CN115786412A - Method for extracting polyunsaturated fatty acid grease - Google Patents
Method for extracting polyunsaturated fatty acid grease Download PDFInfo
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- CN115786412A CN115786412A CN202211464363.1A CN202211464363A CN115786412A CN 115786412 A CN115786412 A CN 115786412A CN 202211464363 A CN202211464363 A CN 202211464363A CN 115786412 A CN115786412 A CN 115786412A
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Abstract
The invention provides an extraction method of polyunsaturated fatty acid grease. The extraction method of the polyunsaturated fatty acid oil comprises the following steps: 1) Breaking the wall of the fermentation liquor of the oil-producing microbial strains to obtain oil-containing emulsion; 2) Contacting a non-polar solvent with the oil-containing emulsion; 3) Then adding short chain monohydric alcohol into the mixture, and stirring the mixture at the speed of 30r/min to 200 r/min; 4) Collecting the polyunsaturated fatty acid oil; the oil content in the oleaginous microorganism is not higher than 20%. The extraction method of the polyunsaturated fatty acid oil provided by the invention can be used for quickly and successfully separating the oil aiming at the oil-producing microbial strains with low oil content, and can also avoid the deterioration of the oil caused by a long-time high-temperature demulsification means.
Description
Technical Field
The invention relates to the technical field of extraction of polyunsaturated fatty acids, and particularly relates to an extraction method of polyunsaturated fatty acid grease.
Background
Omega-3 polyunsaturated fatty acids, especially DHA and EPA, DHA has important effects on brain and visual function development, EPA can help to regulate blood fat, EPA and DHA play positive roles in adult brain function health, cardiovascular health, physiological metabolism and endocrine balance from infant growth, intelligence development and retina formation to middle-aged and old cardiovascular and cerebrovascular health and reduction of adult cancer risk, and Omega-3 has important effects throughout the whole life cycle of human. The compound of EPA and DHA with different proportions can play a diversified role in people with different intakes. For example, high content of EPA is more beneficial to the cardiovascular and cerebrovascular health of the middle-aged and elderly people, and the ratio of EPA to DHA is 1:1-1:2, while DHA: EPA above 4:1 is more beneficial to infant growth and development.
The Omega-3 is mainly derived from fish oil, most of purification and extraction methods are urea inclusion and then molecular distillation, along with the development of the microbial oil industry, various oleaginous microorganisms with different attributes are discovered, various polyunsaturated fatty acid oils with various characteristics can be obtained, such as nannochloropsis, schizochytrium, dinoflagellate, mortierella alpina and the like, and meanwhile, novel strains are continuously developed in the industry according to the characteristics of various algae. At present, strains which can be industrialized through fermentation all have the characteristic of high oil content (45-60%), but the oil content of a small part of strains which can produce special functional mixture ratio is low and is lower than 20%, even lower than 10%, so that the conventional water-enzyme separation process is difficult to adopt in the subsequent process of extracting and separating polyunsaturated fatty acid grease, and the heating, conventional centrifugation including high-speed centrifugation, enzyme assistance and other means mentioned in the prior art can not realize effective demulsification and grease separation due to the small and serious emulsification of oil in fermentation mixed liquor.
Disclosure of Invention
The first purpose of the invention is to provide a method for extracting polyunsaturated fatty acid oil.
It can be known in the art that most of the polyunsaturated fatty acid oil in the market is extracted by oil-producing microorganisms through methods such as fermentation, and taking algae for DHA fermentation production as an example, the algae, especially schizochytrium limacinum, are widely applied to DHA fermentation production, and most of the schizochytrium limacinum has low EPA content. In the continuous research of the applicant, a type of schizochytrium limacinum strain which is fermented to a level higher than that of the conventional EPA is obtained, however, the fermentation characteristics of the type of schizochytrium limacinum strain are greatly different from those of the conventional DHA-fermented schizochytrium limacinum, the content of polyunsaturated fatty acid in the type of schizochytrium limacinum strain is very high, and the oil content is only 1/4-1/2 of that of the conventional DHA-fermented schizochytrium limacinum. The applicants have studied such oleaginous microorganisms for the fermentative production of polyunsaturated fatty acids (e.g., DHA, EPA, etc.) with low oil content and have found that effective demulsification cannot be achieved using conventional methods, thereby resulting in the extraction process of the present invention.
Based on the above, the invention provides an extraction method of polyunsaturated fatty acid oil, which aims at oil-producing microbial strains with low oil content (not higher than 20wt%, preferably not higher than 10 wt%), can be used for rapidly and successfully separating the oil, and simultaneously avoids the oil from being deteriorated by means of long-time high-temperature demulsification.
The extraction method provided by the invention comprises the following steps:
1) Breaking the wall of the fermentation liquid of the oil-producing microbial strains to obtain oil-containing emulsion;
2) Contacting a non-polar solvent with the oil-containing emulsion;
3) Then adding short chain monohydric alcohol into the mixture, and stirring the mixture at the speed of 30r/min to 200 r/min;
4) Collecting the polyunsaturated fatty acid oil;
the oil content in the oleaginous microorganism is no greater than 20%.
In a specific embodiment of the present invention, the wall breaking method in step 1) may be grinding, homogenizing, enzymolysis, etc., aiming to obtain the oil-containing emulsion containing the substances such as oil and fat, cell debris, etc. in step 1). In a preferred embodiment of the present invention, the fermentation broth of the oleaginous microbial species may be subjected to wall breaking using enzymatic methods commonly used in the art, such as aqueous enzymatic methods and the like. In the specific implementation manner of the invention, in the step 1), the fermentation liquid of the oil-producing microbial strain can be stirred and heated to the enzymolysis temperature, the pH value is adjusted to the optimum pH value for enzymolysis, alkaline protease (usually 0.08wt% of the fermentation liquid) is added, and the mixture is stirred and reacted for 6 to 8 hours to carry out enzymolysis and wall breaking, so as to obtain the oil-containing emulsion. Among them, alkaline proteases are those commonly used in the art and commercially available.
In some embodiments, the oil content in the oleaginous microorganism is no greater than 10%.
In a preferred embodiment of the present invention, in step 2), the non-polar solvent is a non-polar solvent immiscible with both water and alcohol, preferably n-hexane. The step 2) of contacting the nonpolar solvent with the oil-containing emulsion specifically includes mixing the nonpolar solvent with the oil-containing emulsion, and sufficiently mixing the nonpolar solvent and the oil-containing emulsion by stirring or the like until the nonpolar solvent and the oil-containing emulsion are uniformly mixed. In the embodiment of the present invention, the stirring speed in this step is not strictly limited, so that the mixed solution is mixed uniformly, and in a specific embodiment, the stirring speed may be about 200 r/min. In a preferred embodiment, the volume ratio of the non-polar solvent to the oil-containing emulsion is at least 1:1.
In the invention, the addition of short-chain monohydric alcohol into the system after the nonpolar solvent is contacted with the oil-containing emulsion in the step 2) is one of the core inventions of the invention. It is preferable to add the short-chain monohydric alcohol directly to the system after the non-polar solvent is contacted with the oil-containing emulsion in step 2), without any additional step between the two steps, i.e., without any additional step between step 2) and step 3). In a preferred embodiment of the present invention, in step 3), the short-chain monohydric alcohol is a monohydric alcohol, which is miscible with water and insoluble in the non-polar solvent, and has a carbon number of not more than 3, and is preferably methanol or ethanol. Wherein, the dosage (volume) of the short chain monohydric alcohol is preferably 70-100% (volume) of the oil-containing emulsion, and is preferably 70%.
In some embodiments, steps 2) and 3) may be repeated as appropriate.
In the scheme provided by the invention, the stirring speed of adding the short-chain monohydric alcohol in the step 3) is also very important, and a large number of experiments show that the stirring speed needs to be 30 r/min-200 r/min to efficiently achieve the purpose of the invention, and the stirring speed is further preferably 60-160 r/min.
The extraction method provided by the invention can be applied to various polyunsaturated fatty acid oils and fats, and is preferably used for EPA oil and fat, DHA oil and ARA oil and fat.
The oil contains abundant polyunsaturated fatty acids including one or more of EPA, ARA, and DHA.
Preferably, the polyunsaturated fatty acid oil contains EPA 5-15%, DHA40-60%, peroxide value 0.04-1, and anisidine value less than 15.
The invention also aims to provide polyunsaturated fatty acid oil, wherein the polyunsaturated fatty acid oil contains 5-15% of EPA, 40-60% of DHA, 0.04-1% of peroxide value and less than 15% of anisidine value.
In the present invention, "%" is a mass percentage unless otherwise specified.
The extraction method of the polyunsaturated fatty acid oil provided by the invention can be used for quickly and successfully separating the oil aiming at the oil-producing microbial strains with low oil content, and can also avoid the deterioration of the oil caused by a long-time high-temperature demulsification means.
Drawings
FIG. 1 is a photograph showing the state of a mixing system after ethanol was added and left to stand in example 1;
FIG. 2 is a photograph showing the state of EPA crude oil obtained in example 1;
FIG. 3 is a photograph showing the state of the mixed system after the centrifugation at high speed in comparative example 2 and the standing.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are provided to illustrate the present invention, but are not intended to limit the scope of the present invention.
The preparation method of the oil-containing emulsion in examples 1 to 3 and comparative examples 1 to 5 of the present invention is:
the oil-producing microorganism is schizochytrium with the oil content of 10% in the thallus after fermentation.
And (2) heating the fermentation liquor of the oil-producing microbial strain to 55 ℃ while stirring, adjusting the pH to 9 with a sodium hydroxide solution, adding alkaline protease according to 0.08 percent of the fermentation liquor, stirring for reaction for 6-8 hours, and performing enzymolysis wall breaking and microscopic examination on the wall breaking condition to obtain the oil-containing emulsion, wherein the oil content of the oil-containing emulsion is 1 percent.
The oil-containing emulsions of example 4 and comparative example 6 were prepared by:
the oil content of the thallus after fermentation is 15 percent. Filtering the mortierella alpina fermentation liquor, and drying in vacuum at 45 ℃ to obtain dry thalli; the water content in the obtained dry microbial cells was 5%. And then shearing and rehydration are carried out: adding 5 times of pure water into the superfine pulverized dry thallus, shearing at 20krpm for rehydration for 15min, wherein the particle size is D50=73 μm; d90=202 μm. The enzymolysis wall breaking comprises the following stages: and (3) a glucanase enzymolysis stage: adjusting the pH value to 4.90, adding beta-glucanase accounting for 3 percent of the mass of the thalli and phospholipase accounting for 3 percent of the mass of the thalli, and stirring in a water bath at 50 ℃ for 22 hours. After the enzymolysis is finished, the temperature is raised to 80 ℃, and the temperature is kept for 30min. And (3) protease enzymolysis stage: cooling to 50 ℃, adjusting the pH value to 7.09, adding protease accounting for 4 percent of the mass of the thalli, and stirring in a water bath at 50 ℃ for 8 hours. After the enzymolysis is finished, the temperature is raised to 80 ℃, and the temperature is kept for 30min. And (3) chitinase enzymolysis stage: cooling to 35 ℃, adjusting the pH value to 6.82, adding chitinase accounting for 5 percent of the mass of the thalli, and stirring in a water bath at 35 ℃ for 13 hours. After the enzymolysis is finished, the temperature is raised to 80 ℃, and the temperature is kept for 30min. The oil content in the finally obtained turbid emulsion was 3%.
Example 1
1) Adding 200mL of normal hexane into 200mL of oil-containing emulsion containing 1% (mass content) of oil, stirring at 200r/min for 10min, and mixing uniformly;
2) Adding 140mL of ethanol, stirring at 60r/min for 5min for demulsification, standing for 10min, after layering is stable (as shown in figure 1), sucking 120mL of supernatant by a vacuum catheter (laboratory glass pipette), supplementing 120mL of normal hexane to the rest part, stirring at 200r/min for 10min, mixing uniformly, adding 20mL of ethanol for demulsification, repeating the operation twice, collecting 300-400mL of supernatant, and desolventizing to obtain 2g of oil (namely polyunsaturated fatty acid oil, also called pufa oil), wherein the oil extraction rate is 100%. The obtained pufa fat had an EPA content of 6.89%, a DHA content of 56.20%, and a peroxide value of 0.05meq/kg.
Example 2
1) Concentrating 600mL of oil-containing 1% oil-containing emulsion to 200mL by using a three-phase centrifuge, adding 200mL of n-hexane, stirring at 200r/min for 10min, and uniformly mixing;
2) Adding 140mL of ethanol, stirring at 60r/min for 5min, demulsifying, standing for 10min, after layering is stable, sucking 120mL of supernatant by using a vacuum catheter (a laboratory glass suction tube), supplementing 120mL of n-hexane, stirring at 200r/min for 10min, uniformly mixing, adding 20mL of ethanol, demulsifying, repeating the operation twice, collecting 300-400mL of supernatant, and desolventizing to obtain 5.8g of oil (namely polyunsaturated fatty acid oil, also called pufa oil), wherein the oil extraction rate is 96.7%. The obtained pufa fat had an EPA content of 6.85%, a DHA content of 8978 zxft Of 8978%, and a peroxide value of 0.04meq/kg.
Example 3
1) Adding 200mL of normal hexane into 200mL of oil-containing emulsion containing 1% of oil, stirring at 200r/min for 10min, and mixing uniformly;
2) Then 140mL of ethanol is added at a speed of 200r/min, stirred for 5-10min and then kept stand for 2h without layering, and layering is only generated after standing overnight (12 h).
Example 4
1) Adding 200mL of n-hexane into 200mL of oil-containing emulsion containing 3% (mass content) of oil, stirring at 200r/min for 10min, and mixing uniformly;
2) Adding 140mL of ethanol, stirring at 60r/min for 5min for demulsification, standing for 10min, after layering is stable (as shown in figure 1), sucking 120mL of supernatant by a vacuum catheter (laboratory glass pipette), supplementing 120mL of normal hexane to the rest part, stirring at 200r/min for 10min, mixing uniformly, adding 20mL of ethanol for demulsification, repeating the operation twice, collecting 300-400mL of supernatant, and desolventizing to obtain 2g of oil (namely polyunsaturated fatty acid oil, also called pufa oil), wherein the oil extraction rate is 100%. The obtained pufa fat had an ARA content of 49.4% and a peroxide value of 0.3meq/kg.
Comparative example 1
1) 200mL of oil-containing 1% oil-containing emulsion is taken, the feed liquid is heated to 90 ℃, and the feed liquid enters a three-phase centrifuge for centrifugal separation while the feed liquid is hot.
The emulsion in this comparative example did not separate out the oil (within 48 h).
Comparative example 2
1) 200mL of an oil-containing 1% emulsion was centrifuged at a high speed (8000 r/min) while it was hot.
In this comparative example, the oil and fat could not be separated from the emulsion, and as shown in FIG. 3, the system was not separated after standing after high-speed centrifugation.
Comparative example 3
This comparative example is the same as example 1, with the only difference that: step 2) was not included in comparative example 3.
As a result, the emulsion could not be separated.
Further heating and standing for 3h at 70 ℃ to separate the grease.
But the yield is 20 percent, and the peroxide value is as high as 8.
Comparative example 4
This comparative example is the same as example 1, with the only difference that: comparative example 4 without step 1) (200 mL of an oil-containing 1% oil-containing emulsion was directly subjected to step 2)).
As a result, the emulsion could not be separated (within 48 h).
Comparative example 5
1) Adding 140mL of ethanol into 200mL of oily 1% emulsion, stirring at 60r/min for 5-10min, and mixing;
2) Then 200mL of n-hexane is added, stirred at 200r/min for 10min and then kept stand.
As a result, the emulsion could not be separated (within 24 h).
Comparative example 6
1) 200mL of an oil-containing emulsion containing 3% of oil was centrifuged at a high speed (8000 r/min) while it was hot.
In the comparative example, the oil could not be separated from the emulsion, and the system after standing after high-speed centrifugation did not separate.
Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for extracting polyunsaturated fatty acid grease is characterized by comprising the following steps:
1) Breaking the wall of the fermentation liquor of the oil-producing microbial strains to obtain oil-containing emulsion;
2) Contacting a non-polar solvent with the oil-containing emulsion;
3) Then adding short chain monohydric alcohol into the mixture, and stirring the mixture at the speed of 30r/min to 200 r/min;
4) Collecting the polyunsaturated fatty acid oil;
the oil content in the oleaginous microorganism is no greater than 20%.
2. The extraction process according to claim 1, wherein in step 2), the non-polar solvent is a non-polar solvent immiscible with both water and alcohol.
3. The extraction method according to claim 2, wherein in the step 2), the nonpolar solvent is n-hexane.
4. The extraction process according to any one of claims 1 to 3, wherein the volume ratio of the non-polar solvent to the oil-containing emulsion is at least 1:1.
5. The extraction method according to any one of claims 1 to 4, wherein in step 3), the short-chain monohydric alcohol is a monohydric alcohol, which is miscible with water and insoluble in the nonpolar solvent, has a carbon number of not more than 3, and is preferably methanol or ethanol.
6. The extraction process according to any one of claims 1 to 5, wherein in step 3), the volume of the short-chain monohydric alcohol is 70% to 100% of the oil-containing emulsion.
7. The extraction method according to any one of claims 1 to 6, wherein the stirring speed in step 3) is 60 to 160r/min.
8. The extraction method according to any one of claims 1 to 7, wherein the polyunsaturated fatty acids in the polyunsaturated fatty acid oil are one or more of EPA, DHA and ARA.
9. A polyunsaturated fatty acid oil characterized in that the polyunsaturated fatty acid oil contains 5-15% of EPA, 40-60% of DHA, 0.04-1% of peroxide value and less than 15% of anisidine value.
10. The polyunsaturated fatty acid oil and fat according to claim 9, which is obtained by the extraction method according to any one of claims 1 to 8.
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| CN202211464363.1A CN115786412A (en) | 2022-11-22 | 2022-11-22 | Method for extracting polyunsaturated fatty acid grease |
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| CN202211464363.1A CN115786412A (en) | 2022-11-22 | 2022-11-22 | Method for extracting polyunsaturated fatty acid grease |
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