Fermentation production method of grease with high DHA or ARA yield and high sn-2 ratio

Abstract

The invention belongs to the technical field of grease production through microbial fermentation, and particularly relates to a fermentation production method of grease with high DHA (docosahexaenoic acid) or ARA (arachidonic acid) yield and high sn-2 proportion, which comprises the steps of strain activation, seed culture, basic fermentation inoculation (seed liquid is inoculated according to 8-12% (v / v) inoculum size in logarithmic phase), fermentation and fermentation. And adding compound amino acid compounded by valine, isoleucine and phenylalanine into the basic culture medium, fermenting for 24-48 hours, supplementing ammonia water-compound organic acid to regulate pH and metabolic flux, fermenting for 48 hours, 72 hours and 96 hours, and supplementing tricarboxylic acid cycle precursor substances to realize staged nutrition regulation. According to the method, the yield of DHA or ARA can be increased by 20% or above, the sn-2 site proportion is increased to 50% or above, the cost of the culture medium is reduced by 35% or above, the method is suitable for large-scale production, and high-quality DHA and ARA raw materials are provided for the fields of food, health care products and medicines.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation production of oil, and particularly relates to a fermentation production method of oil with high DHA or ARA yield and high sn-2 ratio. BACKGROUND

[0002] DHA and ARA are two important polyunsaturated fatty acids, which play a key role in human health. DHA is an important component of human brain and retina, which helps to promote the development of the nervous system of infants; ARA plays an important role in human immune regulation, inflammation response regulation, etc., and both of them are widely used in infant formula food, health food and pharmaceutical preparations. With the continuous growth of market demand for DHA and ARA, how to improve the yield and improve the structure characteristics of oil has become a research hotspot in the industry. At present, microbial fermentation method has become the main technical means for producing DHA and ARA due to its advantages of short production cycle, no limitation of season and climate, high product purity, etc.

[0003] However, there are two major problems in the existing microbial fermentation production method: first, the carbon-nitrogen ratio of the traditional culture medium is easy to be unbalanced, which leads to unreasonable metabolic flow distribution of microorganisms, and low yield of DHA and ARA; second, there is a lack of targeted metabolic regulation in the fermentation process, which makes the sn-2 DHA or ARA ratio in the synthesized oil low, and the sn-2 fatty acid is more easily utilized in the human body during the digestion and absorption process, so the low sn-2 ratio limits the nutritional value and application range of the oil. Therefore, it is of great practical significance and market value to develop a fermentation production method that can simultaneously improve the yield of DHA or ARA and the sn-2 ratio. SUMMARY

[0004] The purpose of the present application is to provide a fermentation production method of oil with high DHA or ARA yield and high sn-2 ratio, which realizes the precise guidance of microbial metabolic flow through the phased nutrition regulation strategy, so as to improve the yield of DHA or ARA and the sn-2 ratio of oil.

[0005] 2.1 Strain selection

[0006] Schizochytrium sp. is selected for DHA fermentation, and Mortierella alpina is selected for ARA fermentation. Both of the two strains have strong polyunsaturated fatty acid synthesis capacity and strong fermentation adaptability, and are suitable for large-scale production.

[0007] 2.2 Preparation of culture medium

[0008] Seed culture medium

[0009] ARA fermentation (Mortierella alpina): glucose 40-60 g / L, yeast extract 20-40 g / L, yeast powder 10-30 g / L, potassium dihydrogen phosphate 1-4 g / L, sodium carbonate 0.05-0.2 g / L.

[0010] DHA fermentation (Schizochytrium sp.): glucose 30-60 g / L, yeast powder 30-60 g / L, potassium dihydrogen phosphate 10-40 g / L, magnesium sulfate 1-5 g / L, potassium chloride 0.2-1 g / L, ammonium sulfate 0.1-5 g / L.

[0011] Basic fermentation medium

[0012] ARA fermentation (Mortierella alpina): glucose 50-80 g / L, yeast powder 30-60 g / L, potassium dihydrogen phosphate 1-5 g / L, sodium carbonate 0.1-0.5 g / L, antifoam agent 0.1-0.5 g / L, and 0.5-2.0 g / L of a compound amino acid (valine: leucine: isophenylalanine = (1-2): (1-2): 1) is added.

[0013] DHA fermentation (Schizochytrium sp.): glucose 40-80 g / L, yeast powder 30-60 g / L, potassium dihydrogen phosphate 10-40 g / L, magnesium sulfate 1-5 g / L, potassium chloride 0.2-1 g / L, ammonium sulfate 0.1-5 g / L, corn steep liquor dry powder 0.5-2 g / L, sodium glutamate 10-40 g / L, antifoam agent 0.1-0.4 g / L, and 0.5-2.0 g / L of a compound amino acid (valine: leucine: isophenylalanine = (1-2): (1-2): 1) is added.

[0014] 2.3 Fermentation process control (supplemental strain activation and inoculation steps)

[0015] Strain activation

[0016] Mortierella alpina (ARA fermentation): A strain glycerol tube stored at -80°C is taken out, and 0.1-0.2 mL of a bacterial solution is taken under sterile conditions and spread on PDA slant medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L) and cultured at 28°C for 3-5 days until the slant is covered with white, fluffy colonies to obtain activated strains.

[0017] Schizochytrium sp. (DHA fermentation): A strain glycerol tube stored at -80°C is taken out, and 0.1-0.2 mL of a bacterial solution is taken under sterile conditions and spread on modified GY slant medium (glucose 20 g / L, yeast powder 10 g / L, sea water crystal 30 g / L, agar 20 g / L) and cultured at 26°C for 3-5 days until orange or orange-red colonies appear on the slant to obtain activated strains.

[0018] Seed culture: Pick 1-2 loops of the activated strain using a sterile inoculation loop and inoculate it into an Erlenmeyer flask containing seed culture medium (the liquid volume should be 20%-30% of the volume of the Erlenmeyer flask). Place the flask in a shaker (200 rpm) or a seed tank and culture it under the corresponding conditions (ARA fermentation 28℃, DHA fermentation 26℃, aeration 80 vvm, stirring 60 rpm) for 18-24 hours until the cells enter the logarithmic growth phase (OD600 value 1.5-2.0). This culture is then used as the fermentation seed liquid.

[0019] Basic fermentation inoculation and start-up: Inoculate the seed culture at a rate of 8%-12% (v / v) into the sterilized and cooled basic fermentation medium through a sterile inoculation pipeline; after inoculation, adjust the initial pH of the fermentation system (8.0 for ARA fermentation, 6.0 for DHA fermentation), turn on aeration (80 vvm) and stirring (60 rpm), record the time, and officially start fermentation.

[0020] Phased feeding regulation

[0021] Fermentation for 24-48 hours: Add 25% (w / w) ammonia water to maintain pH at 5.5-7.0, and at the same time add compound organic acid (pyruvic acid: succinic acid: lactic acid = 1:(0.5-1):(0.5-1)) at a rate of 0.01-0.1 g / L / h.

[0022] After 48 hours of fermentation, add a tricarboxylic acid cycle precursor (α-ketoglutarate: succinic acid: fumaric acid = 1:1:1) at a rate of 0.1-0.3 g / L.

[0023] Fermentation for 72 hours: Add the above-mentioned tricarboxylic acid cycle precursors at a rate of 0.5-0.8 g / L.

[0024] Fermentation for 96 hours: Add the above-mentioned tricarboxylic acid cycle precursors at a rate of 1.0-1.5 g / L.

[0025] Fermentation endpoints: The total fermentation time for ARA was 168 hours, and the total fermentation time for DHA was 132 hours. After fermentation, the cells were collected, the oil was extracted, and relevant indicators were tested.

[0026] The beneficial effects of this invention are:

[0027] 1. Using valine, isoleucine, and phenylalanine in a specific mass ratio as a nitrogen source to replace part of the traditional yeast extract can optimize the carbon flow-directed lipid synthesis pathway and reduce the cost of the basal culture medium by more than 35%.

[0028] 2. pH-metabolic coupling regulation technology in the middle of fermentation: At 24-48 h of fermentation, the pH of the system was controlled at 5.5-7.0 by pulse-adding 25% (w / w) ammonia water, and at the same time, a specific proportion of pyruvic acid, lactic acid and succinic acid compound organic acid was supplemented. Among them, pyruvic acid as the end point of glycolysis and the direct precursor of acetyl-CoA, provides key raw materials for lipid synthesis; lactic acid can inhibit the metabolic "flood discharge" path, forcing carbon flow to preferentially flow to lipid synthesis; succinic acid can not only consume excess NADH to maintain redox balance, but also fix CO2 to improve carbon utilization efficiency, achieving double improvement of carbon efficiency and redox balance.

[0029] 3. Tricarboxylic acid (TCA) cycle activation technology: At 48 h, 72 h and 96 h of fermentation, TCA cycle precursors composed of alpha-ketoglutaric acid, succinic acid and fumaric acid were supplemented in stages, which can induce the up-regulation of lipid synthesis enzyme genes, not only providing sufficient precursor materials and energy for the synthesis of DHA / ARA super-long chain polyunsaturated fatty acids, but also significantly improving the proportion of DHA or ARA at the sn-2 position. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0031] Example 1: ARA fermentation (Mortierella alpina)

[0032] Strain activation: Take the Mortierella alpina strain glycerol tube stored at -80℃, and under sterile operation, take 0.15 mL of bacterial liquid to coat the PDA slant, and cultivate at 28℃ for 4d to obtain activated strain.

[0033] Seed culture: pick the activated strain ring and inoculate into a 2L Erlenmeyer flask containing 500 mL of seed culture medium (glucose 50g / L, yeast extract 30g / L, yeast powder 20g / L, potassium dihydrogen phosphate 2g / L, sodium carbonate 0.1g / L), and cultivate at 28℃, 200rpm shaking bed for 20h, until OD600=1.8, to obtain seed liquid.

[0034] Inoculation and start-up of basic fermentation: Seed liquid was inoculated into a 50 cubic meter fermenter (containing 30 L of basic medium: glucose 65 g / L, yeast powder 45 g / L, potassium dihydrogen phosphate 3 g / L, sodium carbonate 0.3 g / L, defoaming agent 0.3 g / L, and 1.2 g / L of complex amino acids (valine: leucine: isophenylalanine = 1:1:1) at an inoculation amount of 10% (v / v); after inoculation, the initial pH was adjusted to 8.0, and the fermentation was started at 28°C, with aeration of 80 vvm and stirring of 60 rpm.

[0035] Stage-wise feeding: 25% (w / w) ammonia water was added to maintain pH 5.5-7.0 from 24-48 h, and a total feeding amount of 1 g / L of complex organic acids (pyruvic acid: lactic acid: succinic acid = 1:1:1) was added at the same time; 0.1 g / L, 0.5 g / L, and 1 g / L of tricarboxylic acid cycle precursors (alpha-ketoglutaric acid: succinic acid: fumaric acid = 1:1:1) were added at 48 h, 72 h, and 96 h, respectively.

[0036] Fermentation results: The fermentation was ended at 168 h, with a biomass of 52 g / L, a total oil content of 45% (w / w), ARA accounting for 58% of the total fatty acids, and sn-2 position ARA accounting for 60% of the total ARA.

[0037] Example 2: ARA fermentation (Mortierella alpina)

[0038] Strain activation and seed culture: The same as in Example 1, and the seed liquid was cultured to OD600 = 1.9.

[0039] Inoculation and start-up of basic fermentation: Seed liquid was inoculated into a 50 cubic meter fermenter at an inoculation amount of 10% (v / v), and complex amino acids 1.5 g / L (valine: leucine: isophenylalanine = 1:1:1) were added to the basic medium, with the remaining conditions being the same as in Example 1.

[0040] Stage-wise feeding: 25% (w / w) ammonia water was added to maintain pH 5.5-7.0 from 24-48 h, and a total feeding amount of 1.5 g / L of complex organic acids (pyruvic acid: lactic acid: succinic acid = 1:0.5:0.5) was added at the same time; 0.3 g / L, 0.8 g / L, and 1.5 g / L of tricarboxylic acid cycle precursors (alpha-ketoglutaric acid: succinic acid: fumaric acid = 1:1:1) were added at 48 h, 72 h, and 96 h, respectively.

[0041] Fermentation results: The fermentation was ended at 168 h, with a biomass of 54 g / L, a total oil content of 46% (w / w), ARA accounting for 57% of the total fatty acids, and sn-2 position ARA accounting for 62% of the total ARA.

[0042] Example 3: DHA fermentation (S. platensis)

[0043] Strain activation: Take the Schizochytrium sp. glycerol tube stored at -80°C, and under sterile conditions, take 0.15 mL of the bacterial liquid and coat the modified GY slant, and cultivate at 26°C for 4 days to obtain activated strain.

[0044] Seed culture: Take the activated strain ring and inoculate a 2L Erlenmeyer flask containing 500 mL of seed culture medium (glucose 45 g / L, yeast powder 45 g / L, potassium dihydrogen phosphate 25 g / L, magnesium sulfate 3 g / L, potassium chloride 0.6 g / L, ammonium sulfate 2 g / L), and cultivate at 26°C, 200 rpm shaking bed for 22 h, until OD600=1.7, to obtain seed liquid.

[0045] Basic fermentation inoculation and start-up: inoculate the seed liquid into a 50L fermenter (containing 30L basic medium: glucose 60 g / L, yeast powder 45 g / L, potassium dihydrogen phosphate 25 g / L, magnesium sulfate 3 g / L, potassium chloride 0.6 g / L, ammonium sulfate 2 g / L, corn syrup dry powder 1.2 g / L, sodium glutamate 25 g / L, defoaming agent 0.2 g / L, add 1 g / L of complex amino acids (valine: leucine: isophenylalanine = 1:1:1)) at a 10% (v / v) inoculation amount; after inoculation, adjust the initial pH to 6.0, cultivate at 26°C, aeration 80 vvm, stirring 60 rpm, and start fermentation.

[0046] Stage-wise feeding: add 25% (w / w) ammonia water to maintain pH 5.5-7.0, and simultaneously add the compounded organic acid (pyruvic acid: lactic acid: succinic acid = 1:1:1) at a total feeding amount of 1.5 g / L; at 48h, 72h, and 96h, add the tricarboxylic acid cycle precursors (α-ketoglutaric acid: succinic acid: fumaric acid = 1:1:1) at 0.1 g / L, 0.5 g / L, and 1.0 g / L, respectively.

[0047] Fermentation results: end fermentation at 132h, biomass 180 g / L, total oil content 60% (w / w), DHA accounting for 65% of total fatty acids, and sn-2 DHA accounting for 70% of total DHA.

[0048] Example 4: DHA fermentation (S. platensis)

[0049] Strain activation and seed culture: same as Example 3, seed liquid cultivation to OD600=1.8.

[0050] Basic fermentation inoculation and start-up: inoculate the seed liquid into a 50L fermenter at a 10% (v / v) inoculation amount, and the basic medium composition is the same as Example 3, and the remaining conditions are consistent.

[0051] Stage feeding: 24-48h, 25%(w / w) ammonia water was added to maintain pH 5.5-7.0, and the compounded organic acid (pyruvic acid: lactic acid: succinic acid = 1:0.5:0.5) was added, the total amount of 1.0g / L; 48h, 72h, 96h, respectively, the tricarboxylic acid cycle precursor (alpha-ketoglutarate: succinic acid: fumaric acid = 1:1:1) was added 0.3g / L, 0.8g / L, 1.5g / L.

[0052] Fermentation results: 132h, the fermentation was completed, the biomass was 178g / L, the total oil content was 62%(w / w), the DHA accounted for 63% of the total fatty acid, and the sn-2 site DHA accounted for 65% of the total DHA.

[0053] 4. Comparative example

[0054] Comparative example 1: ARA fermentation (without the control measures of the application)

[0055] Conditions: strain activation, seed culture and inoculation method were the same as example 1 (seed liquid was inoculated at 10%(v / v)), but the basic medium did not add compound amino acids, and ammonia water and compounded organic acid were not added during fermentation 24-48h, and succinic acid was not added at 72h.

[0056] Results: 168h, the fermentation was completed, the biomass was 32g / L, the total oil content was 38%(w / w), the ARA accounted for 46% of the total fatty acid, and the sn-2 site ARA accounted for 37% of the total ARA.

[0057] Comparative example 2: ARA fermentation (without the control measures of the application)

[0058] Conditions: strain activation, seed culture and inoculation method were the same as example 2 (seed liquid was inoculated at 10%(v / v)), but the basic medium did not add compound amino acids, and ammonia water and compounded organic acid were not added during fermentation 24-48h, and succinic acid was not added at 72h.

[0059] Results: 168h, the fermentation was completed, the biomass was 35g / L, the total oil content was 38%(w / w), the ARA accounted for 46% of the total fatty acid, and the sn-2 site ARA accounted for 37% of the total ARA.

[0060] Comparative example 3: DHA fermentation (without the control measures of the application)

[0061] Conditions: strain activation, seed culture and inoculation method were the same as example 3 (seed liquid was inoculated at 10%(v / v)), but the basic medium did not add compound amino acids, and ammonia water and compounded organic acid were not added during fermentation 24-48h, and succinic acid was not added at 72h.

[0062] Result: fermentation ended at 168h, biomass 120g / L, total lipid 42%(w / w), DHA 50% of total fatty acid, sn-2 DHA 42% of total DHA.

[0063] Comparative Example 4: DHA fermentation (without the regulatory measures of the present application)

[0064] Condition: strain activation, seed culture and inoculation method are the same as Example 4 (seed liquid is inoculated at 10%(v / v)), but the basic medium does not add complex amino acids, and no ammonia water and compound organic acid is added at 24-48h of fermentation, and no succinic acid is added at 72h.

[0065] Result: fermentation ended at 168h, biomass 126g / L, total lipid 45%(w / w), DHA 52% of total fatty acid, sn-2 DHA 40% of total DHA.

[0066] 1. Strain activation

[0067] Mortierella alpina (ARA fermentation):

[0068] Prepare PDA slant medium in advance, sterilize at 121°C for 20min, and make slant after cooling.

[0069] Open the strain glycerol tube under sterile conditions, use a sterile pipette to take 0.1-0.2mL of bacterial liquid, and evenly spread it on the surface of the slant medium.

[0070] Put the coated slant into a 28°C constant temperature incubator, and incubate statically for 3-5d, until the slant is covered with white fluffy colonies, which is the activated strain, and is stored at 4°C for standby (storage period not more than 7d).

[0071] Schizochytrium sp. (DHA fermentation):

[0072] Prepare modified GY slant medium in advance, sterilize at 121°C for 20min, and make slant after cooling.

[0073] Take 0.1-0.2mL of Schizochytrium sp. glycerol tube bacterial liquid under sterile conditions, and spread it on the modified GY slant, and incubate statically in a 26°C constant temperature incubator for 3-5d.

[0074] When the slant appears orange yellow or orange red, and the surface is smooth, it is the activated strain, and is stored at 4°C for standby (storage period not more than 7d).

[0075] 2. Seed culture

[0076] Seed culture in triangular flask (pilot scale):

[0077] Prepare the medium according to the seed medium formula, and divide it into 2L triangular bottles, each bottle containing 500mL. Sterilize at 121℃ for 20min, and cool to room temperature.

[0078] Use a sterile inoculation loop to pick up 1-2 circles of activated seed, and inoculate the triangular bottle medium. Seal the bottle opening (use a breathable sealing film).

[0079] Place the triangular bottle in a constant temperature shaker. Set the ARA fermentation to 28℃ and 200rpm, and the DHA fermentation to 26℃ and 200rpm. Culture for 18-24h.

[0080] Take samples to detect OD600. When OD600 reaches 1.5-2.0, stop the culture, which is the seed liquid. If it has not reached, extend the culture for 2-4h, and detect OD600 every 2h during this period.

[0081] Seed tank culture (pilot / industrial scale):

[0082] Prepare sufficient medium according to the seed medium formula, and add it to the seed tank. Sterilize at 121℃ for 30min (for tanks with a capacity of >100L), and cool to the set temperature (ARA 28℃, DHA 26℃).

[0083] Turn on the air supply system (80vvm) and stirring system (60rpm) of the seed tank, and supply sterile air for 30min to eliminate miscellaneous bacteria in the tank.

[0084] Under sterile conditions, inoculate the activated seed into the seed tank (if it is a slant seed, first rinse the slant with sterile saline to prepare a bacterial suspension, and then inoculate it). Culture for 18-24h, and detect OD600 to reach 1.5-2.0, which is the seed liquid.

[0085] 3. Basic fermentation preparation

[0086] Accurately weigh each component according to the basic fermentation medium formula, and add it to the fermentation tank. After making up the water, stir evenly, sterilize at 121℃ for 20min (pilot test / pilot test) or 30min (industrial large tank), and cool to the set fermentation temperature (ARA 28℃, DHA 26℃).

[0087] Prepare a concentrated solution of complex amino acids (concentration 100g / L). After sterilization by 0.22μm filter membrane, inoculate the fermentation tank through the sterile feed port after the fermentation medium is cooled and before inoculation, and stir evenly.

[0088] 4. Fermentation process operation

[0089] Inoculation operation:

[0090] Before inoculation, use 75% alcohol to wipe the inoculation port of the fermenter and the interface of the seed liquid container to ensure sterility.

[0091] If it is a triangular flask seed liquid (pilot test), directly open the inoculation port of the fermenter, pour in the seed liquid, and quickly seal the inoculation port; if it is a seed tank seed liquid (pilot test / industrialization), connect the seed tank and the fermenter through a sterile inoculation pipeline, open the pipeline valve, and use the pressure difference to press the seed liquid into the fermenter. After inoculation is completed, close the valve and flush the pipeline with sterile air.

[0092] Inoculum control: adjust according to the fermentation scale, control at 8%-12% (v / v) for small test (500mL triangular flask), pilot test (50L tank) and industrialization (50 cubic tank), preferentially choose 10% (v / v) inoculum to ensure consistent initial cell concentration.

[0093] Fermentation start and parameter monitoring:

[0094] After inoculation, adjust the initial pH of the fermentation system (ARA fermentation 8.0, DHA fermentation 6.0) with 1mol / L hydrochloric acid or sodium hydroxide solution, open the aeration (80vvm) and stirring (60rpm), and record the fermentation start time (0h).

[0095] During fermentation, sample every 4h to detect cell concentration (OD600), residual sugar amount (using DNS method), and detect total oil content and DHA / ARA content every 12h to ensure that the fermentation proceeds as expected.

[0096] Stage-wise feeding operation (same as before, no changes):

[0097] At 24h of fermentation, open the ammonia water supplement system, control the ammonia water supplement rate through the online pH monitor to make the pH of the fermentation system stable at 5.5-7.0; at the same time, open the compounded organic acid supplement pump, supplement the compounded organic acid at a rate of 0.01-0.1g / L / h, and supplement until 48h.

[0098] At 48h, 72h and 96h of fermentation, respectively, add pre-sterilized tricarboxylic acid cycle precursors through the sterile feeding port, with supplementing amounts of 0.1-0.3g / L, 0.5-0.8g / L and 1.0-1.5g / L, respectively.

[0099] 5. Product extraction and detection

[0100] Cell collection: After the fermentation is completed, the fermentation broth is collected by centrifugation (5000 rpm, 10 min), washed with deionized water for 2-3 times, and freeze-dried to a constant weight.

[0101] Oil extraction: Soxhlet extraction method is used to extract oil from the dried cells with n-hexane as the extraction solvent, and the solvent is removed by rotary evaporation to obtain crude oil.

[0102] Index detection: The content of DHA or ARA in the oil is detected by gas chromatography (GC); the proportion of sn-2 DHA or ARA is detected by high performance liquid chromatography (HPLC) combined with evaporative light scattering detector, and the specific detection conditions are as follows:

[0103] Gas chromatography conditions: The chromatographic column is DB-23 capillary column (30 m x 0.25 mm x 0.25 μm), the column temperature program is initial temperature 100℃, holding for 2 min, increasing to 240℃ at 10℃ / min, holding for 15 min; the injection port temperature is 250℃, the detector (FID) temperature is 260℃; the carrier gas is nitrogen with a flow rate of 1.0 mL / min, and the split ratio is 10:1.

[0104] High performance liquid chromatography conditions: The chromatographic column is C18 column (250 mm x 4.6 mm x 5 μm), the mobile phase is acetonitrile-isopropanol-water (80:15:5, v / v / v), the flow rate is 1.0 mL / min, and the column temperature is 30℃; the evaporative light scattering detector drift tube temperature is 40℃, and the carrier gas (nitrogen) pressure is 3.5 bar.

[0105] It can be seen from the comparison of the above examples and comparative examples that:

[0106] 1. The production of ARA and DHA is increased by more than 20% by using the stage-by-stage metabolic regulation method of the present application, wherein the proportion of ARA in total fatty acids is up to 58%, and the proportion of DHA in total fatty acids is up to 65%, which is much higher than the level of 46%-52% in the comparative examples.

[0107] 2. The proportion of sn-2 is greatly improved: the proportion of sn-2 DHA or ARA in the fermentation product is increased to more than 50%, and the highest proportion is 70% (DHA) and 62% (ARA) respectively, which solves the problem of low sn-2 proportion in traditional fermentation oil and poor digestion and absorption efficiency, and improves the nutritional value of the oil.

[0108] 3. Cost reduction: The compound amino acid compounding technology is used in the basic medium to replace part of the high-priced yeast extract, so that the medium cost is reduced by more than 35%, and the carbon utilization efficiency is improved, further reducing the comprehensive cost of large-scale production.

[0109] 4. Good fermentation stability: The fermentation results of examples 1-4 show that the biomass, total oil content, target product content and sn-2 site content are stable, which indicates that the regulation method of the application has strong repeatability and is suitable for industrial large-scale production.

Claims

1. A fermentation production method of high-yield DHA or ARA and high sn-2 ratio oil, characterized in that: The preparation steps include: S1: preparing a basic medium: the basic medium contains 50-80 g / L of a carbon source, 30-60 g / L of a nitrogen source, 1-5.5 g / L of inorganic salts, and 0.1-0.5 g / L of an antifoaming agent; S2: inoculating the seed liquid of the target microorganism in the logarithmic growth phase into the basic medium prepared in step S1 at a volume ratio of 8%-12% inoculation amount, and starting fermentation; S3: during the fermentation for 24-48 h, supplementing an alkaline substance to control the pH of the fermentation system at 5.5-7.0, and supplementing a compounded organic acid at a rate of 0.01-0.1 g / L / h; S4: when the fermentation is performed for 48 h, 72 h, and 96 h, respectively, supplementing a tricarboxylic acid cycle precursor; The basic medium further contains a composite amino acid with an addition amount of 0.5-2.0 g / L. The compounded organic acid is a mixture of pyruvic acid, lactic acid, and succinic acid.

2. The method according to claim 1, wherein the method is characterized by: The composite amino acid is composed of valine, isoleucine, and phenylalanine at a mass ratio of 1-2:1-2:

1. 3.The method according to claim 1, characterized in that: The inorganic salts include potassium dihydrogen phosphate and sodium carbonate.

4. The method according to claim 3, wherein the method is characterized by: The addition amount of potassium dihydrogen phosphate is 1-5 g / L, and the addition amount of sodium carbonate is 0.1-0.5 g / L.

5. The method according to claim 1, wherein the method is characterized by: The microorganism is used for synthesizing DHA when it is a Schizochytrium sp.

6. The method according to claim 1, wherein the method is characterized by: The microorganism is used for synthesizing ARA when it is Mortierella alpina.

7. The method according to claim 1, wherein the method is characterized by: The mass ratio of pyruvic acid, lactic acid, and succinic acid in the compounded organic acid is 1:0.5-1:0.5-1.

8. The method according to claim 1, wherein the method is characterized by: The tricarboxylic acid cycle precursor contains α-ketoglutaric acid, succinic acid, and fumaric acid.

9. The method according to claim 8, wherein the method is characterized by: The supplementing amounts of α-ketoglutaric acid, succinic acid, and fumaric acid are 0.1-0.3 g / L, 0.5-0.8 g / L, and 1.0-1.5 g / L, respectively.