CN117946881A - Meiqi microzyme and application thereof - Google Patents
Meiqi microzyme and application thereof Download PDFInfo
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- CN117946881A CN117946881A CN202410107161.4A CN202410107161A CN117946881A CN 117946881 A CN117946881 A CN 117946881A CN 202410107161 A CN202410107161 A CN 202410107161A CN 117946881 A CN117946881 A CN 117946881A
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- poria cocos
- poria
- fermentation
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- slurry
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Abstract
The invention discloses a Meiqi microzyme and application thereof, wherein after the Meiqi microzyme ferments poria cocos, the Meiqi microzyme enriches the content of various effective components in poria cocos fermentation filtrate, especially can generate more organic acids, and improves the content of total acids in the fermentation filtrate. The poria cocos fermentation filtrate obtained by the invention has high safety, and can be added into cosmetics in high concentration without generating cytotoxicity, so that the poria cocos yeast fermentation filtrate can be used as safe components for tightening, increasing elasticity, controlling oil, removing acne, whitening, resisting inflammation and the like and applied to cosmetics.
Description
Technical Field
The invention relates to a merzizyphus yeast (Metschnikowia sp.) and also relates to application of the merzizyphus yeast (Metschnikowia sp.) in preparation of fermentation products, in particular to application of the merzizyphus yeast (Metschnikowia sp.) in preparation of Chinese herbal medicine poria cocos fermentation filtrate, and also relates to poria cocos fermentation extract obtained by fermentation and application of the poria cocos fermentation extract in cosmetics, belonging to the technical field of cosmetics.
Background
The deep application development of traditional Chinese herbal medicine resources, especially in the non-medical field, has important significance for the utilization of the Chinese herbal medicine resources, and the extraction of active ingredients and the expansion of the utilization range of the active ingredients are one of main research directions.
Poria (Hoelen) (alias: poria, and Poria) belongs to Polyporaceae, is dry sclerotium of Poria Poriacocos Wolf, is produced by pine root, is called Poria, and is first loaded in Shennong Ben Cao Jing, has long and wide application history in China, and has sweet and light taste, and is effective in inducing heart, lung, spleen and kidney meridians; induce diuresis and excrete dampness, invigorate spleen and calm heart, and be combined with proper herbs to exert its unique actions no matter how cold, warm, wind and wet. In the book of Yi Zhong Shen xi, it is recorded that "the stomach is returned with what is called light qi in the book of internal meridians", while "the above-mentioned" internal meridians "in the book of Ji Rou Wu Shu", also called light flavor can nourish spleen yin. The cover has the effect of resolving phlegm-fluid retention in stomach as water source, and is the main drug for excreting dampness and promoting the diuresis, which is infused into spleen and reaches lung, and then down-circulated to triple energizer water channel to enter bladder.
Poria cocos has been studied to contain various chemical components including polysaccharides, triterpenes, sterols, volatile oils, proteins, etc., wherein the main functional components are polysaccharides and triterpenes. Polysaccharides are classified into a large amount of alkali-soluble polysaccharides (about 90% or more of the polysaccharide) and a small amount of soluble polysaccharides. The triterpenes comprise more than 50 triterpenes such as dehydro-mesoporous acid, dehydro-temoic acid, dehydroabietic acid methyl ester, etc., wherein the triterpenes are mainly tetracyclic triterpenes.
At present, the extraction and utilization of the tuckahoe functional components are concentrated on aspects of polysaccharide, triterpene and the like, the functional research is concentrated on two aspects of eating and medical, and the application research on cosmetics is less. In the extraction mode, water extraction and alcohol extraction are studied more, and in recent years, fermentation extraction also occupies a larger proportion. Patent CN109832619a discloses a poria cocos fermented product and a preparation method thereof, wherein lactobacillus brevis, lactobacillus paracasei, lactobacillus plantarum and lactobacillus acidophilus are adopted for fermentation to obtain the poria cocos fermented milk with more poria cocos functional components and good flavor, and the main utilized substances are polysaccharide and triterpene substances, namely, pachymic acid. Patent CN115838676a discloses a method for fermenting poria cocos by using novel lactobacillus plantarum strain and application thereof, wherein the fermented product has one or more of sugar resistance, moisture retention or whitening, and mainly uses substances such as organic acid in poria cocos and fermentation acid production.
At present, most of Poria cocos fermentation strains adopt lactic acid bacteria, less Poria cocos fermentation reports are carried out by adopting saccharomycetes, and meanwhile, the Poria cocos fermentation products are not found to be applied to cosmetics such as tightening and acne removing.
Disclosure of Invention
The invention provides a newly screened Meiqi microzyme (Metschnikowia sp.) which is low in cytotoxicity and high in safety of a fermentation product obtained by fermenting poria cocos, can generate more substances beneficial to skin care in fermentation liquor, has the effects of tightening, increasing elasticity, controlling oil, removing acnes, whitening, resisting inflammation and the like, is high in safety, and can be applied to cosmetics.
The Meiqi microzyme (Metschnikowia sp.) provided by the invention is a strain which is self-separated in a laboratory. The strain is preserved in China general microbiological culture collection center (CGMCC) at 11 and 17 days of 2023, and the address is: the collection number of the microbiological institute of China academy of sciences of No. 3, ganyang area North Star of Beijing city is CGMCC No.29040. The gene sequence of the 18S rDNA of this merthiolase (metschnikowiasp.) is set forth in SEQ ID NO: 1.
The invention also provides a microbial inoculum, which comprises the above-mentioned Meiqijijun microzyme. The microbial inoculum may contain only a microorganism of the genus MeiYeast, or may contain other yeasts.
Further, the microbial inoculum can be a solid preparation or a liquid preparation.
The invention also provides application of the above-mentioned Meiqijimicrozyme (Metschnikowia sp.) or the above-mentioned microbial inoculum in preparation of fermentation products. In this application, fermentation is typically carried out by adding the above-described starter to a substrate to produce a fermentation product.
The use of yeasts in fermentation in various fields is disclosed in the prior art, and the merzizyphus yeast (Metschnikowia sp.) of the present invention or the above-described microbial agents can be used in these previously disclosed fields, and specific fermentation methods can be referred to in the prior art.
Preferably, the merzizyphus (Metschnikowia sp.) or the microbial inoculum described above is used to prepare a Poria cocos fermentation product. The poria cocos fermentation product is obtained by fermenting a slurry of a poria cocos raw material by using a merzizyphus yeast (Metschnikowia sp.) or the microbial inoculum.
The invention also aims to provide a preparation method of the poria cocos fermentation filtrate, which uses specially screened merzizyphus yeast as a fermentation strain to ferment poria cocos, so that more substances beneficial to skin care can be produced in the fermentation liquor, and the fermentation liquor has good application prospect in cosmetics.
The preparation method of the poria cocos fermentation filtrate comprises the step of fermenting poria cocos by adopting the above-mentioned Meiqi microzyme (Metschnikowia sp.). Experiments prove that compared with other types of lactic acid bacteria and saccharomycetes, the Meiqi saccharomycetes (Metschnikowia sp.) CGMCC No.29040 has better advantages in fermenting poria cocos, and more substances beneficial to skin care can be produced in fermentation liquor, so that the obtained fermentation liquor has more advantages in the aspects of enhancing skin elasticity, reducing skin wrinkles, reducing excessive secretion of skin grease, inhibiting growth of acne-related strains, relieving, whitening, resisting inflammation and the like.
Further, the preparation method of the poria cocos fermentation filtrate comprises the following steps:
(1) Mixing Poria powder with water to obtain Poria slurry, and performing ultrasonic and sterilization treatment on Poria slurry;
(2) Inoculating the slurry obtained in the step (1) into Meiqiyeast for fermentation culture to obtain fermented poria cocos slurry; or mixing the slurry obtained in the step (1) with at least one of a carbon source and inorganic salt to obtain a composite culture medium, and then inoculating the Meiqiyeast for fermentation culture to obtain fermented poria cocos slurry;
(3) And (3) carrying out post-treatment on the fermented poria cocos slurry to obtain poria cocos fermented filtrate.
Further, in the step (1), the poria cocos is a poria cocos sclerotium dry product. When in use, the dried tuckahoe sclerotium product is sliced, crushed and sieved to obtain tuckahoe powder with uniform size. Any method that can be implemented to obtain Poria cocos powder is suitable for this step, and is not further limited herein.
Further, in step (1), the content of the dry matter of Poria cocos in the Poria cocos slurry is 5-15wt%, for example, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt% and any value therebetween. Preferably, the poria cocos and the powder are mixed and then kept stand for 1-2 hours, so that the poria cocos fully absorbs water.
Furthermore, in the step (1), the purpose of the ultrasonic treatment is to better disperse the poria cocos powder in water, and the purpose of the ultrasonic treatment is to primarily destroy the lignified structure of the poria cocos powder so as to be beneficial to better precipitation of active ingredients in the poria cocos in the fermentation process. The ultrasound may be performed using ultrasound equipment commonly used in the laboratory, typically for a period of time ranging from 10 to 30 minutes, such as 10min、11min、12min、13min、14min、15min、16min、17min、18min、19min、20min、21min、22min、23min、24min、25min、26min、27min、228min、29min、30min and any value in between.
Further, in the step (1), after the ultrasonic treatment, the poria cocos slurry is sterilized for later fermentation, and the sterilization can be performed by a conventional high-temperature sterilization method, for example, the sterilization is performed at 121 ℃ for about 20min, which is not limited.
Further, in the step (2), the carbon source may be one or more of glucose, sucrose, fructose, maltose, etc., and the inorganic salt may be one or more of dipotassium hydrogen phosphate, sodium chloride, ferrous sulfate, calcium chloride, potassium chloride, etc.
Further, in the step (2), the content of the additional carbon source in the composite medium is low, and is 0.1wt% to 5wt% of the composite medium, for example 0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％、0.6wt％、0.7wt％、0.8wt％、0.9wt％、1wt％、1.1wt％、1.2wt％、1.3wt％、1.4wt％、1.5wt％、1.6wt％、1.7wt％、1.8wt％、1.9wt％、2wt％、2.1wt％、2.2wt％、2.3wt％、2.4wt％、2.5wt％、2.6wt％、2.7wt％、2.8wt％、2.9wt％、3wt％、3.1wt％、3.2wt％、3.3wt％、3.4wt％、3.5wt％、3.6wt％、3.7wt％、3.8wt％、3.9wt％、4wt％、4.1wt％、4.2wt％、4.3wt％、4.4wt％、4.5wt％、4.6wt％、4.7wt％、4.8wt％、4.9wt％、5wt％ and any value in between.
In one embodiment of the invention, the complex medium comprises dipotassium hydrogen phosphate in an amount of 0.01 to 0.5wt%, for example 0.01wt％、0.02wt％、0.03wt％、0.04wt％、0.05wt％、0.06wt％、0.07wt％、0.08wt％、0.09wt％、0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％ and any value in between.
In a specific embodiment of the present invention, the complex medium comprises sodium chloride in an amount of 0.01 to 1wt%, for example 0.01wt％、0.02wt％、0.03wt％、0.04wt％、0.05wt％、0.06wt％、0.07wt％、0.08wt％、0.09wt％、0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％、0.6wt％、0.7wt％、0.8wt％、0.9wt％、1wt％ and any value in between.
In one embodiment of the invention, the complex medium comprises ferrous sulphate in an amount of 0.01 to 0.5wt%, for example 0.01wt％、0.02wt％、0.03wt％、0.04wt％、0.05wt％、0.06wt％、0.07wt％、0.08wt％、0.09wt％、0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％ and any value in between.
In a specific embodiment of the present invention, the complex medium comprises calcium chloride in an amount of 0.01 to 0.5wt%, such as 0.01wt％、0.02wt％、0.03wt％、0.04wt％、0.05wt％、0.06wt％、0.07wt％、0.08wt％、0.09wt％、0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％ and any values therebetween.
In a specific embodiment of the present invention, the complex medium comprises potassium chloride in an amount of 0.01 to 0.5wt%, such as 0.01wt％、0.02wt％、0.03wt％、0.04wt％、0.05wt％、0.06wt％、0.07wt％、0.08wt％、0.09wt％、0.1wt％、0.2wt％、0.3wt％、0.4wt％、0.5wt％ and any values therebetween.
In the step (2), the required amount of the MeiYeast can be obtained by expanding culture at first, then the strain is resuspended by purified water to obtain seed liquid, and the seed liquid is inoculated into a compound culture medium for fermentation. As a medium for the expansion culture, a medium disclosed in patent CN 116286410A can be used, and YPD solid medium is preferable. Preferably, the YPD solid medium comprises the following components: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. In the seed solution, the OD 600 of the strain is about 4.0-5.0. The seed solution is inoculated in the compound culture medium in an amount of about 1-2%.
Further, in the step (2), the culture temperature at the time of fermentation culture is 28℃to 35℃such as 28℃29℃30℃31℃32℃33℃34℃35 ℃.
Further, in the step (2), the fermentation time is generally 24 to 72 hours, for example, 24 hours, 30 hours, 36 hours, 40 hours, 48 hours, 55 hours, 60 hours, 65 hours, 72 hours.
Further, in the step (3), the post-treatment of the fermented poria cocos slurry includes a step of removing insoluble matters from the fermented poria cocos slurry, for example, a step of removing insoluble matters such as solid residues and thalli, and the insoluble matters can be removed by centrifugation, filtration and the like;
Preferably, the centrifugation conditions are 5000-8000rpm;
further preferably, the centrifugation time is 5-20min.
Experiments prove that the poria cocos fermentation filtrate can obviously enhance skin elasticity, reduce skin wrinkles, reduce excessive secretion of skin grease and inhibit growth of related acne strains, can be applied in high concentration, is safe and has no obvious stimulation, and meanwhile has excellent effects of relieving, whitening and resisting inflammation, the effect of the poria cocos fermentation filtrate is obviously superior to products obtained by fermentation of other strains, and the poria cocos fermentation filtrate has good application prospects in cosmetics. Therefore, the poria cocos fermentation filtrate obtained by the method disclosed by the invention is also in the protection scope of the invention.
The invention also provides a cosmetic, which comprises the poria cocos fermented filtrate.
Further, the cosmetic includes, but is not limited to, lotions, skin creams, essences, eye creams, facial masks, soaps, facial washes, body washes, aerosols, sprays.
Further, the amount of the poria cocos fermentation filtrate added to the cosmetic is 0.1% -50% (w/w), for example 0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％, is preferably 2% -30% (w/w).
The cosmetic of the present invention may further comprise an auxiliary material, which may be one or more of a suitable solvent, a propellant, a solubilizing agent, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrator, a permeation enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickener, an inclusion agent, a humectant, an absorbent, a diluent, a flocculant and deflocculant, a filter aid, a release retarder, and the like.
The cosmetic of the present invention may be prepared by a general method, to which one or more diluents or carriers such as aqueous solutions, pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions and the like may be added.
The poria cocos fermentation filtrate is a yeast fermentation product of a poria cocos substrate which is not processed, only a small amount of carbon source and inorganic salt are added except yeast, and the added carbon source is basically consumed after fermentation, so that the consumption of active ingredients in poria cocos by fermentation per se is reduced, and the natural active substances in the poria cocos are maintained to the greatest extent.
The preparation process of the poria cocos fermentation filtrate is simple in components, the contents of various effective components in the poria cocos fermentation filtrate are enriched through special fermentation metabolism of the Meiqi yeast, more organic acids can be produced, and the total acid content in the fermentation filtrate is improved. Experiments prove that the poria cocos fermentation filtrate has the effects of enhancing skin elasticity, reducing skin wrinkles, reducing excessive secretion of skin grease, inhibiting growth of acne-related strains, whitening skin, resisting inflammation and the like, and the effect is obviously superior to that of fermentation filtrate obtained by fermentation of other strains, so that the poria cocos fermentation filtrate is more suitable for being applied to cosmetics.
The poria cocos fermentation filtrate obtained by the invention has high safety, and can be added into cosmetics in high concentration without generating cytotoxicity, so that the poria cocos yeast fermentation filtrate can be used as a safe component for tightening, increasing elasticity, controlling oil, removing acne, whitening and resisting inflammation.
Preservation information
The Meiqi microzyme (Metschnikowia sp.) is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.29040, the preservation date is 2023, 11 and 17, and the preservation address is: north Star way 1, national academy of sciences of China, proc. 3, chaoyang district, beijing.
Detailed Description
The present invention is further described with reference to the following examples, but the present invention is not limited to the following examples, wherein the poria cocos used in the experiments is the dried poria cocos product (Anhui, origin).
EXAMPLE 1 Methodontinuous yeast screening and identification Process
After the soil sample of the orchard (Linxia, gansu province) is collected, the soil sample is diluted and separated by adopting a conventional ten-fold dilution separation method, a flat plate is scratched to obtain single bacterial colonies, and the single bacterial colonies are transferred to a slant for culture, and the bacterial colonies are subjected to microscopic examination. Dilution was again performed to isolate the plates to obtain single colonies, and the above was repeated until a single strain was obtained. Then, through primary screening and secondary screening (poria cocos fermentation culture is carried out, acid production energy of fermentation filtrate is detected), a strain which can grow on a poria cocos-containing culture medium and has highest acid production is screened out, the strain is named as f101, and strain preservation and strain identification are carried out on the strain.
The physiological and biochemical characteristics and genetic characteristics of the strain obtained by final screening are as follows:
(1) Characteristics of the cells: elliptic, spherical or nearly spherical thallus, budding and reproduction;
(2) Colony characteristics: the colony is white, flat, smooth in surface, opaque, creamy and smooth in edge;
(3) Genetic characteristics: the Beijing Liuhua large gene technology Co., ltd.) is entrusted to carry out genome sequencing identification on the strain, and the result shows that the strain is the Meiqijie microzyme (Metschnikowia sp.) and the gene sequence of the 18S rDNA is shown as SEQ ID NO: 1. The strain is preserved with the preservation number of CGMCC No.29040.
Example 2 preparation of Poria fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 5wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20min.
Inoculating single colony of the MeiYeast (Metschnikowia sp.) CGMCC No.29040 into 100mL YPD liquid culture medium, wherein the culture medium comprises the following components: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. After the inoculation, shake flask culture is carried out for 24 hours at 200rpm and 30 ℃, then centrifugation is carried out for 10 minutes at 5000rpm, the supernatant is poured off by the precipitation thallus, and then purified water is used for resuspension, so as to obtain seed liquid, and OD 600 of the seed liquid is about 4.0;
Under the aseptic condition, inoculating the seed liquid into sterilized Poria cocos slurry, wherein the inoculum size is 2% of the volume of the Poria cocos slurry, and culturing in shake flask at 200rpm and 28 ℃ for 48h to obtain Poria cocos yeast fermentation liquor;
And (3) centrifuging the poria cocos yeast fermentation liquor at 5000rpm for 20min, and filtering to remove solid residues and thalli, wherein the obtained supernatant is poria cocos fermentation filtrate for later test.
Example 3 preparation of Poria fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 10% by weight is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20min.
Inoculating single colony of the MeiYeast (Metschnikowia sp.) CGMCC No.29040 into 100mL YPD liquid culture medium, wherein the culture medium comprises the following components: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. After the inoculation, shake flask culture is carried out for 24 hours at 200rpm and 30 ℃, then centrifugation is carried out for 10 minutes at 5000rpm, the supernatant is poured off by the precipitation thallus, and then purified water is used for resuspension, so as to obtain seed liquid, and OD 600 of the seed liquid is about 4.0;
Under the aseptic condition, inoculating the seed liquid into sterilized Poria cocos slurry, wherein the inoculum size is 2% of the volume of the Poria cocos slurry, and culturing in shake flask at 200rpm and 35 ℃ for 24 hours to obtain Poria cocos yeast fermentation liquor;
And (3) centrifuging the poria cocos yeast fermentation liquor at 5000rpm for 20min, filtering to remove solid residues and thalli, obtaining supernatant which is poria cocos fermentation filtrate, diluting the poria cocos fermentation filtrate by 2 times with purified water (so that the extraction ratio of the poria cocos dry matter is 5wt%, namely the content of the poria cocos dry matter raw material is 5 wt%) for later test use.
Example 4 preparation of Poria fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 15wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 30min to primarily destroy the compact structure of the poria cocos powder.
Adding 2wt% glucose, 0.1wt% dipotassium hydrogen phosphate, 0.05wt% sodium chloride, 0.1wt% ferrous sulfate and 0.01wt% potassium chloride into the poria cocos slurry subjected to ultrasonic treatment, and sterilizing at 121 ℃ for 20min to obtain a compound culture medium for later use.
Inoculating single colony of the MeiYeast (Metschnikowia sp.) CGMCC No.29040 into 100mL YPD liquid culture medium, wherein the culture medium comprises the following components: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. After the inoculation, shake flask culture is carried out for 24 hours at 200rpm and 30 ℃, then centrifugation is carried out for 10 minutes at 5000rpm, the supernatant is poured off by the precipitation thallus, and then purified water is used for resuspension, so as to obtain seed liquid, and OD 600 of the seed liquid is about 4.0;
Under the aseptic condition, inoculating the seed liquid into a sterilized composite culture medium, and culturing for 72 hours at 200rpm and 28 ℃ in a shaking bottle, wherein the inoculum size is 2% of the volume of the composite culture medium, so as to obtain a poria cocos yeast fermentation broth;
And (3) centrifuging the poria cocos yeast fermentation liquor at 6500rpm for 10min, filtering to remove solid residues and thalli, obtaining supernatant which is poria cocos fermentation filtrate, and diluting the poria cocos fermentation filtrate by 3 times with purified water (so that the extraction ratio of the poria cocos dry matter is 5wt%, namely the content of the poria cocos dry matter raw material is 5 wt%) for later test use.
Comparative example 1 preparation of Poria Water extraction filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 5wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20 min.
Adding sterilized Poria serous fluid into shake flask, shake flask culturing at 28deg.C for 48 hr to obtain Poria extractive solution;
The poria extract is centrifugated at 5000rpm for 30min, and then filtered to remove solid residues, and the obtained supernatant is the poria water extract filtrate (the extraction ratio of the poria dry matter is 5 wt%).
Comparative example 2 preparation of Poria lactic acid bacteria fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 5wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20min.
Single colonies of lactic acid bacteria (lactobacillus paracasei, ATCC 11578) were inoculated into 100mL of MRS broth, which was formulated as follows: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 1.0mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1000mL of distilled water, 200rpm, shake flask culture at 30 ℃ for 24 hours, centrifugation at 5000rpm for 10 minutes, decanting supernatant of the precipitated thallus, and re-suspending with purified water to obtain seed liquid, wherein OD 600 is about 4.0;
under the aseptic condition, inoculating the seed liquid into sterilized Poria cocos slurry, wherein the inoculum size is 2% of the volume of the Poria cocos slurry, and culturing in shake flask at 200rpm and 35 ℃ for 48h to obtain Poria cocos lactobacillus fermentation liquor;
And (3) centrifuging the poria cocos lactobacillus fermentation liquid at 5000rpm for 20min, and filtering to remove solid residues and thalli, wherein the obtained supernatant is the poria cocos lactobacillus fermentation filtrate for later test.
Comparative example 3 preparation of Poria Yeast fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 5wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20min.
Single colonies of Kluyveromyces (ATCC 36534) were inoculated into 100mL of YPD liquid medium consisting of: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. After the inoculation, shake flask culture is carried out for 24 hours at 200rpm and 30 ℃, then centrifugation is carried out for 10 minutes at 5000rpm, the supernatant is poured off by the precipitation thallus, and then purified water is used for resuspension, so as to obtain seed liquid, and OD 600 of the seed liquid is about 4.0;
Under the aseptic condition, inoculating the seed liquid into sterilized Poria cocos slurry, wherein the inoculum size is 2% of the volume of the Poria cocos slurry, and culturing in shake flask at 200rpm and 28 ℃ for 48h to obtain Poria cocos yeast fermentation liquor;
and (3) centrifuging the poria cocos yeast fermentation liquor at 5000rpm for 20min, and filtering to remove solid residues and thalli, wherein the obtained supernatant is the poria cocos yeast fermentation filtrate for later test.
Comparative example 4 preparation of Poria Yeast fermentation filtrate
Taking dried poria cocos block, slicing, crushing, sieving and collecting poria cocos powder. The poria cocos powder is weighed, purified water is added for constant volume, and poria cocos slurry with the poria cocos dry matter content of 5wt% is prepared.
And standing the poria cocos slurry for 1h to enable the poria cocos powder to fully absorb water, and then carrying out ultrasonic pretreatment on the poria cocos slurry for 10min to primarily destroy the compact structure of the poria cocos powder.
Sterilizing the ultrasonic treated Poria serous fluid at 121deg.C under high pressure for 20min.
Single colony of meiji yeast (CGMCC No: 24953) is inoculated into 100mL YPD liquid culture medium, and the culture medium comprises the following components: yeast extract 10g, peptone 20g, glucose 20g, purified water 1L. After the inoculation, shake flask culture is carried out for 24 hours at 200rpm and 30 ℃, then centrifugation is carried out for 10 minutes at 5000rpm, the supernatant is poured off by the precipitation thallus, and then purified water is used for resuspension, so as to obtain seed liquid, and OD 600 of the seed liquid is about 4.0;
Under the aseptic condition, inoculating the seed liquid into sterilized Poria cocos slurry, wherein the inoculum size is 2% of the volume of the Poria cocos slurry, and culturing in shake flask at 200rpm and 28 ℃ for 48h to obtain Poria cocos yeast fermentation liquor;
and (3) centrifuging the poria cocos yeast fermentation liquor at 5000rpm for 20min, and filtering to remove solid residues and thalli, wherein the obtained supernatant is the poria cocos yeast fermentation filtrate for later test.
Test example 1 evaluation of Total acid in Poria fermentation filtrate
The evaluation of the total acid content in the poria cocos fermentation filtrate is carried out with reference to GB12456-2021 "determination of total acid in food", and the total acid content is calculated by the conversion coefficient of malic acid because the malic acid content in the poria cocos fermentation filtrate is higher.
1. Apparatus and device
Analytical balance (0.1 mg of sensing), basic burette (10 mL, 25 mL), pipette (25 mL, 50 mL), conical flask (250 mL), volumetric flask (50 mL).
2. Reagent preparation
(1) Carbon dioxide free water: boiling water for 15min to remove carbon dioxide, cooling, and sealing.
(2) Phenolphthalein indicator solution (10 g/L): 1g of phenolphthalein was weighed out, dissolved in ethanol (95%) and diluted to 100mL with ethanol (95%).
(3) Sodium hydroxide standard titration solution (0.1 mol/L): the standard titration solution is formulated and calibrated according to the requirements of GB/T5009.1 or purchased with national certification and standard substance certificate granted.
3. Analytical procedure
3.1 Preparation of the solution to be measured
5G or 5mL of the sample (filtrate or dilution of the filtrate prepared in examples and comparative examples, each having a dry matter extraction ratio of 5 wt%) was weighed into a 50mL volumetric flask, and shaken well with carbon dioxide-free water to a scale for measurement.
3.2 Specific analytical procedure
(1) According to the possible content of the total acid in the sample, 25mL or 50mL of the solution to be detected is sucked, the solution to be detected is placed in a 250mL triangular flask, 2-4 drops (10 g/L) of phenolphthalein indicator solution are added, and 0.1mol/L sodium hydroxide standard titration solution is used for titration until reddish color is not faded for 30 s. The volume value of the standard titration solution consuming 0.1mol/L sodium hydroxide is recorded.
(2) Blank experiment: and (3) performing a blank test by replacing the solution to be tested with the same volume of water without carbon dioxide according to the operation, and recording the volume value of the standard titration solution consuming sodium hydroxide.
4. Test data processing
The total acid content is calculated according to formula (1).
X＝(c×(V1-V2)×k×F)/m×1000……………………………………(1)
Wherein:
X-the total acid content in the sample in grams per kilogram/(g/Kg) or grams per liter/(g/L);
c-concentration of sodium hydroxide standard titration solution in moles per liter (mol/L);
V1, the volume of a sodium hydroxide standard titration solution is consumed when the test solution is titrated, and the unit is milliliter (mL);
V2-the volume of sodium hydroxide standard titration solution consumed in milliliters (mL) during blank test;
k-conversion coefficient of acid: malic acid, 0.067;
F, dilution multiple of the test solution;
m-the mass of the sample in grams (g) or the volume of the sample aspirated in milliliters (mL);
1000—a scaling factor.
The calculation results are expressed as arithmetic mean of 3 independent measurement results obtained under repetitive conditions.
5. The pH value and total acid content data of the Poria cocos fermentation filtrate are shown in the following table 1.
TABLE 1 pH and Total acid content of different samples
| PH value of | Total acid content (mg/L) | |
| Example 2 | 3.16 | 420 |
| Example 3 | 2.98 | 491 |
| Example 4 | 2.86 | 530 |
| Comparative example 1 | 4.14 | 238 |
| Comparative example 2 | 3.59 | 329 |
| Comparative example 3 | 3.87 | 290 |
| Comparative example 4 | 3.65 | 312 |
From an analysis of the data in Table 1, it can be seen that the pH values of the Poria cocos fermentation filtrate of examples 2-4 are lower than those of the Poria cocos water extract filtrate of comparative example 1, the Poria cocos lactobacillus fermentation filtrate of comparative example 2, and the Poria cocos yeast fermentation filtrate of comparative examples 3-4, and the corresponding total acid content is higher than that of comparative examples 1-4. The tuckahoe fermentation filtrate prepared by fermenting tuckahoe matrixes by taking the merzizyweed as a fermentation strain is more easy to synthesize organic acid, and is more beneficial to producing the tuckahoe fermentation filtrate taking the organic acid as a main functional component.
Test example 2 safety evaluation of Poria fermentation filtrate
The safety evaluation of the poria cocos fermentation filtrate is carried out by adopting a closed type patch test, and the test method and the evaluation standard refer to the human skin patch test in cosmetic safety technical Specification (2015 edition).
1. Experimental materials
Experimental samples: the poria cocos fermentation filtrate or the diluent of the fermentation filtrate prepared in the examples 2-4, wherein the extraction ratio of the poria cocos dry matter is 5%; poria cocos water extract filtrate prepared in comparative example 1; the poria cocos fermented filtrate prepared in comparative examples 2-4 has the poria cocos dry matter extraction ratio of 5%.
Spot patch sample: 50% of the test sample, the dilution matrix was water.
2. The experimental method comprises the following steps:
(1) Volunteer recruitment
Patch test recruited 30 healthy volunteers, not limited to men and women, aged 20-65 years.
The following should be excluded:
a, antihistamines used for nearly one week or immunosuppressants used in nearly one month;
b any anti-inflammatory agent is applied to the tested part within nearly two months;
c a subject suffering from an inflammatory dermatological condition with clinically unhealed;
d insulin dependent diabetes mellitus patient;
e patients suffering from asthma or other chronic respiratory diseases undergoing treatment;
f receiving anti-cancer chemotherapeutics within approximately 6 months;
g patients suffering from immunodeficiency or autoimmune diseases;
h lactating or pregnant women;
i bilateral mastectomy and bilateral axillary lymphadenectomy;
j effects on skin sites due to scarring, pigment, atrophy, port-marks or other imperfections
A judging unit for judging the test result;
k participated in other clinical trial researchers;
a person with high physical sensitivity;
m non-volunteer participants or those who were unable to complete the prescribed content as required by the experiment.
(2) Enclosed skin patch test
The samples were diluted to the desired concentration (1.0% -100%) with purified water as a control. The package of the plaque tester was torn, and 0.020mL of each of the prepared samples was measured and added to the chamber. The patch tester was applied to the forearm flexor side of the subject, and gently pressed with the palm to apply it uniformly to the skin for 24 hours.
The reaction results were recorded as observed in Table 230 min, 24h and 48h after removal of the plaque assay.
TABLE 2 skin response grading Standard for skin seal Patch test
3. Experimental results:
the subjects observed skin reactions 30min,24h and 48h after removal of the plaque assay. Results 30 subjects who were exposed to the poria extract (1.0% -100% concentration) were all negative. See in particular table 3.
TABLE 3 Patch test results statistics (total number of cases: 30)
Experimental data shows that the poria cocos fermentation filtrate of the embodiment 2-4 has no obvious skin irritation in the patch test process although the total acid content is improved compared with the poria cocos water extract of the comparative example 1, the poria cocos lactobacillus fermentation filtrate of the comparative example 2 and the poria cocos yeast fermentation filtrate of the comparative example 3-4, and the poria cocos fermentation filtrate of the embodiment 2-4 is warm in application process and skin friendly, and can be added into cosmetics by 100%.
Test example 3 Effect of Poria fermented filtrate on skin elasticity, skin texture, and lipid secretion
The effect of Poria cocos fermentation filtrate on skin elasticity, skin texture and oil secretion is evaluated by referring to "test method for skin elasticity influence by T/ZHCA 005-2019 cosmetics" and "test method for oil control efficacy of T/ZHCA 002-2018 cosmetics".
1. Experimental materials
Experimental samples: the poria cocos fermented filtrate prepared in example 4, wherein the extraction ratio of the poria cocos dry matter is 5%.
Test sample: an emulsion (containing 10wt% and 30wt% of poria cocos fermented filtrate) containing poria cocos fermented filtrate (experimental sample) comprises the following components in percentage by weight: xanthan gum 0.2%, glycerol 5%, carbomer 0.1%, aminomethylpropanol 0.05%, shea butter 10%, glyceryl stearate 2.5%, caprylic/capric triglyceride 5%, isononyl isononanoate 5%, dimethyl siloxane 2%, poria fermented filtrate 10wt% or 30wt%, and purified water 100%.
2. Experimental facilities:
Multi-probe skin test System MPA580 (Coura+Khazaka, germany).
3. The experimental method comprises the following steps:
(1) Volunteer recruitment: 24 healthy volunteers, unlimited for men and women, aged 20-65 years, were enrolled under the same conditions as the test example 2 patch test volunteers.
(2) The testing method comprises the following steps:
Volunteers meeting the requirements were randomly divided into 2 groups, with group 1 using an emulsion containing 10% Poria cocos and group 2 using an emulsion containing 30% Poria cocos. After 15 minutes after the volunteers of the left and right half faces of the subject are uniformly cleaned by using the poria containing emulsion and the placebo (the poria containing emulsion) respectively, the skin elasticity of the apple muscle area of the left and right half faces and the skin grease at the forehead of the left and right half faces are detected as initial values, the poria containing emulsion and the placebo are used for the left and right half faces respectively in the morning, evening and keeping the dosage consistent, and all indexes are detected again after 1 week, 2 weeks and 4 weeks of use.
Skin wrinkle texture analysis was performed on the image taken by the VISIA CR using IPP skin analysis-by-synthesis software.
4. Experimental data:
(1) Effect of fermented filtrate emulsion containing Poria on skin elasticity:
The effect of the fermented filtrate emulsion containing Poria on skin elasticity is shown in the following tables 4 and 5, wherein Tx is the skin elasticity value at different times, and T0 is the initial value of skin elasticity.
TABLE 4 Table 4
* Represents p <0.05 compared to placebo
TABLE 5
(2) Effect of Poria cocos fermentation filtrate emulsion on skin texture area
The effect of the Poria cocos fermentation filtrate emulsion on skin texture area is shown in tables 6 and 7 below, wherein Tx is the skin texture area value at different times, and T0 is the initial value of skin texture area.
TABLE 6
TABLE 7
* Represents p <0.05 compared to placebo
(3) Effect of Poria cocos fermented filtrate emulsion on skin oil secretion
Effects of the Poria cocos fermentation filtrate emulsion on skin lipid secretion are shown in tables 8 and 9 below, wherein Tx is the skin lipid content value at different times, and T0 is the initial skin lipid content value.
TABLE 8
TABLE 9
Experimental data show that the poria cocos fermentation filtrate prepared in the embodiment 4 is added into basic emulsion, and in the addition amount of 10% -30%, skin elasticity can be effectively improved, skin texture area can be reduced, and skin grease content can be reduced.
Test example 4 inhibition of Propionibacterium acnes by Poria fermented filtrate
The inhibition effect of the poria cocos fermentation filtrate on propionibacterium acnes is detected and evaluated by referring to a method (a suspension quantification method) for detecting the antibacterial effect of QB/T2738-2012.3 antibacterial daily chemical products.
1. Experimental materials
Experimental samples: a dilution of the Poria fermentation filtrate prepared in example 4;
poria cocos water extract filtrate prepared in comparative example 1;
comparative example 3 Poria cocos yeast fermentation filtrate was prepared.
2. The experimental method comprises the following steps:
suspension quantification method (QB/T2738-2012 7.3)
3. Experimental data:
TABLE 10 bacteriostatic action of Poria fermented filtrate/Water extract filtrate on Propionibacterium acnes
The data in Table 10 shows that the Poria cocos fermented filtrate prepared in example 4 has better short-term (2 h) antibacterial effect and long-term (24 h) antibacterial effect than the Poria cocos water extract filtrate. The antibacterial performance of the poria cocos yeast fermentation filtrate is improved compared with that of the poria cocos water extract, but is obviously lower than that of the poria cocos fermentation filtrate prepared in the embodiment 4.
Test example 5 whitening effect of Poria fermented filtrate
Referring to T/SHRH 027-2019 in vitro test B16 cell melanin synthesis inhibition experiment and Chinese substitution method research center-in vitro detection system based on human melanocytes, whether the sample has whitening effect is examined by examining the influence of the sample on tyrosinase activity.
1. Experimental materials
Experimental samples: a dilution of the Poria fermentation filtrate prepared in example 4;
poria cocos water extract filtrate prepared in comparative example 1;
poria cocos yeast fermentation filtrate prepared in comparative example 3.
2. Experimental method
(1) B16 cells in logarithmic growth phase are inoculated into a 6-hole culture plate at the density of 1X 10 5 cells/mL, 3mL of each hole is inoculated into a carbon dioxide culture box at 37 ℃ and 5% CO 2 for culturing for 24 hours, old culture solution is discarded, 3mL of serum-containing culture medium is added into a negative control group and a model group, 3mL of experimental material solution is added into the experimental group, and Forskolin solution (50 mu M in Forskolin action concentration) is added into each hole of the model group and the experimental group so as to stimulate melanin production, and culturing is continued for 72 hours. The final use concentration of the experimental materials was 1%, 5%.
(2) The old culture solution is discarded in activity detection, the cells are washed twice by PBS, then the cells are lysed by a Tirs-HCL (0.02 mol/L, pH6.8) solution containing 0.1% of triton-100, the cells are treated by ultrasonic for 10min, 0.1% of L-dopa (0.01 mol/L, pH8.0) is added for incubation and culture at 37 ℃ for 60min, the supernatant is centrifugally taken and added into a 96-well plate for 100 mu L/hole, the light absorption value is measured by an enzyme-labeling instrument at the wavelength of 450nm, the ratio of absorbance of an experimental group to absorbance of a model group is used as an index for evaluating the activity of tyrosinase, and if the experimental material has obvious influence on proliferation of B16 cells, the tyrosinase activity is normalized according to the proliferation rate.
3. Experimental data
The inhibition rate of tyrosinase by the experimental materials is shown in table 11.
Table 11 inhibition ratio of the test materials to tyrosinase
| Example 4 | Comparative example 1 | Comparative example 3 | |
| 1％ | 3.15 | 0.73 | 1.05 |
| 5％ | 37.85 | 29.14 | 28.05 |
The data in Table 11 shows that the Poria cocos fermentation filtrate prepared in example 4 has stronger tyrosinase inhibition effect than the Poria cocos water extract filtrate and the Poria cocos yeast fermentation filtrate.
Test example 6 Poria cocos fermented filtrate relieving anti-inflammatory effect
Reference T/SHRH 034-2021 cosmetic soothing efficacy test-in vitro TNF- α inflammatory factor content assay.
1. Experimental materials
Experimental samples: a dilution of the Poria fermentation filtrate prepared in example 4;
poria cocos water extract filtrate prepared in comparative example 1;
poria cocos yeast fermentation filtrate prepared in comparative example 3.
2. Experimental method
The method is characterized in that a mouse macrophage cell line is taken as a model cell, inflammatory factors are secreted by the macrophage through LPS stimulation, the macrophage is treated by an experimental material, and the anti-inflammatory efficacy of the experimental material is examined through quantitative detection of the expression of the inflammatory factors.
2.1 Preparation of solutions
LPS: a mother solution with the concentration of 50 ten thousand units/mL is prepared by using a serum-free 1640 culture solution, filtered and sterilized by a 0.22 mu m filter membrane, stored in a refrigerator at the temperature of minus 20 ℃, and diluted into an action solution with the concentration of 4 ten thousand units/mL before use.
Sample solution to be measured: sample solutions with the final concentrations of 1% and 5% of the experimental materials are prepared by using LPS working solution, and a 0.22 mu m filter membrane is used for filtration sterilization.
2.2 Medicated treatment
Raw264.7 cells were seeded at 1X 10 5/mL in 24-well plates, incubated at 37℃under 5% CO 2 for 24h, samples were added, and incubation was continued for 24h with LPS as model control.
2.3. Detection of
2.3.1 Preparation before detection: the kit should be ready for use after 30 minutes of equilibration at room temperature after removal from the refrigerated environment. Taking cell culture supernatant, centrifuging at 1000rpm for 10min, removing impurities and cell fragments, and taking supernatant for detection. Standards were diluted to 7 different concentrations as indicated.
2.3.2 Sample addition: blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same), standard holes and sample holes to be tested are respectively arranged. And accurately adding 100 mu L of standard substances on the enzyme-labeled coated plate, and adding 100 mu L of samples to be detected into the holes of the samples to be detected. And adding the sample to be detected at the bottom of the ELISA plate hole, and slightly shaking and uniformly mixing without touching the hole wall as much as possible.
2.3.3 Incubation: incubation was carried out for 120min at 37℃after membrane sealing with a sealing plate.
2.3.4 Liquid preparation: the concentrated washing solution was diluted 20 times with distilled water for use.
2.3.5 Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the process for 4 times, and beating.
2.3.6 Adding an antibody working solution: 100. Mu.L of antibody working solution was added to each well.
2.3.7 Incubations for 60 minutes.
2.3.8 Washing: the operation is the same as 2.3.5.
2.3.9 Adding HRP working solution: 100. Mu.L of HRP working solution was added to each well and incubated for 40 minutes.
2.3.10 Washing: the operation is the same as 2.3.5.
2.3.11 Adding TMB working solution: 100. Mu.L of TMB working solution was added to each well and incubated for 15-20min.
2.3.12 Termination: 100. Mu.L of stop solution was added to each well to terminate the reaction (blue turned vertically yellow). The absorbance (OD value) of each well was measured sequentially at the wavelength of blank Kong Diaoling, 450 nm. The measurement should be performed within 15min after the addition of the stop solution.
3. Experimental data
TABLE 12 inhibition of inflammatory factors IL-6, IL-1 beta by experimental materials
The data in Table 12 shows that the Poria cocos fermented filtrate prepared in example 4 has stronger inhibition effect on inflammatory factors IL-6 and IL-1 beta than the Poria cocos water extract filtrate and the Poria cocos yeast fermented filtrate.
Claims (9)
1. A mei-chia yeast (Metschnikowia sp.) characterized by: the preservation number is CGMCC No.29040.
2. A microbial inoculum is characterized in that: comprising the merzizyphus yeast (Metschnikowia sp.) of claim 1.
3. Use of the merthiolase (Metschnikowia sp.) of claim 1 or the microbial inoculum of claim 2 in the preparation of a fermentation product; preferably, the fermentation product is a Poria cocos fermentation product.
4. A preparation method of poria cocos fermentation filtrate is characterized by comprising the following steps: comprises the step of fermenting Poria with Mylabris (Metschnikowia sp.) CGMCC No. 29040.
5. The preparation method according to claim 4, characterized by comprising the steps of:
(1) Mixing Poria powder with water to obtain Poria slurry, and performing ultrasonic and sterilization treatment on Poria slurry;
(2) Inoculating the slurry obtained in the step (1) into Meiqiyeast for fermentation culture to obtain fermented poria cocos slurry; or mixing the slurry obtained in the step (1) with at least one of a carbon source and inorganic salt, and then inoculating with Meiqiyeast for fermentation culture to obtain fermented poria cocos slurry;
(3) And (3) carrying out post-treatment on the fermented poria cocos slurry to obtain poria cocos fermented filtrate.
6. A fermented filtrate of Poria obtained by the process of claim 4 or 5.
7. Use of the merger yeast (Metschnikowia sp.) of claim 1 or the microbial inoculum of claim 2 or the poria cocos fermentation filtrate of claim 6 in the preparation of cosmetics.
8. Use of a merzizyphus yeast (Metschnikowia sp.) according to claim 1 or a microbial inoculum according to claim 2 or a poria cocos fermentation filtrate according to claim 6 in the preparation of a cosmetic, characterized in that: the cosmetic has at least one of the following effects: enhancing skin elasticity, reducing skin wrinkles, inhibiting lipid secretion, inhibiting acne bacillus, relieving, antiinflammatory, and whitening skin.
9. A cosmetic product characterized by: comprising the poria cocos fermented filtrate as claimed in claim 6.
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