CN113337545B - 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, in particular to a schizophyllum commune fermentation product, a preparation method thereof, a skin care product and a schizophyllum commune culture medium. A method for producing a schizophyllum commune fermentation product, comprising: culturing schizophyllum commune in a schizophyllum commune culture medium, and then collecting fermentation products; wherein the schizophyllum commune culture medium contains kudzuvine root. Adding radix Puerariae powder into the culture medium to culture Schizophyllum commune, and collecting fermentation product to obtain fermentation product with positive effects in resisting oxidation and scavenging free radicals.
Description
Technical Field
The application relates to the field of microorganisms, in particular 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 stronger activity, can generate malic acid, can generate a large amount of organic acid during deep fermentation of hyphae, can also generate auxin indoleacetic acid, and can be used in the industries of medicine and health and skin care products.
Disclosure of Invention
It is an aim of embodiments of the present application to provide a schizophyllum commune fermentation product, a method for preparing the same, a skin care product, a schizophyllum commune culture medium, which aims at improving the antioxidant level of the schizophyllum commune fermentation product.
The application provides a preparation method of schizophyllum commune fermentation product, which comprises the following steps:
culturing schizophyllum commune in a schizophyllum commune culture medium, and then collecting fermentation products;
wherein the schizophyllum commune culture medium contains kudzuvine root.
Adding radix Puerariae powder into the culture medium to culture Schizophyllum commune, and collecting fermentation product to obtain fermentation product with positive effects in resisting oxidation and scavenging free radicals.
In some embodiments of the present application, the kudzuvine root is kudzuvine root powder, and the particle size of the kudzuvine root powder is less than or equal to 40 mesh.
In some embodiments of the present application, the amount of kudzuvine root powder in the schizophyllum commune media is 2.0-8.0g/L.
In some embodiments of the present application, the schizophyllum commune is cultivated in a schizophyllum commune medium for a time period of 100-150 hours.
In some embodiments of the present application, the schizophyllum commune is cultivated in a schizophyllum commune medium at a cultivation temperature of 28 ℃ to 30 ℃ and a rotation speed of 140r/min to 170r/min.
In some embodiments of the present application, the schizophyllum commune media comprises 0.1-0.5wt% yeast extract, 0.01-0.15wt% magnesium sulfate, 0.05-0.2wt% potassium dihydrogen phosphate, and 1-4wt% glucose.
In some embodiments of the present application, the step of culturing schizophyllum commune in a schizophyllum commune medium is preceded by the step of:
activating schizophyllum commune strain by a slant culture medium, and culturing for 5-7 days under the conditions of 28-30 ℃ and 140-170 r/min until mycelium grows to fill the slant; transferring the slant strain to liquid culture medium, and performing enlarged culture for 3-4 days to obtain activated schizophyllum commune.
The application also provides a schizophyllum commune fermentation product, which is prepared by the preparation method of the schizophyllum commune fermentation product.
The schizophyllum commune fermentation product provided by the application contains active schizophyllum polysaccharides, combines the antioxidant capacity of the kudzu root matrix, and has better capacity in the aspects of scavenging excessive free radicals and inhibiting related enzyme activities.
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, which contains kudzuvine root, and the content of the kudzuvine root is 2.0-8.0g/L.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered limiting the scope, and that other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard curve of total sugar content measured by colorimetry.
FIG. 2 is a colorimetric calibration curve for reducing sugar content.
Figure 3 shows a DPPH radical scavenging standard curve.
FIG. 4 shows the DPPH radical scavenging capacity of the individual fermentation products.
Figure 5 shows ABTS + & radical clearance standard curves.
FIG. 6 shows the ability of the individual fermentation products to scavenge ABTS+ radicals.
FIG. 7 shows the elastase inhibition by each fermentation product.
FIG. 8 shows tyrosinase inhibition by individual fermentation products.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The fermentation product of schizophyllum commune contains active schizophyllan; the inventors found that the addition of certain substances to the culture medium of schizophyllum commune can provide the fermentation product with the ability of resisting oxidation and scavenging free radicals.
However, not all substances have the functions of promoting oxidation resistance and scavenging free radicals, for example, the addition of licorice to a basal medium is described in the patent application No. CN202110011922.2 to increase the oxidation resistance of the fermentation product and to inhibit elastase and tyrosinase levels. The morinda officinalis which has the functions of enhancing the immunity of the human body, improving osteoporosis, resisting depression and resisting oxidization is added into a culture medium, and the antioxidant capacity, tyrosinase inhibition capacity and elastase inhibition capacity of the obtained fermentation product are not increased but reduced.
The inventors hypothesize that: during fermentation, schizophyllum commune growth metabolism and the like have selectivity to substances in a matrix, and components which have the functions of resisting oxidation and scavenging free radicals can promote the performance of the final fermentation product in resisting oxidation and scavenging free radicals. For some components, not only does not have a promoting effect, but also has a reaction.
Based on the above, the 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 capacity of the schizophyllum commune fermentation product in terms of oxidation resistance and free radical removal.
The following describes the schizophyllum commune fermentation product and preparation method thereof, skin care product and schizophyllum commune culture medium.
The application provides a preparation method of schizophyllum commune fermentation product, which comprises the following steps:
culturing schizophyllum commune in a schizophyllum commune culture medium, and then collecting fermentation products;
wherein the schizophyllum commune culture medium contains kudzuvine root.
In the application, the kudzuvine root refers to a raw material treated by non-chemical treatment means, and the kudzuvine root is crushed or cut by physical means; for example, it may be in the form of powder, block or strip;
in some embodiments of the present application, the method further comprises activating a schizophyllum commune species prior to culturing the schizophyllum commune in the schizophyllum commune medium, specifically comprising: activating schizophyllum commune strain by slant culture medium, culturing for 5-7 days (for example, 5 days, 6 days, 7 days) under the conditions of 28-30deg.C (for example, 28deg.C, 30deg.C) 140r/min-170r/min (for example, 140r/min, 150r/min, 155r/min, 160r/min, 170 r/min) until mycelium grows to fill slant; transferring the slant strain into liquid culture medium, and performing enlarged culture for 3-4 days (for example, 3 days, 3.6 days, and 4 days) to obtain activated Schizophyllum commune.
The activated schizophyllum commune is placed in a schizophyllum commune culture medium for culture.
The schizophyllum commune culture medium contains kudzuvine root, and after research by the inventor, the kudzuvine root is added into the culture medium in a solid or powder form, and fermentation products obtained by adding the schizophyllum commune culture medium in a mode of aqueous extract of the kudzuvine root and then culturing the schizophyllum commune have poor oxidation resistance and other effects.
In some embodiments of the present application, the kudzuvine root is present in the schizophyllum commune media in a powder form, and in some embodiments of the present application, the kudzuvine root has a particle size of less than or equal to 40 mesh. Sieving radix Puerariae powder with 40 mesh sieve, and collecting the undersize.
For example, radix Puerariae is dried, pulverized, sieved with a 40 mesh sieve to obtain radix Puerariae powder, sterilized, and added into Schizophyllum commune culture medium.
In some embodiments of the present application, the amount of Pueraria lobata in the schizophyllum commune media is 2.0-8.0g/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 culture medium for Pueraria lobata and Schizophyllum commune may be other than within the above-described range, if cost, yield, and the like are not considered; accordingly, the particle size of the kudzuvine root powder may be larger than 40 mesh.
In some embodiments of the present application, the schizophyllum commune media comprises 0.1-0.5wt% yeast extract, 0.01-0.15wt% magnesium sulfate, 0.05-0.2wt% potassium dihydrogen phosphate, and 1-4wt% glucose.
As an example, the mass ratio of the yeast extract in the schizophyllum commune culture medium may be 0.1%, 0.5%, 0.2%, 0.3%, 0.4%, and so on.
The mass ratio of the 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 the monopotassium 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% and the like.
The mass ratio of glucose in the schizophyllum commune medium may be 1%, 2%, 3%, 4%, etc.
For example, the basal medium comprises the following components: potassium dihydrogen phosphate 0.1wt%, glucose 3wt%, yeast extract 0.3wt%, magnesium sulfate 0.05wt% and water for the rest, and the pH is natural (about pH 6.0).
Illustratively, in some embodiments of the present application, the schizophyllum commune is cultivated in a schizophyllum commune medium for a time period of 100-150 hours. The time for continuing the cultivation may be, for example, 100h, 110h, 120h, 130h, 140h, 142h, 145h, 150h, etc.
Illustratively, in the examples herein, the schizophyllum commune is cultivated under conditions of 28-31 ℃,160-180r/min, e.g., at 31 ℃,160 r/min.
After the continuous culture is finished, the product is centrifugally separated, and thalli and kudzuvine root powder residues are removed, so that a fermentation product is obtained.
The preparation method of the schizophyllum commune fermentation product provided by the embodiment of the application has at least the following advantages:
adding radix Puerariae powder into the culture medium to culture Schizophyllum commune, and collecting fermentation product to obtain fermentation product with positive effects in resisting oxidation and scavenging free radicals.
The application also provides a schizophyllum commune fermentation product, which is prepared by the preparation method of the schizophyllum commune fermentation product.
As described above, the schizophyllum commune fermentation product provided by the application contains active schizophyllan and combines the antioxidant capacity of the kudzuvine root matrix, thereby showing better capacities in terms of scavenging excessive free radicals and inhibiting the activity of related enzymes.
The application also provides a schizophyllum commune culture medium, which contains kudzuvine root, and the content of the kudzuvine root is 2.0-8.0g/L; as an example, the amount of Pueraria lobata in the schizophyllum commune media may be 2.0g/L, 3.0g/L, 5.0g/L, 6.0g/L, 8.0g/L, etc.
Further, the schizophyllum commune culture medium also comprises 0.1-0.5wt% of yeast extract, 0.01-0.15wt% of magnesium sulfate, 0.05-0.2wt% of monopotassium phosphate, 1-4wt% of glucose and the balance of water.
After the schizophyllum commune culture medium provided by the embodiment of the application is used for culturing the schizophyllum commune, the fermentation product of the schizophyllum commune has better antioxidant capacity and the capacity of scavenging 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 morphology of the skin care product matrix is not limited in this application.
The skin care product provided by the application has better level in the aspects of antioxidation and free radical removal.
The features and capabilities of the present application are described in further detail below in connection with the examples.
Preparing kudzuvine root powder: the kudzuvine root is dried, crushed and sieved by a 40-mesh sieve to obtain kudzuvine root powder, and the kudzuvine root powder is packaged and sterilized for later use.
Activation of schizophyllum commune strain: activating the GIM 5.43 schizophyllum commune strain provided by the Guangdong province microorganism research through a slant culture medium, and culturing for 6 days at 28 ℃ and 160r/min until mycelia grow to fill the slant; transferring the slant strain to a liquid culture medium, and performing enlarged culture for 3 days to obtain fermentation seed liquid; the following examples and comparative examples were conducted using the same schizophyllum commune.
Example 1
The embodiment 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: the pH is natural (about 6.0) with 0.6% kudzu root powder, 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% monobasic potassium phosphate, 3% glucose and the balance water.
2) 10% of schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of medium after seed solution is inoculated) is inoculated into the culture medium, and fermentation is carried out at 28 ℃ and 160 r/min. After 120h of fermentation, the fermentation product is subjected to centrifugal treatment, and thalli and kudzuvine root powder are removed, so that the fermentation product is obtained.
Example 2
The embodiment 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: 0.6% of kudzuvine root powder, 0.05% of magnesium sulfate, 0.1% of monopotassium phosphate, 0.01% of monopotassium phosphate, 3% of glucose and the balance of water, and the pH is natural (about 6.0);
(2) 5% of schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of medium after seed solution is inoculated) is inoculated into the culture medium, and fermentation is carried out at 28 ℃ and 160 r/min. After 120h of fermentation, the fermentation product is subjected to centrifugal treatment, and thalli and kudzuvine root powder are removed, so that the fermentation product is obtained.
Comparative example 1
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) Mixing radix Puerariae powder and water 20 times of the weight of radix Puerariae powder, extracting with ultrasonic assistance at 380W and 60deg.C for 1 hr, centrifuging to obtain supernatant, and constant volume (radix Puerariae powder: water=1:25) with water to obtain radix Puerariae water extract (40 g/L), and sterilizing.
(2) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0) with 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose and the balance water.
(3) And (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 kudzu root water extract in the step (1) into a fermentation system, fermenting for 120 hours, centrifuging the fermentation product, and removing thalli to obtain the fermentation product.
Comparative example 2
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) Mixing radix Puerariae powder and water 20 times of the weight of radix Puerariae powder, extracting with ultrasonic assistance at 380W and 60deg.C for 1 hr, centrifuging to obtain supernatant, and constant volume (radix Puerariae powder: water=1:25) with water to obtain radix Puerariae water extract (40 g/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% potassium dihydrogen phosphate, 3% glucose and the balance water, the pH is natural (about 6.0);
(3) 10% schizophyllum commune (v/v, schizophyllum commune seed liquid/total volume of culture medium after inoculating seed liquid) is inoculated into the culture medium, the kudzu root aqueous extract in the step (1) is added into a fermentation system, the kudzu root aqueous extract is fermented for 120 hours with the addition amount of 15% (v/v), and the fermentation product is subjected to centrifugal treatment to remove thalli, so as to obtain the fermentation product.
Comparative example 3
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0) with 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose and the balance water.
(2) Inoculating 5% schizophyllum commune (v/v, total volume of culture medium after schizophyllum commune seed solution/seed solution is inoculated) into the culture medium for fermentation, fermenting at 28 ℃ under 160r/min for 24h, adding radix puerariae powder into a fermentation system according to the proportion of 6g radix puerariae powder/100 mL culture medium for continuous fermentation until 120h, centrifuging the fermentation product, and removing thalli and radix puerariae powder to obtain the fermentation product.
Comparative example 4
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) Mixing radix Puerariae powder and water 20 times of the weight of radix Puerariae powder, extracting with ultrasonic assistance at 380W and 60deg.C for 1 hr, centrifuging to obtain supernatant, and constant volume (radix Puerariae powder: water=1:25) with water to obtain radix Puerariae water extract (40 g/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% potassium dihydrogen phosphate, 3% glucose and the balance water, the pH is natural (about 6.0);
(3) 10% of schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of culture medium after seed solution is inoculated) is inoculated into the culture medium for fermentation, and fermentation is carried out at 28 ℃ and 160 r/min. Fermenting for 24h, adding the radix puerariae water extract in the step (1) into a fermentation system, wherein the adding amount of the radix puerariae water extract is 15% (v/v), continuing fermenting for 120h, centrifuging 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 prepared mainly 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% potassium dihydrogen phosphate, 3% glucose, and the solid medium contains 3% agar, and has natural pH (about 6.0).
(2) Inoculating 5% Schizophyllum commune (v/v, total volume of culture medium after inoculating seed solution) into the above culture medium, culturing at 28deg.C under 160r/min for 120 hr, centrifuging, and removing thallus to obtain fermentation product.
Comparative example 6
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0), and the solid culture medium contains 3% agar.
(2) 10% schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) is inoculated into the culture medium, and cultured for 120h under the conditions of 28 ℃ and 160r/min, and the fermentation product is subjected to centrifugal treatment to remove thalli, so as to obtain the fermentation product.
Comparative example 7
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0) with 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose and the balance water.
(2) 10% schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) is inoculated into the culture medium, fermentation is carried out for 24 hours, kudzuvine root powder is added into a fermentation system according to the proportion of 6g kudzuvine root powder/100 mL culture medium, fermentation is continued for 120 hours, and the fermentation product is subjected to centrifugal treatment to remove thalli and kudzuvine root powder, thus obtaining the fermentation product.
Comparative example 8
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0) with 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose and the balance water.
(2) 10% schizophyllum commune (v/v, schizophyllum commune seed solution/total volume of culture medium after inoculating seed solution) is inoculated into the culture medium for fermentation, the fermentation is carried out at 28 ℃ and 160r/min for 48 hours, the kudzuvine root powder is added into a fermentation system according to the proportion of 6g kudzuvine root powder/100 mL of culture medium for continuous fermentation until 120 hours, and the fermentation product is subjected to centrifugal treatment to remove thalli and kudzuvine root powder, thus obtaining the fermentation product.
Comparative example 9
The comparative example provides a schizophyllum commune fermentation product which is prepared mainly by the following steps:
(1) The schizophyllum commune culture medium is prepared according to the following mass percentages: the pH is natural (about 6.0) with 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose and the balance water.
(2) 10% schizophyllum commune (v/v, total volume of culture medium after schizophyllum commune seed liquid/seed liquid is inoculated) is inoculated into the culture medium for fermentation, fermentation is carried out at 28 ℃ and 160r/min, the fermentation is carried out for 72 hours, the kudzuvine root powder is added into a fermentation system according to the proportion of 6g kudzuvine root powder/100 mL of culture medium, the fermentation is continued until 120 hours, and the fermentation product is subjected to centrifugal treatment, so as to remove thalli and kudzuvine root powder, thus obtaining the fermentation product.
Test example 1
Determination of schizophyllum commune biomass: taking the fermentation products of the above examples and comparative examples, centrifuging at 15000 Xg and 4 ℃ for 10min, and collecting the precipitate; oven drying at 60deg.C to constant weight, weighing to obtain biomass (if radix Puerariae powder is added, collecting thallus, washing with distilled water, sieving with 40 mesh sieve to avoid influence of residual radix Puerariae powder on biomass), and testing the results shown in Table 1.
TABLE 1
As can be seen from Table 1, the growth of schizophyllum commune mycelium can be promoted to a certain extent after the Chinese medicine kudzu root is added into the culture medium, wherein example 1 has the best effect on the schizophyllum commune mycelium growth, the biomass is 8.79g/L and is higher than that of comparative examples 5 and 6, 31.4 percent and 48.7 percent of biomass is respectively improved, and the biomass of example 1 is higher than that of comparative example 7.
Test example 2
The polysaccharide content of the fermentation products of each of the comparative examples and examples was measured using 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, polysaccharide is firstly hydrolyzed into monosaccharide under the action of concentrated sulfuric acid, and is rapidly dehydrated to generate a furfural derivative which reacts 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 measuring the total sugar content of the fermentation product.
The specific operation steps are as follows:
(1) Adding 1.0mL of to-be-measured liquid with a certain multiple into a 10.0mL test tube with a plug;
(2) 1.0mL of 5% phenol solution was added, and then 5.0mL of concentrated sulfuric acid was rapidly added to the vertical liquid surface, followed by standing for 10min. The reaction solution was thoroughly mixed using a vortex shaker, and then the tube with the stopper was placed in a water bath at 30℃for 20min.
(3) Blank zeroing was performed with the corresponding reagent and absorbance was measured at 490 nm.
Total sugar content (mg/mL) =absorbance corresponds to glucose concentration x dilution N.
The content of reducing sugar in the fermentation product is measured by a DNS method, the reducing sugar can be oxidized into sugar acid and other products under alkaline conditions, and the oxidant 3, 5-dinitrosalicylic acid is reduced into brownish red 3-amino-5-nitrosalicylic acid. The amount of reducing sugar was proportional to the color depth of the reddish brown substance within a certain range, and the absorbance was measured at 550nm by a spectrophotometer. And (3) taking glucose as a standard substance, and measuring the reducing sugar content of the fermentation product.
The specific operation steps are as follows:
(1) Adding 1.0mL of to-be-measured liquid with 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 5min; after cooling, the volume is fixed to 25.0mL by water, and the mixture is fully and uniformly shaken;
(3) Blank zeroing was performed with the corresponding reagent and absorbance was measured at 550 nm.
Reducing sugar content (mg/mL) =absorbance corresponds to glucose concentration x dilution N; fig. 1 is a colorimetric total sugar content standard curve, fig. 2 is a colorimetric reducing sugar content standard curve, and table 2 shows polysaccharide contents of respective examples and comparative examples.
TABLE 2
According to table 2, the addition of the Chinese medicine kudzuvine root as the fermentation substrate can effectively improve the polysaccharide content of the fermentation product, especially, the polysaccharide content of the embodiment 1 is higher, and compared with the comparison of the embodiment 5 and the comparison of the embodiment 6, the polysaccharide yield of the embodiment 1 is respectively improved by 86.0% and 45.9%, which indicates that Ge Genji can promote the growth of schizophyllan to a certain extent and improve the polysaccharide yield of the schizophyllan.
Test example 3
DPPH, also known as 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical of nitrogen centers, and its stability mainly comes from steric hindrance of 3 benzene rings with resonance stabilization, so that unpaired electrons on the nitrogen atom clamped in the middle cannot exert their due electron pairing action. The absolute ethanol solution of the fluorescent dye has purple color, has maximum absorption at 517nm wavelength, and has linear relation between absorbance and concentration. When the free radical scavenger is added into the solution, DPPH can be combined or replaced, so that the amount of free radicals is reduced, the absorbance is reduced, the color of the solution is lightened, and the capability of scavenging the free radicals can be evaluated.
As the stock solution of each fermentation product has stronger oxidation resistance, the free radical clearance rate can reach more than 90 percent. The fermentation products of the comparative examples were thus taken at a concentration of 10% (v/v) and the effect of Trolox on DPPH radical scavenging at various concentrations was determined using a Trolox solution as a positive control. The experimental results are expressed as TEAC values in units of mu mol Trolox equivalents/100 mL, i.e., how much mu mol Trolox the 100mL sample is equivalent to.
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; the reaction was carried out in the dark for 30min, and the absorbance at 517nm was measured.
Wherein:
A 0 : no sample was added and DPPH was added;
A 1 : adding a sample or absolute ethyl alcohol, and adding DPPH;
A 2 : samples or absolute ethanol were added, and DPPH was not added.
Fig. 3 shows a DPPH radical scavenging rate standard curve, fig. 4 shows the DPPH radical scavenging capacity of each fermentation product, and as can be seen from fig. 4, each fermentation product has a certain scavenging capacity for DPPH radicals, and example 1 has the strongest scavenging capacity.
Test example 4
Experiments show that the cleaning capability of the invention to ABTS+ is oxidized into green ABTS+ under the action of proper oxides, when antioxidant substances are added into the reaction, the generation of the ABTS+ is inhibited, and the absorbance is measured at 734nm to measure and calculate the total antioxidant capability of the sample.
As the stock solution of each fermentation product has stronger oxidation resistance, the free radical clearance rate can reach more than 90 percent. Therefore, the effect of Trolox on the elimination of ABTS+ and free radicals at different concentrations was determined by taking each example with a concentration of 10% (v/v) and taking Trolox solution as a positive control. The experimental results are expressed as TEAC values in units of mu mol Trolox equivalents/100 mL, i.e., how much mu mol Trolox the 100mL sample is equivalent to.
The specific experimental steps are as follows:
(1) Taking 7mmol/L of ABTS solution, and diluting 60 times with 10mmol/L of PBS (pH=7.4) to obtain an ABTS working solution;
(2) mu.L of ABTS working solution (or PBS) is sucked, 10 mu.L of sample solution (or PBS) is added, the mixture is shaken for 10s, and the mixture is kept stand at 30 ℃ for 6min, and the absorbance A at 734nm is measured.
Wherein:
A 0 : no sample was added, ABTS was added;
A 1 : adding a sample or PBS, and adding ABTS;
A 2 : samples or PBS were added, and ABTS was not added.
FIG. 5 shows the ABTS+ radical scavenging standard curve, and FIG. 6 shows the ability of the individual fermentation products to scavenge ABTS+ radicals. As can be seen from FIG. 6, the fermentation product has good ability to scavenge ABTS+ free radicals, and example 1 works best.
Test example 5
Experiments show that the hydroxyl radical scavenging capability of the invention is the most active radical with chemical property in active oxygen, can react with any biological macromolecules in living cells almost, has extremely high reaction speed, and is the radical with the greatest harm to organisms. The experiment utilizes salicylic acid 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 if an antioxidant substance is added into the system and the effect of capturing OH by an object to be detected is greater than that of salicylic acid, the OH can be timely removed, so that the generation amount of the colored product is reduced, and the absorbance is reduced.
The specific experimental steps are as follows:
(1) 3.0ml of 2mmol/L FeSO4 solution and 3.0ml of 1mmol/L H were taken 2 O 2 Solution (or distilled water) followed by 3.0mL of 6mmol/L salicylic acid solution and incubation at 37℃for 15min;
(2) 1.0mL of sample (or distilled water) was added, and after incubation at 37℃for 15min, the absorbance was measured at 510 nm.
Wherein:
A 0 : no sample was added, H was added 2 O 2 ；
A 1 : adding sample, adding H 2 O 2 ；
A 2 : add sample, not add H 2 O 2 。
Table 3 shows the hydroxyl radical scavenging capacity of the individual fermentation products.
TABLE 3 Table 3
According to Table 3, each example has a certain capability of scavenging hydroxyl radicals, wherein the fermentation product of example 1 has the best effect of scavenging hydroxyl radicals, and the scavenging rate reaches 44.92 +/-1.22%, which indicates that the addition of the kudzuvine root matrix effectively improves the capability of scavenging 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 effects of ultraviolet irradiation, pressure, pollution and other environmental factors, the synthesis capacity of human fibroblasts is reduced, and the generation of elastase in vivo is promoted. Elastase causes the loss of the epidermal link ancestor, leading to the development of skin aging, wrinkles and photoaging. The inhibition ability of the fermentation product to elastase was tested in this experiment to evaluate the anti-aging efficacy, and the fermentation stock solutions of the examples were treated with a 0.45 μm microporous filter membrane for use.
The specific experimental steps are as follows:
incubating 50. Mu.L of the sample with 100. Mu.L of 0.9U/mL elastase and 100. Mu.L of Tris-HCl buffer at 25℃for 20min; subsequently 50. Mu.L MAAPVN solution was added to start the reaction, and for the sample blank 50. Mu.L Tris-HCl buffer was added instead of MAAPVN. After incubation at 25℃for 60min, absorbance was measured at 410 nm.
Wherein:
A 1 : adding a sample, adding MAAPVN;
A 2 : adding the sample without MAAPVN;
B 1 no sample was added and MAAPVN was added;
B 2 : no sample was added and no maadvn was added.
FIG. 7 shows the elastase inhibition by each fermentation product. As can be seen from FIG. 7, the two-way fermentation product of Schizophyllum commune and radix Puerariae has an inhibitory effect on elastase, wherein example 1 works best, demonstrating that the fermentation product has a certain anti-aging activity.
Test example 7
Melanin is a biological pigment synthesized and secreted by cells, which is insoluble in water and soluble in alkaline solution, belongs to protein derivatives, and can absorb ultraviolet rays to protect skin from various damages caused by ultraviolet rays, including photoaging, inflammation, cancer and the like.
Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, affecting melanin production primarily by affecting the conversion of tyrosine to dopa, and the oxidation of dopa to dopaquinone. Therefore, the whitening efficacy of the whitening agent can be evaluated by measuring the inhibition result of tyrosinase by the whitening agent. Samples of examples 1-8 were subjected to a 0.45 μm microfiltration membrane prior to detection.
The specific experimental steps are as follows:
phosphate buffer solution, different test solutions (the phosphate buffer solution serves as a sample blank) and 500U/mL enzyme solution are sequentially added into a 96-well plate, and finally 1.5mmol/L L-tyrosine is added as a substrate, so that timing is immediately started, and the absorbance at 475nm wavelength is measured when the reaction is performed for 20min.
Wherein:
A 1 : adding a sample and adding L-tyrosine;
A 2 : adding a sample without adding L-tyrosine;
B 1 no sample is added, and L-tyrosine is added;
B 2 : no sample was added and no L-tyrosine was added.
FIG. 8 shows tyrosinase inhibition by individual fermentation products.
As is clear from FIG. 8, each example had a certain inhibitory activity against tyrosinase, and example 1 was found to have the best effect.
In conclusion, the fermentation product provided by the embodiment of the application has better oxidation resistance and has inhibition capability on tyrosinase and elastase.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (3)
1. The preparation method of the schizophyllum commune fermentation product is characterized by mainly comprising the following steps: 1) The schizophyllum commune culture medium is prepared according to the following mass percentages: 0.6% of kudzuvine 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, wherein the pH value is 6.0;
2) Inoculating schizophyllum commune into the culture medium, and fermenting at 28 ℃ and 160 r/min; after 120h of fermentation, centrifuging the fermentation product to remove thalli and radix puerariae powder to obtain the fermentation product; the biomass in the fermentation product was 8.79 mg/mL; the polysaccharide content in the fermentation product is 2.772mg/mL; the content of the schizophyllum commune is 10% (v/v) calculated according to the schizophyllum commune seed liquid volume/the total volume of the culture medium after the schizophyllum commune seed liquid is inoculated;
the preparation method of the kudzuvine root powder comprises the following steps: drying radix Puerariae, pulverizing, sieving with 40 mesh sieve to obtain radix Puerariae powder, packaging, and sterilizing.
2. A schizophyllum commune fermentation product, characterized in that it is produced by the process for producing a schizophyllum commune fermentation product according to claim 1.
3. A skin care product comprising a substrate and the schizophyllum commune fermentation product of claim 2.
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