CN114209617B - Ganoderma lucidum extract fermented by yeast and preparation method and application thereof - Google Patents
Ganoderma lucidum extract fermented by yeast and preparation method and application thereof Download PDFInfo
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- CN114209617B CN114209617B CN202111602728.8A CN202111602728A CN114209617B CN 114209617 B CN114209617 B CN 114209617B CN 202111602728 A CN202111602728 A CN 202111602728A CN 114209617 B CN114209617 B CN 114209617B
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- yeast
- ganoderma lucidum
- extract
- fermentation
- powder
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
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- A—HUMAN NECESSITIES
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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Abstract
The invention provides a yeast fermented ganoderma lucidum extract, which comprises a yeast fermented ganoderma lucidum component and a yeast extract component, in particular to a yeast fermented ganoderma lucidum extract or a powdery product obtained by drying the extract, and also provides a preparation method of the yeast fermented ganoderma lucidum extract and application of the yeast fermented ganoderma lucidum extract in preparing cosmetics. The yeast fermented ganoderma lucidum extract is rich in various nutrient substances, has good effects of whitening, removing freckles, preserving moisture and repairing cell damage, has no skin irritation and good safety, and is suitable for being used as a component of cosmetics.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a yeast fermented ganoderma lucidum extract, a preparation method thereof and application of the extract in preparation of cosmetics.
Background
Animal and vegetable oils and fats extracted from animal and vegetable bodies have been described as skin care cosmetics for a long time in history. After the chemical industry gradually developed, the cosmetic industry also entered the chemical age, and gradually evolved from the second generation cosmetics based on oil and water emulsification technology to the third generation cosmetics added with various animal and plant extracts. In recent years, biological fermentation technology begins to show the horn of the head in the cosmetic industry, due to the consideration of health and product safety, the awareness of green products and the threat awareness of harmful chemicals of people are continuously strengthened, the demand of organic green care products is continuously increased, the market of natural organic care products is continuously increased, and products with green natural components become ideal choices of a plurality of consumers. This has also driven natural cosmetics to become an important area of cosmetic development, where biofermentation technology is of increasing importance in cosmetic applications. The cosmetics naturally fermented by organisms have no essence, pigment, preservative, fluorescent agent, chemical oil, animal oil and chemical additive, and particularly, the components released by natural yeast through a long natural fermentation process are adopted, so that the effects are more remarkable, and the cosmetics are safer and more environment-friendly.
Ganoderma (Ganoderma lucidum) is dried fruiting body of Ganoderma lucidum (Ganoderma lucidum) or Ganoderma sinense (Ganoderma sinense) belonging to Polyporaceae. Has effects of invigorating qi, tranquilizing mind, and relieving cough and asthma. It is mainly used for treating unsteadiness of heart-mind, insomnia, palpitation due to fright, cough and asthma with excessive phlegm, consumptive disease, etc. At present, most of lucid ganoderma is developed into health-care food or medicines, the lucid ganoderma is still rarely developed and applied as cosmetics, and related scientific researches are rarely reported. The existing ganoderma lucidum extract is extracted by fermenting ganoderma lucidum process, and the main components are ganoderma lucidum triterpenoids and ganoderma lucidum polysaccharide. The research on triterpenes in ganoderma lucidum has been increasingly focused after the separation of ganoderic acid A and ganoderic acid B in 1982, which was originally isolated from ganoderma lucidum fruiting bodies. 105 triterpenoids were isolated before 1988, and by the 90 s of the 20 th century, researchers continued to isolate new triterpenoids from fruit bodies or spores. Ganoderan is the most extensively studied class of compounds in ganoderma lucidum, except for triterpenes. The research on ganoderan in the 70 s of the 20 th century mainly focuses on the separation, preparation and pharmacological action of components of ganoderan and polysaccharide complex, and in the 80 s and 90 s mainly focuses on the structure and function relationship of ganoderan and polysaccharide complex. At present, more than 200 kinds of polysaccharides are separated from ganoderma lucidum at home and abroad, and most of ganoderma lucidum polysaccharides contain other monosaccharides such as arabinose, xylose, galactose, fucose, mannose and the like besides glucose. .
There have been studies related to the preparation of ganoderma lucidum extracts by fermentation. The influence of nitrogen sources on the liquid submerged fermentation of ganoderma lucidum mycelia to synthesize ganoderma lucidum triterpenes is studied by von jeikey and the like, and the selection of a proper nitrogen source variety and the determination of the optimal dosage and the composition of a fermentation medium can effectively promote the synthesis of the ganoderma lucidum triterpenes (Jun. Xuan & Shen, 2016, vol.35, no. 6). Dianthus haichiensis and the like research the influence of compound organic nitrogen sources on ganoderma triterpene liquid submerged fermentation, wherein the selection of proper yeast powder as a nitrogen source for ganoderma triterpene liquid submerged fermentation is found to be important (Junzhi journal, 2018, vol. 37, 12 th period). Zusayan et al developed a fermentation process of Ganoderma lucidum extract and evaluated the moisturizing function of the fermented product, wherein Kefir was identified as the strain for Ganoderma lucidum fermentation (Master academic thesis, 2016, university of south China agricultural). Chinese patent application CN202110678737.9 discloses a Ganoderma lucidum fermented product, its preparation method and application in cosmetics. However, many of these studies are still under research, and still have problems of single function, insufficient efficacy, and insufficient nutrition, and the art still expects a ganoderma lucidum extract product for cosmetics having complex functions of whitening, moisturizing, repairing damaged skin, etc., and the product has strong efficacy and is rich in beneficial components for nourishing skin.
The yeast extract (yeast extract), also called yeast extract, is a product obtained by using yeast as a main raw material, performing enzymatic hydrolysis and autolysis under the action of enzyme of the yeast or external enzyme, and then performing separation and extraction. The common extraction method of yeast extract comprises degrading the protein of yeast cells into amino acids and polypeptides, degrading nucleic acids into nucleotides by endogenous enzyme autolysis, exogenous enzyme treatment, chemical treatment, etc., and extracting them with other effective components, such as B vitamins, trace elements (such as selenium, zinc, iron, copper, etc.), and glutathione to obtain water-soluble nutrient concentrate. The yeast extract contains abundant active ingredients such as small molecular peptides, free amino acids, vitamins, nucleic acids, nucleotides and the like, wherein the content of the amino acids is more than 30%, the content of the total proteins is more than 50%, and the content of the nucleotides is more than 10%, and the yeast extract is mainly applied to the fields of foods, seasonings, cosmetics and the like. At present, yeast extracts mainly have three application forms, the first is yeast fermentation product filtrate, for example, a liquid product pitera extracted by SK-II company from special yeast of tectorial spore saccharomyces after fermentation to remove yeast cells, and the yeast extract has the functions of moistening and moisturizing; the second is yeast lysate extract, which is obtained by breaking yeast by wall breaking and refining nutrients, such as yeast lysate extract in the palm bottle series of Yashilandai company. The third is nutrient solution existing in yeast filtrate and yeast extract, and the extract product well utilizes nutrient substances contained in the fermentation product filtrate and thalli, can reduce the acquisition cost of the yeast extract and avoid the waste of useful substances, but also has more defects, such as more product impurities, greater irritation, need of complicated blending process, lower whitening and repairing capability and the like.
Disclosure of Invention
In view of the above technical problems, the inventors of the present invention have conducted extensive studies and provided a technical solution of the present invention, specifically, a yeast fermented ganoderma lucidum extract, a preparation method of the extract, and use of the extract in preparation of cosmetics. The invention can solve the problems existing in the current ganoderma lucidum fermentation product and yeast extract and has good technical effect.
In one aspect, the invention relates to a yeast-fermented ganoderma lucidum extract, which comprises a yeast-fermented ganoderma lucidum component and a yeast extract component, and is a yeast-fermented ganoderma lucidum extract or a powdery product obtained by drying the extract.
In another aspect, the present invention relates to a method for preparing a yeast fermented ganoderma lucidum extract, comprising the steps of:
(1) Strain activation: taking a preserved yeast strain from a low-temperature preservation tube, thawing to obtain a bacterial liquid, taking 1-3 drops of the bacterial liquid, dripping the bacterial liquid into the edge of a solid activated culture medium, inoculating the bacterial liquid by a scribing method, culturing the bacterial liquid at 20-30 ℃ for 2-3 days, selecting a ring of the bacterial liquid, inoculating the bacterial liquid into a triangular flask containing a glucose liquid culture medium, and performing constant-temperature shaking culture at 25-28 ℃ for 12-24 hours at the rotating speed of 100-200rpm to obtain a seed culture liquid;
(2) Shake culture: inoculating the seed culture solution obtained in the step (1) into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 3-8 wt%, and culturing at 20-30 ℃ for 1-2 days at the rotation speed of 150-250rpm;
(3) Middle-stage culture and fermentation: transferring the culture solution obtained in the step (2) into a medium-term culture tank filled with a medium-term culture medium according to the volume ratio of 1: 10-50 for culture and fermentation, culturing at 28-32 ℃ for 3-5 days at the rotating speed of 120-150rpm, and adding the ganoderma lucidum crude extract, the oat beta-glucan and the yeast fermentation auxiliary agent into the medium-term culture medium on the 1 st-2 th day from the start of the medium-term culture and fermentation;
(4) Main fermentation: transferring the culture solution obtained in the step (3) into a main fermentation tank filled with a main fermentation culture medium according to the volume ratio of 1: 50-100, fermenting for 7-15 days, controlling the temperature to be 20-25 ℃, and performing ultrasonic-assisted fermentation; starting to add sodium selenite and zinc sulfate solution into the main fermentation tank at 3-7 days, and adding Ganoderma powder (about 100 micrometers), curcumin, cortex Mori powder, fibroin powder, and lysis product of yeast fermentation product of second split at 5-10 days;
(5) Preparing a yeast fermented ganoderma lucidum extract from the culture solution obtained in the step (4).
According to one aspect of the invention, the yeast species in step (1) is selected from one or more of Saccharomyces rouxii (Metschnikowia reukaufii, CCTCC S2014001), saccharomyces paradoxus (CGMCC 2.5691), victoria virginica (Vischnicozyma victoriae, CGMCC 2.5593).
According to one aspect of the present invention, the preservation temperature of the cryopreservation tube used in step (1) is from-80 to-20 ℃.
According to one aspect of the present invention, the solid activation medium in step (1) is prepared by: dissolving yeast extract 10g, peptone 20g, glucose 20g, and agar powder 20g in 1L deionized water, sterilizing with steam at 121 deg.C for 15 min, cooling to 50 deg.C, and adding 200g/L glucose solution 0.1L.
According to one aspect of the invention, the composition of the shake fermentation medium in step (2) is: 7-15g/L of peptone, 18-26g/L of glucose, 13-15g/L of yeast extract, 4-12g/L of monopotassium phosphate, 4-12g/L of magnesium sulfate, 1-2g/L of potassium chloride, 0.2-0.4g/L of sodium phosphate, 0.01-0.25/L of magnesium chloride, 0.05-0.2g/L of calcium chloride, 0.004-0.01g/L of copper sulfate, 0.005-0.008g/L of sodium molybdate, 0.002-0.006g/L of boric acid, 0.004-0.006g/L of zinc sulfate, 0.3-0.5g/L of tween 80 and pH 7.0. Preferably, the composition of the shake fermentation medium in step (2) is: 8g/L of peptone, 22g/L of glucose, 14g/L of yeast extract, 8g/L of monopotassium phosphate, 8g/L of magnesium sulfate, 1.5g/L of potassium chloride, 0.3g/L of sodium phosphate, 0.1/L of magnesium chloride, 0.1g/L of calcium chloride, 0.005g/L of copper sulfate, 0.006g/L of sodium molybdate, 0.004g/L of boric acid, 0.005g/L of zinc sulfate and 0.4g/L of Tween.
According to one aspect of the present invention, the composition of the medium in the middle stage in step (3) is: 62.5-67.5g/L of sucrose, 35-40g/L of yeast extract, 0.3-0.6g/L of biotin, 10-15g/L of ammonium sulfate, 10-15g/L of corn steep liquor dry powder, 17.5-21.3g/L of casein, 3.3-6.2g/L of magnesium sulfate, 4.2-7.8g/L of potassium dihydrogen phosphate, 3.1-5.6g/L of glutathione and 2.1-3.5g/L of soybean lecithin. Preferably, the composition of the medium in step (3) is: 65g/L of sucrose, 37g/L of yeast extract, 0.4g/L of biotin, 12g/L of ammonium sulfate, 13g/L of corn steep liquor dry powder, 20.4g/L of casein, 4.7g/L of magnesium sulfate, 5.1g/L of monopotassium phosphate, 4.3g/L of glutathione and 5.6g/L of soybean lecithin.
According to one aspect of the present invention, the preparation method of the ganoderma lucidum crude extract in step (3) comprises: crushing ganoderma lucidum into ganoderma lucidum powder of about 100 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1 to 20, stirring under the water bath condition of 75 ℃, extracting for 3 hours, filtering and collecting primary extraction filtrate, repeatedly extracting filter residue once according to the same conditions to obtain secondary extraction filtrate, and combining the primary extraction filtrate and the secondary extraction filtrate to obtain ganoderma lucidum crude extract; the addition amount of the Ganoderma lucidum crude extract per liter of culture medium is 0.2L. The same conditions as described herein mean that the filter residue obtained by separating the first extraction filtrate is added into distilled water according to the feed liquid mass ratio of 1.
According to one aspect of the present invention, the yeast fermentation aid in step (3) is
K. Preferably, the yeast fermentation aid in step (3)
The dosage of K is 50g/L.
According to one aspect of the invention, the amount of oat beta-glucan in step (3) is 2-8g/L, preferably 5g/L.
According to one aspect of the present invention, the composition of the main fermentation medium in step (4) is: 4-7g/L of citric acid, 0.02-0.15g/L of ferrous sulfate, 0.03-0.42g/L of manganese sulfate, 0.01-0.25g/L of cobalt dichloride, 0.02-0.35g/L of boric acid, 0.05-0.5g/L of copper sulfate, 3-5g/L of diammonium sulfate, 5-8g/L of magnesium sulfate, 5-7g/L of calcium dichloride, 15-20g/L of rapeseed oil, 15-25g/L of yeast extract and 12-16g/L of glucose. Preferably, the composition of the main fermentation medium in step (4) is: 5g/L of citric acid, 0.1g/L of ferrous sulfate, 0.24g/L of manganese sulfate, 0.09g/L of cobalt dichloride, 0.21g/L of boric acid, 0.15g/L of copper sulfate, 4g/L of diammonium sulfate, 7g/L of magnesium sulfate, 6g/L of calcium dichloride, 17g/L of rapeseed oil, 20g/L of yeast extract and 14g/L of glucose.
According to one aspect of the invention, the sodium selenite and zinc sulfate solution added in step (4) has a sodium selenite concentration of 0.06-0.1g/L and a zinc sulfate solution concentration of 0.03-0.42g/L, and is added in an amount of 50mL per liter of culture solution. Preferably, the concentration of sodium selenite in the sodium selenite and zinc sulfate solution added in the step (4) is 0.08g/L, and the concentration of the zinc sulfate solution is 0.18g/L.
According to one aspect of the invention, the adding amount of the ganoderma lucidum powder in the step (4) is 12-18g/L, preferably 15g/L; the addition amount of curcumin is 0.1-1.0g/L, preferably 0.5g/L; the addition amount of the cortex mori radicis powder is 12-20g/L, preferably 15g/L; the addition amount of the silk protein powder is 20-40g/L, preferably 30g/L; the addition amount of the yeast lysate of the second split yeast fermentation product is 1-2g/L, preferably 1.5g/L.
According to one aspect of the present invention, the Ganoderma lucidum used in the present invention is Ganoderma lucidum (Ganoderma lucidum) or Ganoderma sinense (Ganoderma sinense); preferably, the ganoderma lucidum used in the invention is ganoderma lucidum.
According to one aspect of the present invention, the specific steps of preparing the yeast-fermented ganoderma lucidum extract in the step (5) are:
a) Separating the thalli in the culture solution in the step (5), and respectively collecting filtrate and thalli;
b) Concentrating the filtrate under negative pressure to 1/5-1/3 volume (preferably 1/4 volume) to obtain filtrate concentrate;
c) Preparing thallus into 20-50% (preferably 40%) by mass slurry with purified water, adding 0.1-0.6% (preferably 0.5%) by mass salt, stirring for 3-5 hr (preferably 4 hr) to remove yeast odor, centrifuging with centrifuge, removing supernatant, and collecting thallus;
d) Diluting thallus with purified water to obtain 20-50 wt% (preferably 40 wt%) slurry, loading into autolysis tank, adding 2-5g/L (preferably 3.125 g/L) sodium chloride, stirring, heating to 45-55 deg.C (preferably 50 deg.C), and holding at constant temperature for 1-3 hr (preferably 2 hr) to promote autolysis;
e) Then adjusting the pH value of the slurry to 4.5-5.5 (preferably 5.0) by using phosphoric acid, and adding a compound hydrolase to carry out enzymolysis for 24-72 hours, wherein the compound hydrolase comprises the following components in percentage by weight: papain 0.2-0.5g/L (preferably 0.25 g/L), pancreatin 0.02-0.05g/L (preferably 0.025 g/L), dextranase 0.2-0.5g/L (preferably 0.25 g/L);
f) Inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/10-1/4 volume (preferably 1/5 volume), and mixing with the concentrated filtrate of step b) to obtain Ganoderma extractive solution fermented by yeast;
g) Optionally, spray drying the yeast fermented ganoderma lucidum extract obtained in step f) to prepare a powdery product.
The ganoderma lucidum powder, the mulberry bark powder and the silk protein powder related to the invention can be obtained by the conventional method known in the field. For example, the ganoderma lucidum powder can be prepared by breaking the walls of the dried ganoderma lucidum solid and pulverizing to micron-sized powder, preferably about 100 microns, or can be prepared by the method disclosed in chinese patent application 201911002877.3. Machines for breaking and pulverizing the walls of dried ganoderma lucidum entities are known in the art, for example, the machines disclosed in chinese patent 201821019801.2 or 201721753452.2. The cortex mori radicis can be pulverized by pulverizing cortex mori radicis to obtain submicron cortex mori radicis powder, for example, a dried cortex mori radicis is pulverized into coarse powder with a particle size of 80 to 200 microns, and then pulverized into fine powder with a particle size of 10 to 30 microns, purified water is added to the fine powder, and then further pulverized into submicron cortex mori radicis powder with a particle size of 0.1 to 0.3 microns, and the submicron cortex mori radicis powder is filtered and dried. The silk protein powder can be obtained by methods known in the art, for example, by the method described in chinese patent CN 01126628.7.
The invention also provides a yeast fermentation ganoderma lucidum extract which is the yeast fermentation ganoderma lucidum extract or a dried powder product thereof prepared by the preparation method.
The invention also provides the application of the yeast fermented ganoderma lucidum extract obtained according to the invention, which can be used for preparing cosmetics. Preferably, the cosmetic is a skin care product. Preferably, the cosmetic is any one selected from the group consisting of a skin lotion, a skin softener, a toner, an astringent, a body lotion, an emulsion, a moisturizing body lotion, a nourishing body lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a mask, a soap, a face wash, a skin lotion, a cleansing cream, a body lotion, or a body milk.
Compared with the prior art, the yeast fermentation ganoderma lucidum extract prepared by the invention is rich in various amino acids, polypeptides, ganoderma lucidum polysaccharide and ganoderma lucidum triterpene compounds, has good effects of whitening, removing freckles, moisturizing and repairing cell damage, has no skin irritation and good safety, and is suitable for being used as a component of cosmetics.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.
Example 1
The ganoderma lucidum used in this example is ganoderma lucidum.
Strain activation: taking a preserved yeast strain (Lukovic yeast, metschnikowia reukaufii, CCTCC S2014001, china center for type culture Collection, wuhan City, hubei province, china) from a low-temperature preservation tube (preservation at-80 ℃), thawing to obtain a bacterial liquid, taking 1 drop of the bacterial liquid, dripping the bacterial liquid into the edge of a solid activated culture medium, inoculating by a streaking method, placing the bacterial liquid in a triangular flask containing a glucose liquid culture medium for 3 days at 20 ℃, selecting a ring of the bacterial liquid to inoculate the bacterial liquid in the triangular flask, and performing shake culture at the constant temperature of 25 ℃ for 24 hours at the rotating speed of 200rpm to obtain a seed culture solution. The solid activation medium is prepared by the following method: dissolving yeast extract 10g, peptone 20g, glucose 20g, and agar powder 20g in 1L deionized water, sterilizing with steam at 121 deg.C for 15 min, cooling to 50 deg.C, and adding 200g/L glucose solution 0.1L.
Shake culture: inoculating the seed culture solution obtained in the strain activation step into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 3 wt%, and culturing at 20 ℃ for 2 days at the rotation speed of 250rpm; wherein the shake fermentation culture medium comprises the following components: 15g/L of peptone, 18g/L of glucose, 13g/L of yeast extract, 12g/L of monopotassium phosphate, 12g/L of magnesium sulfate, 2g/L of potassium chloride, 0.2g/L of sodium phosphate, 0.25/L of magnesium chloride, 0.2g/L of calcium chloride, 0.004g/L of copper sulfate, 0.008g/L of sodium molybdate, 0.002g/L of boric acid, 0.004g/L of zinc sulfate, 0.3g/L of Tween 80 and pH 7.0.
Middle-stage culture and fermentation: transferring the culture solution obtained by shaking culture into a medium-term culture tank filled with medium-term culture medium according to the volume ratio of 10rpm, and adding ganoderma lucidum crude extract, oat beta-glucan and yeast fermentation auxiliary agent into the medium-term culture medium on the 2 nd day after the medium-term culture fermentation begins. Wherein, the medium comprises the following components: 62.5g/L of sucrose, 35g/L of yeast extract, 0.6g/L of biotin, 10g/L of ammonium sulfate, 10g/L of corn steep liquor dry powder, 21.3g/L of casein, 6.2g/L of magnesium sulfate, 4.2g/L of monopotassium phosphate, 3.1g/L of glutathione and 3.5g/L of soybean lecithin. The preparation method of the ganoderma lucidum crude extract comprises the steps of crushing ganoderma lucidum into ganoderma lucidum powder with the particle size of about 100 micrometers, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1; the addition amount of the Ganoderma lucidum crude extract per liter of culture medium is 0.2L. The yeast fermentation auxiliary agent is
K (Raman, quebec, canada) in an amount of 50g/L. The dosage of the oat beta-glucan is 8g/L.
Main fermentation: transferring culture solution obtained by middle-stage culture and fermentation into a main fermentation tank filled with main fermentation medium according to the volume ratio of 1:50, fermenting for 7 days, controlling the temperature at 20 ℃, and performing ultrasonic wave assisted fermentation; starting to add sodium selenite and zinc sulfate solution into the main fermentation tank at 3 days, and adding Ganoderma powder (about 100 micrometers), curcumin, cortex Mori powder, silk protein powder and lysis substance of secondary cracking yeast fermentation product into the main fermentation tank at 5 days; wherein the main fermentation medium comprises the following components: 7g/L of citric acid, 0.02g/L of ferrous sulfate, 0.03g/L of manganese sulfate, 0.25g/L of cobalt dichloride, 0.35g/L of boric acid, 0.5g/L of copper sulfate, 3g/L of diammonium sulfate, 5g/L of magnesium sulfate, 7g/L of calcium dichloride, 15g/L of rapeseed oil, 15g/L of yeast extract and 12g/L of glucose. The concentration of sodium selenite in the added sodium selenite and zinc sulfate solution is 0.1g/L, the concentration of the zinc sulfate solution is 0.42g/L, and the adding amount of each liter of culture solution is 50mL. Wherein the addition amount of the ganoderma lucidum powder is 18g/L, the addition amount of the curcumin is 1.0g/L, the addition amount of the mulberry bark powder is 12g/L, the addition amount of the silk protein powder is 20g/L, and the addition amount of the lysis product of the yeast schizosaccharomyces cerevisiae fermentation product is 1g/L.
Extracting and preparing a yeast extract: separating thallus from the culture liquid after main fermentation, and collecting filtrate and thallus separately. Concentrating the filtrate under negative pressure to 1/5 volume to obtain filtrate concentrate A; mixing thallus with purified water to obtain 20% slurry, adding 0.1% sodium chloride, stirring for 3 hr to remove yeast odor, centrifuging with centrifuge at 2000rpm, removing supernatant, and collecting thallus; diluting thallus with purified water to obtain 20 wt% slurry, loading into an autolysis tank, adding 2g/L sodium chloride, heating to 55 deg.C under stirring, and maintaining the constant temperature for 1 hr to promote autolysis of thallus; then, adjusting the pH of the slurry to 4.5 by using phosphoric acid, and adding a composite hydrolase for enzymolysis for 72 hours, wherein the composite hydrolase comprises the following components in percentage by weight: papain 0.2g/L, pancreatin 0.02g/L, dextranase 0.2g/L; inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/10 volume, mixing with the filtrate concentrate A to obtain yeast fermented Ganoderma extract, reserving half volume of the obtained yeast fermented Ganoderma extract, and spray drying the rest half volume of the yeast fermented Ganoderma extract to obtain powder product.
Example 2
The ganoderma lucidum used in this example is ganoderma lucidum.
Strain activation: taking a preserved yeast strain (Saccharomyces paradoxus, CGMCC 2.5691, china general microbiological culture Collection center, beijing, china) from a low-temperature preservation tube (preservation at minus 80 ℃), thawing to obtain a bacterial solution, dripping 2 drops of the bacterial solution into the edge of a solid activation culture medium, inoculating by a streaking method, placing the bacterial solution in a triangular flask containing a glucose liquid culture medium for 3 days at 25 ℃, selecting a ring of the bacterial solution, inoculating the bacterial solution in the triangular flask, and performing shake culture at the constant temperature of 25 ℃ for 20 hours at the rotation speed of 150rpm to obtain a seed culture solution. The solid activation medium is prepared by the following method: dissolving yeast extract 10g, peptone 20g, glucose 20g, and agar powder 20g in 1L deionized water, sterilizing with steam at 121 deg.C for 15 min, cooling to 50 deg.C, and adding 200g/L glucose solution 0.1L.
Shake culture: inoculating the seed culture solution obtained in the strain activation step into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 5 wt%, and culturing at 25 ℃ for 2 days at the rotation speed of 200rpm; wherein the shake fermentation culture medium comprises the following components: 8g/L peptone, 22g/L glucose, 14g/L yeast extract, 8g/L potassium dihydrogen phosphate, 8g/L magnesium sulfate, 1.5g/L potassium chloride, 0.3g/L sodium phosphate, 0.1/L magnesium chloride, 0.1g/L calcium chloride, 0.005g/L copper sulfate, 0.006g/L sodium molybdate, 0.004g/L boric acid, 0.005g/L zinc sulfate, 0.4g/L tween and pH 7.0.
Middle-stage culture fermentation: transferring the culture solution obtained by shaking culture into a metaphase culture tank filled with a metaphase culture medium according to the volume ratio of 1. Wherein, the medium comprises the following components: 65g/L of sucrose, 37g/L of yeast extract, 0.4g/L of biotin, 12g/L of ammonium sulfate, 13g/L of corn steep liquor dry powder, 20.4g/L of casein, 4.7g/L of magnesium sulfate, 5.1g/L of monopotassium phosphate, 4.3g/L of glutathione and 5.6g/L of soybean lecithin. The preparation method of the ganoderma lucidum crude extract comprises the steps of crushing ganoderma lucidum into ganoderma lucidum powder with the particle size of about 100 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1; the addition amount of the Ganoderma lucidum crude extract per liter of culture medium is 0.2L. The yeast fermentation auxiliary agent is
K (Raman, quebec, canada) in an amount of 50g/L. The dosage of the oat beta-glucan is 8g/L.
Main fermentation: transferring the culture solution obtained by the medium-term culture and fermentation into a main fermentation tank filled with a main fermentation medium according to the volume ratio of 1: 75, fermenting for 10 days, controlling the temperature to be 25 ℃, and performing ultrasonic-assisted fermentation; adding sodium selenite and zinc sulfate solution into the main fermentation tank at 5 th day, and adding Ganoderma powder (about 100 μm), curcumin, cortex Mori powder, silk protein powder, and lysate of fermentation product of yeast schizosaccharomyces at 7 th day; wherein the main fermentation medium comprises the following components: 5g/L of citric acid, 0.1g/L of ferrous sulfate, 0.24g/L of manganese sulfate, 0.09g/L of cobalt dichloride, 0.21g/L of boric acid, 0.15g/L of copper sulfate, 4g/L of diammonium sulfate, 7g/L of magnesium sulfate, 6g/L of calcium dichloride, 17g/L of rapeseed oil, 20g/L of yeast extract and 14g/L of glucose. The concentration of sodium selenite in the added sodium selenite and zinc sulfate solution is 0.08g/L, the concentration of the zinc sulfate solution is 0.18g/L, and the addition amount of each liter of culture solution is 50mL. Wherein the addition amount of the ganoderma lucidum powder is 15g/L, the addition amount of the curcumin is 0.5g/L, the addition amount of the mulberry bark powder is 15g/L, the addition amount of the silk protein powder is 30g/L, and the addition amount of the schizosaccharomyces cerevisiae fermentation product lysate is 1.5g/L.
Extracting and preparing a yeast extract: separating thallus from the culture liquid after main fermentation, and collecting filtrate and thallus separately. Concentrating the filtrate under negative pressure to 1/4 volume to obtain filtrate concentrate A; mixing thallus with purified water to obtain 40% slurry, adding 0.5% sodium chloride, stirring for 4 hr to remove yeast odor, centrifuging with centrifuge at 2000rpm, removing supernatant, and collecting thallus; diluting thallus with purified water to 40 wt% slurry, loading into an autolysis tank, adding sodium chloride at a ratio of 3.125g/L, heating to 50 deg.C under stirring, and holding at the constant temperature for 2 hr to promote autolysis of thallus; then, adjusting the pH of the slurry to 5.0 by using phosphoric acid, and adding a composite hydrolase for enzymolysis for 36 hours, wherein the composite hydrolase comprises the following components in percentage by weight: papain 0.25g/L, pancreatin 0.025g/L and dextranase 0.25g/L; inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/5 volume, mixing with the filtrate concentrate A to obtain Ganoderma extractive solution, retaining half of the Ganoderma extractive solution, and spray drying the rest Ganoderma extractive solution to obtain powder product.
Example 3
The ganoderma lucidum used in this example is ganoderma sinensis.
Strain activation: taking a preserved yeast strain (Vicornia victoriae yeast (Visoniacozyma victoriae, CGMCC 2.5593, china general microbiological culture Collection center, beijing, china) from a low-temperature preservation tube (preservation at minus 20 ℃), thawing to obtain a bacterial solution, dripping 3 drops of the bacterial solution into the edge of a solid activated culture medium, inoculating the bacterial solution by a scribing method, placing the bacterial solution in a triangular flask containing a glucose liquid culture medium for 2 days at 30 ℃, selecting a ring of the bacterial solution, inoculating the bacterial solution into the triangular flask, culturing the bacterial solution at constant temperature of 28 ℃ for 12 hours under shaking at the rotation speed of 100rpm to obtain a seed culture solution, and preparing the solid activated culture medium by dissolving 10g of yeast extract, 20g of peptone, 20g of glucose and 20g of agar powder in 1 liter of deionized water, sterilizing the bacterial solution at high temperature of 121 ℃ for 15 minutes, and adding 0.1 liter of 200g/L of glucose solution when the bacterial solution is cooled to 50 ℃.
Shake culture: inoculating the seed culture solution obtained in the strain activation step into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 8 wt%, and culturing at 30 ℃ for 1 day at the rotation speed of 150rpm; wherein the shake fermentation culture medium comprises the following components: peptone 7g/L, glucose 26g/L, yeast extract 15g/L, potassium dihydrogen phosphate 4g/L, magnesium sulfate 4g/L, potassium chloride 1g/L, sodium phosphate 0.4g/L, magnesium chloride 0.01/L, calcium chloride 0.05g/L, copper sulfate 0.01g/L, sodium molybdate 0.005g/L, boric acid 0.006g/L, zinc sulfate 0.006g/L, tween 80.5g/L, and pH 7.0.
Middle-stage culture and fermentation: transferring the culture solution obtained by shaking culture into a medium-term culture tank filled with a medium-term culture medium according to the volume ratio of 1. Wherein, the medium comprises the following components: 67.5g/L of sucrose, 40g/L of yeast extract, 0.3g/L of biotin, 15g/L of ammonium sulfate, 15g/L of corn steep liquor dry powder, 17.5g/L of casein, 3.3g/L of magnesium sulfate, 7.8g/L of monopotassium phosphate, 5.6g/L of glutathione and 2.1g/L of soybean lecithin. The Ganoderma coarse extractive solution is prepared by pulverizing Ganoderma into Ganoderma powder of about 100 μmAdding ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1; the addition amount of the Ganoderma lucidum crude extract per liter of culture medium is 0.2L. The yeast fermentation auxiliary agent is
K (Raman, quebec, canada) in an amount of 50g/L. The dosage of the oat beta-glucan is 5g/L.
Main fermentation: transferring the culture solution obtained by the medium-term culture fermentation into a main fermentation tank filled with a main fermentation medium according to the volume ratio of 1: 100, fermenting for 15 days, controlling the temperature to be 25 ℃, and performing ultrasonic-assisted fermentation; starting to add sodium selenite and zinc sulfate solution into the main fermentation tank at 7 th day, and adding Ganoderma powder (about 100 micrometers), curcumin, cortex Mori powder, silk protein powder and lysis substance of secondary cracking yeast fermentation product into the main fermentation tank at 10 th day; wherein the main fermentation medium comprises the following components: 4g/L of citric acid, 0.15g/L of ferrous sulfate, 0.42g/L of manganese sulfate, 0.01g/L of cobalt dichloride, 0.02g/L of boric acid, 0.05g/L of copper sulfate, 5g/L of diammonium sulfate, 8g/L of magnesium sulfate, 5g/L of calcium dichloride, 20g/L of rapeseed oil, 25g/L of yeast extract and 16g/L of glucose. The concentration of sodium selenite in the added sodium selenite and zinc sulfate solution is 0.06g/L, the concentration of the zinc sulfate solution is 0.03g/L, and the adding amount of each liter of culture solution is 50mL. Wherein the addition amount of the ganoderma lucidum powder is 12g/L, the addition amount of the curcumin is 0.1g/L, the addition amount of the mulberry bark powder is 20g/L, the addition amount of the silk protein powder is 40g/L, and the addition amount of the schizosaccharomyces cerevisiae fermentation product lysate is 2g/L.
Extracting and preparing a yeast extract: the cells in the culture solution after the main fermentation are separated, and the filtrate and the cells are collected respectively. Concentrating the filtrate under negative pressure to 1/3 volume to obtain filtrate concentrate A; mixing thallus with purified water to obtain 50% slurry, adding 0.6% sodium chloride, stirring for 5 hr to remove yeast odor, centrifuging with centrifuge at 2000rpm, removing supernatant, and collecting thallus; diluting thallus with purified water to 50 wt% slurry, loading into an autolysis tank, adding sodium chloride at a ratio of 5g/L, heating to 45 deg.C under stirring, and maintaining the constant temperature for 3 hr to promote autolysis of thallus; then, adjusting the pH of the slurry to 5.5 by using phosphoric acid, and adding compound hydrolase for enzymolysis for 24 hours, wherein the compound hydrolase comprises the following components in percentage by weight: papain 0.5g/L, pancreatin 0.05g/L, dextranase 0.5g/L; inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/4 volume, mixing with the filtrate concentrate A to obtain yeast fermented Ganoderma extract, reserving half volume of the obtained yeast fermented Ganoderma extract, and spray drying the rest half volume of the yeast fermented Ganoderma extract to obtain powder product.
Comparative examples 1 to 3
Comparative example 1 was conducted under the same conditions as in example 1, except that no sodium selenite and zinc sulfate solution were added in the main fermentation stage.
Comparative example 2 the same conditions as in example 2 were followed, and curcumin, mulberry bark powder, silk protein powder were not added only in the main fermentation stage.
Comparative example 3 was carried out under the same conditions as in example 3, with no addition of the secondary fission yeast fermentation product lysate only during the main fermentation stage.
Example 4
The amino acid components, the ganoderma triterpene compounds and the ganoderma polysaccharide components in the yeast-fermented ganoderma lucidum extracts of examples 1 to 3 of the present invention and comparative examples 1 to 3 were measured in accordance with the following methods.
The amino acid component determination method comprises the following steps: measuring 17 free amino acids in Ganoderma extract solution obtained by yeast fermentation by adopting the amino acid determination method in GB/T5009.124-2016, collecting 2.0mL of refrigerated sample, adding 2.0mL of 5-sulfosalicylic acid with mass concentration of 6g/100mL, reacting for 1h, adding 1mL and 1g/100mL of EDTA-2Na solution, mixing thoroughly, and centrifuging at 4800r/min for 10min. 2.0mL of the above mixture was aspirated, 4.0mL of citric acid buffer solution (pH2.2) was added thereto, mixed well, centrifuged, filtered and directly measured on a machine. The external standard method quantifies the amino acid composition in a sample in mg/100 mL. And (3) testing conditions: ion Exchange Column 2622sc.ph, (4.6 mm × 60 mm); the column temperature is 134 ℃; the double-channel ultraviolet detection wavelength is 440nm and 570nm; the sample size was 20. Mu.L for 148min. And (3) testing conditions: ion Exchange Column 2622sc.ph, (4.6 mm × 60 mm); the column temperature is 134 ℃; the double-channel ultraviolet detection wavelength is 440nm and 570nm; the sample size was 20. Mu.L for 148min. The results are shown in Table 1.1.
TABLE 1.1 amino acid content (g/100 g dry matter) of Ganoderma lucidum extract fermented with yeast
As can be seen, the contents of various amino acids in the yeast-fermented Ganoderma extract solutions of examples 1-3 were significantly higher than those of comparative examples 1-3.
The method for determining the Ganoderma triterpene compounds can be referred to the method disclosed by Feng et al (Feng, J., et al., an unstructured kinetic model for the improvement of the microorganisms by the biochemical process G0119 based on nitrile gene source effect, biotechnology and Bioprocess Engineering,19 (4), 727-732, 2014), and specifically comprises the steps of freeze-drying the Ganoderma extract fermented by yeast to obtain powdery solid, taking 1G of the powdery solid, and extracting the powdery solid with 20mL of 50% (v/v) ethanol for 1 week. After centrifugation the supernatant was taken and dried under vacuum at 50 ℃. The residue was suspended in water and then extracted with chloroform. The triterpene in the chloroform extract was further treated with 5% (w/v) NaHCO 3 And (4) extracting. 2mol/L HCl is added to adjust NaHCO 3 After the pH of the phases was below 3.0, naHCO was extracted again with chloroform 3 Triterpene in the phase. After evaporation of the chloroform at 40 ℃, the triterpene was dissolved in absolute ethanol and the level thereof was determined by a spectrophotometer at 245 nm.
The content of the ganoderma polysaccharide is determined by adopting a phenol-sulfuric acid method, a standard curve (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4 and 0.6 mg/mL) is drawn by taking glucose as a standard product, specifically, 1g of ganoderma extract fermented by yeast is freeze-dried to obtain a powdery solid, 1.0mL of 5% phenol solution is added into a test tube, the powdery solid is shaken uniformly, 5.0mL of concentrated sulfuric acid solution is added along the wall of the test tube, the test tube is shaken for 6 minutes, meanwhile, the test tube is opened, the test tube is placed in a boiling water bath for heating for 10 minutes after being stood for 5 minutes, the test tube is taken out and cooled to the room temperature, a 1cm cuvette is used for measuring the absorbance value at the position with the wavelength of 487nm, and the total polysaccharide content is calculated according to the standard curve.
The content of Ganoderma triterpenes and Ganoderma polysaccharides is shown in Table 1.2, wherein the content is the content of corresponding substances in powder solid obtained by freeze drying per gram of Ganoderma extractive solution fermented by yeast.
TABLE 1.2 Ganoderma lucidum triterpene compound and Ganoderma lucidum polysaccharide content (mg/g) in Ganoderma lucidum extractive solution fermented by yeast
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Ganoderma lucidum triterpene compound | 56.64 | 73.75 | 62.9 | 31.27 | 22.76 | 28.62 |
| Ganoderma lucidum polysaccharide | 108.54 | 133.62 | 129 | 24.48 | 35.59 | 30.14 |
It can be seen that the contents of the components of the ganoderma triterpenoids and ganoderma polysaccharides in the ganoderma lucidum extract liquid fermented by yeast of examples 1-3 are significantly higher than those in comparative examples 1-3.
Example 5
Cosmetics containing the yeast fermented ganoderma lucidum extract of examples 1 to 3 and comparative examples 1 to 3 were prepared and tested for safety according to the following method, specifically, the yeast fermented ganoderma lucidum extract was prepared as an emulsion, comprising the steps of:
(1) Preparing materials: 5% of yeast fermented ganoderma lucidum extract, 0.8% of sorbitan sesquioleate, 3% of tween-80, 4.0% of squalane, 5.0% of dioctyl carbonate, 3.0% of cyclopenta dimethyl silicone, 1.0% of plant bionic sebum, 3.5% of caprylic/capric triglyceride, 5.0% of shea butter, 0.5% of panthenol, 0.35% of xanthan gum, 0.7% of hydroxyethyl cellulose, 0.7% of PEG-40 hydrogenated castor oil, 0.1% of vitamin C, 0.5% of vitamin E, 0.05% of EDTA-2Na, 4.0% of 1, 3-propylene glycol, 0.2% of methyl hydroxybenzoate, 0.1% of propyl hydroxybenzoate, 4.0% of glycerol and 0.06% of essence, and the balance of water.
(2) Adding EDTA-2Na, panthenol, glycerol, xanthan gum, hydroxyethyl cellulose (dispersed with 1, 3-propylene glycol) and deionized water into a water phase pot, heating to 80 deg.C, stirring continuously until dissolved, and dispersing uniformly;
(3) Adding methyl hydroxybenzoate, propyl hydroxybenzoate, squalane, dioctyl carbonate, cyclopentadimethylsiloxane, plant bionic sebum, shea butter, olive oil, caprylic/capric triglyceride, sorbitan sesquioleate, and tween 80 into an oil phase pot, heating to 80 deg.C, and stirring thoroughly to dissolve;
(4) Adding the materials in the oil phase pot into the water phase pot, emulsifying and homogenizing through a homogenizer at the speed of 1000-6000 rpm for 10 minutes, cooling to 40 ℃, adding yeast fermentation lysate for concentration and filtration, and fully stirring;
(5) Continuously cooling to 30 deg.C, adding essence, stirring, and cooling to room temperature.
(6) Vacuumizing, discharging and filling to obtain the emulsion.
And (3) safety testing: selecting common volunteers to form a test population, and dividing into 6 groups; the number of the volunteers in each group is 10, wherein 5 women and 5 men are between 20 and 40 years old, and whether adverse reactions occur on trial of the ganoderma lucidum extract cosmetic fermented by the yeast of the invention by test subjects: the test sites were each determined to have an area of 4X 4cm at a distance of 6cm from the base of the palm on the inner side of the left and right arms of the subject. Before each group of subjects is respectively coated with the emulsion prepared by the method, the experimental part is cleaned, dried and coated on the inner sides of the left arm and the right arm. The medicine is applied once every morning and evening, and the test is continuously carried out for 1 month. The results of the experiment are shown in table 2.
TABLE 2 safety test results of yeast fermented Ganoderma lucidum extracts of examples 1 to 3 and comparative examples 1 to 3
| Ganoderma extract prepared by yeast fermentation | Edema (edema) | Erythema | Peeling off | Intensity of stimulus |
| Example 1 | 0 | 0 | 0 | Has no irritation |
| Example 2 | 0 | 0 | 0 | Has no irritation |
| Example 3 | 0 | 0 | 0 | Has no irritation |
| Comparative example 1 | 0 | 1 | 1 | Slight irritation |
| Comparative example 2 | 1 | 2 | 0 | Slight irritation |
| Comparative example 3 | 2 | 0 | 1 | Slight irritation |
The results in table 2 show that the ganoderma lucidum extract liquid products fermented by the yeast of the embodiments 1 to 3 of the invention have good safety, and particularly, the addition of the ganoderma lucidum powder, the curcumin, the mulberry bark powder and the silk protein powder eliminates the irritation of the products of the invention.
Example 6
The emulsion prepared in example 5 was used to test the whitening and spot-removing effects of the yeast fermented ganoderma lucidum extract of the present invention.
And (3) testing the whitening and freckle removing effects: 180 female volunteers with color spots on the faces are randomly selected to form a test population, the ages of the test population are 20-40 years old, the body is healthy and has no skin disease and allergic history, and the test population is non-sensitive skin, can use cosmetics according to the standard and completes evaluation work according to requirements. 180 subjects are divided into 6 groups randomly, in the experiment, the subjects in each group apply the corresponding emulsion prepared in the example to the face once in the morning and evening, 3mL of the whole face is uniformly applied every time, the face is coated with uniform sun protection after the face is coated in the daytime for 28 days continuously, and other skin care products except for the test emulsion and the sun protection cream are not used for all the subjects.
Facial skin stains were measured with a Visia7 skin tester before the start of the experiment and 28 days later on the same sites. Considering that the change of the left view and the right view of the main view will be slightly different, the average difference percentage is taken for representation, and the final difference percentage of each group is also taken as the average representation. The results of the experiment are shown in table 3.
TABLE 3 test results of whitening and freckle removing efficacies of yeast fermented ganoderma lucidum extracts of examples 1 to 3 and comparative examples 1 to 3
The results show that the yeast fermentation lysate concentrated filtrate emulsions prepared in examples 1-3 have better whitening and freckle removing effects compared with comparative examples 1-3.
Example 7
The yeast-fermented ganoderma lucidum extracts of examples 1 to 3 and comparative examples 1 to 3 were prepared into masks, respectively, and tested for their moisturizing effects.
The preparation method of the facial mask comprises the following steps: according to the mass parts, 0.05 part of xanthan gum, 0.15 part of carbomer, 3 parts of glycerol, 0.1 part of allantoin, 0.05 part of disodium EDTA, 0.12 part of arginine, 2 parts of butanediol, 0.5 part of p-hydroxyacetophenone, 0.5 part of 1, 2-hexanediol, 0.02 part of carboxymethyl chitosan (obtained from MHA Micro), 0.5 part of yeast fermented ganoderma lucidum extract, 0.1 part of bioglycan-1 (obtained from mibelle AG), 0.001 part of sodium hyaluronate (obtained from Ciconfetta) and 0.2 part of Aralia elata kernel oil are taken and dissolved by adding 0.03 part of PEG-40 hydrogenated castor oil, PPG-26-butanol polyether-26, 0.001 part of papaya essence and 0.001 part of red rose essence which are mixed in advance, and the mixture is uniformly stirred.
And (3) testing the moisturizing effect: selecting 60 volunteers with age of 18-35 years and skin color L value close to each other, dividing the volunteers into 6 groups, measuring initial skin hydration degree of each group of volunteers (averaging), preparing facial masks by using the yeast fermented ganoderma lucidum extract in the examples 1-3 and the comparative examples 1-3 respectively, lasting for 4 weeks once a day, testing the skin hydration degree of each group of volunteers in the 2 nd week and the 4 th week (averaging), and evaluating the moisturizing effect of the plant source moisturizing facial mask, wherein the test results are shown in Table 4.
TABLE 4 moisturizing efficacy test results of yeast-fermented Ganoderma lucidum extracts of examples 1-3 and comparative examples 1-3
| Ganoderma extract prepared by yeast fermentation | Degree of skin hydration of 0 week | Degree of hydration of 2 weeks skin | Degree of hydration of 4 weeks skin |
| Example 1 | 46.2 | 63.7 | 77.1 |
| Example 2 | 47.8 | 61.2 | 77.3 |
| Example 3 | 45.6 | 63.5 | 78.2 |
| Comparative example 1 | 45.2 | 58.5 | 63.8 |
| Comparative example 2 | 48.5 | 58.1 | 64.1 |
| Comparative example 3 | 47.8 | 58.5 | 63.7 |
From the results, it was found that the skin hydration degrees of the users who used the masks containing the yeast-fermented ganoderma lucidum extract solutions of examples 1 to 3 were significantly higher than those of comparative examples 1 to 3 through 4-week tests in the case where the initial skin hydration degrees were close, and there was a significant difference therebetween, reflecting that the ganoderma lucidum extract solution fermented with yeast according to the present invention had a good moisturizing effect.
Example 8
The yeast fermented ganoderma lucidum extracts of examples 1-3 and comparative examples 1-3 were tested in vitro for their cell damage repairing effects.
10 μ L of the mixture was mixed at a density of 5X 10 5 Fibroblast cells per mL were seeded in wells of a 96-well plate, 5% CO at 37% 2 The cells were cultured in a constant temperature incubator in IMDM supplemented with 20mM glutamine and 10% fetal calf serum for 24 hours. Adding 1.6mM hydrogen peroxide and 2% of ganoderma lucidum extract obtained by yeast fermentation in examples 1-3 and comparative examples 1-3 into a fresh culture medium respectively by 10 mu L to serve as an experimental group, independently adding 1.6mM hydrogen peroxide to serve as a positive control, taking untreated cells to serve as a negative control, adding 10 mu L of sample liquid and positive and negative control liquid into culture liquid of a corresponding sample group and control group pore plate respectively, continuing to culture for 24h, adding 20 mu L MTT into each pore, removing the culture medium after culturing for 4h, adding 150 mu L DMSO, and detecting the absorbance value under the 490nm wavelength of a microplate reader. Each group was tested 3 times and averaged. Cell viability is expressed as a percentage.
TABLE 5 cell repair results of the yeast fermented Ganoderma lucidum extracts of examples 1 to 3 and comparative examples 1 to 3
From the above results, it can be seen that the fermentation lysate concentrated filtrates of examples 1 to 3 have a stronger effect of repairing damaged cells than those of comparative examples 1 to 3.
It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (5)
1. A yeast fermented Ganoderma extract comprises yeast fermented Ganoderma component and yeast extract component, wherein the yeast fermented Ganoderma extract is yeast fermented Ganoderma extractive solution or powder product of the extractive solution after drying;
the preparation method of the yeast fermented ganoderma lucidum extract comprises the following steps:
(1) Strain activation: taking a preserved yeast strain from a low-temperature preservation tube, thawing to obtain a bacterial liquid, taking 1-3 drops of the bacterial liquid, dripping the bacterial liquid into the edge of a solid activated culture medium, inoculating the bacterial liquid by a scribing method, culturing the bacterial liquid at 20-30 ℃ for 2-3 days, selecting a ring of the bacterial liquid, inoculating the bacterial liquid into a triangular flask containing a glucose liquid culture medium, and performing constant-temperature shaking culture at 25-28 ℃ for 12-24 hours at the rotating speed of 100-200rpm to obtain a seed culture liquid;
(2) Shake culture: inoculating the seed culture solution obtained in the step (1) into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 3-8 wt%, and culturing at 20-30 ℃ for 1-2 days at the rotation speed of 150-250rpm;
(3) Middle-stage culture fermentation: transferring the culture solution obtained in the step (2) into a metaphase culture tank filled with a metaphase culture medium according to the volume ratio of 1: 10-50 for culture fermentation, culturing at 28-32 ℃ for 3-5 days at the rotating speed of 120-150rpm, and adding the ganoderma lucidum crude extract, the oat beta-glucan and the yeast fermentation auxiliary agent into the metaphase culture medium on the 1-2 days from the beginning of the metaphase culture fermentation;
(4) Main fermentation: transferring the culture solution obtained in the step (3) into a main fermentation tank filled with a main fermentation medium according to the volume ratio of 1: 50-100, fermenting for 7-15 days, controlling the temperature to be 20-25 ℃, and performing ultrasonic auxiliary fermentation; starting to add sodium selenite and zinc sulfate solution into the main fermentation tank at 3-7 days, and adding Ganoderma powder, curcumin, cortex Mori powder, fibroin powder and lysate of yeast fermentation product of secondary fission at 5-10 days;
(5) Preparing the yeast fermented ganoderma lucidum extract from the culture solution obtained in the step (4);
wherein, the yeast strain in the step (1) is selected from one or more of Rogomyces rouxii, saccharomyces paradoxus and Victoria, and the preparation method of the solid activated culture medium in the step (1) comprises the following steps: dissolving 10g of yeast extract, 20g of peptone, 20g of glucose and 20g of agar powder in 1 liter of deionized water, carrying out high-temperature steam sterilization at 121 ℃ for 15 minutes, cooling to 50 ℃, and adding 0.1 liter of 200g/L glucose solution;
wherein the shake fermentation culture medium in the step (2) comprises the following components: 7-15g/L peptone, 18-26g/L glucose, 13-15g/L yeast extract, 4-12g/L monopotassium phosphate, 4-12g/L magnesium sulfate, 1-2g/L potassium chloride, 0.2-0.4g/L sodium phosphate, 0.01-0.25/L magnesium chloride, 0.05-0.2g/L calcium chloride, 0.004-0.01g/L copper sulfate, 0.005-0.008g/L sodium molybdate, 0.002-0.006g/L boric acid, 0.004-0.006g/L zinc sulfate, 0.3-0.5g/L tween 80, pH 7.0
Wherein the medium in the step (3) comprises the following components: 62.5-67.5g/L of sucrose, 35-40g/L of yeast extract, 0.3-0.6g/L of biotin, 10-15g/L of ammonium sulfate, 10-15g/L of corn steep liquor dry powder, 17.5-21.3g/L of casein, 3.3-6.2g/L of magnesium sulfate, 4.2-7.8g/L of potassium dihydrogen phosphate, 3.1-5.6g/L of glutathione and 2.1-3.5g/L of soybean lecithin; and the yeast fermentation auxiliary agent is Fermaid K;
the preparation method of the ganoderma lucidum crude extract in the step (3) comprises the steps of crushing ganoderma lucidum into ganoderma lucidum powder of about 100 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1; the adding amount of the ganoderma lucidum crude extract in each liter of culture medium is 0.2L;
the dosage of the oat beta-glucan in the step (3) is 5g/L;
the using amount of the yeast fermentation aid Fermaid K in the step (3) is 50 g/L;
wherein the main fermentation medium in the step (4) comprises the following components: 4-7g/L of citric acid, 0.02-0.15g/L of ferrous sulfate, 0.03-0.42g/L of manganese sulfate, 0.01-0.25g/L of cobalt dichloride, 0.02-0.35g/L of boric acid, 0.05-0.5g/L of copper sulfate, 3-5g/L of diammonium sulfate, 5-8g/L of magnesium sulfate, 5-7g/L of calcium dichloride, 15-20g/L of rapeseed oil, 15-25g/L of yeast extract and 12-16g/L of glucose;
the concentration of sodium selenite in the sodium selenite and zinc sulfate solution added in the step (4) is 0.06-0.1g/L, the concentration of zinc sulfate solution is 0.03-0.42g/L, and the addition amount of each liter of culture solution is 50 mL;
the ganoderma lucidum powder in the step (4) is ganoderma lucidum powder, wherein the ganoderma lucidum powder is about 100 microns, the adding amount of the ganoderma lucidum powder is 12-18g/L, the adding amount of curcumin is 0.1-1.0g/L, the adding amount of mulberry bark powder is 12-20g/L, the adding amount of silk protein powder is 20-40g/L, and the adding amount of the schizosaccharomyces cerevisiae fermentation product lysate is 1-2 g/L;
wherein the specific steps for preparing the yeast fermented ganoderma lucidum extract in the step (5) are as follows:
a) Separating the thalli in the culture solution in the step (5), and respectively collecting filtrate and thalli;
b) Concentrating the filtrate under negative pressure to 1/5-1/3 of the volume to obtain filtrate concentrate;
c) Concocting thallus with purified water to obtain 20-50% slurry, adding 0.1-0.6% salt, stirring for 3-5 hr to remove yeast odor, centrifuging with centrifuge, removing supernatant, and collecting thallus;
d) Diluting thallus with purified water to obtain 20-50 wt% slurry, loading into autolysis tank, adding 2-5g/L sodium chloride, heating to 45-55 deg.C under stirring, and holding at constant temperature for 1-3 hr to promote autolysis;
e) Then, adjusting the pH value of the slurry to 4.5-5.5 by using phosphoric acid, and adding compound hydrolase for enzymolysis for 24-72 hours, wherein the compound hydrolase comprises the following components in percentage by weight: papain 0.2-0.5g/L, pancreatin 0.02-0.05g/L, dextranase 0.2-0.5 g/L;
f) Inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/10-1/4 volume, and mixing with the concentrated filtrate in step b) to obtain Ganoderma extract fermented by yeast.
2. A method of preparing a yeast fermented ganoderma lucidum extract according to claim 1, comprising the steps of:
(1) Strain activation: taking a preserved yeast strain from a low-temperature preservation tube, thawing to obtain a bacterial liquid, taking 1-3 drops of the bacterial liquid, dripping the bacterial liquid into the edge of a solid activated culture medium, inoculating the bacterial liquid by a scribing method, culturing the bacterial liquid at 20-30 ℃ for 2-3 days, selecting a ring of the bacterial liquid, inoculating the bacterial liquid into a triangular flask containing a glucose liquid culture medium, and performing constant-temperature shaking culture at 25-28 ℃ for 12-24 hours at the rotating speed of 100-200rpm to obtain a seed culture liquid;
(2) Shake culture: inoculating the seed culture solution obtained in the step (1) into a triangular flask filled with a shake fermentation culture medium according to the inoculation amount of 3-8 wt%, and culturing at 20-30 ℃ for 1-2 days at the rotation speed of 150-250rpm;
(3) Middle-stage culture and fermentation: transferring the culture solution obtained in the step (2) into a metaphase culture tank filled with a metaphase culture medium according to the volume ratio of 1: 10-50 for culture fermentation, culturing at 28-32 ℃ for 3-5 days at the rotating speed of 120-150rpm, and adding the ganoderma lucidum crude extract, the oat beta-glucan and the yeast fermentation auxiliary agent into the metaphase culture medium on the 1-2 days from the beginning of the metaphase culture fermentation;
(4) Main fermentation: transferring the culture solution obtained in the step (3) into a main fermentation tank filled with a main fermentation medium according to the volume ratio of 1: 50-100, fermenting for 7-15 days, controlling the temperature to be 20-25 ℃, and performing ultrasonic auxiliary fermentation; starting to add sodium selenite and zinc sulfate solution into the main fermentation tank at 3-7 days, and adding Ganoderma powder, curcumin, cortex Mori powder, silk protein powder and lysate of secondary cracking yeast fermentation product into the main fermentation tank at 5-10 days;
(5) Preparing the yeast fermented ganoderma lucidum extract from the culture solution obtained in the step (4);
wherein, the yeast strain in the step (1) is selected from one or more of Rogomyces rouxii, saccharomyces paradoxus and Victoria, and the preparation method of the solid activated culture medium in the step (1) comprises the following steps: dissolving 10g of yeast extract, 20g of peptone, 20g of glucose and 20g of agar powder in 1 liter of deionized water, carrying out high-temperature steam sterilization at 121 ℃ for 15 minutes, cooling to 50 ℃, and adding 0.1 liter of 200g/L glucose solution;
wherein the shake fermentation culture medium in the step (2) comprises the following components: 7-15g/L peptone, 18-26g/L glucose, 13-15g/L yeast extract, 4-12g/L potassium dihydrogen phosphate, 4-12g/L magnesium sulfate, 1-2g/L potassium chloride, 0.2-0.4g/L sodium phosphate, 0.01-0.25/L magnesium chloride, 0.05-0.2g/L calcium chloride, 0.004-0.01g/L copper sulfate, 0.005-0.008g/L sodium molybdate, 0.002-0.006g/L boric acid, 0.004-0.006g/L zinc sulfate, 0.3-0.5g/L tween 80, pH 7.0
Wherein the medium in the step (3) comprises the following components: 62.5-67.5g/L of sucrose, 35-40g/L of yeast extract, 0.3-0.6g/L of biotin, 10-15g/L of ammonium sulfate, 10-15g/L of corn steep liquor dry powder, 17.5-21.3g/L of casein, 3.3-6.2g/L of magnesium sulfate, 4.2-7.8g/L of potassium dihydrogen phosphate, 3.1-5.6g/L of glutathione and 2.1-3.5g/L of soybean lecithin; and the yeast fermentation auxiliary agent is Fermaid K;
the preparation method of the ganoderma lucidum crude extract in the step (3) comprises the steps of crushing ganoderma lucidum into ganoderma lucidum powder of about 100 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1; the adding amount of the ganoderma lucidum crude extract in each liter of culture medium is 0.2L;
the dosage of the oat beta-glucan in the step (3) is 5g/L;
the using amount of the yeast fermentation aid Fermaid K in the step (3) is 50 g/L;
wherein the main fermentation medium in step (4) consists of: 4-7g/L of citric acid, 0.02-0.15g/L of ferrous sulfate, 0.03-0.42g/L of manganese sulfate, 0.01-0.25g/L of cobalt dichloride, 0.02-0.35g/L of boric acid, 0.05-0.5g/L of copper sulfate, 3-5g/L of diammonium sulfate, 5-8g/L of magnesium sulfate, 5-7g/L of calcium dichloride, 15-20g/L of rapeseed oil, 15-25g/L of yeast extract and 12-16g/L of glucose;
the concentration of sodium selenite in the sodium selenite and zinc sulfate solution added in the step (4) is 0.06-0.1g/L, the concentration of zinc sulfate solution is 0.03-0.42g/L, and the addition amount of each liter of culture solution is 50 mL;
the ganoderma lucidum powder in the step (4) is ganoderma lucidum powder, wherein the ganoderma lucidum powder is about 100 microns, the addition amount of the ganoderma lucidum powder is 12-18g/L, the addition amount of curcumin is 0.1-1.0g/L, the addition amount of mulberry bark powder is 12-20g/L, the addition amount of silk protein powder is 20-40g/L, and the addition amount of the schizosaccharomyces cerevisiae fermentation product lysate is 1-2 g/L;
wherein the specific steps for preparing the yeast fermented ganoderma lucidum extract in the step (5) are as follows:
a) Separating the thalli in the culture solution in the step (5), and respectively collecting filtrate and thalli;
b) Concentrating the filtrate under negative pressure to 1/5-1/3 of the volume to obtain filtrate concentrate;
c) Mixing thallus with purified water to obtain 20-50 wt% slurry, adding 0.1-0.6 wt% salt, stirring for 3-5 hr to remove yeast odor, centrifuging with centrifuge, removing supernatant, and collecting thallus;
d) Diluting thallus with purified water to obtain 20-50 wt% slurry, loading into autolysis tank, adding 2-5g/L sodium chloride, heating to 45-55 deg.C under stirring, and holding at constant temperature for 1-3 hr to promote autolysis;
e) Then regulating the pH of the slurry to 4.5-5.5 by using phosphoric acid, and adding a composite hydrolase for enzymolysis for 24-72 hours, wherein the composite hydrolase comprises the following components in percentage by weight: papain 0.2-0.5g/L, pancreatin 0.02-0.05g/L, dextranase 0.2-0.5 g/L;
f) Inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/10-1/4 volume, and mixing with the concentrated filtrate in step b) to obtain Ganoderma extract fermented by yeast.
3. The method according to claim 2, wherein the yeast-fermented ganoderma lucidum extract obtained in f) is spray-dried to prepare a powdered product.
4. Use of a yeast extract for the preparation of a cosmetic, wherein the yeast extract is the yeast-fermented ganoderma lucidum extract according to claim 1 or the yeast-fermented ganoderma lucidum extract obtained by the preparation method according to any one of claims 2 to 3, and the cosmetic is any one selected from a skin lotion, a skin softener, a skin toner, an astringent, a skin lotion, an emulsion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nutrient essence, a mask, a soap, a facial cleanser, a skin lotion, a cleansing cream, a skin lotion, or a body milk.
5. A cosmetic selected from any one of a skin lotion, a skin softener, a skin toner, an astringent, a body lotion, an emulsion, a moisturizing body lotion, a nourishing body lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a mask, a soap, a facial cleanser, a skin cleansing lotion, a cleansing cream, a body lotion or a body milk, characterized by comprising the yeast-fermented Ganoderma lucidum extract according to claim 1 or the yeast-fermented Ganoderma lucidum extract obtained by the production method according to any one of claims 2 to 3.
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Inventor after: Wu Yaqin Inventor after: Shan Yufei Inventor after: Cao Zhengyu Inventor after: Xu Ding Inventor before: Wu Yaqin Inventor before: Shan Yufei Inventor before: Cao Zhengyu |