Mung bean seed fermentation product, skin external preparation containing the same, and preparation method and application thereof

Abstract

The present application discloses a mung bean seed fermentation product, a skin topical preparation containing the same, and a preparation method and application thereof. The preparation method of the mung bean seed fermentation product comprises the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12 to 24 hours, and sterilizing; wherein the lactobacillus includes Lactobacillus casei; the fermentation substrate includes an aqueous extract of mung bean seeds; the aqueous extract of mung bean seeds is prepared by ultrasonic extraction using mung bean seed powder as a raw material and water as an extracting liquid. The mung bean seed fermentation product prepared in the present application has good antioxidant effects, and the preparation process is simple, energy consumption is low, and cost is saved. No organic solvents are used in the preparation process, thereby achieving energy conservation and environmental protection. The prepared mung bean seed fermentation product can be widely used in the field of skin topical preparations, expanding the application field of mung bean seeds, and has high safety of use and ideal beauty effects.

Description

Technical Field

[0001] The present application belongs to the field of fermentation, and in particular relates to a mung bean seed fermentation product, a skin topical preparation containing the same, and a preparation method and application thereof. Background Art

[0002] Mung bean seeds (Vigna radiata (Linn.) Wilczek), also known as green beans, are an annual upright herb belonging to the genus Vigna of the Fabaceae family. Mung bean seeds contain protein, fat, vitamins, folic acid and various minerals, and have high nutritional value. Mung bean seeds are also rich in various active substances such as polypeptides, flavonoids, polysaccharides, etc., which have the effects of clearing heat and detoxifying, and relieving heat. They also have various biological activities such as antioxidant, anti-allergic, anti-tumor and lipid-lowering.

[0003] The main processing and application of mung bean seeds are concentrated in the fields of food and health care products, and are basically based on the application of primary processed products, such as mung bean cake, mung bean soup, mung bean functional beverages and mung bean wine. There are relatively few application studies in the field of skin topicals.

[0004] Therefore, there is an urgent need in the art to develop a method that is simple to operate, low in cost, and can extract active ingredients that can be used in skin topical preparations from mung bean seeds. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects that mung bean seeds are commonly used in the field of food and health products in the prior art, but there are relatively few studies on their application in skin topical preparations. In the face of so many extraction processes, it is difficult to screen out extraction processes suitable for mung bean seeds from which active ingredients with cosmetic effects can be extracted, and a mung bean seed fermentation product is provided, including a skin topical preparation and its preparation method and application. The mung bean seed fermentation product prepared in the present application has good antioxidant and anti-inflammatory effects, and the preparation process is simple, the energy consumption is low, and the cost is saved. No organic solvent is used in the preparation process, and energy saving and environmental protection are achieved. The prepared mung bean seed fermentation product can be widely used in the field of skin topical preparations, expanding the application field of mung bean seeds, and has high safety in use and ideal cosmetic effects.

[0006] This application adopts the following technical solutions to solve the above technical problems:

[0007] The present application provides a method for preparing a mung bean seed fermentation product, which comprises the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12 to 24 hours, and sterilizing; wherein the lactobacillus comprises Lactobacillus paracasei; the fermentation substrate comprises a mung bean seed water extract; the mung bean seed water extract is prepared by using mung bean seed powder as a raw material and water as an extracting liquid through ultrasonic extraction.

[0008] In some embodiments, the particle size of the mung bean seed powder can be conventional in the art, preferably 50-100 mesh, and more preferably 60-80 mesh.

[0009] In some embodiments, the mass ratio of the mung bean seed powder to the water may be 1:(10-50), preferably 1:(10-30), for example 1:20.

[0010] In some embodiments, the temperature of the ultrasonic extraction may be 65-75°C, preferably 70-75°C.

[0011] In some embodiments, the operating frequency of the ultrasonic extraction may be 35-45 KHz, preferably 40 KHz.

[0012] In some embodiments, the ultrasonic power of the ultrasonic extraction may be 280-320W, preferably 300W.

[0013] In some embodiments, the ultrasonic extraction time may be 30 to 60 minutes, preferably 40 to 60 minutes, for example 50 minutes.

[0014] In some embodiments, the fermentation substrate may be further sterilized before use.

[0015] The conditions and methods for sterilizing the fermentation substrate may be conventional conditions and methods for such operations in the art, and may generally be high-temperature sterilization.

[0016] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 115-125° C., more preferably 118-121° C.

[0017] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 25 to 35 minutes, more preferably 28 to 32 minutes, for example 30 minutes.

[0018] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1 to 0.15 MPa, more preferably 0.1 to 0.13 MPa.

[0019] Among them, according to the common practice in the art, the sterilization operation may further include a cooling operation, which can generally be cooling to room temperature.

[0020] In some embodiments, the number of Lactobacillus inoculated in a unit volume of the fermentation substrate may be conventional in the art, preferably 10 4 ~108 CFU / mL, preferably 10 5 ~10 7 CFU / mL.

[0021] In some embodiments, the Lactobacillus paracasei may include the Lactobacillus paracasei deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 20241.

[0022] In some embodiments, the Lactobacillus casei can be added in the form of a Lactobacillus casei bacterial solution according to the conventional art, and the concentration of the Lactobacillus casei in the Lactobacillus casei bacterial solution can be 10 8 ~10 12 CFU / mL, preferably 10 8 ~10 10 CFU / mL.

[0023] In some embodiments, the lactobacillus further comprises Lactobacillus helveticus.

[0024] Wherein, the Lactobacillus helveticus may include the Lactobacillus helveticus deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 20243.

[0025] Wherein, the Lactobacillus helveticus can be added in the form of a Lactobacillus helveticus bacterial solution according to the conventional art, and the concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial solution can be 10 8 ~10 12 CFU / mL, preferably 10 8 ~10 10 CFU / mL.

[0026] When the lactobacillus includes the Lactobacillus paracasei and the Lactobacillus helveticus, the ratio of the number of live bacteria of the Lactobacillus paracasei to the Lactobacillus helveticus may be (0.25-4):1, preferably 1:1.

[0027] In a preferred embodiment, the lactobacillus consists of the Lactobacillus paracasei.

[0028] In another preferred embodiment, the lactobacillus consists of the Lactobacillus paracasei and the Lactobacillus helveticus.

[0029] In some embodiments, the conditions and methods of the fermentation culture may be conventional in the art, and may generally be static fermentation under natural conditions.

[0030] In some embodiments, the fermentation culture time is preferably 14 to 18 hours, more preferably 16 hours.

[0031] In some embodiments, the fermentation culture temperature may be 30-45°C, preferably 37-45°C, such as 43°C.

[0032] In some embodiments, the sterilization conditions and methods may be conventional in the art, and may generally be high temperature sterilization.

[0033] 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 115-125° C., more preferably 118-121° C.

[0034] When the high temperature sterilization method is used for the sterilization, the sterilization time can be the conventional time for such operation in the art, preferably 20 to 35 minutes, more preferably 28 to 32 minutes, for example 30 minutes.

[0035] 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 0.15 MPa, more preferably 0.1 to 0.13 MPa.

[0036] In some embodiments, after the sterilization operation, the process may further include cooling and / or centrifuging to collect the supernatant.

[0037] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0038] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 4000-8000 rpm, more preferably 4800-8000 rpm.

[0039] The centrifugal radius may be a conventional radius for such operations in the art, preferably 8 to 15 cm.

[0040] The centrifugation time may be the conventional time for such operation in the art, preferably 20 to 40 minutes, more preferably 30 to 40 minutes.

[0041] Wherein, the centrifugation operation may further include secondary sterilization and / or mixing with a preservative.

[0042] The secondary sterilization method may be a high temperature sterilization method conventionally used in the art.

[0043] During the secondary sterilization process, the temperature of the secondary sterilization can be a conventional temperature for such operations in the art, preferably 115-125°C, more preferably 118-121°C.

[0044] During the secondary sterilization process, the secondary sterilization time may be the conventional time for such operation in the art, preferably 25 to 35 minutes, more preferably 28 to 32 minutes.

[0045] During the secondary sterilization process, the secondary sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.15 MPa, more preferably 0.1-0.13 MPa.

[0046] During the mixing process with the preservative, the mixing temperature can be a conventional temperature for such operations in the art, preferably 60-75°C.

[0047] In the process of mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional art. When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation may be 0.1% to 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation may be 0.5% to 2%; preferably, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.5%.

[0048] The present application also provides a mung bean seed fermented product, which is prepared by the mung bean seed fermented product preparation method as described above.

[0049] The present application also provides a use of the mung bean seed fermentation product as described above directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0050] In some embodiments, the mung bean seed fermentation product can be used as an antioxidant active ingredient and / or an anti-inflammatory active ingredient in the skin topical preparation.

[0051] Wherein, the antioxidant active ingredient may be an antioxidant active ingredient having a DPPH free radical scavenging effect.

[0052] Among them, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient that has the ability to inhibit the production of inflammatory factor TNF-α. The present application also provides a skin topical preparation, which includes the mung bean seed fermentation product as described above.

[0053] In some embodiments, the skin topical preparation may further include active ingredients conventionally used in the art, generally including at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0054] 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.

[0055] In some embodiments, the mass percentage of the mung bean seed fermentation product in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

[0056] In some embodiments, the room temperature generally refers to 15-40°C.

[0057] 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.

[0058] The reagents and raw materials used in this application are commercially available.

[0059] The positive progressive effects of the present application are as follows: the present application adopts a fermentation process to carry out deep processing of mung bean seeds, thereby reducing waste of resources; the preparation process of mung bean seed fermentation products is simple, easy to operate, and has low energy consumption, which reduces pollution to the environment; under specific fermentation conditions, the active ingredients in the mung bean seeds can be fully released, and the nutritional value and biological activity of the mung bean seeds can be better exerted, with relatively less damage to the active ingredients with beauty effects in the mung bean seeds; the mung bean seed fermentation products can be directly used as cosmetics, or as cosmetic additives or bases, without causing any side effects to the skin, with higher safety, and also with ideal antioxidant and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present application can be better understood by referring to the following description given 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 disclosure and explain the principles and advantages of the present disclosure.

[0061] in:

[0062] Figure 1 It is a comparison chart of DPPH free radical scavenging rates of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3;

[0063] Figure 2 This is a comparison chart of the total sugar content of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3;

[0064] Figure 3 It is a comparison chart of total protein content of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3;

[0065] Figure 4 This is a comparison chart of the total antioxidant capacity of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3;

[0066] Figure 5The figure is a comparison chart of the anti-inflammatory ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3. DETAILED DESCRIPTION

[0067] 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 without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0069] In the following examples and comparative examples, Lactobacillus paracasei was purchased from China Industrial Microorganism Collection Center with a collection number of CICC 20241;

[0070] In the following examples and comparative examples, Lactobacillus helveticus was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 20243.

[0071] Example 1

[0072] (1) crushing and sieving commercially available mung bean seeds to obtain 60-80 mesh powder, weighing 15 g of the mung bean seed powder and 300 g of deionized water, mixing them evenly, and then performing ultrasonic extraction. The temperature of the ultrasonic extraction is 70° C., the ultrasonic power is 300 W, the ultrasonic frequency is 40 KHz, and the ultrasonic time is 50 min. After the ultrasonic extraction is completed, sterilization is performed at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min. After sterilization, the mixture is allowed to stand and cool to room temperature to obtain a fermentation substrate;

[0073] (2) inoculating a Lactobacillus casei bacterial liquid into the fermentation substrate prepared in step (1) for fermentation, wherein the viable bacteria concentration in the Lactobacillus casei bacterial liquid is 10 9 CFU / mL, the inoculation amount of Lactobacillus casei liquid is 0.3mL, and the fermentation culture is statically allowed to proceed for 16 hours under natural conditions, and the fermentation culture temperature is 43°C; after the fermentation culture is completed, sterilization is carried out at a temperature of 121°C and a pressure of 0.12MPa to terminate the fermentation, and the sterilization time is 30min. After sterilization, the sterilized material is centrifuged at a speed of 4800rpm and a centrifugal radius of 15cm for 30min, and the supernatant is collected. After the centrifugation is completed, the supernatant is sterilized for a second time at a temperature of 121°C and a pressure of 0.12MPa for 30min to obtain mung bean seed fermentation product.

[0074] Example 2

[0075] Compared with Example 1, the only difference is that the fermentation time in step (2) is 12 hours, and the other condition parameters are the same as Example 1 to obtain mung bean seed fermentation product.

[0076] Example 3

[0077] Compared with Example 1, the only difference is that the ultrasonic extraction time in step (1) is 60 minutes, and the other condition parameters are the same as Example 1 to obtain mung bean seed fermentation product.

[0078] Example 4

[0079] Compared with Example 1, the only difference is that the bacterial solution inoculated in step (2) is a mixed bacterial solution of Lactobacillus paracasei (CICC20241) and Lactobacillus helveticus (CICC20243), the ratio of the number of live bacteria of the two bacteria in the inoculated mixed bacterial solution is 1:1, the total inoculation amount remains unchanged, and other condition parameters are the same as in Example 1, to obtain mung bean seed fermentation product.

[0080] Comparative Example 1

[0081] Compared with Example 1, the only difference is that in step (1), the fermentation substrate is not subjected to ultrasonic extraction treatment during the preparation process, and other condition parameters are the same as Example 1.

[0082] Comparative Example 2

[0083] Compared with Example 1, the only difference is that no fermentation treatment is performed, specifically the fermentation substrate prepared in step (1) of Example 1.

[0084] Comparative Example 3

[0085] Compared with Example 1, the only difference is that the fermentation culture time is 48 hours, and other condition parameters remain unchanged.

[0086] Effect Example 1

[0087] 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.

[0088] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0089] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);

[0090] (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);

[0091] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0092] (4) After the reaction was protected from light for 30 minutes, the absorbance of tubes A1, A2, and A3 was measured at 517 nm. The scavenging rate was calculated as follows: DPPH free radical scavenging rate = [(A2 + A3) - A1] / A2 × 100%.

[0093] The test solution is the product prepared in Examples 1 to 4 and Comparative Examples 1 to 3. The DPPH free radical scavenging rate of the product prepared in Examples 1 to 4 and Comparative Examples 1 to 3 is tested by the above method. The results are shown in Tables 1 and Figure 1 .

[0094] Table 1

[0095] DPPH free radical scavenging rate (%) Example 1 61.98±1.22 Example 2 52.84±1.77 Example 3 65.37±3.34 Example 4 56.91±1.46 Comparative Example 1 47.65±1.35 Comparative Example 2 35.81±1.0.6 Comparative Example 3 39.52±1.78

[0096] Figure 1 In the table, ns indicates no statistical difference compared with Example 1; **p<0.01 indicates a significant statistical difference compared with Example 1, which is significantly reduced; ***p<0.001 indicates an extremely significant statistical difference compared with Example 1, which is extremely significantly reduced.

[0097] The results show that the DPPH radical scavenging ability of the mung bean seed fermentation products prepared in Examples 1 to 4 of the present application is significantly better than that of the products prepared in Comparative Examples 1 to 3. In addition, statistical analysis shows that the DPPH radical scavenging ability of the products prepared in Examples 1 to 4 is significantly or extremely significantly higher than that of the products prepared in Comparative Examples 1 to 3.

[0098] Effect Example 2

[0099] The total sugar content in the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3 was tested using a total sugar content detection kit with a product number of BC2715 produced by Beijing Solebow Technology Co., Ltd. The results are shown in Tables 2 and Figure 2 .

[0100] The protein content in the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3 was tested using the BCA protein quantitative detection kit with the product number BN27109 produced by Beijing Bairuiji Biotechnology Co., Ltd. The results are shown in Tables 2 and Figure 3 .

[0101] Table 2

[0102]

[0103]

[0104] Figure 2 and Figure 3 In the table, ns indicates no statistical difference compared with Example 1; *p<0.05 indicates a statistical difference compared with Example 1; **p<0.01 indicates a significant statistical difference compared with Example 1, which is significantly reduced; ***p<0.001 indicates an extremely significant statistical difference compared with Example 1, which is extremely significantly reduced.

[0105] The results show that the total sugar content and protein content of the mung bean seed fermented products prepared in Examples 1 to 4 of the present application are higher than those of the products prepared in Comparative Examples 1 to 3. Statistical analysis shows that the total sugar content and protein content of the mung bean seed fermented products prepared in Examples 1 to 4 of the present application are significantly or extremely significantly better than those of the products prepared in Comparative Examples 1 to 3.

[0106] Effect Example 3

[0107] The total antioxidant capacity of the products prepared in Examples 1 to 4 and Comparative Examples 1 to 3 after dilution 5 times was tested using the total antioxidant capacity detection kit (ABTS method) with the product number S0119 produced by Biyuntian Biotechnology Co., Ltd. The results are shown in Tables 3 and Figure 4 .

[0108] Table 3

[0109] serial number Total antioxidant capacity (TEAC / mM) Example 1 0.31±0.02 Example 2 0.29±0.02 Example 3 0.37±0.01 Example 4 0.38±0.02 Comparative Example 1 0.22±0.01 Comparative Example 2 0.23±0.02 Comparative Example 3 0.16±0.02

[0110] Figure 4 In the table, ns means no statistical difference compared with Example 1; **p<0.01 means there is a significant statistical difference compared with Example 1, which is significantly reduced; ***p<0.001 means there is an extremely significant statistical difference compared with Example 1, which is extremely significantly reduced; ## p<0.01, indicating that there is a significant statistical difference compared with Example 1, and the result is significantly increased.

[0111] The results show that the total antioxidant capacity of the mung bean seed fermentation products prepared in Examples 1 to 4 of the present application is better than the total antioxidant capacity of the products prepared in Comparative Examples 1 to 3. Statistical analysis shows that the total antioxidant capacity of the mung bean seed fermentation products prepared in Examples 1 to 4 is significantly or extremely significantly better than that of Comparative Examples 1 to 3.

[0112] Effect Example 4

[0113] Take logarithmic phase HaCaT cells, inoculate the cell suspension on a 6-well cell culture plate at a density of 250,000 / mL, add 2mL of cell suspension to each well, culture for 12 hours, and discard the supernatant. Add 2mL of DMEM solution to the blank group; add 2mL of sample solution diluted with serum-free DMEM medium to the experimental group (the volume of the product prepared in the above embodiment or comparative example accounts for 5% of the total volume after configuration) to treat the cells for 24 hours; add 2mL of DMEM solution to the model group to treat the cells for 24 hours; after the treatment, both the experimental group and the model group are treated with 400μg / mL LPS solution (dissolved in DMEM) for 24 hours, discard the supernatant, wash twice with PBS, treat the cells with cell lysis solution, centrifuge at 10000r / min and 4°C for 10 minutes, and take the supernatant to obtain cell lysis supernatant. Take 20 μL of cell lysate and use BCA kit to detect the total protein content in the sample; the determination of inflammatory factors was carried out according to the instructions of the ELISA kit, and the OD values ​​were measured at 450 nm. According to the OD value, the release level of TNF-α was calculated (the release content of TNF-α was calculated according to the standard curve A, the protein concentration of the lysate obtained by BCA protein measurement was B, and the expression level of TNF-α was calculated as C=A / B). The results are shown in Table 4 and Figure 5 ( Figure 5 ***p<0.001, indicating that there is an extremely significant statistical difference compared with the model group, and a very significant decrease; ns, indicating that there is no significant difference compared with the model group).

[0114] BCA protein quantitative detection kit (Beijing Bairuiji Biotechnology Co., Ltd.) operation steps:

[0115] 1. Sample pretreatment: Prepare lysis solution according to the usage amount at a ratio of RIPA:PMSF of 100:1, remove the culture medium, wash one side with PBS, add 150-250 μL lysis solution according to the amount of cells in each well of the 6-well plate, and pipette several times to make the lysis solution fully contact with the cells;

[0116] 2. Post-processing: Centrifuge the lysed sample at 10,000 rpm for 10 minutes, take the supernatant, and then perform subsequent protein concentration determination;

[0117] 3. According to BCA solution: Cu 2+ The solution is prepared with BCA working solution in a ratio of 50:1;

[0118] 4. Add 20 μL of the protein sample to be tested into a 96-well plate;

[0119] 5. Add 200 μL of the prepared BCA working solution to each well and incubate at 37°C for 20 to 30 minutes.

[0120] 6. Detect absorbance at 562 nm;

[0121] 7. Calculate the protein concentration of the sample based on the standard curve and the dilution factor.

[0122] Human tumor necrosis factor α ELISA kit (Beijing Solebow Technology Co., Ltd.) Detection steps:

[0123] 1. Reagent Preparation

[0124] 1) Reagent warming: First, warm the reagent kit 30 minutes before the experiment. Place the sample to be tested at room temperature. If crystals appear in the concentrated washing solution, place it in a 37°C water bath until all the crystals are dissolved.

[0125] 2) Prepare the washing solution: Calculate the volume of the diluted washing solution in advance, then dilute the 20-fold concentrated washing solution into 1-fold application solution with deionized water. Store the unused concentrated washing solution at 4°C.

[0126] 3) Sample gradient dilution: Add 1 mL of standard / sample diluent (SR1) to the lyophilized standard, let stand for 15 minutes, and gently mix after it is completely dissolved (concentration 1000 pg / mL). Then add 500 μL of standard / sample diluent (SR1) to each of the remaining 6 tubes and dilute 2-fold according to the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, 0 pg / mL. 1000 pg / mL is the highest point concentration of the standard curve, and the standard / sample diluent (SR1) is used as the zero point of the standard curve. The unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or divided into a single use amount as needed and stored in a -80°C refrigerator.

[0127] 4) Biotinylated antibody working solution: Calculate the required amount for the test in advance, dilute the 100-fold antibody concentrate into 1-fold working solution with the detection diluent (SR2) (mix thoroughly before dilution), and add to the reaction wells in 30-well bottles.

[0128] 5) Enzyme conjugate working solution: Prepare the required amount for each test. Use enzyme conjugate diluent (SR3) to dilute the 40-fold concentrated enzyme conjugate into 1-fold working solution (centrifuge before dilution) and use within 30 minutes.

[0129] 6) Washing method: shake off all the liquid in the wells of the ELISA plate, pat dry on absorbent paper, add 300 μL / well of washing solution using a washing bottle, let it stand for 30 seconds, shake off all the liquid in the wells of the ELISA plate, and pat dry on absorbent paper.

[0130] 2. Detection steps

[0131] 1) 30 minutes before the experiment, take out the test kit and return to room temperature. Before adding the standard / sample, wash the plate three times and spin dry;

[0132] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate, incubate at 37°C for 90 min, tap and wash the plate 4 times;

[0133] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, incubate at 37°C for 60 min, tap and wash the plate 4 times;

[0134] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at 37°C for 30 min, tap and wash the plate 5 times;

[0135] 5) Add 50 μL of color development substrate to the reaction wells, seal the plate and incubate in a 37°C incubator in the dark for 15 min;

[0136] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm using an ELISA reader (within 5 min).

[0137] Table 4

[0138] serial number TNF-α(pg / mg) Blank group 1.78±0.28 Model Group 24.35±2.11 Example 1 12.87±1.34 Example 2 14.69±1.41 Example 3 10.88±1.4 Example 4 11.54±2.15 Comparative Example 1 19.31±1.34 Comparative Example 2 22.67±1.49 Comparative Example 3 21.69±2.14

[0139] 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.

[0140] Although the present disclosure has been disclosed above through the description of the specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure 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 by the present disclosure.

Claims

1. A method for preparing a fermented mung bean seed product, characterized in that: The method comprises the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12 to 24 hours, and sterilizing; wherein the lactobacillus comprises Lactobacillus paracasei; the fermentation substrate is mung bean seed water extract; the mung bean seed water extract is prepared by using mung bean seed powder as a raw material and water as an extracting liquid through ultrasonic extraction; Wherein, the particle size of the mung bean seed powder is 50 to 100 mesh; The mass ratio of the mung bean seed powder to the water is 1:(10-50); The temperature of the ultrasonic extraction is 65-75°C; The working frequency of the ultrasonic extraction is 35-45KHz; The ultrasonic power of the ultrasonic extraction is 280-320W; The ultrasonic extraction time is 30 to 60 minutes; The number of Lactobacillus inoculated in the fermentation substrate per unit volume is 10 4 ~10 8 CFU / mL; The Lactobacillus casei includes the Lactobacillus casei that is deposited in the China Industrial Microbiological Culture Collection Center and has a deposit number of CICC20241; The Lactobacillus casei is added in the form of a Lactobacillus casei bacterial liquid, wherein the concentration of the Lactobacillus casei in the Lactobacillus casei bacterial liquid is 10 8 ~10 12 CFU / mL; The temperature of the fermentation culture is 37-45°C.

2. The method for preparing the mung bean seed fermentation product according to claim 1, characterized in that: The fermentation substrate satisfies at least one of the following conditions: The fermentation substrate further includes a sterilization operation before use The sterilization operation also includes cooling to room temperature.

3. The method for preparing the mung bean seed fermentation product according to claim 2, characterized in that: The fermentation substrate satisfies at least one of the following conditions: The particle size of the mung bean seed powder is 60 to 80 meshes; The mass ratio of the mung bean seed powder to the water is 1:(10-30); The temperature of the ultrasonic extraction is 70-75°C; The working frequency of the ultrasonic extraction is 40KHz; The ultrasonic power of the ultrasonic extraction is 300W; The ultrasonic extraction time is 40 to 60 minutes; The sterilization method for the fermentation substrate is a high-temperature sterilization method. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 115-125°C; the sterilization time is 25-35 minutes; and the sterilization pressure is 0.1-0.15Mpa.

4. The method for preparing the mung bean seed fermentation product according to claim 3, characterized in that: The fermentation substrate satisfies at least one of the following conditions: The mass ratio of the mung bean seed powder to the water is 1:20; The ultrasonic extraction time is 50 min; When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 118-121° C.; the sterilization time is 28-32 min; and the sterilization pressure is 0.1-0.13 MPa.

5. The method for preparing the mung bean seed fermentation product according to claim 4, characterized in that: The fermentation substrate satisfies the requirement that the sterilization time is 30 min.

6. The method for preparing the mung bean seed fermentation product according to claim 1, characterized in that: The lactobacillus also includes Lactobacillus helveticus.

7. The method for preparing the mung bean seed fermentation product according to claim 6, characterized in that: The preparation method satisfies at least one of the following conditions: The Lactobacillus helveticus includes the Lactobacillus helveticus that is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC20243; The Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial liquid, wherein the concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial liquid is 10 8 ~10 12 CFU / mL; when the lactobacillus includes the Lactobacillus paracasei and the Lactobacillus helveticus, the ratio of the number of live bacteria of the Lactobacillus paracasei and the Lactobacillus helveticus is (0.25-4):

1.

8. The method for preparing the mung bean seed fermentation product according to claim 7, characterized in that: The preparation method satisfies at least one of the following conditions: The number of Lactobacillus inoculated in the fermentation substrate per unit volume is 10 5 ~10 7 CFU / mL; The concentration of the Lactobacillus casei in the Lactobacillus casei bacterial liquid is 10 8 ~10 10 CFU / mL; The concentration of Lactobacillus helveticus in the Lactobacillus helveticus liquid is 10 8 ~10 10 CFU / mL; When the lactobacillus includes the Lactobacillus paracasei and the Lactobacillus helveticus, the ratio of the number of live bacteria of the Lactobacillus paracasei to the Lactobacillus helveticus is 1:

1.

9. The method for preparing the mung bean seed fermentation product according to claim 1, characterized in that: The preparation method satisfies at least one of the following conditions: The fermentation culture is static fermentation under natural conditions; The fermentation culture time is 14 to 18 hours; The sterilization method is high-temperature sterilization; when the high-temperature sterilization method is used for sterilization, the sterilization temperature is 115-125°C; when the high-temperature sterilization method is used for sterilization, the sterilization time is 20-35 minutes; when the high-temperature sterilization method is used for sterilization, the sterilization pressure is 0.1-0.15MPa.

10. The method for preparing the mung bean seed fermentation product according to claim 9, characterized in that: The preparation method satisfies at least one of the following conditions: The fermentation culture time is 16h; When the high temperature sterilization method is used for the sterilization, the sterilization temperature is 118-121° C.; the sterilization time is 28-32 min; and the sterilization pressure is 0.1-0.13 MPa.

11. The method for preparing the mung bean seed fermentation product according to claim 10, characterized in that: The preparation method satisfies at least one of the following conditions: The fermentation culture temperature is 43°C; When the high temperature sterilization method is used for the sterilization, the sterilization time is 30 minutes.

12. The method for preparing a fermented mung bean seed product according to any one of claims 1 to 11, characterized in that: After the sterilization operation, the process further includes cooling and / or centrifuging and collecting the supernatant.

13. The method for preparing the mung bean seed fermentation product according to claim 12, characterized in that: The cooling is cooling to room temperature; The centrifugal speed is 4000-8000 rpm; The centrifugal radius is 8 to 15 cm; The centrifugation time is 20 to 40 minutes.

14. The method for preparing the mung bean seed fermentation product according to claim 13, characterized in that: The centrifugal speed is 4800-8000 rpm; The centrifugation time is 30 to 40 minutes.

15. The method for preparing the mung bean seed fermented product according to claim 12, characterized in that: The centrifugation operation may be followed by secondary sterilization and / or mixing with a preservative.

16. The method for preparing the mung bean seed fermentation product according to claim 15, characterized in that: The secondary sterilization method is high temperature sterilization; During the secondary sterilization, the temperature of the secondary sterilization is 115-125°C; During the secondary sterilization process, the secondary sterilization time is 25 to 35 minutes; During the secondary sterilization process, the pressure of the secondary sterilization is 0.1-0.15 MPa; During the mixing process with the preservative, the mixing temperature is 60 to 75°C; During the mixing process with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.1% to 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.5% to 2%.

17. The method for preparing a fermented mung bean seed product according to claim 16, wherein: During the secondary sterilization process, the temperature of the secondary sterilization is 118-121°C; During the secondary sterilization process, the secondary sterilization time is 28 to 32 minutes; During the secondary sterilization process, the pressure of the secondary sterilization is 0.1-0.13 MPa; The mass percentage of the p-hydroxyacetophenone in the supernatant obtained after the centrifugation is 0.5%, and the mass percentage of the 1,2-hexanediol in the supernatant obtained after the centrifugation is 0.5%.

18. A fermented product of mung bean seeds, characterized in that: The product is prepared by the method for preparing the mung bean seed fermentation product according to any one of claims 1 to 17.

19. Use of the mung bean seed fermentation product as claimed in claim 18 directly as a product, as an additive or as a base in the preparation of a skin external preparation.

20. The use according to claim 19, characterized in that The mung bean seed fermentation product is used as an antioxidant active ingredient and / or an anti-inflammatory active ingredient in the skin external preparation.

21. The use according to claim 20, characterized in that The antioxidant active ingredient is an antioxidant active ingredient with DPPH free radical scavenging effect; The anti-inflammatory active ingredient is an anti-inflammatory active ingredient that can inhibit the generation of inflammatory factor TNF-α.

22. A skin external preparation, characterized in that: The invention comprises the mung bean seed fermented product as claimed in claim 18.

23. The external skin preparation according to claim 22, wherein 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.

24. The external skin preparation according to claim 23, wherein The skin external preparation includes a facial mask, essence or toner.

25. The external skin preparation according to any one of claims 22 to 24, characterized in that: The mass percentage of the mung bean seed fermentation product in the skin external preparation is 5% to 99%.

26. The external skin preparation according to claim 25, wherein The mass percentage of the mung bean seed fermentation product in the skin external preparation is 60% to 99%.

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