CN103509047A - Extraction process of phosphatidylcholine with antarctic krill as source and preparation method of phosphatidylserine - Google Patents
Extraction process of phosphatidylcholine with antarctic krill as source and preparation method of phosphatidylserine Download PDFInfo
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Abstract
The invention provides an extraction process of phosphatidylcholine with antarctic krill as a source. The extraction process comprises the following steps: collecting heads of the antarctic krill, adding water for decoction, stirring, sending the krill head decoction liquid to a cold pressing machine for solid-liquid separation, to obtain an oily liquid rich in phospholipid and krill heads and other solids; adding absolute ethyl alcohol into the krill heads and other solids, carrying out high shearing extraction, carrying out centrifugal separation, and concentrating to obtain an oily liquid; merging the oily liquids, drying under a reduced pressure, then filtering through a plate frame filter to remove insoluble substances, followed by carrying out membrane separation and concentration, carrying out column chromatography separation and purification, carrying out freeze drying, and thus obtaining the phosphatidylcholine product. The invention also provides a preparation method for phosphatidylserine with the antarctic krill as the source. The preparation method comprises the steps of hydrolyzing the extracted phosphatidylcholine by phospholipase D, and extracting to obtain phosphatidylserine rich in DHA and EPA. The extraction process and the preparation method are environmentally friendly, and are low in cost; and the obtained products have high content and good activity, and can be industrially produced.
Description
Technical field
The present invention relates to a kind of deep processing method from krill, the method that is specifically related to extract phosphatidylcholine from krill He prepares phosphatidylserine.
Background technology
Be rich in the phosphatidylserine (PS) of unsaturated fatty acids docosahexenoic acid (DHA) as preventing in the future senile dementia, the brains such as preventing children hyperkinetic syndrome are healthy and prevent that cardiovascular emerging health care and medical material have wide market outlook.
Krill be one way of life the krill in waters, the Antarctica, be also the most single one of Biological resources of planting of maximum, the procreation of quantity on the earth.The krill oil extracting from krill is rich in the functional components such as phosphatide (mainly take Yelkin TTS as main), and the lipid acid of phosphatide forms take timnodonic acid (EPA) and docosahexenoic acid (DHA) as main, and EPA and DHA content account for the more than 40% of fatty acids in phospholipids total amount.Antarctic krill oil has the cardiovascular disorder of reduction, prevention senile dementia and improves the multiple physiologically actives such as immunity of organisms, and its activity is better than the lecithin product (its lipid acid in forming containing EPA and DHA) in triglyceride type EPA/DHA product in the market and terrestrial plant source, unsaturated fatty acids docosahexenoic acid (DHA) is to be mainly combined in phosphatidyl choline glyceryl 2 upper (Sn2 positions), the phosphatidyl choline PC of unsaturated fatty acids docosahexenoic acid (DHA) combination that namely we mention.DHA forms kephalin, and the basis of brain cell membrane plays a part very importantly to the g and D of the division of brain cell, propagation, nerve conduction, cynapse, be the essential material that human brain forms and IQ is developed.It is on vision, brain activity, metabolism of fat, fetal growth and immunologic function and avoid senile dementia to have very big impact, during shortage, can cause series of symptoms, comprises growth retardation, the skin abnormality scales of skin that peel off, sterile, dysnoesia etc.But common DHA arranges in random mode, in unordered state, cannot guarantee to be absorbed by the body completely, common Sn1, the burned generation heat of DHA meeting on Sn3 position, or in conjunction with the lipid acid on Sn2, form triglyceride, only have the DHA on Sn2 position to be absorbed completely by human body.In krill oil, on the Sn2 position of phosphatide, be DHA, can by people, be absorbed completely, according to our data research, the biological activity of the DHA on Sn2 and bioavailability are 3-5 times of common fish oil DHA activity.Simultaneously by the phosphatidyl choline being rich in krill by the way of biological enzyme, change into phosphatidylserine.Its function of phosphatidylserine is mainly to improve neurocyte function, regulates the conduction of Nerve impulse, promotes brain memory function.Therefore studying for a long period of time and summing up through contriver, the phosphatidylserine of unsaturated fatty acids docosahexenoic acid (DHA) combination of originating from krill oil is due to its comprehensive action mechanism, for improving brain neuroblastoma cell function and brain activity, improve memory and promote infant's brain grow and safeguard that brain health has the problem meaning of great economic worth and biological medicine research.
Under the continuous growth of krill deep processing, how to improve in krill processing and extract the phosphatide of phosphatide and deep processing krill oil in waste, and then to change into phosphatidylserine be that current biological medicine is got important focus.General technical solution has three kinds at present:
The first is existing krill oil finished product of directly buying, carries out the further separating-purifying of phosphatidylcholine.Patent CN102405988A, application Dalian, Ren Wei Liaoning Province marine fishery group company, patent name " extracts the method for high phospholipid content krill oil " from krill; Patent CN102439125A; obligee Shanghai Ying Bei Bioisystech Co., Ltd; patent of invention " a kind of method of preparing the high quality Antarctic krill oil of the phosphatidylserine that is rich in many unsaturated double-bonds fatty acyl group " and Li Bogen (Lipogen) extract the pure Superba krill oil (Superba Krill Oil) from the Aker BioMarine of biotech company of Norway, form the krill oil 10% phosphatidylserine product of originating.The price of this raw material itself is just at the above per kilogram of 200 U.S. dollar at present, more than being about 300-5000 U.S. dollar through the product cost transforming, because this product exists complex manufacturing and loaded down with trivial details, albumen and ionized impurity need to be removed and mobility oily final product krill oil could be formed, especially various countries are more and more stricter for the rules of food raw material at present, finally make satisfactory raw materials cost high, and the extraction that the unsaturated fatty acids that is rich in phosphatide of oily forms the configuration of liquid before phosphatidylserine and product for rear road after catalysis is separated, increase a lot of difficulty, cause product content on the low side, free fatty acid content is high, poor fluidity, the defects such as muddiness and dissolvent residual exceed standard, it not the best industrialization way of obtaining suitability for industrialized production phosphatidylserine.
The second operational path be with an organic solvent or multiple organic solvent extract, at present related genera, like the technique of extracting such as the extracting method (patent No. EP01417211B1) that Canadian Neptune company is representative, adds the mixed solvents such as ethanol and ethyl acetate and carries out the separated and enrichment of impurity and concentrate with acetone.But along with further strictly regulating of our existing environmental protection and Safety production questions, the cost of controlling for the management of sewage disposal and safety in production can be more and more higher, comprise the environmental impact assessment of similar technique and approve and initiate a project on just have very large difficulty.And the existing Japan that much comprises, European Union and China have all proposed a lot of laws and regulations requirements to a lot of solvent extractions, such as acetone can not add and the extraction solvent of healthcare product as food, thereby analogous products have very large rules obstacle on entering part in future world market.Again, due to an organic solvent, particularly portioned product comprises methyl alcohol and acetone etc., will remove a lot of operations of residual increase and difficulty to the later stage.
The third operational path is to use supercritical extraction.CO 2 supercritical is all more feasible means for test and theory stage, but the real a set of equipment that drops into need to could really form industrialization up to the input of more than one hundred million Renminbi.According to the inventor's research, this operational path, there is very large facility investment requirement, even if go into operation, due to the problem of equipment amortization, be equally also expensive, carbon dioxide upercritical fluid extraction is a kind of extensive extraction means simultaneously, the high unsaturated fatty acids of the required phospholipids content of enrichment targetedly, thereby also have its limitation.This operational path can be with reference to CN102358865A, a kind of " method of extracting Euphausia superba oil by using supercritical carbon dioxide " of Shandong Kerui'er Biological Products Co., Ltd.'s application etc.
Therefore, the extraction process of phosphatidylcholine and the preparation method of phosphatidylserine in new environmental protection, lower-cost krill source need to be developed in this area.
Summary of the invention
The extraction process that the object of this invention is to provide the phosphatidylcholine (PC) in a kind of environmental protection, lower-cost krill source.
Second object of the present invention also provides the preparation method of the phosphatidylserine (PS) of a kind of economy, environmental protection.
A first aspect of the present invention, provides the extraction process of the phosphatidylcholine in a kind of krill source, and described extraction process comprises the steps:
(a) collect krill head, add water, decoct, stir, a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtain the oily liquids and the first-class solid of krill that are rich in phosphatide;
(b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step is carried out 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine product again.
In above-mentioned steps (a), krill head in 2h, adds water after thawing, and 40-55 ℃ of decoction, adds ethanol during stirring.The weight percent of described ethanol is 5%.
In above-mentioned steps (b), supernatant liquor, through concentrating under reduced pressure, obtains oily liquids.
In above-mentioned steps (d), food grade organic solvent is ethanol and normal hexane.
Ethanol in above-mentioned steps (d): the volume ratio of normal hexane is 2-4:0.5-1.5.Preferred alcohol: the volume ratio of normal hexane is 3:1.
In above-mentioned steps (d), adopt ultrafiltration membrane separation concentration, solid-liquid ratio W:V is 1:20, and temperature 10-55 ℃ adopts the two membrane separating of bore 100nm → 20nm.
In above-mentioned steps (d), adopt microfiltration membrane or ultra-filtration membrane in membrane sepn, described Mo aperture is 0.02-10 μ m, 0.015 ~ 10MPa exerts pressure on film.
In above-mentioned steps (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
During above-mentioned steps (e) column chromatography, described pillar blade diameter length ratio 1:5-10, described eluent is selected from the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.
Above-mentioned stationary phase is selected 100-200 order silochrom.
A second aspect of the present invention, provides the preparation method of the phosphatidylserine in a kind of krill source, and described preparation method comprises the steps:
(a) collect krill head, boiling, stirs, and a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step extraction 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine again;
(f) in water, add Serine, Phospholipase D, calcium chloride, hydrolytic phosphatide phatidylcholine, generates phosphatidylserine; Remove solvent, obtain the thick product of phosphatidylserine;
(g) the thick product of phosphatidylserine is extracted repeatedly through 2-4 food grade organic solvent, while extracting for the first time, by pH regulator to 3, remove organic solvent, obtain phosphatidylserine product.
In above-mentioned steps (f), phosphatidylcholine is in water during hydrolysis reaction, and temperature is 40-45 ℃, pH5-6, described phosphatidylcholine: Serine: Phospholipase D is 1:7-13:0.03-0.06.
In above-mentioned steps (f), below 50 ℃, adopt decompression method to remove solvent.
In above-mentioned steps (g), while extracting for the first time, the mixture that the food grade organic solvent of use is ethanol and normal hexane, described ethanol and normal hexane volume ratio are 4:1, and adopt edible salt acid for adjusting pH to 3, remove upper strata liquid.
In above-mentioned steps (g), adopt normal hexane to carry out follow-up 1-3 extraction, each extraction finishes to remove upper strata liquid.
In above-mentioned steps (g), repeatedly, after extraction, adopt 90% aqueous ethanolic solution, remove normal hexane, obtain phosphatidylserine product.
In above-mentioned steps (d), food grade organic solvent is ethanol and normal hexane.
In above-mentioned steps (d), ethanol: the volume ratio of normal hexane is 2-4:0.5-1.5.Preferred alcohol: the volume ratio of normal hexane is 3:1.
In above-mentioned steps (a), krill head in 2h, adds water after thawing, and 40-55 ℃ of decoction, adds ethanol during stirring.The weight percent of described ethanol is 5%.
In above-mentioned steps (b), supernatant liquor, through concentrating under reduced pressure, obtains oily liquids.
In above-mentioned steps (d), adopt ultrafiltration membrane separation concentration, solid-liquid ratio W:V is 1:20, and temperature 10-55 ℃ adopts the two membrane separating of bore 100nm → 20nm.
In above-mentioned steps (d), adopt microfiltration membrane or ultra-filtration membrane in membrane sepn, described Mo aperture is 0.02-10 μ m, and 0.015 ~ 10MPa exerts pressure on film.
In above-mentioned steps (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
During above-mentioned steps (e) column chromatography, described pillar blade diameter length ratio 1:5-10, described eluent is selected from the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.
Above-mentioned stationary phase is selected 100-200 order silochrom.
A third aspect of the present invention, provides according to the phosphatidylcholine in the krill source of said extracted technique extraction.
A fourth aspect of the present invention, provides the phosphatidylserine in the krill source of preparing according to above-mentioned preparation method.
" krill head cleans in 2h after thawing " of the present invention refers to: krill head is freezing at once after collection, and while extracting phosphatidylcholine, krill head is boiling in 2h after thawing; Or, after freezing krill is thawed, in 2h, collect krill head, and carry out boiling.
Advantage of the present invention and effect are:
1. the present invention particularly concentrates in time and decocts the krill head of aquatic products company, cold press, high-shear extraction, Plate Filtration, membrane sepn, the pre-treatments such as column chromatography, according to the characteristic of phospholipid prod, specific aim is chosen the combination of multiple physics and nanometer extraction process, in production technique, adopt membrane ultrafiltration technique filter not can polymerization in normal hexane grease etc., greatly reduce the purification of subsequent product and separated load, reduced the impurity upper prop of follow-up resin absorption, improved absorption equivalent, improved the yield of product, quality and the stability of product have further been improved, simultaneously from having saved economically production cost, and greatly reduce the consumption of soda acid and ethanol.And raw material is from aquatic products company waste, and the by product forming in extraction process can also be used as other purposes, has maximum social economic effect.From environmental protection, greatly reduce the discharge of waste water, alleviated environmental protection pressure; From producing, whole system is easy to safeguard, has greatly improved operating environment, has reduced labour intensity.
2. technique of the present invention is compared general solvent-extracted separating-purifying means both at home and abroad, improved the yield of PC, changed an actual industrial difficult problem for PC preparation process low yield and waste water high pollution in the past, compared the industrialization investment of CO 2 supercritical simultaneously, equipment investment only have 3% less than.This technique has effectively prevented oxidation and the degraded of effective constituent activeconstituents in high temperature and batch technology in krill, and the suitability for industrialized production of refining DHA-PS product that makes to obtain high-content is accomplished.
The feature of this technique is that to take first phosphatidylserine (PS) product of preparing high-content unsaturated fatty acids docosahexenoic acid (DHA) be final purpose, by temperature and physics, extract the specific aim array mode of means, phosphatide in the discarded krill head of the extraction krill source mill of the economic environmental protection of energy, and by cold press, high-shear extraction, membrane filtration, the Integrated using of the method such as high speed centrifugation and chromatography, by screening and separating targetedly, remove impurity, the content of unsaturated fatty acids docosahexenoic acid (DHA) phosphatide in mix products can be reached more than 45%.
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, and its specific embodiments is only construed as illustrating, and is not determinate, can not illustrate to limit protection scope of the present invention with following.
Inventive concept is: for the weak point existing in the real industrialization of prior art, provide a kind of krill head is concentrated to pre-treating technology in time, and effectively prevented oxidation and the degraded of effective constituent activeconstituents in high temperature and batch technology in krill, extract to greatest extent separated high-content phosphatidylcholine, the suitability for industrialized production that makes to obtain the krill source phosphatidylserine of high-content can be accomplished.
Experimental technique
Annual late July start krill fish for during the discarded first-class resource of krill of ，Jiang aquatic products processing factory unify to collect, obtain a raw material batch 2000kg left and right, in 15 ℃ of ventilations of room temperature, thaw.The present invention has carried out extraction separating experiment to 3 batch samples, from embodiment 1～3 below, experimental data is known: the high phospholipid complete processing of producing the antarctic krill oil source of DHA-PS is feasible, its final phosphatidylserine PS product that is rich in DHA forming is that content is more than 45%, two class active substances in product, detected, wherein DHA content is up to more than 15%, and PS is up to more than 45%.It has DHA and PS two class active functions simultaneously, can be used as and produce natural health and medical raw material, this raw material with the mixture of these two kinds of one matters of DHA and PS is compared, biological activity has improved 3-5 doubly, is the raw material that has potentiality of process for processing food, medicine and field of health care food.In order to extract to greatest extent separated high-content phosphatidylcholine, should boiling pre-treatment in 2h after krill head thaws; Also can be directly after aquatic products processing factory krill be thawed, 2h is interior by a krill boiling pre-treatment of collecting, then carries out follow-up extraction process.
The preparation 1 of embodiment 1 phosphatidylserine
(1) by the first-class alcohol extracting decoction pot of putting 5 tons of 2000kg krill, pour 2000L water, heat 50 ℃, convenient for follow-up cold press, spray while stirring 5% edible ethanol, edible ethanol weight percent can be 3-6%, general 5% is best, can increase the lubricated of squeezing.Stir 10min, then a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller (model 6YL-165 Xing， Henan chats), cold pressing expeller treatment capacity 1000kg/h, rotating speed 28-35r/min, power 30kw, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(2) solid (1) being obtained is placed in reactor, adds 4 times of quality to the anhydrous ethanol solvent of gained solid, high-shear stir 20min(15 ~ 30min all can), make phospholipid fraction fully be dissolved in centrifugation in ethanol; Throw out is put into reactor and add 4 times of anhydrous ethanol solvent high-shears to gained solid of quality to stir again, centrifugal, twice supernatant liquor concentrating under reduced pressure of gained becomes oily liquids;
(3) the concentrated oily liquids of (1) gained oily liquids and (2) gained is merged, decompression rotation evaporate to dryness, obtains the protein coagulation thing 589kg that contains phosphatide and unsaturated fatty acids.
(4) we detect that in the protein coagulation thing that contains phosphatide and unsaturated fatty acids that (3) obtain, moisture content is gross weight 67%, the protein coagulation thing that takes a morsel, its low-temperature evaporation is dry after, detect dried protein coagulation thing, obtain following index
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 43.9 |
| Crude protein | 47.5 |
| Ash content | 8.6 |
Above detected result tentatively shows, from phosphatide and the unsaturated fatty acids in krill head source, by concentrating, has obtained effective separating-purifying.Further by the composition to lipid acid, analyze, use the gas-chromatography 6890N of U.S. Agilent company to analyze, the analytical column using is J& The 122-7032 of S Scientific DB-Wax.Analyzing total fatty acid content in the protein coagulation thing that gained contains phosphatide and unsaturated fatty acids is 63.5%, and wherein unsaturated fatty acid content is that in 35.5%(the present invention, unsaturated fatty acids mostly is Omega-3 series, DHA7.8% wherein, EPA content 15.0%.
(5) the protein coagulation thing that contains phosphatide and unsaturated fatty acids obtaining in (3) is placed in to 5 tons of dissolving vessels, configuration 3000L food grade organic solvent, ethanol-normal hexane (75:25 volume ratio) wherein, use the press filtration of stainless steel multilayer plate and frame, can use bed filtration plate according to practical condition, what in the present embodiment, use is the standard type of 8 sheet frames, by compression sheet frame, carries out the separated of solid and liquid and purifies.Pass through Plate Filtration, remove insoluble substance, the liquid then separation being obtained carries out ultra-filtration membrane separation, deoil (removing the grease in not polymerization in normal hexane), solid-liquid ratio 1:20(w/v), film pressure 0.1Mpa, adopts the two membrane separating of bore 100nm → 20nm.Prior to 25 ℃, 100nm membrane filtration, phegma and penetrating fluid are crossed 20nm film in 25 ℃, phegma reaches 1:3 with penetrating fluid volume ratio, collect 20nm film phegma, obtain the concentrated solution of unsaturated fatty acids, the concentrated solution evaporation drying that takes a morsel, detects each component concentration in the protein coagulation thing that contains phosphatide and unsaturated fatty acids after concentrating.
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 52.5 |
| Crude protein | 40.4 |
| Ash content | 7.1 |
In the protein coagulation thing that analysis gained contains phosphatide and unsaturated fatty acids, total fatty acid content is 63.5%, and wherein unsaturated fatty acid content is 35.5%(DHA7.8%, EPA content 15.0%).
(6) concentrated solution is used to moving phase normal hexane: Virahol: water (1:1:0.16, v/v/v) dissolves (this ratio is optimum proportion), under room temperature, be stirred to dissolving standby.No. 2 silica gel of gross porosity of learning from else's experience and activating, add moving phase to make pulpous state, and wet method dress post, then uses moving phase balance chromatography column (model Φ 315mm*2000mm).The sample preparing is slowly poured in post, after sample introduction, added moving phase wash-out.Controlling void tower flow velocity is 0.382cm/min, sample introduction concentration is under the room temperature condition of 0.25g concentrated phosphatide (mL/ moving phase), stationary phase can be selected 50-400 order silochrom or macroporous resin, in the present embodiment, stationary phase is selected No. 2 silica gel of 100 ~ 200 order gross porosity, purification rate is 70%, and the suitableeest upper carrying capacity is 0.086gPC(g/ silica gel), through column chromatographic isolation and purification, obtain elutriant, it is more than 80% obtaining PC content.Elutriant can adopt the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.In the present embodiment, adopt normal hexane: Virahol: the above-mentioned elutriant (being phosphatidylcholine mixture) after membrane separation concentration and column chromatographic isolation and purification of elutriant of water (1:1:0.16, v/v/v), through lyophilize, can obtain phosphatidylcholine product.
(7) in water, add Serine, calcium chloride and Phospholipase D, hydrolytic phosphatide phatidylcholine, generates phosphatidylserine, and during hydrolysis reaction, temperature is 40-45 ℃, pH5-6.In the present embodiment, to phosphatidylcholine product 50kg, the Serine 500kg, the calcium chloride 4.2kg reaction that drop into (6) gained in 3000L fermentor tank, add water to 500L, drop into again Phospholipase D (PLD enzyme) 1.0kg, under bath temperature 40-45 ℃, pH5.6 condition, react after 4h, then drop into the PLD enzyme reaction 12h of 0.5kg.The phosphatidylcholine family (PC) of being rich in many unsaturated double-bonds fatty acyl group that (4) are obtained; in water react system, be converted into the phosphatidylserine (PS) that is rich in many unsaturated double-bonds fatty acyl group; mixture after reaction is removed solvent 50 ℃ of following decompressions; obtain high-content phosphatidylserine crude product 46.9kg, phosphatidylserine wherein (PS) content is about 39.8%.
(8) in the 46.9kg product (7) being obtained, drop into 8000L with in upper container, add 1600L normal hexane and ethanol 400L, then the edible hydrochloric acid that adds 10-20L, be adjusted to pH3, fully stirring, layering, remove upper strata liquid, adds 2000L normal hexane in subnatant again, stirring, layering, remove upper strata liquid and repeat 3 times.In solution, add water: ethanol (1:9, v/v) 150-300L, is fully uniformly mixed, regulate below pH9, separated hexane solution, the phosphatidylserine product of removing after normal hexane carries out efficient liquid phase chromatographic analysis, and the content that records phosphatidylserine (PS) is 45.9%.
The preparation 2 of embodiment 2 phosphatidylserines
(1) by the first-class alcohol extracting decoction pot of putting 5 tons of 2050kg krill, pouring 2000L water, heat 55 ℃, is follow-up cold press convenience, spray while stirring 6% ethanol a small amount of, stir 10min, then a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller (model 6YL-165 Xing， Henan chats), cold pressing expeller treatment capacity 1000kg/h, rotating speed 28-35r/min, power 30kw, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(2) solid (1) being obtained is placed in reactor, adds 3 times of quality to the anhydrous ethanol solvent of gained solid, and 15min is stirred in high-shear, makes phospholipid fraction fully be dissolved in centrifugation in ethanol; Throw out is put into reactor and add 3 times of anhydrous ethanol solvent high-shears to gained solid of quality to stir again, centrifugal, twice supernatant liquor concentrating under reduced pressure of gained becomes oily liquids;
(3) the concentrated oily liquids of (1) gained oily liquids and (2) gained is merged, decompression rotation evaporate to dryness, obtains the protein coagulation thing 608kg that contains phosphatide and unsaturated fatty acids.
(4) with embodiment 1, detect that in the protein coagulation thing that contains phosphatide and unsaturated fatty acids that (3) obtain, moisture content is gross weight 62%, the protein coagulation thing that takes a morsel, its low-temperature evaporation is dry after, detect dried protein coagulation thing, obtain following index
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 44.9 |
| Crude protein | 48.2 |
| Ash content | 6.9 |
Further by the composition to lipid acid, analyze, use the gas-chromatography 6890N of U.S. Agilent company to analyze, the analytical column using is J& The 122-7032 of S Scientific DB-Wax.Analyzing total fatty acid content in gained coagulum is 62%, and wherein unsaturated fatty acid content is 37%(DHA8.1%, EPA content 15.6%).
(5) product is placed in to 5 tons of dissolving vessels, configuration 3000L food grade organic solvent, ethanol-normal hexane (8:3) wherein, pass through Plate Filtration, remove insoluble substance etc., and the liquid then separation being obtained carries out ultra-filtration membrane separation, deoils, solid-liquid ratio 1:20(w/v), film pressure 0.1Mpa, adopts the two membrane separating of bore 100nm → 20nm.Prior to 10 ℃, 100nm membrane filtration, phegma and penetrating fluid are in 10 ℃ of mistake 20nm films, and phegma reaches 1:3 with penetrating fluid volume ratio, collects 20nm film phegma, obtains the concentrated solution of unsaturated fatty acids.
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 52.9 |
| Crude protein | 40.6 |
| Ash content | 6.5 |
Analyzing total fatty acid content in gained protein coagulation thing is 62%, and unsaturated fatty acid content is wherein DHA8.1% of 37%(, EPA content 15.6%).
(6) concentrated solution is used to moving phase normal hexane: Virahol: water (1:1:0.16, v/v/v) dissolves, under room temperature, be stirred to dissolving standby.No. 2 silica gel of gross porosity of learning from else's experience and activating, add moving phase to make pulpous state, and wet method dress post, then uses moving phase balance chromatography column.The sample preparing is slowly poured in post, after sample introduction, added moving phase wash-out.Controlling void tower flow velocity is 0.382cm/min, sample introduction concentration is under the room temperature condition of 0.25g concentrated phosphatide (mL/ moving phase), stationary phase is selected No. 2 silica gel of 100 ~ 200 order gross porosity, purification rate is 70%, the suitableeest upper carrying capacity is 0.086gPC(g/ silica gel), obtaining PC content is more than 80% phosphatidylcholine mixture.By the lyophilize of phosphatidylcholine mixture, obtain phosphatidylcholine product.
(7) in water, add Serine, calcium chloride and Phospholipase D, hydrolytic phosphatide phatidylcholine product, generates phosphatidylserine, and during hydrolysis reaction, temperature is 40-45 ℃, pH5-6.In the present embodiment, to dropping into the phosphatidylcholine mixture 50kg of (6) gained in 3000L fermentor tank, Serine 650kg, calcium chloride 20kg reaction, add water to 500L, drop into again Phospholipase D (PLD enzyme) 3kg, at bath temperature 40-45 ℃, pH5-6, in the present embodiment, be adjusted to after pH5.8 reaction 4h, the PLD enzyme reaction 12h that drops into again 0.8kg reacts mixture afterwards and removes solvents 50 ℃ of following decompressions, phosphatidylserine (PS) the crude product 46.3kg of many unsaturated double-bonds fatty acyl group is rich in acquisition, POPS wherein (PS) content is about 39.1%.
(8) in the 46.3kg product (7) being obtained, drop into 8000L with in upper container, add 1600L normal hexane and ethanol 400L, then the edible hydrochloric acid that adds 10-20L, be adjusted to pH3, fully stirring, layering, remove upper strata liquid, adds 2000L normal hexane in subnatant again, stirring, layering, repeat 3 times.In solution, add water: ethanol (1:9, v/v) 150-300L, be fully uniformly mixed, regulate below pH9, separated hexane solution, the product of removing normal hexane carries out efficient liquid phase chromatographic analysis, and the content that records POPS (PS) is 45.0%.
The preparation 3 of embodiment 3 phosphatidylserines
(1) by the first-class alcohol extracting decoction pot of putting 5 tons of 2010kg krill, pouring 2000L water, heat 45 ℃, is follow-up cold press convenience, spray while stirring 3% ethanol a small amount of, stir 10min, then a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller (model 6YL-165 Xing， Henan chats), cold pressing expeller treatment capacity 1000kg/h, rotating speed 28-35r/min, power 30kw, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(2) solid (1) being obtained is placed in reactor, adds 3 times of quality to the anhydrous ethanol solvent of gained solid, and 15min is stirred in high-shear, makes phospholipid fraction fully be dissolved in centrifugation in ethanol; Throw out is put into reactor and add 3 times of anhydrous ethanol solvent high-shears to gained solid of quality to stir again, centrifugal, twice supernatant liquor concentrating under reduced pressure of gained becomes oily liquids;
(3) the concentrated oily liquids of (1) gained oily liquids and (2) gained is merged, decompression rotation evaporate to dryness, obtains the protein coagulation thing 583kg that contains phosphatide and unsaturated fatty acids that contains phosphatide.
(4) with embodiment 1, survey that in the protein coagulation thing that contains phosphatide and unsaturated fatty acids that (3) obtain, moisture content is gross weight 67%, detect dried protein coagulation thing and obtain following index
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 44.0 |
| Crude protein | 47.1 |
| Ash content | 9.1 |
Further by the composition to lipid acid, analyze, use the gas-chromatography 6890N of U.S. Agilent company to analyze, the analytical column using is J& The 122-7032 of S Scientific DB-Wax.Analyzing total fatty acid content in gained coagulum is 64.2%, and wherein unsaturated fatty acid content is 34.8%(DHA7.6%, EPA content 14.6%).
(5) product is dissolved in to 5 tons of dissolving vessels, configuration 3000L food utmost point organic solvent, ethanol-normal hexane (4:1) wherein, use Plate Filtration to remove insoluble substance, isolated liquid carries out ultra-filtration membrane separation again, deoils, solid-liquid ratio 1:20(w/v), film pressure 0.1Mpa, adopts the two membrane separating of bore 100nm → 20nm.Prior to 55 ℃, 100nm membrane filtration, phegma and penetrating fluid are in 55 ℃ of mistake 20nm films, and phegma reaches 1:3 with penetrating fluid volume ratio, collects 20nm film phegma, obtains the concentrated solution of unsaturated fatty acids.
| Composition | After dry, account for the per-cent (%) of dry weight |
| TL | 51.8 |
| Crude protein | 39.9 |
| Ash content | 8.3 |
Analyzing total fatty acid content in gained coagulum is 64.2%, and unsaturated fatty acid content is wherein DHA7.6% of 34.8%(, EPA content 14.6%).
(6) concentrated solution is used to moving phase normal hexane: Virahol: water (1:1:0.16, v/v/v) dissolves, under room temperature, be stirred to dissolving standby.No. 2 silica gel of gross porosity of learning from else's experience and activating, add moving phase to make pulpous state, and wet method dress post, then uses moving phase balance chromatography column.The sample preparing is slowly poured in post, after sample introduction, added moving phase wash-out.Controlling void tower flow velocity is 0.382cm/min, sample introduction concentration is under the room temperature condition of 0.25g concentrated phosphatide (mL/ moving phase), stationary phase is selected No. 2 silica gel of 100 ~ 200 order gross porosity, purification rate is 70%, the suitableeest upper carrying capacity is 0.086gPC(g/ silica gel), obtaining PC content is more than 80% phosphatidylcholine mixture.After lyophilize, obtain phosphatidylcholine product.
(7) to phosphatidylcholine 50kg, the Serine 350kg, the calcium chloride 6.0kg reaction that drop into (6) gained in 3000L fermentor tank, add water to 500L, drop into again PLD enzyme 1.6kg, under bath temperature 40-45 ℃, pH5.6 condition, react after 4h, then drop into the PLD enzyme reaction 12h of 0.8kg.Mixture after reaction is removed solvent 50 ℃ of following decompressions; the phosphatidylcholine family (PC) of being rich in many unsaturated double-bonds fatty acyl group that (4) are obtained; in water react system, be converted into the phosphatidylserine (PS) that is rich in many unsaturated double-bonds fatty acyl group; obtain product 45.7kg, POPS wherein (PS) content is about 39.3%.
(8) in the 45.7kg product (7) being obtained, drop into 8000L with in upper container, add 1600L normal hexane and ethanol 400L, then the edible hydrochloric acid that adds 10-20L, be adjusted to pH3, fully stirring, layering, remove upper strata liquid, adds 2000L normal hexane in subnatant again, stirring, layering, re-extract 3 times.In solution, add water: ethanol (1:9, v/v) 150-300L, be fully uniformly mixed, regulate below pH9, separated hexane solution, the product of removing normal hexane carries out efficient liquid phase chromatographic analysis, and the content that records phosphatidylserine (PS) is 45.1%.The PS product that utilizes the inventive method to obtain, is rich in DHA and EPA, is natural product, active high, can in PS product, add antioxidant as required, so that preservation and later stage are prepared various healthcare products and nutriment, medicine etc.
The extraction process of the phosphatidylcholine in krill of the present invention source, concrete steps are as follows:
(a) collect krill head, add water, decoct, stir, a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtain the oily liquids and the first-class solid of krill that are rich in phosphatide; (b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step is carried out 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine product again.
The preparation method of the phosphatidylserine in krill of the present invention source, concrete steps are as follows:
(a) collect krill head, boiling, stirs, and a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step extraction 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine again;
(f) in water, add Serine, Phospholipase D, calcium chloride, hydrolytic phosphatide phatidylcholine, generates phosphatidylserine; Remove solvent, obtain the thick product of phosphatidylserine;
(g) the thick product of phosphatidylserine is extracted repeatedly through 2-4 food grade organic solvent, while extracting for the first time, by pH regulator to 3, remove organic solvent, obtain phosphatidylserine product.
In above-mentioned steps (f), below 50 ℃, adopt decompression method to remove solvent.
In above-mentioned membrane sepn, can adopt microfiltration membrane or ultra-filtration membrane, described Mo aperture is 0.02-10 μ m, 0.015 ~ 10MPa exerts pressure on film.
In above-mentioned steps (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
During above-mentioned steps (e) column chromatography, described pillar blade diameter length ratio 1:5-10, described eluent is selected from the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.For fish head, include but not limited to calamary head, also can extract phosphatidylcholine, and decompose phosphatidylcholine with reference to technical scheme of the present invention, obtain the phosphatidylserine that is rich in DHA.
The present invention first extracts the 60-70% in a krill phosphatide by cold press, again the krill solid obtaining after cold press is carried out to high-shear extraction, remaining 30-40% in a krill phosphatide is extracted, the relative traditional method of consumption of the dehydrated alcohol that extraction is used like this reduces greatly, and dehydrated alcohol can recycling use.
The present invention is for effective separation of krill waste and refining, and the industrialization that makes scale operation high-content produce phosphatidylserine from krill becomes possibility, makes this product become possibility in the popularization of protective foods biomedicine field.
The above description of this invention is not limited to scope of the present invention.For the person of ordinary skill of the art, can make various corresponding changes according to technical scheme provided by the invention, these changes all should belong to protection scope of the present invention.
Claims (27)
1. an extraction process for the phosphatidylcholine in krill source, is characterized in that, comprises the steps:
(a) collect krill head, add water, decoct, stir, a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtain the oily liquids and the first-class solid of krill that are rich in phosphatide;
(b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step is carried out 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine product again.
2. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 1, is characterized in that, in described step (a), krill head in 2h, adds water after thawing, and 40-55 ℃ of decoction, adds ethanol during stirring.
3. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 1, is characterized in that, in described step (b), supernatant liquor, through concentrating under reduced pressure, obtains oily liquids.
4. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 1, is characterized in that, in described step (d), food grade organic solvent is ethanol and normal hexane.
5. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 4, is characterized in that, in described step (d), and ethanol: the volume ratio of normal hexane is 2-4:0.5-1.5.
6. the extraction process of the phosphatidylcholine in krill source as described in claim as arbitrary in claim 1-5, is characterized in that, in described step (d), employing ultra-filtration membrane is separated, solid-liquid ratio W:V is 1:20, and temperature 10-55 ℃ adopts the two membrane separating of bore 100nm → 20nm.
7. the extraction process of the phosphatidylcholine that claim krill as arbitrary in claim 1-5 is originated, it is characterized in that, in described step (d), adopt microfiltration membrane or ultra-filtration membrane in membrane sepn, described Mo aperture is 0.02-10 μ m, 0.015 ~ 10MPa exerts pressure on film.
8. the extraction process of the phosphatidylcholine that as described in claim as arbitrary in claim 1-5, krill is originated, it is characterized in that, in described step (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
9. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 8, it is characterized in that, during described step (e) column chromatography, described pillar blade diameter length ratio 1:5-10, described eluent is selected from the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.
10. the extraction process of the phosphatidylcholine that krill is originated as claimed in claim 8, is characterized in that, described stationary phase is selected 100-200 order silochrom.
The extraction process of 11. phosphatidylcholines that krill is originated as claimed in claim 7, is characterized in that, in described step (d), adopts ultra-filtration membrane separated, and solid-liquid ratio W:V is 1:20, and temperature 10-55 ℃ adopts the two membrane separating of bore 100nm → 20nm.
The extraction process of 12. phosphatidylcholines that krill is originated as described in claim 9 or 10, is characterized in that normal hexane in described moving phase: Virahol: water volume ratio is 1:1:0.16.
The preparation method of the phosphatidylserine in 13. 1 kinds of krill sources, is characterized in that, comprises the steps:
(a) collect krill head, boiling, stirs, and a krill decoction liquor is delivered to solid-liquid separation in cold pressing expeller, obtains the oily liquids and the first-class solid of krill that are rich in phosphatide;
(b) in the first-class solid of krill, add dehydrated alcohol, high-shear extraction, centrifugation obtains supernatant liquor; This step extraction 1-2 time, merges supernatant liquor, the concentrated oily liquids that obtains;
(c) by oily liquids merging, drying under reduced pressure in step (a) and step (b), obtain the protein coagulation thing that contains phosphatide and unsaturated fatty acids;
(d) the protein coagulation thing that contains phosphatide and unsaturated fatty acids is dissolved in food grade organic solvent, through flame filter press, filters and remove insoluble substance, the liquid obtaining after filtering, again through membrane separation concentration, obtains concentrated solution;
(e) concentrated solution, through column chromatographic isolation and purification, obtains phosphatidylcholine mixture; Through lyophilize, obtain phosphatidylcholine again;
(f) in water, add Serine, Phospholipase D, calcium chloride, hydrolytic phosphatide phatidylcholine, generates phosphatidylserine; Remove solvent, obtain the thick product of phosphatidylserine;
(g) the thick product of phosphatidylserine is extracted repeatedly through 2-4 food grade organic solvent, while extracting for the first time, by pH regulator to 3, remove organic solvent, obtain phosphatidylserine product.
The preparation method of 14. phosphatidylserines that krill is originated as claimed in claim 13, is characterized in that, in described step (f), during hydrolysis reaction, temperature is 40-45 ℃, pH5-6, described phosphatidylcholine: Serine: Phospholipase D is 1:7-13:0.03-0.06.
15. preparation methods of the phosphatidylserines in krill source as claimed in claim 13, is characterized in that, in described step (a) krill head after thawing in 2h, cold press after boiling.
The preparation method of the phosphatidylserine in krill source as described in 16. claims as arbitrary in claim 13-15, it is characterized in that, in described step (g), while extracting for the first time, the mixture that the food grade organic solvent using is ethanol and normal hexane, described ethanol and normal hexane volume ratio are 4:1, and adopt edible salt acid for adjusting pH to 3, remove upper strata liquid.
The preparation method of 17. phosphatidylserines that krill is originated as claimed in claim 16, is characterized in that, in described step (g), adopts normal hexane to carry out follow-up 1-3 extraction, and upper strata liquid is removed in each extraction end.
The preparation method of 18. phosphatidylserines that krill is originated as claimed in claim 16, is characterized in that, in described step (g), repeatedly, after extraction, adopts 90% aqueous ethanolic solution, removes normal hexane, obtains phosphatidylserine product.
The preparation method of 19. phosphatidylserines that krill is originated as described in claim 13-15,17 or 18 arbitrary claims, is characterized in that, in described step (d), food grade organic solvent is ethanol and normal hexane.
The preparation method of 20. phosphatidylserines that krill is originated as claimed in claim 19, is characterized in that, in described step (d), adopt ultrafiltration membrane separation concentration, solid-liquid ratio W:V is 1:20, and temperature 10-55 ℃ adopts the two membrane separating of bore 100nm → 20nm.
The preparation method of 21. phosphatidylserines that krill is originated as claimed in claim 19, is characterized in that, in described step (d), and ethanol: the volume ratio of normal hexane is 2-4:0.5-1.5.
The preparation method of 22. phosphatidylserines that krill is originated as claimed in claim 19, it is characterized in that, in described step (d), in membrane sepn, adopt microfiltration membrane or ultra-filtration membrane, described Mo aperture is 0.02-10 μ m, and 0.015 ~ 10MPa exerts pressure on film.
As described in 23. claims as arbitrary in claim 13-14,17,18,20--22, the preparation method of the phosphatidylserine in krill source, is characterized in that, in described step (a) krill head after thawing in 2h, cold press after boiling.
The preparation method of 24. phosphatidylserines that krill is originated as described in claim 13-15,17 or 18 arbitrary claims, it is characterized in that, in described step (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
25. want the preparation method of the phosphatidylserine in the source of krill as described in 23 as right, it is characterized in that, in described step (e), first concentrated solution is dissolved in the moving phase of normal hexane, Virahol, water composition, stationary phase is selected 50-400 order silochrom or macroporous resin.
The preparation method of 26. phosphatidylserines that krill is originated as claimed in claim 24, it is characterized in that, during described step (e) column chromatography, described pillar blade diameter length ratio 1:5-10, described eluent is selected from the aqueous solution of the aqueous solution of normal hexane, the aqueous solution of Virahol or normal hexane and Virahol.
The preparation method of 27. phosphatidylserines that krill is originated as described in claim 13-15,17,18,20-22, the arbitrary claim of 25-26, is characterized in that, in described step (b), supernatant liquor, through concentrating under reduced pressure, obtains oily liquids.
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