CN115873657B - Schizochytrium limacinum lipid extraction method without using organic solvent and application - Google Patents

Abstract

The invention discloses a schizochytrium limacinum lipid extraction method without using an organic solvent and application thereof, wherein the method comprises the following steps: culturing schizochytrium limacinum strain seed liquid; obtaining third generation seed liquid; inoculating into fermentation medium of schizochytrium, culturing, and collecting fermentation liquid; adjusting the pH of the fermentation liquor; adding wall breaking enzyme for treatment; extracting; adding the composite surfactant into the fermentation broth, uniformly mixing, and centrifuging; standing and layering; centrifuging to obtain supernatant; steaming in a rotary way; drying to obtain total lipid. In the lipid extraction process, the method disclosed by the invention has the advantage that the organic solvent n-hexane is replaced by a compound surfactant, so that the lipid extraction is promoted. The method optimizes the schizochytrium limacinum lipid extraction process, avoids the addition of an organic solvent in the lipid extraction process, improves the lipid safety, expands the market application range of lipid products, and simultaneously has simple and easy operation and stronger universality.

Description

Schizochytrium limacinum lipid extraction method without using organic solvent and application

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a schizochytrium limacinum lipid extraction method and application without using an organic solvent.

Background

Schizochytrium sp is a marine heterotrophic eukaryotic microorganism. Schizochytrium limacinum has the advantages of high growth speed, availability of cheap carbon sources and the like, and has been widely used in scientific research and industrial production. In particular, schizochytrium has a high lipid synthesis and accumulation capacity. And part of wild schizochytrium limacinum has total lipid content reaching more than 55% of Dry Cell Weight (DCW) without fermentation condition optimization or metabolic engineering.

In order to promote economic benefit, the common fermentation carbon source is glucose, and is replaced by an inexpensive carbon source, so that the production cost is reduced; meanwhile, the economic benefit of producing lipid by schizochytrium can be promoted by optimizing fermentation conditions and improving lipid yield by utilizing a metabolic engineering strategy. In addition, lipid extraction is also a key stage in promoting economic benefits. Organic solvents such as: methanol, acetone, n-hexane, chloroform and the like are common organic solvents for extracting lipids from microorganisms. However, this extraction method inevitably has the problem of organic solvent residues, which results in lipid products having certain limitations in the market application.

Surfactants are a class of substances that can significantly reduce the surface tension of a target solution. The surfactant contains two groups of nature: hydrophilic and hydrophobic groups, which can interact with cell membranes, promote cell wall rupture, and thus facilitate extraction of lipids.

Thus, there is a need for one or more related novel schizochytrium lipid extraction methods.

By searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a schizochytrium limacinum lipid extraction method and application without using an organic solvent.

The technical scheme adopted for solving the technical problems is as follows:

a schizochytrium limacinum lipid extraction method without using an organic solvent, the method comprising the steps of:

(1) Taking schizochytrium limacinum fungus preserving tube fungus liquid from a refrigerator at the temperature of minus 80 ℃, adding the schizochytrium limacinum fungus preserving tube fungus liquid into a schizochytrium limacinum fungus seed liquid culture medium, and culturing at the temperature of 28 ℃ and at the speed of 170 rpm;

(2) Transferring 1 mL bacteria liquid to a fresh seed liquid culture medium every 24 h of schizochytrium limacinum seed liquid, and transferring twice continuously to obtain third-generation seed liquid;

(3) Inoculating the third generation seed solution into a fermentation medium of schizochytrium limacinum according to an inoculum size of 1%, culturing at 28 ℃ and 170 rpm for 5 days, and collecting fermentation liquid;

(4) Gradually adding 2M NaOH into the fermentation liquor until the pH value of the fermentation liquor is 10-12;

(5) Adding wall breaking enzyme with a final concentration of 0.3%, and treating at 50deg.C and 170 rpm for 4-6 h;

(6) Carrying out subsequent extraction on the treated fermentation liquor;

(7) Adding the composite surfactant into the fermentation broth instead of normal hexane, uniformly mixing, and centrifuging at 8,000 rpm for 5 min;

(8) Standing for 4-6 h, and layering the two phases;

(9) Centrifuging at 8,000 rpm for 5 min, and collecting supernatant;

(10) Spin-evaporating the supernatant and volatilizing the organic phase;

(11) And after the rotary steaming is finished, placing the rotary steaming bottle in a 60 ℃ oven, drying, and weighing to obtain the total lipid.

Further, the schizochytrium is schizochytrium HX-308 (the strain is a well-known strain in the prior art, for example, disclosed in patent publication No. CN104974944a, and the strain is preserved in China center for type culture collection (CCTCC, the preservation number of which is cctccc No. m 209059).

Further, the seed liquid culture medium is as follows: glucose: 40-60 g/L, yeast extract powder: 4-6 g/L, sodium sulfate: 5-8 g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 8-12g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L.

The fermentation medium is as follows: glucose: 60-100 g/L, yeast extract powder: 5-15 g/L, sodium sulfate: 5-12 g g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 15-20 g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/LCalcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L, vitamin B ₆ :4-10 mg/L, vitamin B ₁₂ ：0.1-0.5 mg/L。

Further, a total lipid of 69.87.+ -. 1.75 g could be obtained per liter of fermentation broth.

Further, the compound surfactant consists of sodium dodecyl benzene sulfonate, betaine and tween 80.

Further, the final concentration of sodium dodecyl benzene sulfonate added in the fermentation broth is 70 mg/L, the final concentration of betaine added in the fermentation broth is 80 mg/L, and the final concentration of Tween 80 added in the fermentation broth is 3 mL/L.

The application of the schizochytrium limacinum lipid extraction method in schizochytrium limacinum lipid extraction.

The beneficial effects obtained by the invention are as follows:

1. in the lipid extraction process, the method disclosed by the invention has the advantage that the organic solvent n-hexane is replaced by a compound surfactant, so that the lipid extraction is promoted. The method optimizes the schizochytrium limacinum lipid extraction process, avoids the addition of an organic solvent in the lipid extraction process, improves the lipid safety, expands the market application range of lipid products, and simultaneously has simple and easy operation and stronger universality.

2. The method of the invention uses a compound surfactant, wherein the compound surfactant comprises three components, namely Sodium Dodecyl Benzene Sulfonate (SDBS), betaine and Tween 80; wherein SDBS is an anionic surfactant, betaine is a zwitterionic surfactant, and Tween 80 is a nonionic surfactant. The three surfactants are all widely available, and the lipid extraction effect of the compound surfactant is better than that of normal hexane (organic solvent). Meanwhile, the problem of organic solvent residue in the lipid production process is avoided, and the application of lipid products in the market is expanded.

3. The invention uses Schizochytrium sp HX-308 as the original strain. Fermenting and culturing HX-308 for 5 days to obtain fermentation liquor. The extraction process of schizochytrium oil is optimized by replacing the extractant n-hexane with the surfactant. By means of optimization tests such as a single factor test and an orthogonal test, the optimal compound surfactant formula is determined as follows: the addition amount of SDBS in the fermentation liquid is 70 mg/L, and the addition amount of betaine in the fermentation liquid is as follows: 80 The addition amount of tween 80 in the fermentation broth is mg/L: 3 mL/L) of 69.87.+ -. 1.75. 1.75 g lipids per liter of fermentation broth.

4. According to the invention, a three-component synergistic effect test is carried out, so that the synergistic effect among three components of sodium dodecyl benzene sulfonate, betaine and tween 80 of the compound surfactant is verified, and the extraction amount of schizochytrium limacinum lipid can be synergistically improved. The invention optimizes the schizochytrium limacinum lipid extraction process, avoids the problem of organic solvent residue, and expands the application of lipid products in the market.

5. In the invention, the compound surfactant is used for replacing normal hexane used in the traditional lipid extraction in the schizochytrium limacinum lipid extraction process, so that the extracted lipid content is increased from 52.73 +/-2.70 g/L to 69.87 +/-1.75 g/L. Meanwhile, the research effectively avoids the residue of organic reagent in the lipid production process, and expands the market application of lipid products.

Drawings

FIG. 1 is a graph showing the effect of different amounts of Sodium Dodecyl Benzene Sulfonate (SDBS) on oil extraction in the present invention;

FIG. 2 is a graph showing the effect of betaine addition on oil extraction;

FIG. 3 is a graph showing the effect of different amounts of Tween 80 on oil extraction in the present invention;

FIG. 4 is a graph showing the effect of different complex surfactants on oil extraction in the present invention;

FIG. 5 is a graph showing the results of verifying whether the synergistic effect of the three components SDBS, betaine, and Tween 80 of the surfactant exists in the present invention.

Detailed Description

The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.

The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.

A schizochytrium limacinum lipid extraction method without using an organic solvent, the method comprising the steps of:

(1) Taking schizochytrium limacinum fungus preserving tube fungus liquid from a refrigerator at the temperature of minus 80 ℃, adding the schizochytrium limacinum fungus preserving tube fungus liquid into a schizochytrium limacinum fungus seed liquid culture medium, and culturing at the temperature of 28 ℃ and at the speed of 170 rpm;

(2) Transferring 1 mL bacteria liquid to a fresh seed liquid culture medium every 24 h of schizochytrium limacinum seed liquid, and transferring twice continuously to obtain third-generation seed liquid;

(3) Inoculating the third generation seed solution into a fermentation medium of schizochytrium limacinum according to an inoculum size of 1%, culturing at 28 ℃ and 170 rpm for 5 days, and collecting fermentation liquid;

(4) Gradually adding 2M NaOH into the fermentation liquor until the pH value of the fermentation liquor is 10-12;

(5) Adding wall breaking enzyme with a final concentration of 0.3%, and treating at 50deg.C and 170 rpm for 4-6 h;

(6) Carrying out subsequent extraction on the treated fermentation liquor;

(7) Adding the composite surfactant into the fermentation broth instead of normal hexane, uniformly mixing, and centrifuging at 8,000 rpm for 5 min;

(8) Standing for 4-6 h, and layering the two phases;

(9) Centrifuging at 8,000 rpm for 5 min, and collecting supernatant;

(10) Spin-evaporating the supernatant and volatilizing the organic phase;

(11) And after the rotary steaming is finished, placing the rotary steaming bottle in a 60 ℃ oven, drying, and weighing to obtain the total lipid.

Preferably, the schizochytrium is schizochytrium HX-308 (this strain is a known strain in the prior art, for example, as disclosed in patent publication No. CN104974944a, which is preserved in the chinese collection of typical cultures (CCTCC, with the preservation number cctccc No. m 209059).

Preferably, the seed liquid culture medium is: glucose: 40-60 g/L, yeast extract powder: 4-6 g/L, sodium sulfate: 5-8 g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 8-12g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L.

The fermentation medium is as follows: glucose: 60-100 g/L, yeast extract powder: 5-15 g/L, sodium sulfate: 5-12 g g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 15-20 g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L, vitamin B ₆ :4-10 mg/L, vitamin B ₁₂ ：0.1-0.5 mg/L。

Preferably, 69.87 + -1.75 g total lipids per liter of fermentation broth can be obtained.

Preferably, the compound surfactant is composed of sodium dodecyl benzene sulfonate, betaine and tween 80.

Preferably, the final concentration of sodium dodecyl benzene sulfonate added in the fermentation broth is 70 mg/L, the final concentration of betaine added in the fermentation broth is 80 mg/L, and the final concentration of tween 80 added in the fermentation broth is 3 mL/L.

The application of the schizochytrium limacinum lipid extraction method in schizochytrium limacinum lipid extraction.

Specifically, the preparation and detection of the correlation are as follows:

the following media were used in the examples:

seed liquid culture medium: glucose: 40-60 g/L, yeast extract powder: 4-6 g/L, sodium sulfate: 5-8 g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 8-12g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L.

Fermentation medium: glucose: 60-100 g/L, yeast extract powder: 5-15 g/L, sodium sulfate: 5-12 g g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 15-20 g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L, vitamin B ₆ :4-10 mg/L, vitamin B ₁₂ ：0.1-0.5 mg/L。

The invention takes the lipid biosynthesized by schizochytrium as a research object. In the lipid extraction process, the extractant n-hexane is replaced by a complex surfactant. Not only promotes the extraction of lipid, but also avoids the residue of organic solvent in the lipid extraction process. Firstly, determining the optimal addition amount of Sodium Dodecyl Benzene Sulfonate (SDBS), betaine and Tween 80 through a single factor test; the optimal proportion of the composite surfactant is further determined through an orthogonal test. In addition, the invention also carries out a synergistic effect test, and determines that the three surfactants have a synergistic effect.

The invention designs and implements single factor tests. In the lipid extraction process, the control test is to extract lipid with n-hexane, and the test group 1 is to replace n-hexane with SDBS, and 30 mg/L,40 mg/L,50 mg/L,60 mg/L,70 mg/L and 80 mg/L are respectively added; test group 2 was prepared by replacing n-hexane with betaine and adding 30 mg/L,40 mg/L,50 mg/L,60 mg/L,70 mg/L,80 mg/L; test group 3 was prepared by replacing n-hexane with tween 80 and adding 1 mL,2 mL,3 mL,4 mL,5 mL,6 mL. Subsequent broth extraction was performed and lipid content was determined.

The present invention devised and implemented orthogonal experiments. According to the single factor test result, determining three factors and three levels of the orthogonal test (SDBS addition amounts are respectively 50 mg/L,60 mg/L and 70 mg/L, betaine addition amounts are respectively 60 mg/L,70 mg/L and 80 mg/L, and Tween 80 addition amounts are respectively 2 mL,3 mL and 4 mL). The fermentation broth is then subjected to lipid extraction and the lipid content is determined.

The invention designs and implements synergistic effect tests. And determining the optimal compound surfactant ratio for promoting lipid extraction according to the orthogonal test result. And further verifying whether the three components have a synergistic effect by using a synergistic effect test. Example 1 is the addition of optimal complex surfactant during lipid extraction; comparative example 1 is the addition of betaine and tween 80 alone; comparative example 2 is the addition of SDBS and tween 80 alone; comparative example 3 is the addition of only SDBS and betaine. Subsequent broth extraction was performed and lipid content was determined.

The preparation method comprises the following steps:

example 1

And (3) determining three groups of optimal adding amounts of the SDBS through a single factor test.

A schizochytrium limacinum lipid extraction method without using an organic solvent, the method comprising the steps of:

(1) Taking a tube of HX-308 bacteria-preserving tube bacterial liquid from a refrigerator at the temperature of minus 80 ℃, adding 50 mL schizochytrium limacinum bacterial seed liquid culture medium, and culturing at the temperature of 28 ℃ and at the speed of 170 rpm;

(2) The schizochytrium limacinum seed solution is transferred to a fresh culture medium from 1 mL every 24 h for two times to obtain a third-generation seed solution;

(3) Taking third generation seed liquid 1 mL in a schizochytrium limacinum fermentation medium of 100 mL, culturing for 5 days under the same culture conditions, and collecting fermentation liquid.

(4) The broth was collected and 2M NaOH was gradually added to the broth to ph=10-12.

(5) Adding wall breaking enzyme with a final concentration of 0.3%, and treating at 50deg.C and 170 rpm for 4-6 h;

(6) The fermentation liquor after treatment is divided into 10 mL parts per part; carrying out subsequent extraction;

(7) The volume ratio of the fermentation liquor to the normal hexane in the control group is 1:1 adding n-hexane into the fermentation broth; the test group replaced n-hexane with different concentrations of SDBS to the fermentation broth. Mixing well, centrifuging (8,000 rpm,5 min);

(8) Standing for 4-6 h, and layering the two phases;

(9) Centrifuging (8,000 rpm,5 min), and collecting supernatant;

(10) Spin-evaporating the supernatant and volatilizing the organic phase;

(11) And (3) after the rotary steaming is finished, placing the rotary steaming bottle in a 60 ℃ oven, drying, and weighing to obtain the total lipid. The results are shown in FIG. 1. The lipid content of the fermentation liquor with the same volume is higher when the adding amount of SDBS is 50 mg/L,60 mg/L and 70 mg/L.

Example 2

And (3) determining three groups of optimal addition amounts of betaine by a single factor test.

Schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. The difference is that the extraction agent n-hexane is replaced by betaine surfactant with different concentrations in the extraction process. The results are shown in FIG. 2. The addition amount of betaine is 60 mg/L,70 mg/L and 80 mg/L, and the lipid content of the fermentation broth with the same volume is higher.

Example 3

And (3) determining three groups of optimal addition amounts of Tween 80 by a single factor test. Schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. The difference is that the extractant n-hexane is replaced by Tween 80 surfactant with different addition amount in the extraction process. The results are shown in FIG. 3. When the addition amount of Tween 80 is 2 ml/L,3 ml/L and 4 ml/L, the lipid content of the fermentation liquid with the same volume is higher.

Example 4

Orthogonal experiments were designed and performed. Three factor three levels were determined according to the one-factor test, as shown in table 1.

Orthogonal experiments were designed as shown in table 2.

Wherein the extractant of the control group of the orthogonal test is n-hexane. Lipid content was determined according to the schizochytrium fermentation and lipid extraction test procedure of example 1. The results are shown in FIG. 4. The added amount of SDBS is 70 mg/L, the added amount of betaine is 80 mg/L, and when the added amount of Tween 80 is 3 mL/L, the lipid content of the fermentation liquid with the same volume is the highest, which is 69.87 +/-1.75 g/L.

Example 5

To verify whether the three components of the composite surfactant sodium dodecyl benzene sulfonate, betaine and tween 80 have a synergistic effect, a synergistic test was designed as follows: schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. Except that n-hexane was replaced with the optimal complex surfactant (SDBS: 70 mg/L, betaine: 80 mg/L, tween 80:3 mL/L, the above-mentioned addition amounts were all the addition amounts in the fermentation broth) obtained by the screening of the present invention during the extraction, and the rest conditions of comparative example 1, comparative example 2, and comparative example 3 were the same as those of example 1, and the lipid content was measured.

Comparative example 1

Schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. The difference is that: in the extraction process, n-hexane is replaced by two components in the optimal composite surfactant obtained by screening according to the invention: betaine (80 mg/L) and Tween 80 (3 mL/L) are added to the fermentation broth, and lipid is extracted and the lipid content is measured.

Comparative example 2

Schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. The difference is that: in the extraction process, n-hexane is replaced by two components in the optimal composite surfactant obtained by screening according to the invention: SDBS (70 mg/L) and Tween 80 (3 mL/L), wherein the addition amounts are the addition amounts in the fermentation broth, and the lipid is extracted and the lipid content is measured.

Comparative example 3

Schizochytrium fermentation conditions and lipid extraction procedure were the same as in example 1. The difference is that: in the extraction process, n-hexane is replaced by two components SDBS (70 mg/L) and betaine (80 mg/L) in the optimal compound surfactant obtained by screening, and the addition amounts are the addition amounts in fermentation liquor, so that lipid is extracted, and the lipid content is measured.

The results are shown in FIG. 5, during schizochytrium lipid extraction. When three components are added simultaneously, the lipid content of the fermentation liquid with the same volume is highest and is 69.87 +/-1.75 g/L; the lipid titers obtained in the three comparative experiments were, however, respectively: 62.43 + -0.49 g/L,66.20 + -1.01 g/L and 65.80 + -1.13 g/L. That is, the three components are proved to have synergistic effect. Therefore, 70 mg/L sodium dodecyl benzene sulfonate, 80 mg/L betaine and 3 mL/L tween 80 are added into the fermentation broth during schizochytrium limacinum lipid extraction, so that the synergistic effect is achieved, and the extraction amount of schizochytrium lipid can be increased.

According to the invention, a formula of the compound surfactant for promoting lipid extraction is obtained through screening by combining a single factor test and an orthogonal test, so that the extracted lipid content is increased from 52.73 +/-2.70 g/L to 69.87 +/-1.75 g/L. The formula of the optimal composite surfactant is as follows: SDBS (70 mg/L), betaine (80 mg/L), tween 80 (3 mL/L). The invention optimizes the lipid extraction process, replaces the organic solvent n-hexane with the surfactant in the lipid extraction process, and avoids the residue of the organic solvent in the extraction process. The invention provides a strategy for effectively promoting lipid extraction, and a formula of the compound surfactant for promoting lipid extraction is obtained through screening, so that the application of a lipid product in the market is expanded.

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims (2)

1. A schizochytrium limacinum lipid extraction method without using an organic solvent, which is characterized in that: the method comprises the following steps:

(1) Taking a tube of HX-308 bacteria-preserving tube bacterial liquid from a refrigerator at the temperature of minus 80 ℃, adding 50 mL schizochytrium limacinum bacterial seed liquid culture medium, and culturing at the temperature of 28 ℃ and at the speed of 170 rpm;

(2) The schizochytrium limacinum seed solution is transferred to a fresh culture medium from 1 mL every 24 h for two times to obtain a third-generation seed solution;

(3) Taking third generation seed liquid 1 mL in a schizochytrium limacinum fermentation medium of 100 mL, culturing for 5 days under the same culture conditions, and collecting fermentation liquid;

(4) Collecting fermentation liquor, and gradually adding 2M NaOH into the fermentation liquor until the pH value of the fermentation liquor is=10-12;

(5) Adding wall breaking enzyme with a final concentration of 0.3%, and treating at 50deg.C and 170 rpm for 4-6 h;

(6) The fermentation liquor after treatment is divided into 10 mL parts per part; carrying out subsequent extraction;

(7) Adding a composite surfactant into the fermentation broth; mixing well, centrifuging at 8,000 rpm for 5 min;

wherein, the addition amount of the compound surfactant is as follows: SDBS:70 mg/L, betaine: 80 mg/L, tween 80:3 mL/L, wherein the addition amounts are all the addition amounts in the fermentation broth;

(8) Standing for 4-6 h, and layering the two phases;

(9) Centrifuging at 8,000 rpm for 5 min, and collecting supernatant;

(10) Spin steaming the supernatant;

(11) After the rotary steaming is finished, placing the rotary steaming bottle in a 60 ℃ oven, drying, and weighing to obtain total lipid;

the seed liquid culture medium is as follows: glucose: 40-60 g/L, yeast extract powder: 4-6 g/L, sodium sulfate: 5-8 g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 8-12g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L;

the fermentation medium is as follows: glucose: 60-100 g/L, yeast extract powder: 5-15 g/L, sodium sulfate: 5-12 g g/L, magnesium sulfate: 2-4 g/L, ammonium sulfate: 4-8 g/L, potassium chloride: 1-2 g/L, calcium chloride: 0.1-0.2 g/L, potassium sulfate: 0.5-1 g/L, potassium dihydrogen phosphate: 0.5-2 g/L, sodium glutamate: 15-20 g/L, zinc sulfate heptahydrate: 1-5 mg/L, cobalt chloride hexahydrate: 0.01-0.1 mg/L, copper sulfate pentahydrate: 2-6 mg/L, nickel sulfate hexahydrate: 1-2 mg/L, ferric sulfate heptahydrate: 8-15 mg/L, calcium pantothenate: 2-4 mg/L, manganese chloride tetrahydrate: 3-5 mg/L, sodium molybdate dihydrate: 0.02-0.04 mg/L, vitamin B ₆ :4-10 mg/L, vitamin B ₁₂ ：0.1-0.5 mg/L。

2. Use of the schizochytrium lipid extraction process of claim 1 in schizochytrium lipid extraction.

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