CN114081862B - Preparation and application of Saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in Pan-Himalaya region - Google Patents
Preparation and application of Saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in Pan-Himalaya region Download PDFInfo
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- CN114081862B CN114081862B CN202010761748.9A CN202010761748A CN114081862B CN 114081862 B CN114081862 B CN 114081862B CN 202010761748 A CN202010761748 A CN 202010761748A CN 114081862 B CN114081862 B CN 114081862B
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Abstract
The invention relates to the field of biological extraction and daily chemical industry, and discloses a highland barley wine yeast-derived saccharomyces cerevisiae fermentation product in a pan-himalayan region, and an extraction method and application thereof. The fermentation product obtained by the saccharomyces cerevisiae can remove free radicals, promote proliferation and activity of dermal fibroblasts and epidermal cells, inhibit tyrosinase, prevent oxidative damage, prevent damage of ultraviolet UVA/UVB to skin and promote collagen secretion, regulate skin flora, and help the steady state maintenance of microbial flora on the surface of a human body, so that the saccharomyces cerevisiae has the effects of maintaining skin health, repairing aged cells, resisting aging and whitening, and protecting human skin cells; the fermentation product can be used as active ingredient for preparing skin external preparation with antiaging, whitening, sun-screening or skin barrier protecting effects.
Description
Technical Field
The invention belongs to the field of biological extraction and daily chemical, and particularly relates to a highland barley wine distiller's yeast-derived saccharomyces cerevisiae fermentation product in a pan-himalayan region, and an extraction method and application thereof.
Background
Highland barley wine is the most important traditional beverage for Tibetan people, has a history of more than 1300 years up to now, and is also an important carrier for Tibetan. Distiller's yeast is a saccharifying, fermenting and aroma-generating agent for brewing wine, and contains various microorganisms and enzymes produced by the microorganisms. The Tibetan yeast is a generic name of a brewing starter prepared by Tibetan people according to the traditional natural inoculation mode, and the yeast produced by Tibetan all places for producing the saccharomyces cerevisiae wine is natural yeast, and has been proved by researches, the number of the saccharomycetes in the Tibetan area is over 96 percent, and the Tibetan yeast has absolute advantages; the proportion of mould and bacteria is small. Therefore, the yeast has important influence on brewing of the Tibetan saccharomyces cerevisiae wine, and is dominant bacteria of Tibetan distiller's yeast. Yeast has wide application, is traditionally used for fermented foods, and has important roles in the cosmetic industry due to the effects of moisturizing, activating and the like of the extract and the oxidation resistance of cell wall polysaccharide. Wild yeast in the polar-like environment of the Himalayan region is suitable for low-temperature, anoxic, strong radiation and other environments. Keratinocytes are the most important cells in the epidermis and participate in the formation of the physical barrier of the skin, preventing invasion of adverse factors such as physical, chemical and microbial factors, and protecting the skin. One of the main characteristics of skin after aging is the thinning of the epidermis and the slow rate of wound healing, which is mainly caused by the reduced regenerative capacity of keratinocytes in the epidermis layer and the reduced proliferative capacity of the keratinocyte system.
Dermal aging is manifested by reduced clearance of the dermis from foreign chemicals, reduced dermis thickness, reduced collagen and elastin synthesis, increased catabolic enzyme activity. These phenomena are all associated with a decrease in the number of fibroblasts and a decrease or abnormality in the secretory synthesis function. Human skin fibroblasts are the most important cells in the dermis reticulation layer of the skin, and are one of the main repair cells after skin aging and cell damage. It not only can promote migration, proliferation and differentiation of epidermal cells, but also can secrete a large amount of collagen, elastin and various cell repair factors, and has strong self-renewal capacity, so that aged skin can be repaired.
Pigmentation is caused by ultraviolet light activating melanogenesis enzymes in melanocytes in the epidermis, producing pigment. In the melanin production process, tyrosinase converts tyrosine into dopa, and dopaquinone is polymerized into macromolecular melanin through non-enzymatic oxidation. Thus, by inhibiting tyrosinase activity, melanin production can be restricted and skin pigmentation can be improved.
The free radicals can cause various irreversible oxidative damages to organisms at the cellular level, the molecular level and even the tissue organ level, accelerate the aging process of organism cells and even the whole organism, and induce various diseases related to aging. Scavenging free radicals or protecting free radicals from oxidative damage again, and protecting skin.
Aging of skin is classified into endogenous aging and exogenous aging, and sunlight, particularly Ultraviolet (UV) irradiation, is a major factor in the formation of exogenous aging, so exogenous aging is also called photoaging. Associated with skin photoaging are mainly UVA (320 nm-400 nm) and small amounts of UVB (280-320 nm). Ultraviolet UVB directly acts on epidermal cells, and the skin barrier function is further affected by the induction of oxidative stress and inflammatory processes, so that photodamage is formed. It would be advantageous to protect against UVA/UVB induced cell damage to prevent skin aging and aging.
The surface flora or surface microecology of the skin is an important biological barrier which is used as an outer layer of the physical barrier of the human skin and plays an important role in the health state of the skin. The surface flora has obvious individual differences according to different sexes, ages, external environments and the like, and unbalance of the surface flora, especially when the proportion of certain conditional pathogenic bacteria in the surface flora is abnormal, various skin diseases are often caused. Staphylococcus epidermidis Staphylococcus epidermidis is one of resident bacteria of human skin, and when the proportion of staphylococcus aureus Staphylococcus aureus is abnormal, skin surface barrier is destroyed, or skin problems such as inflammation are caused. Therefore, the stable maintenance of the microbial flora on the surface of a human body or the adjustment and repair of the unbalanced skin surface microecology can be helped by reducing the S.aureus/S.epididitis value.
The above is a major cause of skin dysfunction, aging or other skin problems, and there is a need to obtain an active substance to solve the above problems.
Disclosure of Invention
The invention aims to provide an application of a saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in a pan-himalayan region.
An application of Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast in Pan-Himalayan region in preparing skin external preparation is provided. Preferably, the external preparation for skin is a cosmetic or skin care product.
The fermentation product is applied to the preparation of skin external preparations with the functions of resisting aging, whitening and preventing sunburn.
The Saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in the Pan-Himalayan area has good free radical scavenging capability, can promote the proliferation of dermal fibroblasts and epidermal cells, has an inhibiting effect on tyrosinase in B16 melanocytes, has the effects of maintaining skin health and repairing aged cells, and has the anti-aging and whitening effects. In addition, it can protect human skin cells, prevent oxidative damage and prevent damage to skin caused by ultraviolet UVA/UVB. The fermentation product can also regulate skin flora, especially reduce the ratio of Staphylococcus aureus (Staphylococcus aureus) to Staphylococcus epidermidis (Staphylococcus epidermidis), and is helpful for maintaining the steady state of microbial flora on human surface, or regulate and repair unbalanced skin surface microecology, and can protect or enhance skin barrier.
The technical scheme of the invention is as follows:
the application of Saccharomyces cerevisiae fermentation product derived from highland barley wine distiller's yeast in Pan-Himalayan area in preparing skin external preparation is provided. Preferably, the external preparation for skin is a cosmetic or skin care product.
The application of Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast in Pan-Himalayan area in preparing skin external preparation with antiaging, whitening, sun-screening or skin protecting effects is provided.
The application of Saccharomyces cerevisiae fermentation product derived from highland barley wine distiller's yeast in Pan-Himalayan region in preparing skin external preparation for scavenging free radicals, inhibiting tyrosinase activity, inhibiting melanin generation, preventing oxidation injury, preventing photoaging, promoting keratinocyte or fibroblast proliferation, promoting collagen secretion, regulating skin surface microorganism flora, and repairing skin external preparation.
The fermentation product is prepared by the following steps: inoculating Saccharomyces cerevisiae from highland barley wine distiller's yeast in Pan-Himalayan area into fermentation medium, and culturing at 5-35 deg.c and 0-300 rpm for 24-72 hr. One preferable scheme is as follows: culturing at 50-300 rpm and 5-35 deg.c for 10-24 hr and 0-100 rpm for 24-48 hr.
Preferably, the fermentation liquor is filtered and decolored, and then is subjected to positive pressure filtration, and the fermentation filtrate is taken. Preferably, the positive pressure filtration is performed with a 0.1 μm to 0.5 μm polyethersulfone resin (PES) or nylon membrane.
The obtained fermentation broth or fermentation filtrate can be further concentrated or dried to obtain concentrated solution or dry powder.
The fermentation product of the saccharomyces cerevisiae from highland barley wine distiller's yeast in the pan-Himalayan region is fermentation liquor, fermentation filtrate, concentrated solution or dry powder of the fermentation liquor or the fermentation filtrate.
Before inoculation, the strain is subjected to activation, purification and expansion culture.
The method for activating the strain comprises the following steps: the preserved strain is selected and inoculated into a liquid seed culture medium, and cultured for 16 to 24 hours at a temperature of between 25 and 30 ℃ and at a speed of between 0 and 300rpm by a shaking table to activate the strain.
The strain purification method comprises the following steps: the activated bacterial liquid is subjected to gradient dilution and plating to obtain single colonies.
The method for the strain expansion culture comprises the following steps: the single colony in the upper plate is picked up and inoculated into liquid YPD culture medium, and cultured for 10 to 16 hours at 25 to 30 ℃ and 0 to 300rpm by a shaking table until reaching OD 600 2 to 6 to obtain zymophyte seed bacterial liquid.
The seed culture medium is YPD culture medium, and the fermentation culture medium is modified YPD culture medium.
Preferably, the collection number of the Saccharomyces cerevisiae from highland barley wine distiller's yeast in the Pan-Himalaya region is CGMCC No.19732, and the patent collection is completed by China general microbiological culture Collection center (CGMCC, address: china academy of sciences of China, 1 st China, 3 rd, which are the areas of North China, which are the areas of Korea, beijing, post code: 100101) on 26 days in 4 months in 2020. The culture method comprises the following steps:
seed culture: culturing in YPD culture medium at 25-30 deg.C and 0-300 rpm for 10-16 hr until reaching OD 600 2-6.
Fermentation culture: inoculating the seed liquid thalli into a fermentation culture medium according to the inoculation amount of 0.1-10.0%, wherein the percentage is the mass percentage; culturing at 5-35 deg.c and 0-300 rpm for 24-72 hr.
The fermentation product of the highland barley wine distiller's yeast source Saccharomyces cerevisiae (CGMCC No. 19732) in the Pan-Himalayan area has better free radical scavenging capability, can promote the proliferation of dermal fibroblasts and epidermal cells, and has an inhibiting effect on tyrosinase in B16 melanocytes, so that the highland barley wine distiller's yeast has the effects of maintaining skin health and repairing aged cells, and has the potential of whitening. Can also protect human skin cells, prevent oxidative damage and prevent ultraviolet UVA/UVB from damaging skin, regulate skin flora, reduce the ratio of staphylococcus aureus to staphylococcus epidermidis, help the steady state maintenance of microbial flora on the surface of a human body, or regulate and repair unbalanced skin surface micro-ecology, and protect skin barrier or prevent inflammation.
Based on the solid content, the yeast fermentation product of highland barley wine yeast in the area of pan-camptotheca has good effect under the condition that the content of the yeast fermentation product is 0.005-2.0%.
Therefore, the Saccharomyces cerevisiae derived from highland barley wine distiller's yeast in the Pan-Himalayan region can be used for preparing skin external preparations.
In addition to Saccharomyces cerevisiae, highland barley wine yeast may also be selected to obtain strains which may be used in cosmetics: candida spp, pichia spp, tectorial spp, rhodotorula spp, kluyveromyces spp, lactobacillus plantarum, lactobacillus casei, lactococcus lactis, leuconostoc spp, aspergillus spp, mucor spp, and the like.
A skin external preparation contains active ingredients of Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast in Pan-Himalayan region. Preferably, the active ingredient is a Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast source in the area of Pan-Himalayan; more preferably, the collection number of the Saccharomyces cerevisiae from highland barley wine distiller's yeast in the Pan-Himalayan region is CGMCC No.19732.
The skin external preparation has the functions of resisting aging, whitening skin, improving skin cell activity, and preventing sunburn or oxidation.
The fermentation product of the Saccharomyces cerevisiae from highland barley wine distiller's yeast in the Pan-Himalayan region is fermentation liquor, fermentation filtrate, concentrated solution or dry powder of the fermentation liquor or the fermentation filtrate. Preferably, the fermentation product of the Saccharomyces cerevisiae from which highland barley wine distiller's yeast is derived in the area of pan-Himalayan is fermentation filtrate, concentrated solution or dry powder of the fermentation filtrate.
A skin external preparation with free radical scavenging, tyrosinase activity inhibiting, melanin generation inhibiting, oxidation injury preventing, antioxidant, photoaging preventing or skin surface microorganism flora regulating, and repairing effects comprises Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast in Pan-Himalayan area as active ingredient. Preferably, the active ingredient is a Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast source in the area of Pan-Himalayan.
A skin external preparation for promoting proliferation of keratinocyte or fibroblast, promoting secretion of collagen, elastin and cell repair factor, or increasing ATP content in skin cell comprises Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast in Pan-Himalayan region as active ingredient. Preferably, the active ingredient is a Saccharomyces cerevisiae fermentation product of highland barley wine distiller's yeast source in the area of Pan-Himalayan.
The invention has the beneficial effects that the yeast from highland barley wine distiller's yeast in the pan-Himalayan area is adopted for fermentation, and the obtained fermentation product can remove free radicals, promote the proliferation of dermal fibroblasts and epidermal cells and inhibit tyrosinase, so that the yeast has the anti-aging effects of resisting oxidation, maintaining skin health and repairing aged cells, and has the potential of whitening skin cells. Can also protect human skin cells, prevent oxidative damage and prevent damage to skin caused by ultraviolet UVA/UVB, regulate skin flora, help maintain the steady state of microbial flora on the surface of a human body, or regulate and repair unbalanced skin surface micro-ecology, and protect or enhance skin barrier. And simultaneously, the ATP content in skin cells can be increased so as to improve the activity of the skin cells. Therefore, the fermentation product or the extract is used as an active ingredient of the skin external agent, can realize the functions of resisting aging, whitening skin, preventing sun or protecting skin barrier, and has good application prospect and market prospect.
Drawings
FIG. 1 shows the effect of Saccharomyces cerevisiae fermentation product on the regulation of skin flora
Detailed Description
EXAMPLE 1 isolation, purification, identification and patent preservation of strains
The strain used in the invention is separated from the traditional self-made highland barley wine distiller's yeast of Tibetan people in the area of pan-himalayan.
1 g of the sample is crushed and then is subjected to gradient dilution to 10 times, 100 times and 1000 times by shaking with 9mL of physiological saline in a constant-temperature shaking incubator for 30min. Each gradient of liquid was plated onto YPD solid medium with antibiotics added, 200 microliters of dilution per plating. Culturing at 28deg.C. Taking a colony with proper growth degree for bacterial detection, and carrying out the next operation if the colony is saccharomycetes.
The method comprises the steps of selecting ideal bacterial colonies by using an inoculation device, marking on a flat plate, marking again after burning and sterilizing, repeating the burning and marking until a proper gradient is marked, burning the inoculation device again, and marking a serial number and inoculation time at the bottom of the flat plate. Culturing under constant temperature. And (5) carrying out monoclonal again after the strain grows to a proper size. The monoclonal strain was picked into liquid YPD for amplification culture. The purified yeast was subjected to 26S rDNA (D1/D2 region) sequencing. The microzyme obtained by preliminary identification is sent to China industry microbiological culture collection center (CICC) for multiphase identification detection such as morphological characteristics, physiological and biochemical characteristics, DNA sequence analysis (26S rDNA) and the like, identified as saccharomyces cerevisiae (Saccharomyces cerevisiae), and reserved strains are put into a refrigerator at the temperature of minus 80 ℃ for preservation.
MEA medium, cultured at 28 ℃ for 72 hours, the colony is flat, white and creamy, thick in texture and irregular in edge. The microcosmic morphology is characterized by elliptic shape of the bacterial cells, single or opposite, and the size is 4-8 multiplied by 3.5-7.5 μm.
The carbon source utilization conditions of the bacteria are as follows:
symbol description: "+", positive; "-", negative; "+ w ", weak positive.
The saccharomyces cerevisiae has the number of JBA-MBY-JT017, and the China general microbiological culture collection center (CGMCC) finishes patent preservation in 26 days of 4 months in 2020, and the preservation number is: CGMCC No.19732.
The culture medium of the filtrate of the fermentation product can be grains such as wheat (barley, wheat, oat, rye, etc.), rice (indica rice, japonica rice, glutinous rice), corn, sorghum or bean products and potatoes and combinations thereof, or semi-synthetic culture medium such as YPD, PDB and modified culture medium thereof, or semi-synthetic culture medium of matching grains with yeast extract, yeast peptone, mineral elements, growth factors, etc.
Seed medium (YPD medium): 1% of Yeast Extract (Yeast Extract), 2% of Peptone (Peptone), 2% of glucose (Dextrose) and the balance of water, wherein the percentages are in mass percent.
Fermentation medium (modified YPD medium): 1% Yeast Extract, 2% Yeast Peptone, 2% glucose (Dextrose) and the balance water, wherein the percentages are mass percentages.
The culture conditions were as follows:
seed culture: culturing in YPD culture medium at 25-30 deg.C and 0-300 rpm for 10-16 hr until reaching OD 600 2-6;
fermentation culture: inoculating the seed liquid thalli into a fermentation culture medium according to the inoculation amount of 0.1-10.0%, wherein the percentage is the mass percentage; culturing at 50-300 rpm and 5-35 deg.c for 10-24 hr, and regulating rotation speed to 0-100 rpm for 24-48 hr.
EXAMPLE 2 preparation of Saccharomyces cerevisiae fermentation product
1) Preparing a culture medium: preparing a seed culture medium (YPD culture medium) and a fermentation culture medium (modified YPD culture medium) according to the growth requirement of saccharomyces cerevisiae, sterilizing at high temperature and high pressure for 20-30 min at 121 ℃, and cooling for later use.
2) Activating strains: picking up the strain preserved at-80 ℃, inoculating the strain into a liquid seed culture medium, and culturing the strain for 16-24 hours at 25-30 ℃ and 150-300 rpm by a shaking table to activate the strain.
3) And (3) strain purification: the bacterial liquid after the activation is diluted in a gradient way and is plated, so that single bacterial colonies are obtained.
4) And (3) strain expansion culture: the single colony in the upper plate is picked up and inoculated into liquid YPD culture medium, and cultured for 10 to 16 hours at 25 to 30 ℃ and 0 to 300rpm by a shaking table until reaching OD 600 2 to 6 to obtain zymophyte seed bacterial liquid.
5) Inoculating and fermenting Saccharomyces cerevisiae: adding the amplified Saccharomyces cerevisiae liquid into the sterilized fermentation medium according to the inoculation amount of 0.1-10.0%, wherein the mass percentage is the percentage. Culturing at 50-300 rpm at 5-35 deg.c in shaking table or fermentation tank for 10-24 hr, and regulating rotation speed to 0-100 rpm for 24-48 hr.
6) Filtering and decoloring: after removing thalli and impurities by a 0.22 mu m hollow fiber membrane, adding 0.1-5.0% of powder or granular active carbon into the filtrate, standing or stirring at 4-60 ℃ for 0.5-20 hours, and carrying out positive pressure filtration on a 0.22 mu m polyether sulfone resin (PES) or nylon membrane to obtain filtrate with the final solid content of about 1-2 wt%, thereby obtaining a Saccharomyces cerevisiae fermentation product for subsequent experiments and detection. Table 1 shows the physicochemical parameters of the fermentation product of Saccharomyces cerevisiae.
TABLE 1 physicochemical parameters of Saccharomyces cerevisiae fermentation products
Example 3 free radical scavenging ability of Saccharomyces cerevisiae fermentation product
1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable nitrogen-centered organic radical. DPPH method was proposed in 1958 and is widely used to measure the anti-aging capacity of biological samples, classified substances and foods. The method is based on the fact that DPPH free radical has single electron, has strong absorption at 517nm, and the alcohol solution is purple. When the free radical scavenger exists, the free radical scavenger is paired with single electron to make its absorption gradually disappear, and its fading degree is quantitatively related to the number of electrons accepted by the free radical scavenger, so that a spectrophotometer can be used for quick quantitative analysis to detect the free radical scavenger, thereby evaluating the anti-aging capability of the sample.
The Saccharomyces cerevisiae fermentation product (solid content 1.16 wt%) of example 2 was diluted, the diluted sample solution contained 2.5% and 25% by volume of filtrate of the fermentation product, respectively, 0.1mL of the diluted sample solution was taken and added into a test tube, then 0.1mL of 30. Mu.g/mL DPPH ethanol solution was added, and the mixture was uniformly mixed, reacted at room temperature in the dark place for 30 minutes at room temperature under the dark place, OD value was measured at 525nm, and the clearance was calculated according to the following formula:
clearance I (%) = [1- (T-T) 0 )/(C-C 0 )]×100％
Wherein: t (T) 0 : absorbance of 0.1ml sample solution plus 0.1ml 95% ethanol; t: absorbance of 0.1mL sample solution plus 0.1mL 30 μg/mL DPPH solution; c (C) 0 : absorbance of 0.1ml water plus 0.1ml 95% ethanol; c: absorbance of 0.1mL water plus 0.1mL 30 μg/mL DPPH solution.
TABLE 2 DPPH clearance of Saccharomyces cerevisiae fermentation products
EXAMPLE 4 cell proliferation of Saccharomyces cerevisiae fermentation product
Keratinocytes are the most important cells in the epidermis and participate in the formation of the physical barrier of the skin, preventing invasion of adverse factors such as physical, chemical and microbial factors, and protecting the skin. One of the main characteristics of skin after aging is the thinning of the epidermis and the slow rate of wound healing, which is mainly caused by the reduced regenerative capacity of keratinocytes in the epidermis layer and the reduced proliferative capacity of the keratinocyte system.
Dermal aging is manifested by reduced clearance of the dermis from foreign chemicals, reduced dermis thickness, reduced collagen and elastin synthesis, increased catabolic enzyme activity. These phenomena are all associated with a decrease in the number of fibroblasts and a decrease or abnormality in the secretory synthesis function. Human skin fibroblasts are the most important cells in the dermis reticulation layer of the skin, and are one of the main repair cells after skin aging and cell damage. It not only can promote migration, proliferation and differentiation of epidermal cells, but also can secrete a large amount of collagen, elastin and various cell repair factors, and has strong self-renewal capacity, so that aged skin can be repaired.
The Saccharomyces cerevisiae fermentation product of example 2 (solid content 1.16 wt%) was added to each serum-free culture solution of human fibroblasts and epidermal cells, and the proliferation effect on human fibroblasts was evaluated by taking deionized water without sample as a blank, taking a medium containing 5% new born calf serum (FCS) as a positive control, culturing for 48 hours, staining the cells by MTT method, measuring absorbance at 550nm with a microplate reader, and the proliferation rate of the blank was 100% and the proliferation effect on human fibroblasts was evaluated by reference to the blank.
The effect of Saccharomyces cerevisiae fermentation product on the growth of fibrast and HaCaT As shown in Table 3 results, the addition of 5% (v/v) of the Saccharomyces cerevisiae fermentation product of example 2 enabled the highest cell viability of fibroblasts and HaCaT to reach 131.3% and 117.0%, respectively.
TABLE 3 proliferation of Saccharomyces cerevisiae fermentation products on human skin cells
Therefore, the product of the fermented Saccharomyces cerevisiae from highland barley wine distiller's yeast in the Pan-Himalayan area can promote the formation of an epidermal barrier and the proliferation of human skin cells, so that the health of the skin state is maintained, the aged skin is repaired, and the highland barley wine distiller's yeast has the potential of resisting aging.
EXAMPLE 5 inhibition of tyrosinase by murine B16 melanoma cells
The skin color is derived from melanin stored within keratinocytes. Generally, people with increased storage melanin are darker in their skin and are also more protected from solar radiation. The amount and quality of melanin is an important factor in determining skin color in skin melanin. Tyrosinase is a copper-containing oxidoreductase with a complex structure, and is widely used in microorganisms, animals, plants and human bodies. Tyrosinase is a key enzyme in the synthesis of melanin by the skin in humans.
Recent studies have demonstrated that pigmentation is caused by ultraviolet light activating melanogenesis enzymes in melanocytes in the epidermis, producing pigment. Melanocytes in basal lamina in the human epidermis are in contact with and associated with surrounding keratinocytes to form "epidermal melanocytes," where melanin is a nitrogen-containing complex synthesized in melanosomes. Generally, the amount and quality of melanin in the skin is an important factor in determining the skin color. In the melanin production process, tyrosinase converts tyrosine into dopa, and dopaquinone is polymerized into macromolecular melanin through non-enzymatic oxidation. Thus, by inhibiting tyrosinase activity, melanin production can be restricted and skin pigmentation can be improved.
The inhibition of tyrosinase by the samples was determined by L-Dopa oxidation. Murine melanoma B16 cells at 1X10 5 Density culture in 96-well plate, after 24h, fermentation product is diluted with culture medium, added into cells for culturing for 48h, the culture solution is removed, 100 μl of PBS buffer containing 1% Triton X-100 is added into each well, then 50 μl of 0.2mg/mL L-DOPA is added, and after 3h treatment at 37deg.C, the absorbance at 490nm is measured. The enzyme activity was calculated as follows: tyrosinase inhibition ratio= [1- (experimental group OD value/control group OD value)]X 100%. The effect of the sample on the viability of B16 cells was also determined using the MTT method.
And determining the content of melanin in the cells by adopting a NaOH cracking method. Murine melanoma B16 cells at 1X10 5 Density culture in 12 hole plate, after 24 hours, the fermentation product is diluted with culture medium, added into cells, cultured for 48 hours, the supernatant is discarded, cells are collected by centrifugation to centrifuge tubes after pancreatin digestion, 150 mu L of 1mol/L NaOH solution (containing 10% DMSO) is added, cells are fully lysed for 1 hour at-80 ℃, and the absorbance at 405nm is measured. The protein concentration of B16 cells was measured by BCA method and used for absorbance correction. Data statistics are expressed as a percentage of the control group.
From the results in Table 4, it is clear that the Saccharomyces cerevisiae fermentation product was able to inhibit tyrosinase activity and melanogenesis of B16 cells.
TABLE 4 inhibition of tyrosinase by Saccharomyces cerevisiae fermentation products on mouse B16 melanoma cells
EXAMPLE 6 protective Effect of Saccharomyces cerevisiae fermentation product on oxidative damage to skin cells
The free radicals can cause various irreversible oxidative damages to organisms at the cellular level, the molecular level and even the tissue organ level, accelerate the aging process of organism cells and even the whole organism, and induce various diseases related to aging. Hydrogen peroxide (hydrogen peroxide, H) 2 O 2 ) Is an important oxygen free radical in cells, and can cause direct oxidative stress reaction and oxidative damage caused by oxidative stress to human skin cells. In vitro utilization of H 2 O 2 Can induce and accelerate aging process of cells caused by oxidative stress, and simulate pathological process of oxidative damage in vivo.
The fermentation product of example 2 was added to each of the culture solutions of human fibroblasts and epidermal cells and cultured for 24 hours. Then is changed to contain hydrogen peroxide (H) 2 O 2 ) 500 mu M starvation medium is treated for 1h and then changed into a medium containing a sample to be tested, and the culture is continued for 24h, and the cell viability is detected by an MTT method. By using a catalyst free of H 2 O 2 The treatment was a blank. VE was used as positive control. The proliferation rate of the blank control was 100%, and the effect of the sample on cell viability was evaluated with reference to the blank control.
As shown in the results of Table 5, the Saccharomyces cerevisiae fermentation product has an obvious protective effect on cell oxidative damage caused by hydrogen peroxide.
TABLE 5 oxidative damage protection of Saccharomyces cerevisiae fermentation products on human skin cells
EXAMPLE 7 protective Effect of Saccharomyces cerevisiae fermentation product on skin cell ultraviolet injury
Aging of skin is classified into endogenous aging and exogenous aging, and sunlight, particularly Ultraviolet (UV) irradiation, is a major factor in the formation of exogenous aging, so exogenous aging is also called photoaging. Associated with skin photoaging are mainly UVA (320 nm-400 nm) and small amounts of UVB (280-320 nm). Many data indicate that 95% of the uv light that is irradiated to the skin is absorbed by keratinocytes, and that exposure of the skin to UVA radiation generates ROS and causes various damage, including oxidative stress. Ultraviolet UVB directly acts on epidermal cells, and the skin barrier function is further affected by the induction of oxidative stress and inflammatory processes, so that photodamage is formed. We therefore established a model of photodamage to skin by irradiation of human immortalized epidermis HaCaT cells and dermis Fibroblast fibroplast with UVA/UVB, and used this model to evaluate the protective effect of actives on skin uv damage.
The fermentation product was added to a culture solution of human fibroblasts and epidermal cells and cultured overnight for 24 hours. Removing culture medium, adding PBS, and irradiating with UVA (365 nm) at a dose of 10J/cm 2 . UVB (312 nm) irradiation at a dose of 40mJ/cm 2 And adding a culture medium containing a sample to be tested, continuously culturing for 24 hours, and detecting the cell viability by using an MTT method. The non-irradiated group served as a blank control and Vc served as a positive control. The proliferation rate of the blank control was 100%, and the effect of the sample on cell viability was evaluated with reference to the blank control.
As can be seen from the results in tables 6 and 7, the Saccharomyces cerevisiae fermentation products had a significant protective effect against UVA/UVB induced cell damage.
TABLE 6 UVA damage protection of Saccharomyces cerevisiae fermentation products on human skin cells
TABLE 7 UVB damage protection effect of Saccharomyces cerevisiae fermentation products on human skin cells
Example 8 Saccharomyces cerevisiae fermentation product regulating action on skin flora
The skin surface and intestinal tract of human body store a large amount of microorganisms, and since birth, the human body is in symbiotic relation with human beings. Wherein, the surface flora or surface microecology of the skin is an important biological barrier which is used as an outer layer of the physical barrier of the human skin and has important effect on the health state of the skin. The surface flora has obvious individual differences according to different sexes, ages, external environments and the like, and unbalance of the surface flora, especially when the proportion of certain conditional pathogenic bacteria in the surface flora is abnormal, various skin diseases are often caused. Staphylococcus epidermidis (s. Epidemic) is one of resident bacteria of human skin, and when the proportion of staphylococcus aureus (s. Aureus) is abnormal, skin surface barrier is destroyed, or skin problems such as inflammation are caused. Therefore, the stable maintenance of the microbial flora on the surface of a human body or the adjustment and repair of the unbalanced skin surface microecology can be helped by reducing the S.aureus/S.epididitis value.
Inoculating the preserved S.aureus and S.epididitis into sterile LB liquid medium for activating recovery (1 CFU/4mL or 50 mu L/mL); culturing at 150-250rpm at 37 deg.C for 8-24 hr to obtain equal OD 600 A bacterial suspension. And respectively adding Saccharomyces cerevisiae fermentation products with different concentrations into sterile LB culture media by using equal OD bacterial suspensions of S.aureus and S.epidrmidis to obtain a Saccharomyces cerevisiae fermentation product culture medium with mass percentages (calculated by dry matter of fermentation products) of 0.05%, 0.1% and 0.5%, wherein the total volume of the test system is 4mL, and inoculating according to the volume ratio of 0.2% -2%. After 8-24 hours of incubation, absorbance OD was measured by turbidimetry 600 Represents the biomass of the microorganism.
The effect of the Saccharomyces cerevisiae fermentation product on the skin flora on the S.aureus/S.epididis ratio is shown in FIG. 1. At the three concentrations, the values of S.aureus/S.epididitis were reduced by 8.59%, 26.22% and 24.71% respectively compared to 1.22 of the blank.
This suggests that Saccharomyces cerevisiae fermentation products can significantly reduce biomass ratios and gradually decrease with increasing concentration, with the potential to adjust the ratio of S.aureus and S.epideris in the skin surface microbial flora to a healthier state.
Example 9 Effect of Saccharomyces cerevisiae fermentation product on intracellular ATP
Adenosine triphosphate (ATP adenosine triphosphate) is formed by connecting adenine, ribose and 3 phosphate groups, and releases more energy during hydrolysis, thus being the most direct energy source in organisms. The energy required by an organism in its vital activity is provided in the form of ATP. As the most important energy molecule, it plays an important role in various physiological processes of cells, and changes in ATP levels affect functions of cells, such as decrease in ATP levels during apoptosis or in toxic states. Thus, it was used to evaluate the effect of Saccharomyces cerevisiae fermentation product on the intracellular ATP content of skin.
The fermentation product was added to human fibroblast culture broth and cultured overnight for 24h. After removal of the medium, 200uL of lysate was added, and after lysing the cells, 12000g was centrifuged for 5min at 4℃to obtain the supernatant. The ATP content of the sample was measured according to the ATP assay kit instructions: taking 100uL of detection working solution into a detection hole, standing for 3-5min at room temperature, and then adding 20uL of cell lysis supernatant or ATP standard solution. After mixing, the intracellular ATP content was calculated by a standard curve after measurement by a chemiluminescent instrument. Meanwhile, protein concentration in the sample was measured using the Biyundian BCA protein concentration measuring kit, and then ATP content was expressed in. Mu. Mol/mg protein.
TABLE 8 influence of Saccharomyces cerevisiae fermentation products on the intracellular ATP content of human skin
**P<0.01
Example 10 determination of type I collagen content secreted by cells from Saccharomyces cerevisiae fermentation product
Skin aging is one of the important external manifestations of body aging, the age is increased, the synthesis capacity of fibroblasts is reduced, if collagen is absent in the skin, the collagen fibers are cured in a linked manner, so that intercellular mucopolysaccharides are reduced, and the skin loses softness, elasticity and luster and is aged. Fibroblasts are the primary cells of the dermis layer and function to synthesize and secrete collagen fibers, elastic fibers and other matrix components that together with fibroblasts act as the main body of the dermis and maintain the elasticity of the dermis layer. Type I collagen (Col I) is the most abundant collagen in skin in the young, and maintains the structural stability of the dermis layer of the skin. Fibroblast synthesis coli gradually decreases with age, causing the skin to show signs of aging. The effect of Saccharomyces cerevisiae fermentation product on skin cells was evaluated by measuring the intracellular level of type I collagen.
The fermentation product was added to human fibroblast culture broth and cultured overnight for 48h. Detection of type i collagen content using ELISA kit: after 40. Mu.L of the cell supernatant (and the standard) was added to the enzyme-labeled coating plate, 10. Mu.L of the biotin-labeled anti-Col-I antibody was added. Mix with gentle shaking, add 50. Mu.L of enzyme-labeled reagent and incubate at 37℃for 30min. The liquid was sucked off, washed 5 times with washing liquid and patted dry. After adding 100. Mu.L of the color-developing agent, 50. Mu.L of stop solution was added to the mixture to develop a color at 37℃for 10 minutes in the absence of light. The absorbance at 450nm was measured. Sample concentrations were calculated from the standard curve. Meanwhile, protein concentration in the sample was measured using the Biyundian BCA protein concentration measuring kit, and then type I collagen content was expressed in ng/mg protein.
TABLE 9 determination of type I collagen content secreted by Saccharomyces cerevisiae fermentation products on cells
*P<0.05
EXAMPLE 11 analytical determination of the composition of Saccharomyces cerevisiae fermentation product
TABLE 10 analysis of Saccharomyces cerevisiae fermentation product composition
Claims (8)
1. A Saccharomyces cerevisiae of highland barley wine distiller's yeast source in Pan-Himalaya area is characterized in that the preservation number is CGMCC No.19732.
2. The use of the fermented product of Saccharomyces cerevisiae from highland barley wine distiller's yeast in the area of pan-himalayan as claimed in claim 1 for preparing skin external preparation with antiaging, whitening, sun-screening, antioxidant, skin cell activity improving, or skin barrier protecting effects.
3. The use of the fermentation product of s.cerevisiae in the region of ubiquity of camptotheca as claimed in claim 1 for the preparation of a skin external preparation for scavenging free radicals, a skin external preparation for inhibiting tyrosinase activity, a skin external preparation for inhibiting melanin formation, a skin external preparation for preventing oxidative damage, a skin external preparation for preventing photoaging, or a skin external preparation for promoting proliferation and viability of keratinocytes or fibroblasts, or a skin external preparation for promoting collagen secretion or a skin external preparation for regulating the functions of microbial flora on the skin surface.
4. The use according to claim 2 or 3, wherein the fermentation product of Saccharomyces cerevisiae from highland barley wine distiller's yeast in the area of pan-Himalayan is a fermentation broth, a fermentation filtrate, a concentrate or a dry powder of the fermentation broth or the fermentation filtrate.
5. The preparation method of the Saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in the Pan-Himalayan area is characterized by comprising the following steps: inoculating the saccharomyces cerevisiae seed bacterial liquid of highland barley wine yeast source in the area of pan-campaigna to a fermentation culture medium, and culturing for 24-72 hours at the temperature of 5-35 ℃ and at the speed of 0-300 rpm.
6. A skin external preparation with the functions of resisting aging, whitening skin, improving skin cell activity, preventing sunburn or resisting oxidation, which is characterized in that a Saccharomyces cerevisiae fermentation product from highland barley wine distiller's yeast in the pan-Himalayan area as described in claim 1 is taken as an active ingredient.
7. A skin external preparation having the functions of scavenging free radicals, inhibiting tyrosinase activity, inhibiting melanin formation, preventing oxidative damage, preventing photoaging or regulating microbial flora on the surface of skin, characterized in that the fermentation product of Saccharomyces cerevisiae from highland barley wine distiller's yeast in Pan-Himalayan area as described in claim 1 is used as an active ingredient.
8. A skin external preparation capable of promoting proliferation of keratinocyte or fibroblast, promoting secretion of collagen, elastin and cell repair factor, or increasing ATP content in skin cells, characterized by comprising Saccharomyces cerevisiae fermentation product derived from highland barley wine distiller's yeast of Himalayan area as described in claim 1 as active ingredient.
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| KR20070067549A (en) * | 2005-12-23 | 2007-06-28 | 주식회사 두산 | Composition for protecting and improving skin comprising the rice bran fermented by lactic acid bacteria |
| CN107308073A (en) * | 2017-06-26 | 2017-11-03 | 上海应用技术大学 | A kind of highland barley fermentation magma and preparation method and application |
| KR101826145B1 (en) * | 2017-07-28 | 2018-02-06 | (주)지에프씨생명과학 | Saccharomyces cerevisiae HEH EHWA and Autolysis Extract Manufactured Using Thereof |
| CN109200214A (en) * | 2018-10-17 | 2019-01-15 | 北京电子科技职业学院 | A kind of highland barley glossy ganoderma fermentation magma and preparation method thereof |
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| KR20070067549A (en) * | 2005-12-23 | 2007-06-28 | 주식회사 두산 | Composition for protecting and improving skin comprising the rice bran fermented by lactic acid bacteria |
| CN107308073A (en) * | 2017-06-26 | 2017-11-03 | 上海应用技术大学 | A kind of highland barley fermentation magma and preparation method and application |
| KR101826145B1 (en) * | 2017-07-28 | 2018-02-06 | (주)지에프씨생명과학 | Saccharomyces cerevisiae HEH EHWA and Autolysis Extract Manufactured Using Thereof |
| CN109200214A (en) * | 2018-10-17 | 2019-01-15 | 北京电子科技职业学院 | A kind of highland barley glossy ganoderma fermentation magma and preparation method thereof |
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