CN113318014B - Ganoderma lucidum fermentation product, preparation method thereof and application thereof in cosmetics - Google Patents
Ganoderma lucidum fermentation product, preparation method thereof and application thereof in cosmetics Download PDFInfo
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Abstract
The invention belongs to the technical field of cosmetic raw materials, and discloses a ganoderma lucidum fermentation product, a preparation method thereof and application thereof in cosmetics. The preparation method comprises the following operation steps: inoculating Ganoderma strain into seed culture medium, culturing Ganoderma seed solution, inoculating Ganoderma seed solution into fermentation culture medium, fermenting, wherein the fermentation process comprises fermentation front stage and fermentation rear stage, and heating for sterilization, filtering to remove residue to obtain Ganoderma fermentation product; the culture condition of the fermentation front section is provided with a culture condition suitable for the growth of the ganoderma lucidum mycelia, and the culture condition of the fermentation rear section deviates from the range suitable for the growth of the ganoderma lucidum mycelia and is added with a stress factor; the stress factor comprises one or two of chitinase and lactic acid bacteria. The Ganoderma fermentation product has antiaging effect on skin, and is effective in scavenging superoxide anion free radical, resisting saccharification, keeping moisture, brightening skin color, eliminating yellow, and tightening skin.
Description
Technical Field
The invention belongs to the technical field of cosmetic raw materials, and particularly relates to a ganoderma lucidum fermentation product, a preparation method thereof and application thereof in cosmetics.
Background
Ganoderma (Ganoderma lucidum) belongs to the phylum Eumycota, Basidiomycetes, Polyporales, Polyporaceae, Ganoderma, is a precious medicinal fungus, has wide application in Asian traditional medicine, and is listed in the United states pharmacopoeia and outline of treatment. The ganoderma lucidum contains various bioactive substances, wherein polysaccharide and triterpenoid substances are main active ingredients, and the content of the polysaccharide and the triterpenoid substances is determined as the quality index of the ganoderma lucidum medicinal material by Chinese pharmacopoeia. Modern researches show that the ganoderan has the effects of regulating immunity, reducing blood sugar, reducing blood fat, resisting oxidation, resisting aging and resisting tumor; the triterpenes can purify blood and protect liver function. In the field of skin care, the ganoderma lucidum extract can play the effects of scavenging free radicals, delaying skin aging, improving skin microcirculation blood perfusion and the like when being applied to cosmetics.
The wild ganoderma lucidum grows at the bottom or stump of the deciduous tree and is very rare; ganoderma lucidum can be artificially cultured to obtain fruiting body, but the production cycle is long, the cultivation cost is high, and the triterpene substance content (usually not more than 0.16%) of the cultured Ganoderma lucidum is usually inferior to that of wild Ganoderma lucidum; ganoderma can be cultured in fermentation tank by liquid fermentation to obtain Ganoderma fermentation product with short production period and low cost, but the content of Ganoderma triterpene is often very low (usually not more than 0.06%) or even zero. The difference of the content of the triterpenes is the main reason of different efficacies of the wild ganoderma, the cultivated ganoderma and the ganoderma fermentation product, the content of the triterpenes in the wild ganoderma is higher, the cultivated ganoderma is lower, and the ganoderma fermentation liquor is very low or even zero. The method has great economic significance for improving the content of triterpenoids in the ganoderma lucidum fermentation liquor, and is also beneficial to protecting wild ganoderma lucidum species resources.
Ganoderan is a primary metabolite of ganoderma lucidum, and its content is positively correlated with the biomass of ganoderma lucidum mycelia. Therefore, during the process of culturing ganoderma lucidum mycelia by liquid fermentation, high-concentration ganoderma lucidum polysaccharide can be produced by only providing proper growth conditions (sufficient nutrition, proper dissolved oxygen, pH value and stirring speed) to improve the biomass of the mycelia. The key point of synthesizing the triterpenoids is to stimulate related metabolic pathways, and the triterpenoids cannot be produced by the lucid ganoderma only by providing proper growth conditions. The idea of stimulating the metabolic pathway of synthesizing triterpenoids from ganoderma lucidum is a microscopic view, each link in the metabolic pathway of the triterpenoids is studied from the molecular level through a metabonomics and genomics method, and key regulating factors in the pathway are found; the other idea is that the growth environments of wild ganoderma, cultivated ganoderma and ganoderma fermentation liquor are observed from a macroscopic view, and the key difference of the wild ganoderma, the cultivated ganoderma and the ganoderma fermentation liquor is analyzed from an ecological level.
From the ecological level, the growing environment of wild ganoderma has many challenges, such as insect bite, invasion of other microorganisms, changes of chills and fever, and the like, while the artificial culture environment does not have the challenges, and the difficulties and comfort are the biggest difference between the challenges. In the plant kingdom, terpene alkaloids with bitter taste are often secondary metabolites that respond to biotic stress; similarly, triterpenes are also secondary metabolites of ganoderma against biotic stress. In CN200910305177.1 patent, Ganoderma lucidum C is prepared from Ganoderma fermentation broth by stimulating Ganoderma mycelia with Catharsii Molossi diethyl ether extract to produce triterpenes, which simulates insect bite in wild environment. Application No. CN201210299040.1 patent application No. Nazel in deep fermentation of Ganoderma lucidum acid uses Nazel to stimulate Ganoderma lucidum mycelium to produce triterpenes, which is nitric oxide in simulated wild environment. CN201310348015.2 patent A method for increasing ganoderic acid content in Ganoderma liquid fermentation mycelium stimulates Ganoderma mycelium with acetic acid to produce triterpenes, which simulates pH change in wild environment. CN201610784316.3 patent of A method for increasing the content of intracellular Ganoderma triterpene in liquid fermentation of Ganoderma mycelia stimulates Ganoderma mycelia to produce triterpene substances by high and low temperature treatment deviating from suitable temperature range, which simulates temperature change in wild environment. CN200810138571.6 patent of Ganoderma mycelia liquid fermentation method for increasing ganoderic acid content with fungal elicitor, uses fungal elicitor to stimulate Ganoderma mycelia to produce triterpenes, which simulates microbial invasion in wild environment. The stress factors used in the above documents to simulate the wild environment all have some effect, but in addition to the two stress factors of pH change and temperature change, other stress factors all use chemicals that are not suitable for use in cosmetics (there may be a safety risk); and if only depending on two stress factors of pH change and temperature change, the concentration of the triterpenoids in the fermentation liquor is still low.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention mainly aims to provide a preparation method of a ganoderma lucidum fermentation product. Inoculating Ganoderma strain into seed culture medium, culturing to obtain Ganoderma seed solution, inoculating Ganoderma seed solution into fermentation culture medium, and fermenting, wherein the fermentation process is divided into two sections; the front section provides culture conditions suitable for the growth of ganoderma lucidum mycelia, and the main purpose is to improve the biomass of the ganoderma lucidum mycelia and produce ganoderma lucidum polysaccharide; the culture conditions of the later stage deviate from the range suitable for the growth of the ganoderma lucidum mycelia and stress factors are added, so that the main purpose is to stimulate the ganoderma lucidum mycelia to produce triterpenes.
The invention also aims to provide a ganoderma lucidum fermentation product prepared by the preparation method.
The invention also aims to provide an application of the ganoderma lucidum fermentation product; the ganoderma lucidum fermentation product contains ganoderma lucidum polysaccharide and ganoderma lucidum triterpene with higher concentration, does not contain unsafe components which can stimulate the skin of a human body, and has the anti-aging effect when being applied to the formula of a skin care product.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a ganoderma lucidum fermentation product comprises the following operation steps: inoculating Ganoderma strain into seed culture medium, culturing Ganoderma seed solution, inoculating Ganoderma seed solution into fermentation culture medium, fermenting, wherein the fermentation process comprises fermentation front stage and fermentation rear stage, and heating for sterilization, filtering to remove residue to obtain Ganoderma fermentation product; the culture condition of the fermentation front section is provided with a culture condition suitable for the growth of the ganoderma lucidum mycelia, and the culture condition of the fermentation rear section deviates from the range suitable for the growth of the ganoderma lucidum mycelia and is added with a stress factor; the stress factor comprises one or two of chitinase and lactic acid bacteria.
The seed culture medium comprises the following components in percentage by weight: 3-5% of malt extract, 1-3% of glucose, 0.1-0.3% of yeast extract, 0-0.2% of monopotassium phosphate, 0-0.1% of magnesium sulfate heptahydrate, 0-0.002% of vitamin B10 and 91.398-95.9% of water.
The culture conditions for culturing the ganoderma lucidum seed liquid are as follows: culturing in a triangular flask at the temperature of 26-30 ℃ and the rotation speed of a shaking table of 150-200 rpm for 3-7 days.
The fermentation medium comprises the following components in percentage by weight: 1-3% of malt flour, 1-3% of malt extract, 1-3% of glucose, 0.4-0.8% of licorice powder, 0.1-0.3% of yeast extract, 0-0.2% of monopotassium phosphate, 0-0.1% of magnesium sulfate heptahydrate, 0-0.002% of vitamin B10 and 89.598-96.5% of water.
The ganoderma lucidum seed liquid is inoculated into a fermentation culture medium, and the inoculation proportion is 1: 20-1: 15.
The culture conditions of the fermentation front stage are as follows: culturing in a fermentation tank at 28-30 ℃, stirring at 100-150 rpm, dissolved oxygen at 60-90% for 36-60 h; the culture conditions of the fermentation later stage are as follows: culturing in a fermentation tank at 32-34 ℃, stirring speed of 100-150 rpm, dissolved oxygen of 20-60% and culturing for 36-60 h.
The chitinase is added after the fermentation front stage is finished, and the adding amount is 0.05 to 0.15 percent of the total weight of the fermentation liquid according to the weight percentage; the lactobacillus is added after the fermentation front section is finished, and the addition amount of the lactobacillus seed liquid is 0.2-0.4% of the total weight of the fermentation liquid according to the weight percentage.
The lactobacillus seed solution is prepared according to the following preparation method: inoculating lactobacillus glycerol strain into MRS broth culture medium, culturing at 36-38 deg.C for 24-72 h; the strain of the Lactobacillus seed liquid is Lactobacillus plantarum (Lactobacillus plantarum)
A Ganoderma lucidum fermented product prepared by the above preparation method is provided.
The application of the Ganoderma fermentation product in preparing cosmetics is provided.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention divides the liquid fermentation process of the ganoderma lucidum into two sections, wherein the front fermentation section provides culture conditions suitable for the growth of ganoderma lucidum mycelia to promote the ganoderma lucidum mycelia to produce ganoderma lucidum polysaccharide; culturing conditions of the fermentation later stage deviate from the proper growth range of the mycelia, and adding a stress factor to promote the ganoderma lucidum mycelia to produce ganoderma lucidum triterpenes; the Ganoderma lucidum fermented product produced by the fermentation process has an effective active matter content not lower than that of Ganoderma lucidum extract extracted from Ganoderma lucidum fruiting body.
(2) In the invention, the ganoderma lucidum strain is cultured by adopting a liquid fermentation method, compared with the cultured ganoderma lucidum, the ganoderma lucidum triterpene produced by liquid fermentation is easy to dissolve in water, while the ganoderma lucidum triterpene produced by the cultured ganoderma lucidum fruiting body is difficult to dissolve in water, because the ganoderma lucidum triterpene produced by the ganoderma lucidum mycelium in a liquid culture medium exists in a terpene glycoside form, and a hydrophobic terpene base is connected with hydrophilic glycosyl.
(3) The invention originally takes chitinase and/or lactic acid bacteria as stress factors, the chitinase simulates insect bite in the wild environment in a fermentation tank, and the lactic acid bacteria simulate microbial invasion in the wild environment. The two stress factors can effectively promote ganoderma lucidum mycelia to produce ganoderma lucidum triterpenes.
(4) The culture medium and the stress factor used in the invention do not contain unsafe components which have irritation to the skin, and the prepared ganoderma lucidum fermentation product can be applied to the formula of cosmetics.
(5) The ganoderma lucidum fermentation product prepared by the invention has an anti-aging effect on skin, and is specifically characterized by eliminating superoxide anion free radicals, resisting saccharification, preserving moisture, brightening skin color, dispelling yellow and tightening skin
Drawings
FIG. 1 is a graph showing the change in water content of stratum corneum.
Fig. 2 is a graph of change in skin color L.
FIG. 3 is a graph showing changes in skin melanin MI.
Fig. 4 is a graph of the change in skin color b.
Fig. 5 is a graph showing changes in skin elasticity.
Fig. 6 is a photograph of a VISIA of a subject.
FIG. 7 is a skin response grading standard chart of skin closed patch test.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
This example is an example of the preparation of a fermented product of Ganoderma lucidum, in which the culture conditions of the post-fermentation stage deviate from the range suitable for the growth of the Ganoderma lucidum mycelia and two stress factors, chitinase and lactic acid bacteria, are added. The process comprises the following steps:
(1) preparing a seed culture medium according to the following formula in percentage by weight: 4% of malt extract, 2% of glucose, 0.2% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate heptahydrate, 10.001% of vitamin B and 93.649% of water; then sterilized at 115 ℃ for 30 min.
(2) Inoculating the ganoderma lucidum strain to the seed culture medium obtained in the step (1), and culturing for 4 days at the temperature of 26-30 ℃ and the rotating speed of a shaking table of 150-200 rpm.
(3) Preparing a fermentation medium according to the following formula in percentage by weight: 2% of malt flour, 2% of malt extract, 2% of glucose, 0.6% of licorice powder, 0.2% of yeast extract, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate heptahydrate, vitamin B10.001% and 93.049% of water; then sterilized at 115 ℃ for 30 min.
(4) Inoculating the ganoderma lucidum seed liquid into the fermentation culture medium obtained in the step (3) for fermentation, wherein the fermentation process is divided into a fermentation front section and a fermentation rear section in sequence;
the conditions of the fermentation front stage were: fermenting for 48 hours at the temperature of 30 ℃, at the stirring speed of 100rpm and with the dissolved oxygen controlled between 60% and 90%;
the conditions of the latter stage of fermentation were: adding chitinase with the addition amount of 0.1 percent of the total weight of the fermentation liquor; adding lactobacillus seed solution in an amount of 0.3 wt% of the total fermentation liquid; the temperature is 30 ℃, the stirring speed is 100rpm, the dissolved oxygen content is controlled between 20 percent and 60 percent, and the fermentation is carried out for 48 hours; the lactobacillus seed solution is prepared according to the following steps: preparing MRS broth culture medium, and sterilizing at 115 deg.C for 30 min; lactobacillus plantarum strain was inoculated into MRS broth and cultured at 37 ℃ for 24 h.
(5) Sterilizing at 95 deg.C for 30min, filtering to remove residue, and collecting filtrate as Ganoderma fermentation product.
The ganoderma lucidum fermentation product prepared in the example is a brown translucent liquid in appearance and has a special smell.
Example 2
This example is an example of the preparation of a fermented product of Ganoderma lucidum, in which the culture conditions of the post-fermentation stage deviate from the range suitable for the growth of the Ganoderma lucidum mycelia and chitinase, a stress factor, is added. The process comprises the following steps:
(1) preparing a seed culture medium according to the following formula in percentage by weight: 4% of malt extract, 2% of glucose, 0.2% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate heptahydrate, 10.001% of vitamin B and 93.649% of water; then sterilized at 115 ℃ for 30 min.
(2) Inoculating the ganoderma lucidum strain to the seed culture medium obtained in the step (1), and culturing for 4 days at the temperature of 26-30 ℃ and the rotation speed of a shaking table of 150-200 rpm.
(3) Preparing a fermentation medium according to the following formula in percentage by weight: 2% of malt flour, 2% of malt extract, 2% of glucose, 0.6% of licorice powder, 0.2% of yeast extract, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate heptahydrate, vitamin B10.001% and 93.049% of water; then sterilized at 115 ℃ for 30 min.
(4) Inoculating the lucid ganoderma seed liquid into the fermentation culture medium obtained in the step (3) for fermentation, wherein the fermentation process is divided into a fermentation front section and a fermentation rear section in sequence;
the conditions of the fermentation front stage were: fermenting for 48 hours at the temperature of 30 ℃, at the stirring speed of 100rpm and with the dissolved oxygen controlled between 60% and 90%;
the conditions of the latter stage of fermentation were: adding chitinase with the addition amount of 0.1 percent of the total weight of the fermentation liquor; the temperature is 30 ℃, the stirring speed is 100rpm, the dissolved oxygen content is controlled between 20 percent and 60 percent, and the fermentation is carried out for 48 hours;
(5) sterilizing at 95 deg.C for 30min, filtering to remove residue, and collecting filtrate as Ganoderma fermentation product.
The ganoderma lucidum fermentation product prepared in the example is a brown translucent liquid in appearance and has a special smell.
Example 3
This example is the preparation of fermented ganoderma products, which is different from the technical scheme of the invention. In this case, the culture conditions of the post-fermentation stage deviate from the range suitable for the growth of the ganoderma lucidum mycelia, but no stress factor is added. The process comprises the following steps:
(1) preparing a seed culture medium according to the following formula in percentage by weight: 4% of malt extract, 2% of glucose, 0.2% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate heptahydrate, 10.001% of vitamin B and 93.649% of water. Then sterilized at 115 ℃ for 30 min.
(2) Inoculating the ganoderma lucidum strain to the seed culture medium obtained in the step (1), and culturing for 4 days at the temperature of 26-30 ℃ and the rotating speed of a shaking table of 150-200 rpm.
(3) Preparing a fermentation medium according to the following formula in percentage by weight: 2% of malt flour, 2% of malt extract, 2% of glucose, 0.6% of licorice powder, 0.2% of yeast extract, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate heptahydrate, vitamin B10.001% and 93.049% of water. Then sterilized at 115 ℃ for 30 min.
(4) Inoculating the ganoderma lucidum seed liquid into the fermentation culture medium obtained in the step (3) for fermentation, wherein the fermentation process is divided into a fermentation front section and a fermentation rear section in sequence;
the conditions of the fermentation front stage were: the temperature is 30 ℃, the stirring speed is 100rpm, the dissolved oxygen is controlled between 60 percent and 90 percent, and the fermentation is carried out for 48 hours.
The conditions of the latter stage of fermentation were: the temperature is 30 ℃, the stirring speed is 100rpm, the dissolved oxygen content is controlled between 20 percent and 60 percent, and the fermentation is carried out for 48 hours.
(5) Sterilizing at 95 deg.C for 30min, filtering to remove residue, and collecting filtrate as Ganoderma fermentation product.
The ganoderma lucidum fermentation product prepared in the example is a brown translucent liquid in appearance and has a special smell.
Example 4
This example is a test of the crude polysaccharide and total terpene content of the fermented Ganoderma product prepared in examples 1, 2 and 3.
The method for testing the content of the crude polysaccharide comprises the following steps:
(1) 10g of sample is taken, 30g of ethanol is added, and the mixture is uniformly mixed and refrigerated at 4 ℃ for 24 h.
(2) The mixture was centrifuged at 3000rpm for 30min and the supernatant was discarded.
(3) The precipitate was dried in an oven at 60 ℃ and the dry weight of the precipitate was weighed.
(4) And (4) calculating. The crude polysaccharide content in the sample was equal to dry weight of precipitate/10 g.
Total terpene content test method:
(1) solution preparation
Vanillin glacial acetic acid solution (5%): 0.5g of vanillin was weighed out and dissolved in 10mL of glacial acetic acid.
Asiaticoside standard solution (0.40 mg/mL): accurately weighing 10mg of asiaticoside reference substance, dissolving in 25mL volumetric flask with anhydrous ethanol, and shaking.
Dilute hydrochloric acid (0.3 mol/L): commercially available 36% -37% concentrated hydrochloric acid 27mL is added with water to 1000mL and shaken up.
(2) Drawing of standard curve
Precisely measuring asiaticoside standard solution (0.40mg/mL)100uL, 200uL, 300uL, 400uL, 500uL and 600uL, respectively placing in a colorimetric tube, and evaporating to dryness in a water bath at 90 deg.C; adding vanillin glacial acetic acid solution 0.2mL and concentrated sulfuric acid 0.8mL, adding plug, mixing, shaking, water bathing at 60 deg.C for 15min, and taking out ice water bathing for 5 min; adding 5mL of glacial acetic acid, mixing, shaking uniformly, and standing for 10 min; taking the accompanied reagent without asiaticoside and sample as blank, and measuring absorbance at 550 nm; and drawing a standard curve by taking the mass (M, mg) of the asiaticoside as a vertical coordinate and the absorbance (A) as a horizontal coordinate.
(3) Sample assay
Sample treatment: weighing 2mL of a sample, placing the sample in a test tube, adding 4mL of dilute hydrochloric acid (0.3mol/L), adding a plug, shaking up, and carrying out water bath at 90 ℃ for 3 hours; after being taken out and cooled to room temperature, 2mL of ethyl acetate is added, the mixture is added with a plug and shaken vigorously for 1min, and the mixture is stood for 10min to separate layers. Note that: each sample was assayed in 3 replicates, starting from sample treatment.
And (3) color development reaction: placing 200uL of ethyl acetate phase in a colorimetric tube, evaporating to dryness in water bath at 90 deg.C, adding vanillin glacial acetic acid solution 0.2mL and concentrated sulfuric acid 0.8mL, adding stopper, mixing, shaking, placing in water bath at 60 deg.C for 15min, and taking out ice water bath for 5 min; adding 5mL of glacial acetic acid, mixing, shaking uniformly, and standing for 10 min; the absorbance was measured at 550nm using the running reagent without asiaticoside and sample as a blank.
(4) Computing
The total terpenes (mg, asiaticoside equivalent) contained in 200uL of the sample were calculated from the standard curve and the absorbance of the sample reaction solution, and converted to concentration (mg/L).
And (3) testing results:
(1) from table 1, it can be found that the content of the crude polysaccharide is the ganoderma lucidum fermentation products prepared in examples 1, 2 and 3 from large to small, which indicates that the technical scheme of the invention is beneficial to improving the content of the ganoderma lucidum polysaccharide in the fermentation products. The content of crude polysaccharide in a commercially available ganoderma lucidum extract is 0.
(2) From table 1, it can be found that the total terpene contents are the ganoderma lucidum fermentation products prepared in examples 1, 2 and 3 respectively from large to small, which indicates that the technical scheme of the invention is beneficial to improving the ganoderma lucidum triterpene content in the fermentation products.
TABLE 1 crude polysaccharide and Total terpene content of Ganoderma lucidum fermentation product
Example 5
This example is a test of superoxide anion radical scavenging activity of the fermented Ganoderma product prepared in examples 1, 2 and 3. Superoxide anion free radical is generated in human bodyThe active oxygen free radical can induce lipid peroxidation in vivo, accelerate aging process from skin to internal organs, and remove superoxide dismutase (SOD) from human body. Superoxide anion radical (. O)2 -) The clearance rate is measured by pyrogallol autoxidation method for characterizing SOD and other antioxidant active targets.
Superoxide anion free radical clearance test method:
(1) solution preparation
Tris-HCl solution (0.05 mol/L): accurately weighing 6.0575g of Tris, dissolving in water, and fixing the volume to 250 mL; 25mL of Tris solution is added with 25mL of 0.1mol/L HCl solution, and water is added to the solution to reach the constant volume of 100 mL.
Pyrogallol solution (3 mmol/L): 0.0378g of pyrogallol is accurately weighed and dissolved with water to a constant volume of 100 mL.
(2) Sample detection
Accurately sucking 0.5mL of sample solution, placing the sample solution into a test tube, adding 4.5mL of Tris-HCl solution and 4mL of deionized water. Shaking, standing at room temperature for 10min, adding 0.3mL pyrogallol solution, mixing, and immediately measuring absorbance at 320 nm. Counting every 30s, recording the value within 3min, and calculating the change SLOPE (the SLOPE function in Excel is SLOPE). Deionized water was used as a blank.
(3) Computing
Superoxide anion radical clearance calculation formula:
in the formula:
Kblank space-blank tube absorbance change slope; kSample(s)-slope of change in absorbance of sample tube.
And (3) testing results:
from table 2, it can be found that the superoxide anion radical scavenging rate of the ganoderma lucidum fermentation products prepared in examples 1 and 2 is greater than that of the ganoderma lucidum fermentation product prepared in example 3, which is deviated from the technical scheme, and is much greater than that of a certain commercial ganoderma lucidum fruit body extract. The ganoderma lucidum fermentation product prepared according to the technical scheme has higher superoxide anion free radical scavenging activity.
TABLE 2 superoxide anion radical scavenging test results
Example 6
This example is a test of the anti-glycation activity of the fermented products of Ganoderma lucidum prepared in examples 1, 2 and 3. The glycation reaction (protein non-enzymatic glycosylation reaction, also called carbonylamino reaction and Maillard reaction) means that saccharides are combined with proteins without the action of enzymes, so that the proteins lose normal structures. After the reaction between sugar and collagen in the dermis, some reversible primary glycosylation products are formed first, and then irreversible advanced glycosylation end products are formed. Glycated collagen is yellow in color and cannot be renewed by degradation in the usual sense of the "dermal matrix remodeling process". Anti-glycation is therefore an important strategy for delaying aging.
The anti-saccharification activity test method comprises the following steps:
(1) solution preparation
PBS buffer (ph 7.4): 8g of NaCl, 0.2g of KCl, 0.2g of KH2PO40.2 g, 1000mL of Na2HPO 4.12H 2O 2.56.56 g and 1000mL of boiled water are dissolved, and 20g of hexanediol and 20g of pentanediol are added after cooling;
and (3) fructose solution: d-fructose is 0.05mol, PBS buffer solution is 100mL, shake and dissolve;
collagen solution: hydrolyzed collagen 2g, PBS buffer 100mL, shake and dissolve.
Sodium carbonate solution: Na2CO39.54g, NaHCO 30.84g and water 1L, and the pH value is 10.8.
NBT color reagent: dissolving 0.03mmol of nitro blue tetrazole in 100mL of sodium carbonate solution, and refrigerating for later use.
(2) Establishing a chemical model
The reagents were added and the procedure was followed as in Table 3.
TABLE 3 procedure for establishing chemical model
C is blank control group. T is a sample group, and the sample solution should be diluted to an appropriate concentration in advance.
Each test should contain multiple sample groups, one blank. Each test should ensure a sample group with a definite anti-glycation efficacy (e.g. 25ug/mL chlorogenic acid in water, or 4mmol/L aminoguanidine in water) as a positive control or control.
(3) Detection of glycosylated primary product Amadori
80uL of each hatching fluid is taken, 1.6mL of NBT solution and 320uL of water are respectively added, the mixture is shaken up and reacted for 15min at 25 ℃, and then the absorbance at 530nm is measured, and the Amadori concentration is increased when the difference (T-T.) is larger.
And (3) testing results:
from table 4, it can be found that the saccharification inhibition ratio of the ganoderma lucidum fermentation products prepared in examples 1 and 2 is greater than that of the ganoderma lucidum fermentation product prepared in example 3, which is different from the technical scheme. The saccharification inhibition rate of the ganoderma lucidum fermentation product prepared in example 1 is greater than that of a commercially available ganoderma lucidum fruit body extract. The ganoderma lucidum fermentation product prepared according to the technical scheme has higher anti-saccharification activity.
TABLE 4 results of the anti-glycation assay
Example 7
This example is a human patch test of the fermented product of Ganoderma lucidum prepared in example 1.
The test method comprises the following steps:
(1) the number of subjects: the number of the people entering the team is not less than 30, 32 people are actually recruited, and the effective data is 32.
(2) Refer to technical Specification for safety of cosmetics 2015 for selection of test subjects for skin spots on human body.
(3) The testing steps are as follows: test samples and qualified plaque test devices are prepared and placed in the plaque test device chamber in an amount of about 0.020g to about 025g (solid or semi-solid) or about 0.020mL to about 0.025mL (liquid). The spot test device with the tested object is applied to the back or the forearm curve side of the tested object by using a low-sensitivity adhesive tape, and is lightly pressed by a palm to be uniformly applied to the skin for 24 hours. Skin reactions were observed at 30min (after disappearance of the indentation), 24h, and 48h after removal of the test article plaque test device, respectively, according to the criteria of table 5 and fig. 7, and the observation results were recorded.
TABLE 5 skin closed Patch test skin response grading Standard
And (3) testing results: the number of patients with grade 2 adverse skin reactions in 32 subjects totaled 2, as detailed in Table 6. According to the explanation of the test result of the skin patch of the human body in the cosmetic hygiene code (2007 edition), the tested object has no adverse skin reaction to the human body.
TABLE 6 test results of skin-enclosed patch test
Example 8
This example is an anti-aging human efficacy test of ganoderma lucidum fermented essence prepared from the ganoderma lucidum fermented product prepared in example 1.
The lucid ganoderma fermentation essence takes lucid ganoderma fermentation products as the only anti-aging functional components, and the formula of the lucid ganoderma fermentation essence comprises the following components in percentage by weight: 83.81% of water, 0.1% of carbomer, 0.09% of triethanolamine, 10% of lucid ganoderma fermentation product, 5% of propylene glycol, 0.5% of p-hydroxyacetophenone and 0.5% of hexanediol.
The test method comprises the following steps:
(1) a detection instrument: a Canfield VISIA facial skin analyzer, a Delfin MoistureMeterDC skin stratum corneum hydration meter, a Delfin SkinColorcatch skin color meter, and a Delfin Elastimeter skin elasticity meter.
(2) Test conditions were as follows: and (3) testing period: 0 week, 2 weeks, 4 weeks, 6 weeks; the number of tested persons: 12 persons, mean age 38.70 ± 6.10 years; testing parts: the face. A photograph of the VISIA of the subject is shown in fig. 6.
And (3) testing results:
(3) after 2 weeks using the ganoderma lucidum fermented essence, the skin elasticity value increased by 6.96% compared to 0 weeks with statistical differences (P < 0.05) (fig. 5). The ganoderma lucidum fermentation essence has the effects of increasing skin elasticity and firming skin after being used for 2 weeks.
(4) After 4 weeks of use of the ganoderma lucidum fermented essence, compared with the skin before use, the moisture content of the skin stratum corneum is respectively increased by 19.01%, the skin color L value is increased by 1.18%, and the skin melanin MI value is reduced by 1.28%, and the skin stratum corneum has statistical differences (P is less than 0.05) (figures 1, 2 and 3). The ganoderma lucidum fermentation essence has the effects of moisturizing and brightening skin after being used for 4 weeks.
(5) After 6 weeks of use of the ganoderma lucidum fermented essence, the skin color b value decreased by 4.71%, the skin elasticity value increased by 8.83%, and both had statistical differences (P < 0.05) (fig. 4, 5), compared to before use. The ganoderma lucidum fermentation essence has the effects of improving skin yellowing, increasing skin elasticity and firming skin after being used for 6 weeks.
In conclusion, the ganoderma lucidum fermentation essence has the effects of moisturizing, brightening skin color, removing yellow and firming skin.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A preparation method of a ganoderma lucidum fermentation product is characterized by comprising the following operation steps: inoculating Ganoderma strain into seed culture medium, culturing Ganoderma seed solution, inoculating Ganoderma seed solution into fermentation culture medium, fermenting, wherein the fermentation process comprises fermentation front stage and fermentation rear stage, and heating for sterilization, filtering to remove residue to obtain Ganoderma fermentation product; the culture condition of the fermentation front section is provided with a culture condition suitable for the growth of the ganoderma lucidum mycelia, and the culture condition of the fermentation rear section deviates from the range suitable for the growth of the ganoderma lucidum mycelia and is added with a stress factor; the stress factors comprise chitinase and lactic acid bacteria; the chitinase is added after the fermentation front stage is finished, and the adding amount of the chitinase is 0.05-0.15 percent of the total weight of the fermentation liquid in percentage by weight; the lactobacillus is added into lactobacillus seed liquid after the fermentation is finished in the front stage, and the addition amount of the lactobacillus seed liquid is 0.2-0.4% of the total weight of the fermentation liquid in percentage by weight.
2. The production method according to claim 1, characterized in that: the seed culture medium comprises the following components in percentage by weight: 3-5% of malt extract, 1-3% of glucose, 0.1-0.3% of yeast extract, 0-0.2% of monopotassium phosphate, 0-0.1% of magnesium sulfate heptahydrate, 0-0.002% of vitamin B10 and 91.398-95.9% of water.
3. The method of claim 1, wherein: the culture conditions for culturing the ganoderma lucidum seed liquid are as follows: culturing in a triangular flask at 26-30 ℃ and 150-200 rpm of shaking table rotation speed for 3-7 days.
4. The method of claim 1, wherein: the fermentation medium comprises the following components in percentage by weight: 1-3% of malt flour, 1-3% of malt extract, 1-3% of glucose, 0.4-0.8% of licorice powder, 0.1-0.3% of yeast extract, 0-0.2% of monopotassium phosphate, 0-0.1% of magnesium sulfate heptahydrate, 0-0.002% of vitamin B10 and 89.598-96.5% of water.
5. The method of claim 1, wherein: inoculating the ganoderma lucidum seed liquid into a fermentation culture medium, wherein the inoculation ratio is 1: 20-1: 15.
6. The production method according to claim 1, characterized in that: the culture conditions of the fermentation front stage are as follows: culturing in a fermentation tank at 28-30 ℃, stirring at 100-150 rpm, dissolved oxygen of 60-90% for 36-60 h; the culture conditions of the fermentation later stage are as follows: culturing in a fermentation tank at 32-34 ℃, stirring at 100-150 rpm, dissolved oxygen of 20-60% and culturing for 36-60 h.
7. The method of claim 1, wherein: the lactobacillus seed solution is prepared according to the following preparation method: inoculating lactobacillus glycerol strain into MRS broth culture medium, culturing at 36-38 deg.C for 24-72 h; the strain of the Lactobacillus seed liquid is Lactobacillus plantarum (Lactobacillus plantarum).
8. A fermented product of Ganoderma lucidum prepared by the method of any one of claims 1 to 7.
9. Use of the ganoderma lucidum fermentation product of claim 8 in the preparation of a cosmetic.
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