Application and method of gingerol in increasing yield of EPA (eicosapentaenoic acid) in schizochytrium limacinum

Abstract

The invention belongs to the technical field of microorganisms, and discloses an application of gingerol in increasing the yield of EPA (Eicosapentaenoic Acid) in schizochytrium limacinum. According to the method, the yield of the EPA in the schizochytrium limacinum is simply, conveniently, economically and efficiently increased in a manner of adding exogenous factors, so that the final EPA yield reaches 4.63 g / L and is increased by more than 6 times compared with the EPA content (about 0.7 g / L) of a control strain, and the method provided by the invention 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 belongs to the Fungi, Oomycetes, Saprolegnes, Thraustochytriaceae, and is commonly found in the ocean. Due to the presence of a large amount of acetyl-CoA precursors in Schizochytrium, it can produce a large amount of polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). In addition, Schizochytrium also produces polyunsaturated fatty acids such as docosapentaenoic acid (EPA) when producing oils.

[0003] Docosapentaenoic acid (EPA) has many important physiological functions. It can reduce the cholesterol and triglyceride content in human blood, reduce blood viscosity, improve blood circulation, and prevent cardiovascular and cerebrovascular diseases such as hypertension and cerebral thrombosis. Therefore, EPA has a wide range of applications in health care products, medical health, mental health and other fields, and has huge market value and market prospects.

[0004] At present, the industrial production of EPA relies on extraction from fish oil. However, the separation and preparation of EPA from fish oil also requires the removal of non-essential components such as pigments, cholesterol, and saturated fatty acids through processes, which results in high production costs. With the increase in people's living needs and the enhancement of health awareness, the market demand for EPA has increased significantly, resulting in a market situation where supply exceeds demand. Therefore, it is urgent to increase the production of EPA. In addition, overfishing can lead to a shortage of fish resources and marine pollution. Therefore, it is very necessary to find an economical, environmentally friendly, and sustainable source of EPA production.

[0005] Microbial fermentation to produce EPA has the advantages of stable yield, reliable quality, low production cost, and no restrictions on environmental conditions. It is an alternative way to produce EPA with great industrial potential. There are a large number of acetyl-CoA precursors in Schizochytrium, which can produce a large number of polyunsaturated fatty acids (PUFAs). Therefore, if Schizochytrium can produce high levels of EPA, it must have great application prospects.

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

[0007] The above-mentioned several related patent publications all focus on using fermentation regulation or exogenous additives to increase the production of squalene and DHA in Schizochytrium, but there is no report on regulating the production of EPA in Schizochytrium by exogenous addition of gingerol. Compared with the exogenous additives mentioned in the above-mentioned patents, gingerol is safer and has lower cost. 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 alpha-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 improving EPA production in Schizochytrium.

[0011] Furthermore, the Schizochytrium adds exogenous substance gingerol during the process of fermenting and producing EPA.

[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 published 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 primary seed solution;

[0015] S2 inoculates the first-level seed solution obtained in S1 into the seed culture medium, and cultures 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 inoculation 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;

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

[0020] The inoculation amount of the secondary seed solution in S3 into the seed culture medium is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200rpm, 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, the inoculation amount is 0.5%-1%, and the culture conditions are 25-30° C., 170-200 rpm, and 72-120 hours.

[0022] Furthermore, the inoculation 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 inoculation amount of the primary seed solution in S2 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and 24 h;

[0024] The inoculum amount 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, an inorganic salt, some trace elements and gingerol;

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

[0029] Further, 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 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, 1.0 g / L ammonium sulfate, and 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 as described above comprises the following steps:

[0034] S1: adjusting the pH of the Schizochytrium culture medium after the 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 bacterial cells with the cell wall-breaking enzyme in a shaker at 120 rpm and 50°C for 2 h. After digestion, 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 the like to obtain a precipitate;

[0037] S4 adds an equal volume of n-hexane as anhydrous ethanol and mixes well. After stratification, take out 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: subjecting the obtained extract to rotary evaporation, and weighing the rotary evaporation bottle before the experiment begins;

[0040] The rotary evaporation bottle after S7 rotary evaporation is placed in an oven to dry to constant weight, and 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 each fatty acid 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 oscillate in an oscillator at room temperature and 1000 r / min 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 r / min 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 (60.0m×0.25mm×0.25μm); detection conditions: injection port temperature 250℃, injection volume 1.0μL, split ratio 69.8:1; chromatographic conditions: initial temperature 100℃, 25℃ / min heating to 196℃. Then, 2℃ / min heating to 220℃ and keeping warm for 21min;

[0045] After the S84 gas phase detection, different fatty acids were identified by comparison with relevant external standards (Sigma, USA). In this gas phase detection, 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 as described above comprises the following steps:

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

[0048] Among them, the volume ratio of the fermentation liquid just fermented: NaCl solution: distilled water is 1:1:1.

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

[0050] 1. The present invention proposes for the first time that adding a new type of exogenous compound can increase the content of EPA in the fermentation process of Schizochytrium. This exogenous compound refers to gingerol. Compared with the control group without gingerol during the fermentation process, the content of EPA produced by Schizochytrium fermentation in the experimental group with different concentrations of gingerol added can be increased. The present invention adds a certain proportion of gingerol to the Schizochytrium fermentation system as an antioxidant, acts as an inhibitor in the Schizochytrium, inhibits the active oxygen in the body, reduces its stress level, thereby reducing the oxidative degradation of oils and fats, and increasing the accumulation of oils and fats. At the same time, the metabolic network is disturbed to reduce the flow of carbon to the biosynthesis of lipids, which can effectively increase the EPA produced by Schizochytrium fermentation, wherein the content of EPA is increased by more than 6.31 times, and the ratio of EPA to biomass (mg / g DCW) is increased by more than 7.22 times.

[0051] 2. The method of the present invention is a method for increasing the yield of EPA by exogenously adding metabolites during the fermentation process. Specifically, in the process of producing 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 yield of EPA in the Schizochytrium fermentation process through simple fermentation regulation. At the same time, gingerol is relatively low in price and easy to obtain, which reduces the cost of the Schizochytrium fermentation process and has great economic benefits.

[0052] 3. The present invention provides an exogenous fermentation regulatory factor for increasing the EPA content of Schizochytrium, namely gingerol. This is the first time that the compound has been used in the fermentation production of EPA by Schizochytrium. The compound has the function of an oxidation inhibitor, which can significantly reduce the oxidative stress of Schizochytrium, reduce the oxidative degradation of oils and fats, increase the accumulation of oils and fats, and effectively adjust the EPA produced by the fermentation of Schizochytrium. The present invention is a method for producing EPA in Schizochytrium, which is simple, efficient, inexpensive and safe.

[0053] 4. The present invention increases the production of EPA 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), indicating 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 strain of Schizochytrium in the present invention;

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

[0056] 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.

[0057] 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.

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

[0059] Preferably, the exogenous substance gingerol is added during the process of the Schizochytrium fermentation to produce EPA.

[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 published 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 primary seed solution;

[0063] S2 inoculates the first-level seed solution obtained in S1 into the seed culture medium, and cultures 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 inoculation 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 inoculation amount of the first-level seed solution in S2 into the seed culture medium is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200rpm, and 20-24h;

[0068] The inoculation amount of the secondary seed solution in S3 into the seed culture medium is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200rpm, 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, the inoculation amount is 0.5%-1%, and the culture conditions are 25-30° C., 170-200 rpm, and 72-120 hours.

[0070] Preferably, the inoculation 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 inoculation amount of the primary seed solution in S2 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and 24 h;

[0072] The inoculum amount 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, an inorganic salt, 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 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, 1.0 g / L ammonium sulfate, and 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 as described above comprises the following steps:

[0082] S1: adjusting the pH of the Schizochytrium culture medium after the 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 bacterial cells with the cell wall-breaking enzyme in a shaker at 120 rpm and 50°C for 2 h. After digestion, 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 the like to obtain a precipitate;

[0085] S4 adds an equal volume of n-hexane as anhydrous ethanol and mixes well. After stratification, take out 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: subjecting the obtained extract to rotary evaporation, and weighing the rotary evaporation bottle before the experiment begins;

[0088] The rotary evaporation bottle after S7 rotary evaporation is placed in an oven to dry to constant weight, and 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 each fatty acid 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 oscillate in an oscillator at room temperature and 1000 r / min 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 r / min 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 (60.0m×0.25mm×0.25μm); detection conditions: injection port temperature 250℃, injection volume 1.0μL, split ratio 69.8:1; chromatographic conditions: initial temperature 100℃, 25℃ / min heating to 196℃. Then, 2℃ / min heating to 220℃ and keeping warm for 21min;

[0093] After the S84 gas phase detection, different fatty acids were identified by comparison with relevant external standards (Sigma, USA). In this gas phase detection, 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 as described above comprises the following steps:

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

[0096] Among them, the volume ratio of the fermentation liquid just fermented: 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 bacterial biomass after fermentation, comprising the following steps:

[0100] S1 Inoculate Schizochytrium CCTCC No.M 209059 into 50mL seed culture medium with an inoculation volume of 1% and culture conditions of 28°C, 170rpm, and 24h to obtain a primary seed culture solution; inoculate 500μL of the primary seed culture solution into a new 50mL seed culture medium and culture conditions of 28°C, 170rpm, and 24h to obtain a secondary seed culture solution; inoculate 500μL of the secondary seed culture solution into a new 50mL seed culture medium and culture at 28°C, 170rpm to obtain a tertiary seed solution in the logarithmic phase.

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

[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 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, 1.0 g / L ammonium sulfate, and 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 original Schizochytrium strain without gingerol fermentation to 4.63 g / L produced by the Schizochytrium strain with exogenous additives fermentation. Figure 1 and Figure 2 shown.

[0108] S3: adjusting the pH of the Schizochytrium culture medium to 10-12 after the fermentation is completed, 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 adds an equal volume of anhydrous ethanol in a volume ratio of 1:1 to precipitate the protein;

[0111] S6 Add n-hexane with an equal volume of anhydrous ethanol and mix well. After stratification, take out 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: subjecting the obtained extract to rotary evaporation, and weighing the rotary evaporation bottle before the experiment begins;

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

[0115] S10 performs methyl esterification on the obtained oil and fat, and analyzes the changes in the content of each fatty acid in the oil and fat;

[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 oscillate in an oscillator at room temperature and 1000 r / min for 4 hours to fully methylate it;

[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, and the mixture was shaken at room temperature and 1000 r / min for 30 min to obtain an extract phase, which was added to a gas phase vial for detection.

[0118] The chromatographic column used in the S103 experiment was DB-23 (60.0m×0.25mm×0.25μm); detection conditions: injection port temperature 250℃, injection volume 1.0μL, split ratio 69.8:1; chromatographic conditions: initial temperature 100℃, 25℃ / min heating to 196℃. Then, 2℃ / min heating to 220℃ and keeping warm for 21min.

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

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

[0121] The detection of other indicators all adopts conventional methods in the prior art.

[0122] Example 2

[0123] Adopt the method among the embodiment 1, the gingerol that adds in the fermentation 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 various indices. 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 Schizochytrium fermentation with the addition of 9 mg / L gingerol was the highest, which was 4.63 g / L. That is, in the oil produced by Schizochytrium fermentation, the proportion of EPA 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 adding gingerol exogenously to make the yield of EPA produced by Schizochytrium fermentation reach 4.63g / L ( Figure 2 ).

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

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

[0128]

[0129]

[0130] Example 3

[0131] The method of Example 1 was adopted, but calcium chloride dihydrate was removed from the fermentation medium, an equivalent amount of zinc sulfate was added, and gingerol was added according to a gradient (0, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L), and fermentation ended when 96 h, and various indexes were detected. The results are shown in Table 2.

[0132] Table 2 Fermentation results of Schizochytrium under 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 was a significant change compared with before the inorganic salt ions were replaced.

[0136] This indicates that inorganic salt ions and gingerol in the fermentation medium have a synergistic effect, which enables Schizochytrium to increase the production of EPA to a greater extent. It also explains the influence of fermentation conditions on EPA production. The synergistic effect of gingerol and inorganic salt ions can make gingerol play a greater role, thereby increasing the production of EPA in Schizochytrium.

[0137] 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. Application of gingerol in increasing the EPA production in Schizochytrium.

2. The use according to claim 1, characterized in that: The exogenous substance gingerol is added during the process of the Schizochytrium fermentation to produce EPA.

3. The use according to claim 1, characterized in that: The schizochytrium is HX-308 schizochytrium HX308 (Schizochytrium sp.), and its preservation number is CCTCC No.M 209059. The strain has been preserved in the China Center for Type Culture Collection CCTCC and has been published in the Chinese invention patent with patent number 201510417269.

4.

4. 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 primary seed solution; S2 inoculates the first-level seed solution obtained in S1 into the seed culture medium, and cultures 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 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.

5. The method according to claim 4, characterized in that: 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; The inoculation amount of the first-level seed solution in S2 into the seed culture medium is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200rpm, and 20-24h; The inoculation amount of the secondary seed solution in S3 into the seed culture medium is 0.5%-1% (v / v, volume ratio), and the culture conditions are 25-30°C, 170-200rpm, and cultured to the logarithmic phase; The third-grade seed liquid in S4 is inoculated into a fermentation medium containing gingerol for fermentation culture, the inoculation amount is 0.5%-1%, and the culture conditions are 25-30° C., 170-200 rpm, and 72-120 hours.

6. The method according to claim 4, characterized in that: The inoculum amount of Schizochytrium strain in S1 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and 24 h; The inoculation amount of the primary seed solution in S2 was 1% (v / v, volume ratio), and the culture conditions were 28°C, 170 rpm, and 24 h; The inoculum amount 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; 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.

7. The method according to any one of claims 4 to 6, characterized in that: The seed culture medium comprises: a nitrogen source, a carbon source, inorganic salts and some trace elements; The fermentation medium comprises: a nitrogen source, a carbon source, an inorganic salt, some trace elements and gingerol; The concentration of gingerol in the fermentation medium is 4-15 mg / L.

8. The method according to claim 7, characterized in that: The seed culture medium has a pH of 6 to 8, and its 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; 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, 1.0 g / L ammonium sulfate, and the solvent is water; The added concentration of gingerol in the fermentation medium is 9 mg / L; The gingerol in the fermentation medium is dissolved in ethyl acetate, sterilized by a membrane, and then added into the fermentation medium.

9. A method for determining the EPA yield in Schizochytrium obtained by the method according to any one of claims 4 to 8, characterized in that: The steps include: S1: adjusting the pH of the Schizochytrium culture medium after the fermentation to 10-12, and adding 0.6% of the Schizochytrium culture medium volume percentage of the cell wall breaking enzyme; S2: Place the bacterial cells with the cell wall-breaking enzyme in a shaker at 120 rpm and 50°C for 2 h. After digestion, observe the enzymatic hydrolysis under a microscope. S3 was added with anhydrous ethanol in a volume ratio of 1:1 to obtain a precipitate; S4 adds an equal volume of n-hexane as anhydrous ethanol and mixes well. After stratification, take out the upper yellow liquid, which is a mixture of n-hexane and oil; S5: repeat step S4 for multiple extractions until the supernatant is colorless and transparent; S6: subjecting the obtained extract to rotary evaporation, and weighing the rotary evaporation bottle before the experiment begins; The rotary evaporation bottle after S7 rotary evaporation is placed in an oven to dry to constant weight, and the obtained oil is the oil produced by fermentation; S8 performs methyl esterification on the obtained oil and fat, and analyzes the changes in the content of each fatty acid in the oil and fat; S81 Weigh 20 μL of oil into a 2 mL centrifuge tube, add 600 μL of 1 M potassium hydroxide-methanol solution, and then oscillate in an oscillator at room temperature and 1000 r / min for 4 hours to fully methylate it; 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 r / min for 30 min to obtain an extract phase; the extract phase was added to a gas phase vial for detection; The chromatographic column used in the S83 experiment was DB-23 (60.0m×0.25mm×0.25μm); detection conditions: injection port temperature 250℃, injection volume 1.0μL, split ratio 69.8:1; chromatographic conditions: initial temperature 100℃, 25℃ / min heating to 196℃. Then, 2℃ / min heating to 220℃ and keeping warm for 21min; After the S84 gas phase detection was completed, different fatty acids were identified by comparison with external standards, and the non-endogenous fatty acid C11:0 was used as the internal standard to estimate the content of individual fatty acids from the peak area on the chromatogram.

10. A method for measuring the biomass of Schizochytrium after fermentation obtained by the method according to any one of claims 4 to 8, characterized in that: The steps include: Take the fermentation liquid that has just been fermented, rotate at 4000r / min for 5min, discard the supernatant, wash it with 2% NaCl solution and distilled water, freeze it in a -80℃ refrigerator for 12h, freeze it in a freeze dryer, and finally weigh and calculate; Among them, the volume ratio of the fermentation liquid just fermented: NaCl solution: distilled water is 1:1:1.

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