A kind of bifid yeast lysate and its preparation method and application
Through the preparation method of difibrillation yeast, using ultrasonic crushing, degreasing, dialysis and dextran gel column elution technology, the obtained difibrillation yeast lysates can effectively reduce tyrosinase activity and inhibit melanin production, solving the problem of current There are shortcomings in this aspect of the technology, and significant effects in skin protection and treatment have been achieved.
Patent Information
- Application Number
- CN202211155839.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-22
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2042-09-22
AI Technical Summary
There is a lack of effective methods in the prior art to reduce tyrosinase activity and inhibit melanin production, especially in skin protection and treatment of skin acne, and this effect of probiotics and prebiotics has not been fully studied and applied.
By a preparation method, dificile yeast is mixed with water and then ultrasonically crushed, lyophilized, degreased, dialyzed, and elutioned using a dextran gel column to obtain dificile lysate dried dificile yeast lysate after fraction 2 and fraction 5. This effect is significantly improved by reducing tyrosinase activity and inhibiting melanin production, especially by the specific (NH4) 2SO4 aqueous solution elution conditions and the mixing of fraction 2 and fraction 5.
The lysate prepared by this method can not only effectively reduce tyrosinase activity, but also significantly inhibit melanin production, and its effect is better than that of the individual primary products, and even higher than traditional arbutin.
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Figure CN115869240B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and in particular to a bifida yeast lysate and a preparation method and application thereof. Background Art
[0002] As the largest organ in the human body, the skin is mainly composed of three layers: the epidermis, dermis, and subcutaneous fat tissue. Although it acts as a physical barrier to isolate the internal environment of the human body from the external environment, it is also subject to a variety of harmful stimuli, such as physical, chemical, and bacterial infections in the external environment. When the external stimuli reach a certain level, it will induce skin oxidation damage, impaired barrier function, skin melanin production, and melanin deposition.
[0003] Studies have found that probiotics on the surface of the skin play a vital role in protecting the skin. Probiotics can competitively exclude pathogens on the surface of the skin. Not only live bacteria, but also prebiotics, which are nutrients for probiotics, have great potential for treating skin acne and healing wounds. Researchers have conducted many discussions on the research of probiotics and prebiotics in the intestines and intestinal immunity, but there are few reports on whether probiotics and the active ingredients in probiotics have the effect of reducing tyrosinase activity and / or inhibiting melanin production in existing technologies. Summary of the invention
[0004] In order to solve at least one of the technical problems existing in the prior art, the present invention first provides a method for preparing a bifid yeast lysate; studies have shown that the bifid yeast lysate prepared by the method has the effect of reducing tyrosinase activity and also has the effect of inhibiting melanin production.
[0005] The technical solution of the present invention is as follows:
[0006] A method for preparing a bifid yeast lysate comprises the following steps:
[0007] (1) Mixing bifid yeast with water, and then subjecting the mixture to ultrasonic disruption and freeze-drying to obtain freeze-dried powder;
[0008] (2) defatting the lyophilized powder with an organic solvent to obtain defatted powder; mixing the defatted powder with water, and then dialyzing the mixture with a dialysis membrane having a pore size of 3 to 5 kD to obtain a first retentate and a first dialysate; then dialyzing the first dialysate with a dialysis membrane having a pore size of 1 to 2 kD to obtain a second retentate and a second dialysate; and lyophilizing the second retentate to obtain a preliminary product of a bifid yeast lysate; namely, the bifid yeast lysate.
[0009] Preferably, the weight amount of water in step (1) is 8 to 15 times the weight of the bifid yeast.
[0010] Most preferably, the weight amount of water used in step (1) is 10 times the weight of the bifid yeast.
[0011] Preferably, the weight amount of water in step (2) is 8 to 15 times the weight of the defatted powder.
[0012] Most preferably, the weight amount of water used in step (2) is 10 times the weight of the defatted powder.
[0013] Preferably, in step (2), the method is firstly dialyzed using a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is then dialyzed using a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; and the second retentate is lyophilized to obtain a primary product of bifid yeast lysate.
[0014] Preferably, the method for preparing bifid yeast lysate further comprises steps (3) and (4); steps (3) and (4) are specifically:
[0015] (3) The initial product of the bifid yeast lysate was loaded onto a dextran gel column and then sequentially treated with 2.0 to 1.8 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 1, and 1.5-1.4 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 2, and 0.9-0.8 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 3, and 0.5-0.4 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 4, and 0.2-0.1 mM (NH 4 ) 2 SO 4 The aqueous solution was used for elution to obtain fraction 5;
[0016] (4) taking fraction 2 and / or fraction 5 and drying them to obtain the bifid yeast lysate.
[0017] The inventors surprisingly found in further research that the bifid yeast lysate obtained by drying fraction 2 or fraction 5 prepared by the above method has better effects on reducing tyrosinase activity and inhibiting melanin production than the initial bifid yeast lysate product prepared in step (2).
[0018] The inventors hereby emphasize that the elution conditions of the dextran gel column play a decisive role in whether the further prepared bifid yeast lysate has a better effect of reducing tyrosinase activity and inhibiting melanin production compared with the initial product of bifid yeast lysate. The inventors have shown through a large number of studies that only the above elution conditions with 1.5-1.4 mM (NH 4 ) 2 SO 4 Fraction 2 obtained by eluting with aqueous solution and 0.2-0.1 mM (NH 4 ) 2 SO 4 Fraction 5 obtained by elution with aqueous solution has better effects on reducing tyrosinase activity and inhibiting melanin production than the initial product of bifid yeast lysate; while the fractions obtained by elution using other elution conditions have no better effects on reducing tyrosinase activity and inhibiting melanin production than the initial product of bifid yeast lysate.
[0019] Preferably, in step (3), the initial product of the bifid yeast lysate is loaded onto a dextran gel column, and then sequentially treated with 1.9 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 1, which was diluted with 1.3 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 2, which was diluted with 0.85 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 3, which was diluted with 0.45 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 4, which was then washed with 0.15 mM (NH 4 ) 2 SO 4 The aqueous solution was used for elution to obtain fraction 5.
[0020] Preferably, fraction 2 and fraction 5 are mixed and then dried to obtain the bifid yeast lysate.
[0021] The inventors surprisingly found in a large number of experiments that the bifida yeast lysate obtained by mixing fraction 2 and fraction 5 obtained by the elution conditions of the above-mentioned dextran gel column of the present invention has a synergistic effect of reducing tyrosinase activity and inhibiting melanin production; its effect of reducing tyrosinase activity and inhibiting melanin production is much higher than that of fraction 2 or fraction 5 alone, and even higher than that of arbutin.
[0022] The present invention also provides a bifid yeast lysate prepared by the method.
[0023] The present invention also provides a use of the bifid yeast lysate in preparing a product having the effects of reducing tyrosinase activity and / or inhibiting melanin production.
[0024] Preferably, the product is a skin care product or a medicine.
[0025] Beneficial effects: The present invention provides a bifid yeast lysate prepared by a new method; studies have shown that the bifid yeast lysate prepared by the method has the effect of reducing tyrosinase activity and inhibiting melanin production. Therefore, the bifid yeast lysate of the present invention as an effective ingredient for developing skin care products and medicines that have the effects of reducing tyrosinase activity and inhibiting melanin production has important application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the effect of the bifid yeast lysate of the present invention on the production of cellular tyrosinase.
[0027] Figure 2 This is the effect of the bifid yeast lysate of the present invention on cell melanin production. DETAILED DESCRIPTION
[0028] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0029] Example 1 Preparation of Bifid Saccharomyces cerevisiae lysate
[0030] (1) Mixing bifid yeast with 10 times the weight of water, and then subjecting the mixture to ultrasonic disruption and freeze-drying to obtain a freeze-dried powder;
[0031] (2) The lyophilized powder is placed in 8 times the weight of isopropanol for defatting for 2 hours to obtain defatted powder; the defatted powder is mixed with 10 times the weight of water, and then dialyzed with a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is dialyzed with a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; the second retentate is lyophilized to obtain a preliminary product of bifid yeast lysate; that is, the bifid yeast lysate.
[0032] Example 2 Preparation of bifid yeast lysate
[0033] (1) Mixing bifid yeast with 10 times the weight of water, and then subjecting the mixture to ultrasonic disruption and freeze-drying to obtain a freeze-dried powder;
[0034] (2) The lyophilized powder is placed in 8 times the weight of isopropanol for defatting for 2 hours to obtain defatted powder; the defatted powder is mixed with 10 times the weight of water, and then dialyzed with a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is dialyzed with a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; the second retentate is lyophilized to obtain a preliminary product of bifid yeast lysate; that is, the bifid yeast lysate.
[0035] (3) The initial product of the bifid yeast lysate was loaded onto a dextran gel column (the amount of dextran gel in the dextran gel column was 50 times the weight of the initial product of the bifid yeast lysate), and then eluted with 1 column volume of the following eluents in sequence: 1.9 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 1, which was diluted with 1.3 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 2, which was diluted with 0.85 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 3, which was diluted with 0.45 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 4, which was then washed with 0.15 mM (NH 4 ) 2 SO 4 The aqueous solution was used for elution to obtain fraction 5;
[0036] (4) taking fraction 2 and drying it to obtain the bifid yeast lysate.
[0037] Example 3 Preparation of Bifid Saccharomyces cerevisiae lysate
[0038] (1) Mixing bifid yeast with 10 times the weight of water, and then subjecting the mixture to ultrasonic disruption and freeze-drying to obtain a freeze-dried powder;
[0039] (2) The lyophilized powder is placed in 8 times the weight of isopropanol for defatting for 2 hours to obtain defatted powder; the defatted powder is mixed with 10 times the weight of water, and then dialyzed with a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is dialyzed with a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; the second retentate is lyophilized to obtain a preliminary product of bifid yeast lysate; that is, the bifid yeast lysate.
[0040] (3) The initial product of the bifid yeast lysate was loaded onto a dextran gel column (the amount of dextran gel in the dextran gel column was 50 times the weight of the initial product of the bifid yeast lysate), and then eluted with 1 column volume of the following eluents in sequence: 1.9 mM (NH4 ) 2 SO 4 The aqueous solution was used to elute fraction 1, which was diluted with 1.3 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 2, which was diluted with 0.85 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 3, which was diluted with 0.45 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 4, which was then washed with 0.15 mM (NH 4 ) 2 SO 4 The aqueous solution was used for elution to obtain fraction 5;
[0041] (4) taking fraction 5 and drying it to obtain the bifid yeast lysate.
[0042] Example 4 Preparation of Bifid Saccharomyces cerevisiae lysate
[0043] (1) Mixing bifid yeast with 10 times the weight of water, and then subjecting the mixture to ultrasonic disruption and freeze-drying to obtain a freeze-dried powder;
[0044] (2) The lyophilized powder is placed in 8 times the weight of isopropanol for defatting for 2 hours to obtain defatted powder; the defatted powder is mixed with 10 times the weight of water, and then dialyzed with a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is dialyzed with a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; the second retentate is lyophilized to obtain a preliminary product of bifid yeast lysate; that is, the bifid yeast lysate.
[0045] (3) The initial product of the bifid yeast lysate was loaded onto a dextran gel column (the amount of dextran gel in the dextran gel column was 50 times the weight of the initial product of the bifid yeast lysate), and then eluted with 1 column volume of the following eluents in sequence: 1.9 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 1, which was diluted with 1.3 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 2, which was diluted with 0.85 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 3, which was diluted with 0.45 mM (NH 4 ) 2 SO 4 The aqueous solution was used to elute fraction 4, which was then washed with 0.15 mM (NH4 ) 2 SO 4 The aqueous solution was used for elution to obtain fraction 5;
[0046] (4) Fraction 2 and fraction 5 are mixed and dried to obtain the bifid yeast lysate.
[0047] Experimental example
[0048] The experimental objects of the following experimental examples are the initial product of the bifid yeast lysate (abbreviated as BL) prepared in Example 1; in Example 2, fraction 2 is dried to obtain the bifid yeast lysate (abbreviated as BBP-1); in Example 3, fraction 5 is dried to obtain the bifid yeast lysate (abbreviated as BBP-2); in Example 4, fraction 2 and fraction 5 are mixed and then dried to obtain the bifid yeast lysate (abbreviated as BPL). The experimental methods and experimental results are as follows:
[0049] Mouse B16 melanoma cells were used as test cells to evaluate the activities of BBP-1, BBP-2, BL and BPL in inhibiting melanocyte proliferation, inhibiting intracellular tyrosinase activity and inhibiting intracellular melanin synthesis.
[0050] Place the culture flask in a 5% CO 2 Culture in a 37℃ incubator. When the melanocytes are spread all over the bottom of the culture bottle, they can be subcultured. Discard the culture medium, add 0.25% trypsin to digest for 2-3 minutes, discard the trypsin, then add 1-3mL culture medium, blow to form a single cell suspension, centrifuge at 1500r / min for 5 minutes, discard the supernatant, take an appropriate amount of cells and inoculate them in a new culture bottle, place it in an incubator for routine culture, subculture once every 3 days, and take the same passage cells for each experiment.
[0051] Experimental Grouping
[0052] Under sterile conditions, BBP-1, BBP-2, BL and BPL were diluted to the following concentrations with cell culture medium: 16, 8, 4, 2, 1 μg / mL. The bacteria were filtered with a 0.22 μm filter membrane and stored at 4°C in the dark. Arbutin was used as a positive control drug, and the simple culture medium was used as a blank control.
[0053] Melanocyte proliferation assay
[0054] The MTT colorimetric method was used to determine the effects of different concentrations of BBP-1, BBP-2, BL, and BPL on the proliferation of melanocytes. The cells in the logarithmic phase were collected and digested with 0.25% trypsin. The cell density was adjusted to 6×10 4 pc / mL, 200 μL per well was inoculated in a 96-well culture plate. 37°C, 5% CO 2After 24 hours of incubation, the cells adhered to the wall, the supernatant was discarded, and 180 μL of the test substance of different concentrations was added according to the experimental groups, and 3 replicates were set for each concentration. After 48 hours of incubation, 20 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 hours. Carefully aspirate the culture medium in the well, then add 150 μL of DMSO, place it on a shaker and oscillate at a low speed for 10 minutes to fully dissolve the crystals, and measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay. Calculate the inhibition rate of melanocyte proliferation in each group.
[0055] Intracellular tyrosinase activity assay
[0056] Take logarithmically growing cells and adjust the cell concentration to 6×10 4 / mL; then add to 24-well plate, 1 mL per well, set up 3 replicates. After 24h cells adhered to the wall, discard the culture medium, add different concentrations of conditioned medium, continue to culture for 48h, discard the culture medium, digest with 0.25% trypsin, centrifuge at 5000r / min for 10min, wash twice with PBS (0.01mol / L) with pH 7.4, add 200μL of 1% TritonX-100 solution each time; quickly freeze at -80℃ for 30min, freeze and thaw repeatedly twice to dissolve the cells; transfer 100μL to 96-well plate, add 20μL of 0.1% L-dopa solution to each well, shake for 5min, put in 37℃ incubator for 1h, and measure A490 value on spectrophotometer. The control group method is the same as above, tyrosinase activity inhibition rate = (1-experimental group A490 value / blank group A490 average value) × 100%.
[0057] Intracellular melanin synthesis assay
[0058] The melanin in melanocytes was measured by NaOH dissolution method, and the treatment was the same as above. After 72 hours of culture, the culture medium was discarded, and the cells were digested with 0.25% trypsin, centrifuged at 1000r / min for 5 minutes, and the supernatant was discarded. 200μL of NaOH (1mol / L) solution containing 10% DMSO was added to the cells under each treatment factor, and the melanin particles were completely dissolved in a 65℃ water bath. The absorbance A value at 490nm was detected by an enzyme-linked immunosorbent assay. Melanin formation rate = A490 value of the experimental group / average value of A490 of the blank group × 100%
[0059] BBP-1, BBP-2, BL, BPL and arbutin control groups can inhibit the tyrosinase activity of melanocytes; among them, the inhibition rate of BPL on the tyrosinase activity of B16 melanocytes under the same concentration conditions (16 μg / mL) is similar to that of arbutin, and is stronger than that of BBP-1, BBP-2, BL ( Figure 1 ).
[0060] The melanin synthesis of cells in the BBP-1, BBP-2, BL, BPL and arbutin groups was reduced. Among them, the inhibition rates of 16μg / mL BBP-1, BBP-2, BL, BPL and arbutin on cell melanin were approximately 33.2%, 31.3%, 25.1%, 60.3%, 54.3% ( Figure 2 ). The above experimental results show that BBP-1, BBP-2, BL and BPL can inhibit the production of melanin by regulating tyrosinase activity without affecting the cell activity of melanocytes.
[0061] The above experimental results show that the bifid yeast lysate prepared by the method has the effect of reducing tyrosinase activity and inhibiting melanin production. The bifid yeast lysate obtained by drying fraction 2 or fraction 5 prepared by the above method of the present invention has better effects on reducing tyrosinase activity and inhibiting melanin production than the initial product of the bifid yeast lysate prepared in step (2). Furthermore, the bifid yeast lysate obtained by mixing fraction 2 and fraction 5 obtained by the elution conditions of the above-mentioned dextran gel column of the present invention has a synergistic effect of reducing tyrosinase activity and inhibiting melanin production; its effect of reducing tyrosinase activity and inhibiting melanin production is much higher than that of fraction 2 or fraction 5 alone, and even higher than arbutin.
Claims
1. A method for preparing a bifid yeast lysate, characterized in that: The following steps are included: (1) Mixing bifid yeast with water, and then ultrasonically disrupting and freeze-drying to obtain freeze-dried powder; (2) Degreasing the lyophilized powder with an organic solvent to obtain defatted powder; mixing the defatted powder with water, and then dialyzing the mixture with a dialysis membrane having a pore size of 3 to 5 kD to obtain a first retentate and a first dialysate; then dialyzing the first dialysate with a dialysis membrane having a pore size of 1 to 2 kD to obtain a second retentate and a second dialysate; and lyophilizing the second retentate to obtain a primary product of bifid yeast lysate; (3) The initial product of the bifid yeast lysate was loaded onto a dextran gel column, and then eluted with a 2.0-1.8 mM (NH4)2SO4 aqueous solution to obtain fraction 1, 1.3 mM (NH4)2SO4 aqueous solution to obtain fraction 2, 0.9-0.8 mM (NH4)2SO4 aqueous solution to obtain fraction 3, 0.5-0.4 mM (NH4)2SO4 aqueous solution to obtain fraction 4, and 0.2-0.1 mM (NH4)2SO4 aqueous solution to obtain fraction 5; (4) Fraction 2 and fraction 5 are mixed and dried to obtain the bifid yeast lysate.
2. The method for preparing a bifida yeast lysate according to claim 1, characterized in that: The weight amount of water used in step (1) is 8 to 15 times the weight of the bifid yeast.
3. The method for preparing a bifida yeast lysate according to claim 1, characterized in that: The weight amount of water in step (2) is 8 to 15 times the weight of the defatted powder.
4. The method for preparing a bifida yeast lysate according to claim 1, characterized in that: In step (2), the first retentate and the first dialysate are dialyzed using a dialysis membrane with a pore size of 4 kD to obtain a first retentate and a first dialysate; the first dialysate is then dialyzed using a dialysis membrane with a pore size of 1 kD to obtain a second retentate and a second dialysate; the second retentate is lyophilized to obtain the initial product of the bifid yeast lysate.
5. The method for preparing a bifida yeast lysate according to claim 1, characterized in that: In step (3), the initial product of the bifid yeast lysate is loaded onto a dextran gel column and then eluted with a 1.9 mM (NH4)2SO4 aqueous solution to obtain fraction 1, 1.3 mM (NH4)2SO4 aqueous solution to obtain fraction 2, 0.85 mM (NH4)2SO4 aqueous solution to obtain fraction 3, 0.45 mM (NH4)2SO4 aqueous solution to obtain fraction 4, and 0.15 mM (NH4)2SO4 aqueous solution to obtain fraction 5.
6. The bifid yeast lysate prepared by the method according to any one of claims 1 to 5.
7. Use of the bifid yeast lysate according to claim 6 in preparing a skin care product having the effects of reducing tyrosinase activity and / or inhibiting melanin production.