A fermentation process for the simultaneous production of superoxide dismutase and silver nanoparticles and product composition

Abstract

The present application belongs to the field of microbial fermentation and its metabolites, and particularly relates to a fermentation method and product composition for simultaneously producing superoxide dismutase and nano silver particles. The present application utilizes the stress tolerance characteristics of Deinococcus radiodurans, controls the concentration of Ag+, improves the enzyme activity of the oxidoreductase of Deinococcus radiodurans including SOD, and at the same time, Ag+ is reduced into nano silver particles (DR-Ag NPs). After the stress of Ag+ on Deinococcus radiodurans, the product is rich in oxidoreductase including SOD and nano silver particles (DR-Ag NPs), and after compounding with hyaluronic acid and the like, a functional cosmetic product can be prepared, which has super strong anti-aging, anti-wrinkle, whitening, antibacterial and anti-inflammatory effects, and at the same time, the use of preservatives is avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial fermentation and its metabolites, and particularly relates to a fermentation method for simultaneously producing superoxide dismutase and nano-silver particles and a product composition. BACKGROUND

[0002] Deinococcus radiodurans (DR) is a microorganism with extremely strong resistance to ionizing radiation, ultraviolet radiation, dryness, high temperature and various chemical mutagenic agents. It is an aerobic bacterium without flagella and spores, and is red and spherical. It is gram-positive, and its optimum growth temperature is 30°C. It is usually cultured using a proteose peptone-glucose-yeast extract medium. Under normal growth conditions, DR bacteria exist in a diplobacillary form during the logarithmic growth phase, and in a tetraplobacillary form during the stationary phase. The cell envelope of DR bacteria is composed of a unique multilayer structure, which can be divided into the following six layers (from outside to inside): polysaccharide layer, surface layer, outer membrane, interstitial layer, peptidoglycan layer and inner membrane, with a total thickness of about 150 nm. The outermost layer is composed of polysaccharide exudates, which 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 protein oxidative damage. Radiation and dryness stress can produce 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 activity in the related pathways such as transcription, translation and DNA repair is necessary for the cell resistance process, an efficient antioxidant system is crucial for the survival of the cell after exposure to oxidative stress.

[0003] Superoxide dismutase (SOD) is an important antioxidant enzyme that exists in all organisms, and its physiological function is to scavenge superoxide free radicals. SOD has a positive effect on the body's protection and anti-aging, anti-inflammatory, anti-tumor, anti-autoimmune disease, anti-shock, anti-oxygen poisoning, beauty and prevention of hair loss, and is highly valued by the medical and biochemical communities. It is also increasingly used in food and cosmetic additives and other fields, and has a wide application prospect. SOD mainly protects cells from oxidative damage by catalyzing the decomposition of superoxide anion free radicals into molecular oxygen and less toxic hydrogen peroxide. According to the difference of protein folding and metal cofactor, SOD can be divided into three families: copper and zinc combined CuZn-SOD type, iron or manganese combined Fe-SOD / Mn-SOD and nickel combined Ni-SOD. CuZn-SOD is most commonly found in eukaryotes. Fe-SOD and Mn-SOD are usually used in prokaryotes and mitochondria. Ni-SOD has only been found in prokaryotes so far. Deinococcus radiodurans contains a cytoplasmic Mn-SOD (DR 1279) and two periplasmic CuZn-SOD (DR 1546 and DR A0202). Among the three SODs in Deinococcus radiodurans, its Mn-SOD can more effectively scavenge superoxide anion free radicals compared to its homologues 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. Studies have reported that under ionizing radiation, ultraviolet light, drying, high temperature and various chemical mutagenic stress conditions, Deinococcus radiodurans produces more oxidoreductases including SOD, and may use a large number of oxidoreductases to protect proteins or nucleic acids from increased ROS.

[0004] The production and preparation process of SOD currently mainly focuses on the extraction and purification of tissues and cells from animals and plants. However, the source of animal and plant tissues is difficult, the SOD content and enzyme activity in the tissues are low, and the current separation and purification process is complex, using methods such as heat denaturation, organic solvent, salting out, ion exchange and gel filtration, and the purification fold and activity recovery rate are low. The use of microorganisms to prepare SOD is increasingly attracting attention, and microorganisms have the advantages of large-scale cultivation, short cultivation period, abundant source, etc.

[0005] Cosmetics can be changed if they are contaminated by microorganisms, and can cause potential harm to users if contaminated by pathogenic bacteria. In order to prevent cosmetics from being contaminated by microorganisms during use and storage, manufacturers need to add a certain amount of preservatives to the cosmetics. There are many preservatives available on the market, but the most commonly used are nipagin ester formaldehyde and formaldehyde releasing agents such as isothiazolinone, imidazole alkyl urea, etc. However, there have been many reports of contact allergies caused by the use of cosmetics, most of which are caused by preservatives and fragrances in cosmetics. Therefore, the use of metal nanoparticles with both efficacy and antibacterial preservative properties is a new strategy for cosmetic preservation.

[0006] The present application utilizes the stress tolerance of Deinococcus radiodurans, controls the concentration of Ag+, and improves the enzyme activity of the oxidoreductase including SOD of Deinococcus radiodurans. At the same time, Ag+ is reduced to 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). After compounding with hyaluronic acid, a functional cosmetic product can be prepared. The oxidoreductase including SOD enzyme has super anti-aging and anti-wrinkle effects, and the DR-Ag NPs can not only resist bacteria and inflammation, but also improve the moisturizing, sunscreen, and whitening effects of cosmetics. The present application utilizes the Ag+ stress tolerance of Deinococcus radiodurans, "one arrow double draw" improves the SOD enzyme activity and produces DR-Ag NPs, which can be used as a cosmetic additive to prepare functional cosmetic products and improve the efficacy of cosmetics. SUMMARY

[0007] The purpose of the present application is to solve the problems of the prior art, provide a fermentation method for simultaneously producing superoxide dismutase and nano silver particles, utilize the Ag+ stress tolerance of Deinococcus radiodurans, "one arrow double draw" improve the SOD enzyme yield and enzyme activity, and produce DR-Ag NPs, which can be used as a cosmetic additive to prepare functional cosmetic products and improve the efficacy of cosmetics.

[0008] The first aspect of the present application is to provide a fermentation method for simultaneously producing superoxide dismutase and nano silver particles, and the specific technical solutions are as follows:

[0009] A fermentation method for simultaneously producing superoxide dismutase and nano silver particles, comprising the following steps:

[0010] (a) inoculate Deinococcus radiodurans in the culture medium to obtain a seed liquid;

[0011] (b) inoculate the seed liquid into the fermentation medium for culture, add Ag+ solution in batches, continue to culture until the absorbance value has no obvious change, and the fermentation culture is completed.

[0012] 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, 30°C.

[0013] Preferably, the inoculation amount of the seed liquid into the fermentation medium is 5% to 10% of the volume of the fermentation medium.

[0014] Preferably, the Ag+ solution is added in two times, the first time is when OD600 = 0.2, and the second time is when OD600 = 0.5 to 0.6.

[0015] Preferably, the Ag+ solution is selected from AgNO3.

[0016] Preferably, the final concentration of the Ag+ solution is 0.01 mM to 1 mM.

[0017] Further preferably, the final concentration of the Ag+ solution is 0.02 mM to 0.06 mM.

[0018] In a preferred embodiment, a fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles includes the following steps:

[0019] (a) inoculating Deinococcus radiodurans in the culture medium to obtain a seed liquid;

[0020] (b) inoculating the seed liquid into the fermentation medium for fermentation culture, when OD600 = 0.2, adding Ag+ solution with a final concentration of 0.01 mM, and continuing the culture; when OD600 = 0.5 to 0.6, adding Ag+ solution with a final concentration of 0.02 to 0.06 mM, and continuing the culture until there is no significant change in the absorbance value, and the fermentation culture is ended.

[0021] In a preferred embodiment, the extraction solution containing the fermentation products superoxide dismutase and silver nanoparticles is obtained by the following method:

[0022] (1) After the fermentation is ended, centrifuging the bacterial liquid at a low speed to obtain the supernatant and the precipitate, respectively, collecting the bacterial cell precipitate, washing the bacterial cell precipitate with phosphate buffer three times, and centrifuging to collect the bacterial cell;

[0023] (2) adding three times of phosphate buffer per gram of the bacterial cell to prepare a bacterial suspension, and treating the bacterial suspension with ultrasonic waves to break the cells;

[0024] (3) centrifuging to collect the supernatant, and the supernatant is the crude enzyme solution of superoxide dismutase;

[0025] (4) centrifuging the supernatant obtained in step (1) at high speed to obtain a precipitate, washing the precipitate with phosphate buffer three times, centrifuging, collecting the nano-silver particles, resuspending the precipitate with the superoxide dismutase crude enzyme solution obtained in step (3) to obtain an extract containing superoxide dismutase and nano-silver particles.

[0026] Preferably, the low-speed centrifugation is 3000-5000 rpm.

[0027] Preferably, the high-speed centrifugation is 8000-12000 rpm.

[0028] The second aspect of the present application is to provide a composition comprising the ferment product superoxide dismutase and nano-silver particle extract of the first aspect.

[0029] Preferably, the composition further comprises hyaluronic acid, water, glycerol, collagen, beeswax, olive oil.

[0030] Preferably, the composition comprises 20-50% of the 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.

[0031] Further preferably, the composition comprises 40% of the 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.

[0032] The form of the composition includes essence water, emulsion, toner, or essence of a mask.

[0033] In a preferred embodiment, the composition is obtained by the following method:

[0034] Weighing the prescribed amount of the superoxide dismutase and nano-silver particle extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil, and water, mixing them uniformly, emulsifying them in a water bath while stirring, and standing to obtain the composition.

[0035] Preferably, the water bath temperature is 45-85℃.

[0036] Preferably, the stirring speed is 50-1200 rpm.

[0037] Preferably, the emulsification time is 5-20 min.

[0038] The present application has the advantages that the present application uses Ag+ to stress Deinococcus radiodurans by the stress resistance of Deinococcus radiodurans, not only improves the enzyme activity of SOD, but also produces nano-silver particles; and obtains a multifunctional cosmetic additive by "one arrow with two targets"; and further prepares a functional composition by compounding the DR-extract with hyaluronic acid, collagen and the like, which has the effects of anti-aging, anti-wrinkle, whitening, antibacterial and anti-inflammatory, and avoids the use of preservatives. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Figure 1 is a transmission electron microscope image of DR-Ag NPs. DETAILED DESCRIPTION

[0040] The present application will be further described and illustrated by specific examples. It should be understood that the following examples are only used to illustrate the present application and are not used to limit the scope of the present application. The materials, raw materials and the like used in the present application are commercially available if not otherwise specified.

[0041] The present application does not limit the type of culture medium used, and any existing culture medium suitable for the growth of Deinococcus radiodurans can be used in the present application. The TGY broth medium and the GY broth medium in the present application are commercially available.

[0042] TGY broth medium: tryptone 5.0 g / L, glucose 1.0 g / L and yeast extract powder 3.0 g / L.

[0043] GY broth medium: tryptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L and glucose 5.0 g / L.

[0044] TGY plate medium: tryptone 5.0 g / L, glucose 1.0 g / L, yeast extract powder 3.0 g / L and 15.0 g / L agarose.

[0045] Example 1

[0046] a, strain screening: inoculate the DR strain ATCC NO. 13939 into 5 ml of TGY broth, and culture at 30℃ with 200 rpm shaking for 48 h. Take a ring of the broth culture and inoculate it on a TGY plate, and culture at 30℃ for 40-48 h, and observe the plate colonies and perform microscopic examination. After identification of DR, inoculate a single colony into 25 ml of GY broth, and culture at 30℃ with 200 rpm shaking overnight, and then store at -70℃ after completion of the culture.

[0047] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.01 mM Ag+ solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.04 mM Ag+ solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0048] Example 2

[0049] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0050] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.01 mM Ag+ solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.04 mM Ag+ solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0051] Example 3

[0052] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0053] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.01 mM Ag+solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.06 mM Ag+solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0054] Example 4

[0055] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0056] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.01 mM Ag+solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.06 mM Ag+solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0057] Example 5

[0058] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0059] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.005 mM Ag+ solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.01 mM Ag+ solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0060] Example 6

[0061] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0062] b. Strain fermentation: The radiation-resistant Deinococcus strain was activated, a single colony was picked and inoculated in TGY medium, and constant temperature and oscillation culture was carried out at 32°C to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and constant temperature and oscillation culture was carried out at 35°C. When OD600nm=0.2, 0.005 mM Ag+ solution was added, and the culture was continued. OD600nm was measured at certain intervals. When OD600nm=0.5, 0.01 mM Ag+ solution was added, and the culture was continued until the absorbance value did not change significantly, and the fermentation culture was completed.

[0063] Comparative Example 1

[0064] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated in 5 ml TGY broth and oscillation culture was carried out at 30°C and 200 rpm for 48 h. A ring of the broth culture was streaked on a TGY plate, and the plate was incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of DR, a single colony was inoculated in 25 ml GY broth, and oscillation culture was carried out at 30°C and 200 rpm overnight. After completion of the culture, the culture was frozen at -70°C.

[0065] b. Strain fermentation: The radiation tolerant Deinococcus strain was activated, and a single colony was picked and inoculated in TGY medium, and incubated at 32°C with constant shaking to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and 0.01 mM Ag+ solution was added. The mixture was incubated at 35°C with constant shaking. OD600nm was measured at intervals. When OD600nm was 0.5, 0.04 mM Ag+ solution was added. The mixture was incubated until OD600nm was stable.

[0066] Comparative Example 2

[0067] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated into 5 ml TGY broth and incubated at 30°C with 200 rpm shaking for 48 h. A loopful of the broth culture was streaked onto a TGY plate and incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of the DR, a single colony was inoculated into 25 ml GY broth and incubated at 30°C with 200 rpm shaking overnight. The culture was stored at -70°C after completion of the incubation.

[0068] b. Strain fermentation: The radiation tolerant Deinococcus strain was activated, and a single colony was picked and inoculated in TGY medium, and incubated at 32°C with constant shaking to obtain seed liquid. The seed liquid was inoculated into a flask containing 250 ml TGY medium at a ratio of 1%, and 0.01 mM Ag+ solution was added. The mixture was incubated at 35°C with constant shaking. OD600nm was measured at intervals. When OD600nm was 0.5, 0.04 mM Ag+ solution was added. The mixture was incubated until OD600nm was stable.

[0069] Comparative Example 3

[0070] a. Strain screening: The DR strain ATCC NO. 13939 was inoculated into 5 ml TGY broth and incubated at 30°C with 200 rpm shaking for 48 h. A loopful of the broth culture was streaked onto a TGY plate and incubated at 30°C for 40-48 h. The plate colonies and microscopic examination were observed. After identification of the DR, a single colony was inoculated into 25 ml GY broth and incubated at 30°C with 200 rpm shaking overnight. The culture was stored at -70°C after completion of the incubation.

[0071] b. Strain fermentation: the radiation-resistant Deinococcus strain was activated, single colonies were picked and inoculated in TGY medium, and incubated at 32°C for constant temperature and shaking. Seed liquid was obtained. The seed liquid was inoculated into a 250ml TGY medium flask at a ratio of 1%, and incubated at 35°C for constant temperature and shaking. The OD600nm was measured at regular intervals until the absorbance value was stable, and the fermentation was completed.

[0072] Verification example

[0073] Fermentation product superoxide dismutase and nano-silver particles were obtained by the following method:

[0074] (1) After fermentation, the bacterial liquid was centrifuged at 3000-5000 rpm, 0-4°C, for 5-10 min to obtain supernatant and precipitate, respectively. The bacterial cell precipitate was washed with 50-60 mM, pH 7.4 phosphate buffer three times, and the bacterial cell precipitate was collected by centrifugation.

[0075] (2) A bacterial suspension was prepared by adding three times the above buffer per gram of bacteria, and the cells were broken by ultrasonic treatment at a power of 400-800w for 5-10 min, with an interval of 2-5s between each treatment.

[0076] (3) Centrifuge at 8000-12000 rpm, 0-4°C for 5-20 min, collect the supernatant, and the supernatant is the crude enzyme solution of superoxide dismutase. The enzyme activity was determined by the pyrogallol autoxidation method, and the results are shown in Table 1. The unit is expressed as the number of enzyme units per milligram of bacterial dry weight.

[0077] (4) The supernatant obtained in step (1) was centrifuged at 8000-12000 rpm to obtain a precipitate, which was washed three times with the above phosphate buffer, centrifuged at 8000-12000 rpm, 0-4°C for 10-20 min, and the nano-silver particle precipitate was collected and resuspended. The OD432nm of the DR-extract was measured at 432 nm by UV spectrophotometry, and the DR-Ag NPs were detected, and the morphology of the DR-Ag NPs was characterized. The results are shown in Figure 1

[0078] Table 1 Specific activity of superoxide dismutase

[0079]

[0080] Preparation of the composition

[0081] 1. Preparation of the extract​

[0082] (1) After the fermentation of Example 1 is completed, centrifuge the bacterial liquid at 3000-5000 rpm, 0-4°C, for 5-10 min to obtain supernatant and precipitate, respectively, collect the bacterial cell precipitate, and wash the bacterial cell precipitate with 50-60 mM phosphate buffer solution, pH 7.4, three times, and centrifuge to collect the bacterial cell;

[0083] (2) Prepare a bacterial suspension by adding three times the above buffer per gram of bacteria, and treat the cells with ultrasonic waves at a power of 400-800 W for 5-10 min, with an interval of 2-5 s between each treatment of 2-3 s;

[0084] (3) Centrifuge at 8000-12000 rpm, 0-4°C, for 5-20 min, and collect the supernatant and precipitate, respectively, and the supernatant is the crude enzyme solution of superoxide dismutase;

[0085] (4) Centrifuge the supernatant obtained in step (1) at 8000-12000 rpm to obtain a precipitate, wash the precipitate with the above phosphate buffer solution three times, centrifuge at 8000-12000 rpm, 0-4°C, for 10-20 min, collect the nano-silver particle precipitate, and resuspend the precipitate with the crude enzyme solution of superoxide dismutase obtained in step (3) to obtain an extract containing superoxide dismutase and nano-silver particles.

[0086] 2. Preparation of the composition

[0087] Weigh the prescription amount of extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil and water according to the component content shown in Table 2, mix them evenly, then place the composition in a water bath according to the emulsification parameters shown in Table 3, emulsify while stirring, and obtain the composition after standing.

[0088] Table 2 Component content of the composition

[0089]

[0090] Table 3 Emulsification parameters of the composition

[0091]

[0092] Verification of the effect of the composition

[0093] 1. Stability evaluation

[0094] The compositions in Examples 7-11 and Comparative Examples 4-6 were stored at -20, 4 and 37°C, respectively, for 180 days, and the stability was evaluated by the clear state of the cosmetic and the presence or absence of precipitation. The results are shown in Table 4.

[0095] Table 4 Stability of the composition

[0096] ;

[0097] 2. Antimicrobial efficacy evaluation

[0098] Different dilutions of gram positive and negative bacteria (Staphylococcus aureus and Escherichia coli) suspensions were each taken 0.1 mL and added to the surface of the respective sterile cooled flat plate medium, evenly coated, cultured, and the bacteria grew throughout the entire culture dish and were evenly distributed. The 102 type Xinhua filter paper was punched into a 8 mm diameter disc, placed in a dry clean test tube, dried at 160°C for 2 h, immersed in the composition of Examples 7-11 and Comparative Examples 4-6, removed after 4 h, and left to dry under sterile conditions for standby use. The filter paper disc was placed on the above-mentioned bacteria-containing plate, three discs per plate, and after 37°C / 24 h culture of the bacteria, the diameter of the inhibition zone was measured, and the diameter of the inhibition zone was used as an index of antibacterial activity, the larger the diameter, the stronger the antibacterial activity. The results are shown in Table 5.

[0099] Table 5. Antibacterial activity

[0100]

[0101] 3. Whitening efficacy evaluation

[0102] The compositions of Example 7 and Comparative Example 4 were respectively applied to the left and right arms, with a coating area of 3 cm x 3 cm and a coating amount of 2.00 ± 0.05 mg / cm 2 After 15 days of continuous application, an area of 3 cm x 3 cm was selected as the negative control, and the Lab color system method was used to measure the color change of the skin color using a universal color difference meter. ΔE is a comprehensive index for evaluating changes in skin color. The larger the ΔE value, the more significant the color difference, and the smaller the value, the closer the color.

[0103] When ΔE is less than 1, the color difference is almost imperceptible, and the human eye cannot distinguish it;

[0104] When ΔE is between 1 and 2, the color change requires an experienced observer to perceive;

[0105] When ΔE is between 2 and 3.5, the color change can be perceived by an average observer;

[0106] When ΔE is between 3.5 and 6, the color change is obvious;

[0107] When ΔE is between 6 and 12, the color change is very significant.

[0108] Table 6. Evaluation of skin color change

[0109]

[0110] 4. Anti-wrinkle efficacy test

[0111] The compositions obtained in Example 7 and Comparative Example 4 were subjected to anti-wrinkle tests. A total of 20 volunteers were divided into two groups of 10, and each group was tested with one of the compositions. After the testers washed their faces, they were kept in a constant temperature and humidity environment (temperature 21-23°C, humidity 55-65%) for 30 minutes, and then photographed using VISACR. Subsequently, the compositions were applied and patted in to be absorbed. The testers used the composite extract for 8 weeks, and after washing their faces at the end of the 8th week, they were photographed using VISACR. Subsequently, the improvement rate of facial wrinkles and the R2 improvement rate before and after use were calculated by the instrument software to reflect the immediate improvement of each composition on facial wrinkles.

[0112] The results are shown in Table 7. The anti-wrinkle efficacy of the composition obtained in Example 7 was significantly better than that of Comparative Example 4. The wrinkle removal rate of Example 7 was more than 45%, indicating that the composition prepared according to the present application has good anti-wrinkle effect.

[0113] Table 7 Anti-wrinkle efficacy

[0114]

Claims

1. A fermentation method for simultaneously producing superoxide dismutase and silver nanoparticles, characterized by, It comprises the following steps: a. The Deinococcus radiodurans strain is inoculated in the culture medium to obtain seed liquid; b. The seed liquid is inoculated in the fermentation medium to culture, and Ag+ solution is added in batches, and the culture is continued until the absorbance value has no obvious change, and the fermentation is completed; The Deinococcus radiodurans strain is selected as ATCC No. 13939, and the Ag+ solution is added in two batches, the first time is to add Ag+ solution with a final concentration of 0.01 mM when OD600=0.2, and the second time is to add Ag+ solution with a final concentration of 0.02 mM-0.06 mM when OD600=0.5-0.

6.

2. The method of claim 1, wherein, The Ag+ solution is selected from AgNO3.

3. A method of extracting the superoxide dismutase and nano silver particles of claim 1, characterized by, It comprises the following steps: (1) After the fermentation is completed, the bacterial liquid is centrifuged at low speed to obtain supernatant and precipitate, and the bacterial cell precipitate is collected and washed with phosphate buffer three times, and the bacterial cell is collected by centrifugation; (2) A bacterial suspension is prepared by adding three times of phosphate buffer per gram of bacterial cell, and the cells are broken by ultrasonic treatment; (3) Centrifugation is performed to collect the supernatant, and the supernatant is the crude enzyme solution of superoxide dismutase; (4) The supernatant obtained in step (1) is centrifuged at high speed to obtain a precipitate, which is washed with phosphate buffer three times, centrifuged, and the nanosilver particles are collected, and the precipitate is resuspended with the crude enzyme solution of superoxide dismutase obtained in step (3) to obtain an extract containing superoxide dismutase and nanosilver particles.

4. A composition comprising the extract of claim 3, wherein, It comprises the following components: extract 20-50%, hyaluronic acid 15-30%, glycerol 0.1-3.0%, collagen 2-10%, beeswax 2-5%, olive oil 2-5%, and water 5-40%.

5. The composition of claim 4, wherein, The composition comprises the following components: extract 40%, hyaluronic acid 20%, glycerol 2%, collagen 5%, beeswax 3%, olive oil 3%, and water 27%.

6. A process for the preparation of a composition according to any one of claims 4-5, characterized in that, It comprises the following steps: the prescribed amount of superoxide dismutase and nanosilver particle extract, hyaluronic acid, glycerol, collagen, beeswax, olive oil and water are weighed and mixed uniformly, then placed in a water bath to emulsify while stirring, and then left to stand to obtain the composition.

7. The method of claim 6, wherein, The water bath temperature is 45-85℃, the stirring speed is 50-1200 rpm, and the emulsification time is 5-20 min.

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