CN114209617A - Yeast fermented ganoderma lucidum extract and preparation method and application thereof - Google Patents
Yeast fermented ganoderma lucidum extract and preparation method and application thereof Download PDFInfo
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- CN114209617A CN114209617A CN202111602728.8A CN202111602728A CN114209617A CN 114209617 A CN114209617 A CN 114209617A CN 202111602728 A CN202111602728 A CN 202111602728A CN 114209617 A CN114209617 A CN 114209617A
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- Prior art keywords
- yeast
- ganoderma lucidum
- extract
- fermentation
- culture
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- 235000001637 Ganoderma lucidum Nutrition 0.000 title claims abstract description 131
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000000605 extraction Methods 0.000 title claims description 26
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- 235000015097 nutrients Nutrition 0.000 claims abstract description 7
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- 238000000855 fermentation Methods 0.000 claims description 111
- 230000004151 fermentation Effects 0.000 claims description 111
- 239000000843 powder Substances 0.000 claims description 78
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- 241000222336 Ganoderma Species 0.000 claims description 43
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K8/9728—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
- A61K8/022—Powders; Compacted Powders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
- A61Q1/02—Preparations containing skin colorants, e.g. pigments
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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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
- A61Q19/10—Washing or bathing preparations
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- A—HUMAN NECESSITIES
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
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 for long time as skin care cosmetics. After the chemical industry has gradually developed, the cosmetic industry has also entered the chemical era and has evolved from the second generation of cosmetics based on oil and water emulsification techniques to the third generation of cosmetics containing 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) of Polyporaceae. Has effects of invigorating qi, tranquilizing mind, relieving cough and asthma. It can be used for treating restlessness of heart-mind, insomnia, palpitation due to fright, cough, asthma, excessive phlegm, and asthenia. 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 fermentation product, a preparation method thereof and application thereof in cosmetics. However, many of these studies are still in the research stage, and still have the problems of single function, insufficient efficacy and insufficient nutrient substances, and the field still expects the ganoderma lucidum extract product for cosmetics with complex functions of whitening, moisturizing, repairing damaged skin and the like, and the product has strong efficacy and is rich in beneficial components for nourishing the skin.
The yeast extract (yeast extract), also called yeast extract, is a product obtained by taking yeast as a main raw material, performing enzymolysis autolysis under the action of enzyme of the yeast or external enzyme, and then performing separation and extraction. The extraction method of yeast extract comprises degrading the protein of yeast cell into amino acids and polypeptides, degrading nucleic acid into nucleotide, 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 protein 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 food, 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 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, higher irritation, need of complicated preparation 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 rotation speed of 100 plus 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-;
(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 and fermentation, culturing for 3-5 days at 28-32 ℃, rotating at the 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 st-2 th day from the beginning of metaphase 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 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; adding sodium selenite and zinc sulfate solution into the main fermentation tank at 3-7 days, and adding Ganoderma powder (about 100 μm), curcumin, cortex Mori powder, fibroin powder, and yeast lysate of secondary fission 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 (Vischniacozyma 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, 800.3-0.5 g/L of tween and pH 7.0. Preferably, the composition of the shake fermentation medium in the step (2) is as follows: 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 800.4 g/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 the middle stage 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 lucid ganoderma into lucid ganoderma powder of about 100 microns, adding the lucid ganoderma powder into distilled water according to the material liquid mass ratio of 1:20, 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 condition to obtain secondary extraction filtrate, and combining the primary extraction filtrate and the secondary extraction filtrate to obtain lucid ganoderma 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 refer to that the filter residue obtained by separating the first extraction filtrate is added into distilled water according to the feed liquid mass ratio of 1:20, stirred under the condition of water bath at 75 ℃, extracted for 3 hours, filtered and collected (i.e. the second extraction filtrate).
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 50 g/L.
According to one aspect of the invention, the amount of oat beta-glucan in step (3) is 2-8g/L, preferably 5 g/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 0.18 g/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 15 g/L; the addition amount of curcumin is 0.1-1.0g/L, preferably 0.5 g/L; the addition amount of the cortex mori radicis powder is 12-20g/L, preferably 15 g/L; the addition amount of the silk protein powder is 20-40g/L, preferably 30 g/L; the addition amount of the yeast lysate of the second split yeast fermentation product is 1-2g/L, preferably 1.5 g/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 present 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% (preferably 40%) slurry, loading into autolysis tank, adding 2-5g/L (preferably 3.125g/L) sodium chloride, stirring while heating to 45-55 deg.C (preferably 50 deg.C) 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 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 0.25g/L), pancreatin 0.02-0.05g/L (preferably 0.025g/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 in 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 wall of the dried Ganoderma lucidum solid and pulverizing to micron-sized powder, preferably about 100 microns Ganoderma lucidum powder, or can be prepared by the method disclosed in Chinese patent application 201911002877.3. Machines for breaking and pulverizing the wall of dried ganoderma lucidum entity are known in the art, such as 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 (Lukov Meiqi yeast, Metschnikowia reukaufii, CCTCC S2014001, China center for type culture Collection, Wuhan City, Hubei province, China) from a low-temperature preservation pipe (preservation at the temperature of minus 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 the bacterial liquid by a scribing method, placing the bacterial liquid in a triangular flask containing a glucose liquid culture medium for 3 days after culturing at the temperature of 20 ℃, selecting a ring of the bacterial liquid culture medium, and performing constant-temperature shaking culture at the 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 250 rpm; 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, 800.3 g/L of tween and pH of 7.0.
Middle-stage culture and fermentation: transferring the culture solution obtained by shake culture into a metaphase culture tank filled with a metaphase culture medium according to the volume ratio of 1:10 for culture and fermentation, culturing at 28 ℃ for 5 days at the rotating speed of 150rpm, and adding the ganoderma lucidum crude extract, the oat beta-glucan and the yeast fermentation auxiliary agent into the metaphase culture medium at the 2 nd day from the beginning of the metaphase culture and fermentation. 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 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1:20, stirring under the condition of water bath at 75 ℃, extracting for 3 hours, filtering and collecting primary extraction filtrate, repeatedly extracting filter residue once according to the same condition to obtain secondary extraction filtrate, and combining the primary extraction filtrate and the secondary extraction filtrate to obtain the ganoderma lucidum crude extract; 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 50 g/L. The dosage of the oat beta-glucan is 8 g/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 1:50, fermenting for 7 days, controlling the temperature to be 20 ℃, and performing ultrasonic-assisted fermentation; adding sodium selenite and zinc sulfate solution into the main fermentation tank at 3 days, and adding Ganoderma powder (about 100 μm), curcumin, cortex Mori powder, silk protein powder, and lysate of fermentation product of yeast schizosaccharomyces 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 50 mL. 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 schizosaccharomyces cerevisiae fermentation product lysate is 1 g/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 1/5 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 compound hydrolase for enzymolysis for 72 hours, wherein the compound hydrolase comprises the following components in percentage by weight: papain 0.2g/L, pancreatin 0.02g/L, dextranase 0.2 g/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 extractive solution, retaining half volume of the obtained yeast fermented Ganoderma extractive solution, and spray drying the rest half volume of the yeast fermented Ganoderma extractive solution 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 the temperature of minus 80 ℃), thawing to obtain a bacterial solution, taking 2 drops of the bacterial solution to drip into the edge of a solid activated culture medium, inoculating by a scribing method, placing the bacterial solution in a triangular flask containing a glucose liquid culture medium for 3 days of culture at the temperature of 25 ℃, picking a ring of the bacterial solution to inoculate in the triangular flask containing the glucose liquid culture medium, and performing constant-temperature shaking culture at the temperature of 25 ℃ for 20 hours at the rotating 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 200 rpm; wherein the shake fermentation culture medium comprises the following components: peptone 8g/L, glucose 22g/L, yeast extract 14g/L, potassium dihydrogen phosphate 8g/L, magnesium sulfate 8g/L, potassium chloride 1.5g/L, sodium phosphate 0.3g/L, magnesium chloride 0.1/L, calcium chloride 0.1g/L, copper sulfate 0.005g/L, sodium molybdate 0.006g/L, boric acid 0.004g/L, zinc sulfate 0.005g/L, Tween 800.4 g/L, and pH 7.0.
Middle-stage culture and fermentation: transferring the culture solution obtained by the shake culture into a medium-term culture tank filled with a medium-term culture medium according to the volume ratio of 1:25 for culture and fermentation, culturing at 30 ℃ for 4 days at the rotating speed of 130rpm, and adding the ganoderma lucidum crude extract, the oat beta-glucan and the yeast fermentation auxiliary agent into the medium-term culture medium at the 2 nd day from the start of the medium-term culture and fermentation. 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 Ganoderma coarse extractive solution is prepared by pulverizing Ganoderma to about 100 μmAdding ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1:20, stirring under the condition of water bath at 75 ℃, extracting for 3 hours, filtering and collecting primary extraction filtrate, repeatedly extracting filter residue once according to the same condition 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 yeast fermentation auxiliary agent is
K (Raman, Quebec, Canada) in an amount of 50 g/L. The dosage of the oat beta-glucan is 8 g/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 adding amount of each liter of culture solution is 50 mL. 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 1.5 g/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 1/4 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 compound hydrolase for enzymolysis for 36 hours, wherein the compound hydrolase comprises the following components in percentage by weight: papain 0.25g/L, pancreatin 0.025g/L, dextranase 0.25 g/L; inactivating enzyme, centrifuging at 4000rpm, collecting supernatant, concentrating under negative pressure to 1/5 volume, mixing with the filtrate concentrate A to obtain yeast fermented Ganoderma extractive solution, retaining half volume of the obtained yeast fermented Ganoderma extractive solution, and spray drying the rest half volume of the yeast fermented Ganoderma extractive solution to obtain powder product.
Example 3
The ganoderma lucidum used in the embodiment is ganoderma sinensis.
Strain activation: taking a preserved yeast strain (Vishnikovian victoria yeast (Visheniazyma victoriae, CGMCC 2.5593, China general microbiological culture Collection center, Beijing, China) from a low-temperature preservation tube (preservation at the temperature of 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, culturing the bacterial solution at the temperature of 30 ℃ for 2 days, selecting a ring of the bacterial solution to be inoculated into a triangular flask containing a glucose liquid culture medium, performing shake culture at the constant temperature of 28 ℃ for 12 hours at the rotating 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, performing high-temperature steam sterilization at the 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 150 rpm; wherein the shake fermentation culture medium comprises the following components: 7g/L of peptone, 26g/L of glucose, 15g/L of yeast extract, 4g/L of monopotassium phosphate, 4g/L of magnesium sulfate, 1g/L of potassium chloride, 0.4g/L of sodium phosphate, 0.01g/L of magnesium chloride, 0.05g/L of calcium chloride, 0.01g/L of copper sulfate, 0.005g/L of sodium molybdate, 0.006g/L of boric acid, 0.006g/L of zinc sulfate, 800.5 g/L of tween and pH 7.0.
Middle-stage culture and fermentation: transferring the culture solution obtained by shake culture into a metaphase culture tank filled with a metaphase culture medium according to the volume ratio of 1:50 for culture and fermentation, culturing at 32 ℃ for 3 days at the rotating speed of 120rpm, and adding the oat beta-glucan and the yeast fermentation auxiliary agent into the metaphase culture medium on the 1 st day from the start of the metaphase culture and fermentation. 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 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 1:20, stirring under the condition of water bath at 75 ℃, extracting for 3 hours, filtering and collecting primary extraction filtrate, repeatedly extracting filter residue once according to the same condition to obtain secondary extraction filtrate, and combining the primary extraction filtrate and the secondary extraction filtrate to obtain the ganoderma lucidum crude extract; 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 50 g/L. The dosage of the oat beta-glucan is 5 g/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 microns), curcumin, cortex Mori powder, silk protein powder and lysate of secondary fission 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 50 mL. 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 2 g/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 0.5 g/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 extractive solution, retaining half volume of the obtained yeast fermented Ganoderma extractive solution, and spray drying the rest half volume of the yeast fermented Ganoderma extractive solution 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 extract solutions of examples 1 to 3 of the present invention and comparative examples 1 to 3 were measured according to the following methods.
The amino acid component determination method comprises the following steps: the 17 free amino acids in the lucid ganoderma extract liquid fermented by the yeast are measured by adopting the amino acid measuring method in GB/T5009.124-2016, 2.0mL of refrigerated sample is taken, 2.0mL of 5-sulfosalicylic acid with the mass concentration of 6g/100mL is added, 1mL and 1g/100mL of EDTA-2Na solution are added after reaction for 1h, the mixture is fully mixed, and the mixture is centrifuged at 4800r/min for 10 min. Sucking 2.0mL of the above mixed solution, adding 4.0mL of citric acid buffer solution with pH of 2.2, mixing uniformly, centrifuging, filtering, and directly measuring on a machine. The external standard method quantifies the amino acid composition in the sample in mg/100 mL. And (3) testing conditions are as follows: ion Exchange Column 2622sc.ph, (4.6mm × 60 mm); the column temperature is 134 ℃; the double-channel ultraviolet detection wavelength is 440nm and 570 nm; the sample size was 20. mu.L for 148 min. And (3) testing conditions are as follows: ion Exchange Column 2622sc.ph, (4.6mm × 60 mm); the column temperature is 134 ℃; the double-channel ultraviolet detection wavelength is 440nm and 570 nm; the sample size was 20. mu.L for 148 min. The results are shown in Table 1.1.
TABLE 1.1 amino acid content (g/100g dry matter) of Ganoderma lucidum extract by yeast fermentation
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 determination of the triterpene compounds in Ganoderma lucidum can be carried out by the method disclosed in Feng et al (Feng, J., et al., An unstructured kinetic model for the improvement of t)riterpenes production by ganoderam lucidum G0119 based on nitrogen source effect, Biotechnology and Bioprocess Engineering,19(4), 727-732, 2014), specifically freeze-drying the yeast fermented Ganoderma lucidum extract to obtain a powdery solid, taking 1G of the powdery solid, and extracting 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) NaHCO3And (4) extracting. 2mol/L HCl is added to adjust NaHCO3After the pH of the phases was below 3.0, NaHCO was extracted again with chloroform3Triterpene 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 lucidum 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 0.6mg/mL) is drawn by taking glucose as a standard product, specifically, 1g of ganoderma lucidum 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 up, 5.0mL of concentrated sulfuric acid solution is added along the wall of the test tube, the test tube is shaken up for 6 minutes, 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, the absorbance value is determined at the position with the wavelength of 487nm by using a 1cm cuvette, 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 yeast fermented ganoderma lucidum extract liquid 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, tween-803, 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 methylparaben, 0.1% of propylhydroxybenzoate, 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 by 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 the test population 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 application is carried out once a day in the 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 randomly divided into 6 groups, in the experiment, the subjects in each group apply the corresponding emulsion prepared in the example once on the face in the morning and evening, 3mL of the whole face is uniformly applied each time, uniform sun protection is applied after the face is applied in the daytime for 28 days, and all the subjects do not use other skin care products except for the test emulsion and the sun protection cream.
Facial skin stains were measured using a Visia7 skin detector 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 is slightly different, the average difference percentage is adopted for representation, and the final difference percentage of each group is also expressed by taking the average. The results of the experiment are shown in table 3.
TABLE 3 test results of whitening and freckle removing efficacy 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 and filtered emulsion prepared in the examples 1 to 3 has better whitening and freckle removing effects compared with the comparative examples 1 to 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 (4) testing the moisturizing effect: selecting 60 volunteers aged 18-35 years and having skin complexion L values close to each other, equally dividing into 6 groups, measuring the initial skin hydration degree (averaging) of each group of volunteers, then respectively using the masks prepared by the yeast fermented ganoderma lucidum extract in the examples 1-3 and the comparative examples 1-3, once per day for 4 weeks, testing the skin hydration degree (averaging) of each group of volunteers in the 2 nd week and the 4 th week, and evaluating the moisturizing effect of the plant-derived moisturizing 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 skin 2 week | Degree of skin hydration of 4 weeks |
| 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 105fibroblasts/mL were seeded in wells of a 96-well plate at 37 ℃ with 5% CO2The culture was carried out in a constant temperature incubator in which IMDM was used as a medium, 20mM glutamine was added, and 10% fetal bovine serum was contained, and the culture was carried out for 24 hours. Adding 1.6mM hydrogen peroxide and 2% of Ganoderma extract solution obtained by fermenting yeast of examples 1-3 and comparative examples 1-3 into fresh culture medium, respectively, each 10 μ L as experimental group, adding 1.6mM hydrogen peroxide alone as positive control, and adding untreated cells as negative control, respectively, adding 10 μ L sample solution and positive and negative control solution into the well plate of corresponding sample group and control groupAnd (3) continuing to culture the cells for 24 hours, adding 20 mu L of MTT into each well, removing the culture medium after culturing for 4 hours, adding 150 mu L of DMSO, and detecting the absorbance value at 490nm 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 Yeast fermented Ganoderma lucidum extracts of examples 1-3 and comparative examples 1-3
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 (10)
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.
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 rotation speed of 100 plus 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-;
(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 and fermentation, culturing for 3-5 days at 28-32 ℃, rotating at the 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 st-2 th day from the beginning of metaphase 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 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).
3. The production method according to claim 2, wherein the yeast species in step (1) is one or more selected from the group consisting of Saccharomyces roukovicensis, Saccharomyces paradoxus, and Saccharomyces victoriae, and the solid-state activation medium in step (1) is produced 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.
4. The method according to claim 2, wherein 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, 800.3-0.5 g/L of tween and pH 7.0.
5. The production method according to claim 2, wherein the composition of the metaphase medium in the 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; and the yeast fermentation auxiliary agent is
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 with the particle size of about 100 microns, adding the ganoderma lucidum powder into distilled water according to the material liquid mass ratio of 1:20, 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 the ganoderma lucidum crude extract; the addition amount of the Ganoderma lucidum crude extract per liter of culture medium is 0.2L.
6. The production method according to claim 2, wherein 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;
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.
7. The preparation method according to claim 2, wherein 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 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 (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% 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 of step b) to obtain Ganoderma extractive solution fermented by yeast.
8. The method according to claim 7, wherein the yeast fermented Ganoderma lucidum extract obtained in f) is spray-dried to prepare a powdery product.
9. 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 8, 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.
10. 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 8.
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