阅读说明：本技术 富含Sn-2位ARA的微生物油脂及其制备方法和应用 (Microbial oil rich in Sn-2 ARA and preparation method and application thereof ) 是由 梁云 曹晟 王身健 于 2019-11-26 设计创作，主要内容包括：本发明涉及微生物技术领域,公开了一种富含Sn-2位ARA的微生物油脂及其制备方法和应用。所述微生物油脂中甘油三酯Sn-2位上ARA的比例不低于23％,所述微生物油脂的制备方法包括将高山被孢霉菌株接种到发酵培养基中发酵制得,所述高山被孢霉菌株为保藏编号为GDMCC No.60734的高山被孢霉(Mortierella alpina)。该微生物油脂中甘油三酯Sn-2位上ARA的比例显著高于甘油三酯Sn-1、Sn-3位上ARA的比例,从而使该微生物油脂中ARA的人体吸收率明显高于常规菌株生产的微生物油脂,促进人体对功能性脂肪酸ARA的吸收与利用。(The invention relates to the technical field of microorganisms, and discloses a microbial oil rich in Sn-2 ARA, and a preparation method and application thereof. The proportion of ARA on the Sn-2 position of triglyceride in the microbial oil is not less than 23 percent, and the preparation method of the microbial oil comprises the step of inoculating a Mortierella alpina strain into a fermentation culture medium for fermentation to obtain the microbial oil, wherein the Mortierella alpina strain is Mortierella alpina (Mortierella alpina) with the preservation number of GDMCC No. 60734. The proportion of ARA on the Sn-2 position of triglyceride in the microbial oil is obviously higher than that of ARA on the Sn-1 position and Sn-3 position of triglyceride, so that the human body absorption rate of the ARA in the microbial oil is obviously higher than that of the microbial oil produced by conventional strains, and the absorption and utilization of the functional fatty acid ARA by a human body are promoted.)

1. A microbial oil rich in ARA at the Sn-2 position, wherein the proportion of ARA at the Sn-2 position of triglyceride in the microbial oil is not less than 23%.

2. The method of producing a microbial oil according to claim 1, wherein the method comprises inoculating a strain of Mortierella alpina (Mortierella alpina) deposited under GDMCC No.60734 to a fermentation medium and fermenting the strain.

3. The method of claim 2, wherein the fermentation medium comprises a carbon source and a nitrogen source, and the fermentation conditions comprise a temperature of 27-31 ℃, a time of 4-8 days, an aeration ratio of 0.5-1.1VVM, a carbon-nitrogen ratio of (3-18): 1. the inoculation amount is 5-10%.

4. The method according to claim 2 or 3, wherein the Mortierella alpina strain is activated, expanded to obtain a seed solution, and then inoculated into the fermentation medium for fermentation.

5. The method according to claim 4, wherein the activation and propagation comprises the following steps:

(1) inoculating the mortierella alpina strain to a PDA slant, culturing until spores are generated and mature, and eluting with sterile water to obtain a spore suspension;

(2) inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium for culture and expansion to obtain shake flask seed liquid;

(3) and (3) inoculating the shake flask seed solution obtained in the step (2) into a first-stage seed tank for culturing for 1-2 days, and then inoculating into a second-stage seed tank for culturing for 1-2 days to form the seed solution.

6. The method of claim 5, wherein step (1) comprises: inoculating the mortierella alpina on a PDA test tube inclined plane, culturing for 5-7 days at 25-30 ℃, transferring the generated spores to a PDA eggplant bottle inclined plane, culturing for 4-6 days at 25-30 ℃ until mature spores are generated, taking hypha and spores on a culture medium, and preparing the spore suspension by using sterile water.

7. The method of claim 5, wherein the step (2) comprises: inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium for culture, wherein the culture temperature is 25-30 ℃, the culture time is 5-8 days, the shaking speed of a shaking table is 180-200r/min, and the carbon source and the nitrogen source in the shake flask seed culture medium are respectively 50-70g/L and 15-25g/L, pH respectively 6.5-7.2.

8. The method of claim 5, wherein in step (3), the culturing conditions of the first and second seed tanks comprise: the temperature is 27-31 deg.C, the time is 1-2 days, the aeration ratio is 0.5-0.8VVM, and the carbon nitrogen ratio is (3-8): 1.

9. The method of claim 3, 7 or 8, wherein the carbon source is glucose or/and starch and the nitrogen source is one or more of peptone, yeast powder, yeast extract and corn steep liquor powder.

10. The method of claim 2, further comprising extracting the fermented product to obtain a lipid product in the fermented product.

11. The triglyceride type microbial oil produced by the method according to any one of claims 2 to 10, which has an ARA content at Sn-2 position on the glycerin skeleton of not less than 23% and an ARA content of the triglyceride type of not less than 38%.

12. Use of the triglyceride-type microbial oil according to claim 11 in food products, preferably infant formulas, health foods and health foods.

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a microbial oil rich in Sn-2 ARA, and a preparation method and application thereof

Background

Lipids are one of the essential nutrients for the human body, not only supply body energy, but also constitute essential raw materials for cells and tissues, and also provide fat-soluble vitamins. However, in order to play these important roles in vivo, they are first digested and absorbed by the intestine, lipid digestion and absorption mainly occur in the small intestine, and lipases of the pancreas and the small intestine and bile salts are involved in digestion and absorption, and the alkaline environment formed by bicarbonate secretion by the pancreas and bile is also an indispensable environmental condition. The efficiency with which lipids are absorbed by the body depends on the various enzymes involved in digestion and their mechanisms of action, as well as the structural form of the lipids. Numerous studies have shown that the structural form of lipids greatly influences the absorption of lipids by the human body. The lipid with triglyceride structure is divided into Sn-1, Sn-2 and Sn-3 fatty acids according to the combination position of the lipid in a glycerol skeleton, pancreatic lipase is a main fat digestive enzyme which plays a role by being adsorbed on an oil-water interface, and simultaneously has a specific ester bond decomposition function, namely, only the ester bonds of Sn-1 and Sn-3 are concentrated and hydrolyzed, so that after the lipid with triglyceride structure is decomposed by the pancreatic lipase, the Sn-1 and Sn-3 fatty acids form free fatty acids, and the Sn-2 fatty acids form monoglyceride, while the free fatty acids are difficult to permeate into bile salt micelle and be absorbed by human body, thereby being easy to combine with calcium and magnesium ions in intestinal tract to form insoluble soap salt and be lost, and the Sn-2 fatty acids form monoglyceride and are easy to permeate into the bile salt micelle and be absorbed by human body, therefore, the Sn-2 site fatty acid has higher human body absorptivity than the Sn-1 and Sn-3 site fatty acids.

Microbial oil, which is one of lipids and contains abundant polyunsaturated fatty acids, has been widely applied to infant food and health food with the enhancement of health consciousness, the nutritional efficacy of the microbial oil is recognized and approved by the public more and more, and the absorption rate of the polyunsaturated fatty acids, i.e. arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are functional factors in the microbial oil, is also concerned by the public more and more. More than 90% of fatty acids in the microbial oil are in a triglyceride structure form, the distribution proportion of the fatty acids in the existing microbial oil on a glycerol skeleton is far lower than that of Sn-2-site fatty acids and Sn-1-3-site fatty acids, and a large amount of Sn-1-3-site fatty acids are lost due to soap salt formation in the digestion process of a human body, so that the health care effect of the microbial oil is limited.

Disclosure of Invention

The invention aims to solve the problem that the absorption rate of fatty acids by a human body is low due to high proportion of fatty acids at Sn-1 and Sn-3 sites in triglyceride in microbial oil in the prior art, and provides the microbial oil rich in Sn-2 site ARA, and the preparation method and the application thereof.

In order to achieve the above object, the present invention provides a microbial oil rich in ARA at Sn-2 position, in which the proportion of ARA at Sn-2 position of triglyceride is not less than 23%, and the content of ARA in triglyceride type is not less than 38%.

The invention further provides a preparation method of the microbial oil, which comprises the step of inoculating the Mortierella alpina strain into a fermentation culture medium for fermentation to obtain the microbial oil, wherein the Mortierella alpina strain is Mortierella alpina (Mortierella alpina) with the preservation number of GDMCC No. 60734.

Preferably, the fermentation medium comprises a carbon source and a nitrogen source, and the fermentation conditions comprise a temperature of 27-31 ℃, a time of 4-8 days, an aeration ratio of 0.5-1.1VVM and a carbon-nitrogen ratio of (3-18): 1. the inoculation amount is 5-10%.

Preferably, the mortierella alpina strain is activated, expanded and cultured into a seed solution, and then inoculated into a fermentation medium for fermentation.

Preferably, the activation and propagation comprises the following steps:

(1) inoculating the mortierella alpina strain to a PDA slant, culturing until spores are generated and mature, and eluting with sterile water to obtain a spore suspension;

(2) inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium for culture and expansion to obtain shake flask seed liquid;

(3) and (3) inoculating the shake flask seeds obtained in the step (2) into a first-stage seed tank for culture, and inoculating the shake flask seeds into a second-stage seed tank for culture for 1-2 days after the shake flask seeds are cultured for 1-2 days to form the seed solution.

Preferably, the step (1) comprises: inoculating the mortierella alpina on a PDA test tube inclined plane, culturing for 5-7 days at 25-30 ℃, transferring the generated spores to a PDA eggplant bottle inclined plane, culturing for 4-6 days at 25-30 ℃ until mature spores are generated, taking hypha and spores on a culture medium, and preparing the spore suspension by using sterile water.

Preferably, the step (2) includes: inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium for culture, wherein the culture temperature is 27-31 ℃, the culture time is 3-6 days, the shaking speed of a shaking table is 180-200r/min, and the carbon source and the nitrogen source in the shake flask seed culture medium are respectively 50-70g/L and 15-25g/L, pH respectively and respectively in the range of 7.5-8.5.

Preferably, in step (3), the culture conditions of the first and second seed tanks include: the temperature is 27-31 deg.C, the time is 1-2 days, the aeration ratio is 0.5-0.8VVM, and the carbon nitrogen ratio is (3-8): 1.

Preferably, the carbon source is glucose or/and starch, and the nitrogen source is one or more of peptone, yeast powder, yeast extract and corn steep liquor powder.

Preferably, the method further comprises extracting the fermented product to obtain a grease product in the fermented product.

The present invention further provides the triglyceride type microbial oil prepared by the above method, wherein the content of ARA at Sn2 position on the glycerol skeleton is not less than 23%, and the content of ARA of triglyceride type is not less than 38%.

The present invention further provides the use of the triglyceride type microbial oil prepared by the above method in food, preferably infant formula, health food and health food.

Through the technical scheme, the microbial oil produced by fermenting the mortierella alpina strain provided by the invention is rich in ARA, the ratio of ARA on the Sn-2 position of triglyceride is obviously higher than the ratio of ARA on the Sn-1 position and the Sn-3 position of triglyceride, the absorptivity of a human body to the ARA in the microbial oil is improved, and thus, the absorption and utilization of the functional fatty acid ARA by the human body are effectively promoted.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The strains of the invention are as follows:

mortierella alpina (Mortierella alpina) was deposited at the microbial culture Collection center of Guangdong province 8/8 in 2019 (address: No. 59, 5 th building of Michelia furiosa No. 100, Guangdong province, microbial research institute, postal code: 510070) (abbreviated as GDMCC in the depository), and the accession number is GDMCC No. 60734.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a microbial oil rich in Sn-2 ARA, wherein the proportion of ARA on Sn-2 of triglyceride in the microbial oil is not less than 23%.

The invention further provides a preparation method of the microbial oil, which comprises the step of inoculating the Mortierella alpina strain into a fermentation culture medium for fermentation to obtain the microbial oil, wherein the Mortierella alpina strain is Mortierella alpina (Mortierella alpina) with the preservation number of GDMCC No. 60734.

The Mortierella alpina provided by the present invention is fermented to produce the ARA-rich microbial oil, and the fermentation method is not particularly required as long as the Mortierella alpina can be proliferated.

Wherein, the temperature, time, ventilation and carbon-nitrogen ratio of the mortierella alpina fermentation are not particularly required, and can be the selection commonly used for obtaining mortierella alpina fermentation products, in order to obtain more fermentation products, preferably, the fermentation medium comprises a carbon source and a nitrogen source, the fermentation conditions comprise the temperature of 27-31 ℃, the time of 4-8 days, the ventilation ratio of 0.5-1.1VVM and the carbon-nitrogen ratio of (3-18): 1. the inoculation amount is 5-10%.

Wherein, in the process of inoculating the mortierella alpina into a fermentation medium for fermentation, a nitrogen source required by the formula is added into the fermentation medium at one time; the carbon source can be continuously supplemented according to the requirement of the carbon-nitrogen ratio in the fermentation process except for the basic adding amount in the fermentation culture medium, and is not supplemented until the residual sugar content in the culture medium is 0 when the fermentation end point is approached, so that the carbon-nitrogen ratio is regulated and controlled to form an auxotrophic nutritional condition, and the level of oil yield of the Mortierella alpina fermentation is ensured.

In order to obtain more fermentation products, the mortierella alpina strain is activated, expanded and cultured into seed liquid, and then inoculated into a fermentation medium for fermentation.

Preferably, the activation and propagation comprises the following steps:

(1) inoculating the mortierella alpina strain on a PDA slant, and culturing until spores are generated and mature to obtain a spore suspension;

(2) inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium to culture so as to obtain shake flask seed liquid;

(3) and (3) inoculating the shake flask seed solution obtained in the step (2) into a first-stage seed tank for culturing for 1-2 days, and then inoculating into a second-stage seed tank for culturing for 1-2 days to form the seed solution.

Further preferably, the step (1) comprises: inoculating the mortierella alpina on a PDA test tube inclined plane, culturing for 5-7 days at 25-30 ℃, transferring the generated spores to a PDA eggplant bottle inclined plane, culturing for 4-6 days at 25-30 ℃ until mature spores are generated, taking hypha and spores on a culture medium, and preparing the spore suspension by using sterile water.

Further preferably, the step (2) includes: inoculating the spore suspension obtained in the step (1) into a shake flask seed culture medium for culture, wherein the culture temperature is 25-30 ℃, the culture time is 3-6 days, the shaking speed of a shaking table is 180-200r/min, and the carbon source and the nitrogen source in the shake flask seed culture medium are respectively 50-70g/L and 15-25g/L, pH respectively 6.8-7.2. Specifically, the inoculation amount in the shake flask seed culture medium is 2-3 triangular flasks per eggplant bottle seed, wherein the specifications of the eggplant bottles and the triangular flasks are both 500 mL.

Specifically, in the step (3), the culture conditions of the first-stage seeding tank and the second-stage seeding tank comprise: the temperature is 27-31 deg.C, the time is 1-2 days, the aeration ratio is 0.5-0.8VVM, and the carbon nitrogen ratio is (3-8): 1.

In the invention, the carbon source can be glucose, starch or any other substance capable of providing a carbon source, and the nitrogen source can be peptone, yeast powder, corn steep liquor powder or any other substance capable of providing a nitrogen source. Preferably, the carbon source is glucose or/and starch, and the nitrogen source is one or more of peptone, yeast powder, yeast extract and corn steep liquor powder.

The present invention may further process the fermented product to obtain the microbial oil, and the processing method is not particularly limited as long as the microbial oil can be separated from the fermented product, and the processing includes extracting the fermented product to obtain the oil product in the fermented product in order to obtain a high yield of the microbial oil.

The invention further provides the triglyceride type microbial oil prepared by the method, wherein the content of Sn-2 ARA on a glycerol skeleton is not less than 23%.

The present invention further provides the use of the triglyceride-type microbial oil prepared by the above method in food, preferably infant formula, health food or health food.

The present invention will be described in detail below by way of examples.

In the following examples, the content of ARA in microbial oil and the fatty acid composition were measured by the method of "determination of fatty acids in food safety national standard for food" GB 5009.168-2016; the content of fatty acid at Sn2 adopts a formula of GB/T24984-2010/ISO 6800: 1997 determination of fatty acid components at 2-position of animal and vegetable oil triglyceride molecule; the proportion of ARA on the Sn-1 and Sn-3 sites of the triglyceride is calculated by adopting a 1, 3-random-2-random distribution theory on the basis of analysis and determination of full-sample fatty acid and Sn-2 site fatty acid; the method for measuring the absorptivity of the human body to the ARA is a method for testing the efficacy of the human body: collecting ARA grease produced by male and female volunteers by eating the method and ARA grease produced by conventional strains, and then extracting blood to measure the content of ARA fatty acid to calculate the absorption rate; the conventional Mortierella alpina strain is from China industrial microbial culture collection management center (CICC), and the strain number is CICC 11092 s; glucose, starch, yeast powder, yeast extract, peptone, corn steep liquor powder, potato and agar are all commercially available products.

The formula of the PDA culture medium in the embodiment of the invention is as follows: 200g of potato, 20g of cane sugar, 15-17g of agar and 1000mL of water.