Portuguese corynespora yeast, fermentation filtrate, preparation method and application thereof

Abstract

The embodiments of the present disclosure disclose Portuguese coryneformis, fermentation filtrate, preparation method and application thereof. The yeast is Portuguese coryneformis P8, which is classified as Clavispora Portuguese P8, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.31344. It has the advantages of fast growth rate, short fermentation cycle, and can enter the fermentation stable period in a short time, as well as the advantages of anti-oxidation, anti-aging, skin repair and anti-damage.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the field of biotechnology, and in particular to Portuguese yeast, fermentation filtrate, and a preparation method and application thereof. Background Art

[0002] Yeast, also known as yeast, is a general term for a group of single-cell fungi. Yeast is rich in natural ingredients such as active sugars, peptides, nucleotides, amino acids, vitamins, etc., so it is widely used in alcohol brewing, bread baking, biomedicine, health foods and cosmetics. At present, the commonly used yeast types include: Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Rhodotorula glutinosus, Candida anonymum, etc.

[0003] However, there are the following technical problems:

[0004] Since there are fewer types of yeast that can be used in cosmetics and the yeast fermentation time is long, the application of yeast in cosmetics is affected.

[0005] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Summary of the invention

[0006] The content of this disclosure is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this disclosure is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0007] Some embodiments of the present disclosure provide Portuguese corynespora yeast, fermentation filtrate, and preparation method and application thereof to solve one or more of the technical problems mentioned in the above background technology section.

[0008] In the first aspect, some embodiments of the present disclosure provide a yeast having antioxidant, anti-aging and skin repair capabilities, wherein the yeast is Portuguese coryneformis P8, which is classified and named Clavispora lusitaniae P8, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.31344.

[0009] In a second aspect, some embodiments of the present disclosure provide a method for preparing a fermentation filtrate, comprising: under sterile conditions, inoculating activated seeds of yeast at an inoculation rate of 10% into a YPD liquid culture medium, wherein the yeast is the Portuguese Corynosporum P8 mentioned above in the first aspect; after the inoculation is completed, culturing the activated seeds inoculated into the YPD liquid culture medium at a temperature of 30°C and a rotation speed of 220 rpm for 8 hours; after the culturing is completed, centrifuging the collected bacterial liquid to obtain a supernatant; filtering the supernatant with a filter membrane to obtain a fermentation filtrate.

[0010] In a third aspect, some embodiments of the present disclosure provide a fermentation filtrate prepared by the fermentation filtrate preparation method as described above in the second aspect.

[0011] In a fourth aspect, some embodiments of the present disclosure provide a use of the fermentation filtrate as described above in the third aspect in the preparation of cosmetics with antioxidant uses, wherein the antioxidant use includes any of the following effects: ABTS free radical scavenging; DPPH free radical scavenging; ROS free radical scavenging.

[0012] In a fifth aspect, some embodiments of the present disclosure provide a use of a fermentation filtrate according to the third aspect in the preparation of a cosmetic having anti-inflammatory uses, wherein the anti-inflammatory uses include the inhibition of the inflammatory factor NO.

[0013] In a sixth aspect, some embodiments of the present disclosure provide a use of the fermentation filtrate as described above in the third aspect in preparing cosmetics with anti-aging uses.

[0014] Optionally, the above anti-aging use includes promoting the synthesis of type I collagen.

[0015] In the seventh aspect, some embodiments of the present disclosure provide a use of the fermentation filtrate as described above in the third aspect in the preparation of cosmetics for skin repair purposes, wherein the above-mentioned skin repair purposes include any of the following effects: cell repair of cells damaged by UVA; cell repair of cells damaged by UVB; cell repair of cells damaged by scratches.

[0016] In an eighth aspect, some embodiments of the present disclosure provide a use of the fermentation filtrate as described above in the third aspect in the preparation of cosmetics for skin repair purposes, wherein the above-mentioned anti-photodamage includes any of the following effects: anti-UVA damage; anti-UVB damage.

[0017] The above-mentioned embodiments of the present disclosure have the following beneficial effects: First, the yeasts with antioxidant, anti-aging and skin repairing abilities of some embodiments of the present disclosure have the advantages of fast growth rate, short fermentation cycle, and can enter the fermentation stable period in a short time. In addition, the yeasts of the present disclosure also have the advantages of antioxidant, anti-aging, skin repair and anti-damage, so that they can be used as effective ingredients to enrich the development of cosmetics containing yeasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and the elements are not necessarily drawn to scale.

[0019] Figure 1 is a schematic diagram of the strain morphology of the strains screened out in some embodiments of the present disclosure;

[0020] Figure 2 is a schematic diagram of a growth curve and a pH value change curve of the Portuguese corynespora yeast P8 in some embodiments of the present disclosure;

[0021] Figure 3 is a comparison chart of DPPH inhibition rates of the Portuguese coryneformis P8 and the model bacterium Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0022] Figure 4 is a comparison chart of DPPH inhibition rates of Corynespora spp. CICC 33497 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0023] Figure 5 is a comparison chart of DPPH inhibition rates of Corynespora spp. CICC 1461 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0024] Figure 6 is a comparison chart of the ABTS inhibition rates of the Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0025] Figure 7 is a comparison chart of the ABTS inhibition rates of the Portuguese corynespora CICC 33497 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0026] Figure 8 is a comparison chart of the ABTS inhibition rates of the Portuguese corynespora CICC 1461 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0027] Fig. 9 is a comparison chart of the ROS scavenging ability of the Portuguese coryneformis P8 and the model bacterium Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0028] Fig.10 is a comparison chart of the ROS scavenging ability of the Portuguese corynespora CICC 33497 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0029] Fig.11 is a comparison chart of the ROS scavenging ability of the Portuguese corynespora CICC 1461 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0030] Fig.12 is a comparison chart of NO inhibition ability of Corynespora spp. P8 of some embodiments of the present disclosure and the model bacteria Corynespora spp. CICC33276 of the present disclosure;

[0031] Fig.13 is a comparison chart of NO inhibition ability of Corynespora spp. CICC 33497 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0032] Fig.14 is a comparison chart of NO inhibition ability of Corynespora spp. CICC 1461 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0033] Fig.15 is a graph showing the β-galactosidase staining results of the Portuguese coryneformis P8 and the model bacterium Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0034] Fig.16 is a β-galactosidase staining result diagram of the Portuguese corynespora CICC 33497 and the model bacteria Portuguese corynespora CICC 33276 of some embodiments of the present disclosure;

[0035] Fig.17 is a β-galactosidase staining result diagram of the Portuguese coryneformis CICC 1461 and the model bacteria Portuguese coryneformis CICC 33276 according to some embodiments of the present disclosure;

[0036] Fig.18 is a graph comparing the expression of type I collagen between the Portuguese coryneformis P8 and the model bacterium Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0037] Fig.19is a graph comparing the expression of type I collagen between the Portuguese corynespora CICC 33497 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0038] Fig. 20 is a comparison chart of the expression of type I collagen between the Portuguese corynespora CICC 1461 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0039] Fig.21 is a comparison chart of the cell viability of the Portuguese coryneformis P8 and the model bacterium Portuguese coryneformis CICC33276 according to some embodiments of the present disclosure;

[0040] Fig. 22 is a comparison chart of cell viability of Corynespora spp. CICC 33497 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0041] Fig.23 is a comparison chart of cell viability of Corynespora spp. CICC 1461 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0042] Fig.24 is another comparison chart of cell viability of the Portuguese corynespora P8 and the model bacterium Portuguese corynespora CICC33276 of some embodiments of the present disclosure;

[0043] Fig.25 is another comparison chart of cell viability of Corynespora spp. CICC 33497 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0044] Fig.26 is another comparison chart of cell viability of Corynespora spp. CICC 1461 and the model bacteria Corynespora spp. CICC 33276 of some embodiments of the present disclosure;

[0045] Fig. 27 is another comparison chart of cell viability of the Portuguese corynespora P8 and the model bacterium Portuguese corynespora CICC33276 of some embodiments of the present disclosure;

[0046] Fig.28 is another comparison chart of cell viability between the Portuguese corynespora CICC 33497 and the model bacteria Portuguese corynespora CICC 33276 of some embodiments of the present disclosure;

[0047] Fig.29is another comparison chart of cell viability between the Portuguese corynespora CICC 1461 and the model bacteria Portuguese corynespora CICC 33276 of some embodiments of the present disclosure;

[0048] Fig.30 is another comparison chart of cell viability of the Portuguese corynespora P8 and the model bacterium Portuguese corynespora CICC33276 of some embodiments of the present disclosure;

[0049] Fig.31 is another comparison chart of cell viability between the Portuguese corynespora CICC 33497 and the model bacterium Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0050] Fig.32 is another comparison chart of cell viability between the Portuguese corynespora CICC 1461 and the model bacteria Portuguese corynespora CICC 33276 according to some embodiments of the present disclosure;

[0051] Fig.33 3 is a comparative chart of the repair ability of scratch damaged cells of Corynosporum portugalense P8, Corynosporum portugalense CICC 33497, Corynosporum portugalense CICC 1461 and the model bacteria Corynosporum portugalense CICC 33276 according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0052] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0053] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0054] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0055] It should be noted that the term “about” used in the present disclosure, when used to define a numerical value, means ±5% of the specified numerical value.

[0056] It should be noted that the term “%” mentioned in the present disclosure represents mass % unless otherwise specified.

[0057] It should be noted that the "normal temperature" mentioned in the present disclosure refers to the ambient temperature, that is, no special heating or cooling treatment has been performed.

[0058] Explanation of terms

[0059] The term "Corynosporum portugalense P8" is classified as Clavispora lusitaniae P8 is deposited in the General Microbiology Center of China Culture Collection of Microorganisms, with the deposit number CGMCC NO.31344. The deposit date is July 18, 2024. The address of the General Microbiology Center of China Culture Collection of Microorganisms is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0060] The Chinese name of the term "Portuguese Corysporum CICC 33497" is Portuguese Corysporum, and the Latin name is Clavispora lusitaniae The strain number is CICC 33497 and was purchased from China Industrial Microbiological Culture Collection Center.

[0061] The Chinese name of the term "Portuguese Corysospora Saccharomyces CICC 1461" is Portuguese Corysospora Saccharomyces, and the Latin name is Clavispora lusitaniae The strain number is CICC 1461 and was purchased from China Industrial Microbiology Culture Collection Center.

[0062] The Chinese name of the term "Portuguese Corysporea Saccharomyces cerevisiae CICC 33276" is Portuguese Corysporea Saccharomyces cerevisiae, and the Latin name is Clavispora lusitaniae The strain number is CICC 33276 and was purchased from China Industrial Microbiology Culture Collection Center.

[0063] The term "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine radical, which is an organic compound.

[0064] Strain screening method description

[0065] Among them, Portuguese coryneformis P8, Portuguese coryneformis CICC 33497, and Portuguese coryneformis CICC1461 were screened by the following steps:

[0066] The first step is to mash the grapes, dissolve them in an appropriate amount of water, and mix them thoroughly.

[0067] In the second step, 0.1 mL of the sample was pipetted into 0.9 mL of sterile saline to obtain 10 -1 diluent.

[0068] Step 3: Pipette 0.1 mL of 10 -1 Dilute in 0.9 mL sterile saline to obtain 10 -2 diluent.

[0069] Step 4: Pipette 0.1 mL of 10 -2 Dilute in 0.9 mL sterile saline to obtain 10 -3 diluent.

[0070] Step 5: Pipette 0.1 mL of 10 -3 Dilute in 0.9 mL sterile saline to obtain 10 -4 diluent.

[0071] Step 6: Pipette 0.1 mL of 10 -4 Dilute in 0.9 mL sterile saline to obtain 10 -5 diluent.

[0072] Step 7: Pipette 0.1 mL of 10 -5 Dilute in 0.9 mL sterile saline to obtain 10 -6 diluent.

[0073] Step 8: Pipette 0.1 mL10 -6 The diluted solution was spread evenly on YPD solid medium with a spreading rod, and then inverted and cultured at a culture temperature of 37°C.

[0074] In the ninth step, after 24 hours, the strains were preliminarily identified by colony morphology and microscopic examination, and the strains meeting the characteristics were selected for streaking purification and culture.

[0075] Among them, the schematic diagram of the strain morphology of the screened strains is as follows Figure 1 As shown in the figure, the selected strains are round, milky white and opaque, with a diameter of about 1 nm to 2 nm, smooth edges, moist, protruding and shiny surfaces. Microscopic examination of the bacteria showed an oval shape, and the individual cells were larger than bacteria.

[0076] Step 10: Select a single colony from the last streaking purification plate and inoculate it into the corresponding 10 mL liquid culture medium and culture it for 24 hours.

[0077] Wherein, the liquid culture medium is YPD liquid culture medium.

[0078] Step 11. After the culture is completed, take 1 mL to 2 mL of the bacterial solution and 40% sterile glycerol cultured for 24 hours into a culture storage tube and store it in a -80℃ refrigerator.

[0079] In the twelfth step, the bacterial solution cultured for 24 hours is centrifuged and the supernatant is discarded as the first bacterial solution.

[0080] The centrifugal speed was 6000 r / min and the centrifugal time was 2 min.

[0081] Step 13: Add 1.5 mL of sterile water to the first bacterial solution, mix thoroughly, and then centrifuge. Discard the supernatant to serve as the second bacterial solution.

[0082] The centrifugal speed was 6000 r / min and the centrifugal time was 3 min.

[0083] Step 14: Add 1.5 mL of sterile water to the second bacterial solution, mix thoroughly, and then centrifuge. Discard the supernatant to serve as the third bacterial solution.

[0084] The centrifugal speed was 6000 r / min and the centrifugal time was 3 min.

[0085] In the fifteenth step, the third bacterial solution was resuspended in 1.5 mL of sterile water and mixed thoroughly, and used as a template for bacterial identification for PCR (Polymerase chain reaction).

[0086] Step 16: sequence the PCR product to obtain the sequencing result.

[0087] Among them, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0088] In the seventeenth step, after splicing the sequencing results, homology analysis was performed in the NCBI (National Center for Biotechnology Information) database using BLAST (Basic Local Alignment Search Tool), and partial sequences with sequence identity ≥ 98% were selected for comparison.

[0089] Among them, according to the comparison results, the strain was determined to be Portuguese coryneformis P8. In addition, Portuguese coryneformis CICC33497 and Portuguese coryneformis CICC 1461 can also repeat the first step to the seventeenth step for repeated screening to determine the strain type, which will not be repeated here.

[0090] Fermentation filtrate preparation method description

[0091] Wherein, the preparation of fermentation filtrate comprises the following steps:

[0092] In the first step, under sterile conditions, the activated yeast seeds were inoculated into YPD liquid medium at a 10% inoculation rate.

[0093] Among them, the above yeast is Portuguese Corynespora P8.

[0094] In the second step, after the inoculation, the activated seeds inoculated into the YPD liquid culture were cultured at a temperature of 30°C and a rotation speed of 220 rpm for 8 hours.

[0095] The third step is to centrifuge the collected bacterial solution after the cultivation is completed to obtain the supernatant.

[0096] The fourth step is to filter the supernatant through a membrane to obtain a fermentation filtrate.

[0097] The supernatant was filtered through a 0.22 μm filter membrane to obtain a fermentation filtrate.

[0098] The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0099] Example 1

[0100] Among them, Example 1 is used to explore the growth characteristics of Portuguese yeast P8.

[0101] The specific steps include:

[0102] In the first step, under sterile conditions, activated seeds of Portuguese yeast P8 were inoculated into YPD liquid culture medium at an inoculation rate of 10%.

[0103] In the second step, after the inoculation, the activated seeds inoculated into the YPD liquid medium were cultured at a temperature of 30° C. and a rotation speed of 220 rpm for 8 hours.

[0104] In the activated seed culture, samples were taken every 2 hours to measure the OD 600 OD and pH 600 The value represents the absorbance value at a wavelength of 600 nm. Each sample was tested 3 times.

[0105] As an example, Figure 2 The growth curve and pH value change curve of Portuguese coryneformis P8 are shown in FIG. Figure 2 The horizontal axis is the culture time and the horizontal axis is OD 600 Value or pH value is the vertical axis, plotted to get, specific, analysis Figure 2 It can be found that the Portuguese coryneformis P8 grows rapidly from 0 to 6 hours, and enters the stable period after 6 hours, which shows that the Portuguese coryneformis P8 has the characteristics of a short fermentation cycle. In addition, the pH value drops to 5.5 from 0 to 6 hours, and then begins to rise again, and the pH value remains at around 6.8 after 16 hours.

[0106] Example 2

[0107] Among them, Example 2 is used to explore the antioxidant activity of Portuguese yeast P8.

[0108] (1) Experiment 1 (DPPH free radical scavenging experiment), specifically including the following steps:

[0109] In the first step, a proper amount of DPPH solid is pipetted and dissolved in methanol, and the solution is diluted with methanol until the absorbance at 517 nm is 1.6.

[0110] In the second step, the reagents were added to the solution, mixed evenly, and reacted in the dark for 30 min, and the absorbance at 517 nm was detected by an ELISA reader.

[0111] Among them, set sample group A s , Blank Group A sb , Sample Group A c , Blank Group A cb . Specifically, the amount of reagent added is shown in Table 1 below:

[0112] .

[0113] Among them, DPPH inhibition rate (%) = [1 - (A s -A sb ) / (A c -A cb )].

[0114] Optionally, the DPPH inhibition rates of Corynosporum portugalense CICC 33497, Corynosporum portugalense CICC 1461, and Corynosporum portugalense CICC 33276 at different sample concentrations (50%, 100%) can also be determined through the first and second steps.

[0115] Specifically, the Portuguese corynespora CICC 33276 was used as the model bacteria, see Figure 3 The DPPH inhibition rate comparison chart of Portuguese corynosporum P8 and the model bacteria Portuguese corynosporum CICC 33276 is shown. Figure 4 The DPPH inhibition rate comparison chart of Portuguese corynosporum CICC 33497 and the model bacteria Portuguese corynosporum CICC 33276 is shown, and Figure 5The DPPH inhibition rate comparison chart of Portuguese corynospora yeast CICC 1461 and the model bacteria Portuguese corynospora yeast CICC 33276 shown in the figure shows that, first, at concentrations of 50% and 100%, the DPPH scavenging ability of Portuguese corynospora yeast P8 is better than that of the model bacteria Portuguese corynospora yeast CICC 33276, especially at a concentration of 100%, the DPPH scavenging ability of Portuguese corynospora yeast P8 is significantly better than that of the model bacteria Portuguese corynospora yeast CICC 33276. Secondly, at a concentration of 50%, the DPPH scavenging ability of Portuguese corynospora yeast CICC 33497 is better than that of the model bacteria Portuguese corynospora yeast CICC 33276. Then, at a concentration of 100%, the DPPH scavenging ability of Portuguese corynospora yeast CICC 33497 is close to that of the model bacteria Portuguese corynospora yeast CICC 33276. In addition, at concentrations of 50% and 100%, the DPPH scavenging ability of the Portuguese yeast CICC 1461 was close to that of the model bacteria Portuguese yeast CICC 33276.

[0116] (2) Experiment 2 (ABTS free radical detection experiment), specifically including the following steps:

[0117] In the first step, the concentrated ABTS solution was diluted until the absorbance at 734 nm was 0.9.

[0118] For example, 7 mM ABTS concentrated solution was prepared by dissolving 7.7 mg ABTS in 2 mL potassium persulfate solution for 16 hours. For example, 2.45 mM potassium persulfate solution was prepared by diluting 6.6 mg potassium persulfate solution to 10 mL.

[0119] In the second step, 50 μL of sample and 150 μL of ABTS solution were added to a 96-well plate, and the absorbance was measured at 734 nm after reacting for 15 min.

[0120] Among them, inhibition rate (%) = (1 - A sample / A control ) × 100%. Among them, A sample Characterize the sample set. A control Characterization of the control group.

[0121] Optionally, the ABTS inhibition rates of Corynosporum portugalense CICC 33497, Corynosporum portugalense CICC 1461, and Corynosporum portugalense CICC 33276 at different sample concentrations (50%, 100%) can also be determined through the first and second steps.

[0122] Specifically, the Portuguese corynespora CICC 33276 was used as the model bacteria, see Figure 6 The ABTS inhibition rate comparison chart of Portuguese corynosporum P8 and the model bacteria Portuguese corynosporum CICC 33276 is shown. Figure 7 The ABTS inhibition rate comparison chart of Portuguese corynosporum CICC 33497 and the model bacteria Portuguese corynosporum CICC 33276 is shown, and Figure 8 The ABTS inhibition rate comparison chart of Portuguese corynospora yeast CICC 1461 and the model bacteria Portuguese corynospora yeast CICC 33276 is shown in the figure. The analysis shows that, firstly, at concentrations of 12.5%, 25% and 50%, the ABTS antioxidant activity of Portuguese corynospora yeast P8 is better than that of the model bacteria Portuguese corynospora yeast CICC 33276. Among them, especially at a concentration of 50%, the ABTS antioxidant activity of Portuguese corynospora yeast P8 is significantly better than that of the model bacteria Portuguese corynospora yeast CICC 33276. Secondly, at concentrations of 12.5%, 25% and 50%, the ABTS antioxidant activity of Portuguese corynospora yeast CICC 33497 is close to the ABTS antioxidant activity of the model bacteria Portuguese corynospora yeast CICC 33276. Then, at a concentration of 12.5%, the ABTS antioxidant activity of C. portugalensis CICC 1461 approached that of the model bacteria C. portugalensis CICC 33276. In addition, at concentrations of 25% and 50%, the ABTS antioxidant activity of C. portugalensis CICC 1461 was superior to that of the model bacteria C. portugalensis CICC 33276.

[0123] (3) Experiment 3 (ROS scavenging experiment), specifically including the following steps:

[0124] In the first step, HFF-1 cells were cultured at 5 × 10 4 The cells were seeded into 6-well plates at a density of 100 / well and cultured in complete medium for 24 hours.

[0125] In the second step, after UVA cell injury of HFF-1 cells, the cells were washed with PBS buffer.

[0126] Among them, HFF-1 cells were set as the control group and the sample group, and the sample concentrations were 0.4%, 2% and 10%, respectively.

[0127] In the third step, 1 mL of DMEM was added to each well, and 1 μL of DCFH-DA fluorescent probe was added to make the final concentration of the fluorescent probe 10 μmol / L.

[0128] In the fourth step, after the HFF-1 cells were cultured in the incubator for 20 minutes, the culture medium was discarded and the cells were washed three times with DMEM to completely remove the fluorescent probe.

[0129] In the fifth step, 1 mL of PBS buffer was added and observed under a fluorescence microscope, and the fluorescence intensity was measured at 488 nm / 525 nm using an ELISA reader.

[0130] Specifically, the relative fluorescence value ratio is used to characterize the ROS scavenging ability, wherein the relative fluorescence value ratio is the ratio of the fluorescence value of the sample group to that of the model group. The lower the fluorescence value ratio, the stronger the ROS scavenging ability. Portuguese Corynosporum CICC 33276 is used as the model bacteria, see Fig. 9 The ROS scavenging ability comparison chart of Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC33276 is shown. Fig.10 The ROS scavenging ability comparison chart of Portuguese coryneformis CICC 33497 and the model bacteria Portuguese coryneformis CICC 33276, and Fig.11 The figure shows a comparison of the ROS scavenging abilities of the Portuguese yeast CICC 1461 and the model bacteria Portuguese yeast CICC 33276. Analysis shows that the ROS scavenging ability of Portuguese yeast P8 is significantly better than that of the model bacteria Portuguese yeast CICC 33276, Portuguese yeast CICC 33497 and Portuguese yeast CICC 1461.

[0131] Example 3

[0132] Among them, Example 3 is used to explore the anti-inflammatory ability of Portuguese yeast P8.

[0133] Experiment 1 (inflammatory factor NO inhibition experiment) specifically includes the following steps:

[0134] In the first step, mouse mononuclear macrophages were digested with trypsin and cultured at a rate of 2 × 10 4 Each well of the plate was inoculated with 2 mL of culture medium and incubated at 37°C in a 5% CO2 incubator for 18 hours.

[0135] Among them, mouse mononuclear macrophages (RAW264.7) were grown in complete medium (high-glucose DMEM medium containing 10% FBS and 1% double antibody).

[0136] In the second step, the culture medium was aspirated and the residual culture medium was washed away with PBS, and 2 mL of DMEM was added to the blank group, 1 μg / mL LPS prepared in DMEM was added to the LPS group, and 1 μg / mL LPS prepared in DMEM and a certain concentration of samples were added to the experimental group, and the cells were cultured together for 24 hours.

[0137] The third step was to take the upper culture medium and centrifuge it at 13,000 rpm for 5 min, then aspirate the supernatant.

[0138] In the fourth step, the NO kit was used to determine the effect of the samples on the levels of LPS-induced inflammatory cytokines.

[0139] Specifically, the Portuguese corynespora CICC 33276 was used as the model bacteria, see Fig.12 The NO inhibition ability comparison chart of Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 is shown. Fig.13 The NO inhibition ability comparison chart of Portuguese coryneformis CICC 33497 and the model bacteria Portuguese coryneformis CICC 33276 is shown, and Fig.14 The comparison chart of NO inhibition ability between Portuguese corynospora CICC 1461 and model bacteria Portuguese corynospora CICC 33276 shows that, firstly, at a concentration of 50%, the NO inhibition ability of Portuguese corynospora P8 is better than that of the model bacteria Portuguese corynospora CICC 33276, indicating that Portuguese corynospora P8 has a certain NO inhibition ability. In addition, when the concentration is equal to 50%, Portuguese corynospora CICC 33497 and Portuguese corynospora CICC 1461 also have a certain NO inhibition ability, but the NO inhibition ability is slightly weaker than that of the model bacteria Portuguese corynospora CICC 33276.

[0140] Example 4

[0141] Among them, Example 4 is used to explore the anti-aging ability of Portuguese yeast P8.

[0142] (1) Experiment 1 (using the β-galactosidase method to verify the anti-aging ability of Portuguese yeast P8), specifically including the following steps:

[0143] In the first step, HaCaT cells were seeded in a 6-well plate for culture, and the culture medium was removed when the cell aggregation degree reached 70%, and the HaCaT cells were washed with PBS buffer (once).

[0144] Second, add 1 mL of β-galactosidase staining fixative to each well and incubate at room temperature for at least 15 minutes to allow cells to fix.

[0145] In the third step, the β-galactosidase staining fixative was removed and the HaCaT cells were washed with PBS buffer (at least 3 times, 3 minutes each time).

[0146] Step 4. After washing, remove the PBS buffer and add 1 mL of staining working solution to each well and incubate overnight at 37°C.

[0147] Step 5: Observe cells using a standard optical microscope.

[0148] Among them, senescent cells appear blue-green.

[0149] Specifically, the smaller the area of ​​blue-green cells is, the stronger the anti-aging ability is. Using Portuguese Corynosporum CICC33276 as a model bacteria, see Fig.15 The β-galactosidase staining results of the Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 are shown in FIG. Fig.16 The β-galactosidase staining results of the Portuguese yeast CICC 33497 and the model bacteria Portuguese yeast CICC 33276 are shown, and Fig.17 The β-galactosidase staining results of the Portuguese coryneformis CICC 1461 and the model bacteria Portuguese coryneformis CICC 33276 are shown in the figure. The analysis found that, first, at concentrations of 0.4%, 2% and 10%, the Portuguese coryneformis CICC 1461 and the model bacteria Portuguese coryneformis CICC 33276 had similar anti-aging abilities. Secondly, at a concentration of 10%, the anti-aging ability of Portuguese coryneformis P8 was significantly better than that of the model bacteria Portuguese coryneformis CICC 33276.

[0150] (2) Experiment 2 (verifying the effect of Portuguese yeast P8 on the expression of type I collagen), specifically including the following steps:

[0151] In the first step, HFF-1 cells were stably passaged twice and then seeded into 96-well plates and incubated at 37°C overnight.

[0152] In the second step, the culture medium in the 96-well plate was discarded, and test samples with concentrations of 0.4%, 2%, and 10% and 100 ng / mL TGF-β1 (transforming growth factor) were selected as positive controls to determine the type I collagen content based on the results of the cell proliferation experiment.

[0153] Among them, the blank control group was the complete culture medium group.

[0154] In the third step, three parallel groups were set up in each group to carry out the drug administration operation. After the drug administration, the 96-well plate was placed in a CO2 incubator for incubation for 24±2 hours.

[0155] The fourth step is to detect the human type I collagen enzyme-linked immunosorbent assay kit after the incubation.

[0156] Specifically, the Portuguese corynespora CICC 33276 was used as the model bacteria, see Fig.18 The comparison of type I collagen expression between the Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 is shown in the figure. Fig.19 The comparison of type I collagen expression between the Portuguese coryneformis CICC 33497 and the model bacteria Portuguese coryneformis CICC 33276 is shown, and Fig. 20 The comparison of type I collagen expression between Portuguese coryspore yeast CICC 1461 and the model bacteria Portuguese coryspore yeast CICC 33276 is shown in the figure. The analysis shows that, firstly, Portuguese coryspore yeast CICC 33497 and Portuguese coryspore yeast CICC 1461 have a certain expression of type I collagen, but it is relatively low. Secondly, at concentrations of 0.4%, 2% and 10%, the expression of type I collagen of Portuguese coryspore yeast P8 is higher than that of the corresponding model bacteria Portuguese coryspore yeast CICC 33276. In addition, compared with the blank control group, the expression of type I collagen of Portuguese coryspore yeast P8 at a concentration of 2% is 2.98 ng / mL, which is significantly higher than the expression of type I collagen of the model bacteria Portuguese coryspore yeast CICC 33276 at the same concentration of 1.64 ng / mL, indicating that Portuguese coryspore yeast P8 has the effect of promoting type I collagen synthesis at a concentration of 2%.

[0157] Example 5

[0158] Example 5 is used to explore the skin repair and skin protection abilities of Portuguese yeast P8.

[0159] (1) Experimental Example 1 (verifying the repair and protection ability of Portuguese coryneformis P8 against UVA damage) specifically includes the following steps:

[0160] In the first step, HFF-1 cells were prepared at a concentration of 1×10 6 The HFF-1 cells were inoculated into 96-well plates at a volume of 100 μL per well and incubated in an incubator at 37°C and 5% CO2.

[0161] The experiment set up a blank control group and a negative control group. Specifically, the negative control group was prepared by diluting the fermentation supernatant with DMEM culture medium to a concentration of 0.8%, 4% and 20% of the sample solution to be tested, respectively.

[0162] In the second step, after 24 hours, the 96-well plate was taken out, the old culture medium was discarded, and PBS was used to wash twice. After washing, 100 μL PBS was added, and the corresponding wells of the blank control group were covered with tin foil and then placed under a UVA light source for irradiation.

[0163] The third step is to accumulate the irradiation energy to 200 mJ / cm 2 After that, the 96-well plate was returned to the incubator for 24 hours.

[0164] In the fourth step, 24 hours after administration, the 96-well plate was removed, the culture medium was removed, and the plate was washed three times with PBS. After washing, 150 μL of 0.5 mg / mL MTT solution was added to each well.

[0165] The MTT solution was prepared based on DMEM.

[0166] Step 5. After 4 h in the incubator, the solution was aspirated and 100 μL DMSO was added, and the absorbance was measured at 490 nm.

[0167] Specifically, the Portuguese coryneformis CICC 33276 was used as a model bacteria. First, from the perspective of cell repair, see Fig.21 The cell viability comparison chart of Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 is shown. Fig. 22 The cell viability comparison chart of Portuguese Corynosporum CICC 33497 and the model bacteria Portuguese Corynosporum CICC 33276 is shown, and Fig.23 The cell viability comparison chart of Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC 33276 is shown. The analysis found that, first, at a concentration of 0.4% to 4%, Portuguese Corynospora P8 had a stronger cell repair ability for HFF-1 cells damaged by UVA than the model bacteria Portuguese Corynospora CICC 33276. Secondly, at a concentration of 10%, Portuguese Corynospora CICC 33497 had a stronger cell repair ability for HFF-1 cells damaged by UVA than the model bacteria Portuguese Corynospora CICC 33276. Then, Portuguese Corynospora CICC 33497 and Portuguese Corynospora CICC 1461 also have certain cell repair capabilities. Second, from the perspective of cell protection, see Fig.24 Another cell viability comparison chart of the Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276, Fig.25 Another comparison of cell viability of the Portuguese yeast CICC 33497 and the model bacteria Portuguese yeast CICC 33276 is shown, and Fig.26Another cell viability comparison chart of Portuguese Corysospora Saccharomyces CICC 1461 and the model bacteria Portuguese Corysospora Saccharomyces CICC 33276 is shown. The analysis found that, first, at a concentration of 0.4% to 10%, Portuguese Corysospora Saccharomyces P8 has a stronger cell protection ability against UVA-damaged HFF-1 cells compared with the model bacteria Portuguese Corysospora Saccharomyces CICC 33276. In particular, at a concentration of 0.4%, Portuguese Corysospora Saccharomyces P8 has a significant cell protection ability against UVA-damaged HFF-1 cells. Secondly, Portuguese Corysospora Saccharomyces CICC 33497 and Portuguese Corysospora Saccharomyces CICC 1461 also have a certain cell protection ability.

[0168] (2) Experimental Example 2 (verifying the repair and protection capabilities of Portuguese coryneformis P8 against UVB damage) specifically includes the following steps:

[0169] In the first step, HaCaT cells were prepared at a concentration of 1×10 6 The cells were suspended in a 96-well plate at a volume of 200 μL per well and incubated in an incubator at 37°C and 5% CO2.

[0170] The experiment set up a blank control group and a negative control group. Specifically, the negative control group was prepared by diluting the fermentation supernatant with DMEM culture medium to a concentration of 0.8%, 4% and 20% of the sample solution to be tested, respectively.

[0171] In the second step, after 24 hours, the 96-well plate was taken out, the old culture medium was discarded, and PBS was used to wash twice. After washing, 100 μL PBS was added, and the corresponding wells of the blank control group were covered with tin foil and then placed under a UVB light source for irradiation.

[0172] The third step is to accumulate the irradiation energy to 200 mJ / cm 2 After that, the 96-well plate was returned to the incubator for 24 hours.

[0173] In the fourth step, 24 hours after administration, the 96-well plate was removed, the culture medium was removed, and the plate was washed three times with PBS. After washing, 150 μL of 0.5 mg / mL MTT solution was added to each well.

[0174] The MTT solution was prepared based on DMEM.

[0175] Step 5. After 4 h in the incubator, the solution was aspirated and 100 μL DMSO was added, and the absorbance was measured at 490 nm.

[0176] Specifically, the Portuguese coryneformis CICC 33276 was used as a model bacteria. First, from the perspective of cell repair, see Fig. 27 Another comparison chart of cell viability between Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 is shown. Fig.28 Another comparison of cell viability of the Portuguese coryneformis CICC 33497 and the model bacteria Portuguese coryneformis CICC 33276 is shown, and Fig.29 Another cell viability comparison chart of Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC33276 is shown. Analysis found that, first, at concentrations of 0.8%, 4% and 20%, Portuguese Corynospora P8 had stronger cell repair capabilities for HaCaT cells damaged by UVB than the model bacteria Portuguese Corynospora CICC 33276. Secondly, at concentrations of 0.8%, 4% and 20%, Portuguese Corynospora CICC 33497 and the model bacteria Portuguese Corynospora CICC 33276 had similar cell repair capabilities for HaCaT cells damaged by UVB. Next, at concentrations of 0.8%, 4% and 20%, Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC 33276 had similar cell repair capabilities for HaCaT cells damaged by UVB. Second, from the perspective of cell protection, see Fig.30 Another cell activity comparison chart of Portuguese coryneformis P8 and the model bacteria Portuguese coryneformis CICC 33276 is shown. Fig.31 Another cell viability comparison chart of the Portuguese coryneformis CICC 33497 and the model bacteria Portuguese coryneformis CICC 33276, and Fig.32 Another cell viability comparison chart of Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC 33276 is shown. Analysis shows that, first, at concentrations of 0.8%, 4%, and 20%, Portuguese Corynospora P8 has a stronger cell protection ability against UVB-damaged HaCaT cells than the model bacteria Portuguese Corynospora CICC 33276. In addition, Portuguese Corynospora CICC 33497 and Portuguese Corynospora CICC 1461 both have a certain cell protection ability, and the cell protection ability increases with increasing concentration.

[0177] (3) Experimental Example 3 (verifying the cell repair ability of Portuguese yeast P8 against scratch-damaged cells) specifically includes the following steps:

[0178] In the first step, place the insert, shiny side up, in the center of each well so that the insert adheres to the bottom of the plate.

[0179] Among them, HaCaT cells were grown in complete medium (high-glucose DMEM medium containing 10% FBS and 1% double antibody). Among them, the cells were digested with trypsin and inoculated into two wells of the insert at 6×105 / well, 70 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0180] In the second step, the plug was removed and the residual culture medium was absorbed with PBS, and 1 mL of DMEM (without double antibody and FBS) was added to the blank group, 1 mL of DMEM (++) was added to the blank control group, and 1 mL of DMEM prepared samples of different concentrations were added to the sample group.

[0181] Three parallel groups were set up in each group and incubated in an incubator for 24 hours.

[0182] In the third step, after incubation for 24 hours, pictures were taken under a microscope and the cell migration area was calculated using ImageJ software.

[0183] For details, see Fig.33 The comparison chart of the repair ability of scratch-damaged cells of Portuguese Corynospora P8, Portuguese Corynospora CICC 33497, Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC 33276 shows that Portuguese Corynospora P8, Portuguese Corynospora CICC 33497, Portuguese Corynospora CICC 1461 and the model bacteria Portuguese Corynospora CICC 33276 all have certain repair ability of scratch-damaged cells. Among them, compared with the model bacteria Portuguese Corynospora CICC 33276, the fermentation supernatant of Portuguese Corynospora P8 has a significant ability to promote the migration of skin cells, and the scratch damage has been completely closed at 4%.

[0184] Example 6

[0185] Among them, Example 6 is used to explore the effect of Portuguese coryneformis P8 on the stability of the mask solution.

[0186] Specifically, the fermentation supernatant of Portuguese corynosporum P8 was added to the facial mask liquid to test the stability of the facial mask liquid. The formula of the facial mask liquid is shown in Table 2 below:

[0187] .

[0188] Specifically, the facial mask liquid was divided into room temperature samples, heat-resistant samples, cold-resistant samples and light-exposed samples. Among them, the room temperature samples were placed away from light. The heat-resistant samples were placed in a constant temperature oven at (50±1°C). The cold-resistant samples were placed in a -20°C refrigerator. The light-exposed samples were placed in an artificial intelligence climate box. After 3 months, the appearance, color, odor, and presence of oil-water separation of the room temperature samples, heat-resistant samples, cold-resistant samples and light-exposed samples were observed. The experimental results are shown in Table 3 below:

[0189] .

[0190] The analysis found that after the fermentation supernatant of Portuguese yeast P8 was mixed with the matrix, there was no discoloration or stratification after being placed at normal temperature, high temperature, low temperature and light conditions for 3 months, and its properties were stable.

[0191] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the embodiments of the present disclosure.

Claims

1. A yeast having antioxidant, anti-aging and skin repairing abilities, wherein: The yeast is Portuguese coryneformis P8, which is classified and named Clavispora lusitaniae P8, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.31344.

2. Use of the yeast as claimed in claim 1 in preparing cosmetics with antioxidant properties, wherein: The antioxidant use includes any of the following effects: ABTS free radical scavenging; DPPH free radical scavenging; ROS free radical scavenging.

3. Use of the yeast as claimed in claim 1 in preparing cosmetics with anti-inflammatory uses, wherein: The anti-inflammatory use is the inhibition of the inflammatory factor NO.

4. A use of the yeast as claimed in claim 1 in the preparation of cosmetics with anti-aging effects, wherein: The anti-aging use is to promote the synthesis of type I collagen.

5. Use of the yeast as claimed in claim 1 in preparing cosmetics for skin repairing purposes, wherein: The skin repair use includes any of the following effects: Cell repair of UVA-damaged cells; Cell repair of UVB-damaged cells; Cellular repair of scratch-damaged cells.

6. Use of the yeast as claimed in claim 1 in preparing cosmetics for anti-photodamage purposes, wherein: The anti-light damage includes any of the following effects: Protects against UVA damage; Resists UVB damage.

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