A composition containing galactosaccharomyces fermenting liquid and its preparation method and application
By scientifically compounding galactosomal yeast fermentation broth with various active ingredients, and adding tannic acid and L-proline during the fermentation process, a composition with highly efficient antioxidant and anti-inflammatory activities was prepared, solving the problem of insufficient activity in existing products. When applied to cosmetics, it achieves a variety of skin care effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Filing Date
- 2026-06-01
- Publication Date
- 2026-07-14
AI Technical Summary
Existing galactosomal yeast fermentation broth products have limited antioxidant and anti-inflammatory activities, lack systematic screening and synergistic effect studies, and lack targeted regulation methods in the fermentation process, making it difficult to meet the needs of high-efficiency skincare.
A combination of galactosomal yeast fermentation broth, plankton extract, European red pine bark extract, black tea leaf extract, nonapeptide-1, multi-amino acid polysaccharide condensate, yeast polypeptides, and other active ingredients was scientifically formulated, and tannic acid and L-proline were added during the fermentation process to prepare a composition with significant antioxidant and anti-inflammatory effects.
It significantly enhances the antioxidant and anti-inflammatory activity of the composition, exerting skin protection through a dual pathway of antioxidant and anti-inflammatory effects, and can be applied to cosmetics with multiple functions such as anti-aging, soothing and repairing, and whitening and brightening.
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Figure CN122376497A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cosmetic preparation technology, specifically relating to a composition containing galactosomal yeast-like fermentation broth, its preparation method, and its application. Background Technology
[0002] In recent years, with the improvement of living standards and the enhancement of skincare awareness, consumers have placed higher demands on the functionality of cosmetics. Antioxidant, anti-inflammatory, anti-aging, and skin barrier repair functions have become key directions in modern skincare product research and development. Long-term exposure of the skin to the external environment (such as ultraviolet rays, pollutants, oxidative stress, etc.) easily induces excessive generation of free radicals, leading to lipid peroxidation, collagen degradation, and the release of inflammatory factors, thereby causing skin aging, pigmentation, and sensitivity. Therefore, developing cosmetic raw materials and compositions with highly effective antioxidant and anti-inflammatory activities has significant market value and application prospects. Galactomyces-like yeast fermentation broth is a type of fermented active ingredient that has received widespread attention in recent years. It is rich in various organic acids, amino acids, vitamins, and polysaccharides, and has the effects of regulating the skin microecology, promoting keratin metabolism, and improving skin texture. However, existing galactosomal yeast fermentation broth products still have certain limitations in practical applications: First, the antioxidant and anti-inflammatory activities of single fermentation broths are limited, making it difficult to meet the needs of high-efficiency skincare; second, commercially available products mostly use simple compounding methods, lacking systematic screening of active ingredients and synergistic effect research; third, existing fermentation processes mostly focus on increasing the yield or basic activity of fermentation broth, lacking targeted induction and regulation methods, such as adding specific precursors or inducers to directionally enhance its functional activity. Summary of the Invention
[0003] The present invention aims to provide a composition containing galactosomal yeast fermentation broth, its preparation method and application. The composition containing galactosomal yeast fermentation broth prepared by the method of the present invention has high antioxidant and anti-inflammatory activity, and can be used to prepare antioxidant cosmetics and skin care products, with good application prospects and market value.
[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A composition containing a galactosomal yeast-like fermentation broth, comprising the following components in parts by weight: 85-95 parts of galactosomal yeast fermentation broth, 3-8 parts of 1,2-hexanediol, 3-6 parts of water, 0.25-0.45 parts of p-hydroxyacetophenone, 0.05-0.1 parts of plankton extract, 0.05-0.1 parts of glycerol, 0.01-0.03 parts of 1,2-pentanediol, 0.008-0.011 parts of 1,3-propanediol, 0.0005-0.001 parts of caprylyl glycol, 0.00015-0.0002 parts of European red pine bark extract, 0.0001-0.00015 parts of black tea leaf extract, 0.00008-0.00012 parts of nonapeptide-1, 0.000012-0.000018 parts of polyamino acid polysaccharide condensate, and 0.000003-0.000007 parts of yeast polypeptides.
[0005] The present invention also provides a method for preparing the composition containing galactosomal yeast-like fermentation broth, comprising the following steps: S1. Prepare galactosomal yeast-like fermentation broth; S2. Preparation of planktonic extract; S3. Add the plankton extract, nonapeptide-1, European red pine bark extract, black tea leaf extract, multi-amino acid polysaccharide condensate, and yeast polypeptides obtained in S2 to the galactosomal yeast-like fermentation broth obtained in S1. Then add 1,2-hexanediol, glycerol, 1,2-pentanediol, p-hydroxyacetophenone, 1,3-propanediol, octyl glycol, and water, and mix well to obtain a composition containing galactosomal yeast-like fermentation broth.
[0006] Preferably, in step S1, the method for preparing the galactosomalid yeast fermentation broth includes the following steps: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slants, incubate in a constant temperature incubator, pick out activated single colonies, inoculate onto malt extract liquid culture medium, and incubate to obtain primary seed culture; S12. Transfer the primary seed culture obtained in S11 to fresh malt extract liquid culture medium and culture to obtain a galactosomal yeast-like seed culture with an effective viable count ≥10⁻⁶. 7 CFU / mL; S13. Transfer the galactosomal yeast seed culture to the fermentation medium, add tannic acid and L-proline to the medium, ferment and culture, centrifuge after fermentation, collect the supernatant, filter, and obtain the galactosomal yeast fermentation broth.
[0007] Preferably, in S11, the temperature in the constant temperature incubator is 25-30℃ and the time is 48-72h; the specific culture conditions for inoculating in malt extract liquid culture medium are: culture at 25-30℃ and 100-150rpm for 24-48h.
[0008] Preferably, in S12, the culture conditions are as follows: culture at 25-30℃ and 100-150rpm for 24-48h.
[0009] Preferably, in S13, tannic acid and L-proline are added to the culture medium, such that the concentration of tannic acid is 1-3 g / L and the concentration of L-proline is 400-600 mg / L. The fermentation culture conditions are specifically: fermentation culture at 28-30℃, pH 5.0, and 100-150 rpm for 36-48 h.
[0010] Preferably, in step S2, the preparation of the plankton extract includes the following steps: S21. Wash the plankton, add deionized water and homogenize to obtain a suspension; S22. Add silica to the suspension obtained in S21, and stir the mixture at 25-30℃ for 30-60 min to obtain a mixed solution. S23. Heat the mixture obtained from S22 in a water bath, adjust the pH to neutral, add lipase, perform enzymatic hydrolysis, raise the temperature, adjust the pH to alkaline, add alkaline protease, and perform enzymatic hydrolysis; after the enzymatic hydrolysis is completed, raise the temperature to inactivate the enzyme, and obtain the enzymatic hydrolysate. S24. Cool the enzymatic hydrolysate obtained in S23, centrifuge, and filter the supernatant to obtain the planktonic extract.
[0011] Preferably, in step S22, the reaction temperature is 25-30°C and the time is 30-60 min.
[0012] Preferably, in step S23, the mixture obtained in step S22 is heated in a water bath to 30-35°C, the pH is adjusted to 7.0-7.5, lipase is added, and enzymatic hydrolysis is performed at 100-150 rpm for 2-4 hours. The temperature is then raised to 55-60°C, the pH is adjusted to 9.0-9.5, alkaline protease is added, and enzymatic hydrolysis is performed for 2-2.5 hours. After enzymatic hydrolysis, the temperature is raised to 85-90°C to inactivate the enzyme for 15-20 minutes to obtain the enzymatic hydrolysate.
[0013] Preferably, in S24, the centrifugation conditions are: 4℃, 8000-10000rpm for 10-15min; the filtration is through a 0.22μm filter membrane.
[0014] The present invention also provides the use of the composition containing galactosomal yeast fermentation broth in the preparation of cosmetics.
[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a composition containing *Galactomyces galactosum* fermentation broth, its preparation method, and its applications. By scientifically compounding the *Galactomyces galactosum* fermentation broth with various active ingredients such as plankton extract, *Pinus tabuliformis* bark extract, *Cotyledon tomentosa* leaf extract, nonapeptide-1, polyamino acid polysaccharide condensates, and yeast polypeptides, and adding tannins and L-proline during fermentation, significant antioxidant and anti-inflammatory effects are achieved. Antioxidant activity tests demonstrate the key role of plankton extract and the addition of tannins and L-proline during fermentation in enhancing the composition's antioxidant activity, and significant synergistic effects exist among the components. Anti-inflammatory activity tests further indicate that the composition of this invention not only possesses excellent direct free radical scavenging ability but also significantly inhibits inflammatory responses at the cellular level, exerting skin protection through a dual pathway of antioxidant and anti-inflammatory effects. This composition has promising application prospects and market value in cosmetics with various functions such as anti-aging, soothing repair, and whitening / brightening.
[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0017] Figure 1 Statistical results of DPPH radical scavenging of the compositions provided in Examples 1-3 and Comparative Examples 1-2; Figure 2 Statistical results of ABTS radical scavenging of the compositions provided in Examples 1-3 and Comparative Examples 1-2; Figure 3 A statistical graph showing the effect of different treatments on interleukin-6 (IL-6) levels; Figure 4 A statistical graph showing the effect of different treatments on tumor necrosis factor-α (TNF-α) levels. Detailed Implementation
[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0020] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.
[0021] The malt extract liquid culture medium consisted of 130.0 g / L malt extract powder and 0.1 g / L chloramphenicol, with the pH adjusted to 5.6 ± 0.2, and sterilized at 115°C for 15 min.
[0022] Preparation of fermentation medium: Weigh 25g glucose, 10g yeast extract, 15g peptone, 1.5g potassium dihydrogen phosphate, and 0.8g magnesium sulfate, add 1L distilled water, stir to dissolve, adjust pH to 5.0±0.1, and sterilize at 121℃ for 15min.
[0023] Example 1 A composition containing galactosomal yeast-like fermentation broth, and a method for preparing it, comprising the following steps: S1. Preparation of galactosomal yeast-like fermentation broth: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slants and incubate at 28℃ for 60 h. Pick activated single colonies and inoculate them into Erlenmeyer flasks containing 100 mL of malt extract liquid culture medium. Incubate at 28℃ and 120 rpm for 36 h to obtain primary seed culture. S12. At an inoculum volume of 4% of the total volume, the primary seed culture obtained in S11 was transferred to fresh malt extract liquid culture medium and cultured at 28℃ and 120 rpm for 36 h to obtain a galactosomal yeast-like seed culture. The viable cell count was ≥10⁻⁶ using the plate count method. 7 CFU / mL; S13. The galactosomal yeast-like seed culture obtained in S12 was transferred to the fermentation medium at an inoculation rate of 8% (v / v). Tannic acid and L-proline were added to the medium to make the concentration of tannic acid 2 g / L and the concentration of L-proline 500 mg / L. The culture was fermented at 28℃, pH 5.0 and 120 rpm for 42 h. After the fermentation was completed, the fermentation broth was centrifuged at 4℃ and 9000 rpm for 12 min to collect the supernatant. The supernatant was then filtered under pressure through a 0.22 μm filter membrane to obtain the galactosomal yeast-like fermentation broth. S2. Preparation of plankton extract: S21. Wash the plankton with deionized water, add deionized water at a solid-liquid ratio of 1:5 (g / mL), and homogenize three times at 10000 rpm for 30 seconds each time to obtain a suspension. S22. Add silica to the suspension obtained in S21, the amount of which is 1.2% (w / v) of the suspension volume, and stir the mixture at 28°C for 45 min to obtain a mixed solution. S23. Heat the mixture obtained in S22 to 32°C in a water bath, adjust the pH to 7.2 with 0.1 mol / L NaOH, add lipase (0.5% of the suspension mass), and enzymatically hydrolyze in a constant temperature water bath shaker at 120 rpm for 3 h. Raise the temperature to 58°C, adjust the pH to 9.2 with 0.1 mol / L NaOH, add alkaline protease (0.6% of the suspension mass), and enzymatically hydrolyze for 2.2 h. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 90°C and maintain it for 18 min to inactivate the enzyme, obtaining the enzymatic hydrolysate. S24. Cool the enzymatic hydrolysate obtained in S23 to room temperature, centrifuge at 9000 rpm for 18 min at 4℃, collect the supernatant, filter it through a 0.22 μm microporous membrane to obtain the planktonic extract. S3. Preparation of the composition: 0.08 parts of plankton extract, 0.0001 parts of nonapeptide-1, 0.00018 parts of European red pine bark extract, 0.00012 parts of black tea leaf extract, 0.000015 parts of polyamino acid polysaccharide condensate, and 0.000005 parts of yeast polypeptides obtained in S2 were added to 90 parts of galactosomal yeast-like fermentation broth obtained in S1. Then, 5 parts of 1,2-hexanediol, 0.08 parts of glycerol, 0.02 parts of 1,2-pentanediol, 0.35 parts of p-hydroxyacetophenone, 0.0095 parts of 1,3-propanediol, 0.0008 parts of octyl glycol, and 4.45928 parts of water were added. The mixture was stirred at 300 rpm for 30 minutes at 25°C until completely dissolved and homogeneous to obtain a composition containing galactosomal yeast-like fermentation broth.
[0024] Example 2 A composition containing galactosomal yeast-like fermentation broth, and a method for preparing it, comprising the following steps: S1. Preparation of galactosomal yeast-like fermentation broth: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slant and incubate at 25℃ for 72h. Pick the activated single colony and inoculate it into an Erlenmeyer flask containing 100mL of malt extract liquid culture medium. Incubate at 25℃ and 100rpm for 48h to obtain the first-stage seed culture. S12. At an inoculum volume of 3% of the total volume, the primary seed culture obtained in S11 was transferred to fresh malt extract liquid culture medium and cultured at 25℃ and 100 rpm for 48 hours to obtain a galactosomal yeast-like seed culture. The effective viable count was ≥10⁻⁶ cells / mL as determined by plate count. 7 CFU / mL; S13. The galactosomal yeast-like seed culture obtained in S12 was transferred to the fermentation medium at an inoculation rate of 5% (v / v). Tannic acid and L-proline were added to the medium to make the concentration of tannic acid 1 g / L and the concentration of L-proline 400 mg / L. The culture was fermented at 28℃, pH 5.0 and 100 rpm for 48 h. After the fermentation was completed, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min to collect the supernatant. The supernatant was then filtered under pressure through a 0.22 μm filter membrane to obtain the galactosomal yeast-like fermentation broth. S2. Preparation of plankton extract: S21. Wash the plankton with deionized water, add deionized water at a solid-liquid ratio of 1:5 (g / mL), and homogenize three times at 10000 rpm for 30 seconds each time to obtain a suspension. S22. Add silica to the suspension obtained in S21, the amount of which is 0.5% (w / v) of the suspension volume, and stir the mixture at 25°C for 60 min to obtain a mixed solution. S23. Heat the mixture obtained in S22 to 30°C in a water bath, adjust the pH to 7.0 with 0.1 mol / L NaOH, add lipase (0.5% of the suspension mass), and enzymatically hydrolyze in a constant temperature water bath shaker at 100 rpm for 4 h; then raise the temperature to 55°C, adjust the pH to 9.0 with 0.1 mol / L NaOH, add alkaline protease (0.6% of the suspension mass), and continue enzymatic hydrolysis for 2.5 h. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 85°C and maintain it for 20 min to inactivate the enzyme, obtaining the enzymatic hydrolysate; S24. Cool the enzymatic hydrolysate obtained in S23 to room temperature, centrifuge at 8000 rpm for 15 min at 4℃, collect the supernatant, filter it through a 0.22 μm microporous membrane to obtain the planktonic extract. S3. Preparation of the mixture: 0.05 parts of plankton extract, 0.00008 parts of nonapeptide-1, 0.00015 parts of European red pine bark extract, 0.00010 parts of black tea leaf extract, 0.000012 parts of polyamino acid polysaccharide condensate, and 0.000003 parts of yeast polypeptides prepared in S2 were added to 85 parts of galactosomal yeast-like fermentation broth obtained in S1. Then, 3 parts of 1,2-hexanediol, 0.05 parts of glycerol, 0.01 parts of 1,2-pentanediol, 0.25 parts of p-hydroxyacetophenone, 0.008 parts of 1,3-propanediol, 0.0005 parts of octyl glycol, and 5.9998 parts of water were added. The mixture was stirred at 300 rpm for 30 minutes at 25°C until completely dissolved and homogeneous to obtain a composition containing galactosomal yeast-like fermentation broth.
[0025] Example 3 A composition containing galactosomal yeast-like fermentation broth, and a method for preparing it, comprising the following steps: S1. Preparation of galactosomal yeast-like fermentation broth: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slant and incubate at 30℃ for 48h. Pick the activated single colony and inoculate it into an Erlenmeyer flask containing 100mL of malt extract liquid culture medium. Incubate at 30℃ and 150rpm for 24h to obtain the first-stage seed culture. S12. At an inoculation rate of 5% of the total volume, the primary seed culture obtained in S11 was transferred to fresh malt extract liquid culture medium and cultured at 30℃ and 150 rpm for 24 h to obtain a galactosomal yeast-like seed culture. The effective viable count was ≥10⁻⁶ cells / mL as determined by the plate count method. 7 CFU / mL; S13. The *Galactomyces galactosum*-like seed culture obtained in S12 was transferred to the fermentation medium at an inoculation rate of 10% (v / v). Tannic acid and L-proline were added to the medium to make the concentration of tannic acid 3 g / L and the concentration of L-proline 600 mg / L. Fermentation was carried out at 30℃, pH 5.0, and 150 rpm for 36 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 10000 rpm for 10 min, and the supernatant was collected. Then, it was filtered under pressure through a 0.22 μm filter membrane to obtain the *Galactomyces galactosum*-like fermentation broth. S2. Preparation of plankton extract: S21. Wash the plankton with deionized water, add deionized water at a solid-liquid ratio of 1:5 (g / mL), and homogenize three times at 10000 rpm for 30 seconds each time to obtain a suspension; S22. Add silica to the suspension obtained in S21, the amount of which is 2.0% (w / v) of the suspension volume, and stir the mixture at 30°C for 30 min to obtain a mixed solution; S23. Heat the mixture obtained in S22 to 35°C in a water bath, adjust the pH to 7.5 with 0.1 mol / L NaOH, add lipase (0.5% of the suspension mass), and hydrolyze in a constant temperature water bath shaker at 150 rpm for 2 hours; then raise the temperature to 60°C, adjust the pH to 9.5 with 0.1 mol / L NaOH, add alkaline protease (0.6% of the suspension mass), and continue hydrolysis for 2 hours. After hydrolysis, rapidly raise the temperature to 90°C and maintain it for 15 minutes to inactivate the enzyme, obtaining the hydrolysate. S24. Cool the enzymatic hydrolysate obtained in S23 to room temperature, centrifuge at 10,000 rpm for 15 min at 4℃, collect the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the planktonic extract; S3. Prepare the composition by mixing; 0.10 parts of plankton extract, 0.00012 parts of nonapeptide-1, 0.00020 parts of European red pine bark extract, 0.00015 parts of black tea leaf extract, 0.000018 parts of polyamino acid polysaccharide condensate, and 0.000007 parts of yeast polypeptides prepared in S2 were added to 95 parts of galactosomal yeast-like fermentation broth obtained in S1. Then, 8 parts of 1,2-hexanediol, 0.10 parts of glycerol, 0.03 parts of 1,2-pentanediol, 0.45 parts of p-hydroxyacetophenone, 0.011 parts of 1,3-propanediol, 0.001 parts of octyl glycol, and 3.05928 parts of water were added. The mixture was stirred at 300 rpm for 30 minutes at 25°C until completely dissolved and homogeneous to obtain a composition containing galactosomal yeast-like fermentation broth.
[0026] Comparative Example 1 A composition containing galactosomal yeast-like fermentation broth, and a method for preparing it, comprising the following steps: S1. Preparation of galactosomal yeast-like fermentation broth: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slants and incubate at 28℃ for 60 h. Pick activated single colonies and inoculate them into Erlenmeyer flasks containing 100 mL of malt extract liquid culture medium. Incubate at 28℃ and 120 rpm for 36 h to obtain primary seed culture. S12. At an inoculum volume of 4% of the total volume, the primary seed culture obtained in S11 was transferred to fresh malt extract liquid culture medium and cultured at 28℃ and 120 rpm for 36 h to obtain a galactosomal yeast-like seed culture. The viable cell count was ≥10⁻⁶ using the plate count method. 7 CFU / mL; S13. The galactosomal yeast-like seed culture obtained in S12 was transferred to the fermentation medium at an inoculation rate of 8% (v / v). Tannic acid and L-proline were added to the medium to make the concentration of tannic acid 2 g / L and the concentration of L-proline 500 mg / L. The culture was fermented at 28℃, pH 5.0 and 120 rpm for 42 h. After the fermentation was completed, the fermentation broth was centrifuged at 4℃ and 9000 rpm for 12 min to collect the supernatant. The supernatant was then filtered under pressure through a 0.22 μm filter membrane to obtain the galactosomal yeast-like fermentation broth. S2. Preparation of the composition: 0.08 parts of deionized water, 0.0001 parts of nonapeptide-1, 0.00018 parts of European red pine bark extract, 0.00012 parts of black tea leaf extract, 0.000015 parts of polyamino acid polysaccharide condensate, and 0.000005 parts of yeast polypeptides were added to 90 parts of the galactosomal yeast-like fermentation broth obtained in S1. Then, 5 parts of 1,2-hexanediol, 0.08 parts of glycerol, 0.02 parts of 1,2-pentanediol, 0.35 parts of p-hydroxyacetophenone, 0.0095 parts of 1,3-propanediol, 0.0008 parts of octyl glycol, and 4.45928 parts of water were added. The mixture was stirred at 300 rpm for 30 minutes at 25°C until completely dissolved and homogeneous to obtain the composition containing the galactosomal yeast-like fermentation broth.
[0027] Comparative Example 2 A composition containing galactosomal yeast-like fermentation broth, and a method for preparing it, comprising the following steps: S1. Preparation of galactosomal yeast-like fermentation broth: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slants and incubate at 28℃ for 60 h. Pick activated single colonies and inoculate them into Erlenmeyer flasks containing 100 mL of malt extract liquid culture medium. Incubate at 28℃ and 120 rpm for 36 h to obtain primary seed culture. S12. At an inoculum volume of 4% of the total volume, the primary seed culture obtained in S11 was transferred to fresh malt extract liquid culture medium and cultured at 28℃ and 120 rpm for 36 h to obtain a galactosomal yeast-like seed culture. The viable cell count was ≥10⁻⁶ using the plate count method. 7 CFU / mL; S13. The galactosomal yeast-like seed culture obtained in S12 was transferred to the fermentation medium at an inoculation rate of 8% (v / v). The culture was fermented at 28℃, pH 5.0, and 120 rpm for 42 h. After the fermentation was completed, the fermentation broth was centrifuged at 4℃ and 9000 rpm for 12 min to collect the supernatant. The supernatant was then filtered under pressure through a 0.22 μm filter membrane to obtain the galactosomal yeast-like fermentation broth. S2. Preparation of plankton extract: S21. Wash the plankton with deionized water, add deionized water at a solid-liquid ratio of 1:5 (g / mL), and homogenize three times at 10000 rpm for 30 seconds each time to obtain a suspension. S22. Add silica to the suspension obtained in S21, the amount of which is 1.2% (w / v) of the suspension volume, and stir the mixture at 28°C for 45 min to obtain a mixed solution. S23. Heat the mixture obtained in S22 to 32°C in a water bath, adjust the pH to 7.2 with 0.1 mol / L NaOH, add lipase (0.5% of the suspension mass), and enzymatically hydrolyze in a constant temperature water bath shaker at 120 rpm for 3 h. Raise the temperature to 58°C, adjust the pH to 9.2 with 0.1 mol / L NaOH, add alkaline protease (0.6% of the suspension mass), and enzymatically hydrolyze for 2.2 h. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 90°C and maintain it for 18 min to inactivate the enzyme, obtaining the enzymatic hydrolysate. S24. Cool the enzymatic hydrolysate obtained in S23 to room temperature, centrifuge at 9000 rpm for 18 min at 4℃, collect the supernatant, filter it through a 0.22 μm microporous membrane to obtain the planktonic extract. S3. Preparation of the composition: 0.08 parts of plankton extract, 0.0001 parts of nonapeptide-1, 0.00018 parts of European red pine bark extract, 0.00012 parts of black tea leaf extract, 0.000015 parts of polyamino acid polysaccharide condensate, and 0.000005 parts of yeast polypeptides obtained in S2 were added to 90 parts of galactosomal yeast-like fermentation broth obtained in S1. Then, 5 parts of 1,2-hexanediol, 0.08 parts of glycerol, 0.02 parts of 1,2-pentanediol, 0.35 parts of p-hydroxyacetophenone, 0.0095 parts of 1,3-propanediol, 0.0008 parts of octyl glycol, and 4.45928 parts of water were added. The mixture was stirred at 300 rpm for 30 minutes at 25°C until completely dissolved and homogeneous to obtain a composition containing galactosomal yeast-like fermentation broth.
[0028] The effects of the compositions containing galactosomal yeast-like fermentation broth provided in Examples 1-3 and Comparative Examples 1-2 were verified.
[0029] The free radical scavenging ability of the above composition was determined using a DPPH free radical scavenging reagent kit (purchased from Jiangsu Edison Biotechnology Co., Ltd.). Specific determination methods were described in the kit instructions. Results are as follows: Figure 1 As shown.
[0030] The specific groupings are as follows: Blank group: 50 μL anhydrous ethanol + 100 μL DPPH working solution + 50 μL anhydrous ethanol; Positive group: 50 μL vitamin C (0.1 mg / mL) solution + 100 μL DPPH working solution + 50 μL anhydrous ethanol; Example 1: 50 μL of the composition from Example 1 (10 mg / mL) + 100 μL of DPPH working solution + 50 μL of anhydrous ethanol; Example 2: 50 μL of the composition from Example 2 (10 mg / mL) + 100 μL of DPPH working solution + 50 μL of anhydrous ethanol; Example 3: 50 μL of the composition from Example 3 (10 mg / mL) + 100 μL of DPPH working solution + 50 μL of anhydrous ethanol; Comparative Example 1: 50 μL Comparative Example 1 composition (10 mg / mL) + 100 μL DPPH working solution + 50 μL anhydrous ethanol; Comparative Example 2: 50 μL of Comparative Example 2 composition (10 mg / mL) + 100 μL of DPPH working solution + 50 μL of anhydrous ethanol.
[0031] Depend on Figure 1 It can be seen that the compositions of Examples 1-3 of the present invention all exhibited high DPPH free radical scavenging ability, significantly higher than that of Comparative Example 1 and Comparative Example 2. Among them, Example 1 had the highest scavenging rate, close to that of the positive control group, while Comparative Example 1 had the lowest scavenging rate. The above results indicate that the addition of planktonic extract and the addition of tannic acid and L-proline during the preparation of galactosomal yeast fermentation broth can significantly enhance the DPPH free radical scavenging ability of the compositions.
[0032] The free radical scavenging ability of the above composition was determined using the ABTS Free Radical Scavenging Ability Kit (purchased from Jiangsu Adison Biotechnology Co., Ltd.). Specific determination methods were described in the kit instructions. Results are as follows: Figure 2 As shown.
[0033] The experimental groups were the same as above. The positive group was: 50 μL Trolox (0.1 mg / mL) solution + 100 μL DPPH working solution + 50 μL anhydrous ethanol.
[0034] The results are as follows Figure 2 As shown, the ABTS radical scavenging rates of Examples 1-3 were significantly higher than those of Comparative Examples 1 and 2. Consistent with the DPPH results, the scavenging abilities of Comparative Examples 1 and 2 were significantly reduced, further confirming the key role of the addition of planktonic extract and the addition of tannic acid and L-proline during the preparation of the galactosomal yeast fermentation broth in enhancing the antioxidant activity of the composition.
[0035] RAW264.7 cells in the logarithmic growth phase were harvested and cultured at a concentration of 5 × 10⁻⁶ cells / year. 3 Cells were seeded into 96-well plates and cultured for 24 h. Then, gradient concentrations of the composition from Example 1 (0, 0.1, 1, 10, 100, 500, 1000 μg / mL) were added, and the plates were cultured for another 24 h. Cell viability was determined using the CCK-8 assay, and a safe concentration range (1, 10, 100 μg / mL) was selected to ensure cell viability >80%. In the formal experiment, RAW264.7 cells were seeded at 5 × 10⁻⁶ wells. 4Cells were seeded into 24-well plates and cultured for 24 hours. Afterward, they were divided into a blank group (no LPS), a model group (LPS, final concentration 1 μg / mL), a positive group (LPS and 1 μmol / L dexamethasone), and low, medium, and high dose groups (LPS and corresponding concentrations of the composition from Example 1). Each group underwent a pretreatment method: pre-incubation for 1 hour with culture medium containing the composition from Example 1 or dexamethasone, followed by incubation with LPS for another 24 hours. Cell culture supernatants were collected from each group. Nitric oxide (NO) content was detected using the Griess reagent colorimetric method (quantified using a sodium nitrite standard curve). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content were detected using a commercially available ELISA kit. All experiments were independently repeated three times, with three replicates per group. Results are shown below. Figures 3-4 .
[0036] Depend on Figures 3-4 It is evident that the composition of Example 1 of this invention, within a concentration range of 10-100 μg / mL, can dose-dependently inhibit the secretion of IL-6 and TNF-α by LPS-induced RAW264.7 cells. The high-dose group (100 μg / mL) showed a higher inhibition rate of IL-6 and TNF-α, comparable to the effect of the positive control dexamethasone, demonstrating that the composition of Example 1 possesses significant in vitro anti-inflammatory activity.
[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A composition containing a galactosomal yeast-like fermentation broth, characterized in that, Includes the following components in parts by weight: 85-95 parts of galactosomal yeast fermentation broth, 3-8 parts of 1,2-hexanediol, 3-6 parts of water, 0.25-0.45 parts of p-hydroxyacetophenone, 0.05-0.1 parts of plankton extract, 0.05-0.1 parts of glycerol, 0.01-0.03 parts of 1,2-pentanediol, 0.008-0.011 parts of 1,3-propanediol, 0.0005-0.001 parts of caprylyl glycol, 0.00015-0.0002 parts of European red pine bark extract, 0.0001-0.00015 parts of black tea leaf extract, 0.00008-0.00012 parts of nonapeptide-1, 0.000012-0.000018 parts of polyamino acid polysaccharide condensate, and 0.000003-0.000007 parts of yeast polypeptides.
2. The method for preparing the composition containing galactosomal yeast-like fermentation broth as described in claim 1, characterized in that, Includes the following steps: S1. Prepare galactosomal yeast-like fermentation broth; S2. Preparation of planktonic extract; S3. Add the plankton extract, nonapeptide-1, European red pine bark extract, black tea leaf extract, multi-amino acid polysaccharide condensate, and yeast polypeptides obtained in S2 to the galactosomal yeast-like fermentation broth obtained in S1. Then add 1,2-hexanediol, glycerol, 1,2-pentanediol, p-hydroxyacetophenone, 1,3-propanediol, octyl glycol, and water, and mix well to obtain a composition containing galactosomal yeast-like fermentation broth.
3. The preparation method according to claim 2, characterized in that, In S1, the method for preparing the galactosomal yeast-like fermentation broth includes the following steps: S11. Inoculate galactosomal yeast-like bacteria onto malt extract agar slants, incubate in a constant temperature incubator, pick out activated single colonies, inoculate onto malt extract liquid culture medium, and incubate to obtain primary seed culture; S12. Transfer the primary seed culture obtained in S11 to fresh malt extract liquid culture medium and culture to obtain a galactosomal yeast-like seed culture with an effective viable count ≥10⁻⁶. 7 CFU / mL; S13. Transfer the galactosomal yeast seed culture to the fermentation medium, add tannic acid and L-proline to the medium, ferment and culture, centrifuge after fermentation, collect the supernatant, filter, and obtain the galactosomal yeast fermentation broth.
4. The preparation method according to claim 3, characterized in that, In S11, the temperature in the constant temperature incubator is 25-30℃ and the time is 48-72h; the specific culture conditions for inoculating in malt extract liquid culture medium are: culture at 25-30℃ and 100-150rpm for 24-48h.
5. The preparation method according to claim 3, characterized in that, In S12, the specific cultivation conditions are: 25-30℃ and 100-150rpm for 24-48h.
6. The preparation method according to claim 3, characterized in that, In S13, tannic acid and L-proline are added to the culture medium, such that the concentration of tannic acid is 1-3 g / L and the concentration of L-proline is 400-600 mg / L. The specific fermentation conditions are: fermentation culture at 28-30℃, pH 5.0, and 100-150 rpm for 36-48 h.
7. The preparation method according to claim 2, characterized in that, In S2, the planktonic extract is prepared, including the following steps: S21. Wash the plankton, add deionized water and homogenize to obtain a suspension; S22. Add silica to the suspension obtained in S21, and stir the mixture at 25-30℃ for 30-60 min to obtain a mixed solution. S23. Heat the mixture obtained from S22 in a water bath, adjust the pH to neutral, add lipase, perform enzymatic hydrolysis, raise the temperature, adjust the pH to alkaline, add alkaline protease, and perform enzymatic hydrolysis; after the enzymatic hydrolysis is completed, raise the temperature to inactivate the enzyme, and obtain the enzymatic hydrolysate. S24. Cool the enzymatic hydrolysate obtained in S23, centrifuge, and filter the supernatant to obtain the planktonic extract.
8. The preparation method according to claim 7, characterized in that, In S22, the reaction temperature is 25-30℃ and the time is 30-60min.
9. The preparation method according to claim 7, characterized in that, In S23, the mixture obtained from S22 is heated in a water bath to 30-35℃, the pH is adjusted to 7.0-7.5, lipase is added, and enzymatic hydrolysis is carried out at 100-150 rpm for 2-4 hours. The temperature is then raised to 55-60℃, the pH is adjusted to 9.0-9.5, alkaline protease is added, and enzymatic hydrolysis is carried out for 2-2.5 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 85-90℃ to inactivate the enzyme for 15-20 minutes to obtain the enzymatic hydrolysate.
10. The use of the composition containing galactosomal yeast fermentation broth as described in claim 1 in the preparation of cosmetics.