CN115836997A - Mung bean seed fermented product, skin external preparation containing mung bean seed fermented product, and preparation method and application of mung bean seed fermented product - Google Patents

Abstract

The application discloses a mung bean seed fermented product, a skin external preparation containing the mung bean seed fermented product, and a preparation method and application of the mung bean seed fermented product. The preparation method of the mung bean seed fermentation product comprises the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12-24 h, and sterilizing; wherein the lactobacillus comprises Lactobacillus paracasei; the fermentation substrate comprises mung bean seed aqueous extract; the mung bean seed water extract is prepared by taking mung bean seed powder as a raw material and water as an extracting solution and performing ultrasonic extraction. The mung bean seed fermentation product prepared by the method has a good antioxidant effect, and is simple in preparation process, low in energy consumption, low in cost, free of any organic solvent in the preparation process, and energy-saving and environment-friendly. The prepared mung bean seed fermented product can be widely applied to the field of skin external preparations, the application field of mung bean seeds is expanded, the use safety is high, and the beautifying effect is ideal.

Description

Mung bean seed fermented product, skin external preparation containing mung bean seed fermented product, and preparation method and application of mung bean seed fermented product

Technical Field

The application belongs to the field of fermentation, and particularly relates to a mung bean seed fermented product, a skin external preparation containing the mung bean seed fermented product, and a preparation method and application of the mung bean seed fermented product.

Background

Semen Phaseoli Radiati (Vigna radiata (Linn.) Wilczek) also called semen Phaseoli Radiati belongs to 1 year old upright herb plant of Vigna of Leguminosae, contains protein, fat, vitamins, folic acid and various minerals, has high nutritive value, contains various active substances such as polypeptide, flavone, polysaccharide, etc., has effects of clearing away heat and toxic materials and relieving summer heat, and also has various biological activities such as oxidation resistance, anti-allergy, anti-tumor and blood lipid reducing.

The main processing and application of mung bean seeds are concentrated in the fields of food and health care products, and basically, primary processed product application is mainly used, for example, the fields of food and health care products such as mung bean cakes, mung bean soup, mung bean functional drinks, mung bean wine and the like, and relatively few application researches are carried out in the field of skin external preparations.

Therefore, there is a need in the art to develop a method for extracting active ingredients from mung bean seeds, which can be used in skin external preparations, with simple operation and low cost.

Disclosure of Invention

The technical problem to be solved by the application is to overcome the defects that mung bean seeds are generally used in the fields of food and health care products in the prior art, the application research in skin external preparations is relatively few, and the extraction process which is suitable for mung bean seeds and can extract active ingredients with beauty treatment effects from mung bean seeds is difficult to screen in the face of a plurality of extraction processes, and the like, so that a mung bean seed fermentation product, a skin external preparation containing the mung bean seed fermentation product, and a preparation method and application of the mung bean seed fermentation product are provided. The mung bean seed fermentation product prepared by the method has good antioxidant effect and anti-inflammatory effect, and is simple in preparation process, low in energy consumption, low in cost, free of any organic solvent in the preparation process and energy-saving and environment-friendly. The prepared mung bean seed fermented product can be widely applied to the field of skin external preparations, the application field of mung bean seeds is expanded, the use safety is high, and the beautifying effect is ideal.

The technical problems are solved by adopting the following technical scheme:

the application provides a preparation method of mung bean seed fermentation, which comprises the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12-24 h, and sterilizing; wherein the Lactobacillus comprises Lactobacillus paracasei (Lactobacillus paracasei); the fermentation substrate comprises mung bean seed aqueous extract; the mung bean seed water extract is prepared by taking mung bean seed powder as a raw material and water as an extracting solution through ultrasonic extraction.

In some embodiments, the particle size of the mung bean seed powder may be conventional in the art, and is preferably 50 to 100 mesh, and more preferably 60 to 80 mesh.

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

in some embodiments, the temperature of the ultrasonic extraction may be 65 to 75 ℃, preferably 70 to 75 ℃.

In some embodiments, the working frequency of the ultrasonic extraction can be 35 to 45KHz, preferably 40KHz.

In some embodiments, the ultrasonic power of the ultrasonic extraction may be 280 to 320W, preferably 300W.

In some embodiments, the time for the ultrasonic extraction may be 30 to 60min, preferably 40 to 60min, for example 50min.

In some embodiments, the fermentation substrate may further comprise a sterilization operation prior to use.

Wherein, the conditions and method for sterilizing the fermentation substrate can be the conditions and method which are conventional in the operation in the field, and generally can be a high-temperature sterilization method.

When the high temperature sterilization method is used to perform the sterilization on the fermentation substrate, the sterilization temperature may be a temperature conventional in such operations in the art, preferably 115 to 125 ℃, more preferably 118 to 121 ℃.

When the fermentation substrate is subjected to said sterilization by said high temperature sterilization, the time for said sterilization may be a time conventional in such operations in the art, preferably from 25 to 35min, more preferably from 28 to 32min, for example 30min.

When the fermentation substrate is subjected to the sterilization by the high-temperature sterilization method, the sterilization pressure may be a pressure that is conventional in such operations in the art, and is preferably 0.1 to 0.15Mpa, more preferably 0.1 to 0.13Mpa.

Wherein, the sterilization operation can be further followed by a cooling operation, generally cooling to room temperature, according to the routine in the field.

In some embodiments, the number of said lactobacillus inoculated per volume of said fermentation substrate may be conventional in the art, preferably 10 ⁴ ～10 ⁸ CFU/mL, more preferably 10 ⁵ ～10 ⁷ CFU/mL。

In some embodiments, the lactobacillus paracasei may include lactobacillus paracasei deposited at the chinese industrial microbial culture collection management center under the collection number CICC 20241.

In some embodiments, the lactobacillus paracasei may be added as is conventional in the art in the form of a lactobacillus paracasei liquid, in which the concentration of the lactobacillus paracasei may be 10 ⁸ ～10 ¹² CFU/mL, preferably 10 ⁸ ～10 ¹⁰ CFU/mL。

In some embodiments, the Lactobacillus further comprises Lactobacillus helveticus (Lactobacillus helveticus).

Wherein, the lactobacillus helveticus can comprise lactobacillus helveticus with the preservation number of CICC20243 which is preserved in China center for culture collection of industrial microorganisms.

The lactobacillus helveticus can be added in the form of lactobacillus helveticus liquid according to the conventional practice in the field, and the concentration of the lactobacillus helveticus in the lactobacillus helveticus liquid can be 10 ⁸ ～10 ¹² CFU/mL, preferably 10 ⁸ ～10 ¹⁰ CFU/mL。

When the lactobacillus comprises the lactobacillus paracasei and the lactobacillus helveticus, the number ratio of the viable bacteria of the lactobacillus paracasei to the lactobacillus helveticus can be (0.25-4): 1, preferably 1:1.

in a preferred embodiment, said lactobacillus consists of said lactobacillus paracasei.

In another preferred embodiment, said lactobacillus consists of said lactobacillus paracasei and said lactobacillus helveticus.

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

In some embodiments, the time period of the fermentation culture is preferably 14 to 18 hours, and more preferably 16 hours.

In some embodiments, the temperature of the fermentation culture may be 30 to 45 ℃, preferably 37 to 45 ℃, e.g., 43 ℃.

In some embodiments, the conditions and methods of sterilization may be conventional in the art, and may generally be autoclaving.

When the sterilization is performed by the high-temperature sterilization method, the sterilization temperature may be a temperature conventional in the operation in the field, preferably 115 to 125 ℃, and more preferably 118 to 121 ℃.

When the sterilization is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the operation of the type in the art, and is preferably 20 to 35min, more preferably 28 to 32min, for example 30min.

When the sterilization is performed by the high-temperature sterilization method, the sterilization pressure may be a pressure that is conventional in such operations in the art, and is preferably 0.1 to 0.15MPa, more preferably 0.1 to 0.13MPa.

In some embodiments, the sterilization may be followed by cooling and/or centrifugation to collect the supernatant.

Wherein the cooling may be to room temperature as is conventional in the art.

Wherein, the rotating speed of the centrifugation can be the rotating speed which is conventional in the operation in the field, preferably 4000-8000 rpm, more preferably 4800-8000 rpm.

Wherein the radius of centrifugation can be the radius conventional in the operation in the field, and is preferably 8-15 cm.

Wherein, the time of centrifugation can be the time which is conventional in the operation in the field, preferably 20-40 min, and more preferably 30-40 min.

Wherein, the operation of centrifugation can further comprise the operation of secondary sterilization and/or mixing with preservative.

The secondary sterilization method may be a high-temperature sterilization method conventionally used in the art.

In the secondary sterilization process, the temperature of the secondary sterilization can be the temperature conventional in the operation in the field, preferably 115-125 ℃, and more preferably 118-121 ℃.

In the secondary sterilization process, the time for the secondary sterilization can be the time conventionally used in the operation in the field, preferably 25-35 min, and more preferably 28-32 min.

In the secondary sterilization process, the pressure of the secondary sterilization can be the pressure conventional in the operation in the field, and is preferably 0.1 to 0.15MPa, and more preferably 0.1 to 0.13MPa.

The temperature of mixing with the preservative may be a temperature conventional to such procedures in the art, preferably 60 to 75 ℃.

During the mixing with the preservative, the preservative may include p-hydroxyacetophenone and/or 1, 2-hexanediol as is conventional in the art. When the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant obtained after the centrifugation can be 0.1% to 0.5%, and the mass percentage of the 1, 2-hexanediol to the supernatant obtained after the centrifugation can be 0.5% to 2%; preferably, the mass percent of the p-hydroxyacetophenone in the supernatant obtained after the centrifugation is 0.5%, and the mass percent of the 1, 2-hexanediol in the supernatant obtained after the centrifugation is 0.5%.

The application also provides the mung bean seed fermented product which is prepared by the preparation method of the mung bean seed fermented product.

The application also provides the application of the mung bean seed leavening directly used as a product, an additive or a base in the preparation of the skin external preparation.

In some embodiments, the mung bean seed ferment may be used as an antioxidant active ingredient and/or an anti-inflammatory active ingredient in the skin external preparation.

Wherein the antioxidant active ingredient is an antioxidant active ingredient having DPPH free radical scavenging activity.

Wherein the anti-inflammatory active ingredient is an anti-inflammatory active ingredient capable of inhibiting the production of inflammatory factor TNF-alpha the application also provides a skin external preparation, which comprises the mung bean seed fermentation product.

In some embodiments, the skin external preparation may further include an active ingredient conventionally used in the art, and may generally include at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergy active ingredient, and an antioxidant active ingredient.

In some embodiments, the topical skin preparations may include, but are not limited to, facial masks, essences, or lotions as is conventional in the art.

In some embodiments, the mung bean seed ferment may be 5 to 99% by weight, preferably 60 to 99% by weight, of the skin external preparation.

In some embodiments, the room temperature is generally 15 to 40 ℃.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred examples of the application.

The reagents and starting materials used in the present application are commercially available.

The positive progress effect of this application lies in: according to the method, the mung bean seeds are deeply processed by adopting a fermentation process, so that the waste of resources is reduced; the preparation process of the mung bean seed leavening is simple and convenient, the operation is simple, the energy consumption is low, the pollution to the environment is reduced, the active ingredients in the mung bean seeds can be fully released under the specific fermentation condition, the nutritional value and the biological activity of the mung bean seeds are better exerted, and the active ingredients with the beauty effect in the mung bean seeds are relatively less damaged; the mung bean seed ferment can be directly used as cosmetics, and can also be used as a cosmetic additive or a base, does not cause any side effect on skin, has higher safety, and also has ideal antioxidant effect and anti-inflammatory effect.

Drawings

The present application may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are incorporated in and form a part of this specification, illustrate preferred embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and advantages of the disclosure.

Wherein:

FIG. 1 is a graph comparing the DPPH radical scavenging rates of products obtained in examples 1 to 4 and comparative examples 1 to 3;

FIG. 2 is a graph comparing the total sugar content of the products obtained in examples 1 to 4 and comparative examples 1 to 3;

FIG. 3 is a graph comparing the total protein content of the products prepared in examples 1 to 4 and comparative examples 1 to 3;

FIG. 4 is a graph comparing the total antioxidant capacity of the products obtained in examples 1 to 4 and comparative examples 1 to 3;

FIG. 5 is a graph comparing the anti-inflammatory ability of the products obtained in examples 1 to 4 and comparative examples 1 to 3.

Detailed Description

The present application is further illustrated by way of the following examples, but is not intended to be limited thereby within the scope of the examples described. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

In the following examples and comparative examples, lactobacillus paracasei was purchased from China center for Industrial microbial Collection with the accession number CICC 20241;

in the following examples and comparative examples, lactobacillus helveticus was purchased from China center for Industrial culture Collection of microorganisms with accession number CICC 20243.

Example 1

(1) Crushing and sieving commercially available mung bean seeds to obtain 60-80 mesh powder, weighing 15g of mung bean seed powder and 300g of deionized water, uniformly mixing, and then carrying out ultrasonic extraction, wherein the ultrasonic extraction temperature is 70 ℃, the ultrasonic power is 300W, the ultrasonic frequency is 40KHz, the ultrasonic time is 50min, after the ultrasonic extraction is finished, sterilizing for 30min under the conditions that the temperature is 121 ℃ and the pressure is 0.12MPa, standing and cooling to room temperature after sterilization to obtain a fermentation substrate;

(2) Inoculating lactobacillus casei-like liquid into the fermentation substrate prepared in the step (1), and fermenting, wherein the viable bacteria concentration in the lactobacillus casei-like liquid is 10 ⁹ CFU/mL, wherein the inoculation amount of the lactobacillus paracasei bacterial liquid is 0.3mL, standing, fermenting and culturing for 16h under natural conditions, wherein the temperature of fermentation and culture is 43 ℃; after the fermentation culture is finished, sterilizing at the temperature of 121 ℃ and under the pressure of 0.12MPa, stopping fermentation, wherein the sterilization time is 30min, cooling to room temperature after sterilization, centrifuging the sterilized materials for 30min under the conditions that the rotating speed is 4800rpm and the centrifugal radius is 15cm, collecting supernatant, and after centrifugation is finished, performing secondary sterilization on the supernatant for 30min under the conditions that the temperature is 121 ℃ and the pressure is 0.12MPa to obtain the mung bean seed fermentation product.

Example 2

Compared with example 1, the difference is that the fermentation time in step (2) is 12h, and the mung bean seed fermentation product is prepared by the same condition parameters as example 1.

Example 3

Compared with example 1, the method is different only in that the ultrasonic extraction time in step (1) is 60min, and other condition parameters are the same as those in example 1 to prepare the mung bean seed fermentation product.

Example 4

Compared with example 1, the difference is that the bacterial liquid inoculated in the step (2) is a mixed bacterial liquid of lactobacillus paracasei (CICC 20241) and lactobacillus helveticus (CICC 20243), the ratio of the number of viable bacteria of the two bacteria in the inoculated mixed bacterial liquid is 1:1, the total bacterial inoculation amount is not changed, and the mung bean seed fermented product is prepared by the same condition parameters as example 1.

Comparative example 1

Compared with the example 1, the method is only different in that the fermentation substrate in the step (1) is not subjected to ultrasonic extraction treatment in the preparation process, and other condition parameters are the same as those in the example 1.

Comparative example 2

The only difference compared to example 1 is that no fermentation treatment, specifically the fermentation substrate obtained in step (1) of example 1, was carried out.

Comparative example 3

Compared with example 1, the difference is only that the time of fermentation culture is 48h, and other condition parameters are not changed.

Effect example 1

DPPH is an early synthesized organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, is very stable in organic solvents, is purple in color, and has a characteristic absorption peak at 517nm, when encountering a radical scavenger, the lone pair of DPPH is paired to discolor it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.

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

(1) Taking the same volume (1 mL) of the solution to be detected and 2X 10 ^-4 mixing (A) with a solution of DPPH in mol/L ₁ A tube);

(2) Taking equal volume (1 mL) of absolute ethanol (solvent of the test substance) and 2X 10 ^-4 mixing (A) with a solution of DPPH in mol/L ₂ A tube);

(3) Mixing the same volume (1 mL) of anhydrous ethanol with the solution to be detected (A) ₃ A tube);

(4) After 30min of reaction in the dark, A was measured at 517nm ₁ Pipe, A ₂ Pipe, A ₃ A tube absorbance value; the clearance calculation formula is: DPPH radical clearance = [ (A) ₂ +A ₃ )-A ₁ ]/A ₂ ×100％。

The solutions to be tested were the products obtained in examples 1 to 4 and comparative examples 1 to 3, and the DPPH radical scavenging rates of the products obtained in examples 1 to 4 and comparative examples 1 to 3 were measured by the above-described method, and the results are shown in Table 1 and FIG. 1.

TABLE 1

	DPPH radical scavenging ratio (%)
		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

In FIG. 1, ns indicates no statistical difference compared to example 1; * P < 0.01, indicating a significant statistical difference, a significant reduction, compared to example 1; * P < 0.001, representing a very significant statistical difference, a very significant reduction, compared to example 1.

The results show that the DPPH free radical scavenging capacity of the mung bean seed fermentation products prepared in the examples 1 to 4 is obviously better than that of the products prepared in the comparative examples 1 to 3. In addition, it was found by statistical analysis that the DPPH radical scavenging ability of the products obtained in examples 1 to 4 was significantly or very significantly higher than that of the products obtained in comparative examples 1 to 3.

Effect example 2

The total sugar content in the products prepared in the above examples 1 to 4 and comparative examples 1 to 3 was measured using a total sugar content detection kit having a product number of BC2715 manufactured by Beijing Soilebao Tech & ltTech & gt, and the results are shown in Table 2 and FIG. 2.

The protein content of the products prepared in the above examples 1 to 4 and comparative examples 1 to 3 was measured using the BCA protein quantitative determination kit having a product number of BN27109, manufactured by Beijing Bairui Biotech Co., ltd., and the results are shown in Table 2 and FIG. 3.

TABLE 2

In FIGS. 2 and 3, ns indicates no statistical difference compared to example 1; * p < 0.05, representing a statistical difference compared to example 1; * P < 0.01, indicating a significant statistical difference, a significant reduction, compared to example 1; * P < 0.001, representing a very significant statistical difference, a very significant reduction, compared to example 1.

The results show that the total sugar content and the protein content of the mung bean seed fermentations prepared in the examples 1-4 are higher than those of the products prepared in the comparative examples 1-3, and statistical analysis shows that the total sugar content and the protein content of the mung bean seed fermentations prepared in the examples 1-4 are remarkably higher than those of the products prepared in the comparative examples 1-3 or are extremely remarkably higher than those of the products prepared in the comparative examples 1-3.

Effect example 3

The total antioxidant capacity of the products prepared in examples 1 to 4 and comparative examples 1 to 3 diluted 5 times was tested using the total antioxidant capacity test kit (ABTS method) with a product number of S0119, produced by the bio-cloud biotechnology limited, and the results are shown in table 3 and fig. 4.

TABLE 3

Numbering	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

In FIG. 4, ns indicates no statistical difference compared to example 1; * P < 0.01, indicating a significant statistical difference, a significant reduction, compared to example 1; * P < 0.001, representing a very significant statistical difference, a very significant reduction compared to example 1; ^## p < 0.01, indicating a significant statistical difference, significantly higher, compared to example 1.

As can be seen from the results, the total antioxidant capacity of the fermented mung bean seeds obtained in examples 1 to 4 of the present application is superior to that of the products obtained in comparative examples 1 to 3. Statistical analysis shows that the total antioxidant capacity of the mung bean seed fermentation products prepared in the examples 1 to 4 is remarkably or extremely remarkably superior to that of the comparative examples 1 to 3.

Effect example 4

Taking log-phase HaCaT cells, inoculating the cell suspension on a 6-well cell culture plate at the density of 25 ten thousand/mL, adding 2mL of cell suspension into each well, culturing for 12h, and sucking and removing supernatant. Adding 2mL of DMEM solution into the blank group; the experimental group added 2mL of sample solution diluted with serum-free DMEM medium (the volume of the product prepared in the above example or comparative example is 5% of the total volume after preparation) to treat the cells for 24h; adding 2mL of DMEM solution into the model group to treat the cells for 24h; after the treatment, the cells of the experimental group and the model group are treated by 400 mug/mL LPS solution (dissolved by DMEM) for 24 hours, supernatant is sucked and discarded, the cells are washed twice by PBS, the cells are treated by cell lysis solution, centrifugation is carried out for 10min at 10000r/min and 4 ℃, and the supernatant is taken to obtain cell lysis supernatant. Taking 20 mu L of cell lysate, and detecting the total protein content in the sample by using a BCA kit; determination of inflammatory factors the experimental procedure was performed according to the ELISA kit instructions, each OD value was determined at 450nm, and based on the OD values, the TNF- α release level was calculated (TNF- α release content a was calculated from the standard curve, protein concentration of lysate was B was calculated by BCA assay, TNF- α expression level was calculated as C = a/B), and the results are shown in table 4 and fig. 5 (in fig. 5, p < 0.001, indicating a very significant statistical difference and a very significant decrease from the model group, and ns, indicating no significant difference from the model group).

The BCA protein quantitative detection kit (Beijing Bairuiji Biotech Co., ltd.) comprises the following steps:

1. sample pretreatment: preparing lysate according to the using amount and the ratio of RIPA to PMSF of 100: 1, removing culture solution, washing one side of the lysate with PBS, adding 150-250 mu L of lysate according to the cell amount of each hole of a 6-hole plate, and blowing the lysate with a pipette to ensure that the lysate is fully contacted with the cells;

2. and (3) post-treatment: centrifuging the cracked sample for 10min under the condition of 10000rpm, and taking the supernatant to perform subsequent protein concentration determination;

3. according to BCA solution: cu ²⁺ Preparing a BCA working solution according to the ratio of 50: 1 of the solution;

4. adding 20 mu L of protein sample to be detected into a 96-well plate;

5. 200 mu L of the prepared BCA working solution is added into each hole, and the mixture is placed for 20-30 min at 37 ℃.

6. Detecting the absorbance at 562 nm;

7. and calculating the protein concentration of the sample according to the standard curve and the dilution factor.

Human tumor necrosis factor alpha ELISA kit (Beijing Soilebao Tech Co., ltd.) detection procedure:

1. reagent preparation

1) And (3) temperature return of the reagent: firstly, the kit is warmed up 30min before the experiment, a sample to be detected is placed at room temperature, if crystallization occurs in the washing solution, the washing solution is concentrated and placed in a water bath at 37 ℃ until the crystallization is completely dissolved.

2) Preparing a washing solution: the usage volume of the diluted washing liquid is calculated in advance, then the 20 times of concentrated washing liquid is diluted into 1 time of application liquid by deionized water, and the concentrated washing liquid which is not used up is stored at 4 ℃.

3) Sample gradient dilution: add 1mL of standard/sample diluent (SR 1) to the lyophilized standard, let stand for 15min, mix gently (1000 pg/mL) after it is completely dissolved. Then 500. Mu.L of each standard/sample dilution (SR 1) was added to each of the remaining 6 tubes, and 2-fold dilutions were made at the following concentrations: 500. 250, 125, 62.5, 31.25, 15.62 and 0pg/mL, wherein 1000pg/mL is the highest concentration of the standard curve, and the standard substance/sample diluent (SR 1) is used as the zero point of the standard curve. The re-dissolved stock solution (with concentration of 1000 pg/mL) of the standard product is discarded or split according to the requirement of one dose, and the stock solution is stored in a refrigerator at the temperature of-80 ℃.

4) Biotinylated antibody working solution: the amount required for the test is calculated in advance, the 100 times antibody concentrated solution is diluted into 1 time application working solution by using detection diluent (SR 2) (fully and uniformly mixed before dilution), and the diluted solution is added into the reaction holes in 30 sub-packages.

5) Enzyme conjugate working solution: prepared according to the required dosage of each test, the 40 times concentrated enzyme conjugate is diluted to 1 time application working solution by using enzyme conjugate diluent (SR 3) (centrifugation before dilution) and used within 30min.

6) The washing method comprises the following steps: and (3) completely throwing off the liquid in the hole of the enzyme label plate, patting the liquid on absorbent paper, adding 300 mu L/hole of washing liquid into a washing bottle, throwing off the liquid in the hole of the enzyme label plate after standing for 30s, and patting the liquid on the absorbent paper.

2. Detection step

1) Taking out the kit 30min before the experiment, recovering to room temperature, washing the plate for three times and drying by spin-drying before adding the standard substance/sample;

2) Adding 100 μ L of standard/sample into the reaction well, sealing the plate, incubating at 37 deg.C for 90min, clapping the plate and washing the plate for 4 times;

3) Adding 100 μ L of biotinylated antibody working solution into the reaction well, sealing the plate, incubating at 37 deg.C for 60min, clapping the plate and washing the plate for 4 times;

4) Adding 100 μ L of the working solution of the enzyme conjugate into the reaction well, sealing the plate, incubating at 37 deg.C for 30min, patting the plate, and washing the plate for 5 times;

5) Adding 50 mu L of chromogenic substrate into the reaction hole, sealing the reaction hole, and then carrying out light-shielding chromogenic reaction for 15min in an incubator at 37 ℃;

6) Add 50. Mu.L of stop solution and immediately measure OD (within 5 min) with a microplate reader at a wavelength of 450 nm.

TABLE 4

Numbering	TNF-α(pg/mg)
		Blank group	1.78±0.28
Model set	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

Finally, it should also be noted that, in the present application, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While the disclosure has been disclosed above by the description of specific embodiments thereof, it should be understood that various modifications, improvements or equivalents of the disclosure may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are intended to be included within the scope of the present disclosure as claimed.

Claims (10)

1. A preparation method of mung bean seed fermentation is characterized by comprising the following steps: inoculating lactobacillus into a fermentation substrate, fermenting and culturing for 12-24 h, and sterilizing; wherein the Lactobacillus comprises Lactobacillus paracasei (Lactobacillus paracasei); the fermentation substrate comprises mung bean seed aqueous extract; the mung bean seed water extract is prepared by taking mung bean seed powder as a raw material and water as an extracting solution through ultrasonic extraction.

2. The method of producing mung bean seed fermented product according to claim 1, wherein the fermentation substrate satisfies at least one of the following conditions:

the particle size of the mung bean seed powder is 50-100 meshes, preferably 60-80 meshes;

the mass ratio of the mung bean seed powder to the water is 1: (10 to 50), preferably 1: (10 to 30), more preferably 1:20;

the temperature of ultrasonic extraction is 65-75 ℃, preferably 70-75 ℃;

the working frequency of the ultrasonic extraction is 35-45 KHz, preferably 40KHz;

the ultrasonic power of the ultrasonic extraction is 280-320W, preferably 300W;

the ultrasonic extraction time is 30-60 min, preferably 40-60 min, and more preferably 50min;

the fermentation substrate further comprises a sterilization operation before use; preferably, the sterilization process performed on the fermentation substrate is a high temperature sterilization process; when the high-temperature sterilization method is adopted to perform the sterilization on the fermentation substrate, the sterilization temperature is 115-125 ℃, and preferably 118-121 ℃; when the high-temperature sterilization method is adopted to perform the sterilization on the fermentation substrate, the sterilization time is 25-35 min, preferably 28-32 min, and more preferably 30min; when the high-temperature sterilization method is adopted to perform the sterilization on the fermentation substrate, the sterilization pressure is 0.1-0.15 MPa, and preferably 0.1-0.13 MPa; the operation of cooling to room temperature is further included after the operation of sterilizing.

3. The method for producing mung bean seed fermented product according to claim 1, wherein the production method satisfies at least one of the following conditions:

the number of said Lactobacillus inoculated per volume of said fermentation substrate is 10 ⁴ ～10 ⁸ CFU/mL, preferably 10 ⁵ ～10 ⁷ CFU/mL；

The lactobacillus paracasei comprises lactobacillus paracasei preserved in China industrial microorganism strain preservation management center with the preservation number of CICC 20241;

the lactobacillus paracasei is added in the form of lactobacillus paracasei liquid, and the concentration of the lactobacillus paracasei in the lactobacillus paracasei liquid is 10 ⁸ ～10 ¹² CFU/mL, preferably 10 ⁸ ～10 ¹⁰ CFU/mL；

The Lactobacillus further comprises Lactobacillus helveticus (Lactobacillus helveticus); preferably, the lactobacillus helveticus comprises lactobacillus helveticus with a preservation number of CICC20243 which is preserved in the china industrial microorganism culture preservation management center; preferably, the lactobacillus helveticus is added in the form of lactobacillus helveticus liquid, and the concentration of the lactobacillus helveticus in the lactobacillus helveticus liquid is 10 ⁸ ～10 ¹² CFU/mL, more preferably 10 ⁸ ～10 ¹⁰ CFU/mL; when the lactobacillus comprises the lactobacillus paracasei and the lactobacillus helveticus, the number ratio of the viable bacteria of the lactobacillus paracasei to the lactobacillus helveticus is (0.25-4): 1, preferably 1:1.

4. the method for producing mung bean seed fermented product according to any one of claims 1 to 3, wherein the production method satisfies at least one of the following conditions:

the fermentation culture is static fermentation under natural conditions;

the fermentation culture time is 14-18 h, preferably 16h;

the temperature of the fermentation culture is 30-45 ℃, preferably 37-45 ℃, and more preferably 43 ℃;

the sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 115-125 ℃, and preferably 118-121 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 20-35 min, preferably 28-32 min, and more preferably 30min; when the high-temperature sterilization method is used for the sterilization, the sterilization pressure is 0.1 to 0.15MPa, preferably 0.1 to 0.13MPa.

5. The method for preparing mung bean seed ferment according to any one of the claims 1 to 3, wherein the sterilization further comprises cooling and/or centrifugation and collection of supernatant;

preferably, the cooling is to room temperature;

preferably, the rotating speed of the centrifugation is 4000-8000 rpm, more preferably 4800-8000 rpm;

preferably, the radius of the centrifugation is 8-15 cm;

preferably, the centrifugation time is 20-40 min, more preferably 30-40 min.

6. The method for producing mung bean seed fermented product according to claim 5, wherein the centrifugation step further comprises the steps of sterilizing the mung bean seed fermented product for a second time and/or mixing the mung bean seed fermented product with a preservative;

preferably, the secondary sterilization method is a high-temperature sterilization method;

preferably, in the secondary sterilization process, the temperature of the secondary sterilization is 115-125 ℃, and more preferably 118-121 ℃;

preferably, in the secondary sterilization process, the time for the secondary sterilization is 25 to 35min, and more preferably 28 to 32min;

preferably, in the secondary sterilization process, the pressure of the secondary sterilization is 0.1-0.15 MPa, and more preferably 0.1-0.13 MPa;

preferably, the mixing temperature is 60-75 ℃ in the mixing process of the preservative;

preferably, the preservative comprises p-hydroxyacetophenone and/or 1, 2-hexanediol during the mixing with the preservative; when the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant obtained after the centrifugation is 0.1-0.5%, and the mass percentage of the 1, 2-hexanediol to the supernatant obtained after the centrifugation is 0.5-2%; preferably, the mass percent of the p-hydroxyacetophenone in the supernatant obtained after the centrifugation is 0.5%, and the mass percent of the 1, 2-hexanediol in the supernatant obtained after the centrifugation is 0.5%.

7. A mung bean seed fermented product, which is produced by the method for producing a mung bean seed fermented product according to any one of claims 1 to 6.

8. Use of the mung bean seed fermented product according to claim 7 for the preparation of a skin external preparation as a product, as an additive or as a base.

9. The use as claimed in claim 8, wherein the mung bean seed fermented product is used as an antioxidant active ingredient and/or an anti-inflammatory active ingredient in the skin external preparation;

preferably, the antioxidant active ingredient is an antioxidant active ingredient having DPPH free radical scavenging action;

preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient having an inhibitory activity against the production of the inflammatory factor TNF- α.

10. A skin external preparation comprising the mung bean seed fermented product according to claim 7;

preferably, 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-allergy active ingredient and an antioxidant active ingredient;

preferably, the skin external agent comprises a mask, essence or toner;

preferably, the mung bean seed leavening accounts for 5 to 99 percent of the mass of the skin external preparation, and more preferably 60 to 99 percent.

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