Schizochytrium limacinum basal culture medium and method for culturing schizochytrium limacinum to produce DHA

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a schizochytrium limacinum basic culture medium and a method for producing DHA (docosahexaenoic acid) by culturing schizochytrium limacinum. The schizochytrium limacinum basic culture medium comprises distillate obtained by performing reduced pressure distillation on waste algae liquid obtained after oil extraction of schizochytrium limacinum. The waste algae liquid distillate obtained after grease extraction of schizochytrium limacinum contains a certain amount of citric acid, malic acid and other organic acid components, and existing researches show that malic acid is a precursor substance for synthesis of DHA from schizochytrium limacinum; a proper amount of citric acid and malic acid are added into the culture medium, so that synthesis of fatty acid and accumulation of DHA by the schizochytrium limacinum can be obviously promoted; the method is simple and easy to implement, reduces the discharge amount of the schizochytrium limacinum waste algae liquid as waste water, realizes the cyclic utilization of the waste algae liquid distillate, saves water resources, and saves the treatment cost of the waste water in the recycling process of the schizochytrium limacinum waste algae liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a basic culture medium for Schizochytrium and a method for culturing Schizochytrium to produce DHA. Background Art

[0002] Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid belonging to the ω-3 fatty acid family. It is a key component of the human brain, retina and other organs. It is particularly important for the vision and brain development of infants and young children. Adults also need an appropriate amount of DHA to maintain brain health and cognitive function. The human body cannot synthesize enough DHA on its own, so it must be taken in through diet. The sources of DHA include some deep-sea fish, but the output of DHA purified from deep-sea fish oil is low and easily affected by seasons, which is far from meeting market demand.

[0003] At present, the production of DHA mostly relies on the fermentation production of Schizochytrium. Schizochytrium, also known as Schizochytrium, is a type of marine fungus belonging to the Thraustochytriaceae family. Compared with other microalgae, Schizochytrium has the characteristics of rapid growth, high oil content, and easy large-scale cultivation. By fermenting Schizochytrium, Schizochytrium algae containing a large amount of oil are obtained, and then the algae are subjected to a wall-breaking treatment, and then the oil is extracted by high-speed centrifugation. During the centrifugation process, waste algae liquid accounting for more than 90% of the volume of the fermentation liquid will be produced, and these waste algae liquids also contain nearly 100g / L dry weight of Schizochytrium residue. Schizochytrium residue is rich in nutrients, containing polysaccharides, proteins, vitamins and other ingredients, and can be used as a feed raw material. CN202110180281.3 discloses a method for preparing a functional feed that uses Schizochytrium residue to improve the quality of grass carp. The Schizochytrium residue in a crushed state after oil extraction is added to the feed at a ratio of 3-9%, which can increase the DHA content of grass carp muscle and the antioxidant capacity of the fish body, and improve the health of grass carp. CN201510476484.1 discloses a method for applying the broken cell wall slurry of Schizochytrium to the cultivation of aquatic fish seedlings. The waste algae liquid of Schizochytrium obtained by three-phase centrifugation is added as a raw material to the feed of fish seedlings, which can increase the average survival rate of fish seedlings by 24.5-34.5%. Compared with traditional feeds, the waste algae liquid as feed has too high water content. Since it is a byproduct after high-speed centrifugation and has high viscosity, it is concentrated to further reduce the water content. However, a large volume of distillate will be produced in this process, and its chemical oxygen demand COD and ammonia nitrogen reach 2864.5 mg / L and 50 mg / L respectively, which are far higher than the national environmental protection standards. The environmental protection treatment cost per ton of COD is 4000-5000 yuan, while the environmental protection treatment cost of ammonia nitrogen is about 4 times that of COD, and the treatment cost is relatively high. Therefore, there is an urgent need for an application of the distillate of the waste algae liquid after the oil is extracted from Schizochytrium, and a method for culturing Schizochytrium to produce DHA using the waste algae liquid distillate of Schizochytrium. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a basic culture medium for Schizochytrium and a method for culturing Schizochytrium to produce DHA, which solves the technical problem of concentrating waste algae liquid by vacuum distillation to produce a large volume of distillate.

[0006] (II) Technical solution

[0007] In order to solve the above problems, the present invention applies the distillate of the waste algae liquid of Schizochytrium to the fermentation medium at a volume ratio of (20-100%) to pure water: (80%-0) for the growth of Schizochytrium and the accumulation of oil. To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, an embodiment of the present invention provides a basic culture medium for Schizochytrium sp., which comprises: a distillate obtained by vacuum distillation of waste algae liquid after oil extraction from Schizochytrium sp.

[0009] By fermenting Schizochytrium algae, Schizochytrium algae bodies containing a large amount of oil are obtained, and then the algae walls are broken, and the oil is extracted by high-speed centrifugation. During the centrifugation process, waste algae liquid accounting for more than 90% of the volume of the fermentation liquid will be produced. However, the waste algae liquid has too high water content as a feed. Since it is already a by-product after high-speed centrifugation and has high viscosity, it needs to be concentrated to further reduce the water content. In this process, a large volume of distillate will be produced. The inventors found that the distillate of the waste algae liquid can be used to prepare a fermentation medium by analyzing the specific components of the distillate.

[0010] The inventors of the present application discovered that the distillate of the waste algae liquid after oil extraction from Schizochytrium contains a certain amount of organic acid components such as citric acid and malic acid. Studies have shown that malic acid is a precursor substance for the synthesis of DHA by Schizochytrium; adding an appropriate amount of citric acid and malic acid to the culture medium can significantly promote the synthesis of fatty acids and the accumulation of DHA by Schizochytrium.

[0011] Optionally, the waste algae liquid after oil extraction from Schizochytrium schizochytrium is distilled under reduced pressure and the distillate is mixed with pure water in a volume ratio of 2-10:8-0.

[0012] In a second aspect, an embodiment of the present invention provides a method for culturing Schizochytrium to produce DHA, comprising the following steps:

[0013] S1. Preparation of culture medium

[0014] Use pure water to prepare the seed culture medium;

[0015] Supplementing the basic culture medium of Schizochytrium provided by the embodiment of the first aspect of the present invention with a carbon source, a nitrogen source and an inorganic salt to prepare a fermentation medium;

[0016] S2. Fermentation culture of Schizochytrium

[0017] Primary seed culture: inoculate the frozen glycerol solution of Schizochytrium into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30° C. for 30-48 hours;

[0018] Secondary seed culture: inoculate the primary seed solution into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30° C. for 24-48 hours;

[0019] Fermentation culture: inoculate the secondary seed liquid into the fermentation medium at a volume percentage of 2-5%, and culture at 150-200rpm and 25-30°C for 48-72h. During the fermentation process, add glucose feed solution to control the glucose concentration at 0-60g / L. When the glucose is exhausted, the fermentation ends.

[0020] S3, extracting oil from the fermentation liquid.

[0021] Optionally, in S1, the carbon source is glucose, the nitrogen source includes one or more of yeast powder, peptone, corn steep liquor, sodium glutamate and glycine, and the inorganic salt includes phosphate, magnesium salt, calcium salt, sodium salt and potassium salt.

[0022] Optionally, in S1, the seed culture medium includes 30-40 g / L glucose, 5-8 g / L yeast powder, 1-3 g / L magnesium sulfate, 1-3 g / L potassium dihydrogen phosphate, 1-3 g / L calcium chloride, 5-10 g / L sodium chloride, 2-5 g / L sodium bicarbonate, 0.1-0.5 g / L potassium chloride, and natural pH;

[0023] The fermentation medium includes 30-50 g / L of glucose, 5-8 g / L of yeast powder, 5-10 g / L of sodium glutamate, 1-3 g / L of magnesium sulfate, 1-3 g / L of potassium dihydrogen phosphate, 1-3 g / L of calcium chloride, 5-10 g / L of sodium chloride, 2-5 g / L of sodium bicarbonate, 0.1-0.5 g / L of potassium chloride, and has a natural pH.

[0024] Optionally, the seed culture medium and the Schizochytrium fermentation culture medium are sterilized at 115-121° C. for 15-30 min and then cooled to room temperature before use.

[0025] Optionally, the concentration of the glucose feed solution is 300-500 g / L, sterilized at 115-121° C. for 15-30 min, and cooled to room temperature before use.

[0026] Optionally, in step S3, after the fermentation is completed, the algal bodies of Schizochytrium in the fermentation broth are enzymatically broken, n-hexane is added and mixed, and then the mixture is centrifuged, the oil layer is heated, and the n-hexane is evaporated and then weighed until a constant weight is reached, the total oil content in the fermentation broth is calculated, and the content of docosahexaenoic acid (DHA) in the oil is determined by gas chromatography.

[0027] Optionally, step S3 includes: using one or more enzymes selected from alkaline protease, cellulase and pectinase to break the algae wall in the fermentation broth; the addition amount is 0.5-2g / L; adjusting the pH of the fermentation broth to 8.0-11.0 with a pH regulator, stirring at a speed of 50-150rpm and a temperature of 40-80°C, and the wall breaking treatment time is 2-4h; during the centrifugation process, the speed is 3000-6000rpm and the time is 3-5min.

[0028] Optionally, the Schizochytrium spp. fermentation culture

[0029] Primary shake flask seed culture: inoculate the frozen glycerol solution of Schizochytrium into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30° C. for 30-48 hours;

[0030] Secondary shake flask seed culture: inoculate the primary shake flask seed solution into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30°C for 24-48 hours;

[0031] Fermentation culture in a 50L fermenter: fill 50-70% of the liquid, access the above-mentioned secondary shake flask seed liquid, the volume percentage of the inoculation amount is 2-5%, the culture temperature is 25-30°C, the initial rotation speed is 80-150rpm, the ventilation volume is 0.5-1.0vvm, the pH is natural, the culture is 15-30h, the nitrogen source is added according to the initial nitrogen source concentration of the culture medium, the glucose concentration is controlled to 0-30g / L by adding glucose, and the culture is 96-120h;

[0032] The fermentation medium in the fermenter is sterilized by steam at 115-123°C for 20-35 min and cooled to 25-30°C.

[0033] During the fermentation and cultivation process of the present invention, samples need to be taken regularly to measure OD650, glucose and glutamate respectively;

[0034] OD650 is measured by spectrophotometer at a wavelength of 650nm. The spectrophotometer is adjusted to a wavelength of 650nm, preheated for 20min, and zeroed with pure water as a blank sample. Then, the absorbance of the Schizochytrium algae solution at a wavelength of 650nm is measured. If the value is greater than 0.8, the Schizochytrium algae solution is diluted and re-measured. The final result = the measurement result × the dilution factor.

[0035] Glucose and glutamate were measured by SBA biosensor analyzer. 25 μL of 50 mg / dL lactic acid and 100 mg / dL glutamate-glucose standard solutions were accurately drawn with a sampling needle for calibration. Multiple sampling measurements were performed until the instrument displayed that the calibration was completed. The algae solution to be tested was centrifuged at 3000-5000 rpm for 2-5 min, the supernatant was taken, and after diluting 100 times, 25 μL was accurately drawn with a sampling needle for injection and detection. The measured values ​​were the concentrations of glutamate and glucose in the algae solution, respectively, in g / L.

[0036] Biomass was determined by dry weight method.

[0037] (III) Beneficial effects

[0038] The waste algae liquid distillate after oil extraction from Schizochytrium in the present invention contains a certain amount of organic acid components such as citric acid and malic acid. Studies have shown that malic acid is a precursor substance for the synthesis of DHA by Schizochytrium; adding an appropriate amount of citric acid and malic acid to the culture medium can significantly promote the synthesis of fatty acids and the accumulation of DHA by Schizochytrium; using the distillate to prepare the culture medium is conducive to the accumulation of Schizochytrium biomass and can reduce the use of culture medium components by a small proportion.

[0039] The invention is simple and easy to implement, reduces the discharge amount of the waste algae liquid of Schizochytrium as wastewater, realizes the recycling of the distillate of the waste algae liquid, saves water resources, and saves the wastewater treatment cost in the process of reusing the waste algae liquid of Schizochytrium.

[0040] The invention reduces production costs while ensuring the quality and yield of DHA, and is a green production process that helps protect the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The fermentation conditions of Schizochytrium truncatum of Example 1 in fermentation medium prepared with the volume ratio of waste algae liquid distillate to pure water being 1:4, 1:1, 1:0 and 0:1 respectively, Figure 1 (a) is the change of glucose, Figure 1 (b) is the change of glutamate, Figure 1 (c) is the change of OD650;

[0042] Figure 2 The biomass of Schizochytrium sp. at the end of fermentation in the fermentation medium prepared with the volume ratio of the waste algae liquid distillate to pure water being 1:4, 1:1, 1:0 and 0:1 respectively;

[0043] Figure 3 The fermentation conditions of Schizochytrium truncatum of Example 2 at 100%, 95% and 85% of the mass of the culture medium of Example 1 are shown in FIG. Figure 3 (a) is the change of glucose, Figure 3 (b) is the change of glutamate, Figure 3 (c) is the change of OD650;

[0044] Figure 4 The biomass of Schizochytrium sp. in Example 2 at the end of fermentation in 100%, 95% and 85% of the mass of the culture medium in Example 1;

[0045] Figure 5 This is the fermentation condition of Schizochytrium sp. in Example 3 in a 50 L fermenter. DETAILED DESCRIPTION

[0046] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0047] Example 1

[0048] The fermentation culture of Schizochytrium in a culture medium prepared with different proportions of waste algae liquid distillate comprises the following steps:

[0049] Prepare seed culture medium: 40g / L glucose, 5g / L yeast powder, 1g / L magnesium sulfate, 2g / L potassium dihydrogen phosphate, 3g / L calcium chloride, 10g / L sodium chloride, 2g / L sodium bicarbonate, 0.2g / L potassium chloride, natural pH, sterilize at 121℃ for 20min, use pure water to prepare seed culture medium, cool and set aside;

[0050] Prepare fermentation medium: 40g / L glucose, 8g / L yeast powder, 8g / L sodium glutamate, 1g / L magnesium sulfate, 2g / L potassium dihydrogen phosphate, 3g / L calcium chloride, 5g / L sodium chloride, 2g / L sodium bicarbonate, 0.2g / L potassium chloride, natural pH, sterilize at 121℃ for 20min, cool to room temperature and set aside; the volume ratio of waste algae liquid distillate to pure water is 1:4, 1:1, 1:0 and 0:1 respectively to prepare fermentation medium;

[0051] Primary shake flask seed culture: 50 mL of seed culture medium was placed in a 250 mL conical flask with baffles, and 5% of the volume percentage of Schizochytrium glycerol cryopreservation solution was added, and then cultured at 150 rpm and 28°C for 36 h to activate the low-temperature frozen Schizochytrium seeds into Schizochytrium seed solution;

[0052] Secondary shake flask seed culture: 50 mL of seed culture medium was placed in a 250 mL conical flask with baffles, and the primary shake flask seed solution was added at a volume percentage of 5%, and then cultured at 150 rpm and 28 °C for 24 h;

[0053] Fermentation culture: 300 mL of fermentation medium was placed in a 1 L conical flask with baffles, and 5% of the volume percentage of the secondary shake flask seed solution was added, and culture was carried out at 150 rpm and 28 ° C. During the fermentation process, glucose feed solution was added twice to control the glucose concentration in the fermentation liquid at 0-60 g / L. The fermentation was terminated after the glucose was exhausted.

[0054] Figure 1 The fermentation conditions of Schizochytrium truncatum of Example 1 in fermentation medium prepared with the volume ratio of waste algae liquid distillate to pure water being 1:4, 1:1, 1:0 and 0:1 respectively, Figure 1 (a) is the change of glucose, Figure 1 (b) is the change of glutamate, Figure 1 (c) shows the changes of OD650.

[0055] As can be seen from the figure, the glucose in the initial culture medium of each group was consumed at 38 hours of culture, and glucose was supplemented at 39 hours and 48 hours respectively, and then all was consumed at 61 hours of culture; at 20 hours of culture, glutamate was basically consumed in the groups with a volume ratio of distillate to pure water of 0:1 and 1:1, and about 2 g / L glutamate remained in other groups; OD650 showed that the cell growth trend of each group was consistent, and at the end of fermentation, the volume ratio of distillate to pure water of 0:1, 1:4, 1:1, and 1:0 groups reached 54.8, 57.8, 59.85, and 56.7, respectively.

[0056] Figure 2 The biomass at the end of fermentation in the fermentation medium prepared for the Schizochytrium in Example 1 was obtained in which the volume ratios of the waste algae liquid distillate to pure water were 1:4, 1:1, 1:0 and 0:1 respectively; at the end of fermentation, the volume ratios of the distillate to pure water in the groups of 0:1, 1:4, 1:1 and 1:0 reached 50.4 g / L, 50.6 g / L, 51.6 g / L and 52.2 g / L respectively, and the dry weight of the biomass increased with the increase of the proportion of distillate in the culture medium. It can be seen that some components in the waste algae liquid distillate can be used by Schizochytrium, which is conducive to the accumulation of biomass.

[0057] Example 2

[0058] Except for glucose, the remaining ingredients are fermented and cultured in a fermentation medium at 85-95% (e.g., 100%, 95% and 85%) of the mass of the culture medium in Example 1, sterilized at 115-121° C. for 15-30 minutes, cooled to room temperature and set aside; wherein the volume ratio of the waste algae liquid distillate to pure water is 1:0, sterilized at 121° C. for 20 minutes and then cooled and set aside;

[0059] Fermentation medium (95% of the mass of the fermentation medium of Example 1): glucose 40 g / L, yeast powder 7.6 g / L, sodium glutamate 7.6 g / L, magnesium sulfate 0.95 g / L, potassium dihydrogen phosphate 1.9 g / L, calcium chloride 2.85 g / L, sodium chloride 4.75 g / L, sodium bicarbonate 1.9 g / L, potassium chloride 0.19 g / L, pH natural;

[0060] Fermentation medium (85% of the fermentation medium mass of Example 1): glucose 40 g / L, yeast powder 6.8 g / L, sodium glutamate 6.8 g / L, magnesium sulfate 0.85 g / L, potassium dihydrogen phosphate 1.7 g / L, calcium chloride 2.55 g / L, sodium chloride 4.25 g / L, sodium bicarbonate 1.7 g / L, potassium chloride 0.17 g / L, pH natural;

[0061] The remaining steps are the same as in Example 1

[0062] Figure 3 The fermentation conditions of Schizochytrium truncatum of Example 2 at 100%, 95% and 85% of the mass of the culture medium of Example 1 are shown in FIG. Figure 3 (a) is the change of glucose, Figure 3 (b) is the change of glutamate, Figure 3 (c) is the change of OD650;

[0063] As can be seen from the figure, at 38h of culture, glucose in the initial culture medium of each group was completely consumed, and glucose was supplemented at 39h and 48h respectively, and then all was consumed at 61h of culture; at 20h of culture, glutamate in the 85% ingredient group had dropped to 0, and there was still a small amount of glutamate in the culture medium of the other two groups; at the end of fermentation, the OD650 of the 100% ingredient group (control group) and the 95% ingredient group were 56.7 and 56.25, respectively, and the 85% ingredient group was lower, at 49.85;

[0064] Figure 4 The biomass of Schizochytrium in Example 2 at the end of fermentation in 100%, 95% and 85% of the mass of the culture medium in Example 1; at the end of fermentation, the biomass of the 100% waste algae liquid distillate group (control group) and the 95% ingredient group were 52.2 g / L and 52.1 g / L, respectively, and the biomass of the 85% ingredient group was lower, at 44.3 g / L.

[0065] Example 3

[0066] like Figure 5 As shown, the growth of glucose, glutamate and biomass in the fermentation medium prepared with the waste algae liquid distillate and pure water in a 50L fermenter at a volume ratio of 1:0. The fermentation culture of Schizochytrium in the medium prepared with the waste algae liquid distillate of the present invention comprises the following steps:

[0067] Prepare fermentation medium: glucose 40g / L, yeast powder 8g / L, sodium glutamate 8g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 2g / L, calcium chloride 3g / L, sodium chloride 5g / L, sodium bicarbonate 2g / L, potassium chloride 0.2g / L, use waste algae liquid distillate to prepare, pH natural, sterilize at 120-123℃ for 30-35min, cool to 28℃ for inoculation;

[0068] Fermentation culture in a 50L fermenter: sample volume 50%, inoculation volume percentage 2%, culture temperature 28°C, initial rotation speed 100rpm, ventilation volume 0.5vvm, pH natural, culture for 15h, add nitrogen source feed solution once according to the initial nitrogen source concentration, control the glucose concentration to 0-30g / L by adding glucose feed solution, and culture for 120h;

[0069] After the fermentation, sodium hydroxide was added to the fermentation liquid to adjust the pH to 9.5±0.05, the temperature was raised to 60±5°C, alkaline protease was added at 0.5‰ (volume-to-mass ratio), the rotation speed was 100 rpm, and the cell wall was broken for 3 hours.

[0070] Use a measuring cylinder to accurately measure 20-50 mL of the fermentation liquid after the cell wall breaking treatment, transfer it to a centrifuge tube, then add 20-30 mL of n-hexane, mix well and centrifuge at 5000 rpm for 5 min, transfer the oil layer after centrifugation to a glass dish dried to constant weight through a rubber-tipped dropper, repeat the extraction with n-hexane 3-10 times until there is no oil color in the n-hexane layer after centrifugation, heat to evaporate the n-hexane, and then weigh until constant weight, and calculate the total oil content in the fermentation liquid.

[0071] The remaining steps are the same as in Example 1

[0072] As can be seen from the figure, in the 50L fermentation tank system, the biomass increased rapidly in the first 95 hours after 120 hours of cultivation, and the biomass tended to be stable after 95 hours. At the end of fermentation, the biomass reached 98g / L. After testing and calculation, the total oil content was 55.28g / L, the DHA content in the oil was 58.82%, and the DHA yield reached 32.5g / L.

[0073] Test example

[0074] Extracting oil from Schizochytrium in Example 1 and Example 2 and determining the DNA content therein comprises the following steps:

[0075] Measure an appropriate volume of fermentation broth and place it in a clean glass beaker, add sodium hydroxide solution to adjust the pH of the fermentation broth to 9.5±0.05, add alkaline protease at 0.5‰ (volume-to-mass ratio), place the beaker on a constant temperature magnetic stirrer, at a temperature of 60±5°C and a rotation speed of 100 rpm, and treat for 3 hours;

[0076] Accurately measure 20-50 mL of the fermentation liquid after the cell wall breaking treatment, transfer it to a centrifuge tube, then add 20-30 mL of n-hexane, mix well and centrifuge at 5000 rpm for 5 min, transfer the oil layer after centrifugation to a glass dish dried to constant weight through a rubber-tipped dropper, repeat the extraction with n-hexane 3-10 times until there is no oil color in the n-hexane layer after centrifugation, heat the glass dish to evaporate the n-hexane, then weigh it until constant weight, and calculate the total oil content in the fermentation liquid.

[0077] The DHA content in the extracted oil was determined by gas chromatography.

[0078] The total fat content and DHA content in the fat of each group are shown in Table 1:

[0079] Table 1 Total fat content and DHA content in fat in each group

[0080]

[0081]

[0082] As can be seen from Table 1, when waste algae liquid distillate is used instead of pure water to prepare the fermentation medium for Schizochytrium fermentation culture, the total oil content, DHA content in the oil and DHA yield gradually increase with the increase of the proportion of distillate. Finally, when 100% distillate is used, the DHA yield is 13.52 g / L, which is 2.9 g / L higher than that of the pure water control group.

[0083] The test results showed that the distillate contained 0.11 mg / L malic acid and 0.1 mg / L citric acid. Schizochytrium increases the production of DHA by regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) during the lipid accumulation stage, and NADPH is controlled by the activity of malic enzyme (ME), which is the only source of NADPH for fatty acid synthase. Malic acid participates in the citric acid / malic acid and transhydrogenase cycles, which can induce structural changes in malic enzyme from dimers to more active tetramers or octamers, enhance the NADPH generation reaction, and provide more precursors for the synthesis of DHA.

[0084] At the same time, it can be seen from Table 1 that the DHA production of the 95% ingredient group and the 100% ingredient group both reached 13g / L, but the DHA production of the 85% ingredient group did not reach 10g / L. This shows that the use of ingredients can be reduced to a certain small proportion when using waste algae liquid distillate to prepare the culture medium.

[0085] Table 2 shows the test results of some items of the waste algae liquid distillate, among which ammonia nitrogen can be used by Schizochytrium for biomass accumulation; chemical oxygen demand COD indicates the amount of reducing substances in water, and reducing substances in water include various organic matter, nitrite, sulfide, ferrous salt, etc.; the total amount of all solutes in water is total dissolved solids TDS. Generally speaking, the higher the conductivity, the higher the TDS, and the higher the salt content in the water. From the results of the embodiment, these components reflected by the distillate COD and TDS can be taken up by Schizochytrium for biomass accumulation, and replace the components of the culture medium in a certain small proportion.

[0086] Table 2 Test results of some items of waste algae liquid distillate

[0087]

[0088] In summary, the present invention is feasible to use the distillate of waste algae liquid of Schizochytrium to prepare the fermentation medium for culturing Schizochytrium to ferment and produce DHA. Schizochytrium can utilize the components in the distillate to accumulate biomass and synthesize DHA. Compared with the pure water group, the DHA yield is significantly improved, and the use of culture medium ingredients can be reduced to a certain small proportion. A 50L fermenter is used for fermentation, and the biomass reaches 98g / L after 120h of culture, and the DHA yield reaches 32.5g / L.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A basic culture medium for Schizochytrium sp., characterized in that: It includes: The distillate of the waste algae liquid after oil extraction from Schizochytrium is distilled under reduced pressure.

2. The Schizochytrium basal culture medium according to claim 1, characterized in that The waste algae liquid after oil extraction from Schizochytrium truncatum is mixed with the distillate obtained by vacuum distillation and pure water in a volume ratio of 2-10:8-0.

3. A method for culturing Schizochytrium to produce DHA, characterized in that: The following steps are involved: S1. Preparation of culture medium Use pure water to prepare the seed culture medium; Supplementing the basic culture medium of Schizochytrium described in any one of claims 1-2 with a carbon source, a nitrogen source and an inorganic salt to prepare a fermentation medium; S2. Fermentation culture of Schizochytrium Primary seed culture: inoculate the frozen glycerol solution of Schizochytrium into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30° C. for 30-48 hours; Secondary seed culture: inoculate the primary seed solution into the seed culture medium, with the volume percentage of the inoculation amount being 2-5%, and culture at 150-200 rpm and 25-30° C. for 24-48 hours; Fermentation culture: inoculate the secondary seed liquid into the fermentation medium at a volume percentage of 2-5%, and culture at 150-200rpm and 25-30°C for 48-72h. During the fermentation process, add glucose feed solution to control the glucose concentration at 0-60g / L. When the glucose is exhausted, the fermentation ends. S3, extracting oil from the fermentation liquid.

4. The method for culturing Schizochytrium to produce DHA according to claim 3, characterized in that: In S1, the carbon source is glucose, the nitrogen source includes one or more of yeast powder, peptone, corn steep liquor, sodium glutamate and glycine, and the inorganic salt includes phosphate, magnesium salt, calcium salt, sodium salt and potassium salt.

5. The method for culturing Schizochytrium to produce DHA according to claim 4 or 5, characterized in that: In S1, The seed culture medium includes 30-40 g / L glucose, 5-8 g / L yeast powder, 1-3 g / L magnesium sulfate, 1-3 g / L potassium dihydrogen phosphate, 1-3 g / L calcium chloride, 5-10 g / L sodium chloride, 2-5 g / L sodium bicarbonate, 0.1-0.5 g / L potassium chloride, and natural pH; The fermentation medium includes 30-50 g / L of glucose, 5-8 g / L of yeast powder, 5-10 g / L of sodium glutamate, 1-3 g / L of magnesium sulfate, 1-3 g / L of potassium dihydrogen phosphate, 1-3 g / L of calcium chloride, 5-10 g / L of sodium chloride, 2-5 g / L of sodium bicarbonate, 0.1-0.5 g / L of potassium chloride, and natural pH.

6. The method for culturing Schizochytrium to produce DHA according to claim 3, characterized in that: The seed culture medium and the Schizochytrium fermentation culture medium are sterilized at 115-121° C. for 15-30 minutes and then cooled to room temperature before use.

7. The method for culturing Schizochytrium to produce DHA according to claim 4, characterized in that: The concentration of the glucose feed solution is 300-500 g / L, sterilized at 115-121° C. for 15-30 min, and then cooled to room temperature before use.

8. The method for culturing Schizochytrium to produce DHA according to claim 4, characterized in that: In step S3, after the fermentation is completed, the algal bodies of Schizochytrium in the fermentation broth are broken by enzymatic method, n-hexane is added and mixed, and then centrifuged, the oil layer is heated, and the n-hexane is volatilized and weighed until constant weight, the total oil content in the fermentation broth is calculated, and the content of docosahexaenoic acid in the oil is determined by gas chromatography.

9. The method for culturing Schizochytrium to produce DHA according to claim 8, characterized in that: Step S3 includes: using one or more enzymes selected from alkaline protease, cellulase and pectinase to break the algae wall in the fermentation liquid; the addition amount is 0.5-2g / L; adjusting the pH of the fermentation liquid to 8.0-11.0 with a pH regulator, stirring at a speed of 50-150rpm and a temperature of 40-80°C, and breaking the wall for 2-4h; during the centrifugation process, the speed is 3000-6000rpm and the time is 3-5min.

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