WO2021217693A1 - 一种水解假丝酵母和日本清酒酵母共生发酵产物的用途 - Google Patents

一种水解假丝酵母和日本清酒酵母共生发酵产物的用途 Download PDF

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WO2021217693A1
WO2021217693A1 PCT/CN2020/088999 CN2020088999W WO2021217693A1 WO 2021217693 A1 WO2021217693 A1 WO 2021217693A1 CN 2020088999 W CN2020088999 W CN 2020088999W WO 2021217693 A1 WO2021217693 A1 WO 2021217693A1
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yeast
candida
japanese sake
sake yeast
hydrolyzed
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French (fr)
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沈琳
陶侃
杨宇阁
郭莉莉
朱蕙林
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杭州谦美化妆品有限公司
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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
    • A61K8/18—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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

Definitions

  • the present invention relates to the technical field of skin care products, in particular to the field of fermented raw materials for skin care products, and in particular to a method and application for symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast.
  • Aging is a spontaneous and inevitable process of living beings over time. Most people’s skin aging basically begins at the age of 25. It is mainly manifested in increased wrinkles, pigmentation and sagging skin. The determinants of skin aging are the influence of the internal and external environment on collagen, elastin, fibrin, and fibrin in the dermis. The destruction of hyaluronic acid and the loss of skin moisture. 80% of the human dermis is collagen. Its three-dimensional spiral structure supports the skin like a spring, making the skin elastic, shiny and smooth.
  • Collagen is produced by fibroblasts in the skin, exists in the extracellular matrix, provides structural stability for the skin, provides durability for connective tissues, and adheres to the tissues to support cell connection, cell proliferation, and induce differentiation of undifferentiated cells. .
  • Elastin is an important component of the skin. Elastin contains only 5% in the skin, but it is the key to stimulate the production of skin collagen and elastic fibers. Elastic fibers determine the firmness and elasticity of the skin, and 90% of elastic fibers are made of elasticity. Protein composition. Fibrin can increase the firmness and elasticity of the skin and molds. Hyaluronic acid has a special water retention effect. It is the best moisturizing substance in nature found so far. It is called an ideal natural moisturizing factor.
  • connective tissue it is widely present in connective tissue, epithelial tissue and nerve tissue. It is the main extracellular matrix. One of the ingredients. Therefore, it can promote the synthesis of skin collagen, elastin, fibrin and hyaluronic acid, which can achieve a certain anti-aging effect.
  • Japanese sake yeast is preserved by the Japan Brewing Association. Association No. 7 yeast (701 or K701), Nagano’s scientific name Saccharomyces cerevisiae Meyen ex ECHansen, is the most widely used association yeast in sake production. It has a very mellow aroma and is widely used. Used for brewing and ordinary brewing. Nagano Prefecture was separated from Showa 21 (1946).
  • Japanese sake yeast extract has a natural, safe and powerful moisturizing effect. Its yeast cells are rich in natural moisturizing factors. It is very similar to the nutrients required by skin cells and can accelerate skin metabolism. It is rich in vitamins and minerals. 100% natural substances, such as substances, amino acids and organic acids, are easily absorbed by human cells.
  • Candida saitoana is an active substance discovered by Japanese biologist Yoshinori Ohsumi in his research on the mechanism of autophagy. With age, the ability of skin cell renewal continues to weaken, hydrolyzing the active ingredients in Candida can make the diseased and damaged cells eliminated through autophagy, so as to achieve cell renewal and repair.
  • Multi-bacterial symbiotic fermentation involves controlled fermentation of multiple bacteria. Not only can more types of beneficial enzymes be fermented, but the number of enzymes produced is multiple times the number of ordinary single bacteria. The more enzymes, the more beneficial to the human body The stronger the catalytic health function.
  • CN108553403B discloses the co-cultivation of yeast and thermophilic bacteria.
  • the active substance content in the fermentation product can be significantly increased, and the fermentation product can be used to prepare anti-inflammatory, skin moisturizing or skin yellowing agents.
  • the fermentation product can be used to prepare anti-inflammatory, skin moisturizing or skin yellowing agents.
  • Candida hydrolyzed yeast and Japanese sake yeast to prepare anti-aging skin care products through symbiotic fermentation.
  • the present invention provides a The symbiotic fermentation composition of Candida hydrolysate and Japanese sake yeast with its own collagen, elastin, fibrin and hyaluronic acid synthesis functions can be used to prepare anti-aging skin care preparations, which is in line with consumers’ concerns about "natural, green and healthy" The expectation of skin care products has a good anti-aging effect.
  • Candida hydrolyzed yeast and Japanese sake yeast can produce some beneficial active substances, the content of active substances is not high, and the activity is not high. If the Candida hydrolyzed yeast and Japanese sake yeast are cultivated symbiotically, the active substances can be significantly increased through a special cultivation method.
  • the present invention provides a use of the symbiotic fermentation product of Candida hydrolyzate and Japanese sake yeast in the preparation of skin care preparations that promote collagen synthesis.
  • the inventors discovered that the symbiotic fermentation product of Candida hydrolysate and Japanese sake yeast can promote the expression of Collagen I, Collagen III and Collagen IV genes, thereby promoting collagen synthesis.
  • Skin type I and type III collagen (Collagen I, Collagen III) is the most important protein component of the extracellular matrix of the skin dermis. It has the functions of maintaining skin moisture, supporting the normal structure of the skin, and maintaining skin toughness. Fibroblast synthesis and secretion.
  • Skin type IV collagen (Collagen IV) is one of the main structural components of the basement membrane zone of the skin. Promote the expression of Collagen I, Collagen III and Collagen IV genes, promote the production of skin collagen and collagen fibers, and achieve skin anti-aging.
  • the method for symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast includes the following steps:
  • the preservation number of Japanese sake yeast is ATCC26422, which was purchased from the No. 7 yeast of the Japan Brewing Association; the preservation number of Candida hydrolyzed yeast is CICC 31239, which was purchased from the Institute of Microbiology, Chinese Academy of Sciences.
  • Japanese sake yeast is Association No. 7 yeast (701 or K701), Nagano scientific name Saccharomyces cerevisiae Meyen ex E.C. Hansen.
  • step (1) the Japanese sake yeast is cultured for at least two times to increase the bacteria, so that the yeast is in the exponential growth phase; in step (2), the Japanese sake yeast culture solution is inoculated with hydrolyzed candida Before yeast, candida hydrolyzed yeast is cultured one or more times for enrichment.
  • the present invention provides the use of the symbiotic fermentation product of Candida hydrolyzed and Japanese sake yeast in the preparation of skin care formulations that promote the synthesis of elastin.
  • Yeast symbiotic fermentation products can promote the expression of elastin (Elastin) gene, thereby promoting elastin synthesis.
  • Elastin is the main component of elastic fibers. Elastin is an important component of the skin. Elastin is only 5% in the skin, but it is the key to stimulate the production of skin collagen and elastic fibers.
  • the collagen which accounts for 70% of the skin, determines the skin's softness and fullness, and the elastic fibers determine the skin. Tighten elasticity, and 90% of elastic fibers are composed of elastin.
  • the elasticity of the skin depends on the elastic fibers in our skin, which are equivalent to countless rubber bands that support the elastic fiber network of the skin tissue.
  • the reduction of elastin is an important cause of skin aging. Promote the expression of Elastin gene, can promote the formation of elastic fibers, but also stimulate the production of skin collagen and elastic fibers, so as to achieve skin anti-aging.
  • the symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast includes the following steps:
  • the preservation number of Japanese sake yeast is ATCC26422, which was purchased from the No. 7 yeast of the Japan Brewing Association; the preservation number of Candida hydrolyzed yeast is CICC 31239, which was purchased from the Institute of Microbiology, Chinese Academy of Sciences.
  • step (1) the Japanese sake yeast is cultured for at least two times to increase the bacteria, so that the yeast is in the exponential growth phase; in step (2), the Japanese sake yeast culture solution is inoculated with hydrolyzed candida Before yeast, candida hydrolyzed yeast is cultured one or more times for enrichment.
  • the present invention provides a use of the symbiotic fermentation product of Candida hydrolyzate and Japanese sake yeast in the preparation of skin care formulations that promote fibrin synthesis.
  • the inventors discovered that the symbiotic fermentation product of Candida hydrolysate and Japanese sake yeast can promote the expression of fibrillin (Fibrillin-1) gene, thereby promoting fibrin synthesis.
  • Fibrin and elastin are the main components of elastic fibers. Fibrin can increase the firmness and elasticity of the skin and molds. Promote the expression of Fibrillin-1 gene, and also promote the formation of elastic fibers, so as to realize the anti-aging of the skin.
  • the symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast includes the following steps:
  • the preservation number of Japanese sake yeast is ATCC26422, which was purchased from the No. 7 yeast of the Japan Brewing Association; the preservation number of Candida hydrolyzed yeast is CICC 31239, which was purchased from the Institute of Microbiology, Chinese Academy of Sciences.
  • step (1) the Japanese sake yeast is cultured for at least two times to increase the bacteria, so that the yeast is in the exponential growth phase; in step (2), the Japanese sake yeast culture solution is inoculated with hydrolyzed candida Before yeast, candida hydrolyzed yeast is cultured one or more times for enrichment.
  • the present invention provides a use of the symbiotic fermentation product of Candida hydrolyzate and Japanese sake yeast in the preparation of a skin care preparation that promotes the synthesis of hyaluronic acid.
  • the inventors found that the symbiotic fermentation product of Candida hydrolysate and Japanese sake yeast can promote the expression of hyaluronic acid synthase (HAS1) gene, thereby promoting fibrin synthesis.
  • HAS1 hyaluronic acid synthase
  • Hyaluronic acid has a special water-retaining effect. It is the best moisturizing substance found in nature and is called an ideal natural moisturizing factor. Hyaluronic acid is widely present in connective tissue, epithelial tissue and nerve tissue. It is one of the main components of extracellular matrix and can be synthesized by the membrane protein hyaluronic acid synthase (HAS1). Hyaluronic acid synthase can extend the hyaluronic acid chain by alternately adding its substrates uridine diphosphate-aN-acetylglucosamine and uridine diphosphate-aD-glucuronic acid, and newly produced polysaccharides enter through the cell membrane. Extracellular matrix, thereby promoting the synthesis of hyaluronic acid. Promote the expression of the HAS1 gene, promote the synthesis of hyaluronic acid, enhance the skin's moisturizing function, and help the skin resist aging.
  • HAS1 membrane protein hyaluronic acid synthase
  • the symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast described in the present invention includes the following steps:
  • the preservation number of Japanese sake yeast is ATCC26422, which was purchased from the No. 7 yeast of the Japan Brewing Association; the preservation number of Candida hydrolyzed yeast is CICC 31239, which was purchased from the Institute of Microbiology, Chinese Academy of Sciences.
  • step (1) the Japanese sake yeast is cultured for at least two times to increase the bacteria, so that the yeast is in the exponential growth phase; in step (2), the Japanese sake yeast culture solution is inoculated with hydrolyzed candida Before yeast, candida hydrolyzed yeast is cultured one or more times for enrichment.
  • Candida hydrolyzate and Japanese sake yeast includes:
  • Step 1 Enrichment culture of Japanese sake yeast and Candida hydrolysate respectively
  • the method for enriching Japanese sake yeast is as follows:
  • the method of enrichment of Candida hydrolyzed bacteria is as follows:
  • Candida hydrolysate CICC31239 cryopreserved liquid on the ultra-clean workbench and put it into the Erlenmeyer flask containing the culture medium with pH 7.8 ⁇ 0.2, and incubate at 150rpm and 25°C for 16h. After the bacteria are in the exponential growth phase, the bacteria liquid Inoculate into the secondary seed pot.
  • Step 2 One fermentation of Japanese sake yeast
  • the secondary seed liquid of Japanese sake yeast was transferred to a fermenter with a pH of 4.8 ⁇ 0.2, and the aeration rate was maintained at 5m 3 /h/50L, 200 rpm, and 25°C for 48 hours.
  • Step 3 Adding Candida hydrolyzed yeast for secondary fermentation
  • the basic fermentation broth formula is: the broth contains beef extract 10g/L, tryptone 5g/L, casein 3g/L, glucose 5g/L, water-soluble starch 1g/L, dihydrogen phosphate Potassium 1.5g/L, anhydrous sodium acetate 3g/L, pH value is adjusted and controlled with acid or alkali, and the rest is water; the seed solution is inoculated into the culture medium at an inoculum of 3-10%.
  • a separation and purification step is further included, wherein the separation and purification includes a centrifugal sterilization step.
  • Candida hydrolyzed yeast and Japanese sake yeast symbiosis fermentation broth is used to prepare skin care products that promote the synthesis of collagen, elastin, fibrin, and hyaluronic acid as solutions, suspensions, facial masks, lotions, creams, One of paste, gel, dry powder, wet powder, spray.
  • the skin care product preparation is an aqueous agent.
  • the optimal addition concentration of the Candida hydrolyzed and Japanese sake yeast symbiotic fermentation broth is 0.625% when preparing a skin care product that promotes the synthesis of collagen, elastin, fibrin, and hyaluronic acid.
  • the anti-aging skin care product formulations of the present invention include fermented products, and within the range that does not impair the effects of the present invention, various ingredients commonly compounded in the field of skin care products and quasi-drugs, such as powders, can be appropriately blended according to needs.
  • the fermentation product of the present invention has a significant improvement effect compared to the separate Japanese sake yeast fermentation product and Candida hydrolysate fermentation product, and the reagent after the fermentation broth is mixed. It can be seen that the co-cultivation of the two bacteria has a mutual promotion effect, The active content or activity is greatly improved.
  • the present invention develops a symbiotic fermentation product of Candida hydrolysate and Japanese sake yeast with anti-aging effects, which can significantly promote the expression of Collagen I, Collagen III and Collagen IV genes, and at the same time significantly promote Elastin, Fibrillin-1 and Has- 1 Gene expression can promote the production of skin collagen and collagen fibers, improve the renewal of human collagen, promote the synthesis of skin elastin and fibrin, and promote the synthesis of skin hyaluronic acid, improve skin moisturizing ability, and the fermentation product It also contains many enzymes and trace elements, which have supplementary and synergistic effects, so as to achieve a better anti-aging effect.
  • the present invention also provides a method for symbiosis culture of Candida hydrolyzed yeast and Japanese sake yeast, through which the symbiosis culture can significantly increase the active substance of the fermentation product.
  • Figure 1 is a sample cell viability curve for the cytotoxicity test of fibroblasts in Example 5
  • Figure 2 is an electron micrograph of the cell morphology test results of the sample obtained from the cytotoxic morphology test of fibroblasts in Example 6
  • Fig. 3 is a curve of gene expression changes in the solvent control group and the positive control group based on the fluorescence quantitative PCR of fibroblasts in Example 7
  • Fig. 4 is a curve of gene expression changes in the solvent control group and sample group 1 based on the fluorescence quantitative PCR of fibroblasts in Example 7
  • Example 1 Symbiotic fermentation of Candida hydrolyzed yeast and Japanese sake yeast
  • the basic fermentation broth formula is: the broth contains beef extract 10g/L, tryptone 5g/L, casein 3g/L, glucose 5g/L, water-soluble starch 1g/L, and potassium dihydrogen phosphate 1.5g/L , Anhydrous sodium acetate 3g/L, pH value is adjusted and controlled with acid or alkali, and the rest is water. Different amounts of culture medium are reduced and enlarged according to the ratio of ingredients.
  • the preservation number of Japanese sake yeast is ATCC26422, purchased from the No. 7 yeast of the Japan Brewing Association; the preservation number of Candida hydrolyzed yeast is CICC 31239, purchased from the Institute of Microbiology, Chinese Academy of Sciences.
  • Step 1 Enrichment culture of Japanese sake yeast and Candida hydrolyzed
  • step (3) Inoculate the 3L strains cultured in step (2) into a 50L first-level seed tank containing 30L of culture solution with a pH of 4.8 ⁇ 0.2, and maintain the aeration rate at 5m 3 /h/50L, 200rpm, 25 Cultivate for 48h at °C.
  • Candida hydrolysate CICC31239 cryopreservation liquid Take 2ml of Candida hydrolysate CICC31239 cryopreservation liquid on the ultra-clean workbench and put it into a 2.5L Erlenmeyer flask containing 2L of culture medium with a pH of 7.8 ⁇ 0.2, culture at 150rpm and 25°C for 16h, the bacteria are in the exponential growth phase Afterwards, 30L of bacterial solution was inoculated into a 500L secondary seed tank containing 300L of culture solution.
  • Step 2 One fermentation of Japanese sake yeast
  • Step 3 Adding Candida hydrolyzed yeast for secondary fermentation
  • the culture broth is centrifuged to sterilize, and the supernatant is retained, which is the symbiotic fermentation product of Candida hydrolysate and Japanese sake yeast.
  • the basic fermentation broth formula is: the broth contains beef extract 10g/L, tryptone 5g/L, casein 3g/L, glucose 5g/L, water-soluble starch 1g/L, and potassium dihydrogen phosphate 1.5g/L , Anhydrous sodium acetate 3g/L, pH value is adjusted and controlled with acid or alkali, and the rest is water. Different amounts of culture medium are reduced and enlarged according to the ratio of ingredients.
  • the preservation number of Japanese sake yeast is ATCC26422, and it was purchased from the No. 7 yeast of the Japan Brewing Association.
  • Step 1 Japanese sake yeast enrichment culture
  • step (3) Inoculate the 3L strains cultured in step (2) into a 50L first-level seed tank containing 30L of culture solution with a pH of 4.8 ⁇ 0.2, and maintain the aeration rate at 5m 3 /h/50L, 200rpm, 25 Cultivate for 48h at °C.
  • Step 2 Japanese sake yeast fermentation culture
  • step 2 Take the fermentation broth of step 2 and perform centrifugal sterilization, and retain the supernatant, which is the fermentation product of Japanese sake yeast.
  • the basic fermentation broth formula is: the broth contains beef extract 10g/L, tryptone 5g/L, casein 3g/L, glucose 5g/L, water-soluble starch 1g/L, and potassium dihydrogen phosphate 1.5g/L , Anhydrous sodium acetate 3g/L, pH value is adjusted and controlled with acid or alkali, and the rest is water. Different amounts of culture medium are reduced and enlarged according to the ratio of ingredients.
  • the Candida hydrolysate strain number is CICC 31239, purchased from Shanghai Institute of Industrial Microbiology.
  • Step 1 Enrichment culture of Candida hydrolyzed
  • Candida hydrolysate CICC31239 cryopreservation liquid Take 2ml of Candida hydrolysate CICC31239 cryopreservation liquid on the ultra-clean workbench and put it into a 2.5L Erlenmeyer flask containing 2L of culture medium with a pH of 7.8 ⁇ 0.2, culture at 150rpm and 25°C for 16h, the bacteria are in the exponential growth phase Afterwards, 30L of bacterial solution was inoculated into a 500L secondary seed tank containing 300L of culture solution.
  • Step 2 Fermentation and cultivation of Candida hydrolyzed
  • Example 4 Reagents obtained by mixing the fermentation products of Examples 2 and 3
  • Cytotoxicity was detected by fibroblast MTT detection method.
  • Human skin fibroblasts in the exponential growth phase were collected, digested and seeded into 96-well plates.
  • the symbiotic fermentation products of Candida hydrolysate and Japanese sake yeast obtained in Example 1 were configured into test substances of different concentrations according to Table 1, and 8% DMSO was used as a positive control.
  • the cell plating rate reaches 40%, the symbiotic fermentation product of Candida hydrolyzate and Japanese sake yeast is added, and then the culture plate is placed in a 37°C, 5% CO 2 incubator for culture. The cells were incubated for 24 hours, the supernatant was discarded, the pre-prepared MTT was added, and the cells were incubated at 37°C in the dark. After 4h, discard the supernatant, add 150 ⁇ L dimethyl sulfoxide to each well, and read the OD value with a microplate reader at 490nm.
  • TGF- ⁇ 1 transforming growth factor- ⁇
  • the experiment set up 4 sample groups, 1 solvent control group and 1 positive control group, with 4 replicate holes in each group.
  • Four sample groups were added with 0.625% of the culture broth of the symbiotic fermentation product of Example 1, the single fermentation broth of Japanese sake yeast in Example 2, the single fermentation broth of Candida hydrolyzate in Example 3, and the culture broth of Example 4
  • the mixture of fermentation products, the solvent control group was added with fresh culture medium, and the positive control group was added with TGF- ⁇ 1 culture medium, and incubated in a 37°C, 5% CO 2 incubator for 24 hours. After the incubation, the culture medium was discarded, and 1 mL of total RNA extraction reagent Trizol was added to each well. After the cells were lysed by pipetting, the samples were collected.
  • RNA was extracted, reverse transcribed into cDNA, and then subjected to fluorescence quantitative PCR detection.
  • the 2- ⁇ CT method was used for calculation of the results, and the T-Test method was used for statistical analysis.
  • TGF- ⁇ 1 fluorescent quantitative PCR detection results of the positive control group are shown in Table 4 and Figure 3
  • fluorescent quantitative PCR detection results of the sample group are shown in Table 5 and Figure 4.
  • the standard deviation of the results obtained in the solvent control group and the positive control group is less than or equal to 0.664%, and the accuracy of the experimental results is very high.
  • the experimental data was analyzed by variance analysis, and it was found that the expression of CollagenI, CollagenIII, CollagenIV, Elastin and Fibrillin1 genes between the solvent control and the positive control group showed a significant difference in the amplification factor (P value less than 0.001) (the specific data is omitted).
  • TGF- ⁇ 1 promotes the formation and reconstruction of the matrix, indicating that this test is effective.
  • sample group 1 has a significant effect on the expression of Collagen I, Collagen III and Collagen IV at the supplemental dosage of 0.625% Candida hydrolyzed and Japanese sake yeast symbiotic fermentation products.
  • the amplification factor reached 1.512, 2.564, and 2.428 times respectively; it significantly promoted the expression of Elastin gene and Fibrillin-1 gene, and the amplification factor reached 2.152 and 1.833 times respectively; it significantly promoted the expression of Has-1 gene,
  • the amplification factor is as high as 11.024 times.
  • the experimental data was analyzed by variance analysis, and it was found that the expression of CollagenI, CollagenIII, CollagenIV, Elastin, Fibrillin1 and Has-1 genes in solvent control compared with sample group 1 showed a significant difference in amplification factor (P value less than 0.002) (specifically) Data omitted). It shows that the symbiotic fermentation product of Candida hydrolyzate and Japanese sake yeast has a superior effect on skin anti-aging under the additive dosage of 0.625%.
  • the sample group 2 has no obvious promotion effect on the expression of Collagen I, Collagen III and Collagen IV at the dosage of 0.625% Japanese sake yeast single fermentation product, but has no obvious effect on the expression of Elastin gene and Fibrillin.
  • the expression of -1 gene and Has-1 gene also had no obvious promoting effect.
  • the experimental data was analyzed by variance analysis, and it was found that the expression of CollagenI, CollagenIII, CollagenIV, Elastin, Fibrillin1 and Has-1 genes in solvent control compared with sample group 2 showed a significant difference in amplification factor (P value less than 0.002) (specifically Data omitted). It shows that adding 0.625% Japanese sake yeast separately fermented product has no obvious effect on skin anti-aging.
  • the sample group 3 has a significant promotion effect on the expression of Collagen I, Collagen III and Collagen IV at a dosage of 0.625% Candida hydrolysate single fermentation product, and the amplification factor reaches 1.305, respectively. 1.882 and 1.989 times; it also significantly promoted the expression of Elastin gene, Fibrillin-1 gene and Has-1 gene, and the amplification factor reached 1.905, 1.692 and 6.003 times, respectively.
  • the standard deviation of the results obtained from the solvent control group and the sample group is less than or equal to 0.754%, and the experimental results are highly accurate.
  • the experimental data was analyzed by variance analysis, and it was found that the expression of CollagenI, CollagenIII, CollagenIV, Elastin, Fibrillin1 and Has-1 genes in solvent control compared with sample group 3 showed a significant difference in amplification factor (P value less than 0.002) (specifically) Data omitted). It shows that adding 0.625% hydrolyzed Candida alone fermented product has certain effect in anti-aging skin, but its effect is obviously not as good as 0.625% hydrolyzed Candida and Japanese sake yeast symbiosis fermentation broth.
  • the experimental data was analyzed by variance analysis, and it was found that the expression of CollagenI, CollagenIII, CollagenIV, Elastin, Fibrillin1 and Has-1 genes in solvent control compared with sample group 4 showed a significant difference in amplification factor (P value less than 0.001) (specifically Data omitted). It shows that adding 0.625% hydrolyzed Candida yeast and Japanese sake yeast fermentation product mixture has certain effects in anti-aging skin, but its effect is obviously not as good as 0.625% hydrolyzed Candida yeast and Japanese sake yeast symbiosis fermentation liquid.
  • the Candida hydrolyzed and Japanese sake yeast symbiosis fermentation broth of the present invention is relatively independent of the fermentation product of Japanese sake yeast and the fermentation product of Candida hydrolysate, and the mixed reagent of the fermentation broth.
  • the promotion effect of Elastin, Fibrillin1 and Has-1 gene expression all have a significant improvement effect. It can be seen that the co-cultivation of the two bacteria has mutual promotion and obvious synergistic effect, and the active content or activity of the active substance is greatly improved.

Abstract

水解假丝酵母和日本清酒酵母共生发酵产物用于制备促进胶原蛋白、弹力蛋白、纤维蛋白、透明质酸合成的护肤品制剂的用途，该发酵产物能显著促进Collagen Ⅰ、CollagenⅢ和Collagen Ⅳ基因的表达，显著促进Elastin和Fibrillin-1基因的表达，显著促进Has-1基因的表达。

Description

一种水解假丝酵母和日本清酒酵母共生发酵产物的用途 技术领域
本发明涉及护肤品技术领域，具体而言，涉及护肤品的发酵原料领域，尤其涉及一种水解假丝酵母和日本清酒酵母共生发酵的方法和用途。
背景技术
衰老是生物随着时间的推移自发的必然过程。大多数人的皮肤衰老基本从25岁就开始了，主要表现在皱纹增多，色素沉着和皮肤松垂等，而皮肤老化的决定因素在于内外环境对真皮层内胶原蛋、弹力蛋白、纤维蛋白、透明质酸的破坏以及皮肤水分的流失等。人真皮层80％是胶原蛋白，它的三维螺旋结构，如弹簧般支撑着皮肤，使皮肤富有弹性、光泽和平滑。胶原蛋白由皮肤中的成纤维细胞生成，存在于细胞外基质中，为皮肤提供结构稳定性，为结缔组织提供耐久性及粘结组织以支持细胞连接、细胞增生和诱导未分化细胞分化等作用。弹力蛋白是肌肤组成的重要成分，弹力蛋白在肌肤中仅含5％，却是激发肌肤胶原蛋白和弹性纤维生成的关键，弹性纤维决定肌肤的紧致弹力，而弹性纤维的90％是由弹力蛋白组成。纤维蛋白可增加皮肤和粘模的紧致度和弹性。透明质酸具有特殊的保水作用，是目前发现的自然界中保湿性最好的物质，被称为理想的天然保湿因子，广泛存在于结缔组织、上皮组织和神经组织中，是细胞外基质的主要成分之一。因此，促进肌肤的胶原蛋白、弹力蛋白、纤维蛋白和透明质酸的合成，能达到一定的抗衰老效果。
近年来研究表明，促进胶原蛋白生成和给皮肤补充胶原蛋白就能减轻皱纹、增加皮肤弹性和强度的作用，从而达到抗衰老的作用。目前市面已开发出的一些含胶原蛋白的护肤品涂抹于皮肤表面之上，由于高分子量的胶原蛋白，难以透皮吸收，抗衰老效果却不理想，并不能改善皮肤的外观，且安全性、功效性也被消费者质疑。据《己使用化妆品原料名称目录(2015版)》，目前可在化妆品中添加的酵母类相关原料名称已经很多，由此可见，在化妆品中添加酵母提取物是潮流趋势，同时，发酵产物是纯生物的过程，没有任何化工成分的添加，符合消费者对于“天然、绿色、健康”护肤品的期待，这一点在护肤品市场上也得到了充分证实。
日本清酒酵母由日本酿造协会保存，协会7号酵母(701 or K701)，长野学名Saccharomyces cerevisiae Meyen ex E.C.Hansen，是在清酒制造中最被广泛使用的协会酵母， 具有很醇的芳香，广泛地被用于呤酿和普通酿造。由昭和21年(1946年)的长野县被分离出来。日本清酒酵母提取物具有天然、安全、强效的保湿功效，其酵母细胞中含有丰富的天然保湿因子，它与肌肤细胞所需的养分非常相近，能够加快肌肤新陈代谢，它含有丰富的维他命、矿物质、氨基酸及有机酸等百分百天然物质，所以极易被人体细胞所吸收。
水解假丝酵母(Candida saitoana)是日本生物学家大隅良典在研究细胞自噬机制中发现的活性物。在随着年龄增长，皮肤细胞更新能力不断减弱的情况下，水解假丝酵母中的活性成分，可以让病变受损的细胞，通过自噬作用消除，以此来达到细胞更新修护。
现有传统技术中，都是单菌种进行分别完成发酵后，再进行两种发酵液进行勾兑。不同的菌群和酶类，溶解到一起发生的抵消反应，对损伤大量的菌群和酶活性，往往实际效果不理想。多菌共生发酵即将多种菌进行可控性发酵，不仅可以发酵出种类更多的有益酶，还能使酶产出的数量是普通单菌种发酵数量的多倍，酶越多，对人体的催化保健功能越强。CN108553403B公开了酵母菌和嗜热菌共同培养，经过特殊的培养方法，可显著提升发酵产物中的活性物质含量，发酵产物并可用于制作消炎、皮肤保湿或皮肤祛黄试剂。但是目前还没有利用水解假丝酵母和日本清酒酵母共生发酵来制备抗衰老护肤品的相关报道。
发明内容
针对现有的抗衰老护肤品，普遍为含胶原蛋白的护肤品，难以更好的透皮吸收，使用周期长，且安全性、功效性得不到保证等问题，本发明提供一种具有促进自身胶原蛋白、弹力蛋白、纤维蛋白和透明质酸合成等功效的水解假丝酵母和日本清酒酵母共生发酵组合物，可用于制备抗衰老护肤品制剂，符合消费者对于“天然、绿色、健康”护肤品的期待，拥有较好的抗衰老效果。
虽然水解假丝酵母和日本清酒酵母单独培养可以产生一些有益的活性物质，但活性物质含量并不高，活性也不高。如果让水解假丝酵母和日本清酒酵母共生培养，经过特殊的培养方法，可以显著提升活性物质。
一方面，本发明提供了一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进胶原蛋白合成的护肤品制剂方面的用途。本发明人发现，所述的水解假丝酵母和日本清酒酵母共生发酵产物能够促进Collagen Ⅰ、CollagenⅢ和CollagenⅣ基因的表达，从而促进胶原蛋白合成。
皮肤Ⅰ、Ⅲ型胶原蛋白(Collagen Ⅰ、CollagenⅢ)是构成皮肤真皮细胞外基质最主要的蛋白成分，具有保持皮肤水分、支撑皮肤正常结构及维持皮肤韧性的功能，主要分布于 真皮层，仅由成纤维细胞合成和分泌。皮肤Ⅳ型胶原蛋白(Collagen Ⅳ)是皮肤基底膜带的主要结构成分之一。促进Collagen Ⅰ、CollagenⅢ和Collagen Ⅳ基因的表达，能促进肌肤胶原蛋白和胶原纤维生成，达到肌肤抗衰老。
为实现该用途，本发明所述的水解假丝酵母和日本清酒酵母共生发酵方法包括如下步骤：
(1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
(2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
日本清酒酵母为协会7号酵母(701 or K701)，长野学名Saccharomyces cerevisiae Meyen ex E.C.Hansen。
进一步地，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
另一方面，本发明提供了一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进弹力蛋白合成的护肤品制剂方面的用途，本发明人发现，所述的水解假丝酵母和日本清酒酵母共生发酵产物能够促进弹力蛋白(Elastin)基因的表达，从而促进弹力蛋白合成。
弹力蛋白是弹性纤维的主要成分。弹力蛋白是肌肤组成的重要成分，弹力蛋白在肌肤中仅含5％，却是激发肌肤胶原蛋白和弹性纤维生成的关键，占肌肤70％的胶原蛋白决定肌肤的柔润饱满，弹性纤维决定肌肤的紧致弹力，而弹性纤维的90％是由弹力蛋白组成。肌肤的弹性取决于我们肌肤里层的弹性纤维，它相当于无数条橡皮筋，支撑起肌肤组织的弹性纤维网。弹力蛋白减少，是肌肤衰老的重要原因。促进Elastin基因的表达，能促进弹力纤维的形成，还能激发肌肤胶原蛋白和弹性纤维生成，从而实现肌肤抗衰老。
为实现该用途，本发明所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
(1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
(2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
进一步地，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
另一方面，本发明提供了一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进纤维蛋白合成的护肤品制剂方面的用途。本发明人发现，所述的水解假丝酵母和日本清酒酵母共生发酵产物能够促进纤维蛋白(Fibrillin-1)基因的表达，从而促进纤维蛋白合成。
纤维蛋白和弹力蛋白都是弹性纤维的主要成分。纤维蛋白可增加皮肤和粘模的紧致度和弹性。促进Fibrillin-1基因的表达，也能促进弹力纤维的形成，从而实现肌肤抗衰老。
为实现该用途，本发明所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
(1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
(2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
进一步地，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
再一方面，本发明提供了一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进透明质酸合成的护肤品制剂方面的用途。本发明人发现，所述的水解假丝酵母和日本清酒酵母共生发酵产物能够促进透明质酸合成酶(HAS1)基因的表达，从而促进纤维蛋白合成。
透明质酸具有特殊的保水作用，是目前发现的自然界中保湿性最好的物质，被称为理想的天然保湿因子。透明质酸广泛存在于结缔组织、上皮组织和神经组织中，是细胞外基质的主要成分之一，可以由膜蛋白透明质酸合成酶(HAS1)合成的。透明质酸合成酶可通过交替反复加入其底物尿苷二磷酸-a-N-乙酰葡糖胺及尿苷二磷酸-a-D-葡萄糖醛酸而延长透明质酸链，新产生的多糖透过细胞膜进入细胞外间质，从而促进透明质酸的合成。促进HAS1基因的表达，能促进透明质酸的合成，提升肌肤保湿功能，帮助肌肤抵抗衰老。
为实现该用途，本发明所述的所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
(1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
(2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
进一步地，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
进一步地，所述的水解假丝酵母和日本清酒酵母共生发酵包括：
步骤1：分别对日本清酒酵母和水解假丝酵母进行增菌培养；
其中，日本清酒酵母增菌方法如下：
在超净工作台上取试管斜面上的日本清酒酵母ATCC26422一环，接入装有pH为4.8±0.2的培养液的三角瓶中，200rpm，25℃培养48h，菌体处于指数生长期；将处于指数生长期的菌液作为一级种子液，按照3-10％的接种量接种于二级种子罐，在25℃，pH为4.8±0.2培养48h。
其中，水解假丝酵母增菌方法如下：
在超净工作台上取水解假丝酵母CICC31239冻存液接入装有pH为7.8±0.2的培养液的三角瓶中，150rpm，25℃培养16h，菌体处于指数生长期后，将菌液接种到二级种子罐中。
步骤2：日本清酒酵母的一次发酵
将日本清酒酵母二级种子液转接至装pH为4.8±0.2的发酵罐中，通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
步骤3：添加水解假丝酵母进行二次发酵
将水解假丝酵母增菌液转移到发酵罐中，150rpm搅拌，用碱液将pH值调节到7.8±0.2，25℃培养24h。
其中，所述的发酵基本培养液配方为：培养液中含有牛肉浸膏10g/L、胰蛋白胨5g/L，酪蛋白3g/L，葡萄糖5g/L，水溶性淀粉1g/L，磷酸二氢钾1.5g/L，无水醋酸钠3g/L， pH值用酸或碱进行调节控制，其余为水；种子液按3-10％的接种量接种至培养基液。
其中，步骤3的二次发酵后，还包括进行分离纯化步骤，其中所述的分离纯化包括离心除菌步骤。
进一步地，所述的水解假丝酵母和日本清酒酵母共生发酵液在制备促进胶原蛋白、弹力蛋白、纤维蛋白、透明质酸合成的护肤品制剂为溶液、悬浮液、面膜、乳液、霜剂、膏状、凝胶、干粉、湿粉、喷剂中的一种。
进一步地，所述的护肤品制剂为水剂。
进一步地，所述的水解假丝酵母和日本清酒酵母共生发酵液在制备促进胶原蛋白、弹力蛋白、纤维蛋白、透明质酸合成的护肤品制剂时的最佳添加浓度为0.625％。
本发明所述的抗衰老护肤品制剂除了包括发酵产物外，在不损本发明效果的范围内，可以根据需要而适宜配合在护肤品品、准药品领域中通常配合的各种成分，例如粉末成分、保湿剂、乳化剂、光稳定剂、增稠剂、增溶剂、金属离子掩蔽剂、色素、pH调节剂、皮肤营养剂、维生素、防腐剂、抗氧化剂、防晒剂、促渗剂、脂质体、皮肤营养组分、抗氧化助剂、香精、香料、色素等中的任意一种或几种的混合。
本发明的发酵产物相对单独的日本清酒酵母发酵产物和水解假丝酵母发酵产物，以及发酵液混合后的试剂，都具有显著的改善作用，可见两种菌共同培养具有相互促进作用，活性物质的活性含量或活性都大大提高。
本发明开发了对一种具有抗衰老功效的水解假丝酵母和日本清酒酵母共生发酵产物，能显著促进进Collagen Ⅰ、CollagenⅢ和Collagen Ⅳ基因的表达，同时显著促进Elastin、Fibrillin-1和Has-1基因的表达，能促进肌肤胶原蛋白和胶原纤维生成，改善人体胶原蛋白的更新，促进肌肤弹力蛋白和纤维蛋白的合成，并促进肌肤透明质酸的合成，提高皮肤保湿能力，且发酵产物中也含有很多酶类和微量元素，有补充增效作用，从而达到更好的抗衰老效果，可用于制备抗衰老护肤品制剂，符合消费者对于“天然、绿色、健康”护肤品的期待。本发明还提供了水解假丝酵母和日本清酒酵母共生培养方法，通过共生培养，可显著提升发酵产物的活性物质。
附图说明
图1为实施例5成纤维细胞的细胞毒性检测的样品细胞活力曲线
图2为实施例6成纤维细胞的细胞毒性形态学检测所得的样品细胞形态学检测结果电子显微镜照片
图3为实施例7基于成纤维细胞的荧光定量PCR的溶剂对照组和阳性对照组基因表达量变化曲线
图4为实施例7基于成纤维细胞的荧光定量PCR的溶剂对照组和样品组1基因表达量变化曲线
具体实施方式
下面结合实施例对本发明作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。
实施例1水解假丝酵母和日本清酒酵母共生发酵
发酵基本培养液配方为：培养液中含有牛肉浸膏10g/L、胰蛋白胨5g/L，酪蛋白3g/L，葡萄糖5g/L，水溶性淀粉1g/L，磷酸二氢钾1.5g/L，无水醋酸钠3g/L，pH值用酸或碱进行调节控制，其余为水。不同量的培养液按照成分比例进行缩小和放大。
本发明所有实施例都采用以上配比的培养基配方作为菌种发酵的培养液。当然，在实际工业生产中，可以根据不同的要求进行配比，这些配方都是培养日本清酒酵母和/或水解假丝酵母的普通配方。
日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
步骤1：分别对日本清酒酵母和水解假丝酵母进行增菌培养
日本清酒酵母增菌：
(1)在超净工作台上取试管斜面上的日本清酒酵母ATCC26422一环，接入装有50ml的pH为4.8±0.2的培养液的250ml三角瓶中，共接2瓶，200rpm，25℃培养48h，菌体处于指数生长期。
(2)将处于指数生长期的菌体80ml接种到装有2L，pH为4.8±0.2的培养液的2.5L三角瓶中，接种量为体积百分数的10％，200rpm，25℃培养48h。
(3)将步骤(2)培养后的3L菌种接种到装有30L，pH为4.8±0.2的培养液的50L一级种子罐中，通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
(4)将30L菌液转移到装有300L培养液的500L二级种子罐中；通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
水解假丝酵母增菌：
在超净工作台上取水解假丝酵母CICC31239冻存液2ml接入装有2L，pH为7.8±0.2 的培养液的2.5L三角瓶中，150rpm，25℃培养16h，菌体处于指数生长期后，将30L菌液接种到装有300L培养液的500L二级种子罐中。
步骤2：日本清酒酵母的一次发酵
将步骤(4)的300L菌液转接至装有2400L，pH为4.8±0.2的培养液的5000L发酵罐中，通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
步骤3：添加水解假丝酵母进行二次发酵
将300L水解假丝酵母增菌液转移到上述5000L发酵罐中，通气量维持在5m 3/h/50L，150rpm搅拌，用碱液将pH值调节到7.8±0.2，25℃培养24h。
步骤4：分离纯化
经过二次发酵后的培养液进行离心除菌，保留上清液，即为水解假丝酵母和日本清酒酵母共生发酵产物。
实施例2日本清酒酵母单独发酵
发酵基本培养液配方为：培养液中含有牛肉浸膏10g/L、胰蛋白胨5g/L，酪蛋白3g/L，葡萄糖5g/L，水溶性淀粉1g/L，磷酸二氢钾1.5g/L，无水醋酸钠3g/L，pH值用酸或碱进行调节控制，其余为水。不同量的培养液按照成分比例进行缩小和放大。
日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母。
步骤1：日本清酒酵母增菌培养
(1)在超净工作台上取试管斜面上的日本清酒酵母ATCC26422一环，接入装有50ml的pH为4.8±0.2的培养液的250ml三角瓶中，共接2瓶，200rpm，25℃培养48h，菌体处于指数生长期。
(2)将处于指数生长期的80ml菌体接种到装有2L，pH为4.8±0.2的培养液的2.5L三角瓶中，接种量为体积百分数的10％，200rpm，25℃培养48h。
(3)将步骤(2)培养后的3L菌种接种到装有30L，pH为4.8±0.2的培养液的50L一级种子罐中，通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
(4)将30L菌液转移到装有300L培养液的500L二级种子罐中；通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
步骤2：日本清酒酵母发酵培养
将300L二级种子罐中的日本清酒酵母培养液转接至装有2700L，pH为4.8±0.2的培养液的5000L发酵罐中，通气量维持在5m 3/h/50L，200rpm，25℃培养48h。
步骤3：分离纯化
取步骤2的发酵培养液，进行离心除菌，保留上清液，即为日本清酒酵母发酵产物。
实施例3水解假丝酵母单独发酵
发酵基本培养液配方为：培养液中含有牛肉浸膏10g/L、胰蛋白胨5g/L，酪蛋白3g/L，葡萄糖5g/L，水溶性淀粉1g/L，磷酸二氢钾1.5g/L，无水醋酸钠3g/L，pH值用酸或碱进行调节控制，其余为水。不同量的培养液按照成分比例进行缩小和放大。
水解假丝酵母菌种编号为CICC 31239，购于上海市工业微生物研究所。
步骤1：水解假丝酵母增菌培养
在超净工作台上取水解假丝酵母CICC31239冻存液2ml接入装有2L，pH为7.8±0.2的培养液的2.5L三角瓶中，150rpm，25℃培养16h，菌体处于指数生长期后，将30L菌液接种到装有300L培养液的500L二级种子罐中。
步骤2：水解假丝酵母发酵培养
将300L水解假丝酵母增菌液转移转接至装有2700L，pH为7.8±0.2的培养液的5000L发酵罐中，150rpm搅拌，25℃培养24h。
步骤3：分离纯化
取步骤2的发酵培养液，进行离心除菌，保留上清液，即为水解假丝酵母发酵产物。实施例4实施例2和3的发酵产物混合获得的试剂
取实施例2和3的发酵产物，按照重量进行1:1比例混合而成的复合试剂。
实施例5成纤维细胞的细胞毒性检测(MTT检测法)
采用成纤维细胞MTT检测法进行细胞毒性检测。收集处于指数生长期的人皮肤成纤维细胞，消化后接种至96孔板。取实施例1获得的水解假丝酵母和日本清酒酵母共生发酵产物，按照表1配置成不同浓度的受试物，并采用8％DMSO作为阳性对照。待细胞铺板率达到40％时，加入水解假丝酵母和日本清酒酵母共生发酵产物，然后将培养板放置在37℃、5％的CO 2培养箱中培养。细胞孵育培养24h，弃掉上清，加入事先配好的MTT，37℃避光孵育。4h后弃掉上清，每孔加150μL二甲基亚砜，酶标仪490nm读取OD值。
表1测试浓度设定表
序号 ① ② ③ ④ ⑤ ⑥ ⑦ ⑧
浓度(v/v) 0.078％ 0.156％ 0.313％ 0.625％ 1.25％ 2.5％ 5％ 10％
根据读取的OD值，代入计算公式：(给药孔OD-调零孔OD)/(对照孔OD-调零孔OD) ×100％＝细胞活力，得到细胞活力检测结果见表2，细胞活力变化趋势如图1所示的细胞活力曲线。
表2样品细胞毒性检测结果
Figure PCTCN2020088999-appb-000001
根据表2和图1所示，当水解假丝酵母和日本清酒酵母共生发酵产物在成纤维细胞上的添加浓度由1.25％增大至2.5％时，成纤维细胞的细胞活力明显下降，由84.5％骤降至41.19％；当共生发酵产物的浓度继续增大，细胞活力不断下降。
标准偏差分析：经过多次重复试验，对实验数据进行方差分析，发现水解假丝酵母和日本清酒酵母共生发酵产物在成纤维细胞上的添加浓度由0.078％-10％范围内变化时，标准偏差≤5.19％，实验结果精确度较高。
方差分析：经过多次重复试验，对实验数据进行方差分析，发现水解假丝酵母和日本清酒酵母共生发酵产物在成纤维细胞上的添加浓度由0.313％-10％范围内变化时，体现的成纤维细胞的细胞活力达到极显著差异(P值小于0.01)(具体数据略)。
实施例6成纤维细胞的细胞毒性形态学检测
根据实施例5的成纤维细胞的细胞毒性检测(MTT检测法)实验结果，选择如表3所示的水解假丝酵母和日本清酒酵母共生发酵产物在成纤维细胞上添加的五个浓度进行形态学检测。
表3样品细胞毒性检测结果
Figure PCTCN2020088999-appb-000002
设立样品组与溶剂对照组，每组设两个复孔。按相应的接种密度接种处于指数生长期的人成纤维细胞至24孔板中，37℃、5％的CO 2培养箱中孵育培养过夜。待24孔板细胞铺板率达到40％时，进行给药，样品组加入不同浓度共生发酵产物，溶剂对照组加入新鲜培养液，37℃、5％的CO 2培养箱中孵育培养24h。孵育结束后，显微镜下观察细胞形态并拍 照，结果如图2所示。根据图2的形态学结果和毒性检测结果，水解假丝酵母和日本清酒酵母共生发酵产物的添加浓度达到1.25％时，细胞形态开始发生变化，出现部分纤维细胞破坏的现象，因此共生发酵产物在成纤维细胞上的最大安全浓度为0.625％。由此可见水解假丝酵母和日本清酒酵母共生发酵液在制备促进胶原蛋白、弹力蛋白、纤维蛋白、透明质酸合成等护肤品制剂时的最佳添加浓度为0.625％。
实施例7基于成纤维细胞的荧光定量PCR检测
以1.8*10 5/孔的接种密度，接种细胞至6孔板中，37℃，5％CO 2培养箱孵育过夜；配制0.625％的发酵产物，以转化生长因子-β(TGF-β1)作为阳性对照。待6孔板中细胞铺板率达到50％左右时，进行给药。
实验设立4个样品组，1个溶剂对照组及1个阳性对照组，每组设4个复孔。4个样品组分别加入含有0.625％的实施例1的共生发酵产物的培养液、实施例2的日本清酒酵母单独发酵培养液、实施例3的水解假丝酵母单独发酵培养液和实施例4的发酵产物混合液，溶剂对照组加入新鲜培养液，阳性对照组加入含TGF-β1的培养液，37℃，5％CO 2培养箱分别孵育培养24h。孵育培养结束后，弃掉培养液，每孔加入1mL总RNA抽提试剂Trizol，吹打裂解细胞后，收样。
提取RNA，反转录至cDNA后，进行荧光定量PCR检测。采用2 -△△CT方法进行结果计算，T-Test方法进行统计分析。
阳性对照组TGF-β1荧光定量PCR检测结果如表4和图3所示，样品组荧光定量PCR检测结果如表5和图4所示。
表4溶剂对照组和阳性对照组基因检测结果
Figure PCTCN2020088999-appb-000003
表5溶剂对照组和样品组1基因检测结果
Figure PCTCN2020088999-appb-000004
表6溶剂对照组和样品组2基因检测结果
Figure PCTCN2020088999-appb-000005
表7溶剂对照组和样品组3基因检测结果
Figure PCTCN2020088999-appb-000006
表8溶剂对照组和样品组4基因检测结果
Figure PCTCN2020088999-appb-000007
由表4和图3可以看出，与溶剂对照相比，阳性对照组加入TGF-β1后，在50ng/mL的暴露剂量下，对CollagenⅠ、CollagenⅢ及CollagenⅣ的表达有显著促进作用，扩增倍数分别达到1.674、1.714和3.147倍；对Elastin基因和Fibrillin1基因的表达有显著促进作用，扩增倍数都分别达到2.1倍；对Has-1基因的表达有显著促进作用，扩增倍数达到7.627倍。经过多次反复试验，溶剂对照组和阳性对照组所得结果的标准偏差≤0.664％，实验结果精确度很高。对实验数据进行方差分析，发现溶剂对照相比和阳性对照组对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin和Fibrillin1基因的表达，体现扩增倍数到极显著差异(P值小于0.001)(具体数据略)。
与文献报道中，TGF-β1促进基质的形成与重建，说明本次检测有效。
由表5和图4可以看出，与溶剂对照相比，样品组1在0.625％水解假丝酵母和日本清酒酵母共生发酵产物的添加剂量下，对CollagenⅠ、CollagenⅢ及CollagenⅣ的表达有显著促进作用，扩增倍数分别达到1.512、2.564、2.428倍；对Elastin基因和Fibrillin-1基因的表达有显著促进作用，扩增倍数分别达到2.152和1.833倍；对Has-1基因的表达有显著促进作用，扩增倍数高达11.024倍。经过多次反复试验，溶剂对照组和样品组所得结果的标准偏差≤1.787％，实验结果精确度很高。对实验数据进行方差分析，发现溶剂对照相比和样品组1对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin、Fibrillin1和Has-1基因的表达，体现扩增倍数到极显著差异(P值小于0.002)(具体数据略)。说明在0.625％的添加剂量下，水解假丝酵母和日本清酒酵母共生发酵产物在肌肤抗衰老方面有比较优越的功效。
同时，由表6可以看出，与溶剂对照相比，样品组2在0.625％日本清酒酵母单独发酵产物的添加剂量下，对CollagenⅠ、CollagenⅢ及CollagenⅣ表达的促进作用不明显，对Elastin基因、Fibrillin-1基因和Has-1基因的表达也无明显促进作用。经过多次反复试验，溶剂对照组和样品组所得结果的标准偏差≤0.159％，实验结果精确度很高。对实验数据进行方差分析，发现溶剂对照相比和样品组2对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin、Fibrillin1和Has-1基因的表达，体现扩增倍数到极显著差异(P值小于0.002)(具体数据略)。说明添加0.625％日本清酒酵母单独发酵产物，在肌肤抗衰老方面功效并不明显。
由表7可以看出，与溶剂对照相比，样品组3在0.625％水解假丝酵母单独发酵产物的添加剂量下，对CollagenⅠ、CollagenⅢ及CollagenⅣ表达具有明显促进作用，扩增倍数分别达到1.305、1.882和1.989倍；对Elastin基因、Fibrillin-1基因和Has-1基因的表达也有明显促进作用，扩增倍数分别达到1.905、1.692和6.003倍。经过多次反复试验，溶剂对照组和样品组所得结果的标准偏差≤0.754％，实验结果精确度很高。对实验数据进行方差分析，发现溶剂对照相比和样品组3对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin、Fibrillin1和Has-1基因的表达，体现扩增倍数到极显著差异(P值小于0.002)(具体数据略)。说明添加0.625％水解假丝酵母单独发酵产物，在肌肤抗衰老方面有一定的功效，但其功效明显不如0.625％水解假丝酵母和日本清酒酵母共生发酵液。
由表8可以看出，与溶剂对照相比，样品组4在0.625％水解假丝酵母和日本清酒酵母的发酵产物混合液，对CollagenⅠ、CollagenⅢ及CollagenⅣ表达具有明显促进作用，扩增倍数分别达到1.313、1.841和1.953倍；对Elastin基因、Fibrillin-1基因和Has-1基因的表达也有明显促进作用，扩增倍数分别达到1.847、1.635和5.083倍。经过多次反复试验，溶剂对照组和样品组所得结果的标准偏差≤0.802％，实验结果精确度很高。对实验数据进行方差分析，发现溶剂对照相比和样品组4对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin、Fibrillin1和Has-1基因的表达，体现扩增倍数到极显著差异(P值小于0.001)(具体数据略)。说明添加0.625％水解假丝酵母和日本清酒酵母的发酵产物混合液，在肌肤抗衰老方面有一定的功效，但其功效明显不如0.625％水解假丝酵母和日本清酒酵母共生发酵液。
综上所述，本发明的水解假丝酵母和日本清酒酵母共生发酵液相对单独的日本清酒酵母发酵产物和水解假丝酵母发酵产物，以及发酵液混合后的试剂，对CollagenⅠ、CollagenⅢ、CollagenⅣ、Elastin、Fibrillin1和Has-1基因表达的促进作用，都具有显著的改善作用，可见两种菌共同培养具有相互促进和明显增效作用，活性物质的活性含量或活性都大大提高。
虽然本发明披露如上，但本发明并非限定于此。任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种更动与修改，因此本发明的保护范围应当以权利要求所限定的范围为准。

Claims (10)

  1. 一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进胶原蛋白合成的护肤品制剂方面的用途，其特征在于，所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
    (1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
    (2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
    其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
  2. 如权利要求1所述的用途，其特征在于，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
  3. 一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进弹力蛋白合成的护肤品制剂方面的用途，其特征在于，所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
    (1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
    (2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
    其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
  4. 如权利要求3所述的用途，其特征在于，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
  5. 一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进纤维蛋白合成的护肤品制剂方面的用途，其特征在于，所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
    (1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
    (2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
    其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
  6. 如权利要求5所述的用途，其特征在于，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
  7. 一种水解假丝酵母和日本清酒酵母共生发酵产物在制备促进透明质酸合成的护肤品制剂方面的用途其特征在于，所述的水解假丝酵母和日本清酒酵母共生发酵包括如下步骤：
    (1)对日本清酒酵母进行低温25℃、pH值为4.0-5.0的酸性条件下的一次或多次的增菌发酵，让其处于指数生长期；
    (2)添加水解假丝酵母，在pH值为7.0-8.5的碱性条件下进行二次发酵，发酵液离心除菌；
    其中，日本清酒酵母的菌种保藏号为ATCC26422，购于日本酿造协会的7号酵母；水解假丝酵母菌种保藏号为CICC 31239，购于中国科学院微生物研究所。
  8. 如权利要求7所述的用途，其特征在于，在步骤(1)中，对日本清酒酵母进行至少两次以上的增菌培养，从而让酵母处于指数生长期；在步骤(2)中，向日本清酒酵母培养液中接种水解假丝酵母前，对水解假丝酵母进行一次或多次增菌培养。
  9. 如权利要求1-8任一项所述的用途，其特征在于，所述的护肤品制剂为溶液、悬浮液、面膜、乳液、霜剂、膏状、凝胶、干粉、湿粉、喷剂中的一种。
  10. 如权利要求1-8任一项所述的用途，其特征在于，所述的水解假丝酵母和日本清酒酵母共生发酵液在制备护肤品制剂时的最佳添加浓度为0.625％。
PCT/CN2020/088999 2020-04-30 2020-05-07 一种水解假丝酵母和日本清酒酵母共生发酵产物的用途 WO2021217693A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN115011492A (zh) * 2021-03-05 2022-09-06 湖州蔻婷生物科技有限公司 一种清酒酵母发酵工艺、发酵产物溶胞物的生产方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105596275A (zh) * 2016-01-06 2016-05-25 名臣健康用品股份有限公司 一种含有双菌发酵豆奶提取物的化妆品基质及应用
CN107822945A (zh) * 2017-09-28 2018-03-23 杭州惠博士健康产业有限公司 一种赛天然酵母组合物、制备方法及在化妆品中应用
CN108653059A (zh) * 2018-06-20 2018-10-16 齐鲁工业大学 一种益生菌发酵制备海藻发酵液的方法及该海藻发酵液在化妆品中的应用
CN110384650A (zh) * 2019-08-29 2019-10-29 湖北省麦诗特生物科技有限公司 一种用于皮肤保湿、屏障保护的益生菌组合物及其在化妆品中的应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9903216D0 (en) * 1999-02-13 1999-04-07 Zylepsis Ltd Preservative compounds,compositions and methods of making and using the same
FR2834718B1 (fr) * 2002-01-15 2004-12-24 Cognis France Sa Substances actives cosmetiques et/ou pharmaceutiques
CN101117622A (zh) * 2006-10-31 2008-02-06 哈尔滨远大牧业有限公司 酵母培养物发酵工艺
FR2920306B1 (fr) * 2007-09-04 2010-07-30 Oreal Utilisation cosmetique d'un lysat de bifidobacterieum species.
KR20100030368A (ko) * 2008-09-10 2010-03-18 주식회사 코리아나화장품 효모 생전환 기술에 의해 얻은 발효 진액을 포함하는 피부 탄력 개선용 화장료 조성물
RU2492850C2 (ru) * 2009-03-30 2013-09-20 Сисеидо Компани, Лтд. Средство для роста фибробластов
FR2949684B1 (fr) * 2009-09-04 2011-12-16 Silab Sa Principe actif issu de candida saitoana et utilisation cosmetique pour la detoxification des cellules de la peau
CN103284940B (zh) * 2013-06-28 2014-08-06 上海生态美日化有限公司 酵母菌发酵中药组合物及其制备方法和应用
CN103468615B (zh) * 2013-09-23 2015-04-15 内蒙古农业大学 一种乳酸菌和酵母菌共生发酵的微生物制剂的生产方法
CN107510633A (zh) * 2017-09-29 2017-12-26 上海雨淑缇化妆品有限公司 一种眼球细胞赋活抗衰精华液及其制备方法
CN108553403B (zh) * 2018-04-20 2019-09-27 上海中翊日化有限公司 一种嗜热栖热菌和酵母菌组合发酵产物的用途
CN108578338B (zh) * 2018-05-16 2020-04-17 江南大学 一种提高中药抗氧化力的发酵方法及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105596275A (zh) * 2016-01-06 2016-05-25 名臣健康用品股份有限公司 一种含有双菌发酵豆奶提取物的化妆品基质及应用
CN107822945A (zh) * 2017-09-28 2018-03-23 杭州惠博士健康产业有限公司 一种赛天然酵母组合物、制备方法及在化妆品中应用
CN108653059A (zh) * 2018-06-20 2018-10-16 齐鲁工业大学 一种益生菌发酵制备海藻发酵液的方法及该海藻发酵液在化妆品中的应用
CN110384650A (zh) * 2019-08-29 2019-10-29 湖北省麦诗特生物科技有限公司 一种用于皮肤保湿、屏障保护的益生菌组合物及其在化妆品中的应用

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