Rosa roxburghii tratt ergothioneine fermentation liquor as well as preparation method and application thereof
Through staged fermentation of complex bacterial species, the combination of prickly pear and ergothioneine solves the problem of lack of combined utilization methods in the prior art, achieves ideal anti-aging and antioxidant effects, and avoids side effects of chemical compositions.
Patent Information
- Application Number
- CN202411809150.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art lacks a method for combining ergothionine with ergothionine, and ergothionine chemical compositions have side effects on the human body.
The staged fermentation is carried out through the complex bacterial strains, the active substances in the prickly pear are released, and ergothionein is added during the secondary fermentation process to improve the synergistic effect of mixed fermentation.
It has achieved ideal anti-aging, antioxidant, promoting cell metabolism and moisturizing effects, and has mild fermentation conditions and has good application prospects.
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Figure CN119606807A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of fermentation technology, and in particular relates to a roxburghii ergothioneine fermentation liquid, and a preparation method and application thereof. Background Art
[0002] Rosa roxburghii Tratt is a wild fruit native to southwest my country. It mainly contains active ingredients such as polysaccharides, flavonoids, superoxide dismutase (SOD), vitamin C (VC) and triterpenes. It has pharmacological effects such as antioxidant, anti-tumor, anti-atherosclerosis, prevention and treatment of diabetes, radiation protection and anti-apoptosis.
[0003] Ergothioneine, a natural antioxidant, is a natural amino acid found in many plants and animals. It cannot be synthesized by the human body and needs to be ingested through food. Ergothioneine has a strong antioxidant property. As a unique cell physiological protector, it plays a variety of physiological functions in the human body, such as removing free radicals in the body, protecting cells from oxidative damage, protecting mitochondrial DNA, reducing cell membrane damage, reducing protein glycation, and delaying skin aging. It can be seen that ergothioneine has many effects on human cells.
[0004] At present, due to the large demand for ergothioneine and the inability to meet the market demand, chemically synthesized ergothioneine is generally used. When using such artificially synthesized substances, it is gradually discovered that they are prone to cause adverse effects on the human body, such as sensitization, redness, irritation and other side effects.
[0005] Therefore, there is an urgent need for a method that effectively combines the advantages of Rosa roxburghii with the effects of ergothioneine and effectively improves the adverse reactions of such chemical synthetics during use. Summary of the invention
[0006] The technical problem to be solved in the present application is: the prior art lacks the method of combining roxburghii and ergothioneine, and ergothioneine chemical synthesis has side effects on human body and other problems, and then the present application provides a roxburghii ergothioneine fermentation liquid, and its preparation method and application. The present application utilizes composite strains, and by the mode of staged fermentation, the active substance in roxburghii is released in a primary fermentation process, ergothioneine is added in a secondary fermentation process, the two are mixed and fermented, the synergistic effect of mixed fermentation is improved, and the obtained product is made to achieve the desired anti-aging, anti-oxidation, promote cell metabolism and moisturizing and other effects, and the fermentation conditions are mild, with good application prospects.
[0007] This application adopts the following technical solutions to solve the above technical problems:
[0008] The present application provides a method for preparing thioneine fermentation liquid of roxburghii, which comprises the following steps: inoculating lactobacillus A into a fermentation substrate, obtaining a preliminary fermentation liquid through a primary fermentation, adding thioneine into the preliminary fermentation liquid, inoculating lactobacillus B through a secondary fermentation, and sterilizing;
[0009] Wherein, the fermentation substrate includes roxburghii and water; the lactobacillus A includes Lactobacillus delbrueckii subspecies bulgaricus, and the lactobacillus B includes Lactobacillus plantarum.
[0010] In some embodiments, the ratio of the viable cell count of the Lactobacillus delbrueckii subspecies bulgaricus to the viable cell count of the Lactobacillus plantarum is 1:(0.2-1), preferably 1:(0.5-1).
[0011] In some embodiments, the Lactobacillus A is added in the form of a Lactobacillus A bacterial liquid according to conventional practice in the art.
[0012] In some embodiments, the Lactobacillus delbrueckii subsp. bulgaricus includes Lactobacillus delbrueckii subsp. bulgaricus deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC20271.
[0013] In some embodiments, the Lactobacillus delbrueckii subspecies bulgaricus is added in the form of a bacterial solution of Lactobacillus delbrueckii subspecies bulgaricus, and the live bacteria concentration of the bacterial solution of Lactobacillus delbrueckii subspecies bulgaricus is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.
[0014] The method for preparing the bacterial liquid of Lactobacillus delbrueckii subspecies bulgaricus comprises: inoculating Lactobacillus delbrueckii subspecies bulgaricus into MRS culture medium and culturing at 37° C. for 24 hours.
[0015] In some embodiments, based on the water in the fermentation substrate, the number of viable bacteria of Lactobacillus delbrueckii subspecies bulgaricus inoculated per unit volume of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.
[0016] In some embodiments, the Lactobacillus B is added in the form of a Lactobacillus B bacterial solution according to conventional practice in the art.
[0017] In some embodiments, the Lactobacillus plantarum includes Lactobacillus plantarum deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 24936.
[0018] In some embodiments, the plant lactobacillus is added in the form of a plant lactobacillus bacterial solution. The live bacteria concentration of the plant lactobacillus bacterial solution is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.
[0019] The preparation method of the Lactobacillus plantarum liquid comprises: inoculating the Lactobacillus plantarum into an MRS culture medium and culturing at 37° C. for 24 hours.
[0020] In some embodiments, based on the water in the fermentation substrate, the number of viable Lactobacillus plantarum inoculated per unit volume of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.
[0021] In some embodiments, the method for preparing the fermentation substrate comprises: mixing the components evenly.
[0022] In some embodiments, the roxburghii roxburghii powder is prepared by crushing and sieving dried roxburghii roxburghii.
[0023] Wherein, the sieved particle size is 50 to 200 meshes.
[0024] In some embodiments, the mass ratio of the roxburghii to the water is 1:(20-200), preferably 1:(50-200), and more preferably 1:100.
[0025] In some embodiments, the fermentation substrate further includes sterilization and cooling operations before use.
[0026] Wherein, the sterilization is a high temperature sterilization method.
[0027] When the high temperature sterilization method is used for the sterilization, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C. When the high temperature sterilization method is used for the sterilization of the fermentation substrate, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.13 MPa. When the high temperature sterilization method is used for the sterilization of the fermentation substrate, the sterilization time can be a conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.
[0028] The cooling is cooling to room temperature.
[0029] In some embodiments, the ergothioneine is ergothioneine in a sterile state.
[0030] In some embodiments, the mass ratio of the ergothioneine to the roxburghii roxburghii is 1:(0.2-5), preferably 1:3.
[0031] In some embodiments, the steps of sterilization and cooling are further performed after adding ergothioneine.
[0032] Wherein, the sterilization is a high temperature sterilization method.
[0033] When the high temperature sterilization method is used for the sterilization, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C. When the high temperature sterilization method is used for the sterilization, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.13 MPa. When the high temperature sterilization method is used for the sterilization, the sterilization time can be a conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.
[0034] The cooling is cooling to room temperature.
[0035] In some embodiments, the primary fermentation is static fermentation, wherein the static fermentation is conventionally performed in a constant temperature incubator in the art.
[0036] In some embodiments, the temperature of the primary fermentation is 30-45°C, preferably 35-40°C, for example 37°C.
[0037] In some embodiments, the time of the primary fermentation is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours.
[0038] In some embodiments, after a fermentation, the method may further include at least one of centrifuging and collecting the supernatant, sterilizing and cooling.
[0039] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.
[0040] The centrifugal rotation speed is a conventional rotation speed for this type of operation in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm.
[0041] Wherein, the radius of the centrifuge is 8 to 15 cm.
[0042] The centrifugation time is conventional in the art for this type of operation, preferably 10 to 40 min, more preferably 20 to 40 min, for example 30 min.
[0043] Wherein, when the high temperature sterilization method is used for the sterilization, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C. When the high temperature sterilization method is used for the sterilization, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.13 MPa. When the high temperature sterilization method is used for the sterilization, the sterilization time may be a conventional time for such operations in the art, preferably 15 to 35 min, more preferably 15 to 25 min, for example 20 min.
[0044] In some embodiments, the secondary fermentation is static fermentation. Preferably, the static fermentation is conventionally performed in a constant temperature incubator in the art.
[0045] In some embodiments, the temperature of the secondary fermentation is 30-45°C, preferably 35-40°C, for example 37°C.
[0046] In some embodiments, the secondary fermentation time is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours.
[0047] In some embodiments, the sterilization is high temperature sterilization.
[0048] When the high temperature sterilization method is used for the sterilization, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C. When the high temperature sterilization method is used for the sterilization, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.13 MPa. When the high temperature sterilization method is used for the sterilization, the sterilization time can be a conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.
[0049] In some embodiments, after the sterilization, the method may further include at least one of cooling, centrifuging and collecting the supernatant, sterilizing, or mixing with a preservative.
[0050] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.
[0051] The centrifugal rotation speed is a conventional rotation speed for this type of operation in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm.
[0052] Wherein, the radius of the centrifuge is 8 to 15 cm.
[0053] The centrifugation time is conventional in the art for this type of operation, preferably 10 to 40 min, more preferably 20 to 40 min, for example 30 min.
[0054] The sterilization method is generally a high temperature sterilization method. The sterilization temperature is a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.
[0055] The sterilization pressure is the conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.13 MPa. The sterilization time is the conventional time for such operations in the art, preferably 15-35 min, more preferably 15-25 min, for example 20 min.
[0056] Wherein, during the mixing process with the preservative, the mixing temperature is a conventional temperature for such operation in the art, preferably 50 to 80°C, more preferably 70 to 80°C.
[0057] Wherein, in the process of mixing with the preservative, the preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional practice in the art.
[0058] When the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization may be 0.5% to 2%, preferably 0.5%.
[0059] When the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5%.
[0060] In some embodiments, the room temperature generally refers to 15-40°C.
[0061] The present application also provides a roxburghii ergothioneine fermentation liquid, which is prepared by the preparation method of the roxburghii ergothioneine fermentation liquid as described above.
[0062] The present application also provides a roxburghii ergothioneine fermentation liquid, which is used directly as a product, as an additive or as a base in the preparation of a skin topical preparation.
[0063] In some embodiments, the ergothioneine fermentation liquid of Rosa roxburghii is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and soothing ingredient in the skin topical preparation.
[0064] The antioxidant active ingredients are antioxidant active ingredients with DPPH free radical scavenging effect and antioxidant active ingredients with hydroxyl free radical scavenging effect.
[0065] Wherein, the anti-aging active ingredient is an anti-aging active ingredient having a cell proliferation effect.
[0066] Wherein, the anti-allergic active ingredient is an anti-allergic active ingredient having the effect of inhibiting hyaluronidase.
[0067] The present application also provides a skin external preparation, which comprises the roxburghii ergothioneine fermentation liquid as described above.
[0068] In some embodiments, the skin topical preparation further includes active ingredients conventionally used in the art, which generally may include at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.
[0069] In some embodiments, the topical skin preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.
[0070] In some embodiments, the mass percentage of the ergothioneine fermentation liquid of Rosa roxburghii to the skin external preparation may be 5% to 99%, preferably 60% to 99%.
[0071] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.
[0072] The reagents and raw materials used in this application are commercially available.
[0073] The positive and progressive effect of the present application is: the present application carries out multi-stage fermentation of ergothioneine and roxburghii by means of step-by-step fermentation, in a primary fermentation process, the active substance in roxburghii is effectively released, ergothioneine is added to the fermentation liquid during the secondary fermentation process, the active substance in the fermentation liquid of ergothioneine is avoided to decompose, and multiple fermentations are performed to improve the release of the active substance in roxburghii, to achieve the desired anti-aging, anti-oxidation, promotion of cell repair and moisturizing effects, and the fermentation conditions are mild, with good application prospects. At the same time, the prepared product can be used directly as cosmetics and can also be added to the basic formula of cosmetics for use, with good compacting, soothing and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0074] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.
[0075] in:
[0076] Figure 1 This is a comparison chart of the DPPH free radical scavenging ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;
[0077] Figure 2 A comparison chart of the hydroxyl radical scavenging abilities of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;
[0078] Figure 3 This is a comparison chart of cell viability after cells were treated with products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;
[0079] Figure 4 This is a comparison chart of intracellular COL-I content after cells were treated with the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4. DETAILED DESCRIPTION
[0080] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions.
[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0082] The Lactobacillus delbrueckii subsp. bulgaricus in the following examples and comparative examples was purchased from the China Industrial Microbiological Culture Collection Administration Center with a collection number of CICC 20271.
[0083] The Lactobacillus plantarum in the following examples and comparative examples was purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 24936.
[0084] The rice wine yeast in the following comparative example was purchased from Beijing Food Brewing Research Institute, and the strain number is AS2.1392.
[0085] The preparation method of Lactobacillus delbrueckii subsp. bulgaricus bacterial solution is as follows: Lactobacillus delbrueckii subsp. bulgaricus is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable cells of the Lactobacillus delbrueckii bacterial solution is 10 10 CFU / mL.
[0086] The preparation method of Lactobacillus plantarum bacterial solution is as follows: Lactobacillus plantarum is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable bacteria in the Lactobacillus plantarum bacterial solution is 10 10 CFU / mL.
[0087] Example 1
[0088] 3 g of roxburghii fruit powder and 300 mL of deionized water were mixed evenly, sterilized in a high-pressure steam sterilizer at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cooled to room temperature after sterilization to obtain a fermentation substrate;
[0089] Inoculate Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid for primary fermentation, the addition amount of Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid is 3mL, access Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid and shake well, stand and ferment in a 37°C constant temperature incubator for 15h, after the primary fermentation is completed, sterilize in a high pressure steam sterilizer under the conditions of 121°C and 0.12MPa for 30min, cool to room temperature after sterilization, after sterilization, cool to room temperature and centrifuge, wherein, the centrifugal speed is 4800r / min, the centrifugal time is 30min, the centrifugal radius is 15cm, and 240g of preliminary fermentation liquid is obtained after centrifugation;
[0090] After centrifugation, sterilize in a high pressure steam autoclave for 30 min at a temperature of 121 ° C and a pressure of 0.12 MPa, cool to room temperature after sterilization, add 1 g of thioneine to the preliminary fermentation liquid, access 2.4 mL of plant lactobacillus liquid, shake well after accessing the liquid, and stand in a 37 ° C constant temperature incubator for 15 h for secondary fermentation;
[0091] After the secondary fermentation, sterilize in a high pressure steam sterilizer at a temperature of 121°C and a pressure of 0.12MPa for 30 minutes. After sterilization, cool to room temperature and centrifuge at a speed of 4800r / min, a centrifugal time of 30 minutes, and a centrifugal radius of 15cm. After centrifugation, sterilize in a high pressure steam sterilizer at a temperature of 121°C and a pressure of 0.12MPa for 30 minutes. After sterilization, add preservatives, and the mixing temperature is 75°C during the mixing process with the preservatives; add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol based on the mass of the fermentation stock solution, and mix evenly.
[0092] The preparation method of Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid is as follows: Lactobacillus delbrueckii subspecies bulgaricus is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable bacteria of the Lactobacillus delbrueckii bacterial liquid is 10 10 CFU / mL.
[0093] The preparation method of Lactobacillus plantarum liquid is as follows: Lactobacillus plantarum is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable bacteria in the Lactobacillus plantarum liquid is 10 10 CFU / mL.
[0094] Example 2
[0095] Compared with Example 1, the only difference is that the time of one fermentation is 12 hours, and other condition parameters are the same as Example 1. The specific operation is as follows:
[0096] 3 g of roxburghii fruit powder and 300 mL of deionized water were mixed evenly, sterilized in a high-pressure steam sterilizer at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cooled to room temperature after sterilization to obtain a fermentation substrate;
[0097] Inoculate Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid for primary fermentation, the addition amount of Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid is 3mL, access Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid and shake well and stand for fermentation in a 37°C constant temperature incubator for 12h, after the primary fermentation is completed, sterilize in a high pressure steam autoclave under the conditions of 121°C and 0.12MPa for 30min, cool to room temperature after sterilization, cool to room temperature after sterilization and centrifuge, centrifuge speed 4800r / min, centrifuge time 30min, centrifuge radius 15cm, and obtain 240g preliminary fermentation liquid after centrifugation;
[0098] After centrifugation, sterilize in a high pressure steam autoclave for 30 min at a temperature of 121 ° C and a pressure of 0.12 MPa, cool to room temperature after sterilization, add 1 g of thioneine to the preliminary fermentation liquid, access 2.4 mL of plant lactobacillus liquid, shake well after accessing the liquid, and stand in a 37 ° C constant temperature incubator for 15 h for secondary fermentation;
[0099] After the secondary fermentation, sterilize at 121°C and 0.12MPa for 30 minutes. After sterilization, cool to room temperature and centrifuge at a speed of 4800r / min, a centrifugal time of 30 minutes, and a centrifugal radius of 15cm. After centrifugation, sterilize again in a high-pressure steam sterilizer at 121°C and 0.12MPa for 30 minutes. After sterilization, add preservatives, and the mixing temperature is 75°C during the mixing process with the preservatives; add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol based on the mass of the fermentation stock solution, and mix evenly.
[0100] The preparation method of Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid is as follows: Lactobacillus delbrueckii subspecies bulgaricus is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable bacteria of the Lactobacillus delbrueckii bacterial liquid is 10 10 CFU / mL.
[0101] The preparation method of Lactobacillus plantarum liquid is as follows: Lactobacillus plantarum is inoculated into MRS medium and cultured at 37°C for 24 hours, wherein the number of viable bacteria in the Lactobacillus plantarum liquid is 10 10 CFU / mL.
[0102] Example 3
[0103] Compared with Example 1, the only difference is that the secondary fermentation time is 12 hours, and other condition parameters are the same as Example 1.
[0104] Example 4
[0105] Compared with Example 1, the difference is only that 1.5g of thioneine is added in the secondary fermentation, and other condition parameters are the same as in Example 1.
[0106] Comparative Example 1
[0107] Compared with Example 1, the only difference is that fermentation is not performed, and other condition parameters are the same as Example 1. The specific preparation method is as follows:
[0108] 3 g of roxburghii fruit powder and 300 mL of deionized water are mixed evenly, sterilized in a high-pressure steam sterilizer at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, cooled to room temperature after sterilization to obtain a fermentation substrate, and then 1 g of ergothioneine is added to the fermentation substrate, and a preservative is added. During the mixing process with the preservative, the mixing temperature is 75° C.; based on the mass of the fermentation stock solution, 0.5% of p-hydroxyacetophenone and 0.5% of 1,2-hexanediol are added, and the mixture is mixed evenly.
[0109] Comparative Example 2
[0110] Compared with Example 1, the only difference is that 3 mL of rice wine yeast liquid is added during one fermentation process, and other condition parameters are the same as Example 1.
[0111] The preparation method of the rice wine yeast liquid is as follows: inoculate the rice wine yeast into the YPD culture medium, the culture temperature is 28°C, the culture time is 48h, and the shaking speed is 180r / min.
[0112] Comparative Example 3
[0113] Compared with Example 1, the difference is that no secondary fermentation is performed, and other condition parameters are the same as Example 1. The specific operation is as follows:
[0114] 3 g of roxburghii fruit powder and 300 mL of deionized water were mixed uniformly, sterilized in a high pressure steam sterilizer at a temperature of 121 ° C and a pressure of 0.12 MPa for 30 min, cooled to room temperature after sterilization, and 1 g of ergothioneine was added to obtain a fermentation substrate;
[0115] The Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid was inoculated for a fermentation, the addition amount of the Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid was 3 mL, and the Lactobacillus delbrueckii subspecies bulgaricus bacterial liquid was inoculated and shaken and allowed to ferment in a 37° C. constant temperature incubator for 15 h. After the first fermentation, the mixture was sterilized in a high pressure steam sterilizer at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cooled to room temperature after sterilization. After the sterilization was completed, the mixture was cooled to room temperature for centrifugation, the centrifugal speed was 4800 r / min, the centrifugal time was 30 min, and the centrifugal radius was 15 cm. After the centrifugation was completed, the mixture was sterilized in a high pressure steam sterilizer at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min. After sterilization, add preservatives to the preliminary fermentation liquid. During the mixing process with the preservatives, the mixing temperature is 75° C. Based on the mass of the fermentation stock liquid, add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol and mix well.
[0116] Comparative Example 4
[0117] Compared with Comparative Example 3, the only difference is that the Lactobacillus delbrueckii subspecies bulgaricus and the Lactobacillus plantarum bacterial solution are inoculated for fermentation, and other condition parameters are the same as those of Comparative Example 3. The specific operations are as follows:
[0118] 3 g of roxburghii fruit powder and 300 mL of deionized water were mixed uniformly, sterilized in a high pressure steam sterilizer at a temperature of 121 ° C and a pressure of 0.12 MPa for 30 min, cooled to room temperature after sterilization, and 1 g of ergothioneine was added to obtain a fermentation substrate;
[0119] The fermentation was carried out by inoculating Lactobacillus delbrueckii subspecies bulgaricus and Lactobacillus plantarum. The addition amount of Lactobacillus delbrueckii subspecies bulgaricus was 1.5 mL, and the addition amount of Lactobacillus plantarum was 1.5 mL. The mixture was then shaken and allowed to stand for fermentation in a 37°C constant temperature incubator for 15 h. After the fermentation was completed, the mixture was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 30 min. The mixture was cooled to room temperature after sterilization. After the sterilization was completed, the preliminary fermentation liquid was cooled to room temperature for centrifugation at a centrifugal speed of 4800 r / min, a centrifugal time of 30 min, and a centrifugal radius of 15 cm. After the centrifugation was completed, the mixture was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 30 min. After sterilization, add preservatives. During the mixing process with the preservatives, the mixing temperature is 75° C. Based on the mass of the fermentation stock solution, add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol and mix well.
[0120] Effect Example 1
[0121] DPPH free radical scavenging experiment
[0122] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.
[0123] The specific experimental steps of the DPPH free radical scavenging experiment are as follows: (dilute the product 100 times)
[0124] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);
[0125] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (tube A2);
[0126] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);
[0127] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm. The clearance rate is calculated as follows: clearance rate = [(A2 + A3) - A1] / A2 × 100%.
[0128] This experiment tested the DPPH free radical scavenging experiment on the embodiments and comparative examples. The results are shown in Tables 1 and Figure 1 .
[0129] Table 1
[0130]
[0131]
[0132] The results show that the DPPH radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation culture steps and fermentation strains have a great influence on the antioxidant properties of the final product.
[0133] Effect Example 2
[0134] Hydroxyl free radical scavenging assay
[0135] The Fenton reaction is used to generate hydroxyl radicals (·OH), and salicylic acid is added to the reaction system. ·OH reacts with salicylic acid to generate a colored compound 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm. The fixed reaction time method is used to measure the absorbance of the reaction solution containing the analyte at 510nm, and compared with the blank solution to determine the scavenging effect of the analyte on ·OH.
[0136] The test solution is the product prepared in the above embodiments and comparative examples diluted 10 times;
[0137] Add reagents to the test tube according to Table 1, and add 9mmol / L FeSO4, 9mmol / L salicylic acid ethanol solution, test solution, appropriate amount of deionized water and 8.8mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 minutes, then take out and measure the absorbance A0 of the blank control and the absorbance A0 of the sample group. x and the background absorbance of the solution without H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group, and the absorbance values were tested and the average values were taken. The results are shown in Table 2. The hydroxyl radical scavenging rate of each group was calculated according to the following formula. The results are shown in Tables 3 and Figure 2 .
[0138] Hydroxyl radical scavenging rate = (A0-(A x -A x0 )) / A0×100%.
[0139] Table 2
[0140]
[0141] Table 3
[0142]
[0143]
[0144] The results show that the hydroxyl radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation process and fermentation strains have a great influence on the antioxidant properties of the final products.
[0145] Effect Example 3
[0146] Cell viability (safety test)
[0147] This experiment uses human skin fibroblasts from the Chinese Science Cell Bank to verify the cytotoxicity of the products obtained in the above examples and comparative examples.
[0148] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; CCK-8 was produced by Beijing Biode Biotech Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bioregi Biotech Co., Ltd.
[0149] Equipment: The manufacturer of WJ-80A-Ⅱ CO2 constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of NUNC 96-well cell culture plate is Thermo Fisher Scientific.
[0150] 1. Experimental steps:
[0151] The products prepared in the above examples and comparative examples were respectively prepared into 1% test solution in the experimental group using serum-free DMEM medium. Human skin fibroblasts were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin) in DMEM medium. The cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When the cell fusion reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cell count was performed on a cell counting plate and the cell suspension concentration was adjusted to 7×10 4 / mL, inoculate the cell suspension into a 96-well plate at a ratio of 100μL per well, and incubate at 37℃, 5% CO2 for 12h. Remove the old culture medium and wash the cells twice with phosphate buffer. Add 100μL of the above-configured, filtered and sterilized test solution of different concentrations to each well in the experimental group, and make 3 replicate wells for each test solution; the control group contains cells, and serum-free DMEM culture medium is added; the blank control group has no cells, and 100μL of PBS is added. Incubate for another 24h at 37℃, 5% CO2. Then add 10μL of CCK-8 solution to each well, incubate for another 3h, measure the absorbance at a wavelength of 450nm, and calculate the cell survival rate of each group. The results are shown in Tables 4 and Figure 3 .
[0152] The cell viability was calculated as follows:
[0153] Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.
[0154] Table 4
[0155] serial number Cell survival rate (%) Example 1 110.2±1.54 Example 2 105.55±2.43 Example 3 107.73±0.94 Example 4 111.76±2.45 Comparative Example 1 93.11±2.25 Comparative Example 2 94.11±2.36 Comparative Example 3 96.57±3.02 Comparative Example 4 95.14±3.28
[0156] The results show that the cell survival rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have a certain effect of promoting cell proliferation, and the fermentation process and fermentation strains have a great influence on the cell survival rate of the final product.
[0157] Effect Example 4
[0158] Intracellular collagen content determination (COL-I content determination)
[0159] This experiment verifies the ability of the products prepared in Example 1 and the comparative example to repair damaged skin by measuring the COL-I content in human skin fibroblasts.
[0160] Human skin fibroblasts in logarithmic phase were taken and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 / mL. 2 mL of cell suspension was added to each well and cultured for 12 hours. Experimental group, blank group and model group were set up. Among them, the experimental group and model group were treated with 18mJ / cm 2 The cells were irradiated with UVA for 40 min. After irradiation, the supernatant in the experimental group, blank group and model group was discarded, 2 mL of DMEM solution was added to the blank group and model group, and 2 mL of the test solution was added to the experimental group (the product prepared in the above embodiment or comparative example was configured into a test solution with a volume percentage of 1% using DMEM, and the test solution needed to be filtered through a 0.22 μm sterile filter membrane before use), and the cells were treated for 24 h after addition, the supernatant was discarded, the cells were washed 2 to 3 times with PBS, and then the cells were treated with cell lysis solution, transferred to a centrifuge tube, centrifuged at 10000 r / min and 4°C for 10 min, and the supernatant was taken to obtain the cell lysate. Take 20 μL of cell lysate and use BCA kit to detect the total protein content in the sample; the collagen content was determined according to the ELISA kit instructions, and the OD values were measured at 450 nm. The collagen expression x was calculated according to the standard curve, and the total protein content b was used for correction to obtain A=x / b, and then the relative expression of collagen I was calculated, and the relative expression of collagen I=A experimental group / model group / A blank group. The results are shown in Table 5 and Figure 4 ( Figure 4 In the table, ###p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns, indicating no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group, **p<0.01, indicating a significant increase compared with the model group, ***p<0.001, indicating an extremely significant increase compared with the model group).
[0161] Table 5
[0162] serial number COL-I content (ng / mg) Blank group 0.476±0.023 Model Group 0.29±0.015 Example 1 0.489±0.016 Example 2 0.463±0.019 Example 3 0.473±0.017 Example 4 0.495±0.015 Comparative Example 1 0.379±0.013 Comparative Example 2 0.385±0.013 Comparative Example 3 0.406±0.017 Comparative Example 4 0.387±0.011
[0163] The results show that after the products prepared in Examples 1 to 4 and Comparative Examples 1 to 4 were used to treat cells, the COL-I content in the groups of Examples 1 to 4 was higher than that in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal cell repair promoting ability, and the fermentation process and fermentation strains have a great influence on the cell repair promoting ability.
[0164] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.
[0165] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.
Claims
1. A method for preparing a thioneine fermentation liquid of roxburghii roxburghii, characterized in that, The method comprises the following steps: inoculating lactobacillus A into a fermentation substrate, performing primary fermentation to obtain a preliminary fermentation liquid, adding ergothioneine into the preliminary fermentation liquid, inoculating lactobacillus B, performing secondary fermentation, and sterilizing; Wherein, the fermentation substrate includes roxburghii and water; the lactobacillus A includes Lactobacillus delbrueckii subspecies bulgaricus, and the lactobacillus B includes Lactobacillus plantarum.
2. the preparation method of thioneine fermentation liquid of roxburghii as claimed in claim 1, is characterized in that, The preparation method of the roxburghii ergothioneine fermentation liquid satisfies at least one of the following conditions: The ratio of the number of live bacteria of the Lactobacillus delbrueckii subspecies bulgaricus to that of the Lactobacillus plantarum is 1:(0.2-1), preferably 1:(0.5-1); The lactobacillus A is added in the form of a lactobacillus A bacterial liquid; The Lactobacillus delbrueckii subsp. bulgaricus includes the Lactobacillus delbrueckii subsp. bulgaricus which is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 20271; The Lactobacillus delbrueckii subspecies bulgaricus is added in the form of a bacterial solution of Lactobacillus delbrueckii subspecies bulgaricus; preferably, the live bacteria concentration of the bacterial solution of Lactobacillus delbrueckii subspecies bulgaricus is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; Based on the water in the fermentation substrate, the number of viable bacteria of Lactobacillus delbrueckii subspecies bulgaricus inoculated per unit volume of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The lactobacillus B is added in the form of a lactobacillus B bacterial liquid; The plant lactobacillus (Lactobacillus plantarum) includes the plant lactobacillus deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 24936; The plant lactobacillus is added in the form of a plant lactobacillus bacterial liquid. Preferably, the plant lactobacillus bacterial liquid has a live bacteria concentration of 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; Based on the water in the fermentation substrate, the number of viable bacteria of the plant lactobacillus inoculated per unit volume of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.
3. the preparation method of thioneine fermentation liquid of roxburghii as claimed in claim 1, is characterized in that, The preparation method of the roxburghii ergothioneine fermentation liquid satisfies at least one of the following conditions: The roxburghii is roxburghii powder, and the preparation method of the roxburghii powder comprises: crushing and sieving the dried roxburghii, preferably, the sieved particle size is 50-200 mesh; The mass ratio of the roxburghii to the water is 1:(20-200), preferably 1:(50-200), and more preferably 1:100; The fermentation substrate also includes a sterilization operation before use. Preferably, the sterilization is a high-temperature sterilization method. When the high-temperature sterilization method is used for the sterilization, the sterilization temperature is 110°C to 125°C, more preferably 115°C to 121°C, for example, 121°C. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example, 0.13 MPa. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35 minutes, more preferably 15 to 25 minutes, for example, 20 minutes. The ergothioneine is ergothioneine in a sterile state; The mass ratio of the ergothioneine to the roxburghii is 1:(0.2-5), preferably 1:3; Also include sterilization, cooling operation after adding the ergothioneine, preferably, the sterilization is a high temperature sterilization method, when the high temperature sterilization method is used to carry out the sterilization, the temperature of the sterilization is 110 ° C ~ 125 ° C, more preferably 115 ° C ~ 121 ° C, such as 121 ° C, when the high temperature sterilization method is used to carry out the sterilization, the pressure of the sterilization is 0.1 ~ 0.14MPa, more preferably 0.1 ~ 0.13MPa, such as 0.13MPa, when the high temperature sterilization method is used to carry out the sterilization, the time of the sterilization is 15 ~ 35min, more preferably 15 ~ 25min, such as 20min; The cooling is cooling to room temperature; The primary fermentation is static fermentation, and preferably, the static fermentation is carried out in a constant temperature incubator; The temperature of the primary fermentation is 30-45°C, preferably 35-40°C, for example 37°C; The time of the primary fermentation is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours; After the primary fermentation, the method further comprises at least one of centrifuging and collecting the supernatant, sterilizing and cooling.
4. the preparation method of thioneine fermentation liquid of roxburghii as claimed in claim 3, is characterized in that, The preparation method of the roxburghii ergothioneine fermentation liquid satisfies at least one of the following conditions: After one fermentation, the centrifugal speed is 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm; After one fermentation, the radius of the centrifuge is 8 to 15 cm; After one fermentation, the centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes, for example 30 minutes; After one fermentation, the sterilization is a high temperature sterilization method. When the high temperature sterilization method is used for the sterilization, the sterilization temperature is 110°C to 125°C, preferably 115°C to 121°C, for example, 121°C; when the high temperature sterilization method is used for the sterilization, the sterilization pressure is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example, 0.13MPa; when the high temperature sterilization method is used for the sterilization, the sterilization time is 15 to 35min, more preferably 15 to 25min, for example, 20min; After the primary fermentation, the cooling is cooling to room temperature.
5. the preparation method of thioneine fermentation liquid of roxburghii as claimed in claim 1, is characterized in that, The preparation method of the roxburghii ergothioneine fermentation liquid satisfies at least one of the following conditions: The secondary fermentation is static fermentation, preferably, the static fermentation is carried out in a constant temperature incubator; The temperature of the secondary fermentation is 30-45°C, preferably 35-40°C, for example 37°C; The secondary fermentation time is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours; The sterilization is high-temperature sterilization. When the high-temperature sterilization method is used for the sterilization, the sterilization temperature is 110°C to 125°C, and more preferably 115°C to 121°C, for example, 121°C; when the high-temperature sterilization method is used for the sterilization, the sterilization pressure is 0.1 to 0.14MPa, and more preferably 0.1 to 0.13MPa, for example, 0.13MPa; when the high-temperature sterilization method is used for the sterilization, the sterilization time is 15 to 35min, and more preferably 15 to 25min, for example, 20min.
6. the preparation method of thioneine fermentation liquid of roxburghii as claimed in claim 5, is characterized in that, After the sterilization, the method further comprises cooling, centrifuging and collecting the supernatant, sterilizing or mixing with a preservative; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugal speed is 3000-9000 rpm, more preferably 4000-6000 rpm, for example 5000 rpm; Preferably, the radius of the centrifuge is 8 to 15 cm; Preferably, the centrifugation time is 10 to 40 min, more preferably 20 to 40 min, for example 30 min; Preferably, the secondary sterilization method is a high temperature sterilization method. Preferably, the temperature of the secondary sterilization is 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C; the pressure of the secondary sterilization is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example 0.13MPa; the time of the secondary sterilization is 15 to 35min, more preferably 15 to 25min, for example 20min; Preferably, during the mixing process with the preservative, the mixing temperature is 50 to 80°C, more preferably 70 to 80°C; Preferably, in the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.1% to 0.6%, and more preferably 0.5%; when the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5% to 1%, and more preferably 0.5%.
7. A roxburghii ergothioneine fermentation liquid, characterized in that, The invention is prepared by the preparation method of thioneine fermentation liquid of roxburghii according to any one of claims 1 to 6.
8. A roxburghii ergothioneine fermentation liquid, used directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the ergothioneine fermentation liquid of Rosa roxburghii is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and anti-allergic active ingredient in the skin topical preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect or an antioxidant active ingredient having a hydroxyl free radical scavenging effect; More preferably, the anti-aging active ingredient is an anti-aging active ingredient having a cell proliferation effect; More preferably, the anti-allergic active ingredient is an anti-allergic active ingredient having hyaluronidase inhibitory effect.
9. A skin external preparation, characterized in that: The invention comprises the roxburghii ergothioneine fermentation liquid according to claim 7.
10. The external skin preparation according to claim 9, wherein The skin external preparation satisfies at least one of the following conditions: The skin external preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; The skin external preparation includes a facial mask, essence or toner; The mass percentage of the roxburghii ergothioneine fermented liquid in the skin external preparation is 5% to 99%, preferably 60% to 99%.