Application and method of tea polyphenol in improving schizochytrium limacinum dha oil yield and oil storage

Abstract

This invention belongs to the field of biochemical technology and discloses the application and method of tea polyphenols in improving the DHA oil yield and oil storage of Schizochytrium. Tea polyphenols are added during the DHA production process of Schizochytrium fermentation. By comparing the differences between tea polyphenol fermentation and conventional fermentation in Schizochytrium, this invention clarifies that the antioxidant tea polyphenols regulate cellular oxidative stress in Schizochytrium, scavenge ROS, and inhibit oil oxidation, thereby delaying oil rancidity and increasing oil synthesis and DHA production. Using Schizochytrium HX-308 as the recipient strain, this invention achieves fermentation conditions with significantly increased oil and DHA content and better oil storage performance by adding the antioxidant tea polyphenols.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, and in particular to the application and method of tea polyphenols in improving the DHA oil yield and oil storage of Schizochytrium. Background Technology

[0002] Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) essential for the development of the human nervous system and retinal tissue. Epidemiological studies have shown that dietary DHA intake offers numerous health benefits, leading to its widespread use in infant formula, nutritional supplements, food supplements, and pharmaceuticals. Traditionally, DHA is extracted from marine fish oil, but this method is unsustainable due to environmental pollution and overfishing, and production cannot meet the growing market demand. Oil-bearing microorganisms are considered potential DHA producers and are receiving increasing attention due to their superiority over fish oil. Schizochytrium, a heterotrophic marine protozoan rich in DHA, is widely used in research and commercial production due to its rapid growth, high DHA content, and ease of cultivation. To date, Schizochytrium has been found to accumulate fatty acids primarily composed of DHA, DPA (docosapentaenoic acid), C16:O, and C14:O. Foods containing omega-3 polyunsaturated fatty acids (PUFAs) such as DHA and EPA (eicosapentaenoic acid) are considered important factors in promoting retinal and brain function development and reducing the risk of cardiovascular disease. Given the significant benefits of omega-3 fatty acids and the increasing demand, increasing the proportion of omega-3 fatty acids, especially DHA, in the total fatty acids of Schizochytrium has become a focus of attention.

[0003] Typically, oleotrophic microorganisms produce small amounts of lipids under optimal growth conditions, while stress promotes lipid production. Stress-induced strategies such as nitrogen consumption, phosphate starvation, and salt stress have been used to improve DHA production in Schizochytrium. However, stress-induced strategies can inhibit microbial growth, thereby reducing the overall productivity of biomass and lipids. Studies have shown that high levels of reactive oxygen species (ROS) can act on DNA, proteins, and lipids, inducing oxidative damage that leads to loss of protein function, even cell death, and PUFA peroxidation.

[0004] Oxidative damage caused by high levels of reactive oxygen species (ROS) to Schizochytrium cells can be mitigated by adding antioxidants such as melatonin, sesamol, or ascorbic acid. Tea polyphenols, a type of polyhydroxyphenol, possess antioxidant and free radical scavenging properties. Studies have shown that tea polyphenols can scavenge ROS and inhibit lipid oxidation, and even small amounts can delay rancidity in lipids, making them widely used as additives in food and pharmaceuticals. Exogenous addition of antioxidants to fermentation media helps reduce intracellular ROS levels, alleviate oxidative damage to cells, and promote lipid synthesis in oily microorganisms. Furthermore, it is noteworthy that if the antioxidant concentration exceeds a certain threshold, it can inhibit cell growth and lipid production. Therefore, the concentration of added antioxidants is crucial for regulating cellular oxidative stress and DHA production in Schizochytrium. However, traditional strategies have focused on enhancing lipid and DHA production in Schizochytrium through chemical regulators, with relatively little research on the effects of chemical regulators on late-stage lipid storage and prevention of DHA degradation. Compared with traditional fermentation technology, this invention effectively inhibits intracellular lipid peroxidation during the fermentation of Schizochytrium by adding appropriate antioxidants, tea polyphenols. This increases DHA lipid yield and results in higher quality lipids during storage. Furthermore, research has shown that adding tea polyphenols significantly increases carotenoid content compared to traditional fermentation, confirming that the lipids produced using this strategy have better storage properties than those produced using traditional methods. The DHA degradation rate of this strategy is also much lower than that of lipids produced using traditional methods, which is significant for lipid quality and storage. However, research indicates that the lipid solubility of tea polyphenols is not significant. Exploring and determining the optimal concentration of tea polyphenols during the fermentation process is of profound significance for significantly improving the quality of lipid products and optimizing production efficiency. This research not only aims to explore the potential of tea polyphenols as a natural additive but also strives to optimize and upgrade the lipid production process through scientific methods. Currently, there are no reports on using exogenous tea polyphenols to regulate Schizochytrium fermentation for lipid production, improve lipid storage, and slow DHA degradation. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application and method for tea polyphenols in increasing the DHA oil yield and oil storage of Schizochytrium.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] Application of a type of tea polyphenol in improving the DHA oil yield and oil storage of Schizochytrium.

[0008] Furthermore, tea polyphenols were added during the fermentation process of Schizochytrium to produce DHA.

[0009] Furthermore, the addition of tea polyphenols during the fermentation process of the Schizochytrium fungus can significantly increase the DHA content and oil yield. Under the optimal tea polyphenol concentration of 2 g / L, the oil yield increased by 42%, and the DHA content increased to 49.34%. The final DHA oil yield was 9.77 g / L, which is 51% higher than that of traditional fermentation without the addition of tea polyphenols. The oil produced by fermentation with added tea polyphenols has better quality when stored at room temperature than that produced by traditional fermentation. Among the oils produced under the same conditions, room temperature storage has the highest economic value when stored under different conditions.

[0010] A method for increasing lipid synthesis and DHA production by adding the antioxidant tea polyphenol to regulate cellular oxidative stress in Schizochytrium involves adding tea polyphenol after 12 hours of Schizochytrium fermentation, with a final concentration of 2 g / L.

[0011] Furthermore, it includes the following steps:

[0012] After the Schizochytrium strain was activated by inoculating it into a seed culture medium, a fermentation strain was obtained. The fermentation strain was then inoculated into a fermentation culture medium for fermentation. After 12 hours of Schizochytrium fermentation, tea polyphenols were added at a final concentration of 2 g / L. The cells were then collected to extract the oil.

[0013] Alternatively, the schistocytic fungus may be selected from oil-producing schistocytic fungus HX-308, ATCC 20888, or schistocytic fungus CCTCCAF 2010001.

[0014] Furthermore, the fermentation strain is obtained by the following method: inoculating Schizochytrium into a seed culture medium and culturing for 24 hours to obtain a primary seed; taking the primary seed and inoculating it into a seed culture medium and culturing for 24 hours to obtain a secondary seed; taking the secondary seed and inoculating it into a seed culture medium and culturing for 24 hours to obtain a tertiary seed, which is used as the fermentation strain;

[0015] The culture conditions are 25–30℃ and shaking culture at 150–250 r / min.

[0016] Furthermore, the specific steps are as follows:

[0017] Single colonies of *Schizochytrium oleiferum* were selected and inoculated into seed culture medium. After culturing at 28°C and 180 rpm for 24 h, they were classified as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation.

[0018] Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of fermentation medium. Add tea polyphenols at a final concentration of 2 g / L after 12 h of fermentation. Culture in a shaker at 28℃ and 180 r / min for 120 h to obtain the fermentation broth.

[0019] After fermentation culture is completed, add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.2% of cell wall breaking enzyme, and shake at 100-200 r / min for 5-15 h at 40-60℃.

[0020] Cool to room temperature and add anhydrous ethanol equal in volume to the cell wall-breaking fermentation broth to inactivate the cell wall-breaking enzyme;

[0021] Add an equal volume of n-hexane to the fermented liquid after cell wall disruption to extract the oil, and repeat the extraction process three times by adding the same volume of n-hexane.

[0022] After extraction, the organic phase was allowed to stand for 5 hours until the upper and lower layers clearly separated. The upper organic phase was then collected. The upper organic phase was removed and placed in a rotary evaporator flask. The organic phase was then evaporated by rotary evaporation at 45°C in a water bath and at 120 rpm. After the organic phase stopped evaporating, the rotary evaporator flask was removed and dried in a 60°C oven until its weight no longer changed. The oil was then weighed to obtain the oil.

[0023] Furthermore, the entire fermentation culture cycle is 48 hours to 120 hours.

[0024] Furthermore, the entire fermentation culture cycle is 48h, 60h, 72h, 84h, 96h, or 120h.

[0025] Furthermore, the Schizochytrium strain was inoculated onto a plate medium and cultured on 500 μg / L G418 resistant plates in a 28°C incubator for 72 h. Then, a single colony was picked and inoculated onto a seed culture medium for activation.

[0026] The plate culture medium has a pH of 6.0–6.5 and comprises: agar 15–20 g / L, glucose 30–60 g / L, yeast extract 8–15 g / L, sodium sulfate 10–15 g / L, magnesium sulfate 2–4 g / L, ammonium sulfate 6–12 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 dichloride 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 0.04 mg / L, and vitamin B6 4–10 mg / L. mg / L, Vitamin B12 0.1-1.5 mg / L, solvent is water;

[0027] Alternatively, the seed culture medium has a pH of 6.0–6.5 and comprises: 40–60 g / L glucose, 4–6 g / L yeast extract, 5–8 g / L sodium sulfate, 2–4 g / L magnesium sulfate, 4–8 g / L ammonium sulfate, 1–2 g / L potassium chloride, 0.1–0.2 g / L calcium chloride, 0.5–1 g / L potassium sulfate, 0.5–2 g / L potassium dihydrogen phosphate, 8–12 g / L monosodium glutamate, 1–5 mg / L zinc sulfate heptahydrate, 0.01–0.1 mg / L cobalt dichloride hexahydrate, 2–6 mg / L copper sulfate pentahydrate, 1–2 mg / L nickel sulfate hexahydrate, 8–15 mg / L ferrous sulfate heptahydrate, 2–4 mg / L calcium pantothenate, 3–5 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and water as the solvent.

[0028] Alternatively, the fermentation medium has a pH of 6.0–6.5 and comprises: 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 dichloride 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 0.04 mg / L, vitamin B6 4–10 mg / L, and vitamin B1. 12 0.1-0.5 mg / L, solvent is water.

[0029] The advantages and positive effects of this invention are as follows:

[0030] 1. Based on the traditional fermentation of Schizochytrium, this invention significantly increases the oil yield and the proportion of DHA in the oil by adding tea polyphenols. The traditional fermentation yield is 13.9 g / L, while the oil yield is 19.8 g / L at the optimal tea polyphenol concentration of 2 g / L, representing a 42% increase in yield. The DHA content is also increased to 49.34%, and the final DHA oil yield is 9.77 g / L, which is 51% higher than the traditional fermentation without the addition of tea polyphenols.

[0031] 2. By comparing the differences in Schizochytrium in tea polyphenol fermentation and traditional fermentation, this invention clarifies that the antioxidant tea polyphenol regulates cellular oxidative stress in Schizochytrium, scavenges ROS and inhibits lipid oxidation, thereby delaying lipid rancidity and increasing lipid synthesis and DHA production.

[0032] 3. By adjusting the concentration of added tea polyphenols, this invention successfully achieved the accumulation of oils and DHA and improved the quality of Schizochytrium.

[0033] 4. This invention regulates cellular oxidative stress in Schizochytrium by adding the antioxidant tea polyphenol, thereby scavenging ROS, inhibiting lipid oxidation, delaying lipid rancidity, and increasing lipid synthesis and DHA production.

[0034] 5. This invention uses Schizochytrium HX-308 as the recipient strain and adjusts the fermentation conditions by adding the antioxidant tea polyphenols, resulting in significantly increased oil and DHA content and better oil storage performance.

[0035] 6. This invention regulates the fermentation of Schizochytrium by adding tea polyphenols, which significantly increases the content of carotenoids and improves the quality of oil compared with traditional fermentation.

[0036] 7. The oil produced by this invention has better storage effect, lower DHA reduction rate, and is more economical. Attached Figure Description

[0037] Figure 1 This is a graph showing the differences in DHA levels during fermentation of tea polyphenols from Schizochytrium HX-308 at different addition times in this invention.

[0038] Figure 2 The figure shows the effect of different concentrations of tea polyphenols on cell growth and lipid synthesis during the fermentation of Schizochytrium HX-308 tea polyphenols in this invention; where (A) biomass, lipid yield and residual sugar; (B) fatty acid composition (SFA and PUFA); (C) DHA yield; (D) carotenoid content;

[0039] Figure 3 This is a comparative analysis diagram of the antioxidant defense system of Schizochytrium cells in the control (0), 0.1, 0.5, 1, 2, 5, and 10 g / L tea polyphenol groups in this invention; where (A) ROS, (B) SOD, (C) CAT, (D) T-AOC, and (E) MDA.

[0040] Figure 4 This diagram illustrates the possible mechanism by which tea polyphenols exert dual effects on oxidative stress and lipid accumulation in Schizochytrium fungi in this invention. Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0042] 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.

[0043] Application of a type of tea polyphenol in improving the DHA oil yield and oil storage of Schizochytrium.

[0044] Preferably, tea polyphenols are added during the fermentation of Schizochytrium to produce DHA.

[0045] Preferably, the addition of tea polyphenols during the fermentation process of the Schizochytrium fungus can significantly increase the DHA content and oil yield. Under the optimal tea polyphenol concentration of 2 g / L, the oil yield increased by 42%, the DHA content increased to 49.34%, and the final DHA oil yield was 9.77 g / L, which is 51% higher than that of traditional fermentation without the addition of tea polyphenols. The oil produced by fermentation with added tea polyphenols has better quality when stored at room temperature than that of oil produced by traditional fermentation. Among the oils produced under the same conditions, room temperature storage has the highest economic value when stored under different conditions.

[0046] A method for increasing lipid synthesis and DHA production by adding the antioxidant tea polyphenol to regulate cellular oxidative stress in Schizochytrium involves adding tea polyphenol after 12 hours of Schizochytrium fermentation, with a final concentration of 2 g / L.

[0047] Preferably, the steps include:

[0048] After the Schizochytrium strain was activated by inoculating it into a seed culture medium, a fermentation strain was obtained. The fermentation strain was then inoculated into a fermentation culture medium for fermentation. After 12 hours of Schizochytrium fermentation, tea polyphenols were added at a final concentration of 2 g / L. The cells were then collected to extract the oil.

[0049] Alternatively, the schistocytic fungus may be selected from oil-producing schistocytic fungus HX-308, ATCC 20888, or schistocytic fungus CCTCCAF 2010001.

[0050] Preferably, the fermentation strain is obtained by the following method: inoculating Schizochytrium into a seed culture medium and culturing for 24 hours to obtain a primary seed; taking the primary seed and inoculating it into a seed culture medium and culturing for 24 hours to obtain a secondary seed; taking the secondary seed and inoculating it into a seed culture medium and culturing for 24 hours to obtain a tertiary seed, which is used as the fermentation strain;

[0051] The culture conditions are 25–30℃ and shaking culture at 150–250 r / min.

[0052] Preferably, the specific steps are as follows:

[0053] Single colonies of *Schizochytrium oleiferum* were selected and inoculated into seed culture medium. After culturing at 28°C and 180 rpm for 24 h, they were classified as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation.

[0054] Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of fermentation medium. Add tea polyphenols at a final concentration of 2 g / L after 12 h of fermentation. Culture in a shaker at 28℃ and 180 r / min for 120 h to obtain the fermentation broth.

[0055] After fermentation culture is completed, add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.2% of cell wall breaking enzyme, and shake at 100-200 r / min for 5-15 h at 40-60℃.

[0056] Cool to room temperature and add anhydrous ethanol equal in volume to the cell wall-breaking fermentation broth to inactivate the cell wall-breaking enzyme;

[0057] Add an equal volume of n-hexane to the fermented liquid after cell wall disruption to extract the oil, and repeat the extraction process three times by adding the same volume of n-hexane.

[0058] After extraction, the organic phase was allowed to stand for 5 hours until the upper and lower layers clearly separated. The upper organic phase was then collected. The upper organic phase was removed and placed in a rotary evaporator flask. The organic phase was then evaporated by rotary evaporation at 45°C in a water bath and at 120 rpm. After the organic phase stopped evaporating, the rotary evaporator flask was removed and dried in a 60°C oven until its weight no longer changed. The oil was then weighed to obtain the oil.

[0059] Preferably, the entire fermentation culture cycle is 48h to 120h.

[0060] Preferably, the entire fermentation culture cycle is 48h, 60h, 72h, 84h, 96h or 120h.

[0061] Preferably, the Schizochytrium strain is inoculated onto a plate medium, cultured on 500 μg / L G418 resistant plates in a 28°C incubator for 72 h, and then a single colony is picked and inoculated onto a seed culture medium for activation.

[0062] The plate culture medium has a pH of 6.0–6.5 and comprises: agar 15–20 g / L, glucose 30–60 g / L, yeast extract 8–15 g / L, sodium sulfate 10–15 g / L, magnesium sulfate 2–4 g / L, ammonium sulfate 6–12 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 dichloride 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 0.04 mg / L, and vitamin B6 4–10 mg / L. mg / L, Vitamin B 12 0.1-1.5 mg / L, solvent is water;

[0063] Alternatively, the seed culture medium has a pH of 6.0–6.5 and comprises: 40–60 g / L glucose, 4–6 g / L yeast extract, 5–8 g / L sodium sulfate, 2–4 g / L magnesium sulfate, 4–8 g / L ammonium sulfate, 1–2 g / L potassium chloride, 0.1–0.2 g / L calcium chloride, 0.5–1 g / L potassium sulfate, 0.5–2 g / L potassium dihydrogen phosphate, 8–12 g / L monosodium glutamate, 1–5 mg / L zinc sulfate heptahydrate, 0.01–0.1 mg / L cobalt dichloride hexahydrate, 2–6 mg / L copper sulfate pentahydrate, 1–2 mg / L nickel sulfate hexahydrate, 8–15 mg / L ferrous sulfate heptahydrate, 2–4 mg / L calcium pantothenate, 3–5 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, and water as the solvent.

[0064] Alternatively, the fermentation medium has a pH of 6.0–6.5 and comprises: 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 dichloride 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 0.04 mg / L, vitamin B6 4–10 mg / L, and vitamin B1. 12 0.1-0.5 mg / L, solvent is water.

[0065] Specifically, the relevant preparation and testing methods are as follows:

[0066] Determination of glucose and monosodium glutamate (MSG) concentrations: Take 1 mL of fermentation broth at a specific time point, centrifuge at 12000 rpm for 1 minute, and then serially dilute the supernatant to 100-fold. Centrifuge the diluted solution at 12000 rpm for 1 minute and detect using an SBA-40ES. Glucose and MSG are measured using different enzyme membranes, calibrated beforehand with standard solutions.

[0067] Determination of cell dry weight (CDW): Take 5 mL of fermentation broth at a specific time point onto filter paper (determine the filter paper weight in advance) / 1 mL of fermentation broth into an EP tube (determine the EP tube weight in advance), filter, let stand to remove liquid interference / centrifuge to remove liquid interference, then wash the cells with deionized water, and finally place the filter paper / EP tube in a 65℃ oven and dry (about 48 hours) to constant weight, and calculate CDW. Determination of oil content: Place a pre-determined mass of rotary evaporator flask for collecting oil in an oven at 65℃ to constant weight, cool, and weigh.

[0068] Determination of fatty acid composition: Approximately 10 mg of oil or 50 mg of wet bacterial cells were placed in a 2 mL EP tube, and 0.6 mL of 1 M sodium hydroxide-methanol solution was added. The mixture was shaken at 1200 rpm for 5 h at room temperature. The reaction was terminated by adding 50 μL of concentrated sulfuric acid. Extraction was performed with 1 mL of n-hexane, followed by shaking at 1200 rpm for 20 min, and then centrifugation at 12000 rpm for 5 min. The upper n-hexane phase was collected for gas chromatography analysis.

[0069] The carotenoid content was determined by high performance liquid chromatography.

[0070] Determination of ROS and total antioxidant activity (T-AOC): The relative ROS content of the samples was determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The DCFH-DA fluorescent probe was dissolved in DMSO to 1 mM and stored at -20℃ for later use. It was then added to cell culture at a volume ratio of 1:100 and incubated in the dark at 30℃ and 170 rpm for 20 minutes to allow the fluorescent probe to fully penetrate the cells. The cell samples were washed twice with PBS buffer to thoroughly remove extracellular DCFH-DA, and then dissolved in PBS. Finally, the relative ROS content in the cells was determined using a multi-mode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The activities of superoxide dismutase (SOD) and catalase (CAT) in Schizochytrium cells were determined using a SOD activity assay kit (Solarbio, China) and a CAT activity assay kit (Solarbio, China), respectively. According to the manufacturer's instructions, the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of cell samples were determined using the T-AOC assay kit (Solarbio, China) and the MDA assay kit (Solarbio, China).

[0071] The oils were stored at -20℃, 4℃, room temperature, 40℃, and 80℃, respectively, and the oils were evaluated at corresponding time intervals (anisidine value, acid value, peroxide value, and fatty acid composition). The anisidine value was determined by spectrophotometry, while the acid value and peroxide value were determined using a kit.

[0072] This invention utilizes Schizochytrium sp. HX-308 and adds the antioxidant tea polyphenol to regulate cellular oxidative stress in Schizochytrium, thereby increasing lipid synthesis and DHA production, laying the foundation for the industrial production and storage of the product.

[0073] Unless otherwise specified, the equipment, reagents, processes, parameters, etc. involved in this invention are all conventional equipment, reagents, processes, parameters, etc., and no further examples will be provided.

[0074] All ranges listed in this invention include all point values ​​within that range.

[0075] In this invention, unless otherwise specified or generally applicable within the field, % refers to mass percentage and ratio refers to mass proportion. The unit of mass is, for example, grams, kilograms, or tons.

[0076] In this invention, "room temperature" refers to the normal ambient temperature, which can be 10 to 30°C.

[0077] The culture media used in the following examples are as follows:

[0078] The plate culture medium has a pH of 6.0–6.5 and includes: agar 15–20 g / L, glucose 30–60 g / L, yeast extract 8–15 g / L, sodium sulfate 10–15 g / L, magnesium sulfate 2–4 g / L, ammonium sulfate 6–12 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 dichloride 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 0.04 mg / L, and vitamin B6 4–10 mg / L. mg / L, Vitamin B 12 0.1-1.5 mg / L, solvent is water.

[0079] The seed culture medium for the seed solution has a pH of 6.0–6.5 and 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 dichloride 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 0.04 mg / L, and water as the solvent.

[0080] The fermentation medium has a pH of 6.0–6.5 and comprises: 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 dichloride 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 0.04 mg / L, vitamin B6 4–10 mg / L, and vitamin B12. 12 0.1-0.5 mg / L, solvent is water.

[0081] Example 1

[0082] Tea polyphenols regulate cellular oxidative stress in Schizochytrium fungi, scavenge ROS and inhibit lipid oxidation, and increase lipid synthesis and DHA production. (Addition time selection)

[0083] Single colonies of *Schizochytrium oleiferum* HX-308 were selected and inoculated into 250 mL Erlenmeyer flasks (containing 50 mL of seed culture medium). After incubation at 28°C and 180 rpm for 24 h, this was the first-generation seed culture. 1 mL of the first-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and incubated at 28°C and 180 rpm for 24 h to obtain the second-generation seed culture. 1 mL of the second-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and incubated at 28°C and 180 rpm for 24 h to obtain the third-generation seed culture, which was used as the fermentation strain.

[0084] 1. Shake-flask fermentation culture: Take 10 mL of tertiary seed culture and inoculate it into a 500 mL Erlenmeyer flask (containing 90 mL of fermentation medium). Add tea polyphenols at a final concentration of 2 g / L at 0 h / 12 ​​h / 24 h / 36 h of fermentation. Culture in a shaker at 28 ℃ and 180 r / min for 120 h. Collect the oil and measure the fatty acid composition.

[0085] 2. Collect the bacterial cells and extract the oil, including the following steps:

[0086] (1) After fermentation culture is completed, add NaOH solution to adjust the pH to 10-13, then add 0.01-0.2% of cell wall breaking enzyme, shake at 100-200 r / min for 5-15 h at 40-60℃.

[0087] (2) Cool to room temperature and add anhydrous ethanol to inactivate the cell-breaking enzyme in the same volume as the fermentation broth after cell wall disruption;

[0088] (3) Add an equal volume of n-hexane to the fermented liquid after cell wall disruption to extract the oil, and repeat the extraction three times by adding the same volume of n-hexane:

[0089] After extraction, the organic phase was allowed to stand for 5 hours. Once the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was then removed and placed in a rotary evaporator flask. The organic phase was then evaporated by rotary evaporation at 45°C in a water bath and at 120 rpm. Once no more organic phase was evaporated, the rotary evaporator flask was removed and dried in a 60°C oven until its weight no longer changed. The oil was then weighed to obtain the oil.

[0090] (4) Gas phase detection and analysis of fatty acids, the specific procedure is as follows:

[0091] 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. Then, 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 oil. The extracted phase was 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 m * 0.22 mm) and a flame ionization detector (FID) was 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 relevant external standards (Sigma, USA). Using non-endogenous fatty acids (C19:0) as internal standards, the content of individual fatty acids was calculated from the peak area on the chromatogram.

[0092] The results are as follows Figure 1 As shown, the proportion of DHA in fermentation without the addition of tea polyphenols is 46%, the proportion of DHA in fermentation with the addition of tea polyphenols at the beginning is 47%, the proportion of DHA in fermentation with the addition of tea polyphenols at a final concentration of 2g / L after 12 hours of fermentation is 49%, the proportion of DHA in fermentation with the addition of tea polyphenols after 24 hours of fermentation is 48%, and the proportion of DHA in fermentation with the addition of tea polyphenols after 36 hours of fermentation is 47%. In summary, the 12-hour addition condition is the optimal condition.

[0093] Example 2: Tea polyphenols regulate cellular oxidative stress in Schizochytrium fungi, scavenge ROS, inhibit lipid oxidation, and increase lipid synthesis and DHA production. (The text then abruptly shifts to a different topic:) Screening for the amount of tea polyphenols added.

[0094] Different masses of tea polyphenols were weighed and added to the fermentation culture medium.

[0095] Seed culture was carried out according to Example 1. After 12 hours of fermentation, 0 / 0.1 / 0.5 / 1 / 2 / 5 / 10 g / L of tea polyphenols were added for fermentation. Biomass, oil yield, oil content and residual sugar; fatty acid composition; fatty acid composition (SFA and PUFA); DHA yield; DHA content; carotenoid content were measured.

[0096] Includes the following steps:

[0097] (1) The specific procedure for determining the concentrations of glucose and monosodium glutamate is as follows: Take 1 mL of fermentation broth at a specific time point, centrifuge at 12000 rpm for 1 minute, and take the supernatant for serial dilution to 100 times. Centrifuge the diluted solution at 12000 rpm for 1 minute and detect it using SBA-40ES. Glucose and monosodium glutamate are measured using different enzyme membranes, and are calibrated with standard solutions in advance.

[0098] (2) Determination of cell dry weight (CDW): The specific procedure is as follows: Take 5 mL of fermentation broth at a specific time point onto filter paper (determine the quality of the filter paper in advance) / 1 mL of fermentation broth onto an EP tube (determine the quality of the EP tube in advance), filter, let stand to remove liquid interference / centrifuge to remove liquid interference, then wash the cells with deionized water, and finally place the filter paper / EP tube in a 65℃ oven and dry (about 48 hours) to constant weight, and calculate CDW. Determination of oil content: Place a pre-determined mass of rotary evaporator flask for collecting oil in an oven at 65℃ to constant weight, cool and weigh.

[0099] (3) Fatty acid composition detection is as described in Example 1 above.

[0100] (4) Take 5 ml of the fermentation broth that has just finished fermentation, centrifuge it, add an equal volume of methanol and soak it several times for extraction. Combine the methanol extracts, remove the methanol using a rotary vacuum evaporator, add carbon tetrachloride to dissolve the concentrate, and filter it through a 0.22 μm organic filter membrane for later use. The content of carotenoids was determined by high performance liquid chromatography (HPLC). Chromatographic column: Amethyst C18-H column (4.6 x 150 mm, 5 μm), detection wavelength 475 nm, column temperature at room temperature, mobile phase flow rate 1.0 mL / min, gradient elution, and corresponding carotenoid standard samples were prepared to obtain the standard curve equation.

[0101] The results are as follows Figure 2 As shown, with increasing concentrations of tea polyphenols, biomass and oil yield first increased and then decreased. 2 g / L of tea polyphenols was the optimal addition for fermentation, at which point the biomass was 60.6 g / L and the oil yield was the highest at 19.8 g / L. Compared to the control group (i.e., no tea polyphenols added), the total oil yield increased by 42%. Table 1 shows the results comparing fermentation with and without tea polyphenols: the group with added tea polyphenols showed increased oil yield and a significant change in fatty acid composition, with a marked increase in the DHA proportion compared to the control group, rising from 46.47% in the control group to 49.34% in the 2 g / L tea polyphenol group. Compared with the control group (0 g / L), the addition of an appropriate concentration of tea polyphenols (2 g / L) significantly increased biomass, oil production, and DHA production. Furthermore, the carotenoid content in the control group (0 g / L) was 47.8 μg / g, while the carotenoid content in the 2 g / L tea polyphenol-added group reached as high as 151 μg / g. The carotenoid content in the polyphenol-added group was also higher than that in the control group, indicating a significant increase in carotenoid content, which is of great importance for oil storage.

[0102] Meanwhile, by comparing Example 1 and Example 2, it can be seen that the two conditions of adding tea polyphenols at a final concentration of 2 g / L and adding tea polyphenols at a time of 12 hours after fermentation have a synergistic effect, which can synergistically improve the yield of the prepared DHA oil and the storage time of the oil and other related indicators.

[0103] Example 3: Data Analysis of the Dual Effects of Tea Polyphenols on the Fermentation Characteristics of Schizochytrium

[0104] Data analysis based on the data measured in Example 2 showed that the dual antioxidant / pro-oxidant activity of the antioxidant tea polyphenols was related to its concentration.

[0105] Figure 2The results showed that as the concentration of added tea polyphenols increased, biomass, total oil production, and DHA production first increased and then decreased. When 2 g / L of tea polyphenols was added, biomass, total oil production, and DHA production all reached their maximum values, at 60.6 g / L, 19.8 g / L, and 9.77 g / L, respectively. However, when 10 g / L was added, the biomass, total oil production, and DHA production were 43 g / L, 13.4 g / L, and 5.81 g / L, respectively, all lower than the corresponding control group (0 g / L). Therefore, the addition of appropriate amounts of antioxidants is beneficial to the production of oil and DHA in Schizochytrium, while high concentrations have a negative effect.

[0106] Example 4: Comparative Analysis of the Antioxidant Defense System of Schizochytrium Cells in Tea Polyphenol Groups

[0107] To mitigate ROS-induced oxidative damage, microorganisms can activate enzymatic and non-enzymatic antioxidant defense mechanisms, including SOD, CAT, and other antioxidant enzymes, as well as intracellular antioxidants. To evaluate the effects of 0, 2, and 10 g / L tea polyphenols on the cellular oxidative defense system, ROS, SOD, CAT, T-AOC, and MDA levels in *Schizochytrium* cells were measured. Other related preparation methods were performed according to Example 2.

[0108] The specific procedures for determining ROS, SOD, CAT, T-AOC, and MDA are as follows: The relative ROS content of the sample was determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The DCFH-DA fluorescent probe was dissolved in DMSO to 1 mM and stored at -20℃ for later use. It was then added to the cell culture at a volume ratio of 1:100 and incubated in the dark at 30℃ and 170 rpm for 20 minutes to allow the fluorescent probe to fully penetrate the cells. The cell samples were washed twice with PBS buffer to thoroughly remove extracellular DCFH-DA, and then dissolved in PBS. 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. The activities of superoxide dismutase (SOD) and catalase (CAT) in *Schizochytrium* cells were determined using a SOD activity assay kit (Solarbio, China) and a CAT activity assay kit (Solarbio, China), respectively. According to the manufacturer's instructions, the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of cell samples were determined using the T-AOC assay kit (Solarbio, China) and the MDA assay kit (Solarbio, China).

[0109] The results are as follows Figure 3As shown, the exogenous addition of the antioxidant tea polyphenols helps reduce intracellular ROS levels. After adding 2 g / L of tea polyphenols, the ROS values ​​were all lower than those of the control group (0 g / L), effectively inhibiting intracellular lipid peroxidation during Schizochytrium fermentation, reducing oxidative damage to cells, and improving DHA lipid yield, quality, and production efficiency. The possible mechanism by which tea polyphenols exert a dual effect on oxidative stress and lipid accumulation in Schizochytrium in this invention can be described as follows: Figure 4 As shown.

[0110] Example 5: Detection of oil storage indicators in different environments produced by normal fermentation and fermentation with the optimal concentration of tea polyphenols under different conditions.

[0111] Oils obtained from normal fermentation and fermentation with the addition of 2 g / L tea polyphenols (other related preparation methods were carried out according to Example 2) were quantitatively stored at -20℃, 4℃, room temperature, 40℃, and 80℃, respectively, using the same fermentation medium. Oil parameters (acid value, peroxide value, fatty acid composition) were measured at 0, 1, 3, 5, 7, 10, 15, 30, 60, 90, 180, and 360 days of storage.

[0112] Acid value and peroxide value were detected using a kit according to the instructions, and fatty acid composition was analyzed by gas chromatography, as described in Example 1.

[0113] The results are shown in Tables 2 and 3. Oils fermented with the optimal concentration of 2 g / L tea polyphenols were more stable under the same storage conditions than oils fermented normally. Both the acid value and peroxide value were significantly lower in the fermented group, indicating superior quality. At -20°C and 80°C, the initial acid values ​​of the oils in the normally fermented group were 0.9 and 1.2 mg(KOH) / g, respectively, increasing to 1.35 and 2.1 mg(KOH) / g after 30 days. In contrast, the initial acid values ​​in the tea polyphenol group were 0.75 and 1.05 mg(KOH) / g, respectively, increasing only to 1.2 and 1.8 mg(KOH) / g after 30 days, showing even lower initial and final acid values. When stored at -4°C, room temperature, and 40°C for 30 days, the acid values ​​of the oils in the normally fermented group were 0.15, 0.15, and 0.3 mg(KOH) / g higher than those in the tea polyphenol group, respectively. In terms of peroxide value, the oil obtained from normal fermentation shows peroxide phenomenon in about 15 days, while the tea polyphenol group shows peroxide phenomenon in about 30 days.

[0114] Regarding the stability of PUFA, the PUFA content of normally fermented oil decreased from 64.2% to 60.9% after 360 days at -20°C, to 56.0% after 360 days at -4°C, to 47.2% after 360 days at room temperature, to 43.9% after 360 days at 40°C, and to 48.2% after 60 days at 80°C. In contrast, the PUFA content of oil obtained with added tea polyphenols was initially 71.4%, decreasing to 70.4% after 360 days at -20°C, to 66.5% after 360 days at -4°C, to 58.1% after 360 days at room temperature, to 50.6% after 360 days at 40°C, and to 55.8% after 60 days at 80°C.

[0115] Regarding the stability of DHA, the DHA content of normally fermented oils decreased from 46.4% to 44.7% after 360 days at -20°C, to 42.0% after 360 days at -4°C, to 32.0% after 360 days at room temperature, to 30.6% after 360 days at 40°C, and to 35.5% after 60 days at 80°C. In contrast, the oils obtained with added tea polyphenols had an initial DHA content of 49.3%, which decreased to 48.7% after 360 days at -20°C, to 48.1% after 360 days at -4°C, to 39.9% after 360 days at room temperature, to 36.7% after 360 days at 40°C, and to 42.3% after 60 days at 80°C.

[0116] Therefore, oils obtained through fermentation under conditions of 2 g / L tea polyphenols exhibit better storage performance, higher PUFA and DHA content, and slower PUFA degradation. Furthermore, optimal storage at room temperature with the addition of antioxidant tea polyphenols yields the highest economic value.

[0117] Table 1. Comparison of fatty acid composition between normal fermentation and fermentation with added tea polyphenols using Schizochytrium.

[0118]

[0119] Table 2 Comparison of storage parameters of oils produced by normal fermentation and fermentation with optimal concentration of tea polyphenols under different environments.

[0120]

[0121]

[0122]

[0123] Table 3. Comparison of PUFA and DHA content in oils produced by normal fermentation and fermentation with optimal concentration of tea polyphenols under different storage environments.

[0124]

[0125]

[0126] Example 6

[0127] This invention replaces *Schizochytrium* HX-308 with *Schizochytrium* ATCC 20888 (purchased from the American Center for Type Culture Collection) and *Schizochytrium* CCTCC AF 2010001 (purchased from the China Center for Type Culture Collection) for testing. Fermentation conditions were the same as for *Schizochytrium* HX-308 (i.e., other related preparation methods were carried out according to Example 2), and the oil and fatty acid composition and lipid storage performance under room temperature conditions were tested. The fermentation and experimental strategies and procedures were the same as in Examples 1-5.

[0128] Table 4. Effects of tea polyphenols on the fermentation performance of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001

[0129]

[0130] The effects of tea polyphenols on the fermentation performance of *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* are shown in Table 4. Compared with normal fermentation, the tea polyphenol group increased the proportions of DHA and PUFA in *Schizochytrium ATCC 20888* from 35.6% and 48.3% to 42.1% and 56.5%, respectively, and the lipid yield also increased from 12.5 g / L to 17.2 g / L. Furthermore, compared with normal fermentation, the tea polyphenol group increased the proportions of DHA and PUFA in *Schizochytrium CCTCC AF 2010001* from 33.2% and 43.2% to 41.2% and 60.2%, respectively, and the lipid yield also increased from 10.2 g / L to 16.7 g / L. Therefore, tea polyphenols significantly improved the DHA, PUFA, and lipid production capacity of Schizochytrium ATCC 20888 and Schizochytrium CCTCAF 2010001.

[0131] Table 5. Effects of tea polyphenols on the storage acid value of fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCAF 2010001* under ambient temperature conditions.

[0132]

[0133]

[0134] Table 5 shows the effect of tea polyphenols on the storage acid value of oils fermented by *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* under normal temperature conditions. The initial acid value of the oils in the tea polyphenol group was 0.2 mg (KOH) / g lower than that in the *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* groups, respectively, compared to the normal fermentation group. After one year of storage, the acid values ​​of the oils fermented by *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* reached 2.85 mg (KOH) / g and 2.45 mg (KOH) / g, respectively, while those in the tea polyphenol group were only 2.45 mg (KOH) / g and 2.35 mg (KOH) / g. Fermentation of tea polyphenols has advantages for the storage of oils from Schizochytrium ATCC 20888 and Schizochytrium CCTCAF 2010001.

[0135] Table 6. Effects of tea polyphenols on the peroxide value of fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCAF 2010001* under ambient temperature conditions.

[0136]

[0137] Table 6 shows the effect of tea polyphenols on the peroxide value of fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* under normal temperature conditions. After one year of storage, the peroxide values ​​of the normally fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* reached 0.6 g / 100 mL and 0.65 g / 100 mL, respectively, while the tea polyphenol group only had 0.5 g / 100 mL and 0.55 g / 100 mL. Tea polyphenol fermentation has a greater advantage in the storage of oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001*.

[0138] Table 7. Effects of tea polyphenols on DHA content during storage of fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCAF 2010001* under ambient temperature conditions.

[0139]

[0140] Table 7 shows the effect of tea polyphenols on the DHA content of oils fermented by *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* during storage under normal temperature conditions. After one year of storage, the DHA content of the normally fermented oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001* decreased from 35.6% and 33.2% to 28.1% and 26.7%, respectively, while the DHA content of the oils fermented with tea polyphenols decreased from 42.1% and 41.2% to 37.5% and 37.1%, respectively. Therefore, tea polyphenol fermentation not only increased the DHA content of oils from *Schizochytrium ATCC 20888* and *Schizochytrium CCTCC AF 2010001*, but also slowed down the degradation of DHA in the oils.

[0141] In summary, this invention provides an application and method for using tea polyphenols to improve the yield and storage of DHA-rich oils from Schizochytrium fungi. This method not only increases the content of DHA and PUFAs in the oils but also increases the oil yield. Furthermore, fermentation with added tea polyphenols improves the quality of the oils, reduces their acid value and peroxide value, and enhances their storage stability. Moreover, fermentation with added tea polyphenols inhibits DHA degradation during storage, significantly improving the economic viability of the oils.

[0142] 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 increasing lipid synthesis and DHA production by adding the antioxidant tea polyphenol to regulate cellular oxidative stress in Schizochytrium fungi, characterized in that: Tea polyphenols were added 12 hours after fermentation with Schizochytrium. The final concentration of tea polyphenols added was 2 g / L. The selected schistocytic fungus is the oil-producing schistocytic fungus HX-308.

2. The method of claim 1, wherein: Includes the following steps: After the Schizochytrium strain was activated by inoculating it into a seed culture medium, a fermentation strain was obtained. The fermentation strain was then inoculated into a fermentation culture medium for fermentation. After 12 hours of Schizochytrium fermentation, tea polyphenols were added at a final concentration of 2 g / L. The cells were then collected to extract the oil.

3. The method of claim 2, wherein: The fermentation strain is obtained by the following method: Schizochytrium is inoculated into a seed culture medium and cultured for 24 hours to obtain a primary seed; the primary seed is inoculated into a seed culture medium and cultured for 24 hours to obtain a secondary seed; the secondary seed is inoculated into a seed culture medium and cultured for 24 hours to obtain a tertiary seed, which is used as the fermentation strain. The culture conditions are 25–30℃ and shaking culture at 150–250 r / min.

4. The method of claim 2, wherein: The specific steps are as follows: Single colonies of *Schizochytrium oleiferum* were selected and inoculated into seed culture medium. After culturing at 28°C and 180 rpm for 24 h, they were classified as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and cultured at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation. Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of fermentation medium. Add tea polyphenols at a final concentration of 2 g / L after 12 h of fermentation. Culture in a shaker at 28℃ and 180 r / min for 120 h to obtain the fermentation broth. After fermentation culture is completed, add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.2% of cell wall breaking enzyme, and shake at 100-200 r / min for 5-15 h at 40-60℃. Cool to room temperature and add anhydrous ethanol equal in volume to the cell wall-breaking fermentation broth to inactivate the cell wall-breaking enzyme; Add an equal volume of n-hexane to the fermented liquid after cell wall disruption to extract the oil, and repeat the extraction process three times by adding the same volume of n-hexane. After extraction, the organic phase was allowed to stand for 5 hours. Once the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was then removed and placed in a rotary evaporator flask. The organic phase was then evaporated by rotary evaporation at 45°C in a water bath and at 120 r / min. Once the organic phase stopped evaporating, the rotary evaporator flask was removed and dried in a 60°C oven until its weight no longer changed. The oil was then weighed to obtain the oil.

5. The method of claim 2, wherein: The entire fermentation culture cycle is 48 hours to 120 hours.

6. The method of claim 5, wherein: The entire fermentation culture cycle is 48h, 60h, 72h, 84h, 96h or 120h.

7. The method according to any one of claims 1 to 6, characterized in that: The Schizochytrium strain was inoculated onto a plate medium and cultured on 500 ug / L G418 resistant plates in a 28°C incubator for 72 h. Single colonies were then picked and inoculated onto seed culture medium for activation. The plate culture medium has a pH of 6.0–6.5 and comprises: agar 15–20 g / L, glucose 30–60 g / L, yeast extract 8–15 g / L, sodium sulfate 10–15 g / L, magnesium sulfate 2–4 g / L, ammonium sulfate 6–12 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 dichloride 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 0.04 mg / L, and vitamin B6 4–10 mg / L. mg / L, Vitamin B 12 0.1-1.5 mg / L, solvent is water; Alternatively, the seed culture medium has a pH of 6.0–6.5 and comprises: 40–60 g / L glucose, 4–6 g / L yeast extract, 5–8 g / L sodium sulfate, 2–4 g / L magnesium sulfate, 4–8 g / L ammonium sulfate, 1–2 g / L potassium chloride, 0.1–0.2 g / L calcium chloride, 0.5–1 g / L potassium sulfate, 0.5–2 g / L potassium dihydrogen phosphate, 8–12 g / L monosodium glutamate, 1–5 mg / L zinc sulfate heptahydrate, 0.01–0.1 mg / L cobalt dichloride hexahydrate, 2–6 mg / L copper sulfate pentahydrate, 1–2 mg / L nickel sulfate hexahydrate, 8–15 mg / L ferrous sulfate heptahydrate, 2–4 mg / L calcium pantothenate, 3–5 mg / L manganese chloride tetrahydrate, and 0.04 mg / L sodium molybdate dihydrate, with water as the solvent. Alternatively, the fermentation medium has a pH of 6.0–6.5 and comprises: 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 dichloride 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 0.04 mg / L, vitamin B6 4–10 mg / L, and vitamin B12. 12 0.1-0.5 mg / L, solvent is water.

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