CN107881119B - Black ganoderma mycelium fermentation liquor and preparation method thereof, and cosmetics with antioxidation and whitening effects - Google Patents
Black ganoderma mycelium fermentation liquor and preparation method thereof, and cosmetics with antioxidation and whitening effects Download PDFInfo
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- CN107881119B CN107881119B CN201711251044.1A CN201711251044A CN107881119B CN 107881119 B CN107881119 B CN 107881119B CN 201711251044 A CN201711251044 A CN 201711251044A CN 107881119 B CN107881119 B CN 107881119B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
Abstract
The invention relates to the technical field of fermentation of ganoderma atrum, in particular to ganoderma atrum mycelium fermentation liquor and a preparation method thereof, and a cosmetic with antioxidant and whitening effects. The method comprises the following steps: the method comprises the following steps of performing slant tube culture on a black ganoderma strain to obtain a mycelium block, inoculating the mycelium block to a plate culture medium, performing mycelium culture, transferring the mycelium block to a malt culture medium, and performing liquid fermentation culture at 25-29 ℃ for 4-7 days. The preparation method of the black ganoderma lucidum mycelium fermentation liquor provided by the invention is simple to operate, and the prepared black ganoderma lucidum mycelium fermentation liquor is high in polysaccharide and triterpene content, and has very high physiological activity and very good health care effect; in addition, the ganoderma atrum hypha fermentation liquor has high tyrosinase activity inhibition rate and oxidation resistance, so that the formation of melanin can be effectively inhibited, and the aging can be delayed.
Description
Technical Field
The invention relates to the technical field of fermentation of ganoderma atrum, in particular to ganoderma atrum mycelium fermentation liquor and a preparation method thereof, and a cosmetic with antioxidant and whitening effects.
Background
Black sesame, also called "Chinese glossy ganoderma", is sweet in taste and neutral in nature. Mainly contains ergosterol, organic acid, glucosamine, polysaccharides, resin, mannitol, polyglycitol, fatty acid, alkaloid, lactone, coumarin, water-soluble protein and multiple enzymes. The application range of the black sesame is very wide, and the black sesame can be taken no matter whether the heart, the lung, the liver, the spleen and the kidney are weak. Scientific research shows that the pharmacological components of ganoderma are very rich, wherein the effective components can be divided into ten major categories, including ganoderma lucidum polysaccharide, ganoderma lucidum polypeptide, three anvils, 16 amino acids (including seven essential amino acids for human body), protein, mannitol, coumarins, alkaloid, organic acid (mainly including fumaric acid), and trace elements of Ge, P, Fe, Ca, Mn, Zn and the like. The Ganoderma extract has skin whitening, moisture keeping, antioxidant, and antiaging effects.
Wild black ganoderma is rare in nature, and the content of ganoderma lucidum polysaccharide is not high enough, so that the cost for developing ganoderma lucidum products is overhigh. Secondly, the artificial cultivation of the ganoderma lucidum has long period, the product quality is easily affected by environmental climate and insect damage, and the popularization is inconvenient. Therefore, the fermentation method for producing the ganoderma lucidum polysaccharide is an effective method. At present, ganoderma active substances such as ganoderma polysaccharide and the like are mainly obtained by taking artificially cultured sporocarp as a raw material and adopting an extraction method, the development is limited by the defects of low yield, difficult large-scale production, great difference in composition and content of active ingredients among different batches and the like. Compared with the ganoderma lucidum fruiting body and the spore powder, the active substances contained in the ganoderma lucidum mycelium have higher activity, shorter liquid fermentation time and less pollution possibility, and are more beneficial to the industrial production of the mycelium. Ganoderan is used as a main bioactive component of ganoderma lucidum, and has been the main research direction of ganoderma lucidum liquid fermentation. And how to improve the yield becomes critical.
Liquid submerged fermentation of edible fungi began in the 40 th of the 20 th century. Compared with the cultivation of the sporocarp, the liquid submerged fermentation of the edible fungi has obvious advantages, the manual cultivation of the sporocarp consumes time and labor, the cost is high, the cultivation is limited by environmental factors, and the quality of the product is difficult to ensure. The liquid submerged fermentation time is short, usually about one week, time and labor are saved, large-scale industrial production can be realized, and the product quality is stable. Meanwhile, some edible fungi which cannot be cultivated at present can also produce mycelium and other metabolites by a liquid submerged fermentation method. Due to the great superiority of liquid fermentation, research and application of liquid inoxidizability are promoted. However, no method for preparing the black ganoderma mycelium fermentation liquor with satisfactory effect exists in the prior art.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preparation method of black ganoderma hypha fermentation liquor, and aims to solve the problems that the black ganoderma culture method in the prior art is long in production period, needs fruiting bodies, is high in cost, is low in tyrosinase activity inhibition rate of black ganoderma hypha protoplasm, is not ideal in antioxidant function, is low in polysaccharide and triterpene content, is difficult to produce in large scale, and is large in difference of active ingredient composition and content among different batches.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention relates to a preparation method of black ganoderma mycelium fermentation liquor, which comprises the following steps:
the method comprises the following steps of performing slant tube culture on a black ganoderma strain to obtain a mycelium block, inoculating the mycelium block to a plate culture medium, performing mycelium culture, transferring the mycelium block to a malt culture medium, and performing liquid fermentation culture at 25-29 ℃ for 4-7 days.
The preparation method of the black ganoderma mycelium fermentation liquor provided by the invention can be used for extracting the effective components in black ganoderma in a land-saving, time-saving and labor-saving manner, can be used for intensive production, and has a simpler extraction process compared with the extraction of fruiting bodies.
The invention also relates to the black ganoderma hypha fermentation liquor prepared by the method.
The invention also relates to a cosmetic with antioxidant and whitening effects, which takes the ganoderma atrum mycelium fermentation liquor or the extract thereof as an active ingredient.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the black ganoderma lucidum mycelium fermentation liquor provided by the invention is simple to operate, and the prepared black ganoderma lucidum mycelium fermentation liquor is high in polysaccharide and triterpene content, and has very high physiological activity and very good health care effect; in addition, the ganoderma atrum hypha fermentation liquor has high tyrosinase activity inhibition rate and oxidation resistance, so that the formation of melanin can be effectively inhibited, and the aging can be delayed; the product is pure fungus extract, is safe and green, and can be used for preventing common skin problems such as skin aging, dim, freckle, yellow and brown spot, sunburn, pigmentation caused by long-term use of cosmetics and wound.
Detailed Description
The invention relates to a preparation method of black ganoderma mycelium fermentation liquor, which comprises the following steps:
the method comprises the following steps of performing slant tube culture on a black ganoderma strain to obtain a mycelium block, inoculating the mycelium block to a plate culture medium, performing mycelium culture, transferring the mycelium block to a malt culture medium, and performing liquid fermentation culture at 25-29 ℃ for 4-7 days.
Preferably, if the fermented solution of the black ganoderma mycelium with the highest polysaccharide content is prepared, carrying out liquid fermentation culture for 4 days; if the preparation aims to obtain the ganoderma lucidum mycelium fermentation liquor with the maximum triterpene content, carrying out liquid fermentation culture for 6 days; if the fermented liquid of the black ganoderma mycelia with the best antioxidant effect is prepared, carrying out liquid fermentation culture for 4 days; and if the fermented solution of the black ganoderma mycelia with the best whitening effect is prepared, carrying out liquid fermentation culture for 7 days.
Specifically, the antioxidant effect means that the antioxidant effect is achieved by the capability of removing DPPH free radicals and superoxide anion free radicals.
Specifically, the whitening effect means that the whitening effect is achieved by inhibiting the activity of tyrosinase and inhibiting the generation of melanin in melanoma cells.
Preferably, in the method for preparing a fermentation broth of mycelia of ganoderma atrum as described above, the plate medium comprises:
25-35 g/L malt extract, 2-4 g/L soybean peptone, 2-4 w/v agar malt extract, 0.2-0.4 w/v soybean peptone, 1.3-1.7 w/v agar, water as solvent, and pH 5.4-5.8;
more preferably, the method for preparing a fermentation broth of mycelia of black sesame as described above, wherein the plating medium comprises:
27-33 g/L malt extract, 2.5-3.5 g/L soybean peptone, 2.5-3.5 w/v agar malt extract, 0.25-0.35 w/v soybean peptone, 1.4-1.6 w/v agar, water as solvent and pH 5.5-5.7;
most preferably, the plating medium comprises:
30g/L malt extract, 3g/L soybean peptone, 3w/v agar malt extract, 0.3w/v soybean peptone, 1.5w/v agar, water as solvent and pH 5.6.
Preferably, in the preparation method of the black ganoderma hypha fermentation liquor, the inoculation amount of the hypha blocks inoculated to the plate culture medium is 0.7-1.3 cm for each plate inoculation2The mycelium block of (1).
Preferably, in the method for preparing the black ganoderma lucidum mycelium fermentation liquor, the mycelium is cultured for 6-8 days at the temperature of 28-32 ℃.
Preferably, in the method for preparing a fermentation broth of mycelia of black sesame as described above, the malt medium includes:
1.5-2.5 w/v% of malt extract powder, 1.5-2.5 w/v% of glucose, 0.07-0.13 w/v% of peptone, 0.07-0.13 w/v% of yeast extract and water as a solvent;
more preferably, in the method for producing a fermentation broth of mycelia of black sesame as described above, the malt medium includes:
1.7-2.3 w/v% of malt extract powder, 1.7-2.3 w/v% of glucose, 0.09-0.11 w/v% of peptone, 0.09-0.11 w/v% of yeast extract and water as a solvent;
most preferably, the method for preparing a fermentation broth of mycelia of black sesame as described above, wherein the malt medium comprises:
2 w/v% of malt extract powder, 2 w/v% of glucose, 0.1 w/v% of peptone, 0.1 w/v% of yeast extract and water as solvent.
Preferably, in the method for preparing the fermentation liquid of the hypha of black sesame, the inoculation amount is 1.3-1.7 cm per 100ml of the culture medium when the black sesame is transferred to a malt culture medium for liquid fermentation culture2The mycelium block of (1).
The invention also relates to the black ganoderma hypha fermentation liquor prepared by the method.
The invention also relates to a cosmetic with antioxidant and whitening effects, which takes the ganoderma atrum mycelium fermentation liquor or the extract thereof as an active ingredient.
Preferably, the cosmetic further comprises at least one of a solvent, an astringent, a stabilizer, a thickener, a conditioner, a humectant, a pH adjuster, a surfactant, an antioxidant, a cooling agent, an emulsifier, a preservative, a colorant, a dispersant, a fragrance;
preferably, the solvent comprises at least one of water, propylene glycol, butylene glycol, glycerol and pentanediol;
preferably, the astringent is zinc oxide or talcum powder;
preferably, the stabilizer comprises hydroxypropyl methyl cellulose, plant carrageenan, octadecanol and behenyl alcohol;
preferably, the thickener comprises at least one of carbomer, SEPINOV EMT-10;
preferably, the conditioning agent comprises at least one of allantoin, vitamin A palmitate, dipotassium glycyrrhizinate, centella asiatica extract, nicotinamide, caprylyl glycine, quaternary ammonium salt-73, salicylic acid, and caprylyl salicylic acid;
preferably, the humectant comprises at least one of ethylhexyl glycerin, butylene glycol, 1, 2-pentanediol, glycerin, propylene glycol, sodium hyaluronate, D-panthenol, PCA-Na;
preferably, the pH regulator comprises at least one of sodium hydroxide, potassium hydroxide, arginine, citric acid and sodium citrate;
preferably, the surfactant comprises at least one of tween-20, PEG-40 hydrogenated castor oil and PEG-60 hydrogenated castor oil;
preferably, the cooling agent comprises at least one of boiling complex, menthol, peppermint essential oil and menthyl lactate.
Preferably, the preparation of the cosmetic comprises aqua, essence, mask, emulsion and cream.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. 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 commercially available.
Examples
1. Mycelium fermentation liquor method
(1) Preparation of media and solutions
Plate culture medium: MEA (Malt Extract agar medium Malt Extract agar): 30g of malt extract, 3g of soybean peptone, 3 w/v% of agar malt extract, 0.3 w/v% of soybean peptone, 1.5 w/v% of agar and H2O1L constant volume, pH5.6, 121 deg.C sterilization for 15 min.
Malt culture medium: malt extract powder 2 w/v%, glucose 2 w/v%, peptone 0.1 w/v%, yeast extract 0.1 w/v%, H2O1L is used for fixing the volume, and is sterilized for 20min at the temperature of 115 ℃.
CYM medium: 20g of glucose, 2g of peptone, 2g of yeast powder and MgSO4﹒7H2O0.5g、K2HPO4 1g、KH2PO40.46g，H2O 1L。
(2) Liquid fermentation culture method of ganoderma atrum strain
Culturing black Ganoderma strain in slant test tube, collecting 1cm of black Ganoderma strain2The mycelia were inoculated into a plate medium and cultured at 30 ℃ for 7 days. Then, 1.5cm of the medium was taken out of the plate medium2The cells were inoculated into malt medium (300ml Erlenmeyer flask, liquid content 100ml) and CYM medium, respectively, and cultured at 27 ℃ for three days, and then recorded while culturing from the fourth day.
2. Determination of polysaccharide yield in black ganoderma liquid fermentation process
(1) And a measuring method
Taking 1ml of sample liquid into a 50ml centrifugal tube, adding 95% ethanol with 3 times volume, and carrying out alcohol precipitation for about 24 hours; centrifuging at 5000r/min for 20min, and discarding the supernatant; drying the precipitate in an ultralow temperature freeze dryer for 2-3 h; and adding 20ml of ultrapure water to dissolve and dilute the precipitate to a proper concentration, and taking 1ml of the solution as a solution to be detected after the precipitate is fully dissolved, wherein the polysaccharide content of the solution is detected by a phenol-sulfuric acid method.
The phenol-sulfuric acid method comprises the following steps: adding 1ml of the solution to be measured into a test tube, adding 1.0ml of 5% phenol solution into the test tube, rapidly adding 5.0ml of concentrated sulfuric acid (vertical to the liquid surface, without contacting the wall of the test tube, so as to be fully mixed with the reaction solution), shaking, standing for reaction for 30min, measuring the absorbance at 490nm, and calculating the polysaccharide content of the solution to be measured by glucose standard.
(2) Analysis of polysaccharide extraction result from fermentation liquor of ganoderma atrum mycelium
Selecting black sesame, taking the content of polysaccharide in the fermentation liquor as a detection index, and screening more appropriate fermentation days. Taking 1.5cm from the plate medium full of mycelia2Inoculated into a liquid medium, cultured at 27 ℃ for three days, and then cultured while recording from the fourth day until the seventh day.
As shown in the following table, polysaccharides and triterpenes can be extracted from the fermentation broth of the mycelia of Ganoderma atrum, and the malt culture medium has better effect than CYM culture medium. Wherein the polysaccharide content in the fermentation liquor of the black ganoderma in the malt culture medium at the 4 th day is the maximum, and can reach 2.8806 mg/ml. The triterpene content in the fermentation liquor at the day 6 can reach 530.3378 μ g/ml at most.
The experimental results are as follows:
mycelium fermentation liquor detection result
3. Measurement of antioxidant Effect of mycelium fermentation liquid
Experimental methods
The antioxidant experiment mainly detects the DPPH free radical clearance rate and superoxide anion free radical clearance rate, and the method comprises the following steps:
(1) and (3) measuring the ability of removing DPPH free radicals, adding 1ml of DPPH with the concentration of 0.2mmol/L into 1ml of solution to be measured, standing for 30min at room temperature, and measuring the change of absorbance at the wavelength of 517 nm. DPPH clearance (%) [1- (Ai-Aj)/a0] × 100% in the formula: ai is the absorbance of 1ml of DPPH +1ml of sample; aj is the absorbance of 1ml of solvent +1ml of sample; a0 Absorbance of 1ml DPPH +1ml solvent
(2) Experiment for scavenging superoxide anion free radical
Adding 5.7ml of 50mmol/L Tris-HCl buffer solution with pH of 8.2 into a 10ml EP tube, adding 0.2ml of sample solution, uniformly mixing, keeping the temperature at 25 ℃ for 10min, adding 0.1ml of 10mmol/L pyrogallol solution preheated at 25 ℃, keeping the total volume at 6ml, rapidly shaking up, and recording the absorbance increase (As) of the solution in 1min at the wavelength of 320nm by using an ultraviolet-visible spectrophotometer. The increase in absorbance per minute in the linear range was calculated. The reagent was taken as above, the sample solution was replaced with equal volume of water, and the increase in absorbance at 320nm for 1min was also measured (A0). The superoxide anion radical clearance (%) - (A0-As)/A0X 100%
Results of the experiment
Mycelium fermentation liquor detection result
The experimental results are as follows:
in the aspect of oxidation resistance, the fermentation medium which takes malt extract powder as a main component is analyzed from the culture medium type, so that the fermentation effect of the fermentation medium after the fermentation of thalli is greatly helped, the effect of the fermentation liquor of the ganoderma atrum mycelium on DPPH self-substrate removal is obviously higher than that of CYM culture medium, and the removal of superoxide anion free radicals fluctuates in different degrees due to different fermentation times. According to analysis on fermentation time, the comprehensive effect of the fermentation liquor is better when the fermentation is carried out to the fourth day;
3. whitening effect of fermentation liquid of mycelia of Ganoderma lucidum
(1) And a detection method
Preparation of Phosphate Buffered Saline (PBS): weighing Na2HPO4﹒12H2O71.63 g, fully dissolving in deionized water to a volumetric flask with the constant volume of 1000ml to prepare 0.2mol/LNaH2PO4Solution: weighing NaH2PO4﹒2H2Dissolving 31.20g of O in deionized water fully, and preparing into 0.2mol/L NaH in a volumetric flask with 1000mL2PO4And (3) solution. In the following ratio of 51: 49, using pH value and measuring and adjusting the pH value to about 6.8 for later use, and preparing the mixture according to the use.
Preparation of tyrosinase solution: weighing a certain amount of tyrosinase according to the activity unit of the tyrosinase being 200U/mL, dissolving the tyrosinase by using a phosphate buffer solution, fixing the volume in a volumetric flask with the required volume, freezing and storing the tyrosinase at the temperature of-20 ℃, and unfreezing the tyrosinase at the temperature of 4 ℃ before use.
Preparing an L-tyrosine solution: precisely weighing 0.0272g of L-tyrosine, pre-dispersing with phosphate buffer solution, and ultrasonically treating for 30min to fix the volume of the L-tyrosine in a volumetric flask with the phosphate buffer solution.
Specific measurement method
And accurately sucking reaction liquids of tyrosinase, PBS and samples in T1, T2, T3 and T4 respectively according to the volumes of the above table by using a micropipette, respectively placing the reaction liquids in 4 PE tubes, uniformly mixing the reaction liquids, carrying out constant-temperature water bath at 37 ℃ for 10min, respectively adding 1ml of L-tyrosine into T2 and T4, reacting the mixture for 10min at 37 ℃, and quickly placing the mixture in ice water for cooling. The absorbances AT1, AT2, AT3 and AT4 were measured AT 475nm with a microplate reader. The tyrosinase inhibiting activity of the samples was calculated according to the following formula:
tyrosinase activity inhibition rate ═ 1- (AT4-AT3)/(AT2-AT1) ] x 100%
Wherein:
AT 1: absorbance at 475nm of the reaction solution without the sample and without the tyrosinase
AT 2: absorbance of the reaction solution without adding sample and tyrosinase at 475nm
AT 3: absorbance at 475nm of the reaction mixture without tyrosinase and with the sample
AT 4: absorbance at 475nm of the reaction mixture containing the sample and the tyrosinase
The experimental results are as follows:
mycelium fermentation liquor detection result
In the fermentation of mycelia, the expression of ganoderma atrum can achieve the best effect, and the culture medium taking malt extract powder as a main raw material has better effect and is generally expressed in the seventh day after fermentation and fungus ball production.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for preparing a black ganoderma mycelium fermentation liquor is characterized by comprising the following steps:
performing slant tube culture on the black ganoderma strain to obtain a mycelium block, inoculating the mycelium block to a plate culture medium for mycelium culture, transferring the mycelium block to a malt culture medium, and performing liquid fermentation culture at 27 ℃ for 4-7 days;
the malt culture medium comprises:
2 w/v% of malt extract powder, 2 w/v% of glucose, 0.1 w/v% of peptone, 0.1 w/v% of yeast extract and water as solvent;
the inoculation amount when the malt culture medium is transferred to a liquid fermentation culture is 1.3-1.7 cm per 100ml of culture medium2The mycelium block of (1).
2. The method for preparing a fermentation broth of mycelia of Ganoderma atrum according to claim 1, wherein the mycelia are inoculated to a plate mediumThe inoculation amount is 0.7-1.3 cm for each plate inoculation2The mycelium block of (1).
3. The method for producing a fermentation broth of mycelia of Ganoderma lucidum according to claim 1 or 2, wherein the mycelia are cultured under the conditions of 28-32 ℃ for 6-8 days.
4. A fermented Ganoderma mycelia solution prepared by the method of any one of claims 1-3.
5. A cosmetic having antioxidant and whitening effects, comprising the mycelia broth of Ganoderma atrum of claim 4 or its extract as an active ingredient.
6. The cosmetic according to claim 5, further comprising at least one of a solvent, an astringent, a stabilizer, a thickener, a conditioner, a humectant, a pH adjuster, a surfactant, an antioxidant, a cooling agent, an emulsifier, a preservative, a colorant, a dispersant, and a fragrance.
7. The cosmetic according to claim 5 or 6, wherein the preparation of the cosmetic comprises a aqua, a serum, a mask, a lotion and a cream.
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| CN106497802B (en) * | 2016-12-20 | 2020-07-31 | 华中农业大学 | Ganoderma lucidum mycelium fermentation process with high intracellular ganoderma lucidum triterpene content |
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