0.02	0.001##
				Pichia pastoris fermentation lysate filtrate (0.5%)	1.62	0.04	0.001**
Pichia pastoris fermentation lysate filtrate (1.0%)	2.30	0.08	0.000**

As a result, compared with the BC group, the LL-37 protein content of the PC (1, 25-dihydroxy vitamin D3) group is obviously increased, which indicates that the positive control detection is effective. Both 0.5% and 1.0% concentrations of pichia pastoris fermentation lysate filtrates resulted in a significant increase in LL-37 protein content compared to the BC group.

The skin micro-ecology is an ecosystem which is composed of various microorganisms such as bacteria, fungi, viruses, mites and arthropods, tissues on the surface of the skin, various secretions of cells, micro-environments and the like. The interaction of skin microorganisms, hosts and the external environment form the skin micro-ecological balance. Research shows that keratinocytes directly kill the alien invasive microbial flora by expressing Antimicrobial polypeptides (AMPs), and provide a life-dependent and friendly place for resident bacteria. The resident bacteria can directly release AMPs and act on pathogenic microorganisms. For example, Staphylococcus epidermidis can secrete antibacterial peptides such as defensins, bacteriocins, LL-37, REG3A, and the like. The resident bacteria also regulate the host immune response by secreting AMPs. Antimicrobial peptides present in the skin in humans belong mainly to two major families: defensins and cathelicidins. Defensins are classified into alpha-defensins and beta-defensins according to their structures, with HBD1 and HBD2 belonging to the beta-defensins. LL-37 is the only human being foundCathelicidin-like antimicrobial peptides in vivo. On the other hand, pathogenic microorganisms can be eliminated by activating various pattern recognition receptors such as TLR2 (Toll-like receptor 2), recognizing exogenous microorganisms, and activating immune response. This experiment is based on the 3D epidermis skin model

Compared with a control group, the content of HBD1, HBD2, TLR2 and LL-37 protein of the pichia pastoris fermentation lysate filtrate is obviously increased at the concentration of 0.5% and 1.0%, and the expression quantity of related target protein is increased in a positive correlation manner along with the increase of the filtrate concentration, so that the pichia pastoris fermentation lysate filtrate prepared by the method can regulate microecology and improve skin barrier by improving the expression of antibacterial peptide, and further achieves the repairing and protecting effect.

Fourth, oil control efficacy detection

In the embodiment, the oil control effect of the sample to be detected is evaluated by detecting the fat drop content of the sebaceous gland cells after the sample acts on the sebaceous gland cells, and Shanxi Boxi general detection science and technology Limited company is entrusted to detect and issue a detection report.

(1) Experimental methods

In the present example, the oil control efficacy was tested by setting a blank group, a positive control group and a sample group, as shown in table 30 below.

TABLE 30 oil control efficacy test protocol

The detection method is briefly described as follows:

SZ95 cells were seeded at a seeding density of 3.5E/well in 24-well plates, incubators (37 ℃ C., 5% CO) ² ) And incubated overnight.

According to the test scheme in table 29, when the plating rate of the cells in the 24-well plate reaches 60% -70%, the administration is performed in groups, each well is loaded with 1mL, and each group is provided with 3 multiple wells. After completion of the administration, the 24-well plate was placed in an incubator (37 ℃ C., 5% CO) ² ) Culturing for 72h, wherein the culture period is dailyLiquid change and administration.

Dyeing with oil red O: the old solution was aspirated, cells were washed 1 time with PBS, 500. mu.L of oil red O working solution (1. mu.g/mL) was added to each well, stained in the dark for 15min (incubation in incubator at 37 ℃) and washed 2 times with PBS.

And (3) photographing and observing by a fluorescence microscope: at Ex: 450nm-500nm, Em: detecting at 515-560 nm, and taking pictures under 200 times of lens.

And (4) result statistical analysis: the results are expressed as Mean ± SD using GraphPad Prism mapping. The comparison between groups was performed by statistical analysis of t-test. Statistical analysis was two-tailed. P <0.05 was considered to have significant differences, and P <0.01 was considered to have very significant differences.

(2) The result of the detection

The results of staining with lipid droplet synthetic oil red O are shown in fig. 20, and the data of the results of staining with lipid droplet synthetic oil red O are summarized in table 31. A histogram of Integrated Optical Density (IOD) values of lipid droplets is shown in fig. 21.

TABLE 31 summary of the results of fat drop synthetic oil red O staining

Remarking: when statistical analysis was performed by the t-test method, the significance of the PC group and the sample group was expressed as "P-value < 0.05", and "P-value < 0.01", compared to the BC group.

The test result shows that compared with the BC group, the synthesis of lipid droplets in the PC group is obviously reduced, which indicates that the positive control is effective; compared with the BC group, the cell lipid droplet synthesis of the sample Pichia pastoris fermentation lysate filtrate is obviously reduced at the concentration of 0.05% and 0.15%, which shows that the Pichia pastoris fermentation lysate filtrate prepared by the invention has the effect of reducing lipid droplet synthesis at the concentration of 0.05% and 0.15%.

Excessive sebum secretion is a major factor inducing various skin problems such as acne, and sebaceous gland cells are combined into a large amount of oil and secreted to the outside of cells in the form of lipid droplets, which affect cell functions and cause various skin problems, and the amount of the lipid droplets is a main index for measuring the oil control degree. Based on sebaceous gland cells SZ95, the lipid content of the sample pichia pastoris fermentation lysate filtrate was significantly reduced at both 0.05% and 0.15% concentrations compared to the control group; the pichia pastoris fermentation lysate filtrate prepared by the method can achieve the oil control effect by reducing the synthesis of lipid droplets.

Example 3: application example of Pichia pastoris fermentation lysate filtrate of the present invention

In this embodiment, the pichia pastoris fermentation lysate filtrate is used as a raw material to prepare a skin care essence, a mask and a skin care cream so as to more intuitively illustrate the application advantages of the pichia pastoris fermentation lysate filtrate, but the fermentation lysate filtrate is not limited to be used for these products, and the pichia pastoris fermentation lysate filtrate can also be used for preparing other cosmetics.

(1) Skin care essence

Taking the lysate filtrate prepared by 10% wet thallus concentration of the fermentation thallus of the engineering bacteria with preservation number of CGMCC NO.7189 in the publication number CN103102407B as an example, the skin care essence is prepared by the following raw materials in percentage by mass, the sum of the total mass percent is 100%, the component proportions are shown as follows, and the preparation is preferably carried out by adding 5% of the Pichia pastoris fermentation lysate filtrate.

The skin care essence comprises 1-10 wt%, preferably 5 wt% of fermentation lysate filtrate, and further comprises 4 wt% of glycerol, 4 wt% of butanediol, 262 wt% of glycerol polyether, 0.02 wt% of EDTA-2Na, 0.05 wt% of sodium hyaluronate, 0.15 wt% of allantoin, 2 wt% of betaine, 0.25 wt% of xanthan gum, 5 wt% of 1, 2-pentanediol and the balance of deionized water.

The preparation method of the skin care essence comprises the following steps: accurately weighing raw materials of each component; at room temperature, uniformly mixing deionized water, glycerol, butanediol and glyceryl polyether-26, adding allantoin and betaine, dissolving and mixing uniformly, heating to 70-75 ℃, adding sodium hyaluronate and xanthan gum, and stirring until completely dissolved; cooling to below 40 deg.C, adding Pichia pastoris fermentation lysate filtrate, and stirring; filtering with 100 mesh filter cloth, and subpackaging in push type vacuum bottles to obtain skin care essence.

(2) Face pack

Taking the preparation of lysate filtrate with 5% wet cell concentration of the fermentation cells of the engineering bacteria with the preservation number of CGMCC NO.7189 in the publication number CN103102407B as an example, the skin care mask solution is prepared by mixing the following raw materials according to the mass percentage shown in the specification, wherein the sum of the total mass percentage is 100%, the components are shown in blind, and the preparation is preferably performed by adding 5% of the Pichia pastoris fermentation lysate filtrate.

The mask comprises 1-10 wt%, preferably 5 wt% of fermentation lysate filtrate, 8 wt% of glycerin, 0.02 wt% of EDTA-2Na, 0.05 wt% of sodium hyaluronate, 0.1 wt% of xanthan gum, 0.12 wt% of carbomer, a proper amount of pH regulator (preferably 0.12 wt%, generally regulated to be neutral), 4 wt% of 1, 2-pentanediol, 0.5 wt% of 1, 2-hexanediol and the balance of deionized water.

The preparation method comprises the following steps:

accurately weighing raw materials of each component for later use; at room temperature, taking part of deionized water and carbomer, and dispersing and stirring uniformly in a container in advance; taking another container, fully dissolving the rest deionized water, glycerol, EDTA-2Na, sodium hyaluronate, xanthan gum, arginine, 1, 2-pentanediol and 1, 2-hexanediol at 70-75 ℃, and uniformly mixing; cooling to 55-60 deg.C, adding pre-dispersed carbomer into the above mixture, mixing and stirring; cooling to 40-50 deg.C, adding pH regulator, mixing and stirring; cooling to 35-40 deg.C, adding Pichia pastoris fermentation lysate filtrate, and stirring; filtering with 100 mesh filter cloth to obtain facial mask solution, mixing with pure cotton membrane cloth, and packaging in facial mask aluminum foil bag to obtain skin caring facial mask.

(3) Skin-care face cream:

taking the lysate filtrate prepared by 5% wet thallus concentration of the fermentation thallus of the engineering bacteria with the preservation number of CGMCC NO.7189 in the publication number CN103102407B as an example, the skin-care cream is prepared by the following raw materials according to the mass percentage, the sum of the total mass percentage is 100%, the component proportions are as shown in the following, and the preferred addition proportion of the Pichia pastoris fermentation lysate filtrate is 5%.

Included in said cream is a fermentation lysate filtrate, said fermentation lysate filtrate being present in an amount of 1-10 wt%, preferably 5 wt%, noted as phase D; also comprises 5 wt% of glycerol, 0.02 wt% of EDTA disodium, 0.15 wt% of xanthan gum, 0.6 wt% of acryloyl dimethyl taurate/VP copolymer, 0.5 wt% of p-hydroxyacetophenone, 0.5 wt% of 1, 2-hexanediol and the balance of deionized water, and the components are mixed to form phase A; cetearyl glucoside/cetearyl alcohol 3 wt%, caprylic capric triglyceride 4 wt%, hydrogenated polyisobutene 3 wt%, dimethicone 1 wt%, mixed as phase B; the pH regulator is C phase, and is generally adjusted to be neutral.

The preparation method comprises the following steps:

putting the phase A components into a water phase pot, heating to 80-85 ℃, and mixing and stirring uniformly; putting the components of the phase B into a water phase pot, heating to 80-85 ℃, and mixing and stirring uniformly; adding the phase B into the phase A, and homogenizing for 5 minutes; stirring and cooling to 60 ℃, and adding the phase C; cooling to 35-40 deg.C, adding phase D, and stirring to obtain skin care cream.

The obtained product was subjected to a population test experiment:

selecting 90 volunteers (all of which are self-describing skin easily reddens and skin easily irritate) with the age of 20-45, randomly dividing into 3 groups, respectively using the products containing the Pichia pastoris lysate filtrate, namely skin care essence, facial mask and skin care cream, and preparing a sample without adding the filtrate by referring to the formula to serve as a blank control, wherein the essence is used in group A, and is applied to the face after cleaning the face 2 times in the morning and evening; the group B uses facial mask 2 times per week, and is applied to face for 10-15 min; and C, using skin cream, cleaning face 2 times in the morning and evening every day, and then applying the face cream to the face, wherein the left face of a testee uses the essence, the facial mask or the facial cream product, the right face uses a control product without filtrate, the application lasts for 4 weeks, other similar skin care products are not used in the period, and the using effects of the sample on relieving skin redness and skin irritation are self-evaluated on the 28 th day.

The scoring standard is as follows:

the skin condition is not improved, even aggravated, and 0 point is obtained; the skin is reddish, easy to be stimulated, does not improve, but is not aggravated, and the score is 1; the skin is reddish and easy to be slightly improved by stimulation, but the effect is not obvious and is divided into 2 points; the skin is reddish and easy to be stimulated and improved, and the score is 3; the skin is reddish and easy to be stimulated, and is obviously improved, and the score is 4.

Test results the average score of each product was: the skin care essence is 3.25 points, the mask is 3.47 points, and the skin care cream is 3.52 points. And (3) displaying a feedback result: none of the product examples containing pichia pastoris lysate filtrate showed any irritation response, and the phenomenon of facial skin redness was significantly alleviated in volunteer response compared to the blank control. The partial photographs of the face of the subject are shown in fig. 22-24, fig. 22 is a comparison photograph before and after the skin care essence is used, fig. 23 is a comparison photograph before and after the mask is used, and fig. 24 is a comparison photograph before and after the skin care cream is used, and the result analysis shows that the product has better effects on relieving the skin and relieving the skin redness.

Claims

1. A method for preparing a fermentation lysate filtrate, wherein the fermentation lysate filtrate is prepared from a strain obtained by fermentation of Pichia pastoris.

2. The method of claim 1, wherein the pichia pastoris is pichia pastoris that expresses recombinant collagen.

3. The method of claim 1, wherein the Pichia pastoris for expressing recombinant collagen is selected from one or more of the strains having the collection number CGMCC NO.7189, CGMCC NO.14057, CGMCC NO.17147, CGMCC NO.17150, CGMCC NO.17148, CGMCC NO.17149, CGMCC NO.20626, CGMCC NO.20627, CGMCC NO.21891 or CGMCC NO. 21892.

4. The method according to claim 1, wherein the microbial cell is obtained by solid-liquid separation of a fermentation product.

5. The method of claim 4, wherein the method comprises dilution of the cells, high pressure homogeneous lysis, centrifugation and filtration.

6. The method according to claim 5, wherein the cell dilution is a cell suspension diluted with pure water to a mass concentration of 5% to 30% (W/W); preferably, the cell dilution is a cell suspension diluted with pure water to a mass concentration of 10% (W/W).

7. The preparation method according to claim 5, wherein the high pressure homogeneous lysis is a lysis solution obtained by crushing the thallus suspension for 1-3 times at a pressure of >100 MPar; preferably, the high pressure homogeneous lysis is to crush the bacterial suspension at a pressure of 1200 MPar.

8. The method according to claim 5, wherein the centrifugation is performed by centrifuging the lysate at 10000g to 20000g to obtain a supernatant.

9. The method of claim 5, wherein said filtering is performed by filtering said supernatant through a 0.22 μm pore size to obtain a fermentation lysate filtrate.

10. A fermentation lysate filtrate prepared by the method of any one of claims 1-9.

11. The fermentation lysate filtrate of claim 10, wherein the fermentation lysate filtrate comprises total protein, recombinant collagen, polysaccharides, amino acids, and multivitamins.

12. A composition comprising a fermentation lysate filtrate prepared by the preparation method of any one of claims 1-9 or the fermentation lysate filtrate of any one of claims 10-11.

13. The composition of claim 12, wherein the amount of fermentation lysate filtrate in said composition is 1-10 wt%, preferably 5 wt%.

14. Use of a fermentation lysate filtrate prepared by a method according to any one of claims 1 to 9 or a fermentation lysate filtrate according to any one of claims 10 to 11 or a composition according to any one of claims 12 to 13 for anti-allergy, soothing, anti-inflammatory, sun protection, skin micro-ecology regulation, oil control, skin barrier repair cosmetic raw materials or products.

15. The use according to claim 14, wherein the anti-allergic, soothing, anti-inflammatory properties include, among others, alleviation of stratum corneum irritation symptoms, amelioration of epidermal tissue damage; reducing the secretion level of an inflammation medium PGE2, relieving the skin inflammation reaction, and reducing the generation of the skin redness; has the effect of relieving skin irritation through a mechanism of inhibiting the expression of TRPV1 protein.

16. Use according to claim 14, characterized in that the sun protection is manifested by the ability to absorb ultraviolet light.

17. The use of claim 14, wherein the modulation of skin micro-ecology is manifested by a boost in the expression of antimicrobial peptides, a modulation of micro-ecology, a boost in the skin barrier, and a restoration efficacy; under the action of the pichia pastoris fermentation lysate filtrate, the contents of HBD1, HBD2, LL-37 and TLR2 proteins are obviously increased.

18. The use of claim 14, wherein the oil control is manifested by inhibition of skin lipid droplet synthesis.

19. Use of a fermentation lysate filtrate prepared by a preparation method according to any one of claims 1 to 9 or a fermentation lysate filtrate according to any one of claims 10 to 11 or a composition according to any one of claims 12 to 13 in the cosmetic field.

20. Use according to claim 19, characterized in that said cosmetic product comprises anti-allergic, soothing, anti-inflammatory, sun-protecting, skin micro-ecological regulating, oil-controlling, skin barrier repairing cosmetic products.

21. A cosmetic comprising the fermentation lysate filtrate produced by the production method of any one of claims 1 to 9 or the fermentation lysate filtrate of any one of claims 10 to 11 or the composition of any one of claims 12 to 13.

22. The cosmetic according to claim 21, wherein the content of the filtrate of the fermentation lysate in the cosmetic is 1 to 10% by weight, preferably 5% by weight.

23. The cosmetic of claim 21, comprising but not limited to skin care serum, mask, skin care cream.