CN115074393B

CN115074393B - Pichia pastoris fermentation lysate filtrate, preparation method and application

Info

Publication number: CN115074393B
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: 2023-11-21
Anticipated expiration: 2042-08-04
Also published as: WO2024027798A1; CN115074393A

Abstract

The invention relates to a pichia pastoris fermentation lysate filtrate, a preparation method and application, belonging to the technical field of cosmetic raw materials; the invention provides a fermented lysate filtrate prepared by fermenting pichia pastoris expressing recombinant collagen; the fermentation lysate filtrate comprises total proteins, recombinant collagen, polysaccharide, amino acids and multiple vitamins; the invention realizes the preparation of the raw material of cosmetics by the pichia pastoris fermentation strain expressing the recombinant human collagen for the first time, reduces the waste treatment pressure caused by the yeast fermentation strain, and changes waste into valuable; the preparation method is a whole-course online and automatic preparation process, and the preparation process is rapid and efficient; meanwhile, the whole preparation process flow can effectively control the breeding of harmful thalli; the lysate filtrate has the effects of relieving skin, enhancing skin microecological barrier, controlling oil, preventing sunburn 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, signaling pathway responses, maintenance, regulation, and repair of normal physiological functions of cells, tissues, and organs. As important natural biological protein, the protein has the unique functional characteristics of good biocompatibility, bioactivity, degradability and the like, and has wide application prospect in various fields of chemical industry, medicine, food, medical cosmetology, cosmetics and the like. The recombinant human collagen produced by the modern genetic engineering technology has stable property, no disease hidden trouble and immunogenicity of animal-derived collagen, and industrial production is not limited by raw material sources, and is now a hotspot in the field and capital attention of national important development support. At present, the largest large-scale production line for producing collagen by fermentation of domestic recombinant collagen production enterprises reaches 20 tons, and the production scale and the production quantity of the large-scale production line can be continuously enlarged and increased in the future.

When recombinant collagen is expressed in pichia pastoris, secretory expression is adopted, and huge amount of fermentation thalli (20-30% of the volume in a general fermentation tank is fermentation thalli) can be produced as waste to be treated. Waste (waste water, thallus and the like) generated in the yeast fermentation industry (such as beer production and bio-pharmaceuticals) is required to be subjected to innocent treatment, and then can be discharged after meeting relevant standards, and the huge pressure is brought to the environmental protection facility treatment capacity of enterprises due to huge volume. At present, further utilization of fermentation waste thalli has been gradually started, such as preparation of single cell protein (protein content is reduced), fermentation into alcohol (utilization of cellulose generated during fermentation, which has little significance on thalli per se), development into organic fertilizer for agricultural production (but needs to be treated in a certain way and a larger place), production of other biomass energy sources, 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 the various utilization modes, but in general, the treated products have limited added value, little 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 innocent treatment.

Microbial lysate and extract have become hot spots in the related art as cosmetic raw materials. In addition to the above uses, yeast cells can be used as a raw material for cosmetics, which is a treatment mode with high added value. In addition to yeast, the yeast can be directly used as a raw material, and various cosmetic raw materials such as yeast derivatives can be prepared.

The skin microecology is an ecological system composed of various microorganisms such as bacteria, fungi, viruses, mites, arthropods and the like, tissues on the surface of the skin, various secretions of cells, microenvironment and the like. In recent years, with the intensive research, skin micro-ecology has also become a focus of attention in the cosmetic world. In cosmetic applications, micro-ecological skin care has also become an important point in the development of many international brand company products. Related products such as the international brands of yashilan indigo, lankomeg, european lycra, baojie and the like are developed and pushed out successively, and the component of the core raw material Pitera of the product is galactose yeast-like fermentation product filtrate, so that the barrier function of skin can be improved by promoting the expression of the tight junction protein and increasing the transmembrane resistance. The star products "small brown bottle" of the yashi blue indigo and the "small black bottle" of the lanoko are the lysate of the fermentation product of the two-split yeast, and the components are not saccharomycetes but are derived from bifidobacteria, and are metabolites, cytoplasmic components, cell wall components and various organelle fragments obtained by culturing and splitting somatic cells. In addition, all international main stream raw material companies focus on microecological skin care, and various metazoan raw materials such as Relipidium of basf are sequentially pushed out, and the main component is hydrolyzed yeast protein, so that the skin colony balance is recovered; the oxygen 229PF of Dissman is mainly composed of yeast lysate, which can reduce Corynebacterium kluyveri, thereby relieving facial redness.

The skin microecology, the physical barrier of the skin and the skin immunity form a first defense system of the organism together, resist the stimulation of various physical and chemical factors of the outside and maintain the normal function 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 repairing raw materials are mainly used for repairing the physical barrier of the stratum corneum layer surface, and lack of cosmetic raw materials with skin barrier repairing effects, which are developed by taking improvement of skin microecological barrier and enhancement of immune barrier as starting points.

In the development of raw materials for cosmetics with skin barrier repair effect, raw materials derived from pichia pastoris are deficient; in addition, the efficacy of the existing yeast cosmetics is mainly focused on the aspects of moisture preservation, aging resistance, sun protection, whitening and the like, and the raw materials for enhancing the skin microecological barrier, the immune barrier and controlling sebum secretion are also deficient as basic efficacy mechanisms.

In addition, the application of collagen in medical and cosmetic applications is generally accepted, but the pichia pastoris fermentation thalli expressing recombinant collagen is used for preparing cosmetic raw materials, especially fermentation lysate filtrate, and related research and development is blank; the preparation mode and efficacy research of the related products are not disclosed in the disclosure; the use of Pichia pastoris for expression of recombinant human collagen as a viable cosmetic material (especially fermentation lysate filtrate) has not been reported; the recombinant collagen production enterprises have no corresponding product release or patent, research literature publication.

In addition, when the extract or lysate filtrate or other cosmetic raw materials are prepared from yeast, the wall breaking and cracking of the yeast are firstly completed, the pichia pastoris has thicker cell wall (70-200 nm), compact structure, high mechanical strength, long treatment time and low efficiency in the conventional treatment mode, and can possibly lead exogenous substances (enzymes). For producing fermentation cells for large-scale industrial production: the period of producing the fermentation thalli is short, the volume is large, harmful thalli are easy to breed in longer treatment time, and pyrogen substances such as endotoxin and the like are produced; the breaking of the thalli during the large-scale production needs to be simple, stable and efficient, and the introduction of exogenous substances is avoided (simultaneously, the cost is also favorably controlled). In addition, the broken pichia pastoris lysate is mixed with insoluble and soluble components, and a set of effective treatment modes are also required for effective separation.

Disclosure of Invention

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

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

In order to achieve the technical aim, the invention firstly provides a preparation method of fermentation lysate filtrate, wherein the fermentation lysate filtrate is prepared by fermenting pichia pastoris.

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

Further, the pichia pastoris for expressing the recombinant collagen is selected from one or more strains with the preservation numbers 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 thalli is obtained by solid-liquid separation of fermentation products.

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

Further, the thalli is diluted into thalli suspension which is diluted to a mass concentration of 5% -30% (W/W) by pure water; preferably, the bacterial cells are diluted with pure water to a bacterial cell suspension having a mass concentration of 10% (W/W).

The high-pressure homogenizing and cracking is carried out, namely, the thallus suspension is crushed for 1 to 3 times at the pressure of more than 100MPar to obtain a cracking solution; preferably, the high pressure homogenisation is carried out such that the bacterial suspension is broken up at a pressure of 1200 MPar.

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

The filtering is to filter the supernatant with a pore size of 0.22 mu m to obtain fermentation lysate filtrate.

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

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

The fermented lysate filtrate has the effects of resisting allergy, relieving inflammation, preventing sun, regulating skin microecology, controlling oil and repairing skin barrier.

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

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

The invention also provides application of the fermentation lysate filtrate and the composition in raw materials of cosmetics for resisting allergy, relieving, resisting inflammation, preventing sun, regulating skin microecology, controlling oil and repairing skin barrier.

The invention also provides the application of the fermentation lysate filtrate and the composition in the field of cosmetics. The cosmetic comprises antiallergic, soothing, antiinflammatory, sun-screening, skin microecological regulating, oil controlling, and skin barrier repairing products.

Wherein the repairing skin barrier comprises enhancing skin microecological barrier and improving skin immune barrier.

Further, the anti-sensitization, relieving and anti-inflammatory mainly comprises improving and relieving the stimulation symptom of the horny layer and improving the damage of the epidermal tissue; reducing the secretion level of inflammatory mediator PGE2, reducing the skin inflammatory response, such as reducing vascular permeability in the skin, thereby reducing the occurrence of redness of the skin; the effect of relieving skin irritation is achieved by a mechanism of inhibiting the expression of TRPV1 protein.

The sun protection is manifested by an ability to absorb ultraviolet light.

The regulation of skin microecology is realized by improving the expression of the antibacterial peptide and the pattern recognition receptor, regulating the microecology and improving the skin barrier so as to achieve the repairing effect; under the action of the Pichia pastoris fermentation lysate filtrate, the protein contents of 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 cosmetic comprises the fermented lysate filtrate, and the cosmetic comprises, but is not limited to, skin care essence, facial mask and face cream.

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

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

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

The invention also provides a skin care cream, which comprises 1-10wt%, preferably 5wt% of fermentation lysate filtrate, and further comprises glycerin, EDTA disodium, xanthan gum, ammonium acryloyldimethyl taurate/VP copolymer, p-hydroxyacetophenone, 1, 2-hexanediol, cetostearyl glucoside/cetostearyl 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 the pichia pastoris fermentation thalli for expressing the recombinant human collagen for the first time, increases an effective and high-added-value utilization way of the recombinant human collagen fermentation thalli, reduces the waste treatment pressure caused by the yeast fermentation thalli, and changes waste into valuable.

(2) The pichia pastoris fermentation strain for expressing recombinant human collagen in the invention prepares lysate filtrate, contains a certain amount of collagen, and does not need to be additionally added.

(3) The preparation method of the fermentation lysate filtrate is a whole-course online and automatic preparation process, pichia pastoris fermentation thalli is physically crushed and cracked by high-pressure homogenization, solid-liquid separation is carried out after the cracking by using a continuous flow centrifuge, lysate filtrate is obtained after filtration, and the preparation process is rapid and efficient, can be prepared in large scale and does not contain any external component of primers; meanwhile, the whole preparation process flow can realize full-automatic operation, materials are conveyed through a full-automatic online pipeline, sterile filtration and filling are completed in a clean area, and the breeding of harmful bacteria can be effectively controlled.

(4) The lysate filtrate prepared by the invention has no safety problem when applied to cosmetics, and besides the efficacy of the traditional yeast-derived cosmetic raw materials, the lysate filtrate has the following functions: the secretion of the antibacterial peptide is promoted, the microecological balance is regulated, and the effect of the skin microecological barrier is enhanced; by inhibiting inflammatory factor expression, improving skin immune barrier and relieving skin; oil control and sun protection effects; these previously have not seen the effects of the materials of the "Pichia pastoris fermentation lysate filtrate", which opens up its range of applications for the preparation of cosmetics.

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

(6) Besides the effects, the prepared Pichia pastoris fermentation lysate filtrate has the effect of reducing the cell lipid secretion through experiments, 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 chart of a process for preparing a filtrate of a fermentation lysate of the present invention.

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

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

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

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

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

FIG. 7 is a 3D-based epidermis model of an embodimentCulturing and then carrying out H&E histomorphogram under microscope after staining, wherein panel a is blank control, B is negative control, i.e. SLS treated group, C is positive control, i.e. SLS and 0.01% dexamethasone are added simultaneously, D is experimental group, i.e. SLS and 2% filtrate are added simultaneously.

FIG. 8 is a bar graph showing the measurement results of PGE2 content in the examples.

FIG. 9 shows the result of immunofluorescence assay of capsaicin receptor (TRPV 1) in the examples.

FIG. 10 is a bar graph of TRPV1 protein versus IOD value in the examples.

FIG. 11 shows UV light ratios of 200-400nm wavelengths in the UV light range of dilutions of different multiples of the fermentation lysate filtrate obtained according to the present invention.

FIG. 12 is a 3D-based epidermal skin modelHBD1 protein staining results of (b).

FIG. 13 is a 3D-based epidermal skin modelHBD1 protein Integrated Optical Density (IOD) value bar graph.

FIG. 14 is a 3D-based epidermal skin modelHBD2 protein staining results of (b).

FIG. 15 is a 3D-based epidermal skin modelHBD2 protein Integrated Optical Density (IOD) value bar graph.

FIG. 16 is a 3D-based epidermal skin modelResults of TLR2 protein staining.

FIG. 17 is a 3D-based epidermal skin modelHistogram of Integrated Optical Density (IOD) values of TLR2 proteins.

FIG. 18 is a 3D-based epidermal skin modelLL-37 protein staining results of (C).

FIG. 19 is a 3D-based epidermal skin modelLL-37 protein Integral Optical Density (IOD) value bar graph.

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

FIG. 21 is a bar graph of lipid drop Integrated Optical Density (IOD) values.

Fig. 22 is a graph showing the effect of volunteers on using Pichia pastoris-containing fermentation lysate filtrate.

Fig. 23 is a graph showing the effect of volunteers using pichia pastoris-containing fermentation lysate filtrate masks.

Fig. 24 is a graph showing the effect of volunteers on using pichia pastoris-containing fermentation lysate filtrate creams.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the following detailed description of the preferred embodiments of the present invention is provided, but the following embodiments do not limit the scope of the present invention.

In the examples of the present invention, processes are not described in any more detail by conventional experimental methods, and those skilled in the art can understand and easily implement the processes according to the product specification or the basic knowledge of the art, so that the detailed description will not be given.

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

the fermentation lysate preparation raw material strains are all from the applicant units of the invention, all have been subjected to corresponding strain preservation, are dedicated pichia pastoris engineering bacteria for expressing recombinant collagen, and are all preserved in the China general microbiological culture Collection center; the strain comprises a strain with the preservation number of CGMCC No.7189 in the publication number of CN103102407B, the preservation number of CGMCC No.14057 in the publication number of CN107090458A, the preservation number of CGMCC No.17147 in the publication number of CN111004319A, the preservation number of CGMCC No.17150 in the publication number of CN110964099A, the preservation number of CGMCC No.17148 in the publication number of CN110606896B, the preservation number of CGMCC No.17149 in the publication number of CN110747198B, the preservation number of CGMCC No.20626 and the preservation number of CGMCC No.20627 in the publication number of CN113185604B, and the preservation numbers of CGMCC No.21891 and CGMCC No.21892 in the publication number of CN 114106150A. The recombinant human collagen expressed by the engineering bacteria has a 6 XHis-Tag at the amino terminal (N terminal) or the carboxyl terminal (C terminal), which is beneficial to detection.

In addition to the above list, other strains producing collagen or common yeast lysate filtrate added with collagen can achieve the technical effect of the invention, and are all within the protection scope of the invention.

The pichia pastoris engineering bacteria are expressed to produce recombinant human collagen as secretion expression: the protein is translated into endoplasmic reticulum, signal peptide is cut off, and then the protein is processed by Golgi apparatus and secreted outside cell, and the translation, processing and secretion are the last three stages of recombinant human collagen expression production, and when the cell expresses protein in large quantity, these three stages are simultaneously carried out, so that although most of recombinant human collagen is secreted outside cell, 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 fermentation tank is fermentation thalli. After the batch fermentation is finished, bacterial liquid separation of bacterial cells and supernatant of fermentation liquid is carried out (a ceramic membrane or a continuous flow centrifuge is mostly used), bacterial slurry with high bacterial cell concentration is produced, the main components are water and wet bacterial cells, the concentration of the wet bacterial cells can reach about 50% (W/W), and the bacterial 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 the weight of the cell (containing water and not dried) to the weight of the liquid of the suspended cell, and the calculation modes and results are given in the specific cases.

Example 1: preparation of lysate filtrate

The yeast cell wall is thicker, the structure is more complex than that of the bacterial cell wall, the property is stable, the structural and component integrity of the yeast cell wall can be maintained after the protoplast dies or apoptosis, the function of a biomembrane system of the die protoplast is lost, enzymes in the lysosome enter cytoplasm to decompose substances in cytoplasm and nucleus greatly, 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 small molecular substances, and the cell wall is affected little and can still maintain the integrity of the protoplast. This is also a great difference between yeast cells and bacterial and animal cells in cell disruption, i.e. only complete lysis of the cell wall can result in a more complete lysate.

The method of the invention can effectively crush the cell walls of the yeast by selecting a high-pressure homogenization method, which is also a physical crushing mode with high speed, high efficiency and no primer exogenous substances. After disruption, the disrupted solution may be separated from insoluble materials (cell wall insoluble glucan, insoluble proteins, etc.) and soluble materials in the lysate using a high-speed continuous flow centrifuge. Taking fermentation thallus with the preservation number of CGMCC No.7189 engineering bacteria in the publication number CN103102407B as an example, the preparation of lysate filtrate is carried out.

(1) Taking fermented bacterial slurry, centrifuging at 12000rpm for 10min, and measuring the wet bacterial concentration of the wet bacterial/bacterial slurry supernatant by a weighing method.

(2) The bacterial slurry is diluted with pure water to obtain bacterial suspension with a wet bacterial concentration of 5% -30% (W/W), and in this example, a wet bacterial concentration (W/W) of 10% is preferable.

(3) The bacterial suspension is input into a high-pressure homogenizer (AH 30-100 plus, an Ta nanotechnology (Suzhou) Co., ltd.), a cold water system is started for refrigeration, the crushing temperature is controlled to be 4-10 ℃, the crushing is carried out at the pressure of more than 100MPar, the lysate is obtained after crushing for 1-3 times, and the lysate is obtained after crushing for 2 times, preferably 120MPar pressure in the embodiment.

(4) The lysate is fed into a high-speed continuous flow centrifuge (GQ 105B, preferably Shanghai mechanical equipment Co., ltd.), a cold water system is started for refrigeration (the temperature is controlled to be 4-10 ℃), and the supernatant is obtained by centrifugation with 10000g-20000g centrifugal force, preferably 10000g centrifugal force in this example.

(5) In a clean shop, the supernatant was fed into a plate-and-frame filter system (CW 300-10, haining City, innovative filtration equipment and plant), and the supernatant was filtered with a 0.45 μm PP filter membrane and a 0.22 μm PES filter membrane in this order to obtain a lysate filtrate.

(6) Adding a small amount of antiseptic components, selecting whether to dilute according to the requirement, and filling into product packages to obtain the finished product.

In this embodiment, the detection of the preparation effect of the obtained fermentation lysate filtrate and the intermediate product in the preparation process specifically includes:

1. and (3) detecting the thallus cracking effect in the step (3):

the effect of pichia pastoris cell lysis can be detected by microscopic examination, and the gram staining method (the gram staining kit is purchased from Beijing Soy Bao technology Co., ltd.) can be used for microscopic examination, and the principle is similar to gram staining: after the crystal violet primary dyeing and the iodine liquid mordant dyeing, a water-insoluble compound of crystal violet and iodine is formed in the cell wall, the cell wall of yeast is dyed into purple, the cell wall of yeast is thicker and denser than the cell wall of gram-positive bacteria, the mesh is reduced due to water loss during the ethanol decolorization treatment, and the ethanol treatment does not contain lipid, so that gaps are not formed during the ethanol treatment, and the compound of crystal violet and iodine can be firmly remained in the wall, so that the cell wall is still purple under the condition of complete cell wall. If the yeast cell wall is broken, the complex of crystal violet and iodine is dissolved out and decolorized when being treated by ethanol, and the complex is colorless, and then the complex is reddish after being counterstained by red dye such as safranine. After staining by gram stain, the unbroken cell wall is purple, both the broken cell wall cells (including the cell morphology types that remain intact) and the released material from the cell are stained red, and the high pressure homogenization break-up product as a whole forms a uniform red background in the field of view.

The experimental method is briefly described as follows:

yeast cells were counted using a hemocytometer plate prior to disruption, total number C.

After cell disruption, the disruption solution was taken, smeared, and gram staining was performed: the same as ordinary gram staining is required, and the application is uniform and not thick, and the ethanol decolorization time is controlled. The slide is counted by observing with a phase contrast microscope or an oil microscope, and the number of purple cells (n 1) and the number of red cells (n 2) in each view under each observation are counted, and different positions on the slide are replaced to obtain a plurality of different views, and the process is repeated for a plurality of times. Calculating the ratio of a=n1/(n1+n2) of the non-broken cells after wall breaking, and taking an average value;

the crushed liquid after cell crushing is also taken, the supernatant is removed after high-speed centrifugation, pure water with the same volume of diluted bacterial slurry is used for re-suspending and precipitating, gram staining or crystal violet staining is used for more convenient observation, a blood cell counting plate is used for counting (preferably a phase contrast microscope is used), and the total number of yeast cells which are still in a more complete cell form after crushing is counted as C'.

Calculating the wall breaking rate, and carrying out statistical analysis:

d (100%): breaking wall rate; c: cell number before disruption; c': total number of cells after disruption; a: the ratio of non-broken cells after breaking the wall.

When 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. Taking the wall breaking rate of the fermentation thalli with the preservation number of CGMCC NO.7189 engineering bacteria in the publication number CN103102407B as an example under different breaking conditions, the method comprises the following steps: 100mpa (3 times): the wall breaking rate is 95.9%;120MPar (2 times): the wall breaking rate is 97.3 percent; 150MPar (2 times) wall breaking rate 99.4%;200MPar (2 times): the wall breaking rate is 99.5%, and the breaking and cracking effects are shown in figure 2. The cracking requirement of pichia pastoris can be completely met after the 120Mpar is crushed for 2 times.

2. Detection of recombinant human collagen, total protein, polysaccharide, amino acid, vitamins in lysate filtrate obtained by the method in this example

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

SDS-PAGE electrophoresis and WB detection results:

taking the prepared lysate filtrate, preparing lysate filtrate by using blank Pichia pastoris (purchased from Simer Feishi technology (China) Co.) which is not transferred into the expressed collagen gene as blank control, and performing SDS-PAGE electrophoresis and Western Blot detection, wherein the result is as follows:

FIG. 3A is an SDS-PAGE electrophoresis of lysate filtrate prepared at 10% wet cell concentration, with proteins of different sizes distributed from top to bottom; FIG. 3B shows Western Blot results (Anti-His, antibodies were purchased from biological engineering (Shanghai) stock, inc., mouse Anti-His monoclonal antibody, cat# D199987) of lysate filtrate prepared at 10% wet cell concentration, and as can be seen from FIG. 3B (ECL chemiluminescent color development, full-automatic chemiluminescent image analysis system Tanon 5200 synthesizes protein molecular mass standard into an image), 6 XHis tag-resistant bands were detected, the size of the target bands were all consistent with the molecular weight in electrophoresis of recombinant type III human collagen purified lyophilized product expressed in the patent of publication No. CN103102407B, indicating that lysate filtrate contained recombinant type III human collagen, whereas blank control (lysate filtrate prepared by lysis of blank Pichia pastoris that had not been transformed to express collagen gene) was free of any bands.

Elisa assay 5D collagen content assay:

and (3) carrying out Elisa detection on lysate filtrate prepared at 10% wet thallus concentration, simultaneously carrying out Elisa standard curve formulation on purified recombinant type III human collagen freeze-dried sponge, and preparing lysate filtrate by using blank pichia pastoris which is not transferred into expressed collagen genes as a blank control, wherein an indirect Elisa method is used, and the experimental steps are briefly described as follows:

(1) The lysate filtrate (10, 100 times) was diluted in gradient with PBS and added to the ELISA plate, and simultaneously the recombinant type III human collagen purified lyophilized sample expressed in the patent publication No. CN103102407B was dissolved in PBS and diluted in gradient and also added to the ELISA plate and allowed to stand at 4℃overnight.

(2) The liquid in the ELISA plate was discarded, and a 5% skim milk powder solution (available from Biotechnology (Shanghai) Co., ltd., in terms of mass to volume ratio, such as 5g of skim milk powder dissolved in 100mL of PBS) diluted with PBS was blocked at 37℃for 2 hours.

(3) After the liquid in the ELISA plate was discarded and washed twice with PBS, a 5% nonfat milk powder solution was added to dilute a primary antibody (Anti-His, antibodies were purchased from biological engineering (Shanghai) Co., ltd., mouse Anti-His monoclonal antibody, cat# D199987) solution at a dilution ratio of 1:5000 Standing at 37 ℃ for 1 hour.

(4) After the liquid in the ELISA plate is discarded and washed twice by PBS, a 5% skimmed milk powder solution is added to dilute HRP-labeled secondary antibody (secondary antibody matched with mouse anti-His monoclonal antibody, product number D199987, purchased from biological engineering (Shanghai) Co., ltd.) solution, wherein the dilution ratio is 1:2000 Standing at 37 ℃ for 1 hour.

(5) Discarding the liquid in the ELISA plate, washing twice with PBS, adding TMB color development solution (TMB color development P0209-100mL, biyunshan) and stopping solution (P0215-100 mL, biyunshan) after the color appears obvious blue, and measuring with an ELISA plate instrument (Varioskan ^TM LUX multifunctional enzyme labeling instrument-VL 0L0TD0, simer Feier technology (China) Co., ltd.) reads absorbance at 450 nm.

(6) The GraphPad Prism 5 fits a standard curve, performs data processing, and calculates the concentration of 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)))

where Y is the protein concentration and X is the absorbance at 450nm at which the sample was detected.

Lysate filtrate Elisa detection results (absorbance at 450 nm) at different concentrations were as follows:

TABLE 1 detection of absorbance at 450nm by Elisa of lysate filtrate of different concentrations

Dilution factor	/	10	100
				Absorbance value of lysate filtrate	0.2379	0.4120	0.1375
Blank control absorbance value	0.0025	0.0036	0.0037

From the curve fitting analysis, a preferred detection range is an OD450 range of between about 0.25 and 1.25. Among the experimental groups, ddH can be effectively calculated ₂ O is the OD450 value of the Elisa experiment after 10 times dilution of the supernatant of the crushing liquid centrifugation. The concentration of recombinant type III human collagen in the lysate filtrate was 11.44. Mu.g/mL. In general, the recombinant collagen content of the lysate filtrate prepared at 10% wet cell concentration is about 10-15 μg/mL, and naturally, the recombinant human collagen content is increased along with the increase of the wet cell concentration. The OD450 of the lysate filtrate prepared by the blank Pichia pastoris which is not transferred to the expressed collagen gene is basically outside the lower limit of the detection effective range, and no collagen is detected.

c. Detection of total protein content in lysate filtrate

Experimental methods refer to the fifth method in the protein content determination method of the pharmacopoeia of the people's republic of China (2020 edition): the total protein content was measured by the Coomassie Brilliant blue method (Bradford method). The lysate filtrate is prepared by fermenting thallus of CGMCC No.7189 engineering bacteria in the patent with the publication number of CN103102407B and 0.2-20% wet thallus concentration, and the Bradford protein concentration measuring reagent is purchased from Biyun Tian biology (P0006), and the experimental steps are as follows:

(1) Protein standard (5 mg/mL BSA) was diluted with pure water to 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5mg/mL.

(2) mu.L of protein standard of different concentrations was added to the protein standard wells of the 96-well plate, while 5. Mu.L of lysate filtrate sample was taken into the sample wells of the 96-well plate.

(3) 250 μ L G of 250 staining solution was added to each well.

(4) With a microplate reader (Varioskan) ^TM LUX multifunctional enzyme-labeled instrument-VL 0L0TD0, simer Feier technology (China) Co., ltd.) for measuring absorbance at 595 nm.

(5) The data are processed to calculate the protein concentration in the sample based on the standard curve and the sample volume used. The standard curve and curve equation are shown in fig. 5. The total protein content (mg/mL) of the lysate filtrate prepared at 0.2% -20% wet cell concentration is shown in Table 2:

TABLE 2 Total protein content (mg/mL) in lysate filtrate prepared at wet cell concentration of 0.2% -20%

Slope of standard curve in Bradford method (R ² )>0.98, with the increase of wet thallus concentration in preparing lysate filtrate, the total protein content in lysate filtrate increases, 0.192-26.9 mg/mL, when the concentration of wet thallus is above 10%, the total protein content in lysate filtrate is greater than 10mg/mL>1 percent, the highest content can reach more than 25mg/mL (2.5 percent), and the total protein content in the filtrate produced by multiple times of cracking with the concentration of 10 percent bacteria is generally 10 to 15mg/mL. Other methods in protein content measurement methods in pharmacopoeia of the people's republic of China (2020 edition) such as BCA method, biuret method and Kjeldahl nitrogen method are also selected according to actual conditions to detect the total protein content.

d. Detection of polysaccharide content in lysate filtrate

The reference documents Jin Fengci and Min Lijing, the phenol-sulfuric acid method for determining the sugar content in lily polysaccharide, the scientific information, 2011 (10)' pages 127-128, the lysate filtrate is prepared by the concentration of 10% -20% wet thalli of fermentation thalli of CGMCC No.7189 engineering bacteria in the patent of publication No. CN103102407B, and the polysaccharide content is detected.

The experimental procedure is briefly described as follows:

(1) Preparation of mannose standard (0.1 mg/mL): precisely weighing 500mg mannose dried to constant weight at 105 ℃, placing in a 500mL volumetric flask, adding water for dissolution, diluting to a scale, shaking uniformly, and obtaining 1mg/mL stock solution for later use. The 10mL of the above solution was removed by a 10mL pipette, and the volume was set to 100mL, to obtain a mannose standard solution of 0.1 mg/mL.

(2) Preparation of 85% phenol solution: heating phenol to 45 ℃ by using a water bath kettle, dissolving 85mL of phenol solution in 15mL of water, and obtaining 85% phenol solution after the phenol solution is completely dissolved. Placing into a refrigerator with the temperature of 4 ℃ and storing in a dark place. Can be used for a long time.

Preparation of 5% phenol solution: taking out 85% of phenol, heating and dissolving in 45 degrees, taking 1mL of solution, and dissolving in 16mL of pure water to obtain 5% of phenol solution. Stored in dark place and used before being prepared.

(3) Standard sample preparation and standard curve drawing: mannose standard solution (0.1 mg/mL) was accurately aspirated and formulated as in Table 3. 0.5mL of phenol solution and 2.5mL of concentrated sulfuric acid (rapid, precise addition) were added, mixed for 20 seconds, left to stand in a water bath at 80℃for 30 minutes, cooled, and absorbance was measured at 490 nm. And drawing a standard curve by taking a blank (blank) reference experiment and taking an absorbance value as an ordinate and mannose concentration as an abscissa, and establishing a standard curve regression equation.

TABLE 3 preparation and detection of Standard samples

(4) And detecting the sample to be detected, wherein the method is the same as that described above.

(5) Calculation of polysaccharide content:

the absorbance of the measured sample is taken into the standard curve equation. Obtaining the corresponding polysaccharide solubility, and dividing the polysaccharide solubility in the sample lysate filtrate to obtain the polysaccharide content.

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

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

the polysaccharide content (mg/mL) in the lysate filtrate prepared was as follows:

when the wet thallus concentration (W/W) of the lysate filtrate is 10%, the polysaccharide content reaches 1.15mg/mL; when the concentration of the wet thalli is 15%, the polysaccharide content reaches 1.45mg/mL; when the concentration of the wet bacterial cells is 20%, the polysaccharide content reaches 1.59mg/mL. The polysaccharide content in the lysate filtrate is generally 1-1.6mg/mL as can be seen from the detection results when other concentrations are combined.

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

The lysate filtrate is prepared with 10% wet thallus concentration of fermentation thallus of CGMCC No.7189 engineering bacteria in publication No. CN 103102407B. The method comprises the steps of determining nicotinic acid and nicotinamide in GB 5009.89-2016 food safety national standard food, determining vitamin B2 in GB 5009.85-2016 food safety national standard food, determining amino acid in GB 5009.124-2016 food safety national standard food, determining choline in GB 5413.20-2013 food safety national standard infant food and dairy product, determining inositol in GB 5009.270-2016 food safety national standard food, determining vitamin B6 in GB 5009.154-2016 food safety national standard food, determining folic acid in GB 5009.210-2016 food safety national standard food, determining folic acid in GB 5009.211-2014 food safety national standard food, determining biotin in GB 5009.259-2016 food safety national standard food, detecting the content of amino acid in lysate filtrate, entrusting detection completion of Shanghai's quality of auspice technical service and detecting report.

The test results are shown in Table 4.

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

The result shows that the lysate prepared at 10% wet thallus concentration contains multiple vitamin B group and amino acids, the total amino acid amount can reach about 0.82%, and the lysate can provide abundant active substance components for cosmetics as raw materials.

3. Safety detection of the lysate filtrate obtained

a. Detection of residual DNA

The DNA in the lysate filtrate is mainly Pichia pastoris genome DNA residue, and the DNA in the lysate filtrate prepared at the concentration of 10% bacteria is detected by a PCR-fluorescent probe method, and the detection report is issued to Huzhou Shen Ke biotechnology limited company. The basic process is briefly described below, and related reagents in the experimental process are produced by Huzhou Shen Ke biotechnology limited company or are commercially available:

(1) Diluting the filtrate sample containing lysate, and purifying by a magnetic bead method;

(2) In vitro amplification of specific fragments of DNA in lysate filtrate samples with primers and probes specific for pichia pastoris (pichia pastoris residual DNA detection kit (PCR-fluorescent probe method), purchased from SK030205P100, shen Ke biotechnology limited, lake) (quantitative PCR instrument 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 fluorescent signal can be detected by using a fluorescent quantitative PCR instrument, and the fluorescent signal data is analyzed by detection software of the PCR instrument.

The detection results are as follows:

TABLE 5 detection of lysate filtrate residual DNA

In the table of the present invention,

detecting the average value: average value of the detected values. CV: CV (Coefficient of Variance) ratio of standard deviation to mean, which is used statistically to characterize the coefficient of variation, the meaning of both CVs is the same. Adding a scalar: refers to the amount of standard added. Adding a mark detection mean value: average values of detection values of the standard and the reference are added.

As a result, the DNA content in the lysate filtrate prepared by the method is detected to be 31.4 mug/mL, which is lower than the DNA residue required in the biopharmaceuticals and is in a safe range.

b. Detection of heavy metals, methanol content and the like

According to the first method related detection method of chapter 1.6 and chapter 2.22 of cosmetic safety technical Specification 2015, the heavy metal and methanol content in lysate filtrate is detected, and the detection report is issued to the Suzhou customs comprehensive technical center.

The test results are shown in Table 6 below:

TABLE 6 detection results of heavy metals and methanol in lysate filtrate

Project	Results unit	Technical requirements	Detection result	Method inspectionConcentration of the product	Single item judgment
						Lead	mg/kg	≤10	＜0.03	0.03	Compliance with
Arsenic (As)	mg/kg	≤2	＜0.001	0.001	Compliance with
						Mercury	mg/kg	≤1	＜0.001	0.001	Compliance with
Cadmium (Cd)	mg/kg	≤5	＜0.001	0.001	Compliance with
						Methanol	mg/kg	≤2000	＜25	25	Compliance 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 an actual dilution effect can be generated in the preparation and use of an actual product, and the safety problem does not exist.

c. Detection of microbial limitation

According to the microbial limit detection method in 2015 edition of cosmetic safety technical Specification, the detection of microbial limit is carried out on lysate filtrate, the detection report is entrusted to the Suzhou customs comprehensive technical center, 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 meet the requirements.

TABLE 7 microbial detection results of lysate filtrate

d. Toxicology detection

And according to the toxicology test method of the 2015 edition of the cosmetic safety technical Specification, the toxicology safety evaluation is carried out on lysate filtrate, and the Ningbo customs technical center is entrusted to complete the detection and give a detection report.

The statistics of the detection results are shown in the following table 8, and the experimental methods listed in the table are all conventional methods specified in the technical Specification of cosmetic safety, and the experiments are all completed in the technical center of Ningbo customs. The result shows that the toxicology detection results of the lysate filtrate prepared by the method meet the requirements, and the safety is ensured.

TABLE 8 results of the toxicological safety of lysate filtrate

(1) Acute oral toxicity test

Sample treatment: weighing 5.0806g of sample (namely lysate filtrate prepared by the invention), placing the sample into a beaker, taking a small amount of pure water, uniformly mixing the sample, transferring the sample into a 20mL volumetric flask, flushing the beaker with the small amount of pure water for many times, transferring the beaker into the volumetric flask, adding the pure water to fix the volume to a scale mark, fully shaking the sample, transferring the sample into a sample tube for identification for later use, and preparing the sample for use. The lavage volume was 2.0mL/100 g.BW.

Experimental animals: 10 SPF grade ICR mice (Shanghai JieJie laboratory animal Co., ltd.) of 6 weeks old, female and male half (female animals are not pregnant and have not born), weight controlled at 18.5 g-21.4 g (the body weight of the homoplasmic individuals does not exceed + -20% of the average)

Animals are adapted to 8 days (Co 60 irradiation of mouse feed, jiangsu province cooperative medical bioengineering Limited liability company; first-class RO ultrafiltration water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) in a barrier environment animal house (barrier environment, 23.0-23.8 ℃ C. And relative humidity of 40.8-59.7%) before the test, and the animals are directly supplied for free drinking through a drinking nozzle, and are fasted overnight before the test.

After the test is started, a primary limit method is adopted, and the gastric lavage dosage is 5080.6mg/kg. The subjects were continuously fasted for 3 hours after exposure to the toxicant, and the symptoms or behavior changes were observed daily, and weighed once a week. The toxication and death of the animals during the toxication process and observation period were observed and recorded, and after the observation period was completed, the surviving animals were sacrificed and subjected to general dissection.

Detection result:

the experimental animals do not see any toxic symptoms and toxic death within 14 days after the infection; the body weight of the male and female animals is not abnormal. At the end of the experimental observation, no abnormal changes were seen in the gross anatomic examination of the test animals. LD50 > 5080.6mg/kg, detailed results are as follows:

TABLE 9 acute oral toxicity test results

It can be seen that under the test conditions, the acute oral LD50 of the test sample to ICR mice is > 5000mg/kg. The samples were classified according to acute oral toxicity, which was of virtually non-toxic grade.

(2) Acute transdermal toxicity test

Sample treatment: the lysate filtrate from the base sample was used for testing.

Experimental animals: 10 SPF-class SD rats (Shanghai Jieshijie laboratory animal Co., ltd.) of 8 weeks old, and the weight of each half of the female animals (female animals were not pregnant and had not born) was controlled at 201.2g to 218.2g.

The animals are adapted to 7 days (Co 60 irradiation of mouse feed, jiangsu province cooperative medical bioengineering Limited liability company) in a barrier environment animal house (barrier environment, 22.0-22.9 ℃ C. And relative humidity of 45.8-55.5%) before the test, and the primary RO ultrafiltration water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) is directly supplied to the animals for free drinking through a drinking nozzle, so that the animals do not fasted before the contamination and are free to drink.

After the test is started, a one-time limit method is adopted, and the contamination dose is 2500mg/kg. Animals were kept in single cages during the trial. The weight range before exposure is 201.2 g-218.2 g, and the body surface area of 10 percent is calculated to be 31.3cm ² ～33.1cm ² . The animal was shaved at 24 hours prior to the test, and the back affected area was 5cm by 7cm. The required sample amount of each animal is weighed to be 0.5g, the animal is uniformly coated on a skin preparation area, then a layer of thin film is used for covering, the animal is fixed by using a non-irritating adhesive tape, the animal is prevented from licking, the fixed object and the covering are taken off after the animal is in closed contact for 24 hours, and the residual sample on the skin is washed off by water. The toxication and death of the animals during the toxication process and observation period were observed and recorded, weighing once a week, the observation period was 14 days, and after the end of the observation period, the surviving animals were sacrificed and subjected to gross dissection.

Test results: the experimental animals do not see any toxic symptoms and toxic death within 14 days after the infection; the body weight of the male and female animals is not abnormal. At the end of the experimental observation, no abnormal changes were seen in the gross anatomic examination of the test animals. LD50 > 2500mg/kg. The results are shown in Table 10 below.

TABLE 10 acute percutaneous toxicity test results

It can be seen that under the present test conditions, the acute percutaneous LD50 of the tested sample on SD rats is > 2180mg/kg, and the sample is of a slightly toxic grade according to the acute skin toxicity classification.

(3) Acute skin irritation test

Experimental animals: 4 rabbits of New Zealand of ordinary grade (Jia Shan Hui Min street Sheng Wang family farm); male and female halves (female animals selected from infertility and non-farrowing); the weight is controlled between 2.27kg and 2.98kg.

Animals are at least suitable for 3d in the environment of an experimental animal house before the experiment, and are fed in a single cage, and the feeding environment temperature is as follows: 22.8-23.6 ℃, relative humidity: 52.9 to 61.4 percent of 4; feeding experimental rabbits with compound feed (maintenance type eastern feed Limited liability company in Tongxiang city); the primary RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking water nozzle.

The experimental method comprises the following steps: the experimental animals were shaved on both sides of the back spine about 24 hours prior to the test, with a hair removal range of about 3cm x 3cm each. 0.5mL of the sample is directly smeared on one side of dehaired skin, the smearing area is 2.5cm multiplied by 2.5cm, then two layers of gauze (2.5 cm multiplied by 2.5 cm) and one layer of cellophane are covered, and then the sample is fixed by using non-irritating adhesive plaster and bandage. The other side skin served as a control. After 4h of the blocking test, the residual test specimen was removed with warm water. Skin reactions at the smears were observed 1h, 24h, 48h and 72h after removal of the test samples, scored for skin reactions, and rated for stimulation intensity based on the highest integrated mean for each observation time point at 24h, 48h and 72 h.

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

TABLE 11 acute skin irritation test results

(4) Multiple skin irritation test

Experimental animals: 4 rabbits of New Zealand of ordinary grade (Jia Shan Hui Min street Sheng Wang family farm); male and female halves (female animals selected from infertility and non-farrowing); the weight is controlled between 2.34kg and 2.92kg.

Animals are at least suitable for 3d in the environment of an experimental animal house before the experiment, and are fed in a single cage, and the feeding environment temperature is as follows: 22.0-23.9 ℃, relative humidity: 40.5 to 58.6 percent; feeding experimental rabbits with compound feed (maintenance type eastern feed Limited liability company in Tongxiang city); the primary RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking water nozzle.

The experimental method comprises the following steps: the experimental animals were shaved off on both sides of the back spine approximately 24 hours before the test, with a hair removal range of 3cm x 3cm each. The application area is 2.5cm multiplied by 2.5cm. 0.5mL of the sample is smeared on one side of the dehaired skin, and the other side of the dehaired skin is used as a control, smeared for 1 time a day and continuously smeared for 14 days. From the next day, the residual test sample was removed with warm water by shearing prior to each application. The results were observed after one hour.

Test results: during the test period, no abnormal symptoms were observed in the experimental animals, and the average integral per animal per day was 0.00 over 14 days. See table 12 below for details. Under the test conditions, the tested sample has no irritation to the skin of the rabbit for a plurality of times.

TABLE 12 results of multiple skin irritation test

(5) Acute eye irritation test

Experimental animals: 3 rabbits of New Zealand of ordinary grade (Jia Shan Hui Min street Sheng Wang family farm); male and female halves (female animals selected from infertility and non-farrowing); the weight is controlled between 2.40kg and 2.61kg.

Animals are at least suitable for 3d in the environment of an experimental animal house before the experiment, and are fed in a single cage, and the feeding environment temperature is as follows: 23.0-24.1 ℃, relative humidity: 49.8% -60.7%; feeding feed: experimental rabbits (maintenance type eastern fodder Limited liability company in Tongxiang city); the primary RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) is directly supplied to animals for free drinking through a drinking water nozzle.

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

Test results: during the test period, the experimental animals do not show abnormal symptoms, and the individual integral average value of the animals is 0.00. See table 13 below for details. Under the test conditions, the acute eye irritation of the tested sample to the rabbit is as follows: has no irritation.

TABLE 13 acute eye irritation test results

* The mean of individual animal integral refers to the mean integral of each animal over four different observation times (24 h, 48h, and 72 h) for cornea, iris, conjunctival congestion, and conjunctival edema (i.e., the sum of the 24h, 48h, and 72h scores for each animal divided by the number of points at observation 3).

(6) Skin phototoxicity test

Sample treatment: the sample lysate filtrate was used for testing. Positive control: 8-methoxy fructus Psoraleae (lot number: L630V07; beijing carboline technologies Co., ltd.), solvent: absolute ethanol, concentration: 0.05%; the dosage is as follows: 2.4000mg.

Experimental animals: 6 guinea pigs of the common class Netherlands and 6 positive control groups each (Simen of Yuyao Zhenfei experiment rabbit farm); the female animals are not limited (female animals are selected from infertility and non-farrowing animals); the weight is controlled between 261.2g and 272.4g.

Animals are acclimatized in the laboratory environment for at least 3 days prior to testing. Temperature of the feeding environment: 23.3-24.0 ℃; relative humidity: 49.5 to 56.7 percent; feeding feed: guinea pig compound feed (maintenance, su state An Mu biotechnology limited); primary RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization). The animal can drink the water directly through the drinking nozzle.

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

The calculation formula of the irradiation time is as follows:

note that: 1mW/cm ² ＝1mJ/cm ² /sec

After the irradiation dose and the average light intensity are input into a UVA-TOXI phototoxic device, the irradiation time is 00:10:64 (namely 664 s) according to a calculation formula;

the test steps are as follows: animals are acclimatized in the laboratory environment for at least 3 days prior to testing. About 24 hours before the test, the skins on two sides of the animal spine are dehaired, and the skin of the test part needs to be intact and free from damage and abnormality. 4 zones of dehairing were prepared, each zone having a dehairing area of about 2cm by 2cm. Animals were fixed and 0.2mL of the test sample was applied to animal dehairing areas 1 and 2 as shown in skin phototoxicity test Table 1, chapter 7 of cosmetic safety Specification (2015). After 30min, the left side (dehairing areas 1 and 3) was covered with aluminum foil, tape-secured and the right side was irradiated with UVA. Skin reactions were observed at 1h, 24h, 48h and 72h, respectively, after the end, and skin reaction scores were determined for each animal according to chapter 7 skin phototoxicity test table 2, cosmetic safety Specification (2015 edition). Positive control groups were performed in the same manner.

Test results: during the test period, no abnormal symptoms appear in the experimental animals, the animal integral of each observation time point is 0, the samples are classified according to skin irritation reaction, and no skin phototoxicity exists in the tested samples. The positive control and test results of the skin phototoxicity test on guinea pigs are shown in Table 14 and the test sample and test results of the skin phototoxicity test on guinea pigs are shown in Table 15. Under the test conditions, the tested sample has phototoxicity test results on guinea pig skin: no skin phototoxicity was seen.

TABLE 14 results of Positive control photo toxicity test on guinea pig skin

Note that: the test areas shown in figure 1 of the skin phototoxicity test are shown in the table heads 1, 2, 3 and 4 of the sixth chapter 7 of the cosmetic safety Specification (2015).

TABLE 15 test results of the phototoxicity of the test samples to guinea pig skin

Note that: the heads 1, 2, 3 and 4 are test areas shown in figure 1 of the skin phototoxicity test in chapter 7 of cosmetic safety Specification (2015).

(7) Skin allergy test

Sample treatment: the sample lysate filtrate was used for testing. Positive control: alpha-hexyl cinnamaldehyde (lot number: LV30V28; beijing carboline technologies Co., ltd.); solvent: absolute ethanol (analytical grade, lot number: 20210119, national drug group chemical Co., ltd.), acetone (analytical grade, lot number: 20170918, national drug group chemical Co., ltd.), millipore pure water.

Induction concentration: 60, evenly mixing 3.6mL of alpha-hexyl cinnamaldehyde with 2.4mL of 80% ethanol solution to prepare the product; excitation concentration: 30, uniformly mixing 3.0mL of alpha-hexyl cinnamaldehyde with 7.0mL of 80% acetone solution to obtain the product; the dosage is as follows: 6.6mL.

Experimental animals: common class guinea pigs (Yuyao city, simen Zhenfei experiment rabbit farm); 20 test groups and positive control groups, and 10 negative control groups; the male and female animals are unlimited (female animals are not pregnant and have not born); the weight is controlled to be 257.8 g-273.4 g.

Animals are acclimatized in the laboratory environment for at least 3 days prior to testing. Temperature of the feeding environment: 22.5-24.1 ℃; relative humidity: 43.4% -61.9%; feed: guinea pig compound feed (maintenance type, an Mu femtocells, su) first-class RO ultrafiltration water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization). The animal can drink the water directly through the drinking nozzle.

The test method comprises the following steps: about 24 hours before the test, the left side of the back of the guinea pig is dehaired to a dehairing range of about 6cm ² 。

Induction contact: 0.2mL of the test sample was applied to 2cm X2 cm dehaired skin of the test group animals, covered with two layers of gauze and one layer of cellophane, and then sealed and fixed with a non-irritating adhesive tape for 6 hours. The 7 th and 14 th steps are repeated once in the same way. The positive control group was operated in the same manner as the test group except that the test sample was not administered to the negative control group.

Excitation contact: after the last induction, 14d, 0.2mL of the test sample was applied to the right 2cm×2cm dehairing area (dehairing 24 hours before contact) on the back of guinea pigs in the test group and the negative control group, then covered with two layers of gauze and one layer of cellophane, and then fixed with a non-irritating adhesive tape for 6 hours. The positive control group was operated in the same manner with 30% alpha-hexyl cinnamaldehyde solution. Skin reactions were observed 24h and 48h after the end of the challenge contact, scored and the sensitization intensity was determined.

Test results: during the test period, no abnormal symptoms appear in the experimental animals, and the sensitization rate of each observation time point is 0%. See tables 16 and 17. Under the test conditions, the test results of the skin allergy reaction of the tested sample on guinea pigs are as follows: skin allergies were not seen.

TABLE 16 weight summary of test animals

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

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

Note that: when the skin reaction integral is 0, 1, 2 and 3 … … in the skin reaction intensity column, the proportion of the number of animals which react to the number of tested animals is represented; the original form in the table refers to the lysate filtrate prepared according to the present invention.

(8) Bacterial back mutation assay

Sample treatment: the concentration of each dosage group is prepared by taking sterile pure water as a solvent.

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

Positive control:

(1) Without S9 (purchased, MOLTOX, usa):

dixon (Dexon): lot number: g1063485; the manufacturing factory: enrenstonfer GmBH, germany; solvent: sterile pure water; concentration: 500 μg/mL; the dosage is as follows: 0.1 mL/dish;

Sodium Azide (SA): lot number: 20120615; the manufacturing factory: shanghai Engelt chemical Co., ltd; solvent: sterile pure water; concentration: 15 μg/mL; the dosage is as follows: 0.1 mL/dish.

(2) Adding S9:

2-aminofluorene (2-AF): lot number: c11449586; the manufacturing factory: shanghai Miclin Biochemical technologies Co., ltd; solvent: dimethyl sulfoxide (DMSO); concentration: 100 μg/mL; the dosage is as follows: 0.1 mL/dish;

1, 8-dihydroxyanthraquinone (1, 8-DHQ): lot number: c10429233; the manufacturing factory: shanghai Miclin Biochemical technologies Co., ltd; solvent: DMSO; concentration: 500 μg/mL; the dosage is as follows: 0.1 mL/dish;

cyclophosphamide (CP): lot number: k1812016; the manufacturing factory: allatin 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 (TA 97a, TA98, TA100, TA102 and TA 1535), purchased from MOLTOX corporation of America, were identified and tested to meet standard requirements.

The test 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, with blank, solvent (negative), positive mutagen and sterile control groups. The strain is subjected to enrichment culture to prepare enrichment liquid, and then the enrichment liquid is subjected to plate doping. In the experiment, 2.0mL of top medium containing 0.5mmol/L histidine and 0.5mmol/L biotin solution is subpackaged in a test tube, the temperature is kept in a water bath at 45 ℃, then 0.1mL of bacteria increasing liquid, 0.1mL of sample to be tested 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, and the mixture is rapidly poured into the bottom medium, and the flat plate is rotated, so that the distribution is uniform. After the condensation and solidification are carried out in a horizontal way, the flat plate is turned over and is inverted to be incubated in an incubator at 37 ℃ for 48 hours. Positive mutagen control, solvent control, blank control, and sterile control were also performed in this manner. The number of revertant colonies per dish was counted.

The number of colony colonies per dish of reverted test subjects, blank control (spontaneous reverting), solvent control and positive mutagen control was recorded and averaged and standard deviation was determined.

Test results: the standard plate incorporation test results are shown in Table 18. The results of solvent control (negative control) show that each group can grow aseptically without adding the test strain salmonella typhimurium, and can be used for the test; the number of the back mutation colonies of the blank control group and the positive mutagen control group is in an acceptable range, and the test is effective;

under the test conditions, the number of recovered colonies detected by the plate doping method of the metabolic activation system with and without the test object in each dosage group is not more than 2 times that of the solvent control group, and the dosage response relation is not shown.

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

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(9) In vivo mammalian cell micronucleus assay

Sample treatment: the sample lysate filtrate is prepared into solutions of each dosage group by taking pure water as a solvent.

Negative control: pure water

Positive control: cyclophosphamide (lot number: F1812016; brand: aladine; solvent: pure water; concentration: 3 mg/mL)

Experimental animals: SPF ICR mice (Shanghai Jieshike laboratory animal Co., ltd.) were 30, and the weight of each half (female animals were not pregnant and had not born) was controlled at 25.0 g-27.4 g, and the body weight of the same sex was not more than.+ -. 20% of the average.

The animals are subjected to quarantine period of 4 days (Co 60 irradiation of mouse feed, jiangsu province cooperative medical bioengineering, inc.) in an animal house (the barrier environment, the temperature of 22.8-23.5 ℃ and the relative humidity of 51.6-57.8%) under the barrier environment before the test, and the first-class RO ultrafiltration water (sodium hypochlorite is added to control the free chlorine content in the water to be 2-3ppm for sterilization) is directly supplied to the animals for free drinking through a drinking nozzle, so that the animals do not fasted before the infection and drink water freely.

The test method comprises the following steps: dose setting: a test group, a negative control group (pure water) and a positive control group (60 mg/kg cyclophosphamide) were set. According to the pre-test results, the final concentration of the test group was set to 1000mg/kg, 2000mg/kg, 5000mg/kg.

The administration mode is as follows: oral lavage; dosing volume: 20 mL/kg.BW.

The test steps are as follows: animal contamination adopts an oral gastric lavage 30h contamination method, namely, the interval between two contamination is 24h, and the materials are obtained 6h after the second contamination. Animals were sacrificed by cervical dislocation and femur removed. Stripping off muscles and wiping off blood stains. Both ends of the femur were cut to expose the medullary cavity. Sucking 0.1mL calf serum by a syringe, flushing the marrow cavity, and performing routine smear with flushing liquid, airing or hot air drying. The dried smear was fixed in methanol for 10min. Dyeing with giemsa application solution for 15min, washing with clear water, and air drying. The procedure for the positive and negative control groups was the same as for the test group. The area with uniform, complete and proper cell distribution and coloring is selected. The number of PCEs (polynuclear erythrocytes) containing micronuclei was counted under an oil microscope. PCE appears gray blue and NCE (mature red blood cells) appears pink. Each animal counts the number of micronuclei contained in 2000 PCEs. Micronucleus cell rate (MN per mill) refers to the PCE number containing micronuclei, expressed in thousandths. 2 or more microkernels are present in 1 PCE, still counting by 1. Microkernel rates between groups were compared using the Boss distribution u test or other suitable significance test methods.

Test results: the test results showed that the number of micronucleated cells in the bone marrow of the mice in the positive control group was significantly increased (P < 0.05) compared to the negative control group, whereas the micronucleus ratio of micronucleated red blood cells in each test group was statistically significant (P > 0.05) compared to the negative control group.

Under the test conditions, the micronucleus test result of the mammalian red blood cells in the tested body is negative, and the sample is a non-mutagenic agent. The detection results of the acute toxicity detection (the acute oral toxicity test is nontoxic, the acute transdermal toxicity test is used for detecting the micro toxicity), the skin irritation/corrosiveness, the skin sensitization, the skin phototoxicity, the genetic toxicity and the like show that the filtrate has good toxicology safety.

TABLE 19 statistics of micronucleus rate for micronucleus test

Note that: p <0.05 compared to the negative control group. Micronucleus rate and PCE/NCE were counted in mice and expressed as mean.+ -. Standard deviation.

e. Human skin patch test

According to the human body safety inspection method in 2015 edition of cosmetic safety technical Specification, the lysate filtrate is subjected to a human body skin patch test, and the method is completed by the English detection technical service (Shanghai) limited company and provides a detection report.

A total of 33 subjects are selected for testing, and the test results show that the 32 subjects have no skin reaction after the spot tester of the tested object is removed for 0.5h, 24h and 48h, and the score grades are all 0; 1 subject was scored on a scale of 1 24 hours after removal of the subject plaque tester, and the results of the specific experiments are shown in table 20 below.

TABLE 20 skin seal Patch test results of lysate filtrate

TABLE 21 skin response grading Standard for skin closed Patch test

Referring to the regulations in the "evaluation point of cosmetic technology", the number of cases of adverse reactions to the skin of class 1 in 30 subjects was not more than 5 (5 cases were excluded), and the specific classification criteria are shown in Table 21 above. The results show that the test result meets the specified safety requirement, and the lysate filtrate prepared by the invention is considered to be safe for being applied to human bodies.

In conclusion, the Pichia pastoris fermentation lysate filtrate prepared by the method disclosed by the invention is subjected to detection of heavy metal content, methanol content, microorganism limit, safety toxicology and human skin patch test according to the method in cosmetic safety technical Specification, and meanwhile, residual DNA detection is performed, so that the safety of the Pichia pastoris fermentation lysate filtrate meets the requirement of being applied to cosmetics.

Example 2: efficacy detection of fermentation lysate filtrate

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

1. Relief efficacy detection

The epidermis is used as a tissue for separating the organism from the external environment, and besides being used as a physical barrier, the keratinocytes can receive the external 'dangerous signal' stimulus and transmit the external 'dangerous signal' stimulus to immune cells in the skin to warn, so that the immune barrier of the skin is formed, and the skin is caused by the disorder of the function and the regulation of the skin immune barrier, so that skin problems and diseases are caused. Some cosmetics or ultraviolet rays with strong skin contact irritation can cause clinical acute injury of skin barrier, dry skin and red skinPlaque phenomenon. The anionic surfactant sodium dodecyl sulfate (Sodium lauryl sulfate, SLS) has amphiphilic (hydrophilic and lipophilic) character and is capable of damaging skin barriers, especially lipid components in the barrier and cell membranes, after contacting the skin at large concentrations. Thus, the experiment uses in vitro recombinant epidermis modelIn order to study objects, an SLS stimulation damage model is adopted, then the lysate filtrate obtained in the example 1 is used for treatment, the change of relevant inflammatory factors and tissue morphology is used as an index, and the change of the contents of tissue morphology, inflammatory mediator PGE2 and capsaicin receptor TRPV-1 is detected to evaluate the soothing efficacy of the lysate filtrate prepared by the invention, and the lysate filtrate is entrusted to detection by Shanxibos general detection technologies, inc. and provides a detection report.

The protocol is shown in table 22 below.

TABLE 22 lysate filtrate soothing efficacy detection packet and detection method

(1) The model was transferred to 6-well plates (0.9 mL of model broth added in advance) according to the test protocol of table 22 above, and test group numbers were noted on the 6-well plates.

(2) Adding 25 mu L of SLS solution with concentration of 0.2% on the surfaces of NC and PC groups, adding 12.5 mu L of SLS solution with concentration of 0.4% on the surfaces of sample groups and 12.5 mu L of sample working solution with corresponding concentration (namely lysate filtrate prepared by the invention), uniformly distributing the samples on the surfaces of the models, and placing the samples on CO ₂ Incubator (37 ℃,5% CO) ₂ ) And incubated for 24h.

(3) After the incubation, the test substance remained on the surface of the model was washed with sterile PBS solution, and the inner and outer residual liquids of the model were gently wiped off with a sterile cotton swab.

(4) Inflammatory mediator (PGE 2) test: after 24h of dosing incubation, the 3D epidermis 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 preservation, and detection analysis is carried out according to the operation instructions of the PGE2 ELISA kit.

(5) Tissue morphology testing: the model for tissue morphology detection (i.e. the epidermis model treated according to table 22) was taken, fixed with 4% paraformaldehyde for 24H, the model was cut off, H & E staining was performed, photographed under a microscope, and pictures were taken and analyzed.

(6) Capsaicin receptor (TRPV 1) test: the model used for detection (i.e., the epidermis model treated according to table 22) was taken, fixed with 4% paraformaldehyde, after 24 hours of fixation, the model was cut off, immunofluorescence detection of capsaicin receptor (TRPV 1) was performed, photographed under a microscope, and pictures were taken and analyzed.

Results statistical analysis: graphPad Prism was used to map, and the results were expressed as mean±sd; the comparison among the groups adopts t-test statistical analysis; statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

Analysis of tissue morphology detection results:

the results of the tissue morphology test are shown in fig. 7, wherein fig. 7 is a tissue morphology chart under a microscope after H & E staining of the epidermis model after the end of the filtrate culture, wherein fig. 7A is a blank control, 7B is a negative control, i.e., SLS-treated group, 7C is a positive control, i.e., SLS and 0.01% dexamethasone are added simultaneously, and 7D is an experimental group, i.e., SLS and 2% filtrate are added simultaneously.

The tissue morphology is a microscopic physiological structure analysis of tissue after HE staining, and the change condition of the skin barrier under different treatment conditions can be seen through the model tissue morphology, when the skin barrier is loosened after injury is caused by SLS stimulation, the thickness of a living cell layer is reduced, and whether the effect of improving the injury is achieved is judged through the morphological change after treatment by adding an active substance. The tissue morphology detection result shows that compared with a blank group, the number of model cells in the negative group after SLS stimulation is reduced, the arrangement is disordered, the stratum corneum is loose, and cavitation phenomenon appears, so that the stimulation condition of the test is effective. Compared with the negative group, the positive control group model has obviously improved loose and cavitation situations of the living stratum corneum, which proves that the positive control detection is effective. Compared with the negative group, the porosity and cavitation of the horny layer after the treatment of the filtrate of the 2% pichia pastoris fermentation lysate are obviously improved, which proves that the 2% filtrate under the in vitro condition is helpful for relieving the stimulation reaction caused by SLS chemical stimulation and improving the epidermal tissue injury caused by the stimulation.

PGE2 test results analysis:

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

TABLE 23 summary of PGE2 content detection results in lysate filtrate

Remarks: when the statistical analysis is performed by the t-test method, compared with the BC group, the significance is expressed as # and the P-value < 0.05 is expressed as # and the P-value < 0.01 is expressed as #; compared with NC groups, significance is expressed as x, P-value < 0.05 is expressed as x, and P-value < 0.01 is expressed as x.

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

Prostanoids play a very important role in the inflammatory response, the most well known of which is PGE2.PGE2 is also currently known as the most widely-synthesized PG in mammals. The primary source of PG in skin is keratinocytes, and PGs synthesized primarily by keratinocytes include PGE2, PGF2a, and PGD2.

The inflammatory response of the skin is often accompanied by one to several important clinical features: erythema, swelling, heat or localized skin temperature elevation, pain, itching, which may be partially accompanied by dander. While four manifestations of inflammatory reactions on the skin (red, swelling, heat, pain) are closely related to PGE 2: PGE2 has vasodilating and vasodilating effects, manifesting as flushing of the skin; under the synergistic effect of histamine and kinin, the vascular permeability is increased, local red and swelling of tissues are caused, local fever is caused, and PGE2 entering blood can be combined with hypothalamic receptors to cause the change of body temperature center, so that systemic fever is caused; in addition, PGE2 may stimulate nerve endings to cause pain. PGE2, a mediator of inflammation, is involved in the initiation and progression of inflammatory responses through a number of pathways during trauma. PGE2 is a strong vasodilator, slows down blood flow and promotes the adherence and adhesion of leukocytes. In addition, PGE2, as a pro-inflammatory mediator, may also mediate the production of other inflammatory mediators, such as IL-6. Experiments show that 2% of the Pichia pastoris fermentation lysate filtrate can obviously reduce the secretion level of PGE2, and the fermentation lysate filtrate disclosed by the invention can relieve skin inflammatory reaction, such as reducing vascular permeability in skin, by mediating the secretion of inflammatory mediator PGE2, so that the skin redness phenomenon is relieved.

TRPV1 test results analysis:

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

TABLE 24 TRPV1 data results summary table

Experimental data show that compared with the BC group, the TRPV1 protein content of the NC group is significantly 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 proves that the positive control detection is effective. Compared with NC group, TRPV1 protein content after 2% pichia pastoris fermentation lysate filtrate action was significantly reduced. It is demonstrated that the filtrate can play a role in relieving inflammation by inhibiting the mechanism of TRPV1 protein expression.

Transient receptor potential vanilloid subtype 1 (transient receptor potential vanilloid type, trpv 1), 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. Is a high-efficiency mediated Ca ²⁺ The cation channels that flow in are widely distributed in cells such as neurons, immune cells, epithelial cells of various organs, and keratinocytes. It was found that TRPV1 channels can be activated by a variety of stimuli, such as various physical and chemical stimuli including high temperature (temperature. Gtoreq.43℃), low pH (acidic environment), voltage, osmolarity, endogenous and exogenous vanilla substances, such as capsaicin, etc. When TRPV1 is activated, calcium ion channels open, calcium ion influx, and intracellular calcium ion concentration increases, causing neurons and their fibers to release neuropeptides such as substance P, neurokinin a, calcitonin gene-related peptide, vasoactive intestinal peptide, and excitatory amino acids such as glutamic acid, 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 the occurrence of pain and itch as well as cutaneous neurogenic inflammation, and thus TRP channels have become important molecular targets for the treatment of inflammatory skin diseases and itch. Therefore, the test selects TRPV1 as a target molecule, and the effect of filtrate on relieving inflammation is evaluated by measuring the change of the expression level of the TRPV1 under the action of chemical stimulation on an epidermis model.

2. Ultraviolet absorption effect detection

The fermentation lysate filtrate obtained by the invention and different multiples thereofIs subjected to absorbance scanning (Varioskan) in the ultraviolet region (200-400 nm) ^TM The LUX multifunctional enzyme-labeled instrument-VL 0L0TD0, semer Feier technology (China) Co., ltd.) was converted to transmittance, and then calculated as absorbance, and the result was shown in FIG. 11.

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

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

The results show 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 sun-proof effect of the lysate filtrate is similar to the effect of common UVB absorbers such as aminobenzoate and derivatives thereof, salicylate and derivatives thereof, cinnamic acid esters and camphor derivatives, and the diluted lysate filtrate can also have good sun-proof skin care effect on ultraviolet rays in a certain wavelength range.

3. Regulation of microecological outcome analysis

In this embodiment based on a 3D epidermal skin modelAnd (3) evaluating the effect of the sample to be tested on the expression level of the skin antibacterial peptide and TOLL-like receptor protein by detecting the change of HBD1, HBD2, LL-37 and TLR2 proteins. The test entrusts Shaanxi Boxi general detection technology Co., ltd to detect and give 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 broth was added in advance) according to the test protocol of table 25 below, and test group numbers were noted on the 6-well plates.

TABLE 25 method for regulating microecology detection of lysate filtrate

Adding working solution with corresponding concentration on the surface of the sample group into a 6-hole plate, and placing the sample group into CO ₂ Incubator (37 ℃, 5% CO) ₂ ) And incubated for 24h. After the incubation, the test substance remained on the surface of the model was washed with a wash bottle containing a sterile PBS solution, and the residual liquid inside and outside the model was wiped off with a sterile cotton swab. And taking a model for immunohistochemical detection, and performing an immunohistochemical test. Results statistics were plotted using GraphPad Prism and the results are expressed as mean±sd. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P (P)<0.05 considered to have significant differences, P<0.01 is considered to have a very significant difference.

(2) Detection result

HBD1 results analysis

The results of staining the HBD1 proteins in each group are shown in FIG. 12, wherein the tan part indicated by the red arrow in the graph is HBD1 protein, and the deeper tan is the higher the HBD1 protein content. The table of HBD1 versus IOD value summary is shown in table 26, and a histogram of HBD1 Integrated Optical Density (IOD) values is shown in fig. 13.

TABLE 26 HBD1 relative IOD value summary Table

Group of	Average of relative IOD	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**

The results showed that the HBD1 protein content of the PC (1, 25-dihydroxyvitamin D3) group was significantly increased compared to the BC group, indicating that the positive control of this test was effective. Compared with the BC group, the HBD1 protein content of the sample Pichia pastoris fermentation lysate filtrate-0.5% and the Pichia pastoris fermentation lysate filtrate-1.0% is obviously increased.

HBD2 results analysis

The results of staining the HBD2 proteins in each group are shown in fig. 14, wherein the tan part indicated by the red arrow in the graph is HBD2 protein, and the deeper tan is the higher HBD2 protein content. The summary table 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 relative IOD value summary table

Group of	Average of relative IOD	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**

The results showed that the HBD2 protein content of the PC (1, 25-dihydroxyvitamin D3) group was significantly increased compared to the BC group, indicating that the positive control of this test was effective. Compared with the BC group, the HBD2 protein content of the sample Pichia pastoris fermentation lysate filtrate-0.5% and the Pichia pastoris fermentation lysate filtrate-1.0% is obviously increased.

TLR2 outcome analysis

The staining results of each group of TLR2 proteins are shown in FIG. 16, wherein the tan part indicated by red arrows in the pictures is TLR2 protein, and the deeper the tan is, the higher the TLR2 protein content is. The summary of TLR2 versus IOD values is shown in table 28, and the histogram of TLR2 Integrated Optical Density (IOD) values is shown in fig. 17.

Table 28.Tlr2 relative IOD value summary table

Group of	Average of relative IOD	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 results showed that the TLR2 protein content of the PC (1, 25-dihydroxyvitamin D3) group was significantly increased compared to the BC group, indicating that the positive control detection was effective. Compared with the BC group, the concentration of 0.5% and 1.0% of the Pichia pastoris fermentation lysate filtrate can obviously increase the TLR2 protein content after the action.

LL-37 results analysis

The results of staining of each group of LL-37 proteins are shown in FIG. 18, where the dark brown portion of the image indicated by the red arrow is LL-37 protein, indicating a higher LL-37 protein content. The 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	Average of relative IOD	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**

The results showed that the LL-37 protein content of the PC (1, 25-dihydroxyvitamin D3) group was significantly increased compared to the BC group, indicating that the positive control test was effective. The Pichia pastoris fermentation lysate filtrate at concentrations of 0.5% and 1.0% both significantly increased the LL-37 protein content compared to the BC group.

The skin microecology is an ecological system composed of various microorganisms such as bacteria, fungi, viruses, mites, arthropods and the like, tissues on the surface of the skin, various secretions of cells, microenvironment and the like. The interaction of skin microorganisms, host and external environment constitutes the skin microecology balance. Studies have shown that keratinocytes directly kill exogenously invading microbiota by expressing antimicrobial polypeptides (Antimicrobial Peptides, AMPs), while providing a life-threatening friendly place for resident bacteria. The resident bacteria can directly release AMPs to act on pathogenic microorganisms. For example, staphylococcus epidermidis can secrete antimicrobial peptides such as defensins, bacteriocins, LL-37, REG3A, and the like. Resident bacteria also regulate the host immune response by secreting AMPs. The antibacterial peptides existing in human body and including skin mainly belong to two main families: defensins and cathelicidins. Defensins are classified into α -defensins and β -defensins according to their structures, wherein HBD1 and HBD2 belong to β -defensins. LL-37 is the only Cathelicidin-based antibacterial peptide found in humans. Alternatively, pathogenic microorganisms can be cleared by activating various pattern recognition receptors, such as TLR2 (Toll-like receptor 2), recognizing exogenous microorganisms, and activating an immune response. The test is based on a 3D epidermal 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 proteins is positively and correlatively increased along with the increase of the concentration of the filtrate, so that the Pichia pastoris fermentation lysate filtrate prepared by the invention can regulate microecology by improving the expression of antibacterial peptide, and further achieve the repairing effect.

4. Oil control efficacy detection

In the embodiment, the oil control efficacy of the sample to be tested is evaluated by detecting the lipid drop content of the sebaceous cells after the sample is acted on based on the sebaceous cells, and the detection is entrusted to the general detection technology company of Shanxi Boxi and provided with a detection report.

(1) Experimental method

In this example, the test set up blank, positive control and sample sets for the oil control efficacy test are shown in table 30 below.

TABLE 30 oil control efficacy detection protocol

The detection method is briefly described as follows:

SZ95 cells were seeded at a seeding density of 3.5E cells/well onto 24 well plate climbs, incubator (37 ℃, 5% CO) ² ) Incubate overnight.

According to the test scheme of Table 29, when the cell plating rate in the 24-well plate reaches 60% -70%, group administration is carried out, 1mL of sample is added to each well, and 3 compound wells are arranged in each group. After the completion of the administration, the 24-well plate was placed in an incubator (37 ℃, 5% CO) ² ) Medium for 72 hours, and daily liquid exchange and administration are carried out during the culture period.

Oil red O staining: the old solution was aspirated, the 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 at 37 ℃ C.) and washed 2 times with PBS.

Photographing and observing by a fluorescence microscope: at Ex:450nm-500nm, em:515nm-560nm, and photographing and observing under a 200-time mirror.

Results statistical analysis: graphPad Prism was used to map and the results were expressed as mean±sd. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

(2) Detection result

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

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

Remarks: when the statistical analysis is performed by the t-test method, the significance of the PC group and the sample group is expressed as a sign, the P-value <0.05 is expressed as a sign, and the P-value <0.01 is expressed as a sign, compared with the BC group.

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

Sebum hypersecretion is a major factor causing various skin problems such as acne, sebaceous gland cells synthesize a large amount of grease and secrete the grease out 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 degree of oil control. Based on sebaceous gland cell SZ95, compared with a control group, the lipid content of the sample Pichia pastoris fermentation lysate filtrate is obviously reduced at the concentration of 0.05% and 0.15%; the Pichia pastoris fermentation lysate filtrate prepared by the method can achieve the oil control effect by reducing lipid drop synthesis.

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

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

(1) Skin care essence

Taking the example of preparing lysate filtrate with 10% wet thallus concentration of fermentation thallus of engineering bacteria with the preservation number of CGMCC No.7189 in the publication No. CN103102407B as the preparation example, preparing a skin care essence, wherein the total mass percentage of the raw materials is 100% as shown in the following list, and the adding proportion of Pichia pastoris fermentation lysate filtrate is preferably 5%.

The skin care essence comprises 1-10wt%, preferably 5wt% of fermentation lysate filtrate, and further comprises 4wt% of glycerin, 4wt% of butanediol, 2wt% of glycerolyether-26, 0.02wt% of EDTA-2Na, 0.05wt% of sodium hyaluronate, 0.15wt% of allantoin, 2wt% of betaine, 0.25wt% of xanthan gum, 5wt% of 1, 2-pentanediol and deionized water.

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

(2) Facial mask

Taking the example of preparing lysate filtrate with the concentration of 5% wet thallus of fermentation thallus of engineering bacteria with the preservation number of CGMCC No.7189 in the publication No. CN103102407B as the preparation example, preparing a skin care mask liquid, wherein the total mass percentage of the raw materials is 100% and the addition proportion of the fermentation lysate filtrate of Pichia pastoris is 5% as shown in the following formula.

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

The preparation method comprises the following steps:

accurately weighing the raw materials of each component for standby; 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 55-60deg.C, adding pre-dispersed carbomer into the above mixture, mixing and stirring; cooling to 40-50deg.C, adding pH regulator, mixing and stirring; cooling to 35-40deg.C, adding Pichia pastoris fermented lysate filtrate, and stirring; filtering with 100 mesh filter cloth to obtain facial mask liquid, matching with pure cotton membrane cloth, and packaging in facial mask aluminum foil bag to obtain skin care facial mask.

(3) Skin care cream:

Taking the example of preparing lysate filtrate with the concentration of 5% wet thallus of fermentation thallus of engineering bacteria with the preservation number of CGMCC No.7189 in the publication No. CN103102407B as the preparation example, preparing the skin care cream, wherein the total mass percentage of the raw materials is 100% and the proportion of the components is as follows, and preferably the adding proportion of Pichia pastoris fermentation lysate filtrate is 5%.

The cream comprises a fermentation lysate filtrate, wherein the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and the fermentation lysate filtrate is marked as a phase D; the composition also comprises 5 weight percent of glycerin, 0.02 weight percent of EDTA disodium, 0.15 weight percent of xanthan gum, 0.6 weight percent of acrylamide dimethyl taurate/VP copolymer, 0.5 weight percent of p-hydroxyacetophenone, 0.5 weight percent of 1, 2-hexanediol and the balance deionized water, and is mixed into phase A; 3wt% of cetostearyl glucoside/cetostearyl alcohol, 3wt% of cetostearyl alcohol, 4wt% of caprylic/capric triglyceride, 3wt% of hydrogenated polyisobutene and 1wt% of polydimethylsiloxane, and mixing the mixture into phase B; the pH regulator is phase C and is generally neutral.

The preparation method comprises the following steps:

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

Crowd test experiments are carried out on the obtained product:

selecting 90 volunteers (all skin-friendly and skin-sensitive people with red skin), randomly dividing the age between 20 and 45 years into 3 groups, respectively using the products containing pichia pastoris lysate filtrate, namely skin care essence, mask and skin care cream, and preparing a sample without filtrate as a blank control by referring to the formula, wherein the group A uses the essence, and is smeared on the face after cleaning the face for 2 times a day; the group B uses the facial mask, uses 2 times a week, applies to the face for 10-15min; the group C uses skin cream, and is smeared on the face after cleaning the face for 2 times a day in the morning and evening, wherein the left face of a subject uses the essence, the mask or the cream product disclosed by the invention, the right face uses a reference substance without filtrate, the skin cream is used for 4 weeks, no other similar skin care products are used in the period, and the use effects of relieving skin redness and relieving skin irritation of the sample are self-evaluated on the 28 th day.

Scoring criteria:

the skin state is not improved, even aggravated, and the score is 0; pi Fufan, no improvement in susceptibility to irritation, but no aggravation, gives a score of 1; pi Fufan is red and is slightly improved in susceptibility to stimulation, but the effect is not obvious, and the score is 2; pi Fufan red and the susceptibility to stimulation are improved to obtain 3 points; pi Fufan red and the susceptibility to stimulation are obviously improved, and the score is 4.

The average of the products of the test results is as follows: the skin care essence is 3.25 minutes, the mask is 3.47 minutes, and the skin care cream is 3.52 minutes. Feedback results show that: the examples of products containing pichia pastoris lysate filtrate do not have any irritation response and the volunteer response is significantly reduced in facial skin redness compared to the placebo. The partial photographs of the face of the subject are shown in fig. 22-24, fig. 22 is a comparison of the skin care essence before and after the use of the facial mask, fig. 23 is a comparison of the facial mask before and after the use of the facial mask, and fig. 24 is a comparison of the facial mask before and after the use of the skin care cream, and analysis of the results shows that the product of the technology has a good effect in relieving the skin and relieving the Pi Fufan red.

Claims

1. The application of the fermented lysate filtrate in preparing cosmetic raw materials or products is characterized in that the cosmetic raw materials or products are anti-inflammatory, sun-proof and oil-control cosmetic raw materials or products; the anti-inflammatory agent mainly reduces the secretion level of inflammatory mediator PGE2, relieves skin inflammatory reaction and reduces the generation of redness phenomenon of skin; the sun protection is manifested by an ability to absorb ultraviolet light; the oil control is characterized by inhibiting skin lipid drop synthesis;

the fermentation lysate filtrate is prepared from bacteria fermented by pichia pastoris, wherein the pichia pastoris is pichia pastoris for expressing recombinant collagen; the preparation method of the fermentation lysate filtrate comprises thallus dilution, high-pressure homogenization cracking, centrifugation and filtration; the thallus is diluted into thallus suspension diluted to a mass concentration of 5% -30% by pure water, the thallus suspension is crushed for 1-3 times by 100-200MPar pressure to obtain a lysate, the lysate is centrifuged at 10000-20000 g to obtain a supernatant, and the supernatant is filtered by 0.22 mu m pore diameter.

2. The use according to claim 1, wherein the pichia pastoris expressing recombinant collagen is selected from one or more of the strains with the preservation numbers 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.

3. The use according to claim 1, wherein the cells are obtained after solid-liquid separation of the fermentation product.

4. The use according to claim 1, wherein the cells are diluted to a cell suspension with pure water to a mass concentration of 10%.

5. The use according to claim 1, wherein the high pressure homogeneous lysis is to break the bacterial suspension at a pressure of 120 mpa.