Fermentation method for simultaneously producing superoxide dismutase and nano-silver particles and product composition

Abstract

The invention belongs to the field of microbial fermentation and metabolites thereof, and particularly relates to a fermentation method for simultaneously producing superoxide dismutase and nano-silver particles and a product composition. According to the invention, the stress tolerance characteristic of deinococcus radiodurans is utilized, and the concentration of Ag < + > is controlled, so that the enzyme activity of oxidation-reduction enzymes including SOD (superoxide dismutase) of the deinococcus radiodurans is improved, and meanwhile, the Ag < + > is reduced into nano-silver particles (DR-Ag NPs). After deinococcus radiodurans is stressed by Ag < + >, the product is rich in oxidoreductase including SOD and nano-silver particles (DR-Ag NPs), the product can be prepared into a functional cosmetic product after being compounded with hyaluronic acid and the like, the functional cosmetic product has super-strong anti-aging, anti-wrinkle, whitening, antibacterial and anti-inflammatory effects and the like, and meanwhile, the use of preservatives is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation and its metabolites, and specifically relates to a fermentation method and a product composition for simultaneously producing superoxide dismutase and silver nanoparticles. Background Art

[0002] Deinococcus radiodurans (DR) is a microorganism with extremely strong resistance to ionizing radiation, ultraviolet rays, drying, high temperature, and various chemical mutagenic stresses. It is an aerobic bacterium without flagella, without spores, and spherical in shape, gram-positive, with an optimal growth temperature of 30 °C, and is usually cultured using a peptone-glucose-yeast extract medium. Under normal growth conditions, DR bacteria exist in the form of dyads during the logarithmic growth phase and often in the form of tetrads during the stationary phase. The cell envelope of DR bacteria is composed of a unique multi-layer structure, which can be divided into the following six layers (from the outside to the inside): polysaccharide layer, surface layer, outer membrane, interstitial layer, peptidoglycan layer, inner membrane, with a total thickness of about 150 nm. Its outermost layer is composed of polysaccharide secretions, and these extracellular polysaccharides can scavenge free reactive oxygen species (ROS) and have antioxidant effects. Therefore, compared with radiation-sensitive species (such as Escherichia coli), Deinococcus radiodurans can effectively prevent oxidative damage to proteins. Stress pressures such as radiation and drying will generate a large amount of reactive oxygen species (ROS) in the cell, but there are enzymatic and non-enzymatic antioxidant systems in Deinococcus radiodurans to scavenge ROS to prevent protein damage. Since the protein activities in related pathways such as transcription, translation, and DNA repair are necessary during the cell's stress resistance process, an efficient antioxidant system is crucial for its survival after exposure to oxidative stress.

[0003] Superoxide dismutase (SOD) is an important antioxidant enzyme widely present in all living organisms. Its physiological function is to scavenge superoxide free radicals. SOD has a positive effect on the body's protection, anti-aging, anti-inflammatory, anti-tumor, anti-autoimmune diseases, anti-shock, anti-oxygen poisoning, beauty care, and prevention of hair loss. It has received great attention in the medical and biochemistry fields and is increasingly used in food and cosmetic additives and other fields, with broad application prospects. SOD mainly protects cells from oxidative damage by catalyzing the decomposition of superoxide anion radicals into molecular oxygen and less toxic hydrogen peroxide. According to the differences in protein folding and metal cofactors, SOD can be divided into three families: copper and zinc-binding CuZn-SOD type, iron or manganese-binding Fe-SOD / Mn-SOD, and nickel-binding Ni-SOD. CuZn-SOD is most commonly found in eukaryotes. Fe-SOD and Mn-SOD are usually found in prokaryotes and mitochondria. Ni-SOD has only been found in prokaryotes until now. Deinococcus radiodurans contains a cytoplasmic-localized Mn-SOD (DR 1279) and two periplasmic CuZn-SODs (DR1546 and DR A0202). Among the three SODs in Deinococcus radiodurans, its Mn-SOD can scavenge superoxide anion radicals more effectively compared to homologs in Escherichia coli and humans. Mn-SOD may play an important role in the antioxidant system of Deinococcus radiodurans. Mn-SOD may also be able to bind to DNA, and the binding of Mn-SOD to DNA can protect DNA from oxidative damage. Some research reports have shown that under the stress of ionizing radiation, ultraviolet rays, drying, high temperature, and various chemical mutagens, Deinococcus radiodurans produces more redox enzymes including SOD and may use a large number of redox enzymes to protect proteins or nucleic acids from the effects of increased ROS.

[0004] The current production and preparation process of SOD mainly focuses on the extraction and purification of tissues and cells from animal and plant sources. However, it is difficult to obtain animal and plant tissues, the SOD content and enzyme activity in tissues are low, and the current separation and purification process is complex, using methods such as heat denaturation, organic solvents, salting out, ion exchange, and gel filtration. The purification multiple and activity recovery rate are both low. The research on producing SOD using microorganisms has received increasing attention. Microorganisms have the advantages of large-scale cultivation, short cultivation cycle, and sufficient sources.

[0005] If cosmetics are contaminated by microorganisms, their components will change. If contaminated by pathogenic bacteria, it will pose potential hazards to users. To prevent cosmetics from being contaminated by microorganisms during use and storage, manufacturers need to add a certain amount of preservatives to cosmetics. There are many preservatives available on the market now. However, the most commonly used ones are parabens, formaldehyde and formaldehyde-releasing agents such as isothiazolinone and imidazolidinyl urea. However, there have always been many reports of contact allergies caused by the use of cosmetics, mostly allergies caused by preservatives and fragrances in cosmetics. Therefore, using metal nanoparticles with both functional and antibacterial and antiseptic properties is a new strategy for cosmetics preservation.

[0006] The present invention utilizes the stress tolerance characteristics of Deinococcus radiodurans, controls the concentration of Ag+, and improves the enzyme activity of the redox enzymes including SOD in Deinococcus radiodurans. At the same time, Ag+ is reduced to silver nanoparticles (DR-Ag NPs). After Deinococcus radiodurans is stressed by Ag+, the product is rich in redox enzymes including SOD and silver nanoparticles (DR-Ag NPs). After being compounded with hyaluronic acid and the like, it can be prepared into a functional cosmetic product. Among them, the redox enzymes including SOD enzyme have super anti-aging and anti-wrinkle effects. DR-Ag NPs can not only antibacterial and anti-inflammatory, but also improve the moisturizing, sunscreen and whitening effects of cosmetics. The present invention utilizes the Ag+-stress tolerance of Deinococcus radiodurans, "killing two birds with one stone" to both improve the SOD enzyme activity and produce DR-Ag NPs, which can be used as a cosmetic additive to prepare a functional cosmetic product and improve the functionality of cosmetics. Summary of the Invention

[0007] The purpose of the present invention is to solve the deficiencies of the prior art, provide a fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles, utilize the Ag+-stress tolerance of Deinococcus radiodurans, "killing two birds with one stone" to both increase the yield and enzyme activity of SOD enzyme and produce DR-Ag NPs, which can be used as a cosmetic additive to prepare a functional cosmetic product and improve the functionality of cosmetics.

[0008] The first aspect of the present invention lies in providing a fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles. The specific technical solution is as follows: A fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles, comprising the following steps: (a) Gradually amplify the Deinococcus radiodurans strain in a culture medium to obtain a seed solution; (b) Inoculate the seed solution into a fermentation medium for cultivation, add the Ag+ solution in batches, and continue cultivation until the absorbance value does not change significantly, and the fermentation cultivation ends.

[0009] In a preferred embodiment, the Deinococcus radiodurans is ATCC No. 13939, purchased from Shanghai Fuxiang Biotechnology Co., Ltd., and the culture conditions are 220 r / min and 30 °C.

[0010] Preferably, the inoculation amount of the seed liquid inoculated into the fermentation medium is 5% - 10% of the volume of the fermentation medium.

[0011] Preferably, the addition of the Ag+ solution in batches is to add the Ag+ solution in two times. The first time is when OD600 = 0.2, add the Ag+ solution; the second time is when OD600 = 0.5 - 0.6, add the Ag+ solution.

[0012] Preferably, the Ag+ solution is selected from AgNO3.

[0013] Preferably, the final concentration of the Ag+ solution is 0.01 mM - 1 mM.

[0014] More preferably, the final concentration of the Ag+ solution is 0.02 mM - 0.06 mM.

[0015] In a preferred embodiment, a fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles includes the following steps: (a) Gradually amplify the Deinococcus radiodurans strain in a medium to obtain a seed liquid; (b) Inoculate the seed liquid into the fermentation medium for fermentation culture. When OD600 = 0.2, add the Ag+ solution with a final concentration of 0.01 mM, and continue culturing; when OD600 = 0.5 - 0.6, add the Ag+ solution with a final concentration of 0.02 - 0.06 mM, and continue culturing until the absorbance value does not change significantly, and the fermentation culture ends.

[0016] In a preferred embodiment, an extract containing the fermentation products superoxide dismutase and silver nanoparticles is obtained by the following method: (1) After the fermentation ends, centrifuge the bacterial liquid at a low speed to obtain the supernatant and the precipitate respectively. Collect the precipitate of the bacterial cells, wash the precipitate of the bacterial cells three times with phosphate buffer, and centrifuge to collect the bacterial cells; (2) Prepare a bacterial suspension by adding three times the phosphate buffer per gram of the bacterial cells, and ultrasonically treat to break the cells; (3) Centrifuge and collect the supernatant. The supernatant is the crude enzyme solution of superoxide dismutase; (4) Centrifuge the supernatant obtained in step (1) at a high speed to obtain a precipitate. Wash the precipitate three times with phosphate buffer, centrifuge, collect the silver nanoparticles, and resuspend the precipitate with the crude enzyme solution of superoxide dismutase obtained in step (3) to obtain an extract containing superoxide dismutase and silver nanoparticles.

[0017] Preferably, the low-speed centrifugation is at 3000 - 5000 rpm.

[0018] Preferably, the high-speed centrifugation is at 8000 - 12000 rpm.

[0019] The second aspect of the present invention lies in providing a composition, which comprises the fermented product superoxide dismutase and the nano-silver particle extract described in the first aspect.

[0020] Preferably, the composition further comprises hyaluronic acid, water, glycerol, collagen, beeswax, and olive oil.

[0021] Preferably, the composition comprises 20 - 50% of superoxide dismutase and nano-silver particle extract, 15 - 30% of hyaluronic acid, 0.1 - 3.0% of glycerol, 2 - 10% of collagen, 2 - 5% of beeswax, 2 - 5% of olive oil, and 5 - 40% of water.

[0022] More preferably, the composition comprises 40% of superoxide dismutase and nano-silver particle extract, 20% of hyaluronic acid, 2% of glycerol, 5% of collagen, 3% of beeswax, 3% of olive oil, and 27% of water.

[0023] The form of the composition includes essence water, emulsion, toner, or the essence of a facial mask.

[0024] In a preferred embodiment, the composition is obtained by the following method: Weigh the prescription amounts of superoxide dismutase and nano-silver particle extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil, and water, mix them evenly, then place them in a water bath and emulsify while stirring, and let it stand to obtain the composition.

[0025] Preferably, the water bath temperature is 45 - 85°C.

[0026] Preferably, the stirring speed is 50 - 1200 rpm.

[0027] Preferably, the emulsification time is 5 - 20 min.

[0028] Advantages of the present invention: With the stress tolerance of Deinococcus radiodurans, Ag+ is used to stress Deinococcus radiodurans, which not only improves the enzyme activity of SOD but also produces nano-silver particles; and a multi-functional cosmetic additive is obtained with "two birds with one stone"; further, the DR-extract is compounded with hyaluronic acid, collagen, etc. to prepare a functional composition, which has the effects of anti-aging, anti-wrinkle, whitening, antibacterial and anti-inflammatory, etc., and at the same time avoids the use of preservatives. Description of the Drawings

[0029] Figure 1, TEM images of DR-Ag NPs. Detailed implementation manners

[0030] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention rather than to limit the scope of the present invention. Materials, raw materials, etc. used in the present invention can be obtained through commercial channels without special instructions.

[0031] The present invention does not limit the types of culture media used, and existing culture media suitable for the growth of Deinococcus radiodurans can be used in the present invention. The TGY broth medium and GY broth medium in the present invention are both commercially available.

[0032] TGY broth medium: Tryptone 5.0 g / L, Glucose 1.0 g / L, and Yeast extract powder 3.0 g / L.

[0033] GY broth medium: Peptone 10.0 g / L, Beef extract 3.0 g / L, Sodium chloride 5.0 g / L, Glucose 5.0 g / L.

[0034] TGY plate medium: Tryptone 5.0 g / L, Glucose 1.0 g / L, Yeast extract powder 3.0 g / L, and Agarose 15.0 g / L.

[0035] Example 1 a. Strain screening: Inoculate the DR strain ATCC NO.13939 into 5 ml of TGY broth, and culture it at 30 °C with shaking at 200 rpm for 48 h. Take a loop of broth culture and streak it on a TGY plate, and culture it at 30 °C for 40 - 48 h, and observe the colonies on the plate and perform microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, and culture it at 30 °C with shaking at 200 rpm overnight, and then store it at -70 °C after culturing.

[0036] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it in a TGY medium, and perform constant-temperature shaking culture at 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and perform constant-temperature shaking culture at 35 °C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.01 mM, and continue to culture. Measure their OD600nm at regular intervals. When OD600nm = 0.5, add an Ag+ solution with a final concentration of 0.04 mM, and continue to culture until the absorbance value does not change significantly, and the fermentation culture ends.

[0037] Example 2 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth, and culture it at 30°C with shaking at 200 rpm for 48 h. Take a loopful of the broth culture and streak it onto a TGY plate, then culture it at 30°C for 40 - 48 h, and observe the colonies on the plate and perform microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it at 30°C with shaking at 200 rpm overnight, and then store it at -70°C after culturing.

[0038] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it into TGY medium, and perform constant-temperature shaking culture at 32°C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and perform constant-temperature shaking culture at 35°C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.01 mM, and continue culturing. Measure their OD600nm at regular intervals. When OD600nm = 0.6, add an Ag+ solution with a final concentration of 0.02 mM, and continue culturing until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0039] Example 3 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth, and culture it at 30°C with shaking at 200 rpm for 48 h. Take a loopful of the broth culture and streak it onto a TGY plate, then culture it at 30°C for 40 - 48 h, and observe the colonies on the plate and perform microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it at 30°C with shaking at 200 rpm overnight, and then store it at -70°C after culturing.

[0040] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it into TGY medium, and perform constant-temperature shaking culture at 32°C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and perform constant-temperature shaking culture at 35°C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.01 mM, and continue culturing. Measure their OD600nm at regular intervals. When OD600nm = 0.5, add an Ag+ solution with a final concentration of 0.06 mM, and continue culturing until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0041] Example 4 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth and culture it with shaking at 30 °C and 200 rpm for 48 h. Take a loopful of the broth culture and streak-inoculate it onto a TGY plate, then culture it at 30 °C for 40 - 48 h, and observe the colonies on the plate and perform microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it with shaking at 30 °C and 200 rpm overnight, and then store it frozen at -70 °C after the culture is completed.

[0042] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate and culture it in TGY medium, and perform constant-temperature shaking culture on it at a temperature of 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and perform constant-temperature shaking culture on it at a temperature of 35 °C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.02 mM, continue the culture, and measure their OD600nm at regular intervals. When OD600nm = 0.5, add an Ag+ solution with a final concentration of 0.04 mM, continue the culture until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0043] Example 5 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth and culture it with shaking at 30 °C and 200 rpm for 48 h. Take a loopful of the broth culture and streak-inoculate it onto a TGY plate, then culture it at 30 °C for 40 - 48 h, and observe the colonies on the plate and perform microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it with shaking at 30 °C and 200 rpm overnight, and then store it frozen at -70 °C after the culture is completed.

[0044] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate and culture it in TGY medium, and perform constant-temperature shaking culture on it at a temperature of 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and perform constant-temperature shaking culture on it at a temperature of 35 °C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.005 mM, continue the culture, and measure their OD600nm at regular intervals. When OD600nm = 0.5, add an Ag+ solution with a final concentration of 0.01 mM, continue the culture until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0045] Example 6 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth and culture it at 30 °C with shaking at 200 rpm for 48 h. Take a loopful of the broth culture and streak it onto a TGY plate, then culture it at 30 °C for 40 - 48 h, and observe the colonies on the plate and conduct microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it at 30 °C with shaking at 200 rpm overnight, and then store it frozen at -70 °C after culturing is completed.

[0046] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it for culture in TGY medium, and conduct constant-temperature shaking culture at 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, and conduct constant-temperature shaking culture at 35 °C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.01 mM, and continue culturing. Measure their OD600nm at regular intervals. When OD600nm = 0.4, add an Ag+ solution with a final concentration of 0.04 mM, and continue culturing until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0047] Comparative Example 1 a. Strain screening: Inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth and culture it at 30 °C with shaking at 200 rpm for 48 h. Take a loopful of the broth culture and streak it onto a TGY plate, then culture it at 30 °C for 40 - 48 h, and observe the colonies on the plate and conduct microscopic examination. After being identified as DR, inoculate a single colony into 25 ml of GY broth, culture it at 30 °C with shaking at 200 rpm overnight, and then store it frozen at -70 °C after culturing is completed.

[0048] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it for culture in TGY medium, and conduct constant-temperature shaking culture at 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1%, add an Ag+ solution with a final concentration of 0.01 mM, and conduct constant-temperature shaking culture at 35 °C. Measure their OD600nm at regular intervals. When OD600nm = 0.5, add an Ag+ solution with a final concentration of 0.04 mM, and continue culturing until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0049] Comparative Example 2 a. Strain screening: Inoculate the DR strain ATCC NO.13939 into 5 ml of TGY broth and culture it at 30 °C with shaking at 200 rpm for 48 h. Inoculate a loopful of the broth culture onto a TGY plate by streaking and culture it at 30 °C for 40 - 48 h. Observe the colonies on the plate and perform microscopy. After being identified as DR, inoculate a single colony into 25 ml of GY broth and culture it overnight at 30 °C with shaking at 200 rpm. After the culture is completed, store it at -70 °C.

[0050] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it into TGY medium for culture. Perform constant-temperature shaking culture at 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1% and perform constant-temperature shaking culture at 35 °C. When OD600nm = 0.2, add an Ag+ solution with a final concentration of 0.04 mM and continue the culture. Measure their OD600nm at regular intervals until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0051] Comparative Example 3 a. Strain screening: Inoculate the DR strain ATCC NO.13939 into 5 ml of TGY broth and culture it at 30 °C with shaking at 200 rpm for 48 h. Inoculate a loopful of the broth culture onto a TGY plate by streaking and culture it at 30 °C for 40 - 48 h. Observe the colonies on the plate and perform microscopy. After being identified as DR, inoculate a single colony into 25 ml of GY broth and culture it overnight at 30 °C with shaking at 200 rpm. After the culture is completed, store it at -70 °C.

[0052] b. Strain fermentation: Activate the Deinococcus radiodurans strain, pick a single colony and inoculate it into TGY medium for culture. Perform constant-temperature shaking culture at 32 °C to obtain a seed solution. Inoculate the seed solution into a triangular flask containing 250 ml of TGY medium at an inoculation ratio of 1% and perform constant-temperature shaking culture at 35 °C. Measure their OD600nm at regular intervals until there is no obvious change in the absorbance value, and the fermentation culture ends.

[0053] Verification Example The fermented products superoxide dismutase and silver nanoparticles are obtained by the following method: (1) After the fermentation ends, centrifuge the bacterial solution at 3000 - 5000 rpm at 0 - 4 °C for 5 - 10 min to obtain the supernatant and precipitate respectively. Collect the precipitate of the bacterial cells, wash the precipitate of the bacterial cells three times with a phosphate buffer solution of 50 - 60 mM, pH 7.4, and centrifuge to collect the bacterial cells; (2) Prepare a cell suspension by adding three times the above buffer per gram of cells, and ultrasonically disrupt the cells at a power of 400 - 800 w for 5 - 10 min, with an interval of 2 - 5 s for every 2 - 3 s of treatment; (3) Centrifuge at 8000 - 12000 rpm for 5 - 20 min under the conditions of 0 - 4 °C, collect the supernatant, and the supernatant is the crude superoxide dismutase enzyme solution. The enzyme activity is measured by the pyrogallol autoxidation method, and the results are shown in Table 1. The unit is expressed as the number of enzyme activity units per milligram of dry cell weight; (4) Centrifuge the supernatant obtained in step (1) at 8000 - 12000 rpm at high speed to obtain a precipitate. Wash the precipitate three times with the above phosphate buffer, centrifuge at 8000 - 12000 rpm, 0 - 4 °C for 10 - 20 min, collect the silver nanoparticle precipitate, and resuspend the precipitate. Use a UV - visible spectrophotometer to measure the OD432nm of the DR - extraction solution at 432 nm to detect DR - Ag NPs, and characterize the morphology of DR - Ag NPs. The results are as Figure 1 shown. The transmission electron microscopy image shows that DR - Ag NPs present a regular spherical morphology, and the particle size is between 20 - 30 nm.

[0054] Table 1 Superoxide dismutase specific activity Preparation of the composition 1. Preparation of the extraction solution (1) After the fermentation in Example 1 is completed, centrifuge the bacterial liquid at 3000 - 5000 rpm under the conditions of 0 - 4 °C for 5 - 10 min to obtain the supernatant and precipitate respectively. Collect the cell precipitate of the bacteria, wash the cell precipitate of the bacteria three times with a phosphate buffer of 50 - 60 mM, pH 7.4, and centrifuge to collect the cell precipitate of the bacteria; (2) Prepare a cell suspension by adding three times the above buffer per gram of cells, and ultrasonically disrupt the cells at a power of 400 - 800 w for 5 - 10 min, with an interval of 2 - 5 s for every 2 - 3 s of treatment; (3) Centrifuge at 8000 - 12000 rpm for 5 - 20 min under the conditions of 0 - 4 °C to collect the supernatant and precipitate respectively. The supernatant is the crude superoxide dismutase enzyme solution; (4) Centrifuge the supernatant obtained in step (1) at 8000 - 12000 rpm at high speed to obtain a precipitate. Wash the precipitate three times with the above phosphate buffer, centrifuge at 8000 - 12000 rpm, 0 - 4 °C for 10 - 20 min, collect the silver nanoparticle precipitate, and resuspend the precipitate with the crude superoxide dismutase enzyme solution obtained in step (3) to obtain an extraction solution containing superoxide dismutase and silver nanoparticles.

[0055] 2. Preparation of the composition Weigh the prescribed amounts of the extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil, and water according to the component contents shown in Table 2, mix them evenly, and then emulsify the composition in a water bath with stirring according to the emulsification parameters shown in Table 3. Let it stand to obtain the composition.

[0056] Table 2 Component Contents of the Composition Table 3 Emulsification Parameters of the Composition Verification of the Composition's Efficacy 1. Stability Evaluation Evaluate the stability of the compositions in Examples 7 - 11 and Comparative Examples 4 - 6 at -20, 4, and 37 °C for 180 days through the clarity state and presence or absence of precipitation of the cosmetics. The results are shown in Table 4.

[0057] Table 4 Stability of the Composition ; 2. Antibacterial and Antiseptic Evaluation Respectively pipette 0.1 mL of suspensions of Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli) at different dilution degrees and add them to the surfaces of their respective suitable sterilized and cooled plate media, spread them evenly, and culture until the bacteria cover the entire petri dish and are evenly distributed. Use a hole punch to make filter paper discs with a diameter of 8 mm from No. 102 Xinhua filter paper, place them in a dry and clean test tube, sterilize them by dry heat at 160 °C for 2 h, immerse them in the compositions of Examples 7 - 11 and Comparative Examples 4 - 6, take them out after 4 h, and let them dry under sterile conditions for later use. Place the filter paper discs on the above-mentioned bacteria-containing petri dishes, three discs per dish. After culturing the bacteria at 37 °C for 24 h, measure the diameter of the inhibition zone, and use the diameter of the inhibition zone as an index of antibacterial activity. The larger the diameter, the stronger the antibacterial activity. The results are shown in Table 5.

[0058] Table 5 Antibacterial Activity 3. Whitening Efficacy Evaluation Apply the compositions in Example 7 and Comparative Example 4 to the left and right arms respectively. The application area is 3 cm × 3 cm, and the application amount is 2.00 ± 0.05 mg / cm 2 , After continuously applying for 15 days, select an unapplied area of 3 cm × 3 cm as the negative control. Using the Lab colorimetric system method, use a general color difference meter to measure the change in skin color chromaticity. ΔE is a comprehensive index for evaluating skin color change. The larger the value of ΔE, the more significant the color difference; the smaller the value, the closer the colors.

[0059] When ΔE is less than 1, the color difference is almost imperceptible and indistinguishable to the human eye; When ΔE is between 1 and 2, the color change can only be detected by an experienced observer; When ΔE is between 2 and 3.5, the color change can be detected by an average observer; When ΔE is between 3.5 and 6, the color change is obvious; When ΔE is between 6 and 12, the color change is very significant.

[0060] Table 6 Evaluation of skin color change 4. Anti-wrinkle efficacy test The anti-wrinkle test was carried out on the compositions obtained in Example 7 and Comparative Example 4. There were 20 volunteers, 10 in each group. Each group tested one composition. After the testers washed their faces, they stayed in a constant temperature and humidity environment (temperature 21 - 23 °C, humidity 55 - 65%) for 30 minutes, then took facial photos using VISA-CR. Subsequently, the composition was applied and gently patted to absorb. The testers used the compound extract for 8 weeks. After 8 weeks, they washed their faces and took facial photos using VISACR. Subsequently, the improvement rate of facial wrinkles and the increase rate of R2 before and after use were calculated by the instrument software to reflect the immediate improvement of facial wrinkles by each composition.

[0061] The results are shown in Table 7. The anti-wrinkle efficacy of the composition obtained in Example 7 is significantly better than that of Comparative Example 4. The wrinkle removal rate of Example 7 reaches more than 45%, indicating that the composition prepared according to the present invention has good anti-wrinkle effect.

[0062] Table 7 Anti-wrinkle efficacy

Claims

1. A fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles, characterized in that, It includes the following steps: a. Gradually amplify the Deinococcus radiodurans strain in a culture medium to obtain a seed solution; b. Inoculate the seed solution into a fermentation medium for cultivation, add the Ag+ solution in batches, and continue cultivation until the absorbance value shows no obvious change, and the fermentation cultivation ends.

2. The method according to claim 1, wherein The adding of the Ag+ solution in batches means adding the Ag+ solution twice. The first time is when OD600 = 0.2, add the Ag+ solution; the second time is when OD600 = 0.5 - 0.6, add the Ag+ solution.

3. The method according to claim 1, wherein The Ag+ solution is selected from AgNO3.

4. The method according to claim 1, wherein The final concentration of the Ag+ solution is 0.01 mM - 1 mM; preferably, the final concentration of the Ag+ solution is 0.02 mM - 0.06 mM.

5. The method according to claim 1, wherein The Deinococcus radiodurans strain is selected as ATCC No. 13939.

6. A method for extracting the superoxide dismutase and silver nanoparticles described in claim 1, characterized in that, It includes the following steps: (1). After fermentation ends, centrifuge the bacterial liquid at a low speed to obtain the supernatant and precipitate respectively. Collect the precipitate of the bacterial cells, wash the precipitate of the bacterial cells three times with phosphate buffer solution, and centrifuge to collect the bacterial cells; (2). Prepare a bacterial suspension by adding three times the phosphate buffer solution per gram of the bacterial cells, and ultrasonically treat to break the cells; (3). Centrifuge to collect the supernatant, and the supernatant is the crude superoxide dismutase solution; (4). Centrifuge the supernatant obtained in step (1) at a high speed to obtain a precipitate. Wash the precipitate three times with phosphate buffer solution, centrifuge, collect the silver nanoparticles, and resuspend the precipitate with the crude superoxide dismutase solution obtained in step (3) to obtain an extract containing superoxide dismutase and silver nanoparticles.

7. A composition comprising the extract according to claim 6, characterized in that, It contains the following components: 20 - 50% of the extract, 15 - 30% of hyaluronic acid, 0.1 - 3.0% of glycerol, 2 - 10% of collagen, 2 - 5% of beeswax, 2 - 5% of olive oil, and 5 - 40% of water.

8. The composition according to claim 7, wherein The composition contains the following components: 40% of the extract, 20% of hyaluronic acid, 2% of glycerol, 5% of collagen, 3% of beeswax, 3% of olive oil, and 27% of water.

9. A method for preparing the composition according to any one of claims 7-8, characterized in that, It includes the following steps: Weigh the prescribed amounts of the superoxide dismutase and silver nanoparticle extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil and water, mix them evenly, then place them in a water bath and emulsify while stirring, and let it stand to obtain the composition.

10. The method according to claim 9, characterized in that, The water bath temperature is 45 - 85 °C; the stirring speed is 50 - 1200 rpm; the emulsification time is 5 - 20 min.

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