A method for high-yield production of EPA by schizochytrium sp. based on stress pre-domestication and compound antioxidant and application

Abstract

The application belongs to the technical field of bio-chemical industry, and discloses a method for high-yield EPA of Schizochytrium sp. based on stress pre-domestication and compound antioxidant and application, wherein the method is to perform oxidative stress pre-acclimatization on Schizochytrium sp. seed liquid, then perform fermentation culture, and add compound exogenous antioxidants propyl gallate and tea polyphenol in the late fermentation stage; the stress pre-acclimatization and the compound antioxidant can interact and cooperatively regulate the metabolic network of Schizochytrium sp., so as to significantly increase the synthesis efficiency and stability of EPA. The stress pre-acclimatization method adopted by the application does not involve genetic engineering technology, is safe, simple and convenient to operate, has no harm to the environment, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, and in particular to a method and application of high EPA production based on stress pre-acclimatization and compound antioxidant Schizochytrium. Background Technology

[0002] Eicosapentaenoic acid (EPA) is an omega-3 series long-chain polyunsaturated fatty acid beneficial to human health, with significant effects in the prevention and adjuvant treatment of cardiovascular and cerebrovascular diseases. Currently, commercially available EPA is mainly derived from deep-sea fish oils. However, due to issues such as the depletion of fishery resources and marine pollution, it suffers from high costs and unstable supply. Developing sustainable alternative sources that do not rely on fishery resources has become a key technical problem that urgently needs to be solved in this field.

[0003] Schizochytrium is a marine fungus with industrial application value and is one of the main species for the industrial production of omega-3 fatty acids. It can directly synthesize EPA through a unique polyketide synthase pathway without relying on desaturase systems, and the EPA content is significantly affected by strain characteristics, culture conditions, and fermentation processes. However, the EPA yield of wild-type Schizochytrium is relatively low, limiting the economic feasibility of its industrial production. Improving the EPA yield of Schizochytrium through fermentation process optimization and metabolic engineering strategies is an important research direction in this field.

[0004] During the fermentation of Schizochytrium, stress strategies such as nitrogen starvation and low temperature to increase fatty acid production induce the accumulation of intracellular reactive oxygen species (ROS). Excessive ROS not only inhibits cell growth and reduces total fatty acid production, but also triggers a chain reaction of lipid peroxidation, leading to the oxidative rancidity of unsaturated fatty acids such as EPA. This results in a significant increase in the peroxide value and anisidine value of the oil, along with the degradation of beneficial nutrients, ultimately causing quality problems such as abnormal color, viscosity, and fishy smell, as well as shortened shelf life.

[0005] Chinese invention patent publication CN 116731871 A discloses a method for continuously acclimating Schizochytrium fungi with light and hydrogen peroxide, which can increase the EPA production of Schizochytrium fungi. Experiments show that under dual-stress fermentation conditions, the EPA content of the acclimated strain is higher than that of the starting strain. While maintaining the dynamic balance between saturated fatty acids and polyunsaturated fatty acids, the DHA synthesis capacity decreases, and the EPA content under dual-stress conditions is higher than that under normal culture conditions. However, white light irradiation can cause cells to initiate a general stress response to cope with stress. Its energy is dispersed in different wavelengths, and only specific wavelengths have a positive effect on metabolic regulation. Other wavelengths have limited effects and may also produce inhibitory effects or side effects (such as heat production). Therefore, this invention selects blue light irradiation, which is highly efficient and has a low thermal effect. Monochromatic blue light has concentrated energy, and most of the photon energy is used to activate specific signaling pathways with less energy loss, which can reduce unnecessary thermal stress.

[0006] Currently, numerous studies have reported on the effects of antioxidants or related additives on microbial lipid yield. For example, Chinese invention patent publication CN 107354121 A discloses that adding salicylic acid to *Phaeodactylum tricornutum* culture can alter the relative content of its neutral lipids; Chinese invention patent publication CN 107354120 A shows that adding methyl jasmonic acid can promote the growth of *Phaeodactylum tricornutum* and increase its biomass. Chinese invention patent publication CN 119265251 A discloses a scheme for treating *Schizochytridactylum* with a combination of gibberellin and chlorpyrifos, which can alter the relative DHA content in the lipids; Chinese invention patent publication CN 110195085 A reports that simultaneously adding appropriate concentrations of indoleacetic acid, gibberellin, and 6-aminopurine to *Chlorella vulgaris* culture has a positive effect on lipid yield. However, these studies mostly focus on regulating the yield of lipids or a specific key product, with insufficient attention paid to the safety of additives, and reports on the effects of additives on lipid quality are relatively scarce.

[0007] Therefore, a safe, low-cost, and high-yield fermentation regulation method is needed to increase the yield of EPA in Schizochytrium. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an efficient and rapid method and application for high EPA production based on stress pre-acclimatization and compound antioxidant Schizochytrium.

[0009] The technical solution adopted by this invention to solve its technical problem is:

[0010] A method for high EPA production by Schizochytrium based on stress pre-acclimatization and compound antioxidants is disclosed. The method involves subjecting Schizochytrium seed culture to oxidative stress pre-acclimatization, followed by fermentation culture. In the later stage of fermentation, compound exogenous antioxidants propyl gallate and tea polyphenols are added. The stress pre-acclimatization and compound antioxidants can interact to synergistically regulate the metabolic network of Schizochytrium, significantly increasing the synthesis efficiency and stability of EPA.

[0011] Furthermore, the oxidative stress pre-adaptation refers to the process of culturing activated Schizochytrium seed culture under blue light irradiation at an intensity of 3000–9000 lx for 10–20 generations with shaking to obtain a strengthened seed culture with enhanced stress resistance.

[0012] Furthermore, the Schizochytrium seed liquid is obtained by activating Schizochytrium seeds through two generations.

[0013] Furthermore, a compound exogenous antioxidant was added at 48 hours of fermentation. The final concentration of propyl gallate was 0.02-0.05 g / L, and the final concentration of tea polyphenols was 0.3-0.5 g / L.

[0014] Furthermore, the final concentration of propyl gallate added is 0.04 g / L, and the final concentration of tea polyphenols added is 0.5 g / L.

[0015] Furthermore, the Schizochytrium fungus selected is Schizochytrium sp HX-308.

[0016] Furthermore, the blue light irradiation intensity is determined by selecting the highest light intensity without affecting the glucose consumption rate.

[0017] Further, the method for culturing the Schizochytrium seed solution includes: Schizochytrium is stored in a 20% glycerol preservation tube at -80℃. When activating the seeds, 1 mL of glycerol-preserved Schizochytrium is inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a constant temperature shaker under the following conditions: 30℃, 170 rpm, and 24 h. Under these conditions, the first generation of seeds is activated. The activated seeds are then transferred at a volume ratio of 1% to a new 500 mL Erlenmeyer flask containing 100 mL of culture medium and cultured under the same conditions to obtain the seed solution.

[0018] Alternatively, the fermentation culture method includes: inoculating the seed culture of Schizochytrium fermentation culture with a bacterial culture that has been cultured for three generations, inoculating it into the fermentation medium at an inoculation rate of 10% by volume, and fermenting it at a temperature of 25-30℃ and a rotation speed of 150-220 rpm for 96-144 hours. After 48 hours from the start of fermentation, add the compound exogenous antioxidants propyl gallate and tea polyphenols to the fermentation broth, so that the final concentration of propyl gallate added during the fermentation culture stage is 0.01-0.05 g / L and the final concentration of tea polyphenols added is 0.1-0.3 g / L. After the fermentation is completed, the fermentation broth is obtained.

[0019] Furthermore, the main components of the Schizochytrium seed culture medium and the Schizochytrium fermentation culture medium are ion solutions (i.e., artificial seawater), glucose solutions, and yeast powder solutions.

[0020] Further, the seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min;

[0021] The fermentation medium formulation includes: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B... 12 0.1-0.5 mg / L, pH 6.0-6.5, sterilized at 121℃ for 20 min.

[0022] Furthermore, the specific steps are as follows:

[0023] (1) Blue light stress: The activated seed liquid of Schizochytrium, which had been cultured in seed medium for two generations, was inoculated into seed medium at a volume of 2% and cultured under blue light stimulation of 6000 lx intensity throughout the process at 170 rpm and 30℃. After 24 h of culture, the seed liquid was inoculated into new seed medium at a volume of 5% and passaged for 20 generations to obtain the 20th generation of blue light stimulation acclimatized strain ALE20;

[0024] The seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min.

[0025] (2) The 20th generation domesticated strain ALE20 was fermented under blue light stimulation, as follows:

[0026] Strain strain ALE20 was inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain tertiary seed culture.

[0027] The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 28℃ and 180 rpm for 120 h. 48 h after the start of fermentation, a compound antioxidant, propyl gallate and tea polyphenols, were added to the fermentation broth. The final concentration of propyl gallate was 0.04 g / L, and the final concentration of tea polyphenols was 0.5 g / L.

[0028] The seed culture medium formula includes: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5, sterilized at 121℃ for 20min before use.

[0029] The fermentation medium formula includes: glucose 80 g / L, yeast extract 10 g / L, sodium sulfate 8 g / L, magnesium sulfate 3 g / L, ammonium sulfate 6 g / L, potassium chloride 1.5 g / L, calcium chloride 0.15 g / L, potassium sulfate 0.8 g / L, potassium dihydrogen phosphate 1.5 g / L, monosodium glutamate 18 g / L, zinc sulfate heptahydrate 3 mg / L, cobalt chloride hexahydrate 0.05 mg / L, copper sulfate pentahydrate 4 mg / L, nickel sulfate hexahydrate 1.5 mg / L, ferrous sulfate heptahydrate 12 mg / L, calcium pantothenate 3 mg / L, manganese chloride tetrahydrate 4 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 7 mg / L, vitamin B... 12 0.3 mg / L, pH 6.0, sterilized at 121℃ for 20 min before use.

[0030] The method described above is applied in the production of EPA.

[0031] The advantages and positive effects of this invention are as follows:

[0032] 1. This invention pre-adapts the strain by subjecting it to blue light stress, effectively activating the intracellular antioxidant defense system of Schizochytrium and laying a metabolic foundation for the subsequent efficient synthesis of EPA.

[0033] 2. The stress preconditioning method used in this invention does not involve genetic engineering technology, the operation process is safe and simple, it is harmless to the environment, and it has good prospects for industrial application.

[0034] 3. The 20 generations of stress-pre-adapted Schizochytrium strain had an EPA content of 4.49%, which is 2.23 times that of the original strain (2.01%), showing a significant improvement in production efficiency.

[0035] 4. This invention effectively removes reactive oxygen species (ROS) accumulated during fermentation by adding a compound antioxidant composed of propyl gallate and tea polyphenols, thus delaying the oxidative rancidity of oils.

[0036] 5. The use of compound antioxidants increased the oil yield to 51.1 g / L, which is more than 100% higher than that of traditional processes, demonstrating significant economic benefits.

[0037] 6. This invention increases the proportion of EPA in total fatty acids to 11.28% through the precise addition of compound antioxidants, which greatly improves the nutritional value of the product.

[0038] 7. The synergistic effect of compound antioxidants significantly improves the quality of oils and fats. The EPA oils and fats produced by this invention have better storage stability and effectively extend the product shelf life.

[0039] 8. In the experiment of producing EPA by fermentation of Schizochytrium, this invention found that pre-adaptation and domestication of the strain before fermentation, combined with a specific compound antioxidant, can synergistically overcome the trade-off between cell growth and product synthesis, ultimately achieving an EPA yield of 11.28%. Moreover, the use of compound antioxidants can reduce the dosage of a single antioxidant by 60%-75%. This method is stable, low-cost, and highly efficient in oil production, making it very suitable for industrial production. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the changes in fatty acid composition after blue light stimulation fermentation of Schizochytrium in this invention.

[0041] Figure 2 This is a graph showing the fermentation results of adding the compound antioxidant at different times after the start of Schizochytrium fermentation in this invention.

[0042] Figure 3 This is a graph showing the change in intracellular reactive oxygen species (ROS) levels over time in the fermentation broth of Schizochytrium in this invention. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments. The embodiments described below are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0044] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0045] A method for high EPA production based on stress pre-acclimatization and the combination of the antioxidant Schizochytrium is disclosed. The method involves pre-acclimatizing Schizochytrium seed culture under oxidative stress, followed by fermentation. During the later stages of fermentation, a combination of exogenous antioxidants, propyl gallate and tea polyphenols, is added. Propyl gallate rapidly captures initial free radicals, inhibiting the increase in peroxide value. Tea polyphenols scavenge free radicals and chelate pro-oxidative metal ions, effectively blocking the formation of secondary oxidation products and controlling the anisidine value. The combined use of these two antioxidants synergistically blocks the entire lipid oxidation process. While scavenging ROS, moderate oxidative stress signals can also synergistically upregulate the differential expression of endogenous antioxidant systems and lipid synthesis-related genes within cells. This positively regulates metabolic flux while protecting existing products, promoting efficient EPA synthesis and stable accumulation, maintaining the content of beneficial byproducts, and comprehensively improving lipid quality. Furthermore, the combined use of propyl gallate and tea polyphenols can significantly reduce the individual concentrations while ensuring antioxidant effects, avoiding the toxicity threshold of single antioxidants and broadening the safe operating range of the fermentation process.

[0046] The stress preconditioning and compound antioxidants can interact to synergistically regulate the metabolic network of Schizochytrium, significantly increasing the synthesis efficiency and stability of EPA.

[0047] Furthermore, the oxidative stress pre-adaptation refers to the process of culturing activated Schizochytrium seed culture under blue light irradiation at an intensity of 3000–9000 lx for 10–20 generations with shaking to obtain a strengthened seed culture with enhanced stress resistance.

[0048] Furthermore, the Schizochytrium seed liquid is obtained by activating Schizochytrium seeds through two generations.

[0049] Furthermore, a compound exogenous antioxidant was added at 48 hours of fermentation. The final concentration of propyl gallate was 0.02-0.05 g / L, and the final concentration of tea polyphenols was 0.3-0.5 g / L.

[0050] Furthermore, the final concentration of propyl gallate added is 0.04 g / L, and the final concentration of tea polyphenols added is 0.5 g / L.

[0051] Furthermore, the Schizochytrium fungus selected is Schizochytrium sp HX-308.

[0052] Furthermore, the blue light irradiation intensity is determined by selecting the highest light intensity without affecting the glucose consumption rate.

[0053] Further, the method for culturing the Schizochytrium seed solution includes: Schizochytrium is stored in a 20% glycerol preservation tube at -80℃. When activating the seeds, 1 mL of glycerol-preserved Schizochytrium is inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a constant temperature shaker under the following conditions: 30℃, 170 rpm, and 24 h. Under these conditions, the first generation of seeds is activated. The activated seeds are then transferred at a volume ratio of 1% to a new 500 mL Erlenmeyer flask containing 100 mL of culture medium and cultured under the same conditions to obtain the seed solution.

[0054] Alternatively, the fermentation culture method includes: inoculating the seed culture of Schizochytrium fermentation culture with a bacterial culture that has been cultured for three generations, inoculating it into the fermentation medium at an inoculation rate of 10% by volume, and fermenting it at a temperature of 25-30℃ and a rotation speed of 150-220 rpm for 96-144 hours. After 48 hours from the start of fermentation, add the compound exogenous antioxidants propyl gallate and tea polyphenols to the fermentation broth, so that the final concentration of propyl gallate added during the fermentation culture stage is 0.01-0.05 g / L and the final concentration of tea polyphenols added is 0.1-0.3 g / L. After the fermentation is completed, the fermentation broth is obtained.

[0055] Furthermore, the main components of the Schizochytrium seed culture medium and the Schizochytrium fermentation culture medium are ion solutions (i.e., artificial seawater), glucose solutions, and yeast powder solutions.

[0056] Further, the seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min;

[0057] The fermentation medium formulation includes: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B... 12 0.1-0.5 mg / L, pH 6.0-6.5, sterilized at 121℃ for 20 min.

[0058] Furthermore, the specific steps are as follows:

[0059] (1) Blue light stress: The activated seed liquid of Schizochytrium, which had been cultured in seed medium for two generations, was inoculated into seed medium at a volume of 2% and cultured under blue light stimulation of 6000 lx intensity throughout the process at 170 rpm and 30℃. After 24 h of culture, the seed liquid was inoculated into new seed medium at a volume of 5% and passaged for 20 generations to obtain the 20th generation of blue light stimulation acclimatized strain ALE20;

[0060] The seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min.

[0061] (2) The 20th generation domesticated strain ALE20 was fermented under blue light stimulation, as follows:

[0062] Strain strain ALE20 was inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain tertiary seed culture.

[0063] The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 28℃ and 180 rpm for 120 h. 48 h after the start of fermentation, a compound antioxidant, propyl gallate and tea polyphenols, were added to the fermentation broth. The final concentration of propyl gallate was 0.04 g / L, and the final concentration of tea polyphenols was 0.5 g / L.

[0064] The seed culture medium formula includes: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5, sterilized at 121℃ for 20min before use.

[0065] The fermentation medium formula includes: glucose 80 g / L, yeast extract 10 g / L, sodium sulfate 8 g / L, magnesium sulfate 3 g / L, ammonium sulfate 6 g / L, potassium chloride 1.5 g / L, calcium chloride 0.15 g / L, potassium sulfate 0.8 g / L, potassium dihydrogen phosphate 1.5 g / L, monosodium glutamate 18 g / L, zinc sulfate heptahydrate 3 mg / L, cobalt chloride hexahydrate 0.05 mg / L, copper sulfate pentahydrate 4 mg / L, nickel sulfate hexahydrate 1.5 mg / L, ferrous sulfate heptahydrate 12 mg / L, calcium pantothenate 3 mg / L, manganese chloride tetrahydrate 4 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 7 mg / L, vitamin B... 12 0.3 mg / L, pH 6.0, sterilized at 121℃ for 20 min before use.

[0066] The method described above is applied in the production of EPA.

[0067] Further, the fatty acid content in the *Schizochytrium* oil in the examples was obtained as follows: After fermentation culture, the pH of the fermentation broth was adjusted to 11-13 with 1 mol / L NaOH. 0.5% (v / v) of a cell-wall-breaking enzyme was added to the broth. After enzymatic hydrolysis at 55°C for 1 hour, the mixture was cooled to room temperature, and an equal volume of anhydrous ethanol was added to inactivate the enzyme. Hexane was added to the broth for extraction, and the mixture was allowed to stand for phase separation. The upper layer, a yellow organic phase, was collected. The hexane phase was removed by rotary evaporation to obtain the microbial oil. After rotary evaporation, the oil was dried in an oven to constant weight and weighed to obtain the oil.

[0068] 20 μL of oil was added to an EP tube containing 1 ml of 1M potassium hydroxide-methanol solution. The mixture was shaken at 20 °C and 1000 rpm for 6 h. The reaction was terminated by adding 50 μL of concentrated sulfuric acid. 1 ml of n-hexane was added, and the mixture was shaken at 20 °C and 1000 rpm for 0.5 h to extract the lipids. The extracted phase was transferred to a liquid chromatography vial for gas chromatography analysis. A GC-2010 (Shimadzu, Japan) gas chromatography system was used, with nitrogen as the carrier gas. The injection volume was 1 μL, and the injection temperature was 250 °C. The column temperature was increased from 100 °C to 200 °C at a rate of 25 °C / min, then increased to 230 °C at a rate of 4 °C / min and held for 9 min. The FID detector temperature was 280 °C. Different fatty acid compositions were identified by comparison with relevant external standards (Sigma, USA). The content of individual fatty acids was estimated from the peak area on the chromatogram using non-endogenous fatty acids (C11:0) as an internal standard.

[0069] Specifically, the relevant preparation and testing methods are as follows:

[0070] In the following examples, Schizochytrium sp. HX-308 is currently deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC M 209059 and has been published in Chinese patent publication CN101575584A.

[0071] Unless otherwise specified, room temperature refers to 25±5℃.

[0072] Biomass determination: Take 1 mL of fermentation broth after fermentation is completed, centrifuge at 4000 r / min for 5 min, discard the supernatant, add 1 mL of 2% NaCl solution to wash once, then wash once with 1 mL of distilled water, dry at 105℃ to constant weight, and finally weigh and calculate.

[0073] ROS determination: The relative ROS content of the samples was determined using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). The DCFH-DA fluorescent probe was dissolved in DMSO to a concentration of 1 mM and stored at -20°C for later use. It was then added to cell culture at a volume ratio of 1:100 and incubated in the dark at 30°C and 170 rpm for 20 minutes to allow the fluorescent probe to fully penetrate the cells. Cell samples were washed twice with 0.01 M PBS buffer to thoroughly remove extracellular DCFH-DA, and then dissolved in 0.01 M PBS buffer. Finally, the relative ROS content within the cells was determined using a multi-mode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0074] The oil was evaluated using indicators (anisidine value, peroxide value, and fatty acid composition). The anisidine value was determined by spectrophotometry, and the peroxide value was determined using a kit.

[0075] Example 1

[0076] The blue light stress conditions were as follows: *Schizochytrium* HX-308 shake flasks were sealed with transparent plastic film and placed in a shaker for culture, while simultaneously applying blue light stimulation at an intensity of 6000 lx, ensuring that the light penetrated the transparent seal and fully acted on the fermentation broth. Activated seed culture, which had been cultured for two generations in seed medium, was inoculated into the seed medium at a 2% inoculum volume. The culture was maintained under light stimulation conditions throughout the entire process at 170 rpm and 30°C. After 24 hours of culture, the culture was subcultured into fresh seed medium at a 5% inoculum volume, continuing for 20 generations.

[0077] The seed culture medium formula is as follows: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5;

[0078] Under blue light stimulation, 20 generations of domesticated Schizochytrium fungi underwent shaker fermentation (170 rpm, 30 °C), resulting in an oil accumulation of 25.5 g·L⁻¹. -1 Compared to the non-blue light control group (all other fermentation conditions were exactly the same as the blue light group), 22.6 g·L -1 The amount of oil accumulation increased significantly by 12.8%. This increase was mainly due to the moderate inhibition of cell growth rate by light, which slowed down glucose consumption and delayed the time when the cells entered the death phase, thus prolonging the efficient oil synthesis stage and resulting in a significantly higher total oil yield after fermentation compared to the control group.

[0079] like Figure 1 As shown, blue light stimulation significantly altered the fatty acid composition of the total oil in Schizochytrium fungi. ALE5, ALE10, and ALE20 represent the 5th, 10th, and 20th generations of strains acclimated to blue light stimulation, respectively. The EPA proportion in the total fatty acids of the 20th generation acclimated strain reached 4.49%, a 1.23-fold increase compared to the 2.01% in the control group (i.e., under non-blue light conditions, the same applies below). Simultaneously, the proportions of DPA and DHA in the 20th generation acclimated strain decreased from 24.71% and 56.24% in the control group to 20.43% and 50.88%, respectively. Not only the 20th generation acclimated strain, but also the EPA proportions of the 5th and 10th generations showed increases compared to the control group, increasing by 5.97% and 87.5%, respectively. This indicates that light stimulation has a specific regulatory effect on the synthesis of polyunsaturated fatty acids.

[0080] As shown in Table 1, blue light stimulation significantly promoted the synthesis and accumulation of various terpenoids in Schizochytrium fungi, including high-value secondary metabolites such as squalene, β-carotene, and astaxanthin. The 10th generation domesticated strain showed a 10.1% increase in squalene, a 12.2% increase in β-carotene, and a 24.2% increase in astaxanthin compared to the control group. The 20th generation domesticated strain showed a 27.4% increase in squalene, a 25.4% increase in β-carotene, and a 19.2% increase in astaxanthin compared to the control group. This further confirms the comprehensive advantages of this invention in enhancing the value of Schizochytrium fungi metabolites.

[0081] Table 1. Effects of blue light acclimatization on the fermentation performance of Schizochytrium.

[0082]

[0083] Example 2

[0084] The 20th generation domesticated strain ALE20, induced by blue light stimulation in Example 1, was fermented. The specific experimental procedure is as follows:

[0085] The seed culture medium formula is as follows: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5. It is sterilized at 121℃ for 20 minutes before use.

[0086] The fermentation medium formula is as follows: glucose 80 g / L, yeast extract 10 g / L, sodium sulfate 8 g / L, magnesium sulfate 3 g / L, ammonium sulfate 6 g / L, potassium chloride 1.5 g / L, calcium chloride 0.15 g / L, potassium sulfate 0.8 g / L, potassium dihydrogen phosphate 1.5 g / L, monosodium glutamate 18 g / L, zinc sulfate heptahydrate 3 mg / L, cobalt chloride hexahydrate 0.05 mg / L, copper sulfate pentahydrate 4 mg / L, nickel sulfate hexahydrate 1.5 mg / L, ferrous sulfate heptahydrate 12 mg / L, calcium pantothenate 3 mg / L, manganese chloride tetrahydrate 4 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 7 mg / L, vitamin B... 12 0.3 mg / L, pH 6.0, sterilized at 121℃ for 20 min before use;

[0087] The domesticated strain ALE20 was inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain tertiary seed culture.

[0088] The third-level seed liquid was inoculated into the fermentation medium at a volume percentage of 10%, and fermented for 120 h at a temperature of 28℃ and a rotation speed of 180 rpm. The compound antioxidants propyl gallate and tea polyphenols were added to the fermentation broth 48 h after the start of fermentation.

[0089] The compound antioxidants propyl gallate and tea polyphenols were added to the fermentation medium at different concentrations (propyl gallate 0.03 g / L + tea polyphenols 0.4 g / L, propyl gallate 0.03 g / L + tea polyphenols 0.5 g / L, propyl gallate 0.03 g / L + tea polyphenols 0.6 g / L, propyl gallate 0.04 g / L + tea polyphenols 0.4 ...4 g / L, propyl gallate 0.03 g / L + tea polyphenols 0.03 g / L + tea polyphenols 0.03 g / L + tea polyphenols 0.03 g / L + tea polyphenols The following solutions were added: propyl gallate 0.04 g / L + tea polyphenols 0.5 g / L; propyl gallate 0.04 g / L + tea polyphenols 0.6 g / L; propyl gallate 0.05 g / L + tea polyphenols 0.4 g / L; propyl gallate 0.05 g / L + tea polyphenols 0.5 g / L; propyl gallate 0.05 g / L + tea polyphenols 0.6 g / L; propyl gallate 0.04 g / L + tea polyphenols 0 g / L; propyl gallate 0 g / L + tea polyphenols 0.5 g / L. Fermentation was stopped after 120 hours, and various indicators were measured. The results are shown in Table 2.

[0090] The cell-wall-breaking enzyme was added to the fermentation broth at a dosage of 3 g / L. Enzymatic hydrolysis was performed for 10 h at pH 12, a rotation speed of 150 rpm, and a temperature of 50℃ to obtain a cell-wall-breaking solution. After cooling, the solution was mixed with ethanol at a volume ratio of 1:1, and then mixed with n-hexane for extraction to obtain the n-hexane phase. The n-hexane phase was then removed by rotary evaporation to obtain microbial oil. After rotary evaporation, the oil was dried in an oven to constant weight and weighed to obtain the oil.

[0091] Fatty acids were analyzed by gas chromatography. 20 μL of oil was added to an EP tube containing 1 ml of 1M potassium hydroxide-methanol solution. The mixture was shaken at 20°C and 1000 r / min for 6 h. The reaction was terminated by adding 50 μL of concentrated sulfuric acid, followed by extraction with 1 ml of n-hexane and shaking at 20°C and 1000 r / min for 0.5 h. The extracted phase was then transferred to a liquid chromatography vial for gas chromatography analysis. A GC-2010 (Shimadzu, Japan) gas chromatography system equipped with a DB-23 capillary column (60 m³) was used for analysis. A 0.22 mm column and a flame ionization detector (FID) were used. Nitrogen was used as the carrier gas. The injection volume was 1 μL, and the injection temperature was 250 °C. The column temperature was increased from 100 °C to 200 °C at a rate of 25 °C / min, then increased to 230 °C at a rate of 4 °C / min and held for 9 min. The FID detector temperature was 280 °C. Different fatty acid compositions were identified by comparison with Sigma standards (related external standards, Sigma, USA). The content of individual fatty acids was calculated from the peak area on the chromatogram using non-endogenous fatty acids (C19:0) as internal standards.

[0092] Other indicators were measured using conventional methods available in the prior art.

[0093] Table 2. The interaction of different antioxidant concentration combinations on fatty acid production in batch cultures of Schizochytrium.

[0094]

[0095] At a concentration of 0.04 g / L propyl gallate and 0.5 g / L tea polyphenols, all indicators reached their peak values. Decreasing tea polyphenols to 0.4 g / L or increasing them to 0.6 g / L significantly reduced oil, DHA, and EPA production, confirming that 0.5 g / L tea polyphenols was the optimal synergistic concentration. Similarly, decreasing propyl gallate to 0.03 g / L or increasing it to 0.05 g / L resulted in significantly lower oil and DHA production compared to the optimal group, indicating that 0.04 g / L propyl gallate was the precise concentration for achieving the best synergistic effect. Compared to other concentration groups, the optimal combination of antioxidants can increase DHA production by 3.8% to 92.9% and polyunsaturated fatty acid content by 3.7% to 19.3%. More importantly, its EPA content and production are significantly increased by 151% and 405% respectively compared to the control group. Compared to other concentration groups, the EPA content increased by a minimum of 13.3% and a maximum of 95.5%, and the EPA production increased by a minimum of 16.8% and a maximum of 208%.

[0096] Data shows that the combination of propyl gallate (0.04 g / L) and tea polyphenols (0.5 g / L) performed best among all tested combinations, with all key indicators reaching their peak values, fully demonstrating the synergistic effect between propyl gallate and tea polyphenols.

[0097] Example 3

[0098] Fermentation was carried out using the method of Example 2, with the only difference being the optimization of the addition time of propyl gallate and tea polyphenols. Tests were conducted at 0h, 24h, 48h, 72h, and 96h, with propyl gallate at 0.04 g / L and tea polyphenols at 0.5 g / L. The results are as follows... Figure 2 As shown, the oil yields obtained by adding a mixed antioxidant consisting of 0.04 g / L propyl gallate and 0.5 g / L tea polyphenols at 0 h, 24 h, 48 h, 72 h, and 96 h were 24.2 g / L, 36.4 g / L, 51.1 g / L, 45.1 g / L, and 38.7 g / L, respectively. The highest oil content (51.1 g / L) was obtained when the mixed antioxidant was added after 48 h of fermentation, representing a 111% increase compared to adding it at the beginning of fermentation, and increases of 40.3%, 13.3%, and 32.0% compared to other addition times, respectively. The oil yield decreased with delayed addition of the mixed antioxidant combination. This is because the mixed antioxidant combination promotes oil synthesis by Schizochytrium, and the later the addition, the shorter the effective period. Conversely, adding it too early inhibits the growth rate of Schizochytrium. Therefore, adding a mixture of propyl gallate and tea polyphenols as antioxidants after 48 hours of fermentation is beneficial for the accumulation of oil in Schizochytrium.

[0099] Example 4

[0100] Fermentation was carried out using the optimal method described in Example 3. At 48 hours, propyl gallate, a single antioxidant, was added to the fermentation medium at a specific concentration gradient (0 g / L, 0.01 g / L, 0.02 g / L, 0.04 g / L, 0.06 g / L, 0.08 g / L, 0.1 g / L, 0.12 g / L). Fermentation was stopped at 120 hours, and various indicators were measured. The results are shown in Table 3.

[0101] Table 3 Fatty acid yield with the addition of a single antioxidant, propyl gallate.

[0102]

[0103] As shown in Table 3, in the group without propyl gallate, 25.5 g / L of oil was obtained after Schizochytrium fermentation, with a DHA content of 50.88% and an EPA content of 4.49%. After adding different concentrations of propyl gallate, the biomass, oil content, and DHA content of Schizochytrium increased in all groups. The group with 0.08 g / L propyl gallate showed the highest oil content, reaching 46.08 g / L. Furthermore, the addition of 0.08 g / L propyl gallate effectively increased the EPA content of the oil produced by Schizochytrium fermentation, reaching 9.45%, more than double that of the control group without propyl gallate. The amount of propyl gallate added is crucial; both excessively low and high concentrations are ineffective, and even minute changes can lead to significant variations in various indicators.

[0104] Example 5

[0105] Fermentation was carried out using the optimal method described in Example 3. At 48 hours, the single antioxidant tea polyphenol was added to the fermentation medium at a specific concentration gradient (0 g / L, 0.4 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.6 g / L, 2 g / L). Fermentation was stopped at 120 hours, and various indicators were measured. The results are shown in Table 4.

[0106] Table 4. Fatty acid yield with the addition of tea polyphenols as a single antioxidant.

[0107]

[0108] As shown in Table 4, in the group without added tea polyphenols, 25.5 g / L of oil was obtained after Schizochytrium fermentation, with a DHA content of 50.88% and an EPA content of 4.49%. After adding different concentrations of tea polyphenols, the biomass, oil content, and DHA content of Schizochytrium increased in all groups. The group with added 1.6 g / L tea polyphenols had the highest oil content, reaching 45.2 g / L. Furthermore, adding 1.6 g / L tea polyphenols effectively increased the EPA content of the oil produced by Schizochytrium fermentation, reaching 8.6%, which is more than 90% higher than the control group without added tea polyphenols. The amount of tea polyphenols added is crucial; both too low and too high concentrations are ineffective, and even slight changes can lead to significant changes in various indicators.

[0109] In the fermentation of Schizochytrium, the addition of either propyl gallate or tea polyphenols alone can promote the fermentation results to a certain extent, but the improvement is limited and a relatively high concentration is required to be effective. However, according to Example 2, the combination of the two at low doses has a synergistic effect. The combined use of propyl gallate and tea polyphenols not only reduces the required concentration but also has a more significant promoting effect.

[0110] As shown in the table, the optimal combination (propyl gallate 0.04 g / L + tea polyphenols 0.5 g / L) achieved a decisive advantage in all core indicators. Compared with the single-factor propyl gallate optimal group (Example 4), its oil yield, DHA yield, EPA yield, and PUFA percentage were 10.9%, 12.4%, 32.4%, and 5.1% higher, respectively; compared with the single-factor tea polyphenol optimal group (Example 5), the leading margin of the above indicators further expanded to 13.1%, 14.2%, 48.1%, and 4.3%. This clearly shows that through the synergistic effect of propyl gallate and tea polyphenols, the performance "ceiling" achievable by a single antioxidant was successfully broken.

[0111] The EPA yield of the optimal combination (11.28 g / L) not only far exceeded the effect of any single additive at its respective optimal concentration, but its increase (150% higher than the control) was also far greater than the simple sum of the increases of the two single-factor optimal groups. This confirms that propyl gallate and tea polyphenols are not simply functionally additive, but rather work through different mechanisms of action. For example, propyl gallate, as a lipid-soluble antioxidant, may be more effective in protecting intracellular lipids from oxidation, while tea polyphenols, as polyphenols, may simultaneously possess antioxidant and regulatory functions on the activity of specific metabolic enzymes, resulting in a strong positive synergy and jointly constructing a more efficient and stable intracellular synthesis environment.

[0112] In summary, the combined formulation of propyl gallate (0.04 g / L) and tea polyphenols (0.5 g / L) is the only and optimal solution for achieving high oil and EPA yields in this technical approach. Its experimental results are significant, far exceeding any single component or other concentration combinations, which is something that those skilled in the art could not easily foresee or obtain through conventional single-factor optimization. The synergistic effect produced by this specific combination is outstanding, bringing unexpected technological advancements and providing a novel and efficient antioxidant application strategy for the efficient production of ω-3 fatty acids via microbial fermentation. It possesses outstanding substantive characteristics and significant progress, fully meeting the inventiveness and practicality requirements for patent authorization. Furthermore, it is evident that 0.04 g / L propyl gallate and 0.5 g / L tea polyphenols in the method of this invention have a synergistic effect, synergistically increasing the yield of oil, DHA, EPA, and PUFA obtained through fermentation.

[0113] Example 6

[0114] To assess the effects of antioxidants on cellular oxidative defense systems, ROS levels in *Schizochytrium* cells were measured. The specific procedure for ROS determination was as follows: The relative ROS content of the samples was determined using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). The DCFH-DA fluorescent probe was dissolved in DMSO to 1 mM and stored at -20°C for later use. It was then added to the cell culture at a 1:100 volume ratio and incubated in the dark at 30°C and 170 rpm for 20 minutes to allow sufficient probe penetration into the cells. The cell samples were washed twice with 0.01 M PBS buffer to thoroughly remove extracellular DCFH-DA, and then dissolved in 0.01 M PBS buffer. Finally, the relative ROS content within the cells was measured using a multi-mode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results are shown below. Figure 3 As shown, the exogenous addition of single antioxidants, propyl gallate and tea polyphenols, both help to reduce intracellular ROS levels. However, the addition of a mixed antioxidant combination of 0.04 g / L propyl gallate and 0.5 g / L tea polyphenols has a more significant effect on reducing ROS levels. Its ROS value is significantly lower than that of the control group, which effectively inhibits intracellular lipid peroxidation during Schizochytrium fermentation, reduces oxidative damage to cells, and improves the yield, quality and oxidative stability of EPA oil.

[0115] The fermentation method is as follows: the strain is inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 hours to obtain primary seed culture; the primary seed culture is inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 hours to obtain secondary seed culture; the secondary seed culture is inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 hours to obtain tertiary seed culture.

[0116] The tertiary seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented for 120 h at a temperature of 28℃ and a rotation speed of 180 rpm. An oxidant was added to the fermentation broth 48 h after the start of fermentation.

[0117] The oils produced by *Schizochytrium* fermentation in Examples 2 (propyl gallate (0.04 g / L) + tea polyphenols (0.5 g / L)), 4 (0.08 g / L propyl gallate), 5 (1.6 g / L tea polyphenols), and the control were tested. Twenty samples were taken from each group, and the peroxide value, anisidine value, and yield of the algal oil in each group were measured. The results are shown in Table 5.

[0118] Table 5. Oxidative stability of lipids produced by Schizochytrium fermentation without and with antioxidants.

[0119]

[0120] As shown in Table 5, the peroxide value of the Schizochytrium algal oil produced in Examples 2 to 5 is ≤0.06 / 100g, and the anisidine value is <2, which is significantly better than the requirement of ≤0.06 peroxide value in the current national standard GB 1903.66-2024, National Food Safety Standard, Food Fortifiers, Docosahexaenoic Acid Oils (Fermentation Method) [S]. The antioxidant capacity of the group with added exogenous antioxidants is better than that of the control group, and the oxidative stability of the group with added mixed antioxidants propyl gallate and tea polyphenols is significantly better than that of other groups. It can also be seen that the final concentration of propyl gallate added in the method of the present invention is 0.04g / L, and the final concentration of tea polyphenols added is 0.5g / L. The two have a synergistic effect, which can synergistically improve the fatty acid yield of the prepared product, especially the rapid accumulation of EPA by the oil-producing Schizochytrium.

[0121] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for high EPA production based on stress pre-acclimatization and compound antioxidant Schizochytrium, characterized in that: The method involves subjecting the Schizochytrium seed culture to oxidative stress pre-acclimatization, followed by fermentation culture, with the addition of compound exogenous antioxidants propyl gallate and tea polyphenols in the later stage of fermentation; the stress pre-acclimatization and compound antioxidants can interact to synergistically regulate the metabolic network of Schizochytrium, significantly increasing the synthesis efficiency and stability of EPA. The oxidative stress pre-adaptation refers to the process of culturing activated Schizochytrium seed culture under blue light irradiation at an intensity of 6000 lx for 10-20 generations with shaking to obtain an enhanced seed culture with improved stress resistance. The Schizochytrium seed liquid was obtained by activating Schizochytrium seeds through three generations. A compound exogenous antioxidant was added at 48 hours of fermentation, with the final concentration of propyl gallate being 0.04 g / L and the final concentration of tea polyphenols being 0.5 g / L. The Schizochytrium strain selected was Schizochytrium sp HX-308. The method for culturing the Schizochytrium seed culture includes: Schizochytrium is stored in a 20% glycerol preservation tube at -80℃. When activating the seeds, 1 mL of the glycerol-preserved Schizochytrium is inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a constant temperature shaker under the following conditions: 30℃, 170 rpm, and 24 h. Under these conditions, the first generation of seeds is activated. The activated seeds are then transferred at a volume ratio of 1% to a new 500 mL Erlenmeyer flask containing 100 mL of the culture medium and cultured under the same conditions to obtain the seed culture. The fermentation culture method includes: inoculating the seed culture of Schizochytrium into the fermentation medium at a volume ratio of 10% with the seed culture of the fungus after three generations of culture; fermenting at a temperature of 25-30℃ and a rotation speed of 150-220 rpm for 96-144 hours; adding the compound exogenous antioxidants propyl gallate and tea polyphenols to the fermentation broth 48 hours after the start of fermentation, so that the final concentration of propyl gallate added during the fermentation culture is 0.04 g / L and the final concentration of tea polyphenols is 0.5 g / L; and the fermentation broth is obtained after the fermentation is completed.

2. The method according to claim 1, characterized in that: The seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min; The fermentation medium formulation includes: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B... 12 0.1-0.5 mg / L, pH 6.0-6.5, sterilized at 121℃ for 20 min.

3. The method according to claim 1, characterized in that: The specific steps are as follows: (1) Blue light stress: The activated seed liquid of Schizochytrium, which had been cultured in seed culture medium for two generations, was inoculated into seed culture medium at a volume of 2%. The culture was carried out under blue light stimulation conditions of 6000 lx intensity, with culture conditions of 170 rpm and 30℃. After 24 h of culture, the seed liquid was inoculated into new seed culture medium at a volume of 5% for passage. This was continued for 20 generations to obtain the 20th generation of domesticated strains under blue light stimulation. The seed culture medium formula includes: glucose 40-60 g / L, yeast extract 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, monosodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5, sterilized at 121℃ for 20 min. (2) The 20th generation of domesticated strains was fermented under blue light stimulation, as follows: The strain was inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 28℃ and 180 rpm for 24 h to obtain tertiary seed culture. The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 28℃ and 180 rpm for 120 h. 48 h after the start of fermentation, a compound antioxidant, propyl gallate and tea polyphenols, were added to the fermentation broth. The final concentration of propyl gallate was 0.04 g / L, and the final concentration of tea polyphenols was 0.5 g / L. The seed culture medium formula includes: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5, sterilized at 121℃ for 20min before use. The fermentation medium formula includes: glucose 80 g / L, yeast extract 10 g / L, sodium sulfate 8 g / L, magnesium sulfate 3 g / L, ammonium sulfate 6 g / L, potassium chloride 1.5 g / L, calcium chloride 0.15 g / L, potassium sulfate 0.8 g / L, potassium dihydrogen phosphate 1.5 g / L, monosodium glutamate 18 g / L, zinc sulfate heptahydrate 3 mg / L, cobalt chloride hexahydrate 0.05 mg / L, copper sulfate pentahydrate 4 mg / L, nickel sulfate hexahydrate 1.5 mg / L, ferrous sulfate heptahydrate 12 mg / L, calcium pantothenate 3 mg / L, manganese chloride tetrahydrate 4 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 7 mg / L, vitamin B... 12 0.3 mg / L, pH 6.0, sterilized at 121℃ for 20 min before use.

4. The application of the method as described in any one of claims 1 to 3 in the production of EPA.

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