CN102887821A - Method for extracting DHA (docosahexaenoic acid) through extraction and separation of marine microalgae fermentation liquid - Google Patents
Method for extracting DHA (docosahexaenoic acid) through extraction and separation of marine microalgae fermentation liquid Download PDFInfo
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Abstract
The invention relates to a method for extracting DHA (docosahexaenoic acid) through the extraction and separation of a marine microalgae fermentation liquid, which comprises the following steps: (1) dehydrating and drying a DHA microalgae fermentation liquid to obtain DHA algae powder; (2) selecting a low-boiling extraction agent to extract the DHA algae powder so as to obtain an extract liquid; (3) performing vacuum concentration on the extract liquid, and removing the solvent to obtain DHA raw oil; and (4) performing decolorization and vapor deodorization on the DHA raw oil, and then concentrating to obtain a DHA algae oil finished product. According to the invention, the total extraction yield of microalgae fermentation DHA production can be improved, the production cost can be lowered, and the discharge amount of three wastes can be decreased; and meanwhile, the extraction agent can be recovered and reutilized, thereby reducing environmental pollution.
Description
Technical field
The present invention relates to the extracting method of microbial oil, specifically, relate to the method for a kind of extracting and separating marine microalgae broth extraction DHA.
Background technology
DHA, full name docosahexenoic acid is commonly called as DHA (docosahexaenoic acid), is a kind of to the very important polyunsaturated fatty acid of human body, belongs to the important member in the Omega-3 unsaturated fatty acids family.DHA is a kind of principal element that nervous system cell is grown and kept, and is brain and amphiblestroid important composition composition, and content is up to 20% in the human brain cortex, proportion is maximum in eye retina, account for 50%, therefore, most important to fetus, baby's intelligence and visual acuity.
Medical research shows, the Main Function of DHA can promote fetal brain development, the maturation of promotion retina light sensation cell, the growth of enhancement brain cell, can also be used for cancer therapy, inflammation-inhibiting, blood fat reducing, prevention cardiovascular disease, the senile dementia etc. of improving.
For raw material makes, with the mixed form existence of timnodonic acid (EPA) and docosahexenoic acid (DHA), we are called Omega-3 or polyenoic acid second fat to early stage DHA product usually mainly with the fish oil that is rich in DHA and EPA (being generally tuna oil).The fish oil DHA production process is simple, low price, longer for some time extensively is used as the nutritional additive in infant and the pregnant woman's food as main DHA source in the past, but because in recent years progressively exhaustion and the atrophy of fishing resources, raw material sources climate and natural environment influence are very large.Along with worldwide pollution aggravation, persistence organic pollutant (POPs) and harm thereof contained in the fish oil progressively cause the public's attention, such as dichlorodiphenyl trichloroethane (DDT), chemical insecticide, polychlorinated biphenyl material (PCBs), Dioxins (Dioxin) and Perchlorobenzene (HCB) etc. is so that the DHA in fish oil source faces increasing predicament.
Opposite with the circumstances of fish oil, the DHA in little algae source is because whole production process can omnidistance be carried out strict Artificial Control, all need strict sterilization from the purebred cultivation of little algae to refining, aseptic technique, and can adopt food safety production and the control and management technology of international endorsement, the DHA purity of producing is high, pollution-free, does not contain fishy smell, and quality far wins fish oil DHA.It all is little algae DHA that DHA supplement in the infant or baby food that little algae DHA also is the unique approval of U.S. FDA, some esbablished corporations such as U.S. Heng Shi, U.S. Hui Shi, good fortune wait the DHA that adds in the product of enterprise near the house.Therefore, little algae DHA replacement fish oil DHA is imperative trend.
At present, the method for the used little algae DHA of separation mainly contains low temperature staging, urea adduct method, solvent method, supercritical gas extraction method etc. in the actual production.Wherein, the low temperature staging need to have large-scale refrigeration equipment, consumes energy high, and extraction efficiency is low; Urea adduct method is a kind of more simple and effective separation method, but when using in actual production, has that solvent loss is large, draining and the problems such as refuse processing that cause because of the urea additive.The supercritical gas extraction method can be isolated highly purified DHA, but the separating effect of the lipid acid that double key number is different to carbon number is identical is relatively poor, and facility investment is large, and technical requirements is high, consumes energy also larger.
The technique of the little algae oil of multiple fermentative Production DHA is also disclosed in the prior art.As yellow and, appoint the road to wait the people quietly and in Chinese invention patent CN101168501A, disclose a kind of also technique of refining DHA enriched fatty acid of from Crypthecodinium cohnii, extracting, first Crypthecodinium cohnii fermented liquid flocculation treatment is carried out solid-liquid separation again, by behind the alkali broken wall again with the cell Mechanical Crushing, then the thalline of fragmentation adopted organic solvent extraction, obtain slightly oil of DHA; The thick oil of DHA is through coming unstuck, obtaining the DHA essential oil behind the alkali refining, decolouring, deodorization.The weak point of this technique is, in the operating process to the thalline then broken wall that flocculates first, because flocculation agent all is the polymer substance of organic or inorganic, thalline in the fermented liquid can be assembled connect and form larger cotton-shaped, and be attached to the thalline top layer and form one deck coating, cause in the subsequent operations process broken wall insufficient, thereby cause the extraction yield of grease on the low side.
Zhan Guanghuang, the people such as Wu Yi disclose a kind of method of extracting the DHA unsaturated fatty acids from dino flagellate fermentation liquor in Chinese invention patent CN101585759, main processes is first dino flagellate fermentation liquor to be regulated pH value with hydrochloric acid soln, the flocculation take noresidue monomer polyacrylamide as flocculation agent, solidifying Plate Filtration obtains filter cake, filter cake adds the shell-broken liquid broken wall that water is made into the compound cellulose lytic enzyme, realize slag with whizzer behind the broken wall, water, the oil phase three phase separation, after repeating 2-5 time substantially without oil phase, merge to be after oil phase is also washed and contain the DHA crude oil, crude oil enters the continuous molecular distillation of Pyatyi, the final unsaturated fatty acids that gets DHA content 40-50%.The present invention adopts rotating speed 8000-12000 rev/min supercentrifuge to separate and directly obtains crude oil, and the refining Pyatyi molecular distillation that adopts of crude oil, can effectively reach the effect of decolouring, depickling, deodorization, but the shortcoming of this technology is to need at first to carry out the biological enzyme broken wall in the preparation process, carry out again the Pyatyi molecular distillation, so that whole extraction process route is long, flow process is complicated, energy consumption is high.
Summary of the invention
For the deficiencies in the prior art, the invention provides the method for a kind of extracting and separating marine microalgae broth extraction DHA, the method can improve the extraction yield of DHA, reduces production costs simultaneously, reduces three wastes discharge amount.
Its technical solution is:
The method of a kind of extracting and separating marine microalgae broth extraction DHA is characterized in that, may further comprise the steps: (1) dehydrates the little algae fermented liquid of DHA, gets DHA algae powder; (2) choose lower boiling extraction agent Extraction of DHA algae powder, be extracted liquid; (3) extraction liquid is carried out vacuum concentration, obtain the DHA crude oil behind the desolvation; (4) the DHA crude oil is decoloured and the steam deodorizing, then the concentrated DHA algae oil finished product that obtains.
Preferably, dehydrating in the step (1) comprises three grades of dehydrations: the one-level dehydration is to add flocculation agent in little algae fermented liquid, and algae liquid is separated with frond; The secondary dehydration is that the frond precipitation that the one-level dehydration obtains is carried out centrifugation, removes flocculation agent and moisture content, obtains mud shape frond; Three grades of dehydrations are that mud shape frond is carried out lyophilize, obtain the algae powder.
Preferably, described flocculation agent is a kind of in the aqueous solution of chitosan, polymerize aluminum chloride, polyacrylamide, alum, Tai-Ace S 150, Calcium Chloride Powder Anhydrous, bodied ferric sulfate or any several mixture, and its add-on is the 1.0-2.0% of fermented liquid quality.
Preferably, by percentage to the quality, the water content of DHA algae powder≤5% described in the step (1).
Preferably, the described lower boiling extraction agent of step (2) is a kind of or any several mixture in 6# organic solvent, sherwood oil, ether, hexane, acetone, chloroform, ethanol, methyl alcohol, ethyl acetate, propyl acetate, butylacetate, the ethyl propionate.
Preferably, the add-on of the described extraction agent of step (2) accounts for 40%～60% of fermented liquid quality, and extraction temperature is 45～60 ℃, and extraction times is 2-4 time.
Preferably, the vacuum tightness≤0.4Kpa of the described vacuum concentration of step (3), and the time that removes of solvent be 1～5 hour.
Preferably, carrying out step (4) before, can carry out purifying to the resulting DHA crude oil of step (3) first: in crude oil, add the NaCl solution that mass percent concentration is respectively 50-85% phosphoric acid solution and 30-70%, add-on is respectively 5%～20% of fermented liquid quality, after stirring under 50～65 ℃ of temperature, left standstill 0.5～1 hour, get upper strata fluid, be washed to neutrality, then adding mass percent concentration is the NaOH solution of 5-10%, stirs 40-50min, and stirring velocity is 30～80r/min, make the neat soap flocculation, then oil is warmed up to 80～100 ℃, when the obvious sedimentation of neat soap, stops to stir, standing sedimentation, separate soap stock, wash then vacuum-drying with 80～100 ℃ hot water again.
Should be pointed out that decolouring, steam deodorizing and the concentration technology of the described DHA crude oil of step of the present invention (4) are those skilled in the art's well-known processes.Preferably, used discoloring agent is atlapulgite, and its add-on accounts for 1～5% of quality of crude oil, and bleaching temperature is 75～85 ℃, and bleaching time is 20～40 minutes; The water vapour deodorization temperature is 75～85 ℃, and vacuum tightness is less than 0.4Kpa, and deodorization time is 0.5～1 hour.
It is that raw material extracts the little algae oil of DHA that the present invention adopts little algae fermented liquid.Wherein said little algae is the little algae of marine microorganism and phycomycetes, such as chrysophyceae, dinoflagellate, hidden algae, diatom, Crypthecodinium cohnii, Marine Chlorella, my Ken Shi kettle algae, thraustochytriale or schizochytrium limacinum etc., wherein preferred Crypthecodinium cohnii fermented liquid, my Ken Shi kettle algae fermented liquid, thraustochytriale fermented liquid or schizochytrium limacinum fermented liquid.Algae and phycomycetes obtain containing the fermented liquid of DHA through behind the normal fermentation slightly for these.
The present invention also has no particular limits described fermenting process, can adopt slant culture that those skilled in the art are familiar with, the cultivation of shaking table liquid kind, seed tank culture, fermentor cultivation etc.
Useful technique effect of the present invention is:
The present invention chooses lower boiling extraction agent, adopt drying, extraction, distillation, decolouring, deodorizing, concentration technology step to extract DHA, wherein extraction step can reduce the generation of mother liquor, reduce the content of DHA in mother liquor, the DHA extraction yield is improved greatly, reduce production costs, reduce three wastes discharge amount; Distilation steps can make lower boiling extraction agent recycling; Decolouring and deodorization step can further improve purity and the quality of DHA finished product.
Embodiment
Embodiment 1
(1) be 90g with the initial total lipid content of Crypthecodinium cohnii (Crypthecodinium cohnii) fermented liquid 1000g() introduce the flocculation sediment tank after, add the 10g chitosan flocculant, collecting precipitation frond after 45 minutes flocculates, through the disc centrifuge centrifugation, remove flocculation agent and moisture, obtain mud shape frond, then it is carried out lyophilize, make little algae dry powder of water content≤5%;
(2) add the 400g6# organic solvent in the little algae dry powder of step (1) gained and carry out the lixiviate extraction, extraction temperature is 45 ℃, and extraction times is four times, is extracted liquid;
(3) step (2) gained extraction liquid is passed through the vacuum concentration desolvation, vacuum tightness is 0.4Kpa, and the time of removing is 1 hour, obtains the DHA crude oil;
(4) adding the 50g mass percent concentration in step (3) the gained crude oil is that 85% phosphoric acid solution and 200g mass percent concentration are 30% NaCl solution, after 50 ℃ of lower low rate mixings are even, left standstill 1 hour, get upper strata fluid, be washed to neutrality, then add mass percent concentration and be 5% NaOH solution, stirred 40 minutes, stirring velocity is 30r/min, makes the neat soap flocculation, then oil is warmed up to 80 ℃, when the obvious sedimentation of neat soap, stop to stir, standing sedimentation separates soap stock, use again 80 ℃ of hot water wash three times, then vacuum-drying;
(5) adding the atlapulgite that accounts for oil body quality 2% in the dried oil body of step (4) gained decolours, bleaching temperature is 75 ℃, and bleaching time is 30 minutes, vacuumizes simultaneously and the appropriateness stirring, remove by filter carclazyte, grease is carried out the deodorizing of water vapour air lift, and the stripping temperature is 75 ℃, and vacuum tightness is 0.4Kpa, deodorization time is 0.5 hour, then keep Slow cooling under the vacuum state, centrifugation gets processed oil 80g, and total fat extraction yield is 89%.
Embodiment 2
(1) be 103g with the initial total lipid content of I Ken Shi kettle algae (Ulkeniaamoeboida) fermented liquid 1000g() introduce the flocculation sediment tank after, add the 20g polyacrylamide flocculant, collecting precipitation frond after 60 minutes flocculates, through the whizzer centrifugation, remove flocculation agent and moisture, obtain mud shape frond, then it is carried out lyophilize, make little algae dry powder of water content≤5%;
(2) add 600g acetone in the little algae dry powder of step (1) gained and carry out the lixiviate extraction, extraction temperature is 60 ℃, and extraction times is twice, is extracted liquid;
(3) step (2) gained extraction liquid is passed through the vacuum concentration desolvation, vacuum tightness is 0.30Kpa, and the time of removing is 5 hours, obtains the DHA crude oil;
(4) adding the 200g mass percent concentration in step (3) the gained crude oil is that 50% phosphoric acid solution and 50g mass percent concentration are 70% NaCl solution, after 65 ℃ of lower low rate mixings are even, left standstill 30 minutes, get upper strata fluid, be washed to neutrality, then add mass percent concentration and be 10% NaOH solution, stirred 50 minutes, stirring velocity is 80r/min, makes the neat soap flocculation, then oil is warmed up to 100 ℃, when the obvious sedimentation of neat soap, stop to stir, standing sedimentation separates soap stock, use again 100 ℃ of hot water wash three times, then vacuum-drying;
(5) adding the atlapulgite that accounts for oil body quality 3% in the dried oil body of step (4) gained decolours, bleaching temperature is 80 ℃, and bleaching time is 45 minutes, vacuumizes simultaneously and the appropriateness stirring, remove by filter carclazyte, grease is carried out the deodorizing of water vapour air lift, and the stripping temperature is 85 ℃, and vacuum tightness is 0.30Kpa, deodorization time is 1 hour, then keep Slow cooling under the vacuum state, the centrifugal processed oil 87g that gets, total fat extraction yield is 84.5%.
Embodiment 3
(1) be 98.3g with the initial total lipid content of thraustochytriale (Thraustochytrids) fermented liquid 1000g() introduce the flocculation sediment tank after, add 15g alum flocculation agent, collecting precipitation frond after 30 minutes flocculates, through the disc centrifuge centrifugation, remove flocculation agent and moisture, obtain mud shape frond, then it is carried out lyophilize, make little algae dry powder of water content≤5%;
(2) add the 500g ethyl acetate in the little algae dry powder of step (1) gained and carry out the lixiviate extraction, extraction temperature is 50 ℃, and extraction times is three times, is extracted liquid;
(3) step (2) gained extraction liquid is passed through the vacuum concentration desolvation, vacuum tightness is 0.35Kpa, and the time of removing is 3 hours, obtains the DHA crude oil;
(4) adding the 150g mass percent concentration in step (3) the gained crude oil is that 65% phosphoric acid solution and 100g mass percent concentration are 60% NaCl solution, after 55 ℃ of lower low rate mixings are even, left standstill 45 minutes, get upper strata fluid, be washed to neutrality, then add mass percent concentration and be 8% NaOH solution, stirred 40 minutes, stirring velocity is 60r/min, makes the neat soap flocculation, then oil is warmed up to 90 ℃, when the obvious sedimentation of neat soap, stop to stir, standing sedimentation separates soap stock, use again 95 ℃ of hot water wash three times, then vacuum-drying;
(5) adding the atlapulgite that accounts for oil body quality 2.5% in the dried oil body of step (4) gained decolours, bleaching temperature is 85 ℃, and bleaching time is 40 minutes, vacuumizes simultaneously and the appropriateness stirring, remove by filter carclazyte, grease is carried out the deodorizing of water vapour air lift, and the stripping temperature is 75 ℃, and vacuum tightness is 0.4Kpa, deodorization time is 0.5 hour, then keep Slow cooling under the vacuum state, centrifugation gets processed oil 86.6g, and total fat extraction yield is 88.1%.
Embodiment 4
(1) be 90g with the initial total lipid content of Crypthecodinium cohnii (Crypthecodinium cohnii) fermented liquid 1000g() introduce the flocculation sediment tank after, add the 10g chitosan flocculant, collecting precipitation frond after 45 minutes flocculates, through the disc centrifuge centrifugation, remove flocculation agent and moisture, obtain mud shape frond, then it is carried out lyophilize, make little algae dry powder of water content≤5%;
(2) add the 400g6# organic solvent in the little algae dry powder of step (1) gained and carry out the lixiviate extraction, extraction temperature is 45 ℃, and extraction times is four times, is extracted liquid;
(3) step (2) gained extraction liquid is passed through the vacuum concentration desolvation, vacuum tightness is 0.4Kpa, and the time of removing is 1 hour, obtains the DHA crude oil;
(4) adding the atlapulgite that accounts for oil body quality 2% in step (3) the gained DHA crude oil decolours, bleaching temperature is 75 ℃, and bleaching time is 30 minutes, vacuumizes simultaneously and the appropriateness stirring, remove by filter carclazyte, grease is carried out the deodorizing of water vapour air lift, and the stripping temperature is 75 ℃, and vacuum tightness is 0.4Kpa, deodorization time is 0.5 hour, then keep Slow cooling under the vacuum state, centrifugation gets processed oil 75.4g, and total fat extraction yield is 83.8%.
Embodiment 5
(1) be 98.3g with the initial total lipid content of thraustochytriale (Thraustochytrids) fermented liquid 1000g() introduce the flocculation sediment tank after, add 15g alum flocculation agent, collecting precipitation frond after 30 minutes flocculates, through the disc centrifuge centrifugation, remove flocculation agent and moisture, obtain mud shape frond, then it is carried out lyophilize, make little algae dry powder of water content≤5%;
(2) add the 500g ethyl acetate in the little algae dry powder of step (1) gained and carry out the lixiviate extraction, extraction temperature is 50 ℃, and extraction times is three times, is extracted liquid;
(3) step (2) gained extraction liquid is passed through the vacuum concentration desolvation, vacuum tightness is 0.35Kpa, and the time of removing is 3 hours, obtains the DHA crude oil;
(4) adding the atlapulgite that accounts for oil body quality 2.5% in the dried oil body of step (3) gained decolours, bleaching temperature is 85 ℃, and bleaching time is 40 minutes, vacuumizes simultaneously and the appropriateness stirring, remove by filter carclazyte, grease is carried out the deodorizing of water vapour air lift, and the stripping temperature is 75 ℃, and vacuum tightness is 0.4Kpa, deodorization time is 0.5 hour, then keep Slow cooling under the vacuum state, centrifugation gets processed oil 81.5g, and total fat extraction yield is 82.9%.
As everyone knows, in the technique of existing little algae broth extraction DHA, total fat extraction yield is generally all below 80%, but can find out from embodiment 1-5, and total fat extraction yield of extraction process of the present invention is all more than 80%.
Claims (8)
1. the method for an extracting and separating marine microalgae broth extraction DHA is characterized in that, may further comprise the steps: (1) dehydrates the little algae fermented liquid of DHA, gets DHA algae powder; (2) choose lower boiling extraction agent Extraction of DHA algae powder, be extracted liquid; (3) extraction liquid is carried out vacuum concentration, obtain the DHA crude oil behind the desolvation; (4) the DHA crude oil is decoloured and the steam deodorizing, then the concentrated DHA algae oil finished product that obtains.
2. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1, it is characterized in that, dehydrating in the step (1) comprises three grades of dehydrations: the one-level dehydration is to add flocculation agent in little algae fermented liquid, and algae liquid is separated with frond; The secondary dehydration is that the frond precipitation that the one-level dehydration obtains is carried out centrifugation, removes flocculation agent and moisture content, obtains mud shape frond; Three grades of dehydrations are that mud shape frond is carried out lyophilize, obtain the algae powder.
3. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 2, it is characterized in that, described flocculation agent is a kind of or any several mixture in chitosan, polymerize aluminum chloride, polyacrylamide, alum, Tai-Ace S 150, Calcium Chloride Powder Anhydrous, the bodied ferric sulfate, and its add-on is the 1.0-2.0% of fermented liquid quality.
4. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1 is characterized in that, by percentage to the quality, and the water content of DHA algae powder≤5% described in the step (1).
5. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1, it is characterized in that, the described lower boiling extraction agent of step (2) is a kind of or any several mixture in 6# organic solvent, sherwood oil, ether, hexane, acetone, chloroform, ethanol, methyl alcohol, ethyl acetate, propyl acetate, butylacetate, the ethyl propionate.
6. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1 is characterized in that, the add-on of the described extraction agent of step (2) accounts for 40%～60% of fermented liquid quality, and extraction temperature is 45～60 ℃, and extraction times is 2-4 time.
7. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1 is characterized in that, the vacuum tightness≤0.4Kpa of the described vacuum concentration of step (3), and the time that removes of solvent be 1～5 hour.
8. the method for a kind of extracting and separating marine microalgae broth extraction DHA according to claim 1, it is characterized in that, carrying out step (4) before, can carry out purifying to the resulting DHA crude oil of step (3) first: in crude oil, add the NaCl solution that mass percent concentration is respectively 50-85% phosphoric acid solution and 30-70%, add-on is respectively 5%～20% of fermented liquid quality, after stirring under 50～65 ℃ of temperature, left standstill 0.5～1 hour, get upper strata fluid, be washed to neutrality, then adding mass percent concentration is the NaOH solution of 5-10%, stir 40-50min, stirring velocity is 30～80r/min, makes the neat soap flocculation, then oil is warmed up to 80～100 ℃, when the obvious sedimentation of neat soap, stop to stir, standing sedimentation separates soap stock, wash then vacuum-drying with 80～100 ℃ hot water again.
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| CN103787864A (en) * | 2014-02-20 | 2014-05-14 | 青岛琅琊台集团股份有限公司 | Method for extracting DHA (Docosahexaenoic acid) from marine microalgae fermentation liquor |
| CN103820326A (en) * | 2014-03-20 | 2014-05-28 | 青岛琅琊台集团股份有限公司 | Method for producing dry algae powder by virtue of cultivation of ocean microalgae |
| CN103922921A (en) * | 2014-04-17 | 2014-07-16 | 青岛琅琊台集团股份有限公司 | Method of extracting ARA (arachidonic acid) from fermentation liquor |
| CN108486177A (en) * | 2018-02-12 | 2018-09-04 | 广州富诺健康科技股份有限公司 | A method of it is prepared using algae oil and is rich in omega-fatty acid phosphatide |
| CN111234912A (en) * | 2018-11-28 | 2020-06-05 | 中国科学院大连化学物理研究所 | Preparation method of microalgae edible oil |
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| CN103922921B (en) * | 2014-04-17 | 2015-11-04 | 青岛琅琊台集团股份有限公司 | The method of ARA is extracted from fermented liquid |
| CN108486177A (en) * | 2018-02-12 | 2018-09-04 | 广州富诺健康科技股份有限公司 | A method of it is prepared using algae oil and is rich in omega-fatty acid phosphatide |
| CN111234912A (en) * | 2018-11-28 | 2020-06-05 | 中国科学院大连化学物理研究所 | Preparation method of microalgae edible oil |
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