American ginseng fermentation extract and application thereof

Abstract

The invention discloses an American ginseng fermentation extract and application thereof, and belongs to the technical field of traditional Chinese medicine fermentation and cosmetics. According to the method, American ginseng is fermented by adopting bacillus mojavensis, and operation and parameters of links such as preparation, fermentation, purification and freeze drying of a fermentation substrate are regulated and controlled, so that various enzymes generated in the metabolic process of the bacillus mojavensis are used for converting and modifying various active ingredients in the American ginseng, and the biological activity of the American ginseng is enhanced. Compared with unfermented American ginseng, the American ginseng fermentation extract prepared by the preparation method disclosed by the invention has the advantages that the soluble solids are obviously increased, the contents of active substances such as saponin and polyphenol are also obviously increased, the antioxidant activity is also obviously improved, and the American ginseng fermentation extract has an obvious improvement effect on oxidative damage cells, effectively improves the skin microenvironment and achieves the purpose of skin care; the important significance is realized on the development of anti-aging cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine fermentation and cosmetics, and more particularly to an American ginseng fermentation extract and application thereof. Background Art

[0002] At present, natural Chinese herbal medicine, as a green, safe and effective ingredient, has been widely used in the development of cosmetics and is loved by consumers.

[0003] American ginseng (Panax quinquefolius L.) is a perennial herbaceous plant of the genus Panax in the Araliaceae family. As a plant with both medicinal and edible properties, it is not only a precious Chinese medicinal material, but can also be used as a daily tonic to regulate the body. Modern pharmacological research has found that the main components of American ginseng include saponins, polyphenols, organic acids, sugars, sterols and other components. The prior art also studies the application of American ginseng in the field of cosmetics. For example, CN104069036A anti-aging cosmetic composition and preparation method thereof discloses the application of a mixture of licorice extract and American ginseng extract in a mass concentration of 1-15% in anti-aging cosmetics, which effectively improves the proliferation activity of skin cells and reduces the symptoms of skin aging; CN106727147A herbal American ginseng skin beauty improvement medicine and cosmetic application and preparation discloses a herbal American ginseng skin beauty improvement Chinese medicine compound fermentation liquid, which uses yeast to ferment a mixture of American ginseng, motherwort, Panax notoginseng, white poria and aloe, and the fermentation liquid is applied to the field of skin beauty improvement cosmetics, but the changes in the content of active ingredients in the samples before and after fermentation are not clear.

[0004] At present, the existing technology for processing American ginseng basically adopts water extraction, alcohol precipitation, concentration, macroporous adsorption resin or ion exchange resin purification, etc., which will introduce ethanol residues in the process. When the extract is used in cosmetics, it may cause skin irritation and other problems, which will have a negative impact on product safety and market reputation. Microbial fermentation of American ginseng can effectively increase the content of effective substances in it, and has the advantages of mild conditions and green environmental protection. At the same time, after fermentation, the granularity of American ginseng powder can be reduced, which improves the comfort of consumers.

[0005] However, the changes in beneficial ingredients in the products of American ginseng fermented by microorganisms of different species are not the same. Therefore, providing a new American ginseng fermentation extract is of great significance for cosmetic development. Summary of the invention

[0006] In view of the above problems, the present invention provides a fermented American ginseng extract and application thereof in cosmetics.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A fermented American ginseng extract is prepared by the following steps:

[0009] (1) Preparation of fermentation substrate: adding ultrapure water to American ginseng powder at a solid-liquid mass ratio of 1:5-20, ultrasonically mixing, adjusting the pH to 7, and then adding ultrapure water to make the volume to a mass concentration of American ginseng of 2.5-10%. After high-temperature and high-pressure steam sterilization, standing and cooling to room temperature, the American ginseng fermentation substrate is obtained; (2) Fermentation: inoculating the seed liquid of Bacillus mohaiwei into the American ginseng fermentation substrate, fermenting and culturing, and obtaining a fermentation liquid; (3) Fermentation: treating the fermentation liquid with a water bath, centrifuging, heat extracting the precipitate, centrifuging again under the same conditions, combining the supernatants after the two centrifugations, filtering through a 0.22 μm membrane, and obtaining a clarified liquid; (4) Freeze-drying the clarified liquid to obtain an American ginseng fermentation extract.

[0010] Preferably, the American ginseng fine powder in step (1) is obtained by crushing American ginseng roots and passing through a 50-200 mesh sieve.

[0011] Preferably, the ultrasonic conditions in step (1) are: frequency 30 kHz, ultrasound 30 min.

[0012] Preferably, the reagent used to adjust the pH in step (1) is NaOH solution.

[0013] Preferably, the conditions for high temperature and high pressure steam sterilization in step (1) are: 103.4 kPa, 121° C., 20 min.

[0014] Specifically, the Bacillus mohaiwei described in step (2) is preferably Bacillus mohaiwei H12, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 23, 2023, with a deposit number of CGMCC No.27186.

[0015] Preferably, the bacterial concentration of the Bacillus mohaiwei seed solution is 5×10 7 -5×10 8 CFU / mL, the inoculation amount of seed liquid is 2%-6%.

[0016] Preferably, the fermentation culture conditions in step (2) are: 30-37°C, 180 rpm, 24-72 h.

[0017] Preferably, the water bath treatment in step (3) is carried out under the conditions of 80-90° C. water bath treatment for 30-60 min.

[0018] Preferably, the centrifugation conditions in step (3) are: centrifugation at 6000-8000 rpm for 15-30 min at room temperature.

[0019] Preferably, the heat extraction in step (3) is as follows: ultrapure water is added to the precipitate at a volume ratio of 1:5-10, and heated at 60-80° C. for 1-2 h.

[0020] Preferably, the freeze-drying in step (4) is freeze-drying at -80°C for 48-72h.

[0021] Another object of the present invention is to provide the American ginseng fermentation extract obtained by the above method.

[0022] Another object of the present invention is to provide the use of the American ginseng fermented extract in anti-aging cosmetics.

[0023] Preferably, the types of anti-aging cosmetics include skin lotion, essence, lotion, face cream, eye cream, face mask or cleanser.

[0024] Another object of the present invention is to provide an anti-aging cosmetic comprising the American ginseng fermented extract.

[0025] Through the above technical scheme, it can be known that the present invention discloses a new American ginseng fermentation extract, which uses Bacillus mohaiwei to ferment American ginseng, regulates the operations and parameters of the preparation, fermentation, purification and freeze-drying of the fermentation substrate, and makes the various enzymes produced in the metabolic process of Bacillus mohaiwei transform and modify the various active ingredients in American ginseng, thereby enhancing its biological activity. The examples prove that compared with unfermented American ginseng, the soluble solids of American ginseng fermented with Bacillus mohaiwei are significantly increased, the content of active substances such as saponins and polyphenols is also significantly increased, and the antioxidant activity of the fermentation product is also significantly improved, which has a significant improvement effect on oxidatively damaged cells. At the same time, the metabolites of Bacillus can also effectively improve the skin microenvironment and achieve the purpose of skin care, which is of great significance for the development of anti-aging cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The American ginseng fermented extract prepared in Example 1;

[0027] Figure 2 : Results of determination of active ingredient contents in the fermentation broth of American ginseng after fermentation by Bacillus mohaiwei at different times, A: total phenols, B: total flavonoids, C: total saponins, * represents p<0.05 compared with unfermented American ginseng;

[0028] Figure 3 :Changes in oxidative activity of American ginseng after fermentation with Bacillus mohaiwei for different time periods, A: ABTS free radical scavenging rate, B: hydroxyl free radical scavenging rate, * represents p<0.05 compared with unfermented American ginseng;

[0029] Figure 4: Determination of the ability of the product of American ginseng fermented by Bacillus mohaiwei to improve cell oxidative damage, A: HSF cell viability determination, B: NIH-3T3 cell viability determination, * indicates p<0.05 compared with the normal group, # indicates p<0.05 compared with the model, Δ indicates p<0.05 compared with the same concentration of unfermented American ginseng;

[0030] Figure 5 : Determination of the content of active substances and antioxidant activity in American ginseng products fermented by different strains, A: Changes in the content of active substances, B: Antioxidant activity, where (W) represents the water extract of the fermentation product, and (M) represents the methanol extract of the fermentation product. * indicates p<0.05, and ** indicates p<0.01. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0033] The Bacillus mohaiwei H12 involved in the embodiment was screened and obtained by the applicant, has been disclosed in CN117511788A, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on April 23, 2023, with a deposit number of CGMCC No. 27186, and can be obtained through the Shaanxi Institute of Microbiology or the corresponding deposit center.

[0034] Lactobacillus pentosus 068-1 was screened and obtained by the applicant, has been disclosed in CN115058367A, and was deposited in the General Microbiology Center of the China Culture Collection Administration on February 23, 2022, with the deposit number CGMCC No. 24424. It can be obtained through the Shaanxi Institute of Microbiology or the corresponding deposit center.

[0035] Bacillus mohaiwei seed solution: After Bacillus mohaiwei H12 was activated for two generations, it was inoculated into YPD medium and cultured in a shaking incubator at 30°C and 180 rpm for 24 h to obtain seed solution. The bacterial concentration of the seed solution was adjusted to 10 8 CFU / mL, 5×10 7 CFU / mL, 5×10 8 CFU / mL, for future use.

[0036] Example 1 Preparation of American ginseng fermented extract

[0037] (1) Weigh 5 g of American ginseng powder, add 50 mL of ultrapure water, and mix them evenly by ultrasonication at a frequency of 30 kHz for 30 min. Adjust the pH to 7.0 with a NaOH solution (0.25 mol / L), and then add ultrapure water to make the volume 100 mL to obtain an American ginseng suspension with a mass concentration of 5%. After high-temperature steam sterilization at 121° C. for 20 min, let it cool at room temperature to obtain an American ginseng fermentation substrate.

[0038] (2) The bacterial concentration was 10 8 CFU / mL of Bacillus mohaiwei seed liquid was inoculated into the American ginseng fermentation substrate at an inoculation rate of 4%, and the fermentation culture was carried out at 30° C. and 180 rpm for 72 hours, and the fermentation was terminated to obtain a fermentation liquid.

[0039] (3) After the fermentation broth was treated in a 90°C water bath for 30 min, it was centrifuged at 6000 rpm for 15 min at room temperature to collect the supernatant; 5 times the volume of ultrapure water was added to the precipitate, heated and extracted at 60°C for 1 h, and centrifuged again at 6000 rpm for 15 min to collect the supernatant, and the supernatants from the two centrifugations were combined and filtered through a 0.22 μm membrane to remove insoluble solids to obtain a transparent clarified liquid.

[0040] (4) The clarified liquid was freeze-dried at -80°C for 48 h to obtain the American ginseng fermentation extract (see schematic diagram). Figure 1 ).

[0041] Example 2

[0042] (1) Weigh 2.5 g of American ginseng powder, add 50 mL of ultrapure water, and perform ultrasonic treatment at a frequency of 30 kHz for 30 min to mix the mixture evenly. Adjust the pH to 7.0 with a NaOH solution (0.25 mol / L), and then add ultrapure water to make the volume 100 mL to obtain an American ginseng suspension with a mass concentration of 2.5%. After high-temperature steam sterilization at 121° C. for 20 min, the mixture was allowed to cool at room temperature to obtain an American ginseng fermentation substrate.

[0043] (2) The bacterial concentration was 5×10 7 CFU / mL of Bacillus mohaiwei seed liquid was inoculated into the American ginseng fermentation substrate at an inoculation rate of 2%, and the fermentation culture was carried out at 32° C. and 180 rpm for 48 hours, and the fermentation was terminated to obtain a fermentation liquid.

[0044] (3) After the fermentation broth was treated in an 80°C water bath for 30 min, it was centrifuged at 6000 rpm for 15 min at room temperature to collect the supernatant; 10 times the volume of ultrapure water was added to the precipitate, heated and extracted at 60°C for 1 h, and centrifuged again at 6000 rpm for 15 min to collect the supernatant, and the supernatants of the two centrifugations were combined and filtered through a 0.22 μm membrane to remove insoluble solids to obtain a transparent clarified liquid.

[0045] (4) freeze-drying the clarified liquid at -80°C for 48 hours to obtain the American ginseng fermentation extract.

[0046] Example 3

[0047] (1) Weigh 10 g of American ginseng powder, add 50 mL of ultrapure water, and mix them evenly by ultrasonication at a frequency of 30 kHz for 30 min. Adjust the pH to 7.0 with a NaOH solution (0.25 mol / L), and then add ultrapure water to make the volume 100 mL to obtain an American ginseng suspension with a mass concentration of 10%. After high-temperature steam sterilization at 121° C. for 20 min, let it cool at room temperature to obtain an American ginseng fermentation substrate.

[0048] (2) The bacterial concentration was 5×10 8 CFU / mL of Bacillus mohaiwei seed liquid was inoculated into American ginseng fermentation substrate at an inoculation rate of 6%, and fermentation culture was carried out at 37° C. and 180 rpm for 72 hours, and fermentation was terminated to obtain fermentation liquid.

[0049] (3) After the fermentation broth was treated in a 90°C water bath for 60 min, it was centrifuged at 8000 rpm for 30 min at room temperature and the supernatant was collected; 5 times the volume of ultrapure water was added to the precipitate, heated and extracted at 80°C for 2 h, and centrifuged again at 6000 rpm for 30 min, and the supernatant was collected. The supernatants from the two centrifugations were combined and filtered through a 0.22 μm membrane to remove insoluble solids to obtain a transparent clarified liquid.

[0050] (4) freeze-drying the clarified liquid at -80°C for 72 hours to obtain the American ginseng fermentation extract.

[0051] Example 4 Safety Testing of American Ginseng Fermented Extract

[0052] 1. Heavy metal detection

[0053] According to the method shown in Chapter 4 of the "Technical Specifications for Safety of Cosmetics (2015 Edition)", the American ginseng fermented extract obtained in Example 1 was tested for the content of four heavy metals: lead, arsenic, mercury, and cadmium. The test results are shown in Table 1.

[0054]

[0055] As can be seen from Table 1, the heavy metal contents in the American ginseng fermentation extract prepared by the present invention meet the requirements of the cosmetic safety technical specifications.

[0056] 2. Microbiological index test

[0057] According to the "Technical Specification for Safety of Cosmetics (2015 Edition)", the American ginseng fermented extract prepared in Example 1 was tested for microbial limits. The results are shown in Table 2.

[0058]

[0059] As can be seen from Table 2, the five microbial indicators in the American ginseng fermentation extract prepared by the present invention are all within the limits specified in the "Technical Specification for Safety of Cosmetics (2015 Edition)" and meet the safety standards.

[0060] 3. Acute eye irritation test

[0061] The American ginseng fermented extract prepared in Example 1 was used as the test sample, and Japanese big-eared white rabbits (female, 2.17-2.37 kg, ordinary grade) were selected as test animals. The two eyes of the test animals were examined within 24 hours before the start of the test, and rabbits with eye irritation symptoms, corneal defects and conjunctival damage were excluded. Three rabbits with normal eyes were randomly selected, and their heads were tilted so that the test eyes were tilted upward, and the lower eyelids were gently pulled open. 0.1g of the test sample was directly placed in the conjunctival sac, and the eyelids were closed for 1s. No rinsing was performed within 24 hours. The other eye was used as a self-control. The damage to the conjunctiva, cornea and iris was checked and recorded at 1h, 24h, 48h, 72h, 4d, and 7d after the sample was given. Scoring was performed according to the "Eye Damage Scoring Standard" table in the "Technical Specifications for Safety of Cosmetics" (2015 Edition). After 24 hours, the eyes of all animals were further examined using sodium fluorescein. If no irritation reaction occurred after 72 hours, the test was terminated. Otherwise, the observation time needed to be extended, not exceeding 21 days. After the test, the eye irritation intensity was graded according to the integrated mean and recovery time of the irritation reaction of the cornea, iris, and conjunctiva of the animals at the observation points of 24 hours, 48 ​​hours, and 72 hours, according to the "Raw Material Eye Irritation Reaction Grading" table. The experimental results are shown in Table 3.

[0062]

[0063]

[0064] It can be seen from Table 3 that under the condition of (no flushing), the American ginseng fermentation extract prepared by the present invention has no acute eye irritation to rabbits.

[0065] 4. Skin irritation test

[0066] The American ginseng fermented extract prepared in Example 1 was used as the test sample, and 4 Japanese big-eared white rabbits (female, 2.17-2.34 kg, ordinary grade) were selected as test animals. 24 hours before the start of the test, the hair on both sides of the rabbit's back spine was removed, and the skin must not be damaged. The hair removal range was about 3 cm × 3 cm on the left and right. Take 0.5 g of the test sample, moisten it with pure water and apply it on one side of the skin with a size of 2.5 cm × 2.5 cm (carefully and slowly apply it to avoid sample loss), and apply the same amount of pure water on the other side as a control, and apply it continuously for 14 days, 1 day / time. Starting from the second day, the hair was sheared before each application, and the residual test sample was removed with water. After 1 hour, the application site was observed and recorded with the naked eye for erythema and edema, and scored according to the "Skin Irritation Reaction Score" table in the "Cosmetic Safety Technical Specifications (2015 Edition)". After the test, the average score of the skin reaction of each animal was calculated, and the skin irritation intensity was graded according to the "Skin Irritation Intensity Grading" table. The test results are shown in Table 4.

[0067]

[0068]

[0069] It can be seen from Table 4 that the American ginseng fermented extract prepared by the present invention has no skin irritation to rabbits.

[0070] 5. Skin phototoxicity test

[0071] The American ginseng fermented extract prepared in Example 1 was used as the test sample, and 6 Hartley guinea pigs were selected as test animals (3 males and 3 females, weighing 264-275g, ordinary grade). 18-24h before the start of the test, according to the "Technical Specifications for Safety of Cosmetics (2015 Edition)" Chapter 6 7 Skin Phototoxicity Test, the hair on both sides of the spine of the guinea pig's back was removed (the hair removal process must not damage the skin, and the skin in the hair removal area must be kept intact), the left side is 1, 3, and the right side is hair removal area 2, 4, each hair removal area is 2cm×2cm. The animal was fixed on the corresponding fixing frame, and 0.2g of the sample was applied to the hair removal area 1 and 2 after moistening with pure water, and the same amount of pure water was applied to the hair removal area 3 and 4 as a control. After 30 minutes, the left side (hair removal area 1 and 3) was covered with aluminum foil, fixed with tape, and placed in the skin phototoxicity instrument, and the irradiation dose was set to 10000mJ / cm 2 UVA irradiation was performed. Skin reactions were observed 1h, 24h, 48h, and 72h after the irradiation, and scores were performed according to "Table 2 Skin Irritation Reaction Scoring Table" in Chapter 6.7 of the "Technical Specifications for Safety of Cosmetics (2015 Edition)". The results are shown in Table 5.

[0072]

[0073]

[0074] The criterion for judging whether the test sample is phototoxic to the skin is: when there is no skin reaction in the unirradiated area after the test sample is applied alone, and the number of animals with a skin reaction score of 2 or more in the irradiated area after the test sample is applied is 1 or more, the test sample is judged to be phototoxic, otherwise it is non-phototoxic. As shown in Table 5, the American ginseng fermented extract prepared by the present invention is non-phototoxic to the guinea pig skin.

[0075] Example 5 Functional detection of American ginseng fermented extract

[0076] According to step (1) of Example 1, a 5% mass concentration of American ginseng culture medium was prepared, the pH of the mixed solution was adjusted to 7, and after high pressure steam sterilization at 121°C for 20 min, 4% of the seed solution of Bacillus mohaiwei H12 was added. The mixture was cultured at 30°C and 180 rpm in a shaking table for 7 days, with the start of the culture being marked as day 0, and the fermentation liquid was sampled every 24 hours for subsequent testing.

[0077] 1. Active ingredient content detection

[0078] The total phenol content in the fermentation products at different time points was determined by the Folin phenol method. Figure 2 A; The content of total flavonoids in the fermentation products at different time points was determined by aluminum salt colorimetric method. The results are shown in Figure 2 B; The vanillin colorimetric method was used to determine the content of total saponins in the fermentation products at different time points. The results are shown in Figure 2 C.

[0079] Depend on Figure 2 B shows that compared with the unfermented American ginseng (initial total flavonoid content 0.013 mg / mL), the total flavonoid content did not change significantly after 1 day of fermentation. On the third day of cultivation, the total flavonoid content increased significantly to 0.061 mg / mL. The total flavonoid content did not change significantly when the fermentation time was extended. Similarly, compared with the total phenol content in unfermented American ginseng (0.024 mg / mL), the total phenol content increased to 0.168 mg / mL after 3 days of fermentation (see Figure 2 A). In addition, the total saponin content increased from 0.643 mg / mL in the initial unfermented state to a maximum of 2.871 mg / mL after fermentation, which is about 4 times the initial value (see Figure 2 C).

[0080] 2. Antioxidant Activity Analysis

[0081] The fermentation broth at different time points was centrifuged and the supernatant was taken. The liquid obtained after filtering with a 0.22 μm membrane was used as the test sample for ABTS free radical scavenging test. The specific operation was as follows: 2.45 mM potassium persulfate solution was used to dissolve ABTS, mixed evenly and kept in dark for 14 h to obtain 7 mM ABTS+ mother solution, the ABTS+ mother solution was diluted with PBS to an absorbance of 0.7, and kept in dark at room temperature for use; 25 μL of the test sample was added to a 96-well plate, and then 175 μL of the ABTS+ mother solution was added, and the reaction was carried out in dark at room temperature for 5 min, and the absorbance was detected at a wavelength of 734 nm to calculate the ABTS free radical scavenging rate. The results are shown in the table. Figure 3 A.

[0082] The hydroxyl radical scavenging activity of the sample was determined by Fenton colorimetry. The test results were as follows: Figure 3 As shown in B.

[0083] from Figure 3 A It can be seen that the scavenging antioxidant activity of unfermented American ginseng is low, with an ABTS free radical scavenging rate of about 50%, and almost no hydroxyl free radical scavenging activity. After fermentation by Bacillus mohaiwei, its antioxidant activity increased significantly, and the ABTS free radical scavenging activity of American ginseng extract after fermentation for 3 days increased to more than 95%. Similarly, the hydroxyl free radical scavenging rate of the fermented American ginseng product increased after 1 day of fermentation, and the hydroxyl free radical scavenging rate reached more than 90% after 3 days of fermentation. In summary, the American ginseng product fermented by Bacillus mohaiwei has good in vitro antioxidant activity (see Figure 3 B).

[0084] 3. Repair ability of oxidatively damaged cells

[0085] The fermented American ginseng product fermented for 3 days was dissolved in cell culture medium (DMEM medium + 10% FBS + 1% penicillin-streptomycin mixture) into 100 mg / mL sample stock solution, filtered through a 0.22 μm membrane, and stored at 4°C for later use. The unfermented American ginseng was dissolved in cell culture medium (DMEM medium + 10% FBS + 1% penicillin-streptomycin mixture) into 100 mg / mL sample solution as a control.

[0086] HSF cells and NIH-3T3 cells were added to 96-well plates at 5000 cells / well, and after the cells adhered to the wall, the supernatant was discarded and the following treatments were performed:

[0087] Normal group: add 200 μL complete culture medium (DMEM+10% FBS+1% penicillin-streptomycin mixture);

[0088] Model group: add 200 μL of 0.98 mM H 2 O 2After pretreatment with the culture medium for 1 hour, the supernatant was removed, the cells were rinsed with PBS, and complete culture medium (DMEM+10% FBS+1% penicillin-streptomycin mixture) was added;

[0089] HSF cell test group: add 200 μL of 0.98 mM H 2 O 2 After pretreatment with the culture medium for 1 h, the supernatant was removed, and the cells were rinsed with PBS. Then, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL of American ginseng fermentation dilutions were added in parallel, respectively, and recorded as F 0.25, F 5, and F 1, respectively;

[0090] HSF cell control group: add 200 μL of 0.98 mM H 2 O 2 After pretreatment with the culture medium for 1 hour, the supernatant was removed, and the cells were rinsed with PBS. Then, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL of unfermented American ginseng dilution were added in parallel, respectively, and recorded as NF 0.25, NF 5, and NF 1, respectively;

[0091] NIH-3T3 cell test group: add 200 μL of 0.98 mM H 2 O 2 After pretreatment with the culture medium for 1 hour, the supernatant was removed, and the cells were rinsed with PBS. Then, 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL of American ginseng fermentation dilutions were added in parallel, respectively, and recorded as F 0.01, F 0.05, and F 0.1;

[0092] NIH-3T3 cell control group: add 200 μL of 0.98 mM HO 2 O 2 After pretreatment with the culture medium for 1 hour, the supernatant was removed, and the cells were rinsed with PBS. Then, unfermented American ginseng dilutions with concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL were added in parallel, respectively, and recorded as NF 0.01, NF 0.05, and NF 0.1;

[0093] After the above treatment groups were cultured at 37°C for 24 h, the cell activity was detected by MTT. Figure 4 AB.

[0094] Depend on Figure 4 As shown in A, H 2 O 2After treatment, the viability of HSF cells decreased to 80%. When treated with unfermented American ginseng, its activity was significantly improved (88%) only when the concentration reached 1 mg / mL. However, the cell viability at three concentrations of fermented American ginseng reached more than 90%, which was significantly different from that of unfermented American ginseng at the same concentration (p<0.05). Figure 4 B), simple H 2 O 2 The cell viability of the induced group was about 40%, and the cell viability of the unfermented American ginseng (NF) treatment did not increase significantly compared with the model group. The products of American ginseng fermented by Bacillus mohaiwei significantly promoted cell proliferation at all experimental concentrations. 2 O 2 Stimulation-induced oxidative damage in both fibroblasts had a good protective effect.

[0095] Example 6 Comparison of the activity of American ginseng products fermented by different strains

[0096] In a solution containing 5% American ginseng substrate, 10 8 CFU / mL of Lactobacillus pentosus 068-1 (strain collection number: CGMCC No. 24424) and Bacillus mohaiwei H12 were fermented under the same conditions for 48 hours, 0.5 g of the fermentation product obtained after freeze-drying was weighed, and 5 mL of water (W) or methanol (M) was added for extraction at the same time, ultrasonic oscillation was performed for 2 hours, and the supernatant was taken after centrifugation at 6000 rpm. The content of polyphenols and flavonoids and the antioxidant activity thereof were determined respectively. The results are shown in Figure 5 AB.

[0097] from Figure 5 As can be seen from A, the total phenol content in the American ginseng fermented by Lactobacillus pentosus 68-1 has no significant change compared with the unfermented American ginseng, and the total flavonoids content has increased slightly. The content of active substances in American ginseng fermented by Bacillus mohaiwei H12 (total phenols: 3.21 mg / g, total flavonoids: 2.40 mg / g) has been significantly increased compared with both the unfermented American ginseng and the American ginseng fermented by Lactobacillus pentosus 68-1.

[0098] from Figure 5 From the antioxidant activity shown in B, the antioxidant activity of the ethanol extract (M) of American ginseng fermented by Lactobacillus pentosus 68-1 and Bacillus mohaiwei H12 was improved, but the activity of the fermentation product of Bacillus mohaiwei H12 was higher (DPPH free radical scavenging rate 29.06%, ABTS free radical scavenging rate 49.45%), which was significantly different from that of the fermentation product of Lactobacillus pentosus 68-1 (p<0.01), and the antioxidant activity of the water-extracted fermentation product (W) of Bacillus mohaiwei H12 was also significantly improved (91.58%).

[0099] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0100] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a fermented American ginseng extract, characterized in that: The following steps are involved: (1) Preparation of fermentation substrate: adding ultrapure water to American ginseng powder at a solid-liquid mass ratio of 1:5-20, ultrasonically mixing, adjusting the pH to 7, and then adding ultrapure water to a fixed volume to a concentration of American ginseng of 2.5%-10%, sterilizing with high-temperature and high-pressure steam, and cooling to room temperature to obtain an American ginseng fermentation substrate; (2) fermentation: inoculating the seed liquid of Bacillus mohaiwei into the American ginseng fermentation substrate, performing fermentation culture, and obtaining a fermentation liquid; (3) Purification: The fermentation broth was treated in a water bath, centrifuged, the precipitate was subjected to heat extraction, and centrifuged again under the same conditions. The supernatants after the two centrifugations were combined and filtered through a 0.22 μm membrane to obtain a clarified solution; (4) freeze-drying the clarified liquid to obtain the American ginseng fermentation extract.

2. The method for preparing a fermented American ginseng extract according to claim 1, characterized in that: The bacterial concentration of the Bacillus mohaiwei seed solution in step (2) is 5×10 7 -5×10 8 CFU / mL, the inoculation amount of seed liquid is 2%-6%.

3. The method for preparing a fermented American ginseng extract according to claim 1, characterized in that: The fermentation culture conditions in step (2) are: 30-37°C, 180rpm, 24-72h.

4. The method for preparing a fermented American ginseng extract according to claim 1, characterized in that: The conditions of the water bath treatment in step (3) are: 80-90° C. water bath treatment for 30-60 min. The centrifugal conditions are: 6000-8000 rpm for 15-30 min at room temperature.

5. The method for preparing a fermented American ginseng extract according to claim 1, characterized in that: The heat extraction in step (3) is as follows: ultrapure water is added to the precipitate at a volume ratio of 1:5-10, and heated at 60-80° C. for 1-2 hours.

6. The method for preparing a fermented American ginseng extract according to claim 1, characterized in that: The freeze drying in step (4) is: freeze drying at -80°C for 48-72h.

7. The American ginseng fermented extract obtained according to any one of claims 1 to 6.

8. Use of the American ginseng fermented extract obtained according to any one of claims 1 to 6 or the American ginseng fermented extract according to claim 7 in anti-aging cosmetics.

9. The use according to claim 8, characterized in that: The types of anti-aging cosmetics include skin lotion, essence, lotion, face cream, eye cream, face mask or cleanser.

10. An anti-aging cosmetic, characterized in that: The anti-aging cosmetic comprises the American ginseng fermented extract obtained by the method according to any one of claims 1 to 6 or the American ginseng fermented extract according to claim 7.

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