Method for producing polyunsaturated fatty acid
By adding rapeseed protein powder to the fermentation medium to activate Schizochytrium seeds, the problem of low EPA and DHA production by Schizochytrium was solved, resulting in a significant increase in EPA and DHA production to meet industrial needs.
Patent Information
- Application Number
- CN202511144149.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-12-19
AI Technical Summary
In existing technologies, the content and yield of polyunsaturated fatty acids EPA and DHA synthesized by Schizochytrium are low, making it difficult to meet the needs of industrial production.
By adding rapeseed protein powder as a nitrogen source to the fermentation medium, combined with appropriate culture temperature and rotation speed, the seeds of Schizochytrium were activated, promoting their high metabolic activity. The polyphenols in the rapeseed protein powder were used to promote the synthesis of EPA and DHA.
It significantly increased the production and proportion of EPA and DHA, meeting the needs of industrial production.
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Figure CN121160809A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present disclosure belongs to the technical field of biological fermentation, and particularly relates to a method for producing polyunsaturated fatty acids. BACKGROUND
[0002] Eicosapentaenoic acid (EPA) in polyunsaturated fatty acids is commonly known as a vascular scavenger, characterized by a 20-carbon chain and 5 double bonds (20:5n-3), and cannot be synthesized by the human body itself and needs to be obtained through diet or supplements. Studies have shown that EPA plays a crucial role in preventing and treating cardiovascular diseases, schizophrenia, maintaining brain development, etc. Docosahexaenoic acid (DHA) in polyunsaturated fatty acids is commonly known as brain gold, and is widely distributed in the retina, cerebral gray matter, myocardium, nervous system and breast milk of the human body.
[0003] In related technologies, Schizochytrium sp. is a kind of heterotrophic marine microorganism with important industrial application value, which has attracted much attention due to its fast growth rate, low cultivation cost and high efficiency in accumulating polyunsaturated fatty acids (PUFA). As an important DHA-producing strain, Schizochytrium sp. has been widely studied. Studies have shown that in addition to mainly synthesizing DHA, the strain also has the potential to synthesize EPA and other omega-3 series PUFAs. When synthesizing DHA and EPA, the activated Schizochytrium sp. is placed in a fermentation medium for cultivation, and then the culture solution after cultivation is extracted to obtain DHA and EPA. The fermentation medium contains glucose, yeast extract and the like.
[0004] However, when producing polyunsaturated fatty acids (including DHA and EPA) according to the above method, the content and yield of DHA and EPA are generally low, which is difficult to meet the needs of industrial production. SUMMARY
[0005] The embodiments of the present disclosure provide a method for producing polyunsaturated fatty acids, which can significantly improve the yield of EPA and DHA. The technical solution is as follows:
[0006] The embodiments of the present disclosure provide a method for producing polyunsaturated fatty acids, which comprises: activating Schizochytrium sp. in a seed culture medium to obtain a Schizochytrium sp. seed culture solution; inoculating the Schizochytrium sp. seed culture solution into a fermentation medium for cultivation, wherein the fermentation medium comprises rapeseed protein powder; and extracting the culture solution in the fermentation medium after cultivation to obtain EPA.
[0007] In a further implementation form of the present disclosure, the protein content in the rapeseed protein powder is not less than 50%, and the concentration of the rapeseed protein powder is 1.0-5.0 g / L.
[0008] In a further implementation form of the present disclosure, the rapeseed protein powder is obtained through purification after coarse grinding of rapeseed meal.
[0009] In a further implementation form of the present disclosure, the fermentation medium further comprises sodium glutamate, yeast extract, anhydrous glucose and sea salt.
[0010] In a further implementation form of the present disclosure, the seed medium comprises sodium glutamate, yeast extract, anhydrous glucose and sea salt.
[0011] In a further implementation form of the present disclosure, the activating the schizochytrium in the seed medium to obtain a schizochytrium seed culture solution comprises: inoculating the schizochytrium into the seed medium, the culture temperature in the seed medium is 25-32℃, the rotation speed is 70-300 rpm, and the culture time is not less than 48 hours.
[0012] In a further implementation form of the present disclosure, the inoculating the schizochytrium seed culture solution into the fermentation medium for culture comprises: inoculating the schizochytrium seed culture solution into the fermentation medium, the culture temperature in the fermentation medium is 25-32℃, the rotation speed is 200-350 rpm, and the culture time is not less than 72 hours.
[0013] In a further implementation form of the present disclosure, the extracting the culture solution in the fermentation medium after culture to obtain EPA and DHA comprises: based on the culture solution in the fermentation medium after culture, collecting freeze-dried bacteria powder or bacteria solution; and extracting EPA and DHA in the freeze-dried bacteria powder or bacteria solution.
[0014] In a further implementation form of the present disclosure, the collecting freeze-dried bacteria powder or bacteria solution based on the culture solution in the fermentation medium after culture comprises: centrifuging the collected culture solution, discarding the supernatant, repeatedly washing and freeze-drying to obtain the freeze-dried bacteria powder.
[0015] In a further implementation form of the present disclosure, the collecting bacteria solution based on the culture solution in the fermentation medium after culture comprises: centrifuging the collected culture solution, discarding the supernatant, repeatedly washing to obtain the bacteria solution.
[0016] The technical scheme provided by the embodiments of the present disclosure has the following beneficial effects:
[0017] When producing polyunsaturated fatty acids, especially EPA and DHA, by the method provided by the embodiments of the present disclosure, since the method first activates the Schizochytrium in the seed culture medium to obtain the Schizochytrium seed culture solution, the Schizochytrium strain in a dormant or preserved state can be restored to high metabolic activity, thereby improving the product yield in the subsequent fermentation process. Then, the method inoculates the Schizochytrium seed culture solution into the fermentation medium for culture, so that the activated Schizochytrium can be fermented in the fermentation medium to provide nutrition and environment for the growth and product synthesis of the Schizochytrium. Moreover, since the fermentation medium includes rapeseed protein powder, which can be directly used by the Schizochytrium as a nitrogen source for cell growth and metabolic product synthesis. In addition to providing a nitrogen source, the rapeseed protein powder also contains a small amount of polyphenols such as meso-zeatin, which has an antioxidant effect, which is conducive to promoting the accumulation of EPA and DHA by the Schizochytrium. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor.
[0019] Figure 1 is a flowchart of a method for producing polyunsaturated fatty acids provided by the embodiments of the present disclosure;
[0020] Figure 2 is a flowchart of another method for producing polyunsaturated fatty acids provided by the embodiments of the present disclosure;
[0021] Figure 3 is a difference control chart of the biomass, total fat, EPA and DHA content in the three test groups;
[0022] Figure 4 is a difference control chart of the fatty acid composition in the three test groups. DETAILED DESCRIPTION
[0023] In order to make the objects, technical solutions and advantages of the present disclosure clearer, the embodiments of the present disclosure will be further described in detail below with reference to the drawings.
[0024] EPA is commonly known as vascular scavenger, characterized by a 20-carbon chain and 5 double bonds (20:5n-3). The human body cannot synthesize itself and needs to obtain it through diet or supplements. Studies have shown that EPA plays a crucial role in preventing and treating cardiovascular diseases, schizophrenia, maintaining brain development, etc.
[0025] DHA is commonly known as brain gold, and its molecular formula is C22 H 32 O2, with a relative molecular mass of 328.5 g / mol. DHA is widely distributed in the human body, including the retina, brain gray matter, myocardium, nervous system, and breast milk. For infants and young children, consuming formula fortified with DHA results in improved intelligence and vision. For adolescents, DHA supplementation helps enhance learning and memory, protects vision, and strengthens brain function. For the elderly, adequate EPA intake helps maintain cardiovascular health, regulates lipid composition, lowers cholesterol and triglyceride levels, enhances blood flow, and effectively prevents thrombosis.
[0026] Currently, EPA and DHA are mainly produced through two pathways: extraction from marine fish and microbial fermentation. Deep-sea fish oil is widely used due to its high content of omega-3 polyunsaturated fatty acids (such as EPA and DHA). However, global overfishing has led to the decline of fishery resources, coupled with marine environmental pollution (such as the accumulation of heavy metals and organic pollutants), resulting in a continuous decline in fish oil production and severely impacting the sustainable supply of EPA. Although some plants (such as flaxseed and walnuts) contain alpha-linolenic acid (ALA), the conversion of ALA to EPA and DHA in the human body is extremely inefficient, making plant sources insufficient to meet demand. Microbial fermentation methods (such as the synthesis of EPA using microalgae, bacteria, and fungi) have become a research hotspot due to their high controllability and environmental friendliness. Some strains (such as Schizochytrium) have shown industrial production potential and are expected to achieve commercial applications in the future.
[0027] Schizochytrium sp., belonging to the order Thraustochytriales, is a unique group of marine heterotrophic microorganisms. It is a group of heterotrophic marine microorganisms with significant industrial application value, attracting considerable attention due to its rapid growth rate and efficient accumulation of polyunsaturated fatty acids (PUFAs). As an important producer of docosahexaenoic acid (DHA), Schizochytrium has been extensively studied. Research indicates that in addition to primarily synthesizing DHA, this strain also possesses the potential to synthesize omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). However, the content and yield of its naturally synthesized EPA are generally low, making it difficult to meet the demands of industrial production.
[0028] Therefore, this disclosure proposes a method to significantly improve the synthesis efficiency of EPA and DHA in Schizochytrium by adding rapeseed protein powder exogenously.
[0029] This disclosure provides a method for producing polyunsaturated fatty acids, such as... Figure 1 As shown, the method includes:
[0030] S101: Schizochytrium is activated in a seed culture medium to obtain a Schizochytrium seed culture solution.
[0031] S102: The Schizochytrium seed culture solution is inoculated into a fermentation culture medium for culture, and the fermentation culture medium includes rapeseed protein powder.
[0032] S103: The culture solution in the fermented culture medium after culture is extracted to obtain EPA and DHA.
[0033] When the method provided by the embodiments of the present disclosure is used to produce polyunsaturated fatty acids (EPA and DHA), since the method first activates Schizochytrium in a seed culture medium to obtain a Schizochytrium seed culture solution, the Schizochytrium strain in a dormant or preserved state can be restored to high metabolic activity, thereby improving the product yield in the subsequent fermentation process. Then, the method inoculates the Schizochytrium seed culture solution into a fermentation culture medium for culture, so that the activated Schizochytrium can be fermented in the fermentation culture medium to provide nutrients and environment for the growth and product synthesis of Schizochytrium. Moreover, since the fermentation culture medium includes rapeseed protein powder, which can be directly used by Schizochytrium as a nitrogen source for cell growth and metabolic product synthesis. In addition to providing a nitrogen source, the rapeseed protein powder also contains a small amount of polyphenols such as mesonine, which have antioxidant effects, and is conducive to promoting the accumulation of EPA and DHA by Schizochytrium.
[0034] On the other hand, the embodiments of the present disclosure also provide another method for producing polyunsaturated fatty acids, as shown in Figure 2 The method comprises:
[0035] S201: Schizochytrium is activated in a seed culture medium to obtain a Schizochytrium seed culture solution.
[0036] The seed culture medium is a shake flask seed culture medium.
[0037] In the embodiments of the present disclosure, the seed culture medium includes sodium glutamate, yeast extract, anhydrous glucose, and sea salt.
[0038] The sodium glutamate provides easily absorbed glutamic acid (amino acid form) for Schizochytrium, which directly participates in cell metabolism. The yeast extract serves as a complex nutrient source, which can provide nitrogen source, growth factors (such as B vitamins B1 and B12), and nucleotides to promote RNA / DNA synthesis and accelerate bacterial proliferation. Anhydrous glucose serves as adenosine triphosphate (ATP) to provide energy to support bacterial growth and maintain metabolism. Sea salt is used to simulate the marine environment and maintain osmotic pressure and ion balance. The sea salt is artificial sea salt.
[0039] In this embodiment of the disclosure, the artificial sea salt includes not only NaCl, but also a variety of mineral elements (such as magnesium, calcium, potassium, etc.), other trace elements (such as Fe, Zn, Cu, Mn, etc.) and vitamins (such as VB1, VB6, VB12, etc.).
[0040] Optionally, the seed culture medium contains 20-50 g / L monosodium glutamate, 5-10 g / L yeast extract, 20-100 g / L anhydrous glucose, and 15-30 g / L sea salt.
[0041] The concentrations of trace elements and minerals can be 1-5 mol / L, and the concentrations of vitamins (VB1, VB6, VB12) can be 0.0001-0.3 mol / L. In this embodiment, the concentrations of vitamins (VB1, VB6, VB12) can be 0.0001-0.3 mol / L.
[0042] This allows for the provision of sufficient carbon and nitrogen sources and nutrients for Schizochytrium by setting appropriate concentrations of monosodium glutamate, yeast extract, anhydrous glucose, and sea salt, thus promoting its growth.
[0043] For example, the monosodium glutamate (MSG) in the seed culture medium can be 30 g / L (i.e., 15 g of MSG added to a 500 mL shake flask seed culture medium), the yeast extract can be 6 g / L (i.e., 3 g of yeast extract added to a 500 mL shake flask seed culture medium), the anhydrous glucose can be 50 g / L (i.e., 25 g of anhydrous glucose added to a 500 mL shake flask seed culture medium), and the artificial sea salt can be 20 g / L (i.e., 10 g of artificial sea salt added to a 500 mL shake flask seed culture medium).
[0044] Optionally, step S201 is implemented in the following way:
[0045] The fissorinomycetes were inoculated into a seed culture medium at a temperature of 25-32°C. The seed culture medium was placed in a shaker at a speed of 70-300 rpm and cultured for at least 48 hours.
[0046] Maintaining the temperature of the seed culture medium at 25-32℃ can prevent the enzyme activity from decreasing and growth from being too low, while also preventing the protein from denaturing due to excessively high temperatures, which could lead to excessive fluidity of the Schizochytrium cell membrane and trigger a heat shock response.
[0047] At the same time, maintaining the rotation speed of the seed culture medium at 70-300 rpm can prevent insufficient dissolved oxygen in the seed culture medium due to low rotation speed, which can lead to hypoxia and stress, and can also prevent damage to the cell membrane due to excessive rotation speed.
[0048] In this embodiment, the step of activating Schizochytrium by inoculating it into the seed culture medium is as follows: Schizochytrium is inoculated into the seed culture medium at an inoculation amount of 0.5-10% (v / v) (for example, the inoculation amount is 2% of the volume of the seed culture medium), the culture temperature is 28°C, the preferred rotation speed is 90-200 (for example, 90) rpm, and the culture is carried out in a shake flask for 48 hours.
[0049] S202: Inoculate the Schizochytrium seed culture medium into the fermentation medium for cultivation.
[0050] The fermentation medium includes rapeseed protein powder.
[0051] Because rapeseed protein powder is rich in amino acids (such as glutamic acid and aspartic acid), it can be directly utilized by Schizochytrium when added to the fermentation medium as a nitrogen source for synthesizing cellular substances and metabolites. Nitrogen is a key element for the growth and lipid synthesis of Schizochytrium. Therefore, sufficient nitrogen source can promote the rapid proliferation of Schizochytrium and lay the foundation for the accumulation of EPA and DHA in Schizochytrium.
[0052] The rapeseed protein powder contains no less than 50% protein. The concentration of the rapeseed protein powder is 1.0-5.0 g / L. For example, the protein content of the rapeseed protein powder is preferably above 80%.
[0053] Optionally, the rapeseed protein powder mentioned above can be obtained by purifying rapeseed meal after coarse grinding. For example, rapeseed meal can be pre-treated by coarse grinding to obtain rapeseed protein powder. The coarsely ground rapeseed meal is dispersed in a solvent (100-500 mmol salt solution, the salt of which is selected from one or more combinations of CaCl2 and MgCl2) and then sieved. After rinsing with the solvent and sieving again, rapeseed protein crude extract and rapeseed dietary fiber can be obtained simultaneously. The rapeseed protein crude extract can then be further processed to obtain rapeseed protein powder.
[0054] In this embodiment, the fermentation medium is a shake flask fermentation medium.
[0055] In this embodiment of the disclosure, the fermentation medium further includes monosodium glutamate, yeast extract, anhydrous glucose, and sea salt.
[0056] Similarly, monosodium glutamate (MSG) provides Schizochytrium with easily absorbed glutamate (in amino acid form), which directly participates in cell metabolism. Yeast extract, as a complex nutrient source, provides both nitrogen and growth factors (such as B vitamins B1 and B12) as well as nucleotides to promote RNA / DNA synthesis and accelerate cell proliferation. Anhydrous glucose serves as ATP to support cell growth and maintain metabolism. Artificial sea salt is used to simulate the marine environment and maintain osmotic pressure and ion balance. The sea salt used can be artificial.
[0057] In this embodiment, the artificial sea salt added to the fermentation culture medium is the same ingredient as the artificial sea salt added to the seed culture medium. Further details will not be provided here.
[0058] Optionally, the fermentation medium may contain rapeseed protein powder (protein content not less than 50%) at a concentration of 1.0-3.0 g / L, monosodium glutamate at 20-50 g / L, yeast extract at 6-12 g / L, anhydrous glucose at 40-60 g / L, and an appropriate amount (e.g., 15-30 g / L) of artificial sea salt. Trace elements and minerals may be present at concentrations of 1-5 mol / L, and vitamins (VB1, VB6, VB12) may be present at concentrations of 0.0001-0.3 mol / L.
[0059] In this embodiment, the vitamins (VB1, VB6, VB12) can all be 0.0001-0.3 mol / L.
[0060] This allows for the provision of sufficient carbon and nitrogen sources and nutrients for Schizochytrium by setting appropriate concentrations of monosodium glutamate, yeast extract, anhydrous glucose, and sea salt, thus promoting its growth.
[0061] For example, the sodium glutamate in the fermentation medium can be 30 g / L (i.e., 15 g of sodium glutamate added in 500 mL shake flask fermentation medium), the yeast extract can be 9 g / L (i.e., 4.5 g of yeast extract added in 500 mL shake flask fermentation medium), the anhydrous glucose can be 50 g / L (i.e., 25 g of anhydrous glucose added in 500 mL shake flask fermentation medium), the artificial sea salt can be 20 g / L (i.e., 10 g of artificial sea salt added in 500 mL shake flask fermentation medium (where VB1, VB6, and VB12 can all be 0.0001-0.3 mol / L), and the rapeseed protein powder (protein content of 50% or more) can be 1.125 or 2.250 g / L.
[0062] Optionally, step S202 is implemented in the following way:
[0063] Inoculate the Schizochytrium seed culture medium into the fermentation medium. The fermentation medium should be cultured at a temperature of 25-32℃ and a rotation speed of 200-350 rpm for at least 72 hours.
[0064] Maintaining the temperature of the seed culture medium at 25-32℃ can prevent enzyme activity from decreasing and growth from being delayed due to excessively low temperatures. It can also prevent protein denaturation caused by excessively high temperatures, which could lead to excessively fluid cell membranes of Schizochytrium and trigger a heat shock response.
[0065] At the same time, keeping the rotation speed of the seed culture medium at 200-350 rpm can accelerate the oxygen dissolution rate and prevent damage to the cell membrane due to excessive rotation speed.
[0066] In this embodiment of the disclosure, the step of activating Schizochytrium by inoculating it into the seed culture medium is as follows: Schizochytrium seed culture solution is inoculated into the fermentation culture medium at an inoculation amount of 3-5% (v / v) (that is, the inoculation amount is 3-5% of the volume of the seed culture medium), the culture temperature in the fermentation culture medium is 28°C, the rotation speed is 300 rpm, and the culture is carried out in shake flasks for 72 hours.
[0067] S203: Based on the culture medium after cultivation, freeze-dried bacterial powder or bacterial solution is collected.
[0068] Optionally, step S203 can be implemented in the following manner:
[0069] The collected culture medium was centrifuged, the supernatant was discarded, and the mixture was washed repeatedly and then freeze-dried to obtain freeze-dried bacterial powder.
[0070] Alternatively, the collected culture medium can be centrifuged, the supernatant discarded, and the mixture washed repeatedly to obtain the bacterial culture.
[0071] S204: Extract EPA and DHA from freeze-dried bacterial powder or bacterial solution.
[0072] Optionally, step S204 can be implemented as follows:
[0073] When the collected material is freeze-dried bacterial powder, it is methylated, and then sulfuric acid methanol is added for vortex mixing. After mixing, it is placed in a metal bath at 90℃±10℃ for 1.5-2 hours and then cooled. Then, hexane and NaCl are added for vortex mixing, and after centrifugation and purification, EPA and DHA are obtained.
[0074] For example, during methyl esterification, 2-10 mg of lyophilized bacterial powder can be placed in a 4 mL bottle, along with 20-100 μL of 1 mg / mL C21:0 internal standard and 500-800 μL of 95% sulfuric acid in methanol. After vortexing and mixing, the mixture can be placed in a metal bath at 90°C or higher for at least 90 minutes, during which time the airtightness is checked.
[0075] After cooling, add 300 μL-500 μL of n-hexane and 500-800 μL of 0.9% NaCl, vortex to mix, and centrifuge at 3500 rpm or higher for at least 10 min. Then, transfer 250 μL of the upper organic phase to a centrifuge tube and centrifuge at 12000 rpm or higher for at least 5 min to purify it. Finally, transfer 200 μL of the purified product to a gas chromatography (GC) sample vial.
[0076] The fatty acid composition of the product was then analyzed using a GC analyzer, and EPA and DHA were extracted.
[0077] In addition, to quantify the yield of EPA and DHA, the freeze-dried bacterial powder can be weighed during EPA and DHA extraction. This is because the EPA or DHA yield can be obtained by dividing the total extracted EPA or DHA by the weight of the freeze-dried bacterial powder.
[0078] When the collected bacterial solution is a bacterial culture, a methyl esterification reaction is performed. First, 2-5 mL of the bacterial culture is placed in a centrifuge tube and centrifuged, removing the supernatant. Then, 300-600 μL of sodium methanol is added and mixed well, followed by 30-60 μL of sulfuric acid and 500-800 μL of n-hexane. The mixture is centrifuged at 4000 rpm for 5 min, and the supernatant is collected. The fatty acid composition of the product is analyzed using a GC analyzer, and EPA and DHA are extracted.
[0079] To further illustrate how the above method of producing EPA and DHA using Schizochytrium can significantly increase the yield of EPA and DHA, the following specific examples will be provided.
[0080] First, the Schizochytrium fungi were inoculated into the seed culture medium at an inoculation rate of 0.5-8% (v / v) to activate them. The seed culture medium was then cultured at a temperature of 28℃ and a rotation speed of 90 rpm for 48 hours in a shake flask to bring the seed culture medium to the optimal state for continuous fermentation, thus obtaining the Schizochytrium fungi seed culture solution.
[0081] The seed culture medium includes: 30 g / L sodium glutamate, 6 g / L yeast extract, 50 g / L anhydrous glucose, and 20 g / L artificial sea salt.
[0082] Then, the *Schizochytrium* seed culture was inoculated into the fermentation medium of the following experimental groups at inoculation rates of 0.5-8% (v / v). The fermentation medium was incubated at 28°C, with a rotation speed of 300 rpm, and in shake flasks for 72 hours.
[0083] The first experimental group served as a blank control group using basal fermentation medium; the second experimental group used 1.125 g / L rapeseed protein powder to replace the yeast extract in the fermentation medium with an equivalent amount of nitrogen; and in the third experimental group, 2.250 g / L rapeseed protein powder was used to replace the yeast extract in the fermentation medium, and this dosage provided twice the total nitrogen content of the yeast extract.
[0084] Table 1: Content of different nutrients in the fermentation medium of each experimental group
[0085]
[0086] It should be noted that, in this embodiment, to reduce costs, when conducting experiments or production, the addition of yeast extract can be omitted while adding rapeseed protein powder to the fermentation medium. This is because the main function of yeast extract is to provide an organic nitrogen source for microorganisms, and rapeseed protein powder can achieve this function perfectly. As shown in the table above, in the second and third experimental groups, after adding different amounts of rapeseed protein powder to the fermentation medium, yeast extract was no longer required.
[0087] Next, the biomass of each fermentation medium was measured.
[0088] When collecting the bacteria, add 10 mL of bacterial culture to a pre-weighed 10 mL centrifuge tube and centrifuge at 4000 rpm for 10 min. Discard the supernatant in the centrifuge tube. Then wash the bacteria twice with deionized water.
[0089] After washing, the bacteria were freeze-dried for 3 days and then weighed.
[0090] The biomass can be obtained by calculating the difference between the final and initial weight of the centrifuge tube and dividing it by the volume of the collected bacterial culture. The biomass of the three experimental groups can be found in [reference needed]. Figure 3 In this embodiment, biomass is fed back using dry cell weight (DCW).
[0091] Alternatively, the collected bacterial solution can be directly subjected to a methyl esterification reaction to extract EPA and DHA.
[0092] Next, fatty acid extraction and gas chromatography detection were performed.
[0093] First, the lyophilized bacterial powder was methylated: 2-10 mg of lyophilized bacterial powder was placed in a 4 mL bottle, along with 20-100 μL of 1 mg / mL LC21:0 internal standard and 500-800 μL of 95% sulfuric acid in methanol. The mixture was vortexed and then incubated in a 90°C metal bath for 90 min, checking for airtightness during the incubation. After cooling, 300 μL of n-hexane and 500 μL of 0.9% NaCl were added, and the mixture was vortexed and centrifuged at 3500 rpm for 10 min to separate the fatty acids. 250 μL of the upper organic phase was transferred to a centrifuge tube and purified by centrifugation at 12000 rpm for 5 min. Another 200 μL of this purified phase was then transferred to a GC sample vial. The fatty acid composition of the product was determined using a GC analyzer.
[0094] Alternatively, wet bacterial methyl esterification: Centrifuge the bacterial culture, discard the supernatant, and add 300-600 μL of sodium ethanol. Then add 30-60 μL of sulfuric acid and 500-800 μL of n-hexane, centrifuge at 4000 rpm for 5 min, and collect the supernatant into a GC bottle. Use a GC analyzer to determine the fatty acid composition of the product.
[0095] The instrument used was a gas chromatograph. The chromatographic column was FFAP, DB-wax, DB-FastFAME, or a similar column.
[0096] Injection settings: injection volume 2 μL, injection temperature 260℃, split ratio 10-50:1;
[0097] Carrier gas: Nitrogen, 60 mL / min;
[0098] Detector temperature: 250℃;
[0099] Column temperature control: Initial temperature 250℃, maintain for 1 min; then increase the temperature to 300℃ at a rate of 3℃ / min and maintain for 10 min.
[0100] The products of each experimental group were obtained by following the above steps, as shown in Table 2 and Table 3 below.
[0101] Table 2: Biomass of Schizochytrium, total oil, EPA and DHA content of rapeseed protein powder at different concentrations
[0102]
[0103] Table 3. Differences in fatty acid composition of Schizochytrium subsp. *Schizochytrium* at different concentrations of rapeseed protein powder.
[0104]
[0105] In the above Cx:y, x represents the total number of carbon atoms in the fatty acid carbon chain, and y represents the total number of double bonds in the carbon chain. Specifically, C14:0 represents myristic acid in saturated fatty acids. C15:0 represents pentadecanoic acid in saturated fatty acids. C16:0 represents palmitic acid in saturated fatty acids. C22:5n6 represents DPAn6 (n6 type eicosapentaenoic acid).
[0106] The data in Table 2 were plotted to obtain... Figure 3 The data in Table 3 were used to draw the graph. Figure 4 .
[0107] See Figure 3 , Figure 3 The horizontal axis in the graph represents three different experimental groups: the blank experimental control group, the second experimental group, and the third experimental group. From... Figure 3 Figure 3As can be seen, after 72 hours of fermentation, the *Schizochytridactylum* in the blank control group had a biomass (DCW) of 22.6 g / L, a total fatty acid (TFA) content of 2.03 g / L, an EPA content of 70 mg / L (EPA accounts for 3.6% of TFA), and a DHA content of 1.0 g / L (DHA accounts for 50.7% of TFA). In contrast, after 72 hours of fermentation, the *Schizochytridactylum* in the second experimental group had a biomass (DCW) of 25.6 g / L, a TFA content of 2.4 g / L, an EPA content of 180 mg / L (EPA content increased by 157.1% compared to the blank group, EPA accounts for 7.8% of TFA), and a DHA content of 1.2 g / L (DHA accounts for 49.1% of TFA). After 72 hours of fermentation, the biomass of *Schizochytridica* in the third experimental group was 31.0 g / L, TFA was 2.96 g / L, and EPA content was 210 mg / L, which was 200% higher than that of the control group. This indicates that the addition of rapeseed protein powder is beneficial to the growth of *Schizochytridica* and the accumulation of oils, especially EPA.
[0108] Furthermore, the addition of rapeseed protein powder can increase the biomass of Schizochytrium and the TFA produced, while also increasing the yield of EPA and its proportion in TFA. Compared with the control group, the biomass increased by 13.3% and 37.2% with the addition of 1.125 g / L and 2.250 g / L rapeseed protein powder, respectively; the TFA increased by 15.4% and 193.6% with the addition of 1.125 g / L and 2.250 g / L rapeseed protein powder, respectively; the EPA content increased by 157.1% and 200% with the addition of 1.125 g / L and 2.250 g / L rapeseed protein powder, respectively; and the DHA content increased by 20% and 180%, respectively. It is evident that the addition of rapeseed protein powder is beneficial to the growth of Schizochytrium and the accumulation of oils, especially DHA and EPA.
[0109] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.
Claims
1. A method for producing polyunsaturated fatty acids, characterized in that, The method includes: Schizochytrium was activated in a seed culture medium to obtain Schizochytrium seed culture solution; The Schizochytrium seed culture solution was inoculated into a fermentation medium for cultivation, the fermentation medium including rapeseed protein powder; The culture broth from the fermentation medium after culturing was extracted to obtain polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid.
2. The method according to claim 1, characterized in that, The rapeseed protein powder has a protein content of not less than 50%, and the concentration of the rapeseed protein powder is 1.0-5.0 g / L.
3. The method according to claim 2, characterized in that, The rapeseed protein powder is obtained by purifying rapeseed meal after coarse grinding.
4. The method according to claim 2, characterized in that, The fermentation medium also includes monosodium glutamate, yeast extract, anhydrous glucose, and sea salt.
5. The method according to any one of claims 1-4, characterized in that, The seed culture medium includes monosodium glutamate, yeast extract, anhydrous glucose, and sea salt.
6. The method according to any one of claims 1-4, characterized in that, The process of activating Schizochytrium in a seed culture medium to obtain Schizochytrium seed culture solution includes: The fissorichytrium is inoculated into the seed culture medium, and the seed culture medium is cultured at a temperature of 25-32℃, a rotation speed of 70-300 rpm, and cultured for no less than 48 hours.
7. The method according to any one of claims 1-4, characterized in that, The step of inoculating the Schizochytrium seed culture medium into a fermentation medium for cultivation includes: The Schizochytrium seed culture solution is inoculated into the fermentation medium, and the fermentation medium is cultured at a temperature of 25-32℃, a rotation speed of 200-350 rpm, and cultured for no less than 72 hours.
8. The method according to any one of claims 1-4, characterized in that, The culture broth extracted from the fermentation medium after culturing yields polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), comprising: Based on the culture medium in the fermented culture medium after cultivation, freeze-dried bacterial powder or bacterial solution is collected. Extract the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid from the freeze-dried bacterial powder or the bacterial solution.
9. The method according to claim 8, characterized in that, The freeze-dried bacterial powder is collected based on the culture broth in the fermentation medium after cultivation, comprising: The collected culture medium was centrifuged, the supernatant was discarded, and the mixture was repeatedly washed and freeze-dried to obtain the freeze-dried bacterial powder.
10. The method according to claim 8, characterized in that, The lyophilized bacterial powder or bacterial solution is collected based on the culture broth in the fermentation medium after cultivation, including: The collected culture medium was centrifuged, the supernatant was discarded, and the mixture was washed repeatedly to obtain the bacterial culture.