Preparation method and application of a fermentation product containing carotenoids

Abstract

This application relates to a preparation method and application of a fermentation product containing carotenoids. This method is obtained by using a two-stage fermentation process of two microorganisms. The strains used in the fermentation are Aspergillus niger YNXC001 CCTCC No: M20242220 and Rhodotorula sp. CCTCC NY 20083087. Among them, Aspergillus niger YNXC001 is isolated from Panax notoginseng and preserved in the China Center for Type Culture Collection. The carotenoid content prepared by the method of this application reaches 85.82 mg / L, and the crude extract of its fermentation product has good anti-allergic efficacy and can be well applied to skin care products with related efficacy.

Description

Technical Field

[0001] This application belongs to the technical field of microbial fermentation, and specifically relates to a preparation method and application of a fermentation product containing carotenoids. Background Art

[0002] Carotenoids are a general term for a class of important natural pigments, which are commonly found in the yellow, orange-red or red pigments of animals, higher plants, fungi and algae. Carotenoids are the main source of vitamin A in the body. Due to their antioxidant, immunomodulatory, anti-cancer, anti-aging and other effects, they are widely used in the cosmetics industry and the food industry.

[0003] Traditional methods for preparing carotenoids include chemical synthesis, biosynthesis, natural extraction and microbial extraction. However, chemical synthesis and biosynthesis use chemical reagents, and the synthesized compounds may cause many safety problems when applied to the cosmetics and food industries. The main extracts of natural extraction are lycopene and β-carotene, and the production cost is high. Therefore, most of the carotenoids on the market are obtained by microbial extraction. Extracting natural carotenoids by microbial fermentation has low cost and avoids the above safety problems.

[0004] Although the existing technology for preparing carotenoids by microbial fermentation is already very developed, the yield of carotenoids prepared by current microbial fermentation is relatively low. Therefore, we urgently need a method for preparing carotenoids that can produce carotenoids in high yield, improve the yield rate, reduce the production cost, and its application in anti-allergy cosmetics. Summary of the Invention

[0005] To solve or partially solve the problems existing in the related technologies, this application provides a preparation method of a fermentation product containing carotenoids, which can improve the yield of carotenoids and has an anti-allergy effect in cosmetics.

[0006] On the one hand, this application provides a preparation method of a fermentation product containing carotenoids, and the method includes the following steps:

[0007] 1) Mix Aspergillus niger YNXC001 and Rhodotorula, and perform secondary fermentation to obtain a fermentation broth;

[0008] 2) After the fermentation is completed, centrifuge the fermentation broth, discard the centrifuged liquid, and retain the centrifuged solid content;

[0009] 3) Perform cell wall breaking and acetone extraction on the centrifuged solid content obtained in step 2) in sequence to obtain a fermentation product containing carotenoids;

[0010] The preservation number of Aspergillus niger YNXC001 is CCTCC No: M20242220; the preservation number of Rhodotorula is CCTCC NY 20083087.

[0011] Furthermore, Aspergillus niger YNXC001 accounts for 25%-40% of the total viable bacteria; Rhodotorula accounts for 60%-75% of the total viable bacteria, and the total viable bacteria concentration is 200-300 billion cfu / g.

[0012] Furthermore, the fermentation medium in the first stage of the secondary fermentation consists of MS medium, sucrose, ammonium sulfate, and corn starch; the temperature in the first stage is 22-30°C, the time is 12-24h, and the rotation speed is 20rpm;

[0013] The addition amounts of each component of the medium are: 30g / L of MS medium, 20g / L of sucrose, 15g / L of ammonium sulfate, and 10g / L of corn starch.

[0014] Furthermore, the fermentation medium in the second stage of the secondary fermentation is composed of sucrose, ammonium sulfate, and corn starch, with the additional addition of fatty acids. The fatty acid is an unsaturated fatty acid, specifically α-linolenic acid, and the addition amount is 0.5g / L; the temperature in the second stage is 30-40°C, the time is 12-24h, the pH is 8-10, and it is statically anaerobically fermented.

[0015] Furthermore, the rotation speed for centrifugation is 4000-8000rpm, the time is 30-60min, the supernatant is discarded, and the bacterial cells are retained, thus obtaining a fermentation product containing carotenoids.

[0016] On the other hand, the present application also provides an application of a fermentation product in the preparation of a cosmetic with anti-allergic efficacy, and the fermentation product is prepared by the above method.

[0017] Beneficial effects

[0018] 1. The present invention uses Aspergillus niger YNXC001 and Rhodotorula to ferment and prepare carotenoids. The reaction conditions are mild, the raw materials are easily available, and the safety of natural strains is guaranteed;

[0019] 2. The carotenoid content obtained by fermenting Aspergillus niger YNXC001 and Rhodotorula in the present application reaches 85.82mg / L, which is significantly higher than the yield of carotenoids prepared by other methods.

[0020] 3. The fermentation product containing carotenoids has good anti-allergic efficacy and has broad prospects in the cosmetic field.

[0021] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. Brief description of the drawings

[0022] Figure 1 Absorbance of each experimental group in Example 4 of this application;

[0023] Note: When comparing the normal group with the model group, # P <0.05, ## P <0.01, ### P <0.001; When comparing the model group with the positive control group and the sample group, ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001. Specific implementation manners

[0024] The embodiments of the present application will be described in more detail below. However, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0025] Example 1

[0026] 1. Selection of strains

[0027] The preservation number of Aspergillus niger YNXC001 is: CCTCC No: M20242220, which was preserved in the China Center for Type Culture Collection on October 16, 2024. The preservation address is: Wuhan University, Wuhan, China. The taxonomic name is: Aspergillus niger YNXC001 Asperillus niger YNXC001. The preservation number of Rhodotorula is CCTCC NY 20083087. Aspergillus niger YNXC001 accounts for 65% of the total viable bacteria; Rhodotorula accounts for 35% of the total viable bacteria, and the total viable bacteria concentration is 200 - 300 billion cfu / g.

[0028] 2. Fermentation of strains

[0029] 1) After mixing the above-mentioned Aspergillus niger YNXC001 and Rhodotorula, secondary fermentation was carried out to obtain a fermentation broth;

[0030] The fermentation medium for the first stage of the secondary fermentation consists of MS medium, sucrose, ammonium sulfate, and corn starch; the temperature for the first stage is 25°C, and the time is 18 h. The temperature for the first stage is natural pH, and the rotation speed is 20 rpm. The addition amounts of each component of the medium are: 30 g / L of MS medium, 20 g / L of sucrose, 15 g / L of ammonium sulfate, and 10 g / L of corn starch.

[0031] The fermentation medium in the second stage of secondary fermentation is based on sucrose, ammonium sulfate, and corn starch, with the additional addition of fatty acid composition; the temperature in the second stage is 35 °C and the time is 16 h. The fatty acid is an unsaturated fatty acid, specifically α-linolenic acid, and the addition amount is 0.5 g / L; the temperature in the second stage is 30 - 40 °C, the time is 12 - 24 h, the pH is controlled at 8 - 10, and static anaerobic fermentation is carried out.

[0032] 2) After the fermentation is completed, the obtained fermentation broth is centrifuged at a rotation speed of 4000 - 8000 rpm for 30 - 60 min, the centrifuged liquid is discarded, and the centrifuged solid content is retained, thus obtaining a fermentation product containing carotenoids.

[0033] 3. Extraction and determination methods of carotenoids

[0034] 1) Determination of cell dry weight: The centrifuged bacterial cells are dried to a constant weight at 60 °C and weighed as a;

[0035] Determination of biomass: Biomass (g / L) = (a × 1000) / volume of bacterial liquid (mL);

[0036] 2) Cell disruption: The bacterial cells are disrupted using a high-pressure homogenizer; the homogenization pressure is 550 - 7000 bar and the time is 5 - 15 min.

[0037] 3) Extraction and determination of carotenoids: Acetone is used to extract carotenoids to obtain a product containing carotenoids. Using acetone as a blank control, the absorbance of the sample is measured at 475 nm using a UV-visible spectrophotometer;

[0038] The carotenoid content is calculated according to the following formula:

[0039] Carotenoid content (μg / g) = AλmaxDV / 0.16a. Where Aλmax is the absorbance at the maximum absorption wavelength of 475 nm, V is the total volume of acetone used for extraction (mL), D is the dilution factor of the extraction solution, a is the dry weight of the bacterial cells (g), and 0.16 is the extinction coefficient of carotenoids. Carotenoid yield (mg / L) = (biomass × carotenoid content) / 1000.

[0040] Example 2

[0041] Except for the following factors, the remaining steps are the same as those in Example 1.

[0042] 1. Selection of bacterial strain

[0043] The preservation number of Aspergillus niger YNXC001 is: CCTCC No: M20242220, and the preservation number of Rhodotorula is CCTCC NY20083087. Aspergillus niger YNXC001 accounts for 55% of the total viable bacteria; Rhodotorula accounts for 45% of the total viable bacteria, and the total viable bacteria concentration is 200 - 300 billion cfu / g.

[0044] 2. Fermentation of strains

[0045] The fermentation temperature in the first stage is 22°C and the time is 24h; the fermentation temperature in the second stage is 40°C and the time is 12h.

[0046] Example 3

[0047] Except for the following factors, the remaining steps are the same as those in Example 1.

[0048] 1. Selection of strains

[0049] The preservation number of Aspergillus niger YNXC001 is: CCTCC No: M20242220, and the preservation number of Rhodotorula is CCTCC NY20083087. Aspergillus niger YNXC001 accounts for 75% of the total viable bacteria; Rhodotorula accounts for 25% of the total viable bacteria, and the total viable bacteria concentration is 200 - 300 billion cfu / g.

[0050] 2. Fermentation of strains

[0051] The fermentation temperature in the first stage is 30°C and the time is 12h; the fermentation temperature in the second stage is 30°C and the time is 24h.

[0052] Comparative Example 1

[0053] Except for the following factors, the remaining steps are the same as those in Example 1.

[0054] Only Rhodotorula (preservation number CCTCC NY 20083087) is used for fermentation.

[0055] Comparative Example 2

[0056] Except for the following factors, the remaining steps are the same as those in Example 1.

[0057] Only Aspergillus niger YNXC001 is used for fermentation.

[0058] Comparative Example 3

[0059] Except for the following factors, the remaining steps are the same as those in Example 1.

[0060] Ordinary Aspergillus niger (a strain from the China Center for Type Culture Collection, preservation number CCTCC AF200036) is used for co-fermentation with Rhodotorula.

[0061] Table 1 Yields of carotenoids prepared by the schemes of different embodiments

[0062]

[0063] In summary, it can be seen from the results of the single-factor experiment (Table 1) that the co-fermentation of Aspergillus niger YNXC001 and Rhodotorula can significantly increase the yield of carotenoids, which is 78.77%-85.82%. In the case of different ratios, the impact on the yield of carotenoids is small. However, when only Rhodotorula, Aspergillus niger YNXC001, or common Aspergillus niger and Rhodotorula are used for fermentation, the yield of carotenoids is significantly lower than that of the fermentation of other bacteria in the above comparative examples. Thus, it shows that the simultaneous fermentation with Aspergillus niger YNXC001 and Rhodotorula can significantly increase the yield of carotenoids.

[0064] Example 4 Application of carotenoids in anti-allergy cosmetics

[0065] Allergens (such as C48 / 80, DNCB) can induce mast cell degranulation, leading to the release of allergic reaction mediators such as histamine, 5-HT, prostaglandin D, and leukotriene, that is, manifested as reactions such as smooth muscle contraction, capillary dilation and enhanced permeability, and increased glandular secretions, causing local or systemic allergies. The transient release of mature tetrameric tryptase after mast cell degranulation will cause a significant increase in serum tryptase levels. Tryptase undergoes a complexation reaction with BAPNA, and the generated complex has a maximum absorption peak at 405 nm. The absorbance value can be detected using an enzyme-linked immunosorbent assay reader, and the absorbance value is proportional to the degree of allergy.

[0066] Anti-allergy active ingredients can stabilize the cell membrane of mast cells, prevent mast cell degranulation, thereby inhibiting the release of allergy reaction mediators such as histamine and slow-reacting substances, and further inhibiting the adverse effects of allergy reaction mediators on tissues. After treating zebrafish with allergens, the anti-allergy efficacy of the test substance can be evaluated by observing the amount of tryptase release inhibited by the test substance.

[0067] The experimental system is wild-type AB strain zebrafish at 2 days post-fertilization (2 dpf). The experimental method refers to the "ZC / SOP-02-028-2022 Standard Operating Procedure for Evaluating the Anti-allergy Efficacy of Samples by Zebrafish".

[0068] Experimental steps:

[0069] (1) Select wild-type AB strain zebrafish at 2 dpf and place them in a 12-well plate, with a fixed volume of 1 mL.

[0070] (2) Use 10 larvae per well (10 zebrafish in each treatment group) for testing, and add 100 μg / mL BAPNA reaction solution, 5 μL per well.

[0071] (3) The normal group was not treated. The positive group was treated with 0.1% (10 mg / mL) sodium cromoglycate. The model group was treated with 0.6 μg / mL DNCB or 1 μg / mL C48 / 80. The group containing the carotenoid fermentation product (prepared by the method of Example 2) was tested at concentrations of 0.05%, 0.1%, and 0.15%.

[0072] (4) After 24 h of reaction, the test liquids in the 12-well plates were transferred to 96-well plates, 200 μL per well.

[0073] (5) The absorbance (OD value) of each experimental group was analyzed using an enzyme-linked immunosorbent assay (ELISA) reader (wavelength 405 nm). Dunnett's T-test was used for statistical analysis with the normal control group as the standard to compare the production content of tryptase in each experimental group. A P value < 0.05 was considered to indicate a significant difference.

[0074] Anti-allergic efficacy (%) = × 100%

[0075] Judgment basis: Statistical analysis with P < 0.05 was considered to indicate a significant difference.

[0076] Detection results:

[0077] 1. The relative content of tryptase in the body of zebrafish in each group.

[0078] It can be seen from Figure 1 that after stimulation with 0.6 μg / mL DNCB, the OD value (relative content of tryptase) of the model group increased significantly. After intervention with 1% sodium cromoglycate and the carotenoid fermentation product sample (0.05%, 0.1%, 0.15%), the OD value decreased compared with the model group (P < 0.05).

[0079] 2. Anti-allergic efficacy

[0080] Table 2 Comparison of anti-allergic efficacy

[0081]

[0082] It can be seen from Table 2 that the carotenoid fermentation product has anti-allergic efficacy in the concentration range of 0.05% - 0.1% (P < 0.05), and the anti-allergic effect is the best at low concentrations. Under the experimental conditions, the carotenoid fermentation product sample produced by Aspergillus niger YNXC001 and Rhodotorula has anti-allergic efficacy in the range of 0.05% - 0.1%.

[0083] In summary, the carotenoid-containing fermentation product prepared in this application has an anti-allergic effect. Therefore, it can be used in cosmetics to prepare anti-allergic cosmetics.

[0084] The embodiments of the present application have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a fermentation product containing carotenoids, characterized in that: The preparation method comprises the following steps: 1) After mixing Aspergillus niger YNXC001 and red yeast, secondary fermentation is performed to obtain a fermentation liquid; The fermentation medium of the first stage of the secondary fermentation is composed of MS medium, sucrose, ammonium sulfate and corn starch; the fermentation medium of the second stage of the secondary fermentation is composed of sucrose, ammonium sulfate and corn starch, and α-linolenic acid is additionally added in an amount of 0.5 g / L; 2) After the fermentation is completed, the fermentation liquid is centrifuged, the centrifuged liquid is discarded, and the centrifuged solids are retained; 3) The centrifuged solids obtained in 2) are sequentially subjected to cell wall breaking and acetone extraction to obtain a fermentation product containing carotenoids; The preservation number of the Aspergillus niger YNXC001 is CCTCC No: M20242220; the preservation number of the red yeast is CCTCCNY 20083087.

2. The preparation method according to claim 1, characterized in that: The Aspergillus niger YNXC001 accounts for 25%-40% of the total number of live bacteria; the red yeast accounts for 60%-75% of the total number of live bacteria, and the total live bacteria concentration is 200-300 billion cfu / g.

3. The preparation method according to claim 1, characterized in that: The temperature of the first stage is 22-30°C, the time is 12-24h, and the speed is 20rpm; The amount of each component added to the culture medium: MS medium 30g / L, sucrose 20g / L, ammonium sulfate 15g / L, corn starch 10g / L.

4. The preparation method according to claim 1, characterized in that: The temperature of the second stage is 30-40°C, the time is 12-24 hours, the pH is 8-10, and the fermentation is carried out under static anaerobic conditions.

5. The preparation method according to claim 1, characterized in that: The centrifugal speed is 4000-8000 rpm, and the time is 30-60 min.

6. Use of a fermentation product in the preparation of anti-allergic cosmetics, characterized in that: The fermentation product is prepared by the preparation method according to claim 1.

Images