A method for improving the stability of a pichia pastoris fermentation lysate filtrate

Abstract

The application discloses a method for improving the stability of Pichia pastoris fermentation cytolysate filtrate, and relates to the technical field of biological fermentation. The method is to obtain the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins by fermenting Pichia pastoris bacteria expressing recombinant heat shock proteins. The Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins obtained by the method has higher stability than the fermentation cytolysate filtrate of common yeast, and can solve the problems of the current natural Pichia pastoris fermentation cytolysate filtrate, such as complex composition and low stability. In addition, the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins has the effects of anti-inflammation, anti-photoaging, repair, promotion of cell proliferation and prevention of hair loss, has no sensitization, and has a wide application prospect in the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation, and particularly relates to a method for improving stability of Pichia pastoris fermentation cytolysate filtrate. BACKGROUND

[0002] Heat shock proteins (HSPs for short) are a group of very conservative protein molecule families produced by all prokaryotic cells and eukaryotic cells suffering from high temperature or other stresses, which mainly play the role of molecular chaperones and are a kind of evolutionarily highly conservative cellular stress proteins. According to the size of molecular weight, they are mainly divided into four families: HSP90 family (83-90KD), HSP70 family (66-78KD), HSP60 family and small molecular HSP family (15-30KD). Heat shock protein 10 is an important member of the small molecular HSP family and is a kind of evolutionarily highly conservative protein, which widely exists in many tissues of mammals. Under a variety of stress conditions, its expression increases, protects cell function as a molecular chaperone, and is also a key signal pathway for regulating cell apoptosis. Under the stimulation conditions of high temperature, ultraviolet rays, drugs, hypoxia and the like, the occurrence of cell apoptosis is inhibited. HSP10 also has an anti-inflammatory effect. Studies have shown that HSP10 may directly and rapidly act on macrophages to increase the secretion of cytokines and produce immunity.

[0003] The natural Pichia pastoris fermentation cytolysate filtrate contains metabolic products, cytoplasmic components, cell wall components and total sugar complexes, and the main components are Pichia pastoris total protein, total sugar, vitamins and minerals and the like. The components are complex, the stability is low, and the components are sensitive to heat, acid, alkali, enzymes and the like, are easy to be inactivated and decomposed, and have high requirements for the storage conditions. At present, no effective method for improving the stability of the fermentation cytolysate filtrate has been found. According to the literature reports, there are protein stabilizers and sugar stabilizers at present. The stability of related substances can be effectively improved by adding the stabilizers. However, the stabilizers are only for proteins or sugars, and the applicability of the stabilizers to the fermentation cytolysate filtrate containing complex components is low. SUMMARY

[0004] The technical problem to be solved by the present application is that the components of the natural Pichia pastoris fermentation cytolysate filtrate are complex and the stability is low.

[0005] In order to solve the above problems, the present application proposes the following technical solutions:

[0006] In a first aspect, the present application provides a method for improving the stability of Pichia pastoris fermentation cytolysate filtrate,

[0007] The method is to obtain the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins by fermenting Pichia pastoris bacteria expressing recombinant heat shock proteins.

[0008] Further, the heat shock protein includes all Pichia pastoris heat shock protein family members, preferably Pichia pastoris heat shock protein 10.

[0009] Further, the content of the heat shock protein in the Pichia pastoris fermentation cell lysate filtrate is 0.300-0.800 mg / ml.

[0010] Further, the method for preparing the Pichia pastoris fermentation cell lysate filtrate overexpressing heat shock protein by fermenting the Pichia pastoris bacteria expressing recombinant heat shock protein comprises:

[0011] The Pichia pastoris heat shock protein amino acid sequence is searched from the NCBI database, the corresponding nucleotide sequence is designed, and a restriction enzyme cleavage site is added at both ends to obtain a heat shock protein gene by full gene synthesis;

[0012] The heat shock protein gene is cloned into a Pichia pastoris universal expression vector to obtain a recombinant expression plasmid;

[0013] The recombinant expression plasmid is transformed into Pichia pastoris cells, and a recombinant genetically engineered strain is obtained by screening;

[0014] The recombinant genetically engineered strain is subjected to methanol induction fermentation to obtain a fermentation broth;

[0015] The fermentation broth is subjected to high-pressure homogenization lysis, centrifugation and filtration to obtain a Pichia pastoris fermentation cell lysate filtrate overexpressing heat shock protein.

[0016] In a second aspect, the present application provides a method for preparing a Pichia pastoris fermentation cell lysate filtrate, which comprises the method of the first aspect.

[0017] In a third aspect, the present application provides a Pichia pastoris fermentation cell lysate filtrate prepared by the method of the second aspect.

[0018] Further, the Pichia pastoris fermentation cell lysate filtrate includes total protein, heat shock protein and total sugar.

[0019] In a fourth aspect, the present application provides a composition comprising the Pichia pastoris fermentation cell lysate filtrate prepared by the method of the second aspect or the Pichia pastoris fermentation cell lysate filtrate of the third aspect.

[0020] Further, the content of the Pichia pastoris fermentation cell lysate filtrate in the composition is 0.1-3.0 wt%, for example, 0.15 wt%, 0.9 wt%, 1.5 wt%, 2.3 wt%, 2.8 wt% or 3.0 wt%.

[0021] The application also provides use of the Pichia pastoris fermentation lysate filtrate prepared by the preparation method of the second aspect or the Pichia pastoris fermentation lysate filtrate of the third aspect or the composition of the fourth aspect in an anti-allergic, soothing, anti-inflammatory, anti-hair loss, skin microecological regulation, oil control, skin barrier repair cosmetic raw material or product.

[0022] Compared with the prior art, the application can achieve the following technical effects:

[0023] The Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins prepared by fermentation of Pichia pastoris bacteria expressing recombinant heat shock proteins has higher stability than ordinary yeast fermentation lysate filtrate, and can solve the problems of complex components and low stability of the natural Pichia pastoris fermentation lysate filtrate. In addition, the Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins also has the effects of anti-inflammatory, anti-photoaging, repair, promotion of cell proliferation, and anti-hair loss, and has no allergenicity, and has a broad application prospect in the field of cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : Fermentation electrophoretogram of heat shock protein 10;

[0025] Figure 2 : Effect of Pichia pastoris fermentation lysate filtrate on TNF-a and IL-1a;

[0026] Figure 3 : Effect of Pichia pastoris fermentation lysate filtrate on zebrafish tail fin area;

[0027] Figure 4 : Effect of Pichia pastoris fermentation lysate filtrate on zebrafish neutrophils;

[0028] Figure 5 : Effect of Pichia pastoris fermentation lysate filtrate on cell migration rate;

[0029] Figure 6 : Effect of Pichia pastoris fermentation lysate filtrate on hair papilla cell proliferation activity. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described clearly and completely in combination with the accompanying drawings of the embodiments of the application, but the following embodiments do not limit the protection scope of the application.

[0031] For adding heat shock proteins in ordinary Pichia pastoris fermentation lysate filtrate, which can achieve the technical effects of the application, are within the protection scope of the application.

[0032] In the embodiments of the present application, the term "ordinary Pichia pastoris fermentation lysate filtrate" refers to the natural Pichia pastoris fermentation lysate filtrate without genetic recombination.

[0033] In the embodiments of the present application, "%" represents mass fraction.

[0034] In the embodiments of the present application, those not described in detail are completed by using conventional experimental methods, and those not described in detail in the embodiments are understood and easily realized by those skilled in the art according to product instructions or basic knowledge in the art, and thus are not described in detail.

[0035] Example 1 Preparation of Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins

[0036] (1) Obtain heat shock protein gene by total gene synthesis

[0037] The amino acid sequence of Pichia pastoris heat shock protein 10 (GenBank No. CCA37067.1) is found in the GeneBank database, with a full length of 104 amino acids and a molecular weight of 11.16 KDa; the amino acid sequence of the Pichia pastoris heat shock protein is as follows:

[0038] MSTLLKSANSLKPLLNRVLVQRAKAATKTASGIYIPEKNVEKLHQATVI

[0039] ATGPGVPNQNGSLEPTIVKAGDNVLIPSFGGSPVKINDEEYLLFSDREILAKIE

[0040] N

[0041] The nucleotide sequence of Pichia pastoris heat shock protein 10 is codon-optimized, and restriction enzyme cleavage sites Xho I and EcoR I are added at both ends, named pHSP10, and the nucleotide sequence thereof is as follows:

[0042] 1 CTCGAGATGT CCACGTTACT GAAGTCAGCA AACTCCTTGA AGCCACTGTT GAACAGAGTT

[0043] 61 TTGGTTCAAC GTGCTAAGGC TGCCACCAAG ACGGCAAGTG GCATTTATAT CCCTGAGAAG

[0044] 121 AATGTTGAAA AACTTCATCA GGCTACTGTA ATCGCTACAG GTCCTGGAGT TCCAAACCAA

[0045] 181 AATGGTTCTC TGGAACCAAC TATCGTGAAA GCAGGTGATA ACGTTTTGAT TCCATCTTTT

[0046] 241 GGTGGTTCCC CAGTCAAGAT AAACGATGAG GAGTATTTGT TATTTTCTGA TAGAGAGATT

[0047] 301 TTGGCTAAGA TTGAAAATTA AGAATTC

[0048] (2) Construction of recombinant vector pPIC9K-pHSP10

[0049] The heat shock protein gene obtained above was connected to the expression vector by enzyme digestion and ligation technique to obtain a recombinant expression plasmid, named pPIC9K-pHSP10.

[0050] Specifically, the vector pPIC9K and pHSP10 were double-digested by Xho I and Eco R I, and the pPIC9K vector was purchased from Invitrogen Company of the United States. The specific band was recovered from a 1.5% agarose gel according to the steps of the product instruction of TIANGEN DNA purification recovery kit (centrifugal column type). The double-digested recovery product pPIC9K was connected to pHSP10 by T4 DNA ligase at 16°C for 12 hours, and was transformed into E. coli DH5α. The positive clones were selected and identified by sequencing.

[0051] (3) Construction of recombinant genetically engineered bacteria

[0052] The pPIC9K-pHSP10 plasmid with correct sequencing was linearized by Sal I endonuclease, and the target band was recovered and electroporated into the competent cells of Pichia pastoris GSS115. The expression strain containing the target gene sequence was obtained by G418 screening and shake flask identification, and the strain was named GS115 / pPIC9K-pHSP10.

[0053] (4) Fermentation of recombinant genetically engineered bacteria

[0054] The above engineered bacteria GS115 / pPIC9K-pHSP10 were inoculated into BSM basic salt medium after activation for fermentation. After fermentation, the fermentation broth was obtained. The fermentation conditions were as follows: temperature 29°C, pH value 5.0, dissolved oxygen controlled at 30%-40%, when the wet bacteria weight of the fermentation broth reached 180-200 mg / mL, the feeding was stopped, starved for 1 hour, methanol induction was started, and the induction fermentation was performed for 40-50 hours. The results are shown in the attachedFigure 1 .

[0055] It should be noted that the vector pPIC9K has an alcohol promoter, and under the induction of methanol, the expression amount of heat shock protein will increase, and the fermentation liquor of overexpressed heat shock protein is obtained.

[0056] The basic salt medium is prepared by dissolving 40 g of glycerol, 18.2 g of K2SO4, 26.7 ml of H3PO4, 0.93 g of CaSO4·2H2O, 14.9 g of MgSO4, and 4.13 g of KOH in 1000 ml of deionized water and mixing uniformly.

[0057] (5) Preparation of cell lysate filtrate

[0058] The fermentation liquor (containing bacteria and supernatant) is subjected to cell wall breaking treatment using a high-pressure homogenizer, the breaking temperature is controlled at 20°C, the breaking pressure is 70-100 MPa, and the breaking is performed 1-3 times to obtain a lysate. In this embodiment, the breaking pressure is preferably 80 MPa, and the breaking is performed 2 times to obtain the lysate.

[0059] The fermentation lysate is centrifuged at 10000 rpm for 30 min to remove residual insoluble substances, the supernatant is filtered using a 0.22 μm microfiltration system to remove residual bacterial fragments and fermentation pigments, the filtrate is collected, and the filtrate is subjected to ultrafiltration desalination using an ultrafiltration membrane with a molecular weight cut-off of 1 KD to obtain the Pichia pastoris fermentation cell lysate filtrate overexpressing heat shock protein.

[0060] Example 2 Stability verification experiment

[0061] (1) The contents of HSP10, total protein, and total sugar in the Pichia pastoris fermentation cell lysate filtrate overexpressing heat shock protein prepared in Example 1 are detected. The specific method is as follows:

[0062] The detection method of HSP10 content is quantitative ELISA kit method, and the universal HSP10 detection kit is purchased from Jianglai Biology. The experimental steps are as follows:

[0063] 1. Dilute the cell lysate filtrate 20000 times with PBS buffer, use PBS buffer as a blank control, dilute the standard according to the kit instructions, and the concentrations are 100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0 ng / ml, respectively.

[0064] 2. Add 100 μl per well of the sample, the different concentration standards provided by the kit, and the blank control to the corresponding wells, cover the plate with a sealing film, and incubate at 37°C for 1 hour.

[0065] 3. Take out the enzyme label board, discard the liquid, do not wash, directly add 100 μl of biotinylated antibody working solution to each hole, cover the sealing film, incubate at 37°C for 1 hour.

[0066] 4. Discard the liquid, add 300 μl of washing liquid to each hole, stand for 1 minute, shake off the washing liquid, pat dry on the water-absorbing paper, repeat the plate washing for 3 times.

[0067] 5. Add 100 μl of enzyme binding working solution to each hole, cover the sealing film, incubate at 37°C for 30 minutes.

[0068] 6. Discard the liquid, wash the plate for 5 times according to the method of step 4.

[0069] 7. Add 90 μl of substrate to each hole, cover the sealing film, incubate at 37°C for 15 minutes.

[0070] 8. Take out the enzyme label board, directly add 50 μl of termination solution to each hole, immediately measure the OD value of each hole at 450 nm wavelength.

[0071] Select 3 batches of Pichia pastoris fermentation lysate overexpressing heat shock protein prepared in Example 1 to detect the content of HSP10 to determine the range value of the content, and the determination results are as follows in Table 1:

[0072] Table 1: HSP10 content detection results of the lysate filtrate prepared in Example 1

[0073]

[0074] As can be seen from the results in Table 1, the content of HSP10 in the Pichia pastoris fermentation lysate overexpressing heat shock protein prepared in Example 1 is between 0.377-0.754 mg / ml.

[0075] The detection method of total protein content is quantified by BCA method, and the reagent kit is purchased from Nanjing Jiancheng Biological Engineering Institute, and the experimental steps are as follows:

[0076] 1. Standard curve drawing: dilute the protein standard solution to concentrations of 0, 25, 50, 100, 250, 500, 1000, and 2500 μg / ml, respectively, take 20 μl of standard solution with different concentrations, add 250 μl of working solution according to the kit instructions, vortex, incubate at 37°C for 30 min, add termination application liquid, mix well, stand for 5 min, measure the absorbance value of each tube at 562 nm, and draw the standard curve with the standard tube concentration as the X axis and the corresponding absorbance value as the vertical coordinate.

[0077] The linear regression equation of the obtained standard curve is: y=0.0002x+0.0003, R 2 =0.9996.

[0078] 2. Take 20 μl of the lysate filtrate, add 250 μl of working solution according to the kit instructions, vortex to mix, incubate at 37℃ for 30 min, add the stop solution, mix well and let stand for 5 min, measure the absorbance value of each tube at 562 nm, and record the results.

[0079] The total protein content of fermentation lysate filtrates of Pichia pastoris overexpressing heat shock protein prepared in Example 1 was determined by analyzing three batches to identify the range of content values. The results are shown in Table 2 below.

[0080] Table 2. Results of total protein content detection in the lysate filtrate prepared in Example 1.

[0081] Sample Average total protein content (%) CV (%) Cytolysate batch 1 16.681 1.225 Cytolysate batch 2 15.425 1.247 Cytolysate batch 3 13.624 0.929

[0082] As shown in Table 2, the total protein content in the fermentation lysate filtrate of Pichia pastoris overexpressing heat shock protein prepared in Example 1 was between 13.624% and 16.681%.

[0083] The total sugar content was determined quantitatively using spectrophotometry. The total sugar content reagent kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. The experimental steps are as follows:

[0084] 1. Standard curve plotting: Using glucose as the standard, with standard concentrations of 1, 0.8, 0.6, 0.5, 0.4, 0.2, and 0.1 mg / ml, a standard curve was plotted with the standard tube concentration as the X-axis and the corresponding absorbance value as the Y-axis.

[0085] The obtained standard curve linear regression equation is: Y = 0.4441x - 0.0462, R0 2 =0.9995.

[0086] 2. Take 0.1 ml of the lysate filtrate, and according to the kit instructions, first add 0.1 ml of Reagent 1 and 0.15 ml of distilled water, heat in a 95°C water bath for 30 min, then add 0.1 ml of Reagent 2, mix well, and bring the volume to 1 ml with distilled water. Centrifuge at 8000 g for 10 min, and collect the supernatant for testing. Note: Reagents 1, 2, and 3 are the reagents listed on the kit.

[0087] 3. Sample addition table

[0088]

[0089]

[0090] Mix well, measure the absorbance at 540 nm, and calculate using the following formula.

[0091]

[0092] in:

[0093] ΔAmeasured = A measured tube - A blank tube, ΔAstandard = A standard tube - A blank tube;

[0094] Vtotal is the total volume of the sample extraction liquid, 1 ml; Vsample is the sample volume, 0.1 ml.

[0095] Three batches of the Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins prepared in Example 1 were selected for total sugar content detection to determine the content range value, and the determination results are as follows in Table 4:

[0096] Table 4

[0097] Sample Average total sugar content (mg / ml) CV (%) Cytolysate batch 1 23.801 0.749 Cytolysate batch 2 19.808 0.341 Cytolysate batch 3 18.611 1.583

[0098] It can be known from the results in Table 1 that the total sugar content in the Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins prepared in Example 1 is between 18.611-23.801 mg / ml.

[0099] (2) The heat stability of the lysate filtrate prepared in Example 1 was investigated by using 42℃ accelerated stability experiment, and the control group was the ordinary Pichia pastoris fermentation lysate filtrate, and the sample group was the lysate filtrate obtained in Example 1.

[0100] The main investigation indexes were the changes of the total protein content and the total sugar content in the lysate filtrate at the preparation initial, 15 days, 30 days, 60 days and 90 days, respectively. The results are as follows in Table 5-1:

[0101] Table 5-1 Results of 42℃ accelerated stability experiment

[0102]

[0103]

[0104] It can be known from the results in Table 5-1 that compared with the control group, the lysate filtrate of Example 1 has obvious advantages in the 42℃ stability experiment. After being placed at 42℃ for 90 days, the total protein content of the control group decreased by 46.2% compared with the initial, and the total sugar content decreased by 58.3% compared with the initial; the total protein content of Example 1 decreased by 5.05% compared with the initial, and the total sugar content decreased by 5.32% compared with the initial. It can be seen that the Pichia pastoris fermentation lysate filtrate overexpressing heat shock proteins prepared by the Pichia pastoris bacteria expressing recombinant heat shock proteins has higher thermal stability than the ordinary yeast fermentation lysate filtrate.

[0105] (3) The stability of the prepared lysate filtrate under acid and alkali conditions was investigated, the control group was the ordinary Pichia pastoris fermentation lysate filtrate, and the sample group was the lysate filtrate obtained in Example 1.

[0106] The main indicators are the changes in total protein content and total sugar content in the lysate filtrate under the conditions of pH 3.5 and pH 9.0 for 15 days, 30 days, 60 days, and 90 days. The results are shown in Table 5-2:

[0107] Table 5-2 Stability test results under different pH conditions

[0108]

[0109] As can be seen from Table 5-2, compared with the control group, the lysate filtrate of Example 1 has obvious advantages in the acid-base stability test: after being placed for 90 days under the condition of pH 3.5, the total protein content of the control group decreased by 33.2% compared with the initial value, and the total sugar content decreased by 28.8% compared with the initial value; the total protein content of Example 1 decreased by 0.8% compared with the initial value, and the total sugar content decreased by 4.1% compared with the initial value. After being placed for 90 days under the condition of pH 9.0, the total protein content of the control group decreased by 54.9% compared with the initial value, and the total sugar content decreased by 17.0% compared with the initial value; the total protein content of Example 1 decreased by 4.4% compared with the initial value, and the total sugar content decreased by 3.1% compared with the initial value. Therefore, the lysate filtrate of overexpressed heat shock protein Pichia pastoris fermented by the Pichia pastoris expressing recombinant heat shock protein provided by the present application has good acid and alkali stability.

[0110] Example 3 Efficacy verification experiment

[0111] In order to verify the efficacy of the lysate filtrate of overexpressed heat shock protein Pichia pastoris fermented by the Pichia pastoris expressing recombinant heat shock protein provided by the present application, the lysate filtrate of overexpressed heat shock protein Pichia pastoris prepared in Example 1 was subjected to anti-photoaging efficacy detection, anti-inflammatory efficacy detection, repair efficacy detection, sensitization test, and anti-hair loss efficacy detection. The specific detection is as follows:

[0112] (1) Anti-photoaging and anti-inflammatory efficacy detection

[0113] Human keratinocytes were subcultured in keratinocyte culture medium. After cell recovery, the cells were cultured in a 37°C, 5% CO2, saturated humidity incubator until the cell fusion rate reached 80-90%. After trypsin digestion, the cells were inoculated into a 96-well plate at a cell inoculation density of 5000 cells / well. After the cells grew to cover about 80% of the well bottom, the original culture medium was discarded, washed once with 200 μL PBS, and just enough PBS was added to cover the bottom of the well to prevent cell dehydration during the operation.

[0114] UV irradiation treatment 102s, total dose 0.15 J / cm 2 UV irradiation treatment 102s, total dose 0.15 J / cm 2 Discard the PBS in the culture hole, replace the blank control group with normal culture medium, add dexamethasone 10 μg / ml to the positive control group culture medium, and add the above-mentioned cell lysate filtrate with a percentage concentration of 1%, 2%, and 3% to the sample group in the normal culture medium, and continue to culture for 72 h. Collect the supernatant in the hole plate, and detect the contents of inflammatory factors TNF-a and IL-1a using an ELISA kit, and the results are shown in Figs. 6 and 7. Figure 2 .

[0115] The results show that the cell lysate filtrate (percentage content 1%, 2%, and 3%) can inhibit the effects of UVB-mediated generation of TNF-a and IL-1a to different extents, indicating that the cell lysate filtrate of Example 1 has anti-photoaging and anti-inflammatory effects.

[0116] (2) Repair efficacy detection

[0117] The zebrafish were divided into three groups, namely a normal control group, a model control group, and a test product group. The normal control group was not treated, and the model control group and the test product group were both quantitatively cut off using a surgical method to complete the modeling process. The test product group was added with 1%, 2%, and 3% of the cell lysate filtrate of Example 1, and was incubated in the dark for a period of time. Then, the zebrafish in each group were photographed under a stereomicroscope and the pictures were saved. The area of the tail fin of the zebrafish was quantitatively analyzed, and the number of neutrophils at the damaged site of the tail fin of the zebrafish was counted. The results are shown in Figs. 8 and 9. Figure 3 , and Figs. 10 and 11. Figure 4 .

[0118] Conclusion: After the comparative experiment of zebrafish, the area of the tail fin of the zebrafish in the test product group was significantly increased compared with the model control group, and the number of neutrophils of the zebrafish in the test product group was significantly reduced compared with the model control group, indicating that the cell lysate filtrate of Example 1 has a repair effect.

[0119] (3) Cell migration test

[0120] 2 x 10 6Personal skin fibroblasts (HFF-1) were inoculated into a 6-well plate and cultured for 24 hours, then a 200 μL pipette tip was used to draw a line, and the cells were washed with PBS for 3 times. Then, the sample group: 1%, 1.5%, 2%, 2.5% overexpressed heat shock protein Pichia pastoris cell lysate filtrate; the control group: 2% commercially available ordinary Pichia pastoris cell lysate filtrate, and a blank control containing only serum-free medium were added for culture. At 0, 6, 20, and 28 hours, photographs were taken under a microscope. The photographs were analyzed using image J and prism 8, and a column chart of cell growth rate and time was drawn, and the results are shown in Figure 5 .

[0121] Conclusion: The overexpressed heat shock protein Pichia pastoris cell lysate filtrate with a content of 2%, 1.5%, and 1% can promote the growth of human fibroblasts within 0-28h, and has a very significant effect (P<0.01) within 6h-28h. Compared with the control group, the sample group has a significantly higher proliferation-promoting effect, indicating that the overexpressed heat shock protein Pichia pastoris cell lysate filtrate has a significantly improved cell migration-promoting effect compared with the commercially available ordinary Pichia pastoris cell lysate filtrate.

[0122] (4) Anti-hair loss efficacy detection

[0123] Blank control group: complete cell culture medium.

[0124] Sample group: complete cell culture medium added with 0.5%, 1%, 1.5%, 2%, and 2.5% overexpressed heat shock protein Pichia pastoris fermentation cell lysate filtrate.

[0125] Control group: 2% commercially available ordinary Pichia pastoris fermentation cell lysate filtrate.

[0126] P1 generation of hair papilla cells were inoculated into a 96-well plate at a density of 3000 cells / well, and the blank control group was cultured using complete cell culture medium; the control group was cultured using complete cell culture medium added with 2% commercially available Pichia pastoris cell lysate filtrate; and the sample group was cultured using complete cell culture medium added with 0.5%, 1%, 1.5%, 2%, and 2.5% overexpressed heat shock protein Pichia pastoris fermentation cell lysate filtrate. After inoculation, they were placed in a 37°C, 5% CO2 cell incubator for 24h of pre-culture, then 10 μL / well of CCK-8 reagent was added, and incubation was carried out in the incubator. The absorbance was measured at 24, 48, 72, 96, and 120 hours of culture, respectively. The absorbance value of the sample at 450 nm was detected in the enzyme marker, and the proliferation growth curve of the hair papilla cells was drawn with the cell culture days as the horizontal axis and the OD value as the vertical axis, and the results are shown in Figure 6 .

[0127] By the attached Figure 6 The results show that the sample group: 0.5%, 1% overexpression of heat shock protein Pichia pastoris lysate filtrate can improve the proliferation activity of hair papilla cells, and 1% content is significant at 72h, which shows that the overexpression of heat shock protein Pichia pastoris fermentation lysate filtrate has obvious effect of promoting the proliferation activity of hair papilla cells compared with ordinary Pichia pastoris lysate filtrate.

[0128] (5) Sensitization test

[0129] Test article: overexpression of heat shock protein Pichia pastoris fermentation lysate filtrate of Example 1.

[0130] Experimental animals: 40 healthy guinea pigs, half male and half female, clean grade, body weight 398±24g at the beginning of the experiment.

[0131] Animal grouping: The guinea pigs were randomly divided into 3 groups according to gender and weight. The first group of 20 was the test group, given 20mg / ml of overexpression of heat shock protein Pichia pastoris lysate filtrate of Example 1; the second group of 10 was the positive control group, given 20mg / ml of 2,4-dinitrochlorobenzene; the third group of 10 was the blank control group.

[0132] Test method: Local occlusion coating method was used. 24 hours before the test, the guinea pigs were shaved on both sides of the spine with an electric shaver, and the area of each side was 3cm×3cm. Induction contact: On the 0th day of the experiment, the overexpression of heat shock protein Pichia pastoris fermentation lysate filtrate was applied to the left side of the guinea pig's hairless area, covered with 2 layers of gauze and a layer of glass paper, and then fixed with non-irritating adhesive tape for 6 hours. The same method was repeated on the 7th and 14th days. The blank control group was not induced to contact, and the positive control group was given 0.5ml of 20mg / ml 2,4-dinitrochlorobenzene on the left side of the hairless area, covered with 2 layers of gauze and a layer of glass paper, and then fixed with non-irritating adhesive tape for 6 hours. The same method was repeated on the 7th and 14th days. Challenge contact: On the 28th day, the guinea pigs were challenged by the same method as the induction contact on the right side of the hairless area. After 6 hours, the test article was removed, and the skin allergic reaction was observed immediately, and again at 24, 48 and 72 hours. The right side of the hairless area of the blank control group was given the overexpression of heat shock protein Pichia pastoris fermentation lysate filtrate, and the right side of the hairless area of the positive control group was given 2% 2,4-dinitrochlorobenzene.

[0133] Evaluation index:

[0134] General condition observation: Observe the changes in diet, excretion, behavior, etc. of the animals and other abnormal conditions.

[0135] Skin reaction score and sensitization reaction determination are shown in Tables 6 and 7, and the test results are shown in Table 8.

[0136] Table 6 Skin reaction score table of sensitization test

[0137] Skin reaction Score Erythema and eschar formation - No erythema 0 Mild erythema (barely visible) 1 Distinct erythema (scattered or patchy) 2 Moderate to severe erythema 3 Severe erythema (purplish red) to mild eschar formation 4 Edema formation - No edema 0 Mild edema (barely visible) 1 Moderate edema (clearly raised contour of skin) 2 Severe edema (raised skin about 1 mm or more than 1 mm) 3 Maximum score 7

[0138] Table 7 Sensitization intensity

[0139] Sensitization rate % Sensitization intensity 0～8 Weak 9～28 Light 29～64 Medium strong 65～80 Strong 81～100 Very strong

[0140] Table 8 Sensitization test results of Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins

[0141]

[0142]

[0143] It can be seen from the results in Table 8 that the test group and the blank control group did not have skin erythema and edema after 24, 48 and 72 hours of provocation, the average value of skin irritation was 0, and the sensitization rate was 0, which indicated that the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins prepared in Example 1 had no sensitization.

[0144] Example 4

[0145] The present embodiment provides the use of the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins prepared in Example 1 for preparing an anti-photoaging essence, which is prepared from the following raw materials in a mass ratio:

[0146] A component: deionized water 66.2%, sodium carboxymethyl cellulose 1%, allantoin 0.3%; B component: rose water 20%, Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins 5%, coenzyme Q10 3%, ascorbic acid glucoside 0.5%; C component: 3% hexylene glycol, 1% pentylene glycol. After the A component is swelled overnight, it is incubated at 80°C for 20 minutes, the prepared B component is added, and the C component is added after being fully mixed to obtain the anti-photoaging essence.

[0147] When used, an appropriate amount is taken after cleansing and evenly applied to the face, and used once in the morning and once in the evening.

[0148] Example 5

[0149] The present embodiment provides the use of the Pichia pastoris fermentation cytolysate filtrate overexpressing heat shock proteins prepared in Example 1 for preparing a repair gel cosmetic, which is prepared from the following raw materials in a mass ratio:

[0150] A component: deionized water 80.45%, glycerin 3%, carbomer 940 0.2%, polyglutamic acid sodium 0.15%, allantoin 0.2%; B component: deionized water 10%, squalane 0.3%, Pichia pastoris fermentation lysate overexpressing heat shock protein 3%, sea buckthorn oil 2.5%; C component: arginine 0.2%. After swelling overnight, the A component is heated at 80℃ for 20 minutes, the prepared B component is added, and after being fully mixed, the C component is added and stirred uniformly to obtain the repair gel.

[0151] In use, an appropriate amount is evenly applied to the face after cleansing, and is used before going to bed at night.

[0152] Example 6

[0153] The present example provides the use of the Pichia pastoris fermentation lysate overexpressing heat shock protein prepared in Example 1 for preparing an anti-hair loss hair care essence made from the following mass ratio of raw materials:

[0154] A component: white oil 20%, glycerin 5%, acetylated lanolin 5%; B component: deionized water 67.4%, Pichia pastoris fermentation lysate overexpressing heat shock protein 1.5%, rosemary essential oil 0.05%, cedar essential oil 0.05%; C component: phenoxyethanol 0.5%, ethylhexylglycerin 0.5%. The A component is heated to 80℃ for 20 minutes, cooled to about 40℃, the prepared B component is added, and after being fully mixed, the C component is added and stirred uniformly to obtain the anti-hair loss hair care essence.

[0155] In use, the hair tips are applied after shampooing, and are rinsed off after staying for two or three minutes.

[0156] In the above examples, the description of each example focuses on different aspects, and the parts not described in detail in a certain example can be referred to the relevant description of other examples.

[0157] The above is a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for improving the stability of Pichia pastoris fermentation lysate filtrate, characterized in that, Fermentation broth was obtained by fermentation of Pichia pastoris expressing recombinant heat shock protein; The fermentation broth was subjected to high-pressure homogenization and lysis, followed by centrifugation and filtration to obtain a fermentation lysate filtrate of Pichia pastoris overexpressing heat shock proteins. The heat shock protein content in the Pichia pastoris fermentation lysate filtrate was 0.300-0.800 mg / ml; The heat shock protein is Pichia pastoris heat shock protein 10; The method for preparing the fermentation lysate filtrate of Pichia pastoris expressing recombinant heat shock protein includes: The amino acid sequence of Pichia pastoris heat shock protein 10 was found in the NCBI database, the corresponding nucleotide sequence was designed, restriction endonuclease sites were added to both ends, and the heat shock protein gene was synthesized in its entirety. The heat shock protein gene was cloned into a Pichia pastoris universal expression vector to obtain a recombinant expression plasmid, wherein the universal expression vector was pPIC9K. The recombinant expression plasmid was transformed into Pichia pastoris cells, and recombinant genetically engineered strains were obtained by screening. The recombinant genetically engineered strain was subjected to methanol-induced fermentation to obtain the fermentation broth; The fermentation broth was subjected to high-pressure homogenization and lysis at a temperature of 20°C and a pressure of 70-100 MPa. After centrifugation and filtration, the fermentation lysate filtrate of Pichia pastoris overexpressing heat shock protein was obtained.

2. A method for preparing a filtrate of Pichia pastoris fermentation lysate, characterized in that, Includes the method described in claim 1.

3. A filtrate of Pichia pastoris fermentation lysate prepared by the method of claim 2.

4. The Pichia pastoris lysate filtrate as described in claim 3, characterized in that, The fermentation lysate filtrate of Pichia pastoris contains total protein, heat shock protein, and total sugar.

5. A composition comprising the Pichia pastoris fermentation lysate filtrate prepared by the method of claim 2 or the Pichia pastoris fermentation lysate filtrate of any one of claims 3-4.

6. The composition according to claim 5, characterized in that, The content of Pichia pastoris fermentation lysate filtrate in the composition is 0.1-3.0 wt%.

7. The use of the Pasteurella fermentation lysate filtrate prepared by the preparation method according to claim 2, or the Pasteurella fermentation lysate filtrate according to any one of claims 3-4, or the composition according to any one of claims 5-6, in cosmetic raw materials or products for anti-allergy, anti-inflammatory, anti-hair loss, and skin barrier repair purposes.

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