Application and method of a gingerol in improving production of EPA in schizochytrium

Abstract

The application belongs to the technical field of microorganisms, and discloses application of gingerol in improving EPA yield in schizochytrium. The application improves the EPA yield in schizochytrium by adding an exogenous factor, and the final EPA yield reaches 4.63 g / L, which is more than 6 times of the EPA content (about 0.7 g / L) of a control strain, so that the method provided by the application has very high application potential and value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular to an application and method of gingerol in improving the EPA yield in Schizochytrium. Background Art

[0002] Schizochytrium is a fungus belonging to the phylum Fungi, class Oomycetes, order Saprolegnes, and family Thraustochytridaceae. It is commonly found in the ocean. Due to the presence of a large amount of acetyl-CoA precursors, Schizochytrium is able to produce large amounts of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). Furthermore, during oil production, Schizochytrium also produces polyunsaturated fatty acids such as docosapentaenoic acid (EPA).

[0003] Docosapentaenoic acid (EPA) has many important physiological functions, including reducing blood cholesterol and triglyceride levels, lowering blood viscosity, improving circulation, and preventing cardiovascular and cerebrovascular diseases such as hypertension and cerebral thrombosis. Therefore, EPA has a wide range of applications in health supplements, medical health, and mental health, and possesses enormous market value and prospects.

[0004] Currently, industrial production of EPA relies on extraction from fish oil. However, the isolation and preparation of EPA from fish oil requires a process to remove non-essential components such as pigments, cholesterol, and saturated fatty acids, resulting in high production costs. With increasing demands for quality and health awareness, the market demand for EPA has increased significantly, leading to a supply shortage and an urgent need to increase EPA production. Furthermore, overfishing can lead to fish stock depletion and marine pollution. Therefore, finding an economical, environmentally friendly, and sustainable source of EPA is crucial.

[0005] Microbial fermentation for EPA production offers advantages such as stable yield, reliable quality, low production costs, and freedom from environmental constraints, making it a promising alternative pathway for EPA production. Schizochytrium contains a large amount of acetyl-CoA precursors, enabling the production of large quantities of polyunsaturated fatty acids (PUFAs). Therefore, if Schizochytrium could achieve high EPA production, it would have significant potential for future applications.

[0006] Currently, patents with authorization number CN104450809B increase squalene production in Schizochytrium by exogenously adding sesamol during the fermentation process; patent publication number CN107177640B increases oil production in Schizochytrium by exogenously adding para-benzoic acid derivatives during the fermentation process; patent authorization number CN104480152B increases DHA production in Schizochytrium by changing the carbon source during fermentation; and patent publication number CN201610287253.0 discloses a method of increasing squalene production in Schizochytrium by adding an exogenous regulatory factor, alcohol, through fermentation regulation. Currently, there are no reports on regulating EPA content in Schizochytrium strains by exogenously adding gingerol.

[0007] The aforementioned patent publications all focus on increasing squalene and DHA production in Schizochytrium through fermentation manipulation or exogenous additives. However, there are currently no reports on regulating EPA production in Schizochytrium through exogenous gingerol addition. Compared to the exogenous additives mentioned in the aforementioned patents, gingerol is safer and less expensive. Summary of the Invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application and method of α-lipoic acid in increasing the EPA yield in Schizochytrium.

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

[0010] Application of gingerol in increasing EPA production in Schizochytrium.

[0011] Furthermore, the exogenous substance gingerol is added during the process of fermentation and production of EPA by Schizochytrium.

[0012] Furthermore, the schizochytrium is HX-308 schizochytrium HX308 (Schizochytrium sp.), whose preservation number is CCTCC No. M 209059. The strain has been deposited in the China Center for Type Culture Collection CCTCC and has been disclosed in the Chinese invention patent No. 201510417269.4.

[0013] A method for increasing EPA production by culturing Schizochytrium using a fermentation medium containing gingerol comprises the following steps:

[0014] S1: inoculating the Schizochytrium strain into a seed culture medium and culturing it to obtain a first-level seed solution;

[0015] S2: inoculate the first-level seed solution obtained in S1 into the seed culture medium and culture it again to obtain the second-level seed solution;

[0016] S3: inoculating the secondary seed solution obtained in S2 into the seed culture medium and continuing to culture to obtain the tertiary seed solution;

[0017] S4 inoculates the third-level seed liquid obtained in S3 into a fermentation medium containing gingerol to perform fermentation and culture to produce EPA and DHA.

[0018] Furthermore, the inoculum amount of the Schizochytrium strain in S1 was 0.5%-1% (v / v, volume ratio), and the culture conditions were 25-30° C., 170-200 rpm, and 20-24 h;

[0019] The inoculum size of the first-stage seed solution in S2 is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200 rpm, and 20-24 h;

[0020] The inoculum size of the secondary seed solution in S3 is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200 rpm, and cultured to the logarithmic phase;

[0021] The third-grade seed liquid in S4 is inoculated into a fermentation medium containing gingerol for fermentation culture, with an inoculation amount of 0.5%-1% and culture conditions of 25-30° C., 170-200 rpm, and 72-120 hours.

[0022] Furthermore, the inoculum amount of the Schizochytrium strain in S1 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and 24 h;

[0023] The inoculum size of the primary seed solution in S2 was 1% (v / v), and the culture conditions were 28°C, 170 rpm, and 24 h;

[0024] The inoculum size of the secondary seed solution in S3 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and cultured to the logarithmic phase;

[0025] The third-grade seed liquid in S4 was inoculated into a fermentation medium containing gingerol for fermentation culture. The inoculation amount was 1% (v / v, volume ratio) and the culture conditions were 26° C., 170 rpm, and 96 h.

[0026] Furthermore, the seed culture medium includes: a nitrogen source, a carbon source, inorganic salts and some trace elements;

[0027] The fermentation medium comprises: a nitrogen source, a carbon source, inorganic salts, some trace elements and gingerol;

[0028] The concentration of gingerol in the fermentation medium is 4-15 mg / L.

[0029] Furthermore, the pH of the seed culture medium is 6-8, and the ingredients include: 30.0 g / L glucose, 10.0 g / L yeast extract, 12.0 g / L sodium sulfate, 0.17 g / L calcium chloride dihydrate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, and the solvent is water;

[0030] The fermentation medium has a pH of 6 to 8 and includes the following ingredients: 80.0 g / L glucose, 5.0 g / L sodium glutamate, 5.0 g / L yeast powder, 12.0 g / L sodium sulfate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.17 g / L calcium chloride dihydrate, and 1.0 g / L ammonium sulfate; the solvent is water.

[0031] The added concentration of gingerol in the fermentation medium is 9 mg / L;

[0032] The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

[0033] A method for determining the EPA yield in Schizochytrium obtained by the method described above comprises the following steps:

[0034] S1: adjusting the pH of the Schizochytrium culture medium after fermentation to 10-12, and adding 0.6% of the Schizochytrium culture medium volume percentage of the cell wall breaking enzyme;

[0035] S2: Place the bacteria with cell wall-breaking enzyme in a shaker at 120 rpm and 50°C for 2 h. After the digestion is complete, observe the enzymatic hydrolysis under a microscope.

[0036] S3 adds anhydrous ethanol in an equal volume to the culture medium in a volume ratio of 1:1 to precipitate proteins and obtain a precipitate;

[0037] S4 Add n-hexane with an equal volume of anhydrous ethanol and mix well. After stratification, remove the upper yellow liquid, which is a mixture of n-hexane and oil.

[0038] S5: Repeat step S4 for multiple extractions until the supernatant is colorless and transparent;

[0039] S6: The obtained extract is subjected to rotary evaporation. The weight of the rotary evaporation bottle is weighed before the experiment begins;

[0040] After S7 rotary evaporation, the rotary evaporation bottle is placed in an oven and dried to constant weight. The obtained oil is the oil produced by fermentation;

[0041] S8 performs methyl esterification on the obtained oil and fat, and analyzes the changes in the content of various fatty acids in the oil and fat;

[0042] S81 Weigh 20 μL of oil into a 2 mL centrifuge tube, add 600 μL of 1 M potassium hydroxide-methanol solution, and then shake in a shaker at room temperature and 1000 rpm for 4 hours to fully methylate it.

[0043] S82 added 40 μL of concentrated sulfuric acid to terminate the reaction, added 1 mL of n-hexane containing an internal standard, and oscillated at room temperature and 1000 rpm for 30 min to obtain an extract phase; the extract phase was added to a gas phase vial for detection;

[0044] The chromatographic column used in the S83 experiment was DB-23 (60.0 m × 0.25 mm × 0.25 μm); the detection conditions were: injection port temperature 250°C, injection volume 1.0 μL, split ratio 69.8:1; chromatographic conditions were: initial temperature 100°C, heating at 25°C / min to 196°C, then heating at 2°C / min to 220°C and holding for 21 min;

[0045] After the S84 gas phase test, different fatty acids were identified by comparison with relevant external standards (Sigma, USA). In this gas phase test, the non-endogenous fatty acid C11:0 was used as the internal standard, and the content of individual fatty acids was estimated from the peak area on the chromatogram.

[0046] A method for measuring the biomass of Schizochytrium after fermentation obtained by the method described above comprises the following steps:

[0047] Take 1 mL of the fermentation broth that has just been fermented, spin at 4000 r / min for 5 min, discard the supernatant, wash once with 1 mL of 2% NaCl solution and 1 mL of distilled water, freeze in a -80°C refrigerator for 12 h, then freeze-dry in a freeze dryer, and finally weigh and calculate;

[0048] The volume ratio of the fermentation liquid just after fermentation: NaCl solution: distilled water is 1:1:1.

[0049] The advantages and positive effects achieved by the present invention are:

[0050] 1. This invention proposes for the first time that adding a novel exogenous compound, gingerol, can increase EPA content during Schizochytrium fermentation. Compared to a control group without gingerol, experimental groups treated with varying concentrations of gingerol consistently increased EPA content during Schizochytrium fermentation. By adding a certain proportion of gingerol to the Schizochytrium fermentation system, this invention acts as an antioxidant and inhibitor within the Schizochytrium, inhibiting reactive oxygen species and reducing stress levels, thereby reducing oxidative degradation of lipids and increasing lipid accumulation. Furthermore, by perturbing the metabolic network and reducing carbon flux toward lipid biosynthesis, it effectively increases EPA production during Schizochytrium fermentation. The EPA content increased by over 6.31 times, and the ratio of EPA to biomass (mg / g DCW) increased by over 7.22 times.

[0051] 2. The present invention provides a method for increasing EPA production by exogenously adding metabolites during fermentation. Specifically, during the production of EPA by Schizochytrium, a certain amount of gingerol is exogenously added to increase the EPA produced by Schizochytrium fermentation. The present invention can significantly increase the EPA production during Schizochytrium fermentation through simple fermentation regulation. At the same time, gingerol is relatively inexpensive and easily available, which reduces the cost of the Schizochytrium fermentation process and has great economic benefits.

[0052] 3. This invention provides gingerol, an exogenous fermentation regulator for increasing EPA content in Schizochytrium. This is the first application of this compound in the fermentation production of EPA in Schizochytrium. This compound acts as an oxidation inhibitor, significantly reducing oxidative stress in Schizochytrium, minimizing oxidative degradation of oils and fats, increasing oil accumulation, and effectively regulating EPA production in Schizochytrium. This invention provides a simple, efficient, inexpensive, and safe method for producing EPA in Schizochytrium.

[0053] 4. The present invention increases the EPA production in Schizochytrium in a simple, convenient, economical and efficient manner by adding exogenous factors, so that the final EPA production reaches 4.63 g / L, which is more than 6 times the EPA content of the control strain (about 0.7 g / L). This shows that the method provided by the present invention has very high application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a graph showing the EPA production results of the wild-type Schizochytrium strain of the present invention;

[0055] Figure 2 This is a graph showing the EPA production results of the Schizochytrium strain fermented with exogenous additives in the present invention. DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0057] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0058] Application of gingerol in increasing EPA production in Schizochytrium.

[0059] Preferably, the exogenous substance gingerol is added during the process of fermentation and production of EPA by Schizochytrium.

[0060] Preferably, the Schizochytrium sp. is HX-308 Schizochytrium sp., whose deposit number is CCTCC No. M 209059. The strain has been deposited in the China Center for Type Culture Collection CCTCC and has been disclosed in the Chinese invention patent No. 201510417269.4.

[0061] A method for increasing EPA production by culturing Schizochytrium using a fermentation medium containing gingerol comprises the following steps:

[0062] S1: inoculating the Schizochytrium strain into a seed culture medium and culturing it to obtain a first-level seed solution;

[0063] S2: inoculate the first-level seed solution obtained in S1 into the seed culture medium and culture it again to obtain the second-level seed solution;

[0064] S3: inoculating the secondary seed solution obtained in S2 into the seed culture medium and continuing to culture to obtain the tertiary seed solution;

[0065] S4 inoculates the third-level seed liquid obtained in S3 into a fermentation medium containing gingerol to perform fermentation and culture to produce EPA and DHA.

[0066] Preferably, the inoculum amount of the Schizochytrium strain in S1 is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30° C., 170-200 rpm, and 20-24 h;

[0067] The inoculum size of the first-stage seed solution in S2 is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200 rpm, and 20-24 h;

[0068] The inoculum size of the secondary seed solution in S3 is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200 rpm, and cultured to the logarithmic phase;

[0069] The third-grade seed liquid in S4 is inoculated into a fermentation medium containing gingerol for fermentation culture, with an inoculation amount of 0.5%-1% and culture conditions of 25-30° C., 170-200 rpm, and 72-120 hours.

[0070] Preferably, the inoculum amount of the Schizochytrium strain in S1 is 1% (v / v, volume ratio), and the culture conditions are 28° C., 170 rpm, and 24 h;

[0071] The inoculum size of the primary seed solution in S2 was 1% (v / v), and the culture conditions were 28°C, 170 rpm, and 24 h;

[0072] The inoculum size of the secondary seed solution in S3 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and cultured to the logarithmic phase;

[0073] The third-grade seed liquid in S4 was inoculated into a fermentation medium containing gingerol for fermentation culture. The inoculation amount was 1% (v / v, volume ratio) and the culture conditions were 26° C., 170 rpm, and 96 h.

[0074] Preferably, the seed culture medium comprises: a nitrogen source, a carbon source, inorganic salts and some trace elements;

[0075] The fermentation medium comprises: a nitrogen source, a carbon source, inorganic salts, some trace elements and gingerol;

[0076] The concentration of gingerol in the fermentation medium is 4-15 mg / L.

[0077] Preferably, the pH of the seed culture medium is 6-8, and the ingredients include: 30.0 g / L glucose, 10.0 g / L yeast extract, 12.0 g / L sodium sulfate, 0.17 g / L calcium chloride dihydrate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, and the solvent is water;

[0078] The fermentation medium has a pH of 6 to 8 and includes the following ingredients: 80.0 g / L glucose, 5.0 g / L sodium glutamate, 5.0 g / L yeast powder, 12.0 g / L sodium sulfate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.17 g / L calcium chloride dihydrate, and 1.0 g / L ammonium sulfate; the solvent is water.

[0079] The added concentration of gingerol in the fermentation medium is 9 mg / L;

[0080] The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

[0081] A method for determining the EPA yield in Schizochytrium obtained by the method described above comprises the following steps:

[0082] S1: adjusting the pH of the Schizochytrium culture medium after fermentation to 10-12, and adding 0.6% of the Schizochytrium culture medium volume percentage of the cell wall breaking enzyme;

[0083] S2: Place the bacteria with cell wall-breaking enzyme in a shaker at 120 rpm and 50°C for 2 h. After the digestion is complete, observe the enzymatic hydrolysis under a microscope.

[0084] S3 adds anhydrous ethanol in an equal volume to the culture medium in a volume ratio of 1:1 to precipitate proteins and obtain a precipitate;

[0085] S4 Add n-hexane with an equal volume of anhydrous ethanol and mix well. After stratification, remove the upper yellow liquid, which is a mixture of n-hexane and oil.

[0086] S5: Repeat step S4 for multiple extractions until the supernatant is colorless and transparent;

[0087] S6: The obtained extract is subjected to rotary evaporation. The weight of the rotary evaporation bottle is weighed before the experiment begins;

[0088] After S7 rotary evaporation, the rotary evaporation bottle is placed in an oven and dried to constant weight. The obtained oil is the oil produced by fermentation;

[0089] S8 performs methyl esterification on the obtained oil and fat, and analyzes the changes in the content of various fatty acids in the oil and fat;

[0090] S81 Weigh 20 μL of oil into a 2 mL centrifuge tube, add 600 μL of 1 M potassium hydroxide-methanol solution, and then shake in a shaker at room temperature and 1000 rpm for 4 hours to fully methylate it.

[0091] S82 added 40 μL of concentrated sulfuric acid to terminate the reaction, added 1 mL of n-hexane containing an internal standard, and oscillated at room temperature and 1000 rpm for 30 min to obtain an extract phase; the extract phase was added to a gas phase vial for detection;

[0092] The chromatographic column used in the S83 experiment was DB-23 (60.0 m × 0.25 mm × 0.25 μm); the detection conditions were: injection port temperature 250°C, injection volume 1.0 μL, split ratio 69.8:1; chromatographic conditions were: initial temperature 100°C, heating at 25°C / min to 196°C, then heating at 2°C / min to 220°C and holding for 21 min;

[0093] After the S84 gas phase test, different fatty acids were identified by comparison with relevant external standards (Sigma, USA). In this gas phase test, the non-endogenous fatty acid C11:0 was used as the internal standard, and the content of individual fatty acids was estimated from the peak area on the chromatogram.

[0094] A method for measuring the biomass of Schizochytrium after fermentation obtained by the method described above comprises the following steps:

[0095] Take 1 mL of the fermentation broth that has just been fermented, spin at 4000 r / min for 5 min, discard the supernatant, wash once with 1 mL of 2% NaCl solution and 1 mL of distilled water, freeze in a -80°C refrigerator for 12 h, then freeze-dry in a freeze dryer, and finally weigh and calculate;

[0096] The volume ratio of the fermentation liquid just after fermentation: NaCl solution: distilled water is 1:1:1.

[0097] Specifically, the relevant preparation and detection are as follows:

[0098] Example 1

[0099] A method for culturing Schizochytrium to increase EPA production by exogenously adding gingerol, and a method for measuring EPA production and post-fermentation bacterial biomass, comprising the following steps:

[0100] S1: Inoculate Schizochytrium CCTCC No.M 209059 into 50 mL of seed culture medium with an inoculation volume of 1%. The culture conditions are 28°C, 170 rpm, and 24 h to obtain a first-level seed culture solution. Inoculate 500 μL of the first-level seed culture solution into a new 50 mL of seed culture medium. The culture conditions are 28°C, 170 rpm, and 24 h to obtain a second-level seed culture solution. Inoculate 500 μL of the second-level seed culture solution into a new 50 mL of seed culture medium and culture at 28°C, 170 rpm to obtain a third-level seed solution in the logarithmic phase.

[0101] S2: inoculating the logarithmic phase tertiary seed liquid into a fermentation medium containing gingerol at an inoculum size of 1% (v / v, volume ratio) in a shake flask at 26° C. and 170 rpm for 96 h to obtain an EPA-containing oil;

[0102] The pH of the seed culture medium is 6-8, and the ingredients include: 30.0 g / L glucose, 10.0 g / L yeast extract, 12.0 g / L sodium sulfate, 0.17 g / L calcium chloride dihydrate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, and the solvent is water;

[0103] The fermentation medium has a pH of 6 to 8 and includes the following ingredients: 80.0 g / L glucose, 5.0 g / L sodium glutamate, 5.0 g / L yeast powder, 12.0 g / L sodium sulfate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.17 g / L calcium chloride dihydrate, and 1.0 g / L ammonium sulfate; the solvent is water.

[0104] 4-15 mg / L of gingerol was added to the fermentation medium, and the optimal concentration was 9 mg / L.

[0105] The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

[0106] Meanwhile, fermentation medium without gingerol addition was used as a control group.

[0107] The final oil test results showed that the proportion of EPA in the oil produced by Schizochytrium fermentation increased from 734 mg / L produced by the Schizochytrium strain without gingerol to 4.63 g / L produced by the Schizochytrium strain with exogenous additives. Figure 1 and Figure 2 shown.

[0108] S3: adjusting the pH of the Schizochytrium culture medium after fermentation to 10-12, and adding 0.6% (v / v, volume ratio) of the cell wall-breaking enzyme;

[0109] S4: Place the bacterial cells with the cell wall-breaking enzyme into a conical flask and incubate in a shaker at 120 rpm and 50°C for 2 h. After the digestion is complete, observe the enzymatic hydrolysis under a microscope.

[0110] S5: add an equal volume of anhydrous ethanol in a volume ratio of 1:1 to precipitate the protein;

[0111] S6 Add an equal volume of n-hexane and anhydrous ethanol and mix well. After stratification, remove the upper yellow liquid, which is a mixture of n-hexane and oil.

[0112] S7: Repeat step S4 for multiple extractions until the supernatant is colorless and transparent;

[0113] S8: The obtained extract is subjected to rotary evaporation. The weight of the rotary evaporation bottle is weighed before the experiment begins;

[0114] The rotary evaporation bottle after S9 rotary evaporation is placed in an oven and dried to constant weight. The obtained oil is the oil produced by fermentation;

[0115] S10 performs methyl esterification on the obtained oil and analyzes the changes in the content of various fatty acids in the oil;

[0116] S101 Weigh 20 μL of oil into a 2 mL centrifuge tube, add 600 μL of 1 M potassium hydroxide-methanol solution, and then shake in a shaker at room temperature and 1000 rpm for 4 hours to fully methylate the oil.

[0117] S102 was added with 40 μL of concentrated sulfuric acid to terminate the reaction, and 1 mL of n-hexane containing an internal standard was added. The mixture was shaken at room temperature and 1000 rpm for 30 minutes to obtain an extract. The extract was added to a gas phase vial for detection.

[0118] The S103 experiment used a DB-23 column (60.0 m × 0.25 mm × 0.25 μm). The detection conditions were: an inlet temperature of 250°C, an injection volume of 1.0 μL, and a split ratio of 69.8:1. The chromatographic conditions were: an initial temperature of 100°C, ramped to 196°C at 25°C / min, then ramped to 220°C at 2°C / min and held for 21 min.

[0119] After the S104 gas phase test, different fatty acids were identified by comparison with relevant external standards (Sigma, USA). In this gas phase test, a non-endogenous fatty acid (C11:0) was used as the internal standard, and the content of individual fatty acids was estimated from the peak area on the chromatogram.

[0120] S11 Determination of biomass: Take 1 mL of the fermentation broth that has just been fermented, spin at 4000 r / min for 5 min, discard the supernatant, wash once with 1 mL of 2% NaCl solution and 1 mL of distilled water, freeze in a -80°C refrigerator for 12 h, then freeze-dry in a freeze dryer, and finally weigh and calculate.

[0121] The detection of other indicators all adopts conventional methods in the existing technology.

[0122] Example 2

[0123] Adopt the method among the embodiment 1, the gingerol that adds in the middle of fermention medium adds according to certain gradient concentration (0,1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L, 6mg / L, 7mg / L, 8mg / L, 9mg / L, 10mg / L, 11mg / L, 12mg / L, 13mg / L, 14mg / L, 15mg / L), finishes when fermenting to 96h, and carries out the detection of every index. The results are shown in Table 1.

[0124] As shown in Table 1, after adding different concentrations of exogenous gingerol, the biomass after fermentation decreased. The EPA content produced by the Schizochytrium fermentation with the addition of 9 mg / L gingerol was the highest, which was 4.63 g / L. That is, the proportion of EPA in the oil produced by the Schizochytrium fermentation increased from 734 mg / L produced by the Schizochytrium strain without adding gingerol to 4.63 g / L produced by the Schizochytrium strain with the addition of exogenous additives. This can effectively increase the content of EPA produced by Schizochytrium fermentation, among which the content of EPA increased by more than 6.31 times, and the proportion of EPA in biomass increased by more than 7.22 times.

[0125] At present, the yield of EPA produced by the control bacteria Schizochytrium is generally 0.734g / L ( Figure 1 ), and the present invention uses a simple, convenient, economical and efficient method of exogenous addition of gingerol to achieve a yield of EPA of 4.63 g / L ( Figure 2 ).

[0126] The amount of gingerol added in the present invention is very important. Both too low and too high a content will lead to unsatisfactory fermentation results. Too low a content may have no effect on the fermentation of the strain or may only increase the EPA yield to a limited extent. Too high a content will greatly affect the biomass and oil content.

[0127] Table 1 Fermentation results of Schizochytrium at different gingerol addition amounts

[0128]

[0129]

[0130] Example 3

[0131] Adopt the method for embodiment 1, but remove calcium chloride dihydrate in fermention medium, add equivalent zinc sulfate, carry out gingerol and add according to gradient (0,1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L, 6mg / L, 7mg / L, 8mg / L, 9mg / L, 10mg / L, 11mg / L, 12mg / L, 13mg / L, 14mg / L, 15mg / L) again, finish when fermenting to 96h, and carry out the detection of various indices. The results are shown in Table 2.

[0132] Table 2 Fermentation results of Schizochytrium at different gingerol addition amounts

[0133]

[0134]

[0135] As shown in Table 2, when one of the inorganic salt ions was replaced, the biomass of Schizochytrium decreased compared with the previous fermentation medium, and the EPA production was significantly reduced compared with the previous medium. In addition, the oil produced by Schizochytrium fermentation also decreased to a certain extent. For example, when the concentration of exogenously added gingerol was 9 mg / L, when the inorganic salt ions were not replaced, the biomass of Schizochytrium fermentation was 104.35 g / L, the oil content was 69.64 g / L, and the EPA production was 4.63 g / L. After replacing the inorganic salt ions, the biomass of Schizochytrium fermentation was 95.59 g / L, the oil content was 59.63 g / L, and the EPA production was 3.69 g / L, which were significant changes compared with before the inorganic salt ions were replaced.

[0136] This study demonstrates that inorganic salt ions and gingerol in the fermentation medium produce a synergistic effect, enabling Schizochytrium to significantly increase EPA production. It also demonstrates the influence of fermentation conditions on EPA production, demonstrating that the synergistic effect of gingerol and inorganic salt ions can maximize the effectiveness of gingerol, thereby increasing EPA production in Schizochytrium.

[0137] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand 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 gingerol in increasing EPA production in Schizochytrium sp., characterized in that: The exogenous substance gingerol is added during the fermentation process of EPA production by Schizochytrium; The fermentation medium used in the fermentation production has a pH of 6-8 and includes the following ingredients: 80.0 g / L glucose, 5.0 g / L sodium glutamate, 5.0 g / L yeast powder, 12.0 g / L sodium sulfate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.17 g / L calcium chloride dihydrate, and 1.0 g / L ammonium sulfate; the solvent is water; The added concentration of gingerol in the fermentation medium is 8-10 mg / L; The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

2. The use according to claim 1, characterized in that: The Schizochytrium sp. is HX-308 Schizochytrium sp., and its preservation number is CCTCC No. M 209059. The strain has been deposited in the China Center for Type Culture Collection CCTCC.

3. A method for increasing EPA production by culturing Schizochytrium using a fermentation medium containing gingerol, characterized in that: The steps include: S1: inoculating the Schizochytrium strain into a seed culture medium and culturing it to obtain a first-level seed solution; S2: inoculate the first-level seed solution obtained in S1 into the seed culture medium and culture it again to obtain the second-level seed solution; S3: inoculating the secondary seed solution obtained in S2 into the seed culture medium and continuing to culture to obtain the tertiary seed solution; S4: inoculating the third-grade seed liquid obtained in S3 into a fermentation medium containing gingerol to perform fermentation and culture to produce EPA and DHA; The pH of the fermentation medium is 6-8, and the ingredients include: 80.0 g / L glucose, 5.0 g / L sodium glutamate, 5.0 g / L yeast powder, 12.0 g / L sodium sulfate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.17 g / L calcium chloride dihydrate, and 1.0 g / L ammonium sulfate, and the solvent is water; The added concentration of gingerol in the fermentation medium is 8-10 mg / L; The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

4. The method according to claim 3, wherein: The inoculum size of the Schizochytrium strain in S1 was 0.5%-1% (v / v), and the culture conditions were 25-30°C, 170-200 rpm, and 20-24 h; The inoculum size of the first-stage seed solution in S2 to the seed culture medium is 0.5%-1% (v / v), and the culture conditions are 25-30°C, 170-200 rpm, and 20-24 h; The inoculum size of the secondary seed solution in S3 is 0.5%-1% (v / v) and the culture conditions are 25-30°C, 170-200 rpm, and cultured to the logarithmic phase; The third-grade seed liquid of S4 is inoculated into a fermentation medium containing gingerol for fermentation culture, with an inoculation amount of 0.5%-1% and culture conditions of 25-30° C., 170-200 rpm, and 72-120 hours.

5. The method according to claim 3, wherein: The inoculum size of the Schizochytrium strain in S1 was 1% (v / v), and the culture conditions were 28°C, 170 rpm, and 24 h; The inoculum size of the primary seed solution in S2 was 1% (v / v), and the culture conditions were 28°C, 170 rpm, and 24 h; The inoculum size of the secondary seed solution in S3 was 1% (v / v), and the culture conditions were 28°C, 170 rpm, and cultured to the logarithmic phase; The third-grade seed liquid of S4 was inoculated into a fermentation medium containing gingerol for fermentation culture. The inoculation amount was 1% (v / v) and the culture conditions were 26°C, 170 rpm, and 96 h.

6. The method according to any one of claims 3 to 5, characterized in that: The seed culture medium includes: a nitrogen source, a carbon source, inorganic salts and some trace elements; The concentration of gingerol in the fermentation medium is 9 mg / L.

7. The method according to claim 6, characterized in that: The pH of the seed culture medium is 6-8, and the ingredients include: 30.0 g / L glucose, 10.0 g / L yeast extract, 12.0 g / L sodium sulfate, 0.17 g / L calcium chloride dihydrate, 0.50 g / L potassium chloride, 2.0 g / L magnesium sulfate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, and the solvent is water.

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