A strain of Schizochytrium and its application in fermentation production of algae oil rich in Sn-2 DHA

Abstract

The invention discloses a schizochytrium and an application thereof in fermentation production of algae oil rich in Sn-2 DHA, belonging to the technical field of bioengineering. The schizochytrium strain is schizochytrium (Schizochytrium sp.) TKD-2212, and its preservation number is CCTCC No.M20221978; the algae oil rich in Sn-2 DHA is obtained by fermentation of schizochytrium (TKD-2212), and comprises the following production steps: activating the schizochytrium rich in Sn-2 DHA algae oil to obtain seed liquid; the seed liquid is subjected to primary, secondary and tertiary fermentation to produce fermentation liquid; the fermentation liquid is subjected to cell wall breaking and extraction treatment to obtain crude DHA algae oil; the crude DHA algae oil is then subjected to acidification degumming, decolorization and deodorization treatment to obtain DHA algae oil; and finally, the DHA algae oil is subjected to alcoholysis and solvent extraction to obtain the algae oil rich in Sn-2 DHA. The DHA content of the algae oil produced by the present invention is 40-65%, wherein the proportion of DHA at the Sn-2 position is 35-50%, which is more similar to natural DHA and is more easily absorbed by the human body than ordinary DHA.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a schizochytrium strain and application thereof in fermentation production of Sn-2 position DHA-rich algae oil. Background Art

[0002] Docosahexaenoic acid is also known as DHA, and its molecular formula is C 22 H 32 O2, belongs to the ω-3 series of unsaturated fatty acids. DHA is mainly distributed in the gray matter of the brain, nervous system, retinal system, myocardium, eosinophils and breast milk in the human body, and has important physiological functions. Studies have shown that the special structure of DHA triglycerides at the Sn-2 position is more conducive to human absorption. After the triglyceride structure of oil is decomposed by pancreatic lipase, the fatty acids at the Sn-1 and Sn-3 positions form free fatty acids, which are difficult to penetrate into bile salt micelles and be absorbed by the human body, and are easily combined with calcium and magnesium ions in the intestine to form insoluble soap salts and lost; while the fatty acids at the Sn-2 position form monoglycerides, which are easy to penetrate into bile salt micelles and be absorbed by the human body, so the fatty acids at the Sn-2 position have a higher human absorption rate than the fatty acids at the Sn-1 and Sn-3 positions.

[0003] Schizochytrium, also known as Schizochytrium, is a marine fungus belonging to the phylum Myxomycetes, class Myxomycetes, order Thraustochytriales, and family Thraustochytriaceae. It is widely used in scientific research and commercial production due to its fast growth rate and easy cultivation. At present, the main source of DHA obtained by microbial methods is the high-density fermentation culture of Schizochytrium, which can accumulate a large amount of fat in its body, and 70% exists in the form of triglycerides, of which DHA can account for 45-55% of the total fatty acids.

[0004] At present, the distribution ratio of DHA oils and fats from microorganisms on the glycerol skeleton is the content of Sn-2, which is much lower than the content of Sn-1 and Sn-3. A large amount of DHA in Sn-1 and Sn-3 forms soap salts and is lost during human digestion, and the health care effect of microbial oils and fats is limited. In order to improve the utilization rate of DHA algae oil by humans, the present invention produces an algae oil rich in DHA in Sn-2, which is more similar to natural DHA than ordinary commercial algae oil, increases the content of DHA in Sn-2 in algae oil, and is more easily absorbed by human intestinal mucosa. Summary of the invention

[0005] The purpose of the present invention is to provide a Schizochytrium strain and its application in the fermentation production of Sn-2 DHA-rich algae oil to solve the problems in the background technology.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A strain of Schizochytrium sp. TKD-2212, which was deposited in the China Center for Type Culture Collection on December 14, 2022, with the deposit address located at Wuhan University, Wuhan, China, and the deposit number is CCTCC No.M20221978.

[0008] Schizochytrium colony and cell morphology: Schizochytrium sp. TKD-2212 was cultured on seawater medium at 25°C for 7 days. The colony diameter was 3-5 mm, white, and light brown in the later stage. The cell wall was thin, spherical, transparent, and the cell diameter was 6.0-18.2 μm.

[0009] Results of Schizochytrium rRNA gene sequencing (18S rRNA sequence fragment):

[0010] >rRNA_Scaffold34_21600-23382_DIR+ / molecule=18s_rRNA / score=1330.9

[0011]

[0012] A Schizochytrium strain and its application in fermentation production of Sn-2 DHA-rich algae oil, comprising the following steps:

[0013] Step 1: activating Schizochytrium with Sn-2 DHA-rich algae oil to obtain Schizochytrium seed liquid;

[0014] Step 2: subjecting the Schizochytrium seed liquid to a first-stage seed tank expansion culture and a second-stage seed tank expansion culture and then to a third-stage fermentation to produce a Schizochytrium fermentation liquid;

[0015] Step 3: crushing the Schizochytrium fermentation broth by an enzymatic method and then extracting it to obtain crude DHA algae oil;

[0016] Step 4: The crude DHA algae oil is then subjected to acidification, degumming and deodorization treatment to obtain refined DHA algae oil;

[0017] Step 5: The DHA algae oil is purified by alcoholysis and solvent extraction to obtain the Sn-2 position-rich DHA algae oil.

[0018] Furthermore, the method for screening strains in step 1 is: obtaining Schizochytrium sp. from the natural environment through screening, mutagenesis, and breeding, conducting directed domestication after whole-genome sequencing, screening out the Schizochytrium sp. TKD-2212 strain, sealing it with glycerol, and storing it at -80°C.

[0019] The activation method is as follows: the schizochytrium is inoculated into the seed culture medium at an inoculation rate of 8%, and the algae species are obtained by constant temperature culture at 30°C for 36 hours, and then the inoculation rate is inoculated into the fermentation culture medium at a inoculation rate of 5% from the seed culture medium, and the temperature is controlled at 30°C and the rotation speed is 180rpm to obtain the seed liquid. The preparation process of the seed culture medium includes: 10g / L glucose, 15g / L yeast powder, 5g / L peptone, pH 6.5-7.0, and 115°C high pressure steam sterilization for 30min. The preparation process of the fermentation culture medium includes: 40g / L glucose, 15g / L yeast powder, 5g / L peptone, pH 6.5-7.0, and 115°C high pressure steam sterilization for 30min.

[0020] Furthermore, in step 2, the fermentation temperature is controlled at 20°C-28°C and the rotation speed is 160-200rpm, and a 3L seed tank is used for fermentation. During the fermentation process, when the glucose concentration in the fermentation system is lower than 5g / L, high-concentration glucose is added at a rate of 0.8-1.2g / L / h to supplement the carbon source.

[0021] Furthermore, in step 3, the specific enzyme activity used was 3.5×10 5U / g alkaline protease, the enzyme is an extracellular enzyme, simple to culture, high yield, suitable for large-scale production applications.

[0022] The enzymatic crushing and extraction method is as follows: 1.5%-2.5% alkaline protease is used to crush the Schizochytrium fermentation liquid at 50°C-60°C and pH 9-10, and stirred for 3h-5h. The fermentation liquid after enzymolysis is added with an equal volume of n-hexane and stirred at room temperature for 2h-3h, and separated by high-speed centrifugation (6000rpm, 5min). The precipitate is repeatedly extracted with n-hexane 2-3 times, and the supernatant oil phase is combined to form a mixed liquid of algae oil and n-hexane. The mixed liquid is evaporated under reduced pressure (50-55°C, -2.8~1.2kPa) to evaporate the n-hexane, and obtain crude algae oil rich in DHA.

[0023] Furthermore, the use of 85% phosphoric acid in step 4 can transform phospholipids and phospholipid metal complexes into hydrated phospholipids, thereby effectively reducing the content of colloids and trace metals in oils and fats.

[0024] The acid degumming and deodorizing method is as follows: add 0.1%-0.3% phosphoric acid (mass concentration 85%) of the mass of primary algae oil into a 300-400Pa vacuum tank, stir for 15 minutes (60rpm), let stand for 15 minutes and then centrifuge (6000rpm, 10min) to remove gums and aldehydes and ketones in the algae oil, collect the supernatant to obtain refined algae oil rich in DHA.

[0025] Furthermore, the immobilized lipase used in step five is acyl hydrolase Lipozyme RM IM (Novozymes, Denmark), which is a Sn-1,3 specific lipase. It can retain the DHA at the Sn-2 position in the algae oil enzymatic hydrolysis reaction, and hydrolyze the fatty acids at the Sn-1 and Sn-3 positions into free fatty acids, thereby enriching more DHA at the Sn-2 position; high-purity Sn-2 DHA algae oil can be obtained after extraction and purification with ethanol and n-hexane solvents.

[0026] The specific method for enriching Sn-2 DHA algae oil is as follows: the amount of anhydrous ethanol added is 3 to 5 times the mass of DHA algae oil, and after mixing, 8 to 12% of the mass of the algae oil is added with immobilized lipase, and the reaction is carried out at a temperature of 25 to 35°C and a stirring speed of 300 to 400 rpm for 8 to 10 hours. After the reaction, the immobilized lipase is separated by centrifugation (5000 rpm, 10 min), and the supernatant is rotary evaporated to remove ethanol, and the obtained algae oil is rich in Sn-2 DHA. In addition to Sn-2 DHA monoglyceride, the product generated by the alcoholysis reaction also contains a large amount of fatty acid ethyl ester, which is purified by solvent extraction: the algae oil is dissolved in 85% ethanol solution (the volume ratio of algae oil to ethanol is 1:10), and then the ethanol system is washed three times with n-hexane equal to the volume of the ethanol solution, and the ethanol phase is taken. After the ethanol is removed by rotary evaporation, the algae oil rich in Sn-2 DHA monoglyceride is obtained.

[0027] The beneficial effects of the present invention are as follows: the method selects a Schizochytrium TKD-2212, and a high yield of DHA is achieved after primary and secondary expansion culture and tertiary fermentation; the Sn-1,3-position acyl-specific selective lipase is used to carry out alcoholysis reaction on the degummed and deodorized algae oil to reduce the content of non-Sn-2 fatty acids, and the non-aqueous phase (anhydrous ethanol) condition is used to reduce the migration of Sn-2 position DHA to Sn-1 and Sn-3 positions, and combined with solvent extraction purification, the enrichment of Sn-2 position DHA in the algae oil is achieved.

[0028] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0030] Figure 1 This is a microscopic image of Schizochytrium in Example 1 of the present invention;

[0031] Figure 2 This is the phylogenetic tree diagram of Schizochytrium TKD-2212;

[0032] Figure 3 A shows the total fatty acid composition of Sn-2 DHA-rich algae oil. Figure 3 B is the liquid chromatography of the fatty acid composition at the Sn-2 position. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] Schizochytrium sp. TKD-2212 was obtained from the natural environment through screening, mutagenesis and breeding. After full genome sequencing, targeted domestication was performed to screen out the Schizochytrium sp. TKD-2212 strain (see Figure 1 ) and then sealed with glycerol and stored at -80°C.

[0036] like Figure 1 As shown, the colony and cell morphology of Schizochytrium sp. TKD-2212 was cultured on seawater medium at 25°C for 7 days. The colony diameter was 3-5 mm, white, and light brown in the later stage. The cell wall was thin, spherical, transparent, and the cell diameter was 6.0-18.2 μm.

[0037] The Schizochytrium sp. TKD-2212 was deposited in the China Center for Type Culture Collection on December 14, 2022. The deposit address is located at Wuhan University, Wuhan, China, and the deposit number is CCTCC No.M20221978.

[0038] The results of rRNA gene sequencing of Schizochytrium schizochytrium TKD-2212 (18S rRNA sequence fragment, see Figure 2 ) is as follows:

[0039] Results of Schizochytrium rRNA gene sequencing (18S rRNA sequence fragment):

[0040] >rRNA_Scaffold34_21600-23382_DIR+ / molecule=18s_rRNA / score=1330.9

[0041]

[0042] Example 2

[0043] A Schizochytrium sp. and its application in fermentation production of Sn-2 DHA-rich algae oil, using Schizochytrium sp. TKD-2212 to produce Sn-2 DHA-rich algae oil, specifically comprising the following steps:

[0044] The process of producing Sn-2 DHA algae oil rich in Schizochytrium TKD-2212 is as follows: screening Schizochytrium TKD-2212 strains rich in Sn-2 DHA algae oil, activating the Schizochytrium strains to obtain Schizochytrium seed liquid; subjecting the Schizochytrium seed liquid to primary seed tank expansion culture and secondary seed tank expansion culture and then to tertiary fermentation to produce Schizochytrium fermentation liquid; using an enzymatic method to crush the Schizochytrium fermentation liquid and then subjecting it to extraction treatment to obtain crude DHA algae oil, and then subjecting it to acid degumming and deodorization treatment to obtain refined DHA algae oil, and finally subjecting it to alcoholysis and solvent extraction to obtain Sn-2 DHA algae oil.

[0045] The above-mentioned Schizochytrium sp. TKD-2212 strain screening method is: Schizochytrium is obtained from the natural environment through screening, mutagenesis, and breeding, and directed domestication is performed after full genome sequencing. After the Schizochytrium sp. TKD-2212 strain is screened, it is sealed with glycerol and stored at -80°C.

[0046] The method for preparing the Schizochytrium TKD-2212 seed liquid is as follows: the Schizochytrium is inoculated into the seed culture medium at an inoculation rate of 8%, and the algae species are obtained by constant temperature culture at 30°C for 36 hours, and then the inoculation rate is inoculated into the fermentation culture medium at a inoculation rate of 5% from the seed culture medium, and the temperature is controlled at 30°C and the rotation speed is 180rpm to obtain the seed liquid. The preparation process of the seed culture medium includes: 10g / L glucose, 15g / L yeast powder, 5g / L peptone, pH 6.5-7.0, and high-pressure steam sterilization at 115°C for 30min. The preparation process of the fermentation culture medium includes: 40g / L glucose, 15g / L yeast powder, 5g / L peptone, pH 6.5-7.0, and high-pressure steam sterilization at 115°C for 30min.

[0047] The above-mentioned seed liquid expansion culture method is: the seed liquid is fermented in 3L, 5L, 10L primary, secondary seed tanks and tertiary fermentation tanks, the fermentation temperature is controlled at 20°C and the rotation speed is 180rpm. During the fermentation process, when the glucose concentration in the system is lower than 5g / L, high-concentration glucose is added at a rate of 1.2g / L / h to supplement the carbon source.

[0048] The crude DHA algae oil preparation method is as follows: using 2% alkaline protease to break up the Schizochytrium fermentation broth at 55° C. and pH 9.5, then adding n-hexane with an equal volume of the fermentation broth and stirring at room temperature for 3 hours to extract 3 times to obtain the crude DHA algae oil.

[0049] The method for preparing the refined DHA algae oil is as follows: adding 0.3% of the oil weight of 85% phosphoric acid to the crude algae oil in a 400Pa vacuum tank to remove the gum substances and aldehydes and ketones in the algae oil to obtain the refined algae oil.

[0050] The method for preparing the Sn-2 DHA-rich algae oil is as follows: adding 10% of the volume of refined algae oil to immobilized lipase at 25°C and performing alcoholysis at a speed of 350 rpm to obtain a crude Sn-2 DHA-rich algae oil, and then extracting with ethanol and n-hexane solvents three times to obtain the product of the present invention (such as Figure 3 A shows the total fatty acid composition of Sn-2 DHA-rich algae oil. Figure 3 B is the liquid chromatogram of the fatty acid composition at the Sn-2 position; the peak in the box diagram is the Sn-2 peak).

[0051] Example 3

[0052] A Schizochytrium sp. and its application in fermentation production of Sn-2 DHA-rich algae oil, using Schizochytrium sp. TKD-2212 to produce Sn-2 DHA-rich algae oil, specifically comprising the following steps:

[0053] The specific steps of producing Sn-2 DHA-rich algae oil by Schizochytrium sp. TKD-2212 are consistent with the steps in Example 2.

[0054] The above-mentioned Schizochytrium TKD-2212 strain screening and seed liquid preparation method is consistent with the method in Example 2.

[0055] The above-mentioned seed liquid expansion culture method is: the seed liquid is fermented in 3L, 5L, 10L primary, secondary seed tanks and tertiary fermentation tanks, the fermentation temperature is controlled at 22°C and the rotation speed is 160rpm. During the fermentation process, when the glucose concentration in the system is lower than 5g / L, high-concentration glucose is added at a rate of 0.8g / L / h to supplement the carbon source.

[0056] The crude DHA algae oil preparation method is as follows: using 1.5% alkaline protease to break up the Schizochytrium fermentation broth at 50° C. and pH 9, then adding n-hexane with an equal volume of the fermentation broth and stirring at room temperature for 2 hours to extract twice to obtain the crude DHA algae oil.

[0057] The method for preparing the refined DHA algae oil is as follows: adding 0.2% of the oil weight of 85% phosphoric acid to the crude algae oil in a 350Pa vacuum tank to remove the gum substances and aldehydes and ketones in the algae oil to obtain the refined algae oil.

[0058] The method for preparing the Sn-2 DHA-rich algae oil is as follows: adding 8% immobilized lipase by volume of refined algae oil at 20°C and performing alcoholysis at a speed of 300 rpm to obtain crude Sn-2 DHA-rich algae oil, followed by extraction twice with ethanol and n-hexane solvents to obtain the product of the present invention.

[0059] Example 4

[0060] A Schizochytrium sp. and its application in fermentation production of Sn-2 DHA-rich algae oil, using Schizochytrium sp. TKD-2212 to produce Sn-2 DHA-rich algae oil, specifically comprising the following steps:

[0061] The specific steps of producing Sn-2 DHA-rich algae oil by Schizochytrium sp. TKD-2212 are consistent with the steps in Example 2.

[0062] The above-mentioned Schizochytrium TKD-2212 strain screening and seed liquid preparation method is consistent with the method in Example 2.

[0063] The above-mentioned seed liquid expansion culture method is: the seed liquid is fermented in 3L, 5L, 10L primary, secondary seed tanks and tertiary fermentation tanks, the fermentation temperature is controlled at 24°C and the rotation speed is 170rpm. During the fermentation process, when the glucose concentration in the system is lower than 5g / L, high-concentration glucose is added at a rate of 0.9g / L / h to supplement the carbon source.

[0064] The crude DHA algae oil preparation method is as follows: using 1.8% alkaline protease to break up the Schizochytrium fermentation broth at 53° C. and pH 9.3, then adding n-hexane with an equal volume of the fermentation broth and stirring at room temperature for 3 hours to extract twice to obtain the crude DHA algae oil.

[0065] The method for preparing the refined DHA algae oil is as follows: adding 0.25% of 85% phosphoric acid by weight to the crude algae oil in a 330Pa vacuum tank to remove the gum substances and aldehydes and ketones in the algae oil to obtain the refined algae oil.

[0066] The method for preparing the Sn-2 DHA-rich algae oil is as follows: adding 9% of the volume of refined algae oil with immobilized lipase at 23°C and performing alcoholysis at a rotation speed of 330 rpm to obtain crude Sn-2 DHA-rich algae oil, and then extracting with ethanol and n-hexane solvents three times in succession to obtain the product of the present invention.

[0067] Example 5

[0068] A Schizochytrium sp. and its application in fermentation production of Sn-2 DHA-rich algae oil, using Schizochytrium sp. TKD-2212 to produce Sn-2 DHA-rich algae oil, specifically comprising the following steps:

[0069] The specific steps of producing Sn-2 DHA-rich algae oil by Schizochytrium sp. TKD-2212 are consistent with the steps in Example 2.

[0070] The above-mentioned Schizochytrium TKD-2212 strain screening and seed liquid preparation method is consistent with the method in Example 2.

[0071] The above-mentioned seed liquid expansion culture method is: the seed liquid is fermented in 3L, 5L, 10L primary, secondary seed tanks and tertiary fermentation tanks, the fermentation temperature is controlled at 26°C and the rotation speed is 190rpm. During the fermentation process, when the glucose concentration in the system is lower than 5g / L, high-concentration glucose is added at a rate of 1.0g / L / h to supplement the carbon source.

[0072] The crude DHA algae oil production method is as follows: using 2.3% alkaline protease to break up the Schizochytrium fermentation broth at 58° C. and pH 9.8, then adding n-hexane with an equal volume of the fermentation broth and stirring at room temperature for 2 hours to extract 3 times to obtain the crude DHA algae oil.

[0073] The method for preparing the refined DHA algae oil is as follows: adding 0.1% of the oil weight of 85% phosphoric acid to the crude algae oil in a 380Pa vacuum tank to remove the gum substances and aldehydes and ketones in the algae oil to obtain the refined algae oil.

[0074] The method for preparing the Sn-2 DHA-rich algae oil is as follows: adding 11% of the volume of refined algae oil with immobilized lipase at 28°C and performing alcoholysis at a rotation speed of 380 rpm to obtain crude Sn-2 DHA-rich algae oil, and then extracting twice with ethanol and n-hexane solvents to obtain the product of the present invention.

[0075] Example 6

[0076] A Schizochytrium sp. and its application in fermentation production of Sn-2 DHA-rich algae oil, using Schizochytrium sp. TKD-2212 to produce Sn-2 DHA-rich algae oil, specifically comprising the following steps:

[0077] The specific steps of producing Sn-2 DHA-rich algae oil by Schizochytrium sp. TKD-2212 are consistent with the steps in Example 2.

[0078] The above-mentioned Schizochytrium TKD-2212 strain screening and seed liquid preparation method is consistent with the method in Example 2.

[0079] The above-mentioned seed liquid expansion culture method is: the seed liquid is fermented in 3L, 5L, 10L primary, secondary seed tanks and tertiary fermentation tanks, the fermentation temperature is controlled at 28°C and the rotation speed is 200rpm. During the fermentation process, when the glucose concentration in the system is lower than 5g / L, high-concentration glucose is added at a rate of 1.1g / L / h to supplement the carbon source.

[0080] The crude DHA algae oil preparation method is as follows: using 2.5% alkaline protease to break up the Schizochytrium fermentation broth at 60° C. and pH 10, then adding n-hexane with an equal volume of the fermentation broth and stirring at room temperature for 3 hours to extract 3 times to obtain the crude DHA algae oil.

[0081] The method for preparing the refined DHA algae oil is as follows: adding 0.15% of the oil weight of 85% phosphoric acid to the crude algae oil in a 400Pa vacuum tank to remove the gum substances and aldehydes and ketones in the algae oil to obtain the refined algae oil.

[0082] The method for preparing the Sn-2 DHA-rich algae oil is as follows: adding 12% of the volume of refined algae oil with immobilized lipase at 30°C and performing alcoholysis at a speed of 400 rpm to obtain crude Sn-2 DHA-rich algae oil, and then extracting with ethanol and n-hexane solvents three times to obtain the product of the present invention.

[0083] The present invention uses Schizochytrium sp. TKD-2212 to produce algae oil rich in Sn-2 DHA. Compared with common Schizochytrium sp., the algae oil produced by TKD-2212 has a higher content of Sn-2 DHA and is more easily absorbed by human intestinal mucosa. The comparison between Schizochytrium sp. TKD-2212 and common Schizochytrium sp. is shown in Table 1.

[0084] Table 1

[0085]

[0086]

[0087] The present invention optimizes the fermentation process of Schizochytrium during the seed liquid expansion culture process, controls the fermentation temperature, detects the glucose concentration in the fermentation system, and uses a method of adding high-concentration glucose to supplement the carbon source when the glucose concentration is lower than 5 g / L, thereby increasing the biomass of Schizochytrium and the production capacity of DHA algae oil, and a lower temperature is conducive to the accumulation of DHA.

[0088] The total DHA content of the Sn-2 DHA algae oil prepared according to Example 2-6 was calculated compared with the ordinary DHA algae oil. The specific results are shown in Table 2.

[0089] Table 2

[0090]

[0091] The present invention uses alkaline protease to extract algae oil, and the oil yield and DHA content in the algae oil produced by other methods are higher. In addition, the enzyme is an extracellular enzyme, which is simple to cultivate and has a large yield, and is suitable for large-scale production applications.

[0092] The unsaturated fatty acid content of the Sn-2 DHA algae oil prepared according to Example 2-6 was calculated compared with the ordinary DHA algae oil. The specific results are shown in Table 3.

[0093] Table 3

[0094]

[0095] The present invention uses immobilized lipase to perform alcoholysis on algae oil and then extracts it to obtain enriched Sn-2 DHA algae oil. The ratio of Sn-2 DHA to total DHA content is calculated. The specific embodiment results are shown in Table 4.

[0096] Table 4

[0097]

[0098] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. Use of a Schizochytrium sp. strain in fermentation production of Sn-2 DHA-rich algae oil, characterized in that: The steps include: Step 1: activating Schizochytrium with Sn-2 DHA-rich algae oil to obtain Schizochytrium seed liquid; Step 2: subjecting the Schizochytrium seed liquid to a first-stage seed tank expansion culture and a second-stage seed tank expansion culture and then to a third-stage fermentation to produce a Schizochytrium fermentation liquid; Step 3: The Schizochytrium fermentation liquid is crushed by an enzymatic method and then subjected to extraction to obtain crude DHA algae oil. The specific enzymatic method is as follows: the Schizochytrium fermentation liquid is crushed by using 1.5%-2.5% alkaline protease at 50°C-60°C and pH 9-10, and then an equal volume of n-hexane is added to the fermentation liquid and stirred for extraction at room temperature for 2h-3h, and the supernatant is separated by centrifugation, and extracted 2-3 times to obtain crude DHA algae oil; Step 4: The crude DHA algae oil is then subjected to acidification, degumming and deodorization treatment to obtain refined DHA algae oil; specifically, 0.1%-0.3% of the mass of the primary algae oil and 85% phosphoric acid are added into a 300-400Pa vacuum tank, stirred at 60rpm for 15min, allowed to stand for 15min and then centrifuged to remove the gums and aldehydes and ketones in the algae oil, and the supernatant is collected to obtain refined DHA-rich algae oil; Step 5: After alcoholysis and solvent extraction purification of DHA algae oil, Sn-2 DHA-rich algae oil is obtained, wherein the alcoholysis reaction uses immobilized lipase, and 8-12% of the volume of algae oil is added to the non-aqueous phase under 20-30° C. and a speed of 300-400 rpm to alcoholyze to obtain crude Sn-2 DHA-rich algae oil, and then ethanol and n-hexane solvents are used for extraction and purification 2-3 times to obtain the product; The immobilized lipase used in the alcoholysis reaction was acyl hydrolase Lipozyme RM IM, which is a Sn-1,3 specific lipase; The classification name of the schizochytrium is Schizochytrium sp. TKD-2212. The schizochytrium was deposited in the China Center for Type Culture Collection on December 14, 2022. The deposit address is located at Wuhan University, Wuhan, China, and the deposit number is CCTCC No.M20221978.

2. The use according to claim 1, characterized in that: The activation method in step 1 is: inoculating the Schizochytrium into a seed culture medium at an inoculation rate of 8%, culturing at a constant temperature of 30° C. for 36 hours to obtain algae seeds, inoculating the seed culture medium into a fermentation culture medium at an inoculation rate of 5%, controlling the temperature at 30° C. and the rotation speed at 180 rpm to obtain a seed solution; The preparation process of the seed culture medium includes: 10 g / L glucose, 15 g / L yeast powder, 5 g / L peptone, pH 6.5-7.0, and 115°C high-pressure steam sterilization for 30 minutes; the preparation process of the fermentation culture medium includes: 40 g / L glucose, 15 g / L yeast powder, 5 g / L peptone, pH 6.5-7.0, and 115°C high-pressure steam sterilization for 30 minutes.

3. The use according to claim 1, characterized in that: In the step 2, the fermentation temperature is controlled at 20°C-28°C and the rotation speed is 160-200rpm. 3L, 5L, and 10L seed tanks are used for fermentation. During the fermentation process, when the glucose concentration in the fermentation system is lower than 5g / L, high-concentration glucose is added at a rate of 0.8-1.2g / L / h to supplement the carbon source.

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