CN117051048A - Lysate of fermentation product of saccharomyces cerevisiae, and preparation method and application thereof - Google Patents
Lysate of fermentation product of saccharomyces cerevisiae, and preparation method and application thereof Download PDFInfo
- Publication number
- CN117051048A CN117051048A CN202211636605.0A CN202211636605A CN117051048A CN 117051048 A CN117051048 A CN 117051048A CN 202211636605 A CN202211636605 A CN 202211636605A CN 117051048 A CN117051048 A CN 117051048A
- Authority
- CN
- China
- Prior art keywords
- lysate
- saccharomyces cerevisiae
- fermentation product
- fermentation
- bifidobacterium longum
- Prior art date
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Classifications
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- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a lysate of a fermentation product of saccharomyces cerevisiae, a preparation method and application thereof. The lysate of the fermentation product of the saccharomyces cerevisiae is prepared from a bifidobacterium longum fermentation product; the bifidobacterium longum is bifidobacterium longum Bifidobacterium longum HGCW-18, and the preservation number is CCTCC NO: m20221015. The invention selects the bifidobacterium longum HGCW-18 obtained by screening to prepare the lysate of the fermentation product of the two yeasts, the lysate of the fermentation product of the two yeasts has better moisturizing, crease-resistant and barrier repairing effects than the lysate of the fermentation product of the two yeasts prepared by bifidobacterium longum in the prior art, and the lysate of the fermentation product of the two yeasts has high lysis safety and is suitable for being applied to cosmetics.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a lysate of a fermentation product of saccharomyces cerevisiae, a preparation method and application thereof.
Background
Bifidobacteria are an important flora colonizing the intestinal tract of humans and animals. The bifidobacterium longum is used as an important probiotics of bifidobacterium, is widely planted in intestinal tracts of human bodies, has a plurality of potential probiotics functions on human bodies, can synthesize and secrete various polypeptide substances and enzymes such as antibacterial peptides, protease, lipase, amylase and the like, and promotes the decomposition of nutrient substances so as to be beneficial to the absorption and utilization of the human bodies.
The Bifidobacterium longum contains multiple nutritional components such as amino acids, polypeptides, vitamin B, vitamin C, vitamin K, folic acid, etc. Cell lysates obtained by fermentation with bifidobacteria are also known as lysates of the fermentation products of the genus Saccharomyces, and the presence of nutrients in bifidobacteria gives the lysates of the fermentation products of the genus Saccharomyces a variety of efficacy. For example, the lysate of the fermentation product of the saccharomyces cerevisiae has anti-aging effect, can neutralize free radicals, improve wrinkles and fine lines, increase skin elasticity, enable skin to be smooth and fine, and also can reduce and prevent skin color darkness caused by the early aging process of the skin. For example, the polysaccharide substances contained in the lysate of the fermentation product of the saccharomyces cerevisiae are natural moisturizers, and certain polysaccharide components have anti-inflammatory effects. For example, the skin brightening effect can promote metabolism of melanocyte, accelerate cutin shedding, promote synthesis of epidermal protein, improve skin texture and brighten skin color.
However, bifidobacterium longum is a strict anaerobic fermentation strain, effective substances of a lysate of a fermentation product of the bifidobacterium longum obtained by a lysis technology in the prior art are fewer, and the skin care effect of the lysate of the fermentation product of the bifidobacterium longum is still poorer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a lysate of a fermentation product of a saccharomyces cerevisiae, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a lysate of a fermentation product of Saccharomyces cerevisiae, prepared by fermentation of Bifidobacterium longum; the Bifidobacterium longum is Bifidobacterium longum sp.HGCW-18, and the preservation number is CCTCC NO: m20221015.
The invention selects the bifidobacterium longum HGCW-18 obtained by screening to prepare the lysate of the fermentation product of the two yeasts, the lysate of the fermentation product of the two yeasts has better moisturizing, crease-resistant and barrier repairing effects than the lysate of the fermentation product of the two yeasts prepared by bifidobacterium longum in the prior art, and the lysate of the fermentation product of the two yeasts has high lysis safety and is suitable for being applied to cosmetics.
In a second aspect, the invention provides a method for preparing a lysate of a fermentation product of Saccharomyces cerevisiae, comprising the steps of:
(1) Inoculating the Bifidobacterium longum sp.HGCW-18 into a solid culture medium for activation to obtain a single colony; inoculating the single colony into a liquid culture medium for static culture to obtain seed liquid;
(2) Inoculating the obtained seed liquid into a fermentation medium for static culture to obtain fermentation liquid;
(3) Centrifuging the fermentation liquor, removing supernatant, washing precipitate with water, and performing pyrolysis to obtain wall-broken thalli; and centrifuging and filtering the obtained wall-broken thalli to obtain a lysate of a fermentation product of the saccharomyces cerevisiae.
As a preferred embodiment of the method for producing a lysate of a fermentation product of Saccharomyces cerevisiae of the present invention, in the step (1), the liquid medium is MRS medium, and the solid medium is TPY solid medium or MRS solid medium; the temperature of the activation and the static culture is 32-40 ℃ and the time is 35-70 h.
As a preferred embodiment of the method for producing a lysate of a fermentation product of Saccharomyces cerevisiae of the present invention, in step (2), the fermentation medium comprises a nitrogen source and a carbon source; the nitrogen source is at least one of soybean peptone, tryptone, fish meal, soybean powder and ammonium salt; the carbon source is at least one of glucose, maltose, sucrose, high fructose syrup and hydrolyzed starch. The adding amount of the nitrogen source in the fermentation culture medium is 0.5-10% by mass percent, and the adding amount of the carbon source in the fermentation culture medium is 0.2-4%; preferably, the nitrogen source is added to the fermentation medium in an amount of 1% -4%, and the carbon source is added to the fermentation medium in an amount of 0.5% -1%.
As a preferred embodiment of the method for producing a lysate of a fermentation product of Saccharomyces cerevisiae of the present invention, in the step (2), the mass ratio of the nitrogen source to the carbon source is 2 to 8:1, a step of; preferably 4:1; the temperature of the static culture is 32-40 ℃ and the time is 35-70 h.
As a preferred embodiment of the method for preparing a lysate of a fermentation product of Saccharomyces cerevisiae, in the step (3), the rotation speed is 8000 rpm-25000 rpm and the time is 5-30 min when the supernatant is removed by centrifugation of the fermentation liquid; the cracking temperature is 0-20 ℃, the pressure is 35-100 Mpa, and the time is 10-50 min; the rotational speed of the wall-broken thalli is 8000 rpm-25000 rpm, and the time is 10 min-40 min; the filtration is carried out by adopting a ceramic membrane, and the ceramic membrane is a filter membrane with the thickness of 0.1-1.5 mu m.
In a third aspect, the invention provides a yeast fermentation product lysate concentrate comprising said yeast fermentation product lysate.
As a preferred embodiment of the lysate essence of the fermentation product of the saccharomyces cerevisiae, the added amount of the lysate of the fermentation product of the saccharomyces cerevisiae in the essence is 3-20% by mass percent.
In a fourth aspect, the invention provides the use of a lysate of a fermentation product of Saccharomyces cerevisiae in cosmetics.
As a preferred embodiment of the application of the present invention, the cosmetic is any one of skin-softening lotion, emulsion, cream, facial mask, gel, powder, spray, essence, washing and caring cosmetics.
Compared with the prior art, the invention has the beneficial effects that:
the invention selects the bifidobacterium longum HGCW-18 obtained by screening to prepare the lysate of the fermentation product of the two yeasts, the lysate of the fermentation product of the two yeasts has better moisturizing, crease-resistant and barrier repairing effects than the lysate of the fermentation product of the two yeasts prepared by bifidobacterium longum in the prior art, and the lysate of the fermentation product of the two yeasts has high lysis safety and is suitable for being applied to cosmetics.
Drawings
FIG. 1 is a colony morphology of isolated bifidobacterium longum HGCW-18;
FIG. 2 is a gram stain of bifidobacterium longum HGCW-18 under a microscope;
FIG. 3 is an agarose gel electrophoresis of the 16S rDNA PCR product of bifidobacterium longum HGCW-18;
FIG. 4 is a graph showing the statistical rate of change of moisture content of the stratum corneum before and after the use of the essence;
FIG. 5 is a statistical plot of the rate of change of skin transdermal moisture loss before and after the use of the serum;
FIG. 6 is a statistical plot of the rate of change of skin elasticity R2 before and after application of the serum;
FIG. 7 is a graph showing statistics of the rate of change of the area ratio of wrinkles before and after the use of the serum;
FIG. 8 is a statistical chart of fluorescent quantitative PCR detection.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: isolation and characterization of bifidobacterium longum HGCW-18
1. Isolation and purification and morphological identification
Collecting cheese products as samples, and separating microorganisms in the cheese products by using a TPY solid culture medium. Culturing the separated microorganism in a workstation at 37 ℃ for 48 hours, picking dominant bacterial colonies on a flat plate, streaking and separating to obtain pure bacterial colonies, carrying out morphological observation through gram staining, inoculating the pure bacterial colonies into a TPY solid culture medium, carrying out anaerobic culture at 37 ℃ for 48 hours, and preserving at 4 ℃ after the culture is finished. A single strain HGCW-18 was obtained.
After the strain HGCW-18 is purified, single colony is formed in TPY solid culture medium, its form is shown in figure 1, and its form is almost identical, most of colony form is round, white and its surface roughness is drier. Can grow in the environment of 30-45 ℃, is negative in contact with enzyme, motility, nitrate reduction, indole and H2S production and gelatin liquefaction tests,
the results of gram staining are shown in FIG. 2, and the morphology of purple rod-shaped cells was observed under a microscope, and one of the lactic acid bacteria was determined.
2. Thin layer chromatography detection and identification
The strain HGCW-18 is identified by detecting the production of organic acids during the glycolysis of different sugar sources by thin layer chromatography using the characteristic that lactic acid and acetic acid are mainly produced by the glycolysis process of lactobacillus.
Culturing the strain HGCW-18 separated in the above steps in culture medium containing different sugar sources at 37deg.C for 24 hr, centrifuging at 5000rpm for 20min, collecting supernatant, and detecting by thin layer chromatography with mixed solution of methylation and bromophenol blue dissolved in 70% alcohol as indicator.
The results are shown in Table 1.
TABLE 1 thin layer chromatography assay for strain HGCW-18
3. 16S rDNA identification
The bacterial 16S rDNA sequence is highly conserved among different species, with the 16S rDNA sequence being different among different species. Therefore, by PCR amplification and sequencing of the 16S rDNA sequence, the bacterial species can be determined by comparison with the known sequences in GenBank, and they can be classified into specific genera or species. Identification of 16S rDNA (16S rRNA is a subunit of ribosomal RNA, and 16S rDNA is a gene encoding 16S rRNA) refers to the identification of bacterial species by sequencing the bacterial 16S rDNA sequence.
Extracting and purifying the DNA of the bacterial strain HGCW-18 by using a bacterial genome DNA extraction kit, and amplifying the extracted genome DNA as a 16S rDNA-PCR template.
Primer information is shown in Table 2.
Table 2 general primers
The components of the amplification system are shown in Table 3.
TABLE 3PCR amplification System
| 1×TSE101 gold medal mix | 45μL |
| F(10P) | 2μL |
| R(10P) | 2μL |
| DNA template | 1μL |
The above amplification system was amplified according to the amplification procedure shown in Table 4.
TABLE 4PCR amplification procedure
The amplified PCR products were subjected to agarose gel electrophoresis (2. Mu.L of sample+6. Mu.L of bromophenol blue) at 300V for 12 minutes to obtain an identification gel. The electrophoretogram is shown in fig. 3.
The PCR products were subjected to a first generation of sequencing (27F/1492R as sequencing primer) and the sequencing results were subjected to homology alignment with the GenBank gene library by BLAST program. The 16S rDNA homology analysis results show that the homology of the strain with known bifidobacterium longum in GenBank database is up to 100%.
The isolated lactobacillus is identified as bifidobacterium longum Bifidobacterium longum which is preserved in China Center for Type Culture Collection (CCTCC) at the time of 7 th month 4 of 2022 by combining morphological identification, detection identification by a thin layer chromatography and 16S rDNA homology analysis, and the preservation number is CCTCC NO: m20221015, which is classified and named as Bifidobacterium longum sp.HGCW-18, and the preservation unit address is Jiuqiu 299 Wuhan university in Wuhan district of Wuhan, hubei province, china.
Example 2: preparation of lysate of fermentation product of Saccharomyces cerevisiae
(1) Preparation of Bifidobacterium longum seed liquid
Inoculating bifidobacterium longum HGCW-18 into a TPY solid culture medium, and culturing and activating for 48 hours at 37 ℃ to obtain bifidobacterium longum with single colony; inoculating single colony bifidobacterium longum into 100mL MRS culture medium, and standing and culturing at 37 ℃ for 48 hours to obtain bifidobacterium longum seed liquid.
(2) Preparation of Bifidobacterium longum fermentation broth
Inoculating the bifidobacterium longum seed liquid into a fermentation medium according to the inoculum size of 1%, and standing and culturing for 48 hours at 37 ℃ to obtain bifidobacterium longum fermentation liquid; the fermentation medium contains 2% of soybean peptone and 0.5% of glucose by mass percent.
(3) Preparation of lysate of fermentation product of Saccharomyces cerevisiae
Centrifuging the obtained Bifidobacterium longum fermentation broth at 15000rpm for 10min, discarding supernatant, washing precipitate with deionized water, centrifuging at 15000rpm for 10min, subjecting the washed precipitate to aseptic and high pressure cracking at 15deg.C under 50Mpa for 30min to obtain wall-broken thallus, centrifuging the obtained wall-broken thallus at 15000rpm for 20min, and filtering supernatant with 0.2 μm filter membrane to obtain lysate of fermentation product of Saccharomyces cerevisiae.
Example 3: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: in the step (2), the soybean peptone content in the fermentation medium was 1%.
Example 4: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: in the step (2), the soybean peptone content in the fermentation medium was 3%.
Example 5: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: in the step (2), the soybean peptone content in the fermentation medium was 4%.
Comparative example 1: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: bifidobacterium longum is commercially available as bifidobacterium longum ATCC55814, available from beijing deposit biotechnology limited.
Comparative example 2: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: bifidobacterium longum is commercially available as bifidobacterium longum ATCC55816, available from beijing deposit biotechnology limited.
Comparative example 3: preparation of lysate of fermentation product of Saccharomyces cerevisiae
The only difference from example 2 is that: the bifidobacterium longum is a commercial bifidobacterium longum cic 6068 available from Shanghai deposit biotechnology limited.
Example 6: preparation of lysate essence of fermentation product of Saccharomyces cerevisiae
The lysate of the fermentation products of the examples 2-5 are respectively prepared into lysate essence containing 3% -20% of the fermentation products of the Saccharomyces cerevisiae.
The formulation of the serum is shown in table 5.
Table 5 formulation of essence
The preparation steps of the essence are as follows:
(1) Respectively premixing components A, B, C, D, E according to the formula of table 5 for later use;
(2) Stirring and heating the component A to 60-80 ℃, adding the component B, continuously heating to 75-85 ℃ to completely dissolve each component, and preserving heat for 10-30 min to achieve the purposes of sterilization and defoaming to obtain a mixed solution I;
(3) Heating the component C to 75-85 ℃, and preserving heat for 10-30 min to achieve the aim of sterilization and defoaming;
(4) Slowly adding the mixed solution I into the preheated component C at 75-85 ℃, homogenizing for 2-25 min until the mixed solution I is completely and uniformly emulsified to obtain a mixed solution II;
(4) Cooling to below 45deg.C, adding component D and component E into the mixed solution II, and stirring to obtain essence.
Example 7: preparation of lysate essence of fermentation product of Saccharomyces cerevisiae
The lysates of the fermentation products of examples 2 to 5 and comparative examples 1 to 3 were prepared as lysates of the fermentation products of Saccharomyces cerevisiae in an amount of 10% as a sample for testing.
The formulation of the serum is shown in table 6.
Table 6 formulation of essence
The preparation steps of the essence are as follows:
(1) Respectively premixing components A, B, C, D, E according to the formula of Table 6 for later use;
(2) Stirring and heating the component A to about 70 ℃, adding the component B, continuously heating to 82 ℃ to completely dissolve each component, and preserving heat for 20min to obtain a mixed solution I;
(3) Heating the component C to 82 ℃, and preserving heat for 15min;
(4) Slowly adding the mixed solution I into the preheated component C at 82 ℃, homogenizing for 5min until the mixed solution I is completely and uniformly emulsified to obtain a mixed solution II;
(5) Cooling to 40 ℃, adding the component D and the component E into the mixed solution II, and uniformly stirring to obtain the essence.
Test example 1: skin stratum corneum moisture content test
Test sample: 10% of the lysate essence of the fermentation product of Saccharomyces cerevisiae (examples 2 to 5 and comparative examples 1 to 3).
The testing steps are as follows: 210 healthy volunteers aged 18-45 years were selected, 30 volunteers per sample. The essences of examples 2 to 5 and comparative examples 1 to 3 were distributed on the left and right sides of the volunteers according to a random distribution table of test areas, and were continuously applied to the volunteers for 28 days, 2 times a day (each time in the morning and evening), and 0.2g each time. The test was performed before use, 2 weeks after use, and 4 weeks.
Test instrument: skin moisture tester Cornmeometer CM825 (manufactured by CK, germany).
Rate of change= (average after use-average before use)/average before use x 100%.
The higher the value tested, the higher the moisture content of the skin cuticle, the more obvious the moisturizing effect of the essence.
The test results are shown in Table 7 and FIG. 4.
TABLE 7 results summary of moisture content of skin stratum corneum before and after 2 and 4 weeks of application of essence
Test results show that the essence prepared by using the lysate of the fermentation products of the two yeasts of examples 2-5 has better moisturizing effect compared with the lysate of the fermentation products of the two yeasts obtained by fermenting the bifidobacterium longum in the prior art. As is clear from comparative examples 2 to 4, when the soybean peptone content in the fermentation medium is 2% (example 2), the moisture content change rate of the skin horny layer is the highest, and the moisturizing effect of the essence is the best.
Test example 2: skin transdermal water diversion test
Test sample: same as in test example 1.
The testing steps are as follows: same as in test example 1.
Test instrument: skin moisture loss tester Tewameter TM300 (manufactured by CK company, germany).
Rate of change= (average after use-average before use)/average before use x 100%.
The lower the number tested, the less skin water loss per unit time, the better the skin barrier.
The test results are shown in Table 8 and FIG. 5.
TABLE 8 results summary of skin transdermal moisture loss before and after 2 and 4 weeks of use with serum
The test results show that the lysates of the Saccharomyces cerevisiae fermentation products of examples 2 to 5 have better barrier repair effects than the conventional lysates of the Saccharomyces cerevisiae fermentation products. When the content of soybean peptone in the fermentation medium was 2% (example 2), the rate of change of the percutaneous water loss amount per unit time of skin was the largest, i.e., the skin barrier repairing effect was the best.
Test example 3: skin elasticity test
Test sample: same as in test example 1.
The testing steps are as follows: same as in test example 1.
Test instrument: skin elasticity tester MPA580 (manufactured by CK company, germany).
Rate of change= (average after use-average before use)/average before use x 100%.
The higher the skin elasticity R2 value, the better the skin elasticity.
The test results are shown in table 9 and fig. 6.
Table 9 summary of results of skin elasticity R2 after 2 and 4 weeks before application of the serum
The test results show that the lysates of the fermentation products of examples 2 to 5 have a better effect of improving skin elasticity than the lysates of the conventional fermentation products of Saccharomyces cerevisiae. When the content of soybean peptone in the fermentation medium was 2% (example 2), the skin elasticity R2 value was the highest, indicating that the skin elasticity was the best.
Test example 4: anti-wrinkle Performance test
Test sample: same as in test example 1.
The testing steps are as follows: same as in test example 1.
Test instrument: face imager VISIA-CR (manufactured by Canfield Scientific company, usa).
Rate of change= (average after use-average before use)/average before use x 100%.
The lower the skin wrinkle area ratio, the less obvious the wrinkles are, and the better the anti-wrinkle effect of the product is.
The test results are shown in table 10 and fig. 7.
Table 10 summary of results of wrinkle area ratios after 2 and 4 weeks before use of the serum
Test results show that the lysate of the fermentation products of the two-way yeast of examples 2-5 has better anti-wrinkle effect. When the content of soybean peptone in the fermentation medium was 2% (example 2), the skin wrinkle area ratio was the largest in the change rate, indicating that the anti-wrinkle effect of the product was the best.
Test example 5: repair barrier test
And carrying out experiments on fluorescence quantitative PCR (polymerase chain reaction) of the barrier repair related genes of the lysate of the fermentation product of the saccharomyces cerevisiae, and evaluating the barrier repair capability of the lysate of the fermentation product of the saccharomyces cerevisiae.
Test sample: the aqueous solutions of lysates of the fermentation products of the yeasts of examples 2 to 5 and those of comparative examples 1 to 3 (concentrations are shown in Table 11).
TABLE 11 cytotoxicity test concentration gradient setting table
Test cells: keratinocytes (purchased from Guangdong Boxi Biotechnology Co., ltd.).
The testing steps are as follows: first, a sample cytotoxicity test was performed based on keratinocytes, and the maximum safe dose of each sample on keratinocytes was determined. Secondly, based on the safe dosing, carrying out a fluorescent quantitative PCR test to evaluate whether the sample has an improvement effect on barrier repair at the gene expression level, and carrying out statistical analysis on the result, wherein the method comprises the following steps:
(1) Cytotoxicity test
Cell inoculation: according to 4X 10 4 Cell/well seeding Density cells were seeded into 96-well plates in an incubator (37 ℃ C., 5% CO) 2 95% relative humidity) was cultured overnight.
Experimental grouping: the experiment sets a blank control group, a positive control group and a sample group. In the sample group, 8 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.
Preparing liquid: test object working solutions were prepared according to the cytotoxicity test concentration gradient setting table (see table 11).
Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of cell culture medium was added to each well of the blank control group; 200. Mu.L of cell culture medium containing 4% DMSO was added to each well of the positive control group; 200 mu L of cell culture solution containing the test substance with corresponding concentration is added into each hole of the sample group; zero-well cell-free inoculation was performed, and only 200. Mu.L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO) 2 95% relative humidity).
And (3) testing: after cell culture for 24h, the supernatant was discarded, MTT working solution (0.5 mg/mL, as prepared) was added, incubated at 37℃for 4h in the absence of light, after incubation was completed, the supernatant was discarded, 150. Mu.L of cell culture solution containing 4% DMSO was added to each well, and OD was read at 490 nm.
Cell relative viability calculation: according to the formula, the relative cell viability= (dosing well OD-zeroing well OD)/(blank pair white pair OD-zeroing well OD) ×100%.
The cytotoxicity test was performed on keratinocytes with 8 dosing concentrations from high to low, and the cell relative viability results are shown in table 12.
TABLE 12 results of cell relative viability of samples at different concentrations
The concentration with cell viability above 80 was selected as the maximum safe dosing concentration, and as can be seen from the table, when the maximum safe dosing concentration is 10%, the lysate solution of the fermentation product of the Saccharomyces cerevisiae was selected for testing in examples 2 to 5, and the cells still remained high in activity and still within the safe concentration range.
(2) Fluorescent quantitative PCR test
Cell inoculation: according to 2.5X10 5 Density of wells cells were seeded into 6-well plates in incubator (37 ℃, 5% CO) 2 95% relative humidity) was cultured overnight.
Preparing liquid: the test working fluid was prepared according to the test group of fluorescent quantitative PCR tests (see Table 13).
Table 13 fluorescent quantitative PCR test packets
Administration: according to the test group of Table 10, 3 duplicate wells were set in each group, and when the plating rate of cells in the 6-well plate reached 40% -50%, the group administration was performed in an incubator (37 ℃ C., 5% CO) 2 95% relative humidity) for 24h.
Collecting cells: after incubation of the samples for 24h, washing was performed with PBS solution, 1 mL/well, and washing was performed twice. After washing, 1mL RNAiso Plus was added to each well, and after cell lysis by blowing, the cells were collected in 1.5mL enzyme-free EP tubes and stored in a-80℃refrigerator.
Fluorescent quantitative PCR test: RNA extraction operation was performed according to RNAiso Plus instructions, reverse transcription operation was performed according to a reverse transcription kit, and fluorescence quantitative PCR reaction system configuration and on-machine detection were performed according to SYBR Premix Ex TaqTMII fluorescent dye (TaKaRa DRR 081A) product instructions.
And (3) calculating results: and (5) carrying out result calculation by adopting a 2-AACT method.
The results of the fluorescent quantitative PCR test are shown in Table 14, and the trend of the gene change is shown in FIG. 8.
TABLE 14 summary of fluorescent quantitative PCR test results
Compared with a blank control group, the samples of the comparative examples 1 to 3 of examples 2 to 5 have remarkable promotion effect on the expression of barrier-related genes TGM1, FLG, IVL and ABCA12, which indicates that the lysate of the fermentation product of the saccharomyces cerevisiae provided by examples 2 to 5 has better barrier repair effect. Among them, the lysate of the fermentation product of Saccharomyces cerevisiae of example 2 has the strongest promoting effect on the expression of barrier-related genes TGM1, FLG, IVL and ABCA12, and has the best barrier repair effect.
Test example 6: human use evaluation
Test sample: same as in test example 1.
The testing steps are as follows: 210 healthy volunteers aged 18-45 years were selected, 30 volunteers per sample. The volunteers take out the product according to the custom amount, evenly apply the product on facial skin, continuously use the product for 4 weeks, and use the product 2 times per day. Skin image photographing was performed on the face before the sample was used and after the 4 th week of use, and the data were recorded as: before use, after 4 weeks. The volunteers evaluate the initial skin condition of the facial skin by questionnaire before use, and evaluate the feeling of use and effect of the sample after the 4 th week of use.
The skin condition change scores are shown in table 15.
TABLE 15 skin State Change score (full score 4 points)
The test results show that the skin state of the essence containing the lysate of the fermentation products of the two yeasts of examples 2 to 5 changes more remarkably after use, and the skin state of the volunteers became better. Example 2 the skin state changes of the lysates of the fermentation products of the two yeasts were scored the highest and the skin state changes after use were optimal.
The evaluation of the effect after 4 weeks is shown in Table 16.
Table 16 shows the effect evaluation (number of people, number of people ratio) for 4 weeks
The test results show that the essence containing the lysate of the fermentation products of the saccharomyces cerevisiae in examples 2-5 has better effects of shrinking pores and brightening skin. Wherein the effect evaluation of the lysate of the fermentation product of Saccharomyces cerevisiae of example 2 was relatively optimal for 4 weeks.
The overall effect evaluation using 4 weeks is shown in table 17.
Table 17 shows the overall effect evaluation (number of people, number of people ratio) for 4 weeks
The test results show that the overall effect of the essence containing the lysates of the fermentation products of the Saccharomyces cerevisiae of examples 2 to 5 is better evaluated. Wherein example 2 the total effect evaluation of the two yeast fermentation product lysate was relatively optimal for 4 weeks.
Test example 7: safety test
Eye irritation test reference SN/T2329-2009 cosmetic eye irritation corrosive chick embryo chorioallantoic membrane test.
The ability of the nanoemulsion to cause changes in chick embryo chorioallantoic membrane toxicity was tested using chick embryo chorioallantoic membrane experiments, describing the potential eye irritation elements and processes for the nanoemulsion. Chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chick embryo. The chick embryo chorioallantoic membrane has abundant blood vessels on the surface and can be regarded as a complete organism, and the experiment utilizes the characteristics of complete, clear and transparent blood vessel system of the hatched chick embryo chorioallantoic membrane, a certain amount of nano emulsion IS contacted with the chick embryo chorioallantoic membrane, the chick embryo chorioallantoic membrane IS acted for a set time, the toxicity effect index (such as bleeding, coagulation and vascular dissolution) of the chorioallantoic membrane IS finally observed, the grading (IS) IS carried out, and the mathematical average value IS calculated to evaluate the eye irritation of the nano emulsion.
The stimulus score (IS) was calculated using the reaction time method, the calculation formula IS as follows:
wherein: the average time in seconds for onset of bleeding observed on sec H (bleeding time) -CAM membrane; sec L (vascular hemolysis time) -the average time in seconds at which onset of vascular lysis is observed on the CAM membrane; the average time in seconds for onset of clotting is observed on the sec C (clotting time) CAM membrane.
The results of the stimulation scoring classification are shown in Table 18.
TABLE 18 stimulation score stimulation classification
| Stimulation scoring | Irritation classification |
| IS<1 | No irritation |
| 1≤IS<5 | Light irritation |
| 5≤IS<9 | Moderate irritation |
| IS≥10 | Strong irritation/corrosiveness |
The specific experimental method is as follows:
the test subjects were 9 day old chick embryos.
Samples were the lysates (20% aqueous solutions) of the fermentation products of the Saccharomyces cerevisiae of examples 2 to 5 and comparative examples 1 to 3, respectively. The negative control group was normal saline. The positive control group was 1% sodium dodecyl sulfate solution (SDS solution).
Preparation of chick embryo: eggs of 0 day old were placed in an incubator for 9 days (temperature of the incubator is 37.5 ℃ C. And relative humidity is 55% to 70%). Selecting chicken embryos with good vascular development, marking the positions of air chambers on the surfaces of eggshells, and distributing eggs required by samples to be tested (6 chicken embryos for one sample, 1 chicken embryo for negative control and 1 chicken embryo for positive control).
Testing: the marked eggshell portion is removed with a tool. The egg membrane was wetted by adding 0.9% sodium chloride solution (physiological saline) dropwise to the solution via a pipette, and the sodium chloride solution was poured out. Carefully remove the inner membrane by forceps, ensure that the blood vessel is not damaged, observe the chick embryo without obvious bleeding or turbidity, and then judge that the chick embryo can be used for experiments. 300. Mu.L of the test substance was applied to the chorioallantoic membrane, and after 3 minutes, the test substance was washed with physiological saline for 30 seconds. Immediately observe chorioallantoic membrane reaction, record chick embryo status by photographing, and end observation. The results are shown in Table 19.
TABLE 19 results of irritation test
| Name of the name | IS value | Irritation classification |
| Example 2 | 0.3 | No irritation |
| Example 3 | 0.4 | No irritation |
| Example 4 | 0.3 | No irritation |
| Example 5 | 0.3 | No irritation |
| Comparative example 1 | 0.6 | No irritation |
| Comparative example 2 | 0.5 | No irritation |
| Comparative example 3 | 0.5 | No irritation |
The results show that the lysates of the fermentation products of the two yeasts of examples 2 to 5 and comparative examples 1 to 3 are free of eye irritation and have high safety. The lower IS value of the lysates of the Saccharomyces cerevisiae fermentation products of examples 2-5 compared to the IS values of comparative examples 1-3, indicates that the lysates of the Saccharomyces cerevisiae fermentation products of examples 2-5 are less toxic.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A lysate of a fermentation product of a saccharomyces cerevisiae, characterized in that it is prepared by fermentation of bifidobacterium longum; the bifidobacterium longum is bifidobacterium longum Bifidobacterium longum HGCW-18, and the preservation number is CCTCC NO: m20221015.
2. A method of preparing a lysate of a fermentation product of saccharomyces cerevisiae according to claim 1, comprising the steps of:
(1) Inoculating the bifidobacterium longum Bifidobacterium longum HGCW-18 into a solid culture medium for activation to obtain single bacterial colony; inoculating the single colony into a liquid culture medium for static culture to obtain seed liquid;
(2) Inoculating the obtained seed liquid into a fermentation medium for static culture to obtain fermentation liquid;
(3) Centrifuging the fermentation liquor, removing supernatant, washing precipitate with water, and performing pyrolysis to obtain wall-broken thalli; and centrifuging and filtering the obtained wall-broken thalli to obtain a lysate of a fermentation product of the saccharomyces cerevisiae.
3. The method for producing a lysate of a fermentation product of Saccharomyces cerevisiae according to claim 2, wherein in the step (1), the liquid medium is MRS medium, and the solid medium is TPY solid medium or MRS solid medium; the temperature of the activation and the static culture is respectively 32-40 ℃ and the time is respectively 35-70 h.
4. The method of claim 2, wherein in step (2), the fermentation medium comprises a nitrogen source and a carbon source; the nitrogen source is at least one of soybean peptone, tryptone, fish meal, soybean powder and ammonium salt; the carbon source is at least one of glucose, maltose, sucrose, high fructose syrup and hydrolyzed starch; the adding amount of the nitrogen source in the fermentation medium is 0.5-10% by mass percent, and the adding amount of the carbon source in the fermentation medium is 0.2-4% by mass percent.
5. The method for producing a lysate of a fermentation product of Saccharomyces cerevisiae according to claim 2, wherein in the step (2), the mass ratio of the nitrogen source to the carbon source is 2-8: 1, a step of; the temperature of the static culture is 32-40 ℃ and the time is 35-70 h.
6. The method for producing a lysate of a fermentation product of Saccharomyces cerevisiae according to claim 2, wherein in the step (3), the rotation speed is 8000rpm to 25000rpm for 5min to 30min when the supernatant is removed by centrifugation of the fermentation liquid; the cracking temperature is 0-20 ℃, the pressure is 35-100 Mpa, and the time is 10-50 min; the rotational speed of the wall-broken thalli is 8000 rpm-25000 rpm, and the time is 10 min-40 min; the filtration is carried out by adopting a ceramic membrane, and the ceramic membrane is a filter membrane with the thickness of 0.1-1.5 mu m.
7. A saccharomyces cerevisiae fermentation product lysate concentrate comprising the saccharomyces cerevisiae fermentation product lysate of claim 1.
8. The extract of a lysate of a fermentation product of Saccharomyces cerevisiae according to claim 7, wherein the lysate of a fermentation product of Saccharomyces cerevisiae is added to the extract in an amount of 3-20% by mass.
9. Use of a lysate of a fermentation product of Saccharomyces cerevisiae according to claim 1 in cosmetics.
10. The use according to claim 9, wherein the cosmetic is any one of a smoothing toner, an emulsion, a cream, a face mask, a gel, a powder, a spray, an essence, a wash and care, and a makeup cosmetic.
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