CN111235035A

CN111235035A - Schizochytrium limacinum mutant strain, and method and application thereof in preparation of docosahexaenoic acid grease

Info

Publication number: CN111235035A
Application number: CN201911399018.2A
Authority: CN
Inventors: 汪志明; 陆姝欢; 余超; 李慕梓; 杨艳红; 李翔宇
Original assignee: Cabio Biotech Wuhan Co Ltd
Current assignee: Cabio Biotech Wuhan Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-06-05

Abstract

The invention relates to a schizochytrium limacinum mutant strain, a method for preparing docosahexaenoic acid grease by using the same and application thereof, wherein the strain is preserved in a China center for type culture collection (CCTCCM 2019990) 12, 2 and 2019 at the preservation address of Wuhan university in Wuhan city, Hubei province, China. According to the invention, the schizochytrium limacinum mutant strain CCTCCM2019990 is obtained by screening through high-throughput culture and the sedimentation characteristic of the schizochytrium limacinum by combining ultraviolet mutagenesis with an ion beam mutagenesis technology, and the mutant strain is used for producing docosahexaenoic acid grease, so that the obtained docosahexaenoic acid grease is low in long-chain saturated fatty acid content, the lowest content of C18:0 is 0.4 wt%, the lowest content of C16:0 is 12.5 wt%, and the highest content of DHA can reach 57.08 wt%, and the intake of diabetes mellitus crowds is facilitated.

Description

Schizochytrium limacinum mutant strain, and method and application thereof in preparation of docosahexaenoic acid grease

Technical Field

The invention relates to the field of microorganisms, in particular to a schizochytrium limacinum mutant strain and a method and application thereof for preparing docosahexaenoic acid grease.

Background

Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia occurring in diabetes over a long period of time can lead to chronic damage to, and dysfunction of, various tissues, particularly the eyes, kidneys, heart, blood vessels, nerves. The prevalence of diabetes is higher in the elderly, men, urban residents, residents in economically developed areas, overweight and obese people.

Diabetes mellitus is divided into type 1 diabetes mellitus and type 2 diabetes mellitus, insulin resistance is one of the main pathophysiological characteristics of type 2 diabetes mellitus, and numerous researches show that n-3 polyunsaturated fatty acid including DHA can regulate immunity and reduce the release of inflammatory factors through various molecular mechanisms, so that the lipid metabolism of type 2 diabetes mellitus is improved, the insulin sensitivity is improved, and diabetes mellitus and complications are favorably prevented and treated.

At present, the main source of DHA is oil produced by marine microorganisms, and the related marine microorganisms comprise thraustochytrium, schizochytrium and the like. Microbial oils contain, in addition to polyunsaturated fatty acids, a significant amount of long chain saturated fatty acids, especially palmitic acid C16:0, typically in amounts of greater than 2 wt%, and in certain microbial oils C16:0 up to 50 wt%. There is evidence that excessive intake of long chain saturated fatty acids can cause lipid accumulation in the body to cause lipotoxicity, resulting in insulin resistance in skeletal muscle, adipose tissue and liver. In addition, studies have been made to restore insulin sensitivity by knocking out mouse long chain fatty acid elongase 6, which causes the proportion and change of fatty acid composition, indicating that long chain fatty acid elongase may be a potential target for the treatment of insulin resistance, diabetes, cardiovascular diseases and other metabolic diseases. These studies suggest that both endogenous and exogenous long chain fatty acids negatively impact type 2 diabetes.

Because the diabetes risk people have a high-oil dietary habit, the people who take DHA and reduce the intake of long-chain saturated fatty acid become urgent needs of the people, and the high-content DHA grease can reduce C16 as far as possible under the condition of meeting the normal intake of DHA: 0 and other long chain saturated fatty acids. Therefore, the development of a DHA microbial oil rich in DHA and low in long-chain saturated fatty acid, especially C16:0 palmitic acid, is urgently needed.

Disclosure of Invention

In order to solve the problems, the invention provides a schizochytrium limacinum mutant strain, and a method and application thereof for preparing docosahexaenoic acid grease.

In A first aspect, the present invention provides A Schizochytrium sp CABIO-A-2-II strain, which has the following deposit information: the preservation number is CCTCCM 2019990; the classification is named as: schizochytrium sp; the preservation unit is China center for type culture Collection; the preservation address of Wuhan university in Wuhan city, Hubei province, China, the zip code: 430072; the preservation date is 12 months and 2 days in 2019.

The invention adopts ultraviolet mutagenesis combined with ion beam mutagenesis technology, obtains A schizochytrium mutant strain through high-throughput culture and the sedimentation characteristic of the schizochytrium, and determines that the schizochytrium mutant strain CABIO-A-2-II accords with the characteristic of the schizochytrium through culture characteristic, micro morphological characteristic, physiological and biochemical characteristic and genetic characteristic.

The physiological characteristics of the schizochytrium limacinum mutant strain CABIO-A-2-II provided by the invention are as follows:

(1) the vegetative cells are single cells, are nearly spherical, have the diameter of 4-11 mu m, and are proliferated in a typical binary division mode;

(2) in liquid culture, the cells generally aggregate into large aggregates;

(3) the optimum culture temperature is 25 ℃.

The invention further provides A microbial inoculum containing the Schizochytrium limacinum mutant strain CABIO-A-2-II.

In A second aspect, the invention provides A method for producing docosahexaenoic acid grease by using the schizochytrium limacinum mutant strain CABIO-A-2-II or microbial inoculum, which comprises the following steps: fermenting the Schizochytrium limacinum mutant strain CABIO-A-2-II or microbial inoculum, collecting fermentation product, and separating and purifying.

As A preferred embodiment, the invention provides A method for producing docosahexaenoic acid grease by using the Schizochytrium limacinum mutant strain CABIO-A-2-II or microbial inoculum, which comprises the following specific steps:

(1) activating and culturing seeds: inoculating the Schizochytrium limacinum mutant strain CABIO-A-2-II or the microbial inoculum into an activation culture medium, and performing shake culture to obtain A seed activation culture solution;

(2) seed amplification culture: inoculating the seed culture solution cultured by shaking in the step (1) into an amplification culture medium for shaking culture to obtain a seed amplification culture solution;

(3) fermentation culture: inoculating the seed culture solution cultured by shaking in the step (2) into a fermentation bottle filled with a fermentation culture medium for fermentation culture;

or according to the size of the final fermentation tank, carrying out amplification culture on the seed culture solution cultured in the shake flask in the step (2) step by step to obtain a seed amplification culture solution, and then inoculating the seed amplification culture solution into the fermentation tank for fermentation culture.

(4) And (3) carrying out post-treatment on the fermentation liquor to obtain the docosahexaenoic acid grease.

Further, the step (1) is as follows: inoculating the Schizochytrium limacinum mutant strain CABIO-A-2-II into an activation culture medium for culturing at the temperature of 25-30 ℃, with the shaking table shaking rotation speed of 150-: 10-20g/L of glucose, 20-30g/L of sodium glutamate, 5-10g/L of yeast extract, 10-20g/L of sodium chloride, 0.5-1g/L of magnesium sulfate and natural pH.

Further, the step (2) is as follows: when the bacterial concentration of the seed activation culture solution in the step (1) reaches 3-6% (volume concentration), inoculating the seed activation culture solution into an amplification culture medium according to the inoculation amount of 5-20% (volume ratio) for culture, wherein the culture temperature is 25-30 ℃, the culture time is 32-48h, the shaking table shaking rotation speed is 150-: 30-50g/L of glucose, 20-40g/L of sodium glutamate, 5-10g/L of yeast extract, 10-20g/L of sodium chloride, 5-10g/L of magnesium sulfate, 5-10g/L of monopotassium phosphate, 0.5-1g/L of calcium chloride and natural pH.

Further, the step (3) may also be: and (3) carrying out stepwise amplification culture on the seed culture solution cultured in the shake flask in the step (2) according to the size of a final fermentation tank to obtain the seed amplification culture solution, inoculating the seed amplification culture solution into the fermentation tank for fermentation culture when the bacteria concentration of the seed amplification culture solution reaches 3-6% (volume concentration), wherein the inoculation amount is 10-20% (volume ratio), the temperature of the fermentation tank is 25-30 ℃, the stirring speed is 100-250 r/min, the ventilation amount is 0.5-2vvm (L/L.min), namely the required air introduction amount per minute in each liter of fermentation liquor is 1-2L, the tank pressure is 0.05-0.1MPa, and the culture is carried out for 96-120h, wherein the fermentation culture medium in the fermentation tank is: 40-60g/L of glucose, 4-6g/L of yeast extract, 20-40g/L of sodium glutamate, 1-5g/L of sodium chloride, 1-5g/L of monopotassium phosphate, 5-10g/L of magnesium sulfate, 0.5-1g/L of calcium chloride, 0.5-1g/L of sodium bicarbonate, 5-10g/L of sodium sulfate, 5-20g/L of ammonium sulfate, 0.5-1g/L of potassium chloride and natural pH.

In a more preferred embodiment, the concentration of the carbon source in the fermentation broth in step (3) can be controlled to be 5 to 20g/L by feeding glucose during the fermentation.

In a more preferred embodiment, the pH of the fermentation broth in step (3) can be controlled between 6 and 8 by adding citric acid or hydrochloric acid during the fermentation.

Further, the post-treatment in the step (4) is to perform wall breaking on thalli in the fermentation liquor by using alkaline protease, and then adding an extracting agent for extraction.

The bacterial concentration is the proportion of the volume of the lower layer of bacteria in the total system volume after the culture solution is centrifuged.

The docosahexaenoic acid oil obtained by the method contains C16:0 less than 15.50%, long-chain saturated fatty acid C16:0 and C18:0 less than 16.01 wt%, and DHA higher than 50.40%.

In A third aspect, the invention provides the application of the schizochytrium limacinum mutant strain CABIO-A-2-II and A microbial inoculum in preparation of docosahexaenoic acid grease.

Further, the content of C16:0 in the docosahexaenoic acid oil is less than 15.50 wt%, preferably less than 15.00 wt%, more preferably less than 14.12 wt%, and still more preferably less than 12.50 wt%.

Further, the content of C18:0 in the docosahexaenoic acid oil is less than 0.51 wt%, preferably less than 0.41 wt%, and more preferably less than 0.40 wt%.

Further, the sum of the contents of long-chain saturated fatty acids C16:0 and C18:0 in the docosahexaenoic acid oil is less than 16.01 wt%, preferably less than 15.41 wt%, more preferably less than 14.52 wt%, and even more preferably less than 12.90 wt%.

Furthermore, the DHA content of the docosahexaenoic acid grease is higher than 50.40 wt%, preferably higher than 52.37 wt%, further preferably higher than 54.90 wt%, and further preferably higher than 57.08 wt%.

The invention further provides the application of the schizochytrium limacinum mutant strain CABIO-A-2-II, the microbial inoculum and the docosahexaenoic acid grease in preparing food, feed, cosmetics or medicines.

The invention further provides a microbial oil, wherein the content of docosahexaenoic acid oil in the microbial oil is at least 50%, and the content of long-chain saturated fatty acid C16:0 in the docosahexaenoic acid oil is less than 15.50 wt%, preferably less than 15.00 wt%, further preferably less than 14.12 wt%, and further preferably less than 12.50 wt%.

The invention provides a schizochytrium limacinum mutant strain, a method for preparing docosahexaenoic acid grease by using the same and application thereof, and the schizochytrium limacinum mutant strain has the following beneficial effects:

the invention adopts ultraviolet mutagenesis combined with ion beam mutagenesis technology, obtains A schizochytrium mutant strain CABIO-A-2-II by screening through high-throughput culture and the sedimentation characteristic of the schizochytrium, and utilizes the mutant strain to produce the long-chain saturated fatty acid C16 in the docosahexaenoic acid grease: 0 and C18: the content of 0 is less than 16.01 wt%, and is stable and appropriate with the fermentation state, further, less than 15.41 wt%, also less than 14.52 wt%, and as low as 12.90 wt%, and the content of DHA is at least higher than 50.40 wt%, and as high as 57.08 wt%. The invention has the advantages that the microbial oil produced by the mutant strain has low content of long-chain saturated fatty acid, and is more beneficial to the intake of diabetes patients.

Drawings

FIG. 1 is A diagram showing the results of microscopic observation of the growth and propagation process of Schizochytrium sp CABIO-A-2-II according to example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 Breeding of Schizochytrium limacinum mutant strains

The embodiment provides a method for breeding schizochytrium limacinum mutant strains, which comprises the following specific steps:

(1) schizochytrium limacinum obtained by screening and separating from seawater is used as an original strain, and the strain is obtained from a yellow sea water area of Shandong province.

(2) Inoculating the strain to an activation culture medium for culturing at 28 ℃ at the shaking table rotation speed of 200r/min for 48h to logarithmic phase.

(3) And (3) injecting 1ml of the activated seed culture solution obtained in the step (2) onto a sterile culture dish, and irradiating by using an ultraviolet lamp, wherein the irradiation distance of the ultraviolet lamp is 30 cm, the irradiation time is 60 seconds, and the power of the ultraviolet lamp is 30 watts.

(4) The bacteria liquid after ultraviolet mutagenesis is dried by sterile wind to become bacterial plaque. Aseptically transferring the culture dish containing bacterial plaque into high-energy particle beam injection machine, and allowing the culture dish to pass through high-energy N with energy of 20KeV⁺Ion beam implantation mutagenesis, N⁺The ion beam implantation dosage is 100 x 10¹⁷ions/cm²。

(5) And (3) eluting the mycoderm subjected to mutagenesis treatment by using sterile water, diluting, coating on an activation medium flat plate for culturing, wherein the solid activation medium is as follows: 10g/L of glucose, 25g/L of sodium glutamate, 10g/L of yeast extract, 20g/L of sodium chloride, 0.5g/L of magnesium sulfate, 20g/L of agar powder and natural pH. Collecting the mutagenized single colony, inoculating the single colony into a porous plate filled with a fermentation culture medium by using a toothpick point for culturing at the culture temperature of 28 ℃ and the shaking speed of a shaking table of 200r/min for 96 h; transferring the fermentation liquor in the porous plate into a glass test tube, standing and settling, and observing the layering condition; selecting single colony fermentation liquor with slow cell sedimentation for detection and analysis; and (3) carrying out vacuum drying on the screened cells, taking a 20mg sample for fatty acid component analysis, and taking a single colony with the long-chain saturated fatty acid content of less than 20 wt% and the DHA content of more than 35 wt% in the fatty acid component as a starting strain for next step of mutagenesis. The fermentation medium is as follows: 40-60g/L of glucose, 4-6g/L of yeast extract, 20-40g/L of sodium glutamate, 1-5g/L of sodium chloride, 1-5g/L of monopotassium phosphate, 5-10g/L of magnesium sulfate, 0.5-1g/L of calcium chloride, 0.5-1g/L of sodium bicarbonate, 5-10g/L of sodium sulfate, 5-20g/L of ammonium sulfate, 0.5-1g/L of potassium chloride and natural pH.

In the embodiment, A mutant strain with low content of long-chain saturated fatty acid is obtained through multiple mutagenesis and screening, the strain is subjected to continuous subculture for 10 times, the lowest content of palmitic acid C16:0 can reach 12.50 wt%, the lowest content of stearic acid C18:0 can reach 0.40 wt%, and the highest content of DHA can reach 56.68 wt%, the mutant strain is the Schizochytrium mutant strain (Schizochytrium sp.) CABIO-A-2-II, and the strain is stored in the ChinA center for type culture collection in 12 months and 2 days in 2019; the preservation address is Wuhan university in Wuhan city of Hubei province in China; and E, postcode: 430072, with a collection number of CCTCCM2019990, and is classified and named as: schizochytrium sp.

The schizochytrium limacinum strain CABIO-A-2-II has the following characteristics:

(1) the vegetative cells are unicellular, nearly spherical, and have a diameter of 4-11 μm, and are proliferated in a typical binary division manner, and the growth state under a microscope is shown in figure 1;

(2) in liquid culture, the cells generally aggregate into large aggregates;

(3) the optimum culture temperature is 25 ℃.

Example 2 production of docosahexaenoic acid Using Schizochytrium limacinum mutant strains

In this example, the starting strain obtained by screening and separating in step (1) of example 1 was used to produce docosahexaenoic acid by the following specific method:

(1) activating and culturing seeds: inoculating the starting strain into an activation culture medium for culturing at the culture temperature of 28 ℃, the shaking rotation speed of a shaking table of 200r/min for 48h, wherein the activation culture medium is as follows: 10g/L of glucose, 25g/L of sodium glutamate, 10g/L of yeast extract, 20g/L of sodium chloride, 0.5g/L of magnesium sulfate and natural pH.

(2) Seed amplification culture: inoculating the activated seed culture solution cultured by shaking in the step (1) into an amplification culture medium according to the inoculation amount of 10% (volume ratio) for culturing, wherein the culture temperature is 28 ℃, the culture time is 48h, the shaking rotation speed of the shaking table is 200r/min, and the amplification culture medium in the shaking table is as follows: 40g/L glucose, 25g/L sodium glutamate, 10g/L yeast extract, 10g/L sodium chloride, 5g/L magnesium sulfate, 1g/L potassium dihydrogen phosphate, 0.5g/L calcium chloride and natural pH.

(3) And (3) fermentation bottle culture: inoculating the expanded seed culture solution cultured by shaking in the step (2) into a fermentation bottle filled with a fermentation culture medium according to the inoculation amount of 10% (volume ratio), and carrying out fermentation culture at the culture temperature of 28 ℃ for 120h at the shaking rotation speed of 220 r/min, wherein the fermentation culture medium is: 40g/L glucose, 4g/L yeast extract, 30g/L sodium glutamate, 5g/L sodium chloride, 1g/L monopotassium phosphate, 5g/L magnesium sulfate, 0.5g/L calcium chloride, 0.5g/L sodium bicarbonate, 8g/L sodium sulfate, 6g/L ammonium sulfate, 0.5g/L potassium chloride and natural pH.

(4) And (3) post-treatment: and (2) breaking the wall of fermentation liquor obtained by fermentation culture with alkaline protease, adding an extracting agent n-hexane for extraction, transferring a solid phase obtained by separation after extraction into an extraction container for repeated extraction, ending the extraction process until no oil exists in the extraction liquor, adding 200 ml of n-hexane during the first extraction, adding 150 ml of n-hexane each time, filtering and separating mixed oil after each extraction, and desolventizing to obtain the microbial oil.

(5) Gas chromatographic analysis: performing gas chromatography analysis on the microbial oil obtained in the step (4) to obtain the results shown in the table 1:

TABLE 1 DHA oil and fat obtained in comparative example 2

Fatty acid composition	Content (wt%)
		14:0	5.37％
16:0	28.50％
		18:0	0.91％
18:1	0.40％
		18:2	0.91％
20:4n6	2.50％
		20:5n3(EPA)	1.79％
22:5n6	17.36％
		22:5n3	1.30％
22:6n3(DHA)	39.90％

In the DHA oil prepared by the starting strain in this example, the content of palmitic acid C16:0 was 28.50 wt%, the content of stearic acid C18:0 was 0.91 wt%, the content of long chain fatty acid was 29.41 wt%, and the content of DHA was 39.9 wt%.

Example 3 production of docosahexaenoic acid Using Schizochytrium limacinum mutant strains

In this example, the method for producing docosahexaenoic acid by using the schizochytrium limacinum mutant obtained in example 1 is as follows:

(1) activating and culturing seeds: the schizochytrium limacinum mutant strain CABIO-A-2-II in example 1 is inoculated into an activation culture medium for culturing, the culture temperature is 28 ℃, the shaking table shaking speed is 200r/min, and the culture time is 48h, wherein the activation culture medium is as follows: 10g/L of glucose, 25g/L of sodium glutamate, 10g/L of yeast extract, 20g/L of sodium chloride, 0.5g/L of magnesium sulfate and natural pH.

(5) Gas chromatographic analysis: performing gas chromatography analysis on the microbial oil obtained in the step (4) to obtain the results shown in the table 2:

table 2 content of each fatty acid in DHA oil in example 3

In the DHA grease prepared by the mutant strain in the embodiment, the content of palmitic acid C16:0 is 15.5 wt%, the content of stearic acid C18:0 is 0.51 wt%, and the content of DHA reaches 50.4 wt%.

Example 4 production of docosahexaenoic acid Using Schizochytrium limacinum mutant strains

(2) Seed amplification culture: inoculating the activated seed culture solution cultured by shaking in the step (1) into an amplification culture medium according to the inoculation amount of 10% (volume ratio) for culturing, wherein a seed tank is a 10L seed amplification fermentation tank, the culture temperature is 28 ℃, the culture time is 48h, the shaking rotation speed of the shaking table is 200r/min, and the amplification culture medium in the seed tank is as follows: 40g/L glucose, 25g/L sodium glutamate, 10g/L yeast extract, 10g/L sodium chloride, 5g/L magnesium sulfate, 1g/L potassium dihydrogen phosphate, 0.5g/L calcium chloride and natural pH.

(3) And (3) fermentation bottle culture: inoculating the expanded seed culture solution cultured by shaking in the step (2) into a 50L fermentation tank filled with 30L of fermentation medium according to the inoculation amount of 10% (volume ratio), fermenting and culturing, wherein the culture temperature is 28 ℃, the culture time is 120h, the stirring speed is 220 r/min, the carbon source concentration in the fermentation broth is controlled to be 20g/L by feeding glucose in the fermentation process, the pH of the fermentation broth is controlled to be 7.2-7.8 by adding citric acid, and the fermentation medium is: 40g/L glucose, 4g/L yeast extract, 30g/L sodium glutamate, 5g/L sodium chloride, 1g/L monopotassium phosphate, 5g/L magnesium sulfate, 0.5g/L calcium chloride, 0.5g/L sodium bicarbonate, 8g/L sodium sulfate, 6g/L ammonium sulfate, 0.5g/L potassium chloride and natural pH.

(5) Gas chromatographic analysis: performing gas chromatography analysis on the microbial oil obtained in the step (4) to obtain the results shown in the table 3:

table 3 content of each fatty acid in DHA oil of example 4

Fatty acid composition	Content (wt%)
		14:0	5.27％
16:0	15.00％
		18:0	0.41％
18:1	1.22％
		18:2	2.43％
20:4n6	3.67％
		20:5n3(EPA)	1.95％
22:5n6	15.00％
		22:5n3	1.57％
22:6n3(DHA)	52.37％

In the DHA oil prepared by the mutant strain in the embodiment, the content of palmitic acid C16:0 is 15.0 wt%, the content of stearic acid C18:0 is 0.41 wt%, and the content of DHA reaches 52.37 wt%.

Example 5 production of docosahexaenoic acid Using Schizochytrium limacinum mutant strains

(2) Seed amplification culture: inoculating the activated seed culture solution cultured by shaking in the step (1) into an amplification culture medium according to the inoculation amount of 10 percent (volume ratio) for culture, and sequentially inoculatingSelecting a volume of 100L, 1.7m³，12m³The seed tank is used for gradually expanding and culturing the seed liquid, the culture temperature is 28 ℃, the culture time is 48h, the shaking rotating speed of a shaking table is 200r/min, and the expanding culture medium in the seed tank is as follows: 40g/L glucose, 25g/L sodium glutamate, 10g/L yeast extract, 10g/L sodium chloride, 5g/L magnesium sulfate, 1g/L potassium dihydrogen phosphate, 0.5g/L calcium chloride and natural pH.

(3) And (3) fermentation bottle culture: inoculating the expanded seed culture solution cultured by shaking in the step (2) to a container containing 30m of the culture solution according to the inoculation amount of 10 percent (volume ratio)³50m of fermentation Medium³Performing fermentation culture in a fermentation tank at the culture temperature of 28 ℃ for 120h at the stirring speed of 220 r/min, controlling the carbon source concentration in the fermentation broth to be 10g/L by feeding glucose in the fermentation process, and controlling the pH of the fermentation broth to be 6.8-7.2 by adding citric acid, wherein the fermentation culture medium is as follows: 40g/L glucose, 4g/L yeast extract, 30g/L sodium glutamate, 5g/L sodium chloride, 1g/L monopotassium phosphate, 5g/L magnesium sulfate, 0.5g/L calcium chloride, 0.5g/L sodium bicarbonate, 8g/L sodium sulfate, 6g/L ammonium sulfate, 0.5g/L potassium chloride and natural pH.

(5) Gas chromatographic analysis: performing gas chromatography analysis on the microbial oil obtained in the step (4) to obtain the results shown in the table 4:

table 4 content of each fatty acid in DHA oil of example 5

In the DHA oil prepared by the mutant strain in the embodiment, the content of palmitic acid C16:0 is 14.12 wt%, the content of stearic acid C18:0 is 0.40 wt%, and the content of DHA reaches 54.90 wt%. Example 6 production of docosahexaenoic acid Using Schizochytrium limacinum mutant strains

(2) Seed amplification culture: inoculating the activated seed culture solution cultured by shaking in the step (1) into an amplification culture medium according to the inoculation amount of 10% (volume ratio) for culturing, and sequentially selecting 10L, 50L and 5m³，50m³The seed tank is used for gradually expanding and culturing the seed liquid, the culture temperature is 28 ℃, the culture time is 48 hours, the stirring is 200r/min, and the expanding culture medium in the seed tank is as follows: 40g/L glucose, 25g/L sodium glutamate, 10g/L yeast extract, 10g/L sodium chloride, 5g/L magnesium sulfate, 1g/L potassium dihydrogen phosphate, 0.5g/L calcium chloride and natural pH.

(3) And (3) fermentation bottle culture: inoculating the expanded seed culture solution cultured by shaking in the step (2) to a container with a volume of 130m according to an inoculation amount of 10 percent (volume ratio)³200m of fermentation medium³Fermenting and culturing in a fermentation bottle at the culture temperature of 28 ℃ for 120h under stirring at 220 r/min, wherein the carbon source concentration in the fermentation broth is controlled to be 5g/L by feeding glucose in the fermentation process, the pH of the fermentation broth is controlled to be 6.3-6.8 by adding citric acid, and the fermentation medium is: 40g/L glucose, 4g/L yeast extract, 30g/L sodium glutamate, 5g/L sodium chloride, 1g/L monopotassium phosphate, 5g/L magnesium sulfate, 0.5g/L calcium chloride, 0.5g/L sodium bicarbonate, 8g/L sodium sulfate, 6g/L ammonium sulfate, 0.5g/L potassium chloride and natural pH.

(5) Gas chromatographic analysis: the microbial oil obtained in step (4) was subjected to gas chromatography analysis, and the results shown in table 5 were obtained:

TABLE 5 content of fatty acids in DHA oil and fat in example 6

Fatty acid composition	Content (wt%)
		14:0	5.50％
16:0	12.50％
		18:0	0.40％
18:1	1.90％
		18:2	2.20％
20:4n6	3.01％
		20:5n3(EPA)	1.50％
22:5n6	12.23％
		22:5n3	1.25％
22:6n3(DHA)	57.08％

In the DHA oil prepared by the mutant strain in the embodiment, the content of palmitic acid C16:0 is 12.50 wt%, the content of stearic acid C18:0 is 0.40 wt%, and the content of DHA reaches 57.08 wt%.

In the above examples, it can be seen that the long-chain fatty acid in docosahexaenoic acid produced by the schizochytrium limacinum mutant strain CABIO-A-2-II is very obviously reduced, the fatty acid composition is optimized in A small range by optimizing the culture conditions, the minimum content of palmitic acid C16:0 can reach 12.50 wt%, the minimum content of stearic acid C18:0 can reach 0.40 wt%, and the maximum content of DHA can reach 57.08 wt%.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A Schizochytrium sp CABIO-A-2-II is characterized in that the preservation number of the Schizochytrium sp CABIO-A-2-II is CCTCCM 2019990.

2. A microbial preparation comprising the Schizochytrium sp CABIO-A-2-II mutant strain according to claim 1.

3. A method for producing docosahexaenoic acid oil, which comprises the following steps: fermenting the Schizochytrium sp (CABIO-A-2-II) strain of claim 1 or the microbial inoculum of claim 2, collecting the fermentation product, and separating and purifying.

4. Use of A mutant Schizochytrium sp (Schizochytrium sp) cab io-A-2-ii according to claim 1 or A microbial preparation according to claim 2 for the preparation of docosahexaenoic acid oil.

5. Use according to claim 4, wherein the docosahexaenoic acid oil/fat has a C16:0 content of less than 15.50 wt.%, preferably less than 15.00 wt.%, more preferably less than 14.12 wt.%, even more preferably less than 12.50 wt.%.

6. Use according to claim 5, wherein the docosahexaenoic acid oil/fat has a content of C18:0 of less than 0.51 wt.%, preferably less than 0.41 wt.%, more preferably less than 0.40 wt.%.

7. Use according to claim 5 or 6, wherein the sum of the contents of long-chain saturated fatty acids C16:0 and C18:0 in the docosahexaenoic acid oil is less than 16.01 wt.%, preferably less than 15.41 wt.%, more preferably less than 14.52 wt.%, even more preferably less than 12.90 wt.%.

8. Use according to any of claims 5 to 7, wherein the docosahexaenoic acid lipid has a DHA content higher than 50.40 wt%, preferably higher than 52.37 wt%, further preferably higher than 54.90 wt%, and even further preferably higher than 57.08 wt%.

9. Use of A mutant strain of Schizochytrium (Schizochytrium sp) CABIO-A-2-ii according to claim 1 or A bacterial agent according to claim 2 or A docosahexaenoic acid oil or fat as described in any one of claims 4 to 8 for the preparation of A food product, feed, cosmetic or pharmaceutical.

10. Microbial oil, characterized in that the content of docosahexaenoic acid oil in the microbial oil is at least 50%, and the content of long-chain saturated fatty acid C16:0 in the docosahexaenoic acid oil is less than 15.50 wt%, preferably less than 15.00 wt%, further preferably less than 14.12 wt%, and further preferably less than 12.50 wt%.