CN114891846A - Dischizosaccharomyces cerevisiae fermentation lysate and application thereof in whitening and anti-aging cosmetics - Google Patents

Abstract

The application discloses a saccharomyces bifidus fermentation lysate and application thereof in whitening and anti-aging cosmetics. The split yeast fermentation lysate comprises a split yeast polysaccharide having terminal glucose residues and terminal galactose residues, the molecular structure of the polysaccharide comprising predominantly glucose and galactose units and comprising glucosamine and galactosamine. The polysaccharide is obtained by extracting and purifying the fermentation liquor of the saccharomyces bifidus, has obvious antioxidant and tyrosinase activity inhibiting performances, also has the effects of promoting the growth and proliferation of fibroblasts, and has whitening and anti-aging effects.

Description

Dischizosaccharomyces cerevisiae fermentation lysate and application thereof in whitening and anti-aging cosmetics

Technical Field

The application relates to the technical field of yeast schizosaccharomyces, in particular to a yeast schizosaccharomyces fermentation lysate and application thereof in whitening and anti-aging cosmetics.

Background

The secondary fission yeast fermentation product filtrate and the secondary fission yeast fermentation lysate are commonly used functional components in the current cosmetics, and are recorded in the catalog of the names of used cosmetic raw materials 2021, and the fermentation strain is bifidobacterium, especially bifidobacterium longum. Bifidobacterium is a gram-positive, immotile, rod-shaped, sometimes bifurcated, strictly anaerobic bacterium. The fermentation product of the yeast schizosaccharomyces cerevisiae contains various metabolites including polysaccharide, organic acid, amino acid, polypeptide, protein, nucleic acid, vitamin and other frof, and the ingredients can provide various nutrients for human skin and have good biological effects on the skin.

Disclosure of Invention

Based on this, the inventors of the present application creatively found 5 polysaccharides, S1, S2, S3, S4 and S5, in the lysate of schizosaccharomyces cerevisiae, and carried out a preliminary structural analysis on the five schizosaccharomyces cerevisiae fermentation polysaccharides, and found that S1, S4 and S5 are polysaccharides containing amide groups, and S2 and S3 contain uronic acid.

Further, in vitro tests on the obtained schizosaccharomyces cerevisiae fermentation lysate discover that the schizosaccharomyces cerevisiae fermentation lysate provided by the embodiments 1 and 2 has lower DPPH free radical clearance rate IC50, hydroxyl free radical clearance rate IC50, in vitro tyrosinase inhibition rate IC50 and in vitro hyaluronidase inhibition rate IC50, which indicates that the schizosaccharomyces cerevisiae fermentation lysate provided by the embodiments can inhibit tyrosinase activity and melanin synthesis, can promote fibroblast growth and cell proliferation, and has whitening and anti-aging effects.

Animal experiments further prove that the yeast-bifida fermented polysaccharide based on S1, S4 and S5 has no obvious stimulation and side effect on skin, can effectively reduce melanin synthesis, and has obvious whitening and anti-aging effects.

In a first aspect, the present examples disclose a split yeast fermentation lysate comprising at least one selected from the group consisting of S1, S4, and S5, S1 having a molecular weight of 62.5kD, S4 having a molecular weight of 72.4kD, and S5 having a molecular weight of 76.9 kD; the S1 molecule comprises 32.5% of glucose, 34.7% of galactose, 10.8% of fucose, 9.2% of mannose, 6.4% of fructose, 3.5% of galactosamine and 2.9% of glucosamine in mol percentage; the S4 molecule comprises 25.4% of glucose, 40.8% of galactose, 7.8% of rhamnose, 11.4% of fucose, 9.6% of mannose, 2.7% of galactosamine and 2.3% of glucosamine according to molar ratio; the S5 molecule contained, in mole percent, 27.4% glucose, 38.3% galactose, 7.9% mannose, 8.2% fructose, 3.1% galactosamine, and 2.8% glucosamine.

In the examples of the present application, the glycosidic bond of S1 includes, in mole percentage, 7.25% → 1) -D-Glcp- (2,4,6 →, 4.36% → 1) -D-Glcp- (4 →, 3.14% → 1) -D-Glcp- (6 →, 2.88% → 4) -D-GlcAp- (1 →, 4.39% → 4) -D-GlcAp- (3 →, 6.14% →3) - β -GalNAc- (1 →, 7.25% → 4) -a-GlcA- (1 →, 18.6% → 1) -D-Galp- (3 →, 2.8% → 1) -D-Galp- (6 →, 12.3% → 1) -D-Galp- (6 →, and,

4.5％→4)-β-GalA-(1→、6.48％→1)-D-Fucp-(3→、4.31％→2)-D-Fucp-(4→、5.55％→1)-D-Manp-(2,4→、3.65％→1)-D-Manp-(3,6→、3.92％→1)-L-Frup-(3→、2.48％→2)-L-Frup-(4→；

The glycosidic bond of S4 includes, in mole percent, 8.15% → 1) -D-Glcp- (2,4,6 →, 5.68% → 1) -D-Glcp- (4 →, 4.17% → 1) -D-Glcp- (6 →, 4.11% → 4) -D-GlcAp- (1 →, 2.19% → 4) -D-GlcAp- (3 →, 2.03% → 3) - β -GalNAc- (1 →, 1.36% → 4) - α -GlcA- (1 →, 22.34% → 1) -D-Galp- (3 →, 8.42% → 1) -D-Galp- (6 →, 7.94% → 1) -D-Galp- (6 →, 4.79% > -4) - β -GalA- (1 →, 3.15-Galp- (2 → L) -Galp- (1 →, 7.94% → 1) -D-Galp- (6 →, 4.79% > -GalA- (1 →, and 3.15-L → 2 → L → 1 →, 4.58% → 2,4) -L-Rhap- (1 →, 7.92% → 1) -D-Fucp- (3 →, 3.47% → 2) -D-Fucp- (4 →, 6.08% → 1) -D-Manp- (2,4 → and 3.52% → 1) -D-Manp- (3,6 →;

the glycosidic bond possessed by S5 includes, in mole percent, 8.31% → 1) -D-Glcp- (2,4,6 →, 3.68% → 1) -D-Glcp- (4 →, 5.18% → 1) -D-Glcp- (6 →, 2.61% → 4) -D-Glcp- (1 →, 4.58% → 4) -D-Glcp- (3 →, 3.97% → 3) - β -GalNAc- (1 →, 1.86% → 4) -a-GlcA- (1 →, 23.28% → 1) -D-Galp- (3 →, 9.21% → 1) -D-Galp- (6 →, 6.35% → 1) -D-Galp- (6 →, 2.55% > -4) - β -GalA- (1 →, 5.24-Galp- (2 → 2.55% → 4) - β -GalA- (1 → and 5.24-Xylp (1), 3 →, 7.06% → 1) -D-Xylp- (3 →, 4.39% → 1) -D-Manp- (2,4 →, 3.51% → 1) -D-Manp- (3,6 →, 4.36% → 1) -L-Frup- (3 → and 3.84% → 2) -L-Frup- (4 →.

In the present example, S1 further has terminal glucose residues and terminal galactose residues, S4 further has terminal glucose residues and terminal galactose residues, and S5 further has terminal glucose residues and terminal galactose residues.

In a second aspect, the examples herein disclose a split yeast fermentation lysate comprising, by weight, 14.8-16.4% S1, 12.3-12.6% S4, and 11.1-12.5% S5.

In a third aspect, the present application discloses a method for preparing a fermentation lysate of a split yeast, comprising the steps of:

obtaining activated bacteria liquid of the secondary fission yeast;

carrying out amplification and fermentation culture on the activated bacterial liquid to obtain fermentation liquid;

filtering the fermentation liquor to obtain filtrate and thalli;

and performing wall breaking treatment on the thalli to obtain wall breaking liquid, mixing the wall breaking liquid with the thalli, and drying to obtain the secondary cracking yeast fermentation lysate.

In the embodiment of the application, an amplification medium and a fermentation medium are used for amplification and fermentation, and the amplification medium and/or the fermentation medium comprise 10g/L casein peptone, 5g/L yeast extract, 5g/L beef extract, 5g/L soybean peptone, 10g/L glucose and 0.15g/L NH ₄ C1, 0.5g/L manganese sulfate monohydrate, 0.01% v/v Tween 80, 50mg/L cysteine salt, 25mg/L cysteine, 0.05mg/L calcium chloride dihydrate, 0.15mg/L magnesium sulfate heptahydrate, 0.25m g/L potassium dihydrogen phosphate, 0.25g/L dipotassium hydrogen phosphate, 2.5mg/L sodium bicarbonate and 2.5mg/L sodium chloride.

In the examples herein, the expansion and fermentation uses an expansion medium and a fermentation medium, the expansion medium and/or the fermentation medium comprising 38g/L peptone, 96g/L glucose, 19g/L yeast extract, 0.06g/L xylose, 0.15mg/L sodium chloride, 96mg/L cysteine hydrochloride, 56mg/L cysteine, 0.15mg/L calcium chloride, 0.15mg/L magnesium sulfate, 0.15mg/L dipotassium hydrogen phosphate and 0.77g/L sodium bicarbonate.

In the embodiment of the application, the content of the crude polysaccharide in the activated bacterium liquid is not less than 0.5 g/L.

In a fourth aspect, the embodiment of the application discloses a whitening and anti-aging essence which comprises at least one of S1, S4 or S5, butanediol, propylene glycol, disodium EDTA and water, wherein the contents of S1, S4 or S5 are not less than 5.6 wt%, the content of butanediol is 0.25-3.5 wt%, the content of propylene glycol is 0.5-1.75 wt%, and the content of disodium EDTA is 0.05-0.25 wt%, and the whitening and anti-aging essence is prepared by dissolving in deionized water.

In a fifth aspect, the application discloses application of the saccharomyces cerevisiae fermented polysaccharide in preparing whitening and/or anti-aging cosmetics.

Drawings

FIG. 1 is an infrared spectrum of the methylation product of 5 yeast schizosaccharomyces zymolysis polysaccharides provided in the examples of the present application.

FIG. 2 is a GC-MS chromatogram of S1 provided in an example of the present application.

FIG. 3 is a GC-MS chromatogram of S2 provided in an example of the present application.

FIG. 4 is a GC-MS chromatogram of S3 provided in an example of the present application.

FIG. 5 is a GC-MS chromatogram of S4 provided in an example of the present application.

FIG. 6 is a GC-MS chromatogram of S5 provided in an example of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Culturing and fermenting schizosaccharomyces

1. Bacterial strains

Schizosaccharomyces, B71914, bright boat organism.

2. Activation of bacterial strains

Transferring the secondary fission yeast preserved at the temperature of minus 80 ℃ to an MRS solid plate, carrying out anaerobic culture at the temperature of 37 ℃ for 24h to obtain a plate bacterial colony, transferring the plate bacterial colony to an MRS liquid culture medium again, and carrying out anaerobic culture at the temperature of 37 ℃ for 24h to obtain activated bacterial liquid.

(1) Observation of colony morphology

And (4) observing the strain morphology of the plate bacterial colony, and measuring the biomass and crude polysaccharide of the activated liquid bacterial liquid.

(2) Biological quantity measurement

Collecting activated liquid bacterial liquid, centrifuging for 10min at 5000r/min, discarding supernatant, washing thallus with distilled water for 2 times, oven drying in 70 deg.C oven to constant weight, and weighing.

(3) Crude polysaccharide assay

Collecting supernatant obtained by centrifuging activated bacterial liquid for 10min at 5000r/min, precipitating with 3 times volume of 95% ethanol, centrifuging at 10000r/min for 5min, collecting precipitated extracellular polysaccharide precipitate in a 15mL centrifuge tube, centrifuging at 10000r/min for 2min, collecting precipitate, and removing supernatant; dissolving the collected precipitate in a small amount of deionized water, then adding 4 times of 80% (m/v) trichloroacetic acid, stirring at room temperature for 45min, standing at 4 ℃ for 30min, centrifuging at 10000r/min for 15min again, removing the precipitate and collecting the supernatant; the supernatant was concentrated by a rotary evaporator, and 3 times the volume of absolute ethanol (95%) was added to the resulting concentrate, and the mixture was allowed to stand overnight at 4 ℃ to remove proteins and cells from the crude polysaccharide.

Sealing the centrifuge tube with sealing film, ventilating the sealing film, and freeze drying at-80 deg.C for 36 hr to obtain extracellular polysaccharide powder. Taking out, grinding the polysaccharide into powder by using a grinding bowl, collecting the extracellular polysaccharide powder in a 2mL centrifuge tube, and placing the centrifuge tube in a refrigerator at 4 ℃ for storage for later use.

3. Expanding culture

In some examples, the activated bacterial suspension is inoculated into an expansion medium and cultured at 37 ℃ for 3 days to obtain an expanded culture solution. In some embodiments, the expanded broth is also transferred to a fermentation medium and incubated at 37 ℃ for 7d to harvest the final fermentation broth. In some embodiments, the activated bacteria solution having a crude polysaccharide content of not less than 0.5g/L is inoculated into an amplification medium or a fermentation medium. In a comparative example 1, an activated bacterial broth having a crude polysaccharide content of 0.4g/L was inoculated into an amplification medium or a fermentation medium.

In some embodiments, the activated bacteria solution having a crude polysaccharide content of not less than 0.5g/L is inoculated into an amplification medium or a fermentation medium. Both the scale-up culture and the fermentation culture are carried out under anaerobic conditions, for example, a gas atmosphere containing 10% carbon dioxide, 10% hydrogen, and 80% mixed gas (the composition is 5% hydrogen, 10% carbon dioxide, and the remaining gas is nitrogen).

In one example 1, the amplification medium and/or the fermentation medium comprises 10g/L casein peptone, 5g/L yeast extract, 5g/L beef extract, 5g/L soy peptone, 10g/L glucose, 0.15g/L NH ₄ C1, 0.5g/L manganese sulfate monohydrate, 0.01% v/v Tween 80, 50mg/L cysteine salt, 25mg/L cysteine, 0.05mg/L calcium chloride dihydrate, and sulfur heptahydrateMagnesium 0.15mg/L, potassium dihydrogen phosphate 0.25m g/L, dipotassium hydrogen phosphate 0.25g/L, sodium bicarbonate 2.5mg/L, sodium chloride 2.5 mg/L. The expanded medium and/or fermenter medium can be boiled under anaerobic conditions and cooled, added with cysteine, adjusted to pH 6.8 with 2M NaOH before autoclaving, and then autoclaved at 115 deg.C for 15min in an autoclave.

In one example 2, the amplification medium and/or the fermentation medium comprises 38g/L peptone, 96g/L glucose, 19g/L yeast extract, 0.06g/L xylose, 0.15mg/L sodium chloride, 96mg/L cysteine hydrochloride, 56mg/L cysteine, 0.15mg/L calcium chloride, 0.15mg/L magnesium sulfate, 0.15mg/L dipotassium hydrogen phosphate, and 0.77g/L sodium bicarbonate. The expanded medium and/or fermenter medium can be boiled under anaerobic conditions and cooled, cysteine can be added, the pH of the medium can be adjusted to 7.2 with 2M NaOH before autoclaving, and then autoclaving at 115 deg.C for 15min in an autoclave.

In the expanding and/or fermenting culture process of the comparative example 1, the activated bacterium liquid provided by the comparative example 1 is adopted as a seed.

In an expansion and/or fermentation culture process of comparative example 2, both the expansion or fermentation media used were MRS media.

Preparation of a Yeast fermentation lysate

In some embodiments, a method of producing a split yeast fermentation lysate comprises: collecting culture solution for amplification culture or fermentation culture, and filtering with ceramic membrane made of ZrO ₂ The aperture of the membrane is 50nm, cross-flow filtration is carried out, and the concentrated solution and the filtrate are obtained, wherein the thalli are contained in the concentrated solution; placing the concentrated solution into ion beam equipment, bombarding thallus with nitrogen ion beam, wherein the energy of the ion is 10 keV-25 keV, and the amount of the implanted ion is 1 × 10 ¹⁸ ～1×10 ¹⁹ N+/cm ² Injecting for 15s, stopping injecting for 3s, and repeatedly operating for 30-60 min to complete the wall-breaking treatment of the secondary fission yeast to obtain wall-breaking liquid; mixing the wall breaking solution and the filtrate, and freeze-drying to obtain the secondary fission yeast fermentation lysate.

Analysis of polysaccharide component in fermentation lysate of split yeast

In one embodiment, the polysaccharide component in the fermentation lysate of the yeast schizolysis is separated, after the fermentation lysate of the yeast schizolysis is precipitated by 3 times of 95 percent ethanol, the centrifugation at 10000r/min is carried out for 5min, the precipitated extracellular polysaccharide is collected and precipitated in a 15mL centrifuge tube, and the centrifugation at 10000r/min is carried out for 2min, and the supernatant is removed by the collection and precipitation; dissolving the collected precipitate in a small amount of deionized water, then adding 4 times of 80% (m/v) trichloroacetic acid, stirring at room temperature for 45min, standing at 4 ℃ for 30min, centrifuging at 10000r/min for 15min again, removing the precipitate and collecting the supernatant; concentrating the supernatant with a rotary evaporator, adding 3 times of anhydrous ethanol (95%) into the obtained concentrated solution, standing overnight at 4 deg.C to remove protein and cells in the crude polysaccharide, sealing the centrifuge tube with a sealing membrane, ventilating the sealing membrane, and freeze-drying at-80 deg.C for 36 hr to obtain crude polysaccharide. In some embodiments, the crude yeast split yeast fermentation polysaccharide is further purified. The treatment step comprises purifying the polysaccharide crude product by gel chromatography column, such as SephadexG-50, collecting eluate, lyophilizing to obtain refined lyophilized product of yeast fermentation polysaccharide, and determining molecular weight and structure.

In one embodiment, the molecular weight of the collected refined freeze-dried product is measured by using a water-soluble gel chromatographic column, and the measuring method is as follows:

detection conditions are as follows: water-soluble gel chromatography columns (Waters); the mobile phase was 0.1N sodium nitrate, column temperature 30 ℃, differential refractometer detection (WatersArc), sample size 20. mu.L, and all test results were integrated by Breeze software (Waters Chromatography, Inc). Standard curve: preparing standard dextran (molecular weight of 1000,5000,12000,80,150,270,670kDa, Nanjing Torreya Biotech Co., Ltd.) into 2% solution, centrifuging at 13000 r/min for 10min, collecting supernatant for preparing standard curve, sampling 20 μ L to obtain chromatogram, and drawing with elution volume (mL) of eluate with peak as abscissa and logarithm of dextran weight average molecular weight of 1gMw as ordinateAnd (3) calculating the molecular weight of the sample according to the molecular weight standard curve, and finally obtaining a standard equation as follows: y is 13.562 e-0.078X, R ² ＝0.991。

And (3) sample determination: dissolving the lyophilized powder purified by gel chromatography with mobile phase to obtain 10mg/mL solution, filtering with 0.45 μm filter membrane, detecting by sampling under the above conditions, substituting the retention time into standard curve, and calculating to obtain corresponding molecular weight.

The embodiment of the application obtains 5 schizosaccharomyces cerevisiae S1, S2, S3, S4 and S5 with the molecular weight of 30-80 kD, and the molecular weight of the schizosaccharomyces cerevisiae is 62.5kD, 41.7kD, 38.5kD, 72.4kD and 76.9kD in sequence. In addition, the present examples also analyzed the components and contents of S1 to S5 in the split yeast polysaccharides obtained in example 1, example 2, comparative example 1, and comparative example 2, respectively, and the results are shown in table 1. As can be seen from Table 1, the split yeast polysaccharides in the split yeast fermentation lysates prepared in examples 1 and 2 mainly contained S1, S4 and S5, whereas the split yeast polysaccharides in the split yeast fermentation lysates prepared in comparative examples 1 and 2 mainly contained S1, S2 and S3, and comparative example 2 provided lower contents of S1, S2 and S3.

TABLE 1

Structural analysis of Yeast polysaccharide

1. Analysis of monosaccharide composition of polysaccharide by fermentation of yeast

Qualitative and quantitative analysis is carried out on the composition types and concentrations of monosaccharides in the yeast schizosaccharomyces zymophyte polysaccharide with the molecular weight of 30-80 kD by adopting an ion chromatography method:

pretreatment: accurately preparing 2mol/L trifluoroacetic acid containing 1mg/mL of a schizosaccharomyces cerevisiae fermentation polysaccharide sample, treating for 6h at 110 ℃ to obtain a hydrolysis solution of the schizosaccharomyces cerevisiae fermentation polysaccharide, carrying out rotary concentration at 40 ℃, adding a 1.5-time volume of a silicon etherifying reagent (pyridine: hexamethyldisilazane: trimethylchlorosilane: 10:2:1 volume ratio), carrying out water bath heating treatment for 1h at 80 ℃, carrying out silicon etherifying, blowing dry by nitrogen after the reaction is finished, fixing the volume by 1mL of n-hexane, and detecting by GC-MS/MS.

And (3) chromatographic detection conditions: TSQ Fortis ^TM Plus triple quadrupole mass spectrometer (Saimerfi), TG-5MS AMINE Thermo GC column (TG-5MS AMINE, Thermo Fisher Scientific); an Organomation N-EV AP nitrogen blower (Organomation Co., USA). Chromatographic parameters: mass spectrum scanning mode, positive ion mode; the scanning range is 50-500; the gas-phase carrier gas is nitrogen, and the flow rate is 1.5 mL/min; the sample injection mode is that the sample is not divided and the sample injection amount is 1 mu L. The temperature raising procedure of the gas phase is that the temperature is raised from 80 ℃ to 150 ℃ at a speed of 10 ℃/min, the temperature is maintained at 150 ℃ for 2min, then the temperature is raised to 180 ℃ at a speed of 1.5 ℃/min, and then the temperature is raised to 210 ℃ at a speed of 10 ℃ for 20min, and the temperature is maintained at 210 ℃ for 20 min.

And (3) standard substance: respectively preparing 200 mu g/mL of aqueous solution of glucose, galactose, xylose, rhamnose, fucose, arabinose, mannose, fructose, galactosamine, glucosamine and glucuronic acid; preparing a mixed standard solution with the concentration of 5 mu g/mL; taking 10 mu g inositol as internal standard from 5, 1, 0.5, 0.1 and 0.05mL solution respectively, and drying by a nitrogen blower. The silicon etherification derivatization was performed in the same manner as the samples, and the GC-MS/MS detection was performed under the above conditions.

2. Structural analysis of polysaccharide by fermentation of yeast

Periodate oxidation analysis of zymosan from Spirodiaecium was performed using methods such as those disclosed in "Niu, YG., Wang, H.Y, Xie, Z.H., where, M.A., Gao, X.D., et al, structural analysis and biological activity of a polysaccharide from the microorganisms of Aspergillus membrane aceus (Fisch) by. var. mongolicus (by.) Hsiao [ J ] Food Chemistry,2011,128(3): 620-. The methylation analysis of the yeast secondary fission fermentation polysaccharide was performed as follows.

(1) Preparation of sodium hydroxide-dimethyl sulfoxide suspension (0.025g/mL)

Mixing 0.2mL of newly configured sodium hydroxide solution (50%) with 0.2mL of methanol, adding 6mL of DMSO solution, violently oscillating on a vortex oscillator, carrying out ultrasonic bath for 3-5 min, then centrifuging at 10,000r/min, collecting NaOH precipitate at 20 ℃, repeating the operation for three times, and finally adding 4mL of DMSO solution into the sodium hydroxide precipitate.

(2) Methylation of samples

Placing 2mg of a dried polysaccharide sample into a 10mL rotary evaporation bottle, adding 0.5mL of dimethyl sulfoxide solution, carrying out ultrasonic water bath for 2min, standing for 30min, adding 0.6mL of sodium hydroxide-dimethyl sulfoxide suspension and 0.6mL of methyl iodide, covering a cover tightly, continuously oscillating and mixing on a vortex mixer, adding 4mL of water to terminate the reaction, adding an appropriate amount of chloroform to oscillate, standing for layering, removing an upper-layer water phase, repeating the operation for a plurality of times, carrying out reduced pressure concentration on an organic phase at the temperature lower than 40 ℃, and carrying out infrared spectrum detection to determine whether the sample is completely methylated or not. Adding the sample containing complete methylation into formic acid solution, sealing and reacting at 100 ℃ for 3h, concentrating under reduced pressure below 40 ℃, adding 2M trifluoroacetic acid with the same volume, reacting at 100 ℃ for 6h, and concentrating under reduced pressure below 40 ℃.

Adding 3mL of deionized water into the reaction bottle after evaporation to dryness, adding a proper amount of sodium borohydride, carrying out intermittent oscillation reaction for 3 hours, removing redundant sodium borohydride by using acetic acid, adding a proper amount of methanol, and concentrating under reduced pressure. Adding 4mL of acetic anhydride, carrying out sealing reaction for 1h at 100 ℃, cooling, adding a proper amount of toluene, carrying out vacuum concentration, repeating the reaction for a plurality of times, adding a proper amount of chloroform, adding a proper amount of distilled water, fully shaking, standing for layering, removing an upper aqueous solution, and repeating the extraction operation for a plurality of times. Drying chloroform layer with appropriate amount of anhydrous sodium sulfate, filtering with organic phase microporous membrane, removing anhydrous sodium sulfate, concentrating the filtrate under reduced pressure, adding 0.5mL chloroform, and analyzing by gas chromatography and mass spectrometry.

GC-MS analysis: the gas chromatograph was connected to an HP-5 capillary column (30 m.times.0.25 mm.times.0.25 μm, Agilent) at a column flow rate of 1 mL/min. Temperature programming, namely, the initial temperature of an HP-S capillary column is 120 ℃, the temperature is raised to 240 ℃ at 10 ℃ lmin, and the temperature is kept for 6.5 min. Injecting 1 mu L of sample, the split ratio is 1:30, the injection port temperature is 250 ℃, the detector temperature is 250 ℃, the hydrogen is 35mL/min, the air is 350mL/min, and the tail gas is blown for 30 mmL/min. The gas chromatography-mass spectrometry (GC-MS) is connected with an HP-5 capillary column (30m multiplied by 0.25mm multiplied by 0.25 mu m), the temperature programming is carried out on the gas chromatography, the interface temperature is 250 ℃, the ion source temperature is 250 ℃, the mass number scanning range is 40-500 amu, and the scanning time is 0.28 s.

4. Results

The statistics of the monosaccharide composition detection results for each of the zymosan S1 to S5 are shown in table 2, and "-" in table 2 indicates no detection.

Table 2 mol% w/w

Components	S1	S2	S3	S4	S5
						Glucose Glc	32.5	29.3	26.4	25.4	27.4
Galactose Gal	34.7	12.4	9.8	40.8	38.3
						Xylose Xyl	－	15.6	16.7	－	12.3
Rhamnose Rha	－	13.7	15.3	7.8	－
						Fucose Fuc	10.8	2.6	2.5	11.4	－
Ara arabinose	－	10.9	－	－	－
						Mannose Man	9.2	－	12.4	9.6	7.9
Fructose Fru	6.4	8.7	9.3	－	8.2
						Galactosamine (GalNAc)	3.5	3.1	3.8	2.7	3.1
Glucosamine (GlcNAc)	2.9	1.2	1.7	2.3	2.8
						Glucuronic acid (GlcA)	－	2.5	2.1	－	－

Periodic acid oxidation analysis shows that the consumption of periodic acid and the generation of formic acid of S1-S5 after being oxidized by periodic acid are both more than 2, and the molar quantity of the consumed periodic acid is less than 2, which indicates that glucan bond types contained in the composition comprise type I: 1 →,1 → 2 or 1 → 6 linkage type; type II: 1 → 4, 1 → 2,6 or 1 → 4,6 linkage type; type III: 1 → 3,4, 1 → 3,6, 1 → 3,4,6, 1 → 2,3,6, 1 → 2,4 or 1 → 2,4, 6.

The methylation products of S1-S5 shown in FIG. 1 are 3200-3600 ^-1 Peak of hydroxyl radicalDisappeared at 2930 ^-1 A methyl peak appears at 1050 ^-1 The vibration peak is caused by the stretching vibration of ether bond in adjacent pyranose ring and the bending of O-H bond at 840 ^-1 The oscillation peaks that appear are caused by the characteristic denaturation of the carbon-hydrogen bonds in the alpha-adjacent pyranose ring; and S1, S4, and S5 at 1720 ^-1 Has no vibration peak caused by furfural, and is at 1650 ^-1 There occurs vibration caused by expansion and contraction of C ═ O of the amide group. From this, it is again demonstrated that S1, S4 and S5 are polysaccharides containing amide groups, and S2 and S3 contain uronic acid, which corresponds to the monosaccharide composition described above.

Completely methylating The completely reduced S1-S5 to obtain sugar alcohol acetate derivatives (PMAAs), measuring by GC-MS to obtain a total ion flow diagram and a corresponding mass spectrogram of The PMAAs of S1-S5, comparing The mass spectrogram corresponding to The peak of The obtained PMAAs with a standard Spectral library (The CCRC Spectral Database for PMAA) summarized by sugar compound research of The university of Georgia and a chemical Database of The national academy of sciences to determine The type of methylated sugar residues, and simultaneously calculating The relative mole percentage of each methylated sugar residue according to The peak area of The chromatographic peak to finally obtain The type and The mole percentage of The glycosidic bond of The two polysaccharide components, wherein The specific statistical result is shown in Table 3.

In Table 3, "-" indicates no detection. As can be seen from Table 3, S1, S4 and S5 all contained terminal glucose and terminal galacturonic acid. Wherein the glycosidic bond of S1 includes → 1) -D-Glcp- (2,4,6 → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 4) -D-GlcAp- (1 →, → 4) -D-GlcAp- (3 →, → 3) - β -GalNAc- (1 →, → 4) - α -GlcA- (1 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 4) - β -GalA- (1 →, → 1) -D-Fucp- (3 →, → 2) -D-Fucp- (4 → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -L-Frup- (3 →, → 2) -L-Frup- (4 →. The glycosidic bond of S2 includes → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 4) -D-GlcAp- (1 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 1) -D-Xylp → (2,3 →, → 2) -D-Xylp → (3,4 → 1) -D-Xylp- (3 →,2 → L-Rhap- (1 →, → 2,4) -L-Rhap- (1 → 1) -D-fuc- (3 → 2) -D-Fucp- (4 →, → 3,5) -L-Araf- (1 → 2,4) -L-Fucp- (1 → D-Fucp- (3 → 2, 5) -L-Araf- (1 → 3,5) -L-D-Fucp → 3 → 1 → 3, 2,3 → 3, and, → 5) -L-Araf- (1 →, → 1) -L-Frup- (3 → and → 2) -L-Frup- (4 →.

The glycosidic bond of S3 includes → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (6 →, → 4) -D-GlcAp- (3 →, → 3) - β -GalNAc- (1 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 1) -D-Xylp → (2,3 →, → 2) -D-Xylp → (3,4 →, → 1) -D-Xylp- (3 →, → 2) -L-Rhap- (1 → 2,4) -L-Rhap- (1 → D-fuc- (3 → 2) -D-Fucp- (4 → 1) -D-mancp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -L-Frup- (3 → and → 2) -L-Frup- (4 →.

The glycosidic bond of S4 includes → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 4) -D-GlcAp- (1 →, → 4) -D-GlcAp- (3 →, → 3) - β -GalNAc- (1 →, → 4) -a-GlcA- (1 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 4) - β -GalA- (1 →, → 2) -L-Rhap- (1 →, → 2,4) -L-Rhap- (1 → D-Fucp- (3 → 1 → 4) - β -GalA- (1 →, → 2,4) -L-Rhap- (1 → D-Fucp- (3 → 4) -L-Rhap- (1 → D-Fucp- (3 → D → 3 → D-Glcp, → 2) -D-Fucp- (4 →, → 1) -D-Manp- (2,4 → and → 1) -D-Manp- (3,6 →.

The glycosidic bond of S5 includes → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 4) -D-Glcp- (1 →, → 4) -D-Glcp- (3 →, → 3) - β -GalNAc- (1 →, → 4) -a-GlcA- (1 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 4) - β -GalA- (1 →, → 1) -D-Xylp → (2,3 → 1) -D-Xylp- (3 → 1) -D-mangp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -L-Frup- (3 → and → 2) -L-Frup- (4 →.

The molar percentages of glycosidic linkages in each molecule in tables 3S 1-S5

Glycosidic linkages	S1	S2	S3	S4	S5
						Terminal-Glc	0.00085	－	－	0.00085	0.00085
→1)-D-Glcp-(2,4,6→	7.25	18.26	17.52	8.15	8.31
						→1)-D-Glcp-(4→	4.36	4.15	－	5.68	3.68
→1)-D-Glcp-(6→	3.14	6.67	5.73	4.17	5.18
						→4)-D-GlcAp-(1→	2.88	3.91		4.11	2.61
→4)-D-GlcAp-(3→	4.39	－	4.54	2.19	4.58
						→3)-β-GalNAc-(1→	6.14		2.41	2.03	3.97
→4)-ɑ-GlcA-(1→	7.25	－	－	1.36	1.86
						Terminal-GalA	0.00027	－	－	0.00027	0.00027
→1)-D-Galp-(3→	18.6	6.79	3.67	22.34	23.28
						→1)-D-Galp-(6→	2.8	－	－	8.42	9.21
→1)-D-Galp-(6→	12.3	8.72	6.93	7.94	6.35
						→4)-β-GalA-(1→	4.5	－	－	4.79	2.55
→1)-D-Xylp→(2,3→	－	9.45	10.69	－	5.24
						→2)-D-Xylp→(3,4→	－	2.83	2.15	－	－
→1)-D-Xylp-(3→	－	3.32	3.85	－	7.06
						→1)-L-Rhap-(1→	－	－	－	－	－
→2)-L-Rhap-(1→	－	6.63	5.92	3.15	－
						→2,4)-L-Rhap-(1→	－	7.08	9.37	4.58	－
→1)-D-Fucp-(3→	6.48	1.39	0.68	7.92	－
						→2)-D-Fucp-(4→	4.31	1.21	1.82	3.47	－
→3,5)-L-Araf-(1→	－	3.27	－	－	－
						→5)-L-Araf-(1→	－	7.69	－	－	－
→1)-D-Manp-(2,4→	5.55	－	1.36	6.08	4.39
						→1)-D-Manp-(3,6→	3.65	－	11.04	3.52	3.51
→1)-L-Frup-(3→	3.92	2.36	2.06	－	4.36
						→2)-L-Frup-(4→	2.48	6.34	7.24	－	3.84

In vitro performance analysis

1. Test article

The split yeast fermentation lysates provided in example 1, example 2, comparative example 1 and comparative example 2 were used as test samples, and the compositions and contents of the split yeast polysaccharides contained therein are shown in Table 1.1 mg/mL, 5mg/mL, 20mg/mL, 50mg/mL and 100mg/mL were prepared with deionized water, respectively.

2. Antioxidant property

(1) DPPH radical scavenging Capacity test

Formulation 10 ^-4 mixing the volume of the DPPH solution with the same volume with the samples with different concentrations to serve as a test group; uniformly mixing DPPH solution with the same volume and the same concentration with deionized water with the same volume to obtain a blank group; the same volume of absolute ethyl alcohol and the same volume of the test sample with different concentrations are mixed uniformly to be used as a control group. After the three groups of solutions are respectively reacted for 30min, the absorbance at 517nm is respectively detected, and the concentration IC50-1 of the test solution with the DPPH free radical clearance rate of 50 percent is calculated, wherein the DPPH free radical clearance rate is (blank group A + control group A-test group A)/blank group A multiplied by 100 percent.

(2) Measurement of hydroxyl radical scavenging ability

Mixing equal volumes of 2mM ferrous sulfate solution, 1mM hydrogen peroxide solution and 6mM salicylic acid, placing in 37 deg.C water bath for 15min, taking out, and dividing into test group and control group. Adding test solutions with different concentrations into the test group to react, wherein the volume of the test solutions accounts for 10% of the total solution, continuing heating in water bath for 15min, taking out, and measuring the absorbance at 510nm, and recording as the test group A. Adding deionized water with the same volume into the mixed solution of the control group, continuing heating in water bath for 15min, taking out, measuring the absorbance at 510nm, and marking as A control group. The absorbance of the sample at 510nm was recorded as A blank. The concentration of the test solution IC50-2 was calculated to have a hydroxyl radical scavenging rate of 50%, where hydroxyl radical scavenging rate is (a blank + a control-a test group)/a blank × 100%.

3. In vitro tyrosinase inhibition assay

Tyrosinase activity in vitro was measured by the dopa rate oxidation method. A, B, C and D were set, and after mixing the amounts and substances added as shown in Table 3, and carrying out a water bath reaction at 37 ℃ and then detecting the absorbance at 475nm, respectively, the concentration of the test solution IC50-3 at which the tyrosinase activity inhibition rate was 50% was calculated, where the tyrosinase activity inhibition rate was [ (A1-A2) - (A3-A4) ]/(A1-A2) × 100%. Wherein the concentration of PBS is 25mM, and the pH value is 6.8; the liquid adding sequence is to add PBS, test sample dissolved oxygen and tyrosinase solution (500U/mL) in sequence, then to add 0.98 g/L-dopa solution quickly after water bath for 10min at 37 deg.C, and then to detect the light absorption value immediately.

TABLE 3 in vitro tyrosinase inhibiting ability test solution addition

Group of	PBS(mL)	Test solution (mL)	Tyrosinase solution (mL)	L-dopa solution
					A	3.3	0	0.3	0.4
B	3.6	0	0	0.4
					C	2.6	0.7	0.3	0.4
D	2.9	0.7	0	0.4

4. In vitro hyaluronidase inhibition assay

The fermentation lysates of the yeasts for the second split yeast provided in example 1, example 2, comparative example 1 and comparative example 2 were prepared to 1mg/mL, 5mg/mL, 20mg/mL, 50mg/mL and 100mg/mL using an acetate buffer solution having a pH of 5.6, respectively, as a test solution.

The test is divided into: test, control, blank 1 and blank 2 groups. In the test group, 0.1mL of 0.25mM calcium chloride solution and 0.5mL of hyaluronidase solution (500U/mL) are kept at 37 ℃ for 20 min; adding 0.5mL of daily solution of the test sample, and keeping the temperature at 37 ℃ for 20 min; adding 0.5mL sodium hyaluronate solution, keeping the temperature at 37 ℃ for 30min, and standing at normal temperature for 5 min; adding 0.1mL of 0.4M sodium hydroxide solution and 0.5mL of acetylacetone solution, heating in a boiling water bath for 15min, and immediately cooling with ice water for 5 min; adding 1.0mL of an Ellisib reagent, diluting with 3.0mL of absolute ethanol, standing for 20min for color development, and measuring the absorbance value of the reaction system at 555nm by using a spectrophotometer and recording as C. In the control group, the test solution was replaced with an acetic acid buffer solution, and the absorbance was designated as A. In blank 1, the sample solution and the enzyme solution were replaced with acetic acid buffer solution, and the absorbance was recorded as B. In blank 2, the hyaluronidase solution was replaced with acetic acid buffer solution, and the absorbance was recorded as D.

The concentration of the test solution IC50-4 was calculated to have an in vitro hyaluronidase inhibition of 50%, where the in vitro hyaluronidase inhibition is [ (a-B) - (C-D) ]/(a-B) × 100%.

5. B16-F10 test

(1) B16-F10 cell viability assay

The test is divided into a test group, a control group and a blank group. A single cell suspension of B16 cells (melanoma cells from mice, Shanghai-Yigao Biotech Co., Ltd.) was prepared and adjusted to a concentration of 1X 10 ⁵ one/mL was inoculated into 96-well plates, 100mL per well, in 5% CO ₂ And after the culture is carried out for 24 hours in an incubator at 37 ℃, the culture is divided into a test group and a control group. The test group was replaced with a DMEM complete medium containing a test solution at a different concentration, and the control group was replaced with a DMEM complete medium. Placing in 5% CO ₂ And culturing at 37 ℃ for 24 h. The old medium was discarded, washed three times with PBS, 100mL of a serum-free medium solution containing 0.5mg/mL of MTT was added to each well, and the mixture was placed in% CO ₂ Incubating at the constant temperature of 37 ℃ for 4h, discarding the old culture medium, adding 150mL of DMSO solution to dissolve for 10min, and measuring the absorbance value at 570nm by using a microplate reader. The wells to which the MTT solution was added were used as a blank. Calculating the concentration IC50-5 of the test solution with cell survival rate of 50%, wherein the cell survival rate isThe ratio of (test absorbance-blank absorbance)/(control absorbance-blank absorbance) x 100%.

(2) B16-F10 cell melanin synthesis inhibition assay

The test is divided into a test group, a control group and a blank group. B16 cells in logarithmic growth phase were digested with 0.25 wt% trypsin and then prepared in DMEM complete medium at a density of 5X 10 ⁴ Cell suspension/mL, inoculated in 6-well culture plate, 2mL per well, placed in 5% CO ₂ And after the culture is carried out for 24 hours in an incubator at 37 ℃, the culture is divided into a test group and a control group. The test group was replaced with a fresh medium using DMEM complete medium containing the sample solution at a sample solution concentration at which the cell survival rate was 50%, and the control group was replaced with a fresh medium using DMEM complete medium. At 37 ℃ with 5% CO ₂ After culturing in an incubator for 72h, adding 400 mu L of 1mol/L solution containing 10 wt% DMSO NaOH, keeping the temperature at 80 ℃ for 2h, and measuring the absorbance value of 100 mu L at 490nm of an enzyme-labeling instrument, namely the absorbance value of the test group. In the blank group, no test solution was added and the novel medium was replaced. The relative melanin content was calculated after treatment of the test article solution with a cell viability of 50%. Untreated cells were set as a control and the relative melanin content (test absorbance-blank absorbance)/(control absorbance-blank absorbance) x 100%.

6. Fibroblast assay

(1) Effect on fibroblast survival

Collecting fibroblast (human skin fibroblast, BNCC341540, Hainan engineering research center for engineering and technology of strain of industrial microorganism) in logarithmic growth phase, plating to obtain cell density of 5000 cells/well, and culturing at 37 deg.C and 5% CO ₂ Culturing, growing the cells to a certain density, changing the culture solution, adding samples with different volume fractions, and taking the culture solution without the samples as a control. Continuing to culture for 24h, adding 20 μ L of 0.5% MTT solution (5g/L) into each well, culturing for 4h, discarding the culture solution, washing with PBS for 2-3 times, adding 150 μ L of dimethyl sulfoxide into each well, and shaking on a shaking table at low speed for 10min to dissolve the crystal completely. The absorbance of each well was measured at 490 nm. Cell viability ═ cell viability (assay well absorbance-blank control absorbance)/(fines)Absorbance of cell control-absorbance of blank control) × 100%.

(2) Effect on fibroblast type I collagen Synthesis

And (3) centrifuging the fibroblast culture solution acted in the cell survival test for 20min at a speed of 2000-3000 r/min, and storing the supernatant. Detection is carried out by using a Col-I detection kit (Shanghai Ribank Biotech Co., Ltd.), and the operation is carried out according to the steps of the instruction.

7. Test results

TABLE 4(mg/mL)

Detailed description of the preferred embodiments	IC50-1	IC50-2	IC50-3	IC50-4
					Example 1	2.73±0.38b	3.19±0.52b	15.32±2.15b	29.12±3.25
Example 2	2.59±0.82b	3.04±0.68b	13.95±1.48b	27.83±4.18
					Comparative example1	56.12±12.68a	73.19±18.22a	537.59±34.36a	－
Comparative example 2	71.29±10.76a	75.05±13.48a	－

TABLE 5(mg/mL)

Table 4 shows the DPPH free radical clearance rate IC50, hydroxyl free radical clearance rate IC50, in vitro tyrosinase inhibition rate IC50 and in vitro hyaluronidase inhibition rate IC50, which were detected as test samples, respectively, from the fermentation lysates of the yeast schizosaccharomyces cerevisiae provided in example 1, example 2, comparative example 1 and comparative example 2, respectively, wherein "-" indicates no detection. Table 4 also performed multiple comparisons and significant difference labeling for each column of data.

As can be seen from Table 4, the split yeast fermentation lysates provided in examples 1 and 2 had lower DPPH free radical clearance rate IC50, hydroxyl free radical clearance rate IC50, in vitro tyrosinase inhibition rate IC50 and in vitro hyaluronidase inhibition rate IC50, indicating that they had significant antioxidant, anti-tyrosinase and anti-hyaluronidase inhibitory activities, and also had melanin synthesis inhibitory and anti-inflammatory effects.

Table 5 shows the effect of the fermentation lysates of the yeasts of the second split yeast, respectively provided in example 1, example 2, comparative example 1 and comparative example 2, on the survival of B16-F10 cells and the production of melanin thereof, respectively, and the effect on the survival of human fibroblasts and the expression of Col-I, respectively, as a test sample. Table 5 also performed multiple comparisons and significant difference labeling for each column of data. The results show that the split yeast fermentation lysates provided in examples 1 and 2 can not only inhibit the growth of B16-F10 and the synthesis of melanin thereof, but also promote the proliferation of fibroblasts and the expression of Col-I, which suggests that the split yeast fermentation lysates provided in the examples of the present application can not only inhibit tyrosinase activity and inhibit the synthesis of melanin, but also promote the growth of fibroblasts and promote the proliferation of cells, and have whitening and anti-aging effects.

Dynamic effect test

1. Test article

The fermentation lysates of the yeasts of the second split yeast provided in example 1, example 2, comparative example 1 and comparative example 2 were prepared in 50mg/mL solutions in PBS at pH 6.8.

The whitening and anti-aging essence is prepared by taking saccharomyces cerevisiae fermented polysaccharide as a base, and comprises at least one of S1, S4 or S5, butanediol, propylene glycol, disodium EDTA and water, wherein the contents of S1, S4 or S5 are not less than 5.6 wt%, the content of butanediol is 0.25-3.5 wt%, the content of propylene glycol is 0.5-1.75 wt%, the content of disodium EDTA is 0.05-0.25 wt%, and the whitening and anti-aging essence is prepared by dissolving in deionized water.

In an example 3, the whitening and anti-aging essence comprises 5.6 wt% of S1, 3.5 wt% of butylene glycol, 0.5 wt% of propylene glycol, and 0.05 wt% of disodium EDTA, and is dissolved in deionized water.

In example 4, the whitening and anti-aging essence comprises 5.6 wt% of S4, 0.25 wt% of butylene glycol, 1.75 wt% of propylene glycol, and 0.25 wt% of disodium EDTA, and is prepared by dissolving in deionized water.

In an example 5, the whitening and anti-aging essence comprises 5.6 wt% of S5, 0.25 wt% of butylene glycol, 1.75 wt% of propylene glycol, and 0.25 wt% of disodium EDTA, which are dissolved in deionized water.

In an example 6, the whitening anti-aging essence comprises 3.5 wt% of the saccharomyces cerevisiae fermentation lysate provided in example 1, 0.25 wt% of butanediol, 1.75 wt% of propylene glycol, and 0.25 wt% of disodium EDTA, and is prepared by dissolving in deionized water.

In example 7, the whitening anti-aging essence comprises 3.5 wt% of the split yeast fermentation lysate provided in example 2, 0.25 wt% of butanediol, 1.75 wt% of propylene glycol, and 0.25 wt% of disodium EDTA, and is prepared by dissolving in deionized water.

2. Skin irritation test

According to the technical specification for cosmetic safety (2015 edition), white adult hairless guinea pigs were selected as test animals, qualified patch materials (samming and biotechnology limited) were selected, 20 μ L of PBS solution containing 50mg/mL of the split yeast fermentation lysate provided in examples 1 to 7, comparative example 1 and comparative example 2 and having pH of 6.8 was put into a patch tester, and no control wells were added. The spot tester with the tested object is pasted on the back hairless area of the tested animal by using a non-irritating adhesive tape, the spot tester is lightly pressed by hands and hands to be uniformly pasted on the skin, and the spot tester is continuously kept on the skin for 24 hours. The interval is 30min after the plaque remover is removed, and the skin reaction is observed after the indentation disappears. The observation was done 24 and 48h after the patch test. Judging the result, wherein the 0-grade reaction is that the tested part has no reaction; grade 1 reaction, light erythema on the skin; grade 2 reaction, erythema, infiltration or pimple on the skin; grade 3 reaction, edema erythema and pimple on skin; grade 4 response, marked redness and swelling of the skin with pimples or scaring.

3. Ultraviolet irradiation test

The hairless guinea pigs were divided into a model group and a test group, and placed in an environment at a distance of about 30cm from a UVB ultraviolet lamp tube, irradiated at a wavelength of 310nm for 10min each time, irradiated 1 time every 8 hours, and continuously irradiated 5 times to serve as model mice. Before each ultraviolet irradiation, the test group smears the essence respectively provided in examples 1-7 and comparative examples 1-2 on the back skin, 2.5mL each time, smears the essence for 1 time every 8 hours, and then performs ultraviolet irradiation; after the application is continuously carried out for 5 times and continuously for 14 days, a skin biopsy of a guinea pig is stained, subjected to histological observation, photomicrograph and optical density analysis, the number of melanocyte-containing cells and dopa-positive cells in basal cells are counted, and the ratio of the melanocyte-containing cells to the dopa-positive cells is calculated as a pigment index.

4. Results

TABLE 6

As can be seen from Table 6, the split yeast fermentation lysates provided in examples 1 and 2, respectively, had no significant side effects on hairless guinea pig skin, whereas the test products provided in comparative examples 1 and 2 had different degrees of adverse reactions and were not suitable for cosmetic applications.

Table 6 also shows that the split yeast fermentation lysates provided in examples 1 to 2, and the intervention results of the whitening and anti-aging essence liquids provided in examples 3 to 7 on the skin of guinea pigs irradiated with ultraviolet light, multiple comparisons and significant difference marks are performed on the skin pigment indexes, and as a result, the examples 1 to 7 have a better intervention effect of reducing pigment synthesis as a test product, while the comparative examples 1 to 2 have no significant effect, and the whitening and anti-aging essence liquids provided in examples 3 to 7 have a better whitening effect.

In summary, in the embodiments of the present application, the schizosaccharomyces cerevisiae is fermented and cultured, the fermentation product is filtered, the bacterial wall is broken, the wall-broken liquid is mixed with the filtrate, and the filtrate is dried to obtain the schizosaccharomyces cerevisiae fermentation lysate, 5 types of schizosaccharomyces cerevisiae polysaccharides S1, S2, S3, S4 and S5 with molecular weight of 30 to 80kD are separated from the lysate, and the schizosaccharomyces cerevisiae fermentation polysaccharide is subjected to primary structural analysis, and is found to be a polysaccharide containing amide groups in S1, S4 and S5, while S2 and S3 contain uronic acid.

Further, in vitro tests on the obtained schizosaccharomyces cerevisiae fermentation lysate discover that the schizosaccharomyces cerevisiae fermentation lysate provided by the embodiments 1 and 2 has lower DPPH free radical clearance rate IC50, hydroxyl free radical clearance rate IC50, in vitro tyrosinase inhibition rate IC50 and in vitro hyaluronidase inhibition rate IC50, which indicates that the schizosaccharomyces cerevisiae fermentation lysate provided by the embodiments can inhibit tyrosinase activity and melanin synthesis, can promote fibroblast growth and cell proliferation, and has whitening and anti-aging effects.

Animal experiments further prove that the saccharomyces cerevisiae fermentation polysaccharide taking S1, S4 and S5 in the saccharomyces cerevisiae fermentation lysate as the basis has no obvious stimulation and side reaction effects on skin, can effectively reduce melanin synthesis, and has obvious whitening and anti-aging effects.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims (10)

1. A split yeast fermentation lysate comprising a polysaccharide selected from at least one of S1, S4 and S5, S1 having a molecular weight of 62.5kD, S4 having a molecular weight of 72.4kD, S5 having a molecular weight of 76.9kD,

the S1 molecule comprises 32.5% of glucose, 34.7% of galactose, 10.8% of fucose, 9.2% of mannose, 6.4% of fructose, 3.5% of galactosamine and 2.9% of glucosamine in mol percentage;

the S4 molecule comprises 25.4% of glucose, 40.8% of galactose, 7.8% of rhamnose, 11.4% of fucose, 9.6% of mannose, 2.7% of galactosamine and 2.3% of glucosamine according to molar ratio;

the S5 molecule contained, in mole percent, 27.4% glucose, 38.3% galactose, 7.9% mannose, 8.2% fructose, 3.1% galactosamine, and 2.8% glucosamine.

2. The secondary fission yeast fermentation polysaccharide according to claim 1, wherein the glycosidic bond of S1 comprises, in mole percent, 7.25% → 1) -D-Glcp- (2,4,6 →, 4.36% → 1) -D-Glcp- (4 →, 3.14% → 1) -D-Glcp- (6 →, 2.88% → 4) -D-GlcAp- (1 →, 4.39% → 4) -D-GlcAp- (3 →, 6.14% → 3) - β -GalNAc- (1 →, 7.25% → 4) -alpha-GlcA- (1 →, 18.6% → 1) -D-Galp- (3 →, 2.8% → 1) -D-Galp- (6 →, 12.3% → 1) -D-Galp- (6% >, 4.5% → 4) - β -Galp- (1 → 1) -D-Galp- (6 →, 4.5% → 1 → A → 1) -D-Galp- (1 → 1), 6.48% → 1) -D-Fucp- (3 →, 4.31% → 2) -D-Fucp- (4 →, 5.55% → 1) -D-Manp- (2,4 →, 3.65% → 1) -D-Manp- (3,6 →, 3.92% → 1) -L-Frup- (3 →, 2.48% → 2) -L-Frup- (4 →;

the glycosidic bond of S4 includes, in mole percent, 8.15% → 1) -D-Glcp- (2,4,6 →, 5.68% → 1) -D-Glcp- (4 →, 4.17% → 1) -D-Glcp- (6 →, 4.11% → 4) -D-GlcAp- (1 →, 2.19% → 4) -D-GlcAp- (3 →, 2.03% → 3) - β -GalNAc- (1 →, 1.36% → 4) - α -GlcA- (1 →, 22.34% → 1) -D-Galp- (3 →, 8.42% → 1) -D-Galp- (6 →, 7.94% → 1) -D-Galp- (6 →, 4.79% > -4) - β -GalA- (1 →, 3.15-Galp- (2 → L) -Galp- (1 →, 7.94% → 1) -D-Galp- (6 →, 4.79% > -GalA- (1 →, and 3.15-L → 2 → L → 1 →, 4.58% → 2,4) -L-Rhap- (1 →, 7.92% → 1) -D-Fucp- (3 →, 3.47% → 2) -D-Fucp- (4 →, 6.08% → 1) -D-Manp- (2,4 → and 3.52% → 1) -D-Manp- (3,6 →;

the glycosidic bond possessed by S5 includes, in mole percent, 8.31% → 1) -D-Glcp- (2,4,6 →, 3.68% → 1) -D-Glcp- (4 →, 5.18% → 1) -D-Glcp- (6 →, 2.61% → 4) -D-Glcp- (1 →, 4.58% → 4) -D-Glcp- (3 →, 3.97% → 3) - β -GalNAc- (1 →, 1.86% → 4) -a-GlcA- (1 →, 23.28% → 1) -D-Galp- (3 →, 9.21% → 1) -D-Galp- (6 →, 6.35% → 1) -D-Galp- (6 →, 2.55% > -4) - β -GalA- (1 →, 5.24-Galp- (2 → 2.55% → 4) - β -GalA- (1 → and 5.24-Xylp (1), 3 →, 7.06% → 1) -D-Xylp- (3 →, 4.39% → 1) -D-Manp- (2,4 →, 3.51% → 1) -D-Manp- (3,6 →, 4.36% → 1) -L-Frup- (3 → and 3.84% → 2) -L-Frup- (4 →.

3. A split yeast fermentation lysate according to claim 2 wherein S1 further has terminal glucose residues and terminal galactose residues, S4 further has terminal glucose residues and terminal galactose residues, and S5 further has terminal glucose residues and terminal galactose residues.

4. A split yeast fermentation lysate comprising, by weight, 14.8-16.4% S1, 12.3-12.6% S4, and 11.1-12.5% S5.

5. A method of producing a split yeast fermentation lysate comprising the steps of:

obtaining activated bacterium liquid of the secondary fission yeast;

carrying out amplification and fermentation culture on the activated bacterial liquid to obtain fermentation liquid;

filtering the fermentation liquor to obtain filtrate and thalli;

and performing wall breaking treatment on the thalli to obtain wall breaking liquid, mixing the wall breaking liquid with the thalli, and drying to obtain the secondary cracking yeast fermentation lysate.

6. The method of claim 5, wherein the expanding and fermenting step uses an expanding medium and a fermenting medium, and the expanding medium and/or the fermenting medium comprises 10g/L casein peptone, 5g/L yeast extract, 5g/L beef extract, 5g/L soybean peptone, 10g/L glucose, 0.15g/L NH ₄ C1, 0.5g/L manganese sulfate monohydrate, 0.01% v/v Tween 80, 50mg/L cysteine salt, 25mg/L cysteine, 0.05mg/L calcium chloride dihydrate, 0.15mg/L magnesium sulfate heptahydrate, 0.25m g/L potassium dihydrogen phosphate, 0.25g/L dipotassium hydrogen phosphate, 2.5mg/L sodium bicarbonate and 2.5mg/L sodium chloride.

7. The process according to claim 5, wherein the expanding and fermenting uses an expanding medium and a fermentation medium, and the expanding medium and/or the fermentation medium comprises 38g/L peptone, 96g/L glucose, 19g/L yeast extract, 0.06g/L xylose, 0.15mg/L sodium chloride, 96mg/L cysteine hydrochloride, 56mg/L cysteine, 0.15mg/L calcium chloride, 0.15mg/L magnesium sulfate, 0.15mg/L dipotassium hydrogen phosphate and 0.77g/L sodium bicarbonate.

8. The method according to claim 5, wherein the content of the crude polysaccharide in the activated bacterium liquid is not less than 0.5 g/L.

9. The whitening and anti-aging essence comprises at least one of S1, S4 or S5, butanediol, propylene glycol, disodium EDTA and water, wherein the content of S1, S4 or S5 is not less than 5.6 wt%, the content of butanediol is 0.25-3.5 wt%, the content of propylene glycol is 0.5-1.75 wt%, the content of disodium EDTA is 0.05-0.25 wt%, and the whitening and anti-aging essence is prepared by dissolving in deionized water.

10. Use of the split yeast fermentation lysate of any one of claims 1 to 3 for the preparation of a whitening and/or anti-aging cosmetic.

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