Method for producing sn-2DHA algal oil through schizochytrium limacinum fermentation

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a method for producing DHA algal oil through schizochytrium limacinum fermentation. The method comprises the following steps: fermenting schizochytrium limacinum in a culture medium containing glucose; the fermentation is divided into first-stage fermentation, second-stage fermentation and third-stage fermentation; in the second-stage fermentation process, glucose and acetate are supplemented into the culture medium; in the third-stage fermentation process, acetate is supplemented into the culture medium, so that the total carbon source in the culture medium is 2-5g / L; the stopping time of the first-stage fermentation is 22-26 hours, and the stopping time of the second-stage fermentation is 46-50 hours. According to the method, the yield of sn-2DHA can be effectively increased while the fermentation period is shortened.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for producing sn-2DHA algal oil by fermentation of Schizochytrium. Background Technology

[0002] Traditional Schizochytrium fermentation often uses a single sugar source (such as glucose or sucrose) as a carbon source. In the early stages of fermentation, it can quickly provide energy and carbon skeletons for the cells. However, when the substrate concentration is too high, it often leads to an increase in osmotic pressure, substrate inhibition, and metabolic imbalance, which restricts cell growth. On the other hand, as the fermentation process progresses, the sugar source is gradually depleted, and the cells cannot obtain enough carbon source in time to maintain continuous proliferation and lipid accumulation, thus limiting the DHA yield. Summary of the Invention

[0003] The purpose of this invention is to overcome the problem of limited DHA yield in the existing technology and to provide a method for producing sn-2DHA algal oil by fermentation of Schizochytrium. This method can ferment and produce algal oil containing sn-2DHA, and at the same time, it can effectively improve the yield of sn-2DHA while shortening the fermentation cycle.

[0004] To achieve the above objectives, the present invention provides a method for producing sn-2DHA algal oil by fermentation of Schizochytrium, the method comprising: placing Schizochytrium in a culture medium containing glucose for fermentation; the fermentation being divided into a first stage fermentation, a second stage fermentation, and a third stage fermentation; during the second stage fermentation, adding glucose and acetate to the culture medium; during the third stage fermentation, adding acetate to the culture medium so that the total carbon source in the culture medium is 2-5 g / L; the first stage fermentation is stopped at a time of 22-26 h, and the second stage fermentation is stopped at a time of 46-50 h.

[0005] Preferably, during the first stage of fermentation, the dissolved oxygen in the culture medium is controlled at 30% to 50% air saturation at 28°C.

[0006] During the second and third stages of fermentation, the dissolved oxygen in the culture medium was controlled at 28°C to be between 10% and 50% air saturation.

[0007] Preferably, the concentration of glucose in the glucose-containing culture medium is 20-40 g / L.

[0008] More preferably, the glucose-containing culture medium also contains acetate, and the mass ratio of the acetate to the glucose is 1:1.5-2.5.

[0009] More preferably, the glucose-containing culture medium also contains yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, and vitamin B1.

[0010] More preferably, in the culture medium, the concentration of the yeast extract is 15-25 g / L, the concentration of the peptone is 4-6 g / L, the concentration of the potassium dihydrogen phosphate is 0.8-1.2 g / L, the concentration of the magnesium sulfate is 1.5-2.5 g / L, the concentration of the sodium chloride is 15-25 g / L, and the concentration of the vitamin B1 is 0.008-0.012 g / L.

[0011] Preferably, the fermentation conditions include at least the following: a stirring rate of 300-600 rpm, a sterile air flow rate of 0.8-1.2 vvm, and an initial pH of 6.4-6.6.

[0012] Preferably, the glucose and the acetate are added to the culture medium in solution form.

[0013] More preferably, the concentration of glucose in the solution is 250-400 g / L; and the concentration of acetate in the solution is 200-500 g / L.

[0014] Preferably, the acetate is potassium acetate and / or sodium acetate.

[0015] Preferably, during the second stage of fermentation, the total carbon source in the culture medium is controlled to be 5-10 g / L.

[0016] Preferably, during the second stage of fermentation, the mass ratio of the added glucose to the added acetate is 1.5-2.5:1.

[0017] More preferably, during the second stage of fermentation, the mass ratio of the added glucose to the added acetate is 1.8-2.2:1.

[0018] Preferably, during the second stage of fermentation, glucose and acetate are added to the culture medium in the following manner: when the dissolved oxygen in the culture medium at 28°C is less than or equal to 30% air saturation, glucose and acetate are added to the culture medium; when the dissolved oxygen in the culture medium at 28°C is greater than 45% air saturation, the addition of glucose and acetate to the culture medium is stopped.

[0019] Preferably, during the third stage of fermentation, the acetate is added when the pH of the culture medium is greater than or equal to 6.6 and the carbon source is less than 2 g / L.

[0020] More preferably, the timing for stopping the addition of acetate is when the solubility of acetate in the culture medium is greater than or equal to 1.1 g / L.

[0021] The method provided by this invention, through the above technical solution, involves fermenting Schizochytrium fungi in a glucose-containing culture medium. Glucose and acetate are added to the medium 22-26 hours after fermentation begins, and acetate is added again 46-20 hours after fermentation begins, ensuring a total carbon source concentration of 2-5 g / L in the medium. This method enables the production of algal oil containing sn-2DHA, while simultaneously shortening the fermentation cycle and effectively increasing the yield of sn-2DHA. Furthermore, the method provided by this invention effectively reduces the consumption of external alkali, thereby lowering the salt load and processing costs in post-treatment. Attached Figure Description

[0022] Figure 1 This is a graph showing the relationship between DHA production and time in Example 1;

[0023] Figure 2 This is a graph showing the relationship between DHA production and time in Example 2. Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] As mentioned above, the present invention provides a method for producing sn-2DHA algal oil by fermentation of Schizochytrium, the method comprising: placing Schizochytrium in a culture medium containing glucose for fermentation.

[0026] The fermentation is divided into a first stage fermentation, a second stage fermentation, and a third stage fermentation. During the second stage fermentation, glucose and acetate are added to the culture medium. During the third stage fermentation, acetate is added to the culture medium so that the total carbon source in the culture medium is 2-5 g / L. The first stage fermentation is stopped at 22-26 h, and the second stage fermentation is stopped at 46-50 h.

[0027] The study found that fermenting *Schizochytrium* in a glucose-containing medium, with the addition of glucose and acetate 22-26 hours after fermentation and further addition of acetate 46-20 hours after fermentation to maintain a total carbon source of 2-5 g / L, promoted rapid cell growth, quickly entering the logarithmic growth phase, and enhanced lipid synthesis, thereby increasing the yield of sn-2DHA while shortening the fermentation cycle. The added glucose and sodium acetate are food additives, non-toxic, and ensure food safety. Furthermore, the method provided by this invention effectively reduces the consumption of external alkali, thus lowering the salt load and processing costs in post-treatment.

[0028] The fermentation cycle of the method provided by this invention is only about 100 hours, which effectively shortens the fermentation cycle compared to ordinary fermentation (cycle of more than 114 hours).

[0029] According to the present invention, the carbon source is a carbon source as conventionally understood in the art, that is, the content of carbon-containing substances, such as glucose and acetate mentioned above. The total carbon source is all carbon-containing substances. The glucose and acetate mentioned above are commercially available.

[0030] Preferably, during the first stage of fermentation, the dissolved oxygen (DO) in the culture medium at 28°C is controlled to be 30% to 50% air saturation; during the second and third stages of fermentation, the dissolved oxygen in the culture medium at 28°C is controlled to be 10% to 50% air saturation. Controlling the dissolved oxygen at 28°C within the above ranges during the first, second, and third stages of fermentation further improves the yield of sn-2DHA. Further preferably, to further improve the DHA yield, during the first stage of fermentation, the dissolved oxygen in the culture medium at 28°C is controlled to be 40% to 50% air saturation; during the second and third stages of fermentation, the dissolved oxygen in the culture medium at 28°C is controlled to be 20% to 50% air saturation.

[0031] According to the present invention, dissolved oxygen levels are monitored and controlled during fermentation using an online dissolved oxygen sensor (dissolved oxygen electrode).

[0032] Preferably, the dissolved oxygen in the culture medium can be controlled as follows: when the dissolved oxygen content in the culture medium is low, it can be increased by increasing the stirring rate and / or by aeration, whereby the aeration can be increased by increasing the oxygen content in the introduced gas or by increasing the aeration rate; when the dissolved oxygen content in the culture medium is high, it can be decreased by increasing the feeding rate. These methods enable precise feeding, thereby further improving the yield of sn-2DHA.

[0033] According to the present invention, the glucose content is calculated after monitoring the glucose concentration using a biochemical sensor (glucose oxidase electrode). Experience shows that glucose consumption is positively correlated with sodium acetate consumption. After multiple measurements, the positive correlation coefficient was confirmed. The amount of sodium acetate consumed was determined based on the amount of glucose consumed, and then the sodium acetate content was determined based on the original content, the amount added, the amount consumed, and the positive correlation coefficient.

[0034] Preferably, during the second stage of fermentation, the pH of the culture medium is controlled to be 6.4-6.6, which can be 6.4, 6.5, 6.6, or any value within any two of these ranges. This method can further improve the accuracy of feeding and further increase the yield of sn-2DHA.

[0035] Preferably, during the third stage of fermentation, the pH of the culture medium is controlled to be 6.4-6.6, which can be 6.4, 6.5, 6.6, or any value within any two of these ranges. This method can further improve the accuracy of feeding and further increase the yield of sn-2DHA.

[0036] According to the present invention, the pH of the culture medium is tested under the following conditions: real-time monitoring is performed at 28°C using an online pH sensor (pH electrode).

[0037] Preferably, the pH of the culture medium can be controlled as follows: when the pH is greater than 6.6, the pH of the culture medium can be lowered by pausing feeding or reducing the feeding rate; when the pH is less than 6.4, the pH of the culture medium can be increased by adjusting with alkali solution.

[0038] Preferably, during the third stage of fermentation, acetate is fed in a pulsed manner. This feeding method can better promote lipid synthesis.

[0039] To further improve the yield of sn-2DHA, preferably, the glucose concentration in the glucose-containing culture medium is 20-40 g / L, which can be 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or any value within any two of these ranges. More preferably, the glucose concentration in the glucose-containing culture medium is 20-30 g / L.

[0040] Preferably, the glucose-containing culture medium further contains acetate, and the mass ratio of acetate to glucose is 1:1.5-2.5, which can be 1:1.5, 1:1.7, 1:1.9, 1:2.1, 1:1.3, 1:2.5, or any value within any two of these ranges. The interaction between specific amounts of glucose and acetate can further improve the yield of DHA. Further preferably, considering the potential for further improving DHA yield, the glucose-containing culture medium also contains yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, and vitamin B1. More preferably, in the culture medium, the concentration of the yeast extract is 15-25 g / L, which can be any value within any two of the ranges formed by 15 g / L, 17 g / L, 19 g / L, 21 g / L, 23 g / L, and 25 g / L; the concentration of the peptone is 4-6 g / L, which can be 4 g / L, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L, and so on. The concentrations are 5 g / L, 5.2 g / L, 5.4 g / L, 5.6 g / L, 5.8 g / L, 6 g / L, or any value within any two of the above ranges; the concentration of potassium dihydrogen phosphate is 0.8-1.2 g / L, and can be 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, 1 g / L, 1.05 g / L, 1.1 g / L, 1.15 g / L, etc. The concentration of magnesium sulfate is 1.5-2.5 g / L, which can be 1.5 g / L, 1.7 g / L, 1.9 g / L, 2.1 g / L, 2.3 g / L, or 2.5 g / L, and any value within any two of the above ranges; the concentration of sodium chloride is 15-25 g / L, which can be 15 g / L, 17 g / L, 19 g / L, 21 g / L, 23 g / L, or 25 g / L, and any value within any two of the above ranges; the concentration of vitamin B1 is 0.008-0.012 g / L, which can be 0.008 g / L, 0.009 g / L, 0.01 g / L, 0.011 g / L, or 0.012 g / L, and any value within any two of the above ranges.

[0041] Preferably, the glucose-containing culture medium also contains FeSO4·7H2O, ZnSO4·7H2O, and CuSO4. More preferably, the concentration of FeSO4·7H2O is 1.8-2.2 g / L, the concentration of ZnSO4·7H2O is 3.5-4.5 g / L, and the concentration of CuSO4 is 2-3 g / L.

[0042] Preferably, the fermentation conditions include at least the following: a stirring rate of 300-600 rpm, which can be 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, or any value within any two of these ranges; a sterile air flow rate of 0.8-1.2 vvm, which can be 0.8 vvm, 0.9 vvm, 1 vvm, 1.1 vvm, 1.2 vvm, or any value within any two of these ranges; and an initial pH of 6.4-6.6, which can be 6.4, 6.5, 6.6, or any value within any two of these ranges. Under these conditions, the fermentation effect is better, thereby further improving the yield of sn-2DHA.

[0043] Controlling the amount of sterile air introduced within the above range ensures dissolved oxygen mass transfer and uniform cell suspension, thereby further improving the fermentation effect of Schizochytrium and increasing the yield of DHA.

[0044] Preferably, the fermentation conditions further include a temperature of 20-30℃, which can be 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, or any value within any two of the above ranges. At the above fermentation temperatures, better fermentation results are achieved, thereby further increasing the yield of DHA. Considering the potential to further increase the yield of sn-2DHA, it is further preferred that the fermentation temperature be 25-28℃.

[0045] Preferably, the glucose and acetate are added to the culture medium in solution form, which allows for better control of the amount of glucose and acetate added, thereby further improving the yield of sn-2DHA. Specifically, solutions containing glucose and solutions containing acetate can be prepared separately, or solutions containing both glucose and acetate can be prepared. More preferably, in the solution, the concentration of glucose is 250-400 g / L; and the concentration of acetate is 200-500 g / L.

[0046] To further improve the yield of DHA, preferably, the acetate is potassium acetate and / or sodium acetate.

[0047] Preferably, during the second stage of fermentation, the total carbon source in the culture medium is controlled to be 5-10 g / L, which can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any value within any two of these ranges. Controlling the total carbon source in the culture medium to 5-10 g / L during the second stage of fermentation can further improve the yield of sn-2DHA.

[0048] Preferably, during the second stage of fermentation, the mass ratio of the added glucose to the added acetate is 1.5-2.5:1, which can be 1:1.5, 1:1.7, 1:1.9, 1:2.1, 1:1.3, 1:2.5, or any value within any two of these ranges. Controlling the mass ratio of the added glucose to acetate within the above range can further improve the yield of sn-2DHA. Further preferably, considering the potential for further improving the yield of sn-2DHA, during the second stage of fermentation, the mass ratio of the added glucose to the added acetate is 1.8-2.2:1.

[0049] Preferably, during the second stage of fermentation, glucose and acetate are added to the culture medium as follows: when the dissolved oxygen in the culture medium at 28°C is less than or equal to 30% air saturation, glucose and acetate are added; when the dissolved oxygen in the culture medium at 28°C is greater than 45% air saturation, the addition of glucose and acetate is stopped. This method can further improve the yield of sn-2DHA.

[0050] Preferably, during the third stage of fermentation, the acetate is added when the pH of the culture medium is greater than or equal to 6.6 and the carbon source concentration is less than 2 g / L. This indicates that the cell metabolism is primarily used for lipid synthesis, and initiating feeding at this point can further improve the yield of sn-2DHA and the carbon source conversion rate. Further preferably, to further improve the carbon source conversion rate, the acetate addition is stopped when the acetate concentration in the culture medium is greater than or equal to 1.1 g / L. This method also prevents high osmotic pressure or metabolic inhibition caused by high acetate concentrations.

[0051] The final solubility of acetate in the culture medium was determined by gas chromatography.

[0052] Preferably, after fermentation is complete, the feeding, aeration, and stirring systems are shut off, the contents of the fermenter are removed, and the bacterial cells are collected by centrifugation. The collected bacterial cells are first washed and dried, and then subjected to acidic methanolization treatment.

[0053] According to the present invention, gas chromatography-flame ionization detection (GC-FID) is used to quantitatively analyze DHA production, thereby evaluating the accumulation effect of DHA and the carbon source utilization efficiency during fermentation.

[0054] According to a particularly preferred embodiment of the present invention, a method for improving DHA production by Schizochytrium is provided, comprising the following steps: placing Schizochytrium in a culture medium containing glucose for fermentation; after fermentation, shutting off the feeding, aeration, and stirring systems; removing the contents of the fermenter; and centrifuging to collect the bacterial sediment; the fermentation conditions are: temperature 20-30℃, stirring rate 300-600 rpm, sterile air flow rate 0.8-1.2 vvm, and initial pH 6.4-6.6;

[0055] The fermentation process is divided into three stages: a first stage, a second stage, and a third stage. During the first stage, the dissolved oxygen in the culture medium is controlled at 30% to 50% air saturation at 28°C. During the second stage, the dissolved oxygen in the culture medium is controlled at 10% to 50% air saturation at 28°C, and glucose and acetate in solution form at a mass ratio of 1.5-2.5 are added to the culture medium. The pH of the culture medium is controlled at 6.4-6.6, and the total carbon source is 5-10 g / L. During the third stage, the culture medium is controlled... When the dissolved oxygen level at 28°C is 10% to 50% air saturation, the pH of the culture medium is greater than or equal to 6.6, and the carbon source is less than 2 g / L, an acetate-containing solution is added to the culture medium using a pulse feeding method to bring the total carbon source in the culture medium to 2-5 g / L. The pH of the culture medium is controlled at 6.4-6.6, and the acetate concentration in the culture medium is greater than or equal to 1.1 g / L. Then, the addition of the acetate-containing solution to the culture medium is stopped. The first stage of fermentation is stopped at 22-26 hours, and the second stage of fermentation is stopped at 46-50 hours.

[0056] The glucose-containing culture medium contains: glucose 20-40 g / L, yeast extract 15-25 g / L, peptone 4-6 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, magnesium sulfate 1.5-2.5 g / L, sodium chloride 15-25 g / L, vitamin B1 0.008-0.012 g / L, FeSO4·7H2O 1.8-2.2 g / L, ZnSO4·7H2O 3.5-4.5 g / L, and CuSO4 2-3 g / L; the culture medium also contains acetate, with an acetate-to-glucose mass ratio of 1:1.5-2.5; the acetate is sodium acetate and / or potassium acetate; in the solution, the glucose concentration is 400-500 g / L; in the solution, the acetate concentration is 200-400 g / L;

[0057] During the second stage of fermentation, glucose and acetate are added to the culture medium as follows: when the dissolved oxygen in the culture medium at 28°C is less than or equal to 30% air saturation, glucose and acetate are added to the culture medium; when the dissolved oxygen in the culture medium at 28°C is greater than 45% air saturation, the addition of glucose and acetate to the culture medium is stopped.

[0058] The method provided by the preferred embodiments of the present invention enables rapid cell growth, quickly entering the logarithmic growth phase, and promotes lipid synthesis, thereby accelerating oil synthesis and effectively increasing the yield of sn-2DHA while shortening the fermentation cycle. The added glucose and sodium acetate are food additives, non-toxic, and ensure food safety. Furthermore, the method provided by the present invention effectively reduces the consumption of external alkali, thereby reducing the salt load and processing costs in post-processing.

[0059] The present invention will be described in detail below through examples. In the following examples, the Schizochytrium (Schizochytrium HX-308 (accession number CCTCCM209059)) was derived from the laboratory modification and has been described in the patent application with publication number CN114561434A.

[0060] Example 1

[0061] (1) The Schizochytrium, which was frozen at -80℃, was revived on a plate and a single colony was picked and inoculated into 250mL of seed culture medium (120mL). The culture medium was formulated with 5g / L tryptone, 5g / L yeast extract, 20g / L NaCl and 2g / L methionine. The pH was adjusted to 6.8±0.1. After sterilization at 121℃ for 20min, it was cultured in a shaker at 180rpm and 28℃ for 24h to obtain the first-grade seed liquid.

[0062] (2) Inoculate the primary seed culture at a ratio of 5% (v / v) into 1L of seed culture medium with the same formulation (in a 2L Erlenmeyer flask), and incubate at 180 rpm and 28℃ for 24 hours until OD reaches 100%. 600 ≈4.5, to obtain active secondary seed liquid.

[0063] (3) The culture medium for fermentation was prepared in a 50L tank with a working volume of 30L. The culture medium consisted of: glucose 26.7g / L, anhydrous sodium acetate 13.3g / L, malt extract powder 20g / L, peptone 5g / L, magnesium sulfate 2g / L, KH₂PO₄ 1g / L, NaCl 20g / L, vitamin B1 0.01g / L, FeSO₄·7H₂O 1.98g / L, ZnSO₄·7H₂O 4.0g / L, and CuSO₄ 2.5g / L. The overall pH was adjusted to 6.5±0.1. All components were sterilized at 121℃ for 30min and then cooled to room temperature for later use.

[0064] The fermenter parameters were set as follows: temperature 28±0.5℃, stirring speed 400rpm, air intake 1.0vvm sterile air, pH maintained at 6.5±0.1 (automatically adjusted with 2M NaOH or 2M HCl), DO maintained at 50% air saturation, and oxygenation or stirring accelerated when the air saturation dropped to 30%.

[0065] During inoculation, 10% (v / v) of secondary seed liquid is introduced into the fermenter. After completion, maintain a 0-24h batch growth period (during which no additional feeding is done, only ensuring the above parameters remain stable) to allow the cells to accumulate and enter the exponential growth phase.

[0066] The feeding phase begins after 24 hours. The feeding solution is a 500 g / L high-concentration mixed solution (glucose and sodium acetate in a 2:1 mass ratio). Feeding begins at 24 hours. During the feeding process, when the dissolved oxygen in the culture medium at 28°C is less than or equal to 30% air saturation, the high-concentration mixed solution is added to the culture medium. When the dissolved oxygen in the culture medium at 28°C is greater than 45% air saturation, the addition of the high-concentration mixed solution is stopped. During this process, the total carbon source content in the culture medium is 5-10 g / L. At 48 hours, when the pH of the culture system reaches above 6.6 and the carbon source is below 2 g / L, the feeding solution is replaced with a 500 g / L sodium acetate solution. Both the high-concentration mixed solution and the sodium acetate solution are added continuously in stages, and the total carbon source in the culture medium is controlled to be 2-5 g / L. Regarding online monitoring, if DO > 50% air saturation during the feeding process, the feeding rate can be appropriately increased; if DO < 10% air saturation, the stirring rate or oxygenation should be increased; when pH > 6.6, feeding can be temporarily stopped or the feeding rate reduced; when pH < 6.4, the pH will be automatically adjusted back to the set range using acid or alkali solutions.

[0067] If the total carbon source is >5 g / L, the feeding rate can be slowed down accordingly; if the total carbon source is <2 g / L, it indicates that the carbon source is limited, and the feeding rate can be increased appropriately.

[0068] During sampling and analysis, 5 mL samples were taken from the container at nine time points: 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h. After sampling, the OD of 1 mL of the sample was measured first. 600 The stem cell quality was calculated based on the standard curve, and then DHA was extracted and measured from the remaining stem cells.

[0069] The sample was centrifuged (10000 rpm, 4℃, 10 min) to collect the bacterial cells. After lyophilization, about 100 mg of lyophilized powder was weighed and added to 10 mL of chloroform:methanol (volume ratio 2:1) mixed solvent. The mixture was ultrasonically extracted for 30 min and then filtered to collect the organic phase.

[0070] Half of the organic phase was measured and 1 mL of 0.5 M MeOH-KOH was added. The mixture was methylated in a water bath at 70 °C for 35 min. After adjusting the pH to neutral or weakly acidic with boric acid, 1 mL of n-hexane was added to extract the supernatant. The precipitate was dried under nitrogen and redissolved in 1 mL of n-hexane for GC analysis. The GC conditions were: HP-88 capillary column (100 m × 0.25 mm, 0.2 μm), temperature program: 120 °C (hold for 1 min) → 240 °C (hold for 10 min) at 10 °C / min, nitrogen as carrier gas, injection port temperature 250 °C, and FID detector temperature 260 °C. DHA was quantified using the external standard method.

[0071] The other half of the organic phase was rotary evaporated to obtain an oil sample containing DHA. 1 mg of the oil was weighed and placed in a 5 mL centrifuge tube. 7 mM Tris buffer (pH = 7.6), 110 μL of bile salt solution (0.1 wt%), 5 μL of CaCl2 (2.5 wt%), and 1 mg of porcine pancreatic lipase were added sequentially. The mixture was reacted in a 37°C shaking water bath for 20 minutes. After the reaction, the mixture was cooled to room temperature, and 1 mL of anhydrous diethyl ether was added. The mixture was vortexed for 3 minutes, followed by centrifugation at 6000 rpm for 5 minutes. The upper organic phase was collected and concentrated to dryness using a nitrogen blowdown apparatus. 1 μL of n-hexane was added to dissolve the phase completely, and the solution was spotted. Thin-layer chromatography was performed using n-hexane / anhydrous diethyl ether / acetic acid (50:60:1, v / v / v) as the developing solvent. The mixture was placed in an iodine bath for 3 minutes to identify and scrape off the sn-2 glycerol monoester band, which was then extracted with 25 μL of anhydrous diethyl ether. After methylation, gas chromatography analysis was performed to determine the content of the product sn-2DHA based on the elution peaks. Figure 1 As shown.

[0072] Substrate detection was performed using HPLC to determine the concentrations of glucose and sodium acetate in the supernatant. Results showed that at the 96-hour endpoint, the remaining substrate concentrations were approximately 5 g / L glucose and 2 g / L sodium acetate.

[0073] Example 2

[0074] DHA was produced by fermentation according to the method in Example 1, except that the culture medium contained 20 g / L glucose and 20 g / L sodium acetate; and the mass ratio of glucose to sodium acetate in the added 500 g / L high-concentration mixed solution was 1:1.

[0075] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 3 g / L and the sodium acetate was approximately 2.5 g / L.

[0076] The relationship between DHA production and time is as follows: Figure 2 As shown.

[0077] Example 3

[0078] DHA was produced by fermentation according to the method of Example 1, except that the mass ratio of glucose to sodium acetate in the added 500 g / L high-concentration mixed solution was 1.8:1.

[0079] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 4.5 g / L and the sodium acetate was approximately 2.3 g / L.

[0080] Example 4

[0081] DHA was produced by fermentation according to the method of Example 1, except that the mass ratio of glucose to sodium acetate in the added 500 g / L high-concentration mixed solution was 2.2:1.

[0082] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 5.1 g / L and the sodium acetate was approximately 2.1 g / L.

[0083] Example 5

[0084] DHA was produced by fermentation according to the method of Example 1, except that the mass ratio of glucose to sodium acetate in the added 500 g / L high-concentration mixed solution was 1.5:1.

[0085] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 4.1 g / L and the sodium acetate was approximately 3 g / L.

[0086] Example 6

[0087] DHA was produced by fermentation according to the method of Example 1, except that the mass ratio of glucose to sodium acetate in the added 500 g / L high-concentration mixed solution was 2.5:1.

[0088] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 5.3 g / L and the sodium acetate was approximately 3 g / L.

[0089] Example 7

[0090] The fermentation process was carried out according to the method of Example 1 to produce DHA. The difference was that in step (3), the fermentation tank parameters were set as follows: temperature 28±0.5℃, stirring speed 400rpm, air intake 1.0vvm sterile air, pH maintained at 6.5±0.1 (automatically adjusted with 2M NaOH or 2MHCl), DO maintained at 50% air saturation, and oxygenation or stirring was increased when the air saturation dropped to 20%.

[0091] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 4.7 g / L and the sodium acetate was approximately 2.4 g / L.

[0092] Example 8

[0093] The method of Fermentation to produce DHA was followed according to Example 1. The difference was that in step (3), the feeding stage started after 24 hours. The feeding solution was a 500 g / L high-concentration mixed solution (glucose and sodium acetate in a mass ratio of 2:1). The feeding time started from 24 hours and the continuous feeding rate was 200 mL / h. At 48 hours, when the pH of the culture system reached above 6.6 and the carbon source was below 2 g / L, the feeding solution was replaced with a 500 g / L sodium acetate solution. Both the high-concentration mixed solution and the sodium acetate solution were added continuously in stages.

[0094] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 5.9 g / L and the sodium acetate was approximately 3.3 g / L.

[0095] Example 9

[0096] The fermentation process for DHA production followed the method described in Example 1, except that a feeding phase began after 24 hours. The feed solution was a 500 g / L high-concentration mixed solution (glucose and sodium acetate in a 2:1 mass ratio). Feeding began after 24 hours. During the feeding process, when the dissolved oxygen in the culture medium at 28°C was less than or equal to 30% air saturation, the high-concentration mixed solution was added to the culture medium. When the dissolved oxygen in the culture medium at 28°C was greater than 45% air saturation, the addition of the high-concentration mixed solution was stopped. At 40 hours, when the pH of the culture system reached above 6.6 and the carbon source was 2.3 g / L, the feed solution was replaced with a 500 g / L sodium acetate solution. Both the high-concentration mixed solution and the sodium acetate solution were added continuously in stages.

[0097] At 96 hours after the endpoint, the remaining glucose in the substrate was approximately 4.9 g / L and the sodium acetate was approximately 2.3 g / L.

[0098] Example 10

[0099] The fermentation process for producing DHA followed the method in Example 1, except that the culture medium in step (3) was as follows: culture medium: glucose 24 g / L, anhydrous potassium acetate 16 g / L, malt extract powder 15 g / L, peptone 4 g / L, magnesium sulfate 2.5 g / L, KH2PO4 1.2 g / L, NaCl 25 g / L, vitamin B1 0.008 g / L, FeSO4·7H2O 2.2 g / L, ZnSO4·7H2O 3.5 g / L, CuSO4 2.0 g / L.

[0100] Example 11

[0101] The fermentation process for producing DHA followed the method in Example 1, except that the culture medium in step (3) was as follows: culture medium: glucose 24 g / L, anhydrous sodium acetate 16 g / L, malt extract powder 25 g / L, peptone 6 g / L, magnesium sulfate 1.5 g / L, KH2PO4 0.8 g / L, NaCl 15 g / L, vitamin B1 0.012 g / L, FeSO4·7H2O 1.8 g / L, ZnSO4·7H2O 4.5 g / L, CuSO4 3.0 g / L.

[0102] Comparative Example 1

[0103] DHA was produced by fermentation according to the method in Example 1, except that the 500 g / L high-concentration mixed solution was replaced with a 500 g / L glucose solution, and the sodium acetate solution was replaced with a glucose solution.

[0104] Comparative Example 2

[0105] DHA was generated by fermentation according to the method of Example 1, except that in step (3), the 500 g / L high concentration mixed solution was replaced with a 500 g / L glucose solution.

[0106] Comparative Example 3

[0107] DHA was generated by fermentation according to the method of Example 1, except that in step (3), the sodium acetate solution was replaced with a glucose solution.

[0108] Comparative Example 4

[0109] DHA was produced by fermentation according to the method in Example 1, except that the 500 g / L high-concentration mixed solution was replaced with a 500 g / L sodium acetate solution, and the 500 g / L sodium acetate solution was replaced with a 500 g / L high-concentration mixed solution (the mass ratio of glucose to sodium acetate was 2:1).

[0110] Comparative Example 5

[0111] DHA was produced by fermentation according to the method in Example 1, except that the sodium acetate in the 500 g / L high-concentration mixed solution was replaced with cane molasses, and the sodium acetate in the 500 g / L sodium acetate solution was replaced with cane molasses.

[0112] Test Example 1

[0113] The fermentation cutoff time, sn-2DHA yield, oil yield, DHA yield, cell dry weight and intracellular lipid content of the above-mentioned embodiments and comparative examples are shown in Table 1.

[0114] Wherein, DHA yield = DHA production / oil production

[0115] Taking Example 1 as an example, the oil yield reached 67 g / L, the DHA yield was 42.9 g / L, and the DHA recovery rate was 64%.

[0116] The standard curve method was used to measure the dry weight of bacterial cells.

[0117] After measuring the OD600 of culture broth of Schizochytrium at different growth stages, a known volume (V) of sample was vacuum filtered through a 0.45 μm filter membrane, washed three times with distilled water to remove culture medium residue, and dried at 105℃ to constant weight (about 4-6 hours). The weight gain of the filter membrane was then measured.

[0118] Dry weight (g / L) = (W2 - W1) / V × 1000

[0119] (W1 = original weight of filter membrane, W2 = weight of filter membrane + bacterial weight)

[0120] Then, establish a standard curve: dry weight (g / L) = k × OD600 + b. Determine the values ​​of k and b, and calculate the corresponding dry weight based on the OD600 data.

[0121] Intracellular lipid content was determined using the Bligh & Dyer method, as detailed below:

[0122] 1. Take a known dry weight of biomass, grind or homogenize it to fully break down the tissue / cells.

[0123] 2. Add methanol to the extraction system at a volume ratio of 2:1:0.8 (methanol:chloroform:water) and vortex mix thoroughly for 1 minute.

[0124] 3. Add chloroform and water to make the total volume ratio 1:1:0.9 (methanol:chloroform:water); commonly, add 2mL of chloroform and 2mL of water, and vortex again. Centrifuge (1000-3000×g, 5-10min) to separate into two phases: upper methanol / water phase and lower chloroform phase (containing lipids).

[0125] 4. Transfer the entire lower chloroform phase into a pre-weighed, dried, and cooled-to-room-temperature weighing bottle. Chloroform can be added to the aqueous phase for a second or third extraction. Combine the chloroform phases to improve the recovery rate.

[0126] 5. Evaporate chloroform under reduced pressure in a fume hood or rotary evaporator; further dry the remaining lipids to constant weight in an oven (60°C) or under a nitrogen stream.

[0127] 6. Record the lipid mass after evaporation and calculate the intracellular lipid content according to the following formula.

[0128] Intracellular lipid content = (net lipid mass / stem cell weight) × 100%

[0129] Table 1

[0130]

[0131] As can be seen from the results in Table 1, the intracellular lipid content, DHA yield, and cell dry weight of Examples 1-11 are all higher than those of Comparative Examples 1-5, indicating that the method provided by the present invention has a high sn-2DHA yield and intracellular lipid content, providing an efficient operating scheme for industrial production.

[0132] Furthermore, compared to Comparative Example 1, the intracellular sn-2DHA yield in Example 1 increased by approximately 5.6 g / L, a growth of about 15%, indicating that this technical solution can effectively improve sn-2DHA production. Throughout the experiment, the amount of sodium hydroxide required in Comparative Example 1 was approximately 1.5 mol / L, while in Example 1 it was approximately 0.87 mol / L. Compared to Comparative Example 1, the sodium hydroxide consumption decreased by 42%, reducing the burden on downstream extraction and wastewater treatment, and also lowering treatment costs and environmental impact. Comparative Example 1 reached its maximum yield after 114 hours but still required 12-24 hours of follow-up treatment to ensure saturated oil accumulation. Example 1 reached its maximum yield at 96 hours, and further fermentation could not significantly increase the yield; therefore, fermentation was terminated at approximately 100 hours. Thus, the method provided by this invention saves 14 hours of fermentation time compared to conventional methods, which is particularly important after large-scale scaling, enabling higher equipment utilization and lower production costs.

[0133] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for producing sn-2DHA algal oil by fermentation of Schizochytrium, characterized in that, The method includes: fermenting Schizochytrium in a culture medium containing glucose; The fermentation is divided into a first stage fermentation, a second stage fermentation, and a third stage fermentation. During the second stage fermentation, glucose and acetate are added to the culture medium. During the third stage fermentation, acetate is added to the culture medium so that the total carbon source in the culture medium is 2-5 g / L. The first stage fermentation is stopped at 22-26 h, and the second stage fermentation is stopped at 46-50 h.

2. The method according to claim 1, characterized in that, During the first stage of fermentation, the dissolved oxygen in the culture medium was controlled at 30% to 50% air saturation at 28°C. During the second and third stages of fermentation, the dissolved oxygen in the culture medium was controlled at 28°C to be between 10% and 50% air saturation.

3. The method according to claim 1, characterized in that, The glucose concentration in the glucose-containing culture medium is 20-40 g / L; Preferably, the glucose-containing culture medium also contains acetate, and the mass ratio of acetate to glucose is 1:1.5-2.5; Preferably, the glucose-containing culture medium also contains yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride and vitamin B1; Preferably, in the culture medium, the concentration of the yeast extract is 15-25 g / L, the concentration of the peptone is 4-6 g / L, the concentration of the potassium dihydrogen phosphate is 0.8-1.2 g / L, the concentration of the magnesium sulfate is 1.5-2.5 g / L, the concentration of the sodium chloride is 15-25 g / L, and the concentration of the vitamin B1 is 0.008-0.012 g / L.

4. The method according to claim 1, characterized in that, The fermentation conditions include at least the following: a stirring rate of 300-600 rpm, a sterile air flow rate of 0.8-1.2 vvm, and an initial pH of 6.4-6.

6.

5. The method according to claim 1, characterized in that, The glucose and the acetate were added to the culture medium in solution form; Preferably, the concentration of glucose in the solution is 250-400 g / L; In the solution, the concentration of acetate is 200-500 g / L.

6. The method according to claim 1, characterized in that, The acetate is potassium acetate and / or sodium acetate.

7. The method according to claim 1, characterized in that, During the second stage of fermentation, the total carbon source in the culture medium is controlled to be 5-10 g / L.

8. The method according to any one of claims 1 to 7, characterized in that, During the second stage of fermentation, the mass ratio of the added glucose to the added acetate is 1.5-2.5:1, preferably 1.8-2.2:

1.

9. The method according to any one of claims 1 to 7, characterized in that, During the second stage of fermentation, glucose and acetate are added to the culture medium as follows: when the dissolved oxygen in the culture medium at 28°C is less than or equal to 30% air saturation, glucose and acetate are added to the culture medium; when the dissolved oxygen in the culture medium at 28°C is greater than 45% air saturation, the addition of glucose and acetate to the culture medium is stopped.

10. The method according to any one of claims 1 to 7, characterized in that, During the third stage of fermentation, the acetate is added when the pH of the culture medium is greater than or equal to 6.6 and the carbon source is less than 2 g / L. Preferably, the addition of acetate is stopped when the solubility of acetate in the culture medium is greater than or equal to 1.1 g / L.

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