Preparation method and application of fermentation product containing carotenoid

Abstract

The invention relates to a preparation method and application of a fermentation product containing carotenoid. According to the method, the Pseudo-ginseng is prepared by using a graded fermentation process of two microorganisms, the fermented strains are Aspergillus niger YNXC001 CCTCC No: M20242220 and Rhodotorula glutinis CCTCC NY 20083087, and the Aspergillus niger YNXC001 is separated from the plant Pseudo-ginseng and is preserved in the China Center for Type Culture Collection. The carotenoid content prepared by the method reaches 85.82 mg / L, and the crude extract of the fermentation product of the carotenoid has a good anti-allergy effect and can be well applied to skin care products with related effects.

Description

Technical Field

[0001] The present application belongs to the field of microbial fermentation technology, and specifically relates to a method for preparing a fermentation product containing carotenoids and its application. Background Art

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

[0003] Traditional methods for preparing carotenoids include chemical synthesis, biosynthesis, natural extraction and microbial extraction. Chemical synthesis and biosynthesis use chemical reagents, and the synthesized compounds will bring many safety issues when applied to the cosmetics and food industries. The main extracts of natural extraction are lycopene and B2 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 is low in cost and avoids the above-mentioned safety issues.

[0004] Although the technology of preparing carotenoids by microbial fermentation is already very advanced, the current yield of carotenoids prepared by microbial fermentation is low. Therefore, we urgently need a method for preparing carotenoids that can produce carotenoids in high yield, improve yield, reduce production costs, and apply them in anti-allergic cosmetics. Summary of the invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a method for preparing a fermentation product containing carotenoids, which can increase the yield of carotenoids and play an anti-allergic effect in cosmetics.

[0006] In one aspect, the present application provides a method for preparing a fermentation product containing carotenoids, the method comprising the following steps: 1) After mixing Aspergillus niger YNXC001 and red yeast, secondary fermentation is performed to obtain a fermentation liquid; 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 CCTCC NY 20083087.

[0007] Furthermore, Aspergillus niger YNXC001 accounted for 25%-40% of the total live bacteria; red yeast accounted for 60%-75% of the total live bacteria, and the total live bacteria concentration was 200-300 billion cfu / g.

[0008] Furthermore, the fermentation medium of the first stage of the secondary fermentation is composed of MS medium, sucrose, ammonium sulfate, and corn starch; the temperature of the first stage is 22-30° C., the time is 12-24 hours, and the rotation speed is 20 rpm; 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.

[0009] Furthermore, the fermentation medium of the second stage of the secondary fermentation is composed of sucrose, ammonium sulfate, and corn starch, with additional fatty acids added. The fatty acids are unsaturated fatty acids, specifically α-linolenic acid, and the addition amount is 0.5 g / L; the temperature of the second stage is 30-40°C, the time is 12-24h, the pH is 8-10, and static anaerobic fermentation is performed.

[0010] Furthermore, the centrifugal speed is 4000-8000 rpm, the time is 30-60 min, the supernatant is discarded, and the bacterial cells are retained to obtain a fermentation product containing carotenoids.

[0011] On the other hand, the present application also provides an application of a fermentation product in the preparation of cosmetics with anti-allergic effects, wherein the fermentation product is prepared by the above method.

[0012] Beneficial Effects 1. The present invention uses Aspergillus niger YNXC001 and red yeast to ferment and prepare carotenoids. The reaction conditions are mild, the raw materials are easily available, and the safety of natural strains is guaranteed; 2. The carotenoid content obtained by fermentation of Aspergillus niger YNXC001 and red yeast in the present application reaches 85.82 mg / L, which is significantly higher than the yield of carotenoids prepared by other methods.

[0013] 3. Fermentation products containing carotenoids have good anti-allergic effects and have broad prospects for application in the cosmetics field.

[0014] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The absorbance of each experimental group in Example 4 of the present application; Note: Compared with the normal group and the model group, # P <0.05,## P <0.01, ### P <0.001; compared with the positive control group and sample group, ns P >0.05,* P <0.05,** P <0.01,*** P <0.001. DETAILED DESCRIPTION

[0016] 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.

[0017] Example 1 1. Selection of strains The deposit number of Aspergillus niger YNXC001 is: CCTCC No: M20242220, and it was deposited in the China Center for Type Culture Collection on October 16, 2024, with the deposit address: Wuhan University, Wuhan, China, and the classification name is: Aspergillus niger YNXC001 Aspergillus niger YNXC001. The deposit number of red yeast is CCTCC NY 20083087. Aspergillus niger YNXC001 accounts for 65% of the total number of live bacteria; red yeast accounts for 35% of the total number of live bacteria, and the total live bacteria concentration is 200-300 billion cfu / g.

[0018] 2. Fermentation of strains 1) After mixing the Aspergillus niger YNXC001 and red yeast, perform secondary fermentation to obtain a fermentation liquid; The fermentation medium of the first stage of secondary fermentation consists of MS medium, sucrose, ammonium sulfate, and corn starch; the temperature of the first stage is 25°C, and the time is 18 hours. The temperature of the first stage is natural pH, and the rotation speed is 20rpm. The amount of each component of the medium added: MS medium 30g / L, sucrose 20g / L, ammonium sulfate 15g / L, corn starch 10g / L.

[0019] The fermentation medium of the second stage of secondary fermentation is composed of sucrose, ammonium sulfate, corn starch, and additional fatty acids; the temperature of the second stage is 35°C, and the time is 16 hours. The fatty acid is an unsaturated fatty acid, specifically α-linolenic acid, and the addition amount is 0.5g / L; the temperature of the second stage is 30-40°C, the time is 12-24 hours, the pH is controlled at 8-10, and the anaerobic fermentation is allowed to stand.

[0020] 2) After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000-8000 rpm for 30-60 min, the centrifuged liquid is discarded, and the centrifuged solids are retained to obtain a fermentation product containing carotenoids.

[0021] 3. Extraction and determination of carotenoids 1) Determination of dry cell weight: The centrifuged cells were dried at 60°C to a constant weight, which was weighed as a; Determination of biomass: biomass (g / L) = (a × 1000) / bacterial liquid volume (mL); 2) Cell disruption: Use a high-pressure homogenizer to break the bacterial cell wall; the homogenization pressure is 550-7000 bar, and the time is 5-15 min.

[0022] 3) Extraction and determination of carotenoids: Acetone was used to extract carotenoids to obtain a product containing carotenoids. Acetone was used as a blank control, and the absorbance of the sample was measured at 475nm using a UV-visible spectrophotometer; The carotenoid content was calculated according to the following formula: Carotenoid content (μg / g) = AλmaxDV / 0.16a. Aλmax is the absorbance at the maximum absorption wavelength of 475nm, V is the total volume of acetone used for extraction (mL), D is the dilution factor of the extract, a is the dry weight of the bacteria (g), and 0.16 is the extinction coefficient of carotenoids. Carotenoid yield (mg / L) = (biomass × carotenoid content) / 1000.

[0023] Example 2 Except for the following factors, the remaining steps are the same as those in Example 1.

[0024] 1. Selection of strains The preservation number of Aspergillus niger YNXC001 is CCTCC No: M20242220, and the preservation number of red yeast is CCTCC NY20083087. Aspergillus niger YNXC001 accounts for 55% of the total number of live bacteria; red yeast accounts for 45% of the total number of live bacteria, and the total live bacteria concentration is 200-300 billion cfu / g.

[0025] 2. Fermentation of strains The fermentation temperature of the first stage is 22°C and the time is 24 hours; the fermentation temperature of the second stage is 40°C and the time is 12 hours.

[0026] Example 3 Except for the following factors, the remaining steps are the same as those in Example 1.

[0027] 1. Selection of strains The preservation number of Aspergillus niger YNXC001 is CCTCC No: M20242220, and the preservation number of red yeast is CCTCC NY20083087. Aspergillus niger YNXC001 accounts for 75% of the total number of live bacteria; red yeast accounts for 25% of the total number of live bacteria, and the total live bacteria concentration is 200-300 billion cfu / g.

[0028] 2. Fermentation of strains The fermentation temperature of the first stage is 30℃ and the time is 12h; the fermentation temperature of the second stage is 30℃ and the time is 24h.

[0029] Comparative Example 1 Except for the following factors, the remaining steps are the same as those in Example 1.

[0030] Only red yeast (accession number CCTCC NY 20083087) was used for fermentation.

[0031] Comparative Example 2 Except for the following factors, the remaining steps are the same as those in Example 1.

[0032] Only Aspergillus niger YNXC001 was used for fermentation.

[0033] Comparative Example 3 Except for the following factors, the remaining steps are the same as those in Example 1.

[0034] The fermentation was carried out with common Aspergillus niger (strain of China Center for Type Culture Collection, collection number CCTCC AF200036) and red yeast.

[0035] Table 1 Yields of carotenoids prepared by different embodiments 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 red yeast can significantly increase the yield of carotenoids by 78.77%-85.82%, and the different ratios have little effect on the yield of carotenoids. However, if only red yeast, Aspergillus niger YNXC001 or common Aspergillus niger and red yeast are used for fermentation, the yield of carotenoids is significantly lower than that of the fermentation of other bacteria in the above comparative example. This shows that fermentation with Aspergillus niger YNXC001 and red yeast at the same time can significantly increase the yield of carotenoids.

[0036] Example 4 Application of carotenoids in anti-allergic cosmetics Allergens (such as C48 / 80, DNCB) can induce mast cell degranulation, leading to the release of allergic reaction active mediators such as histamine, 5-HT, prostaglandin D, and leukotrienes, which manifest as smooth muscle contraction, capillary dilation and increased permeability, increased glandular secretions, and other reactions, causing local or systemic allergies. The instantaneous release of mature tetrameric tryptase after mast cell degranulation will lead to a significant increase in serum tryptase levels. Tryptase reacts with BAPNA to form a complex with a maximum absorption peak at 405nm. The absorbance value can be detected using an enzyme marker, and the absorbance value is proportional to the degree of allergy.

[0037] Anti-allergic ingredients can stabilize the cell membrane of mast cells and prevent mast cell degranulation, thereby inhibiting the release of allergic reaction mediators such as histamine and slow-reacting substances, and further inhibiting the adverse effects of allergic reaction mediators on tissues. After zebrafish are treated with allergens, the anti-allergic effect of the test substance can be evaluated by observing the amount of tryptase release inhibited by the test substance.

[0038] The experimental system is wild-type AB strain zebrafish, and the zebrafish are 2 days post-fertilization (2dpf). The experimental method refers to "ZC / SOP-02-028-2022 Standard Operating Procedure for Evaluating the Anti-allergic Efficacy of Zebrafish Samples".

[0039] Experimental steps: (1) Select 2 dpf wild-type AB zebrafish and place them in a 12-well plate and adjust the volume to 1 mL.

[0040] (2) Ten zebrafish were used in each well (10 zebrafish in each treatment group) for testing, and 100 μg / mL BAPNA reaction solution was added to each well, 5 μL / well.

[0041] (3) The normal group received no treatment, the positive group received 0.1% (10 mg / mL) sodium cromoglycate, the model group received 0.6 μg / mL DNCB or 1 μg / mL C48 / 80, and the carotenoid fermentation product sample (carotenoid fermentation product prepared by the method of Example 2) group received 0.05%, 0.1%, and 0.15% concentrations for the experiment.

[0042] (4) After 24 h of reaction, transfer the test liquid in the 12-well plate to the 96-well plate, 200 μL / well.

[0043] (5) The absorbance (OD value) of each experimental group was analyzed using an ELISA instrument (wavelength 405 nm). Dunnett's T-test was used for statistical analysis. The normal control group was used as the standard to compare the production content of Tryptase in each experimental group. P < 0.05 was considered to be significantly different.

[0044] Anti-allergic effect (%) = ×100% Judgment basis: Statistical analysis P < 0.05, judged to be significant difference.

[0045] Test results: 1. Relative content of tryptase in each group of zebrafish.

[0046] Depend on Figure 1 It can be seen 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 fermentation product samples containing carotenoids (0.05%, 0.1%, 0.15%), the OD value decreased compared with the model group (P<0.05).

[0047] 2. Anti-allergic effect Table 2 Comparison of anti-allergic effects As can be seen from Table 2, carotenoid-containing fermentation products have anti-allergic effects in the concentration range of 0.05%-0.1% (P<0.05), and the anti-allergic effect is best at low concentrations. Under the experimental conditions, the carotenoid-containing fermentation product samples produced by Aspergillus niger YNXC001 and red yeast fermentation have anti-allergic effects in the range of 0.05%-0.1%.

[0048] In summary, the carotenoid-containing fermentation product prepared in the present application has anti-allergic effect, and therefore can be used in cosmetics to prepare anti-allergic cosmetics.

[0049] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection 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 persons of 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; 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 fermentation medium of the first stage of the secondary fermentation is composed of MS medium, sucrose, ammonium sulfate and corn starch; the temperature of the first stage is 22-30°C, the time is 12-24h, and the rotation 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 fermentation medium of the second stage of the secondary fermentation is composed of sucrose, ammonium sulfate, and corn starch, with additional fatty acids added. The fatty acids are unsaturated fatty acids, specifically α-linolenic acid, and the addition amount is 0.5 g / L. The temperature of the second stage is 30-40°C, the time is 12-24h, the pH is 8-10, and static anaerobic fermentation is performed.

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