Schizochytrium limacinum strain for producing high-content EPA (eicosapentaenoic acid) grease and application thereof

Abstract

The invention provides a schizochytrium limacinum strain Schizochytrium sp. ABBS-E11 with high EPA (Eicosapentaenoic Acid) content, and a preparation method of the schizochytrium limacinum strain. The strain is preserved in the China Center for Type Culture Collection, the preservation number of the strain is CCTCC NO: M 20251165, and the preservation date is May 23, 2025. The mutant strain is obtained through separation, mutagenesis and screening, and compared with schizochytrium limacinum in the prior art, the EPA content in a product is remarkably increased, high biomass and titer are kept, and the absolute yield of EPA is increased. The invention further provides a method for producing grease rich in EPA through fermentation of the schizochytrium limacinum mutant strain. According to the method, a standard fermentation process is adopted, no special medicine or reagent needs to be added, the safety performance is guaranteed, and the method has important significance on industrial application. Moreover, the grease rich in EPA prepared by adopting the strain also contains more than 35% of DHA and lower DPA, so that the grease not only has double nutritional values, but also is convenient to separate and purify, and has huge market value.

Description

Technical Field

[0001] The present invention belongs to the field of marine microbial technology and relates to the field of genetic engineering, and specifically relates to a Schizochytrium strain capable of producing high-content eicosapentaenoic acid (EPA) oil, a construction method and an application thereof. Background Art

[0002] Eicosapentaenoic acid (EPA) is an important ω-3 polyunsaturated fatty acid with significant anti-inflammatory, lipid-lowering, and cardiovascular health-promoting properties. Currently, the primary source of EPA is deep-sea fish oil. However, due to limited fish resources, environmental pollution, and the potential for heavy metal contamination during the fish oil extraction process, fish oil-derived EPA has certain limitations. Therefore, alternative microbial resources rich in EPA, such as microalgae and fungi, are needed to replace fish oil. Currently reported microalgae and fungi capable of producing EPA primarily come from the genera Nannochloropsis, Mortierella, Pythium, and Schizochytrium. Compared to other microalgae and fungi, Schizochytrium (also known as Schizochytrium) offers advantages such as rapid production, high intracellular lipid content, and a rich supply of polyunsaturated fatty acids (PUFAs). Consequently, Schizochytrium is currently widely used in the fermentation production of docosahexaenoic acid (DHA), with products found in infant formula, health foods, and specialty pharmaceuticals. Although the PUFA produced by Schizochytrium contains a large amount of DHA and can also synthesize a certain amount of EPA, the EPA production of the strains obtained by natural isolation is too low to be worth large-scale industrial production.

[0003] Based on the aforementioned technical background, researchers have conducted several studies on Schizochytrium to improve EPA production. Among them, the Schizochytrium mutant strains obtained through mutagenesis screening (non-genetic modification) had the highest EPA production. The strain obtained through ultraviolet mutagenesis technology and selective culture medium screening, which is patented in invention patent ZL202411073290.2, contained a high-EPA-producing Schizochytrium. The EPA content of this strain was as high as 10.11%, but due to the decrease in biomass, the titer was only 5.06g / L, which was difficult to meet the needs of industrial production. Among the genetically modified engineered strains, invention patent application CN202411387574.9 provides an engineered Schizochytrium strain that produces EPA through an extended desaturation pathway. The engineered strain had an oil content of 74.25g / L, an EPA content of 10.51%, and a titer of up to 7.80g / L. Although this strain achieves a high EPA content and titer, due to the introduction of exogenous genes through genetic engineering, especially genes from pathogens (such as Fusarium and Saprolegnia), there may be potential safety issues and it is difficult to be accepted by consumers.

[0004] In summary, existing Schizochytrium strains generally have low EPA content (mostly less than 10%). Although certain special methods can increase EPA content, these often lead to decreased biomass and titer (mostly no more than 6g / L), affecting their industrial application value. Other methods for increasing EPA content require the addition of chemical reagents unsuitable for food production, thus affecting the safety and application value of the product. Therefore, developing a Schizochytrium strain that can achieve high EPA content, maintain high biomass and titer, and meet food safety standards has important market and application prospects. Summary of the Invention

[0005] In response to the current state of EPA production by Schizochytrium strains, the present invention provides a high-EPA-content Schizochytrium sp. ABBS-E11 strain. This strain, obtained through isolation, mutagenesis, and screening, not only efficiently synthesizes EPA, increasing the EPA content in the product while significantly boosting the absolute EPA yield, but also meets food safety production standards, making it of great significance for industrial applications.

[0006] The technical solution of the present invention:

[0007] The present invention provides a Schizochytrium sp. mutant strain ABBS-E11 that efficiently produces EPA. The strain, deposited with the China Center for Type Culture Collection at Wuhan University in Wuchang District, Wuhan City, Hubei Province, is CCTCC No. M 20251165 and was deposited on May 23, 2025. The mutant strain was obtained by the inventors through isolation, mutagenesis, and screening. Compared with Schizochytrium sp. strains in the prior art, it not only significantly increases the EPA content in the product, but also maintains high biomass and titer, thereby increasing the absolute EPA yield.

[0008] A bacterial agent comprising the Schizochytrium mutant strain described above.

[0009] A method for producing EPA-rich oil comprises fermenting the aforementioned Schizochytrium mutant strain or a bacterial agent containing the Schizochytrium mutant strain to obtain EPA-rich oil.

[0010] The aforementioned Schizochytrium mutant strain is used in the production of EPA-rich oils. This strain is obtained through isolation, mutagenesis, and screening. Furthermore, the production of EPA-rich oils using this strain does not require the addition of any chemical reagents unsuitable for food production. Therefore, when used to produce EPA-rich oils, this strain not only achieves high yields but also ensures safety, making it of great significance for industrial application.

[0011] A method for producing EPA-rich oils and fats, employing the aforementioned Schizochytrium mutant strain or inoculum to ferment and produce the EPA-rich oils and fats. The fermentation process specifically comprises: high-density fermentation in a liquid culture medium, followed by sequential cell fragmentation and separation; the inoculum concentration of the Schizochytrium mutant strain in the liquid culture medium is 10-15%. The fermentation process is carried out under the following conditions: culturing at 22-30°C, a pH of 6-7, and a dissolved oxygen content of no less than 20% for 4-6 days. The liquid culture medium comprises: 60-120 g / L glucose, 3-20 g / L yeast extract, 3-20 g / L corn steep liquor, 2-8 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate, 1-4 g / L sodium citrate, 5-30 g / L sea crystals, 1-30 mg / L vitamin B1, 1-30 mg / L vitamin B6, 1-10 mg / L vitamin B12, and 1-10 mg / L biotin. The crushing step is enzymatic hydrolysis, and the separation step is centrifugation. The production method can not only efficiently synthesize EPA, but also maintain a high DHA content and a low DPA (docosapentaenoic acid) content, meeting the needs of industrial application.

[0012] Preferably, the fermentation step further includes a sugar supplementation operation to ensure that the glucose concentration is 2-20 g / L.

[0013] Beneficial effects of the present invention:

[0014] (1) The present invention provides a Schizochytrium mutant strain that efficiently produces EPA; the mutant strain was obtained by the inventors through isolation, mutagenesis, and screening. Compared with the Schizochytrium in the prior art, the mutant strain not only significantly increases the EPA content in the product (11.5%), but also maintains a high biomass and titer (9.2 g / L), thereby achieving an increase in the absolute production of EPA.

[0015] (2) In the process of using the strain to prepare EPA-rich oil, a standard fermentation process is adopted without the addition of special drugs or reagents; therefore, the strain is used to prepare EPA-rich oil, which not only has a high yield but also has guaranteed safety performance, which is of great significance for industrial application.

[0016] (3) The strain is used to prepare EPA-rich oil, which not only significantly improves the EPA content compared with the existing technology, but also contains more than 35% DHA, which has dual nutritional value; moreover, the DPA content in the oil is low (the DPA / DHA ratio is less than 10%), which facilitates the subsequent separation and purification of DHA and has huge market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attachment Figure 1 This is a microscopic morphological diagram of the Schizochytrium mutant strain of the present invention;

[0018] Attachment Figure 2 The fermentation stability results of the Schizochytrium ABBS-E11 strain after ten passages in Example 3 are shown;

[0019] Attachment Figure 3 This is a gas chromatogram of oil extraction and determination after batch fed-batch fermentation of Schizochytrium ABBS-E11 in a 5 L fermentor in Example 4. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the embodiments.

[0021] Example 1: Mutagenesis and screening of Schizochytrium cells with high EPA content

[0022] Schizochytrium was isolated from water samples collected from the mangrove region of Wenzhou, Zhejiang Province, using a pine pollen fishing method. The method involved mixing a 10 g / L pine pollen suspension with a seawater sample. After allowing the mixture to stand for 30 minutes, the floating pine pollen was collected and spread onto enrichment plates (containing 1 g / L glucose, 0.1 g / L yeast extract, 1 g / L gelatin hydrolysate, 0.1 g / L peptone, 15 g / L sea crystal, 12 g / L agar, pH 6.5, and 50 mg / L penicillin G and streptomycin sulfate). The cells were incubated at 25°C in the dark for 48 hours. A single colony was streaked and purified to obtain a wild-type Schizochytrium strain.

[0023] Strains identified as Schizochytrium by microscopic observation were collected and subjected to multiple rounds of "heavy ion irradiation mutagenesis - low temperature adaptive evolution - ARTP mutagenesis - low temperature adaptive evolution" and single-cell high-throughput screening. The specific steps for each round are as follows:

[0024] 1. Heavy ion irradiation mutagenesis: Take the bacteria in the logarithmic growth phase (OD600≈1.0), wash them with PBS buffer, and irradiate them with heavy ion beam. 12 C 6+ Irradiation mutagenesis: The ion beam energy is 80 MeV / u and the radiation intensity is 280 Gy. The survival rate of irradiation mutagenesis is controlled at 10-20%.

[0025] 2. Low-temperature adaptive evolution: The irradiated bacteria were inoculated into liquid culture medium (60 g / L glucose, 20 g / L yeast extract, 15 g / L peptone, 8 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 1 g / L sodium citrate, 5 g / L sea crystal, 1 mg / L vitamin B1, 30 mg / L vitamin B6, 10 mg / L biotin) and cultured at 5°C with shaking at 150 rpm for 10 generations, with each generation lasting 24 h.

[0026] 3. ARTP mutagenesis: After cold acclimation, treat the cells using an atmospheric pressure room temperature plasma (ARTP) mutagenizer (power 100W, helium flow rate 10L / min) for 60 seconds, spread onto solid culture medium, and incubate at 25°C for 48 hours. Analyze the survival rate of the cells after mutagenesis. The survival rate of the cells after mutagenesis should be controlled at 10-20%.

[0027] 4. Low-temperature adaptive evolution: The irradiated bacteria were inoculated into liquid culture medium (same as step 2) and cultured at 5°C with shaking at 150 rpm for 10 generations, each generation for 24 h.

[0028] 5. High-throughput screening: The bacterial solution after multiple rounds of mutagenesis and evolution was inoculated into a liquid culture medium (same as step 2), revived and cultured at 25°C for 12 hours, and then sorted by a single-cell Raman sorter, and the one in ten thousand strains with the highest PUFA content were screened and collected. The collected cells were spread on a solid culture medium (the liquid culture medium in step 2 was supplemented with 12 g / L agar) plate and cultured at 25°C for 2 days. The obtained single colonies were inoculated in 96-well plates for culture, and then the oil was extracted to determine the fatty acid composition. It was unexpectedly found that the EPA content in one of the strains reached 9%. After passage and purification, the strain was named ABBS-E11 and preserved in the China Center for Type Culture Collection at Wuhan University, Wuchang District, Wuhan City, Hubei Province. Its preservation number is CCTCC NO: M 20251165, and the preservation date is May 23, 2025.

[0029] The results were observed using a microscope. Figure 1 .like Figure 1 As shown, the ABBS-E11 strain obtained by the aforementioned screening has a spherical or ellipsoidal bacterial body with a diameter of 5-20 microns, and the cells mainly reproduce by division.

[0030] Example 2: Shake flask fermentation of Schizochytrium mutant bacteria

[0031] The ABBS-E11 strain screened in Example 1 was inoculated into a seed medium (30 g / L glucose, 10 g / L yeast extract, 5 g / L peptone, 20 g / L sea crystal, pH 6.0) and cultured at 25°C, 200 rpm, and shaken for 24 h. A 15% inoculum was then transferred to a fermentation medium (120 g / L glucose, 5 g / L yeast extract, 10 g / L sodium glutamate, 20 g / L sea crystal), with 50 mL of liquid added to each 250 mL shake flask, and cultured under the same conditions for 72 h.

[0032] After fermentation, the biomass (dry weight), residual sugar concentration and oil content were determined as follows:

[0033] (1) Biomass: The cells were collected by centrifugation (8000 rpm, 10 min) and freeze-dried for 24 h to a constant weight. The final biomass was 25 g / L.

[0034] (2) Oil extraction: After grinding the cells with liquid nitrogen, the cells were extracted with chloroform-methanol (2:1, v / v). The solvent was removed by rotary evaporation, and the oil yield was calculated by weighing. The result was 12 g / L. This calculation showed that the oil content was 48%.

[0035] (3) Fatty acid analysis: The extracted oil was methylated and then tested by GC-FID. The results showed that EPA accounted for 9.2%, DHA 38.5%, and DPA only 4.5%.

[0036] Normally, the DPA and DHA content of Schizochytrium is fixed, with a DHA / DPA ratio of about 3-5, and DPA accounting for 20-30% of PUFA. Although DPA is also a polyunsaturated fatty acid, its nutritional value has been less studied and it is usually not used as an effective nutrient. Therefore, when concentrating and preparing high-purity DHA or EPA in actual production, it is usually necessary to remove it. The present application found that the DHA / DPA ratio in the oil produced by the ABBS-E11 strain reached more than 7. Considering that the strain also produces high concentrations of EPA, the content of DPA in PUFA (i.e., DHA+EPA+DPA) is less than 10%, which achieves a significant reduction in DPA content compared to the prior art.

[0037] In summary, the oil produced by the ABBS-E11 strain described in the present application not only has high EPA and DHA content, but also has a low DPA content, which is very beneficial for the subsequent preparation of high-purity DHA or EPA.

[0038] Example 3: Verification of the stability of Schizochytrium mutants

[0039] This example verifies the stability of the Schizochytrium mutant strain ABBS-E11 obtained in Example 1. The specific operation is: the ABBS-E11 strain is passaged 10 times using the seed culture medium (same as in Example 2), and after each passage, it is cultured in the fermentation medium, and the oil is extracted to determine the fatty acid composition. The results are detailed in Figure 2 Among them, 1-10 are each generation, oil content is the percentage of oil in dry biomass, and EPA, DPA, and DHA are the percentages of these three fatty acids in the total fatty acids.

[0040] Depend on Figure 2After 1-10 passages, the ABBS-E11 strain fermentation culture yielded a biomass of 24-27 g / L and a lipid content of 47-54%. The EPA content of the total fatty acids was 8-9%, the DHA content was 35-40%, and the DPA content was 4-5%. These results are almost identical to those of the ABBS-E11 strain fermentation culture in Example 3 (biomass 25 g / L, lipid yield 12 g / L, EPA 9.2%, DHA 38.5%, and DPA only 4.5%). This demonstrates that the Schizochytrium mutant strain has good genetic stability across generations, can meet the needs of industrial production, and has practical application value.

[0041] Example 4: Fed-batch fermentation of Schizochytrium mutants in a 5 L fermentor

[0042] In this example, the ABBS-E11 strain was used for batch fed-batch fermentation in a 5 L fermenter. The specific steps were as follows:

[0043] (1) Seed solution preparation: The ABBS-E11 strain was inoculated into a liquid seed culture medium (glucose 40 g / L, yeast extract 10 g / L, corn steep liquor 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, sodium citrate 1 g / L, sea crystal 15 g / L, vitamin B1 1 mg / L, vitamin B6 30 mg / L, vitamin B12 1 mg / L, biotin 10 mg / L) and cultured in a shaking incubator at 25°C and 200 rpm for 72 h to obtain a seed solution.

[0044] (2) Fermentation culture: The fermentation medium was sterilized, and then 10% of the seed solution prepared in step (1) was inoculated into a 5L fully automatic fermenter for batch fed fermentation. The initial fermentation medium composition was as follows: glucose 120 g / L, yeast extract 10 g / L, corn steep liquor 10 g / L, sea crystal 15 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, sodium citrate 1 g / L, sea crystal 15 g / L, vitamin B1 10 mg / L, vitamin B6 15 mg / L, vitamin B12 8 mg / L, biotin 8 mg / L. Fermentation conditions: temperature 25°C, pH 6.5 (adjusted by 2M NaOH / HCl), dissolved oxygen 30% (maintained by a stirring rate of 200-600 rpm and a ventilation volume of 1-2 vvm). During the fermentation process, when the residual sugar dropped below 10 g / L, 800 g / L of glucose solution was added to maintain the residual sugar at 2-10 g / L. Fermentation was completed after 144 hours.

[0045] After the fermentation was completed, the biomass, oil yield and fatty acid analysis were determined using the method described in Example 2. The biomass was determined to be 166 g / L and the oil yield was 81.2 g / L. The fatty acid composition of the oil was determined by gas chromatography, and the results are detailed in Figure 3 .Depend on Figure 3 It can be seen that the EPA content in the oil is 11.6%, the DPA content is 2.8%, and the DHA content is 37%. Calculation shows that the EPA titer of the Schizochytrium mutant strain in a 5L fermenter batch fed-batch fermentation culture is 9.4g / L.

[0046] Example 5: Fed-batch fermentation of Schizochytrium mutants in a 50 L fermentor

[0047] In this example, the ABBS-E11 strain was used to conduct an amplification experiment in a 50 L fermenter. The specific steps are as follows:

[0048] Fermentation was carried out in a 50L fermentor (working volume 30L) using the same initial culture medium and feeding scheme as in Example 4, with three batches of fermentation. The specific conditions for batch 1 fermentation were: 25°C, pH = 6.5, and 30% dissolved oxygen for 4 days; the specific conditions for batch 2 fermentation were: 30°C, pH = 6, and 25% dissolved oxygen for 5 days; and the specific conditions for batch 3 fermentation were: 22°C, pH = 7, and 40% dissolved oxygen for 6 days. After fermentation, the oil was extracted by enzymatic hydrolysis and centrifugation, and the EPA, DHA, and DPA yields in the product were detected. The results are detailed in Table 1.

[0049] Table 1. Experimental results of fed-batch fermentation in 50 L fermenter

[0050]

[0051] As can be seen from Table 1, the dry biomass of the three batches of fed-batch fermentation cultures was 156-167 g / L, with an average of 161.7 g / L; the oil yield was 77.8-82.5 g / L, with an average of 80.1 g / L; the EPA content was 11.2-11.7% on average, with an average of 11.5%; the EPA titer was 8.7-9.6 g / L, with an average of 9.2 g / L; the DHA content was 36.7-38.9%, with an average of 37.7%; and the DPA content was 2.7-2.9%, with an average of 2.8%. In summary, (1) the results of the three batches of fed-batch fermentation were relatively consistent, indicating that the Schizochytrium mutant strain described in this application has good stability for industrial production; (2) in the products of the three batches of fed-batch fermentation, not only the EPA content (11.5%) and EPA titer (9.2 g / L) were significantly improved compared with the prior art, but also the DHA content was stable at more than 35%, and the DPA content was less than 3%, producing unexpected technical effects.

[0052] In summary, the Schizochytrium mutant strain provided by the present invention, after using a standard fermentation process, has an average EPA content of 11.5%, an oil yield of 80 g / L, and an EPA titer of 9.2 g / L. This not only significantly increases the EPA content in the product, but also maintains high biomass and titer, achieving an increase in absolute EPA production. Furthermore, the prepared product also contains over 35% DHA, possessing dual nutritional value. The low DPA content (DPA / DHA ratio less than 10%) facilitates subsequent separation and purification of DHA, and has significant market value.

Claims

1. A Schizochytrium mutant strain that efficiently produces EPA oil, characterized by: The Schizochytrium sp. mutant strain was named Schizochytrium sp. ABBS-E11 and was deposited in the China Center for Type Culture Collection at Wuhan University, Wuchang District, Wuhan City, Hubei Province. Its deposit number is CCTCC NO: M 20251165 and the deposit date is May 23, 2025.

2. A bacterial agent, characterized in that: The bacterial agent comprises the Schizochytrium mutant strain according to claim 1.

3. Use of the Schizochytrium mutant according to claim 1 in preparing EPA-rich oils and fats.

4. A method for producing EPA-rich oil, characterized in that: The EPA-rich oil is obtained by fermentation using the Schizochytrium mutant strain according to claim 1 or the bacterial agent according to claim 2.

5. The production method according to claim 4, characterized in that: The fermentation production specifically comprises: high-density fermentation in a liquid culture medium, and sequentially crushing and separating the bacterial cells.

6. The production method according to claim 4, characterized in that: The inoculation amount of the Schizochytrium mutant strain in the liquid culture medium is 10-15%.

7. The production method according to any one of claims 4 to 6, characterized in that: The specific conditions of the fermentation are: culturing for 4-6 days at 22-30° C., pH=6-7, and dissolved oxygen not less than 20%.

8. The production method according to claim 7, characterized in that: The liquid culture medium comprises: 60-120 g / L of glucose, 3-20 g / L of yeast extract, 3-20 g / L of corn steep liquor, 2-8 g / L of potassium dihydrogen phosphate, 1-5 g / L of magnesium sulfate, 1-4 g / L of sodium citrate, 5-30 g / L of sea crystal, 1-30 mg / L of vitamin B1, 1-30 mg / L of vitamin B6, 1-10 mg / L of vitamin B12, and 1-10 mg / L of biotin.

9. The production method according to claim 7, characterized in that: The crushing step is enzymatic hydrolysis, and the separation step is centrifugation.

10. The production method according to claim 7, characterized in that: The fermentation step also includes a sugar supplementation operation to ensure that the concentration of glucose is 2-10 g / L.

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