Pseudomonas oryzae P.0719 and application thereof

Abstract

The invention relates to pseudomonas oryzae P.0719 and application thereof, and belongs to the technical field of microorganisms. The invention provides a pseudomonas oryzae P.0719 strain capable of producing protease. The pseudomonas oryzae P.0719 strain can cause skin aging; meanwhile, according to the growth characteristics of the strain, multiple types of raw materials are screened, growth of the strain is accurately inhibited, the raw materials are added into a skin care product, the effect of reducing the abundance of human face pseudomonas is achieved through human body testing, and therefore skin aging caused by metabolite of the pseudomonas is reduced. Part of polyol compounds and polypeptide raw materials can inhibit pseudomonas oryzae P.0719, and meanwhile, it is accidentally found that the raw materials can also promote proliferation of propionibacterium acnes sourced from healthy skin, so that improvement of an aging skin flora structure can be remarkably promoted.

Description

Technical Field

[0001] The invention relates to a strain of Pseudomonas oryzae P.0719 and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] As people become more aware of the relationship between skin microorganisms and body health, it has been proposed that skin microorganisms are the biological barrier of the skin and play an important role in maintaining body health.

[0003] With age, the physiological characteristics of the skin change accordingly, such as a decrease in sebum content and transepidermal water loss (TEWL), a decrease in skin elasticity, an increase in skin roughness, an increase in wrinkles, etc., and the skin shows signs of aging. Similarly, along with the changes in the physiological characteristics of the skin, the skin flora also changes with age, showing a certain regularity. Skin aging is closely related to intestinal and skin flora. Skin microorganisms and hosts have formed a mutually dependent and mutually beneficial relationship in long-term coexistence. The skin flora can inhibit the growth of pathogenic bacteria, regulate cytokine production, maintain the integrity of the skin barrier, inhibit local inflammation, and maintain tissue homeostasis. It can be seen that the skin flora is an important part of the body's health. In 2023, Cell published an article proposing 12 major markers of aging, pointing out that ecological disorder is a major factor in accelerating aging. The microecology or flora affects the aging of the body, and anti-aging strategies targeting the microbiome have been gradually proposed.

[0004] With the in-depth study of intestinal and skin flora, it is found that their impact on skin health is far beyond our imagination. In the future, intervention based on the microbiome will be an important way to diagnose and treat skin problems or diseases. On the product application side, international brands such as L'Oreal and Unilever have proposed skin problem solutions targeting the skin microbiome. The main abundance strains that cause skin problems have received widespread attention, such as acne-causing Propionibacterium acnes, Malassezia that causes dandruff, and Staphylococcus epidermidis associated with atopic dermatitis (AD). Low-abundance flora also play an important role in maintaining skin health. In the investigation of big data on skin flora of different problem skins or populations, many literatures have proposed a certain correlation between low-abundance skin flora and physiological parameters. In the analysis of skin flora, the impact of low-abundance flora on the skin has not yet attracted enough attention.

[0005] In the present invention, a low-abundance strain of Pseudomonas is obtained from healthy skin. Currently, there is no relevant report on the research of skin-derived microorganisms of this genus. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a strain of Pseudomonas oryzae P.0719 and its application. Specifically, the present invention isolates a low-abundance bacterium from healthy skin, Pseudomonas oryzae P.0719, and finds that the bacterium can produce protease, causing collagen degradation, thereby causing skin aging, and can also promote the expression of inflammatory factors, and has a pro-inflammatory effect; the present invention provides multiple types of raw materials that can significantly inhibit the bacteria, and these multiple types of raw materials can be applied to products, which can reduce the abundance of Pseudomonas on the human face. The present invention unexpectedly found that while some raw materials can inhibit the growth of Pseudomonas, they can also promote the proliferation of Propionibacterium acnes, which can significantly promote the improvement of the flora structure of aging skin.

[0007] The technical solution of the present invention is as follows:

[0008] A strain of Pseudomonas oryzihabitans P.0719 was deposited in the General Microbiology Center of China Culture Collection Administration on January 18, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29673.

[0009] In the present invention, the above-mentioned Pseudomonas oryzae P.0719 is isolated from healthy human facial skin. The colonies on the R2A blood plate are yellow, round, convex, dry and wrinkled, with neat edges and no hemolysis zone. Under an optical microscope, the bacteria are observed to be short round rods with a size of 0.5-1.0 μm×1.5-5.0 μm, and the bacteria exist in a dispersed state.

[0010] In the present invention, the above-mentioned Pseudomonas oryzae P.0719 can produce protease, and the components in its metabolites can cause collagen degradation, thereby causing skin aging; at the same time, it can also promote the expression of inflammatory factors and has a pro-inflammatory effect.

[0011] The above-mentioned Pseudomonas oryzae P.0719 is used as a marker in screening raw materials for cosmetics or skin care products.

[0012] Preferably according to the present invention, the raw materials of cosmetics or skin care products can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

[0013] Further preferably, the raw materials of the cosmetics or skin care products include: probiotic fermented brown algae extract, 9-β-D-ribofuranosyl adenine, 17kDa collagen, lactobacillus fermentation filtrate, dipropylene glycol, 1,3-butylene glycol, PEG / PPG-17 / 6 copolymer or PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

[0014] Further preferably, the cosmetic or skin care product raw materials can also promote the proliferation of Propionibacterium acnes and improve the flora structure of aging skin.

[0015] Further preferably, the raw materials of the cosmetics or skin care products include: probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol or PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

[0016] The use of raw materials screened according to the above applications in the preparation of cosmetics or skin care products.

[0017] Preferably according to the present invention, the cosmetics or skin care products can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

[0018] A cosmetic or skin care product containing a probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol. The cosmetic or skin care product can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

[0019] Preferably, according to the present invention, the cosmetics or skin care products can also promote the proliferation of Propionibacterium acnes and improve the flora structure of aging skin.

[0020] Beneficial effects:

[0021] This patent provides a strain of Pseudomonas oryzae P.0719 that can produce protease, which can cause skin aging; at the same time, according to the growth characteristics of the strain, multiple types of raw materials are screened to accurately inhibit its growth, and this type of raw material is added to skin care products. After human testing, it has the effect of reducing the abundance of Pseudomonas on the human face, thereby reducing the aging caused by its metabolites to the skin. While some polyol compounds and polypeptide raw materials inhibit Pseudomonas oryzae P.0719, it is unexpectedly found that the raw materials can also promote the proliferation of Propionibacterium acnes from healthy skin, so it can significantly promote the improvement of the structure of the aging skin flora. Experiments have confirmed that after the late logarithmic phase of Pseudomonas oryzae P.0719, the pH value of the fermentation broth shows an upward trend, the pH can reach 7.6, and the fermentation broth is alkaline; protease is produced during the fermentation process, and after 18-20 hours of culture, the crude enzyme amount produced is about 0.3-0.5g / L; on fibroblasts, the fermentation supernatant of the strain can significantly inhibit the expression of type III collagen, with an inhibition rate of 27.94%, and promote the production of cellular ROS, with a promotion rate of 98%; on keratinocytes, it promotes the expression of inflammatory factors, among which the promotion rates of IL-1, IL-6 and TNF-α are 22.1%, 18.4% and 13.9% respectively; the multi-type raw materials provided in this patent can significantly inhibit the growth of the strain, with an inhibition rate of 20%-80%; some raw materials can also promote the proliferation of Propionibacterium acnes from healthy skin and improve the flora structure of aging skin. In summary, the present invention is a new skin care direction in resisting skin aging, and has significant effects and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the colony morphology of Pseudomonas oryzae P.0719 on R2A blood agar plate.

[0023] Figure 2 This is a picture of the bacterial morphology of Pseudomonas oryzae P.0719 under a microscope.

[0024] Figure 3 This is the phylogenetic tree of Pseudomonas oryzae P.0719.

[0025] Figure 4 This is the growth curve of Pseudomonas oryzae P.0719.

[0026] Figure 5 This is the pH change curve of the bacterial solution of Pseudomonas oryzae P.0719.

[0027] Figure 6 This is a picture of the hydrolysis circle of Pseudomonas oryzae P.0719 on the gelatin medium plate.

[0028] Figure 7This is the SDS-PAGE gel electrophoresis diagram of the reaction solution between the fermentation supernatant of Pseudomonas oryzae P.0719 and type III collagen.

[0029] Figure 8 This is a bar graph showing the residual protein content in the reaction solution between the fermentation supernatant of Pseudomonas oryzae P.0719 and type III collagen.

[0030] Fig. 9 This is a bar graph showing the expression of inflammatory factors in HaCaT cells under the action of fermentation supernatant of Pseudomonas oryzae P.0719.

[0031] Fig.10 This is a bar graph of the expression of type III collagen in HSF cells under the action of the fermentation supernatant of Pseudomonas oryzae P.0719.

[0032] Fig.11 This is a ROS staining image of HSF cells under the action of fermentation supernatant of Pseudomonas oryzae P.0719.

[0033] Fig.12 This is a bar graph of the relative content of ROS in HSF cells under the action of fermentation supernatant of Pseudomonas oryzae P.0719.

[0034] Fig.13 This is a diagram of the skin flora structure before and after using the essence.

[0035] Fig.14 This is a comparison of the abundance of skin flora before and after using the essence. DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or according to the product specifications. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0037] The Pseudomonas oryzihabitans P.0719 of the present invention was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on January 18, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29673.

[0038] The sources of different types of raw materials in the examples are as follows:

[0039] Table 1. Raw materials and their sources

[0040] raw material source Probiotic fermented brown algae extract Shandong Jiuxin Information Technology Co., Ltd. 9-β-D-ribofuranosyladenine Jiangsu Sulu Biotechnology Co., Ltd. 17kDa collagen Hebei Naco Biotechnology Co., Ltd. Lactobacillus fermentation filtrate Shandong Freda Biotechnology Co., Ltd. Dipropylene glycol SK PICGLOBAL CO.,LTD. 1,3-Butanediol Wanhua Chemical Group Co., Ltd. PEG / PPG-17 / 6 copolymer Dow Chemical (Shanghai) Co., Ltd. PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol NOF Corporation Acrylate copolymer Evonik Operations GmbH Tromethamine Yalu Chemical Trading (Shanghai) Co., Ltd. 1,2-Hexanediol Dezhixin Flavors & Fragrances (Nantong) Co., Ltd. p-Hydroxyacetophenone Dezhixin Flavors & Fragrances (Nantong) Co., Ltd.

[0041] Example 1: Correlation analysis between Pseudomonas and skin physiological parameters

[0042] The skin physiological parameters of the female population aged 25-65 years were subjected to Spearman correlation analysis with skin Pseudomonas spp. The Spearman correlation coefficient (r) and significance (p) results are shown in Table 2.

[0043] Table 2. Correlation coefficients (r) and significance (p) between skin-derived Pseudomonas and physiological parameters

[0044]

[0045]

[0046] Note: *p<0.05, **p<0.01, ***p<0.001, r(-) indicates negative correlation.

[0047] From Table 2, it can be seen that there is an association between Pseudomonas and skin physiological parameters, among which it is positively correlated with skin water content, gloss, firmness (F4) and wrinkles, and negatively correlated with transepidermal water loss, sebum content, melanin and hemoglobin. In particular, the abundance of Pseudomonas is significantly positively correlated with skin firmness (F4) and wrinkles.

[0048] Example 2: Isolation and identification of Pseudomonas oryzae P.0719

[0049] Select an area of ​​approximately 3cm×3cm on the cheek skin on both sides of the face of a healthy adult, moisten a sterile cotton swab in the wetting solution for 15-30s, rub it back and forth 50 times in the selected area, place the sterile cotton swab in the sampling tube with sterile tweezers, cut off the sterile cotton swab head with the sample with sterile scissors in the clean bench, place it in a centrifuge tube (EP tube) containing 5mL of TSB liquid culture medium (containing 300μg / mL vancomycin and 5μg / mL amphotericin B), and enrich and culture it at 37℃ and 180r / min for 24h. Take 0.5 mL of the above culture medium and add 4.5 mL of sterile water to perform concentration gradient dilution (10-fold gradient dilution), select the appropriate dilution, aspirate 0.1 mL of the dilution and spread it on R2A blood agar plates (containing 300 μg / mL vancomycin and 5 μg / mL amphotericin B), spread 2 plates for each dilution, and place them in a 32°C incubator for aerobic culture for 24 h.

[0050] According to the colony characteristics of the strain on the R2A blood plate, single colonies were picked and streaked on new R2A blood plates, purified and cultured at 32℃ for 24h, and the colony morphology of the strain on the plate was observed. After multiple streaking purifications, the colony morphology of the selected strain P.0719 on the R2A blood plate was as follows: Figure 1As shown in the figure, the colonies of the screened strain P.0719 are yellow, round, convex, dry and wrinkled, with neat edges and no hemolytic zone.

[0051] Pick a small amount of purified bacteria from the R2A blood plate and smear it on a slide, perform Gram staining, and observe the morphological characteristics of the bacteria under an optical microscope. The results are as follows: Figure 2 As shown. Under an optical microscope, the bacteria were observed to be short round rods with a size of 0.5-1.0μm×1.5-5.0μm, and were dispersed.

[0052] Pick a single colony from the purified R2A blood plate and inoculate it into TSB liquid culture medium. After culturing at 37°C for 18 hours, centrifuge the bacterial solution (5000g, 10min) to collect the bacteria. Use a genomic DNA extraction kit to extract the strain DNA, and use two synthetic universal primers (16s 27F: 5'-GAGAGTTTGATCCTGGCTCAG-3'; 16s 1492R: 5'-CGGCTACCTTGTTACGACTT-3') for PCR amplification. The PCR amplification product is recovered using a gel recovery kit (BioFlux), purified and sequenced, and the sequencing results are compared by BLAST in the NCBI database to determine the species of strain P.0719. A phylogenetic tree was constructed based on the sequence alignment results ( Figure 3 ), and the final screening strain P.0719 was identified as Pseudomonas oryzihabitans.

[0053] The above-mentioned Pseudomonas oryzihabitans P.0719 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 18, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29673.

[0054] Example 3: Characteristics of Pseudomonas oryzae P.0719

[0055] 1. Strain growth curve and pH change of bacterial solution

[0056] Pick a single colony of Pseudomonas oryzae P.0719, inoculate it into TSB liquid medium, culture it at 32°C, and measure the OD of the bacterial solution every 2 hours. 600 , measure the pH of the bacterial solution every four hours, and the results are as follows Figure 4 and Figure 5The results showed that Pseudomonas oryzae P.0719 was cultured in TSB liquid medium, and the strain grew rapidly, entering the logarithmic phase at 4 hours and the stable phase at 18 hours; after the late logarithmic phase (after 16 hours), the pH value of the bacterial solution showed an upward trend, and the pH reached 7.6 after 24 hours of cultivation, and the bacterial solution was alkaline.

[0057] 2. Detection of enzyme production by strains

[0058] Sterilize the prepared gelatin medium at 121℃ for 15min, cool to an appropriate temperature, pour into a plate, and place overnight for use. Pick a single colony of Pseudomonas oryzae P.0719, streak it onto the prepared gelatin medium, and incubate it inverted in a 37℃ incubator overnight. Then take out the plate and evenly apply 35% trichloroacetic acid solution. The results are as follows: Figure 6 It can be seen that there is a transparent ring around the colony of Pseudomonas oryzae P.0719. The reason is that protease can degrade gelatin, and 35% trichloroacetic acid solution can denature gelatin. Therefore, when gelatin is degraded by protease, the culture medium around the colony will form a transparent ring visible to the naked eye under the action of trichloroacetic acid. Therefore, it is preliminarily judged that Pseudomonas oryzae P.0719 can produce protease.

[0059] A single colony of Pseudomonas oryzae P.0719 was selected and inoculated into TSB liquid medium. The culture was shaken for 16-18 h (37 ° C, 180 r / min) to obtain seed solution. The seed solution was diluted with TSB liquid medium to 1×10 7 CFU / mL bacterial concentration, set aside. Inoculate the seed liquid in the sterilized TSB liquid medium (1400mL), the inoculation amount is 2% (v / v), 180r / min, 37℃ culture for 18-20h, and obtain the fermentation liquid. Centrifuge the fermentation liquid (8000-10000r / min, 4℃, 10min) to obtain the fermentation supernatant, which is the crude enzyme liquid. Slowly add ammonium sulfate to the beaker containing the crude enzyme liquid until the saturation is 20%, observe whether there is precipitation, stand at 4℃ overnight, centrifuge to take the supernatant, and then continue to slowly add ammonium sulfate to the supernatant until the saturation is 80%, stand at 4℃ overnight, centrifuge to take the precipitate, and then re-dissolve the precipitate with a Tris-HCl buffer solution with a concentration of 50mM. Put the re-dissolved supernatant into a dialysis bag and fully dialyze it in a 4℃ refrigerator and freeze-dry it to obtain a crude enzyme, about 0.5g.

[0060] 3. Activity detection of strain fermentation supernatant

[0061] A single colony of Pseudomonas oryzae P.0719 was selected and inoculated into TSB liquid medium. The culture was shaken for 16-18 h (37 ° C, 180 r / min) to obtain seed solution. The seed solution was diluted with TSB liquid medium to 1×10 7CFU / mL bacterial concentration, set aside. Inoculate the seed liquid into the sterilized TSB liquid medium (1400mL), the inoculation amount is 2% (v / v), and culture at 180r / min and 37°C for 18-24h to obtain the fermentation liquid. Centrifuge at 8000-10000r / min and filter through a 0.22μm filter to remove the bacteria, and obtain the fermentation supernatant of Pseudomonas oryzae P.0719.

[0062] (1) Degradation of collagen

[0063] The reaction solution for each lane was prepared as shown in Table 3, and the reaction was carried out in a shaking water bath at 37°C for 24 h. Then, SDS-PAGE protein loading buffer (5×) was added, and the reaction was carried out in a metal bath at 100°C for 10 min. 4-20% SDS-PAGE gel (purchased from Changzhou Boyi Biotechnology Co., Ltd., catalog number: ET12420Gel) was used to run the gel at 160 V for 45 min, and the gel was washed twice with buffer and stained overnight in Coomassie Brilliant Blue dye. After elution for 2 h, the gel was photographed and the protein content change was calculated using BLT BioAnaly analysis software.

[0064] Table 3. SDS-PAGE gel electrophoresis lane reaction solution composition

[0065]

[0066] The results are as follows Figure 7 , Figure 8 As shown, compared with the reaction without Pseudomonas oryzae P.0719 fermentation supernatant (lane 5), the use of different contents of Pseudomonas oryzae P.0719 fermentation supernatant will reduce the content of type III collagen, and the higher the content of Pseudomonas oryzae P.0719 fermentation supernatant, the greater the degradation effect on type III collagen. The degradation of type III collagen by the Pseudomonas oryzae P.0719 fermentation supernatant in lane 4 reached 36.2%.

[0067] (2) Inducing the expression of inflammatory factors

[0068] HaCaT cells (purchased from Hunan Fenghui Biotechnology Co., Ltd.) were cultured at 1×10 4Cells were inoculated into 6-well plates at a density of 100 cells / well and incubated in a 37°C, 5% CO2 incubator for 24 h. When the cell plating rate in the 6-well plate reached about 50%, new culture medium was replaced, and a blank control group (BC), a positive control group (PC) and a sample group were set up. The blank control group used high-glucose DMEM culture medium, the positive control group used high-glucose DMEM culture medium containing 80 μM dexamethasone, and the sample group used high-glucose DMEM culture medium containing 0.625% and 2.5% fermentation supernatant of Pseudomonas oryzae P.0719 and 0.625% and 2.5% TSB culture medium, respectively. The plates were placed in a 37°C, 5% CO2 incubator and cultured for 24 h. After the culture was completed, the culture supernatant was collected, and the expression levels were detected by ELISA kits for IL-1, IL-6 and TNF-α.

[0069] The results are as follows Fig. 9 As shown, compared with the blank control group, the fermentation supernatant of Pseudomonas oryzae P.0719 can upregulate the expression of inflammatory factors IL-1, IL-6 and TNF-α. When the content of fermentation supernatant was 2.5%, the promotion rates reached 22.1%, 18.4% and 13.9%, respectively, leading to the occurrence of inflammation.

[0070] (3) Effects on the production of type III collagen by fibroblasts

[0071] HSF cells (purchased from Guangdong Boxi Biotechnology Co., Ltd.) were cultured at 1×10 4 Cells were inoculated into 6-well plates at a density of 100 cells / well and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cell plating rate in the 6-well plate reached about 50%, new culture medium was replaced, and a blank control group (BC), a positive control group (PC) and a sample group were set up. The blank control group used low-glucose DMEM culture medium, the positive control group used low-glucose DMEM culture medium containing TGF-β1 (100 ng / mL), and the sample group used low-glucose DMEM culture medium containing 1% and 5% fermentation supernatant of Pseudomonas oryzae P.0719 and 1% and 5% TSB culture medium, respectively. The plates were placed in a 37°C, 5% CO2 incubator and cultured for 24 hours. After the culture was completed, the culture supernatant was collected, and the expression level was detected by Human COL3 (Collagen TypeШ) ELISA Kit.

[0072] The results are as follows Fig.10 As shown in the figure, compared with the BC group, the expression of type III collagen was upregulated to a certain extent after HSF cells were treated with TSB medium, but the expression of type III collagen was significantly downregulated in the sample group with the addition of the fermentation supernatant of Pseudomonas oryzae P.0719, and the inhibition rate reached 27.94% when the fermentation supernatant content was 5%. This shows that the fermentation supernatant of Pseudomonas oryzae P.0719 can inhibit the expression of type III collagen.

[0073] (4) Producing ROS

[0074] HSF cells were cultured at 1×10 5 Cells were inoculated into 6-well plates at a density of cells / well and incubated in a 37°C, 5% CO2 incubator for 24 h. When the cell plating rate in the 6-well plate reached about 80%, new culture medium was replaced, and a blank control group (BC), a positive control group (PC) and a sample group were set up. Among them, the blank control group used low-glucose DMEM culture medium, the positive control group used low-glucose DMEM culture medium containing VE (7 μg / mL), and the sample group used low-glucose DMEM culture medium containing 1% and 5% fermentation supernatant of Pseudomonas oryzae P.0719 and 1% and 5% TSB culture medium, respectively. The plates were placed in a 37°C, 5% CO2 incubator and cultured for 24 h. After the culture was completed, the cells were stained, and the changes in ROS were detected using a reactive oxygen species detection kit (Biyuntian, S0033M).

[0075] The results are as follows Fig.11 and Fig.12 As shown, compared with the BC group, treatment of HSF cells with the fermentation supernatant of Pseudomonas oryzae P.0719 significantly stimulated and upregulated the expression of ROS in HSF cells, with a promotion rate of 98%, indicating that the fermentation supernatant of Pseudomonas oryzae P.0719 would stimulate skin cells to produce more oxidative by-products and accelerate oxidative aging.

[0076] Example 4: Effects of different ingredients on the growth of Pseudomonas oryzae P.0719

[0077] A single colony of Pseudomonas oryzae P.0719 was selected and inoculated into TSB liquid medium. The culture was shaken for 16-18 h (37 ° C, 180 r / min) to obtain seed solution. The seed solution was diluted with TSB liquid medium to 1×10 7 The bacterial concentration of CFU / mL is set aside.

[0078] Take different types of raw materials, dilute each raw material with TSB liquid culture medium, and obtain four or five concentrations of samples to be tested for each raw material. The sample concentrations are detailed in Table 4. Set up the sample group (100 μL seed solution + 100 μL series concentration raw material solution), control group 1 (100 μL series concentration raw material solution + 100 μL TSB culture medium), control group 2 (100 μL seed solution + 100 μL TSB culture medium), and blank group (200 μL TSB culture medium) in a 96-well plate. After the addition of samples, place the 96-well plate in a 37°C constant temperature incubator for 16-18 hours, use an enzyme marker to measure the OD value at 600 nm, and calculate the inhibition rate:

[0079] Inhibition rate (%) = [1-(OD 样品组-OD 对照组1 ) / (OD 对照组2 -OD 空白组 )]×100%

[0080] The calculated inhibition rates of different types of raw materials on Pseudomonas oryzans P.0719 are shown in Table 4. The inhibition ability of different types of raw materials on Pseudomonas oryzans P.0719 increases with the increase of raw material concentration. Among them, nitrogen-containing components such as 9-β-D-ribofuranosyladenine and 17kDa collagen have the effect of promoting the proliferation of Pseudomonas oryzans P.0719 at low concentrations. Only when the addition amount reaches a certain concentration, the inhibition ability on Pseudomonas oryzans P.0719 begins to gradually increase, especially 17kDa collagen. The original protein can have a certain inhibitory effect on it only when the addition amount is above 0.01%; when the addition amount of dipropylene glycol and 1,3-butylene glycol is above 2%, the inhibition rate is greater than 50%, among which 1,3-butylene glycol has an inhibition rate of 84.18% when it is 4%; PEG / PPG-17 / 6 copolymer and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol are water-soluble oils. When the addition amount is higher than 0.1%, their inhibitory ability to Pseudomonas oryzae P.0719 gradually increases, and at 2%, the inhibition rate reaches more than 50%.

[0081] Table 4. Inhibition rate of different types of raw materials on Pseudomonas oryzae P719

[0082]

[0083]

[0084] Example 5: Effects of different ingredients on the growth of Propionibacterium acnes

[0085] Preparation of Propionibacterium acnes seed solution: inoculate the powder of skin bacteria Propionibacterium acnes CCSM0331 (existing strain, deposit number is CCTCC NO: M2022781) on a TSA medium plate with a disposable sterile inoculation loop, place it in a 37°C constant temperature incubator for aerobic culture for 16-20 hours, pick a single colony and streak it on a second-generation TSA medium plate, place it in a 37°C constant temperature incubator for aerobic culture for 16-20 hours, pick a purified single colony from the second-generation plate and inoculate it into TSB liquid culture medium, place it in a 37°C shaker for shaking culture for 16-20 hours, and wait until OD 600 When the value is between 0.5-1.0, the seed liquid of the fungus is obtained and is ready for use.

[0086] Take the raw materials of probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, dilute each raw material with TSB liquid medium, and obtain five concentrations of samples to be tested for each raw material. The sample concentrations are detailed in Table 5. Set up a sample group (100μL seed solution + 100μL series concentration raw material solution) and a blank control group (100μL seed solution + 100μL TSB medium) in a 96-well plate. After the sample is added, place the 96-well plate in a 37°C constant temperature incubator for 16-20h, use an ELISA reader to measure the OD value at 600nm, and calculate the relative proliferation rate. The calculation formula is as follows:

[0087] Relative proliferation rate (%) = (OD 样品组 -OD 空白对照组 ) / OD 空白对照组 ×100%.

[0088] The calculated relative proliferation rates of different types of raw materials on Propionibacterium acnes CCSM0331 are shown in Table 5. Raw materials of different concentrations have a certain effect on promoting the proliferation of Propionibacterium acnes, and the promoting effect increases with the increase of concentration; among them, 17kDa collagen has a certain promoting effect at a lower concentration, and the proliferation rate reaches 48.38% at an addition amount of 0.05%; dipropylene glycol in polyols and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol in water-soluble oils can also promote the proliferation of Propionibacterium acnes to a certain extent; the proliferation rate of probiotic fermented brown algae extract on Propionibacterium acnes reaches 48.95% at 4%.

[0089] The above four raw materials inhibited Pseudomonas oryzae P.0719 at a certain concentration and promoted the growth of Propionibacterium acnes.

[0090] Table 5 Effects of different types of raw materials on the growth of Propionibacterium acnes

[0091]

[0092] Example 6: Effects of Screened Ingredients on Human Facial Skin Flora

[0093] Probiotic fermented brown algae extract, 17 kDa collagen, dipropylene glycol and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol were used as skin care ingredients to prepare essence. The formula of the essence is shown in Table 6.

[0094] The method for preparing the essence is as follows:

[0095] Take 60g of purified water and 4.0g of dipropylene glycol, mix them evenly and heat them to 80°C, add 0.25g of acrylate copolymer under stirring at 300r / min, continue stirring until completely dissolved, add 0.18g of tromethamine and stir to dissolve evenly; cool the above material to 60-65°C, add 0.5g of p-hydroxyacetophenone and 0.5g of 1,2-hexanediol, stir to dissolve evenly; after the material body continues to cool to 40°C, add 2.0g of probiotic fermented brown algae extract, 0.010g of 17kDa collagen and 2.0g of PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, continue stirring until dissolved and mixed evenly, add purified water to 100g to obtain the essence.

[0096] Table 6. Essence formula

[0097]

[0098]

[0099] Volunteers aged 28-35 were recruited as test subjects. The volunteers were asked to use the essence for 28 consecutive days. The effects of the ingredients in the essence on the skin flora were evaluated by measuring the changes in the volunteers' skin flora before and after use. The facial flora of the volunteers was collected before and after use, and the structure and abundance of the skin flora were analyzed. Fig.13 and Fig.14 As shown, it was found that after using the essence containing probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, the abundance of Pseudomonas in the volunteers was significantly reduced, the abundance of Cutibacterium increased, and the structure of the skin flora was regulated.

Claims

1. A strain of Pseudomonas oryzihabitans P.0719 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 18, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29673.

2. Use of the Pseudomonas oryzae P.0719 described in claim 1 as a marker in screening raw materials for cosmetics or skin care products.

3. The use according to claim 2, characterized in that The raw materials of cosmetics or skin care products can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

4. The use according to claim 3, characterized in that The raw materials of the cosmetics or skin care products include: probiotic fermented brown algae extract, 9-β-D-ribofuranosyl adenine, 17kDa collagen, lactobacillus fermentation filtrate, dipropylene glycol, 1,3-butylene glycol, PEG / PPG-17 / 6 copolymer or PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

5. The use according to claim 3, characterized in that The cosmetic or skin care product raw materials can also promote the proliferation of Propionibacterium acnes and improve the flora structure of aging skin.

6. The use according to claim 5, characterized in that The raw materials of the cosmetics or skin care products include: probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol or PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

7. Use of the raw materials screened by the application according to claim 2 in the preparation of cosmetics or skin care products.

8. The use according to claim 7, characterized in that The cosmetics or skin care products can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

9. A cosmetic or skin care product containing a probiotic fermented brown algae extract, 17kDa collagen, dipropylene glycol and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, wherein the cosmetic or skin care product can inhibit the growth of Pseudomonas oryzae P.0719, reduce the abundance of Pseudomonas on the human face, and thus reduce skin aging caused by its metabolites.

10. The cosmetic or skin care product according to claim 9, characterized in that: The cosmetics or skin care products can also promote the proliferation of Propionibacterium acnes and improve the flora structure of aging skin.

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