Medium for the directed accumulation of the astaxanthin precursor beta-carotene, method of preparation and apparatus

Abstract

The application discloses astaxanthin precursor beta-carotene directional accumulation culture medium, a preparation method and equipment, relates to the field of microbial fermentation culture medium preparation, and comprises the following components: a carbon source, a nitrogen source, inorganic salt, a beta-carotene synthesis precursor supply agent, a membrane lipid fluidity regulator, an oxidation-reduction auxiliary factor, a product in-situ capture agent, and the balance is water; through optimizing the accurate proportioning of the composite carbon source, the nitrogen source and the inorganic salt, the growth metabolism law of astaxanthin-producing microorganisms can be adapted, the microbial proliferation rate and the secondary metabolism intensity are balanced, a stable and sufficient basic nutrition environment is provided for beta-carotene synthesis, the metabolic disorder problem caused by a single nutrient source is avoided, the carotenoid synthesis metabolic pathway is continuously activated while the efficient growth of the bacteria is ensured, the bio-synthesis efficiency of beta-carotene is directionally strengthened by introducing the beta-carotene synthesis precursor supply agent, and the in-situ synthesis flux is improved.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation culture medium preparation technology, and in particular to a culture medium for the directional accumulation of astaxanthin precursor β-carotene, its preparation method, and equipment. Background Technology

[0002] β-carotene is a core precursor for the microbial synthesis of astaxanthin, and its intracellular accumulation directly determines the final yield and production efficiency of astaxanthin. Currently, the culture medium formulations for industrial and laboratory fermentation preparation of β-carotene are relatively traditional, often employing a combination of a single carbon source, conventional inorganic nutrients, and common nitrogen sources, resulting in a crude nutrient ratio and limited functional components.

[0003] Existing culture media can only meet the basic growth and metabolic needs of microorganisms and cannot specifically enhance the β-carotene synthesis pathway. This results in problems such as insufficient supply of precursor substances, limited cell membrane permeability, imbalance of intracellular redox environment, and easy degradation and loss of synthesized products. Furthermore, conventional culture media exhibit poor dispersibility of functional adjuvants and lack in-situ product immobilization mechanisms, easily leading to low β-carotene synthesis efficiency, low intracellular accumulation, and severe product loss, significantly limiting the production capacity and purity of subsequent astaxanthin synthesis.

[0004] Furthermore, traditional culture medium preparation processes only employ simple mixing and sterilization, resulting in poor compatibility and low activity retention of various functional components. They lack dedicated processes for modifying, dispersing, encapsulating, and aseptically and precisely formulating functional auxiliaries, and there is no supporting integrated preparation equipment. This leads to poor batch production stability and low repeatability, making it difficult to achieve targeted, efficient, and large-scale accumulation of β-carotene, and thus failing to meet the needs of high-purity astaxanthin industrial production. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a culture medium, preparation method, and equipment for the targeted accumulation of β-carotene, a precursor of astaxanthin. The technical solution adopted is as follows:

[0006] The astaxanthin precursor β-carotene directional accumulation medium, by mass fraction of the total culture medium, comprises the following components: carbon source 3.5%–4.0%, nitrogen source 0.5%–1.5%, inorganic salts 0.2%–0.6%, β-carotene synthesis precursor supplier 0.05%–0.2%, membrane lipid fluidity regulator 0.1%–0.4%, redox cofactor 0.0005%–0.002%, wherein the redox cofactor is pyrroloquinoline quinone, which is introduced as an in-situ product capture agent by adding a 0.1%–0.5% (w / w) pyrroloquinoline quinone aqueous solution stock solution, with the remainder being water;

[0007] The carbon source is a mixture of glucose and sucrose; the nitrogen source is a mixture of yeast extract and ammonium sulfate; the inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate.

[0008] The β-carotene synthesis precursor supplier is an ester formed by geraniol and 5-aminolevulinic acid in a molar ratio of 1:0.8 to 1:1.2;

[0009] The membrane lipid fluidity regulator is a complex formed by β-sitosterol and propylene glycol alginate in a mass ratio of 1:3 to 1:6.

[0010] The redox cofactor is pyrroloquinoline quinone;

[0011] The in-situ trapping agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides at a mass ratio of 1:0.4 to 1:1.

[0012] Optionally, the mass ratio of glucose to sucrose in the carbon source is 2:1 to 5:1; the mass ratio of yeast extract to ammonium sulfate in the nitrogen source is 2:1 to 4:1; and the mass ratio of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate in the inorganic salt is 10:4:2:0.5 to 10:6:3:1.

[0013] Optionally, the esterification site of the β-carotene synthesis precursor donor is the carboxyl group of 5-aminolevulinic acid and the hydroxyl group of geranyl geraniol; the ester is prepared under the catalysis of immobilized lipase, the enzyme being Novozym435, the reaction temperature being 30℃~40℃, and the reaction time being 10h~14h; the ester is added in the form of anhydrous ethanol mother liquor, the mass concentration of which is 10%~20%.

[0014] Optionally, the membrane lipid flow modifier is added in the form of a nano-dispersion. The nano-dispersion is prepared by adding anhydrous ethanol solution of β-sitosterol dropwise into an aqueous solution of propylene glycol alginate, followed by ultrasonic and high-pressure homogenization. The high-pressure homogenization pressure is 60 MPa to 100 MPa, and the cycles are repeated 2 to 4 times. The average particle size of the dispersion is 50 nm to 200 nm. The redox cofactor pyrroloquinoline quinone is pre-prepared into an aqueous stock solution with a mass concentration of 0.1% to 0.5%, and then the stock solution is added to the culture medium system to control the mass fraction of pyrroloquinoline quinone in the final culture medium to be 0.0005% to 0.002%.

[0015] Optionally, the in-situ capturing agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides, wherein the medium-chain triglycerides are caprylic / capric triglycerides; the inclusion complex is prepared by stirring and freeze-drying in a saturated aqueous solution, with the stirring temperature being 40℃~50℃ and the time being 3h~5h, and the mixture being freeze-dried until the moisture content is less than or equal to 5%.

[0016] A method for preparing a culture medium for the targeted accumulation of astaxanthin precursor β-carotene, characterized in that: the method for preparing the culture medium for the targeted accumulation of astaxanthin precursor β-carotene includes the following steps:

[0017] Step 1: Dissolve the carbon source, nitrogen source and inorganic salts in water, sterilize, and obtain the basic culture medium;

[0018] Step 2: Geraniol and 5-aminolevulinic acid are esterified under the catalysis of immobilized lipase to obtain the β-carotene synthesis precursor supply mother liquor.

[0019] Step 3: β-sitosterol and propylene glycol alginate are subjected to solvent displacement and high-pressure homogenization to obtain a membrane lipid flow modifier dispersion;

[0020] Step 4: Gamma-cyclodextrin and medium-chain triglycerides are mixed, encapsulated, and freeze-dried to obtain the in-situ product trapping agent;

[0021] Step 5: Under aseptic conditions, add the mother liquor obtained in Step 2, the dispersion obtained in Step 3, the inclusion complex obtained in Step 4, and the aqueous solution of pyrroloquinoline quinone to the basic culture medium obtained in Step 1, adjust the pH value to the set value, and filter aseptically to obtain the astaxanthin precursor β-carotene directional accumulation culture medium.

[0022] Optionally, in step 2, the molar ratio of geraniol to 5-aminolevulinic acid is 1:0.8 to 1:1.2, the immobilized lipase is Novozym435, the enzyme dosage is 5% to 10% of the total substrate mass, the reaction solvent is a mixed solvent of tert-butanol / n-hexane with a volume ratio of 1:3 to 1:5, the reaction temperature is 30℃ to 40℃, the stirring speed is 150 rpm to 250 rpm, and the reaction time is 10 h to 14 h. After the reaction, the enzyme is removed by filtration, the filtrate is concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume, and diluted with anhydrous ethanol to a mass concentration of 10% to 20% to obtain the precursor supply mother liquor.

[0023] Optionally, in step 3, β-sitosterol is first dissolved in anhydrous ethanol to prepare a 5%–10% alcohol phase, and propylene glycol alginate is dissolved in deionized water to prepare a 1%–2% aqueous phase. The alcohol phase is then added dropwise to the aqueous phase at 0.3 mL / min–0.8 mL / min at 40℃–50℃, and simultaneously sonicated for 20 min–30 min. Subsequently, high-pressure homogenization is performed at a pressure of 60 MPa–100 MPa, and the mixture is cycled 2–4 times to obtain a nano-dispersion with an average particle size of 50 nm–200 nm.

[0024] Optionally, in step 4, γ-cyclodextrin is dissolved in deionized water at 40℃~50℃ to form a saturated solution. Medium-chain triglycerides are added at a mass ratio of γ-cyclodextrin to medium-chain triglycerides of 1:0.4~1:1. The mixture is stirred at 800rpm~1000rpm for 3h~5h to incorporate the γ-cyclodextrin. Then, the mixture is cooled to 4℃ and allowed to stand for 8h~16h. After filtration, the mixture is freeze-dried until the moisture content is less than or equal to 5% to obtain the inclusion complex.

[0025] In step 5, the mixing temperature is 20-30℃, the stirring speed is 150-250rpm, the stirring time is 20-40min, the pH value is adjusted to 6.5-7.0, and the pore size of the terminal sterile filter membrane is 0.22μm-0.45μm.

[0026] An apparatus for preparing a culture medium for the directional accumulation of β-carotene, a precursor of astaxanthin, is characterized in that the apparatus comprises a reaction vessel, an immobilized enzyme reactor, a high-pressure homogenizer, a freeze dryer, a sterile mixing tank, a sterile filtration unit, and a controller;

[0027] The reactor is used to prepare the basal culture medium; the immobilized enzyme reactor is used to esterify geraniol with 5-aminolevulin; the high-pressure homogenizer is used to prepare membrane lipid flowability regulator nano-dispersions; the freeze dryer is used to dry the product in-situ trapping agent; the aseptic mixing tank is used to mix the basal culture medium with β-carotene synthesis precursor supplier, membrane lipid flowability regulator, redox cofactor and product in-situ trapping agent; the aseptic filtration unit is used for terminal sterilization; the controller is electrically connected to the reactor, immobilized enzyme reactor, high-pressure homogenizer and aseptic mixing tank respectively, and is used to control the feeding sequence, temperature, stirring speed and homogenization pressure.

[0028] In summary, the present invention has at least one of the following beneficial technical effects:

[0029] This invention provides a culture medium, preparation method, and equipment for the targeted accumulation of β-carotene, a precursor to astaxanthin. By optimizing the precise ratio of composite carbon sources, nitrogen sources, and inorganic salts, it can adapt to the growth and metabolic patterns of astaxanthin-producing microorganisms, balancing the microbial proliferation rate and secondary metabolic intensity. This provides a stable and sufficient basic nutrient environment for β-carotene synthesis, avoiding metabolic disorders caused by a single nutrient source. It ensures efficient cell growth while continuously activating the carotenoid synthesis metabolic pathway. A specially formulated β-carotene precursor supplier is introduced, precisely supplementing key substrates in the synthesis pathway through specific esterification products. This overcomes the technical bottleneck of precursor material scarcity in traditional fermentation processes, directionally enhancing the biosynthetic efficiency of β-carotene and significantly increasing the intracellular basic synthesis flux. Combined with a nanoscale membrane lipid fluidity regulator, it effectively improves microbial cell membrane permeability, promotes intracellular nutrient transport and orderly accumulation of metabolites, stabilizes cell membrane structure, and reduces the inhibitory effect of cell stress damage on β-carotene synthesis during fermentation.

[0030] Adding specific redox cofactors precisely regulates the intracellular redox microenvironment, alleviates the degradation and damage of β-carotene caused by oxidative stress, maintains high activity of enzymes related to anabolic metabolism, and ensures the continuous and efficient operation of the anabolic pathway.

[0031] By employing a proprietary inclusion-type in-situ product capture agent, intracellularly synthesized β-carotene can be immobilized in situ, reducing extracellular leakage and natural degradation of the product, significantly increasing the intracellular directional accumulation of β-carotene, and providing sufficient precursor reserves for the subsequent efficient synthesis of astaxanthin. Attached Figure Description

[0032] Figure 1 This is a schematic flowchart of the method for preparing the astaxanthin precursor β-carotene-oriented accumulation culture medium of the present invention. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings.

[0034] This invention discloses a culture medium, preparation method, and equipment for the directional accumulation of β-carotene, the precursor of astaxanthin.

[0035] Example 1

[0036] The astaxanthin precursor β-carotene directional accumulation medium, by mass fraction of the total culture medium, comprises the following components: carbon source 3.5%–4.0%, nitrogen source 0.5%–1.5%, inorganic salts 0.2%–0.6%, β-carotene synthesis precursor supplier 0.05%–0.2%, membrane lipid fluidity regulator 0.1%–0.4%, redox cofactor 0.0005%–0.002%, wherein the redox cofactor is pyrroloquinoline quinone, which is introduced as an in-situ product capture agent by adding a 0.1%–0.5% (w / w) pyrroloquinoline quinone aqueous solution stock solution, with the remainder being water;

[0037] The carbon source is a mixture of glucose and sucrose; the nitrogen source is a mixture of yeast extract and ammonium sulfate; the inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate.

[0038] The β-carotene synthesis precursor supplier is an ester formed by geraniol and 5-aminolevulinic acid in a molar ratio of 1:0.8 to 1:1.2;

[0039] The membrane lipid fluidity regulator is a complex formed by β-sitosterol and propylene glycol alginate in a mass ratio of 1:3 to 1:6.

[0040] The redox cofactor is pyrroloquinoline quinone;

[0041] The in-situ trapping agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides at a mass ratio of 1:0.4 to 1:1.

[0042] By employing the above technical solution, the carbon source is a mixture of glucose and sucrose, the nitrogen source is a mixture of yeast extract and ammonium sulfate, and the inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate. The β-carotene synthesis precursor supplier is an esterified product formed by geraniol and 5-aminolevulinic acid at a molar ratio of 1:0.8 to 1:1.2. The membrane lipid fluidity regulator is a complex formed by β-sitosterol and propylene glycol alginate at a mass ratio of 1:3 to 1:6. The redox cofactor is pyrroloquinoline quinone. The product in-situ trapping agent is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides at a mass ratio of 1:0.4 to 1:1. In this culture medium, the carbon source provides the carbon skeleton and energy required for cell growth and carotenoid synthesis, the nitrogen source provides the nitrogen element required for amino acid and nucleic acid synthesis, and the inorganic salts maintain osmotic pressure and supply the metal ions required for enzyme activity. β-Carotene precursor suppliers directly replenish the skeletal source of geranylide pyrophosphate in the carotenoid synthesis pathway, while 5-aminolevulinic acid promotes cytochrome synthesis, enhances electron transport in the respiratory chain and NADPH regeneration, providing reducing power for β-carotene accumulation. Membrane lipid fluidity regulators optimize the membrane microenvironment by altering the cell membrane lipid composition, facilitating the transport and storage of hydrophobic β-carotene within the membrane. Pyrroloquinoline quinone acts as a redox cofactor to maintain intracellular redox balance. In-situ product traps form nanoscale lipid reservoirs by encapsulating medium-chain triglycerides with γ-cyclodextrin, selectively capturing β-carotene during fermentation and reducing feedback inhibition of the synthesis pathway.

[0043] Example 2

[0044] The mass ratio of glucose to sucrose in the carbon source is 2:1 to 5:1; the mass ratio of yeast extract to ammonium sulfate in the nitrogen source is 2:1 to 4:1; and the mass ratio of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate in the inorganic salt is 10:4:2:0.5 to 10:6:3:1.

[0045] By employing the above technical solutions, glucose, as a fast-acting carbon source, can rapidly initiate cell growth, while sucrose, as a slow-acting carbon source, continuously provides the carbon skeleton in the middle and late stages of fermentation. Maintaining a ratio of 2:1 to 5:1 between glucose and sucrose avoids carbon metabolism inhibition caused by excessive glucose concentration, while ensuring sufficient carbon source during β-carotene synthesis. Yeast extract provides amino acids, vitamins, and growth factors, while ammonium sulfate provides inorganic nitrogen. A mass ratio of 2:1 to 4:1 balances rapid cell proliferation and the initiation of secondary metabolism. Among the inorganic salts, potassium dihydrogen phosphate provides phosphorus and acts as a buffer system to maintain pH stability; magnesium sulfate provides magnesium ions to activate various enzymes in the carotenoid synthesis pathway; and calcium chloride and zinc sulfate provide calcium and zinc ions, with zinc ions being an important cofactor for enzymes related to β-carotene synthesis.

[0046] Example 3

[0047] The esterification sites of the β-carotene synthesis precursor donor are the carboxyl group of 5-aminolevulinic acid and the hydroxyl group of geranyl geraniol; the esterified product is prepared under the catalysis of immobilized lipase, the enzyme being Novozym435, the reaction temperature being 30℃~40℃, and the reaction time being 10h~14h; the esterified product is added in the form of anhydrous ethanol mother liquor, with a mother liquor mass concentration of 10%~20%.

[0048] By employing the above technical solution, the esterified product was prepared under the catalysis of an immobilized lipase. The product was then separated and purified to remove N-acylation byproducts. Novozym435 was selected as the immobilized lipase, the reaction temperature was 30℃–40℃, and the reaction time was 10–14 h. The esterified product was added in the form of anhydrous ethanol mother liquor, with a mass concentration of 10%–20%. Geranilide and 5-aminolevulinic acid are linked by an ester bond. This improves the lipophilicity and slow-release properties of geranilide, preventing the rapid release of free geranilide into the fermentation system and its toxicity to cell membranes. Furthermore, 5-aminolevulinic acid, as a precursor for porphyrin synthesis, promotes heme and cytochrome synthesis, enhances oxidative phosphorylation efficiency, and increases ATP and NADPH supply levels, thereby driving the β-carotene synthesis pathway towards product accumulation. The immobilized lipase-catalyzed esterification reaction conditions are mild, avoiding the damage to the double bond structure of geranilide caused by high-temperature chemical esterification. Adding anhydrous ethanol stock solution facilitates uniform dispersion in the aqueous culture medium during the aseptic mixing stage.

[0049] Example 4

[0050] The membrane lipid flow modifier is added in the form of a nano-dispersion, which is prepared by adding anhydrous β-sitosterol ethanol solution dropwise to propylene glycol alginate aqueous solution and then homogenizing by ultrasound and high pressure. The high pressure homogenization pressure is 60MPa to 100MPa, and the cycle is 2 to 4 times. The average particle size of the dispersion is 50nm to 200nm. The redox cofactor pyrroloquinoline quinone is pre-prepared into an aqueous stock solution with a mass concentration of 0.1% to 0.5%, and then the stock solution is added to the culture medium system to control the mass fraction of pyrroloquinoline quinone in the final culture medium to be 0.0005% to 0.002%.

[0051] By employing the above technical solutions, β-sitosterol can be embedded in the phospholipid bilayer of microbial cell membranes, regulating the orderly arrangement and fluidity of membrane lipids, and improving the distribution and storage capacity of hydrophobic β-carotene within the membrane. Propylene glycol alginate, as an amphiphilic carrier, can enhance the dispersion stability of β-sitosterol in the aqueous phase. High-pressure homogenization controls the particle size of the complex to 50 nm–200 nm, which is beneficial for forming a uniform dispersion in the culture medium and avoids aggregation and precipitation. Pyrroloquinoline quinone is added in the form of a low-concentration aqueous solution, which avoids excessively high local concentrations that could cause oxidative stress to the cells, while fully utilizing its redox cofactor function to promote the intracellular NADH and NADPH redox cycle. This invention controls the amount of membrane lipid fluidity regulator added, achieving membrane regulation while maintaining the culture medium viscosity within a range that does not significantly affect dissolved oxygen transfer.

[0052] Example 5

[0053] The in-situ capture agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides, wherein the medium-chain triglycerides are caprylic / capric triglycerides. The inclusion complex is prepared by stirring and freeze-drying in a saturated aqueous solution. The stirring temperature is 40℃~50℃ and the time is 3h~5h. The product is then freeze-dried until the moisture content is less than or equal to 5%.

[0054] By employing the above-mentioned technical solution, the lumen of γ-cyclodextrin is hydrophobic, enabling it to encapsulate medium-chain triglycerides to form a nanoscale lipid reservoir. During fermentation, β-carotene, due to its hydrophobicity, can migrate into this lipid reservoir and be stably captured, reducing the extracellular and intracellular concentration of free β-carotene and thus weakening the feedback inhibition of the product on key enzymes in the carotenoid synthesis pathway. Caprylic / capric triglycerides are selected as the medium-chain triglycerides, possessing low viscosity and good biocompatibility, which facilitates the dissolution and migration of β-carotene. Freeze-drying to a moisture content of ≤5% improves the storage stability of the inclusion complex and facilitates accurate weighing and addition during aseptic mixing.

[0055] Example 6

[0056] A method for preparing a culture medium for the targeted accumulation of astaxanthin precursor β-carotene, characterized in that: the method for preparing the culture medium for the targeted accumulation of astaxanthin precursor β-carotene includes the following steps:

[0057] Step 1: Dissolve the carbon source, nitrogen source and inorganic salts in water, sterilize, and obtain the basic culture medium;

[0058] Step 2: Geraniol and 5-aminolevulinic acid are esterified under the catalysis of immobilized lipase to obtain the β-carotene synthesis precursor supply mother liquor.

[0059] Step 3: β-sitosterol and propylene glycol alginate are subjected to solvent displacement and high-pressure homogenization to obtain a membrane lipid flow modifier dispersion;

[0060] Step 4: Gamma-cyclodextrin and medium-chain triglycerides are mixed, encapsulated, and freeze-dried to obtain the in-situ product trapping agent;

[0061] Step 5: Under aseptic conditions, add the mother liquor obtained in Step 2, the dispersion obtained in Step 3, the inclusion complex obtained in Step 4, and the aqueous solution of pyrroloquinoline quinone to the basic culture medium obtained in Step 1, adjust the pH value to the set value, and filter aseptically to obtain the astaxanthin precursor β-carotene directional accumulation culture medium.

[0062] By adopting the above technical solution, the stepwise preparation of functional components avoids the damage to the precursor supply, membrane lipid flow regulator, and product in-situ capture agent caused by high-temperature sterilization of the basal culture medium, while also preventing mutual interference between the functional components during the preparation process. Finally, the components are mixed under aseptic conditions and terminally filtered aseptically to ensure the sterility of the culture medium and the integrity of the functional components' activity.

[0063] Example 7

[0064] In step 2, the molar ratio of geraniol to 5-aminolevulinic acid is 1:0.8 to 1:1.2, the immobilized lipase is Novozym435, the enzyme dosage is 5% to 10% of the total substrate mass, the reaction solvent is a mixed solvent of tert-butanol / n-hexane with a volume ratio of 1:3 to 1:5, the reaction temperature is 30℃ to 40℃, the stirring speed is 150 rpm to 250 rpm, and the reaction time is 10 h to 14 h. After the reaction, the enzyme is removed by filtration, the filtrate is concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume, and diluted with anhydrous ethanol to a mass concentration of 10% to 20% to obtain the precursor supply mother liquor.

[0065] By employing the above technical solution, after the reaction, the enzyme is removed by filtration, and the filtrate is concentrated under reduced pressure to 1 / 5 to 1 / 10 of its original volume. It is then diluted with anhydrous ethanol to a mass concentration of 10% to 20% to obtain the precursor supply mother liquor. A mixed solvent of tert-butanol / n-hexane can simultaneously dissolve hydrophilic 5-aminolevulinic acid and hydrophobic geraniol, improving substrate contact efficiency. The immobilized lipase-catalyzed esterification reaction exhibits regioselectivity, primarily catalyzing the dehydration of the carboxyl group of 5-aminolevulinic acid to the hydroxyl group of geraniol, thus avoiding side reactions. Reduced pressure concentration removes most of the organic solvent, and dilution with anhydrous ethanol to 10% to 20% yields a mother liquor that is easy to aseptically filter and add. Furthermore, the anhydrous ethanol can be diluted to a level that is non-toxic to the bacteria in subsequent mixing steps.

[0066] Example 8

[0067] In step 3, β-sitosterol is first dissolved in anhydrous ethanol to prepare a 5%–10% alcohol phase, and propylene glycol alginate is dissolved in deionized water to prepare a 1%–2% aqueous phase. The alcohol phase is then added dropwise to the aqueous phase at 0.3 mL / min–0.8 mL / min at 40℃–50℃, and simultaneously sonicated for 20 min–30 min. Subsequently, high-pressure homogenization is performed at a pressure of 60 MPa–100 MPa for 2–4 cycles to obtain a nano-dispersion with an average particle size of 50 nm–200 nm.

[0068] By employing the above technical solution, the slow droplet addition of the alcohol phase to the aqueous phase avoids the large-scale precipitation and aggregation of β-sitosterol due to sudden solvent changes. Simultaneous ultrasound provides localized high shear force, enabling β-sitosterol and propylene glycol alginate to form primary complex particles in the aqueous phase. High-pressure homogenization further refines the particles and homogenizes their size distribution, allowing the nano-dispersion to remain stably suspended in the culture medium for extended periods. This facilitates sufficient contact with the microbial cell membrane during fermentation and allows the nanoparticles to regulate membrane lipid fluidity.

[0069] Example 9

[0070] In step 4, γ-cyclodextrin is dissolved in deionized water at 40℃~50℃ to form a saturated solution. Medium-chain triglycerides are added at a mass ratio of γ-cyclodextrin to medium-chain triglycerides of 1:0.4~1:1. The mixture is stirred at 800rpm~1000rpm for 3h~5h to incorporate the γ-cyclodextrin. Then, the mixture is cooled to 4℃ and allowed to stand for 8h~16h. After filtration, the mixture is freeze-dried until the moisture content is less than or equal to 5% to obtain the inclusion complex.

[0071] In step 5, the mixing temperature is 20-30℃, the stirring speed is 150-250rpm, the stirring time is 20-40min, the pH value is adjusted to 6.5-7.0, and the pore size of the terminal sterile filter membrane is 0.22μm-0.45μm.

[0072] By employing the above technical solution, the inclusion rate of γ-cyclodextrin for medium-chain triglycerides can be improved through stirring in a saturated aqueous solution. Cooling and allowing the mixture to stand promotes precipitation of the inclusion complex from the aqueous phase. Filtering followed by freeze-drying yields a solid inclusion complex, facilitating long-term storage and precise addition. Step 5 involves mixing at a low temperature of 20–30°C to avoid hydrolysis of the precursor's ester bonds or aggregation of the membrane lipid flow modifier nanodispersions caused by excessively high temperatures. A stirring speed of 150–250 rpm ensures uniform dispersion of all functional components without excessive foaming. The terminal sterile filter membrane has a pore size of 0.22 μm–0.45 μm, effectively trapping bacteria and fungi while allowing the nanodispersions, inclusion complexes, and dissolved precursors to pass through, ensuring sterility of the culture medium and preventing loss of functional components.

[0073] Example 10

[0074] An apparatus for preparing a culture medium for the directional accumulation of β-carotene, a precursor of astaxanthin, is characterized in that the apparatus comprises a reaction vessel, an immobilized enzyme reactor, a high-pressure homogenizer, a freeze dryer, a sterile mixing tank, a sterile filtration unit, and a controller;

[0075] The reactor is used to prepare the basal culture medium; the immobilized enzyme reactor is used to esterify geraniol with 5-aminolevulin; the high-pressure homogenizer is used to prepare membrane lipid flowability regulator nano-dispersions; the freeze dryer is used to dry the product in-situ trapping agent; the aseptic mixing tank is used to mix the basal culture medium with β-carotene synthesis precursor supplier, membrane lipid flowability regulator, redox cofactor and product in-situ trapping agent; the aseptic filtration unit is used for terminal sterilization; the controller is electrically connected to the reactor, immobilized enzyme reactor, high-pressure homogenizer and aseptic mixing tank respectively, and is used to control the feeding sequence, temperature, stirring speed and homogenization pressure.

[0076] By employing the above technical solution, the reaction vessel is used to prepare the basal culture medium, the immobilized enzyme reactor is used to esterify geraniol with 5-aminolevulinic acid, the high-pressure homogenizer is used to prepare membrane lipid flow regulator nanodispersions, the freeze dryer is used to dry the in-situ product trap, the aseptic mixing tank is used to mix the basal culture medium with β-carotene synthesis precursor supplier, membrane lipid flow regulator, redox cofactor, and in-situ product trap, and the aseptic filtration unit is used for terminal sterilization. The controller is electrically connected to the reaction vessel, immobilized enzyme reactor, high-pressure homogenizer, and aseptic mixing tank to control the feeding sequence, temperature, stirring speed, and homogenization pressure. By setting the feeding sequence through the controller, the basal culture medium can be sterilized and the functional components prepared first, and then the functional components can be added to the aseptic mixing tank in the set order, avoiding cross-contamination and inactivation of functional components. The controller provides closed-loop control of the temperature, stirring speed, and reaction time of the immobilized enzyme reactor, which can improve the esterification reaction conversion rate and batch consistency. The controller sets the homogenization pressure and cycle number of the high-pressure homogenizer, ensuring stable control of the nanoparticle size of the membrane lipid flow modifier dispersion within the target range. The aseptic filtration unit connects to the outlet of the aseptic mixing tank, enabling terminal sterilization filtration of the mixed culture medium to ensure the final product is sterile and ready for direct fermentation inoculation.

[0077] The following specific embodiments illustrate the implementation principle of the present invention:

[0078] Preferred Implementation Case 1: Application in Pharfogel's Red yeast fermentation. The culture medium, by mass fraction of total culture medium, consisted of 3.5% carbon source, 1.0% nitrogen source, 0.4% inorganic salts, 0.10% β-carotene synthesis precursor supplier, 0.25% membrane lipid flow regulator, 0.001% redox cofactor, 1.0% product in-situ capture agent, and the balance being water. The mass ratio of glucose to sucrose in the carbon source was 3:1; the mass ratio of yeast extract to ammonium sulfate in the nitrogen source was 3:1; and the mass ratio of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate in the inorganic salts was 10:5:2.5:0.8. The β-carotene synthesis precursor supplier was an esterified product formed by geraniol and 5-aminolevulinic acid in a molar ratio of 1:1, added as a 15% anhydrous ethanol mother liquor. The mass ratio of β-sitosterol to propylene glycol alginate in the membrane lipid flow regulator was 1:4, and the average particle size of the nano-dispersion was 120 nm. Redox cofactors were added in a 0.3% aqueous solution. The mass ratio of γ-cyclodextrin to medium-chain triglycerides in the in-situ scavenging agent of the product was 1:0.7. Fermentation conditions were: temperature 22℃, pH 6.8, dissolved oxygen 30%, agitation speed 400 rpm, inoculum size 10%, and fermentation time 120 h.

[0079] Preferred Implementation Case 2: Application in the fermentation of genetically engineered Saccharomyces cerevisiae. The culture medium, by mass fraction of total mass, consisted of 4.0% carbon source, 0.8% nitrogen source, 0.5% inorganic salts, 0.15% β-carotene synthesis precursor supplier, 0.30% membrane lipid flow regulator, 0.0015% redox cofactor, 1.2% product in-situ capture agent, and the balance being water. The mass ratio of glucose to sucrose in the carbon source was 4:1; the mass ratio of yeast extract to ammonium sulfate in the nitrogen source was 2.5:1; and the mass ratio of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate in the inorganic salts was 10:4.5:2.2:0.6. The β-carotene synthesis precursor supplier was an esterified product formed by geraniol and 5-aminolevulinic acid at a molar ratio of 1:1.1, added as a 18% anhydrous ethanol mother liquor. The mass ratio of β-sitosterol to propylene glycol alginate in the membrane lipid flow regulator was 1:5, and the average particle size of the nano-dispersion was 80 nm. Redox cofactors were added in a 0.4% aqueous solution. The mass ratio of γ-cyclodextrin to medium-chain triglycerides in the in-situ product trap was 1:0.6. Fermentation conditions were: temperature 30℃, pH 6.5, dissolved oxygen 20%, rotation speed 350 rpm, inoculum size 8%, and fermentation time 96 h.

[0080] Comparative Example of Traditional Culture Medium: The traditional culture medium was YM medium, using *Phaefflera rubiginosa* and *Saccharomyces cerevisiae* (a genetically engineered strain producing β-carotene), commonly used in the field, as model strains. The composition, by mass fraction of the total culture medium, was: glucose 2.0%, peptone 2.0%, yeast extract 1.0%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, with the remainder being water. No β-carotene precursor suppliers, membrane lipid flow regulators, redox cofactors, or in-situ product traps were added. Fermentation was carried out using the same strains and under the same fermentation conditions.

[0081] To verify the effect of the culture medium of the present invention on the targeted accumulation of β-carotene, *Phaefflera heliotropium* and genetically engineered *Saccharomyces cerevisiae* were used as production strains. Under the same fermentation tank, inoculum size, temperature, pH, dissolved oxygen, and rotation speed conditions, the preferred embodiments of the present invention were compared with conventional culture media. The conventional culture media for each strain used the exact same fermentation conditions as the preferred embodiments, differing only in the culture medium formulation. The detection indicators included biomass, β-carotene yield, β-carotene content, the proportion of β-carotene to total carotenoids, NADPH / NADP+ ratio, and production intensity.

[0082] The comparison results between the culture medium of the preferred implementation case 1 and the traditional culture medium are shown in Table 1:

[0083] Table 1

[0084] detection indicators Traditional culture medium The preferred culture medium in Implementation Case 1 Biomass g / L 8.5 10.2 β-carotene production (mg / L) 12.5 68.4 β-carotene content (mg / gDCW) 1.47 6.71 % of total carotenoids by percentage of β-carotene 65 92 NADPH / NADP+ ratio 1.2 2.8 Production intensity (mg / L / h) 0.104 0.570

[0085] Therefore, in the application scenario of *Phaefflera rubrum*, the β-carotene yield of the culture medium of this invention increased from 12.5 mg / L to 68.4 mg / L, which is 5.47 times that of the traditional culture medium; the β-carotene content increased from 1.47 mg / gDCW to 6.71 mg / gDCW; the proportion of β-carotene in total carotenoids increased from 65% to 92%; the NADPH / NADP+ ratio increased from 1.2 to 2.8; and the production intensity increased from 0.104 mg / L / h to 0.570 mg / L / h. The results indicate that geraniol-5-aminolevulinate supplements the precursor carbon skeleton and enhances reducing power supply; β-sitosterol and propylene glycol alginate nanodispersion optimizes the membrane lipid microenvironment; and γ-cyclodextrin and medium-chain triglyceride inclusion complexes capture products in situ, reducing feedback inhibition. These three factors synergistically and significantly enhance the directed accumulation of β-carotene in *Phaefflera rubrum*.

[0086] The comparison results between the culture medium of the preferred implementation case 2 and the traditional culture medium are shown in Table 2:

[0087] Table 2

[0088] detection indicators Traditional culture medium The preferred culture medium in Implementation Case 2 Biomass g / L 12.3 14.1 β-carotene production (mg / L) 8.2 52.7 β-carotene content (mg / gDCW) 0.67 3.74 % of total carotenoids by percentage of β-carotene 58 90 NADPH / NADP+ ratio 0.9 2.4 Production intensity (mg / L / h) 0.085 0.549

[0089] Therefore, in the application scenario of genetically engineered Saccharomyces cerevisiae, the β-carotene yield of the culture medium of this invention increased from 8.2 mg / L to 52.7 mg / L, which is 6.43 times that of the traditional culture medium; the β-carotene content increased from 0.67 mg / gDCW to 3.74 mg / gDCW; the proportion of β-carotene in total carotenoids increased from 58% to 90%; the NADPH / NADP+ ratio increased from 0.9 to 2.4; and the production intensity increased from 0.085 mg / L / h to 0.549 mg / L / h. The results indicate that the precursor supplier, membrane lipid fluidity regulator, redox cofactor, and product in-situ trapping agent also play a synergistic role in Saccharomyces cerevisiae, effectively driving the metabolic flux from cell growth to directional accumulation of β-carotene, and significantly increasing the product content per unit cell and the production intensity of the fermentation process.

[0090] To verify the synergistic effect among the β-carotene synthesis precursor supplier, membrane lipid fluidity regulator, redox cofactor, and product in-situ trap in the culture medium of this invention, *Phaefflera heptaphylla* was used as the test strain. The fermentation conditions were kept identical to those in the preferred embodiment (temperature 22℃, pH 6.8, dissolved oxygen 30%, rotation speed 400 rpm, inoculum size 10%, fermentation time 120 h). A blank substrate control group, a single-component addition control group, a single-component deficiency control group, and a complete formulation experimental group were set up. Each group had three biological replicates. The experimental results were taken as mean ± standard deviation and statistically significant were analyzed using SPSS software. The results are shown in Table 3.

[0091] Table 3 Comparison of fermentation effects of Paffir yeast under different combinations of functional components

[0092] A Basic nutrient culture medium (carbon source + nitrogen source + inorganic salts, without the four functional components) 14.1±0.7 1.72±0.09 67±2 B Add only β-carotene synthesis precursor supply 28.6±1.2 3.15±0.13 76±2 C Add only membrane fluidity modifier 21.3±0.9 2.33±0.11 71±3 D Add only redox cofactors 17.5±0.8 1.94±0.08 69±2 E Add only product in-situ trapping agent 23.7±1.0 2.58±0.10 73±2 F The β-carotene precursor donor was removed, while the other three functional components were retained. 39.2±1.4 4.13±0.15 81±2 G The membrane lipid fluidity modifier was removed, while the other three functional components were retained. 44.5±1.6 4.67±0.17 84±3 H The redox cofactor was removed, while the other three functional components were retained. 49.7±1.8 5.12±0.16 86±2 I The in-situ trapping agent of the product was removed, while the other three functional components were retained. 42.1±1.5 4.41±0.14 82±3 J Complete formulation experimental group (preferred implementation case 1) 68.4±2.1 6.71±0.20 92±2

[0093] As shown in Table 3, the results indicate that: 1. Although the β-carotene production in group BE, which only added a single functional component, was higher than that in the blank base group A, the increase was limited; when the production gains of groups B, C, D, and E were simply added together, the theoretical cumulative production was approximately 57.0 mg / L, which was still significantly lower than the 68.4 mg / L of the complete formula experimental group J (P < 0.05). This proves that the combination of the four functional components in this invention is not a simple additive effect, but rather has a significant synergistic effect.

[0094] 2. In group FI, which omits any one of the functional components, the yield, intracellular content, and proportion of β-carotene in total carotenoids all decreased significantly compared to the complete formulation J. This indicates that all four functional components are indispensable for achieving the targeted accumulation effect of this invention: the β-carotene synthesis precursor supplier provides the substrate for the synthesis pathway; the membrane lipid fluidity regulator optimizes the cell membrane transport and storage environment; the redox cofactor maintains intracellular reducing power; and the product in situ trapping agent alleviates product feedback inhibition. The four components work together to achieve efficient and targeted intracellular accumulation of β-carotene.

[0095] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A culture medium for the directional accumulation of β-carotene, a precursor of astaxanthin, characterized in that, The culture medium comprises the following components by mass fraction: 3.5%–4.0% carbon source, 0.5%–1.5% nitrogen source, 0.2%–0.6% inorganic salts, 0.05%–0.2% β-carotene synthesis precursor supplier, 0.1%–0.4% membrane lipid fluidity regulator, and 0.0005%–0.002% redox cofactor. The redox cofactor is pyrroloquinoline quinone, which is introduced as an in-situ product capture agent by feeding a 0.1%–0.5% (w / w) pyrroloquinoline quinone aqueous solution mother liquor, with the remainder being water. The carbon source is a mixture of glucose and sucrose; the nitrogen source is a mixture of yeast extract and ammonium sulfate; the inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate. The β-carotene synthesis precursor supplier is an ester formed by geraniol and 5-aminolevulinic acid in a molar ratio of 1:0.8 to 1:1.2; The membrane lipid fluidity regulator is a complex formed by β-sitosterol and propylene glycol alginate in a mass ratio of 1:3 to 1:

6. The redox cofactor is pyrroloquinoline quinone; The in-situ trapping agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides at a mass ratio of 1:0.4 to 1:

1.

2. The astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 1, characterized in that, The mass ratio of glucose to sucrose in the carbon source is 2:1 to 5:1; the mass ratio of yeast extract to ammonium sulfate in the nitrogen source is 2:1 to 4:1; and the mass ratio of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and zinc sulfate in the inorganic salt is 10:4:2:0.5 to 10:6:3:

1.

3. The astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 2, characterized in that, The esterification sites of the β-carotene synthesis precursor donor are the carboxyl group of 5-aminolevulinic acid and the hydroxyl group of geranyl geraniol; the esterified product is prepared under the catalysis of immobilized lipase, the enzyme being Novozym435, the reaction temperature being 30℃~40℃, and the reaction time being 10h~14h; the esterified product is added in the form of anhydrous ethanol mother liquor, with a mother liquor mass concentration of 10%~20%.

4. The astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 3, characterized in that, The membrane lipid flow modifier is added in the form of a nano-dispersion, which is prepared by adding anhydrous β-sitosterol ethanol solution dropwise to propylene glycol alginate aqueous solution and then homogenizing by ultrasound and high pressure. The high pressure homogenization pressure is 60MPa to 100MPa, and the cycle is 2 to 4 times. The average particle size of the dispersion is 50nm to 200nm. The redox cofactor pyrroloquinoline quinone is pre-prepared into an aqueous stock solution with a mass concentration of 0.1% to 0.5%, and then the stock solution is added to the culture medium system to control the mass fraction of pyrroloquinoline quinone in the final culture medium to be 0.0005% to 0.002%.

5. The astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 4, characterized in that, The in-situ capturing agent of the product is an inclusion complex formed by γ-cyclodextrin and medium-chain triglycerides, wherein the medium-chain triglycerides are caprylic / capric triglycerides. The inclusion complex is prepared by stirring and freeze-drying in a saturated aqueous solution. The stirring and inclusion temperature is 40℃~50℃ and the time is 3h~5h. The product is then freeze-dried until the moisture content is less than or equal to 5%.

6. A method for preparing a culture medium for the directional accumulation of β-carotene, a precursor of astaxanthin, characterized in that: The method for preparing the astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 5 comprises the following steps: Step 1: Dissolve the carbon source, nitrogen source and inorganic salts in water, sterilize, and obtain the basic culture medium; Step 2: Geraniol and 5-aminolevulinic acid are esterified under the catalysis of immobilized lipase to obtain the β-carotene synthesis precursor supply mother liquor. Step 3: β-sitosterol and propylene glycol alginate are subjected to solvent displacement and high-pressure homogenization to obtain a membrane lipid flow modifier dispersion; Step 4: Gamma-cyclodextrin and medium-chain triglycerides are mixed, encapsulated, and freeze-dried to obtain the in-situ product trapping agent; Step 5: Under aseptic conditions, add the mother liquor obtained in Step 2, the dispersion obtained in Step 3, the inclusion complex obtained in Step 4, and the aqueous solution of pyrroloquinoline quinone to the basic culture medium obtained in Step 1, adjust the pH value to the set value, and filter aseptically to obtain the astaxanthin precursor β-carotene directional accumulation culture medium.

7. The method for preparing the astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 6, characterized in that: In step 2, the molar ratio of geraniol to 5-aminolevulinic acid is 1:0.8 to 1:1.2, the immobilized lipase is Novozym435, the enzyme dosage is 5% to 10% of the total substrate mass, the reaction solvent is a mixed solvent of tert-butanol / n-hexane with a volume ratio of 1:3 to 1:5, the reaction temperature is 30℃ to 40℃, the stirring speed is 150 rpm to 250 rpm, and the reaction time is 10 h to 14 h. After the reaction, the enzyme is removed by filtration, the filtrate is concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume, and diluted with anhydrous ethanol to a mass concentration of 10% to 20% to obtain the precursor supply mother liquor.

8. The method for preparing the astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 7, characterized in that: In step 3, β-sitosterol is first dissolved in anhydrous ethanol to prepare a 5%–10% alcohol phase, and propylene glycol alginate is dissolved in deionized water to prepare a 1%–2% aqueous phase. The alcohol phase is then added dropwise to the aqueous phase at 0.3 mL / min–0.8 mL / min at 40℃–50℃, and the mixture is simultaneously sonicated for 20 min–30 min. Subsequently, high-pressure homogenization was carried out at a pressure of 60 MPa to 100 MPa for 2 to 4 cycles to obtain nano-dispersions with an average particle size of 50 nm to 200 nm.

9. The method for preparing the astaxanthin precursor β-carotene-directed accumulation culture medium according to claim 8, characterized in that: In step 4, γ-cyclodextrin is dissolved in deionized water at 40℃~50℃ to form a saturated solution. Medium-chain triglycerides are added at a mass ratio of γ-cyclodextrin to medium-chain triglycerides of 1:0.4~1:

1. The mixture is stirred at 800rpm~1000rpm for 3h~5h to incorporate the γ-cyclodextrin. Then, the mixture is cooled to 4℃ and allowed to stand for 8h~16h. After filtration, the mixture is freeze-dried until the moisture content is less than or equal to 5% to obtain the inclusion complex. In step 5, the mixing temperature is 20-30℃, the stirring speed is 150-250rpm, the stirring time is 20-40min, the pH value is adjusted to 6.5-7.0, and the pore size of the terminal sterile filter membrane is 0.22μm-0.45μm.

10. An apparatus for preparing the directional accumulation culture medium for the astaxanthin precursor β-carotene as described in claim 1, characterized in that, The equipment includes a reaction vessel, an immobilized enzyme reactor, a high-pressure homogenizer, a freeze dryer, a sterile mixing tank, a sterile filtration unit, and a controller; The reactor is used to prepare the basal culture medium; the immobilized enzyme reactor is used to esterify geraniol with 5-aminolevulin; the high-pressure homogenizer is used to prepare membrane lipid flowability regulator nano-dispersions; the freeze dryer is used to dry the product in-situ trapping agent; the aseptic mixing tank is used to mix the basal culture medium with β-carotene synthesis precursor supplier, membrane lipid flowability regulator, redox cofactor and product in-situ trapping agent; the aseptic filtration unit is used for terminal sterilization; the controller is electrically connected to the reactor, immobilized enzyme reactor, high-pressure homogenizer and aseptic mixing tank respectively, and is used to control the feeding sequence, temperature, stirring speed and homogenization pressure.

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