Schizochytrium limulus oil accumulation and EPA preparation process promoted by p-aminobenzoic acid

Abstract

This invention belongs to the field of EPA preparation technology, specifically a process for promoting lipid accumulation in Schizochytrium fungi and preparing EPA using para-aminobenzoic acid. The process includes steps such as strain selection and fermentation initiation, time-sequential synergistic regulation, cell collection, cell wall disruption, lipid extraction, crude lipid purification, and regulator recovery and reuse. It employs time-sequential addition of para-aminobenzoic acid and a small-molecule sulfur donor to synergistically regulate Schizochytrium metabolism; optimizes fermentation conditions by combining mixed carbon sources, segmented dissolved oxygen, and pH regulation; protects the EPA structure using low-temperature composite cell disruption, supercritical extraction, and tandem purification processes; and designs a regulator recovery process for recycling. This invention solves the problems of existing technologies such as single regulatory effect, low EPA purity, easy oxidation, and raw material waste, significantly increasing cell biomass, lipid yield, and EPA ratio, improving regulator utilization, and offering a green, low-cost, and highly feasible process suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of EPA preparation technology, specifically a process for promoting oil accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid. Background Technology

[0002] Eicosapentaenoic acid (EPA) has many important physiological functions and is widely used in various fields. Current EPA preparation mainly relies on deep-sea fish oil extraction and microbial fermentation. However, deep-sea fish oil extraction is limited by resources and pollution, making it difficult to meet the demand for large-scale, high-quality production. Microbial fermentation has become the mainstream method, and Schizochytrium strains are the most promising strains for application due to their strong lipid synthesis capabilities and ability to selectively enrich EPA.

[0003] Currently, among the methods to increase the production of oil and EPA in Schizochytrium, gene editing technology is complex and costly, while exogenous regulators are more feasible for industrialization. However, existing regulators have problems such as single regulatory effect and poor synergy, and cannot achieve simultaneous increase in oil and EPA.

[0004] There are no reports of p-aminobenzoic acid being used to regulate the synthesis of EPA in Schizochytrium oils. Furthermore, existing processes suffer from low utilization of regulators, waste of raw materials, environmental pollution, unreasonable carbon metabolic flow during fermentation, severe EPA oxidative degradation, and low purity and structural damage in purification processes. Therefore, there is an urgent need to develop an efficient, green, and scalable EPA preparation process. Summary of the Invention

[0005] To address the shortcomings of existing technologies, and considering the limitations of deep-sea fish oil extraction, the singular effect of regulators in microbial fermentation, the inability to simultaneously enhance oil and EPA production, low regulator utilization, raw material waste, environmental pollution, and difficulty in ensuring product quality during fermentation and purification, this invention provides a process for promoting oil accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid. Through precise process design, a synergistic regulation system, and a closed-loop recovery scheme, this process achieves efficient, green, and large-scale EPA preparation while reducing production costs, ensuring product quality, and solving existing technical challenges in this field.

[0006] The technical solution adopted by this invention to solve its technical problem is: a process for promoting the accumulation of oil in Schizochytrium fungi and preparing EPA using para-aminobenzoic acid, comprising the following steps: Step 1: Select Schizochytrium as the fermentation strain, prepare the fermentation medium and inoculate it with Schizochytrium to start the fermentation culture; Step 2: Para-aminobenzoic acid and small molecule sulfur donors were added to the fermentation system in a time-sequential and segmented manner for synergistic regulation. Para-aminobenzoic acid was used to reshape the central carbon metabolism flow to enhance lipid synthesis, while small molecule sulfur donors were used to supplement the sulfur-dependent reducing power required for EPA synthesis. The synergistic effect of para-aminobenzoic acid and small molecule sulfur donors achieved a simultaneous and significant increase in lipid biomass and EPA ratio. Step 3: After the fermentation culture is completed, collect the bacterial cells in the fermentation system; Step 4: The collected bacterial cells are subjected to cell wall disruption to release intracellular lipids; Step 5: Extract the oil from the broken-cell system to obtain crude oil; Step 6: Purify the crude oil to obtain high-purity EPA product.

[0007] Furthermore, in step 2, the small molecule sulfur donor is at least one of L-cysteine, glutathione, or sodium thiosulfate. The combination of the small molecule sulfur donor and p-aminobenzoic acid can repair intracellular oxidative stress and enhance desaturase activity, forming a synergistic metabolic regulatory combination.

[0008] Furthermore, para-aminobenzoic acid was added once during the mid-logarithmic growth phase, while the small-molecule sulfur donor was added in a gradient feed method during the rapid oil accumulation phase. The timing of the para-aminobenzoic acid addition node and the small-molecule sulfur donor feed node was matched to achieve segmented and targeted regulation of cell growth, oil synthesis and EPA enrichment.

[0009] Furthermore, the final concentration of para-aminobenzoic acid is 40–120 mg / L, and the final concentration of small molecule sulfur donor is 5–30 mg / L. The ratio of para-aminobenzoic acid concentration to small molecule sulfur donor concentration is well matched, so that the carbon metabolic flow is maximized to fatty acid synthesis and EPA polyunsaturated fatty acid generation.

[0010] Furthermore, the fermentation process in step 2 employs a two-stage dissolved oxygen control: during the growth phase, dissolved oxygen is maintained at 30-40% to promote the transmembrane absorption and intracellular transport of para-aminobenzoic acid; during the synthesis of oils and EPA, dissolved oxygen is reduced to 10-20% to reduce EPA oxidative degradation and improve desaturation efficiency.

[0011] Furthermore, the fermentation medium uses a glycerol-sodium acetate mixed carbon source, with sodium acetate acting as a cotransporter to improve the membrane permeability and effective intracellular concentration of para-aminobenzoic acid, while glycerol provides a continuous carbon skeleton supply for the synthesis of oils and EPA.

[0012] Furthermore, the entire fermentation process in step 2 is controlled by segmented constant pH. The pH is controlled at 6.2-6.5 during the growth period and adjusted to 5.8-6.1 during the oil synthesis period to stabilize the dissociation state of para-aminobenzoic acid and improve the utilization rate of small molecule sulfur donors.

[0013] Furthermore, in step 4, after the bacterial cells are collected, they are subjected to low-temperature high-pressure homogenization combined with complex enzymatic hydrolysis to break the cell wall. The cell-breaking enzyme is a complex preparation of lysozyme and lipase, which improves the intracellular lipid release rate while protecting the structural stability of EPA.

[0014] Furthermore, in step 5, the oil purification adopts a series process of urea inclusion and vacuum molecular distillation, wherein the material-to-liquid ratio of urea inclusion is 1:3 to 1:6, the vacuum degree of molecular distillation is controlled at 1 to 10 Pa, and the distillation temperature is 80 to 120°C.

[0015] Furthermore, it also includes a recovery step for p-aminobenzoic acid and small-molecule sulfur donors, including the following: D1. Collect the fermentation system after fermentation is completed, perform solid-liquid separation, and obtain the supernatant; D2, the supernatant is concentrated by passing it through a nanofiltration membrane to recover the unused p-aminobenzoic acid and small molecule sulfur donor in the supernatant, and a recovery solution is obtained. D3, the recovered liquid is filtered through a 0.22μm filter membrane to remove residual bacterial fragments and suspended impurities in the recovered liquid; D4. The actual concentrations of p-aminobenzoic acid and small-molecule sulfur donors in the recovered liquid after filtration were determined by high-performance liquid chromatography (HPLC). D5, 30%-70% of the qualified recovered liquid is reused in the next batch of fermentation. If it is reused in the initial culture medium, the recovered liquid is added to the newly prepared initial culture medium and new para-aminobenzoic acid and small molecule sulfur donor are added. If it is reused in the fed-batch medium, the recovered liquid is used as the feed medium for small molecule sulfur donors and is mixed with new small molecule sulfur donors before use. D6 ensures that the final concentration of p-aminobenzoic acid in the reused fermentation system is maintained at 40–120 mg / L and the final concentration of small molecule sulfur donor is maintained at 5–30 mg / L.

[0016] The beneficial effects of this invention are as follows: 1. The present invention describes a process for promoting lipid accumulation and EPA preparation in Schizochytrium using para-aminobenzoic acid. Through the time-segmented synergistic regulation of para-aminobenzoic acid and small molecule sulfur donors, combined with a glycerol-sodium acetate mixed carbon source, segmented dissolved oxygen, and pH regulation, the biomass, lipid yield, and EPA ratio of Schizochytrium are simultaneously increased. Compared with the prior art, the biomass is increased by more than 22.4%, the lipid yield is increased by more than 25.4%, and the EPA ratio is increased by more than 41.4%, effectively overcoming the bottleneck of the prior art where the regulation effect is singular and it is difficult to balance product yield and quality.

[0017] 2. The process for promoting oil accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid described in this invention, through a combination of low-temperature high-pressure homogenization combined with complex enzymatic hydrolysis to break down cell walls, supercritical CO2 extraction, and urea inclusion and vacuum molecular distillation purification, effectively protects the structural stability of EPA, reduces EPA oxidation loss, and improves oil release rate and product purity. This results in a reduction of EPA oxidation loss rate by more than 60.4%, an increase in oil release rate by more than 20.0%, and an EPA purity of more than 92.3%, solving the problems of easy oxidation, low purity, and incomplete oil release of EPA in existing technologies.

[0018] 3. The process for promoting oil accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid described in this invention significantly improves the utilization rate of para-aminobenzoic acid and other regulators through nanofiltration membrane concentration, filtration purification, concentration detection, and recycling of regulators, while reducing raw material consumption and environmental pollution. Compared with existing technologies, the utilization rate of regulators is increased by more than 36.5%, and raw material consumption is reduced by more than 20.3%, achieving green and low-cost processes, making it more suitable for large-scale industrial application. Attached Figure Description

[0019] The invention will now be further described with reference to the accompanying drawings.

[0020] Figure 1 This is a flowchart of the preparation process of the present invention. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: As Figure 1 As shown, the core technical solution of this invention is to simultaneously improve the accumulation of oil and EPA synthesis in Schizochytrium by synergistically regulating the time sequence of para-aminobenzoic acid and small molecule sulfur donors, combined with fermentation parameter optimization, efficient cell wall disruption extraction, precise purification and recycling of regulators.

[0023] The present invention provides a process for promoting lipid accumulation in Schizochytrium fungi and preparing EPA using para-aminobenzoic acid, comprising the following steps: Step 1: Strain selection and fermentation start-up: First, Schizochytrium was selected as the fermentation strain. Schizochytrium is a microorganism that can efficiently synthesize lipids. Compared with yeast and Escherichia coli commonly used in existing technologies, it contains a rich fatty acid synthase system and can directionally synthesize EPA. Moreover, the culture conditions are mild, making it suitable for large-scale industrial cultivation without the need for complex culture equipment. In this step, conventional commercial strains of Schizochytrium can be used, or high-yield strains obtained through simple mutagenesis, such as ultraviolet mutagenesis, can be selected. This eliminates the need for complex gene editing operations and reduces the difficulty of implementation. Subsequently, a fermentation medium was prepared. The basic formula of the fermentation medium was: glucose 10-20 g / L, peptone 5-10 g / L, yeast extract 3-5 g / L, sea salt 20-30 g / L, potassium dihydrogen phosphate 1-2 g / L, and magnesium sulfate 0.5-1 g / L, with the pH adjusted to 6.2-6.5. This basic formula is the optimal growth formula for Schizochytrium, which can ensure rapid cell proliferation and lay the foundation for subsequent oil and EPA synthesis. In some existing technologies, some culture medium formulas are simple, only adding glucose as a carbon source, resulting in slow cell growth and weak metabolic capacity. This basic formula provides sufficient nitrogen source and growth factors by adding peptone and yeast extract, thereby improving cell activity. After preparation, the fermentation medium is placed in a fermenter and sterilized at 121℃ and 0.1MPa for 20 minutes. The purpose of sterilization is to remove contaminants from the medium and prevent them from competing with Schizochytrium for nutrients, which could lead to fermentation failure. This is a routine operation for microbial fermentation and can also be completed using a conventional high-pressure steam sterilizer. After sterilization, the medium is cooled to 28℃. An inoculum of 5%-10% is then added to the fermentation medium. The inoculum is the volume ratio of Schizochytrium seed liquid to the fermentation medium. This ratio ensures that Schizochytrium adapts quickly to the fermentation environment and avoids slow cell growth due to insufficient inoculum or rapid consumption of nutrients due to excessive inoculum. The activated Schizochytrium seed liquid is then inoculated into the fermentation medium to start the fermentation culture. The fermenter speed is controlled at 180-220 r / min, the initial dissolved oxygen is maintained at 30-40%, and the temperature is controlled at 28℃ to begin the first stage (growth period) of fermentation. By optimizing the culture medium formulation and inoculation parameters, we can ensure the rapid and stable proliferation of Schizochytrium, forming sufficient cell biomass to provide ample cell biomass for subsequent oil and EPA synthesis, thus solving the problem of low product yield caused by slow cell growth and insufficient biomass in existing technologies.

[0024] Step 2: Time-series segmented coordinated regulation: This step differs from the existing technology's single-dose, one-time addition of a single regulator. It employs a time-sequential, segmented addition of para-aminobenzoic acid and a small-molecule sulfur donor to synergistically regulate the metabolic processes of Schizochytrium. The specific working process is as follows: First, the roles of the two regulators are clarified: para-aminobenzoic acid (PABA) is an aromatic compound that can participate in the central carbon metabolism of Schizochytrium as a metabolic precursor. By upregulating the activity of acetyl-CoA carboxylase and fatty acid synthase, it shifts the carbon metabolic flow from cell proliferation to lipid synthesis, thereby increasing the total amount of lipids. Small-molecule sulfur donors, such as L-cysteine, can provide sulfur to supplement the sulfur-dependent reducing power required for EPA synthesis. Reducing power is the "energy" for the synthesized products in the process of microbial metabolism. As a polyunsaturated fatty acid, EPA requires a large amount of reducing power for synthesis. In the current technology, the supply of reducing power is insufficient, resulting in low EPA synthesis efficiency. At the same time, it can repair the intracellular oxidative stress that para-aminobenzoic acid may cause. That is, excessive addition of para-aminobenzoic acid may lead to oxidative damage to the cells. Small-molecule sulfur donors can alleviate this damage and ensure normal cell metabolism. Secondly, the timing of the addition is crucial: After fermentation begins, the growth phase (approximately 12-18 hours) begins. When the Schizochytrium reaches the mid-logarithmic growth phase, the biomass reaches 50%-60% of its peak, and the metabolic activity is at its strongest. This is the optimal time to add the regulator, which can be determined by measuring the OD600 value of the fermentation broth. An OD600 value between 1.5 and 2.0 indicates the mid-logarithmic growth phase. Non-professionals can use a spectrophotometer to measure this. Add p-aminobenzoic acid to the fermentation system in one step, with the final concentration controlled at 40-120 mg / L. This concentration is the optimal result obtained through multiple trials and optimizations. Concentration is crucial; too low a concentration will not provide effective regulation, while too high a concentration will inhibit cell growth. After addition, fermentation continues, entering the rapid oil accumulation phase (approximately 24-36 hours of fermentation, at which point cell proliferation slows down and large amounts of oil synthesis begin). A gradient feed method is used to add the small molecule sulfur donor, with the feed time lasting 8-12 hours, and the final concentration controlled at 5-30 mg / L. Gradient feed means adding every 2 hours, with each addition being 1 / 4-1 / 5 of the total amount. This avoids excessively high concentrations caused by a single addition, ensuring that the small molecule sulfur donor continues to function and synergizes with para-aminobenzoic acid.

[0025] In this step, the synergistic mechanism of para-aminobenzoic acid and the small molecule sulfur donor is as follows: para-aminobenzoic acid is responsible for "opening up the source," converting more nutrients into oils; the small molecule sulfur donor is responsible for "enhancing the effect," significantly increasing the proportion of EPA in the converted oils while protecting the bacteria from damage. When used alone, para-aminobenzoic acid can only increase the total amount of oils without significantly increasing the proportion of EPA, and the small molecule sulfur donor can only maintain the normal growth of the bacteria without increasing the yield of oils and EPA. However, when the two are used in sequence, the effects of increasing the total amount of oils and increasing the proportion of EPA are achieved, which solves the defect of the single effect of the regulator in the existing technology.

[0026] Step 3: Bacterial cell collection: When fermentation reaches 48-72 hours, the oil and EPA content in the fermentation broth reaches its peak. This can be determined by testing the oil content of the fermentation broth. Fermentation can be stopped when the oil content reaches more than 40% of the dry weight of the cells. After stopping the fermentation, place the fermentation system in a high-speed centrifuge and centrifuge at 5000-8000 rpm for 10-15 minutes. The purpose of centrifugation is to separate the Schizochytrium cells from the fermentation supernatant. High-speed centrifugation ensures that the cells are fully precipitated, avoiding oil loss due to cell residue. Alternatively, a conventional high-speed centrifuge can be used. After centrifugation, discard the supernatant, which will be used to recover the regulator. Collect the cell precipitate at the bottom and wash it 2-3 times with physiological saline (0.9% sodium chloride solution) to remove residual fermentation medium and impurities on the cell surface, obtaining pure cells.

[0027] By centrifuging and washing, pure bacterial cells are obtained, avoiding impurities from affecting the subsequent oil extraction efficiency and product purity, thus solving the problems of low oil extraction rate and excessive impurities caused by incomplete bacterial cell separation in existing technologies.

[0028] Step 4: Cell disruption: The lipids and EPA in Schizochytrium are mainly stored intracellularly. Cell wall disruption is necessary to break the cell wall and release the intracellular lipids. The specific process is as follows: The washed bacterial cell precipitate was added to a buffer solution, specifically a phosphate buffer solution with a pH of 6.0-6.5 and a concentration of 0.1 mol / L. The buffer solution maintains the osmotic pressure of the bacterial cells, preventing excessive cell rupture during cell disruption that could lead to EPA oxidation. The bacterial cells were mixed with the buffer solution at a volume ratio of 1:5 to 1:8 to prepare a bacterial suspension. Subsequently, a low-temperature, high-pressure homogenization combined with enzymatic hydrolysis was used to disrupt the cell wall. The low temperature was controlled at 4-8℃. This low temperature protects the EPA structure from oxidation. Existing technologies often use high-temperature cell disruption, which can lead to EPA oxidation and degradation, reducing product quality. High-pressure homogenization... The pressure is controlled at 30-50 MPa, and homogenization is performed 2-3 times, with each homogenization lasting 5-10 minutes. The cell walls of the bacteria are broken by high-pressure impact. After homogenization, a compound preparation of lysozyme and lipase, i.e., cell wall breaking enzyme, is added to the bacterial suspension. The concentration of lysozyme is 100-200 U / mL, and the concentration of lipase is 50-100 U / mL. Enzymatic hydrolysis is carried out at 30-35℃ for 30-60 minutes. Lysozyme can further decompose the peptidoglycan of the bacterial cell wall, and lipase can assist in the decomposition of intracellular fat droplets, promoting the release of lipids.

[0029] The combined use of low-temperature high-pressure homogenization and complex enzymatic hydrolysis can not only efficiently break down the cell walls of bacteria and release intracellular lipids, but also maximize the protection of the structural stability of EPA and prevent EPA oxidation and degradation. At the same time, it can improve the lipid release rate. Compared with existing single cell wall breaking methods, such as high-pressure homogenization alone, the lipid release rate is increased by more than 20%, which solves the problems of low cell wall breaking efficiency and easy oxidation of EPA in existing technologies.

[0030] Step 5: Oil extraction: The bacterial suspension after cell wall disruption contains a large amount of oil. Crude oil is extracted using supercritical CO2 extraction. Supercritical CO2 extraction is a green and efficient extraction method. Compared with commonly used organic solvent extraction technologies, it leaves no solvent residue and does not damage the EPA structure, making it suitable for high-quality EPA extraction. It can also be performed using small-scale supercritical extraction equipment. The specific operation is as follows: The cell-wall-broken bacterial suspension is placed in a supercritical extraction vessel. The extraction temperature is controlled at 35-45℃, the extraction pressure at 30-40MPa, the CO2 flow rate at 10-20L / h, and the extraction time at 2-3 hours. After extraction, CO2 and oil are separated by vacuum separation. CO2 can be recovered and reused. The separated oil is collected as crude oil, which contains EPA, other fatty acids, and a small amount of impurities.

[0031] Supercritical CO2 extraction is used, which has high extraction efficiency and no solvent residue. At the same time, it protects the EPA structure from damage and achieves a crude oil purity of over 80%. This solves the problems of solvent residue and easy oxidation of EPA in existing organic solvent extraction technologies.

[0032] Step 6: Purification of crude oil: Crude oil contains impurities such as saturated fatty acids and monounsaturated fatty acids, which need to be purified to obtain high-purity EPA products. This invention uses a combination of urea inclusion and vacuum molecular distillation, and the specific working process is as follows: Step 1, urea inclusion complex: Crude oil and urea-ethanol solution are mixed at a ratio of 1:3 to 1:6, where the ratio is the volume ratio of crude oil to urea-ethanol solution. This ratio ensures that saturated fatty acids are fully included. The concentration of the urea-ethanol solution is 10-15 g / 100 mL. The mixture is stirred at 50-60°C for 30-60 minutes to allow the urea to fully dissolve and combine with the saturated fatty acids in the crude oil to form an inclusion complex. The mixture is then cooled to 0-4°C and allowed to stand for 2-4 hours. The inclusion complex precipitates out and is removed by filtration. The filtered oil solution is collected. At this point, the saturated fatty acids in the oil are removed, and the EPA ratio is increased. The second step is vacuum molecular distillation: the urea-encapsulated oil solution is placed in a molecular distillation apparatus, and the vacuum degree is controlled at 1-10 Pa. High vacuum can reduce the distillation temperature and avoid EPA oxidation. The distillation temperature is 80-120℃ and the distillation rate is 5-10 mL / h. Molecular distillation can separate EPA from other polyunsaturated fatty acids (such as DHA). The light fraction after distillation is collected, which is the high-purity EPA product.

[0033] By combining urea inclusion with vacuum molecular distillation, impurities in crude oil can be precisely removed, achieving an EPA purity of over 92% with no solvent residue and no damage to the EPA structure. Compared to existing single purification methods, such as urea inclusion alone, the EPA purity is increased by more than 15%, solving the problems of low EPA purity and high impurity content in existing technologies.

[0034] Selection of small molecule sulfur donors: At least one of L-cysteine, glutathione, or sodium thiosulfate is selected as the small molecule sulfur donor, with L-cysteine ​​being preferred. L-cysteine ​​has the highest sulfur utilization rate and is easily absorbed by Schizochytrium, and has the best synergistic effect with para-aminobenzoic acid. When small molecule sulfur donors are used in combination with para-aminobenzoic acid, not only can the reducing power required for EPA synthesis be supplemented, but the intracellular oxidative stress caused by para-aminobenzoic acid can also be repaired, making the cell metabolism more stable. Compared with the addition of para-aminobenzoic acid alone, the EPA synthesis rate is increased by more than 30%.

[0035] The timing and concentration of regulator addition: Para-aminobenzoic acid (P-aminobenzoic acid) is added once during the mid-logarithmic growth phase, when the bacterial metabolic activity is strongest, allowing for rapid absorption of P-aminobenzoic acid and initiation of lipid synthesis. Small molecule sulfur donors are added in a gradient during the rapid lipid accumulation phase, ensuring a continuous supply and avoiding excessively high or low concentrations due to a single addition. This precise matching with the P-aminobenzoic acid addition point enables segmented and targeted regulation of bacterial growth, lipid synthesis, and EPA enrichment. The final concentration of P-aminobenzoic acid is 40-120 mg / L, and the final concentration of small molecule sulfur donors is 5-30 mg / L. This concentration ratio has been optimized through multiple experiments to ensure that the carbon metabolic flow is maximized towards fatty acid synthesis and EPA generation, preventing the carbon flow from being biased towards bacterial proliferation or byproduct synthesis. Compared with existing arbitrary concentration ratios, lipid yield is increased by more than 25%, and EPA ratio is increased by more than 18%.

[0036] Fermentation dissolved oxygen regulation: A two-stage dissolved oxygen control method is adopted. During the growth period, the dissolved oxygen is maintained at 30-40%. At this time, the dissolved oxygen is sufficient to promote the transmembrane absorption and intracellular transport of para-aminobenzoic acid, ensuring that para-aminobenzoic acid can exert its effects quickly. During the oil and EPA synthesis period, the dissolved oxygen drops to 10-20%. At this time, the low dissolved oxygen can reduce the oxidative degradation of EPA. EPA is an unsaturated fatty acid, and high dissolved oxygen can easily lead to its oxidation. At the same time, it can increase the activity of desaturase, which is a key enzyme in EPA synthesis. Low dissolved oxygen can activate its activity. The dissolved oxygen switching point corresponds precisely to the regulator addition point. That is, the dissolved oxygen is switched at the beginning of the rapid oil accumulation period, further amplifying the synergistic effect and reducing EPA oxidation loss by more than 40%.

[0037] Selection of fermentation carbon source: The fermentation medium uses a mixed carbon source of glycerol and sodium acetate, with a mass ratio of glycerol to sodium acetate of 3:1-5:1. Glycerol provides a continuous carbon skeleton for the synthesis of lipids and EPA. Glycerol can be rapidly metabolized by Schizochytrium and converted into acetyl-CoA as a raw material for lipid synthesis. Sodium acetate, as a cotransporter, can improve the membrane permeability and effective intracellular concentration of para-aminobenzoic acid. Sodium acetate can regulate the permeability of the bacterial cell membrane, making it easier for para-aminobenzoic acid to enter the cell. The mixed carbon source and the dual regulator work together to ensure sufficient intracellular acetyl-CoA supply and balanced reducing power. Compared with a single carbon source system, the total lipid content is increased by more than 25%.

[0038] Fermentation pH control: The entire fermentation process employs segmented constant pH control. During the growth phase, the pH is maintained at 6.2-6.5, which is suitable for the growth of Schizochytrium and promotes the absorption of para-aminobenzoic acid. During the oil synthesis phase, the pH is lowered to 5.8-6.1. This pH stabilizes the dissociation state of para-aminobenzoic acid, preventing its inactivation due to excessively high or low pH. It also improves the utilization rate of small-molecule sulfur donors, which are more easily dissociated and absorbed by the cells under weakly acidic conditions. This avoids pH fluctuations that could lead to regulator failure or metabolic disorders, ensuring a stable fermentation process and keeping product yield and purity fluctuations within 5%.

[0039] Details of the cell wall breaking process: The cell wall breaking enzyme is a compound preparation of lysozyme and lipase, with a mass ratio of 2:1-3:1. Lysozyme is responsible for decomposing the bacterial cell wall, while lipase is responsible for assisting in the decomposition of intracellular lipid droplets, thus synergistically improving the cell wall breaking efficiency. The low-temperature high-pressure homogenization pressure is controlled at 30-50MPa and the temperature is controlled at 4-8℃, which ensures thorough cell wall breaking and protects the EPA structure from oxidation. Compared with a single cell wall breaking method, the oil release rate is increased by more than 20%, and the EPA oxidation loss is reduced by more than 30%.

[0040] Purification process details: The feed-to-liquid ratio for urea inclusion is 1:3-1:6. If the feed-to-liquid ratio is too low, the inclusion of saturated fatty acids will be incomplete; if the feed-to-liquid ratio is too high, EPA will be lost. The molecular distillation vacuum is controlled at 1-10 Pa, and the distillation temperature is 80-120℃. High vacuum can reduce the distillation temperature and avoid EPA oxidation. Precise temperature control can achieve effective separation of EPA from other fatty acids, ensuring that the purity of EPA reaches more than 92% and there is no cis-trans isomerization. That is, the cis structure of EPA is the key to its physiological function, and isomerization will lead to a decrease in its activity.

[0041] Regulator recovery and reuse process: To improve the utilization rate of regulators, reduce production costs, and minimize environmental pollution, this invention designs a process for the recovery and reuse of p-aminobenzoic acid and small molecule sulfur donors. The specific working process is as follows: D1: Supernatant collection: After fermentation, during the cell collection process in step 3, the supernatant obtained by centrifugation is collected separately. The supernatant contains unused para-aminobenzoic acid, small molecule sulfur donors, and a small amount of cell fragments and suspended impurities. It is placed in a sterile container for later use to avoid waste.

[0042] D2: Nanofiltration membrane concentration and recovery: The collected supernatant is concentrated using a nanofiltration membrane with a molecular weight cutoff of 100-200 Da. This molecular weight cutoff can precisely retain p-aminobenzoic acid and small molecule sulfur donors, while allowing water and small molecule impurities to pass through. The concentration pressure is controlled at 0.5-1.0 MPa, and the concentration temperature is controlled at 25-30℃. The solution is concentrated to 1 / 5-1 / 3 of its original volume, resulting in a recovery solution containing p-aminobenzoic acid and small molecule sulfur donors. At this point, the concentration of the regulator in the recovery solution is significantly increased, and it can be directly reused.

[0043] D3: Filtration and purification of the recovered liquid: The concentrated recovered liquid is filtered through a 0.22μm filter membrane. The 0.22μm filter membrane can remove a small amount of residual bacterial fragments and suspended impurities in the recovered liquid. If these impurities are carried into the next batch of fermentation, they will affect the growth of Schizochytrium and the effect of the regulator. After filtration, a pure recovered liquid is obtained.

[0044] D4: Concentration detection of the recovered solution: The actual concentrations of p-aminobenzoic acid and small molecule sulfur donor in the filtered recovered solution were detected by high performance liquid chromatography (HPLC). HPLC is an accurate concentration detection method and can also be performed using conventional HPLC equipment. Detection conditions: mobile phase: methanol-water = 30:70, flow rate: 1.0 mL / min, detection wavelength: 280 nm. The detection results were recorded for subsequent concentration adjustment for reuse.

[0045] D5: Recycled liquid reuse: Recycled liquid is reused at a rate of 30%-70%, which is the volume ratio of the recycled liquid to the initial culture medium or fed broth for the next batch of fermentation. This ratio avoids the accumulation of small amounts of metabolic byproducts in the recycled liquid, ensuring fermentation stability. Recycled liquid that passes the test is reused for the next batch of fermentation. There are two specific reuse methods: (1) Reuse in the initial culture medium: Add the recovered liquid to the newly prepared initial fermentation culture medium to replace part of the deionized water. Based on the concentration of the regulator in the recovered liquid detected in step 4, add new para-aminobenzoic acid and small molecule sulfur donor to ensure that after the addition, the final concentration of para-aminobenzoic acid in the next batch of fermentation system is maintained at 40-120 mg / L and the final concentration of small molecule sulfur donor is maintained at 5-30 mg / L. (2) Recycled liquid: The recycled liquid is used as the feed medium for small molecule sulfur donors. It is mixed with new small molecule sulfur donors in a gradient feed method and fed into the fermentation system during the rapid accumulation period of oil. Similarly, new small molecule sulfur donors are added according to the detected concentration of regulator in the recycled liquid to ensure that the concentration of regulator in the fermentation system after feed meets the limit.

[0046] D6: Confirmation of reuse concentration: After the recovered liquid is reused, the concentrations of para-aminobenzoic acid and small molecule sulfur donor in the fermentation system are checked again 1-2 hours after the fermentation starts to ensure that the concentrations are maintained within the specified range. If the concentrations are insufficient, a small amount of new regulator is added to ensure that the fermentation proceeds normally.

[0047] The technical benefits of this recycling process are: it enables the recycling of para-aminobenzoic acid and small-molecule sulfur donors, significantly improves the utilization rate of regulators, reduces raw material consumption while maintaining the same level of oil and EPA synthesis, reduces the emission of regulators in fermentation waste liquid, reduces environmental pollution, solves the problems of low regulator utilization, raw material waste, and environmental pollution in existing technologies, and simplifies operation, making it suitable for large-scale industrial applications.

[0048] To further verify the technical effect of the present invention, the following prior art is proposed as a comparative example, and experiments are conducted to verify the technical effect of the present invention: Comparative Example: This comparative example uses the conventional process for preparing EPA by fermentation of Schizochytrium in the prior art. The specific steps are as follows, maintaining the same basic conditions as Example 1, such as strain, basic culture medium, and culture temperature. The steps are as follows: Step 1: Strain selection and fermentation start-up: The same Schizochytrium strain as in Example 1 was selected, and a basic fermentation medium was prepared with the same formula as in Example 1: 15 g / L glucose, 8 g / L peptone, 4 g / L yeast extract, 25 g / L sea salt, 1.5 g / L potassium dihydrogen phosphate, and 0.8 g / L magnesium sulfate, with the pH adjusted to 6.3. The medium was sterilized at 121°C and 0.1 MPa for 20 minutes, cooled to 28°C, and then inoculated with the activated Schizochytrium seed liquid at an 8% inoculum rate. Fermentation was started, with the fermenter speed at 180-220 r / min. Dissolved oxygen was maintained at 30-40% and the temperature at 28°C throughout the process, without segmented dissolved oxygen control. Glucose was used as the sole carbon source without any mixed carbon source optimization.

[0049] Step 2: Addition of a single regulator: Instead of using the time-sequential synergistic regulation of p-aminobenzoic acid and small molecule sulfur donors, a single regulator, p-aminobenzoic acid, was added only once during the middle of the logarithmic growth phase, 12 hours after fermentation started. The final concentration was the same as in Example 1, i.e., 80 mg / L, without the addition of small molecule sulfur donors. The pH was maintained at 6.3 throughout the fermentation process without segmented pH regulation.

[0050] Step 3: Bacterial cell collection: The procedure was the same as in Example 1: after fermentation for 72 hours, fermentation was stopped, centrifuged at 5000 r / min for 10 minutes, the bacterial precipitate was collected, and washed twice with 0.9% physiological saline to obtain pure bacterial cells.

[0051] Step 4: Cell disruption: The existing single high-pressure homogenization cell disruption method is adopted, without low-temperature control and compound enzymatic hydrolysis: the bacterial cells are mixed with 0.1 mol / L phosphate buffer at pH 6.3 at a volume ratio of 1:6 to prepare a bacterial suspension. The suspension is homogenized twice at 30 MPa pressure at room temperature for 8 minutes each time. No lysozyme and lipase compound preparation is added, and subsequent oil extraction is carried out directly.

[0052] Step 5: Oil extraction: The existing organic solvent extraction method was used to replace the supercritical CO2 extraction in Example 1: hexane was added to the bacterial suspension after cell wall disruption, with a volume ratio of bacterial suspension to hexane of 1:2. The mixture was stirred and extracted at 35°C for 2 hours. After standing and separating, the upper organic phase was collected, and hexane was removed by rotary evaporation to obtain crude oil.

[0053] Step 6: Purification of crude oil: The existing single urea inclusion purification method is adopted: crude oil is mixed with 12g / 100mL urea-ethanol solution at a volume ratio of 1:4, stirred at 55℃ for 40 minutes, cooled to 2℃ and allowed to stand for 3 hours, the inclusion complex is removed by filtration, and the oil solution is collected as EPA product, without subsequent molecular distillation steps.

[0054] Step 7: Regulator treatment: After fermentation, the supernatant obtained by centrifugation is directly discharged without the recovery and reuse of para-aminobenzoic acid, and there is no process for recovering any regulators, resulting in waste of raw materials and environmental pollution.

[0055] Apart from the differences mentioned above, the comparative examples maintained the same operating conditions as Example 1, such as strain activation, fermentation time, and centrifugation parameters, to ensure the fairness and accuracy of the experimental comparison.

[0056] Experimental Design: Experimental objective: To compare the differences between Example 1 (the process of this invention) and the comparative example (the prior art process) in key indicators such as Schizochytrium mycelium biomass, oil yield, EPA ratio, EPA purity, regulator utilization rate, raw material consumption, and EPA oxidation loss, and to verify the technical effectiveness and innovation of the process of this invention.

[0057] Experimental materials and equipment: Experimental materials: Schizochytrium strain, para-aminobenzoic acid, L-cysteine ​​(small molecule sulfur donor), glycerol, sodium acetate, urea, ethanol, n-hexane, lysozyme, lipase, physiological saline, potassium dihydrogen phosphate, magnesium sulfate, etc. Experimental equipment: 50L fermenter, high-speed centrifuge, low-temperature high-pressure homogenizer, supercritical CO2 extraction equipment, molecular distillation equipment, high-performance liquid chromatograph (HPLC), spectrophotometer, rotary evaporator, sterile operating table, high-pressure steam sterilizer, etc.

[0058] Experimental grouping: Two experimental groups were set up, with three parallel samples in each group. The parallel samples were operated on in exactly the same way to ensure the repeatability of the experimental data. Experimental Group 1: The process of the present invention described in Example 1 was used to fully execute the entire process of strain selection, time-series synergistic regulation, cell collection, low-temperature compound cell disruption, supercritical extraction, tandem purification, and regulator recovery and reuse. Experimental Group 2: Using the existing technology and process described in the comparative example, the process included strain selection, single regulation, cell collection, single cell disruption at room temperature, organic solvent extraction, single purification, and direct discharge of the regulator.

[0059] Testing indicators and testing methods: Cell biomass: The OD600 value of the fermentation broth was measured using a spectrophotometer and converted using the dry weight method. A linear correlation was found between the OD600 value and the cell dry weight. Standard curve: y = 0.38x + 0.02, R0 2 =0.996; Oil yield: The oil content in the dry weight of the bacterial cells was determined by Soxhlet extraction, and the oil yield was calculated. EPA ratio: The percentage of EPA in crude oil by mass was determined by gas chromatography (GC). EPA purity: The purity of EPA in the final product was determined by HPLC. The detection conditions were: mobile phase methanol-water = 30:70, flow rate 1.0 mL / min, and detection wavelength 280 nm. Regulator utilization rate: The content of unused p-aminobenzoic acid in the supernatant after fermentation was detected by HPLC, and the utilization rate was calculated as follows: Utilization rate = (addition amount - residual amount) / addition amount × 100%; Raw material consumption: Calculate the amount of p-aminobenzoic acid and small molecule sulfur donor required to produce 1L of EPA product, and calculate the reduction rate of raw material consumption; EPA oxidation loss: The content of EPA oxidation products and peroxides during cell disruption, extraction, and purification was detected, and the oxidation loss rate was calculated. Oil release rate: Calculate the percentage of oil released after cell wall disruption to the total oil content of the bacteria.

[0060] Experimental period: The single-group experiment cycle is 72 hours, fermentation is 48-72 hours, and with the subsequent cell wall breaking, extraction, purification and recycling steps, the total cycle is 96 hours; parallel samples are carried out simultaneously, and the average value of 3 parallel samples is taken as the final experimental data after the experiment, with the error controlled within ±2%.

[0061] Experimental Data and Analysis:

[0062] The experimental data above show that, compared with the comparative example, Example 1 has significantly improved in all key indicators. The specific analysis is as follows: Cell biomass and oil yield: The cell biomass of Example 1 reached 18.6 g / L, an increase of 22.4% compared with the comparative example; the oil yield reached 8.9 g / L, an increase of 25.4% compared with the comparative example. This is mainly due to the use of glycerol-sodium acetate mixed carbon source, segmented pH and dissolved oxygen regulation, and the synergistic effect of para-aminobenzoic acid and small molecule sulfur donors in this invention, which provide sufficient carbon source and suitable metabolic environment for cell growth and oil synthesis, and solve the problems of slow cell growth and low oil yield in the prior art.

[0063] EPA ratio and purity: The EPA ratio in Example 1 reached 28.7%, which is 41.4% higher than that in the comparative example; the EPA purity reached 92.3%, which is 21.8% higher than that in the comparative example. The core reason is that the present invention adopts time-sequential segmented synergistic regulation, redirection of carbon flow of para-aminobenzoic acid, supplementation of reducing power of L-cysteine, and tandem purification process of urea inclusion and vacuum molecular distillation, which not only improves the synthesis efficiency of EPA, but also accurately removes impurities, thus solving the defects of low EPA ratio and insufficient purity in the prior art.

[0064] Regulator utilization rate and raw material consumption: In Example 1, the utilization rate of p-aminobenzoic acid reached 82.6%, which is 36.5% higher than that of the comparative example; the raw material consumption was reduced by 20.3%, which is attributed to the regulator recycling process designed in this invention, which realizes the recycling of p-aminobenzoic acid and small molecule sulfur donors, avoids raw material waste, reduces production costs, and reduces environmental pollution, thus solving the problems of low regulator utilization rate and serious pollution in the prior art.

[0065] EPA oxidation loss and oil release rate: The EPA oxidation loss rate in Example 1 was only 3.8%, which was 60.4% lower than that in the comparative example; the oil release rate reached 94.2%, which was 20.0% higher than that in the comparative example. This is mainly due to the low temperature and high pressure homogenization combined with enzymatic hydrolysis and cell wall breaking method adopted in this invention, as well as the supercritical CO2 extraction process, which not only protects the structural stability of EPA and avoids its oxidative degradation, but also achieves efficient release of intracellular oil, thus solving the problems of easy oxidation and low cell wall breaking efficiency of EPA in the prior art.

[0066] Experimental results show that the process of this invention achieves simultaneous improvement in lipid accumulation and EPA synthesis in Schizochytrium by synergistic regulation of p-aminobenzoic acid and small molecule sulfur donors, optimization of fermentation parameters, efficient cell wall disruption extraction, precise purification, and recovery and reuse of regulators. It is significantly superior to existing technologies and comparative methods in key indicators such as cell biomass, lipid yield, EPA ratio, EPA purity, and regulator utilization rate. It also has the advantages of low raw material consumption, low environmental pollution, and strong feasibility, making it suitable for large-scale industrial application.

[0067] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid, characterized in that, Includes the following steps: Step 1: Select Schizochytrium as the fermentation strain, prepare the fermentation medium and inoculate it with Schizochytrium to start the fermentation culture; Step 2: Para-aminobenzoic acid and small molecule sulfur donors are added to the fermentation system in a time-sequential, segmented manner for synergistic regulation. Para-aminobenzoic acid is used to reshape the central carbon metabolism flow to enhance lipid synthesis, while small molecule sulfur donors are used to supplement the sulfur-dependent reducing power required for EPA synthesis. Step 3: After the fermentation culture is completed, collect the bacterial cells in the fermentation system; Step 4: The collected bacterial cells are subjected to cell wall disruption to release intracellular lipids; Step 5: Extract the oil from the broken-cell system to obtain crude oil; Step 6: Purify the crude oil to obtain high-purity EPA product.

2. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using p-aminobenzoic acid according to claim 1, characterized in that, In step 2, the small molecule sulfur donor is at least one of L-cysteine, glutathione, or sodium thiosulfate.

3. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid according to claim 2, characterized in that, Para-aminobenzoic acid was added once during the mid-logarithmic growth phase, while the small molecule sulfur donor was added in a gradient feed during the rapid accumulation phase of the oil. The timing of the addition nodes of para-aminobenzoic acid and the feed nodes of the small molecule sulfur donor were matched.

4. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid according to claim 3, characterized in that, The final concentration of p-aminobenzoic acid is 40–120 mg / L, and the final concentration of the small molecule sulfur donor is 5–30 mg / L. The ratio of p-aminobenzoic acid concentration to small molecule sulfur donor concentration is appropriate.

5. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid according to claim 1, characterized in that, The fermentation process in step 2 employs two-stage dissolved oxygen control: dissolved oxygen is maintained at 30-40% during the growth period; and dissolved oxygen is reduced to 10-20% during the oil and EPA synthesis period.

6. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid according to claim 1, characterized in that, The fermentation medium uses a glycerol-sodium acetate mixed carbon source, with sodium acetate acting as a cotransporter and glycerol providing a continuous carbon skeleton supply for the synthesis of oils and EPA.

7. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using p-aminobenzoic acid according to claim 1, characterized in that, In step 2, the entire fermentation process is controlled by a segmented constant pH. The pH is controlled at 6.2-6.5 during the growth period and adjusted to 5.8-6.1 during the oil synthesis period.

8. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using p-aminobenzoic acid according to claim 1, characterized in that, In step 4, after the bacterial cells are collected, they are subjected to low-temperature high-pressure homogenization combined with complex enzymatic hydrolysis to break the cell walls. The cell-breaking enzyme is a complex preparation of lysozyme and lipase.

9. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using para-aminobenzoic acid according to claim 1, characterized in that, In step 5, the oil purification process employs a series of processes including urea encapsulation and vacuum molecular distillation. The urea encapsulation material-to-liquid ratio is 1:3 to 1:6, the vacuum degree of molecular distillation is controlled at 1 to 10 Pa, and the distillation temperature is 80 to 120°C.

10. The process for promoting lipid accumulation in Schizochytrium and preparing EPA using p-aminobenzoic acid according to claim 1, characterized in that, It also includes a recovery step for para-aminobenzoic acid and small-molecule sulfur donors, including the following: D1. Collect the fermentation system after fermentation is completed, perform solid-liquid separation, and obtain the supernatant; D2, the supernatant is concentrated by passing it through a nanofiltration membrane to recover the unused p-aminobenzoic acid and small molecule sulfur donor in the supernatant, and a recovery solution is obtained. D3, the recovered liquid is filtered through a 0.22μm filter membrane to remove residual bacterial fragments and suspended impurities in the recovered liquid; D4. The actual concentrations of p-aminobenzoic acid and small molecule sulfur donor in the filtered recovery liquid were determined by high performance liquid chromatography. D5, 30%-70% of the qualified recovered liquid is reused in the next batch of fermentation. If it is reused in the initial culture medium, the recovered liquid is added to the newly prepared initial culture medium and new para-aminobenzoic acid and small molecule sulfur donor are added. If it is reused in the fed-batch medium, the recovered liquid is used as the feed medium for small molecule sulfur donors and is mixed with new small molecule sulfur donors before use. D6 ensures that the final concentration of p-aminobenzoic acid in the reused fermentation system is maintained at 40–120 mg / L and the final concentration of small molecule sulfur donor is maintained at 5–30 mg / L.

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