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CN115006310B - Mung bean sprout fermentation product, external skin preparation containing mung bean sprout fermentation product, and preparation method and application of external skin preparation - Google Patents

Mung bean sprout fermentation product, external skin preparation containing mung bean sprout fermentation product, and preparation method and application of external skin preparation Download PDF

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CN115006310B
CN115006310B CN202210792878.8A CN202210792878A CN115006310B CN 115006310 B CN115006310 B CN 115006310B CN 202210792878 A CN202210792878 A CN 202210792878A CN 115006310 B CN115006310 B CN 115006310B
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mung bean
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temperature
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ferment
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CN115006310A (en
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王海涛
由士权
祖士高
耿继满
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Heze Yixin Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

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Abstract

The invention discloses mung bean sprouting fermentation product, an external skin preparation containing the mung bean sprouting fermentation product, and a preparation method and application of the external skin preparation. The preparation method of the mung bean sprouting fermentation product comprises the following steps: inoculating lactobacillus into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises mung bean sprouting water extract; the preparation method of the mung bean sprout water extract comprises the steps of taking mung bean sprouts as raw materials, taking water as an extracting agent, and leaching for 20-60 min at the temperature of 80-95 ℃. The mung bean sprouting fermentation product has ideal oxidation resistance, simple preparation process, mild fermentation conditions, low energy consumption and cost saving, does not adopt any organic reagent in the preparation process, has higher safety, meets the requirements of modern skin external agents on functionality and safety, and realizes energy conservation and environmental protection; can be widely applied to the field of external skin preparations, and expands the application field of mung bean sprouting.

Description

Mung bean sprout fermentation product, external skin preparation containing mung bean sprout fermentation product, and preparation method and application of external skin preparation
Technical Field
The invention belongs to the field of fermentation, and particularly relates to mung bean sprouting fermentation product, an external skin preparation containing the mung bean sprouting fermentation product, and a preparation method and application of the external skin preparation.
Background
Mung bean Vigna radiata (linn.) is also known as green bean, belonging to the genus 1 year old standing herb of the genus Vigna of the family leguminosae. The chemical composition of mung bean mainly comprises protein, fat, vitamins, folic acid, polyphenol and flavonoid substances. The active substances of mung beans are changed in the germination process, the change is mainly concentrated in flavone, phenols and vitamins, the substances have the functions of resisting oxidization and delaying aging, and the flavone and the phenols are one of hot spots for researching natural antioxidants in recent years, so that the mung beans have good free radical scavenging effect.
At present, mung bean sprouts are mostly applied to the fields of fresh food, feed and the like, and the research of mung bean sprouts in the field of cosmetics is relatively less; the processing of mung bean sprouts still stays in the primary processing stage, and the deep processing by taking mung bean sprouts as raw materials is still yet to be developed; at present, the extraction of active substances is concentrated on special parts such as hypocotyls, and the whole components in mung bean sprouts cannot be extracted, so that the phenomena of low resource utilization rate, serious waste and the like of mung bean sprouts are caused. The prior art also reports that the organic solvent is adopted to extract one active ingredient in mung bean sprouts, so that not only can the environment be polluted, but also other active ingredients in mung bean sprouts can be lost.
Therefore, there is a need in the art to develop mung bean sprout extract which can efficiently utilize the active ingredients in mung bean sprouts, has low preparation cost, can be used in the field of external skin preparations, and has cosmetic activity.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, the processing of mung bean sprouts remains in the primary processing stage, the extraction of active substances is concentrated on special parts such as hypocotyls, and the like, and the full components in mung bean sprouts cannot be extracted, so that the mung bean sprouts have low resource utilization rate and serious waste; the extraction process uses organic solvent, pollutes the environment, and has the defects of fresh report of application in the cosmetic field, etc., thereby providing mung bean sprouting fermentation product, skin external agent containing the mung bean sprouting fermentation product, and a preparation method and application thereof. The mung bean sprouting fermentation product has ideal oxidation resistance, simple preparation process, mild fermentation conditions, low energy consumption and cost saving, does not adopt any organic reagent in the preparation process, has higher safety, meets the requirements of modern skin external agents on functionality and safety, and realizes energy conservation and environmental protection; can be widely applied to the field of external skin preparations, and expands the application field of mung bean sprouting.
The invention adopts the following technical scheme to solve the technical problems:
the invention provides a preparation method of mung bean sprouting fermentation product, which comprises the following steps: inoculating lactobacillus into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises mung bean sprouting water extract; the preparation method of the mung bean sprout water extract is that mung bean sprouts are taken as raw materials, water is taken as an extracting agent, and leaching is carried out for 20-60 min at the temperature of 80-95 ℃.
In some embodiments, the mung bean sprouting method may be conventional in the art, and may be generally prepared by sprouting mung beans.
Wherein the conditions and methods of the germination treatment may be those conventional in the art, and may generally be performed in a germination machine.
Wherein the temperature of the germination treatment may be a temperature conventional in the art for such operations, preferably 25 to 30 ℃, more preferably 28 ℃.
The germination treatment may be performed in a manner conventional in the art, preferably for 48 to 96 hours, more preferably for 60 to 80 hours, for example, 72 hours.
In the preparation process of the mung bean sprouting water extract, the soaking in advance can further comprise a homogenizing operation.
In the preparation process of the mung bean sprouting water extract, the mass ratio of mung bean sprouting to water can be 1: (10-30), preferably 1:20.
in the preparation process of the mung bean sprouting water extract, the leaching temperature is preferably 90-95 ℃.
During the preparation of the mung bean sprouting aqueous extract, the leaching can be carried out under stirring conditions which are conventional in the art, and the stirring speed can be 200-400 rpm, preferably 280-320 rpm, for example 300rpm.
In the preparation process of the mung bean sprouting water extract, the leaching time is preferably 30-60 min, for example 40min.
In some embodiments, the fermentation substrate may also be subjected to procedures conventional in the art including sterilization prior to use.
Wherein the conditions and methods of sterilization may be those conventional in the art for such procedures and may generally be high temperature sterilization.
When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization temperature may be a temperature which is conventional in the art for such operations, preferably 115 to 125 ℃, more preferably 118 to 121 ℃.
When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the art for such an operation, preferably 30 to 40 minutes, more preferably 35 minutes.
When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization pressure may be a pressure which is conventional in such an operation in the art, preferably 0.1 to 0.15Mpa, more preferably 0.1 to 0.13Mpa, for example 0.12Mpa.
Wherein the sterilization operation may further comprise a cooling operation, typically to room temperature, as is conventional in the art.
In some embodiments, the lactobacillus may include lactobacillus helveticus (Lactobacillus helveticus) and/or lactobacillus plantarum subspecies plantarum (Lactobacillus plantarum subsp.
Wherein the lactobacillus helveticus (Lactobacillus helveticus) can comprise lactobacillus helveticus with the preservation number of CICC 20243 which is preserved in China center for type culture Collection of industrial microorganisms.
Wherein the lactobacillus plantarum subspecies (Lactobacillus plantarum subsp. Plantarum) can comprise lactobacillus plantarum subspecies deposited with the China center for Industrial microorganism culture Collection, with a deposit number of CICC 20261.
Wherein when the lactobacillus includes lactobacillus helveticus and lactobacillus plantarum subspecies plantarum, the ratio of viable bacteria numbers of the lactobacillus helveticus and the lactobacillus plantarum subspecies plantarum can be (0.1-10): 1, preferably (0.25 to 4): 1, more preferably (0.3 to 3): 1.
when the Lactobacillus helveticus is used, the Lactobacillus helveticus may be added as a Lactobacillus helveticus bacterial solution, the concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial solution may be 10, according to the conventional method in the art 9 ～10 11 CFU/mL, preferably 10 10 CFU/mL。
When the Lactobacillus plantarum subspecies are used, the Lactobacillus plantarum subspecies may be added as a Lactobacillus plantarum subspecies solution, in which the Lactobacillus plantarum subspecies concentration may be 10, as is conventional in the art 9 ～10 11 CFU/mL, preferably 10 10 CFU/mL。
In some embodiments, the amount of Lactobacillus inoculated per unit mass of the fermentation substrate may be conventional in the art, preferably 6X 10 6 CFU/mL～6×10 8 CFU/mL, more preferably 6X 10 7 CFU/mL～6×10 8 CFU/mL。
In some embodiments, the fermentation time may be 12 to 24 hours, preferably 14 to 18 hours, more preferably 16 hours.
In some embodiments, the temperature of the fermentation culture may be 30 to 45 ℃, preferably 35 to 45 ℃.
In some embodiments, the conditions and methods of sterilization may be conventional in the art, and may generally be high temperature sterilization.
When the sterilization is performed by the high temperature sterilization method, the sterilization temperature may be a temperature which is conventional in such an operation in the art, preferably 115 to 125 ℃, 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 art for such an operation, preferably 30 to 40 minutes, more preferably 35 minutes.
When the sterilization is performed by the high temperature sterilization method, the pressure of the sterilization may be a pressure which is conventional in such an operation in the art, preferably 0.1 to 0.15MPa, more preferably 0.1 to 0.13MPa, for example 0.12MPa.
In some embodiments, the sterilization may be followed by cooling and/or centrifugation to collect supernatant.
Wherein the cooling may be to room temperature, as is conventional in the art.
The rotational speed of the centrifugation may be a rotational speed conventional in the art, preferably 4000 to 8000rpm, more preferably 4800 to 8000rpm.
Wherein the radius of the centrifugation may be a radius conventional in this type of operation in the art, preferably 8-15 cm.
The centrifugation time may be a time conventional in the art, preferably 20 to 40min, more preferably 30min.
Wherein the centrifugation may be followed by a secondary sterilization and/or mixing with a preservative.
The secondary sterilization method may be a high temperature sterilization method conventionally used in the art.
When the secondary sterilization is performed by the high temperature sterilization method, the temperature of the secondary sterilization may be a temperature which is conventional in the art for such operations, preferably 115 to 125 ℃, more preferably 118 to 121 ℃.
When the secondary sterilization is performed by the high temperature sterilization method, the time for the secondary sterilization may be a time conventional in the art for such an operation, preferably 30 to 40 minutes, more preferably 35 minutes.
When the secondary sterilization is performed by the high temperature sterilization method, the pressure of the secondary sterilization may be a pressure which is conventional in such an operation in the art, preferably 0.1 to 0.15MPa, more preferably 0.1 to 0.13MPa, for example 0.12MPa.
The temperature of the mixing during mixing with the preservative may be a temperature conventional in the art for such operations, preferably 60-75 ℃.
In 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 fluid prepared after the centrifugation can be 0.1-1%, and the mass percentage of the 1, 2-hexanediol to the supernatant fluid prepared after the centrifugation can be 0.5-2%; preferably, the p-hydroxyacetophenone accounts for 0.5% of the supernatant obtained after the centrifugation, and the 1, 2-hexanediol accounts for 0.8% of the supernatant obtained after the centrifugation.
The invention also provides a mung bean sprouting fermentation product, which is prepared by the preparation method of the mung bean sprouting fermentation product.
The invention also provides an application of the mung bean sprouting fermentation product in preparing external skin preparations, wherein the mung bean sprouting fermentation product is directly used as a product, an additive or a substrate.
In some embodiments, the mung bean sprout fermentation may be used as an antioxidant active ingredient in the external skin preparation.
Wherein the antioxidant active ingredient may be an antioxidant active ingredient having DPPH radical scavenging action and/or having hydroxy radical scavenging action.
The invention also provides a skin external agent which comprises the mung bean sprouting fermentation product.
In some embodiments, the external skin 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-allergic active ingredient, and an anti-oxidation active ingredient.
In some embodiments, the skin external preparation may include, but is not limited to, a mask, essence, or toner as is conventional in the art.
In some embodiments, the mung bean sprouting ferment may be 5-99% by mass, preferably 60-99% by mass of the skin external agent.
In some embodiments, the room temperature generally refers to 15-40 ℃.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the mung bean sprouting fermentation product prepared by the method has ideal oxidation resistance, simple preparation process, mild fermentation conditions, low energy consumption, cost saving, no use of any organic reagent, high safety, and suitability for the requirements of modern external skin preparations for functionality and safety, can be widely applied to the field of external skin preparations, and expands the application field of mung bean sprouting.
Drawings
The present disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are included to provide a further illustration of the preferred embodiments of the disclosure and to explain the principles and advantages of the disclosure, are incorporated in and form a part of the specification along with the detailed description that follows. Wherein:
FIG. 1 is a graph showing comparison of cell viability after treatment of human skin fibroblasts using the products prepared in examples 1 to 4 or comparative examples 1 to 4;
FIG. 2 is a graph showing the DPPH radical scavenging ability of the products obtained in examples 1 to 4 or comparative examples 1 to 4;
FIG. 3 is a graph showing the total antioxidant capacity of the products obtained in examples 1 to 4 or comparative examples 1 to 4;
FIG. 4 is a graph showing the comparison of the scavenging ability of the hydroxyl radicals of the products obtained in examples 1 to 4 or comparative examples 1 to 4.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
In the following examples and comparative examples, lactobacillus helveticus (Lactobacillus helveticus) was purchased from the chinese industrial microorganism strain collection management center under the collection number cic 20243;
in the following examples and comparative examples, lactobacillus plantarum subspecies (Lactobacillus plantarum subsp. Plantarum) were purchased from the chinese industrial microbiological bacterial deposit management center under the deposit number cic 20261;
in the following comparative examples, streptococcus thermophilus (Streptococcus thermophilus) was Streptococcus thermophilus model HH-ST08, manufactured by Zhengzhou and He Biotechnology Co., ltd.
Example 1
(1) Screening clean and symmetric mung beans without damage and discoloration, rinsing the mung beans for 3 times, spreading the cleaned mung beans in a germination machine for germination treatment, and culturing for 3 days at 28 ℃ to obtain mung bean germination; weighing 15g of mung bean sprouts and 300mL of deionized water for homogenating, leaching for 40min at 90 ℃ after homogenating, wherein the rotating speed of a stirring paddle is 300rpm during leaching, and preparing mung bean sprout water extract; sterilizing at 121deg.C under 0.12Mpa for 35min, sterilizing, and cooling to room temperature to obtain fermentation substrate;
(2) The viable cell count of Lactobacillus helveticus (CICC 20243) and Lactobacillus plantarum subspecies (CICC 20261) were each 10 10 CFU/mL bacterial liquid; inoculating 1.35mL of lactobacillus helveticus bacterial liquid and 0.45mL of lactobacillus plantarum subspecies liquid into the fermentation substrate prepared in the step (1), and culturing for 16 hours in a shaking incubator at a constant temperature of 30 ℃ with a shaking table rotating speed of 180r/min; sterilizing at 121deg.C and 0.12MPa for 35min after fermentation, cooling to room temperature after sterilization, centrifuging at 4800r/min for 30min; and (3) obtaining supernatant after centrifugation, and performing secondary sterilization on the supernatant, wherein the temperature of the secondary sterilization is 121 ℃, the pressure of the secondary sterilization is 0.12MPa, and the time of the secondary sterilization is 35min, so as to obtain the mung bean sprouting fermentation product.
Example 2
The difference from example 1 is that the lactobacillus strain solution introduced in the step (2) is 0.45mL of lactobacillus helveticus strain solution and 1.35mL of lactobacillus plantarum subspecies strain solution, and other condition parameters are the same as in example 1.
Example 3
The difference from example 1 is that the lactobacillus strain solution introduced in the step (2) was 1.8mL of lactobacillus helveticus strain solution, and other conditions and parameters were the same as those in example 1.
Example 4
The difference from example 1 is that the lactobacillus strain solution introduced in the step (2) is 1.8mL of lactobacillus plantarum subspecies plantarum strain solution, and other conditions and parameters are the same as in example 1.
Comparative example 1
In comparison with example 1, only the difference was that the hot water extraction was not performed in step (1), and 15g of mung bean sprouts and 300mL of water were homogenized and sterilized to obtain fermentation substrates, and other conditions and parameters were the same as in example 1.
Comparative example 2
The only difference compared to example 1 is that no fermentation treatment is performed, specifically the fermentation substrate obtained in step (1) of example 1.
Comparative example 3
Compared with example 1, the difference is only that in the process of preparing mung bean sprouting water extract in the step (1), the leaching time is 70min, and other condition parameters are the same as in example 1.
Comparative example 4
The difference from example 1 is that in the step (2), the inoculated fermentation broth is Streptococcus thermophilus (HH-ST 08) broth, and the viable count in the Streptococcus thermophilus broth is 10 10 CFU/mL was added in an amount of 1.8mL, and other conditions were the same as in example 1.
Effect example 1 human skin fibroblast toxicity test
The experiment adopts human skin fibroblasts from Chinese scientific cell bank to verify cytotoxicity of the products prepared in the above examples 1-4 and comparative examples 1-4.
Reagent: 0.25% (containing EDTA) trypsin manufacturer is GIBCO company, U.S.A.; the DMEM medium manufacturer is GIBCO, usa; the manufacturer of the double antibody is Corning company in the United states; the manufacturer of CCK-8 is Beijing Bayer Di Biotechnology Co., ltd; the manufacturer of the fetal bovine serum is GIBCO company in the United states; the manufacturer of phosphate buffer is Beijing Bai Rui Biotech Co.
The device comprises: WJ-80A-II type CO 2 The manufacturer of the constant temperature incubator is Shanghai Saint Corp instruments and equipment limited company; the manufacturer of the Sunrise enzyme-labeled instrument is Diken trade company; the TL80-2 medical centrifuge manufacturer is Jiangsu Tianli medical instruments limited company; the manufacturer of NUNC 96 well cell culture plates is sameidie science and technology.
1. The experimental steps are as follows:
the serum-free DMEM media for the products prepared in examples 1 to 4 and comparative examples 1 to 4 were prepared as experimental group test solutions having a volume percentage of 5%. Human skin fibroblasts were cultured in a medium containing 10% fetal bovine serum and 1% diabody (1X 10) 5 U/L penicillin, 100mg/L streptomycin) in DMEM medium. Cell growth at 37℃with 5% CO 2 When the cell fusion reached 85% or more in an incubator with saturated humidity, cells in the logarithmic growth phase were digested with 0.05% pancreatin, and the digestion reaction was terminated with DMEM containing serum. Cell counting plate counts, cell suspension concentration was adjusted to 7X 10 4 Each mL of the cell suspension was inoculated into a 96-well plate at a rate of 100. Mu.L per well, 37℃and 5% CO 2 Incubate under conditions for 12h. Old culture medium was removed and cells were washed twice with phosphate buffer. Adding 100 mu L of the filtered and sterilized experimental group to-be-tested liquid in each hole of the experimental group, and making 6 compound holes for each to-be-tested liquid; the control group contains cells, and serum-free DMEM medium is added; the blank group was cell-free and 100. Mu.L of PBS was added. Then at 37 ℃ and 5% CO 2 Incubate under conditions for 24h. Then 10. Mu.L of CCK-8 solution was added to each well, incubated for 3 hours, absorbance was measured at a wavelength of 450nm, and the cell viability of each group was calculated, and the results are shown in Table 1 and FIG. 1.
The cell viability was calculated as follows:
cell viability (%) = (a experimental group-a blank)/(a control group-a blank) ×100%.
TABLE 1
The results show that when the products prepared in the examples 1-4 of the invention with the same concentration are used for treating human skin fibroblasts, the cell survival rate is obviously higher than that of the comparative examples 1-4.
Effect example 2
DPPH is an early synthetic organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, which is very stable in organic solvents, purple in color, and has a characteristic absorption peak at 517nm, when a radical scavenger is encountered, the lone pair of electrons of DPPH are paired to fade 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 DPPH free radical scavenging experiment comprises the following specific experimental steps:
(1) Equal volumes (1 mL) of the test solutions (products obtained in examples 1 to 4 or comparative examples 1 to 4) were taken together with 2X 10 - 4 mixing the DPPH solution of mol/L (A) 1 A tube);
(2) Taking equal volume (1 mL) of absolute ethanol (solvent of the object to be detected) and 2X 10 -4 mixing the DPPH solution of mol/L (A) 2 A tube);
(3) Mixing the same volume (1 mL) of absolute ethanol with the liquid to be measured (A) 3 A tube);
(4) After reaction in the dark for 30min, A was measured at 517nm 1 Tube A 2 Tube A 3 Tube absorbance values; the clearance rate calculation formula is: clearance = [ (A) 2 +A 3 )-A 1 ]/A 2 ×100％。
The products prepared in examples 1 to 4 and comparative examples 1 to 4 were subjected to DPPH radical scavenging test, and the results are shown in Table 2 and FIG. 2.
TABLE 2
As can be seen from the results of FIG. 2 and Table 2, the DPPH radical scavenging ability of the mung bean sprout fermentations prepared in examples 1-4 of the present invention was significantly higher than that of the products prepared in comparative examples 1-4. In fig. 2, ns represents no statistical difference from example 1; * p < 0.05, indicating a statistical difference compared to example 1; * P < 0.01, indicating significant statistical differences compared to example 1, significant reductions; * P < 0.001, indicating a very significant statistical difference compared to example 1, a very significant decrease.
Effect example 3
The products prepared in examples 1 to 4 and comparative examples 1 to 4 were tested for total antioxidant capacity using the total antioxidant capacity test kit (ABTS method) of the product number S0119 manufactured by the company of the bio-technology limited of bi yun, and the results are shown in table 3 and fig. 3.
TABLE 3 Table 3
Total antioxidant capacity (TEAC/mM)
Example 1 0.375±0.007
Example 2 0.384±0.01
Example 3 0.318±0.014
Example 4 0.36±0.007
Comparative example 1 0.282±0.012
Comparative example 2 0.253±0.005
Comparative example 3 0.243±0.006
Comparative example 4 0.252±0.014
As can be seen from the results of FIG. 3 and Table 3, the total antioxidant capacity of the mung bean sprouting fermented products prepared in examples 1 to 4 of the present invention is significantly higher than that of the products prepared in comparative examples 1 to 4. In fig. 3, ns represents no statistical difference from example 1; * p < 0.05, indicating a statistical difference compared to example 1; * P < 0.01, indicating significant statistical differences compared to example 1, significant reductions; * P < 0.001, indicating a very significant statistical difference compared to example 1, a very significant decrease.
Effect example 4
Hydroxyl free radicals (OH) are generated by Fenton reaction, salicylic acid is added into a reaction system, the OH reacts with the salicylic acid, and the generated colored compound 2, 3-dihydroxybenzoic acid has characteristic absorption at 510 nm. Measuring absorbance of the reaction solution containing the test object at 510nm by using a fixed reaction time method, comparing with a blank solution, measuring the cleaning effect of the test object on OH, adding the reagent into a test tube according to the table 4, sequentially adding 9mmol/L FeSO 4 9mmol/L salicylic acid ethanol solution, samples (products prepared in examples 1-4 or comparative examples 1-4), a proper amount of deionized water and 8.8mmol/L H 2 O 2 A solution. Shaking upHeating in 37deg.C water bath for 15min, taking out, and measuring absorbance A of blank control 0 (the configuration mode is shown in group A in Table 4), absorbance A is added to the sample x (see Table 4 for configuration, group B) and no H 2 O 2 Solution background absorbance A of (2) x0 (see Table 4 for configuration, panel C). Determination A 0 When the reference solution is a system without adding hydrogen peroxide; the radical scavenging rate of each group was calculated according to the following formula, and the results are shown in Table 5 and FIG. 4.
The hydroxyl radical clearance calculation formula is:
clearance= (a) 0 －(A x －Ax 0 ))/A 0 ×100％。
TABLE 4 Table 4
TABLE 5
As can be seen from the results of FIGS. 4 and 5, the hydroxyl radical scavenging ability of the mung bean sprout ferments prepared in examples 1 to 4 of the present invention was significantly higher than that of the products prepared in comparative examples 1 to 4. In fig. 4, p < 0.05, showing a statistical difference compared to example 1; * P < 0.01, indicating significant statistical differences compared to example 1, significant reductions; * P < 0.001, indicating a very significant statistical difference compared to example 1, a very significant decrease; ## p < 0.01, indicating a significant statistical difference compared to example 1, a significant increase.
Finally, it is also noted that 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 by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.

Claims (28)

1. The preparation method of the mung bean sprouting fermentation product is characterized by comprising the following steps of: inoculating lactobacillus into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises mung bean sprouting water extract; the preparation method of the mung bean sprout water extract comprises the steps of taking mung bean sprouts as a raw material, taking water as an extracting agent, and leaching for 20-60 min at the temperature of 80-95 ℃;
the lactobacillus includes lactobacillus helveticus (Lactobacillus helveticus) and lactobacillus plantarum subspecies plantarum (Lactobacillus plantarum subsp. Plantarum);
the lactobacillus helveticus (Lactobacillus helveticus) comprises lactobacillus helveticus with the preservation number of CICC 20243 which is preserved in China center for type culture Collection of industrial microorganisms;
the lactobacillus plantarum subspecies (Lactobacillus plantarum subsp. Plantarum) comprise lactobacillus plantarum subspecies with the preservation number of CICC 20261 which are preserved in China center for type culture Collection of industrial microorganisms;
the ratio of the number of viable bacteria of the Lactobacillus helveticus and the Lactobacillus plantarum subspecies plantarum is (0.1-10): 1.
2. the method for preparing mung bean sprout ferment according to claim 1, wherein the method for preparing mung bean sprout ferment satisfies at least one of the following conditions:
the mung bean sprouting preparation method comprises the step of sprouting mung beans; in the preparation process of the mung bean sprouting water extract, the soaking step further comprises the operation of homogenizing in advance;
in the preparation process of the mung bean sprouting water extract, the mass ratio of mung bean sprouting to water is 1: (10-30);
in the preparation process of the mung bean sprouting water extract, the leaching temperature is 90-95 ℃;
in the preparation process of the mung bean sprouting water extract, the leaching is carried out under the condition of stirring, and the stirring rotating speed is 200-400 rpm;
in the preparation process of the mung bean sprouting water extract, the leaching time is 30-60 min.
3. The method for preparing mung bean sprout ferment according to claim 2, wherein the method for preparing mung bean sprout ferment satisfies at least one of the following conditions:
in the preparation process of mung bean germination, the temperature of the germination treatment is 25-30 ℃;
in the preparation process of mung bean germination, the germination treatment time is 48-96 hours;
in the preparation process of the mung bean sprouting water extract, the mass ratio of mung bean sprouting to water is 1:20, a step of;
in the preparation process of the mung bean sprouting water extract, the leaching is carried out under the condition of stirring, and the stirring rotating speed is 280-320 rpm.
4. The method for preparing mung bean sprout ferment according to claim 3, wherein the method for preparing mung bean sprout ferment satisfies at least one of the following conditions:
in the preparation process of mung bean germination, the temperature of the germination treatment is 28 ℃;
in the preparation process of mung bean germination, the germination treatment time is 60-80 hours;
in the preparation process of the mung bean sprouting water extract, the leaching is carried out under the condition of stirring, and the stirring rotating speed is 300rpm.
5. The method for producing a fermented mung bean sprout according to claim 4, wherein the germination treatment time is 72 hours during the germination of mung bean sprouts.
6. The method of preparing mung bean sprout fermentation according to claim 1, wherein the fermentation substrate further comprises a sterilization operation prior to use.
7. The method for producing a mung bean sprouted fermentation according to claim 6, wherein said method for sterilizing said fermentation substrate is a high-temperature sterilization method; when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization temperature is 115-125 ℃; when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization time is 30-40 min; when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.15 MPa.
8. The method for producing a mung bean sprouting fermentation according to claim 7, characterized in that when said fermentation substrate is subjected to said sterilization by said high-temperature sterilization method, the temperature of said sterilization is 118 to 121 ℃; when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization time is 35min; when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.13 MPa.
9. The method of claim 6, wherein said sterilizing said fermentation substrate is followed by cooling to room temperature.
10. The method for preparing mung bean sprout ferment according to claim 1, wherein the method for preparing mung bean sprout ferment satisfies at least one of the following conditions;
the ratio of the number of viable bacteria of the Lactobacillus helveticus and the Lactobacillus plantarum subspecies plantarum is (0.25-4): 1, a step of; the Lactobacillus helveticus is added in the form of Lactobacillus helveticus bacterial liquid, and the concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial liquid is 10 9 ～10 11 CFU/mL；
The Lactobacillus plantarum subspecies are added in the form of Lactobacillus plantarum subspecies bacterial liquid, and the concentration of the Lactobacillus plantarum subspecies in the Lactobacillus plantarum subspecies bacterial liquid is 10 9 ～10 11 CFU/mL；
The amount of the lactobacillus inoculated in the fermentation substrate per unit mass is 6×10 6 CFU/mL～6×10 8 CFU/mL。
11. The method for preparing mung bean sprout ferment according to claim 10, wherein the method for preparing mung bean sprout ferment satisfies at least one of the following conditions:
the ratio of the number of viable bacteria of the Lactobacillus helveticus and the Lactobacillus plantarum subspecies plantarum is (0.3-3): 1, a step of;
the Lactobacillus helveticus is added in the form of Lactobacillus helveticus bacterial liquid, and the concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial liquid is 10 10 CFU/mL；
The Lactobacillus plantarum subspecies are added in the form of Lactobacillus plantarum subspecies bacterial liquid, and the concentration of the Lactobacillus plantarum subspecies in the Lactobacillus plantarum subspecies bacterial liquid is 10 10 CFU/mL；
The amount of the lactobacillus inoculated in the fermentation substrate per unit mass is 6×10 7 CFU/mL～6×10 8 CFU/mL。
12. The method for producing a mung bean sprout ferment according to any one of claims 1 to 11, wherein the method for producing a mung bean sprout ferment satisfies at least one of the following conditions:
the fermentation culture time is 12-24 hours;
the temperature of the fermentation culture is 30-45 ℃;
the sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is adopted for the sterilization, the temperature of the sterilization is 115-125 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 30-40 min; when the high-temperature sterilization method is adopted for the sterilization, the pressure of the sterilization is 0.1-0.15 MPa.
13. The method of preparing mung bean sprout ferment according to claim 12, wherein the method of preparing mung bean sprout ferment satisfies at least one of the following conditions:
the fermentation culture time is 14-18 h;
the temperature of the fermentation culture is 35-45 ℃;
when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 118-121 ℃;
when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 35min;
when the high-temperature sterilization method is adopted for the sterilization, the pressure of the sterilization is 0.1-0.13 MPa.
14. The method for producing a mung bean sprouting fermentation according to claim 13, wherein the fermentation culture time is 16 hours.
15. The method for preparing mung bean sprouting fermented product according to claim 1, further comprising the operation of cooling and/or centrifuging and collecting supernatant after said sterilization operation.
16. The method of preparing mung bean sprout ferment according to claim 15, wherein the method of preparing mung bean sprout ferment satisfies at least one of the following conditions:
the cooling is to cool to room temperature;
the rotational speed of the centrifugation is 4000-8000 rpm;
the radius of the centrifugation is 8-15 cm;
the centrifugation time is 20-40 min.
17. The method of preparing mung bean sprout ferment according to claim 16, wherein the method of preparing mung bean sprout ferment satisfies at least one of the following conditions:
the rotational speed of the centrifugation is 4800-8000 rpm;
the centrifugation time was 30min.
18. The method for preparing a germinated mung bean fermentation according to any one of claims 15 to 17, wherein the centrifugation step is further followed by secondary sterilization and/or mixing with a preservative.
19. The method of preparing mung bean sprout ferment according to claim 18, wherein the method of preparing mung bean sprout ferment satisfies at least one of the following conditions:
the secondary sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization is 115-125 ℃; when the high-temperature sterilization method is adopted for the secondary sterilization, the time of the secondary sterilization is 30-40 min; when the high-temperature sterilization method is adopted for the secondary sterilization, the pressure of the secondary sterilization is 0.1-0.15 MPa;
in the process of mixing with the preservative, the mixing temperature is 60-75 ℃;
during mixing with the preservative, the preservative comprises p-hydroxyacetophenone and/or 1, 2-hexanediol.
20. The method of preparing a mung bean sprout ferment according to claim 19, wherein the method of preparing a mung bean sprout ferment satisfies at least one of the following conditions:
when the high-temperature sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization is 118-121 ℃; when the high-temperature sterilization method is adopted for the secondary sterilization, the time of the secondary sterilization is 35min; when the high-temperature sterilization method is adopted for the secondary sterilization, the pressure of the secondary sterilization is 0.1-0.13 MPa;
when the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant fluid prepared after centrifugation is 0.1-1%, and the mass percentage of the 1, 2-hexanediol to the supernatant fluid prepared after centrifugation is 0.5-2%.
21. The method for preparing mung bean sprout fermentation according to claim 20, wherein when the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after the centrifugation is 0.5%, and the mass percentage of 1, 2-hexanediol in the supernatant obtained after the centrifugation is 0.8%.
22. A mung bean sprout ferment prepared by the method of preparing a mung bean sprout ferment according to any one of claims 1 to 21.
23. Use of a mung bean sprout ferment according to claim 22 as a direct product, as an additive or as a substrate in the preparation of a skin external agent.
24. The use according to claim 23, wherein the mung bean sprouted fermentation is used as an antioxidant active ingredient in the external skin preparation.
25. The use according to claim 24, wherein the antioxidant active ingredient is an antioxidant active ingredient having DPPH radical scavenging action and/or having hydroxy radical scavenging action.
26. A skin external preparation comprising the mung bean sprouting fermented product according to claim 22.
27. The skin external preparation according to claim 26, 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 anti-oxidation active ingredient;
the skin external agent comprises a facial mask, essence or toner;
the mung bean sprouting fermentation product accounts for 5-99% of the mass of the skin external agent.
28. The external preparation for skin according to claim 26 or 27, wherein the mung bean sprouting fermentation product accounts for 60-99% by mass of the external preparation for skin.
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