A polyunsaturated fatty acid oil product and a method for its production

Abstract

This invention relates to the field of microbial fermentation engineering, specifically to a polyunsaturated fatty acid oil product and its preparation method. The oil product provided by this invention contains fermentation products from a strain with accession number CCTCC NO: M 2021564. The astaxanthin content in the oil product is not less than 1.0%, and the DHA:astaxanthin ratio is (10:1)–(45:1). This invention employs ion beam implantation mutagenesis combined with flow cytometry to efficiently obtain target mutant strains. High-astaxanthin mutants are screened from a large number of Schizochytrium mutant strains. The mutant strains obtained by this invention can be rapidly industrialized with low cost and high production efficiency. The Schizochytrium mutant strains provided by this invention can be used to produce oil products with high astaxanthin content as well as deeply processed products.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, specifically to a polyunsaturated fatty acid oil product and its preparation method. Background Technology

[0002] Polyunsaturated fatty acids have functions such as regulating blood lipids, enhancing immunity, clearing blood clots, and improving inflammation. Among them, docosahexaenoic acid (DHA), a representative component, is an essential building block for brain cell formation, development, and movement. Human memory and cognitive functions rely on DHA for maintenance and improvement. Supplementing with DHA can promote full brain cell development, delay cognitive decline, reduce forgetfulness, and prevent Alzheimer's disease. Astaxanthin (AST) is a carotenoid with strong antioxidant activity. It is fat-soluble, and its antioxidant capacity is 10 times that of β-carotene, 200 times that of tea polyphenols, and more than 500 times higher than that of natural vitamin E. Studies have shown that consuming natural astaxanthin alone can significantly improve subjects' abilities to recognize, remember, and retrieve words and geometric shapes. 。 As research into carotenoids and polyunsaturated fatty acids deepens, the application range of polyunsaturated fatty acid oils containing astaxanthin is rapidly expanding.

[0003] Currently available natural oil sources rich in polyunsaturated fatty acids and astaxanthin are Haematococcus pluvialis and Antarctic krill. Haematococcus pluvialis can produce oil through photosynthetic autotrophy, but due to its high cost, low production efficiency, susceptibility to pollution, and low content of polyunsaturated fatty acids, it cannot be used on a large scale. On the other hand, the source of Antarctic krill raw materials is quite limited, and pre-processing and other procedures will lose a large amount of polyunsaturated fatty acids. Furthermore, the overfishing of Antarctic krill will cause environmental damage.

[0004] Schizochytrium is a type of marine fungus rich in oils. Its intracellular oils contain various unsaturated fatty acids. Studies have found that Schizochytrium cells possess an intracellular astaxanthin biosynthetic pathway, allowing the oils to contain small amounts of astaxanthin, which is beneficial for the oil's antioxidant properties. Current technology records that under continuous fluorescence, heterotrophic culture of Schizochytrium can synthesize DHA and astaxanthin, with the astaxanthin content in the fermented polyunsaturated fatty acids being approximately 0.1%-0.2%. Because the conversion of the precursor acetyl-CoA to astaxanthin within Schizochytrium cells involves as many as 14 enzymatic reactions, and the main destination of acetyl-CoA is the production of fatty acids, which competes with the astaxanthin metabolic pathway, the astaxanthin content in Schizochytrium is currently limited. Simply relying on fermentation to regulate and increase this content has limited potential, thus restricting the further value enhancement and application development of microbial oils produced by Schizochytrium. Summary of the Invention

[0005] The purpose of this invention is to provide a polyunsaturated fatty acid oil product and its preparation method.

[0006] In a first aspect, the present invention provides a polyunsaturated fatty acid oil product, wherein the oil product is a fermented oil-containing schistocytic trophoblast, and the astaxanthin content in the oil is above 1.0%; and / or the DHA:astaxanthin ratio in the oil is (10:1)-(45:1).

[0007] In the oil products provided by the present invention, the astaxanthin content in the oil is above 1.5%.

[0008] Furthermore, the astaxanthin content in the oil can be above 2.5%.

[0009] In the above-mentioned oil products, the DHA content in the oil is above 20%; preferably, the DHA content in the oil is above 35%.

[0010] In the oil products provided by the present invention, the Schizochytrium body is Schizochytrium with the preservation number CCTCC NO: M2021564, that is, the above oil products are obtained by fermentation of Schizochytrium with the preservation number CCTCC NO: M 2021564.

[0011] Secondly, the present invention provides a Schizochytrium sp. CABIO-A-2-III, which is now deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M 2021564 and deposit date May 19, 2021.

[0012] The Schizochytrium mutant strain provided by this invention was obtained by ion beam implantation mutagenesis combined with flow cytometry sorting. Specifically, the acquisition of the Schizochytrium mutant strain includes:

[0013] (a) Mutagenesis

[0014] (1) The starting strain is Schizochytrium isolated from seawater. The specific properties of this strain are disclosed in Example 2 of patent CN111235035A.

[0015] (2) Inoculate the bacterial strain into the activation medium and culture at a temperature of 28°C. Shake the incubator at a speed of 200 r / min for 48 h until the logarithmic growth phase.

[0016] (3) Take 1 mL of the activated seed culture medium from step (2) above and air dry it under sterile conditions to form a bacterial plaque. Aseptically transfer the culture dish containing the bacterial plaque into a high-energy particle beam injector and pass it through a high-energy N2 with an energy of 20 keV. + Ion beam implantation mutagenesis, N+ Ion beam implantation dose 10 17 ions / cm 2 .

[0017] (4) The bacterial film after the above mutagenesis treatment was washed off with sterile water and inoculated into a low nitrogen and low sugar Schizochytrium culture medium. The culture temperature was 28℃, the shaking speed of the shaker was 200r / min, and the culture was carried out for 60h until the stationary phase.

[0018] (II) Screening

[0019] The culture medium in (4) above was sterilely centrifuged, physiological saline was added to adjust the cell density and 1 mg / L Nile Red was added for staining. The mixture was then dispensed into the sample tubes of the flow cytometer. The algal cell resuspension was detected by the flow cytometer equipped with an argon ion laser in the FL1 channel for single-cell sorting, and the single colony with the strongest fluorescence signal superposition was selected.

[0020] The above steps were repeated multiple times. Due to the powerful and efficient sorting function of the flow cytometer, manual operation was greatly reduced, and strains with high oil content and high astaxanthin concentration, CABIO-A-2-Ⅲ, could be screened from a large number of mutant strains.

[0021] The Schizochytrium sp. CABIO-A-2-Ⅲ obtained by mutagenesis in this invention is now deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, China, accession number CCTCC NO: M2021564, deposit date May 19, 2021.

[0022] Fermentation using the Schizochytrium provided in this invention results in oil accounting for more than 40% of the oil-containing Schizochytrium cells in the fermentation product, ensuring oil yield. The astaxanthin content in the oil product is higher than 1%, and can reach as high as 2.5% or more. The content of the main polyunsaturated fatty acid DHA is not significantly affected, reaching more than 35%. The ratio of DHA to astaxanthin in the oil is (10:1) to (45:1).

[0023] Thirdly, this invention provides a method for obtaining oil products through fermentation. The oil is obtained directly from the aforementioned polyunsaturated fatty acid oil products through cell wall disruption and extraction; alternatively, the oil can be obtained by activating and inoculating the aforementioned Schizochytrium fungi, followed by cell wall disruption and extraction. In the method for obtaining oil products through fermentation provided by this invention, the Schizochytrium fungi inoculation amount is 8-12% of the fermentation medium volume; the culture temperature is 25-29℃; and the culture time is 110-130 hours.

[0024] The oil product prepared using the method provided by this invention contains at least 1.0% astaxanthin and at least 20% DHA. Preferably, the astaxanthin content is at least 1.5%; preferably, DHA accounts for at least 35% of the total fatty acid content. Fermentation using the Schizochytrium provided by this invention yields a DHA:astaxanthin ratio of (5:1) to (45:1) in the fermentation product.

[0025] As understood by those skilled in the art, the present invention also claims protection for the use of the above-mentioned oil products, the above-mentioned Schizochytrium fungus, or the above-mentioned methods in the production of mycelial powder, food, pharmaceuticals, cosmetics, food additives, or feed additives.

[0026] Specifically, the present invention provides a bacterial powder containing either a fermented oil product of the aforementioned Schizochytrium or the aforementioned Schizochytrium. After fermentation using a Schizochytrium mutant strain obtained through mutagenesis screening according to the present invention, the bacterial powder contains 24% DHA and 2.4% astaxanthin.

[0027] This invention also claims protection for food, health products, cosmetics, food additives, or feed additives containing the above-mentioned oil products or the above-mentioned Schizochytrium or oils obtained by the above-mentioned methods.

[0028] The beneficial effects of this invention are at least as follows:

[0029] (1) The present invention obtains target mutant strains efficiently by combining ion beam injection mutagenesis with flow cytometry sorting method;

[0030] (2) After fermentation using the Schizochytrium mutant strain obtained by mutagenesis screening in this invention, an oil product with high astaxanthin content is obtained, wherein the astaxanthin content can be as high as 2.5% or more, and the ratio of DHA to astaxanthin is (10:1)-(45:1).

[0031] (3) In the fermentation products obtained by the mutagenesis screening of the present invention, the total oil accounts for more than 40% of the cell, and the astaxanthin content in the oil can reach more than 2.5%; the DHA content can reach more than 35%.

[0032] (4) Using the Schizochytrium mutant strain obtained by the present invention, fermentation products can be obtained based on fermentation culture, and different forms of products can be prepared, such as astaxanthin powder, astaxanthin-containing microbial oil, etc. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all experimental materials, reagents, instruments, etc. used in the examples of this invention are commercially available; unless otherwise specified, all technical means in the examples of this invention are conventional means well known to those skilled in the art.

[0035] The detection method for DHA content in this invention is the same as that in GB 26400-2011; the detection method for astaxanthin is the same as that in GB / T31520-2015.

[0036] Example 1

[0037] This embodiment provides a method for mutagenesis screening of Schizochytrium mutant strains, the steps of which are as follows:

[0038] (a) Mutagenesis

[0039] (1) The starting strain is Schizochytrium isolated from seawater. The specific properties of this strain are disclosed in Example 2 of patent CN111235035A.

[0040] (2) Inoculate the bacterial strain into the activation medium and culture at a temperature of 28°C. Shake the incubator at a speed of 200 r / min for 48 h until the logarithmic growth phase.

[0041] (3) Take 1 mL of the activated seed culture medium from step (2) above and air dry it under sterile conditions to form a bacterial plaque. Aseptically transfer the culture dish containing the bacterial plaque into a high-energy particle beam injector and pass it through a high-energy N2 with an energy of 20 keV. + Ion beam implantation mutagenesis, N + The ion beam implantation dose is 10 17 ions / cm 2 .

[0042] (4) The bacterial film after the above mutagenesis treatment was washed off with sterile water and inoculated into a low nitrogen and low sugar Schizochytrium culture medium. The culture temperature was 28℃, the shaking speed of the shaker was 200r / min, and the culture was carried out for 60h until the stationary phase.

[0043] (II) Screening

[0044] Centrifuge the culture medium in (4) aseptically, and add physiological saline to adjust the cell density to 1×10⁻⁶. 5 -1×10 6 The concentration of CFU / mL (cells / mL) was adjusted and 1 mg / L of Nile Red was added for staining. The mixture was then dispensed into the sample tubes of the flow cytometer. The flow cytometer, equipped with an argon ion laser, was used to detect the algal cell resuspension in the FL1 channel for single-cell sorting. The single colony with the strongest fluorescence signal was selected for further verification.

[0045] The above steps were repeated multiple times. Due to the powerful and efficient sorting function of the flow cytometer, manual operation was greatly reduced, and strains with high oil content and high astaxanthin concentration, CABIO-A-2-Ⅲ, could be screened from a large number of mutant strains.

[0046] Under an optical microscope, the morphology and structure of the strains screened in this example were observed. The bacterial cells were spherical with a diameter of 15 μm, consistent with the morphology and structure of the original strain. *Schizochytrium sp.* CABIO-A-2-III is now deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, accession number CCTCC NO: M 2021564, deposited on May 19, 2021.

[0047] Example 2

[0048] The *Schizochytrium* strain obtained through mutagenesis screening in Example 1 was used to ferment and produce an oil product with high astaxanthin content. The steps are as follows:

[0049] (1) Seed activation culture: The Schizochytrium mutant strain obtained in Example 1 was inoculated into the activation culture medium and cultured at a temperature of 28°C. The shaking speed of the shaker was 200 r / min, and the culture time was 48 h. The activation culture medium consisted of 10 g / L glucose, 25 g / L sodium glutamate, 10 g / L yeast extract, 20 g / L sodium chloride, 0.5 g / L magnesium sulfate, and natural pH.

[0050] (2) Seed expansion culture: The activated seed culture solution from the shaking culture in step (1) above was inoculated into the expansion culture medium at an inoculation rate of 10% (volume ratio) for culture. The culture temperature was 28℃, the culture time was 48 h, and the shaking speed of the shaker was 200 rpm. The expansion culture medium in the shake flask was: glucose 40 g / L, sodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 10 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.5 g / L, and the pH was natural.

[0051] (3) Fermentation in a fermentation bottle: The expanded seed culture broth from step (2) was inoculated at a rate of 10% (volume ratio) into a 1L fermentation bottle (1 / 4 full) containing fermentation medium. The fermentation temperature was 28℃, the fermentation time was 120 h, and the shaking speed on the shaker was 220 rpm. The fermentation medium consisted of: 30 g / L glucose, 4 g / L yeast extract, 30 g / L monosodium glutamate, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.5 g / L sodium bicarbonate, 8 g / L sodium sulfate, 6 g / L ammonium sulfate, 0.5 g / L potassium chloride, and natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by adding glucose continuously.

[0052] (4) Post-processing of fermentation products: After the fermentation broth obtained from the fermentation culture is broken by enzymatic method, for details, refer to the cell breaking method in paragraph 76 of the instruction manual in CN111378699A. Under nitrogen protection, hexane is added as an extractant for extraction. The solid phase obtained after extraction is transferred to an extraction vessel for repeated extraction until no oil is present in the extract. The extraction process is then ended. The mixed oil obtained after each extraction is filtered and desolventized to obtain microbial oil.

[0053] In this embodiment, the oil accounts for 45% of the dry matter, the oil content in the fermentation volume reaches 40 g / L, the astaxanthin content in the oil reaches 1.2%, the DHA content in the oil reaches 46.6%, and the DHA:astaxanthin content ratio is 39:1.

[0054] Example 3

[0055] The *Schizochytrium* strain obtained through mutagenesis screening in Example 1 was used to produce an oil product containing DHA and astaxanthin through fermentation. The fermentation steps were the same as in Example 2, except that the inoculum size was 8% (volume ratio), the fermentation temperature in the fermentation flask was 27°C, the culture time was 110 h, and the fermentation medium consisted of 45 g / L glucose, 6 g / L yeast extract, 25 g / L monosodium glutamate, 6 g / L sodium chloride, 0.5 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate, 1.0 g / L calcium chloride, 0.5 g / L sodium bicarbonate, 5 g / L sodium sulfate, 4 g / L ammonium sulfate, and 0.1 g / L potassium chloride, with a natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by feeding glucose continuously.

[0056] In this embodiment, the oil accounted for 43% of the dry matter, the oil content in the fermentation volume reached 39 g / L, the astaxanthin content in the oil reached 1.5%, the DHA content in the oil reached 43.5%, and the DHA:astaxanthin content ratio was 29:1.

[0057] Example 4

[0058] The *Schizochytrium* strain obtained through mutagenesis screening in Example 1 was used to produce an oil product containing DHA and astaxanthin through fermentation. The fermentation steps were the same as in Example 2, except that the inoculum size was 12% (volume ratio), the fermentation temperature in the fermentation flask was 29°C, the culture time was 120 h, and the fermentation medium consisted of 45 g / L glucose, 8 g / L yeast extract, 25 g / L monosodium glutamate, 3 g / L sodium chloride, 0.1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.8 g / L sodium bicarbonate, 2 g / L sodium sulfate, 8 g / L ammonium sulfate, and 0.1 g / L potassium chloride, with a natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by feeding glucose continuously.

[0059] In this embodiment, the oil accounts for 40% of the dry matter, the oil content in the fermentation volume reaches 35 g / L, the astaxanthin content in the oil reaches 2.5%, the DHA content in the oil reaches 35%, and the DHA:astaxanthin content ratio is 14:1.

[0060] Example 5

[0061] This embodiment provides a method for fermenting the Schizochytrium fungi obtained by mutagenesis screening in Example 1 on a 50L tank. The steps are as follows:

[0062] (1) The seed activation and seed expansion steps are the same as in Example 2.

[0063] (2) Fermentation tank culture: The expanded seed culture broth from the shaking culture was inoculated at a rate of 10% (volume ratio) into a 50L fermentation tank containing 30L of fermentation medium for fermentation culture. The culture temperature was 28℃, the culture time was 120 h, and the stirring speed was 220 rpm. The fermentation medium consisted of: glucose 40 g / L, yeast extract 4 g / L, monosodium glutamate 30 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, calcium chloride 0.5 g / L, sodium bicarbonate 0.5 g / L, sodium sulfate 8 g / L, ammonium sulfate 6 g / L, potassium chloride 0.5 g / L, and natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by feeding glucose continuously.

[0064] (3) Post-fermentation treatment: After the fermentation broth obtained from the fermentation culture is broken by enzymatic method, for details, refer to the cell breaking method in paragraph 76 of the instruction manual in CN111378699A. Under nitrogen protection, hexane is added as an extractant for extraction. The solid phase obtained after extraction is transferred to an extraction container for repeated extraction. This process is repeated until there is no oil in the extract. After each extraction, the mixed oil obtained by filtration is desolventized to obtain microbial oil.

[0065] In this embodiment, the oil accounted for 43% of the dry matter, the oil content in the fermentation volume reached 42 g / L, the astaxanthin content in the oil reached 2.7%, the DHA content in the oil reached 39%, and the DHA:astaxanthin ratio was 14.4:1.

[0066] Comparative Example 1

[0067] This comparative example uses Schizochytrium without mutagenesis or screening treatment for fermentation culture, and the steps are as follows:

[0068] (1) Seed activation culture: Unmutated Schizochytrium was inoculated into activation culture medium and cultured at 28℃ with a shaking speed of 200r / min for 48h. The activation culture medium consisted of 10g / L glucose, 25g / L sodium glutamate, 10g / L yeast extract, 20g / L sodium chloride, 0.5g / L magnesium sulfate, and natural pH.

[0069] (2) Seed expansion culture: The activated seed culture solution from the shaking culture in step (1) above was inoculated into the expansion culture medium at an inoculation rate of 10% (volume ratio) for culture. The culture temperature was 28℃, the culture time was 48 h, and the shaking speed of the shaker was 200 rpm. The expansion culture medium in the shake flask was: glucose 40 g / L, sodium glutamate 25 g / L, yeast extract 10 g / L, sodium chloride 10 g / L, magnesium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 0.5 g / L, and the pH was natural.

[0070] (3) Fermentation in a fermentation bottle: The expanded seed culture broth from step (2) was inoculated at a rate of 10% (volume ratio) into a 1L (1 / 4 full) fermentation bottle containing fermentation medium. The fermentation temperature was 28℃, the fermentation time was 120 h, and the shaking speed on the shaker was 220 rpm. The fermentation medium consisted of: 40 g / L glucose, 4 g / L yeast extract, 30 g / L monosodium glutamate, 5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.5 g / L sodium bicarbonate, 8 g / L sodium sulfate, 6 g / L ammonium sulfate, 0.5 g / L potassium chloride, and natural pH. During fermentation, the glucose concentration in the fermentation broth was controlled at 5 g / L by adding glucose continuously.

[0071] (4) Post-processing: After the fermentation broth obtained from the fermentation culture is broken by enzymatic method, for details, refer to the cell breaking method in paragraph 76 of the instruction manual in CN111378699A. Under nitrogen protection, hexane is added as an extractant for extraction. The solid phase obtained after extraction is transferred to the extraction vessel for repeated extraction. This process is repeated until there is no oil in the extract. After each extraction, the mixed oil obtained by filtration is desolventized to obtain microbial oil.

[0072] The oil obtained above accounted for 48% of the dry matter, 43 g / L of the fermentation volume, 0.0002% of the astaxanthin in the oil, 41.4% of the DHA in the oil, and the ratio of DHA to astaxanthin was 207000:1.

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A Schizochytrium sp. with a preservation number of CCTCC NO: M 2021564.

2. Use of the Schizochytrium sp. of claim 1 in the preparation of oil with high astaxanthin content.

3. Use of the Schizochytrium sp. of claim 1 in the production of bacterial powder, food, medicine, cosmetics, food additive or feed additive.

4. A bacterial powder, characterized by, The bacterial powder contains the Schizochytrium sp. of claim 1.