Application and method of tea polyphenol in improvement of schizochytrium limacinum DHA grease yield and grease storage

Abstract

The invention belongs to the technical field of biochemical engineering, and discloses application and a method of tea polyphenol in improvement of schizochytrium limacinum DHA (docosahexaenoic acid) grease yield and grease storage, and tea polyphenol is added in the process of producing DHA by fermentation of schizochytrium limacinum. By comparing the difference of schizochytrium limacinum in tea polyphenol fermentation and traditional fermentation, it is clear that the antioxidant tea polyphenol regulates cell oxidative stress in schizochytrium limacinum, removes ROS and inhibits grease oxidation, so that grease rancidity is delayed, and grease synthesis and DHA production are increased. According to the invention, schizochytrium limacinum HX-308 is used as a receptor strain, and an antioxidant tea polyphenol is added for regulation, so that fermentation conditions under which the content of grease and DHA is obviously increased and the grease storage effect is better are obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular to an application and method of tea polyphenols in improving the DHA oil production of Schizochytrium and oil storage. Background Art

[0002] Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid (PUFA) that is essential for the development of the human nervous system and retinal tissue. Epidemiological studies have shown that dietary intake of DHA has many health benefits, so DHA has been widely used in infant formula, nutritional products, food supplements and medicines. DHA is traditionally extracted from marine fish oil, but this method is not sustainable due to environmental pollution and overfishing, and the yield cannot meet the growing market demand for DHA. Oil-containing microorganisms are considered potential producers of DHA and have received increasing attention due to their advantages over fish oil. Schizochytrium is a heterotrophic marine protist rich in DHA (docosahexaenoic acid). It is widely used in scientific research and commercial production due to its fast growth rate, rich DHA content and easy cultivation. So far, Schizochytrium has been found to accumulate fatty acids mainly in the form of DHA, DPA (docosapentaenoic acid), C16:0 and C14:0. Foods containing ω-3 polyunsaturated fatty acids (PUFAs) such as DHA and EPA (eicosapentaenoic acid) are considered to be important factors in promoting the development of retinal and brain functions and reducing the risk of cardiovascular diseases. Considering the huge benefits and growing demand for ω-3 fatty acids, increasing the proportion of ω-3 fatty acids, especially DHA, in the total fatty acids of Schizochytrium has become a focus of attention.

[0003] Typically, under optimal growth conditions, oil-containing microorganisms produce a small amount of oil, while stressful environments promote oil production. Stress-induced strategies such as nitrogen depletion, phosphate starvation, and salt stress have been used to improve DHA production in Schizochytrium. However, stress-induced strategies inhibit microbial growth and thus reduce the overall productivity of biomass and oil. According to studies, high levels of ROS can act on DNA, proteins, and oils and induce oxidative damage, leading to loss of protein function or even cell death and PUFA peroxidation.

[0004] Oxidative damage to Schizochytrium cells caused by high levels of ROS can be alleviated by adding antioxidants such as melatonin, sesamol or ascorbic acid. Tea polyphenols are a type of polyhydroxyphenolic substance that has antioxidant and free radical scavenging effects. Studies have shown that tea polyphenols can scavenge ROS and inhibit oil oxidation, and a small amount of addition can delay rancidity in oils and fats, and are widely used as additives in food and medicine. Exogenous addition of antioxidants to fermentation media helps to reduce intracellular ROS levels, reduce oxidative damage to cells and promote oil synthesis in oily microorganisms. In addition, it is worth noting that if the concentration of antioxidants exceeds a certain threshold, it will inhibit cell growth and oil production. Therefore, the concentration of added antioxidants is very important for regulating cellular oxidative stress and DHA production in Schizochytrium. However, the traditional strategy is to focus on enhancing the production of oil and DHA in Schizochytrium through chemical regulators, and there is a relative lack of research on the effects of chemical regulators on the later storage of oils and the prevention of DHA degradation. Compared with the traditional fermentation technology, the present invention can effectively inhibit the intracellular lipid peroxidation during the fermentation of Schizochytrium by adding appropriate antioxidant tea polyphenols, which can increase the DHA oil production on the one hand, and the produced oil has higher quality in storage on the other hand. In addition, the study also found that the carotenoid content was significantly improved compared with the traditional fermentation by adding tea polyphenols for fermentation, and confirmed that the oil of the strategy of the present invention has better storage properties than the oil produced by the traditional strategy, and the degradation rate of DHA of the strategy of the present invention is much lower than that of the oil DHA of the traditional production strategy, which is of great significance to the quality and storage of oil. However, the study shows that the fat solubility of tea polyphenols is not significant. Exploring and determining the optimal addition concentration of tea polyphenols in the fermentation process of oil production has far-reaching significance for significantly improving the quality of oil products and optimizing production efficiency. This study not only aims to explore the potential of tea polyphenols as a natural additive, but also strives to optimize and upgrade the oil production process through scientific methods. At present, there is no report on the research of regulating the production of oil by Schizochytrium fermentation, improving the storage effect of oil and slowing down the degradation of DHA by exogenous addition of tea polyphenols. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application and method of tea polyphenols in improving the DHA oil production and oil storage of Schizochytrium.

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

[0007] Application of tea polyphenols in improving DHA oil production and oil storage of Schizochytrium.

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

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

[0010] A method for regulating cellular oxidative stress in Schizochytrium by adding antioxidant tea polyphenols to increase oil synthesis and DHA production. Tea polyphenols are added after Schizochytrium fermentation for 12 hours, and the final concentration of tea polyphenols is 2g / L.

[0011] Further, the following steps are included:

[0012] The Schizochytrium strain is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation culture, tea polyphenols are added after the Schizochytrium fermentation for 12 hours, the final concentration of the tea polyphenols is 2 g / L, and the bacteria are collected to extract oil;

[0013] Alternatively, the Schizochytrium is selected from the oil-producing Schizochytrium HX-308 Schizochytrium, ATCC 20888 or Schizochytrium CCTCCAF 2010001.

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

[0015] The culture conditions are 25-30°C and shaking at 150-250 r / min.

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

[0017] A single colony of oil-producing Schizochytrium was selected and inoculated into a seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain first-generation seeds; 1 mL of the first-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain second-generation seeds; 1 mL of the second-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain third-generation seeds, which were used as fermentation strains;

[0018] Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of the fermentation medium. After 12 hours of fermentation, add tea polyphenols with a final concentration of 2 g / L. Cultivate at 28°C and 180 r / min shaking for 120 hours to obtain the fermentation solution.

[0019] After the fermentation culture is completed, NaOH solution is added to the fermentation liquid to adjust the pH to 10-13, and then a wall-breaking enzyme with a final concentration of 0.01-0.2% is added, and the mixture is shaken at 40-60° C. and 100-200 r / min for 5-15 hours;

[0020] After cooling to room temperature, anhydrous ethanol with the same volume as the fermentation liquid after cell wall breaking was added to inactivate the cell wall breaking enzyme;

[0021] Add the same volume of n-hexane as the cell-broken fermentation liquid to extract the oil, and repeat the same volume of n-hexane extraction 3 times:

[0022] The extracted organic phase was allowed to stand for 5 hours, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation bottle, and the organic phase was rotary evaporated in a 45°C water bath at 120r / min. After the organic phase no longer evaporated, the rotary evaporation bottle was removed and dried in a 60°C oven until the weight no longer changed, and the oil was weighed.

[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] Further, the Schizochytrium strain was inoculated into a plate culture medium, and after being cultured on a 500ug / L G418 resistant plate in a 28° constant temperature incubator for 72h, a single colony was picked and inoculated into a seed culture medium for activation;

[0026] The pH value of the plate culture medium is 6.0-6.5, and the plate culture medium comprises: 15-20 g / L agar, 30-60 g / L glucose, 8-15 g / L yeast extract, 10-15 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 6-12 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 sodium 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 vitamin B 64-10 mg / L, Vitamin B 12 0.1-1.5mg / L, solvent is water;

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

[0028] Alternatively, the pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and vitamin B 6 4-10mg / L, Vitamin B 12 0.1-0.5mg / L, the solvent is water.

[0029] The advantages and positive effects achieved by the present invention are:

[0030] 1. Based on the traditional fermentation of Schizochytrium, the present invention significantly improves the oil yield and the proportion of DHA in the oil by adding tea polyphenols. The traditional fermentation oil yield is 13.9 g / L. Under the fermentation with the optimal tea polyphenol addition concentration of 2 g / L, the oil yield is 19.8 g / L, the yield is increased by 42%, and the DHA content is increased to 49.34%. The final DHA oil yield is 9.77 g / L, which is 51% higher than that of the traditional fermentation without adding tea polyphenols.

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

[0032] 3. The present invention successfully achieves the accumulation of oil and DHA and improves the quality by adjusting the concentration of added tea polyphenols.

[0033] 4. The present invention regulates the cellular oxidative stress in Schizochytrium by adding the antioxidant tea polyphenols, removes ROS and inhibits oil oxidation, delays oil rancidity, and increases oil synthesis and DHA production.

[0034] 5. The present invention uses Schizochytrium HX-308 as a receptor strain and obtains fermentation conditions with significantly increased oil and DHA content and better oil storage effect by adding antioxidant tea polyphenols for regulation.

[0035] 6. The present invention adjusts Schizochytrium fermentation by adding tea polyphenols, and compared with traditional fermentation, the carotenoid content is significantly increased, and the oil quality is significantly improved.

[0036] 7. The oil produced by the present invention has better storage effect, lower DHA reduction rate and is more economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a diagram showing the difference of DHA in the fermentation of Schizochytrium HX-308 tea polyphenols at different addition times in the present invention;

[0038] Figure 2 The figure is a graph showing the effect of different concentrations of tea polyphenols on cell growth and oil synthesis in the tea polyphenol fermentation of Schizochytrium HX-308 in the present invention; wherein, (A) biomass, oil yield and residual sugar; (B) fatty acid composition (SFA and PUFA); (C) DHA yield; (D) carotenoid content;

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

[0040] Figure 4 This is a possible mechanism diagram of the dual effects of tea polyphenols on oxidative stress and lipid accumulation in Schizochytrium in the present invention. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0043] Application of tea polyphenols in improving DHA oil production and oil storage of Schizochytrium.

[0044] Preferably, tea polyphenols are added during the process of producing DHA by Schizochytrium fermentation.

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

[0046] A method for regulating cellular oxidative stress in Schizochytrium by adding antioxidant tea polyphenols to increase oil synthesis and DHA production. Tea polyphenols are added after Schizochytrium fermentation for 12 hours, and the final concentration of tea polyphenols is 2g / L.

[0047] Preferably, the method comprises the following steps:

[0048] The Schizochytrium strain is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation culture, tea polyphenols are added after the Schizochytrium fermentation for 12 hours, the final concentration of the tea polyphenols is 2 g / L, and the bacteria are collected to extract oil;

[0049] Alternatively, the Schizochytrium is selected from the oil-producing Schizochytrium HX-308 Schizochytrium, ATCC 20888 or Schizochytrium CCTCCAF 2010001.

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

[0051] The culture conditions are 25-30°C and shaking at 150-250 r / min.

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

[0053] A single colony of oil-producing Schizochytrium was selected and inoculated into a seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain first-generation seeds; 1 mL of the first-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain second-generation seeds; 1 mL of the second-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain third-generation seeds, which were used as fermentation strains;

[0054] Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of the fermentation medium. After 12 hours of fermentation, add tea polyphenols with a final concentration of 2 g / L. Cultivate at 28°C and 180 r / min shaking for 120 hours to obtain the fermentation solution.

[0055] After the fermentation culture is completed, NaOH solution is added to the fermentation liquid to adjust the pH to 10-13, and then a wall-breaking enzyme with a final concentration of 0.01-0.2% is added, and the mixture is shaken at 40-60° C. and 100-200 r / min for 5-15 hours;

[0056] After cooling to room temperature, anhydrous ethanol with the same volume as the fermentation liquid after cell wall breaking was added to inactivate the cell wall breaking enzyme;

[0057] Add the same volume of n-hexane as the cell-broken fermentation liquid to extract the oil, and repeat the same volume of n-hexane extraction 3 times:

[0058] The extracted organic phase was allowed to stand for 5 hours, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation bottle, and the organic phase was rotary evaporated in a 45°C water bath at 120r / min. After the organic phase no longer evaporated, the rotary evaporation bottle was removed and dried in a 60°C oven until the weight no longer changed, and the oil was weighed.

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

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

[0061] Preferably, the Schizochytrium strain is inoculated on a plate culture medium, cultured on a 500ug / L G418 resistant plate in a 28° constant temperature incubator for 72 hours, and then a single colony is picked and inoculated on a seed culture medium for activation;

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

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

[0064] Alternatively, the pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and vitamin B 6 4-10mg / L, Vitamin B 12 0.1-0.5mg / L, the solvent is water.

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

[0066] Determination of glucose and sodium glutamate concentrations: Take 1 mL of fermentation broth at a specific time point, centrifuge at 12,000 rpm for 1 minute, and take the supernatant for gradient dilution to 100 times, centrifuge at 12,000 rpm for 1 minute, and detect with SBA-40ES. Glucose and sodium glutamate were determined with different enzyme membranes and calibrated with standard solutions in advance.

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

[0068] Determination of fatty acid composition: Take about 10 mg of oil or about 50 mg of wet bacteria in a 2 mL EP tube, add 0.6 mL of 1 M sodium hydroxide-methanol solution, and shake at 1200 rpm for 5 hours at room temperature. Add 50 μL of concentrated sulfuric acid to terminate the reaction. Add 1 mL of n-hexane for extraction, shake at 1200 rpm for 20 minutes, then centrifuge at 12000 rpm for 5 minutes, and take the upper n-hexane phase for gas phase detection.

[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°C for later use. Then it was added to the cell culture at a volume ratio of 1:100 and placed in the dark at 30°C and 170 rpm for 20 minutes to allow the fluorescent probe to fully enter the cells. The cell samples were washed twice with PBS buffer to fully remove the extracellular DCFH-DA, and then the cell samples were dissolved in PBS. Finally, the relative content of intracellular ROS was determined using a multifunctional microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The superoxide dismutase (SOD) and catalase (CAT) activities of Schizochytrium cells were determined using the SOD activity assay kit (Solarbio, China) and the CAT activity assay kit (Solarbio, China), respectively. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of the cell samples were determined using a T-AOC assay kit (Solarbio, China) and an MDA assay kit (Solarbio, China) according to the manufacturer’s instructions.

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

[0072] The invention adopts Schizochytrium sp. HX-308 and adjusts the cellular oxidative stress in Schizochytrium sp. by adding antioxidant tea polyphenols, thereby increasing oil synthesis and DHA production, and laying a foundation for industrial production and storage of the product.

[0073] Unless otherwise specified, the equipment, reagents, processes, parameters, etc. involved in the present invention are conventional equipment, reagents, processes, parameters, etc. and are not given as embodiments.

[0074] All ranges recited herein include all point values ​​within the range.

[0075] In the present invention, unless otherwise specified in the art, % refers to mass percentage, and ratio refers to mass ratio. The unit of mass is, for example, gram, kilogram or ton.

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

[0077] The culture medium used in the following examples is as follows:

[0078] The pH value of the plate culture medium is 6.0-6.5, and includes: agar 15-20g / L, glucose 30-60g / L, yeast extract 8-15g / L, sodium sulfate 10-15g / L, magnesium sulfate 2-4g / L, ammonium sulfate 6-12g / L, potassium chloride 1-2g / L, calcium chloride 0.1-0.2g / L, potassium sulfate 0.5-1g / L, potassium dihydrogen phosphate 0.5-2g / L, sodium glutamate 8-12g / L, zinc sulfate heptahydrate 1-5mg / L, cobalt dichloride hexahydrate 0.01-0.1mg / L, copper sulfate pentahydrate 2-6mg / L, nickel sulfate hexahydrate 1-2mg / L, ferrous sulfate heptahydrate 8-15mg / L, calcium pantothenate 2-4mg / L, manganese chloride tetrahydrate 3-5mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B 6 4-10 mg / L, Vitamin B 12 0.1-1.5mg / L, the solvent is water.

[0079] The pH value of the seed culture medium of the seed liquid is 6.0-6.5, and the seed liquid comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract powder, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water.

[0080] The fermentation medium has a pH value of 6.0-6.5 and comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and vitamin B 6 4-10 mg / L, Vitamin B 12 0.1-0.5mg / L, the solvent is water.

[0081] Example 1

[0082] Tea polyphenols regulate cellular oxidative stress in Schizochytrium, scavenge ROS and inhibit lipid oxidation, increase lipid synthesis and DHA production. Screening of addition time

[0083] Select a single colony of oil-producing Schizochytrium HX-308 and inoculate it in a 250mL conical flask (containing 50mL of seed culture medium). After culturing at 28°C and 180r / min shaking for 24h, it is the first-generation seed. Take 1mL of the first-generation seed culture solution and inoculate it in a 250mL conical flask (containing 50mL of seed culture medium). Take 1mL of the second-generation seed culture solution and inoculate it in a 250mL conical flask (containing 50mL of seed culture medium). Take 1mL of the second-generation seed culture solution and inoculate it in a 250mL conical flask (containing 50mL of seed culture medium). Take 28°C and 180r / min shaking for 24h to obtain the third-generation seed, which is used as the fermentation strain.

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

[0085] 2. Collecting the cells to extract oil, including the following steps:

[0086] (1) After the fermentation culture is completed, NaOH solution is added to the fermentation broth to adjust the pH to 10-13, and then a cell wall-breaking enzyme is added at a final concentration of 0.01-0.2%, and the mixture is shaken at 40-60° C. and 100-200 r / min for 5-15 hours;

[0087] (2) cooling to room temperature, and adding anhydrous ethanol in an equal volume to the fermentation liquid after cell wall breaking to inactivate the cell wall breaking enzyme;

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

[0089] The extracted organic phase was allowed to stand for 5 hours, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation bottle, and the organic phase was rotary evaporated in a 45°C water bath at 120r / min. After the organic phase no longer evaporated, the rotary evaporation bottle was removed and dried in a 60°C oven until the weight no longer changed, and the oil was weighed;

[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 1 M potassium hydroxide-methanol solution, and the mixture was shaken at 20 °C and 1000 r / min for 6 h. 50 μL of concentrated sulfuric acid was added to terminate the reaction, and 1 ml of n-hexane was added to extract the oil at 20 °C and 1000 r / min for 0.5 h. The extract was placed in a liquid phase vial for gas phase detection. The analysis was performed using a GC-2010 (Shimadzu, Japan) gas phase system equipped with a DB-23 capillary column (60 m*0.22 mm) and a flame ionization detector (FID). Nitrogen was used as a 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, and then increased to 230 °C at a rate of 4 °C / min and maintained 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 acid (C19:0) as the internal standard, 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 fermented without adding tea polyphenols was 46%, the proportion of DHA fermented with tea polyphenols added at the beginning was 47%, the proportion of DHA added with a final concentration of 2g / L tea polyphenols after fermentation for 12h was 49%, the proportion of DHA added with tea polyphenols after fermentation for 24h was 48%, and the proportion of DHA added with tea polyphenols after fermentation for 36h was 47%. In summary, the addition condition of 12h is the best.

[0093] Example 2 Tea polyphenols regulate cellular oxidative stress in Schizochytrium, remove ROS and inhibit lipid oxidation, increase lipid synthesis and DHA production Addition amount screening

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

[0095] The seed liquid culture was carried out in the early stage according to Example 1. 0 / 0.1 / 0.5 / 1 / 2 / 5 / 10 g / L tea polyphenols were added for fermentation after 12 h of fermentation. The biomass, oil yield, oil content and residual sugar; fatty acid composition; fatty acid composition (SFA and PUFA); DHA yield; DHA content; and carotenoid content were detected.

[0096] The following steps are involved:

[0097] (1) Determination of glucose and sodium glutamate concentrations. The specific procedure is as follows: 1 mL of fermentation broth at a specific time point is taken, centrifuged at 12,000 rpm for 1 minute, and the supernatant is gradient diluted to 100 times, the dilution is centrifuged at 12,000 rpm for 1 minute, and detected with SBA-40ES. Glucose and sodium glutamate are determined using different enzyme membranes, which are calibrated with standard solutions in advance.

[0098] (2) Determination of cell dry weight (CDW), the specific procedure is as follows: take the fermentation broth (5mL) at a specific time point onto filter paper (determine the mass of the filter paper in advance) / fermentation broth (1mL) into an EP tube (determine the mass of the EP tube in advance), filter, let stand to remove liquid interference / centrifuge to remove liquid interference, then wash the bacteria with deionized water, finally place the filter paper / EP tube in a 65℃ oven and dry (about 48h) to constant weight, calculate CDW. Determination of oil content: Place a rotary evaporator with a predetermined mass of oil collection in an oven at 65℃ and dry to constant weight, then weigh after cooling.

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

[0100] (4) Take 5 ml of the fermentation liquid that has just been fermented, centrifuge it, add an equal volume of methanol to soak and extract it several times, combine the methanol extracts, remove the methanol by rotary vacuum evaporator, add carbon tetrachloride to dissolve the concentrate, filter it through a 0.22 μm organic filter membrane, and set it aside. Use high performance liquid chromatography to determine the carotenoid content. Chromatographic column: Amethyst C18-H column (4.6X150mm, 5μm), detection wavelength 475nm, column temperature is room temperature, mobile phase flow rate is 1.0mL / min, use gradient elution, prepare the corresponding carotenoid standard sample, and obtain the standard curve equation.

[0101] The results are as follows Figure 2 As shown in Table 1, with the increase of tea polyphenol concentration, biomass and oil yield first increased and then decreased. 2g / L tea polyphenol is the best addition amount for fermentation. At this time, the biomass is 60.6g / L, the oil production is the highest, 19.8g / L, and the total oil yield is increased by 42% compared with the control group (i.e., without adding tea polyphenol). The results of fermentation with tea polyphenol and normal fermentation (i.e., without adding tea polyphenol) are compared, as shown in Table 1: the oil yield of Schizochytrium in the tea polyphenol group increased, the fatty acid composition changed greatly, and the proportion of DHA was significantly higher than that of the control group, and the proportion of DHA increased from 46.47% in the control group to 49.34% in the 2g / L tea polyphenol group. Compared with no addition, the addition of appropriate concentration of tea polyphenols (2g / L) significantly increased biomass, oil yield and DHA yield. It was found that the carotenoid content of the control group (0g / L) was 47.8μg / g, while the carotenoid content of the group adding 2g / L tea polyphenols was as high as 151μg / g. The carotenoid content after adding tea polyphenols was higher than that of the control group. The carotenoid content was significantly improved after adding tea polyphenols, which is of great significance for oil storage.

[0102] At the same time, by comparing Example 1 and Example 2, it can be seen that the final concentration of tea polyphenols added in the method of the present invention is 2 g / L and the addition time of tea polyphenols is 12 hours after fermentation. There is a synergistic effect between the two conditions, which can synergistically improve the DHA oil yield 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] According to the data analysis in Example 2, the dual anti-oxidation / pro-oxidation activity of the antioxidant tea polyphenols is related to its added concentration.

[0105] Figure 2 The results showed that with the increase of tea polyphenols concentration, biomass, total oil yield and DHA yield showed a trend of first increasing and then decreasing. When 2g / L of tea polyphenols was added, the biomass, total oil yield and DHA yield all reached the maximum value, which were 60.6g / L, 19.8g / L and 9.77g / L respectively, while at 10g / L, the biomass, total oil yield and DHA yield were 43g / L, 13.4g / L and 5.81g / L respectively, which were all lower than the corresponding control group (0g / L). Therefore, the addition of an appropriate amount of antioxidants is helpful for the oil and DHA production of 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] In order to reduce the oxidative damage caused by ROS, microorganisms can activate enzymatic and non-enzymatic antioxidant defense mechanisms, including SOD, CAT and other antioxidant enzymes, as well as intracellular antioxidants. In order 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 in Schizochytrium cells were measured. The remaining relevant preparation methods were carried out according to Example 2.

[0108] The specific procedures for the determination of ROS and 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°C for later use. Then it was added to the cell culture at a volume ratio of 1:100 and placed in the dark at 30°C and 170 rpm for 20 minutes to allow the fluorescent probe to fully enter the cells. The cell samples were washed twice with PBS buffer to fully remove the extracellular DCFH-DA, and then the cell samples were dissolved in PBS. Finally, the relative content of intracellular ROS was determined using a multifunctional microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The superoxide dismutase (SOD) and catalase (CAT) activities of Schizochytrium cells were determined using the SOD activity assay kit (Solarbio, China) and the CAT activity assay kit (Solarbio, China), respectively. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of the cell samples were determined using a T-AOC assay kit (Solarbio, China) and an MDA assay kit (Solarbio, China) according to the manufacturer’s instructions.

[0109] The results are as follows Figure 3 As shown in the results, exogenous addition of antioxidant tea polyphenols helps to reduce the intracellular ROS level. After adding 2g / L tea polyphenols, the ROS value is lower than that of the control group (0g / L), which effectively inhibits intracellular lipid peroxidation during the fermentation of Schizochytrium, reduces oxidative damage to cells, and improves DHA oil yield, quality and production efficiency. The possible mechanism of the dual effects of tea polyphenols on oxidative stress and lipid accumulation in Schizochytrium can be as follows: Figure 4 shown.

[0110] Example 5 Storage index detection of oil produced by normal fermentation of Schizochytrium and fermentation with optimal concentration of tea polyphenols in different environments

[0111] The oils obtained by fermentation with the same fermentation medium under normal fermentation conditions and fermentation conditions with the addition of 2g / L tea polyphenols (the rest of the relevant preparation methods are carried out according to Example 2) were quantitatively stored at -20°C, 4°C, room temperature, 40°C, and 80°C, and the oil indices (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] The acid value and peroxide value were detected using a kit according to the instructions, and the fatty acid composition was analyzed by gas phase detection, as described in Example 1.

[0113] The results are shown in Table 2 and Table 3. The oil produced by adding the optimum concentration of 2g / L tea polyphenols is more stable than the oil fermented normally under the same storage environment. Both the acid value and the peroxide value are much lower than the normal fermentation group, and the quality performance is more outstanding. Under -20° and 80° conditions, the acid value of the oil in the normal fermentation group started at 0.9 and 1.2 mg(KOH) / g, and increased to 1.35 and 2.1 mg(KOH) / g respectively after 30 days; while the acid value of the tea polyphenol group started at 0.75 and 1.05 mg(KOH) / g, and only increased to 1.2 and 1.8 mg(KOH) / g respectively after 30 days, with lower initial and final acid values. When stored at -4°, room temperature and 40° for 30 days, the acid value of the oil in the normal fermentation group was 0.15, 0.15 and 0.3 mg(KOH) / g higher than that in the tea polyphenol group, respectively. In terms of peroxide value, the oil obtained by normal fermentation showed peroxidation in about 15 days, while the tea polyphenols group showed peroxidation in about 30 days.

[0114] In terms of PUFA stability, the PUFA content of normal fermented oil decreased from 64.2% to 60.9% after 360 days at -20°, to 56.0% after 360 days at -4°, to 47.2% after 360 days at room temperature, to 43.9% after 360 days at 40°, and to 48.2% after 60 days at 80°; while the oil obtained with the addition of tea polyphenols had an initial PUFA content of 71.4%, which decreased to 70.4% after 360 days at -20°, to 66.5% after 360 days at -4°, to 58.1% after 360 days at room temperature, to 50.6% after 360 days at 40°, and to 55.8% after 60 days at 80°.

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

[0116] Therefore, the oil obtained by fermentation under 2g / L tea polyphenols has better storage effect, higher PUFA and DHA content, and lower PUFA degradation rate. In addition, it is best stored under normal temperature with antioxidant tea polyphenols and has the highest economic value.

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

[0118]

[0119] Table 2 Comparison of storage index parameters of oil produced by normal fermentation of Schizochytrium and fermentation with optimal concentration of tea polyphenols in different environments

[0120]

[0121]

[0122]

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

[0124]

[0125]

[0126] Example 6

[0127] The present invention tests the replacement of Schizochytrium HX-308 with Schizochytrium ATCC 20888 (purchased from the American Type Culture Collection) and Schizochytrium CCTCC AF 2010001 (purchased from the China Type Culture Collection). The fermentation conditions are the same as those of Schizochytrium HX-308 (i.e., the rest of the relevant preparation methods are carried out according to Example 2), and the oil, fatty acid composition and lipid storage performance at room temperature are tested respectively. The fermentation and experimental strategies and steps are the same as those of 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 polyphenols group increased the proportion of DHA and PUFA of Schizochytrium ATCC 20888 from 35.6% and 48.3% to 42.1% and 56.5, and the lipid yield was also increased from 12.5 g / L to 17.2 g / L. In addition, compared with normal fermentation, the tea polyphenols group increased the proportion of DHA and PUFA of Schizochytrium CCTCCAF 2010001 from 33.2% and 43.2% to 41.2% and 60.2, and the lipid yield was also increased from 10.2 g / L to 16.7 g / L. Therefore, tea polyphenols significantly increased the DHA, PUFA and lipid production capacity of Schizochytrium ATCC 20888 and Schizochytrium CCTCCAF 2010001.

[0131] Table 5 Effects of tea polyphenols on storage acid value of oils fermented by Schizochytrium ATCC 20888 and Schizochytrium CCTCCAF 2010001 under normal temperature

[0132]

[0133]

[0134] 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 is shown in Table 5. The initial acid value of oils in the tea polyphenol group was 0.2 mg (KOH) / g and 0.05 mg (KOH) / g lower than that in the normal fermentation group in Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001, respectively. After the oils were stored for 1 year, the acid value of 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 that of the tea polyphenol group was only 2.45 mg (KOH) / g and 2.35 mg (KOH) / g. Tea polyphenol fermentation has more advantages for oil storage of Schizochytrium ATCC 20888 and Schizochytrium CCTCCAF 2010001.

[0135] Table 6 Effects of tea polyphenols on storage peroxide value of fermented oils of Schizochytrium ATCC 20888 and Schizochytrium CCTCCAF 2010001 under room temperature

[0136]

[0137] The effect of tea polyphenols on the storage peroxide value of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 fermented oil under normal temperature conditions is shown in Table 6. After the oil was stored for one year, the peroxide value of the normal fermented oil of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 reached 0.6g / 100mL and 0.65g / 100mL, respectively, while that of the tea polyphenol group was only 0.5g / 100mL and 0.55g / 100mL. Tea polyphenol fermentation has more advantages for the storage of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 oil.

[0138] Table 7 Effects of tea polyphenols on DHA content during storage of Schizochytrium ATCC 20888 and Schizochytrium CCTCCAF 2010001 fermented oils at room temperature

[0139]

[0140] The effect of tea polyphenols on the DHA content of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 fermented oils during storage under normal temperature conditions is shown in Table 7. After the oils were stored for one year, the DHA content of Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 fermented oils decreased from 35.6% and 33.2% to 28.1% and 26.7%, respectively, while the DHA content of the tea polyphenol group fermented oils 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 Schizochytrium ATCC 20888 and Schizochytrium CCTCC AF 2010001 oils, but also slowed down the degradation of DHA in the oils.

[0141] In summary, the present invention provides an application and method of tea polyphenols in increasing the DHA oil production of Schizochytrium and oil storage, which not only increases the content of DHA and PUFA in Schizochytrium oil, but also increases the oil production. In addition, adding tea polyphenols for fermentation also improves the quality of the oil, reduces the acid value and peroxide value of the oil, and improves the storage stability of the oil. Moreover, adding tea polyphenols for fermentation also inhibits the degradation of DHA in the oil during storage, greatly improving the economy of the oil.

[0142] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An application of tea polyphenols in improving the DHA oil production and oil storage of Schizochytrium.

2. The use according to claim 1, characterized in that: Tea polyphenols are added during the production of DHA by Schizochytrium fermentation.

3. The use according to claim 1 or 2, characterized in that: The addition of tea polyphenols during the fermentation process of the Schizochytrium can greatly increase the DHA content and oil yield. Under the fermentation condition of 2 g / L of the optimum tea polyphenol addition concentration, the oil yield is increased by 42%, the DHA content is increased to 49.34%, and the final DHA oil yield is 9.77 g / L, which is 51% higher than that of the traditional fermentation without adding tea polyphenols. The oil produced by the fermentation with the addition of tea polyphenols has better quality when stored at room temperature than the traditional fermented oil. The oil produced under the same conditions and stored under different conditions has the highest economic value when stored at room temperature.

4. A method for regulating cellular oxidative stress in Schizochytrium by adding antioxidant tea polyphenols to increase lipid synthesis and DHA production, characterized in that: Tea polyphenols were added after 12 hours of Schizochytrium fermentation, and the final concentration of tea polyphenols was 2 g / L.

5. The method according to claim 4, characterized in that: The steps include: The Schizochytrium strain is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation culture, tea polyphenols are added after the Schizochytrium fermentation for 12 hours, the final concentration of the tea polyphenols is 2 g / L, and the bacteria are collected to extract oil; Alternatively, the Schizochytrium is selected from the oil-producing Schizochytrium HX-308 Schizochytrium, ATCC 20888 or Schizochytrium CCTCCAF 2010001.

6. The method according to claim 5, characterized in that: The fermentation strain is obtained by the following method: inoculating Schizochytrium into a seed culture medium, culturing for 24 hours to obtain primary seeds; taking the primary seeds and inoculating them into a seed culture medium, culturing for 24 hours to obtain secondary seeds; taking the secondary seeds and inoculating them into a seed culture medium, culturing for 24 hours to obtain tertiary seeds as the fermentation strain; The culture conditions are 25-30°C and shaking at 150-250 r / min.

7. The method according to claim 5, characterized in that: The specific steps are as follows: A single colony of oil-producing Schizochytrium was selected and inoculated into a seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain first-generation seeds; 1 mL of the first-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain second-generation seeds; 1 mL of the second-generation seed culture solution was inoculated into 50 mL of the seed culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 24 hours to obtain third-generation seeds, which were used as fermentation strains; Take 10 mL of the third-level seed culture solution and inoculate it into 90 mL of the fermentation medium. After 12 hours of fermentation, add tea polyphenols with a final concentration of 2 g / L. Cultivate at 28°C and 180 r / min shaking for 120 hours to obtain the fermentation solution. After the fermentation culture is completed, NaOH solution is added to the fermentation liquid to adjust the pH to 10-13, and then a wall-breaking enzyme with a final concentration of 0.01-0.2% is added, and the mixture is shaken at 40-60° C. and 100-200 r / min for 5-15 hours; After cooling to room temperature, anhydrous ethanol with the same volume as the fermentation liquid after cell wall breaking was added to inactivate the cell wall breaking enzyme; Add the same volume of n-hexane as the cell-broken fermentation liquid to extract the oil, and repeat the same volume of n-hexane extraction 3 times: The extracted organic phase was allowed to stand for 5 hours, and after the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was taken out and placed in a rotary evaporation bottle, and the organic phase was rotary evaporated in a 45°C water bath at 120r / min. After the organic phase no longer evaporated, the rotary evaporation bottle was removed and dried in a 60°C oven until the weight no longer changed, and the oil was weighed.

8. The method according to claim 5, characterized in that: The entire fermentation culture period is 48 hours to 120 hours.

9. The method according to claim 8, characterized in that: The entire fermentation culture cycle is 48h, 60h, 72h, 84h, 96h or 120h.

10. The method according to any one of claims 4 to 9, characterized in that: The Schizochytrium strain was inoculated into a plate culture medium, and after being cultured on a 500ug / L G418 resistant plate in a 28° constant temperature incubator for 72 hours, a single colony was picked and inoculated into a seed culture medium for activation; The pH value of the plate culture medium is 6.0-6.5, and the plate culture medium comprises: 15-20 g / L agar, 30-60 g / L glucose, 8-15 g / L yeast extract, 10-15 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 6-12 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 sodium 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 4-10 g vitamins B6. mg / L, Vitamin B 12 0.1-1.5mg / L, solvent is water; Alternatively, the seed culture medium has a pH value of 6.0-6.5 and comprises: 40-60 g / L of glucose, 4-6 g / L of yeast extract, 5-8 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 8-12 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, and the solvent is water; Alternatively, the pH value of the fermentation medium is 6.0-6.5, and the fermentation medium comprises: 60-100 g / L of glucose, 5-15 g / L of yeast extract, 5-12 g / L of sodium sulfate, 2-4 g / L of magnesium sulfate, 4-8 g / L of ammonium sulfate, 1-2 g / L of potassium chloride, 0.1-0.2 g / L of calcium chloride, 0.5-1 g / L of potassium sulfate, 0.5-2 g / L of potassium dihydrogen phosphate, 15-20 g / L of sodium glutamate, 1-5 mg / L of zinc sulfate heptahydrate, 0.01-0.1 mg / L of cobalt dichloride hexahydrate, 2-6 mg / L of copper sulfate pentahydrate, 1-2 mg / L of nickel sulfate hexahydrate, 8-15 mg / L of ferrous sulfate heptahydrate, 2-4 mg / L of calcium pantothenate, 3-5 mg / L of manganese chloride tetrahydrate, 0.04 mg / L of sodium molybdate dihydrate, 4-10 mg / L of vitamin B6, and 10 mg / L of vitamin B6. 12 0.1-0.5mg / L, the solvent is water.

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