CN113337545A - Schizophyllum commune fermentation product, preparation method thereof, skin care product and schizophyllum commune culture medium - Google Patents
Schizophyllum commune fermentation product, preparation method thereof, skin care product and schizophyllum commune culture medium Download PDFInfo
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Abstract
The application relates to the field of microorganisms, and particularly relates to a schizophyllum commune fermentation product, a preparation method thereof, a skin care product and a schizophyllum commune culture medium. The preparation method of the Schizophyllum commune fermentation product comprises the following steps: culturing Schizophyllum commune in Schizophyllum commune culture medium, and collecting fermentation product; wherein the schizophyllum commune culture medium contains kudzu root. Pueraria powder is added into a culture medium to culture Schizophyllum commune, and a fermentation product is collected, wherein the obtained fermentation product has positive effects on oxidation resistance and free radical removal.
Description
Technical Field
The application relates to the field of microorganisms, and particularly relates to a schizophyllum commune fermentation product, a preparation method thereof, a skin care product and a schizophyllum commune culture medium.
Background
The Schizophyllum commune contains cellulase with strong activity, and can produce malic acid, and can produce a large amount of organic acid during submerged fermentation of mycelia, and can also produce auxin indoleacetic acid, and the fermentation product of Schizophyllum commune can be used in medicine, health and skin care product industries.
Disclosure of Invention
The embodiments of the present application aim to provide a fermentation product of schizophyllum commune, a preparation method thereof, a skin care product, and a culture medium of schizophyllum commune, which aim to improve the oxidation resistance level of the fermentation product of schizophyllum commune.
The application provides a preparation method of a Schizophyllum commune fermentation product, which comprises the following steps:
culturing Schizophyllum commune in Schizophyllum commune culture medium, and collecting fermentation product;
wherein the schizophyllum commune culture medium contains kudzu root.
Pueraria powder is added into a culture medium to culture Schizophyllum commune, and a fermentation product is collected, wherein the obtained fermentation product has positive effects on oxidation resistance and free radical removal.
In some embodiments of the present application, the kudzu root is kudzu root powder having a particle size of 40 mesh or less.
In some embodiments of the present application, the kudzu root powder is present in the schizophyllum commune medium in an amount of 2.0-8.0 g/L.
In some embodiments of the present application, the time for culturing Schizophyllum commune in the Schizophyllum commune medium is 100-150 h.
In some embodiments of the present application, the Schizophyllum commune is cultured in a Schizophyllum commune culture medium at a temperature of 28 deg.C-30 deg.C and at a rotation speed of 140r/min-170 r/min.
In some embodiments of the present application, the Schizophyllum commune medium comprises yeast extract 0.1-0.5 wt%, magnesium sulfate 0.01-0.15 wt%, potassium dihydrogen phosphate 0.05-0.2 wt%, and glucose 1-4 wt%.
In some embodiments of the present application, the step of culturing Schizophyllum commune in a Schizophyllum commune medium further comprises:
activating the Schizophyllum commune strain by a slant culture medium, and culturing at 28-30 deg.C and 140-170 r/min for 5-7 days until the slant is full of mycelia; transferring the slant strain to liquid culture medium, and performing amplification culture for 3-4 days to obtain activated Schizophyllum commune.
The application also provides a Schizophyllum commune fermentation product, and the Schizophyllum commune fermentation product is prepared by the preparation method of the Schizophyllum commune fermentation product.
The Schizophyllum commune fermentation product provided by the application contains Schizophyllum commune polysaccharide serving as an active ingredient, and shows better capabilities in the aspects of removing excessive free radicals and inhibiting related enzyme activities by combining the antioxidant capacity of the radix puerariae matrix.
The application also provides a skin care product which comprises a substrate and the Schizophyllum commune fermentation product.
The application also provides a schizophyllum commune culture medium, wherein the schizophyllum commune culture medium contains 2.0-8.0g/L of kudzu root.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a standard curve of total sugar content by colorimetry.
FIG. 2 is a standard curve for determining reducing sugar content by colorimetry.
Figure 3 shows a DPPH radical scavenging standard curve.
FIG. 4 shows the DPPH radical scavenging ability of the individual fermentation products.
FIG. 5 shows the ABTS +. free radical clearance standard curve.
FIG. 6 shows the ABTS +. free radical scavenging ability of the individual fermentation products.
FIG. 7 shows the elastase inhibition rate for each fermentation product.
FIG. 8 shows tyrosinase inhibition rates for individual fermentation products.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The fermentation product of Schizophyllum commune contains Schizophyllum commune polysaccharide as active ingredient; the inventor finds that the addition of certain substances into the culture medium of the schizophyllum commune can enable the fermentation product to have the capabilities of resisting oxidation and scavenging free radicals.
However, not all substances have the effects of promoting oxidation resistance and scavenging free radicals, and for example, the patent with the application number of CN202110011922.2 describes that the addition of licorice to a basic culture medium can increase the oxidation resistance of a fermentation product and inhibit the levels of elastase and tyrosinase. Morinda officinalis with effects of enhancing immunity of human body, improving osteoporosis, resisting depression and oxidation is added into culture medium, and the obtained fermentation product has oxidation resistance, tyrosinase inhibition ability and elastase inhibition ability not only not increased, but also decreased.
The inventor guesses that: during the fermentation process, the growth and metabolism of the schizophyllum commune are selective to substances in the substrate, and the components which do not have the effects of resisting oxidation and scavenging free radicals can promote the performances of the final fermentation product in the aspects of resisting oxidation and scavenging free radicals. Some ingredients have not only a promoting effect but also a counteracting effect.
Based on the above, the present application provides a Schizophyllum commune fermentation product, a preparation method thereof, a skin care product and a Schizophyllum commune culture medium, which aim to increase the capacities of the Schizophyllum commune fermentation product in oxidation resistance and free radical scavenging.
The fermentation product of Schizophyllum commune of the embodiments of the present application, its preparation method, skin care product, and culture medium of Schizophyllum commune will be described in detail below.
The application provides a preparation method of a Schizophyllum commune fermentation product, which comprises the following steps:
culturing Schizophyllum commune in Schizophyllum commune culture medium, and collecting fermentation product;
wherein the schizophyllum commune culture medium contains kudzu root.
In the present application, pueraria lobata refers to a raw material processed by a non-chemical processing means, which is crushed or cut by a physical means; for example, it may be in the form of powder, block or strip;
in some embodiments of the present application, the culturing of the schizophyllum commune in the schizophyllum commune medium further comprises activating the schizophyllum commune species, including: activating Schizophyllum commune strain with slant culture medium, and culturing at 28-30 deg.C (such as 28 deg.C, 29 deg.C, 30 deg.C) for 5-7 days (such as 5 days, 6 days, 7 days) at 140-170 r/min (such as 140r/min, 150r/min, 155r/min, 160r/min, 170r/min) until mycelia overgrow the slant; transferring slant strain to liquid culture medium, and performing amplification culture for 3-4 days (such as 3 days, 3.6 days, and 4 days) to obtain activated Schizophyllum commune.
And culturing the activated schizophyllum commune in a schizophyllum commune culture medium.
The research of the inventor shows that the radix puerariae is added into the culture medium in a solid or powder form, and the fermentation product obtained by culturing the schizophyllum commune after adding the radix puerariae into the schizophyllum commune culture medium in a water extract mode of the radix puerariae is poor in the aspects of oxidation resistance and the like.
In some embodiments of the present application, the kudzu root is present in a powdered form in the schizophyllum commune medium, and in some embodiments of the present application, the kudzu root has a particle size of less than or equal to 40 mesh. Sieving radix Puerariae powder with 40 mesh sieve, and collecting the sieved material.
For example, radix Puerariae is dried, pulverized, sieved with a 40 mesh sieve to obtain radix Puerariae powder, sterilized, and added to a Schizophyllum commune culture medium.
In some embodiments of the present application, the kudzu root is present in the schizophyllum commune medium in an amount of 2.0-8.0 g/L. For example, it may be 2.0g/L, 3.0g/L, 4.0g/L, 5.0g/L, 6.0g/L, 7.0g/L, 8.0g/L, etc.
In other embodiments of the present application, the amount of the pueraria lobata and the schizophyllum commune medium may be out of the above range, regardless of the cost and yield; accordingly, the particle size of the pueraria powder may be larger than 40 mesh.
In some embodiments of the present application, the Schizophyllum commune medium comprises yeast extract 0.1-0.5 wt%, magnesium sulfate 0.01-0.15 wt%, potassium dihydrogen phosphate 0.05-0.2 wt%, and glucose 1-4 wt%.
Illustratively, the mass ratio of the yeast extract in the schizophyllum commune medium may be 0.1%, 0.5%, 0.2%, 0.3%, 0.4%, etc.
The mass ratio of magnesium sulfate in the schizophyllum commune culture medium can be 0.01%, 0.06%, 0.07%, 0.09%, 0.1%, 0.12%, 0.15% and the like.
The mass ratio of potassium dihydrogen phosphate in the schizophyllum commune culture medium can be 0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, etc.
The mass ratio of glucose in the schizophyllum commune medium can be 1%, 2%, 3%, 4%, etc.
For example, the basal medium comprises the following components: potassium dihydrogen phosphate 0.1 wt%, glucose 3 wt%, yeast extract 0.3 wt%, magnesium sulfate 0.05 wt%, and water in balance, and has a natural pH (about pH 6.0).
Illustratively, in some embodiments of the present application, the time for culturing Schizophyllum commune in the Schizophyllum commune medium is 100-. The time for continuing the culture may be, for example, 100h, 110h, 120h, 130h, 140h, 142h, 145h, 150h, or the like.
Illustratively, in the examples of the present application, Schizophyllum commune is cultured under conditions of 28-31 deg.C and 160-180r/min, such as 31 deg.C and 160 r/min.
And after the continuous culture is finished, performing centrifugal separation on the product, and removing thalli and kudzu powder residues to obtain a fermentation product.
The preparation method of the Schizophyllum commune fermentation product provided by the embodiment of the application at least has the following advantages:
pueraria powder is added into a culture medium to culture Schizophyllum commune, and a fermentation product is collected, wherein the obtained fermentation product has positive effects on oxidation resistance and free radical removal.
The application also provides a Schizophyllum commune fermentation product, and the Schizophyllum commune fermentation product is prepared by the preparation method of the Schizophyllum commune fermentation product.
In light of the above, the schizophyllum commune fermentation product provided by the application contains schizophyllan as an active ingredient, and shows better capabilities in the aspects of removing excessive free radicals and inhibiting related enzyme activities by combining the antioxidant capacity of the pueraria lobata matrix.
The application also provides a schizophyllum commune culture medium, wherein the schizophyllum commune culture medium contains kudzu root, and the content of the kudzu root is 2.0-8.0 g/L; illustratively, the concentration of kudzu root in the schizophyllum commune medium can be 2.0g/L, 3.0g/L, 5.0g/L, 6.0g/L, 8.0g/L, and the like.
Furthermore, the schizophyllum commune culture medium also comprises 0.1 to 0.5 weight percent of yeast extract, 0.01 to 0.15 weight percent of magnesium sulfate, 0.05 to 0.2 weight percent of potassium dihydrogen phosphate, 1 to 4 weight percent of glucose and the balance of water.
After the culture medium for the schizophyllum commune provided by the embodiment of the application is used for culturing the schizophyllum commune, a fermentation product of the schizophyllum commune has excellent antioxidant capacity and capacity of removing excessive free radicals.
The application also provides a skin care product, which comprises a skin care product substrate and the Schizophyllum commune fermentation product; the form of the skin care product matrix is not limited in this application.
The skin care product provided by the application has better levels in the aspects of oxidation resistance and free radical scavenging.
The features and properties of the present application are described in further detail below with reference to examples.
Preparation of kudzu root powder: drying radix Puerariae, pulverizing, sieving with 40 mesh sieve to obtain radix Puerariae powder, packaging, and sterilizing.
And (3) activation of the Schizophyllum commune strain: activating a GIM 5.43 Schizophyllum commune strain provided by Guangdong province microbial research through a slant culture medium, and culturing for 6 days at 28 ℃ and 160r/min until hyphae grows over the slant; transferring the slant strain to a liquid culture medium, and performing amplification culture for 3 days to obtain a fermented seed solution; the following examples and comparative examples were selected from the same Schizophyllum commune.
Example 1
This example provides a Schizophyllum commune fermentation product, which is prepared by the following steps:
1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.6% of kudzu root powder, 0.3% of yeast extract, 0.05% of magnesium sulfate, 0.1% of monopotassium phosphate, 3% of glucose and the balance of water, and the pH is natural (about 6.0).
2) Inoculating 10% Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) into the above culture medium, and fermenting at 28 deg.C and 160 r/min. After fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Example 2
This example provides a Schizophyllum commune fermentation product, which is prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.6% kudzu root powder, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 0.01% monopotassium phosphate, 3% glucose and the balance water, the pH is natural (about 6.0);
(2) inoculating 5% Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) into the above culture medium, and fermenting at 28 deg.C and 160 r/min. After fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Comparative example 1
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) mixing radix Puerariae powder and 20 times of water, ultrasonic extracting at 380W and 60 deg.C for 1 hr, centrifuging, collecting supernatant, adding a certain amount of water to desired volume (radix Puerariae powder: water: 1:25) to obtain radix Puerariae water extractive solution (40g/L), and sterilizing.
(2) The schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, and the pH is natural (about 6.0).
(3) Inoculating 5% of Schizophyllum commune (v/v, Schizophyllum commune seed liquid/total volume of culture medium after inoculating seed liquid) into the culture medium, adding the water extract of radix Puerariae in step (1) into a fermentation system, fermenting for 120h with the water extract of radix Puerariae added in an amount of 15% (v/v), centrifuging the fermentation product, and removing thallus to obtain the fermentation product.
Comparative example 2
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) mixing radix Puerariae powder and 20 times of water, ultrasonic extracting at 380W and 60 deg.C for 1 hr, centrifuging, collecting supernatant, adding a certain amount of water to desired volume (radix Puerariae powder: water: 1:25) to obtain radix Puerariae water extractive solution (40g/L), and sterilizing.
(2) The schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, the pH is natural (about 6.0);
(3) inoculating 10% of Schizophyllum commune (v/v, Schizophyllum commune seed liquid/total volume of culture medium after inoculating seed liquid) into the culture medium, adding the water extract of radix Puerariae in step (1) into a fermentation system, fermenting for 120h with the water extract of radix Puerariae in an amount of 15% (v/v), centrifuging the fermentation product, and removing thallus to obtain the fermentation product.
Comparative example 3
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, and the pH is natural (about 6.0).
(2) Inoculating 5% of Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of the culture medium after inoculating the seed solution) into the culture medium, fermenting at 28 deg.C for 24h at 160r/min, adding radix Puerariae powder into the fermentation system according to the ratio of 6g radix Puerariae powder/100 mL culture medium, continuing fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Comparative example 4
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) mixing radix Puerariae powder and 20 times of water, ultrasonic extracting at 380W and 60 deg.C for 1 hr, centrifuging, collecting supernatant, adding a certain amount of water to desired volume (radix Puerariae powder: water: 1:25) to obtain radix Puerariae water extractive solution (40g/L), and sterilizing;
(2) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, the pH is natural (about 6.0);
(3) inoculating 10% Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) into the above culture medium, fermenting at 28 deg.C and 160 r/min. And (2) fermenting for 24h, adding the water extract of the kudzuvine root obtained in the step (1) into a fermentation system, continuing fermenting for 120h, performing centrifugal treatment on the fermentation product, and removing thalli to obtain the fermentation product.
Comparative example 5
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose, 3% agar in solid medium, and natural pH (about 6.0).
(2) Inoculating 5% Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) into the above culture medium, culturing at 28 deg.C and 160r/min for 120 hr, centrifuging the fermentation product, and removing thallus to obtain fermentation product.
Comparative example 6
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose, natural pH (about 6.0), solid medium containing 3% agar.
(2) Inoculating 10% Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) into the above culture medium, culturing at 28 deg.C and 160r/min for 120 hr, centrifuging the fermentation product, and removing thallus to obtain fermentation product.
Comparative example 7
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, and the pH is natural (about 6.0).
(2) Inoculating 10% of Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of the culture medium after inoculating the seed solution) into the culture medium, fermenting for 24h, adding radix Puerariae powder into the fermentation system according to the ratio of 6g radix Puerariae powder/100 mL culture medium, continuing fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Comparative example 8
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, and the pH is natural (about 6.0).
(2) Inoculating 10% of Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of the culture medium after inoculating the seed solution) into the culture medium, fermenting at 28 deg.C and 160r/min for 48h, adding radix Puerariae powder into the fermentation system according to the ratio of 6g radix Puerariae powder/100 mL culture medium, continuing fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Comparative example 9
The comparative example provides a Schizophyllum commune fermentation product, which is mainly prepared by the following steps:
(1) the schizophyllum commune culture medium is prepared according to the following mass percentages: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monopotassium phosphate, 3% glucose and the balance water, and the pH is natural (about 6.0).
(2) Inoculating 10% of Schizophyllum commune (v/v, Schizophyllum commune seed solution/total volume of the culture medium after inoculating the seed solution) into the culture medium, fermenting at 28 deg.C and 160r/min for 72h, adding radix Puerariae powder into the fermentation system according to the ratio of 6g radix Puerariae powder/100 mL culture medium, continuing fermenting for 120h, centrifuging the fermentation product, and removing thallus and radix Puerariae powder to obtain the fermentation product.
Test example 1
Measurement of Schizophyllum commune Biomass: taking the fermentation products of the above examples and comparative examples, centrifuging at 15000 Xg and 4 deg.C for 10min, and collecting the precipitate; oven drying at 60 deg.C to constant weight, weighing to obtain biomass (if adding radix Puerariae powder, collecting thallus, washing with distilled water, and sieving with 40 mesh sieve to avoid influence of the rest radix Puerariae powder on biomass), and test results are shown in Table 1.
TABLE 1
As can be seen from Table 1, after the traditional Chinese medicine kudzu root is added into the culture medium, the growth of the schizophyllum commune mycelium can be promoted to a certain extent, wherein the example 1 has the best growth effect on the schizophyllum commune mycelium, the biomass is 8.79g/L, the biomass is respectively increased by 31.4% and 48.7% when being higher than that of the comparative example 5 and the comparative example 6, and the biomass of the example 1 is higher than that of the comparative example 7.
Test example 2
The polysaccharide contents of the fermentation products of the respective comparative examples and examples were measured by the sulfuric acid-phenol method and the DNS method.
The total sugar content of the fermentation product is determined by a sulfuric acid-phenol method, the polysaccharide is firstly hydrolyzed into monosaccharide under the action of concentrated sulfuric acid, and is quickly dehydrated to generate furfural derivatives, the furfural derivatives react with phenol to generate orange yellow solution, and the maximum absorption is realized at 490 nm. And (3) taking glucose as a standard substance, and determining the total sugar content of the fermentation product.
The specific operation steps are as follows:
(1) adding 1.0mL of a solution to be detected which is diluted by a certain multiple into a 10.0mL test tube with a plug;
(2) 1.0mL of 5% phenol solution was added, 5.0mL of concentrated sulfuric acid was quickly added to the vertical liquid surface, and the mixture was left to stand for 10 min. The reaction solution was mixed well using a vortex shaker, and then the stoppered test tube was placed in a water bath at 30 ℃ for 20 min.
(3) Blank zeroing with the corresponding reagents and measuring absorbance at 490 nm.
Total sugar content (mg/mL) is absorbance versus glucose concentration x dilution factor N.
The content of reducing sugar in the fermentation product is determined by a DNS method, the reducing sugar can be oxidized into sugar acid and other products under the alkaline condition, and the oxidant 3, 5-dinitrosalicylic acid is reduced into brownish red 3-amino-5-nitro salicylic acid. Within a certain range, the amount of reducing sugar is in direct proportion to the color depth of the red brown substance, and the absorbance of the red brown substance is measured at 550nm by using a spectrophotometer. And (3) measuring the content of reducing sugar in the fermentation product by taking glucose as a standard substance.
The specific operation steps are as follows:
(1) adding 1.0mL of a solution to be detected which is diluted by a certain multiple into a 10mL test tube with a plug;
(2) adding 3.0mL of DNS solution, and developing after boiling water bath for 5 min; after cooling, the volume is determined to be 25.0mL by water, and the mixture is fully shaken up;
(3) blank zeroing with the corresponding reagent and measuring absorbance at 550 nm.
Reducing sugar content (mg/mL) is absorbance corresponding to glucose concentration × dilution factor N; FIG. 1 is a standard curve for measuring total sugar content by colorimetry, FIG. 2 is a standard curve for measuring reducing sugar content by colorimetry, and Table 2 shows the polysaccharide contents of respective examples and comparative examples.
TABLE 2
According to table 2, the content of polysaccharide in the fermentation product can be effectively increased by adding the traditional Chinese medicine radix puerariae as the fermentation substrate, particularly, the polysaccharide content in the example 1 is higher, compared with the polysaccharide content in the comparative examples 5 and 6, the polysaccharide yield in the example 1 is respectively increased by 86.0% and 45.9%, which shows that the radix puerariae substrate can promote the growth of schizophyllum commune to a certain extent and increase the polysaccharide yield.
Test example 3
DPPH, also known as 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical with a nitrogen center, and the stability of DPPH is mainly caused by steric hindrance of 3 benzene rings with resonance stabilization, so that unpaired electrons on the nitrogen atom in the middle cannot play the role of electron pairing. Its absolute ethyl alcohol solution is purple, and has maximum absorption at wavelength of 517nm, and its absorbance and concentration are in linear relation. When a radical scavenger is added thereto, DPPH may be combined with or substituted for the radical scavenger, whereby the radical scavenging ability can be evaluated by decreasing the number of radicals, decreasing the absorbance, and decreasing the color of the solution.
Because each fermentation product stock solution has strong inoxidizability, the clearance rate of free radicals can reach more than 90 percent. Therefore, 10% (v/v) of the fermentation product of each comparative example of the example was taken and a positive control Trolox solution was used to determine the effect of Trolox on DPPH radical scavenging at different concentrations. The results are expressed as TEAC values in μmol Trolox equivalents/100 mL, i.e., 100mL of sample is equivalent to how much μmol Trolox has antioxidant capacity.
The specific experimental steps are as follows:
respectively sucking 2.0mL of sample solution (or absolute ethyl alcohol) and 2.0mL of LDPPH solution into a test tube with a plug, and uniformly mixing; and (5) keeping the reaction in the dark for 30min, and measuring the light absorption value at the wavelength of 517 nm.
In the formula:
A0: no sample was added, DPPH was added;
A1: adding sample or absolute ethyl alcohol, and adding DPPH;
A2: samples or absolute ethanol were added without DPPH.
FIG. 3 shows a standard curve of DPPH radical scavenging rate, FIG. 4 shows the DPPH radical scavenging ability of each fermentation product, and it can be seen from FIG. 4 that each fermentation product has a certain scavenging ability to DPPH radicals, and example 1 has the strongest scavenging ability.
Test example 4
Experiments examine the cleaning capacity of the invention to ABTS +, ABTS is oxidized into green ABTS + under the action of proper oxide, when antioxidant substances are added into the reaction, the generation of ABTS + is inhibited, and the total antioxidant capacity of the sample can be measured and calculated by measuring the absorbance at 734 nm.
Because each fermentation product stock solution has strong inoxidizability, the clearance rate of free radicals can reach more than 90 percent. Therefore, the removal effect of Trolox on ABTS +. free radicals at different concentrations was determined by taking 10% (v/v) concentration of each example and using Trolox solution as a positive control. The results are expressed as TEAC values in μmol Trolox equivalents/100 mL, i.e., 100mL of sample is equivalent to how much μmol Trolox has antioxidant capacity.
The specific experimental steps are as follows:
(1) taking a 7mmol/L ABTS solution, and diluting the ABTS solution by 60 times with 10mmol/L PBS (pH 7.4) to obtain an ABTS working solution;
(2) the sample solution (or PBS) was added to 190. mu.L of ABTS working solution (or PBS), followed by shaking for 10 seconds, standing at 30 ℃ for 6min, and measuring the absorbance A at 734 nm.
In the formula:
A0: no sample was added, ABTS was added;
A1: adding sample or PBS, adding ABTS;
A2: sample or PBS was added, no ABTS was added.
FIG. 5 shows the ABTS +. free radical scavenging standard curve, and FIG. 6 shows the ABTS +. free radical scavenging capacity of the individual fermentation products. As can be seen from FIG. 6, the fermentation product has a good ability to scavenge ABTS +. free radicals, and the best effect is obtained in example 1.
Test example 5
The experiment researches the scavenging ability of the invention to the hydroxyl radical, the hydroxyl radical is the most active radical in active oxygen with chemical properties, can almost react with any biomacromolecule in living cells, has extremely high reaction speed, and is the radical which has the greatest harm to organisms. In the experiment, salicylic acid is utilized to effectively capture OH in a reaction system and generate a colored substance 2, 3-dihydroxybenzoic acid. The colored substance has a strong absorption peak at 510nm, and can remove OH in time if antioxidant is added into the system and the function of the substance to be detected for capturing OH is greater than that of salicylic acid, so that the absorbance is reduced due to the reduction of the generation amount of the colored product.
The specific experimental steps are as follows:
(1) 3.0mL of a 2mmol/L FeSO4 solution, 3.0mL of a 1mmol/L H solution2O2Solution (or distilled water) followed by 3.0mL of 6mmol/L salicylic acid solution and incubation at 37 ℃ for 15 min;
(2) after adding 1.0mL of the sample (or distilled water) and incubating at 37 ℃ for 15min, the absorbance was measured at 510 nm.
In the formula:
A0: without addition of sample, H2O2；
A1: adding sample, adding H2O2；
A2: sample addition, no H addition2O2。
Table 3 shows the hydroxyl radical scavenging ability of each fermentation product.
TABLE 3
According to table 3, each example has a certain scavenging ability for hydroxyl radicals, wherein the fermentation product of example 1 has the best scavenging effect for hydroxyl radicals, and the scavenging rate reaches 44.92 ± 1.22%, which indicates that the addition of pueraria lobata substrate effectively improves the scavenging ability for hydroxyl radicals of the fermentation product.
Test example 6
The elastic fiber is composed of elastin and microfibril, and is distributed in dermis and subcutaneous tissue to make skin elastic. Due to the action of some environmental factors such as ultraviolet irradiation, pressure, pollution and the like, the synthetic capacity of human fibroblasts is reduced, and the production of elastase in vivo is promoted. Elastase causes the loss of epidermal junction progenitors, resulting in the development of skin aging, wrinkles and photoaging. The experiment tests the inhibition ability of the fermentation product on elastase to evaluate the anti-aging effect, and the fermentation stock solution of each example is processed by a 0.45 mu m microporous filter membrane for later use.
The specific experimental steps are as follows:
incubating 50 μ L of the sample with 100 μ L of 0.9U/mL elastase and 100 μ L of Tris-HCl buffer for 20min at 25 ℃; the reaction was then started by adding 50. mu.L of MAAPVN solution and for the sample blank 50. mu.L of Tris-HCl buffer was added instead of MAAPVN. After incubation at 25 ℃ for 60min, the absorbance was measured at 410 nm.
In the formula:
A1: adding the sample, adding MAAPVN;
A2: sample addition, no MAAPVN addition;
B1no sample added, MAAPVN added;
B2: no sample was added, no MAAPVN was added.
FIG. 7 shows the elastase inhibition rate for each fermentation product. As can be seen from FIG. 7, the biforked fermentation product of Schizophyllum commune and radix Puerariae has inhibitory activity on elastase, wherein the best effect is shown in example 1, which indicates that the fermentation product has certain anti-aging activity.
Test example 7
Melanin is a biological pigment which is synthesized and secreted by cells, is insoluble in water and soluble in alkaline solution, belongs to protein derivatives, and can absorb ultraviolet rays so as to protect various injuries of skin caused by ultraviolet rays, including photoaging, inflammation, cancer and the like.
Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, which affects melanin production mainly by affecting tyrosine conversion to dopa, and oxidation of dopa to dopaquinone. Therefore, the whitening efficacy of the whitening agent can be evaluated by measuring the inhibition result of the whitening agent on tyrosinase. Before the detection, the samples of examples 1-8 were passed through a 0.45 μm microfiltration membrane.
The specific experimental steps are as follows:
phosphate buffer, different test solutions (the phosphate buffer serves as a sample blank) and 500U/mL enzyme solution are sequentially added into a 96-well plate, finally 1.5mmol/L L-tyrosine is added into a substrate, timing is started immediately, and the light absorption value at the wavelength of 475nm is measured when the reaction is carried out for 20 min.
In the formula:
A1: adding a sample, and adding L-tyrosine;
A2: sample was added without addition of L-tyrosine;
B1adding L-tyrosine without adding sample;
B2: no sample was added, no L-tyrosine was added.
FIG. 8 shows tyrosinase inhibition rates for individual fermentation products.
As can be seen from FIG. 8, each example has a certain inhibitory activity against tyrosinase, and example 1 is the best.
In conclusion, the fermentation product provided by the embodiment of the application has better inoxidizability and has inhibition capability on tyrosinase and elastase.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A preparation method of a Schizophyllum commune fermentation product is characterized by comprising the following steps:
culturing Schizophyllum commune in Schizophyllum commune culture medium, and collecting fermentation product;
wherein the schizophyllum commune culture medium contains kudzu root.
2. The method for preparing a Schizophyllum commune fermentation product according to claim 1, wherein the radix Puerariae is radix Puerariae powder with particle size less than or equal to 40 mesh.
3. The method of claim 1, wherein the amount of kudzu vine root in the Schizophyllum commune culture medium is 2.0-8.0 g/L.
4. The method for preparing a fermentation product of Schizophyllum commune as defined in claim 1, wherein the time for culturing Schizophyllum commune in the Schizophyllum commune culture medium is 100-150 h.
5. The method for preparing a fermentation product of Schizophyllum commune according to claim 1, wherein the Schizophyllum commune is cultured in the Schizophyllum commune culture medium at a temperature of 28-30 deg.C and at a rotation speed of 140-170 r/min.
6. The method of producing a Schizophyllum commune fermentation product of any of claims 1-5, wherein the Schizophyllum commune culture medium comprises yeast extract 0.1-0.5 wt%, magnesium sulfate 0.01-0.15 wt%, potassium dihydrogen phosphate 0.05-0.2 wt%, and glucose 1-4 wt%.
7. A method of producing a fermentation product of Schizophyllum commune according to any one of claims 1-5, wherein the step of culturing Schizophyllum commune in a Schizophyllum commune culture medium further comprises:
activating the Schizophyllum commune strain by a slant culture medium, and culturing at 28-30 deg.C and 140-170 r/min for 5-7 days until the slant is full of mycelia; transferring the slant strain to liquid culture medium, and performing amplification culture for 3-4 days to obtain activated Schizophyllum commune.
8. A Schizophyllum commune fermentation product obtained by the method of any one of claims 1-7.
9. A skin care product comprising a substrate and the Schizophyllum commune fermentation product of claim 8.
10. A schizophyllum commune culture medium is characterized by comprising 2.0-8.0g/L of kudzu vine root.
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