阅读说明：本技术 一种微生物油脂提取方法及其所得微生物油脂 (Microbial oil extraction method and microbial oil obtained by same ) 是由 汪志明 陆姝欢 杨刚 马凡提 李翔宇 于 2020-05-18 设计创作，主要内容包括：本发明属于微生物油脂提取技术领域,具体涉及一种微生物油脂提取方法及其所得微生物油脂。所述微生物油脂提取方法包括：对菌体采用干法破壁,并以极性Et(30)≥38的极性溶剂进行提取；其中所述菌体的含水率为3％-10％。本发明所述方法不仅能够显著提高难破壁的微生物的破壁率及油脂提取率,同时所得油脂产品的过氧化值和茴香胺值均相对较低,具有良好的品质；此外,该方法工艺简单,能耗低,更有利于实现工业化生产。(The invention belongs to the technical field of microbial oil extraction, and particularly relates to a microbial oil extraction method and microbial oil obtained by the same. The microbial oil extraction method comprises the following steps: breaking the wall of the thallus by a dry method, and extracting by a polar solvent with the polarity Et (30) being more than or equal to 38; wherein the water content of the thallus is 3% -10%. The method disclosed by the invention can obviously improve the wall-breaking rate of the microorganisms difficult to break the wall and the oil extraction rate, and simultaneously, the peroxide value and the anisidine value of the obtained oil product are relatively low, so that the oil product has good quality; in addition, the method has simple process and low energy consumption, and is more favorable for realizing industrial production.)

1. A microbial oil extraction method is characterized by comprising the following steps: breaking cell wall by dry method, and adding polar E_t(30) Extracting with polar solvent of 38 or more; the water content of the thallus is 3% -10%.

2. The method for extracting microbial oil according to claim 1, wherein the water content of the thalli is 3% -5%;

and/or the thallus is obtained by drying wet thallus obtained by filtering or centrifuging microorganism fermentation liquor at the temperature of less than 60 ℃;

and/or the particle size of the thallus is below 10 meshes.

3. The microbial oil extraction method of claim 1 or 2, wherein the dry wall breaking is micronization wall breaking;

preferably, the superfine grinding wall breaking is grinding type superfine grinding wall breaking, and the operating conditions are as follows: the pressure is 0.1-1MPa, the rotating speed is 10-60r/min, and the gap between the rollers is less than 1 mm.

4. The method for extracting microbial oils and fats according to claim 3, wherein said polar solvent is 38. ltoreq. E_t(30) Extracting the oil by extracting the wall-broken materials when the value is less than or equal to 52;

preferably, said 38 ≦ E_t(30) The polar solvent with the value less than or equal to 52 is one or more of ethyl acetate, isopropanol, chloroform, acetone, acetic acid or acetonitrile; further preferably isopropyl alcohol and/or ethyl acetate;

or the like, or, alternatively,

the polar solvent is E_t(30) When the polar solvent is more than 52, preparing emulsion from the materials after wall breaking, demulsifying, separating and extracting the grease;

preferably, said E_t(30) The polar solvent with the purity of more than 52 is water and/or ethanol; further preferably, the mass concentration of the material in the emulsion is between 10 and 20%.

5. The microbial oil extraction method of claim 4, wherein in the demulsification process, the pH of the system is controlled to be 7-10, the emulsion is heated to 50-60 ℃ for 4-12 hours, and then heated to 90-100 ℃ for 5-10 min.

6. The microbial oil extraction method of claim 5, wherein the demulsifying further comprises: adding alkaline protease to carry out enzymolysis reaction;

preferably, the adding amount of the alkaline protease is 0.5-1% of the mass of the dry thallus;

preferably, the conditions of the enzymatic hydrolysis reaction are as follows: the pH value of the system is 7-10, the temperature is 50-60 ℃, and the time is 5-7 h.

7. The microbial oil extraction method of claim 6, wherein the demulsifying further comprises: adding auxiliary enzyme into the system;

preferably, the helper enzyme is one or more of pectinase, beta-glucanase or phospholipase; further preferred are pectinase and beta-glucanase.

8. The method for extracting microbial oil according to claim 7, wherein the auxiliary enzymes are added in two ways: one is that the auxiliary enzyme is added after the enzymolysis reaction of the alkaline protease is finished, the pH value of the system is controlled to 6-8, and the enzymolysis reaction is carried out for 4-6 h;

alternatively, the auxiliary enzyme and the alkaline protease are added simultaneously, and the pH of the system is controlled to 7-9;

preferably, the helper enzyme is added simultaneously with the alkaline protease.

9. A microbial oil and fat extraction method according to any one of claims 1 to 8, wherein the microorganism is selected from one or more of dinoflagellates, Chlamydomonas wukenensis, Schizochytrium, Thraustochytrium, yeast, and Mortierella alpina.

10. A microbial oil obtainable by the process of any one of claims 1 to 9; the polyunsaturated fatty acid in the microbial oil is one or more of omega-3 fatty acid, omega-6 fatty acid, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid or stearidonic acid;

preferably, in the microbial oil, the content of triglyceride is more than 90%, the anisidine value of the oil is less than or equal to 5, and the peroxide value is less than or equal to 3;

more preferably, in the microbial oil, the anisidine value of the oil is less than or equal to 4.

Technical Field

The invention belongs to the technical field of microbial oil extraction, and particularly relates to a microbial oil extraction method and microbial oil obtained by the same.

Background

The traditional extraction process of microbial oil is to separate thallus from fermentation liquor, dry the thallus and extract intracellular lipid by using alkane solvent (i.e. non-polar solvent). The method has high extraction rate, but needs to use a large amount of volatile and combustible alkane solvents, so that the operation condition is dangerous, expensive explosion-proof equipment is needed, a solvent recovery process is needed, the cost is increased, and the complexity of the process is increased; moreover, microorganisms are heated due to the drying of thalli, and the quality of oil and fat is reduced if the microorganisms are not properly operated; in addition, the obtained oil product still has a part of alkane solvent, which has a great influence on the product quality.

In order to avoid the above problems, the technicians try to extract the oil from the microorganisms by using polar solvent instead of non-polar solvent, but the fermentation liquid is subjected to wall breaking treatment, such as CN1416469A, CN107523417A, CN105960235A, etc.

However, for some thalli (such as mortierella alpina, dinoflagellate, ophiocordyceps wukensonii, etc.) with complex cell wall thickness or cell wall composition, the conventional wall-breaking method has poor wall-breaking effect; the subsequent oil extraction rate is very low, and the oil quality is seriously damaged by adopting an extreme wall-breaking means (such as wall breaking under extreme chemical conditions).

Therefore, how to improve the wall-breaking rate and the oil extraction rate of the microorganisms difficult to break the wall, realize industrialized extraction, and ensure the quality of the oil becomes one of the technical problems to be solved urgently in the industry.

Disclosure of Invention

In order to solve the technical problems, the invention provides a novel microbial oil extraction method. The method can not only obviously improve the wall-breaking rate of microorganisms difficult to break the wall and the oil extraction rate, but also relatively lower peroxide value and anisidine value of the obtained oil product and has good quality; in addition, the method has simple process and low energy consumption, and is more favorable for realizing industrial production.

The microbial oil extraction method comprises the following steps: breaking cell wall by dry method, and adding polar E_t(30) Extracting with polar solvent of not less than 38(ii) a Wherein the water content of the thallus is 3% -10%.

In the prior art, the dried thalli are extracted by adopting an alkane solvent (namely a nonpolar solvent), although the extraction rate is high, the oil quality is poor, and solvent residues exist; the polar solvent extraction technology can avoid partial defects of a non-polar solvent, but the fermentation liquor needs to be subjected to wet wall breaking treatment; however, for some thalli (such as mortierella alpina, dinoflagellate, ophiocladium wukensonii and the like) with complex cell wall thickness or cell wall composition, the wall breaking effect is poor, and even the wall can not be broken, so that the subsequent oil extraction rate is very low; and the quality of the grease is seriously damaged by adopting an extreme wall breaking means (such as wall breaking under extreme chemical conditions).

The dry wall breaking method has the advantages of high wall breaking rate, no solvent residue and the like, and is commonly used in the field of traditional Chinese medicine preparation; the technicians try to use the method for breaking the cell wall of the thalli with complicated cell wall thickness or cell wall composition so as to solve the problem of difficult wall breaking; experiments show that although the wall breaking rate can be obviously improved by adopting the dry wall breaking method, the cell carbonization or gelatinization of thalli is easy to occur due to overhigh temperature in the wall breaking process because of strong wall breaking capability of the thalli, and part of grease also undergoes Maillard reaction, so that the color of the grease is darker, the subsequent grease refining is difficult, higher oil extraction rate cannot be obtained, and the quality of the grease can be reduced.

Therefore, the technicians of the invention further research and discover that when the water content of the thalli is between 3% and 10%, the thalli has proper brittleness, and the dry-method wall breaking is adopted, so that the high wall breaking rate can be realized, the wall breaking time can be shortened, and the phenomenon that the thalli is carbonized or gelatinized at high temperature for a long time is avoided, so that the oil extraction rate is obviously improved, and the oil quality is ensured; the purpose of extracting the oil of the microorganisms which are difficult to break the wall (such as mortierella alpina, dinoflagellate, wuken's chytrid, schizochytrium or thraustochytrium and the like) by the polar solvent is really realized.

Preferably, the water content of the thallus is controlled between 3% and 5%.

According to some embodiments of the invention, the water content of the biomass is obtained by drying wet biomass (60-70% water content) obtained by filtering or centrifuging a microbial fermentation broth at a temperature below 60 ℃.

In the present invention, the specific drying process can be selected as required. In a more preferred embodiment, the water content of the cells is obtained by the following method: firstly, filtering or centrifuging microorganism fermentation liquor to obtain wet thalli (with the water content of 60-70%) and controlling water by means of extrusion and the like until the water content is below 40%; and drying the wet thallus at 50-60 deg.C under vacuum to reach target water content. Researches show that by controlling the water content in the thalli in stages, the finally obtained thalli can be ensured to have good brittleness, the wall breaking rate and the oil extraction rate are improved, and the influence on the quality of oil products caused by the change of lipid structures can be avoided. The drying mode can be freeze drying, boiling drying, vacuum drying and other conventional modes.

According to some embodiments of the present invention, the particle size of the bacteria is controlled to be less than 10 mesh. Research shows that reasonable control of the thallus granularity is not only beneficial to reducing the wall breaking difficulty, improving the wall breaking rate and shortening the wall breaking time, but also beneficial to protecting the stability of the lipid structure and shortening the whole extraction time.

According to some embodiments of the invention, the dry wall breaking is preferably micronization wall breaking; the granularity of the material treated by the technology is not more than 20 mu m, which is beneficial to improving the subsequent oil extraction rate; and the wall breaking efficiency is high, the wall breaking time is greatly shortened, the overhigh peroxide value and anisidine value of the grease can be avoided, and the grease quality is more favorably improved.

In addition, in order to obtain better wall breaking effect, the superfine grinding wall breaking is preferably carried out under the conditions of vacuum or nitrogen-filled environment and temperature less than 45 ℃; researches show that under the protection of inert gas atmosphere or vacuum and other conditions, the temperature is kept at a lower temperature, so that the instrument is more friendly, and the wall breaking effect is further improved.

Further, the superfine grinding wall breaking is preferably carried out in a grinding type superfine grinding wall breaking mode; the pressure condition of the grinding type superfine grinding wall breaking is 0.1-1MPa, and the appropriate pressure condition can be adjusted according to the specific types of the thalli; for example, for dinoflagellate, schizochytrium, thraustochytrium, yeast and other cells with thicker cell walls and/or spherical thallus, the wall breaking is required under the condition of higher pressure, such as 0.5-1 MPa; the pressure selection range of the irregular filamentous fungi such as Mortierella alpina is wider, and the pressure can be within the range of 0.1-1 MPa.

In one embodiment, the conditions of the grinding type ultrafine grinding are as follows: the pressure is 0.1-1MPa, the rotating speed is 10-60r/min, and the gap between the rollers is less than 1 mm.

According to some embodiments of the invention, the polar solvent is E_t(30) The value range is 38 ≤ E_t(30) Polar solvent having a value of 52 or less and E_t(30)>52 are two broad classes of polar solvents.

E_T(30) Is the most well established and commonly used empirical parameter for polarity at present, and is characterized by the electron transition energy of the absorption band of the ultraviolet-visible spectrum (UV-Vis) of pyridinium-N-phenoxyinner salt dye (Reichardt's dye) in a solvent, as proposed by Reichardt and Dimroth et al. The molecule has a pi-absorption band with intramolecular Charge Transfer (CT) characteristic, so that the application range is wide. More than 300 pure solvents of E have been measured using the pyridinium-N-phenoxyinner salt dye method_T(30) The value, and thus the polarity of the solvent, has been recognized by researchers in the field.

According to some embodiments of the invention, E of the polar solvent_t(30) The value is 38. ltoreq. E_t(30) When the value is less than or equal to 52, the polar solvent can be directly extracted into the grease through the material after the wall is broken by extraction; the extraction conditions were: extracting at 20-50 deg.C for 10-60min for 1-2 times; the volume amount of the polar solvent is 5-10 times of that of the thallus.

Said E_t(30) The value is 38. ltoreq. E_t(30) The polar solvent having a value of 52 or less is preferably one or more of Ethyl acetate, i-propanol (isopropanol), Chloroform (Chloroform), Acetone (Acetone), Acetic acid (Acetic acid) or Acetonitrile (Acetonitrile); further preferred is isopropanol and/or ethyl acetate.

According to some embodiments of the invention, said E_t(30)>Polar solvent with the solvent content of more than 52, the extraction steps are as follows: by breaking cell wallPreparing emulsion, demulsifying, separating and extracting oil. Preferably, said E_t(30)>When the polar solvent above 52 is water and/or ethanol, the mass concentration of the materials is controlled to be between 10 and 20 percent, and the proper fluidity is more beneficial to improving the oil extraction rate.

According to some embodiments of the invention, in the demulsification process, the pH of the system is controlled to be 7-10, the emulsion is heated to 50-60 ℃ and kept for 4-12h, stirring operation can be assisted, and then the temperature is increased to 90-100 ℃ and kept for 5-10 min; researches show that the demulsification effect is better improved by reasonably controlling the temperature by stages, and meanwhile, the reduction of the grease quality caused by long-time high temperature is avoided.

In order to obtain better demulsification effect, the demulsification also comprises the following steps: adding alkaline protease to carry out enzymolysis reaction; the alkaline protease can destroy protein in the material, so that the emulsion is broken, and two phases are separated.

Preferably, the conditions of the enzymatic hydrolysis reaction are as follows: controlling the pH value of the system to 7-10, the temperature to 50-60 ℃ and the time to 5-7 h; researches show that the enzymolysis effect is better under the condition, and the oil extraction rate is more favorably improved.

Preferably, the adding amount of the alkaline protease is 0.5-1% of the mass of the dry thallus.

According to some embodiments of the invention, the demulsifying further comprises: adding auxiliary enzyme into the system; the auxiliary enzyme is one or more of pectinase, beta-glucanase or phospholipase; pectinases and beta-glucanases are preferred.

Wherein the addition amount of the pectinase is 0.1-0.5% of the mass of the dry thalli; the addition amount of the beta-glucanase is 0.1 to 0.5 percent of the mass of the dry thalli; the addition amount of the phospholipase is 0.1-0.5% of the mass of the dry thallus. Researches show that the addition of the enzyme can improve the oil extraction rate of the oil, and simultaneously, the peroxide value and the anisidine value of the oil product are remarkably reduced, and the product quality is remarkably improved.

The addition modes of the auxiliary enzyme are divided into two modes: one is that the auxiliary enzyme is added after the enzymolysis reaction of the alkaline protease is finished, and the pH value of the system is controlled to 6 to 8 for enzymolysis reaction for 4 to 6 hours; the other is that the auxiliary enzyme and the alkaline protease are added simultaneously, and the pH of the system is controlled to be 7-9; preferably, it is added simultaneously with the alkaline protease.

According to some embodiments of the invention, the extracting comprises: and centrifuging the reaction liquid obtained after the enzymolysis reaction, and cooling the oil phase to below 35 ℃ after separation to obtain the microbial oil product. Wherein the rotation speed of the centrifugation is 6000-; the centrifugation time is 3-5 min.

In addition, in order to further improve the oil extraction rate, the extraction method further comprises the step of performing wet physical wall breaking on the emulsion before the emulsion is broken; the wet physical wall breaking comprises stirring, high-speed shearing, colloid mill shearing, wet grinding, high-pressure homogenizing and the like.

According to some embodiments of the invention, the extracting of the grease further comprises: and centrifuging the demulsified two-phase system, separating, and cooling the oil phase to below 35 ℃ to obtain the microbial oil product. Wherein the rotation speed of the centrifugation is 6000-; the centrifugation time is 3-5 min.

Further, in some embodiments, the microbial oil can also be recovered from the surface of the separated composition by, for example, decanting, skimming, vacuuming, pumping, sucking, drawing, siphoning, or otherwise.

The extraction method is suitable for most microorganisms, is particularly suitable for thick cell wall and/or spherical thalli such as dinoflagellates, wuken's kettles, schizochytrium, thraustochytrids, yeasts and the like, and mould irregular filamentous fungi such as mortierella alpina and the like, and can obviously improve the oil extraction rate of the microorganisms.

The invention also provides microbial oil obtained by the method, which contains not less than 70% of polyunsaturated fatty acid; the polyunsaturated fatty acid is selected from one or more of omega-3 fatty acid, omega-6 fatty acid, docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, arachidonic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid or stearidonic acid;

preferably, the microbial oil is for crude oil.

Preferably, in the microbial oil, the content of triglyceride is more than 90%, the anisidine value of the oil is less than or equal to 5, and the peroxide value is less than or equal to 3.

More preferably, in the microbial oil, the content of triglyceride is more than 90%, the anisidine value of the oil is less than or equal to 4, and the peroxide value is less than or equal to 3.

Preferably, the microbial oil comprises arachidonic acid (ARA) in an amount of not less than 37% by triglyceride, anisidine value (AnV). ltoreq.4, and peroxide value (POV). ltoreq.3.

Preferably, the microbial oil comprises docosahexaenoic acid (DHA) not less than 35%, anisidine (AnV) not more than 5%, and a peroxide number (POV) not more than 3, calculated as triglyceride.

The invention has the following beneficial effects:

the invention firstly provides a grease extraction mode combining dry wall breaking (especially superfine grinding wall breaking) and polar solvent extraction. The method can remarkably improve the cell wall thickness, the wall breaking rate (more than 98%) of the complex microorganisms and the oil extraction rate; moreover, the obtained crude oil has good indexes, and the pressure of the subsequent refining process is reduced.

Drawings

FIG. 1 is a microscopic image of the microbial oil product and emulsion obtained by the method described in example 1.

FIG. 2 is a microscopic image of the oil product and emulsion of the product obtained by acid-method wall-breaking in the prior art.

Detailed Description

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

Example 1

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) superfine grinding and wall breaking:

firstly, 5000g of mortierella alpina fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65 percent, and the wet thalli are dried in vacuum at the temperature of 60 ℃ to obtain 900g of dry thalli; the water content in the obtained thallus is 4.8 percent;

micronizing the obtained thallus at 30r/min under 0.4MPa for 10 min; the wall breaking rate is 98% by microscopic examination;

(2) extraction:

extracting with water as polar solvent; adding water into the material obtained by wall breaking to dilute the material to a mass concentration of 15% of cell fragments to obtain emulsion;

demulsifying: adjusting pH of the emulsion to 8, stirring at 55 deg.C for 6 hr, and boiling at 95 deg.C for 5 min;

separation: centrifuging and separating the two-phase system obtained in the step (2); the centrifugal speed is 8000r, and the centrifugal time is 3 min; and (3) cooling the separated oil phase to below 35 ℃ in a heat exchange manner to obtain the microbial oil, wherein the content of triglyceride is 93%, and a microscopic image is shown in figure 1.

FIG. 1 is a microscopic image of the microbial oil product and emulsion obtained by the method described in example 1.

FIG. 2 is a microscopic image of the oil product and emulsion of the product obtained by acid-method wall-breaking in the prior art.

Example 2

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) superfine grinding and wall breaking:

firstly, 5000g of mortierella alpina fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65 percent, and the wet thalli are dried in vacuum at the temperature of 60 ℃ to obtain 900g of dry thalli; the water content in the obtained thallus is 8 percent;

micronizing the obtained thallus at 30r/min under 0.3MPa for 20 min; the wall breaking rate is 98% by microscopic examination;

(2) same as in example 1.

Example 3

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) same as example 1;

(2) extraction:

extracting with water as polar solvent;

adding water into the wall-broken material to dilute the wall-broken material to a mass concentration of 20% of cell fragments to obtain an emulsion;

demulsifying: 9g of alkaline protease was added to the emulsion, the pH was adjusted to 9, the mixture was stirred at 55 ℃ for 6 hours, and then incubated at 90 ℃ for 5 min.

Extraction: the centrifugal speed is 6000 r/min.

Example 4

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) same as example 1;

(2) extraction:

extraction with water as polar solvent: adding water into the wall-broken material to dilute the wall-broken material to a mass concentration of 20% of cell fragments to obtain an emulsion;

demulsifying: adding 9g of alkaline protease into the emulsion, adjusting the pH to 9, and stirring at 55 ℃ for 6 hours; adding 0.9g of pectinase and 0.9g of beta-glucanase, adjusting the pH value to 6, and reacting for 6 hours; finally boiling at 95 deg.C for 5 min.

Extraction: the centrifugal speed is 6000 r/min.

Example 5

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) same as example 1;

(2) extraction:

extraction with water as polar solvent: adding water into the wall-broken material to dilute the wall-broken material to a mass concentration of 20% of cell fragments to obtain an emulsion;

demulsifying: 9g of alkaline protease, 0.9g of pectinase and 0.9g of beta-glucanase are simultaneously added into the emulsion, stirred and reacted for 6 hours under nitrogen, the pH value is adjusted to 8, and the reaction is carried out for 6 hours at 55 ℃. Finally boiling at 95 deg.C for 5 min.

Extraction: the centrifugal speed is 6000 r/min.

Example 6

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) same as example 1;

(2) extraction:

extracting with water as polar solvent; diluting the obtained material with water to a mass concentration of 15% containing cell debris, shearing with a shearing machine at 20000r, mixing for 30min, and circulating with a sand mill for 10 times to obtain emulsion.

Demulsifying: same as in example 4

Separation: the rotating speed is 8000 r/min.

Example 7

The embodiment provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) same as example 1;

(2) extraction:

extracting by taking isopropanol as a solvent; wherein the volume of isopropanol used in single extraction is 5 times of that of thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Example 8

The embodiment provides a method for extracting dinoflagellate grease, which comprises the following steps:

(1) micronizing for breaking cell wall

Firstly, 5000g of dinoflagellate fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65%, and the wet thalli is dried in vacuum at the temperature of 60 ℃ to obtain 1500g of dry thalli; the water content in the obtained thallus is 5 percent;

micronizing the obtained thallus at 30r/min under 0.8MPa for 10 min; the wall breaking rate is 98% by microscopic examination;

(2) extraction:

extracting with water as polar solvent; adding water into the material obtained by wall breaking to dilute the material to a mass concentration of 15% of cell fragments to obtain emulsion;

demulsifying: adjusting pH of the emulsion to 9, stirring at 55 deg.C for 6 hr, and boiling at 95 deg.C for 5 min;

separation: centrifuging and separating the two-phase system obtained in the step (2); the centrifugal speed is 8000r, and the centrifugal time is 3 min; and (3) cooling the separated oil phase to below 35 ℃ in a heat exchange manner to obtain the microbial oil, wherein the content of triglyceride of the microbial oil is 95%.

Example 9

The embodiment provides a method for extracting dinoflagellate grease, which comprises the following steps:

(1) micronizing for breaking cell wall

Firstly, 5000g of dinoflagellate fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65%, and the wet thalli is dried in vacuum at the temperature of 60 ℃ to obtain 1500g of dry thalli; the water content in the obtained thallus is 5 percent;

micronizing the obtained thallus at 30r/min under 0.8MPa for 10 min; the wall breaking rate is 98% by microscopic examination;

(2) extraction:

extracting by taking isopropanol as a solvent; wherein the volume of isopropanol used in single extraction is 5 times of that of thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Example 10

The embodiment provides a method for extracting dinoflagellate grease, which comprises the following steps:

(1) superfine grinding and wall breaking:

firstly, 5000g of dinoflagellate fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65%, and the wet thalli is dried in vacuum at the temperature of 60 ℃ to obtain 1500g of dry thalli; the water content in the obtained thallus is 5 percent;

micronizing the obtained thallus at 30r/min under 0.3MPa for 20 min; wall breaking rate by microscopic examination is 93%;

(2) extraction:

the same as in example 9.

Example 11

The embodiment provides a method for extracting dinoflagellate grease, which comprises the following steps:

(1) micronizing for breaking cell wall

Firstly, 5000g of dinoflagellate fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65%, and the wet thalli is dried in vacuum at the temperature of 60 ℃ to obtain 1500g of dry thalli; the water content in the obtained thallus is 5 percent;

micronizing the obtained thallus at 30r/min under 0.8MPa for 10 min; the wall breaking rate is 98% by microscopic examination;

(2) extraction:

extracting by using ethyl acetate as a solvent; wherein the volume of ethyl acetate used in single extraction is 7 times of that of the thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Example 12

The embodiment provides a method for extracting mortierella alpina grease, which comprises the following steps:

(1) and (3) breaking the wall by a dry method: breaking the wall of the dry thallus by a ball mill for 240min, wherein the wall breaking rate reaches 86%;

(2) extraction:

extracting with isopropanol as polar solvent; wherein the volume of the isopropanol used in single extraction is 5 times of that of the dry thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Example 13

The embodiment provides a method for extracting mortierella alpina grease, which comprises the following steps:

(1) and (3) breaking the wall by a dry method: breaking the cell wall of the dry thallus by a high-speed shearing grinder for 240min, wherein the wall-breaking rate reaches 78%;

(2) extraction:

extracting with isopropanol as polar solvent; wherein the volume of the isopropanol used in single extraction is 5 times of that of the dry thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Comparative example 1

The comparative example provides a conventional acid-method solvent-free extraction method of Mortierella alpina oil

(1) Acid method wall breaking: 1000g of mortierella alpina fermentation liquor is firstly subjected to solid concentration measurement of 12%, the pH value of the fermentation liquor is adjusted to 0.7 by hydrochloric acid, the fermentation liquor is uniformly mixed by a shearing machine, and then the fermentation liquor is stirred and reacted for 28 hours at 90 ℃.

(2) And (3) temperature rising demulsification: adjusting the pH value to 8, heating the reacted solution to 95 ℃, and preserving the temperature for 5 min.

(3) Centrifugal separation: and (4) carrying out heat preservation and centrifugation on the heated solution for 5min at the rotating speed of 8000r.

(4) Taking the grease layer, and rapidly cooling to below 35 ℃.

Comparative example 2

The comparative example provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) and (3) wet wall breaking: adding water into 900g of dry thalli to dilute the thalli by 20 percent, and circulating the thalli for 3 times by a high-pressure homogenizer under the pressure of 50 MPa;

(2) extraction:

demulsifying: adding 9g of alkaline protease into the wall-broken material, adjusting pH to 9, stirring at 55 deg.C for 6 hr, and boiling at 95 deg.C for 5 min;

separation: centrifuging at 8000r for 3min to separate oil.

Comparative example 3

The comparative example provides an extraction method of mortierella alpina grease, which comprises the following steps: and (3) directly extracting the dry thallus without wall breaking by using isopropanol, wherein the volume of the isopropanol used in single extraction is 5 times that of the dry thallus, the temperature is 50 ℃, stirring is carried out for 30min, and extraction is carried out for 2 times.

Comparative example 4

The comparative example provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) and (3) breaking the wall by a dry method: diluting the dry thallus with water to 20%, and performing colloid mill to break cell wall for 160min until the material particle size is 20 μm;

(2) extraction:

extracting with isopropanol as polar solvent; wherein the volume of the isopropanol used in single extraction is 5 times of that of the dry thallus, the temperature is 50 ℃, the stirring is carried out for 30min, and the extraction is carried out for 2 times.

Comparative example 5

The comparative example provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) superfine grinding and wall breaking:

firstly, 5000g of mortierella alpina fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65 percent, and the wet thalli are dried in vacuum at the temperature of 60 ℃ to obtain 900g of dry thalli; the water content in the obtained thallus is 2 percent;

micronizing the obtained thallus at 30r/min under 0.3MPa for 10 min; the wall breaking rate is 98% by microscopic examination;

(2) same as in example 1

Comparative example 6

The comparative example provides an extraction method of mortierella alpina grease, which comprises the following steps:

(1) superfine grinding and wall breaking:

firstly, 5000g of mortierella alpina fermentation liquor is filtered or centrifuged to obtain wet thalli with the water content of 65 percent, and the wet thalli are dried in vacuum at the temperature of 60 ℃ to obtain 900g of dry thalli; the water content in the obtained thallus is 17 percent;

micronizing the obtained thallus at 30r/min under 0.3MPa for 30 min; wall breaking rate under microscopic examination is 60%;

(2) same as in example 1.

Comparative example 7

The embodiment provides a method for extracting dinoflagellate grease, which comprises the following steps: and (3) directly extracting the dry thallus without wall breaking by using isopropanol, wherein the volume of the isopropanol used in single extraction is 5 times that of the dry thallus, the temperature is 50 ℃, stirring is carried out for 30min, and extraction is carried out for 2 times.

Effect verification

The results of examination of the products obtained by the methods described in examples 1 to 13 and comparative examples 1 to 7 are shown in tables 1 and 2.

TABLE 1

TABLE 2

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