WO2024027798A1 - Pichia pastoris fermentation lysate filtrate, preparation method therefor, and use thereof - Google Patents

Pichia pastoris fermentation lysate filtrate, preparation method therefor, and use thereof Download PDF

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WO2024027798A1
WO2024027798A1 PCT/CN2023/111026 CN2023111026W WO2024027798A1 WO 2024027798 A1 WO2024027798 A1 WO 2024027798A1 CN 2023111026 W CN2023111026 W CN 2023111026W WO 2024027798 A1 WO2024027798 A1 WO 2024027798A1
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skin
lysate filtrate
fermentation
test
filtrate
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PCT/CN2023/111026
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French (fr)
Chinese (zh)
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李佳佳
纪白慧
钱松
曹凯杰
陈聚印
石叶飞
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江苏创健医疗科技股份有限公司
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Publication of WO2024027798A1 publication Critical patent/WO2024027798A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a Pichia pastoris fermentation lysate filtrate, a preparation method and an application, and belongs to the technical field of cosmetic raw materials.
  • Collagen plays an important role in cell migration, cell metabolism, signaling pathway response, maintenance and regulation of normal physiological functions of cells, tissues, and organs, and damage repair.
  • Recombinant human collagen produced with modern genetic engineering technology has stable properties and does not have the disease risks and immunogenicity of animal-derived collagen. Factory production is not restricted by the source of raw materials. It has become a key area of national development support and a hot spot of capital attention. . At present, the largest large-scale production line for fermentation and production of collagen by domestic recombinant collagen manufacturers has reached 20 tons. In the future, its production scale and quantity will continue to expand and rise.
  • fermentation waste bacteria have gradually begun to be further utilized, such as preparing single-cell proteins (the protein content will be reduced), fermenting into alcohol (using the fiber produced during fermentation, which has little significance for the bacteria themselves), Developed as organic fertilizer for agricultural production (but requires certain processing and a larger site), used to produce other biomass energy, used to make animal feed, prepare yeast extract, yeast extract, etc., extract glucan sugar, preparing yeast extract, and more.
  • Yeast fermentation cells can be utilized in the above various ways, but in general, the added value of the processed products is limited, the market space is not large or there are many substitute products, and there are certain requirements for production equipment, production sites, and the environment, resulting in most Production companies would rather discharge the bacteria after harmless treatment.
  • yeast cells can also be used as raw materials for cosmetics, which is a high value-added treatment method.
  • yeast directly as a raw material it can also be prepared into various forms of cosmetic raw materials such as yeast derivatives.
  • the skin microecology is an ecosystem composed of various microorganisms such as bacteria, fungi, viruses, mites, and arthropods, as well as tissues, cells, various secretions, and the microenvironment on the skin surface.
  • various microorganisms such as bacteria, fungi, viruses, mites, and arthropods
  • tissues, cells, various secretions, and the microenvironment on the skin surface In recent years, with the deepening of research, skin microecology has also become the focus of attention in the cosmetics industry. In cosmetic applications, microecological skin care has also become the focus of product development for many international brand companies. International brands such as Estee Lauder, Lanc consultants, L'Oréal, and Procter & Gamble have successively developed and launched related products.
  • Procter &Gamble'sSK-II's FairyWater
  • the core raw material of which is Pitera is the filtrate of the fermentation product of galactosaccharomyces-like bacteria. Improves the barrier function of the skin by promoting the expression of tight junction proteins and increasing transmembrane resistance.
  • the core raw materials used in Estee Lauder's star products "Little Brown Bottle” and Lanc consultants's "Little Black Bottle” are lysates of the fermentation product of Saccharomyces bifidum. Although their names contain yeast, they are not Saccharomyces genus.
  • Bifidobacterium is obtained by culturing and lysing bacterial cells to obtain metabolites, cytoplasmic components, cell wall components and various organelle fragments.
  • major international mainstream raw material companies have also focused on micro-ecological skin care, and have successively launched various postbiotic raw materials, such as BASF's Relipidium, which is mainly composed of hydrolyzed yeast protein and has the effect of restoring the balance of skin flora; DSM's OXY 229PF
  • the main ingredient is yeast Bacterial lysate, which reduces Corynebacterium cruzi and thereby relieves facial redness.
  • Skin microecology the skin's own physical barrier and skin immunity together form the body's first defense system, resisting stimulation from various external physical and chemical factors and maintaining the body's normal functions. Therefore, the integrity of the skin barrier is of great significance to healthy skin, and the development of safer and more effective skin barrier repair raw materials has become a problem that needs to be solved in the development of current skin care products.
  • Existing cosmetic barrier repair raw materials mostly repair the physical barrier at the stratum corneum level, and there is a lack of cosmetic raw materials with skin barrier repair effects developed based on improving the skin's microecological barrier and enhancing the immune barrier.
  • raw materials derived from Pichia pastoris are lacking; and the functions of existing yeast cosmetics are mostly focused on moisturizing, anti-aging, sun protection, whitening, etc. to enhance skin micro-organisms.
  • Raw materials with ecological barrier, immune barrier, and control of sebum secretion as the basic functional mechanisms are also lacking.
  • the first thing to be completed is the lysis of the yeast cell wall.
  • the cell wall of Pichia pastoris is relatively thick (70nm-200nm) and has a dense structure, which is mechanically The intensity is high, the conventional treatment method takes a long time, is low in efficiency, and may also induce exogenous substances (enzymes).
  • the production cycle of fermentation bacteria is short, the volume is large, and the long processing time is easy to breed harmful bacteria and produce endotoxins and other pyrogenic substances; in large-scale production, the bacteria The crushing needs to be simple, stable and efficient to avoid the introduction of exogenous substances (which also helps control costs).
  • the broken Pichia pastoris lysate is a mixture of insoluble and soluble components, and effective separation also requires an effective treatment method.
  • the purpose of the present invention is to overcome some technical problems existing in the prior art, mainly to fill in the existing technology "preparation of fermentation lysate filtrate using Pichia pastoris expressing recombinant collagen" and its preparation Gaps in methods, product efficacy and applications solve the technical problems of long processing time, low efficiency, and the possibility of introducing exogenous substances in the preparation method of lysate filtrate in the existing technology.
  • the present invention provides a Pichia pastoris fermentation lysate filtrate, preparation method and application.
  • the present invention first provides a method for preparing a fermentation lysate filtrate.
  • the fermentation lysate filtrate is prepared from cells fermented by Pichia pastoris.
  • Pichia pastoris is Pichia pastoris expressing recombinant collagen.
  • Pichia pastoris expressing recombinant collagen is selected from the group consisting of CGMCC NO.7189, CGMCC NO.14057, CGMCC NO.17147, CGMCC NO.17150, CGMCC NO.17148, CGMCC NO. One or more strains of 17149, CGMCC NO.20626, CGMCC NO.20627, CGMCC NO.21891 or CGMCC NO.21892.
  • the bacterial cells are obtained by solid-liquid separation of fermentation products.
  • the preparation method of the fermentation lysate filtrate includes bacterial cell dilution, high-pressure homogenization lysis, centrifugation and filtration.
  • the bacterial cells are diluted with pure water to a bacterial cell suspension with a mass concentration of 5% to 30% (W/W); preferably, the bacterial cells are diluted with pure water to a mass concentration of 5% to 30% (W/W). 10% (W/W) bacterial cell suspension.
  • the high-pressure homogeneous lysis is to crush the bacterial cell suspension 1-3 times at a pressure of >100MPar to obtain a lysate; preferably, the high-pressure homogeneous lysis is to crush the bacterial cell suspension at a pressure of 1200MPar broken.
  • the centrifugation is centrifuging the lysate at a speed of 10,000g-20,000g to obtain a supernatant.
  • the filtration is to filter the supernatant with a pore size of 0.22 ⁇ m to obtain the fermentation lysate filtrate.
  • the present invention also provides fermentation lysate filtrate prepared by the preparation method.
  • the fermentation lysate filtrate includes total protein, recombinant collagen, polysaccharides, amino acids, and multiple vitamins.
  • the total protein content accounts for 0.1 to 2.5%, preferably 1% (10 mg/mL); the recombinant collagen content is 1 to 15 ⁇ g/mL. mL, generally above 10 ⁇ g/mL, preferably 10-15 ⁇ g/mL; polysaccharide content is about 0.1-1.5 mg/mL, preferably 1 mg/mL; total amino acid content is about 0.2% to 1%, preferably 0.6% to 0.82% .
  • the fermentation lysate filtrate of the present invention has the effects of anti-allergy, soothing, anti-inflammatory, sun protection, regulating skin microecology, controlling oil, and repairing skin barrier.
  • the present invention also provides a composition, which includes the fermentation lysate filtrate.
  • the content of the fermentation lysate filtrate in the composition is 1-10 wt%, preferably, the content is 5 wt%.
  • the present invention also provides applications of the fermentation lysate filtrate and the composition in anti-allergic, soothing, anti-inflammatory, sunscreen, skin microecological regulation, oil control, and skin barrier repair cosmetic raw materials.
  • the present invention also provides applications of the fermentation lysate filtrate and the composition in the field of cosmetics.
  • the cosmetics include anti-allergic, soothing, anti-inflammatory, sunscreen, skin microecological regulation, oil control, and skin barrier repair products.
  • the skin barrier repair category includes enhancing the skin's microecological barrier and improving the skin's immune barrier.
  • the anti-allergic, soothing and anti-inflammatory mainly include the ability to improve and relieve the symptoms of stratum corneum irritation and improve epidermal tissue damage; reduce the secretion level of inflammatory mediator PGE2 and alleviate the skin inflammatory reaction, such as reducing the vascular permeability in the skin, thereby Relieves skin redness; soothes skin irritation through the mechanism of inhibiting TRPV1 protein expression.
  • the sun protection is represented by the ability to absorb ultraviolet rays.
  • the regulation of skin microecology is manifested by increasing the expression of antimicrobial peptides and pattern recognition receptors, regulating the microecology and improving the skin barrier, thereby achieving a repair effect; under the action of the lysate filtrate of Pichia pastoris fermentation, HBD1, HBD2, LL-37, and TLR2 protein levels increased significantly.
  • the oil control is manifested by inhibiting the synthesis of skin lipid droplets.
  • the present invention also provides a cosmetic, which includes the fermentation lysate filtrate.
  • the cosmetic includes but is not limited to skin care essence, facial mask, and facial cream.
  • the content of the fermentation lysate filtrate in the cosmetics is 1-10 wt%, preferably, the content is 5 wt%.
  • the present invention also provides a skin care essence, the skin care essence includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and further Including deionized water, glycerin, butylene glycol, glycerol polyether-26, EDTA-2Na, sodium hyaluronate, allantoin, betaine, xanthan gum, and 1,2-pentanediol.
  • the skin care essence includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and further Including deionized water, glycerin, butylene glycol, glycerol polyether-26, EDTA-2Na, sodium hyaluronate, allantoin, betaine, xanthan gum, and 1,2-pentanediol.
  • the present invention also provides a facial mask.
  • the facial mask includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes deionized water, glycerol, EDTA-2Na, Sodium hyaluronate, xanthan gum, carbomer, pH adjuster, 1,2-pentanediol, 1,2-hexanediol.
  • the present invention also provides a skin care cream, which includes fermentation lysate filtrate.
  • the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerin, disodium EDTA, and xanthan gum.
  • Capric triglyceride hydrogenated polyisobutylene, polydimethylsiloxane, deionized water.
  • the present invention realizes for the first time the preparation of cosmetic raw materials by Pichia pastoris fermentation cells expressing recombinant human collagen, adding an effective and high value-added utilization method of recombinant human collagen fermentation cells, reducing the It eliminates the waste disposal pressure caused by yeast fermentation cells and turns waste into treasure.
  • the lysate filtrate prepared from the fermentation cells of Pichia pastoris expressing recombinant human collagen in the present invention contains a certain amount of collagen without additional addition.
  • the preparation method of the fermentation lysate filtrate in the present invention is a fully online, automated preparation process.
  • the Pichia pastoris fermentation cells are physically fragmented and lysed by high-pressure homogenization, and a continuous flow centrifuge is used to lyse the solids after lysis.
  • the liquid is separated, and the lysate filtrate is obtained after filtration.
  • the preparation process is fast and efficient, can be prepared on a large scale, and does not introduce any foreign ingredients.
  • the entire preparation process can be fully automated, and the materials are fully automated online pipeline transmission in the clean area. Complete sterile filtration and filling, which can effectively control the growth of harmful bacteria.
  • the lysate filtrate prepared by the present invention has no safety issues when used in cosmetics.
  • it also has the following functions: by promoting the secretion of antibacterial peptides, regulating the microecological balance, and enhancing the skin's microecological barrier.
  • Efficacy by inhibiting the expression of inflammatory factors, improving the skin immune barrier and soothing the skin; oil control and sun protection effects; these effects have not been seen in the raw material of "Pichia pastoris fermentation lysate filtrate" before, and have opened up its uses Application range in the preparation of cosmetics.
  • the prepared Pichia pastoris fermentation lysate filtrate has a dual barrier repair effect on the skin microecological barrier and immune barrier, which explains to a certain extent the mechanism of its efficacy in cosmetics.
  • Figure 1 is a flow chart of the fermentation lysate filtrate preparation method of the present invention.
  • Figure 2 shows the results of Gram staining of the lysate of Pichia pastoris after high-pressure homogenization under different parameter conditions in the example.
  • A1 is the lysate after lysing 3 times under 100MPar conditions
  • B1 is lysing 2 times under 120MPar conditions
  • C1 is the lysate after 2 times of lysis at 150MPar
  • D1 is the lysate after 2 times of lysis at 200MPar.
  • Figure 3 is the SDS-PAGE (A, loading volume: 10 ⁇ L) and WB chart (B) of the lysate filtrate prepared in Example 1.
  • lane 1 is the lysate filtrate, and the loading volume is 10 ⁇ L.
  • Lane 2 is a blank control
  • lane 3 is recombinant type III human collagen, and the loading amount is 2.5 ⁇ g.
  • Lane 4 is recombinant type III human collagen, and the loading amount is 1 ⁇ g.
  • Figure 4 is a standard curve fitted during the calculation of the concentration of recombinant collagen in the sample in the example.
  • Figure 5 shows the standard curve and curve equation fitted during the calculation of the total protein content in the sample in the example.
  • Figure 6 shows the standard curve and curve equation fitted during the calculation of the polysaccharide content in the sample in the example.
  • Figure 7 is an embodiment based on a 3D epidermal skin model.
  • Picture A is the blank control
  • B is the negative control, that is, the SLS treatment group
  • C is the positive control, that is, SLS and 0.01% dexamethasone are added at the same time
  • D is the experimental group. , that is, add SLS and 2% filtrate at the same time.
  • Figure 8 is a histogram of PGE2 content detection results in the embodiment.
  • FIG 9 is the immunofluorescence detection result of capsaicin receptor (TRPV1) in the Example.
  • Figure 10 is a histogram of relative IOD values of TRPV1 protein in Examples.
  • Figure 11 shows the ultraviolet rate of the fermentation lysate filtrate obtained by the present invention and its dilutions at different times in the ultraviolet region of 200-400nm wavelength.
  • Figure 12 is a 3D epidermal skin model. HBD1 protein staining results.
  • Figure 13 is a 3D epidermal skin model. Histogram of HBD1 protein integrated optical density (IOD) values.
  • Figure 14 is a 3D epidermal skin model. HBD2 protein staining results.
  • Figure 15 is a 3D epidermal skin model. Histogram of HBD2 protein integrated optical density (IOD) values.
  • Figure 16 is a 3D epidermal skin model. TLR2 protein staining results.
  • Figure 17 is a 3D epidermal skin model. Histogram of integrated optical density (IOD) values of TLR2 protein.
  • Figure 18 is a 3D epidermal skin model. LL-37 protein staining results.
  • Figure 19 is based on the 3D epidermal skin model Histogram of integrated optical density (IOD) values of LL-37 protein.
  • Figure 20 shows the results of synthetic Oil Red O staining of lipid droplets; the scale bar in the figure is 50 ⁇ m.
  • Figure 21 is a histogram of lipid droplet integrated optical density (IOD) values.
  • Figure 22 shows the effect of volunteers using the essence of Pichia pastoris fermentation lysate filtrate.
  • Figure 23 is a picture of the effect of volunteers using a facial mask containing Pichia pastoris fermentation lysate filtrate.
  • Figure 24 is a picture of the effect of volunteers using a facial cream containing Pichia pastoris fermentation lysate filtrate.
  • the raw material strains involved in the preparation of fermentation lysates in the present invention are all from the applicant's unit, and have made corresponding strain deposits. They are all exclusive Pichia pastoris engineering strains that express recombinant collagen, and are all deposited in China.
  • General Microbiology Center of the Microbial Culture Collection and Management Committee including the publication number CN103102407B, the preservation number is CGMCC NO.7189, the publication number CN107090458A, the preservation number is CGMCC NO.14057, the publication number CN111004319A, the preservation number is CGMCC NO.17147, the publication number
  • the accession number in CN110964099A is CGMCC NO.17150, the accession number in Publication No.
  • CN110606896B is CGMCC NO.17148
  • the accession number in Publication No. CN110747198B is CGMCC NO.17149
  • the accession number in Publication No. CN113185604B is CGMCC NO.20626
  • the accession number is The strains deposited in CGMCC NO. 20627 and Publication No. CN114106150A are CGMCC NO. 21891 and CGMCC NO. 21892.
  • the recombinant human collagen expressed by these engineered bacteria has a 6 ⁇ His-Tag tag at the amino terminus (N terminus) or carboxyl terminus (C terminus), which facilitates detection.
  • the expression and production of recombinant human collagen by the engineered strain of Pichia pastoris is secretory expression: after the protein is translated, it enters the endoplasmic reticulum. After excision of the signal peptide, it is processed by the Golgi apparatus and secreted outside the cell. Translation, processing, and secretion are The last three stages of recombinant human collagen expression and production are carried out simultaneously when cells express a large amount of protein. Therefore, although most recombinant human collagen is secreted extracellularly, there is still collagen in Pichia pastoris cells. Retain. For large-scale fermentation production of recombinant human collagen, generally 20 to 30% of the volume in the fermentation tank is fermentation bacteria.
  • the bacterial cells and the supernatant of the fermentation broth are separated (ceramic membranes or continuous flow centrifuges are often used) to produce a bacterial slurry with a high concentration of bacterial cells.
  • the main components are water and wet bacteria.
  • the wet bacterial cell concentration can reach about 50% (W/W) and can be directly used as raw material for the preparation of dissolved filtrate.
  • the wet bacterial cell concentration described in the present invention is the ratio of the weight of the (water-containing but not dry) bacterial cells/the weight of the suspended bacterial cell liquid. The calculation methods and results in specific cases are given in the article.
  • Yeast cell wall is thicker, more complex in structure than bacterial cell wall, and stable in nature. It can maintain the integrity of its structure and components after the death or apoptosis of protoplasts. The function of the biofilm system of dead protoplasts is lost and dissolved.
  • the enzymes in the enzyme body enter the cytoplasm and decompose a large number of substances in the cytoplasm and nucleus. Various small molecule substances escape through the cell wall, but macromolecular substances (such as nucleic acids, proteins, etc.) can only be degraded into small molecule substances. Only then can it pass through the cell wall, and the cell wall is slightly affected and can still maintain its integrity. This is also a major difference between yeast cells and bacterial and animal cells during cell disruption. That is, when cells are disrupted, only complete lysis of the cell wall can obtain a relatively complete lysate.
  • the present invention chooses to use a high-pressure homogenization method to effectively break the cell wall of yeast. This is also a physical breakage method that is fast, efficient and does not induce foreign substances.
  • a high-speed continuous flow centrifuge can be used to separate the insoluble matter (cell wall insoluble glucan, insoluble protein, etc.) and soluble matter in the lysate.
  • the lysate filtrate was prepared.
  • the effect of Pichia pastoris cell lysis can be detected by microscopic examination.
  • Gram staining method can be used (Gram staining kit purchased from Beijing Solebao Technology Co., Ltd.). The principle is also the same as that of Gram staining. Lang's staining is similar: after initial dyeing with crystal violet and mordant with iodine solution, a water-insoluble complex of crystal violet and iodine is formed in the cell wall, staining the cytoplasm purple.
  • the yeast cell wall is thicker and more dense than the cell wall of Gram-positive bacteria. Dense. During ethanol decolorization, the mesh will shrink due to water loss. In addition, it does not contain lipids, so there will be no gaps after ethanol treatment.
  • the crystal violet and iodine complex can be firmly retained in the wall, so When the cell wall is intact, it remains purple. If the yeast cell wall is ruptured and treated with ethanol, the crystal violet and iodine complex will be dissolved and decolorized, and it will remain colorless. It will then turn red after being counterstained with red dyes such as safranin. After Gram staining, cells with unbroken cell walls are purple, cells with ruptured cell walls (including cell types that still maintain a relatively complete morphology) and substances released from the cells are dyed red, and the high-pressure homogenized broken products are overall in A uniform red background is formed within the field of view.
  • D (100%): wall breaking rate; C: number of cells before breaking; C’: total number of cells after breaking; a: proportion of unbroken cells after breaking.
  • the recombinant human collagen expressed by the engineering bacteria used in this example has a 6 ⁇ His-Tag tag at the amino terminus (N terminus) or carboxyl terminus (C terminus), which facilitates the detection of collagen in the filtrate and can be carried out using Western Blot. Intuitive qualitative detection and quantitative detection using Elisa.
  • the deposit number in the publication number CN103102407B is CGMCC NO.7189 Taking Chengji as an example, the lysate filtrate prepared by the fermentation bacteria with a wet bacterial concentration of 10% was used for corresponding testing.
  • Figure 3A is an SDS-PAGE electrophoresis diagram of the lysate filtrate prepared with a wet bacterial cell concentration of 10%. Proteins of different sizes are distributed from top to bottom;
  • Figure 3B is a lysate prepared with a wet bacterial cell concentration of 10%.
  • GraphPad Prism 5 fits the standard curve, performs data processing, and calculates the concentration of recombinant collagen in the sample.
  • the fitted standard curve is shown in Figure 4.
  • the abscissa is the logarithm of the collagen concentration, and the ordinate is the absorbance value at 450nm.
  • Y is the protein concentration
  • X is the absorbance light value at 450nm of the detected sample.
  • the better detection range is that the OD450 range is approximately between 0.25 and 1.25.
  • What can be effectively calculated in the experimental group is the OD450 value of the Elisa experiment after diluting the centrifugation supernatant of the broken liquid 10 times with ddH 2 O.
  • the concentration of recombinant type III human collagen in the lysate filtrate was 11.44 ⁇ g/mL.
  • the recombinant collagen content of the lysate filtrate prepared with a wet bacterial cell concentration of 10% is approximately 10-15 ⁇ g/mL.
  • the OD450 of the lysate filtrate prepared from the blank Pichia pastoris without the collagen gene expression was basically outside the lower limit of the effective detection range, and no collagen could be detected.
  • the experimental method refers to the fifth method of protein content determination in the "Pharmacopoeia of the People's Republic of China" (2020 edition): Coomassie Brilliant Blue method (Bradford method) to detect the total protein content.
  • the lysate filtrate was prepared from the fermentation cells of CGMCC NO.7189 engineering bacteria in the patent with the publication number CN103102407B, with a wet cell concentration of 0.2% to 20%.
  • the Bradford protein concentration determination reagent was purchased from Beyotime Biotechnology (P0006). Experimental steps A brief description is as follows:
  • microplate reader (Varioskan TM LUX multifunctional microplate reader-VL0L0TD0, Thermo Fisher Scientific (China) Co., Ltd.) to measure the absorbance value at 595 nm.
  • the slope (R 2 ) of the standard curve in the Bradford method is >0.98.
  • the total protein content in the lysate filtrate increases, ranging from 0.192 to 26.9 mg/mL.
  • the wet bacterial cell concentration is above 10%, the total protein content in the lysate filtrate is greater than 10 mg/mL (>1%), and can reach a maximum of more than 25 mg/mL (2.5%).
  • the total protein content in the filtrate produced by lysis is generally 10 to 15 mg/mL.
  • Mannose standard solution 0.1 mg/mL: Precisely weigh 500 mg of mannose dried to constant weight at 105°C and place it in a 500 mL volumetric flask. Add water to dissolve and dilute to the mark. Shake well to obtain 1 mg/mL. Reserve solution for later use. Pipette 10 mL of the upper solution with a 10 mL pipette and adjust the volume to 100 mL to obtain a 0.1 mg/mL mannose standard solution.
  • Preparation of 5% phenol solution Take out 85% phenol in a water bath and heat it to dissolve at 45 degrees, take 1 mL of the solution, and then dissolve it in 16 mL of pure water to obtain a 5% phenol solution. Store away from light, use freshly prepared.
  • Standard sample preparation and standard curve drawing Accurately draw mannose standard solution (0.1 mg/mL) and prepare according to Table 3. Add 0.5 mL of phenol solution and 2.5 mL of concentrated sulfuric acid (quick and precise addition), mix for 20 seconds, place in a water bath at 80°C for 30 minutes, cool, and measure the absorbance value at a wavelength of 490 nm. Use a blank (blank) reference experiment, draw a standard curve with the absorbance value as the ordinate and the mannose concentration as the abscissa, and establish a standard curve regression equation.
  • W the polysaccharide content of the sample
  • W1 the solubility of the polysaccharide calculated under the standard curve regression equation
  • W2 the solubility of the polysaccharide in the sample lysate filtrate.
  • the polysaccharide content (mg/mL) in the prepared lysate filtrate is as follows:
  • the polysaccharide content in the general lysate filtrate is 1-1.6mg/mL.
  • the lysate filtrate was prepared with a 10% wet cell concentration of fermentation cells of CGMCC No. 7189 engineering bacteria in Publication No. CN103102407B.
  • the lysate filtrate prepared with a wet bacterial cell concentration of 10% contains a variety of B vitamins and amino acids, and the total amino acid content can reach about 0.82%. As a raw material, it can provide rich active ingredients for cosmetics.
  • the DNA in the lysate filtrate is mainly the genomic DNA residue of Pichia pastoris.
  • the PCR-fluorescent probe method was used to detect the DNA in the lysate filtrate prepared with a bacterial concentration of 10%.
  • Huzhou Shenke Biotechnology Co., Ltd. was commissioned Complete and issue test report. The basic process is briefly described as follows. The relevant reagents during the experiment are produced by Huzhou Shenke Biotechnology Co., Ltd. or are commercially available unless otherwise specified:
  • Pichia pastoris-specific primers and probes Pichia pastoris residual DNA detection kit (PCR-fluorescent probe method), purchased from Huzhou Shenke Biotechnology Co., Ltd. SK030205P100
  • lyse Specific fragments of DNA in the filtrate samples were amplified in vitro (quantitative PCR instrument FQD-96A, LineGene 9600Plus quantitative PCR system);
  • the specific probe will be released from the template DNA, and then fluorescence quantification will be used.
  • the PCR instrument can detect the fluorescence signal, and the fluorescence signal data is analyzed through the detection software of the PCR instrument.
  • Detection mean the average value of detection values.
  • CV Coefficient of Variance
  • Added standard amount refers to the amount of standard added.
  • Average value of spiked test the average value of the added standard and reference test values.
  • the results show that the DNA content in the lysate filtrate prepared by the present invention was detected to be 31.4 ⁇ g/mL, which is lower than the DNA residual amount required in biological drugs and is within a safe range.
  • the results show that the heavy metal and methanol contents in the lysate filtrate prepared by the present invention meet the safety requirements.
  • the lysate filtrate in the present invention is a cosmetic raw material, and will produce an actual dilution effect during the preparation and use of actual products. There are no security issues.
  • the lysate filtrate was tested for microbial limits, and the Suzhou Customs Comprehensive Technology Center was entrusted to complete and issue a test report. The results all met the safety requirements.
  • the test results are as follows As shown in Table 7, the results show that the microbial results in the lysate filtrate prepared by the present invention meet the requirements.
  • Test sample processing Weigh 5.0806g sample (i.e., the lysate filtrate prepared by the present invention) and place it in a beaker. Take a small amount of pure water, mix the sample evenly, and transfer it to a 20mL volumetric flask. Rinse the beaker multiple times with a small amount of pure water. Transfer it together into a volumetric flask, add pure water to adjust the volume to the mark, shake well and transfer it into a sample tube to mark for later use. The sample is ready for use.
  • the intragastric administration volume is 2.0mL/100g ⁇ BW.
  • mice 10 6-week-old SPF grade ICR mice (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half female (female animals are not pregnant and have never given birth), and the weight is controlled between 18.5g and 21.4g (the same sex weight) Not more than ⁇ 20% of the mean)
  • the animals were adapted to the barrier environment animal room (barrier environment, 23.0°C to 23.8°C, relative humidity of 40.8% to 59.7%) for 8 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.; Level 1 RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization) is directly provided to the animals for free drinking through the drinking spout). The animals fasted overnight before exposure.
  • the one-time limited method was adopted, and the intragastric dose was 5080.6 mg/kg.
  • the intragastric dose was 5080.6 mg/kg.
  • the experimental animals did not show any poisoning symptoms or death from poisoning within 14 days of exposure; there were no abnormalities in the body weight of male and female animals. After the experimental observation was completed, no abnormal changes were found in the gross anatomical examination of the test animals. LD50>5080.6mg/kg, the detailed results are as follows:
  • the acute oral LD50 of the test sample to ICR mice is >5000mg/kg. According to the acute oral toxicity classification, this sample belongs to the actual non-toxic grade.
  • Test sample processing Use the original sample lysate filtrate for testing.
  • mice 10 8-week-old SPF grade SD rats (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half female (female animals are not pregnant and have never given birth), and the weight is controlled between 201.2g and 218.2g.
  • the animals were adapted to the barrier environment animal room (barrier environment, 22.0°C to 22.9°C, relative humidity of 45.8% to 55.5%) for 7 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.; Level 1 RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization) is directly provided to the animals through the drinking spout. The animals do not fast before being exposed to the poison and can drink water freely.
  • the one-time limit method was adopted, and the dose was 2500mg/kg.
  • animals were kept in individual cages.
  • the body weight before exposure ranged from 201.2g to 218.2g, and the calculated 10% body surface area was 31.3cm 2 to 33.1cm 2 .
  • 24 hours before the test the coat of the infected area on the back of the animal was shaved, and the skin preparation area was 5cm ⁇ 7cm.
  • Fixtures and coverings are washed with water to remove any remaining test sample from the skin. Observe and record the poisoning process and the poisoning and death of animals during the observation period. Weigh once a week. The observation period is 14 days. After the observation period, the surviving animals are killed and gross dissection is performed.
  • Test results The experimental animals did not show any poisoning symptoms or death from poisoning within 14 days of exposure; there were no abnormalities in the body weight of male and female animals. After the experimental observation was completed, no abnormal changes were found in the gross anatomical examination of the test animals. LD50>2500mg/kg. The results are shown in Table 10 below.
  • the acute percutaneous LD50 of the test sample to SD rats is >2180 mg/kg. According to the acute skin toxicity classification, the sample is slightly toxic.
  • Test sample processing Use the original sample lysate filtrate for testing.
  • mice 4 ordinary-grade New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.27kg and 2.98kg.
  • the animals were adapted to the environment of the experimental animal room for at least 3 days. They were raised in a single cage. The environment temperature was 22.8°C to 23.6°C, and the relative humidity was 52.9% to 61.4%.
  • the feeding feed was experimental rabbit compound feed (maintenance type, Tongxiang Dongfang). Feed Co., Ltd.); first-level RO ultrafiltered water (adding sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
  • Test results During the test, none of the experimental animals showed abnormal symptoms, and the most severe symptoms at each observation time point (24h, 48h and 72h) The mean high skin irritation score is 0.00. Under the conditions of this test, the acute skin irritation of the test sample to rabbits is: non-irritating.
  • Test sample processing Use the original sample lysate filtrate for testing.
  • mice 4 ordinary-grade New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.34kg and 2.92kg.
  • the animals were adapted to the experimental animal room environment for at least 3 days, and were raised in single cages.
  • the breeding environment temperature was: 22.0°C ⁇ 23.9°C, and the relative humidity was: 40.5% ⁇ 58.6%;
  • the feeding feed was experimental rabbit compound feed (maintenance type, Tongxiang Dongfang Feed Co., Ltd.); first-level RO ultrafiltered water (added with sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
  • test results During the test period, no experimental animals showed abnormal symptoms, and the average score of each animal per day within 14 days was 0.00. See Table 12 below for details. Under the conditions of this test, the test sample is non-irritating to the skin of rabbits.
  • Test sample processing Use the original sample lysate filtrate for testing.
  • mice 3 ordinary New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.40kg and 2.61kg.
  • the animals were adapted to the environment of the experimental animal room for at least 3 days and were raised in single cages.
  • the breeding environment temperature was: 23.0°C ⁇ 24.1°C, and the relative humidity was: 49.8% ⁇ 60.7%;
  • the feeding feed was: experimental rabbit compound feed (maintenance type, Tongxiang Dongfang Feed Co., Ltd.); first-level RO ultrafiltered water (adding sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
  • Test method Examine both eyes of the test animals within 24 hours before the start of the test (including examination using 2% fluorescein sodium solution). Animals with symptoms of eye irritation, corneal defects, and conjunctival damage should not be used for testing. Gently open the lower eyelid of one eye of the rabbit, drop 0.1 mL of the test sample into the conjunctival sac, and passively close the upper and lower eyelids for 1 s to prevent the loss of the test sample. The other eye was left untreated for self-control. Do not rinse eyes within 24 hours after instilling the test sample. The eyes of the animals were examined at 1h, 24h, 48h, 72h, and on the 4th and 7th days after instillation of the test sample. If no irritation reaction occurs within 72 hours, the test can be terminated.
  • Test results During the test, the experimental animals showed no abnormal symptoms, and the average individual points of the animals were all 0.00. See Table 13 below for details. Under the conditions of this test, the acute eye irritation of the test sample to rabbits is: non-irritating.
  • the average animal score refers to the average score of cornea, iris, conjunctival congestion and conjunctival edema at three different observation times (24h, 48h and 72h) for each animal (i.e. the 24h, 48h and 72h scores of each animal) The sum is divided by the number of points at the time of observation 3).
  • Test sample processing Use the original sample cell lysate filtrate for testing.
  • Positive control 8-methoxypsoralen (batch number: L630V07; Beijing Bailingwei Technology Co., Ltd.), solvent: absolute ethanol, concentration: 0.05%; dosage: 2.4000 mg.
  • mice 6 ordinary-grade Dutch guinea pig test group and 6 positive control group (Jianfei Experimental Rabbit Breeding Farm, Simen Town, Yuyao City); there is no limit on male or female (female animals are selected as non-pregnant and non-pregnant animals); the weight is controlled at 261.2 g ⁇ 272.4g.
  • the animals were adapted to the environment of the experimental animal room for at least 3 days before the test. Breeding environment temperature: 23.3°C ⁇ 24.0°C; relative humidity: 49.5% ⁇ 56.7%; feeding feed: guinea pig compound feed (maintenance type, Suzhou Anmufei Biotechnology Co., Ltd.); first-level RO ultrafiltered water (add sodium hypochlorite, The free chlorine content in the water is controlled at 2-3ppm (sterilization). The animals can drink directly through the drinking spout.
  • the irradiation time is 00:10:64 according to the calculation formula (that is, the irradiation time is 664s);
  • Test procedures The animals must adapt to the environment of the experimental animal room for at least 3 days before the test. About 24 hours before the test, the skin on both sides of the animal's spine should be hairless. The skin at the test site must be intact and free of damage and abnormalities. Prepare 4 hair removal areas, each with a hair removal area of approximately 2cm x 2cm. Fix the animal, and apply 0.2 mL of the test sample on the animal hair removal areas 1 and 2 as shown in Table 1 of Chapter 6, 7 Skin Phototoxicity Test of the "Technical Specifications for Safety of Cosmetics" (2015 Edition). After 30 minutes, the left side (hair removal areas 1 and 3) was covered with aluminum foil and fixed with tape, and the right side was irradiated with UVA.
  • test results During the test, no experimental animals showed abnormal symptoms, and the animal points at each observation time point were all 0. According to the skin irritation response classification, no skin phototoxicity was found in the test sample.
  • the phototoxicity test results of the positive control substance on guinea pig skin are shown in Table 14 below, and the phototoxicity test results of the test sample on guinea pig skin are shown in Table 15. Under this test condition, the test results of the test sample's phototoxicity to guinea pig skin: no skin phototoxicity was found.
  • Table headers 1, 2, 3, and 4 are the test areas shown in Figure 1 of Chapter 6, 7. Skin Phototoxicity Test, of "Cosmetic Safety Technical Specifications” (2015).
  • Test sample processing Use the original sample cell lysate filtrate for testing.
  • Positive control ⁇ -hexylcinnamic aldehyde (batch number: LV30V28; Beijing Bailingwei Technology Co., Ltd.); solvent: absolute ethanol (analytically pure, batch number: 20210119, Sinopharm Chemical Reagent Co., Ltd.), acetone (analytically pure, batch number: 20170918, Sinopharm Chemical Reagent Co., Ltd.), Millipore Pure water.
  • Induction concentration 60%, prepared by mixing 3.6mL ⁇ -hexylcinnamic aldehyde and 2.4mL 80% ethanol solution; excitation concentration: 30%, prepared by mixing 3.0mL ⁇ -hexylcinnamic aldehyde and 7.0mL 80% acetone solution; Dosage: 6.6mL.
  • mice ordinary Dutch guinea pigs (Jianfei Experimental Rabbit Breeding Farm, Simen Town, Yuyao City); 20 test groups and 20 positive control groups each, and 10 negative control groups; male or female (female animals are not pregnant and have never given birth) ); weight is controlled between 257.8g and 273.4g.
  • the animals were adapted to the environment of the experimental animal room for at least 3 days before the test. Breeding environment temperature: 22.5°C ⁇ 24.1°C; relative humidity: 43.4% ⁇ 61.9%; feed: guinea pig compound feed (maintenance type, Suzhou Anmufei Biotechnology Co., Ltd.), first-level RO ultrafiltered water (add sodium hypochlorite, The free chlorine content in the water is controlled at 2-3ppm (sterilization). The animals can drink directly through the drinking spout.
  • Test method About 24 hours before the test, remove the hair on the left side of the guinea pig's back, and the area of hair removal is about 6cm 2 .
  • Induced contact Apply 0.2 mL of the test sample on the 2cm ⁇ 2cm hairless skin of the animals in the test group, cover it with two layers of gauze and one layer of cellophane, and then seal and fix it with non-irritating tape for 6 hours. Repeat steps 7d and 14d in the same way.
  • the positive control group was treated with 60% ⁇ -hexylcinnamic aldehyde solution in the same manner, and the negative control group was treated in the same manner as the test group except that no test sample was given.
  • Provocative exposure 14 days after the last induction, apply 0.2 mL of the test sample to the 2cm ⁇ 2cm hair removal area on the right side of the back of the guinea pigs in the test group and the negative control group (hair removal 24 hours before contact), and then cover it with two layers of gauze and one layer of cellophane. , and then fixed with non-irritating tape for 6 hours.
  • the positive control group used 30% ⁇ -hexylcinnamic aldehyde solution in the same manner. Observe the skin reaction 24h and 48h after the provocative exposure, score and determine the sensitization intensity.
  • test results During the test, no experimental animals showed abnormal symptoms, and the sensitization rate at each observation time point was 0%. See Table 16 and Table 17. Under this test condition, the test results of the guinea pig skin allergy test of the test sample were: no skin allergy was observed.
  • Test sample processing Use sterile pure water as the solvent to prepare the concentrations of each dosage group.
  • Negative control sterile pure water, dimethyl sulfoxide (DMSO, colorless clear liquid)
  • Dexon Batch number: G1063485; Manufacturer: Dr. Enrenstorfer GmBH, Germany; Solvent: sterile pure water; Concentration: 500 ⁇ g/mL; Dosage: 0.1mL/dish;
  • SA Sodium azide
  • 2-Aminofluorene (2-AF) Batch number: C11449586; Manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.; Solvent: dimethyl sulfoxide (DMSO); Concentration: 100 ⁇ g/mL; Dosage: 0.1mL/dish;
  • 1,8-Dihydroxyanthraquinone (1,8-DHQ): Batch number: C10429233; Manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.; Solvent: DMSO; Concentration: 500 ⁇ g/mL; Dosage: 0.1mL/dish;
  • Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) were purchased from the American MOLTOX company. After identification, the test strains met the standard requirements.
  • Test steps Select four dosage groups of 12.5 ⁇ L/dish, 25.0 ⁇ L/dish, 50.0 ⁇ L/dish and 100.0 ⁇ L/dish, and set up blank control, solvent control (negative control), positive mutagen control and sterile control group. .
  • the bacterial strain is enriched and cultured to prepare an enrichment liquid, which is then incorporated into the plate.
  • 2.0 mL of the top medium containing 0.5 mmol/L histidine and 0.5 mmol/L biotin solution was divided into test tubes and incubated in a 45°C water bath. Then, 0.1 mL of enrichment solution and 0.1 mL of enrichment solution were added to each tube.
  • Test results The standard plate incorporation test results are shown in Table 18. The results of the solvent control (negative control) showed that without the addition of the test strain Salmonella typhimurium, no colonies grew in each group and could be used for the test; the number of reverse mutation colonies in the blank control and positive mutagen control groups were within an acceptable range. Within, this test is valid;
  • Test sample processing The sample lysate filtrate is prepared into solutions for each dose group using pure water as the solvent.
  • mice 30 SPF grade ICR mice (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half male (female animals are not pregnant and have never given birth), the weight is controlled between 25.0g and 27.4g, and the weight of the same sex does not exceed the average. ⁇ 20%.
  • the animals were kept in a barrier environment animal room (barrier environment, 22.8°C to 23.5°C, relative humidity of 51.6% to 57.8%) for a quarantine period of 4 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.;
  • the first-level RO ultrafiltered water sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization
  • the animals do not fast before being exposed to the poison and can drink water freely.
  • Test method Dosage setting: Set up test substance group, negative control group (pure water) and positive control group (60mg/kg cyclophosphamide). Based on the preliminary test results, the final concentrations of the test substance groups were set to 1000 mg/kg, 2000 mg/kg, and 5000 mg/kg.
  • Administration method Oral gavage; Administration volume: 20mL/kg ⁇ BW.
  • Test procedures The animals were exposed to the poison by oral gavage for 30 hours, that is, the interval between two exposures was 24 hours, and the samples were taken 6 hours after the second exposure. The animals were sacrificed by cervical dislocation, and the femurs were removed. Peel off the muscles and wipe away the blood. Cut off both ends of the femur to expose the medullary cavity. Use a syringe to absorb 0.1 mL of calf serum, rinse the bone marrow cavity, use regular smear with flushing solution, and air dry or blow dry with hot air. The dried smears were fixed in methanol for 10 min. Dye with Giemsa application solution for 15 minutes, then rinse with water and dry.
  • the operating procedures of the positive and negative control groups were the same as those of the experimental group. Select an area where the cells are evenly distributed, intact, and properly colored. Count the contents under an oil lens The number of PCE (polychromatic erythrocytes) with micronuclei. PCE is gray-blue, and NCE (mature red blood cells) is pink. The number of micronuclei contained in 2000 PCEs was counted for each animal. Micronucleus cell rate (MN ⁇ ) refers to the number of PCEs containing micronuclei, expressed in parts per thousand. If 2 or more micro-cores appear in 1 PCE, they are still counted as 1. Use the Poisson distribution u test or other appropriate significance test methods to compare the micronucleus rates between groups.
  • Test results The test results showed that compared with the negative control group, the number of polychromatic cells containing micronuclei in the bone marrow of the mice in the positive control group was significantly increased (P ⁇ 0.05), while the number of polychromatic erythrocyte micronuclei in each test group was significantly higher (P ⁇ 0.05). Compared with the negative control group, the difference was not statistically significant (P>0.05).
  • Acute toxicity test (acute oral toxicity test detects non-toxicity, acute dermal toxicity test detects slight toxicity), skin irritation/corrosion, skin sensitization, skin phototoxicity and genetic toxicity, etc. The results show that the filtrate has good toxicological safety.
  • test results showed that 0.5h, 24h, and 48h after removing the test substance spot tester, the scores of 32 subjects were all 0, and no skin reaction occurred; 1 subject The tester's score level is 1 24 hours after removing the test object spot tester.
  • the specific experimental results are shown in Table 20 below.
  • the number of grade 1 adverse skin reactions among 30 subjects shall not be more than 5 (excluding 5 cases).
  • the specific grading standards are shown in Table 21 above. The results show that the test results meet the prescribed safety requirements. Accordingly, it is believed that the lysate filtrate prepared in the present invention is safe to be applied to the human body.
  • Pichia pastoris fermentation lysate filtrate prepared by the present invention was tested for heavy metal content, methanol content, microbial limits, safety toxicology, and human skin patching according to the method in the "Cosmetic Safety Technical Specifications" Experimental testing and residual DNA testing were conducted to prove that its safety meets the requirements for application in cosmetics.
  • Example 1 the lysate filtrate prepared in Example 1 was used as an example to conduct multiple efficacy evaluations.
  • the epidermis is a tissue that separates the body from the external environment.
  • keratinocytes can receive stimulation from external "danger signals" and transform them into early warning cells for immune cells in the skin, forming an immune barrier for the skin.
  • Disorders in the regulation of the skin's immune barrier function can cause Causes inflammatory reactions in the skin, causing skin problems and diseases.
  • Skin contact with some highly irritating cosmetics or ultraviolet rays can cause clinical acute damage to the skin barrier, resulting in dry skin and erythema.
  • the anionic surfactant sodium lauryl sulfate (SLS) has amphiphilic (hydrophilic and lipophilic) characteristics and can damage the skin barrier, especially the lipid components in the barrier, after contacting the skin at a large concentration. and cell membranes. Therefore, this experiment used an in vitro reconstituted epidermal model As the research object, an injury model caused by SLS stimulation was used, and then the lysate filtrate obtained in Example 1 was used for treatment. Related inflammatory factors and tissue morphology changes were used as indicators, and tissue morphology, inflammatory mediator PGE2 and capsaicin receptors were detected. To evaluate the soothing effect of the lysate filtrate prepared by the present invention based on changes in the body TRPV-1 content, Shaanxi Boxi General Testing Technology Co., Ltd. was entrusted to conduct the test and issue a test report.
  • PGE2 test After administration and incubation for 24 hours, collect the 3D epidermal skin model culture fluid in EP tubes. After collection, the samples for ELISA testing are frozen and stored in a -80°C refrigerator. According to PGE2 Perform detection and analysis according to the operating instructions of the ELISA kit.
  • Tissue morphology test Take the model for tissue morphology detection (i.e., the epidermal model processed according to Table 22), fix it with 4% paraformaldehyde, and after 24 hours of fixation, remove the model ring and perform H&E staining. Detect, take photos and observe under a microscope, collect pictures and analyze them.
  • Capsaicin receptor (TRPV1) test Take the model used for testing (i.e., the epidermal model processed according to Table 22), fix it with 4% paraformaldehyde, and after fixing for 24 hours, remove the model ring. Carry out immunofluorescence detection of capsaicin receptor (TRPV1), take pictures and observe them under a microscope, collect pictures and analyze them.
  • FIG. 7 is the tissue morphology diagram of the epidermal model after H&E staining under the microscope after the filtrate culture is completed.
  • Figure 7A is the blank control
  • 7B is the negative control, that is, the SLS treatment group
  • 7C is the positive Control, that is, SLS and 0.01% dexamethasone are added at the same time
  • 7D is the experimental group, that is, SLS and 2% filtrate are added at the same time.
  • Histological morphology is an analysis of the microphysiological structure of the tissue after HE staining. Through the histological morphology of the model, we can see the changes in the skin barrier under different treatment conditions. When damage is caused by SLS stimulation, the skin barrier becomes loose and the viable cell layer The thickness is reduced and the morphological changes after treatment by adding active substances are used to determine whether it has the effect of improving this damage. The tissue morphology test results showed that compared with the blank group, the number of cells in the negative group model after SLS stimulation was reduced, the arrangement was disordered, the stratum corneum was loose, and vacuoles appeared, indicating that the stimulation conditions of this test were effective.
  • the porosity and vacuoles in the active stratum corneum of the positive control group model were significantly improved, indicating that the positive control test was effective.
  • the looseness and vacuoles of the stratum corneum were significantly improved after treatment with 2% Pichia pastoris fermentation lysate filtrate, indicating that 2% filtrate under in vitro conditions can help relieve chemical stimulation caused by SLS. It can improve the irritation reaction caused by stimulation and improve the damage of epidermal tissue caused by stimulation.
  • the PGE2 test results are shown in Table 23, and the histogram of the PGE2 content test results is shown in Figure 8.
  • PGE2 is also the most commonly synthesized and widely distributed PG in mammals.
  • the main source of PG in the skin is keratinocytes.
  • the main PGs synthesized by keratinocytes include PGE2, PGF2a and PGD2.
  • PGE2 has the effect of dilating and relaxing blood vessels, manifesting as skin flushing; in substances such as histamine and kinin, Under the synergistic effect, it causes an increase in vascular permeability, causing local redness and swelling of tissues, and can cause local fever.
  • PGE2 entering the blood can also combine with receptors in the hypothalamus to cause changes in the body temperature center, thereby causing systemic fever; In addition, PGE2 can stimulate nerve endings and cause pain.
  • PGE2 as an inflammatory mediator, participates in the occurrence and development of inflammatory responses through multiple pathways. PGE2 is a strong vasodilator, slowing down blood flow and promoting the attachment and adhesion of white blood cells.
  • PGE2 as a pro-inflammatory mediator can also mediate the production of other inflammatory mediators and inflammatory factors such as IL-6.
  • the TTRPV1 immunofluorescence detection results showed that compared with the BC group, the fluorescence intensity of model TRPV1 in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the fluorescence intensity of model TRPV1 in the PC group was significantly reduced, indicating that this positive control test was effective. Compared with the NC group, the fluorescence intensity of the model TRPV1 in the Pichia pastoris fermentation lysate filtrate treatment group with a sample concentration of 2% was significantly reduced.
  • the TRPV1 immunofluorescence detection results are shown in Figure 9, the TRPV1 data results are shown in Table 24, and the TRPV1 protein relative IOD value histogram is shown in Figure 10.
  • TRPV1 Transient receptor potential vanilloid type 1
  • capsaicin receptor is an important nociceptor expressed on peripheral primary afferent neurons and is involved in acute The development of inflammatory pain sensitivity. It is a cation channel that can efficiently mediate the influx of Ca 2+ and is widely distributed in cells such as neurons, immune cells, epithelial cells of various organs, and keratinocytes. Studies have found that TRPV1 channels can be activated by a variety of stimuli, such as various physical and chemical stimuli: high temperature environment (temperature ⁇ 43°C), low pH value (acidic environment), voltage, osmolarity, endogenous and Exogenous vanilla substances, such as capsaicin, etc.
  • stimuli such as various physical and chemical stimuli: high temperature environment (temperature ⁇ 43°C), low pH value (acidic environment), voltage, osmolarity, endogenous and Exogenous vanilla substances, such as capsaicin, etc.
  • TRPV1 When TRPV1 is activated, calcium ion channels open, calcium ions flow in, and the concentration of calcium ions in the cytoplasm 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 glutamate and aspartate.
  • neuropeptides such as substance P, neurokinin A, calcitonin gene-related peptide, Vasoactive intestinal peptide and excitatory amino acids such as glutamate and aspartate.
  • TRPV1 On the skin, TRPV1 is expressed in skin sensory nerves, keratinocytes and mast cells.
  • the activated TRP channels not only mediate temperature perception and regulation, but are also involved in pain and itch sensation and the occurrence of skin neurogenic inflammation. Therefore, TRP channels have become a therapeutic Important molecular targets in inflammatory skin diseases and pruritus. Therefore, TRPV1 was selected as
  • the fermentation lysate filtrate obtained in the present invention and its different multiple dilutions were subjected to absorbance scanning in the ultraviolet region (200-400nm) (Varioskan TM LUX multi-function microplate reader-VL0LOTD0, Thermo Fisher Scientific (China) Co., Ltd.), after converting it into transmittance, it is calculated as absorption rate, and the results are shown in Figure 11.
  • UVC area 200-280nm
  • UVB area 280-320nm
  • UVA area 320-400nm
  • UVA 98% of the ultraviolet rays that reach the earth's surface are UVA, which has the strongest penetrability and can reach directly into the dermis, which is the main cause of skin tanning.
  • UVB 98% of the ultraviolet rays that reach the earth's surface are UVB, which has the strongest penetrability and can reach directly into the dermis, which is the main cause of skin tanning.
  • UVB less than 2%
  • UVC Although generally absorbed by the ozone layer, short-wave ultraviolet rays are the most harmful to the human body, and long-term exposure can cause skin cancer.
  • the test results show that the average absorption rate of the fermentation lysate filtrate of the present invention in the UVC area is close to 100.00%, the average absorption rate in the UVB area is >95%, and the average absorption rate in the UVA area is >80%.
  • the ultraviolet absorption capacity of the diluted lysate filtrate also decreases.
  • the absorption rate of the 50% concentration filtrate in the UVC area does not decrease significantly, but the absorption rate of ultraviolet rays in the UVB area and UVA area There is a significant decrease, but they are all above 40%; the average absorption rate of the lysate filtrate with a concentration of 25% in the UVC area is >90%, and the average absorption rate of UVB decreases from 89% to 65% with the increase of ultraviolet wavelength.
  • the average absorption rate for the UVA area is >27%; the lysate filtrate diluted 10 times, the average absorption rate for the UVC area is >90%, the average absorption rate for UVB ultraviolet wavelengths increases from 87% to 34%, and for the UVA area
  • the average absorption rate is >20%; as the dilution factor increases, its UV absorption effect gradually decreases, but when diluted to 1%, the average absorption rate in the UVC area is >60%, and the average absorption rate for UVB is also maintained at 20%. about.
  • the average absorption rate for the UVC area can reach 74%-99%, the average absorption rate for UVB decreases from 67% to 23% with the increase of ultraviolet wavelength, and the average absorption rate for the UVA area Absorption rate >15%.
  • the sunscreen effect of the lysate filtrate is similar to that of commonly used UVB absorbers such as aminobenzoates and their derivatives, salicylates and their derivatives, cinnamic acid esters and camphor derivatives.
  • the cell filtrate also has good sun protection and skin care effects on ultraviolet rays within a certain wavelength range.
  • HBD1 protein staining results of each group are shown in Figure 12.
  • the tan part pointed by the red arrow in the picture is HBD1 protein.
  • the summary table of HBD1 relative IOD values is shown in Table 26, and the HBD1 integrated optical density (IOD) value histogram is shown in Figure 13.
  • HBD2 protein staining results of each group are shown in Figure 14.
  • the tan part pointed by the red arrow in the picture is HBD2 protein.
  • the summary table of HBD2 relative IOD values is shown in Table 27, and the HBD1 integrated optical density (IOD) value histogram is shown in Figure 15.
  • TLR2 protein staining results of each group are shown in Figure 16.
  • the tan part pointed by the red arrow in the picture is the TLR2 protein.
  • a summary table of TLR2 relative IOD values is shown in Table 28, and a histogram of TLR2 integrated optical density (IOD) values is shown in Figure 17.
  • the LL-37 protein staining results of each group are shown in Figure 18.
  • the tan part pointed by the red arrow in the picture is the LL-37 protein.
  • the summary table of relative IOD values of LL-37 is shown in Table 29, and the histogram of the integrated optical density (IOD) value of LL-37 is shown in Figure 19.
  • the skin microecology is an ecosystem composed of various microorganisms such as bacteria, fungi, viruses, mites, and arthropods, as well as tissues, cells, various secretions, and the microenvironment on the skin surface.
  • the interaction between skin microorganisms, the host and the external environment constitutes the skin's microecological balance.
  • keratinocytes directly kill foreign invading microbial flora by expressing antimicrobial peptides (AMPs), and at the same time provide a friendly place for resident bacteria to thrive. Resident bacteria can directly release AMPs to act on pathogenic microorganisms.
  • Staphylococcus epidermidis can secrete defensins, bacteriocins, LL-37, REG3A and other antimicrobial peptides. Resident bacteria can also regulate the host's immune response by secreting AMPs.
  • the antimicrobial peptides present in the skin of the human body mainly belong to two major families: defensins and cathelicidin. Defensins are divided into ⁇ -defensins and ⁇ -defensins according to their different structures, among which HBD1 and HBD2 belong to ⁇ -defensins.
  • LL-37 is the only cathelicidin antimicrobial peptide found in the human body.
  • pathogenic microorganisms can be eliminated by activating a variety of pattern recognition receptors such as TLR2 (Toll-like receptor 2) to recognize exogenous microorganisms and activate immune responses.
  • TLR2 Toll-like receptor 2
  • This test is based on a 3D epidermal skin model Compared with the control group, the HBD1, HBD2, TLR2, and LL-37 protein contents of the Pichia pastoris fermentation lysate filtrate at 0.5% and 1.0% concentrations increased significantly, and as the filtrate concentration increased, the correlation Expression of target protein The amount also increases in a positive correlation, indicating that the Pichia pastoris fermentation lysate filtrate prepared in the present invention can regulate the microecology and improve the skin barrier by increasing the expression of antibacterial peptides, thereby achieving a repair effect.
  • TLR2 Toll-like receptor 2
  • This example is based on sebaceous gland cells.
  • the oil control effect of the sample to be tested is evaluated.
  • Shaanxi Boxi General Testing Technology Co., Ltd. is entrusted to conduct the test and issue a test report.
  • test for the detection of oil control efficacy, the test set up a blank group, a positive control group and a sample group, as shown in Table 30 below.
  • the detection method is briefly described as follows:
  • SZ95 cells were seeded onto a 24-well plate at a seeding density of 3.5E cells/well and incubated overnight in an incubator (37°C, 5% CO 2 ).
  • Oil Red O staining Aspirate the old solution, wash the cells once with PBS, add 500 ⁇ L Oil Red O working solution (1 ⁇ g/mL) to each well, stain for 15 min in the dark (incubate at 37°C in an incubator), and wash twice with PBS.
  • test results showed that compared with the BC group, the synthesis of lipid droplets in the PC group was significantly reduced, indicating that the positive control was effective; compared with the BC group, the sample Pichia pastoris fermentation lysate filtrate at 0.05% and 0.15% At the concentration of 0.05% and 0.15%, the synthesis of cellular lipid droplets is significantly reduced, indicating that the Pichia pastoris fermentation lysate filtrate prepared in the present invention has the effect of reducing the synthesis of lipid droplets at concentrations of 0.05% and 0.15%.
  • Example 3 Application example of Pichia pastoris fermentation lysate filtrate of the present invention
  • the Pichia pastoris fermentation lysate filtrate of the present invention is used as raw material to prepare skin care essence, facial mask, and skin care cream, in order to more intuitively illustrate the application advantages of the fermentation lysate filtrate of the present invention.
  • the fermentation lysate filtrate of the present invention is not limited to use in these products.
  • the Pichia pastoris fermentation lysate filtrate of the present invention can also be used to prepare other cosmetics.
  • lysate filtrate with a 10% wet bacterial cell concentration from the fermentation bacterial cell of the engineering bacterium CGMCC NO.7189 in Publication No. CN103102407B as an example
  • a skin care essence is prepared.
  • Each raw material is prepared with the mass fraction listed below. The total The sum of the mass percentages is 100%, and the proportions of each component are as follows.
  • the preferred addition ratio of the Pichia pastoris fermentation lysate filtrate is 5% for preparation.
  • the skin care essence includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerol 4wt%, butylene glycol 4wt%, glycerol polyether-262wt%, EDTA-2Na 0.02wt%, sodium hyaluronate 0.05wt%, allantoin 0.15wt%, betaine 2wt%, xanthan gum 0.25wt%, 1,2-pentanediol 5wt%, the rest is deionized water.
  • the preparation method of the skin care essence accurately weigh the raw materials of each component; mix deionized water, glycerin, butylene glycol, and glycerol polyether-26 at room temperature, and then add allantoin and betaine thereto, Dissolve and mix evenly, raise the temperature to 70-75°C, add sodium hyaluronate and xanthan gum, and stir until completely dissolved; cool down to below 40°C, add the filtrate of Pichia pastoris fermentation lysate, and stir evenly; 100 mesh Filter with a filter cloth and put into press-type vacuum bottles to get the skin care essence.
  • a skin care facial mask liquid is prepared.
  • Each raw material is prepared according to the mass fraction as shown below. The sum of the total mass percentages is 100%, and the proportions of each component are as shown in (b).
  • the preferred addition ratio of the Pichia pastoris fermentation lysate filtrate is 5% for preparation.
  • the facial mask includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerin 8wt%, EDTA-2Na 0.02wt%, sodium hyaluronate 0.05wt%, Xanthan gum 0.1wt%, carbomer 0.12wt%, appropriate amount of pH regulator (preferably 0.12wt%, generally adjusted to neutral), 1,2-pentanediol 4wt%, 1,2-hexanediol 0.5wt %, the others are deionized water.
  • pH regulator preferably 0.12wt%, generally adjusted to neutral
  • 1,2-pentanediol 4wt% 1,2-hexanediol 0.5wt %
  • the others are deionized water.
  • a skin care cream is prepared.
  • Each raw material is prepared according to the mass fraction as shown below. The total mass The sum of the percentages is 100%, and the proportion of each component is as follows. It is preferable that the addition ratio of the Pichia pastoris fermentation lysate filtrate is 5%.
  • the cream includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, recorded as phase D; it also includes glycerin 5wt%, EDTA disodium 0.02wt%, xanthan gum 0.15wt%, ammonium acryloyldimethyltaurate/VP copolymer 0.6wt%, p-hydroxyacetophenone 0.5wt%, 1,2-hexanediol 0.5wt%, the others are deionized water, the mixture is A Phase; cetearyl glucoside/cetearyl alcohol 3wt%, cetearyl alcohol 3wt%, caprylic capric triglyceride 4wt%, hydrogenated polyisobutylene 3wt%, polydimethylsiloxane 1wt% , mixed into phase B; the pH adjuster is phase C, generally adjusted to neutral.
  • phase A Put the components of phase A into the water phase pot, heat to 80-85°C, mix and stir evenly; then put the ingredients of phase B into the water phase pot, heat to 80-85°C, mix and stir evenly; then Add phase B to phase A and homogenize for 5 minutes; stir and cool to 60°C and add phase C; when cooled to 35-40°C, add phase D and stir evenly to obtain a skin care cream.
  • the subjects use the essence, facial mask or cream product of the present invention on the left face and the right face Use the control substance that does not contain filtrate for 4 weeks. During this period, do not use other similar skin care products. On the 28th day, self-evaluate the effectiveness of the above sample in relieving skin redness and relieving skin irritation.
  • the skin condition does not improve or even worsens, it will be scored as 0 points; if the skin is red and susceptible to irritation, it will not improve but is not aggravated, it will be scored as 1 point; if the skin is red and susceptible to irritation, it will slightly improve, but the effect is not obvious, it will be scored as 2 points; If the skin's redness and susceptibility to irritation is improved, it will be scored 3 points; if the skin's redness and susceptibility to irritation is significantly improved, it will be scored 4 points.

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Abstract

The present invention provides a Pichia pastoris fermentation lysate filtrate, a preparation method therefor, and use thereof, and belongs to the technical field of cosmetic raw materials. Provided is a fermentation lysate filtrate prepared by using Pichia pastoris expressing recombinant collagen and performing fermentation. The fermentation lysate filtrate comprises total protein, recombinant collagen, polysaccharides, amino acids, and various vitamins. Pichia pastoris fermentation bacteria expressing recombinant human collagen are used for preparing cosmetic raw materials for the first time, so that the waste treatment pressure caused by yeast fermentation bacteria is reduced, and waste is turned into wealth. The preparation method for the Pichia pastoris fermentation lysate filtrate is a whole-process online and automatic preparation process, and the preparation process is rapid and efficient. Meanwhile, the breeding of harmful bacteria can be effectively controlled during the whole preparation process flow. The lysate filtrate has the effects of soothing skin, enhancing a skin micro-ecological barrier, controlling oil, screening sunlight, and the like at the same time, thereby widening the application range thereof in preparing cosmetics.

Description

一种巴斯德毕赤酵母发酵溶胞物滤液、制备方法及应用A kind of Pichia pastoris fermentation lysate filtrate, preparation method and application 技术领域Technical field
本发明涉及一种巴斯德毕赤酵母发酵溶胞物滤液、制备方法及应用，属于化妆品原料技术领域。The invention relates to a Pichia pastoris fermentation lysate filtrate, a preparation method and an application, and belongs to the technical field of cosmetic raw materials.
背景技术Background technique
胶原蛋白(collagen)，在细胞迁移、细胞代谢、信号通路应答、细胞、组织、器官的正常生理功能的维护、调节及损伤修复方面有重要作用。作为重要的天然生物蛋白，有良好的生物相容性、生物活性和可降解性等独特功能特征，在化工、医药、食品、医学美容、化妆品等众多领域具有广阔的应用前景。以现代基因工程技术生产的重组人胶原蛋白，性质稳定，无动物源胶原的疾病隐患、免疫原性，工厂化生产不受原料来源限制，现已成为国家重点发展扶持的领域和资本关注的热点。目前，国内重组胶原蛋白生产企业发酵生产胶原蛋白最大的规模化生产线已达到20吨级，未来其生产规模、数量会继续扩大、上升。Collagen plays an important role in cell migration, cell metabolism, signaling pathway response, maintenance and regulation of normal physiological functions of cells, tissues, and organs, and damage repair. As an important natural biological protein, it has unique functional characteristics such as good biocompatibility, bioactivity and degradability, and has broad application prospects in many fields such as chemical industry, medicine, food, medical cosmetology, cosmetics, etc. Recombinant human collagen produced with modern genetic engineering technology has stable properties and does not have the disease risks and immunogenicity of animal-derived collagen. Factory production is not restricted by the source of raw materials. It has become a key area of national development support and a hot spot of capital attention. . At present, the largest large-scale production line for fermentation and production of collagen by domestic recombinant collagen manufacturers has reached 20 tons. In the future, its production scale and quantity will continue to expand and rise.
重组胶原蛋白于毕赤酵母中表达时采用分泌表达，会产生巨量发酵菌体(一般发酵罐中20～30％体积为发酵菌体)作为废弃物需处理。酵母发酵工业中(例如啤酒生产、生物制药)产生的废弃物(废水、菌体等)，均需要进行无害化处理后，满足相关的标准后方能排放，因为体量巨大，给企业环保设施处理能力带来了巨大的压力。现在，发酵废弃菌体已逐步开始进行了进一步的利用，如制备单细胞蛋白(蛋白质含量会有所降低)、发酵为酒精(利用发酵时产生的纤维质，于菌体本身意义不大)、开发为有机肥料用于农业生产(但需要进行一定处理，也需要较大场地)、用于生产其它的生物质能源、用于制作动物饲料、制备酵母提取物、酵母浸膏等、提取葡聚糖、制备酵母提取物等等。酵母发酵菌体有以上多种利用方式，但总体而言，处理后产品附加值有限、市场空间不大或可替代产品多，且对生产设备、生产场所、环境均有一定的要求，导致多数生产企业宁愿进行无害化处理后将菌体排放掉。When recombinant collagen is expressed in Pichia pastoris, it uses secretion expression, which will produce a huge amount of fermentation cells (generally 20-30% of the volume in the fermentation tank is fermentation cells) as waste that needs to be disposed. The waste (wastewater, bacteria, etc.) generated in the yeast fermentation industry (such as beer production, biopharmaceuticals) needs to be harmlessly treated and must meet relevant standards before it can be discharged. Because of its huge volume, it requires environmental protection facilities of the enterprise. Processing power puts a huge strain on it. Nowadays, fermentation waste bacteria have gradually begun to be further utilized, such as preparing single-cell proteins (the protein content will be reduced), fermenting into alcohol (using the fiber produced during fermentation, which has little significance for the bacteria themselves), Developed as organic fertilizer for agricultural production (but requires certain processing and a larger site), used to produce other biomass energy, used to make animal feed, prepare yeast extract, yeast extract, etc., extract glucan sugar, preparing yeast extract, and more. Yeast fermentation cells can be utilized in the above various ways, but in general, the added value of the processed products is limited, the market space is not large or there are many substitute products, and there are certain requirements for production equipment, production sites, and the environment, resulting in most Production companies would rather discharge the bacteria after harmless treatment.
以微生物溶胞物、提取物作为化妆品原料已成为相关领域的热点。除去以上几种用途外，酵母菌体还可用于作为化妆品的原料，这是一种附加值高的处理方式。除了可以直接以酵母作为原料，还可制备成为酵母衍生物等多种形式的化妆品原料。Using microbial lysates and extracts as cosmetic raw materials has become a hot topic in related fields. In addition to the above uses, yeast cells can also be used as raw materials for cosmetics, which is a high value-added treatment method. In addition to using yeast directly as a raw material, it can also be prepared into various forms of cosmetic raw materials such as yeast derivatives.
皮肤微生态是由细菌、真菌、病毒、螨虫和节肢动物等各种微生物与皮肤表面的组织、细胞各种分泌物、微环境等共同组成的生态***。近年来，随着研究的深入，皮肤微生态也成为化妆品界关注的焦点。在化妆品应用上，微生态护肤也成为许多国际品牌公司产品开发的重点。如雅诗兰黛、兰蔻、欧莱雅、宝洁等国际品牌都陆续开发和推出相关产品，如宝洁旗下的SK-II的“神仙水”，其核心原料Pitera的成分即为半乳糖酵母样菌发酵产物滤液，可以通过促进紧密连接蛋白的表达和增加跨膜电阻来提高皮肤的屏障功能。而雅诗兰黛的明星产品“小棕瓶”、兰蔻的“小黑瓶”使用的核心原料均为二裂酵母发酵产物溶胞物，其成分为虽然名字中带有酵母，但本身并非酵母菌属，而是来源于双歧杆菌，为经培养、裂解菌体细胞得到代谢产物、细胞质成分、细胞壁组分及多种细胞器片段。此外，各大国际主流原料公司也纷纷聚焦于微生态护肤，相继推出各种后生元原料，如巴斯夫的Relipidium，主要成分为水解酵母蛋白，具有恢复皮肤菌落平衡的功效；帝斯曼的OXY 229PF主要成分为酵母 菌溶胞物，它能够减少克氏棒状杆菌，从而缓解面部发红。The skin microecology is an ecosystem composed of various microorganisms such as bacteria, fungi, viruses, mites, and arthropods, as well as tissues, cells, various secretions, and the microenvironment on the skin surface. In recent years, with the deepening of research, skin microecology has also become the focus of attention in the cosmetics industry. In cosmetic applications, microecological skin care has also become the focus of product development for many international brand companies. International brands such as Estee Lauder, Lancôme, L'Oréal, and Procter & Gamble have successively developed and launched related products. For example, Procter &Gamble'sSK-II's"FairyWater", the core raw material of which is Pitera, is the filtrate of the fermentation product of galactosaccharomyces-like bacteria. Improves the barrier function of the skin by promoting the expression of tight junction proteins and increasing transmembrane resistance. The core raw materials used in Estee Lauder's star products "Little Brown Bottle" and Lancôme's "Little Black Bottle" are lysates of the fermentation product of Saccharomyces bifidum. Although their names contain yeast, they are not Saccharomyces genus. Rather, it is derived from Bifidobacterium and is obtained by culturing and lysing bacterial cells to obtain metabolites, cytoplasmic components, cell wall components and various organelle fragments. In addition, major international mainstream raw material companies have also focused on micro-ecological skin care, and have successively launched various postbiotic raw materials, such as BASF's Relipidium, which is mainly composed of hydrolyzed yeast protein and has the effect of restoring the balance of skin flora; DSM's OXY 229PF The main ingredient is yeast Bacterial lysate, which reduces Corynebacterium cruzi and thereby relieves facial redness.
皮肤微生态、皮肤自身的物理屏障和皮肤免疫三者共同形成了机体的第一道防御体系，抵抗外界的各种理化因素的刺激，维持机体的正常功能。因此，皮肤屏障的完整对健康的皮肤有着重要意义，开发更安全有效的皮肤屏障修复原料也成为当下护肤品开发需要解决的问题。现有的化妆品屏障修复原料多针对角质层层面进行物理屏障的修复，缺少以改善皮肤微生态屏障和增强免疫屏障为出发点而开发的具有皮肤屏障修复作用的化妆品原料。Skin microecology, the skin's own physical barrier and skin immunity together form the body's first defense system, resisting stimulation from various external physical and chemical factors and maintaining the body's normal functions. Therefore, the integrity of the skin barrier is of great significance to healthy skin, and the development of safer and more effective skin barrier repair raw materials has become a problem that needs to be solved in the development of current skin care products. Existing cosmetic barrier repair raw materials mostly repair the physical barrier at the stratum corneum level, and there is a lack of cosmetic raw materials with skin barrier repair effects developed based on improving the skin's microecological barrier and enhancing the immune barrier.
有皮肤屏障修复作用的化妆品原料开发中，巴斯德毕赤酵母来源的原料是欠缺的；并且现有酵母类化妆品的功效，多集中于保湿、抗衰防晒、美白等方面，以增强皮肤微生态屏障、免疫屏障、控制皮脂分泌为基本功效机理的原料也是欠缺的。In the development of cosmetic raw materials with skin barrier repair effects, raw materials derived from Pichia pastoris are lacking; and the functions of existing yeast cosmetics are mostly focused on moisturizing, anti-aging, sun protection, whitening, etc. to enhance skin micro-organisms. Raw materials with ecological barrier, immune barrier, and control of sebum secretion as the basic functional mechanisms are also lacking.
另外，胶原蛋白在医美、化妆品上的应用已被普遍接受，但表达重组胶原蛋白的巴斯德毕赤酵母发酵菌体用于制备化妆品原料，尤其是发酵溶胞物滤液，尚无先例，相关研发处于空白；相关产品的制备方式、功效研究也未见公开资料披露；以表达重组人胶原蛋白的巴斯德毕赤酵母制备的化妆品原料(尤其是发酵溶胞物滤液)是否可行、是否有更好、更多的功效也未见诸报道；重组胶原蛋白生产企业也未有相应的产品推出或专利、研究文献发表。In addition, the application of collagen in medical beauty and cosmetics has been generally accepted, but there is no precedent for Pichia pastoris fermentation cells expressing recombinant collagen to be used to prepare cosmetic raw materials, especially fermentation lysate filtrate. Relevant research and development is in the blank; the preparation methods and efficacy studies of related products have not been disclosed in public information; whether cosmetic raw materials (especially fermentation lysate filtrate) prepared from Pichia pastoris expressing recombinant human collagen are feasible and There have been no reports of better or more effective effects; recombinant collagen production companies have not launched corresponding products or published patents or research documents.
此外，在以酵母菌体制备提取物或溶胞物滤液或其它种类化妆品原料时，首先要完成的是酵母菌的破壁裂解，毕赤酵母细胞壁比较厚(70nm-200nm)且结构致密，机械强度大，常规的处理方式处理时间长、效率低，还有可能引物外源性物质(酶类)。对于规模化工业生产的产生发酵菌体而言：发酵菌体产生的周期短、体量大，较长的处理时间容易滋生有害菌体，产生内毒素等热原物质；规模化生产时菌体的破碎需要简单、稳定、高效，避免外源性物质导入(同时也利于控制成本)。另外，破碎后的毕赤酵母溶胞物是不溶性、可溶性成份混杂在一起的，如何进行有效的分离也需要一套有效处理方式。In addition, when preparing extracts or lysate filtrate or other types of cosmetic raw materials from yeast cells, the first thing to be completed is the lysis of the yeast cell wall. The cell wall of Pichia pastoris is relatively thick (70nm-200nm) and has a dense structure, which is mechanically The intensity is high, the conventional treatment method takes a long time, is low in efficiency, and may also induce exogenous substances (enzymes). For the production of fermentation bacteria in large-scale industrial production: the production cycle of fermentation bacteria is short, the volume is large, and the long processing time is easy to breed harmful bacteria and produce endotoxins and other pyrogenic substances; in large-scale production, the bacteria The crushing needs to be simple, stable and efficient to avoid the introduction of exogenous substances (which also helps control costs). In addition, the broken Pichia pastoris lysate is a mixture of insoluble and soluble components, and effective separation also requires an effective treatment method.
发明内容Contents of the invention
本发明的目的在于，克服现有技术中存在的一些技术问题，主要是填补现有技术中“以表达重组胶原蛋白的巴斯德毕赤酵母制备发酵溶胞物滤液”这一产品、其制备方法、产品功效和应用方面的空白，解决现有技术中溶胞物滤液制备方法存在的处理时间长、效率低、有可能引入外源性物质等的技术问题。The purpose of the present invention is to overcome some technical problems existing in the prior art, mainly to fill in the existing technology "preparation of fermentation lysate filtrate using Pichia pastoris expressing recombinant collagen" and its preparation Gaps in methods, product efficacy and applications solve the technical problems of long processing time, low efficiency, and the possibility of introducing exogenous substances in the preparation method of lysate filtrate in the existing technology.
为此，本发明提供一种巴斯德毕赤酵母发酵溶胞物滤液、制备方法及应用。To this end, the present invention provides a Pichia pastoris fermentation lysate filtrate, preparation method and application.
为达到上述技术目的，本发明首先提供一种发酵溶胞物滤液的制备方法，所述发酵溶胞物滤液由巴斯德毕赤酵母菌发酵后的菌体制备获得。In order to achieve the above technical objectives, the present invention first provides a method for preparing a fermentation lysate filtrate. The fermentation lysate filtrate is prepared from cells fermented by Pichia pastoris.
进一步的，所述巴斯德毕赤酵母菌为表达重组胶原蛋白的巴斯德毕赤酵母菌。Further, the Pichia pastoris is Pichia pastoris expressing recombinant collagen.
进一步的，所述表达重组胶原蛋白的巴斯德毕赤酵母菌选自保藏编号为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或CGMCC NO.21892的菌株中的一种或多种。Further, the Pichia pastoris expressing recombinant collagen is selected from the group consisting of CGMCC NO.7189, CGMCC NO.14057, CGMCC NO.17147, CGMCC NO.17150, CGMCC NO.17148, CGMCC NO. One or more strains of 17149, CGMCC NO.20626, CGMCC NO.20627, CGMCC NO.21891 or CGMCC NO.21892.
其中，所述菌体为发酵产物进行固液分离后获得。Wherein, the bacterial cells are obtained by solid-liquid separation of fermentation products.
所述发酵溶胞物滤液的制备方法包括菌体稀释、高压均质裂解、离心及过滤。 The preparation method of the fermentation lysate filtrate includes bacterial cell dilution, high-pressure homogenization lysis, centrifugation and filtration.
进一步的，所述菌体稀释为以纯水稀释至质量浓度为5％～30％(W/W)的菌体悬液；优选的，所述菌体稀释为以纯水稀释至质量浓度为10％(W/W)的菌体悬液。Further, the bacterial cells are diluted with pure water to a bacterial cell suspension with a mass concentration of 5% to 30% (W/W); preferably, the bacterial cells are diluted with pure water to a mass concentration of 5% to 30% (W/W). 10% (W/W) bacterial cell suspension.
所述高压均质裂解为将所述菌体悬液以＞100MPar的压力破碎1-3次后获得裂解液；优选的，所述高压均质裂解为将所述菌体悬液以1200MPar的压力破碎。The high-pressure homogeneous lysis is to crush the bacterial cell suspension 1-3 times at a pressure of >100MPar to obtain a lysate; preferably, the high-pressure homogeneous lysis is to crush the bacterial cell suspension at a pressure of 1200MPar broken.
所述离心为将所述裂解液以10000g-20000g的速度离心，获得上清液。The centrifugation is centrifuging the lysate at a speed of 10,000g-20,000g to obtain a supernatant.
所述过滤为将所述上清液以0.22μm孔径过滤，即得发酵溶胞物滤液。The filtration is to filter the supernatant with a pore size of 0.22 μm to obtain the fermentation lysate filtrate.
本发明还提供所述制备方法制备得到的发酵溶胞物滤液，所述发酵溶胞物滤液中包括总蛋白、重组胶原蛋白、多糖、氨基酸、多种维生素。The present invention also provides fermentation lysate filtrate prepared by the preparation method. The fermentation lysate filtrate includes total protein, recombinant collagen, polysaccharides, amino acids, and multiple vitamins.
根据本发明的实施例，以在发酵溶胞物滤液中所占质量体积分量计，总蛋白质含量占0.1～2.5％，优选为1％(10mg/mL)；重组胶原蛋白含量为1～15μg/mL，一般在10μg/mL以上，优选为10-15μg/mL；多糖含量约在0.1-1.5mg/mL，优选为1mg/mL；氨基酸总量约0.2％～1％，优选0.6％～0.82％。According to embodiments of the present invention, based on the mass volume component of the fermentation lysate filtrate, the total protein content accounts for 0.1 to 2.5%, preferably 1% (10 mg/mL); the recombinant collagen content is 1 to 15 μg/mL. mL, generally above 10 μg/mL, preferably 10-15 μg/mL; polysaccharide content is about 0.1-1.5 mg/mL, preferably 1 mg/mL; total amino acid content is about 0.2% to 1%, preferably 0.6% to 0.82% .
本发明所述发酵溶胞物滤液具有抗敏、舒缓、抗炎、防晒、调节皮肤微生态、控油、修复皮肤屏障的功效。The fermentation lysate filtrate of the present invention has the effects of anti-allergy, soothing, anti-inflammatory, sun protection, regulating skin microecology, controlling oil, and repairing skin barrier.
本发明还提供一种组合物，所述组合物中包括所述的发酵溶胞物滤液。The present invention also provides a composition, which includes the fermentation lysate filtrate.
进一步的，所述组合物中发酵溶胞物滤液的含量为1-10wt％，优选的，含量为5wt％。Further, the content of the fermentation lysate filtrate in the composition is 1-10 wt%, preferably, the content is 5 wt%.
本发明还提供所述发酵溶胞物滤液、所述组合物在抗敏类、舒缓类、抗炎类、防晒类、调节皮肤微生态类、控油类、修复皮肤屏障类化妆品原料中的应用。The present invention also provides applications of the fermentation lysate filtrate and the composition in anti-allergic, soothing, anti-inflammatory, sunscreen, skin microecological regulation, oil control, and skin barrier repair cosmetic raw materials.
本发明还提供所述发酵溶胞物滤液、所述组合物在化妆品领域中的应用。所述化妆品包括抗敏类、舒缓类、抗炎类、防晒类、调节皮肤微生态类、控油类、修复皮肤屏障类产品。The present invention also provides applications of the fermentation lysate filtrate and the composition in the field of cosmetics. The cosmetics include anti-allergic, soothing, anti-inflammatory, sunscreen, skin microecological regulation, oil control, and skin barrier repair products.
其中所述修复皮肤屏障类包括增强皮肤微生态屏障、提高皮肤免疫屏障。The skin barrier repair category includes enhancing the skin's microecological barrier and improving the skin's immune barrier.
进一步的，所述抗敏、舒缓、抗炎主要包括能改善缓解角质层刺激症状、改善表皮组织损伤；降低炎症介质PGE2分泌水平，缓解皮肤炎症反应，如降低皮肤中的血管通透性，从而缓解皮肤的泛红现象的产生；通过抑制TRPV1蛋白表达的机制起到舒缓皮肤刺激的功效。Further, the anti-allergic, soothing and anti-inflammatory mainly include the ability to improve and relieve the symptoms of stratum corneum irritation and improve epidermal tissue damage; reduce the secretion level of inflammatory mediator PGE2 and alleviate the skin inflammatory reaction, such as reducing the vascular permeability in the skin, thereby Relieves skin redness; soothes skin irritation through the mechanism of inhibiting TRPV1 protein expression.
所述防晒表现为对紫外线吸收的能力。The sun protection is represented by the ability to absorb ultraviolet rays.
所述调节皮肤微生态表现为通过提升抗菌肽和模式识别受体的表达，调节微生态，提升皮肤屏障，进而达到修护功效；巴斯德毕赤酵母发酵溶胞物滤液作用下，HBD1、HBD2、LL-37、TLR2蛋白含量显著上升。The regulation of skin microecology is manifested by increasing the expression of antimicrobial peptides and pattern recognition receptors, regulating the microecology and improving the skin barrier, thereby achieving a repair effect; under the action of the lysate filtrate of Pichia pastoris fermentation, HBD1, HBD2, LL-37, and TLR2 protein levels increased significantly.
所述控油表现为抑制皮肤脂滴合成。The oil control is manifested by inhibiting the synthesis of skin lipid droplets.
本发明还提供一种化妆品，所述化妆品中包括所述的发酵溶胞物滤液，所述化妆品包括但不限于护肤精华液、面膜、面霜。The present invention also provides a cosmetic, which includes the fermentation lysate filtrate. The cosmetic includes but is not limited to skin care essence, facial mask, and facial cream.
进一步的，所述化妆品中发酵溶胞物滤液的含量为1-10wt％，优选的，含量为5wt％。Further, the content of the fermentation lysate filtrate in the cosmetics is 1-10 wt%, preferably, the content is 5 wt%.
根据本发明的实施例，本发明还提供一种护肤精华液，所述护肤精华液中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选为5wt％，还包括去离子水、甘油、丁二醇、甘油聚醚-26、EDTA-2Na、透明质酸钠、尿囊素、甜菜碱、黄原胶、1，2-戊二醇。 According to an embodiment of the present invention, the present invention also provides a skin care essence, the skin care essence includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and further Including deionized water, glycerin, butylene glycol, glycerol polyether-26, EDTA-2Na, sodium hyaluronate, allantoin, betaine, xanthan gum, and 1,2-pentanediol.
本发明还提供一种面膜，所述面膜中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选为5wt％，还包括去离子水、甘油、EDTA-2Na、透明质酸钠、黄原胶、卡波姆、pH调节剂、1，2-戊二醇、1，2-己二醇。The present invention also provides a facial mask. The facial mask includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes deionized water, glycerol, EDTA-2Na, Sodium hyaluronate, xanthan gum, carbomer, pH adjuster, 1,2-pentanediol, 1,2-hexanediol.
本发明还提供一种护肤面霜，所述面霜中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选为5wt％，还包括甘油、EDTA二钠、黄原胶、丙烯酰二甲基牛磺酸铵/VP共聚物、对羟基苯乙酮、1，2-己二醇、鲸蜡硬脂基葡糖苷/鲸蜡硬脂醇、鲸蜡硬脂醇、辛酸癸酸甘油三酯、氢化聚异丁烯、聚二甲基硅氧烷、去离子水。The present invention also provides a skin care cream, which includes fermentation lysate filtrate. The content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerin, disodium EDTA, and xanthan gum. , Ammonium acryloyldimethyltaurate/VP copolymer, p-hydroxyacetophenone, 1,2-hexanediol, cetearyl glucoside/cetearyl alcohol, cetearyl alcohol, caprylic acid Capric triglyceride, hydrogenated polyisobutylene, polydimethylsiloxane, deionized water.
本发明的有益效果：Beneficial effects of the present invention:
(1)本发明首次实现了表达重组人胶原蛋白的巴斯德毕赤酵母发酵菌体制备化妆品原料，增加了一种重组人胶原蛋白发酵菌体的有效的、高附加值的利用途径，减轻了酵母发酵菌体带来的废弃物处理压力，变废为宝。(1) The present invention realizes for the first time the preparation of cosmetic raw materials by Pichia pastoris fermentation cells expressing recombinant human collagen, adding an effective and high value-added utilization method of recombinant human collagen fermentation cells, reducing the It eliminates the waste disposal pressure caused by yeast fermentation cells and turns waste into treasure.
(2)本发明中的表达重组人胶原蛋白的巴斯德毕赤酵母发酵菌体制备溶胞物滤液，含有一定量的胶原蛋白，不用额外添加。(2) The lysate filtrate prepared from the fermentation cells of Pichia pastoris expressing recombinant human collagen in the present invention contains a certain amount of collagen without additional addition.
(3)本发明中发酵溶胞物滤液的制备方法为全程在线、自动化的制备工艺，以高压均质对毕赤酵母发酵菌体进行物理性破碎裂解，使用连续流离心机进行裂解后的固液分离，过滤后获得溶胞物滤液，制备过程快速高效、可规模化制备、不引物任何外来成份；同时整个制备工艺流程可实现全自动操作，物料为全自动在线式管道传输，洁净区内完成无菌过滤和灌装，可有效控制有害菌体的滋生。(3) The preparation method of the fermentation lysate filtrate in the present invention is a fully online, automated preparation process. The Pichia pastoris fermentation cells are physically fragmented and lysed by high-pressure homogenization, and a continuous flow centrifuge is used to lyse the solids after lysis. The liquid is separated, and the lysate filtrate is obtained after filtration. The preparation process is fast and efficient, can be prepared on a large scale, and does not introduce any foreign ingredients. At the same time, the entire preparation process can be fully automated, and the materials are fully automated online pipeline transmission in the clean area. Complete sterile filtration and filling, which can effectively control the growth of harmful bacteria.
(4)本发明制备的溶胞物滤液应用于化妆品无安全性问题，除去传统酵母来源化妆品原料功效外，同时具有了：通过促进抗菌肽的分泌，调节微生态平衡，增强皮肤微生态屏障的功效；通过抑制炎症因子表达，提高皮肤免疫屏障，舒缓皮肤的功效；控油、防晒功效；这些之前未曾见诸于“巴斯德毕赤酵母发酵溶胞物滤液”原料的效果，开拓了其用于制备化妆品的应用范围。(4) The lysate filtrate prepared by the present invention has no safety issues when used in cosmetics. In addition to the efficacy of traditional yeast-derived cosmetic raw materials, it also has the following functions: by promoting the secretion of antibacterial peptides, regulating the microecological balance, and enhancing the skin's microecological barrier. Efficacy; by inhibiting the expression of inflammatory factors, improving the skin immune barrier and soothing the skin; oil control and sun protection effects; these effects have not been seen in the raw material of "Pichia pastoris fermentation lysate filtrate" before, and have opened up its uses Application range in the preparation of cosmetics.
(5)制备的巴斯德毕赤酵母发酵溶胞物滤液，同时具有于皮肤微生态屏障和免疫屏障上双重屏障修复作用，一定程度上阐释了其应用于化妆品产生功效的机理。(5) The prepared Pichia pastoris fermentation lysate filtrate has a dual barrier repair effect on the skin microecological barrier and immune barrier, which explains to a certain extent the mechanism of its efficacy in cosmetics.
(6)制备的巴斯德毕赤酵母发酵溶胞物滤液，除去上述功效外，还以实验验证了其同时具有降低细胞脂质分泌的作用，进一步开拓了其用于制备化妆品的应用范围(控油型化妆品)。(6) The prepared Pichia pastoris fermentation lysate filtrate, in addition to the above effects, has also been experimentally verified to have the effect of reducing cellular lipid secretion, further expanding its application range for the preparation of cosmetics ( Oil control cosmetics).
附图说明Description of the drawings
图1为本发明的发酵溶胞物滤液制备方法流程图。Figure 1 is a flow chart of the fermentation lysate filtrate preparation method of the present invention.
图2为实施例中不同参数条件下高压匀质后毕赤酵母后的裂解液革兰氏染色的结果，图中，A1为100MPar条件下裂解3次后裂解液，B1为120MPar条件下裂解2次后裂解液，C1为150MPar条件下裂解2次后裂解液，D1为200MPar条件下裂解2次后裂解液。Figure 2 shows the results of Gram staining of the lysate of Pichia pastoris after high-pressure homogenization under different parameter conditions in the example. In the figure, A1 is the lysate after lysing 3 times under 100MPar conditions, and B1 is lysing 2 times under 120MPar conditions. C1 is the lysate after 2 times of lysis at 150MPar, and D1 is the lysate after 2 times of lysis at 200MPar.
图3为实施例1中制备的溶胞物滤液的SDS-PAGE(A，上样量10μL)图和WB图(B)，图3B中，泳道1为溶胞物滤液，上样量10μL，泳道2为空白对照，泳道3为重组III型人胶原蛋白，上样量2.5μg，泳道4为重组III型人胶原蛋白，上样量1μg。Figure 3 is the SDS-PAGE (A, loading volume: 10 μL) and WB chart (B) of the lysate filtrate prepared in Example 1. In Figure 3B, lane 1 is the lysate filtrate, and the loading volume is 10 μL. Lane 2 is a blank control, lane 3 is recombinant type III human collagen, and the loading amount is 2.5 μg. Lane 4 is recombinant type III human collagen, and the loading amount is 1 μg.
图4为实施例中计算样品中重组胶原蛋白浓度过程中拟合的标准曲线。Figure 4 is a standard curve fitted during the calculation of the concentration of recombinant collagen in the sample in the example.
图5为实施例中计算样品中总蛋白含量过程中拟合的标准曲线及曲线方程。 Figure 5 shows the standard curve and curve equation fitted during the calculation of the total protein content in the sample in the example.
图6为实施例中计算样品中多糖含量过程中拟合的标准曲线及曲线方程。Figure 6 shows the standard curve and curve equation fitted during the calculation of the polysaccharide content in the sample in the example.
图7为实施例基于3D表皮皮肤模型培养后进行H&E染色后显微镜下的组织形态图，其中图A为空白对照，B为阴性对照，即SLS处理组，C为阳性对照，即同时添加SLS和0.01％***，D为实验组，即同时添加SLS和2％滤液。Figure 7 is an embodiment based on a 3D epidermal skin model. The tissue morphology diagram under the microscope after H&E staining after culture. Picture A is the blank control, B is the negative control, that is, the SLS treatment group, C is the positive control, that is, SLS and 0.01% dexamethasone are added at the same time, and D is the experimental group. , that is, add SLS and 2% filtrate at the same time.
图8为实施例中PGE2含量检测结果柱状图。Figure 8 is a histogram of PGE2 content detection results in the embodiment.
图9为实施例中辣椒素受体(TRPV1)的免疫荧光检测结果。Figure 9 is the immunofluorescence detection result of capsaicin receptor (TRPV1) in the Example.
图10为实施例中TRPV1蛋白相对IOD值柱状图。Figure 10 is a histogram of relative IOD values of TRPV1 protein in Examples.
图11为本发明得到的发酵溶胞物滤液及其不同倍数的稀释液紫外光区200-400nm波长紫外线率。Figure 11 shows the ultraviolet rate of the fermentation lysate filtrate obtained by the present invention and its dilutions at different times in the ultraviolet region of 200-400nm wavelength.
图12为基于3D表皮皮肤模型的HBD1蛋白染色结果。Figure 12 is a 3D epidermal skin model. HBD1 protein staining results.
图13为基于3D表皮皮肤模型的HBD1蛋白积分光密度(IOD)值柱状图。Figure 13 is a 3D epidermal skin model. Histogram of HBD1 protein integrated optical density (IOD) values.
图14为基于3D表皮皮肤模型的HBD2蛋白染色结果。Figure 14 is a 3D epidermal skin model. HBD2 protein staining results.
图15为基于3D表皮皮肤模型的HBD2蛋白积分光密度(IOD)值柱状图。Figure 15 is a 3D epidermal skin model. Histogram of HBD2 protein integrated optical density (IOD) values.
图16为基于3D表皮皮肤模型的TLR2蛋白染色结果。Figure 16 is a 3D epidermal skin model. TLR2 protein staining results.
图17为基于3D表皮皮肤模型的TLR2蛋白积分光密度(IOD)值柱状图。Figure 17 is a 3D epidermal skin model. Histogram of integrated optical density (IOD) values of TLR2 protein.
图18为基于3D表皮皮肤模型的LL-37蛋白染色结果。Figure 18 is a 3D epidermal skin model. LL-37 protein staining results.
图19为基于3D表皮皮肤模型的LL-37蛋白积分光密度(IOD)值柱状图。Figure 19 is based on the 3D epidermal skin model Histogram of integrated optical density (IOD) values of LL-37 protein.
图20为脂滴合成油红O染色结果；图中标尺大小为50μm。Figure 20 shows the results of synthetic Oil Red O staining of lipid droplets; the scale bar in the figure is 50 μm.
图21为脂滴积分光密度(IOD)值柱状图。Figure 21 is a histogram of lipid droplet integrated optical density (IOD) values.
图22为志愿者使用含巴斯德毕赤酵母发酵溶胞物滤液精华液效果图。Figure 22 shows the effect of volunteers using the essence of Pichia pastoris fermentation lysate filtrate.
图23为志愿者使用含巴斯德毕赤酵母发酵溶胞物滤液面膜效果图。Figure 23 is a picture of the effect of volunteers using a facial mask containing Pichia pastoris fermentation lysate filtrate.
图24为志愿者使用含巴斯德毕赤酵母发酵溶胞物滤液面霜效果图。Figure 24 is a picture of the effect of volunteers using a facial cream containing Pichia pastoris fermentation lysate filtrate.
具体实施方式Detailed ways
为了使本领域技术人员更好的理解本发明的技术方案，下面对本发明的较佳实施例进行详细的阐述，但是如下实施例并不限制本发明的保护范围。In order to enable those skilled in the art to better understand the technical solution of the present invention, preferred embodiments of the present invention are described in detail below, but the following examples do not limit the scope of the present invention.
本发明的实施例中，没有多作说明的都是采用常规实验方法完成，实施例中所涉及过程没有多作说明的都是本领域技术人员根据产品说明书或本领域基础知识可以理解并且容易实现的，因此不再详细描述。In the embodiments of the present invention, if there is no further explanation, they are all completed by conventional experimental methods. If there is no further explanation of the processes involved in the embodiments, those skilled in the art can understand and easily implement them based on the product instructions or basic knowledge in the field. , so will not be described in detail.
本发明中涉及的原料来源：Sources of raw materials involved in the present invention:
本发明中所涉及的发酵溶胞物制备原料菌株，均来自于本发明申请人单位，均已做相应的菌株保藏，均为表达重组胶原蛋白的专属的毕赤酵母工程菌，均保藏于中国微生物菌种保藏管理委员会普通微生物中心；包括公开号CN103102407B中保藏编号为CGMCC NO.7189、公开号CN107090458A中保藏编号为CGMCC NO.14057、公开号CN111004319A中的保藏编号为CGMCC NO.17147、公开号CN110964099A中的保藏编号为CGMCC NO.17150、公开号CN110606896B中的保藏编号为CGMCC NO.17148、公开号CN110747198B中的保藏编号为CGMCC NO.17149、公开号CN113185604B中的保藏编号为CGMCC NO.20626与保藏编号为 CGMCC NO.20627、公开号CN114106150A中的保藏编号为CGMCC NO.21891与CGMCC NO.21892的菌株。这些工程菌表达的重组人胶原蛋白在氨基端(N端)或羧基端(C端)有6×His-Tag标签，利于检测。The raw material strains involved in the preparation of fermentation lysates in the present invention are all from the applicant's unit, and have made corresponding strain deposits. They are all exclusive Pichia pastoris engineering strains that express recombinant collagen, and are all deposited in China. General Microbiology Center of the Microbial Culture Collection and Management Committee; including the publication number CN103102407B, the preservation number is CGMCC NO.7189, the publication number CN107090458A, the preservation number is CGMCC NO.14057, the publication number CN111004319A, the preservation number is CGMCC NO.17147, the publication number The accession number in CN110964099A is CGMCC NO.17150, the accession number in Publication No. CN110606896B is CGMCC NO.17148, the accession number in Publication No. CN110747198B is CGMCC NO.17149, the accession number in Publication No. CN113185604B is CGMCC NO.20626 and The accession number is The strains deposited in CGMCC NO. 20627 and Publication No. CN114106150A are CGMCC NO. 21891 and CGMCC NO. 21892. The recombinant human collagen expressed by these engineered bacteria has a 6×His-Tag tag at the amino terminus (N terminus) or carboxyl terminus (C terminus), which facilitates detection.
除上述所列之外，其他产胶原蛋白的菌株，或普通酵母类溶胞物滤液中添加胶原蛋白，能达到本发明技术效果的，均在本发明保护范围之内。In addition to those listed above, other collagen-producing strains, or the addition of collagen to the filtrate of common yeast lysates, which can achieve the technical effects of the present invention, are all within the scope of the present invention.
巴斯德毕赤酵母工程菌表达生产重组人胶原蛋白为分泌表达：蛋白质被翻译后会进入内质网，切除信号肽后，再经高尔基体加工后分泌于胞外，翻译、加工、分泌是重组人胶原蛋白表达生产最后三个阶段，而在细胞大量表达蛋白质时，这三个阶段是同时进行的，所以虽然多数重组人胶原蛋白分泌于胞外，但毕赤酵母胞仍内有胶原蛋白留存。规模化发酵生产重组人胶原蛋白，一般发酵罐中20～30％体积为发酵菌体。在分批发酵结束后，会进行菌体与发酵液上清的菌液分离(多使用陶瓷膜或连续流离心机)，产生含菌体浓度很高的菌浆，主要成份是水和湿菌体，湿菌体浓度可达到50％(W/W)左右，可用直接作为原料用于溶物滤液的制备。The expression and production of recombinant human collagen by the engineered strain of Pichia pastoris is secretory expression: after the protein is translated, it enters the endoplasmic reticulum. After excision of the signal peptide, it is processed by the Golgi apparatus and secreted outside the cell. Translation, processing, and secretion are The last three stages of recombinant human collagen expression and production are carried out simultaneously when cells express a large amount of protein. Therefore, although most recombinant human collagen is secreted extracellularly, there is still collagen in Pichia pastoris cells. Retain. For large-scale fermentation production of recombinant human collagen, generally 20 to 30% of the volume in the fermentation tank is fermentation bacteria. After the batch fermentation is completed, the bacterial cells and the supernatant of the fermentation broth are separated (ceramic membranes or continuous flow centrifuges are often used) to produce a bacterial slurry with a high concentration of bacterial cells. The main components are water and wet bacteria. The wet bacterial cell concentration can reach about 50% (W/W) and can be directly used as raw material for the preparation of dissolved filtrate.
本发明中所述的湿菌体浓度为(含水非干燥)菌体重量/悬浮菌体液体重量的比值，文中给出的具体情况下的计算方式、结果。The wet bacterial cell concentration described in the present invention is the ratio of the weight of the (water-containing but not dry) bacterial cells/the weight of the suspended bacterial cell liquid. The calculation methods and results in specific cases are given in the article.
实施例1：溶胞物滤液的制备Example 1: Preparation of lysate filtrate
酵母菌细胞壁较厚，结构比细菌细胞壁更为复杂，且性质稳定，可以在原生质体死亡或凋亡之后仍保持其结构和成分的完整性，已死亡的原生质体的生物膜***功能丧失，溶酶体中的酶进入胞浆中大量分解胞质内、细胞核中的物质，各种小分子物质通过细胞壁后逸出，但大分子物质(如核酸、蛋白质等)只能待降解为小分子物质后才能通过细胞壁，而细胞壁受到的影响很小，仍能保持其完整性。这也是细胞破碎时酵母细胞与细菌、动物细胞一大不同点，即细胞破碎时，只有细胞壁彻底的裂解方能获得较为完全的溶胞物。Yeast cell wall is thicker, more complex in structure than bacterial cell wall, and stable in nature. It can maintain the integrity of its structure and components after the death or apoptosis of protoplasts. The function of the biofilm system of dead protoplasts is lost and dissolved. The enzymes in the enzyme body enter the cytoplasm and decompose a large number of substances in the cytoplasm and nucleus. Various small molecule substances escape through the cell wall, but macromolecular substances (such as nucleic acids, proteins, etc.) can only be degraded into small molecule substances. Only then can it pass through the cell wall, and the cell wall is slightly affected and can still maintain its integrity. This is also a major difference between yeast cells and bacterial and animal cells during cell disruption. That is, when cells are disrupted, only complete lysis of the cell wall can obtain a relatively complete lysate.
本发明选择使用高压力均质的方法可有效破碎酵母的细胞壁，这也是一种快速、高效、不引物外源物质的物理破碎方式。破碎后，对破碎液可使用高速连续流离心机进行裂解液中不溶物(细胞壁不溶性的葡聚糖、不溶性的蛋白质等)和可溶物进行分离。以公开号CN103102407B中保藏编号为CGMCC NO.7189工程菌的发酵菌体为例，进行溶胞物滤液的制备。The present invention chooses to use a high-pressure homogenization method to effectively break the cell wall of yeast. This is also a physical breakage method that is fast, efficient and does not induce foreign substances. After crushing, a high-speed continuous flow centrifuge can be used to separate the insoluble matter (cell wall insoluble glucan, insoluble protein, etc.) and soluble matter in the lysate. Taking the fermentation cell of the engineering bacterium CGMCC NO.7189 in Publication No. CN103102407B as an example, the lysate filtrate was prepared.
(1)取发酵后的菌浆，12000rpm离心10min，称重法，测定其湿菌体/菌浆上清的湿菌体浓度。(1) Take the fermented bacterial slurry, centrifuge it at 12,000 rpm for 10 minutes, and measure the wet bacterial cell concentration/the wet bacterial cell concentration of the bacterial slurry supernatant by weighing.
(2)以纯水稀释菌浆，稀释得到湿菌体浓度为5％～30％(W/W)的菌体悬液，本实施例中优选10％湿菌体浓度(W/W)。(2) Dilute the bacterial slurry with pure water to obtain a bacterial cell suspension with a wet bacterial cell concentration of 5% to 30% (W/W). In this embodiment, a wet bacterial cell concentration of 10% (W/W) is preferred.
(3)将菌体悬液输入高压均质机(AH30-100plus，安拓思纳米技术(苏州)有限公司)，开启冷水***制冷，控制破碎温度在4-10℃，以＞100MPar的压力，破碎1-3次后即获得裂解液，本实施例中优选120MPar压力、破碎2次后即获得裂解液。(3) Enter the bacterial suspension into a high-pressure homogenizer (AH30-100plus, Antos Nanotechnology (Suzhou) Co., Ltd.), turn on the cold water system for refrigeration, control the crushing temperature at 4-10°C, and use a pressure of >100 MPar. The lysate is obtained after crushing 1-3 times. In this embodiment, the preferred pressure is 120MPar and the lysate is obtained after crushing 2 times.
(4)将裂解液输入高速度连续流离心机(GQ105B，佳毅(上海)机械设备有限公司)，开启冷水***制冷(控制温度在4-10℃)，以10000g-20000g离心力离心，获得上清液，本实施例中优选10000g离心力。(4) Input the lysate into a high-speed continuous flow centrifuge (GQ105B, Jiayi (Shanghai) Machinery Equipment Co., Ltd.), turn on the cold water system refrigeration (control the temperature at 4-10°C), and centrifuge at 10000g-20000g centrifugal force to obtain the upper For clear liquid, 10000g centrifugal force is preferred in this embodiment.
(5)洁净车间内，将上清液输入板框过滤器***(CW300-10，海宁市创伟过滤设备器材厂)，以0.45μm的PP滤膜和0.22μmPES滤膜先后将上清液过滤，获得溶胞物滤液。 (5) In the clean workshop, input the supernatant into the plate and frame filter system (CW300-10, Haining Chuangwei Filtration Equipment Factory), and filter the supernatant successively with 0.45 μm PP filter membrane and 0.22 μm PES filter membrane. , to obtain the lysate filtrate.
(6)加入少量防腐成分，根据需要选择是否稀释，灌装至产品包装中，为成品。(6) Add a small amount of antiseptic ingredients, choose whether to dilute it as needed, and fill it into product packaging to complete the product.
本实施例中对得到的发酵溶胞物滤液制备效果及制备过程中间产物进行了检测，具体的包括：In this example, the preparation effect of the obtained fermentation lysate filtrate and the intermediate products in the preparation process were tested, specifically including:
一、对步骤(3)中菌体裂解效果检测：1. Detection of bacterial cell lysis effect in step (3):
毕赤酵母菌体裂解的效果可以通过镜检的方式进行检测，可使用革兰氏染色方法(革兰氏染色试剂盒购自北京索莱宝科技有限公司)进行镜检，其原理也与革兰氏染色类似：结晶紫初染和碘液媒染后，在细胞壁内形成了不溶于水的结晶紫与碘的复合物，将细胞质染成紫色，酵母细胞壁比革兰氏阳性菌细胞壁更厚更致密，乙醇脱色处理时，因失水反而使网孔缩小，再加上它不含类脂，故乙醇处理不会出现缝隙，因此能把结晶紫与碘复合物牢牢留在壁内，所以细胞壁完整的情况下，其仍呈紫色。而如果酵母细胞壁破裂，经乙醇处理时，则会将结晶紫与碘复合物溶出脱色后仍呈无色，再经番红等红色染料复染后呈红色。革兰氏染色法染色后，细胞壁未破裂的呈紫色，细胞壁破裂的细胞(包括仍保持较完整的细胞形态类型)和从胞内释放的物质均被染成红色，高压匀质破碎产物整体在视野内形成均匀的红色背景。The effect of Pichia pastoris cell lysis can be detected by microscopic examination. Gram staining method can be used (Gram staining kit purchased from Beijing Solebao Technology Co., Ltd.). The principle is also the same as that of Gram staining. Lang's staining is similar: after initial dyeing with crystal violet and mordant with iodine solution, a water-insoluble complex of crystal violet and iodine is formed in the cell wall, staining the cytoplasm purple. The yeast cell wall is thicker and more dense than the cell wall of Gram-positive bacteria. Dense. During ethanol decolorization, the mesh will shrink due to water loss. In addition, it does not contain lipids, so there will be no gaps after ethanol treatment. Therefore, the crystal violet and iodine complex can be firmly retained in the wall, so When the cell wall is intact, it remains purple. If the yeast cell wall is ruptured and treated with ethanol, the crystal violet and iodine complex will be dissolved and decolorized, and it will remain colorless. It will then turn red after being counterstained with red dyes such as safranin. After Gram staining, cells with unbroken cell walls are purple, cells with ruptured cell walls (including cell types that still maintain a relatively complete morphology) and substances released from the cells are dyed red, and the high-pressure homogenized broken products are overall in A uniform red background is formed within the field of view.
实验方法步骤简述如下：The experimental method steps are briefly described as follows:
破碎前使用血球计数板对酵母细胞进行计数，总数计为C。Use a hemocytometer to count the yeast cells before disruption, and the total number is counted as C.
细胞破碎后，取破碎液，涂片，进行革兰氏染色：要求与普通革兰氏染色相同，注意涂布要均匀且不要厚，乙醇脱色时间要控制。相差显微镜或油镜观察，进行玻片计数，对每次观察下每个视野下紫色的细胞数(n1)和红色细胞数(n2)进行计数，更换玻片上不同位置以获取多个不同视野，重复多次。计算破壁后未破壁细胞的比例a＝n1/(n1+n2)，取平均值；After the cells are disrupted, take the disrupted fluid, smear, and perform Gram staining: the requirements are the same as ordinary Gram staining. Pay attention to the coating to be even and not thick, and the ethanol destaining time to be controlled. Observe with a phase contrast microscope or oil microscope, count the slides, count the number of purple cells (n1) and the number of red cells (n2) in each field of view under each observation, and replace different positions on the slide to obtain multiple different fields of view. Repeat multiple times. Calculate the proportion of unbroken cells after breaking the wall a=n1/(n1+n2), and take the average;
同样取细胞破碎后破碎液，高速离心后去上清，以稀释菌浆相同体积的纯水重悬沉淀，革兰氏染色或只以结晶紫染色，更利于观察，使用血球计数板进行计数(最好使用相差显微镜)，计破碎后仍呈较完整细胞形态的酵母细胞总数目，计为C’。Similarly, take the lysate after cell disruption, centrifuge it at high speed, remove the supernatant, resuspend the pellet in the same volume of pure water as the diluted bacterial slurry, and stain with Gram or only crystal violet, which is more convenient for observation. Use a hemocytometer for counting ( It is best to use a phase contrast microscope), count the total number of yeast cells that still show a relatively complete cell shape after being broken, and count it as C'.
破壁率计算，统计分析：
Wall breaking rate calculation and statistical analysis:
D(100％)：破壁率；C：破碎前细胞数目；C’：破碎后细胞总数目；a：破壁后未破壁细胞的比例。D (100%): wall breaking rate; C: number of cells before breaking; C’: total number of cells after breaking; a: proportion of unbroken cells after breaking.
酵母破碎时，以要求细胞壁破裂为最为根本的要求，革兰氏染色法是一种极为直观、有效的检测方法，清晰明确、分辨性高。以公开号CN103102407B中保藏编号为CGMCC NO.7189工程菌的发酵菌体在不同破碎条件下破壁率为例得到：100MPar(3次)：破壁率95.9％；120MPar(2次)：破壁率97.3％；150MPar(2次)：破壁率99.4％；200MPar(2次)：破壁率99.5％，其破碎裂解效果如图2所示。120Mpar破碎2次已完全可满足毕赤酵母的裂解需求。When yeast is broken, the most fundamental requirement is to break the cell wall. Gram staining is an extremely intuitive and effective detection method that is clear and highly resolving. Taking the wall-breaking rate of the fermentation bacterial cell with the deposit number CGMCC NO.7189 in Publication No. CN103102407B as an example under different crushing conditions: 100MPar (3 times): wall-breaking rate 95.9%; 120MPar (2 times): wall-breaking The rate is 97.3%; 150MPar (2 times): the wall breaking rate is 99.4%; 200MPar (2 times): the wall breaking rate is 99.5%. The crushing and cracking effect is shown in Figure 2. 120Mpar broken twice can fully meet the lysis needs of Pichia pastoris.
二、对本实施例中方法得到的溶胞物滤液中重组人胶原蛋白、总蛋白、多糖、氨基酸、维生素的检测2. Detection of recombinant human collagen, total protein, polysaccharides, amino acids, and vitamins in the lysate filtrate obtained by the method in this example
本实施例所采用的工程菌表达的重组人胶原蛋白在氨基端(N端)或羧基端(C端)有6×His-Tag标签，利于检测滤液中的胶原蛋白的检测可使用Western Blot进行直观的定性检测、使用Elisa进行定量检测。以公开号CN103102407B中保藏编号为CGMCC NO.7189工 程菌为例，采用发酵菌体10％湿菌体浓度制备的溶胞物滤液，进行相应检测。The recombinant human collagen expressed by the engineering bacteria used in this example has a 6×His-Tag tag at the amino terminus (N terminus) or carboxyl terminus (C terminus), which facilitates the detection of collagen in the filtrate and can be carried out using Western Blot. Intuitive qualitative detection and quantitative detection using Elisa. The deposit number in the publication number CN103102407B is CGMCC NO.7189 Taking Chengji as an example, the lysate filtrate prepared by the fermentation bacteria with a wet bacterial concentration of 10% was used for corresponding testing.
a.SDS-PAGE电泳及WB检测结果：a.SDS-PAGE electrophoresis and WB detection results:
取制备的溶胞物滤液，以未转入表达胶原蛋白基因的空白巴斯德毕赤酵母菌(购自赛默飞世尔科技(中国)有限公司)制备溶胞物滤液为空白对照，进行SDS-PAGE电泳及Western Blot检测，结果如下：Take the prepared lysate filtrate and use the blank Pichia pastoris that has not been transformed with collagen gene expression (purchased from Thermo Fisher Scientific (China) Co., Ltd.) to prepare the lysate filtrate as a blank control. SDS-PAGE electrophoresis and Western Blot detection, the results are as follows:
图3A为以10％湿菌体浓度制备的溶胞物滤液进行SDS-PAGE电泳图，从上到下分布着不同的大小的蛋白质；图3B为以10％湿菌体浓度制备的溶胞物滤液Western Blot结果(Anti-His，抗体购自生工生物工程(上海)股份有限公司，小鼠抗Hi s单克隆抗体，货号D199987)，从图3B(ECL化学发光显色，全自动化学发光图像分析***Tanon 5200将蛋白质分子质量标准合成于图像)中可以看到，6×His标签可检测到抗6×His标签的条带，目的条带大小均与公开号CN103102407B的专利中所表达的重组III型人胶原蛋白纯化冻干品的电泳中分子量一致，说明溶胞物滤液含有重组III型人胶原蛋白，而空白对照(未转入表达胶原蛋白基因的空白巴斯德毕赤酵母菌裂解制备的溶胞物滤液)则无任何条带显现。Figure 3A is an SDS-PAGE electrophoresis diagram of the lysate filtrate prepared with a wet bacterial cell concentration of 10%. Proteins of different sizes are distributed from top to bottom; Figure 3B is a lysate prepared with a wet bacterial cell concentration of 10%. Western Blot results of the filtrate (Anti-His, the antibody was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., mouse anti-His monoclonal antibody, product number D199987), from Figure 3B (ECL chemiluminescence color development, fully automatic chemiluminescence image The analysis system Tanon 5200 synthesizes the protein molecular mass standard in the image) It can be seen that the 6×His tag can detect the anti-6×His tag band, and the size of the target band is the same as the recombinant expressed in the patent No. CN103102407B The molecular weights of the purified freeze-dried products of type III human collagen in electrophoresis were consistent, indicating that the lysate filtrate contained recombinant type III human collagen, while the blank control (blank Pichia pastoris without transfection of collagen gene expression was prepared by lysis lysate filtrate), no bands appeared.
b.Elisa检测5D胶原蛋白含量检测：b. Elisa detection of 5D collagen content detection:
取以10％湿菌体浓度制备的溶胞物滤液进行Elisa检测，同时取纯化后的重组III型人胶原蛋白冻干海绵进行Elisa标准曲线制定，以未转入表达胶原蛋白基因的空白巴斯德毕赤酵母菌制备溶胞物滤液为空白对照，使用间接Elisa法，实验步骤简述如下：Take the lysate filtrate prepared with 10% wet bacterial cell concentration for Elisa detection. At the same time, take the purified recombinant type III human collagen freeze-dried sponge to develop the Elisa standard curve. Use the blank Bath that has not been transformed into the collagen gene expression. The lysate filtrate prepared by Pichia pastoris was used as a blank control, and the indirect Elisa method was used. The experimental steps are briefly described as follows:
(1)以PBS梯度稀释溶胞物滤液(10、100倍)，加入酶标板中，同时以PBS溶解及梯度稀释公开号CN103102407B的专利中所表达的重组III型人胶原蛋白纯化冻干品样品，也加入酶标板中，4℃静置过夜。(1) Gradiently dilute the lysate filtrate (10, 100 times) with PBS, add it to the enzyme plate, and simultaneously dissolve and gradiently dilute the purified lyophilized recombinant type III human collagen expressed in the patent with Publication No. CN103102407B with PBS Samples were also added to the enzyme plate and left to stand at 4°C overnight.
(2)弃酶标板中液体，以PBS稀释的5％脱脂奶粉溶液(购自生工生物工程(上海)股份有限公司，这里为质量体积比，如5g的脱脂奶粉溶解于100mL的PBS中)，37℃中封闭处理2小时。(2) Discard the liquid in the enzyme plate and use 5% skimmed milk powder solution diluted with PBS (purchased from Sangon Bioengineering (Shanghai) Co., Ltd., here is the mass to volume ratio, such as 5g of skimmed milk powder dissolved in 100mL of PBS) , sealed at 37°C for 2 hours.
(3)弃酶标板中液体，PBS清洗两次后，加入5％脱脂奶粉溶液稀释一抗(Anti-His，抗体购自生工生物工程(上海)股份有限公司，小鼠抗His单克隆抗体，货号D199987)溶液，稀释比例为1∶5000，37℃中静置1小时。(3) Discard the liquid in the enzyme plate, wash it twice with PBS, add 5% skim milk powder solution to dilute the primary antibody (Anti-His, the antibody was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., mouse anti-His monoclonal antibody , Cat. No. D199987) solution, the dilution ratio is 1:5000, and let stand at 37°C for 1 hour.
(4)弃酶标板中液体，PBS清洗两次后，加入5％脱脂奶粉溶液稀释HRP标记的二抗(购自生工生物工程(上海)股份有限公司，小鼠抗Hi s单克隆抗体配套的二抗，货号D199987)溶液，稀释比例为1∶2000，37℃中静置1小时。(4) Discard the liquid in the enzyme plate, wash twice with PBS, add 5% skim milk powder solution to dilute HRP-labeled secondary antibody (purchased from Sangon Bioengineering (Shanghai) Co., Ltd., mouse anti-His monoclonal antibody package Secondary antibody (Cat. No. D199987) solution, the dilution ratio is 1:2000, and let stand at 37°C for 1 hour.
(5)弃酶标板中液体，PBS清洗两次后，加入TMB显色液(TMB显色P0209-100mL，碧云天生物)，待出现明显蓝色后，加入终止液(P0215-100mL，碧云天生物)，于酶标仪(VarioskanTM LUX多功能酶标仪-VL0L0TD0，赛默飞世尔科技(中国)有限公司)读取450nm的吸光度值。(5) Discard the liquid in the enzyme plate, wash it twice with PBS, add TMB chromogenic solution (TMB chromogenic P0209-100mL, Beyotime Biotech), wait until obvious blue color appears, add stop solution (P0215-100mL, Beyotime Biotech) Yuntian Biotechnology), read the absorbance value at 450 nm on a microplate reader (Varioskan TM LUX multifunctional microplate reader-VL0L0TD0, Thermo Fisher Scientific (China) Co., Ltd.).
(6)GraphPad Prism 5对标准曲线进行拟合，进行数据处理，计算样品中重组胶原蛋白浓度。拟合的标准曲线如图4所示，横坐标是胶原蛋白浓度的对数，纵坐标是450nm的吸光度值。曲线拟合方程为：
Y＝0.0007221+(1.372-0.0007221)/(1+10^((-0.1103-X)*1.341)))(6) GraphPad Prism 5 fits the standard curve, performs data processing, and calculates the concentration of recombinant collagen in the sample. The fitted standard curve is shown in Figure 4. The abscissa is the logarithm of the collagen concentration, and the ordinate is the absorbance value at 450nm. The curve fitting equation is:
Y＝0.0007221+(1.372-0.0007221)/(1+10^((-0.1103-X)*1.341)))
其中，Y是蛋白质浓度，X是检测到样品的450nm的吸度光值。Among them, Y is the protein concentration, and X is the absorbance light value at 450nm of the detected sample.
不同浓度的溶胞物滤液Elisa检测结果(450nm吸光度值)如下： The Elisa detection results (450nm absorbance value) of lysate filtrate at different concentrations are as follows:
表1.不同浓度的溶胞物滤液Elisa检测450nm吸光度值
Table 1. Absorbance values at 450nm detected by Elisa of lysate filtrate at different concentrations
从曲线拟合上分析，较好的检测范围为OD450范围约在0.25～1.25之间。实验组中可以有效计算的是ddH2O为破碎液离心上清稀释10倍后Elisa实验OD450值。溶胞物滤液中重组III型人胶原蛋白浓度为11.44μg/mL。总体而言，10％湿菌体浓度制备的溶胞物滤液，重组胶原蛋白含量约为10～15μg/mL，当然随着湿菌体浓度的增长，重组人胶原蛋白含量也会升高。而未转入表达胶原蛋白基因的空白巴斯德毕赤酵母菌制备溶胞物滤液的OD450则基本处于检测有效范围下限之外，检测不到胶原蛋白。From the analysis of curve fitting, the better detection range is that the OD450 range is approximately between 0.25 and 1.25. What can be effectively calculated in the experimental group is the OD450 value of the Elisa experiment after diluting the centrifugation supernatant of the broken liquid 10 times with ddH 2 O. The concentration of recombinant type III human collagen in the lysate filtrate was 11.44 μg/mL. In general, the recombinant collagen content of the lysate filtrate prepared with a wet bacterial cell concentration of 10% is approximately 10-15 μg/mL. Of course, as the wet bacterial cell concentration increases, the recombinant human collagen content will also increase. However, the OD450 of the lysate filtrate prepared from the blank Pichia pastoris without the collagen gene expression was basically outside the lower limit of the effective detection range, and no collagen could be detected.
c.溶胞物滤液中总蛋白含量的检测c. Detection of total protein content in lysate filtrate
实验方法参考《中华人民共和国药典》(2020版)蛋白质含量测定法中第五法：考马斯亮蓝法(Bradford法)进行总蛋白质含量的检测。溶胞物滤液以公开号为CN103102407B的专利中CGMCC NO.7189工程菌的发酵菌体，0.2％～20％湿菌体浓度制备，Bradford蛋白浓度测定试剂购自碧云天生物(P0006)，实验步骤简述如下：The experimental method refers to the fifth method of protein content determination in the "Pharmacopoeia of the People's Republic of China" (2020 edition): Coomassie Brilliant Blue method (Bradford method) to detect the total protein content. The lysate filtrate was prepared from the fermentation cells of CGMCC NO.7189 engineering bacteria in the patent with the publication number CN103102407B, with a wet cell concentration of 0.2% to 20%. The Bradford protein concentration determination reagent was purchased from Beyotime Biotechnology (P0006). Experimental steps A brief description is as follows:
(1)以纯水稀释蛋白标准(5mg/mL BSA)至0、0.125、0.25、0.5、0.75、1、1.5mg/mL。(1) Dilute the protein standard (5 mg/mL BSA) with pure water to 0, 0.125, 0.25, 0.5, 0.75, 1, and 1.5 mg/mL.
(2)取5μL不同浓度蛋白标准加到96孔板的蛋白标准孔中，同时取5μL溶胞物滤液样品到96孔板的样品孔中。(2) Add 5 μL of protein standards of different concentrations to the protein standard wells of the 96-well plate, and at the same time add 5 μL of lysate filtrate sample to the sample wells of the 96-well plate.
(3)各孔加入250μL G250染色液。(3) Add 250μL G250 staining solution to each well.
(4)用酶标仪(VarioskanTM LUX多功能酶标仪-VL0L0TD0，赛默飞世尔科技(中国)有限公司)测定595nm吸光度值。(4) Use a microplate reader (Varioskan TM LUX multifunctional microplate reader-VL0L0TD0, Thermo Fisher Scientific (China) Co., Ltd.) to measure the absorbance value at 595 nm.
(5)处理数据，根据标准曲线和使用的样品体积计算出样品中的蛋白浓度。标准曲线及曲线方程如图5所示。0.2％～20％湿菌体浓度制备的溶胞物滤液中总蛋白含量(mg/mL)结果如表2：(5) Process the data and 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 Figure 5. The results of total protein content (mg/mL) in the lysate filtrate prepared at a wet bacterial cell concentration of 0.2% to 20% are shown in Table 2:
表2.0.2％～20％湿菌体浓度制备的溶胞物滤液中总蛋白含量(mg/mL)
Table 2. Total protein content (mg/mL) in lysate filtrate prepared from 0.2% to 20% wet bacterial cell concentration
Bradford法中标准曲线的斜率(R2)＞0.98，随着制备溶胞物滤液时湿菌体浓度的增加，溶胞物滤液中总蛋白含量随之增加，为0.192～26.9mg/mL，当湿菌体浓度在10％以上时，溶胞物滤液中总蛋白含量均大于10mg/mL(＞1％)，最高可达到到25mg/mL(2.5％)以上，而多次以10％菌浓度裂解生产的滤液中总蛋白含量一般为10～15mg/mL。也根据实际情况选取《中华人民共和国药典》(2020版)蛋白质含量测定法中其它方法比如BCA法、双缩脲法、凯氏定氮法进行总蛋白含量的检测。The slope (R 2 ) of the standard curve in the Bradford method is >0.98. As the concentration of wet bacteria increases when preparing the lysate filtrate, the total protein content in the lysate filtrate increases, ranging from 0.192 to 26.9 mg/mL. When the wet bacterial cell concentration is above 10%, the total protein content in the lysate filtrate is greater than 10 mg/mL (>1%), and can reach a maximum of more than 25 mg/mL (2.5%). However, many times with a bacterial concentration of 10% The total protein content in the filtrate produced by lysis is generally 10 to 15 mg/mL. Other methods such as BCA method, biuret method, and Kjeldahl nitrogen determination method in the "Pharmacopoeia of the People's Republic of China" (2020 edition) protein content determination method were also selected according to the actual situation to detect the total protein content.
d.溶胞物滤液中多糖含量的检测d. Detection of polysaccharide content in lysate filtrate
参考文献“金凤慈与闵莉静，苯酚-硫酸法测定百合多糖中糖含量.科技信息，2011(10)”第127-128页中的方法，溶胞物滤液以公开号CN103102407B的专利中CGMCC NO.7189工程菌的发酵菌体10％-20％湿菌体浓度制备，检测多糖含量。Refer to the method on pages 127-128 of "Jin Fengci and Min Lijing, phenol-sulfuric acid method for determination of sugar content in lily polysaccharides. Science and Technology Information, 2011 (10)", and the lysate filtrate was published in CGMCC NO. 7189 of the patent No. CN103102407B. The fermentation cells of the engineered bacteria are prepared with a wet cell concentration of 10%-20%, and the polysaccharide content is detected.
实验步骤简述如下： The experimental steps are briefly described as follows:
(1)甘露糖标准液(0.1mg/mL)的制备：精密称取105℃干燥至恒重的甘露糖500mg置于500mL容量瓶中，加水溶解并稀释至刻度摇匀，得1mg/mL的储备溶液，备用。用10mL移液管移取上溶液10mL，定容至100mL，得0.1mg/mL的甘露糖标准液。(1) Preparation of mannose standard solution (0.1 mg/mL): Precisely weigh 500 mg of mannose dried to constant weight at 105°C and place it in a 500 mL volumetric flask. Add water to dissolve and dilute to the mark. Shake well to obtain 1 mg/mL. Reserve solution for later use. Pipette 10 mL of the upper solution with a 10 mL pipette and adjust the volume to 100 mL to obtain a 0.1 mg/mL mannose standard solution.
(2)85％的苯酚溶液的制备：将苯酚用水浴锅加热至45度，取85mL的苯酚溶液，溶于15mL的水中，待溶解完，得到85％的苯酚溶液。放入冰箱4度冰箱，避光保存。长期使用。(2) Preparation of 85% phenol solution: Heat the phenol in a water bath to 45 degrees, take 85 mL of the phenol solution, and dissolve it in 15 mL of water. After the dissolution is completed, an 85% phenol solution is obtained. Store in the refrigerator at 4 degrees Celsius and away from light. long-term use.
5％的苯酚溶液的制备：取出85％的苯酚水浴45度加热溶解，取1mL的溶液，再溶解于16mL纯水中既得5％的苯酚溶液。避光保存，现配先用。Preparation of 5% phenol solution: Take out 85% phenol in a water bath and heat it to dissolve at 45 degrees, take 1 mL of the solution, and then dissolve it in 16 mL of pure water to obtain a 5% phenol solution. Store away from light, use freshly prepared.
(3)标准样品制备与标准曲线绘制：精确吸取甘露糖标准液(0.1mg/mL)，按照表3进行配制。加入苯酚液0.5mL和(快速，精密加入)浓硫酸2.5mL，混匀20秒，在80℃水浴下放置30min后，冷却，于波长490nm处测定吸光度值。以空白(空白)参比实验，以吸光度值为纵坐标，甘露糖浓度为横坐标绘制标准曲线，建立标准曲线回归方程。(3) Standard sample preparation and standard curve drawing: Accurately draw mannose standard solution (0.1 mg/mL) and prepare according to Table 3. Add 0.5 mL of phenol solution and 2.5 mL of concentrated sulfuric acid (quick and precise addition), mix for 20 seconds, place in a water bath at 80°C for 30 minutes, cool, and measure the absorbance value at a wavelength of 490 nm. Use a blank (blank) reference experiment, draw a standard curve with the absorbance value as the ordinate and the mannose concentration as the abscissa, and establish a standard curve regression equation.
表3.标准样品的配制和检测
Table 3. Preparation and detection of standard samples
(4)待测样品检测，方法同上。(4) Detect the sample to be tested, the method is the same as above.
(5)多糖含量的计算：(5) Calculation of polysaccharide content:
将测得样品的吸光度带入标准曲线方程。得到相应的多糖溶度，再除以样品溶胞物滤液中多糖的溶度，既得多糖的含量。
Insert the measured absorbance of the sample into the standard curve equation. The corresponding polysaccharide solubility is obtained, and then divided by the solubility of the polysaccharide in the sample lysate filtrate to determine the polysaccharide content.
W：样品多糖的含量；W1：在标准曲线回归方程式下，计算得到的多糖的溶度；W2：样品溶胞物滤液中多糖的溶度。W: the polysaccharide content of the sample; W1: the solubility of the polysaccharide calculated under the standard curve regression equation; W2: the solubility of the polysaccharide in the sample lysate filtrate.
标准曲线及曲线方程如图6所示：The standard curve and curve equation are shown in Figure 6:
所制备得到溶胞物滤液中多糖含量(mg/mL)如下：The polysaccharide content (mg/mL) in the prepared lysate filtrate is as follows:
制备溶胞物滤液时的湿菌体浓度(W/W)为10％时，多糖含量达到1.15mg/mL；湿菌体浓度15％时，多糖含量达到1.45mg/mL；湿菌体浓度20％时，多糖含量达到1.59mg/mL。综合其他浓度时检测结果可知，一般溶胞物滤液中的多糖含量在1-1.6mg/mL。When the wet bacterial cell concentration (W/W) when preparing the lysate filtrate is 10%, the polysaccharide content reaches 1.15mg/mL; when the wet bacterial cell concentration is 15%, the polysaccharide content reaches 1.45mg/mL; the wet bacterial cell concentration is 20 %, the polysaccharide content reached 1.59 mg/mL. Combining the test results with other concentrations, it can be seen that the polysaccharide content in the general lysate filtrate is 1-1.6mg/mL.
e.溶胞物滤液中氨基酸、维生素含量的检测e. Detection of amino acid and vitamin content in lysate filtrate
溶胞物滤液以公开号CN103102407B中CGMCC NO.7189工程菌的发酵菌体10％湿菌体浓度制备。以“GB 5009.89-2016食品安全国家标准食品中烟酸和烟酰胺的测定”第二法、“GB 5009.85-2016食品安全国家标准食品中维生素B2的测定”第一法、“GB 5009.124-2016食品安全国家标准食品中氨基酸的测定”、“GB 5413.20-2013食品安全国家标准婴幼儿食品和乳品中胆碱的测定”第二法、“GB 5009.270-2016食品安全国家标准食品中肌醇的测 定”第二法、“GB 5009.154-2016食品安全国家标准食品中维生素B6的测定”第一法、“GB5009.210-2016食品安全国家标准食品中泛酸的测定”第一法、“GB 5009.211-2014食品安全国家标准食品中叶酸的测定”、“GB 5009.259-2016食品安全国家标准食品中生物素的测定”检测溶胞物滤液中氨基酸、维生素的含量，委托上海天祥质量技术服务有限公司检测完成并出具检测报告。The lysate filtrate was prepared with a 10% wet cell concentration of fermentation cells of CGMCC No. 7189 engineering bacteria in Publication No. CN103102407B. Use the second method of "GB 5009.89-2016 National Food Safety Standard for Determination of Niacin and Niacinamide in Foods", "GB 5009.85-2016 National Food Safety Standard for Determination of Vitamin B2 in Foods" First Method, and "GB 5009.124-2016 Food "National Food Safety Standard for Determination of Amino Acids in Foods", "GB 5413.20-2013 National Food Safety Standard for Determination of Choline in Foods for Infants and Young Children and Dairy Products" Second Method, "GB 5009.270-2016 National Food Safety Standard for Determination of Inositol in Foods" The second method of "GB 5009.154-2016 National Food Safety Standard for Determination of Vitamin B6 in Foods", the first method of "GB 5009.210-2016 National Food Safety Standard for Determination of Pantothenic Acid in Foods", "GB 5009.211- "2014 National Food Safety Standard Determination of Folic Acid in Foods" and "GB 5009.259-2016 National Food Safety Standard Determination of Biotin in Foods" were used to detect the contents of amino acids and vitamins in the lysate filtrate, and were commissioned by Shanghai Tianxiang Quality Technical Service Co., Ltd. Complete and issue test report.
检测结果如表4所示。The test results are shown in Table 4.
表4.溶胞物滤液中氨基酸、微生物检测结果
Table 4. Test results of amino acids and microorganisms in lysate filtrate
结果显示，以10％湿菌体浓度制备的的溶胞滤液中含有多种维生素B族、氨基酸，氨基酸总量可达到约0.82％，作为原料可为化妆品提供丰富的活性物质成份。The results show that the lysate filtrate prepared with a wet bacterial cell concentration of 10% contains a variety of B vitamins and amino acids, and the total amino acid content can reach about 0.82%. As a raw material, it can provide rich active ingredients for cosmetics.
三、对得到的溶胞物滤液的安全性检测3. Safety testing of the obtained lysate filtrate
a.残留DNA的检测a. Detection of residual DNA
溶胞物滤液中的DNA主要是巴斯德毕赤酵母基因组DNA残留，以PCR-荧光探针法检测以10％菌浓度制备的溶胞物滤液中的DNA，委托湖州申科生物技术有限公司完成并出具检测报告。基本过程简述如下，实验过程中相关试剂无特殊说明均为湖州申科生物技术有限公司生产或或市售可购买得到：The DNA in the lysate filtrate is mainly the genomic DNA residue of Pichia pastoris. The PCR-fluorescent probe method was used to detect the DNA in the lysate filtrate prepared with a bacterial concentration of 10%. Huzhou Shenke Biotechnology Co., Ltd. was commissioned Complete and issue test report. The basic process is briefly described as follows. The relevant reagents during the experiment are produced by Huzhou Shenke Biotechnology Co., Ltd. or are commercially available unless otherwise specified:
(1)含有溶胞物滤液样品进行稀释后，以磁珠法纯化；(1) The filtrate sample containing lysates is diluted and then purified by magnetic bead method;
(2)以巴斯德毕赤酵母特异性的引物和探针(毕赤酵母残留DNA检测试剂盒(PCR-荧光探针法)，购自湖州申科生物技术有限公司SK030205P100)，对溶胞物滤液样品中DNA的特异性片段进行体外扩增(定量PCR仪FQD-96A，LineGene 9600Plus定量PCR***)；(2) Using Pichia pastoris-specific primers and probes (Pichia pastoris residual DNA detection kit (PCR-fluorescent probe method), purchased from Huzhou Shenke Biotechnology Co., Ltd. SK030205P100), lyse Specific fragments of DNA in the filtrate samples were amplified in vitro (quantitative PCR instrument FQD-96A, LineGene 9600Plus quantitative PCR system);
(3)在PCR扩增过程中，特异性探针会从模板DNA上释放出来，随之利用荧光定量 PCR仪就能检测出荧光信号，荧光信号数据通过PCR仪所具有的检测软件进行分析。(3) During the PCR amplification process, the specific probe will be released from the template DNA, and then fluorescence quantification will be used. The PCR instrument can detect the fluorescence signal, and the fluorescence signal data is analyzed through the detection software of the PCR instrument.
检测结果如下：The test results are as follows:
表5.溶胞物滤液残留DNA检测结果
Table 5. Residual DNA detection results of lysate filtrate
表中，In the table,
检测均值：检测值的平均值。CV：CV(Coefficient of Variance)，标准差与均值的比率，统计学中常用于表征变异系数的，两个CV的含义都是一样的。加标量：指的加入的标准品的量。加标检测均值：加入标准品、参照品检测值的平均值。Detection mean: the average value of detection values. CV: CV (Coefficient of Variance), the ratio of the standard deviation to the mean, is often used to characterize the coefficient of variation in statistics. The meaning of both CVs is the same. Added standard amount: refers to the amount of standard added. Average value of spiked test: the average value of the added standard and reference test values.
结果可见，本发明制备的溶胞物滤液中的DNA含量检测为31.4μg/mL，低于生物药品中要求的DNA残留量，处于安全范围。The results show that the DNA content in the lysate filtrate prepared by the present invention was detected to be 31.4 μg/mL, which is lower than the DNA residual amount required in biological drugs and is within a safe range.
b.重金属、甲醇含量等的检测b. Detection of heavy metals, methanol content, etc.
依据《化妆品安全技术规范》2015版第四章1.6和2.22第一法相关检测方法，对溶胞物滤液中重金属、甲醇含量进行检测，委托苏州海关综合技术中心完成并出具检测报告。According to the relevant detection methods of Method 1 of Chapter 4, 1.6 and 2.22, of the 2015 edition of the "Cosmetic Safety Technical Specifications", the heavy metal and methanol content in the lysate filtrate was tested, and the Suzhou Customs Comprehensive Technology Center was entrusted to complete and issue a test report.
检测结果如下表6所示：The test results are shown in Table 6 below:
表6.溶胞物滤液重金属、甲醇检测结果
Table 6. Test results of heavy metals and methanol in lysate filtrate
结果可见，本发明制备的溶胞物滤液中重金属、甲醇含量，结果均符合安全性要求，本发明中溶胞物滤液为化妆品原料，在实际产品的配制使用中，会产生实际的稀释效果，不存在安全性问题。The results show that the heavy metal and methanol contents in the lysate filtrate prepared by the present invention meet the safety requirements. The lysate filtrate in the present invention is a cosmetic raw material, and will produce an actual dilution effect during the preparation and use of actual products. There are no security issues.
c.微生物限度的检测c. Testing of microbial limits
依据《化妆品安全技术规范》2015版中微生物限度检测方法，对溶胞物滤液进行微生素限度的检测，委托苏州海关综合技术中心完成并出具检测报告，结果均符合安全性要求，检测结果如下表7所示，结果可见，本发明制备的溶胞物滤液中微生物结果均符合要求。According to the microbial limit testing method in the 2015 version of the "Cosmetic Safety Technical Specifications", the lysate filtrate was tested for microbial limits, and the Suzhou Customs Comprehensive Technology Center was entrusted to complete and issue a test report. The results all met the safety requirements. The test results are as follows As shown in Table 7, the results show that the microbial results in the lysate filtrate prepared by the present invention meet the requirements.
表7.溶胞物滤液微生物检测结果

Table 7. Microbial detection results of lysate filtrate

d.毒理学检测 d. Toxicological testing
依据《化妆品安全技术规范》2015版中毒理学试验方法，对溶胞物滤液进行毒理学安全性评价，委托宁波海关技术中心完成检测并出具检测报告。According to the toxicological test methods of the 2015 version of the "Cosmetic Safety Technical Specifications", the toxicological safety evaluation of the lysate filtrate was conducted, and the Ningbo Customs Technology Center was entrusted to complete the test and issue a test report.
检测结果统计如下表8所示，表中所列实验方法均为《化妆品安全技术规范》中规定的常规方法，实验均在宁波海关技术中心完成。结果可见，本发明制备的溶胞物滤液中毒理学检测结果均符合要求，安全性有保障。The statistics of the test results are shown in Table 8 below. The experimental methods listed in the table are all conventional methods stipulated in the "Cosmetic Safety Technical Specifications", and the experiments were all completed at the Ningbo Customs Technology Center. The results show that the toxicology test results of the lysate filtrate prepared by the present invention meet the requirements and the safety is guaranteed.
表8.溶胞物滤液毒理学安全性检测结果
Table 8. Toxicological safety test results of lysate filtrate
(1)急性经口毒性试验(1) Acute oral toxicity test
受试样品处理：称取5.0806g样品(即本发明制备的溶胞物滤液)置于烧杯中取少量纯水将样品混匀后转入20mL容量瓶内，以少量纯水多次冲洗烧杯一并转入容量瓶中，加入纯水定容至刻度线，充分摇匀后转入样品管标识备用，样品现配现用。灌胃体积2.0mL/100g·BW。Test sample processing: Weigh 5.0806g sample (i.e., the lysate filtrate prepared by the present invention) and place it in a beaker. Take a small amount of pure water, mix the sample evenly, and transfer it to a 20mL volumetric flask. Rinse the beaker multiple times with a small amount of pure water. Transfer it together into a volumetric flask, add pure water to adjust the volume to the mark, shake well and transfer it into a sample tube to mark for later use. The sample is ready for use. The intragastric administration volume is 2.0mL/100g·BW.
实验动物：6周龄SPF级ICR小鼠(上海杰思捷实验动物有限公司)10只，雌雄各半(雌性动物未孕和未曾产仔)，体重控制在18.5g～21.4g(同性别体重不超过均值的±20％)Experimental animals: 10 6-week-old SPF grade ICR mice (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half female (female animals are not pregnant and have never given birth), and the weight is controlled between 18.5g and 21.4g (the same sex weight) Not more than ±20% of the mean)
试验前动物在屏障环境动物房(屏障环境、23.0℃～23.8℃、相对湿度为40.8％～59.7％)中适应8天(Co60辐照鼠料，江苏省协同医药生物工程有限责任公司；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用)，染毒前动物禁食过夜。Before the test, the animals were adapted to the barrier environment animal room (barrier environment, 23.0°C to 23.8°C, relative humidity of 40.8% to 59.7%) for 8 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.; Level 1 RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization) is directly provided to the animals for free drinking through the drinking spout). The animals fasted overnight before exposure.
试验开始后，采用一次限量法，灌胃剂量为5080.6mg/kg。染毒后继续禁食3小时，每天观察中毒症状或行为变化，每周称重一次。观察并记录染毒过程和观察期内动物的中毒和死亡情况，观察周期14天，观察期结束后，处死存活动物并进行大体解剖。After the start of the test, the one-time limited method was adopted, and the intragastric dose was 5080.6 mg/kg. Continue to fast for 3 hours after exposure to the poison, observe poisoning symptoms or behavioral changes every day, and weigh once a week. Observe and record the poisoning process and the poisoning and death of animals during the observation period. The observation period is 14 days. After the observation period, the surviving animals are killed and gross dissection is performed.
检测结果：Test results:
实验动物在染毒14天内未见任何中毒症状和中毒死亡；雌雄动物的体重未见异常。实验观察结束，对受试动物进行大体解剖检查也未见异常变化。LD50＞5080.6mg/kg，详细结果如下：The experimental animals did not show any poisoning symptoms or death from poisoning within 14 days of exposure; there were no abnormalities in the body weight of male and female animals. After the experimental observation was completed, no abnormal changes were found in the gross anatomical examination of the test animals. LD50>5080.6mg/kg, the detailed results are as follows:
表9.急性经口毒性试验结果
Table 9. Acute oral toxicity test results
可见，在本试验条件下，受试样品对ICR小鼠的急性经口LD50＞5000mg/kg。根据急性经口毒性分级，该样品属于实际无毒级。 It can be seen that under the conditions of this test, the acute oral LD50 of the test sample to ICR mice is >5000mg/kg. According to the acute oral toxicity classification, this sample belongs to the actual non-toxic grade.
(2)急性经皮毒性试验(2) Acute dermal toxicity test
受试样品处理：用原样品溶胞物滤液测试。Test sample processing: Use the original sample lysate filtrate for testing.
实验动物：8周龄SPF级SD大鼠(上海杰思捷实验动物有限公司)10只，雌雄各半(雌性动物未孕和未曾产仔)，体重控制在201.2g～218.2g。Experimental animals: 10 8-week-old SPF grade SD rats (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half female (female animals are not pregnant and have never given birth), and the weight is controlled between 201.2g and 218.2g.
试验前动物在屏障环境动物房(屏障环境、22.0℃～22.9℃、相对湿度为45.8％～55.5％)中适应7天(Co60辐照鼠料，江苏省协同医药生物工程有限责任公司；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用，染毒前动物不禁食，自由饮水。Before the test, the animals were adapted to the barrier environment animal room (barrier environment, 22.0°C to 22.9°C, relative humidity of 45.8% to 55.5%) for 7 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.; Level 1 RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization) is directly provided to the animals through the drinking spout. The animals do not fast before being exposed to the poison and can drink water freely.
试验开始后，采用一次限量法，染毒剂量为2500mg/kg。试验期间动物单笼饲养。染毒前体重范围201.2g～218.2g，计算10％的体表面积为31.3cm2～33.1cm2。试验前24小时，剃除动物背部染毒区域的被毛，备皮区域为5cm×7cm。称取每只动物所需受试样量为0.5g，均匀涂敷于备皮区域，然后用一层薄胶片覆盖，再用无刺激胶布固定，防止动物舔食，封闭接触24小时后取下固定物和覆盖物，用水洗去皮肤上残留的受试样品。观察并记录染毒过程和观察期内动物的中毒和死亡情况，每周称重一次，观察周期为14天，观察期结束后，处死存活动物并进行大体解剖。After the test started, the one-time limit method was adopted, and the dose was 2500mg/kg. During the experiment, animals were kept in individual cages. The body weight before exposure ranged from 201.2g to 218.2g, and the calculated 10% body surface area was 31.3cm 2 to 33.1cm 2 . 24 hours before the test, the coat of the infected area on the back of the animal was shaved, and the skin preparation area was 5cm × 7cm. Weigh 0.5g of the required test sample for each animal, apply it evenly on the skin preparation area, then cover it with a thin layer of film, and then fix it with non-irritating tape to prevent animals from licking it. Close the contact and remove it after 24 hours. Fixtures and coverings are washed with water to remove any remaining test sample from the skin. Observe and record the poisoning process and the poisoning and death of animals during the observation period. Weigh once a week. The observation period is 14 days. After the observation period, the surviving animals are killed and gross dissection is performed.
试验结果：实验动物在染毒14天内未见任何中毒症状和中毒死亡；雌雄动物的体重未见异常。实验观察结束，对受试动物进行大体解剖检查也未见异常变化。LD50＞2500mg/kg。结果见下表10。Test results: The experimental animals did not show any poisoning symptoms or death from poisoning within 14 days of exposure; there were no abnormalities in the body weight of male and female animals. After the experimental observation was completed, no abnormal changes were found in the gross anatomical examination of the test animals. LD50>2500mg/kg. The results are shown in Table 10 below.
表10.急性经皮毒性试验结果
Table 10. Acute dermal toxicity test results
可见，在本试验条件下，受试样品对SD大鼠的急性经皮LD50＞2180mg/kg，根据急性皮肤毒性分级，该样品为微毒级。It can be seen that under the conditions of this test, the acute percutaneous LD50 of the test sample to SD rats is >2180 mg/kg. According to the acute skin toxicity classification, the sample is slightly toxic.
(3)急性皮肤刺激性试验(3) Acute skin irritation test
受试样品处理：用原样品溶胞物滤液测试。Test sample processing: Use the original sample lysate filtrate for testing.
实验动物：普通级新西兰兔4只(嘉善县惠民街道生旺家庭农场)；雌雄各半(雌性动物选用未孕和未曾产仔)；体重控制在2.27kg～2.98kg。Experimental animals: 4 ordinary-grade New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.27kg and 2.98kg.
试验前动物在实验动物房环境中至少适应3d，单笼饲养，饲养环境温度：22.8℃～23.6℃，相对湿度：52.9％～61.4％4；饲养饲料实验兔配合饲料(维持型，桐乡市东方饲料有限责任公司)；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用。Before the test, the animals were adapted to the environment of the experimental animal room for at least 3 days. They were raised in a single cage. The environment temperature was 22.8°C to 23.6°C, and the relative humidity was 52.9% to 61.4%. The feeding feed was experimental rabbit compound feed (maintenance type, Tongxiang Dongfang). Feed Co., Ltd.); first-level RO ultrafiltered water (adding sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
实验方法：试验前约24h将实验动物背部脊柱两侧被毛剪掉，去毛范围各约3cm×3cm。取受试样品0.5mL直接涂在一侧去毛皮肤上，涂抹面积为2.5cm×2.5cm，然后用二层纱布(2.5cm×2.5cm)和一层玻璃纸覆盖，再用无刺激性胶布和绷带加以固定。另一侧皮肤作为对照。封闭试验4h后用温水清除残留受试样品。于清除受试样品后的1h、24h、48h和72h观察涂抹部位皮肤反应，进行皮肤反应评分，并根据24h、48h和72h各观察时点最高积分均值，进行刺激强度分级。Experimental method: About 24 hours before the test, the hair on both sides of the back spine of the experimental animals was cut off, and the hair removal range was about 3cm × 3cm. Take 0.5mL of the test sample and apply it directly on one side of the depilated skin. The application area is 2.5cm×2.5cm. Then cover it with two layers of gauze (2.5cm×2.5cm) and a layer of cellophane, and then use non-irritating tape. and bandage to secure. The skin on the other side served as a control. After 4 hours of sealing test, remove the remaining test sample with warm water. Observe the skin reaction at the application site at 1h, 24h, 48h and 72h after removing the test sample, and score the skin reaction. The irritation intensity will be graded based on the highest mean score at each observation time point at 24h, 48h and 72h.
试验结果：试验期间，实验动物均未出现异常症状，各观察时点(24h、48h和72h)最 高皮肤刺激积分均值为0.00。在本试验条件下，受试样品对家兔急性皮肤刺激性为：无刺激性。Test results: During the test, none of the experimental animals showed abnormal symptoms, and the most severe symptoms at each observation time point (24h, 48h and 72h) The mean high skin irritation score is 0.00. Under the conditions of this test, the acute skin irritation of the test sample to rabbits is: non-irritating.
表11.急性皮肤刺激性试验结果
Table 11. Acute skin irritation test results
(4)多次皮肤刺激性试验(4) Multiple skin irritation tests
受试样品处理：用原样品溶胞物滤液测试。Test sample processing: Use the original sample lysate filtrate for testing.
实验动物：普通级新西兰兔4只(嘉善县惠民街道生旺家庭农场)；雌雄各半(雌性动物选用未孕和未曾产仔)；体重控制在2.34kg～2.92kg。Experimental animals: 4 ordinary-grade New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.34kg and 2.92kg.
试验前动物在实验动物房环境中至少适应3d，单笼饲养，饲养环境温度：22.0℃～23.9℃，相对湿度：40.5％～58.6％；饲养饲料实验兔配合饲料(维持型，桐乡市东方饲料有限责任公司)；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用。Before the test, the animals were adapted to the experimental animal room environment for at least 3 days, and were raised in single cages. The breeding environment temperature was: 22.0°C ~ 23.9°C, and the relative humidity was: 40.5% ~ 58.6%; the feeding feed was experimental rabbit compound feed (maintenance type, Tongxiang Dongfang Feed Co., Ltd.); first-level RO ultrafiltered water (added with sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
实验方法：试验前约24h将实验动物背部脊柱两侧被毛剪掉，去毛范围各为3cm×3cm。涂抹面积2.5cm×2.5cm。取受试样品0.5mL涂抹在一侧去毛皮肤上，另一侧去毛皮肤作为对照，每天涂抹1次，连续涂抹14d。从第二天开始，每次涂抹前剪毛，用温水清除残留受试样品。一小时后观察结果。Experimental method: About 24 hours before the test, the hair on both sides of the back spine of the experimental animals was cut off, and the hair removal range was 3cm × 3cm each. The application area is 2.5cm×2.5cm. Take 0.5 mL of the test sample and apply it on one side of the depilated skin, and the other side of the depilated skin as a control. Apply once a day for 14 days. Starting from the next day, clip the hair before each application and remove the remaining test sample with warm water. Observe the results after one hour.
试验结果：试验期间，实验动物均未出现异常症状，14天内每天每只动物平均积分为0.00。详见下表12。在本试验条件下，受试样品对家兔多次皮肤刺激性为无刺激性。Test results: During the test period, no experimental animals showed abnormal symptoms, and the average score of each animal per day within 14 days was 0.00. See Table 12 below for details. Under the conditions of this test, the test sample is non-irritating to the skin of rabbits.
表12.多次皮肤刺激性试验结果
Table 12. Multiple skin irritation test results
(5)急性眼刺激性试验(5)Acute eye irritation test
受试样品处理：用原样品溶胞物滤液测试。Test sample processing: Use the original sample lysate filtrate for testing.
实验动物：普通级新西兰兔3只(嘉善县惠民街道生旺家庭农场)；雌雄各半(雌性动物选用未孕和未曾产仔)；体重控制在2.40kg～2.61kg。 Experimental animals: 3 ordinary New Zealand rabbits (Shengwang Family Farm, Huimin Street, Jiashan County); half male and half female (female animals were selected as non-pregnant and non-pregnant animals); body weight was controlled between 2.40kg and 2.61kg.
试验前动物在实验动物房环境中至少适应3d，单笼饲养，饲养环境温度：23.0℃～24.1℃，相对湿度：49.8％～60.7％；饲养饲料：实验兔配合饲料(维持型，桐乡市东方饲料有限责任公司)；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用。Before the test, the animals were adapted to the environment of the experimental animal room for at least 3 days and were raised in single cages. The breeding environment temperature was: 23.0°C ~ 24.1°C, and the relative humidity was: 49.8% ~ 60.7%; the feeding feed was: experimental rabbit compound feed (maintenance type, Tongxiang Dongfang Feed Co., Ltd.); first-level RO ultrafiltered water (adding sodium hypochlorite to control the free chlorine content in the water to 2-3ppm for sterilization) can be directly supplied to animals for free drinking through the drinking spout.
试验方法：在试验开始前的24h内对试验动物的两只眼睛进行检查(包括使用2％荧光素钠溶液检查)。有眼睛刺激症状、角膜缺陷和结膜损伤的动物不能用于试验。轻轻拉开家兔一侧眼睛的下眼睑，将受试样品0.1mL滴入结膜囊中，使上、下眼睑被动闭合1s，以防止受试样品丢失。另一侧眼睛不处理作自身对照。滴入受试样品后24h内不冲洗眼睛。在滴入受试样品后1h、24h、48h、72h以及第4d和第7d对动物眼睛进行检查。如果72h未出现刺激反应，即可终止试验。Test method: Examine both eyes of the test animals within 24 hours before the start of the test (including examination using 2% fluorescein sodium solution). Animals with symptoms of eye irritation, corneal defects, and conjunctival damage should not be used for testing. Gently open the lower eyelid of one eye of the rabbit, drop 0.1 mL of the test sample into the conjunctival sac, and passively close the upper and lower eyelids for 1 s to prevent the loss of the test sample. The other eye was left untreated for self-control. Do not rinse eyes within 24 hours after instilling the test sample. The eyes of the animals were examined at 1h, 24h, 48h, 72h, and on the 4th and 7th days after instillation of the test sample. If no irritation reaction occurs within 72 hours, the test can be terminated.
试验结果：试验期间，实验动物未出现异常症状，动物个体积分均值均为0.00。详见下表13。在本试验条件下，受试样品对家兔急性眼刺激性为：无刺激性。Test results: During the test, the experimental animals showed no abnormal symptoms, and the average individual points of the animals were all 0.00. See Table 13 below for details. Under the conditions of this test, the acute eye irritation of the test sample to rabbits is: non-irritating.
表13.急性眼刺激性试验结果
Table 13. Acute eye irritation test results
*动物个体积分均值指的是每只动物在三个不同观察时间(24h、48h和72h)角膜、虹膜、结膜充血和结膜水肿四方面的平均积分(即每只动物的24h、48h和72h评分之和除以观察时点数3)。*The average animal score refers to the average score of cornea, iris, conjunctival congestion and conjunctival edema at three different observation times (24h, 48h and 72h) for each animal (i.e. the 24h, 48h and 72h scores of each animal) The sum is divided by the number of points at the time of observation 3).
(6)皮肤光毒性试验(6) Skin phototoxicity test
受试样品处理：用原样品胞溶物滤液测试。阳性对照：8-甲氧基补骨脂(批号：L630V07；北京百灵威科技有限公司)，溶剂：无水乙醇，浓度：0.05％；用量：2.4000mg。Test sample processing: Use the original sample cell lysate filtrate for testing. Positive control: 8-methoxypsoralen (batch number: L630V07; Beijing Bailingwei Technology Co., Ltd.), solvent: absolute ethanol, concentration: 0.05%; dosage: 2.4000 mg.
实验动物：普通级荷兰种豚鼠试验组和阳性对照组各6只(余姚市泗门镇建飞实验兔养殖场)；雌雄不限(雌性动物选用未孕和未曾产仔)；体重控制在261.2g～272.4g。Experimental animals: 6 ordinary-grade Dutch guinea pig test group and 6 positive control group (Jianfei Experimental Rabbit Breeding Farm, Simen Town, Yuyao City); there is no limit on male or female (female animals are selected as non-pregnant and non-pregnant animals); the weight is controlled at 261.2 g～272.4g.
试验前动物在实验动物房环境中至少适应3d。饲养环境温度：23.3℃～24.0℃；相对湿度：49.5％～56.7％；饲养饲料：豚鼠配合饲料(维持型，苏州安慕飞生物科技有限公司)；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)。经饮水嘴直接供动物自由饮用。The animals were adapted to the environment of the experimental animal room for at least 3 days before the test. Breeding environment temperature: 23.3℃～24.0℃; relative humidity: 49.5%～56.7%; feeding feed: guinea pig compound feed (maintenance type, Suzhou Anmufei Biotechnology Co., Ltd.); first-level RO ultrafiltered water (add sodium hypochlorite, The free chlorine content in the water is controlled at 2-3ppm (sterilization). The animals can drink directly through the drinking spout.
试验方法：照射剂量：10000mJ/cm2，平均光强度：15.1mW/cm2 Test method: Irradiation dose: 10000mJ/cm 2 , average light intensity: 15.1mW/cm 2
照射时间计算公式为：
The calculation formula of irradiation time is:
注：1mW/cm2＝1mJ/cm2/secNote: 1mW/cm 2 =1mJ/cm 2 /sec
在UVA-TOXI光毒仪里输入上述照射剂量和平均光强度后，按计算公式得出照射时间为00：10：64(即照射时间为664s)；After inputting the above irradiation dose and average light intensity into the UVA-TOXI photopoison meter, the irradiation time is 00:10:64 according to the calculation formula (that is, the irradiation time is 664s);
试验步骤：试验前动物在实验动物房环境中至少适应3d。试验前约24h，将动物脊柱两侧皮肤去毛，试验部位皮肤需完好，无损伤及异常。备4块去毛区，每块去毛面积约为2cm×2cm。将动物固定，按《化妆品安全技术规范》(2015年版)第六章7皮肤光毒性试验表1所示，在动物去毛区1和2涂敷0.2mL受试样品。30min后，左侧(去毛区1和3)用铝箔复盖，胶带固定，右侧用UVA进行照射。结束后分别于1h、24h、48h和72h观察皮肤反应，根据《化妆品安全技术规范》(2015年版)第六章7皮肤光毒性试验表2判定每只动物皮肤反应评分。阳性对照组按同样方法进行。Test procedures: The animals must adapt to the environment of the experimental animal room for at least 3 days before the test. About 24 hours before the test, the skin on both sides of the animal's spine should be hairless. The skin at the test site must be intact and free of damage and abnormalities. Prepare 4 hair removal areas, each with a hair removal area of approximately 2cm x 2cm. Fix the animal, and apply 0.2 mL of the test sample on the animal hair removal areas 1 and 2 as shown in Table 1 of Chapter 6, 7 Skin Phototoxicity Test of the "Technical Specifications for Safety of Cosmetics" (2015 Edition). After 30 minutes, the left side (hair removal areas 1 and 3) was covered with aluminum foil and fixed with tape, and the right side was irradiated with UVA. Observe the skin reaction at 1h, 24h, 48h and 72h after the end, and determine the skin reaction score of each animal according to Table 2 of Chapter 6, 7 Skin Phototoxicity Test of "Safety Technical Specifications for Cosmetics" (2015 Edition). The positive control group was carried out in the same way.
试验结果：试验期间，实验动物均未出现异常症状，各观察时点的动物积分均为0，根据皮肤刺激反应分级，该受试样品未见皮肤光毒性。阳性对照物对豚鼠皮肤光毒性试验结果见下表14，受试样品对豚鼠皮肤光毒性试验结果见表15。在本试验条件下，受试样品对豚鼠皮肤光毒性试验结果：未见皮肤光毒性。Test results: During the test, no experimental animals showed abnormal symptoms, and the animal points at each observation time point were all 0. According to the skin irritation response classification, no skin phototoxicity was found in the test sample. The phototoxicity test results of the positive control substance on guinea pig skin are shown in Table 14 below, and the phototoxicity test results of the test sample on guinea pig skin are shown in Table 15. Under this test condition, the test results of the test sample's phototoxicity to guinea pig skin: no skin phototoxicity was found.
表14.阳性对照物对豚鼠皮肤光毒性试验结果
Table 14. Phototoxicity test results of positive control substances on guinea pig skin
注：表头中1、2、3、4为《化妆品安全技术规范》(2015年)第六章7、皮肤光毒性试验之图1所示试验区。Note: 1, 2, 3, and 4 in the header are the test areas shown in Figure 1 of Chapter 6, 7. Skin Phototoxicity Test, of the "Safety Technical Specifications for Cosmetics" (2015).
表15.受试样品对豚鼠皮肤光毒性试验结果
Table 15. Phototoxicity test results of test samples on guinea pig skin
注：表头1、2、3、4为《化妆品安全技术规范》(2015年)第六章7、皮肤光毒性试验之图1所示试验区。Note: Table headers 1, 2, 3, and 4 are the test areas shown in Figure 1 of Chapter 6, 7. Skin Phototoxicity Test, of "Cosmetic Safety Technical Specifications" (2015).
(7)皮肤***反应试验(7)Skin allergy test
受试样品处理：用原样品胞溶物滤液测试。阳性对照：α-己基肉桂醛(批号：LV30V28；北京百灵威科技有限公司)；溶剂：无水乙醇(分析纯，批号：20210119，国药集团化学试剂有限公司)、丙酮(分析纯，批号：20170918，国药集团化学试剂有限公司)、Millipore 纯水。Test sample processing: Use the original sample cell lysate filtrate for testing. Positive control: α-hexylcinnamic aldehyde (batch number: LV30V28; Beijing Bailingwei Technology Co., Ltd.); solvent: absolute ethanol (analytically pure, batch number: 20210119, Sinopharm Chemical Reagent Co., Ltd.), acetone (analytically pure, batch number: 20170918, Sinopharm Chemical Reagent Co., Ltd.), Millipore Pure water.
诱导浓度：60％，取3.6mLα-己基肉桂醛与2.4mL 80％乙醇溶液混匀配制得到；激发浓度：30％，取3.0mLα-己基肉桂醛与7.0mL 80％丙酮溶液混匀配制得到；用量：6.6mL。Induction concentration: 60%, prepared by mixing 3.6mL α-hexylcinnamic aldehyde and 2.4mL 80% ethanol solution; excitation concentration: 30%, prepared by mixing 3.0mL α-hexylcinnamic aldehyde and 7.0mL 80% acetone solution; Dosage: 6.6mL.
实验动物：普通级荷兰种豚鼠(余姚市泗门镇建飞实验兔养殖场)；试验组和阳性对照组各20只，阴性对照组10只；雌雄不限(雌性动物未孕和未曾产仔)；体重控制在257.8g～273.4g。Experimental animals: ordinary Dutch guinea pigs (Jianfei Experimental Rabbit Breeding Farm, Simen Town, Yuyao City); 20 test groups and 20 positive control groups each, and 10 negative control groups; male or female (female animals are not pregnant and have never given birth) ); weight is controlled between 257.8g and 273.4g.
试验前动物在实验动物房环境中至少适应3d。饲养环境温度：22.5℃～24.1℃；相对湿度：43.4％～61.9％；饲料饲料：豚鼠配合饲料(维持型，苏州安慕飞生物科技有限公司)，一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)。经饮水嘴直接供动物自由饮用。The animals were adapted to the environment of the experimental animal room for at least 3 days before the test. Breeding environment temperature: 22.5°C ~ 24.1°C; relative humidity: 43.4% ~ 61.9%; feed: guinea pig compound feed (maintenance type, Suzhou Anmufei Biotechnology Co., Ltd.), first-level RO ultrafiltered water (add sodium hypochlorite, The free chlorine content in the water is controlled at 2-3ppm (sterilization). The animals can drink directly through the drinking spout.
试验方法：试验前约24h，将豚鼠背部左侧去毛，去毛范围约为6cm2。Test method: About 24 hours before the test, remove the hair on the left side of the guinea pig's back, and the area of hair removal is about 6cm 2 .
诱导接触：将0.2mL受试样品涂在试验组动物2cm×2cm去毛皮肤上，以二层纱布和一层玻璃纸覆盖，再以无刺激胶布封闭固定6h。第7d和第14d以同样方法重复一次。阳性对照组用60％α-己基肉桂醛溶液同法操作，阴性对照组除不给予受试样品外与试验组同法操作。Induced contact: Apply 0.2 mL of the test sample on the 2cm × 2cm hairless skin of the animals in the test group, cover it with two layers of gauze and one layer of cellophane, and then seal and fix it with non-irritating tape for 6 hours. Repeat steps 7d and 14d in the same way. The positive control group was treated with 60% α-hexylcinnamic aldehyde solution in the same manner, and the negative control group was treated in the same manner as the test group except that no test sample was given.
激发接触：末次诱导后14d，将0.2mL受试样品涂于试验组和阴性对照组豚鼠背部右侧2cm×2cm去毛区(接触前24h脱毛)，然后用二层纱布和一层玻璃纸覆盖，再以无刺激胶布固定6h。阳性对照组用30％α-己基肉桂醛溶液同法操作。激发接触结束后24h和48h观察皮肤反应，评分并判定致敏强度。Provocative exposure: 14 days after the last induction, apply 0.2 mL of the test sample to the 2cm × 2cm hair removal area on the right side of the back of the guinea pigs in the test group and the negative control group (hair removal 24 hours before contact), and then cover it with two layers of gauze and one layer of cellophane. , and then fixed with non-irritating tape for 6 hours. The positive control group used 30% α-hexylcinnamic aldehyde solution in the same manner. Observe the skin reaction 24h and 48h after the provocative exposure, score and determine the sensitization intensity.
试验结果：试验期间，实验动物均未出现异常症状，各观察时点的致敏率均为0％。见表16、表17。在本试验条件下，受试样品对豚鼠皮肤***反应试验结果为：未见皮肤***反应。Test results: During the test, no experimental animals showed abnormal symptoms, and the sensitization rate at each observation time point was 0%. See Table 16 and Table 17. Under this test condition, the test results of the guinea pig skin allergy test of the test sample were: no skin allergy was observed.
表16.试验动物体重汇总表
Table 16. Summary table of test animal body weight
表17.受试样品或阳性对照对豚鼠皮肤***反应试验结果(BT法)
Table 17. Results of test samples or positive controls on guinea pig skin allergy test (BT method)
注：在皮肤反应强度栏中填写皮肤反应积分为0、1、2、3……时，表示发生反应的动物数占受试动物数的比例；表中的原样指的是本发明制备的溶胞物滤液。Note: When filling in the skin reaction score as 0, 1, 2, 3... in the skin reaction intensity column, it indicates the proportion of the number of animals that reacted to the number of animals tested; the original in the table refers to the solution prepared by the present invention. Cell filtrate.
(8)细菌回复突变试验(8) Bacterial reverse mutation test
受试样品处理：以无菌纯水为溶剂分别配制成各剂量组浓度。 Test sample processing: Use sterile pure water as the solvent to prepare the concentrations of each dosage group.
阴性对照：无菌纯水、二甲基亚砜(DMSO，无色澄清液体)Negative control: sterile pure water, dimethyl sulfoxide (DMSO, colorless clear liquid)
阳性对照：Positive control:
(1)不加S9(购买，美国MOLTOX)：(1) Without S9 (purchase, US MOLTOX):
敌克松(Dexon)：批号：G1063485；生产厂家：德国Dr.Enrenstorfer GmBH公司；溶剂：无菌纯水；浓度：500μg/mL；用量：0.1mL/皿；Dexon: Batch number: G1063485; Manufacturer: Dr. Enrenstorfer GmBH, Germany; Solvent: sterile pure water; Concentration: 500μg/mL; Dosage: 0.1mL/dish;
叠氮钠(SA)：批号：20120615；生产厂家：上海埃彼化学试剂有限公司；溶剂：无菌纯水；浓度：15μg/mL；用量：0.1mL/皿。Sodium azide (SA): Batch number: 20120615; Manufacturer: Shanghai Abbey Chemical Reagent Co., Ltd.; Solvent: sterile pure water; Concentration: 15μg/mL; Dosage: 0.1mL/dish.
(2)加S9：(2)Add S9:
2-氨基芴(2-AF)：批号：C11449586；生产厂家：上海麦克林生化科技有限公司；溶剂：二甲基亚砜(DMSO)；浓度：100μg/mL；用量：0.1mL/皿；2-Aminofluorene (2-AF): Batch number: C11449586; Manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.; Solvent: dimethyl sulfoxide (DMSO); Concentration: 100μg/mL; Dosage: 0.1mL/dish;
1，8-二羟基蒽醌(1，8-DHQ)：批号：C10429233；生产厂家：上海麦克林生化科技有限公司；溶剂：DMSO；浓度：500μg/mL；用量：0.1mL/皿；1,8-Dihydroxyanthraquinone (1,8-DHQ): Batch number: C10429233; Manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.; Solvent: DMSO; Concentration: 500μg/mL; Dosage: 0.1mL/dish;
环磷酰胺(CP)：批号：K1812016；生产厂家：阿拉丁试剂(上海)有限公司；溶剂：无菌纯水；浓度：2000μg/mL；用量：0.1mL/皿。Cyclophosphamide (CP): Batch number: K1812016; Manufacturer: Aladdin Reagent (Shanghai) Co., Ltd.; Solvent: sterile pure water; Concentration: 2000μg/mL; Dosage: 0.1mL/dish.
实验菌株：鼠伤寒沙门氏菌株(TA97a、TA98、TA100、TA102和TA1535)，购自美国MOLTOX公司，经鉴定，试验菌株符合标准要求。Experimental strains: Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) were purchased from the American MOLTOX company. After identification, the test strains met the standard requirements.
试验步骤：选择12.5μL/皿、25.0μL/皿、50.0μL/皿和100.0μL/皿四个剂量组，同时设空白对照、溶剂对照(阴性对照)、阳性诱变剂对照和无菌对照组。菌株经增菌培养制得增菌液后进行平板掺入。实验时，将含0.5mmol/L组氨酸、0.5mmol/L生物素溶液的2.0mL顶层培养基分装于试管中，于45℃水浴保温，然后每管依次加入增菌液0.1mL、受试样品0.1mL和磷酸盐缓冲液0.5mL或者S9混合液0.5mL(需代谢活化时)，充分混匀，迅速倾入底层培养基上，转动平板，使其分布均匀。水平放置待冷凝固化后，将平板翻转，倒置于37℃培养箱中孵育48h。阳性诱变剂对照、溶剂对照、空白对照和无菌对照也依此法操作。计数每皿回变菌落数。Test steps: Select four dosage groups of 12.5μL/dish, 25.0μL/dish, 50.0μL/dish and 100.0μL/dish, and set up blank control, solvent control (negative control), positive mutagen control and sterile control group. . The bacterial strain is enriched and cultured to prepare an enrichment liquid, which is then incorporated into the plate. During the experiment, 2.0 mL of the top medium containing 0.5 mmol/L histidine and 0.5 mmol/L biotin solution was divided into test tubes and incubated in a 45°C water bath. Then, 0.1 mL of enrichment solution and 0.1 mL of enrichment solution were added to each tube. Mix 0.1 mL of test sample and 0.5 mL of phosphate buffer or 0.5 mL of S9 mixture (when metabolic activation is required), quickly pour it into the bottom culture medium, and rotate the plate to make it evenly distributed. Place it horizontally until condensation solidifies, turn the plate over, and place it upside down in a 37°C incubator for 48 hours. The positive mutagen control, solvent control, blank control and sterility control are also operated in this way. Count the number of colonies per dish.
记录受试物各剂量组、空白对照(自发回变)、溶剂对照以及阳性诱变剂对照的每皿回变菌落数，并求平均值和标准差。Record the number of reversion colonies per dish in each dose group of the test substance, blank control (spontaneous reversion), solvent control and positive mutagen control, and calculate the average value and standard deviation.
试验结果：标准平板掺入试验结果见表18。溶剂对照(阴性对照)结果表明在不添加试验菌株鼠伤寒沙门氏菌条件下，各组均无菌落生长，可用于试验；空白对照和阳性诱变剂对照组的回复突变菌落数在可被接受的范围内，本试验有效；Test results: The standard plate incorporation test results are shown in Table 18. The results of the solvent control (negative control) showed that without the addition of the test strain Salmonella typhimurium, no colonies grew in each group and could be used for the test; the number of reverse mutation colonies in the blank control and positive mutagen control groups were within an acceptable range. Within, this test is valid;
本试验条件下，受试物各剂量组在加与不加代谢活化***平板掺入法检测的回复菌落数均未超过溶剂对照组的2倍以上，也未呈现剂量反应关系。Under the conditions of this test, the number of recovered colonies detected by the plate incorporation method of each dosage group of the test substance with or without metabolic activation system did not exceed 2 times that of the solvent control group, and there was no dose-response relationship.
表18.鼠伤寒沙门氏菌回复突变试验结果(个/皿，x±s)

Table 18. Salmonella typhimurium reverse mutation test results (pieces/dish, x±s)

(9)体内哺乳动物细胞微核试验(9) In vivo mammalian cell micronucleus test
受试样品处理：样品溶胞物滤液以纯水为溶剂配制成各剂量组溶液。Test sample processing: The sample lysate filtrate is prepared into solutions for each dose group using pure water as the solvent.
阴性对照：纯水Negative control: pure water
阳性对照：环磷酰胺(批号：F1812016；品牌：阿拉丁；溶剂：纯水；浓度：3mg/mL)Positive control: cyclophosphamide (batch number: F1812016; brand: Aladdin; solvent: pure water; concentration: 3mg/mL)
实验动物：SPF级ICR小鼠(上海杰思捷实验动物有限公司)30只，雌雄各半(雌性动物未孕和未曾产仔)，体重控制在25.0g～27.4g，同性别体重不超过均值的±20％。Experimental animals: 30 SPF grade ICR mice (Shanghai Jiesijie Experimental Animal Co., Ltd.), half male and half male (female animals are not pregnant and have never given birth), the weight is controlled between 25.0g and 27.4g, and the weight of the same sex does not exceed the average. ±20%.
试验前动物在屏障环境动物房(屏障环境、22.8℃～23.5℃、相对湿度为51.6％～57.8％)中检疫期为4天(Co60辐照鼠料，江苏省协同医药生物工程有限责任公司；一级RO超滤水(加入次氯酸钠，将水中游离氯含量控制在2-3ppm除菌)经饮水嘴直接供动物自由饮用，染毒前动物不禁食，自由饮水。Before the test, the animals were kept in a barrier environment animal room (barrier environment, 22.8°C to 23.5°C, relative humidity of 51.6% to 57.8%) for a quarantine period of 4 days (Co60 irradiated rat feed, Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd.; The first-level RO ultrafiltered water (sodium hypochlorite is added to control the free chlorine content in the water to 2-3ppm for sterilization) is directly provided to the animals through the drinking spout. The animals do not fast before being exposed to the poison and can drink water freely.
试验方法：剂量设置：设受试物组、阴性对照组(纯水)和阳性对照组(60mg/kg环磷酰胺)。根据预试验结果，受试物组的终浓度设定为1000mg/kg、2000mg/kg、5000mg/kg。Test method: Dosage setting: Set up test substance group, negative control group (pure water) and positive control group (60mg/kg cyclophosphamide). Based on the preliminary test results, the final concentrations of the test substance groups were set to 1000 mg/kg, 2000 mg/kg, and 5000 mg/kg.
给药方式：经口灌胃；给药体积：20mL/kg·BW。Administration method: Oral gavage; Administration volume: 20mL/kg·BW.
试验步骤：动物染毒采用经口灌胃30h染毒法，即两次染毒间隔24h，第二次染毒后6h取材。用颈椎脱臼法处死动物，取股骨。剥除肌肉，擦净血污。切断股骨两端，暴露骨髓腔。用注射器吸取0.1mL小牛血清，冲洗骨髓腔用冲洗液常规涂片，晾干或热风吹干。将已干的涂片，在甲醇中固定10min。用姬姆萨应用液染色15min，然后用清水冲洗，晾干。阳性与阴性对照组的操作程序同试验组。选择细胞分布均匀、完整、着色适当的区域。在油镜下计数含 微核的PCE(嗜多染红细胞)数。PCE呈灰蓝色，NCE(成熟红细胞)呈粉红色。每只动物计数2000个PCE中含有的微核数。微核细胞率(MN‰)指含有微核的PCE数，以千分率表示。1个PCE中出现有2个或多个微核，仍按1个计数。采用波松分布u检验或其他适当的显著性试验方法，对各组间的微核率进行比较。Test procedures: The animals were exposed to the poison by oral gavage for 30 hours, that is, the interval between two exposures was 24 hours, and the samples were taken 6 hours after the second exposure. The animals were sacrificed by cervical dislocation, and the femurs were removed. Peel off the muscles and wipe away the blood. Cut off both ends of the femur to expose the medullary cavity. Use a syringe to absorb 0.1 mL of calf serum, rinse the bone marrow cavity, use regular smear with flushing solution, and air dry or blow dry with hot air. The dried smears were fixed in methanol for 10 min. Dye with Giemsa application solution for 15 minutes, then rinse with water and dry. The operating procedures of the positive and negative control groups were the same as those of the experimental group. Select an area where the cells are evenly distributed, intact, and properly colored. Count the contents under an oil lens The number of PCE (polychromatic erythrocytes) with micronuclei. PCE is gray-blue, and NCE (mature red blood cells) is pink. The number of micronuclei contained in 2000 PCEs was counted for each animal. Micronucleus cell rate (MN‰) refers to the number of PCEs containing micronuclei, expressed in parts per thousand. If 2 or more micro-cores appear in 1 PCE, they are still counted as 1. Use the Poisson distribution u test or other appropriate significance test methods to compare the micronucleus rates between groups.
试验结果：试验结果表明与阴性对照组比较，阳性对照组小鼠骨髓中含微核的嗜多染细胞数显著升高(P＜0.05)，而各受试物组的嗜多染红细胞微核率与阴性对照组比较，差异无统计学意义(P＞0.05)。Test results: The test results showed that compared with the negative control group, the number of polychromatic cells containing micronuclei in the bone marrow of the mice in the positive control group was significantly increased (P<0.05), while the number of polychromatic erythrocyte micronuclei in each test group was significantly higher (P<0.05). Compared with the negative control group, the difference was not statistically significant (P>0.05).
在本试验条件下，该受试物体内哺乳动物红细胞微核试验结果为阴性，该样品为非致突变剂。急性毒性检测(急性经口毒性试验检测无毒，急性经皮毒性试验检测微毒性)、皮肤刺激性/腐蚀性、皮肤致敏性、皮肤光毒性及遗传毒性等多项检测结果表明滤液具有良好的毒理学安全性。Under the conditions of this test, the test result of mammalian erythrocyte micronucleus in the subject was negative, and the sample was a non-mutagenic agent. Acute toxicity test (acute oral toxicity test detects non-toxicity, acute dermal toxicity test detects slight toxicity), skin irritation/corrosion, skin sensitization, skin phototoxicity and genetic toxicity, etc. The results show that the filtrate has good toxicological safety.
表19.微核试验微核率统计结果
Table 19. Statistical results of micronucleus rate in micronucleus test
注：与阴性对照组比较，P＜0.05。微核率和PCE/NCE以小鼠为单位统计，表示为均值±标准差。Note: Compared with the negative control group, P<0.05. The micronucleus rate and PCE/NCE were calculated in mouse units and expressed as mean ± standard deviation.
e.人体皮肤斑贴试验e. Human skin patch test
依据《化妆品安全技术规范》2015版中人体安全性检验方法，对溶胞物滤液进行人体皮肤斑贴试验，委托英格尔检测技术服务(上海)有限公司完成并出具检测报告。According to the human safety test method in the 2015 version of the "Cosmetic Safety Technical Specifications", a human skin patch test was conducted on the lysate filtrate, and Ingels Testing Technology Service (Shanghai) Co., Ltd. was entrusted to complete and issue a test report.
共选取33名受试者进行了测试，试验结果显示32名受试者在去除受试物斑试器后0.5h、24h、48h，评分等级均为0，均未出现皮肤反应；1名受试者在去除受试物斑试器后24h，评分等级为1，具体实验结果见下表20所示。A total of 33 subjects were selected for the test. The test results showed that 0.5h, 24h, and 48h after removing the test substance spot tester, the scores of 32 subjects were all 0, and no skin reaction occurred; 1 subject The tester's score level is 1 24 hours after removing the test object spot tester. The specific experimental results are shown in Table 20 below.
表20.溶胞物滤液皮肤封闭型斑贴试验测试结果
Table 20. Lysate filtrate skin closed patch test test results
表21.皮肤封闭型斑贴试验皮肤反应分级标准
Table 21. Skin occlusion patch test skin reaction grading criteria
参照《化妆品技术审评要点》中的规定，30例受试者中出现1级皮肤不良反应的人数不得多于5例(不含5例)，具体分级标准见上表21。结果显示，本次测试结果符合规定的安全性要求，据此认为，本发明制备的溶胞物滤液应用于人体是安全的。According to the regulations in the "Key Points for Technical Review of Cosmetics", the number of grade 1 adverse skin reactions among 30 subjects shall not be more than 5 (excluding 5 cases). The specific grading standards are shown in Table 21 above. The results show that the test results meet the prescribed safety requirements. Accordingly, it is believed that the lysate filtrate prepared in the present invention is safe to be applied to the human body.
综上所述，本发明制备所得的巴斯德毕赤酵母发酵溶胞物滤液，根据《化妆品安全技术规范》中的方法进行重金属含量、甲醇含量、微生物限度、安全毒理学、人体皮肤斑贴试验检测，同时进行了残留DNA检测，证明其安全性符合应用于化妆品的要求。In summary, the Pichia pastoris fermentation lysate filtrate prepared by the present invention was tested for heavy metal content, methanol content, microbial limits, safety toxicology, and human skin patching according to the method in the "Cosmetic Safety Technical Specifications" Experimental testing and residual DNA testing were conducted to prove that its safety meets the requirements for application in cosmetics.
实施例2：发酵溶胞物滤液的功效性检测Example 2: Efficacy test of fermentation lysate filtrate
本实施例中，以实施例1中制备的溶胞物滤液为例，进行多项功效性评测。In this example, the lysate filtrate prepared in Example 1 was used as an example to conduct multiple efficacy evaluations.
一、舒缓功效检测1. Soothing efficacy test
表皮作为分隔机体与外界环境的组织，除了作为物理屏障外，角质形成细胞能接受外界“危险信号”刺激并转化传递给皮肤内免疫细胞预警，形成皮肤的免疫屏障，皮肤免疫屏障功能调节紊乱会导致皮肤炎症性反应，引发皮肤问题和疾病。皮肤接触刺激性较强的一些化妆品或紫外线等，会导致皮肤屏障发生临床型的急性损伤，导致皮肤干燥，出现红斑现象。阴离子表面活性剂十二烷基硫酸钠(Sodium lauryl sulfate，SLS)具有双亲性(亲水性和亲脂性)特征，在较大浓度下接触皮肤后能够损伤皮肤屏障，尤其是屏障中的脂质成分以及细胞膜。因此本实验以体外重组的表皮模型为研究对象，采用SLS刺激造成损伤模型，再用实施例1的得到的溶胞物滤液进行处理，以相关炎症因子和组织形态变化为指标，通过检测组织形态、炎性介质PGE2以及辣椒素受体TRPV-1的含量的变化来评价本发明制备的溶胞物滤液的舒缓功效，委托陕西博溪通用检测科技有限公司进行检测并出具检测报告。The epidermis is a tissue that separates the body from the external environment. In addition to serving as a physical barrier, keratinocytes can receive stimulation from external "danger signals" and transform them into early warning cells for immune cells in the skin, forming an immune barrier for the skin. Disorders in the regulation of the skin's immune barrier function can cause Causes inflammatory reactions in the skin, causing skin problems and diseases. Skin contact with some highly irritating cosmetics or ultraviolet rays can cause clinical acute damage to the skin barrier, resulting in dry skin and erythema. The anionic surfactant sodium lauryl sulfate (SLS) has amphiphilic (hydrophilic and lipophilic) characteristics and can damage the skin barrier, especially the lipid components in the barrier, after contacting the skin at a large concentration. and cell membranes. Therefore, this experiment used an in vitro reconstituted epidermal model As the research object, an injury model caused by SLS stimulation was used, and then the lysate filtrate obtained in Example 1 was used for treatment. Related inflammatory factors and tissue morphology changes were used as indicators, and tissue morphology, inflammatory mediator PGE2 and capsaicin receptors were detected. To evaluate the soothing effect of the lysate filtrate prepared by the present invention based on changes in the body TRPV-1 content, Shaanxi Boxi General Testing Technology Co., Ltd. was entrusted to conduct the test and issue a test report.
实验方案如下表22所示。The experimental protocol is shown in Table 22 below.
表22.溶胞物滤液舒缓功效检测分组及检测方法
Table 22. Soothing efficacy test groups and test methods of lysate filtrate
(1)根据上表22测试方案，将模型转移到6孔板中(提前添加0.9mL模型培养液)，在6孔板上标注测试组编号。(1) According to the test plan in Table 22 above, transfer the model to a 6-well plate (add 0.9mL model culture medium in advance), and mark the test group number on the 6-well plate.
(2)NC和PC组表面添加25μL浓度为0.2％的SLS溶液，样品组表面添加12.5μL浓度为0.4％的SLS溶液和12.5μL相应浓度的样品工作液(即本发明制备的溶胞物滤液)，将样品 均匀分布于模型表面，置于CO2培养箱(37℃，5％CO2)中孵育24h。(2) Add 25 μL of SLS solution with a concentration of 0.2% to the surface of the NC and PC groups, and add 12.5 μL of SLS solution with a concentration of 0.4% to the surface of the sample group and 12.5 μL of the sample working solution of the corresponding concentration (i.e., the lysate filtrate prepared by the present invention) ), put the sample Distribute evenly on the model surface and incubate in a CO 2 incubator (37°C, 5% CO 2 ) for 24 hours.
(3)孵育结束后，用无菌PBS溶液清洗模型表面残留的受试物，用无菌棉签轻轻拭去模型内、外残留液体。(3) After the incubation, clean the remaining test substance on the surface of the model with sterile PBS solution, and gently wipe away the remaining liquid inside and outside the model with a sterile cotton swab.
(4)炎性介质(PGE2)测试：给药孵育24h后，收集3D表皮皮肤模型培养液于EP管中，收集完后将用于ELISA检测的样品置于-80℃冰箱冷冻保存，根据PGE2 ELISA试剂盒的操作说明书进行检测分析。(4) Inflammatory mediator (PGE2) test: After administration and incubation for 24 hours, collect the 3D epidermal skin model culture fluid in EP tubes. After collection, the samples for ELISA testing are frozen and stored in a -80°C refrigerator. According to PGE2 Perform detection and analysis according to the operating instructions of the ELISA kit.
(5)组织形态测试：取用于组织形态检测的模型(即按表22处理的表皮模型)，用4％的多聚甲醛进行固定处理，固定24h后，将模型环切取下，进行H&E染色检测，显微镜下拍照观察，采集图片并分析。(5) Tissue morphology test: Take the model for tissue morphology detection (i.e., the epidermal model processed according to Table 22), fix it with 4% paraformaldehyde, and after 24 hours of fixation, remove the model ring and perform H&E staining. Detect, take photos and observe under a microscope, collect pictures and analyze them.
(6)辣椒素受体(TRPV1)测试：取用于检测的模型(即按表22处理的表皮模型)，用4％的多聚甲醛进行固定处理，固定24h后，将模型环切取下，进行辣椒素受体(TRPV1)的免疫荧光检测，显微镜下拍照观察，采集图片并分析。(6) Capsaicin receptor (TRPV1) test: Take the model used for testing (i.e., the epidermal model processed according to Table 22), fix it with 4% paraformaldehyde, and after fixing for 24 hours, remove the model ring. Carry out immunofluorescence detection of capsaicin receptor (TRPV1), take pictures and observe them under a microscope, collect pictures and analyze them.
结果统计分析：应用GraphPad Prism作图，结果表示为Mean±SD；各组间比较采用t-test统计分析；统计分析均为双尾。P＜0.05认为具有显著差异，P＜0.01认为具有极显著差异。Statistical analysis of results: GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD; comparisons between groups were analyzed using t-test statistical analysis; statistical analyzes were all two-tailed. P＜0.05 was considered to be significantly different, and P＜0.01 was considered to be extremely significant difference.
组织形态检测结果分析：Analysis of tissue morphology test results:
组织形态测试结果如图7所示，图7为滤液培养结束后表皮模型进行H&E染色后显微镜下的组织形态图，其中图7A为空白对照，7B为阴性对照，即SLS处理组，7C为阳性对照，即同时添加SLS和0.01％***，7D为实验组，即同时添加SLS和2％滤液。The tissue morphology test results are shown in Figure 7. Figure 7 is the tissue morphology diagram of the epidermal model after H&E staining under the microscope after the filtrate culture is completed. Figure 7A is the blank control, 7B is the negative control, that is, the SLS treatment group, and 7C is the positive Control, that is, SLS and 0.01% dexamethasone are added at the same time, and 7D is the experimental group, that is, SLS and 2% filtrate are added at the same time.
组织形态是经过HE染色后的组织微观生理学结构分析，通过模型组织学形态，可以看出不同处理条件下皮肤屏障的变化情况，当经SLS刺激引起损伤后，皮肤屏障变得疏松，活细胞层厚度降低，通过添加活性物进行处理后的形态变化，判断是否具有改善这种损伤的效用。组织形态检测结果显示，与空白组相比，经SLS刺激后的阴性组模型细胞数量减少，排列紊乱，角质层疏松，出现空泡现象，说明本次测试刺激条件有效。与阴性组相比，阳性对照组模型活角质层疏松和空泡情况明显改善，说明本次阳性对照检测有效。而与阴性组相比，2％巴斯德毕赤酵母发酵溶胞物滤液处理后角质层疏松程度和和空泡有明显改善，说明体外条件下2％的滤液有助于缓解因SLS化学刺激引起的刺激反应，改善刺激产生的表皮组织损伤。Histological morphology is an analysis of the microphysiological structure of the tissue after HE staining. Through the histological morphology of the model, we can see the changes in the skin barrier under different treatment conditions. When damage is caused by SLS stimulation, the skin barrier becomes loose and the viable cell layer The thickness is reduced and the morphological changes after treatment by adding active substances are used to determine whether it has the effect of improving this damage. The tissue morphology test results showed that compared with the blank group, the number of cells in the negative group model after SLS stimulation was reduced, the arrangement was disordered, the stratum corneum was loose, and vacuoles appeared, indicating that the stimulation conditions of this test were effective. Compared with the negative group, the porosity and vacuoles in the active stratum corneum of the positive control group model were significantly improved, indicating that the positive control test was effective. Compared with the negative group, the looseness and vacuoles of the stratum corneum were significantly improved after treatment with 2% Pichia pastoris fermentation lysate filtrate, indicating that 2% filtrate under in vitro conditions can help relieve chemical stimulation caused by SLS. It can improve the irritation reaction caused by stimulation and improve the damage of epidermal tissue caused by stimulation.
PGE2测试结果分析：PGE2 test result analysis:
PGE2测试结果如表23所示，PGE2含量检测结果柱状图如图8所示。The PGE2 test results are shown in Table 23, and the histogram of the PGE2 content test results is shown in Figure 8.
表23.溶胞物滤液中PGE2含量检测结果汇总表
Table 23. Summary of PGE2 content detection results in lysate filtrate
备注：用t-test方法进行统计分析时，与BC组相比，显著性以#表示，P-value＜0.05表示为#，P-value＜0.01表示为##；与NC组相比，显著性以*表示，P-value＜0.05表示为*，P-value＜0.01表示为**。 Note: When using the t-test method for statistical analysis, compared with the BC group, significance is represented by #, P-value <0.05 is represented by #, P-value <0.01 is represented by ##; compared with the NC group, significance The sex is represented by *, P-value＜0.05 is represented by *, and P-value＜0.01 is represented by **.
实验结果显示，与BC组相比，NC组的PGE2分泌水平极显著升高，表明SLS刺激可以极显著提高PGE2的分泌量，说明本次测试SLS刺激条件有效。与NC组相比，PC组的PGE2分泌水平极显著降低，表明本次阳性对照检测有效。与NC组相比，2％巴斯德毕赤酵母发酵溶胞物滤液的PGE2分泌水平极显著降低。The experimental results showed that compared with the BC group, the secretion level of PGE2 in the NC group was significantly increased, indicating that SLS stimulation can significantly increase the secretion of PGE2, indicating that the SLS stimulation conditions in this test were effective. Compared with the NC group, the PGE2 secretion level in the PC group was extremely significantly reduced, indicating that this positive control test was effective. Compared with the NC group, the PGE2 secretion level of the 2% Pichia pastoris fermentation lysate filtrate was extremely significantly reduced.
***素类在炎症反应中扮演着极为重要的角色，其中最熟知的为PGE2。PGE2也是目前已知在哺乳动物体内合成最多、分布最为广泛的PG。皮肤中PG主要来源是角质形成细胞，角质形成细胞主要合成的PG包括PGE2、PGF2a和PGD2。Prostaglandins play an extremely important role in inflammatory reactions, the most well-known of which is PGE2. PGE2 is also the most commonly synthesized and widely distributed PG in mammals. The main source of PG in the skin is keratinocytes. The main PGs synthesized by keratinocytes include PGE2, PGF2a and PGD2.
皮肤的炎症反应常常伴有以下一个到数个重要的临床特征：红斑、肿胀、发热或局部皮温升高、疼痛、瘙痒，部分可伴有鱗屑。而出现的炎症反应在皮肤上的四大表现症状(红、肿、热、痛)都与PGE2密切相关：PGE2具有扩张和舒张血管的作用，表现为皮肤潮红；在组胺及激肽等物质的协同作用下，引起血管通透性的增加，而致组织局部红、肿，可致局部发热，进入血液的PGE2也可与下丘脑的受体结合引发体温中枢的改变，从而引起全身发热；此外，PGE2可以刺激神经末梢引起疼痛。在创伤过程中，PGE2作为一种炎症介质通过多个途径参与炎症反应的发生和发展。PGE2是强烈的血管扩张剂，使血流减慢，促进了白细胞的贴壁、粘附。此外，PGE2作为前炎症介质也可介导如IL-6等其他炎症介质及炎症因子的产生。实验表明2％巴斯德毕赤酵母发酵溶胞物滤液的能显著降低PGE2分泌水平，说明本发明所述的发酵溶胞物滤液可能通过介导炎症介质PGE2的分泌缓解皮肤炎症反应，如降低皮肤中的血管通透性，从而缓解皮肤泛红现象的产生。Inflammatory reactions of the skin are often accompanied by one or more of the following important clinical features: erythema, swelling, heat or local increase in skin temperature, pain, itching, and sometimes scales. The four major symptoms of inflammatory reaction on the skin (redness, swelling, heat, and pain) are closely related to PGE2: PGE2 has the effect of dilating and relaxing blood vessels, manifesting as skin flushing; in substances such as histamine and kinin, Under the synergistic effect, it causes an increase in vascular permeability, causing local redness and swelling of tissues, and can cause local fever. PGE2 entering the blood can also combine with receptors in the hypothalamus to cause changes in the body temperature center, thereby causing systemic fever; In addition, PGE2 can stimulate nerve endings and cause pain. During trauma, PGE2, as an inflammatory mediator, participates in the occurrence and development of inflammatory responses through multiple pathways. PGE2 is a strong vasodilator, slowing down blood flow and promoting the attachment and adhesion of white blood cells. In addition, PGE2, as a pro-inflammatory mediator, can also mediate the production of other inflammatory mediators and inflammatory factors such as IL-6. Experiments show that 2% Pichia pastoris fermentation lysate filtrate can significantly reduce the secretion level of PGE2, indicating that the fermentation lysate filtrate of the present invention may alleviate skin inflammatory reactions by mediating the secretion of inflammatory mediator PGE2, such as reducing Improves blood vessel permeability in the skin, thereby alleviating skin redness.
TRPV1测试结果分析：Analysis of TRPV1 test results:
TTRPV1免疫荧光检测结果显示，与BC组相比，NC组模型TRPV1的荧光强度显著上升，说明本次测试刺激条件有效。与NC组相比，PC组模型TRPV1的荧光强度显著降低，说明本次阳性对照检测有效。与NC组相比，样品浓度为2％的巴斯德毕赤酵母发酵溶胞物滤液处理组模型TRPV1的荧光强度显著降低。TRPV1免疫荧光检测结果如图9所示，TRPV1数据结果如表24所示，TRPV1蛋白相对IOD值柱状图如图10所示。The TTRPV1 immunofluorescence detection results showed that compared with the BC group, the fluorescence intensity of model TRPV1 in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the fluorescence intensity of model TRPV1 in the PC group was significantly reduced, indicating that this positive control test was effective. Compared with the NC group, the fluorescence intensity of the model TRPV1 in the Pichia pastoris fermentation lysate filtrate treatment group with a sample concentration of 2% was significantly reduced. The TRPV1 immunofluorescence detection results are shown in Figure 9, the TRPV1 data results are shown in Table 24, and the TRPV1 protein relative IOD value histogram is shown in Figure 10.
表24.TRPV1数据结果汇总表
Table 24. Summary of TRPV1 data results
备注：用t-test方法进行统计分析时，与BC组相比，显著性以#表示，P-value＜0.05表示为#，P-value＜0.01表示为##；与NC组相比，显著性以*表示，P-value＜0.05表示为*，P-value＜0.01表示为**。Note: When using the t-test method for statistical analysis, compared with the BC group, significance is represented by #, P-value <0.05 is represented by #, P-value <0.01 is represented by ##; compared with the NC group, significance The sex is represented by *, P-value＜0.05 is represented by *, and P-value＜0.01 is represented by **.
实验数据显示，与BC组相比，NC组的TRPV1蛋白含量显著上升，说明本次测试刺激条件有效。与NC组相比，PC组的TRPV1蛋白含量显著下降，说明本次阳性对照检测有效。与NC组相比，2％巴斯德毕赤酵母发酵溶胞物滤液作用后的TRPV1蛋白含量显著下降。说明滤液可通过抑制TRPV1蛋白表达的机制起到舒缓炎症的作用。Experimental data showed that compared with the BC group, the TRPV1 protein content in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the TRPV1 protein content in the PC group decreased significantly, indicating that this positive control test was effective. Compared with the NC group, the TRPV1 protein content after the action of 2% Pichia pastoris fermentation lysate filtrate significantly decreased. This shows that the filtrate can relieve inflammation by inhibiting the expression of TRPV1 protein.
瞬时受体电位香草酸亚型1(transient receptor potential vanilloid type 1，TRPV1)，也称辣椒素受体，是表达于外周初级传入神经元上的重要伤害感受器，参与急性 炎症痛敏的形成。是一种可高效介导Ca2+流入的阳离子通道，广泛分布于神经元、免疫细胞、多种脏器上皮细胞及角质形成细胞等细胞中。研究发现，TRPV1通道可以被多种刺激所激活，如各种物理和化学刺激因素：高温环境(温度≥43℃)、低pH值(酸性环境)、电压、摩尔渗透压浓度、内源性和外源性香草类物质，如辣椒素等。当TRPV1被激活后，钙离子通道开放，钙离子内流，胞质内钙离子浓度升高，引起神经元及其纤维释放神经肽如P物质、神经激肽A、降钙素基因相关肽、血管活性肠肽和兴奋性氨基酸，如谷氨酸、天门冬氨酸。在皮肤上，TRPV1表达于皮肤感觉神经、角质形成细胞及肥大细胞，激活的TRP通道不但介导温度感知和调节，而且还参与痛痒觉以及皮肤神经源炎症的发生，因此，TRP通道已成为治疗炎症性皮肤病和瘙痒的重要分子靶标。故本次试验选择TRPV1作为靶分子，通过测定其在表皮模型上化学刺激作用下表达量的变化来评价滤液对炎症的舒缓作用。Transient receptor potential vanilloid type 1 (TRPV1), also known as capsaicin receptor, is an important nociceptor expressed on peripheral primary afferent neurons and is involved in acute The development of inflammatory pain sensitivity. It is a cation channel that can efficiently mediate the influx of Ca 2+ and is widely distributed in cells such as neurons, immune cells, epithelial cells of various organs, and keratinocytes. Studies have found that TRPV1 channels can be activated by a variety of stimuli, such as various physical and chemical stimuli: high temperature environment (temperature ≥43°C), low pH value (acidic environment), voltage, osmolarity, endogenous and Exogenous vanilla substances, such as capsaicin, etc. When TRPV1 is activated, calcium ion channels open, calcium ions flow in, and the concentration of calcium ions in the cytoplasm 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 glutamate and aspartate. On the skin, TRPV1 is expressed in skin sensory nerves, keratinocytes and mast cells. The activated TRP channels not only mediate temperature perception and regulation, but are also involved in pain and itch sensation and the occurrence of skin neurogenic inflammation. Therefore, TRP channels have become a therapeutic Important molecular targets in inflammatory skin diseases and pruritus. Therefore, TRPV1 was selected as the target molecule in this experiment, and the soothing effect of the filtrate on inflammation was evaluated by measuring the changes in its expression under chemical stimulation on the epidermal model.
二、紫外线吸收效果检测2. Ultraviolet absorption effect detection
对本发明得到的发酵溶胞物滤液及其不同倍数的稀释液(纯水稀释)进行紫外光区(200-400nm)吸光度扫描(VarioskanTM LUX多功能酶标仪-VL0L0TD0，赛默飞世尔科技(中国)有限公司)，换算成透过率后，计算为吸收率，结果如图11所示。The fermentation lysate filtrate obtained in the present invention and its different multiple dilutions (pure water dilution) were subjected to absorbance scanning in the ultraviolet region (200-400nm) (Varioskan TM LUX multi-function microplate reader-VL0LOTD0, Thermo Fisher Scientific (China) Co., Ltd.), after converting it into transmittance, it is calculated as absorption rate, and the results are shown in Figure 11.
紫外线一般分为UVC区(200-280nm)、UVB区(280-320nm)和UVA区(320-400nm)。到达地球表面的紫外线98％为UVA，穿透性最强，可直达真皮层，是皮肤晒黑的主因；另外，只有不足2％为UVB，是造成皮肤晒伤与红斑的主要元凶，而UVC虽一般被臭氧层吸收，但短波紫外线对人体危害性最大，长期照射会造成皮肤癌。试验结果显示，本发明的发酵溶胞物滤液对UVC区平均吸收率接近100.00％，对UVB的平均吸收率＞95％，对UVA区的平均吸收率＞80％。Ultraviolet rays are generally divided into UVC area (200-280nm), UVB area (280-320nm) and UVA area (320-400nm). 98% of the ultraviolet rays that reach the earth's surface are UVA, which has the strongest penetrability and can reach directly into the dermis, which is the main cause of skin tanning. In addition, less than 2% is UVB, which is the main culprit of skin sunburn and erythema, while UVC Although generally absorbed by the ozone layer, short-wave ultraviolet rays are the most harmful to the human body, and long-term exposure can cause skin cancer. The test results show that the average absorption rate of the fermentation lysate filtrate of the present invention in the UVC area is close to 100.00%, the average absorption rate in the UVB area is >95%, and the average absorption rate in the UVA area is >80%.
随着稀释倍数的增加，稀释后的溶胞物滤液对紫外线吸收能力也在下降，50％浓度的滤液在UVC区的吸收率并未明显降低，但在UVB区、UVA区对紫外线的吸收率明显下降，但均在在40％以上；25％浓度的溶胞物滤液，对UVC区平均吸收率＞90％，对UVB的平均吸收率随着紫外线波长的增加从89％下降至65％，对UVA区平均吸收率＞27％；稀释10倍的溶胞物滤液，对UVC区平均吸收率＞90％，对UVB的平均吸收率紫外线波长的增加从87％下降至34％，对UVA区平均吸收率＞20％；随着稀释倍数的增加其对紫外线吸收的效果逐渐降低，但稀释至1％时，对UVC区平均吸收率＞60％，对UVB的平均吸收率也维持在20％左右。未来最有可能使用的5％的稀释浓度，对UVC区平均吸收率可达到74％-99％，对UVB的平均吸收率随着紫外线波长的增加从67％下降至23％，对UVA区平均吸收率＞15％。As the dilution factor increases, the ultraviolet absorption capacity of the diluted lysate filtrate also decreases. The absorption rate of the 50% concentration filtrate in the UVC area does not decrease significantly, but the absorption rate of ultraviolet rays in the UVB area and UVA area There is a significant decrease, but they are all above 40%; the average absorption rate of the lysate filtrate with a concentration of 25% in the UVC area is >90%, and the average absorption rate of UVB decreases from 89% to 65% with the increase of ultraviolet wavelength. The average absorption rate for the UVA area is >27%; the lysate filtrate diluted 10 times, the average absorption rate for the UVC area is >90%, the average absorption rate for UVB ultraviolet wavelengths increases from 87% to 34%, and for the UVA area The average absorption rate is >20%; as the dilution factor increases, its UV absorption effect gradually decreases, but when diluted to 1%, the average absorption rate in the UVC area is >60%, and the average absorption rate for UVB is also maintained at 20%. about. At the 5% dilution concentration that is most likely to be used in the future, the average absorption rate for the UVC area can reach 74%-99%, the average absorption rate for UVB decreases from 67% to 23% with the increase of ultraviolet wavelength, and the average absorption rate for the UVA area Absorption rate >15%.
结果表明，本发明的发酵溶胞物滤液可吸收绝大部分的中短波紫外线，有效抵御紫外线对皮肤的伤害。溶胞物滤液的防晒效果与氨基苯甲酸酯及其衍生物、水杨酸酯及其衍生物、肉桂酸酯类和樟脑类衍生物等常用UVB吸收剂的作用相类似，稀释后的溶胞物滤液也能对一定波长范围内紫外线具有良好的防晒护肤作用。The results show that the fermentation lysate filtrate of the present invention can absorb most of the medium and short-wave ultraviolet rays and effectively resist the damage of ultraviolet rays to the skin. The sunscreen effect of the lysate filtrate is similar to that of commonly used UVB absorbers such as aminobenzoates and their derivatives, salicylates and their derivatives, cinnamic acid esters and camphor derivatives. The cell filtrate also has good sun protection and skin care effects on ultraviolet rays within a certain wavelength range.
三、调节微生态结果分析3. Analysis of the results of regulating microecology
本实施例中基于3D表皮皮肤模型通过检测HBD1、HBD2、LL-37、TLR2蛋白变化，评价待测样品对皮肤抗菌肽及TOLL样受体蛋白水平表达量的作用。此项测试委托陕西博溪通用检测科技有限公司进行检测并出具检测报告。In this embodiment, based on the 3D epidermal skin model By detecting changes in HBD1, HBD2, LL-37, and TLR2 proteins, the effect of the test sample on the level expression of skin antimicrobial peptides and TOLL-like receptor proteins was evaluated. This test was entrusted to Shaanxi Boxi General Testing Technology Co., Ltd. to conduct the test and issue a test report.
(1)试验方法：(1)Test method:
根据下表25测试方案将模型转移到6孔板中(提前添加0.9mL相应分组的 EpiGrowth培养液)，在6孔板上标注测试组编号。Transfer the model to a 6-well plate according to the test protocol in Table 25 below (add 0.9mL of the corresponding group in advance EpiGrowth culture medium), mark the test group number on the 6-well plate.
表25.溶胞物滤液调节微生态检测方法
Table 25. Lysate filtrate adjusted microecological detection method
样品组表面添加相应浓度的工作液于6孔板中，置于CO2培养箱(37℃、5％CO2)中孵育24h。孵育结束后，用装有无菌PBS溶液的洗瓶清洗模型表面残留的受试物，用无菌棉签拭去模型内、外残留液体。取用于免疫组化检测的模型，进行免疫组化测试。结果统计应用GraphPad Prism作图，结果表示为Mean±SD。各组间比较采用t-test统计分析。统计分析均为双尾。P＜0.05认为具有显著差异，P＜0.01认为具有极显著差异。Add corresponding concentration of working solution to the surface of the sample group in a 6-well plate, place it in a CO 2 incubator (37°C, 5% CO 2 ) and incubate for 24 hours. After the incubation, use a washing bottle filled with sterile PBS solution to clean the remaining test substance on the surface of the model, and use a sterile cotton swab to wipe away the remaining liquid inside and outside the model. Take the model used for immunohistochemical detection and perform immunohistochemical testing. The statistical results were graphed using GraphPad Prism, and the results were expressed as Mean±SD. Comparisons between groups were analyzed using t-test statistics. Statistical analyzes were all two-tailed. P＜0.05 was considered to be significantly different, and P＜0.01 was considered to be extremely significant difference.
(2)检测结果(2)Test results
a.HBD1结果分析a.HBD1 result analysis
各组HBD1蛋白染色结果见图12，图片中红色箭头所指的棕褐色部分为HBD1蛋白，棕褐色越深表明HBD1蛋白含量越高。HBD1相对IOD值汇总表如表26所示，HBD1积分光密度(IOD)值柱状图如图13所示。The HBD1 protein staining results of each group are shown in Figure 12. The tan part pointed by the red arrow in the picture is HBD1 protein. The darker the tan, the higher the HBD1 protein content. The summary table of HBD1 relative IOD values is shown in Table 26, and the HBD1 integrated optical density (IOD) value histogram is shown in Figure 13.
表26.HBD1相对IOD值汇总表
Table 26. Summary table of HBD1 relative IOD values
结果可见，与BC组相比，PC(1，25-二羟基维生素D3)组的HBD1蛋白含量显著上升，说明本次测试阳性对照有效。与BC组相比，样品巴斯德毕赤酵母发酵溶胞物滤液-0.5％、巴斯德毕赤酵母发酵溶胞物滤液-1.0％的HBD1蛋白含量显著上升。The results showed that compared with the BC group, the HBD1 protein content of the PC (1,25-dihydroxyvitamin D3) group increased significantly, indicating that the positive control of this test was effective. Compared with the BC group, the HBD1 protein content of the samples Pichia pastoris fermentation lysate filtrate-0.5% and Pichia pastoris fermentation lysate filtrate-1.0% increased significantly.
b.HBD2结果分析b.HBD2 result analysis
各组HBD2蛋白染色结果见图14，图片中红色箭头所指的棕褐色部分为HBD2蛋白，棕褐色越深表明HBD2蛋白含量越高。HBD2相对IOD值汇总表如表27所示，HBD1积分光密度(IOD)值柱状图如图15所示。The HBD2 protein staining results of each group are shown in Figure 14. The tan part pointed by the red arrow in the picture is HBD2 protein. The darker the tan, the higher the HBD2 protein content. The summary table of HBD2 relative IOD values is shown in Table 27, and the HBD1 integrated optical density (IOD) value histogram is shown in Figure 15.
表27.HBD2相对IOD值汇总表
Table 27. Summary table of HBD2 relative IOD values
结果可见，与BC组相比，PC(1，25-二羟基维生素D3)组的HBD2蛋白含量显著上升，说明本次测试阳性对照有效。与BC组相比，样品巴斯德毕赤酵母发酵溶胞物滤液-0.5％、巴 斯德毕赤酵母发酵溶胞物滤液-1.0％的HBD2蛋白含量显著上升。The results showed that compared with the BC group, the HBD2 protein content in the PC (1,25-dihydroxyvitamin D3) group increased significantly, indicating that the positive control of this test was effective. Compared with the BC group, the samples Pichia pastoris fermentation lysate filtrate-0.5%, P. The HBD2 protein content of Pichia pastoris fermentation lysate filtrate-1.0% increased significantly.
c.TLR2结果分析c.TLR2 result analysis
各组TLR2蛋白染色结果见图16，图片中红色箭头所指的棕褐色部分为TLR2蛋白，棕褐色越深表明TLR2蛋白含量越高。TLR2相对IOD值汇总表如表28所示，TLR2积分光密度(IOD)值柱状图如图17所示。The TLR2 protein staining results of each group are shown in Figure 16. The tan part pointed by the red arrow in the picture is the TLR2 protein. The darker the tan, the higher the TLR2 protein content. A summary table of TLR2 relative IOD values is shown in Table 28, and a histogram of TLR2 integrated optical density (IOD) values is shown in Figure 17.
表28.TLR2相对IOD值汇总表
Table 28. Summary of TLR2 relative IOD values
结果可见，与BC组相比，PC(1，25-二羟基维生素D3)组的TLR2蛋白含量显著上升，说明本次阳性对照检测有效。与BC组相比，浓度为0.5％和1.0％的巴斯德毕赤酵母发酵溶胞物滤液作用后均能使TLR2蛋白含量显著上升。The results showed that compared with the BC group, the TLR2 protein content in the PC (1,25-dihydroxyvitamin D3) group increased significantly, indicating that this positive control test was effective. Compared with the BC group, Pichia pastoris fermentation lysate filtrate at concentrations of 0.5% and 1.0% could significantly increase the TLR2 protein content.
d.LL-37结果分析d.LL-37 result analysis
各组LL-37蛋白染色结果见图18，图片中红色箭头所指的棕褐色部分为LL-37蛋白，棕褐色越深表明LL-37蛋白含量越高。LL-37相对IOD值汇总表如表29所示，LL-37积分光密度(IOD)值柱状图如图19所示。The LL-37 protein staining results of each group are shown in Figure 18. The tan part pointed by the red arrow in the picture is the LL-37 protein. The darker the tan, the higher the LL-37 protein content. The summary table of relative IOD values of LL-37 is shown in Table 29, and the histogram of the integrated optical density (IOD) value of LL-37 is shown in Figure 19.
表29.LL-37相对IOD值汇总表
Table 29. LL-37 relative IOD value summary table
结果可见，与BC组相比，PC(1，25-二羟基维生素D3)组的LL-37蛋白含量显著上升，说明本次阳性对照检测有效。与BC组相比，0.5％和1.0％浓度的巴斯德毕赤酵母发酵溶胞物滤液均能使LL-37蛋白含量显著上升。The results showed that compared with the BC group, the LL-37 protein content in the PC (1,25-dihydroxyvitamin D3) group increased significantly, indicating that the positive control test was effective. Compared with the BC group, both 0.5% and 1.0% concentrations of P. pastoris fermentation lysate filtrate could significantly increase the LL-37 protein content.
皮肤微生态是由细菌、真菌、病毒、螨虫和节肢动物等各种微生物与皮肤表面的组织、细胞各种分泌物、微环境等共同组成的生态***。皮肤微生物、宿主及外环境三者相互作用构成了皮肤微生态平衡。研究表明，角质形成细胞通过表达抗菌多肽(Antimicrobial Peptides，AMPs)，直接杀死外来入侵的微生物群系，同时又为常驻菌提供了赖以生息的友好场所。常驻菌能直接释放AMPs，作用于病原微生物。例如，表皮葡萄球菌可分泌防御素、细菌素、LL-37、REG3A等抗菌肽。常驻菌也能通过分泌AMPs，调节宿主的免疫应答。人体内存在于皮肤的抗菌肽主要属于两大家族：防御素类和Cathelicidin类。防御素类根据其结构的不同分为α-防御素和β-防御素，其中HBD1和HBD2属于β-防御素。LL-37是发现的唯一存在于人体中的Cathelicidin类抗菌肽。另一方面，可通过激活多种模式识别受体如TLR2(Toll样受体2)，识别外源性微生物，激活免疫应答来清除病原微生物。本试验基于3D表皮皮肤模型与对照组相比，巴斯德毕赤酵母发酵溶胞物滤液在0.5％和1.0％浓度下，HBD1、HBD2、TLR2、LL-37蛋白含量显著上升，且随着滤液浓度的增大，相关目标蛋白的表达 量也呈正相关递增，说明本发明制备的巴斯德毕赤酵母发酵溶胞物滤液可通过提升抗菌肽的表达，调节微生态，提升皮肤屏障，进而达到修护功效。The skin microecology is an ecosystem composed of various microorganisms such as bacteria, fungi, viruses, mites, and arthropods, as well as tissues, cells, various secretions, and the microenvironment on the skin surface. The interaction between skin microorganisms, the host and the external environment constitutes the skin's microecological balance. Studies have shown that keratinocytes directly kill foreign invading microbial flora by expressing antimicrobial peptides (AMPs), and at the same time provide a friendly place for resident bacteria to thrive. Resident bacteria can directly release AMPs to act on pathogenic microorganisms. For example, Staphylococcus epidermidis can secrete defensins, bacteriocins, LL-37, REG3A and other antimicrobial peptides. Resident bacteria can also regulate the host's immune response by secreting AMPs. The antimicrobial peptides present in the skin of the human body mainly belong to two major families: defensins and cathelicidin. Defensins are divided into α-defensins and β-defensins according to their different structures, among which HBD1 and HBD2 belong to β-defensins. LL-37 is the only cathelicidin antimicrobial peptide found in the human body. On the other hand, pathogenic microorganisms can be eliminated by activating a variety of pattern recognition receptors such as TLR2 (Toll-like receptor 2) to recognize exogenous microorganisms and activate immune responses. This test is based on a 3D epidermal skin model Compared with the control group, the HBD1, HBD2, TLR2, and LL-37 protein contents of the Pichia pastoris fermentation lysate filtrate at 0.5% and 1.0% concentrations increased significantly, and as the filtrate concentration increased, the correlation Expression of target protein The amount also increases in a positive correlation, indicating that the Pichia pastoris fermentation lysate filtrate prepared in the present invention can regulate the microecology and improve the skin barrier by increasing the expression of antibacterial peptides, thereby achieving a repair effect.
四、控油功效检测4. Oil control efficacy test
本实施例中是基于皮脂腺细胞，通过检测样品作用后皮脂腺细胞的脂滴含量，评价待测样品的控油功效，委托陕西博溪通用检测科技有限公司进行检测并出具检测报告。This example is based on sebaceous gland cells. By detecting the lipid droplet content of sebaceous gland cells after the sample is processed, the oil control effect of the sample to be tested is evaluated. Shaanxi Boxi General Testing Technology Co., Ltd. is entrusted to conduct the test and issue a test report.
(1)实验方法(1) Experimental methods
本实施例中对控油功效的检测，试验设置空白组、阳性对照组和样品组，具体的如下表30所示。In this embodiment, for the detection of oil control efficacy, the test set up a blank group, a positive control group and a sample group, as shown in Table 30 below.
表30.控油功效检测方案
Table 30. Oil control efficacy test plan
检测方法简述如下：The detection method is briefly described as follows:
按3.5E个/孔的接种密度接种SZ95细胞至24孔板爬片，培养箱(37℃、5％CO2)中孵育过夜。SZ95 cells were seeded onto a 24-well plate at a seeding density of 3.5E cells/well and incubated overnight in an incubator (37°C, 5% CO 2 ).
根据表29测试方案，待24孔板中细胞铺板率达到60％～70％时，进行分组给药，每孔加样1mL，每组设3个复孔。给药完成后将24孔板放置在培养箱(37℃、5％CO2)中培养72h，培养期间进行每天换液和给药。According to the test plan in Table 29, when the cell plating rate in the 24-well plate reaches 60% to 70%, drug administration will be carried out in groups. 1 mL of sample will be added to each well, and each group will have 3 duplicate wells. After the administration was completed, the 24-well plate was placed in an incubator (37°C, 5% CO 2 ) and cultured for 72 hours. During the culture period, medium replacement and administration were performed every day.
油红O染色：吸弃旧液，用PBS清洗细胞1次，每孔加入500μL油红O工作液(1μg/mL)，避光染色15min(孵育箱37℃孵育)，PBS洗涤2次。Oil Red O staining: Aspirate the old solution, wash the cells once with PBS, add 500 μL Oil Red O working solution (1 μg/mL) to each well, stain for 15 min in the dark (incubate at 37°C in an incubator), and wash twice with PBS.
荧光显微镜拍照观察：在Ex：450nm-500nm，Em：515nm-560nm检测，200倍镜下拍照观察。Fluorescence microscope photography and observation: Detect at Ex: 450nm-500nm, Em: 515nm-560nm, take photos and observe under 200x microscope.
结果统计分析：应用GraphPad Prism作图，结果表示为Mean±SD。各组间比较采用t-test统计分析。统计分析均为双尾。P＜0.05认为具有显著差异，P＜0.01认为具有极显著差异。Statistical analysis of results: GraphPad Prism was used for graphing, and the results were expressed as Mean±SD. Comparisons between groups were analyzed using t-test statistics. Statistical analyzes were all two-tailed. P＜0.05 was considered to be significantly different, and P＜0.01 was considered to be extremely significant difference.
(2)检测结果(2)Test results
脂滴合成油红O染色结果如图20所示，脂滴合成油红O染色结果数据汇总如表31所示。脂滴积分光密度(IOD)值柱状图如图21所示。The results of lipid droplet synthetic Oil Red O staining are shown in Figure 20, and the data summary of lipid droplet synthetic Oil Red O staining results are shown in Table 31. The histogram of lipid droplet integrated optical density (IOD) values is shown in Figure 21.
表31.脂滴合成油红O染色结果数据汇总表
Table 31. Data summary table of lipid droplet synthetic Oil Red O staining results
备注：用t-test方法进行统计分析时，PC组、样品组与BC组相比，显著性以*表示，P-value＜0.05表示为*，P-value＜0.01表示为**。 Note: When using the t-test method for statistical analysis, the significance of the PC group and sample group compared with the BC group is represented by *, P-value <0.05 is represented by *, and P-value <0.01 is represented by **.
试验结果显示，与BC组相比，PC组脂滴合成明显减少，说明本次阳性对照有效；与BC组相比，样品巴斯德毕赤酵母发酵溶胞物滤液在0.05％和0.15％的浓度下，细胞脂滴合成明显减少，说明本发明制备的巴斯德毕赤酵母发酵溶胞物滤液在0.05％和0.15％的浓度下，均有减少脂滴合成的作用。The test results showed that compared with the BC group, the synthesis of lipid droplets in the PC group was significantly reduced, indicating that the positive control was effective; compared with the BC group, the sample Pichia pastoris fermentation lysate filtrate at 0.05% and 0.15% At the concentration of 0.05% and 0.15%, the synthesis of cellular lipid droplets is significantly reduced, indicating that the Pichia pastoris fermentation lysate filtrate prepared in the present invention has the effect of reducing the synthesis of lipid droplets at concentrations of 0.05% and 0.15%.
皮脂分泌过多是引发多种皮肤问题例如痤疮的主要因素，皮脂腺细胞会合成大量的油脂并以脂滴形式分泌到细胞外，影响细胞功能，导致多种皮肤问题产生，脂滴量的多少是衡量控油程度的主要指标。基于皮脂腺细胞SZ95，与对照组相比，样品巴斯德毕赤酵母发酵溶胞物滤液在0.05％和0.15％的浓度下，脂质含量均显著下降；说明本发明制备的巴斯德毕赤酵母发酵溶胞物滤液可通过降低脂滴合成达到控油功效。Excessive sebum secretion is the main factor causing various skin problems such as acne. Sebaceous gland cells will synthesize a large amount of oil and secrete it out of the cells in the form of lipid droplets, which affects cell function and leads to various skin problems. The amount of lipid droplets is The main indicator to measure the degree of oil control. Based on sebaceous gland cells SZ95, compared with the control group, the lipid content of the sample Pichia pastoris fermentation lysate filtrate decreased significantly at the concentrations of 0.05% and 0.15%; indicating that the Pichia pastoris prepared by the present invention Yeast fermentation lysate filtrate can achieve oil control effects by reducing lipid droplet synthesis.
实施例3：本发明的巴斯德毕赤酵母发酵溶胞物滤液应用实例Example 3: Application example of Pichia pastoris fermentation lysate filtrate of the present invention
本实施例中，以本发明的巴斯德毕赤酵母发酵溶胞物滤液为原料，制备了护肤精华液、面膜、护肤面霜，以便更直观的说明本发明发酵溶胞物滤液的应用优势，但本发明的发酵溶胞物滤液不限于用于这几种产品，本发明的巴斯德毕赤酵母发酵溶胞物滤液还可用于制备其他化妆品。In this example, the Pichia pastoris fermentation lysate filtrate of the present invention is used as raw material to prepare skin care essence, facial mask, and skin care cream, in order to more intuitively illustrate the application advantages of the fermentation lysate filtrate of the present invention. However, the fermentation lysate filtrate of the present invention is not limited to use in these products. The Pichia pastoris fermentation lysate filtrate of the present invention can also be used to prepare other cosmetics.
(1)护肤精华液(1) Skin care essence
以公开号CN103102407B中保藏编号为CGMCC NO.7189工程菌的发酵菌体10％湿菌体浓度制备溶胞物滤液为例，制备一种护肤精华液，各原料如下所列的质量分数配制，总质量百分比之和为100％，各成份配比如下所示，优选巴斯德毕赤酵母发酵溶胞物滤液添加比例为5％进行制备。Taking the preparation of lysate filtrate with a 10% wet bacterial cell concentration from the fermentation bacterial cell of the engineering bacterium CGMCC NO.7189 in Publication No. CN103102407B as an example, a skin care essence is prepared. Each raw material is prepared with the mass fraction listed below. The total The sum of the mass percentages is 100%, and the proportions of each component are as follows. The preferred addition ratio of the Pichia pastoris fermentation lysate filtrate is 5% for preparation.
所述护肤精华液中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选5wt％，还包括甘油4wt％、丁二醇4wt％、甘油聚醚-262wt％、EDTA-2Na 0.02wt％、透明质酸钠0.05wt％、尿囊素0.15wt％、甜菜碱2wt％、黄原胶0.25wt％、1，2-戊二醇5wt％，其他为去离子水。The skin care essence includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerol 4wt%, butylene glycol 4wt%, glycerol polyether-262wt%, EDTA-2Na 0.02wt%, sodium hyaluronate 0.05wt%, allantoin 0.15wt%, betaine 2wt%, xanthan gum 0.25wt%, 1,2-pentanediol 5wt%, the rest is deionized water.
所述护肤精华液的制备方法：精确称取各组分原料；室温下，将去离子水、甘油、丁二醇、甘油聚醚-26混合均匀，再将尿囊素、甜菜碱加入其中，溶解混合均匀，升温至70-75℃，加入透明质酸钠、黄原胶，搅拌至完全溶解；降温至40℃以下，加入巴斯德毕赤酵母发酵溶胞物滤液，搅拌均匀；100目滤布过滤，并分装于按压式真空瓶中，即得护肤精华液。The preparation method of the skin care essence: accurately weigh the raw materials of each component; mix deionized water, glycerin, butylene glycol, and glycerol polyether-26 at room temperature, and then add allantoin and betaine thereto, Dissolve and mix evenly, raise the temperature to 70-75°C, add sodium hyaluronate and xanthan gum, and stir until completely dissolved; cool down to below 40°C, add the filtrate of Pichia pastoris fermentation lysate, and stir evenly; 100 mesh Filter with a filter cloth and put into press-type vacuum bottles to get the skin care essence.
(2)面膜(2)Face mask
以公开号CN103102407B中保藏编号为CGMCC NO.7189工程菌的发酵菌体5％湿菌体浓度制备溶胞物滤液为例，制备一种护肤面膜液，各原料按如下所示的质量分数配制，总质量百分比之和为100％，各成份配比如瞎所示，优选巴斯德毕赤酵母发酵溶胞物滤液添加比例为5％进行制备。Taking the preparation of lysate filtrate with a concentration of 5% wet bacterial cell from the fermentation bacterial cell of the engineering strain CGMCC NO.7189 in Publication No. CN103102407B as an example, a skin care facial mask liquid is prepared. Each raw material is prepared according to the mass fraction as shown below. The sum of the total mass percentages is 100%, and the proportions of each component are as shown in (b). The preferred addition ratio of the Pichia pastoris fermentation lysate filtrate is 5% for preparation.
所述面膜中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选5wt％，还包括甘油8wt％、EDTA-2Na 0.02wt％、透明质酸钠0.05wt％、黄原胶0.1wt％、卡波姆0.12wt％、pH调节剂适量(优选0.12wt％，一般调节至中性)、1，2-戊二醇4wt％、1，2-己二醇0.5wt％，其他为去离子水。The facial mask includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, and also includes glycerin 8wt%, EDTA-2Na 0.02wt%, sodium hyaluronate 0.05wt%, Xanthan gum 0.1wt%, carbomer 0.12wt%, appropriate amount of pH regulator (preferably 0.12wt%, generally adjusted to neutral), 1,2-pentanediol 4wt%, 1,2-hexanediol 0.5wt %, the others are deionized water.
制备方法：Preparation:
精确称取各组分原料，备用；室温下，取部分去离子水与卡波姆预先在容器中分散搅拌均匀；取另一容器，将余下去离子水、甘油、EDTA-2Na、透明质酸钠、黄原胶、精氨酸、1， 2-戊二醇、1，2-己二醇，70-75℃下充分溶解，混合均匀；降温至55-60℃，将预先分散好的卡波姆投入上述混合物中，混合搅拌均匀；降温至40-50℃，加入pH调节剂，混合搅拌均匀；降温至35-40℃，加入巴斯德毕赤酵母发酵溶胞物滤液，搅拌均匀；100目滤布过滤，得面膜液，搭配纯棉膜布，灌装于面膜铝箔袋中，即得护肤面膜。Accurately weigh the raw materials of each component and set aside; at room temperature, take part of the deionized water and carbomer and disperse and stir evenly in a container; take another container and mix the remaining deionized water, glycerin, EDTA-2Na, and hyaluronic acid Sodium, xanthan gum, arginine, 1, Fully dissolve 2-pentanediol and 1,2-hexanediol at 70-75°C and mix evenly; cool down to 55-60°C, add the pre-dispersed carbomer into the above mixture, mix and stir evenly; cool down to 40-50°C, add pH regulator, mix and stir evenly; cool to 35-40°C, add Pichia pastoris fermentation lysate filtrate, stir evenly; filter with 100 mesh filter cloth to obtain facial mask liquid, mix with pure The cotton film cloth is filled into the mask aluminum foil bag to obtain a skin care mask.
(3)护肤面霜：(3) Skin care cream:
以公开号CN103102407B中保藏编号为CGMCC NO.7189工程菌的发酵菌体5％湿菌体浓度制备溶胞物滤液为例，制备一种护肤面霜，各原料按如下所示质量分数配制，总质量百分比之和为100％，各成份配比如下所示，优选巴斯德毕赤酵母发酵溶胞物滤液添加比例为5％进行。Taking the preparation of lysate filtrate with a concentration of 5% wet bacterial cell from the fermentation bacterial cell of the engineering strain CGMCC NO.7189 in Publication No. CN103102407B as an example, a skin care cream is prepared. Each raw material is prepared according to the mass fraction as shown below. The total mass The sum of the percentages is 100%, and the proportion of each component is as follows. It is preferable that the addition ratio of the Pichia pastoris fermentation lysate filtrate is 5%.
所述面霜中包括发酵溶胞物滤液，所述发酵溶胞物滤液含量为1-10wt％，优选5wt％，记为D相；还包括甘油5wt％、EDTA二钠0.02wt％、黄原胶0.15wt％、丙烯酰二甲基牛磺酸铵/VP共聚物0.6wt％、对羟基苯乙酮0.5wt％、1，2-己二醇0.5wt％，其他为去离子水，混合为A相；鲸蜡硬脂基葡糖苷/鲸蜡硬脂醇3wt％、鲸蜡硬脂醇3wt％、辛酸癸酸甘油三酯4wt％、氢化聚异丁烯3wt％、聚二甲基硅氧烷1wt％，混合为B相；pH调节剂为C相，一般调节至中性。The cream includes fermentation lysate filtrate, the content of the fermentation lysate filtrate is 1-10wt%, preferably 5wt%, recorded as phase D; it also includes glycerin 5wt%, EDTA disodium 0.02wt%, xanthan gum 0.15wt%, ammonium acryloyldimethyltaurate/VP copolymer 0.6wt%, p-hydroxyacetophenone 0.5wt%, 1,2-hexanediol 0.5wt%, the others are deionized water, the mixture is A Phase; cetearyl glucoside/cetearyl alcohol 3wt%, cetearyl alcohol 3wt%, caprylic capric triglyceride 4wt%, hydrogenated polyisobutylene 3wt%, polydimethylsiloxane 1wt% , mixed into phase B; the pH adjuster is phase C, generally adjusted to neutral.
制备方法：Preparation:
将A相各组分投入至水相锅中，加热到80-85℃，混合搅拌均匀；再将B相各组分投入至水相锅中，加热到80-85℃，混合搅拌均匀；然后将B相加入A相均质5分钟；搅拌冷却到60℃加入C相；待冷却到35-40℃，加入D相，搅拌均匀得到护肤面霜。Put the components of phase A into the water phase pot, heat to 80-85°C, mix and stir evenly; then put the ingredients of phase B into the water phase pot, heat to 80-85°C, mix and stir evenly; then Add phase B to phase A and homogenize for 5 minutes; stir and cool to 60°C and add phase C; when cooled to 35-40°C, add phase D and stir evenly to obtain a skin care cream.
对得到的产品进行人群测试实验：Conduct crowd testing experiments on the resulting product:
选取90位志愿者(均为自述皮肤易泛红、易受刺激皮肤者)，年龄在20-45岁之间，随机分成3组，分别使用上述含有巴斯德毕赤酵母溶胞物滤液的产品，即护肤精华液、面膜、护肤面霜，并参照上述配方制备不添加滤液的样品作为空白对照，其中A组使用精华液，每天早晚2次洁面后涂抹于面部；B组使用面膜，每周使用2次，贴敷于面部10-15min；C组使用护肤霜，每天早晚2次洁面后涂抹于面部，其中受试者左脸使用本发明所述的精华液、面膜或面霜产品，右脸使用不含有滤液的对照品，使用4周，在此期间不使用其他同类护肤产品，在第28天对上述样品缓解皮肤泛红、缓解皮肤刺激的使用效果进行自我评估。Select 90 volunteers (all of whom reported that their skin is prone to redness and irritation), aged between 20-45 years old, and were randomly divided into 3 groups. They used the above-mentioned filtrate containing Pichia pastoris lysate respectively. Products, namely skin care essence, facial mask, skin care cream, and refer to the above formula to prepare samples without adding filtrate as a blank control. Group A uses the essence and applies it to the face after cleansing twice a day, morning and evening; Group B uses a facial mask, every week Use it twice and apply it on the face for 10-15 minutes; Group C uses skin care cream and applies it to the face after cleansing twice a day, morning and evening. The subjects use the essence, facial mask or cream product of the present invention on the left face and the right face Use the control substance that does not contain filtrate for 4 weeks. During this period, do not use other similar skin care products. On the 28th day, self-evaluate the effectiveness of the above sample in relieving skin redness and relieving skin irritation.
评分标准：Grading:
皮肤状态无改善，甚至加重，得0分；皮肤泛红、易受刺激无改善，但也没有加重，得1分；皮肤泛红、易受刺激轻微改善，但效果不明显，得2分；皮肤泛红、易受刺激得到改善，得3分；皮肤泛红、易受刺激明显改善，得4分。If the skin condition does not improve or even worsens, it will be scored as 0 points; if the skin is red and susceptible to irritation, it will not improve but is not aggravated, it will be scored as 1 point; if the skin is red and susceptible to irritation, it will slightly improve, but the effect is not obvious, it will be scored as 2 points; If the skin's redness and susceptibility to irritation is improved, it will be scored 3 points; if the skin's redness and susceptibility to irritation is significantly improved, it will be scored 4 points.
测试结果各产品的平均分为：护肤精华液为3.25分，面膜为3.47分，护肤面霜为3.52分。反馈结果显示：含有巴斯德毕赤酵母溶胞物滤液的产品实施例均无任何刺激反应，并且有志愿者反应相较于空白对照，脸部皮肤泛红现象明显缓解。受试者面部局部照片如图22-24所示，图22为使用护肤精华液前后的对比照，图23为使用面膜前后的对比照，图24为使用护肤面霜前后的对比照，结果分析表明，本技术产品在舒缓皮肤和缓解皮肤泛红方面有较好的效果。 The average score of each product in the test results is: skin care essence is 3.25 points, facial mask is 3.47 points, and skin care cream is 3.52 points. Feedback results showed that none of the product examples containing Pichia pastoris lysate filtrate had any irritation reaction, and some volunteers responded that facial skin redness was significantly relieved compared to the blank control. Partial photos of the subject's face are shown in Figures 22-24. Figure 22 is a comparison photo before and after using skin care essence. Figure 23 is a comparison photo before and after using facial mask. Figure 24 is a comparison photo before and after using skin care cream. The analysis of the results shows that , this technology product has a better effect in soothing the skin and relieving skin redness.

Claims (19)

  1. 一种发酵溶胞物滤液的制备方法，其特征在于，所述发酵溶胞物滤液由巴斯德毕赤酵母菌发酵后的菌体制备获得，所述巴斯德毕赤酵母菌为表达重组胶原蛋白的巴斯德毕赤酵母菌，所述制备方法包括菌体稀释、高压均质裂解、离心及过滤；所述菌体稀释为以纯水稀释至质量浓度为5％～30％(W/W)的菌体悬液，所述高压均质裂解为将所述菌体悬液以>100MPar的压力破碎1-3次后获得裂解液，所述离心为将所述裂解液以10000g-20000g的速度离心，获得上清液，所述过滤为将所述上清液以0.22μm孔径过滤。A method for preparing fermentation lysate filtrate, characterized in that the fermentation lysate filtrate is prepared from cells fermented by Pichia pastoris, and the Pichia pastoris is an expression recombinant Pichia pastoris of collagen, the preparation method includes bacterial cell dilution, high-pressure homogenization lysis, centrifugation and filtration; the bacterial cell dilution is diluting with pure water to a mass concentration of 5% to 30% (W /W) bacterial cell suspension, the high-pressure homogeneous lysis is to crush the bacterial cell suspension 1-3 times at a pressure of >100MPar to obtain a lysate, and the centrifugation is to crush the lysate liquid at a pressure of 10000g- Centrifuge at a speed of 20,000g to obtain a supernatant. The filtration is to filter the supernatant with a pore size of 0.22 μm.
  2. 根据权利要求1所述的制备方法，其特征在于，所述表达重组胶原蛋白的巴斯德毕赤酵母菌选自保藏编号为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或CGMCC NO.21892的菌株中的一种或多种。The preparation method according to claim 1, characterized in that the Pichia pastoris expressing recombinant collagen is selected from the group consisting of CGMCC NO.7189, CGMCC NO.14057, CGMCC NO.17147, CGMCC NO. One or more strains of .17150, CGMCC NO.17148, CGMCC NO.17149, CGMCC NO.20626, CGMCC NO.20627, CGMCC NO.21891 or CGMCC NO.21892.
  3. 根据权利要求1所述的制备方法，其特征在于，所述菌体为发酵产物进行固液分离后获得。The preparation method according to claim 1, characterized in that the bacterial cells are obtained by solid-liquid separation of fermentation products.
  4. 根据权利要求1所述的制备方法，其特征在于，所述菌体稀释为以纯水稀释至质量浓度为10％(W/W)的菌体悬液。The preparation method according to claim 1, characterized in that the bacterial cells are diluted into a bacterial cell suspension diluted with pure water to a mass concentration of 10% (W/W).
  5. 根据权利要求1所述的制备方法，其特征在于，所述高压均质裂解为将所述菌体悬液以1200MPar的压力破碎。The preparation method according to claim 1, characterized in that the high-pressure homogeneous lysis involves crushing the bacterial cell suspension at a pressure of 1200 MPar.
  6. 一种权利要求1-5任一项所述制备方法制备得到的发酵溶胞物滤液。A fermentation lysate filtrate prepared by the preparation method according to any one of claims 1 to 5.
  7. 根据权利要求6所述的发酵溶胞物滤液，其特征在于，所述发酵溶胞物滤液中包括总蛋白、重组胶原蛋白、多糖、氨基酸、多种维生素。The fermentation lysate filtrate according to claim 6, wherein the fermentation lysate filtrate includes total protein, recombinant collagen, polysaccharides, amino acids, and multiple vitamins.
  8. 一种组合物，所述组合物包括权利要求1-5任一项所述制备方法制备的发酵溶胞物滤液或权利要求6-7任一项所述的发酵溶胞物滤液。A composition comprising the fermentation lysate filtrate prepared by the preparation method of any one of claims 1-5 or the fermentation lysate filtrate of any one of claims 6-7.
  9. 根据权利要求8所述的组合物，其特征在于，所述组合物中发酵溶胞物滤液的含量为1-10wt％，优选的，含量为5wt％。The composition according to claim 8, wherein the content of the fermentation lysate filtrate in the composition is 1-10 wt%, preferably, the content is 5 wt%.
  10. 权利要求1-5任一项所述制备方法制备的发酵溶胞物滤液或权利要求6-7任一项所述的发酵溶胞物滤液或权利要求8-9任一项所述组合物在抗敏类、舒缓类、抗炎类、防晒类、调节皮肤微生态类、控油类、修复皮肤屏障类化妆品原料或产品中的应用。The fermentation lysate filtrate prepared by the preparation method of any one of claims 1-5 or the fermentation lysate filtrate of any one of claims 6-7 or the composition of any one of claims 8-9 is in Applications in cosmetic raw materials or products such as anti-allergic, soothing, anti-inflammatory, sunscreen, regulating skin microecology, oil control, and repairing skin barrier cosmetics.
  11. 根据权利要求10所述的应用，其特征在于，所述抗敏、舒缓、抗炎主要包括能缓解角质层刺激症状、改善表皮组织损伤；降低炎症介质PGE2分泌水平，缓解皮肤炎症反应，减少皮肤的泛红现象的产生；通过抑制TRPV1蛋白表达的机制起到舒缓皮肤刺激功效。The application according to claim 10, characterized in that the anti-allergic, soothing and anti-inflammatory properties mainly include relieving stratum corneum irritation symptoms and improving epidermal tissue damage; reducing the secretion level of inflammatory mediator PGE2, alleviating skin inflammatory reactions and reducing skin inflammation. The occurrence of redness; it has the effect of soothing skin irritation through the mechanism of inhibiting the expression of TRPV1 protein.
  12. 根据权利要求10所述的应用，其特征在于，所述防晒表现为对紫外线吸收的能力。The application according to claim 10, characterized in that the sun protection is represented by the ability to absorb ultraviolet rays.
  13. 根据权利要求10所述的应用，其特征在于，所述调节皮肤微生态表现为通过提升抗菌肽 的表达，调节微生态，提升皮肤屏障，进而达到修护功效；巴斯德毕赤酵母发酵溶胞物滤液作用下，HBD1、HBD2、LL-37、TLR2蛋白含量显著上升。The application according to claim 10, characterized in that the regulation of skin microecology is performed by increasing antimicrobial peptides expression, regulate microecology, improve skin barrier, and achieve repair effect; under the action of Pichia pastoris fermentation lysate filtrate, HBD1, HBD2, LL-37, and TLR2 protein contents increased significantly.
  14. 根据权利要求10所述的应用，其特征在于，所述控油表现为抑制皮肤脂滴合成。The application according to claim 10, characterized in that the oil control is characterized by inhibiting the synthesis of skin lipid droplets.
  15. 权利要求1-5任一项所述制备方法制备的发酵溶胞物滤液或权利要求6-7任一项所述的发酵溶胞物滤液或权利要求8-9任一项所述组合物在化妆品领域中的应用。The fermentation lysate filtrate prepared by the preparation method of any one of claims 1-5 or the fermentation lysate filtrate of any one of claims 6-7 or the composition of any one of claims 8-9 is in Applications in the field of cosmetics.
  16. 根据权利要求15所述的应用，其特征在于，所述化妆品包括抗敏类、舒缓类、抗炎类、防晒类、调节皮肤微生态类、控油类、修复皮肤屏障类化妆品。The application according to claim 15, characterized in that the cosmetics include anti-allergic, soothing, anti-inflammatory, sunscreen, skin microecological regulation, oil control, and skin barrier repair cosmetics.
  17. 一种化妆品，所述化妆品包括1-5任一项所述制备方法制备的发酵溶胞物滤液或权利要求6-7任一项所述的发酵溶胞物滤液或权利要求8-9任一项所述组合物。A cosmetic, said cosmetics comprising the fermentation lysate filtrate prepared by the preparation method according to any one of 1-5 or the fermentation lysate filtrate according to any one of claims 6-7 or any one of claims 8-9 The composition described in the item.
  18. 根据权利要求17所述的化妆品，所述化妆品中发酵溶胞物滤液的含量为1-10wt％，优选的，含量为5wt％。According to the cosmetics according to claim 17, the content of the fermentation lysate filtrate in the cosmetics is 1-10wt%, preferably, the content is 5wt%.
  19. 根据权利要求17所述的化妆品，所述化妆品包括但不限于护肤精华液、面膜、护肤面霜。 The cosmetics according to claim 17, including but not limited to skin care essence, facial mask, and skin care cream.
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