阅读说明：本技术 一种微生物来源的dha油脂及其制备方法、以及功能食品 (Microbial-derived DHA grease, preparation method thereof and functional food ) 是由 杨启伟 谭少伟 蔡双山 潘其军 汪益红 刘慧芳 胡锐 于 2021-01-26 设计创作，主要内容包括：本发明公开一种微生物来源的DHA油脂及其制备方法、以及功能食品,所述微生物来源的DHA油脂包括不饱和脂肪酸成分和饱和脂肪酸成分,所述饱和脂肪酸成分包括棕榈酸,所述不饱和脂肪酸成分包括第一不饱和脂肪酸和第二不饱和脂肪酸,所述第一不饱和脂肪酸为二十二碳六稀酸；其中,以甘油三脂计,所述饱和脂肪酸占所述微生物来源的DHA油脂总脂肪酸的含量不高于60％,所述二十二碳六稀酸占所述微生物来源的DHA油脂总脂肪酸的含量不低于35％,所述第二不饱和脂肪酸中任意一种不饱和脂肪酸占所述微生物来源的DHA油脂总脂肪酸的含量均不高于1％。本发明提供的微生物来源的DHA油脂的抗氧化性能明显改善。(The invention discloses a microbial DHA grease, a preparation method thereof and a functional food, wherein the microbial DHA grease comprises an unsaturated fatty acid component and a saturated fatty acid component, the saturated fatty acid component comprises palmitic acid, the unsaturated fatty acid component comprises a first unsaturated fatty acid and a second unsaturated fatty acid, and the first unsaturated fatty acid is docosahexaenoic acid; wherein, the content of the saturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 60%, the content of the docosahexamine in the total fatty acid of the DHA oil derived from the microorganism is not lower than 35%, and the content of any one of the unsaturated fatty acids in the second unsaturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 1%. The DHA grease from the microorganisms provided by the invention has obviously improved oxidation resistance.)

1. A DHA oil derived from microorganisms is characterized by comprising an unsaturated fatty acid component and a saturated fatty acid component, wherein the saturated fatty acid component comprises palmitic acid, the unsaturated fatty acid component comprises a first unsaturated fatty acid and a second unsaturated fatty acid, and the first unsaturated fatty acid is docosahexaenoic acid;

wherein, the content of the saturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 60%, the content of the docosahexamine in the total fatty acid of the DHA oil derived from the microorganism is not lower than 35%, and the content of any one of the unsaturated fatty acids in the second unsaturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 1%.

2. The DHA oil of claim 1, wherein the second unsaturated fatty acid comprises at least one of an omega-3 type unsaturated fatty acid, an omega-6 type unsaturated fatty acid, and an omega-9 type unsaturated fatty acid.

3. The microbial-derived DHA oil according to claim 2, wherein the second unsaturated fatty acid comprises eicosatetraenoic acid and eicosapentaenoic acid.

4. The microbial-origin DHA oil according to claim 1, wherein the palmitic acid accounts for not less than 28% of the total fatty acids of the microbial-origin DHA oil in terms of triglyceride.

5. The microbial-origin DHA oil according to claim 1, wherein the sum of the docosahexaenoic acid and the palmitic acid is not less than 70% of the total fatty acids of the microbial-origin DHA oil in terms of triglyceride.

6. The DHA oil of claim 1, wherein the saturated fatty acid component further comprises at least one of tridecanoic acid, tetradecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid, and tetracosanoic acid.

7. The microbial-origin DHA oil according to claim 1, wherein the saturated fatty acid component comprises tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid and tetracosanoic acid, and the content of the tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid and tetracosanoic acid in the microbial-origin DHA oil is 3.4-3.6%, 6.5-7.0%, 28-35.2%, 1.6-1.9%, 1.8-2.0%, 0.28-0.32%, 0.88-1.0% and 0.6-0.7%, respectively;

the unsaturated fatty acid components comprise arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, and the contents of the eicosatetraenoic acid, the eicosapentaenoic acid and the docosahexaenoic acid in the total fatty acid of the DHA grease from the microorganism are respectively 0.35-0.95%, 0.4-1.0% and 35-46.5%.

8. A method for preparing DHA oil derived from microorganisms according to any one of claims 1 to 7, characterized by comprising the following steps:

fermenting microorganisms to obtain DHA grease from the microorganisms, wherein vegetable oil is added as a defoaming agent in the fermentation process.

9. The method for producing a DHA oil derived from a microorganism according to claim 8, wherein the step of obtaining the DHA oil derived from a microorganism by fermenting a microorganism, comprises:

activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture at the temperature of 22-29 ℃ and the speed of 200rpm for 36-48 hours to obtain shake-flask seed liquid;

inoculating the shake flask seed solution into a first-stage seed tank, culturing for h under the conditions of 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the first-stage seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain first-stage fermentation liquor;

inoculating the primary fermentation liquid into a secondary seed tank, culturing for 12-24 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the secondary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain secondary fermentation liquid;

inoculating the secondary fermentation liquor into a tertiary seed tank, culturing for 72-120 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the tertiary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain tertiary fermentation liquor;

and breaking the walls of the thalli in the third-stage fermentation liquor and extracting oil components in the broken walls of the thalli to obtain the DHA oil from the microorganisms.

10. A functional food comprising the DHA oil-and-fat derived from the microorganism according to any one of claims 1 to 7.

Technical Field

The invention relates to the technical field of functional grease, and particularly relates to microbial-derived DHA grease, a preparation method thereof and functional food.

Background

With the change of consumption concept and the improvement of living standard, the health care consciousness of people is increasingly strengthened. The importance of polyunsaturated fatty acids (PUFAs) in human health has become increasingly important. Among them, docosahexaenoic acid (abbreviated as DHA) is favored by people for its important physiological functions in human and animal bodies, plays an important role in the development of the visual system and the nervous system of infants, and also has the effects of reducing cholesterol, preventing senile dementia and the like.

The traditional DHA is usually obtained from deep sea fish oil, and has the problems of unstable yield, low yield, high cost and the like, so the research of producing DHA by fermenting marine microorganisms is proposed, and the commonly used microorganisms comprise schizochytrium, crypthecodinium and the like. However, the existing DHA grease prepared by microbial fermentation has the problems of low DHA content and unsatisfactory antioxidant performance, and needs to be further improved.

Disclosure of Invention

The invention mainly aims to provide a DHA grease from microorganisms, a preparation method thereof and a functional food, and aims to improve the oxidation resistance of the DHA grease.

In order to achieve the above object, the present invention provides a DHA oil of microbial origin, comprising an unsaturated fatty acid component and a saturated fatty acid component, wherein the saturated fatty acid component comprises palmitic acid, the unsaturated fatty acid component comprises a first unsaturated fatty acid and a second unsaturated fatty acid, and the first unsaturated fatty acid is docosahexaenoic acid;

wherein, the content of the saturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 60%, the content of the docosahexamine in the total fatty acid of the DHA oil derived from the microorganism is not lower than 35%, and the content of any one of the unsaturated fatty acids in the second unsaturated fatty acid in the total fatty acid of the DHA oil derived from the microorganism is not higher than 1%.

Optionally, the second unsaturated fatty acid comprises at least one of an omega-3 type unsaturated fatty acid, an omega-6 type unsaturated fatty acid, and an omega-9 type unsaturated fatty acid.

Optionally, the second unsaturated fatty acid comprises arachidonic acid and eicosapentaenoic acid.

Optionally, the palmitic acid content is not less than 28% of the total fatty acids of the microbial source DHA oil based on triglycerides.

Optionally, the sum of the docosahexaenoic acid and the palmitic acid accounts for not less than 70% of the total fatty acids of the microbial-derived DHA oil based on triglycerides.

Optionally, the saturated fatty acid component further comprises at least one of tridecanoic acid, tetradecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid, and tetracosanoic acid.

Optionally, the saturated fatty acid component comprises tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid and tetracosanoic acid, wherein the tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid and tetracosanoic acid respectively account for 3.4-3.6%, 6.5-7.0%, 28-35.2%, 1.6-1.9%, 1.8-2.0%, 0.28-0.32%, 0.88-1.0% and 0.6-0.7% of the total fatty acids of the microbial-derived DHA fat;

the unsaturated fatty acid components comprise arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, and the contents of the eicosatetraenoic acid, the eicosapentaenoic acid and the docosahexaenoic acid in the total fatty acid of the DHA grease from the microorganism are respectively 0.35-0.95%, 0.4-1.0% and 35-46.5%.

Further, the invention also provides a preparation method of the DHA grease from the microorganisms, which comprises the following steps:

fermenting microorganisms to obtain DHA grease from the microorganisms, wherein vegetable oil is added as a defoaming agent in the fermentation process.

Optionally, the step of fermenting the microorganism to obtain the DHA oil derived from the microorganism comprises:

activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture at the temperature of 22-29 ℃ and the speed of 200rpm for 36-48 hours to obtain shake-flask seed liquid;

inoculating the shake flask seed solution into a first-stage seed tank, culturing for 12-24 h at 22-29 ℃ and 200rpm, and controlling aeration flow and rotation speed during the culture period to maintain the dissolved oxygen condition in the first-stage seed tank at 20-90% and 0.01-0.03 MPa to obtain first-stage fermentation liquor;

inoculating the primary fermentation liquid into a secondary seed tank, culturing for 72-120 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the secondary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain secondary fermentation liquid;

inoculating the secondary fermentation liquor into a tertiary seed tank, culturing for 72-120 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the tertiary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain tertiary fermentation liquor;

and breaking the walls of the thalli in the third-stage fermentation liquor and extracting oil components in the broken walls of the thalli to obtain the DHA oil from the microorganisms.

Furthermore, the invention also provides a functional food, which comprises the DHA oil derived from the microorganisms.

In the technical scheme provided by the invention, the microbial-derived DHA grease comprises palmitic acid, docosahexaenoic acid and second unsaturated fatty acids, wherein the content of the docosahexaenoic acid in total fatty acids of the microbial-derived DHA grease is not less than 35% and the content of any unsaturated fatty acid in the second unsaturated fatty acids in total fatty acids of the microbial-derived DHA grease is not more than 1% in terms of triglyceride; therefore, the antioxidant performance of the DHA grease from the microorganisms provided by the invention is obviously improved compared with that of the DHA grease prepared by the existing microbial method, and the oxidation induction period of the DHA grease at 90 ℃ is more than 24 h.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a schematic flow chart of an embodiment of a method for preparing DHA oil from microorganisms according to the present invention.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to improve the oxidation resistance of DHA grease prepared by traditional microbial fermentation, the invention provides the DHA grease from microorganisms, which comprises an unsaturated fatty acid component and a saturated fatty acid component, wherein the saturated fatty acid component comprises palmitic acid (namely hexadecanoic acid), the unsaturated fatty acid component comprises a first unsaturated fatty acid and a second unsaturated fatty acid, the first unsaturated fatty acid is docosahexa-acid, and the second unsaturated fatty acid is unsaturated fatty acid except docosahexa-acid; wherein, calculated as triglyceride (calculated as the content of all oils and fats related to unsaturated fatty acid, saturated fatty acid and the like in the following text, the content of the saturated fatty acid in the total fatty acid of the DHA oil and fat derived from the microorganism is not higher than 60%, the content of the docosahexa-acid in the total fatty acid of the DHA oil and fat derived from the microorganism is not lower than 35%, and the content of any unsaturated fatty acid in the second unsaturated fatty acid in the total fatty acid of the DHA oil and fat derived from the microorganism is not higher than 1%.

In the technical scheme provided by the invention, the microbial-derived DHA grease comprises palmitic acid, docosahexaenoic acid and second unsaturated fatty acids, wherein the content of the docosahexaenoic acid in total fatty acids of the microbial-derived DHA grease is not less than 35% and the content of any unsaturated fatty acid in the second unsaturated fatty acids in total fatty acids of the microbial-derived DHA grease is not more than 1% in terms of triglyceride; therefore, the antioxidant performance of the DHA grease from the microorganisms provided by the invention is obviously improved compared with that of the DHA grease prepared by the existing microbial method, and the oxidation induction period of the DHA grease at 90 ℃ is more than 24 h.

The second unsaturated fatty acid is an unsaturated fatty acid other than docosahexaenoic acid, and in the embodiment of the present invention, at least one of an ω -3 type unsaturated fatty acid, an ω -6 type unsaturated fatty acid, and an ω -9 type unsaturated fatty acid may be specifically selected, and any one of the above-mentioned types of unsaturated fatty acids may be selected, or a mixture of two or more of them may be selected. In some embodiments provided herein, the second unsaturated fatty acid comprises arachidonic acid and eicosapentaenoic acid.

Further, in a specific embodiment of the present invention, the palmitic acid accounts for not less than 28% of the total fatty acids of the DHA oil of microbial origin, based on triglycerides. Therefore, the total content of the docosahexamine acid and the palmitic acid is not less than 63%, the oxidation resistance of the DHA grease from the microorganisms is ensured, and the nutrition is more balanced. Still further, in some embodiments provided by the present invention, the sum of the docosahexaenoic acid and the palmitic acid accounts for not less than 70% of the total fatty acids of the DHA oil derived from the microorganism, which is more favorable for further improvement of the antioxidant property of the DHA oil.

In addition to the palmitic acid, the saturated fatty acid component may include other saturated fatty acids, as long as the total content of the saturated fatty acid component is not more than 60%, and in the embodiment of the present invention, at least one selected from tridecanoic acid, tetradecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid and tetracosanoic acid is included, but not limited to, any one of the above saturated fatty acids, or a mixture of two or more thereof, which falls within the protection scope of the present invention.

Preferably, in an embodiment of the microbial DHA grease provided by the present invention, the saturated fatty acid component includes tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid, and tetracosanoic acid, and the content of the tridecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, heneicosanoic acid, and tetracosanoic acid in the total fatty acids of the microbial DHA grease is 3.4-3.6%, 6.5-7.0%, 28-35.2%, 1.6-1.9%, 1.8-2.0%, 0.28-0.32%, 0.88-1.0%, and 0.6-0.7, respectively; the unsaturated fatty acid components comprise arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, and the contents of the eicosatetraenoic acid, the eicosapentaenoic acid and the docosahexaenoic acid in the total fatty acid of the DHA grease from the microorganism are respectively 0.35-0.95%, 0.4-1.0% and 35-46.5%. According to the DHA grease from the microorganisms provided by the embodiment of the invention, the proportion of each grease component is strictly controlled, so that the sum of the total content of the docosahexaenoic acid and the palmitic acid is higher than 70%, the content of each unsaturated fatty acid is not more than 1%, and the total content of the saturated fatty acid is not more than 50%, so that the antioxidant property of the DHA grease from the microorganisms is optimal.

Based on the microbial DHA grease, the invention also provides a preparation method of the microbial DHA grease, and the DHA grease is prepared by a microbial fermentation process. Specifically, in an embodiment of the method for preparing DHA oil derived from microorganisms provided by the present invention, the method for preparing DHA oil derived from microorganisms includes the following steps:

fermenting the microorganisms to obtain DHA-containing grease, wherein vegetable oil is added as a defoaming agent in the fermentation process.

The antifoaming agent may be selected from vegetable oils such as palmitic acid and soybean oil, and the addition amount thereof may be 0.5 to 2%, preferably 1%, of the total mass of the fermented product. The DHA-containing grease is prepared by microbial fermentation, and the fermentation raw materials are all natural except inorganic salts and do not contain any chemical synthesis position, so that the DHA-containing grease obtained by fermentation is higher in safety when used as a food raw material.

Specifically, in this embodiment, the preparation method of the DHA oil derived from microorganisms includes:

s11, activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture at the temperature of 22-29 ℃ and the speed of 200rpm for 36-48 hours to obtain shake-flask seed liquid;

s12, inoculating the shake flask seed liquid into a primary seed tank, culturing for 12-24 h at 22-29 ℃ and 200rpm, and controlling aeration flow and rotation speed during the culture period to maintain the dissolved oxygen condition in the primary seed tank at 20-90% and 0.01-0.03 MPa to obtain primary fermentation liquid;

s13, inoculating the primary fermentation liquid into a secondary seed tank, culturing for 12-24 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the secondary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain secondary fermentation liquid;

s14, inoculating the secondary fermentation liquid into a tertiary seed tank, culturing for 72-120 h at 22-29 ℃ and 200rpm, and controlling the aeration flow and the rotation speed during the culture period to maintain the dissolved oxygen condition in the tertiary seed tank at 20-90% and the air pressure of 0.01-0.03 MPa to obtain tertiary fermentation liquid;

and S15, breaking the walls of the thalli in the tertiary fermentation liquid and extracting grease components in the broken thalli to obtain the DHA grease from the microorganisms.

Wherein the culture medium used for the subculture shake flask culture contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate and the balance of water, wherein the pH value is 5.0-8.0;

the components of the expanding culture medium used for fermentation culture in the first-stage seed tank and the second-stage seed tank are the same, and the expanding culture medium contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 5.0-8.0.

The schizochytrium limacinum is subjected to activation and shake flask culture, then sequentially passes through a first-stage seed tank, a second-stage seed tank and a third-stage seed tank to be subjected to gradual amplification fermentation culture, and during the amplification culture, parameters such as aeration flow, rotation speed and the like are controlled to obtain more dissolved oxygen and maintain the high-pressure condition of carbon dioxide, so that the rapid propagation of microorganisms is promoted. The matching of the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has various modes, including but not limited to 1m³、10m³And 50m³1m of a combination of³、10m³And 100m³Or 5m, or³、50m³And 200m³Combinations of (a) and (b). According to the preparation method of the DHA grease from the microorganisms, provided by the invention, the fermentation process and the formula are controlled, so that the content of saturated fatty acid and unsaturated fatty acid in the prepared DHA grease is distributed according to expectation, the preparation process is simple and feasible, the industrial production is easy to realize, no chemical synthesized substance is contained, and the food safety of the prepared product is higher.

In addition, the invention also provides a functional food, which comprises the DHA grease from the microorganism, and the specific composition of the DHA grease from the microorganism refers to the above examples. Specifically, the functional food includes, but is not limited to, functional foods containing DHA grease such as infant milk powder, pregnant woman milk powder, DHA supplement and the like. It can be understood that, since the functional food of the present invention adopts all the technical solutions of all the embodiments, at least all the beneficial effects brought by the technical solutions of the embodiments are achieved, and no further description is provided herein.

The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.

Example 1

(1) Activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture for 42h at the temperature of 25 ℃ and the speed of 200rpm to obtain shake-flask seed liquid; wherein, the subculture medium contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, the balance of water and the pH value of 7.0;

(2) inoculating the shake flask seed solution into a first-level seed tank (1 m)³) Performing shake culture at 25 deg.C and 200rpm for 18h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the primary seed tank at 50% and air pressure of 0.02MPa to obtain primary fermentation broth;

(3) inoculating the obtained primary fermentation liquid into a secondary seed tank (10 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 18h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the secondary seed tank at 50% and air pressure of 0.02MPa to obtain secondary fermentation liquid;

(4) inoculating the obtained secondary fermentation liquid into a third seed tank (50 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 108h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the three-stage seed tank at 50% and air pressure of 0.02MPa to obtain three-stage fermentation liquid;

the culture medium in the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has the same components and contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 7.0;

(5) and breaking the walls of the thalli in the obtained tertiary fermentation liquor, and extracting grease components in the broken walls of the thalli to obtain the DHA grease from the microorganisms.

The fatty acid composition of the DHA oil derived from microorganisms and the oxidation induction period at 90 ℃ thereof are shown in table 1 below.

Example 2

(1) Activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture at the temperature of 22 ℃ and the speed of 200rpm for 36 hours to obtain shake-flask seed liquid; wherein, the subculture medium contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, the balance of water and the pH value of 7.0;

(2) inoculating the shake flask seed solution into a first-level seed tank (1 m)³) Performing shake culture at 22 deg.C and 200rpm for 12h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the primary seed tank at 30% and air pressure of 0.01MPa to obtain primary fermentation liquid;

(3) inoculating the obtained primary fermentation liquid into a secondary seed tank (10 m)³) Fermenting and culturing at 252 deg.C and 160rpm for 12h, and controlling aeration flow and rotation speed during the culture period to maintain the dissolved oxygen condition in the secondary seed tank at 30% and air pressure of 0.01MPa to obtain secondary fermentation liquid;

(4) inoculating the obtained secondary fermentation liquid into a tertiary seed tank (100 m)³) Fermenting and culturing at 22 deg.C and 160rpm for 72h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the three-stage seed tank at 30% and air pressure of 0.01MPa to obtain three-stage fermentation liquid;

the culture medium in the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has the same components and contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 7.0;

(5) and breaking the walls of the thalli in the obtained tertiary fermentation liquor, and extracting grease components in the broken walls of the thalli to obtain the DHA grease from the microorganisms.

The fatty acid composition of the DHA oil derived from microorganisms and the oxidation induction period at 90 ℃ thereof are shown in table 1 below.

Example 3

(1) Activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture for 48 hours at the temperature of 25 ℃ and the speed of 200rpm to obtain shake-flask seed liquid; wherein, the subculture medium contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, the balance of water and the pH value of 7.0;

(2) inoculating the shake flask seed solution into a first-stage seed tank (5 m)³) Performing shake culture at 25 deg.C and 200rpm for 24h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the primary seed tank at 20% and air pressure of 0.02MPa to obtain primary fermentation broth;

(3) inoculating the obtained primary fermentation liquid into a secondary seed tank (50 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 24h, and controlling aeration flow and rotation speed during the culture period to maintain the dissolved oxygen condition in the secondary seed tank at 20% and air pressure of 0.02MPa to obtain secondary fermentation liquid;

(4) inoculating the obtained secondary fermentation liquid into a third seeding tank (200 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 90 hr, and controlling aeration flow and rotation speed during culture period to maintain dissolved oxygen condition in the three-stage seed tank at 20% and air pressure of 0.02MPa to obtain three-stage fermentation liquid；

The culture medium in the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has the same components and contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 7.0;

(5) and breaking the walls of the thalli in the obtained tertiary fermentation liquor, and extracting grease components in the broken walls of the thalli to obtain the DHA grease from the microorganisms.

The fatty acid composition of the DHA oil derived from microorganisms and the oxidation induction period at 90 ℃ thereof are shown in table 1 below.

Example 4

(1) Activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture for 39h at the temperature of 25 ℃ and the speed of 200rpm to obtain shake-flask seed liquid; wherein, the subculture medium contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, the balance of water and the pH value of 7.0;

(2) inoculating the shake flask seed solution into a first-level seed tank (1 m)³) Performing shake culture at 25 deg.C and 200rpm for 16h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the primary seed tank at 70% and air pressure of 0.03MPa to obtain primary fermentation broth;

(3) inoculating the obtained primary fermentation liquid into a secondary seed tank (10 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 16h, and controlling aeration flow and rotation speed during culture period to make the dissolved oxygen condition in the secondary seed tankMaintaining at 70% and air pressure of 0.03MPa to obtain secondary fermentation liquid;

(4) inoculating the obtained secondary fermentation liquid into a third seed tank (50 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 96 hr, and controlling aeration flow and rotation speed during culture period to maintain dissolved oxygen condition in the three-stage seed tank at 70% and air pressure of 0.03MPa to obtain three-stage fermentation liquid;

the culture medium in the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has the same components and contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 7.0;

(5) and breaking the walls of the thalli in the obtained tertiary fermentation liquor, and extracting grease components in the broken walls of the thalli to obtain the DHA grease from the microorganisms.

The fatty acid composition of the DHA oil derived from microorganisms and the oxidation induction period at 90 ℃ thereof are shown in table 1 below.

Example 5

(1) Activating the schizochytrium limacinum, inoculating the activated schizochytrium limacinum to a subculture medium, and performing shake-flask culture for 45 hours at the temperature of 25 ℃ and the speed of 200rpm to obtain shake-flask seed liquid; wherein, the subculture medium contains the following components: 5g/L glucose, 5g/L glycerol, 1g/L yeast extract, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, the balance of water and the pH value of 7.0;

(2) inoculating the shake flask seed solution into a first-level seed tank (1 m)³) In the culture, the medium is subjected to shake-flask culture at 25 ℃ and 200rpm for 20 hours, during which aeration flow rate androtating speed to maintain the dissolved oxygen condition in the primary seed tank at 90% and the air pressure at 0.02MPa to obtain primary fermentation liquid;

(3) inoculating the obtained primary fermentation liquid into a secondary seed tank (10 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 20h, and controlling aeration flow and rotation speed during the culture period to maintain dissolved oxygen condition in the secondary seed tank at 90% and air pressure of 0.02MPa to obtain secondary fermentation liquid;

(4) inoculating the obtained secondary fermentation liquid into a third seed tank (50 m)³) Fermenting and culturing at 25 deg.C and 160rpm for 120h, and controlling aeration flow and rotation speed during culture period to maintain dissolved oxygen condition in the three-stage seed tank at 90% and air pressure of 0.02MPa to obtain three-stage fermentation liquid;

the culture medium in the first-stage seed tank, the second-stage seed tank and the third-stage seed tank has the same components and contains the following components: 20g/L glucose, 10g/L glycerol, 1g/L yeast extract, 1g/L soybean peptone, 1g/L corn steep liquor, 15g/L sodium chloride, 1g/L magnesium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L potassium chloride, 0.01g/L calcium chloride, 0.5g/L ammonium sulfate, 0.01g/L sodium bicarbonate, 0.01mg/L manganese chloride, 0.5mg/L ferrous chloride, 5mg/L cyanocobalamin, 0.1mg/L zinc chloride, 0.01mg/L cobalt chloride, 0.01mg/L copper sulfate, 0.1mg/L nickel sulfate hexahydrate, 0.1mg/L pantothenic acid, 0.1mg/L biotin, and the balance of water, wherein the pH value is 7.0;

(5) and breaking the walls of the thalli in the obtained tertiary fermentation liquor, and extracting grease components in the broken walls of the thalli to obtain the DHA grease from the microorganisms.

The fatty acid composition of the DHA oil derived from microorganisms and the oxidation induction period at 90 ℃ thereof are shown in table 1 below.

Table 1 fatty acid composition and antioxidant performance comparison of DHA oil and fat provided in example and comparative example

As can be seen from table 1 (in table 1, comparative example 1 is a commercially available microbial DHA oil), the microbial DHA oil prepared in the embodiment of the present invention has more types of fatty acids, wherein the content of docosahexaenoic acid reaches more than 35%, which is significantly higher than that in comparative example 1; meanwhile, the content of palmitic acid is not less than 28%, the total content of palmitic acid and docosahexaenoic acid is not less than 70%, and the content of unsaturated fatty acids except docosahexaenoic acid is not more than 1%, so that the oxidation induction period (at 90 ℃) of the DHA grease of the embodiment of the invention reaches more than 24 h.

The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.