Deinococcus radiodans and saussurea involucrata co-fermentation product as well as preparation method and application thereof

Abstract

The invention provides a deinococcus radiodans and saussurea involucrata co-fermentation product as well as a preparation method and application thereof, and belongs to the technical field of cosmetic raw material development. According to the invention, deinococcus radiodurans and saussurea involucrata are subjected to co-fermentation, such that a deinococcus radiodurans and saussurea involucrata co-fermentation product is obtained. An anti-oxidation experiment shows that the expression of catalase genes and glutathione peroxidase genes after UVB irradiation is remarkably reduced, and the expression of the catalase genes and the glutathione peroxidase genes is remarkably increased after the co-fermentation product of the deinococcus radiodurans and the saussurea involucrata provided by the invention is added, so that the co-fermentation product has an obvious anti-oxidation effect; a cDNA repair experiment shows that the content of pyrimidine dimer is increased after UVB irradiation, the content of pyrimidine dimer is remarkably reduced after the deinococcus radiodurans and saussurea involucrata co-fermentation product is added, and it is proved again that the product has the excellent anti-oxidation effect, and the application prospect is wide. The deinococcus radiodurans and saussurea involucrata co-fermentation product can be used as an anti-ultraviolet radiation cosmetic raw material, and has a very good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw material development, and in particular to a co-fermentation product of Deinococcus radiatus and Saussurea involucrata and a preparation method and application thereof. Background Art

[0002] Deinococcus radiodurans (DR) is a microorganism with strong tolerance to extreme environments such as ionizing radiation, oxidative damage, and dryness stress. The special structure of the unique cell wall S-layer of Deinococcus radiodurans is related to its extreme resistance. The resistance of Deinococcus radiodurans to extreme environments mainly focuses on resistance to ionizing radiation, resistance to oxidative stress, resistance to long-term dry environments, and resistance to ultraviolet radiation (100-295nm). There is a special tetraterpenoid carotenoid in Deinococcus radiodurans - deinoxanthin, which can remove active oxygen to protect the bacteria. Deinoxanthin has a higher ability to remove peroxides than carotene and lutein, and has a significant protective effect on the DNA and protein of the bacteria. The application prospects of deoxyxanthin in the fields of cosmetics, skin care, etc. are mainly reflected in the series of side effects caused by anti-ultraviolet rays, such as increased melanin, decreased antioxidant enzyme activity, decreased cell vitality, collagen loss, increased ROS, etc. It even has a certain healing effect on wounds. Most of its effects are better than Vc. It has broad application prospects in the fields of sun protection and skin care.

[0003] Chinese patent CN111686069A discloses an anti-blue light repair composition containing camellia extract and its application, wherein the composition comprises the following components in percentage by weight: Deinococcus radiodurans Adenosine Glucosylrutin Bilberry Fruit Extract Dextran Dipotassium glycyrrhizinate The composition can weaken the pigment-inducing effect of blue light on the skin in multiple ways, effectively resist subsequent pigmentation, reduce the damage of blue light to the skin, keep the skin elastic and shiny, and is safe and efficient. The prepared anti-blue light repair composition can be widely used in cosmetics such as toners, lotions, creams and other products that repair dry, dull, loose and other photo-aging skin problems.

[0004] However, the existing technology still lacks more products with better efficacy developed based on radioresistant Deinococcus radiodurans. Summary of the invention

[0005] The purpose of the present invention is to provide a co-fermentation product of Deinococcus radiodurans and Saussurea involucrata, which can be used as an effective raw material for anti-ultraviolet radiation cosmetics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a co-fermentation product of Deinococcus radiatus and Saussurea involucrata, comprising the following steps:

[0008] The extract of Saussurea involucrata is mixed with the basic culture medium to obtain a co-fermentation culture medium;

[0009] inoculating Deinococcus radiatus into a co-fermentation medium and fermenting to obtain a fermentation liquid;

[0010] The fermentation liquid is subjected to crushing treatment, ultrasonic treatment, water bath treatment and centrifugation treatment in sequence to obtain a supernatant, which is the co-fermentation product of the Deinococcus radiatus and Saussurea involucrata.

[0011] Preferably, the volume ratio of the Saussurea involucrata extract to the basal culture medium is 1:2-6.

[0012] Preferably, the Saussurea involucrata extract is prepared by a preparation method comprising the following steps:

[0013] The lyophilized powder of callus tissue of Saussurea tianshanica is subjected to low-temperature grinding, ultrasonic-assisted alcohol extraction and evaporation concentration in sequence to obtain Saussurea tianshanica extract.

[0014] Preferably, the basal medium comprises TGY medium;

[0015] And / or, the extract of Saussurea tianshanica is filtered through a filter membrane before being mixed with the basic culture medium;

[0016] And / or, the pore size of the filter membrane is below 0.22 μm;

[0017] and / or, the inoculation amount of the inoculation is 0.5-2%;

[0018] And / or, the fermentation temperature is 27-32°C;

[0019] And / or, the fermentation time is 16 to 24 hours.

[0020] Preferably, the crushing process adopts high-pressure homogenization crushing;

[0021] The processing parameters of the high-pressure homogenization crushing are 1500-2000Mpa, 2-6°C;

[0022] And / or, the ultrasonic treatment time is 20 to 40 minutes;

[0023] And / or, the temperature of the water bath treatment is 50-70°C;

[0024] And / or, the water bath treatment time is 20 to 40 minutes;

[0025] And / or, the rotation speed of the centrifugal treatment is 2000-6000 rpm

[0026] And / or, the centrifugal treatment time is 5 to 20 minutes.

[0027] The present invention also provides a co-fermentation product of Deinococcus radiatus and Saussurea involucrata obtained by the preparation method of the claim.

[0028] The present invention also provides the use of the co-fermentation product of Deinococcus radiatus and Saussurea involucrata in the preparation of antioxidant products.

[0029] The present invention also provides the use of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata in the preparation of anti-ultraviolet radiation products.

[0030] The present invention also provides the use of the co-fermentation product of Deinococcus radiatus and Saussurea involucrata in the preparation of skin repair products.

[0031] Preferably, the skin repair product is a skin repair product for ultraviolet damage.

[0032] Beneficial effects of the present invention:

[0033] The present invention obtains a co-fermentation product of Deinococcus radioresistance and Saussurea involucrata by co-fermenting Deinococcus radioresistance and Saussurea involucrata. Antioxidant experiments show that the expression of catalase gene and glutathione peroxidase gene is significantly reduced after UVB irradiation, and significantly increased after adding the co-fermentation product of Deinococcus radioresistance and Saussurea involucrata provided by the present invention, indicating that it has obvious antioxidant effect; cDNA repair experiments show that the content of pyrimidine dimers increases after UVB irradiation, and significantly decreases after adding the co-fermentation product of Deinococcus radioresistance and Saussurea involucrata provided by the present invention, which once again proves that the product of the present invention has excellent antioxidant effect. The co-fermentation product of Deinococcus radioresistance and Saussurea involucrata in the present invention can be used as a raw material for anti-ultraviolet radiation cosmetics, and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Comparative photos of the appearance of the co-fermentation product lysate (left), mixed solution (middle) and radiodurans lysate (right);

[0035] Figure 2 The cytotoxicity test results of the co-fermentation products of the present invention are

[0036] Figure 3The experimental results of the effects of the co-fermentation products of the present invention on the expression of CAT gene;

[0037] Figure 4 The experimental results of the effects of the co-fermentation products of the present invention on the expression of GPX gene;

[0038] Figure 5 This is the result of the DNA repair effect experiment of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata.

[0039] Figure 6 These are the experimental results of the effect of the seabuckthorn co-fermentation product control group on the expression of CAT gene. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Sources of biological materials in the embodiments of the present invention:

[0042] Deinococcus radiodurans: purchased from Beijing Biobo Biotechnology Co., Ltd., platform number: Bio-096064, specification: lyophilized, Latin genus name: Deinococcus Radiodurans.

[0043] Example 1

[0044] Tianshan Snow Lotus Plant Extract:

[0045] 1. Pre-cool the mortar and pestle before grinding (add liquid nitrogen to the mortar for pre-cooling, the amount of liquid nitrogen added should be at least two-thirds of the volume of the mortar, and stir lightly with the pestle. Repeat the operation twice after the liquid nitrogen evaporates). Take an appropriate amount of freeze-dried powder of Saussurea involucrata callus in liquid nitrogen and grind it into powder (the powder can pass through a 60-mesh sieve). Store it at -20°C for future use.

[0046] 2. Take the treated powder and add 50% anhydrous ethanol at a (m / m) material-liquid ratio of 1:10. Under the condition of ultrasonic power of 2000W, action temperature of 40℃, action time of 30min, repeat the extraction 3 times, filter and combine the extracts with a Buchner funnel, and then filter through a 0.22μm filter membrane.

[0047] 3. Remove the solvent from the solution after pressure filtration by rotary evaporator: the first step is rotary evaporation conditions: 40°C, rotation speed 100rpm, the liquid in the rotary evaporation bottle does not overflow as the standard, and the degree of rotary evaporation is judged according to the volume of anhydrous ethanol in the collection bottle (specific judgment criteria: the volume of liquid in the rotary evaporation bottle is significantly reduced compared with that before rotary evaporation or the liquid in the collection bottle is about one third of the initial rotary evaporation liquid volume); the second step is to increase the rotary evaporation temperature to 48°C and rotary evaporation for 30 minutes; the third step is to determine the anhydrous ethanol content with reference to the alcohol meter comparison table. If the anhydrous ethanol content is within the range of 3-5°, continue rotary evaporation at 48°C for 30 minutes.

[0048] Note: Storage conditions of Tianshan Saussurea callus freeze-dried powder concentrate: -20℃ (completely thawed before preparation);

[0049] Preparation of co-fermentation products of Deinococcus radiodurans and Saussurea involucrata:

[0050] Live Deinococcus radiodurans bacteria: Activate the bacteria using TGY liquid medium;

[0051] Co-fermentation medium preparation:

[0052] The plant extract was filtered through a 0.22 μm filter membrane and then mixed with the sterilized TGY medium at a ratio of 1:4 in an ultra-clean bench.

[0053] Co-fermentation culture: activate the radioresistant Deinococcus radiodurans (OD 600 =1.7) was inoculated into the co-fermentation medium at an inoculation content of 1% and fermented at 30°C for 20 h.

[0054] Fermentation broth treatment: The fermentation broth was directly subjected to high-pressure homogenization (1800 MPa 4°C), repeated three times, ultrasonicated for 30 min, water bathed at 60°C for 30 min, centrifuged at 4000 rpm for 10 min, and the supernatant was collected and recorded as the co-fermentation product lysate in subsequent experiments.

[0055] Comparative Example 1

[0056] Preparation of Deinococcus radiodurans lysate

[0057] Live Deinococcus radiodurans: Use TGY solid or liquid culture medium to activate the strain;

[0058] The activated Deinococcus radiodurans was inoculated into TGY liquid medium at 1% inoculation content and fermented at 30°C for 20 hours;

[0059] The fermentation broth was centrifuged at 4000 rpm for 10 min, the bacteria were washed three times with pure water of one sixth of the fermentation broth volume, resuspended with pure water of one half of the fermentation broth volume, and high-pressure homogenized at 1800 MPa and 4°C, repeated three times, centrifuged at 4000 rpm for 5 min, and the supernatant was collected.

[0060] Comparative Example 2

[0061] The only difference from Example 1 is that the Saussurea involucrata is replaced by Hippophae rhamnoides.

[0062] Comparative Example 3

[0063] Live Deinococcus radiodurans bacteria: Activate the bacteria using TGY liquid medium;

[0064] The activated Deinococcus radiodurans (OD 600 =1.7) was inoculated into TGY medium at an inoculum content of 1% and fermented at 30°C for 20h. The fermented liquid was directly subjected to high-pressure homogenization (1800mpa 4°C), which was repeated three times. The mixture was ultrasonicated for 30min, water bathed at 60°C for 30min, and centrifuged at 4000rpm for 10min. The supernatant (radioresistant Deinococcus lysate) was collected and mixed with the Saussurea involucrata plant extract (same as in Example 1) in a mixing ratio of 4:1 to obtain a lysate-Saussurea involucrata mixed solution.

[0065] Experimental Example 1: Appearance Comparison

[0066] The appearance of the radioresistant Deinococcus radiodurans lysate, the co-fermentation product lysate prepared in Example 1, and the lysate-Saussurea involucrata mixed solution prepared in Comparative Example 3 were compared. The results are as follows: Figure 1 shown.

[0067] Depend on Figure 1 It can be seen that the color of the co-fermentation product prepared by the present invention is obviously darker than that of the mixed solution. It can be inferred that after the co-fermentation of Deinococcus radiodurans and Saussurea involucrata, some changes have occurred in the composition.

[0068] Experimental Example 2 Cytotoxicity test of lysate of co-fermentation product of Deinococcus radiodurans and Saussurea involucrata A sample group and a blank control group were set up for comparative experiments:

[0069] The blank control (BC) group was cultured in serum-free medium;

[0070] The cell-free control (NCC) group was cultured in serum-free medium without inoculation of cells;

[0071] The experimental groups were cultured in serum-free medium containing 0.05%, 0.1%, 0.5%, and 1% of the lysate of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata;

[0072] The cell experiment was set up with 6 replicates (n=6);

[0073] Human keratinocytes (HaCaT) in logarithmic growth phase were taken and cultured at 1*10 4Each well was inoculated into a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h;

[0074] 100 μL of sample solution of corresponding concentration was added to each well, and 100 μL of serum-free medium was added to each well of the blank control group, and incubated for 24 h;

[0075] Add 10 μL CCK-8 reagent to each well, incubate at 37°C for 1 h, and measure the OD value at 450 nm. Figure 2 .

[0076] The results showed that the lysate of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata had no cytotoxicity at a concentration of 1%.

[0077] Experimental Example 3 Determination of the Antioxidant Efficacy of Lysate of Co-fermentation Product of Deinococcus radiodurans and Saussurea involucrata The expression of antioxidant-related genes CAT and GPX was determined by RT-qPCR. The main steps are as follows:

[0078] Blank control (BC) group: cultured in serum-free medium;

[0079] Model (NC) group: UVB (60mJ / cm 2 ) were treated and then cultured with serum-free medium;

[0080] Experimental group: UVB (60mJ / cm 2 ) were treated and then cultured in serum-free medium containing sample solutions of different concentrations, the samples were 0.1%, 0.5% radioresistance Deinococcus fermentation product lysate, 0.1%, 0.5% radioresistance Deinococcus and Saussurea involucrata co-fermentation product lysate, 0.1% radioresistance Deinococcus and Hippophae rhamnoides co-fermentation product lysate;

[0081] 1) HaCaT cells in logarithmic growth phase were cultured at 1*10 6 Cells / well were seeded in 6-well plates and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0082] 2) Abandon the culture medium and UVB (60mJ / cm 2 ) treatment (the control group did not require UVB treatment);

[0083] 3) The experimental group was added with samples of various concentrations, and the control group and model group were added with serum-free culture medium, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0084] 4) Discard the supernatant, wash twice with PBS, and collect the cells;

[0085] 5) Extract RNA, reverse transcribe it into cDNA and perform RT-qPCR.

[0086] Effects of the lysate of co-fermentation of Deinococcus radiodurans and Saussurea involucrata on the expression of antioxidant-related genes CAT and GPX Figures 3-4 , Figure 6 After UVB irradiation, the gene expressions of CAT and GPX were significantly decreased, and increased after drug administration. The most significant upregulation of CAT was in 0.1% DR+Saussurea involucrata, and the most significant upregulation of GPX was in 0.5% DR+Saussurea involucrata.

[0087] Experimental Example 4 Determination of DNA repair efficacy of lysate from co-fermentation product of Deinococcus radiodurans and Saussurea involucrata

[0088] Sample solution preparation

[0089] The sample stock solution was diluted to 0.1% and 0.5% using DMEM basal medium.

[0090] CPD detection reagent preparation

[0091] 1x Wash Buffer: Dilute 10x Wash Buffer Concentrate to 1x with deionized water and stir until homogeneous;

[0092] Anti-CPD antibody and secondary antibody, HRP conjugate: dilute the antibody 1:100 and the secondary antibody 1:1000 in assay diluent immediately before use;

[0093] Denaturing Solution A: Dilute Denaturing Solution A 1:100 with 70% ethanol immediately before use;

[0094] Denaturing Solution B: Dilute Denaturing Solution B 1:100 with DPBS (containing magnesium and calcium) immediately before use.

[0095] Model drug administration:

[0096] HaCaT cells in logarithmic growth phase were cultured at a rate of 1-3*10 4 Cells / well were seeded in a 96-well plate and cultured in a 37°C 5% CO2 incubator for 24 h;

[0097] The culture medium was discarded and UVB (60 mJ / cm 2 ) treated cells (blank control group did not need to be treated with UVB);

[0098] After the end, the 96-well plate was taken out, the sample group was added with the sample, the blank control group and the negative control group were added with serum-free culture medium, the positive control group was added with the corresponding positive drug, and the cells were cultured in a 37° C. 5% CO 2 incubator for 24 h.

[0099] CPD content detection:

[0100] Wash each well of the 96-well plate with 100 μL DPBS (containing calcium and magnesium), aspirate each well, and add 100 μL 75% methanol / 25% acetic acid to each well. Incubate at room temperature for 30 min;

[0101] Aspirate each well and add 100 μL of 70% ethanol to each well. Incubate at room temperature for 30 min;

[0102] Aspirate each well and add 100 μL of denaturing solution A (see reagent preparation) to each well. Incubate at room temperature for 5 minutes;

[0103] Wash gently three times with 200 μL DPBS (containing magnesium and calcium);

[0104] Aspirate each well and add 100 μL of denaturing solution B (see reagent preparation) to each well. Incubate at room temperature for 10 min;

[0105] Aspirate each well and add 200 μL of detection diluent to each well. Incubate at room temperature for 30 minutes;

[0106] Aspirate each well and add 100 μL of diluted anti-CPD antibody (see Reagent Preparation) to each well.

[0107] Incubate on an orbital shaker for 1 h;

[0108] Wash the microplate four times with 250 μL of 1× wash buffer per well, aspirating thoroughly before each wash. After the last wash, empty the wells and place the microplate on an absorbent pad or paper towel to remove excess 1× wash buffer;

[0109] Add 100 μL of diluted secondary antibody and HRP conjugate (see Reagent Preparation) to each well. Incubate on an orbital shaker for 1 h;

[0110] Wash the microplate 4 times as in step 2 above. Proceed immediately to the next step;

[0111] Heat the substrate solution to room temperature. Add 100 μL of substrate solution to each well, including the blank well. Incubate on an orbital shaker at room temperature for 10 min.

[0112] Add 100 μL of stop solution to each well, including the blank well, to stop the enzyme reaction, and read the results immediately;

[0113] The absorbance of each microwell was read on a microplate reader at 450 nm as the detection wavelength.

[0114] The results of DNA repair in the lysate of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata are shown in Figure 5 After UVB irradiation, the CPD content increased significantly, and decreased significantly after drug administration, among which 0.5% DR + Tianshan Saussurea had the most significant effect.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a co-fermentation product of Deinococcus radiatus and Saussurea involucrata, characterized in that: The following steps are involved: The extract of Saussurea involucrata is mixed with the basic culture medium to obtain a co-fermentation culture medium; inoculating Deinococcus radiatus into a co-fermentation medium and fermenting to obtain a fermentation liquid; The fermentation liquid is subjected to crushing treatment, ultrasonic treatment, water bath treatment and centrifugation treatment in sequence to obtain a supernatant, which is the co-fermentation product of the Deinococcus radiatus and Saussurea involucrata.

2. The preparation method according to claim 1, characterized in that: The volume ratio of the Tianshan Saussurea extract to the basic culture medium is 1:2-6.

3. The preparation method according to claim 2, characterized in that: The inosculate honeysuckle extract is prepared by a preparation method comprising the following steps: subjecting in sequence the inosculate honeysuckle callus freeze-dried powder to low-temperature grinding, ultrasonic-assisted alcohol extraction and evaporation concentration to obtain the inosculate honeysuckle extract.

4. The preparation method according to claim 1, characterized in that: The basal medium includes TGY medium; And / or, the extract of Saussurea tianshanica is filtered through a filter membrane before being mixed with the basic culture medium; And / or, the pore size of the filter membrane is less than 0.22 μm; and / or, the inoculation amount of the inoculation is 0.5-2%; And / or, the fermentation temperature is 27-32°C; And / or, the fermentation time is 16 to 24 hours.

5. The preparation method according to claim 1, characterized in that: The crushing process adopts high-pressure homogenization crushing; The processing parameters of the high-pressure homogenization crushing are 1500-2000Mpa, 2-6°C; And / or, the ultrasonic treatment time is 20 to 40 minutes; And / or, the temperature of the water bath treatment is 50-70°C; And / or, the water bath treatment time is 20 to 40 minutes; And / or, the rotation speed of the centrifugal treatment is 2000-6000 rpm And / or, the centrifugal treatment time is 5 to 20 minutes.

6. A co-fermentation product of Deinococcus radiatus and Saussurea involucrata obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the co-fermentation product of Deinococcus radiatus and Saussurea involucrata according to claim 6 in the preparation of antioxidant products.

8. Use of the co-fermentation product of Deinococcus radiodurans and Saussurea involucrata as claimed in claim 6 in the preparation of anti-ultraviolet radiation products.

9. Use of the co-fermentation product of Deinococcus radiatus and Saussurea involucrata according to claim 6 in the preparation of skin repair products.

10. The use according to claim 9, characterized in that: The skin repair product is a skin repair product for ultraviolet damage.

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