CN113913315B - Yeast with whitening and freckle removing effects and application thereof - Google Patents

Yeast with whitening and freckle removing effects and application thereof Download PDF

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CN113913315B
CN113913315B CN202111452337.2A CN202111452337A CN113913315B CN 113913315 B CN113913315 B CN 113913315B CN 202111452337 A CN202111452337 A CN 202111452337A CN 113913315 B CN113913315 B CN 113913315B
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whitening
yeast
freckle
removing effects
freckle removing
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CN113913315A (en
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刘琳
赵裕栋
于忠国
黄琳
陈慧敏
周鑫杰
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Guangzhou Jichuang Biomedical Research Institute Co ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N1/18—Baker's yeast; Brewer's yeast
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi

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Abstract

The invention provides yeast with whitening and freckle removing effects and application thereof. The yeast with the whitening and freckle removing effects is named Saccharomyces cerevisiae and is stored in the Guangdong province microorganism strain collection center, the storage number is GDMCC NO.61987, the storage date is 2021, 10 months and 28 days, and the storage address is 5 building 59 of the Hirship No. 100 university in Guangzhou City. The invention also provides a screening method and a culturing method of the yeast with the whitening and freckle-removing effects, a yeast extract with the whitening and freckle-removing effects prepared by using the yeast extract, and a whitening and freckle-removing essence. The yeast with the whitening and freckle removing effects is easy to culture, has high yield, and can inhibit the activity of tyrosinase and the generation of melanin; the cosmetic containing the yeast extract has remarkable whitening effect and extremely high application value.

Description

Yeast with whitening and freckle removing effects and application thereof
Technical Field
The invention belongs to the technical field of microorganism directional mutation breeding technology and microorganism efficacy, and particularly relates to yeast with whitening and freckle removing effects and application thereof.
Background
Along with the development of economy and the improvement of living standard of people, the whitening cosmetic becomes one of the most active fields in the domestic skin care product market, and the research and development of the whitening skin care product and the evaluation of efficacy naturally become the focus of attention of researchers and consumers. As consumer's consumption ideas gradually tend to be rational, traditional hiding physical whitening products cannot meet consumer demands, and development of an effective whitening and freckle-removing raw material is a future development direction.
The color of human skin depends on the content and distribution of melanin, and the effect of whitening skin can be achieved by inhibiting melanin generation, blocking melanin transportation and other ways. Along with the rapid development of biomedical technology in the fields of foods, medicines and the like, molecular pharmacological technology is promoted and applied in the fields of cosmetic safety, efficacy evaluation and the like. Therefore, efficacy evaluation by in vitro cell experiments is an important means for examining the efficacy of cosmetic raw materials. The cell experiment can avoid experimental errors caused by individual differences of animals in animal experiments, so that the experiment is more repeatable. The establishment of the in-vitro cell level evaluation system of the whitening cosmetics provides a screening basis for the research and development and application of whitening and freckle-removing raw materials.
At present, the raw materials for whitening and removing freckles on the market are various, and common physical raw materials only have a covering function, have poor compatibility with skin and cannot be used for a long time. The production process of the chemical raw materials is complex, the introduced purification reagent is excessive, the preparation process is complex, and certain potential safety hazard exists. Biological materials are distinguished by their good biocompatibility, wherein yeast extract-based materials have advantages that are not comparable to common materials. The research shows that the yeast extract can obviously reduce melanin, and the human body test result shows that the yeast can effectively improve hereditary skin allergy, reduce percutaneous water loss of skin, be comparable to vitamin safety, have the effect of improving skin conditions (such as improving skin roughness and the like), and meanwhile, subject evaluation shows that the yeast extract has good effect of brightening skin.
Compared with the traditional raw materials, the yeast fermentation component has high activity, small molecular weight and easy absorption. The yeast extract has good biocompatibility, can inhibit the expression of tyrosinase, inhibit the generation of melanin, block the transportation of melanin and accelerate the metabolism speed of epidermal cells. The yeast extract can block the signal path in the melanin generation process, so that the problem is solved from the root, and the yeast extract plays an important role in whitening skin and removing spots. However, yeast extracts have a certain problem in practical application: 1. yeast extract has various effects, but has no very definite emphasis, and cannot meet the accurate classification and positioning of the user on the cosmetic demands; 2. the yeast extract has complex components, and most manufacturers cannot clarify key components, action mechanisms and proportion contents; 3. most of the raw materials applied to the field of cosmetics at present have complex preparation processes and high cost.
Therefore, how to provide a yeast raw material with definite functions, reduce the complicated steps of the purification and extraction process and reduce the cost, so as to solve the defects of high cost, inaccurate efficacy and the like of the existing cosmetic raw material in actual production, and the like, which is a problem to be solved.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the yeast with the whitening and freckle removing effects and the application thereof, wherein the yeast has good effects of inhibiting tyrosinase activity and melanin content, can lighten skin color, whiten and remove freckles, and has practical application value.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a yeast with whitening and freckle removing effects, which is named Saccharomyces cerevisiae JC-101-998, is deposited in the Guangdong province microorganism strain collection, and has a deposit number of GDMCC No.61987, a deposit date of 2021, 10 months and 28 days, and a deposit address of Guangzhou City first China center 100 No. 59 building.
In the invention, a strain library is established by mutagenesis of saccharomyces cerevisiae, molecular sieve is utilized for preliminary screening, and high-flux re-screening detection is carried out by tyrosinase activity inhibition experiments, melanin content inhibition experiments and the like, so that the mechanism is clear, the efficacy is clear, and the obtained yeast strain has good whitening and freckle removing effects. The yeast strain also has good biological activity, can be produced in a large scale through fermentation, and creates conditions for the development and preparation of subsequent related products.
In a second aspect, the present invention provides a method for screening yeasts having whitening and freckle removing effects according to the first aspect, the screening method comprising:
after mutagenesis, primary screening and secondary screening, the yeast with the whitening and freckle removing effects is obtained.
Preferably, the primary screening comprises room temperature atmospheric pressure plasma mutagenesis (ARTP).
Preferably, the primary screening comprises molecular sieve high throughput screening.
Preferably, the rescreening comprises high throughput rescreening by tyrosinase activity inhibition assay and/or melanin content inhibition assay.
In the invention, the screening method is constructed on the basis of tyrosinase inhibition activity and melanocyte melanin content inhibition experiments, has definite efficacy and clear mechanism, avoids the problems of complex components and fuzzy efficacy of the prior yeast extract, and lays a foundation for related application.
In a third aspect, the present invention provides a method for culturing yeast with whitening and freckle removing effects according to the first aspect, the method comprising:
inoculating the yeast with whitening and freckle removing effects into YPD culture medium, and culturing.
Preferably, the culture is shake culture at a temperature of 25 to 32℃such as 25℃and 26℃and 27℃and 28℃and 29℃and 30℃and 31℃and 32℃and at a shaking speed of 100 to 200rpm such as 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm or 200rpm, and at a time of 3 to 5 days such as 3 days, 3.5 days, 4 days, 4.5 days or 5 days, and other specific values within the range of values may be selected and will not be described in detail.
Preferably, the concentration of the glucose in the YPD medium is 25-50 g/L, for example, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50g/L, etc., and other specific values in the numerical range can be selected, which will not be described in detail herein.
Preferably, the concentration of the yeast powder in the YPD medium is 15-30 g/L, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L or 30g/L, etc., and other specific values in the numerical range are selected and are not repeated herein.
Preferably, the concentration of the peptone in the YPD medium is 20-35 g/L, for example, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 32g/L, 33g/L, 34g/L or 35g/L, etc., and other specific values within the numerical range are selected and are not repeated herein.
Preferably, the concentration of the magnesium sulfate in the YPD medium is 1.5-4 g/L, for example, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L or 4g/L, and other specific values in the numerical range can be selected, and will not be described in detail herein.
Preferably, the concentration of the sodium dihydrogen phosphate in the YPD medium is 1.5-5 g/L, for example, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L or 5g/L, etc., and other specific values in the numerical range can be selected, which will not be described in detail herein.
Preferably, the concentration of the disodium hydrogen phosphate in the YPD medium is 1.5-5 g/L, for example, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L or 5g/L, etc., and other specific values in the numerical range can be selected, which will not be described in detail herein.
Preferably, the concentration of the glycerol in the YPD medium is 0.5-2 g/L, for example, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L or 2g/L, etc., and other specific values within the range of values are selected and are not repeated herein.
Preferably, the uracil concentration in the YPD medium is 15-25 mg/L, for example, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L or 25mg/L, etc., and other specific values in the numerical range can be selected, and will not be described in detail herein.
Preferably, the concentration of vitamin B1 in the YPD medium is 0.001-0.003 g/L, for example, 0.001g/L, 0.002g/L or 0.003g/L, and other specific values within the numerical range can be selected, and will not be described in detail herein.
Preferably, the pH of the YPD medium is 6 to 7, for example, 6, 6.5 or 7, and other specific values within the range of values are selected, which will not be described in detail herein.
As a preferred embodiment, the YPD medium of the present invention comprises: 25-50 g/L of glucose, 15-30 g/L of yeast powder, 20-35 g/L of peptone, 1.5-4 g/L of magnesium sulfate, 1.5-5 g/L of sodium dihydrogen phosphate, 1.5-5 g/L of disodium hydrogen phosphate, 0.5-2 g/L of glycerin, 15-25 mg/L of uracil and 10.001-0.003 g/L of vitamin B, and the pH value is 6-7.
In the invention, the culture conditions and the culture medium components of the yeast strain are optimized, so that the proliferation and division of the yeast are promoted, the yield of thalli is improved, and conditions are created for the industrialized production and development of subsequent strains.
In a fourth aspect, the invention provides an application of the yeast with whitening and freckle removing effects in the first aspect in preparing a product with whitening and freckle removing effects.
In a fifth aspect, the present invention provides a yeast extract with whitening and freckle removing effects, which is prepared by using the yeast with whitening and freckle removing effects of the first aspect as a raw material through the processes of fermentation culture, thallus collection, thallus homogenate preparation and purification.
Preferably, the method of fermentation culture is the same as the culture method described in the third aspect.
Preferably, the preparation method of the bacterial cell homogenate comprises ice bath high-pressure homogenate.
Preferably, the purification is preceded by a filtration step.
Preferably, the purification comprises molecular sieve purification.
In a sixth aspect, the invention provides a whitening and freckle-removing essence, which contains the yeast extract with whitening and freckle-removing effects in the fifth aspect.
Preferably, the whitening and freckle-removing essence further comprises a plant extract, a humectant and a biological polypeptide.
Preferably, the plant extract comprises an alga extract.
Preferably, the humectant comprises mannitol.
Preferably, the biological polypeptide comprises acetyl tetrapeptide-5 and fibronectin.
Preferably, the mass fraction of the yeast extract with the whitening and freckle-removing effects in the whitening and freckle-removing essence is 1% -5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, etc., and other specific point values within the numerical range can be selected, so that no further description is given here.
Preferably, the mass fraction of the seaweed extract in the whitening and freckle-removing essence is 1% -2%, for example, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc., and other specific values within the numerical range are selectable, so that no further description is given here.
Preferably, the mass fraction of mannitol in the whitening and freckle-removing essence is 1% -2%, for example, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, etc., and other specific values within the numerical range are selectable, so that no further description is given here.
Preferably, the mass fraction of the acetyl tetrapeptide-5 in the whitening and freckle-removing essence is 0.05% -0.2%, for example, may be 0.05%, 0.1%, 0.15% or 0.2%, and other specific point values in the numerical range are selectable, and will not be described in detail herein.
Preferably, the mass fraction of the fibronectin in the whitening and freckle-removing essence is 0.15% -0.5%, for example, may be 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%, etc., and other specific point values within the numerical range are selectable, so that no further description is given here.
As a preferable technical scheme, the whitening and freckle-removing essence comprises, by mass, 1% -5% of yeast extract, 1% -2% of seaweed extract, 1% -2% of mannitol, 0.05% -0.2% of acetyl tetrapeptide-5, 0.15% -0.5% of fibronectin and water, wherein the yeast extract has the whitening and freckle-removing effects.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention carries out mutagenesis on industrial saccharomyces cerevisiae strains by using a normal pressure room temperature plasma mutagenesis technology, so as to obtain a mutation library with higher mutation rate, and further enrich the diversity and stability of the strains; the molecular sieve high-flux primary screening saccharomyces cerevisiae strain mutation library is utilized, and then the primary screening yeast extract is subjected to high-flux secondary screening by using a tyrosinase activity inhibition experiment and a melanin content inhibition experiment, so that the effect is clear, the mechanism is clear, and the yeast strain with the whitening and freckle removing effects is obtained;
(2) The invention improves the yield of yeast thallus by optimizing the culture conditions of the yeast and the culture medium used, has simple and convenient operation and does not need professional instruments and equipment, thereby providing conditions for the industrialized production of related products;
(3) The yeast extract prepared by using the yeast strain with the whitening and freckle removing effects can inhibit the activity of tyrosinase and inhibit the generation of melanin, and has good effect of brightening skin color for an ultraviolet-induced human skin blackening model after being prepared into the whitening and freckle removing essence, obvious effect, great value and wide application prospect.
Drawings
FIG. 1 is a graph showing the results of culturing the yeast strain JC-101 of example 1 of the present invention, wherein the results of culturing the A-activated original strain JC-101 (scale bar=10 mm); pictures of culture results of the strain after B-ARTP mutagenesis (scale bar=10 mm); c-culturing result pictures (proportion scale=10mm) of the yeast strains with whitening and freckle removing effects; d-morphological pictures of the yeast strains with whitening and freckle removing effects obtained by screening (scale = 5 μm);
FIG. 2 is a photograph showing the result of tyrosinase activity inhibition assay in example 1 of the present invention;
FIG. 3 is a graph showing the results of the melanin content-inhibiting experiment in example 1 of the present invention;
FIG. 4 is a photograph showing the result of SDS-PAGE in example 3 of the present invention, wherein lane 1-cell homogenate, lane 2-yeast extract, lane 3-flow-through liquid during purification, and lane Marker-standard protein molecular weight Marker;
FIG. 5 is a graph showing the result of HPLC detection in example 3 of the present invention;
FIG. 6 is a graph showing the results of the variation of the ITA DEG value of the skin at different experimental periods in example 4 of the present invention;
FIG. 7 is a graph showing the results of the variation of MI values of the skin at different experimental periods in example 4 of the present invention;
FIG. 8 is a graph showing the results of the variation of the ITA DEG value of the skin at different experimental periods in example 5 of the present invention;
FIG. 9 is a graph showing the results of the variation of MI values of the skin at different experimental periods in example 5 of the present invention;
FIG. 10 is a graph showing the results of the variation of the ITA DEG value of the skin at different experimental periods in example 6 of the present invention;
FIG. 11 is a graph showing the results of the variation of MI values of the skin at different experimental periods in example 6 of the present invention.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Materials and methods:
yeast with whitening and freckle removing effects is from an industrial Saccharomyces cerevisiae strain JC-101, which is named Saccharomyces cerevisiae JC-101-998 and is deposited in the Guangdong province microbiological bacterial culture collection center (GDMCC) at 10-28 days 2021, and has the address of Guangzhou Hirudo 100 # college No. 59 building 5, the mail code 510070 and the deposit number of GDMCC No.61987.
Industrial Saccharomyces cerevisiae strain JC-101 is maintained in Guangzhou and Church Biotechnology Co.
Yeast powder was purchased from Siemens technology.
Peptones were purchased from BD company in the united states.
Trypsin was purchased from sameimer feishi technologies limited.
Coomassie brilliant blue was purchased from beggaboolone immunotechnology limited.
Example 1
In the embodiment, the industrial saccharomyces cerevisiae strain (Saccharomyces cerevisiae) JC-101 is taken as an original strain, and yeasts with whitening and freckle removing effects are screened through mutagenesis, preliminary screening and secondary screening, and the steps are as follows:
(1) Activating and fermenting strains:
inoculating industrial Saccharomyces cerevisiae JC-101 preserved at-80deg.C onto YPD solid culture medium slant for activating, and culturing in 30 deg.C incubator for 5d, wherein the culture result is shown in figure 1A; picking single colony, inoculating into YPD liquid culture medium, and shake culturing at 30deg.C and 150rpm for 5 days;
the JC-101 after activation is inoculated into a fermentation culture medium and is cultured in a shaking way at 30 ℃ and 150rpm until the bacterial cells grow to the logarithmic phase.
The formulation of YPD medium is as follows: 30g/L glucose, 20g/L yeast powder, 20g/L peptone, 1.5g/L magnesium sulfate, 1.5g/L sodium dihydrogen phosphate, 3g/L disodium hydrogen phosphate, 1g/L glycerol, 20mg/L uracil and 1.001 g/L vitamin B (agar 20g/L is added to the solid medium), and the pH value is 6.5.
The preparation method of the YPD medium is as follows: all the components except glucose were dissolved in 500mL of water, the volume was set to 850mL, and after autoclaving, 150mL of a 20% mass fraction of filtered sterilized glucose solution was added.
(2) Mutagenesis:
the mutagenesis is carried out by a room temperature and normal pressure plasma mutagenesis technology, and the steps are as follows:
taking 1mL of bacterial liquid cultured to mid-log phase, placing the bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 8000rpm for 5min at 4 ℃, and discarding the supernatant;
washing with physiological saline containing 5% glycerol for 3 times, sucking 10 μl of the bacterial suspension, placing on a cell counting plate, counting the number of bacterial cells with a microscope, and diluting the bacterial cell concentration to 1×10 according to the counting result 6 ～1×10 7 /mL；
Uniformly coating 10 mu L of bacterial suspension on the surface of a sterile slide, then placing the slide on a carrier of an ARTP mutation breeding instrument for mutation treatment,
the ARTP mutagenesis treatment conditions are as follows:
high purity helium (99.99%): the flow rate is 15L/min, the input power is 120W, the irradiation distance is 2mm, the treatment temperature is lower than 40 ℃, and the mutagenesis is performed for 100s;
after the bacterial body mutagenesis is completed, the slide glass is placed into an EP tube filled with 1mL of physiological saline by using tweezers, the full shaking is continued for 1min, bacterial bodies attached to the slide glass are fully eluted, bacterial suspension is formed, and 10 mu L of bacterial liquid is directly taken from a control group and added into 1mL of physiological saline.
(3) And (3) primary screening:
the primary screening is carried out by molecular sieve high-flux screening, and the steps are as follows:
200. Mu.L of the mutagenized yeast suspension was pipetted onto the surface of a non-resistant YPD plate and incubated at 30℃for 72h, the results of which are shown in FIG. 1, panel B. Selecting strains with large colony diameter and growth advantage, placing the strains in a non-resistant YPD liquid culture medium, and culturing at 30 ℃ for 48 hours;
taking 1mL of bacterial liquid for strain conservation and strain label marking, and preparing the rest bacterial liquid for bacterial homogenate, wherein the steps are as follows:
preparing a heavy suspension (20 mM Tris-HCl and 0.1M NaCl, pH 7.0), adding the heavy suspension according to the ratio of thallus wet weight (g) to heavy suspension (mL) =1:15, and re-suspending for 3min at 10000 rpm;
high pressure homogenization at 800bar was performed 2 times using a tissue triturator: inserting a probe into the homogenate, setting the power to be 10 percent, the ultrasonic time to be 3 seconds, the interval time to be 5 seconds, the ultrasonic time to be 20 minutes and the ultrasonic times to be 3 times, wherein the process is carried out in an ice bath;
after the thalli are crushed, 17000g of the thalli are centrifugated for 30min at the temperature of 4 ℃ by using a large-sized low-temperature centrifuge, and the supernatant fluid is collected to obtain thalli homogenate;
the cell homogenate was filtered through a 0.45 μm filter and purified using a Superdex (TM) 30prep grade molecular sieve under the following conditions:
the 9V bed was equilibrated with 20mM PB buffer (18 ℃, pH 7.0) at a linear flow rate of 0.80cm/h (volumetric flow rate 4.4 mL/min);
loading the filtered thallus homogenate according to 10% (C/V) loading amount, flushing a column bed by using PB buffer solution (18 ℃ C., pH value is 7.0 and electric conductance is 1 ms/cm) at a linear flow rate of 0.8cm/h, and performing gradient elution to obtain better purity;
during elution, the detector indication rises to more than 70 to start collection, and the segmented receiving is carried out: 70-200 is the first bottle elution, 200 is more than-peak tip-drop back 200 is the second bottle elution, 200 is dropped to 70 is the third bottle elution, ultraviolet is dropped to below 70, collection is stopped, and the obtained eluent is the yeast extract;
the collection amount of the primary screening is ranked at the front according to the value of collection amount (volume multiplied by purity) of the collection liquid, and the collection amount of the Saccharomyces cerevisiae strain after ARTP mutagenesis is ranked at the front.
(4) And (3) re-screening:
the high-throughput re-screening is carried out through tyrosinase activity inhibition experiments and melanin content inhibition experiments, and the steps are as follows:
(1) tyrosinase activity inhibition assay:
taking B16 cells in logarithmic phase, preparing into single cell suspension of 7X10 3 Amount of cells/well was inoculated into 96-well cell culture plates (edge wells were filled with sterile PBS) and cells were placed at 37℃in 5% CO 2 Incubating for 1d in a cell incubator until the cell monolayer is fully paved at the bottom of the hole;
respectively adding cell culture medium containing three concentration gradient yeast extracts of high concentration (2%), medium concentration (0.2%) and low concentration (0.02%), adding cell holes of equal amount of PBS solution as negative control, adding cell holes of equal amount of kojic acid solution as positive control, respectively setting 3 repeated holes for each group, and continuing at 37deg.C and 5% CO 2 Incubating in a cell incubator, discarding the culture medium after 48 hours, and washing 2 times with PBS with pH of 7.4;
50 mu L of TritonX-100 solution with 1% concentration is added into each hole, the mixture is rapidly frozen at the temperature of minus 80 ℃ for 30min, cells are completely broken by melting at room temperature, 10 mu L of L-dopa solution with 1% concentration after preheating at 37 ℃ is added into the mixture, incubation is carried out for 2h at 37 ℃, the mixture is placed into an enzyme-labeling instrument to measure absorbance at the wavelength of 490nm, and tyrosinase activity inhibition rate is calculated by the following formula:
tyrosinase activity inhibition ratio (%) =1-average absorbance value of experimental group/average absorbance value of negative control group×100%.
The results of the test are shown in FIG. 2, and it is shown that the changes of the levels of the tyrosinase and the yeast extracts with different concentrations show a dose-effect relationship, and the change levels have statistical significance (p < 0.05), so that the yeast extracts have tyrosinase inhibition effect. And (3) setting the inhibition rate of the negative control group as a threshold value, and screening out strains with the inhibition rate larger than the threshold value, namely the yeast strains with tyrosinase inhibition efficacy.
(2) Melanin content inhibition experiments:
taking log to growPhase B16 cells were prepared as single cell suspensions at 2X 10 5 The amount of individual/well was seeded on 6-well cell culture plates;
adding cell culture medium containing three concentration gradient yeast extracts of high concentration (2%), medium concentration (0.2%) and low concentration (0.02%) respectively, adding cell holes of equal amount of PBS solution as negative control, adding cell holes of equal amount of 7% sodium ascorbate solution as positive control, setting 3 repeated holes for each group respectively, placing cells at 37deg.C and 5% CO 2 Incubating in a cell incubator, discarding the culture medium after 72 hours, and washing 2 times with PBS with pH of 7.4;
adding 0.25% trypsin to digest and collect cells, sonicating cells, centrifuging at 1000rpm for 10min;
washing the centrifuged precipitate with 10% trichloroacetic acid and ethanol, centrifuging at 1200g for 5min, removing supernatant, adding 1mol/L NaOH (containing 10% DMSO), and maintaining the temperature in 80deg.C water bath for 2 hr;
the absorbance values of each group were measured at 470nm using an enzyme-linked immunosorbent assay and the relative melanin synthesis content was calculated by the following formula:
relative melanin synthesis content (%) = [ (average absorbance value of experimental group/cell density of experimental group)/(average absorbance value of negative control group/cell density of negative control group) ]x100%.
The test results are shown in FIG. 3, and the changes of the relative content of the yeast extract and the melanin synthesis at different concentrations show a dose-effect relationship, and the change level has a statistical significance (p < 0.05), so that the yeast extract has the effect of inhibiting the melanin generation. And (3) setting the relative melanin synthesis content of the negative control group as a threshold value, and screening out strains with inhibition rate larger than the threshold value, namely yeast strains with whitening and freckle removing effects for inhibiting melanin generation.
Screening the yeast strain with tyrosinase inhibition effect and melanogenesis inhibition effect, namely the yeast strain with whitening and freckle removing effects.
Through the operation, a strain of yeast with whitening and freckle removing effects is successfully screened, the culture result is shown as a graph C in fig. 1, a bacterial colony is white and round, the central bulge is opaque, the surface is smooth, the edge is neat, the diameter is 1-2 mm, the morphological picture is shown as a graph D in fig. 1, cells are spherical or oval, and single or multiple cells exist, and the diameter is 5-10 mu m. Subsequent testing of the strain was performed.
Example 2
In this example, the yeast strains with whitening and freckle removing effects screened in example 1 were detected, and the steps are as follows:
cell biomass measurement
The yeast strain was subjected to fermentation culture in the same manner as in example 1;
taking 50mL of yeast fermentation culture solution, placing the yeast fermentation culture solution into a 100mL centrifuge tube, centrifuging at 8000rpm at 4 ℃ for 15min, and discarding the supernatant; washing with distilled water, centrifuging, repeating for 3 times, and discarding supernatant;
the bacterial precipitate is baked to constant weight in a dryer at 60 ℃, cooled to normal temperature in the dryer and weighed.
The result of the biomass measurement of the cells shows that the biomass of the cells of the yeast strain is 5-10 g/L.
Yeast extract polypeptide assay
A yeast extract was prepared in the same manner as in example 1;
precisely measuring 1mL of the solution to be measured, and calculating the polypeptide content in the yeast extract solution to be 2-4 g/L according to a protein content measurement method (second method of appendix VIIM of second edition 2010 of Chinese pharmacopoeia) and a linear regression equation.
Diluting the yeast extract solution by taking water as blank correction, and respectively detecting the absorbance of the diluted solution at the wavelengths of 260nm and 280nm to ensure that the indication is between 0.2 and 0.8;
concentration and yield were calculated according to the formula:
C＝(1.45×OD 280 -0.74×OD 260 ) X dilution factor, wherein, C: sample concentration (mg/mL); OD (optical density) 280 : an ultraviolet absorbance at 280 nm; OD (optical density) 260 : an ultraviolet absorbance at 260 nm;
yield = amount of protein loaded/amount of protein loaded x 100%.
Wherein the amount of protein in the sample is the amount of protein in the final yeast extract collection; the amount of the protein to be loaded is the amount of the protein of the molecular sieve to be loaded on the bacterial homogenate.
The protein yield is calculated to be 30% -40% according to the formula.
Genetic stability detection
The strain is continuously passaged for 8 times, each generation of strain is used for fermentation culture, and the biomass of thalli and the content of polypeptide in a fermentation product are detected.
The results show that after 8 times of continuous passage, the biomass of thallus and the content of polypeptide in each generation of strain fermentation products are stable, and the yeast strain has good stability.
According to the detection result, the screened yeast strain has the effects of whitening and removing freckles and has good stability. Yeast was designated Saccharomyces cerevisiae JC-101-998 and deposited at the collection of microorganisms and cell cultures (GDMCC) of Guangdong province, 10 th month of 2021, under accession number GDMCC No.61987, as identified by the name of 30 th floor of the university of Hirscho 100 in Guangzhou City, post code 510070.
Example 3
In this example, the extract of yeast strain JC-101-998 was assayed as follows:
extracts of strain JC-101-998 were prepared in the same manner as in example 1.
SDS-PAGE electrophoresis detection of yeast extracts
Mixing thallus homogenate, purified flow-through liquid and yeast extract with loading buffer solution respectively, treating in boiling water for 10min, centrifuging at 10000rpm for 3min at 4deg.C, and loading 10 μl of supernatant;
in electrophoresis, the voltage of the sample is 90V when the gel is concentrated, the voltage is 120V when the gel is separated, and the current is set to be 50mA;
the preparation method of the 4X loading buffer solution is as follows: 0.303g of Tris, 2mg of bromophenol blue, 0.8g of SDS, 4mL of glycerol and 189 mu L of hydrochloric acid are weighed, and the mixture is complemented to 10mL by water;
the preparation method of the SDS-PAGE electrode buffer is as follows: weighing 3g of Tris, 14.4g of glycine and 1g of SDS, and fixing the volume to 1L by using water;
after electrophoresis, taking out the separation gel, placing the separation gel in coomassie brilliant blue staining solution R250, and shaking for 15min at 50rpm for staining; after dyeing, taking out the separating gel, placing the separating gel into a decoloring solution, and decoloring by shaking for 3 hours at 50 rpm;
the preparation method of the coomassie brilliant blue staining solution comprises the following steps: 2.5g of Coomassie brilliant blue R250.5 g, 250mL of absolute ethyl alcohol and 80mL of 10% trichloroacetic acid solution are weighed, and water is used for constant volume to 1L;
the preparation method of the coomassie brilliant blue decolorization solution comprises the following steps: 500mL of absolute ethanol and 100mL of 10% trichloroacetic acid solution were weighed and the volume was fixed to 1L using water.
As shown in FIG. 4, the results of SDS-PAGE show that the yeast extract was highly enriched in about 16 kDa.
High performance liquid chromatography detection of yeast extracts
The purity of the yeast extract was measured using high performance liquid chromatography under the following conditions:
chromatographic column:C18，5μm，4.6×150mm，/>
mobile phase: 0.1% aqueous trifluoroacetic acid solution 0.1% trifluoroacetic acid in acetonitrile=85:15;
flow rate: 1.0mL/min; sample injection amount: 10. Mu.L; column temperature: 30 ℃; wavelength: 215nm.
The results of the measurements are shown in FIG. 5, and the results are counted as shown in Table 1.
TABLE 1
Sequence number Peak area (mV x min) Proportion (%) Retention time (min) Degree of polymerization
1 10958170 75.88 20.397 More than ten peptides
2 1261089 8.73 19.799 Decapeptides below
3 971624 6.73 19.290 Pentapeptides or more
4 586642 4.06 18.813 Tetrapeptides, pentapeptides
5 663893 4.59 18.389 Dipeptide, tripeptide
6 172 0.01 - Amino acids
As can be seen from FIG. 5 and Table 1, the results of high performance liquid chromatography show that 80% or more of the yeast extract is mainly decapeptides, and the remainder is small molecule active peptides and other trace substances.
Example 4
The embodiment provides a whitening and freckle-removing essence, which comprises, by mass, 4% of an extract of JC-101-998, 1.5% of a seaweed extract, 1.5% of mannitol, 0.01% of acetyl tetrapeptide-5 and 0.3% of fibronectin, and the balance of water.
Wherein the extract of JC-101-998 was prepared in the same manner as in example 1.
Filtering and subpackaging the whitening and freckle-removing essence in an ultra-clean workbench, and testing the efficacy of freckle removal and whitening after aseptic experiments, stability and safety detection.
30 healthy male or female volunteers 18-60 years old were enrolled and randomized into three groups of 10 volunteers each. The three groups are respectively subjected to experiments of negative control, whitening and freckle removing essence and positive control, the arm positions are used as test areas, and the skin color ITA degree value of the test areas is in the range of 20-41 degrees. Ultraviolet irradiation is carried out for 1 time per day at the same irradiation point by using a sunlight simulator according to the dose of 0.75 times of MED, and continuous irradiation is carried out for 4 days, so that the skin enters the blackening period, and a human skin blackening model is established.
Skin color detection was performed on the skin color of the test area on day 5 after the end of irradiation, followed by application of the sample with distilled water as a negative control and 7% ascorbic acid solution as a positive control. Samples were continuously applied for 4 weeks, and skin colors were instrumentally measured and recorded at 1, 2, 3 and 4 weeks after application, respectively.
Measuring the L, a and b values of each test area by using a skin colorimeter respectively, testing each area for 3 times, recording and calculating an ITA degree value, wherein the larger the ITA degree value is, the lighter the representing skin color is, and otherwise, the darker the skin color is; the MI values of each test area were measured separately using a skin melanin detector, each test area was tested three times, and recorded. The smaller the MI value, the lower the skin melanin content, whereas the higher the melanin content.
The results of the variation of skin ITA DEG values at different experimental periods are shown in FIG. 6, and the results of the variation of skin MI values are shown in FIG. 7. The graph shows that before and after the whitening and freckle-removing essence is smeared, the ITA degree difference or MI difference is obviously improved compared with a negative control, and the improvement level has statistical significance (p is less than 0.05), so that the whitening and freckle-removing essence has the effects of whitening and freckle removing, and the strain is verified to have the effects of whitening and freckle removing.
Example 5
The embodiment provides a whitening and freckle-removing essence, which comprises, by mass, 5% of an extract of JC-101-998, 1% of a seaweed extract, 1% of mannitol, 0.05% of acetyl tetrapeptide-5, 0.15% of fibronectin and the balance of water.
Wherein the extract of JC-101-998 was prepared in the same manner as in example 1.
Filtering and subpackaging the whitening and freckle-removing essence in an ultra-clean workbench, and testing the efficacy of freckle removal and whitening after aseptic experiments, stability and safety detection.
Example 6
The embodiment provides a whitening and freckle-removing essence, which comprises, by mass, 1% of an extract of JC-101-998, 2% of a seaweed extract, 2% of mannitol, 0.2% of acetyl tetrapeptide-5, 0.5% of fibronectin and the balance of water.
Wherein the extract of JC-101-998 was prepared in the same manner as in example 1.
Filtering and subpackaging the whitening and freckle-removing essence in an ultra-clean workbench, and testing the efficacy of freckle removal and whitening after aseptic experiments, stability and safety detection.
The same test experiment was performed on the spot-removing and whitening essences prepared in example 5 and example 6, wherein the change of the skin ITA ° value in the different experimental periods of example 5 is shown in fig. 8, the change of the skin MI value is shown in fig. 9, and the change of the skin ITA ° value in the different experimental periods of example 6 is shown in fig. 10, and the change of the skin MI value is shown in fig. 11. The results show that the ITA degree difference and the MI difference of the experimental group are obviously improved compared with the negative control and the positive control, and the improvement level also has statistical significance, so that the corresponding products also have the effects of whitening and removing freckles.
In conclusion, the yeast strain with whitening and freckle removing effects is successfully screened by the normal-pressure room-temperature plasma mutagenesis technology, the molecular sieve primary screening and the tyrosinase activity inhibition experiment and the melanin content inhibition experiment secondary screening. The yeast strain can be cultured on a large scale through fermentation, the operation is simple, the production efficiency is high, and the yield is further improved through optimizing the culture conditions and the used culture medium; the yeast extract prepared by the yeast strain has good whitening and freckle removing effects, can inhibit the activity of tyrosinase and the generation of melanin, can be prepared into cosmetics, can obviously lighten skin color after being used, and has the value of being applied to production practice.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (12)

1. The yeast with the whitening and freckle removing effects is named as Saccharomyces cerevisiae (Saccharomyces cerevisiae) JC-101-998, is deposited in the Guangdong province microorganism strain collection, and is deposited with the number GDMCC NO.61987, the date of deposition is 2021, 10 months and 28 days, and the deposited address is 5 th floor of Guangzhou national institute of first-class No. 100, no. 59.
2. A method for culturing yeast having whitening and freckle removing effects according to claim 1, wherein the method comprises:
inoculating the yeast with whitening and freckle removing effects into YPD culture medium, and culturing.
3. The method for culturing yeast with whitening and freckle removing effects according to claim 2, wherein the culturing is shake culturing, the temperature of the shake culturing is 25-32 ℃, the shake rotating speed of the shake culturing is 100-200 rpm, and the time of the shake culturing is 3-5 d.
4. The method for culturing yeast having whitening and freckle removing effects according to claim 2, wherein the YPD medium comprises: 25-50 g/L of glucose, 15-30 g/L of yeast powder, 20-35 g/L of peptone, 1.5-4 g/L of magnesium sulfate, 1.5-5 g/L of sodium dihydrogen phosphate, 1.5-5 g/L of disodium hydrogen phosphate, 0.5-2 g/L of glycerin, 15-25 mg/L of uracil and 0.001-0.003 g/L of vitamin B, and the pH value is 6-7.
5. The use of the yeast with whitening and freckle removing effects as claimed in claim 1 for preparing products with whitening and freckle removing effects.
6. The yeast extract with the whitening and freckle removing effects is characterized in that the yeast extract with the whitening and freckle removing effects is prepared by taking the yeast with the whitening and freckle removing effects as a raw material through the processes of fermenting, culturing, collecting thalli, preparing thalli homogenate and purifying;
the fermentation culture method is the same as the culture method as defined in any one of claims 2 to 4;
the preparation method of the bacterial homogenate comprises ice bath high-pressure homogenate;
the purification is preceded by a filtration step;
the pore diameter of the filter membrane used for filtering is 0.45 mu m;
the purification includes molecular sieve purification;
the molecular sieve comprises Superdex TM 30prep grade molecular sieve.
7. The whitening and freckle-removing essence is characterized by comprising the yeast extract with whitening and freckle-removing effects as claimed in claim 6.
8. The whitening and freckle removing essence according to claim 7, further comprising a plant extract, a humectant and a biological polypeptide.
9. The whitening and freckle removing essence according to claim 8, wherein the plant extract comprises seaweed extract.
10. The whitening and freckle removing essence according to claim 8, wherein the moisturizing agent comprises mannitol.
11. The whitening and freckle removing essence according to claim 8, wherein the biological polypeptide comprises acetyl tetrapeptide-5 and fibronectin.
12. The whitening and freckle removing essence according to claim 7, wherein the whitening and freckle removing essence comprises, by mass, 1% -5% of yeast extract, 1% -2% of seaweed extract, 1% -2% of mannitol, 0.05% -0.2% of acetyl tetrapeptide-5, 0.15% -0.5% of fibronectin and water.
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