Culture medium for efficiently synthesizing eumelanin and application thereof

Abstract

This invention discloses a culture medium for the efficient synthesis of eumelanin and its applications, belonging to the fields of bioengineering and microbial fermentation technology. This culture medium is optimized for the tyrosine metabolic pathway in eumelanin synthesis and includes three formulations: a basic type, a shake-flask type, and an industrial fermenter type. Through the synergistic supply of a dual carbon source of glucose and glycerol, precise ratio of compound nitrogen sources, and specific trace element combinations, it significantly improves the bioavailability of L-tyrosine and tyrosinase activity. Specifically, the basic culture medium achieves an eumelanin yield of 1.53 g / L, a 168% increase compared to LB medium; the shake-flask type achieves a yield of 2.48 g / L; and the industrial fermenter type uses corn steep liquor instead of a high-cost refined nitrogen source, reducing costs by 65%, with a yield of 16.89 g / L in a 5L tank. This invention also discloses the corresponding fermentation process, which achieves efficient and stable synthesis of eumelanin without the need for additional exogenous phenolic inducers, making it suitable for large-scale production in fields such as cosmetics and biomedical materials.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and microbial fermentation technology, and in particular to a culture medium for the efficient synthesis of eumelanin and its application. Background Technology

[0002] Eumelanin is a natural heterogeneous high-molecular-weight pigment that is widely present in living organisms. With its broad-spectrum ultraviolet absorption, excellent free radical scavenging ability, mixed ion-electron conductivity and good biocompatibility, it has been applied in many high-value-added fields such as functional cosmetics, biomedical materials, and green bioelectronic devices.

[0003] Current methods of eumelanin production have significant limitations:

[0004] Natural extraction method: mainly separated from animal tissues such as cuttlefish ink sac, the raw material source is scarce, the extraction cost is high and the batch stability is poor, which cannot meet the needs of large-scale industrial production.

[0005] Conventional fermentation methods: Mostly use general-purpose culture media such as LB, which are not designed for the special metabolic needs of eumelanin synthesis. On the one hand, L-tyrosine has poor water solubility and is easy to precipitate, with a bioavailability of less than 30%. On the other hand, the carbon and nitrogen metabolic flux is not directed towards product synthesis. The fermentation yields reported in the present paper are generally lower than 0.6 g / L, and the yield decreases significantly after being scaled up to a fermenter.

[0006] Industrialization bottleneck: Existing optimized formulations mostly rely on expensive, refined nitrogen sources (yeast extract, peptone), resulting in high production costs. Furthermore, there is a lack of feed conversion strategies suitable for high-density fermentation, making large-scale implementation difficult. To address these issues, a solution is proposed below. Summary of the Invention

[0007] The purpose of this invention is to provide a culture medium for the efficient synthesis of eumelanin and its application, including three types of formulations: basic, shake flask, and industrial fermenter. The carbon-nitrogen ratio, precursor concentration, and trace element composition are optimized for different fermentation scenarios, which significantly improves the yield and conversion rate of eumelanin.

[0008] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0009] A culture medium for the efficient synthesis of eumelanin, based on a 1L volume, consists of the following components:

[0010] Carbon source: 12g glucose, 12g glycerol;

[0011] Nitrogen source: 8g yeast extract, 10g peptone, 5g ammonium sulfate;

[0012] Eumelanin synthesis precursor: L-tyrosine 2g;

[0013] Inorganic salts: 3g potassium dihydrogen phosphate, 6g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.1g calcium chloride, 0.5g sodium chloride;

[0014] 10 mL of trace element solution;

[0015] The pH is 7.0, and the remainder is water.

[0016] Preferably, the trace element solution, based on a 1L volume, consists of the following components: 40mg ZnCl2, 200mg FeCl3·6H2O, 10mg CuCl2, and 10mg MnCl2·4H2O.

[0017] A culture medium for the efficient synthesis of eumelanin, specifically designed for shake-flask fermentation, comprises the following components per 1L volume:

[0018] Carbon source: 20g glucose, 15g glycerol;

[0019] Nitrogen source: 8g yeast extract, 10g peptone, 5g ammonium sulfate;

[0020] Eumelanin synthesis precursor: L-tyrosine 3g;

[0021] Inorganic salts: 3g potassium dihydrogen phosphate, 6g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.1g calcium chloride, 0.5g sodium chloride;

[0022] 10 mL of trace element solution;

[0023] The pH is 6.8-7.2, and the remainder is water.

[0024] A culture medium for the efficient synthesis of eumelanin, specifically designed for industrial fermenters, comprises the following components per 1L volume:

[0025] Inexpensive nitrogen source: 20g corn steep liquor, 15g (NH4)2SO4;

[0026] Carbon source: 2% glycerol;

[0027] Eumelanin synthesis precursor: L-tyrosine 4g;

[0028] Buffer salts: 5g KH2PO4, 7g K2HPO4;

[0029] 10 mL of trace element solution;

[0030] The pH is 7.0, and the remainder is water.

[0031] Preferably, the trace element solution, based on a 1L volume, consists of the following components: 40mg ZnCl2, 200mg FeCl3·6H2O, 10mg CuCl2, and 10mg MnCl2·4H2O.

[0032] Preferably, when the culture medium is used for recombinant microbial fermentation to produce eumelanin, no additional exogenous phenolic inducers are required.

[0033] The beneficial effects of this invention are as follows: the yield of eumelanin in the basal culture medium can reach 1.53 g / L, which is 168% higher than that of LB culture medium; the yield of the shake flask-specific culture medium reaches 2.48 g / L, which is one of the highest levels currently achieved in shake flasks; the yield of the culture medium in the industrial fermenter reaches 16.89 g / L, with a cost reduction of 65%; and the combination of trace elements significantly improves tyrosinase activity, increasing the precursor utilization rate to over 80%. Attached Figure Description

[0034] Figure 1 The effect of the basic culture medium of this invention on eumelanin production;

[0035] Figure 2 This invention illustrates the effect of different culture media on eumelanin production. Detailed Implementation

[0036] The following description is only a preferred embodiment of the present invention, and the scope of protection is not limited to this embodiment. Any technical solution that falls within the scope of the present invention should be protected by the present invention.

[0037] Example 1

[0038] Basic culture medium fermentation

[0039] Seed culture preparation: Strawberry strains were streaked onto LB solid medium (containing 0.1% kanamycin) and incubated overnight at 37°C. Single colonies were then inoculated into 50 mL of LB liquid medium (containing 0.1% kanamycin) and incubated at 37°C and 230 rpm for 10 h until OD500 reached. 600 ≈2.0.

[0040] Fermentation culture: Inoculate the seed culture into the basic culture medium (500mL Erlenmeyer flask, 100mL liquid volume) at a 1% inoculation rate, and culture at 37℃ and 230rpm with shaking. After 6h, add 0.25mM IPTG to induce the expression of eumelanin synthase, and continue to culture for 48h.

[0041] Results: The fermentation broth was centrifuged to collect the cells, and eumelanin was extracted by acid hydrolysis. The yield was 1.53 g / L, which was 168% higher than that of the control LB medium (0.57 g / L).

[0042] Example 2

[0043] Shake flask fermentation with special culture medium

[0044] Seed liquid preparation: Same as in Example 1.

[0045] Fermentation culture: Inoculum size 1%, culture medium is shake flask special medium, culture conditions are the same as in Example 1, only the concentration of precursor L-tyrosine is adjusted to 3g / L.

[0046] Results: After 48 hours of fermentation, the yield of eumelanin was 2.48 g / L, the product purity was ≥98%, and there was no visible precursor precipitate.

[0047] Example 3

[0048] Industrial fermenter culture medium scale-up production

[0049] Seed culture preparation: Same as in Example 1, but the inoculum volume was increased to 8% for a 5L fermenter.

[0050] Fermentation conditions: 2.5L liquid volume, 37℃ temperature, pH 6.5 (adjusted with automatic ammonia addition), aeration rate 0.6m³ / h, stirring speed 400-800rpm. When OD... 600 When the concentration reaches 10, add 0.25 mM IPTG for induction. Use exponentially fed glycerol to maintain a glycerol concentration of 5-10 g / L.

[0051] Results: OD after 72 hours of fermentation 600 The nitrogen source cost was 116.4 g / L, and the eumelanin yield reached 16.89 g / L, with nitrogen source cost reduced by 65% ​​compared to conventional formulas.

[0052] Example 4

[0053] The effect of trace element combination on yield

[0054] Zn was removed separately in shake flask-specific culture medium. 2+ Fe 3+ Cu 2+ Mn 2+ Other conditions were the same as in Example 2. The results showed that the lack of Fe... 3+ or Cu 2+ The hourly yield decreased by more than 40%, demonstrating the crucial role of the micronutrient combination in tyrosinase activity.

[0055] The specific embodiments described above further illustrate the technical problems, technical solutions, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for the efficient synthesis of eumelanin, characterized in that, Based on a volume of 1L, it consists of the following components: Carbon source: 12g glucose, 12g glycerol; Nitrogen source: 8g yeast extract, 10g peptone, 5g ammonium sulfate; Eumelanin synthesis precursor: L-tyrosine 2g; Inorganic salts: 3g potassium dihydrogen phosphate, 6g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.1g calcium chloride, 0.5g sodium chloride; 10 mL of trace element solution; The pH is 7.0, and the remainder is water.

2. The culture medium according to claim 1, characterized in that, The trace element solution, based on a 1L volume, consists of the following components: 40mg ZnCl2, 200mg FeCl3·6H2O, 10mg CuCl2, and 10mg MnCl2·4H2O.

3. A culture medium for the efficient synthesis of eumelanin, characterized in that, This is a culture medium specifically for shake flask fermentation, composed of the following components per 1L volume: Carbon source: 20g glucose, 15g glycerol; Nitrogen source: 8g yeast extract, 10g peptone, 5g ammonium sulfate; Eumelanin synthesis precursor: L-tyrosine 3g; Inorganic salts: 3g potassium dihydrogen phosphate, 6g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.1g calcium chloride, 0.5g sodium chloride; 10 mL of trace element solution; The pH is 6.8-7.2, and the remainder is water.

4. A culture medium for the efficient synthesis of eumelanin, characterized in that, This is a culture medium specifically designed for industrial fermenters, and it consists of the following components per 1L volume: Inexpensive nitrogen source: 20g corn steep liquor, 15g (NH4)2SO4; Carbon source: 2% glycerol; Eumelanin synthesis precursor: L-tyrosine 4g; Buffer salts: 5g KH2PO4, 7g K2HPO4; 10 mL of trace element solution; The pH is 7.0, and the remainder is water.

5. The culture medium for efficient synthesis of eumelanin according to claim 3 or 4, characterized in that, The trace element solution, based on a 1L volume, consists of the following components: 40mg ZnCl2, 200mg FeCl3·6H2O, 10mg CuCl2, and 10mg MnCl2·4H2O.

6. The culture medium for efficient synthesis of eumelanin according to claim 1, 3, or 4, characterized in that, When the culture medium is used for recombinant microbial fermentation to produce eumelanin, no additional exogenous phenolic inducers are required.

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