CN1757395A - Pure ursine fat, and its prepn. method - Google Patents

Abstract

A purified fur seal fat is prepared from natural fur seal fat through esterifying, and multi-stage molecular distilling for purifying the esterified fur seal fat. Its advantages are high contents of EPA, DPA and DHA, and no specific odor.

Description

A kind of pure ursine fat and preparation method thereof

Technical field

The present invention relates to a kind of pure ursine fat and preparation method thereof.

Background technology

Fur seal is the deep-sea mammal, lives in the Arctic Circle with in interior-50 ℃ high and cold waters, is food with famous and precious morrhua, and subcutaneous have a thick fat deposit, is rich in ω-3 polyenoic fatty acid in the body.With Canada's northern pollution-free marine site fur seal fat is the Adeps Phocae vitulinae that raw material extracts, wherein not only contain three kinds of important ω-3 polyenoic fatty acids: EPA (Eic-osapen-taenoic Acid, eicosapentaenoic acid), DPA (Docosapentaenoic Acid, clupanodonic acid) and DHA (Docosahexaenoic Acid, docosahexenoic acid), and total content more than 20%.Medical circle is verified through years of researches, and these three kinds of fatty acids have multiple unique biological active to human body: EPA has the blood circulation of improvement, vessel softening, adjustment blood fat, brings high blood pressure down and blood glucose and antiinflammatory action; DHA and DPA have the nutrition brain, promote fetus and child's brain development, vision protection, adjusting immunity and antitumaous effect that wherein DHA is known as " NAOHUANGJIN " again.People find that again they have therapeutical effect to some diseases in the recent period: EPA has been developed the medicine as treatment arteriosclerosis and hyperlipidemia; Find that also Adeps Phocae vitulinae has treatment and preventive effect to type ii diabetes, fatty liver and prostatitis.

Human body self can not synthesize ω-3 polyenoic fatty acid, can only absorb from the external world, though also also have ω-3 polyenoic fatty acid in the cold water fish oil of deep-sea, it is compared with Adeps Phocae vitulinae except that content is generally low, also has following significant difference:

1. the fur seal and the mankind are all mammal, the chemical constitution of glyceride is to human similar in its body, ω-3 polyenoic fatty acid generally is positioned at 1,3 of triglyceride, and fish is rudimentary cold blooded animal, ω-3 polyenoic fatty acid is positioned at 2 of triglyceride, could utilize through after human liver's metabolism.Therefore ω-3 polyenoic fatty acid of Adeps Phocae vitulinae has better bioavailability by contrast, and can not increase burden of liver.

2. the Squalene that also contains 2-3% in the Adeps Phocae vitulinae, this also is not have in the fish oil, this material effectively suppresses the absorption of bad cholesterol in the biology and quickens its metabolism, also has protective effect on cancer risk, in addition skin is also had the effect of moistening.

3. not only be rich in EPA and DHA in the Adeps Phocae vitulinae, and be rich in DPA, and DPA content is very low in most of fish oil.

4. contain cholesterol in the Adeps Phocae vitulinae hardly, and most of fish oil always contains more cholesterol.

EPA in the human body, DHA, DPA mainly contain two sources: the one, from meals, obtain, and this is topmost source; The 2nd, by alpha-linolenic acid (LinolenicAcid, LA, 18:3-3) through the effect metabolism of desaturase (desaturating enzyme) and chain elongation enzyme (elongating enzyme), i.e. LA → EPA → DPA → DHA.But this synthesis capability of human body is extremely limited, and must be with LA exist for the basis in a large number.

-3 fatty acids and-6 fatty acids are competed desaturation thromboxane (TX2) and four unsaturated leukotrienes (LT4) in the metabolism in vivo, the two unsaturated eicosanoids (eicosanoids) of general designation, mainly contain vasoconstriction and the effect of coagulant collection, have vasodilation and the effect of anticoagulant collection simultaneously concurrently; And-3 fatty acid metabolisms generate three unsaturated prostaglandins (PG3), three unsaturated thromboxanes (TX3) and five unsaturated leukotrienes (LT5), these speciality are all local hormone, though its half-life is very lacked (about 1～2 minute), but has multiple physiologically active, and act on extremely strongly, mainly contain the effect of the two unsaturated eicosanoids of vasodilation, anticoagulant collection and antagonism.Fatty acid suppresses the arachidonic free and inflammatory mediators such as generation PG2, TX2 of membrane phospholipid simultaneously-3.Therefore its artery sclerosis disease incident is obviously on the low side in the crowd of high-3 fatty acid diet.

The physiological function of EPA, DHA, DPA:

EPA claims again " blood vessel street cleaner ", can not enter brain by blood brain barrier, mainly acts on cardiovascular system, has following physiological effect:

Improve the blood circulation people: promote the endothelial cell growth, anticoagulant increases the hemocyte deformability, and blood viscosity lowering has preventive effect to brain blood myocardial infarction etc.

Adjust blood fat: triglyceride reducing (TG), T-CHOL (TC), very low density lipoprotein (VLDL) (VLDL) and low density lipoprotein, LDL (LDL), high density lipoprotein increasing (HDL) prevents and treats moving fat sclerosis, hyperlipidemia.

Bring high blood pressure down: EPA can reduce the reaction of endogenous vaso-active substance to blood vessel, and its metabolite PG13, PGD3 also can directly act on blood vessel wall, and PGD3 also can influence the release and the function of norepinephrine, thereby makes blood pressure drops.

Antiinflammatory action: (arachidonic acid, metabolism AA) reduce the generation of diene PG and tetraene LT but the EPA accumulation increases the competitive inhibition arachidonic acid.Known PGE2 and LTB4 are important endogenous inflammatory mediators, reduce their content and can alleviate the local inflammation damage.

Blood sugar lowering: but animal experiment shows EPA blood sugar lowering concentration, and its mechanism may be to work by the release that influences insulin, raising insulin level.

Based on above effect, EPA has been developed the medicine as treatment arteriosclerosis and hyperlipidemia.

DHA claims again " NAOHUANGJIN ", is easy to enter brain by blood brain barrier, mainly makes in nervous system.It has the effect of EPA basically, just than a little less than the EPA effect slightly.The following physiological effect of Ta Hai Restrain-anger in addition:

Nutrition brain: have 10% to be DHA in the thin lipid of human brain, the extension of its formation to brain cell, growth promoter and brain cell projection, growth all play an important role, so it can improve learning capacity, improve memory, prevent presenile dementia, also have town's table and urge the eye effect.

Vision protection: DHA is the important composition composition of retina and optic nerve, and it can improve vision, prevents and treat visual disturbance.

Promote fetus and child's brain development: DHA can enter liver and the brain of fetus by Placenta Hominis, fetus from later stage of pregnancy to birth 6 months, brain and retinal development are the fastest, need competent DHA.If it is on the low side that DHA takes in, the baby due body weight may be on the low side, and premature labor easily.DHA content is very high in the lacto, about 7-22mg/100ml.

Regulate immunity: but DHA also metabolism generate immunologic active materials such as PG, TX, LT, participate in immunity of organism and regulate, can anti-atopic dermatitis, bronchial asthma, Hay Fever, and can prevent and treat wind-warm syndrome.

Antitumaous effect: zoology test data and Epidemiological study data show that heavy dose of DHA has function of tumor inhibition, but its anticancer propagation, and control cancer cell shifts, and is especially obvious to breast carcinoma, and colon cancer and pulmonary carcinoma are also had effect.

DPA has with DHA and similarly acts on, and also has many effects in addition, do not study fully as yet at present clear, but noticeable be that DPA content was also very high during human milk was analyzed, and was the important composition composition of human brain tissue and neurocyte equally.The research of Tokyo hospital and some other universities is thought: EPA is to the growth promoting function of endothelial cell, is the effect that is metabolized to behind the DPA in fact.

Therefore, Adeps Phocae vitulinae has higher using value with respect to fish oil, can help people to defeat human " the modern civilization disease " of long-term puzzlement such as diabetes, fatty liver, coronary heart disease.

But, the Adeps Phocae vitulinae fatty acid is formed more complicated, major part is a monounsaturated fatty acid in its early-products, accounts for 60%, and satisfied fatty acid accounts for 13%, we were concerned about has active three kinds of ω-3 polyenoic fatty acid (the polyunsaturated fatty acid of important biomolecule, PUFA)-and EPA, DHA, DPA total content just about 21%, be to improve PUFA content, improve the Adeps Phocae vitulinae quality, we must carry out purification to its early-products, to satisfy the injection requirement.

In the separation process of natural product, isolation technics commonly used has: steam distillation, adsorption resin method, supercritical fluid extraction and molecularly distilled.Preceding two kinds of methods are fit to the rough of product, and then two kinds of method persons are that rerum natura under utilizing specific condition is carried out isolating.Supercritical fluid extraction is suitable for the last stage of separation process, promptly from natural material required composition is extracted.Molecular distillation (Molecular Distillation, abbreviation MD) also claims short-path distillation (Short-Path Distillation), it is a kind of special liquid-liquid separation technology, its principle is under XHV, according to the difference of mixture molecular motion mean free path, under away from the temperature of its boiling point with its separation.

The MD technology is specially adapted to the separation of high boiling point, thermal sensitivity and readily oxidizable substance system.This technology has been widely used in food, daily use chemicals, petrochemical industry and pharmaceutical industry at present.

The molecule that the outstanding feature of MD technology is a distilling material is not subjected to the influence of intermolecular impact force by evaporating surface to the stroke of cryosurface, and the distance between the two sides is less than the molecular motion mean free path of distilling material.This technology has three big advantages: vapo(u)rizing temperature is low, working vacuum degree height, material heated time short.Molecular distillation apparatus can be divided into falling film type (falling-film evaporator), scraped film type (wiped-filmevaporator) and centrifugal (centrifugal evaporator) three kinds according to the design difference that forms the evaporation liquid film.

Along with the development of people to molecular distillation research, dissimilar molecular stills also occurs in succession, as E type V-type, M type, centrifugal, wipe membrane type, vertical etc.The appearance of these molecular stills has further reduced the heated time of material, has improved separation efficiency.

The molecular still development mainly contains two kinds of structural formula forms: centrifugal thin-film formula and rotor scraped film type so far.Both main distinctions are that material forms the difference of thin film mode.For centrifugal thin-film formula molecular distillation, because liquid film forms under centrifugal force and viscous force effect, centrifugal acceleration is generally greater than acceleration of gravity, so material is bigger along the evaporating surface flowing velocity, film thickness is generally 0.05mm.The volatile components of material is very short between the stopping time on the heating surface (being generally 0.05～1.5 second), take place to decompose and polymeric danger very little, efficient is very high, is particularly suitable for the separation of very hot quick material and concentrated.Weak point is the structure more complicated, and fair speed running structure is arranged, and maintenance is difficult, invests relatively large.Rotor scraped film type molecular distillation is to make liquid stream film forming by the epitrochanterian film device of scraping, and scrapes film thickness and is generally 0.1～0.25mm, about 5～15 seconds of residence time of material.It is simple in structure, and the processing system is easy, and operating parameter is controlled easily, and is easy to maintenance, and counter investment is less, wide application, the national conditions of suitable China.Fig. 1 is the composition sketch map of scraped film type molecular still:

In view of molecular distillation fundamental difference on principle is distilled in routine, thereby it possesses the incomparable advantage of many conventional distillations:

1. operative temperature is low

Molecular distillation technique realizes that the separation of mixed material is owing to be subjected to the result of thermo-molecular effusion liquid level, just can realize below far below the material boiling point, does not need boiling.And conventional distillation or rectification under vacuum are operated under fluidized state, add big many of the resistance ratios molecular distillation of its pedal or filler, so its operative temperature is higher than the molecule distillation.As certain mixture operative temperature when the vacuum distilling is 260 ℃, and in molecular distillation only be 150 ℃ just passable.Usually the molecular distillation operative temperature is lower than 300 ℃.

2. distill the vacuum height

Because molecular distillation apparatus particular structure form, its internal pressure is minimum, can obtain very high vacuum, and its work absolute pressure is generally the 0.1Pa order of magnitude.Reduce the boiling point that operating pressure just reduces material, the protection material is avoided heat damage, it is very favourable to avoid air oxidation.Conventional various vacuum stills are because there is pressure drop in steam between from the evaporating surface to the cryosurface, and limit operation pressure generally is limited in about 2kPa, as vacuum distillator etc. intermittently.

3. the distilling material heated time is short

The length of distilling material heated time is relevant with molecular distillation column length, knifing speed (with centrifugal rotational speed), material viscosity, circumference load etc.By the molecular distillation principle as can be known, the required distance of heating surface and cryosurface is less than the motion mean free path of light molecule, and the light molecule of being overflowed by liquid level almost just reaches cryosurface without collision, so heated time is very short.In addition, mixed liquor is film like, makes liquid level and heating surface and area almost equal, and heat transfer efficiency is very high, and material heated time in still-process is just shorter like this.For vacuum distilling, heated time is about 1 hour, and molecular distillation only is about 15 seconds.

4. separation degree height

Molecular distillation usually is used for separating the routine distillation and is difficult for material separately, yet with regard to the isolating material of the equal energy of these two kinds of methods, the separation degree of molecular distillation is higher.

The above-mentioned advantage of molecular distillation has determined it than conventional art clear superiority to be arranged in actual applications:

1. for some isolating high boiler material of ability under high-temperature condition, conventional distillating method can't use because of equipment, encapsulant are difficult to reach requirement.At this moment adopt molecular distillation just very suitable, concentrate the separation of monoglyceride etc. as natural cod-liver oil.

2. for some heat-sensitive substances, conventional distillation procedure temperature height, heated time is long, can cause some decomposition of components or polymerization in the material, adopts molecular distillation technique, can guarantee the natural quality of material.As the low terpene chemical compound of taking off of natural perfume material, deodorize, decolouring and purification, the separation of some medicines in the medical industries, concentrated, the plasticizer purification in the chemical industry etc.

3. but the molecular distillation utmost point removes the lower-molecular substance (as organic solvent, stink etc.) in the liquid effectively, and this is unusual effective method for adopting behind the solvent extraction precipitation of liquid;

4. molecular distillation can selectively steam the purpose product, removes other impurity, can separate two or more materials simultaneously by multiple stage separation;

5. compare with traditional Chemical Decomposition method,,, reduce simultaneously to the pollution of environment with to the harm of operator's health, and can not bring new impurity and noxious substance into so save a large amount of solvents because molecular distillation is pure physical process.

6. though the in the initial stage of that one-time investment is bigger, follow-up production cost (comprising material, water power, the labour force) is smaller.Separation to high value material and extensive low value material is purified all very suitable.

There is abundant marine animal oil resource in China, but utilization rate is not high, especially it is backward relatively to extract deep process technology, with traditional Chemical Decomposition method, not only consume a large amount of solvents, environment is polluted, operator's health is worked the mischief, and can in product, bring new impurity and noxious substance into.The product Adeps Phocae vitulinae that makes simultaneously has special fishy smell, and its physical behavior is relatively poor.And in existing molecular distillation separating and purifying technology, the Adeps Phocae vitulinae raw material is being carried out in the step of esterification, the esterification process of a lot of bibliographical informations is two step esterifications, complex steps, though what have is a step esterification, condition is violent, and sample is had destruction.Simultaneously not only yield is lower for resulting end product, and have in the product active three kinds of ω-3 polyenoic fatty acids of important biomolecule (polyunsaturated fatty acid, PUFA)-content of EPA, DHA, DPA is also very low.Therefore, need improve the technology of existing separation and purification Adeps Phocae vitulinae, just can obtain purity and yield can both satisfactory Adeps Phocae vitulinae product.

Summary of the invention

The object of the present invention is to provide a kind of pure ursine fat, the total content of three kinds of ω-3 polyenoic fatty acids-eicosapentaenoic acid (EPA), clupanodonic acid (DPA) and docosahexenoic acid (DHA) significantly improves in this pure ursine fat, the special fishy smell of Adeps Phocae vitulinae is removed totally substantially simultaneously, and physical behavior is greatly improved.

The content of three kinds of ω-3 polyenoic fatty acids is respectively in a kind of pure ursine fat provided by the present invention: EPA 10-20%, DHA25-30%, DPA 15-20%.

Preferably, the content of three kinds of ω-3 polyenoic fatty acids is respectively EPA14-16% in the pure ursine fat, DHA26-28%, DPA 17-19%.

Preferred, the content of three kinds of ω-3 polyenoic fatty acids is respectively in the pure ursine fat: the content of EPA is 15-16%, and the content of DHA is 26-27%, and the content of DPA is 18-19%.

Another object of the present invention is to provide a kind of preparation method of pure ursine fat, this method is earlier with natural Adeps Phocae vitulinae esterification, uses the Adeps Phocae vitulinae of multiple-grade molecular distillation purification technique after to esterification then and carries out purification and obtained pure ursine fat.

The preparation method of a kind of pure ursine fat provided by the present invention may further comprise the steps:

1) esterification treatment of dog oil

Get the raw material Adeps Phocae vitulinae, mix mutually with the alkali metal salt equal-volume of the alcohol of 0.2-1.0mol/L, adjust pH is to 6.5-7.5;

2) molecular distillation purification

A) degassing: will enter molecular distillation apparatus after the preheating of esterification Adeps Phocae vitulinae, vapo(u)rizing temperature 75-85 ℃, get heavy ends, repeat to distill 1-5 time;

B) first step is separated: get the distillation of step heavy ends sample introduction, vapo(u)rizing temperature 60-110 ℃, repeat to distill 1-5 time;

C) second step separated: get step heavy ends sample introduction again, 100-120 ℃ of distillation collected light component and promptly got pure ursine fat.

Specifically, the preparation method of a kind of pure ursine fat of the present invention may further comprise the steps:

1) esterification treatment of Adeps Phocae vitulinae

Get the raw material Adeps Phocae vitulinae, mix with the alkali metal salt face of the alcohol of isopyknic 0.5-1.0mol/L, adjust pH is to 6.5-7.5, and the polyenoic fatty acid in the Adeps Phocae vitulinae is esterified;

2) molecular distillation purification

A) degassing: material is entered molecular distillation apparatus after 50-70 ℃ of preheating, feed rate 650-750ml/h, 80 ℃ of vapo(u)rizing temperatures, absolute pressure 0.04～the 0.06mbar of system, knifing spinner velocity 110-170rpm, polyene fatty acid ethylester enters heavy ends, repeats to distill 1-5 time;

B) first step is separated: get the distillation of step heavy ends sample introduction, and 60-110 ℃ of preheating, feed rate 90-1800ml/h, vapo(u)rizing temperature 60-110 ℃, the absolute pressure 0.001～0.03mbar of system, knifing spinner velocity 100-500rpm repeats to distill 1-5 time;

C) second step separated: get step heavy ends sample introduction again, and 15-70 ℃ of preheating, feed rate 90～1800ml/h, 100～120 ℃ of distillations, the absolute pressure 0.001～0.03mbar of system, knifing spinner velocity 100-500rpm collects light component and promptly gets pure ursine fat.

Because the fatty acid in the natural Adeps Phocae vitulinae all exists with the form of mixing triglyceride, the very high difficult vaporization of its boiling point, and the Adeps Phocae vitulinae complicated component, 1,2 of triglyceride with 3 on may be respectively saturation and the great different fatty acids of carbon chain lengths difference.For purification three kinds of ω-3 polyenoic fatty acids wherein, must earlier it be disintegrated down from triglyceride.Can reach this purpose by transesterificationization, and generate boiling point lower, be easy to vaporize and more stable monoester form.

Preferably, the alkali metal salt of the alcohol in the described esterification treatment is alcoholic acid sodium salt or the potassium salt of 0.5-0.8mol/L.

Also can contain the 0.001%-0.005% antioxidant in the alkali metal salt of alcohol, described antioxidant is a 2,6 ditertiary butyl p cresol or to hydroxyl tert-butyl group methoxybenzene, and antioxidant preferred content in the alkali metal salt of alcohol is 0.003%-0.005%.

When regulating pH value, can add the acid commonly used of this areas such as glacial acetic acid, acetic acid, it is good regulating pH value 6.8-7.2.

Preferably, the preheat temperature that outgases in the described distillation is 55-65 ℃, and feed rate is 680-720ml/h, and vapo(u)rizing temperature 78-82 ℃, system's absolute pressure is 0.04-0.05mbar, knifing spinner velocity 130-150rpm.

Preferably, the isolating preheat temperature of the first step is 60-70 ℃ in the described distillation, and feed rate is 200-300ml/h, and vapo(u)rizing temperature 70-90 ℃, system's absolute pressure is 0.001-0.01mbar, knifing spinner velocity 100-200rpm; Repeat to distill 1-3 time.

Preferably, in the described distillation second the step isolating preheat temperature be 50-70 ℃, feed rate is 100-200ml/h, vapo(u)rizing temperature is 110-120 ℃, the absolute pressure 0.001-0.02mbar of system, knifing spinner velocity 100-200rpm repeats to distill 1-3 time.

Preferred, the alkali metal salt of the alcohol that esterification is used is a Sodium ethylate, and its molar concentration is 0.5mol/L.The pH value of esterification is 7.1.

Preferred, the preparation method of pure ursine fat may further comprise the steps:

1) esterification treatment of Adeps Phocae vitulinae:

Get the raw material Adeps Phocae vitulinae, with contain 0.005%2, isopyknic Sodium ethylate of the 0.5mol/L of 6-ditertbutylparacresol mixes mutually, fills nitrogen under the room temperature and stirs 10 minutes adding glacial acetic acid and regulate pH value to 7.1, the decompression rotary evaporation is removed ethanol, removes the bottom small amount of solid and promptly gets the ethyl ester Adeps Phocae vitulinae;

2) molecular distillation purification

A) degassing: material is entered molecular distillation apparatus after 60 ℃ of preheatings, feed rate 720ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.040mbar of system, knifing spinner velocity 140rpm, polyene fatty acid ethylester enters heavy ends;

B) first step is separated: get step heavy ends sample introduction, 60 ℃ of preheatings, feed rate 210ml/h, 80 ℃ of distillations, the absolute pressure 0.005mbar of system, knifing spinner velocity 140rpm; Get heavy ends sample introduction again, vapo(u)rizing temperature changes 85 ℃ into, feed rate 90ml/h, and other are the same;

C) second step separated: get step heavy ends sample introduction again, and 60 ℃ of preheatings, 120 ℃ of distillations, the absolute pressure 0.012mbar of system, knifing spinner velocity 140rpm, feed rate 150ml/h collects light component and promptly gets pure ursine fat.

The present invention provides the assay method of effective ingredient in a kind of fatty oil simultaneously, may further comprise the steps:

1) derivatization treatment of fatty oil:

Get fatty oil 80-120 μ l, place the 10-20ml measuring bottle, the alkali metal salt 4-8ml that adds 0.2-1.0mol/L alcohol, shake up, 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, add 0.2-0.5ml acid cessation reaction again, with the pure standardize solution that contains the 0.001%-0.005% antioxidant, 0.2 μ m-0.3 μ m membrane filtration gets the fatty oil need testing solution that derivation process is crossed;

2) the chromatograph sample introduction is analyzed

Get the fatty oil after the derivation process, enter chromatograph of liquid, described chromatographic chromatographic condition is:

Mobile phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient eluting,

Flow velocity: 1.1-1.5ml/min,

Column temperature: 15-45 ℃,

Detect wavelength: 202～230nm,

Sample size: 5～50 μ l;

3) record chromatogram is with the content of external standard method fatty acid.

Wherein, described fatty oil is the fatty oil that fish oil, vegetable oil or fur seal wet goods contain three kinds of polyenoic fatty acid EPA, DPA and DHA, the effective ingredient of being measured in the fatty oil is meant three kinds of ω-3 polyenoic fatty acids: EPA-Eicosapentaenoic Acid, eicosapentaenoic acid; DPA-Docosapentaenoic Acid, clupanodonic acid and DHA-Docosahexaenoic Acid, docosahexenoic acid, this method can be measured one or more in these three kinds of fatty acids.This method especially is fit to the mensuration of these three kinds of ω-3 polyenoic fatty acids in the Adeps Phocae vitulinae.

The alkali metal salt of the alcohol described in the derivative reaction can be Feldalat NM, Sodium ethylate or Feldalat KM etc., is preferably Feldalat NM, Feldalat NM at 15 days with the basic no change of interior catalysis derivatization ability.Its concentration is 0.2-1.0mol/L, is preferably 0.5-0.8mol/L.

Described antioxidant is 2,6 ditertiary butyl p cresol, to hydroxyl tert-butyl group methoxybenzene etc., the used acid of cessation reaction is glacial acetic acid, formic acid, trifluoroacetic acid or sulphuric acid etc., the used alcohol of standardize solution is methanol, ethanol etc.

Preferably, derivatization treatment of the present invention was reacted 5-30 minute down at 0-60 ℃, and for the ease of operation, derivative reaction more preferably at room temperature carries out, and reacts 15 minutes.

Described derivatization treatment method is preferably: get fatty oil 100-120 μ l, place the 10-200ml measuring bottle, add the Feldalat NM 4-6ml of 0.5-0.8mol/L, shake up room temperature reaction 15-30 minute, to little oil droplet complete obiteration, add 0.2-0.4ml glacial acetic acid cessation reaction again, with containing 0.003%-0.005%2, the methanol constant volume of 6-ditertbutylparacresol, 0.2l μ m membrane filtration gets the fatty oil need testing solution that derivation process is crossed.

The used chromatographic column of chromatograph of the present invention can be: Allsphere Octyl (C8), 3 μ m, 100 , 4.6mm * 150mm; Waters SymmetryShield RP-18,5 μ m, 100 , 3.9mm * 150mm and LichrospherRP-18,5 μ m, 100 , 4.6mm * 250mm.With the RP-18 type, 5 μ m, 100 , 4.6mm * 250mm model is for well.As the Waters high performance liquid chromatograph, Lichrospher high performance liquid chromatograph, Mightsil high performance liquid chromatograph.Be preferably, chromatograph of liquid is the RP-18 type, 5 μ m, 100 , 4.6mm * 250mm model.

When selecting chromatographic chromatographic condition, find to detect wavelength between 202～230nm all can, wherein the three kinds of fatty acids in 210nm place absorb by force and stable, precision also meets the pharmacopeia requirement, so the detection wavelength is selected 210nm for use.

The present invention selects in methanol-water system, acetonitrile tetrahydrofuran water system or three kinds of systems of acetonitrile methanol water system any one to carry out the constant gradient eluting as mobile phase; Through debugging repeatedly, the change ratio finds that finally the acetonitrile methanol water system is best, is that 7: 1: 2 flow velocitys are increased to 1.1-1.5ml/min gradually in its ratio, finds that at last every index reaches best when flow velocity is 1.2ml/min.So mobile phase is preferably the acetonitrile methanol water system, three's ratio is 7: 1: 2, and flow velocity is 1.2ml/min, and column temperature is chosen in 15-45 ℃ simultaneously.

Described derivatization treatment method most preferably is: get fatty oil 120 μ l, place the 100ml volumetric flask, add 0.5mol/L Feldalat NM 4ml, shake up, room temperature reaction 15～30 minutes is to little oil droplet complete obiteration, add 0.2ml glacial acetic acid cessation reaction again, with the methanol constant volume that contains 0.005%BHT, 0.2 μ m membrane filtration gets the fatty oil need testing solution that derivation process is crossed.

The present invention also provides the assay method of effective ingredient in a kind of Adeps Phocae vitulinae simultaneously, and described assay method may further comprise the steps:

1) derivatization treatment of Adeps Phocae vitulinae:

Get Adeps Phocae vitulinae 80-120 μ l, place the 10-200ml measuring bottle, add 0.2-1.0mol/L Feldalat NM 4-8ml, shake up,, add 0.2-0.5ml glacial acetic acid cessation reaction again 0-60 ℃ of extremely little oil droplet complete obiteration of reaction down, with containing 0.001%-0.005%2, the pure standardize solution of 6-ditertbutylparacresol, 0.2 μ m-0.3 μ m membrane filtration gets the Adeps Phocae vitulinae need testing solution that derivation process is crossed;

2) the chromatograph sample introduction is analyzed

Get the Adeps Phocae vitulinae need testing solution after the derivation process, inject chromatograph of liquid, described chromatographic chromatographic condition is:

Mobile phase: methanol-water system, acetonitrile tetrahydrofuran water system or acetonitrile methanol water system, the constant gradient eluting,

Flow velocity: 1.1-1.5ml/min,

Column temperature: 15-45 ℃,

Detect wavelength: 202-230nm,

Sample size: 5～50 μ l;

3) record chromatogram is with the content of external standard method fatty acid.

Wherein, the effective ingredient of being measured in the Adeps Phocae vitulinae is meant three kinds of ω-3 polyenoic fatty acids: EPA-Eicosapentaenoic Acid, eicosapentaenoic acid; DPA-Docosapentaenoic Acid, clupanodonic acid and DHA-Docosahexaenoic Acid, one or more in the docosahexenoic acid.

Described derivatization treatment method is preferably: get Adeps Phocae vitulinae 120 μ l, place the 100ml volumetric flask, add 0.5mol/L Feldalat NM 4ml, shake up, room temperature reaction 15～30 minutes adds 0.2ml glacial acetic acid cessation reaction again, with containing 0.005%2, the methanol constant volume of 6-ditertbutylparacresol, 0.2 μ m membrane filtration gets the Adeps Phocae vitulinae need testing solution that derivation process is crossed.

The acid number of the prepared a kind of pure ursine fat of the present invention is 0.1-0.5, and iodine number is 250-300, and peroxide content is 0.10-0.15.

A kind of pure ursine fat of the present invention and preparation method thereof is the result that the technology of the present invention personnel grope Adeps Phocae vitulinae purification and preparation method thereof for a long time, has the following advantages:

1. the esterification process of Adeps Phocae vitulinae of the present invention was groped to draw through the long period, and original esterification process mostly is two step esterifications, complex steps; Though the method that has is a step esterification, condition is violent, and sample is had destruction.Esterification process of the present invention not only can be complete with the sample esterification under temperate condition, and one the step finish, easy and simple to handle.

2. the present invention has set up the multiple-grade molecular distillation purification process of Adeps Phocae vitulinae first through to the groping of distil process condition, and successfully the content of three kinds of fatty acids in the Adeps Phocae vitulinae is brought up to about 60% from more than 20%, and the while total recovery significantly improves.

3. molecular distillation method used in the present invention is a kind of Physical Separation Technology of very environmental protection; compare with traditional Chemical Decomposition method; it has saved a large amount of solvents, has reduced to the pollution of environment with to operator's harm, and can not bring new impurity and noxious substance into.It can be used for suitability for industrialized production after amplifying in addition, has wide DEVELOPMENT PROSPECT.Simultaneously, but the molecular distillation utmost point removes lower-molecular substance (as organic solvent, stink etc.) and pigment in the liquid effectively, therefore is removed substantially totally with its special fishy smell of the purified pure ursine fat of this method, and physical behavior is greatly improved.

4. there is abundant marine animal oil resource in China, and therefore, exploitation molecular distillation separation purifying technique is made with extra care similar products and had great importance.The present invention has carried out desk study to this field, has proved the feasibility of molecular distillation pure ursine fat, and provides important parameter for commercial production in the future.

Description of drawings

Fig. 1 is the composition sketch map of scraped film type molecular still;

Fig. 2 investigates figure for the esterification completeness;

Fig. 3 is EPAE content in the heavy ends;

Fig. 4 is an EPAE content in the light component;

Fig. 5 is DHAE content in the heavy ends;

Fig. 6 is a DHAE content in the light component;

Fig. 7 is DPAE content in the heavy ends;

Fig. 8 is a DPAE content in the light component;

Fig. 9 is EPAE yield in the heavy ends;

Figure 10 is an EPAE yield in the light component;

Figure 11 is DHAE yield in the heavy ends;

Figure 12 is a DHAE yield in the light component;

Figure 13 is DPAE yield in the heavy ends;

Figure 14 is a DPAE yield in the light component;

The specific embodiment

Below be specific embodiments of the invention, described embodiment is used to describe the present invention, rather than restriction the present invention.

Embodiment 1

1. the esterification treatment of Adeps Phocae vitulinae:

Get Adeps Phocae vitulinae 500ml, mix mutually, add about glacial acetic acid adjust pH to 6.5, promptly get the Adeps Phocae vitulinae of ethyl esterization with the 500ml Sodium ethylate of the 0.5mol/L that contains 0.001%BHT.

2. molecular distillation

A) degassing: material is entered molecular distillation apparatus after 50 ℃ of preheatings, feed rate 650ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.050mbar of system, knifing spinner velocity 110rpm, polyene fatty acid ethylester enters heavy ends,

B) first step is separated: get the distillation of step heavy ends sample introduction, 60 ℃ of preheatings, feed rate 90ml/h, 60 ℃ of distillations, the absolute pressure 0.001mbar of system, knifing spinner velocity 100rpm; Redistillation is four times under the similarity condition;

C) second step separated: get step heavy ends sample introduction again, and 15 ℃ of preheatings, feed rate 90ml/h, 100 ℃ of distillations, the absolute pressure 0.001mbar of system, knifing spinner velocity 100rpm collects light component and promptly gets pure ursine fat.

The content of three kinds of ω-3 polyenoic fatty acids is respectively in the prepared pure ursine fat product: the content of EPA is 9.89%, and the content of DHA is 25.02%, and the content of DPA is 15.15%, acid number is 0.12, iodine number is 249, and peroxide value is 0.10, and total recovery is 51.45%.

Embodiment 2

1. the esterification treatment of Adeps Phocae vitulinae:

Get the 500ml Adeps Phocae vitulinae, mix mutually, add about glacial acetic acid adjust pH to 7.1, promptly get the Adeps Phocae vitulinae of ethyl esterization with the 500ml potassium ethoxide of the 0.5mol/L that contains 0.003%BHT.

2. molecular distillation

A) degassing: material is entered molecular distillation apparatus after 60 ℃ of preheatings, feed rate 720ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.040mbar of system, knifing spinner velocity 140rpm, polyene fatty acid ethylester enters heavy ends;

B) first step is separated: get the distillation of step heavy ends sample introduction, 60 ℃ of preheatings, feed rate 210ml/h, 80 ℃ of distillations, the absolute pressure 0.005mbar of system, knifing spinner velocity 140rpm gets heavy ends sample introduction again, vapo(u)rizing temperature changes 85 ℃ into, feed rate 90ml/h, and other are the same;

C) second step separated: get step heavy ends sample introduction again, and 60 ℃ of preheatings, 120 ℃ of distillations, the absolute pressure 0.012mbar of system, knifing spinner velocity 140rpm, feed rate 150ml/h collects light component and promptly gets pure ursine fat.

The content of three kinds of ω-3 polyenoic fatty acids is respectively in the prepared pure ursine fat product: the content of EPA is 15.5%, and the content of DHA is 26.94%, and the content of DPA is 18.59%, acid number is 0.3, iodine number is 286, and peroxide value is 0.12, and total recovery is 57.98%.

Embodiment 3

1. the esterification treatment of Adeps Phocae vitulinae:

Get the 500ml Adeps Phocae vitulinae, mix mutually, add about glacial acetic acid adjust pH to 7.5, promptly get the Adeps Phocae vitulinae of ethyl esterization with the 500ml potassium ethoxide of the 0.7mol/L that contains 0.005%BHT.

2. molecular distillation

A) degassing: material is entered molecular distillation apparatus after 70 ℃ of preheatings, feed rate 750ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.060mbar of system, knifing spinner velocity 170rpm, PUFAE enters heavy ends,

B) first step is separated: get the distillation of step heavy ends sample introduction, 110 ℃ of preheatings, feed rate 900ml/h, 110 ℃ of distillations, the absolute pressure 0.03mbar of system, knifing spinner velocity 300rpm; Get heavy ends sample introduction again, condition is the same to be repeated to distill twice again.

C) second step separated: get step heavy ends sample introduction again, and 70 ℃ of preheatings, feed rate 900ml/h, 120 ℃ of distillations, the absolute pressure 0.03mbar of system, knifing spinner velocity 300rpm collects light component and promptly gets pure ursine fat.

The content of three kinds of ω-3 polyenoic fatty acids is respectively in the prepared pure ursine fat product: the content of EPA is 19.2%, and the content of DHA is 29.65%, and the content of DPA is 19.86%, acid number is 0.5, iodine number is 297, and peroxide value is 0.15, and total recovery is 59.89%.

Embodiment 4

1. the esterification treatment of Adeps Phocae vitulinae:

Get the 500ml Adeps Phocae vitulinae, mix mutually, add about glacial acetic acid adjust pH to 7.5, promptly get the Adeps Phocae vitulinae of ethyl esterization with the 500ml potassium ethoxide of the 0.7mol/L that contains 0.005% pair of hydroxyl tert-butyl group methoxybenzene.

2. molecular distillation

A) degassing: material is entered molecular distillation apparatus after 70 ℃ of preheatings, feed rate 750ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.060mbar of system, knifing spinner velocity 170rpm, PUFAE enters heavy ends,

B) first step is separated: get the distillation of step heavy ends sample introduction, 110 ℃ of preheatings, feed rate 1800ml/h, 110 ℃ of distillations, the absolute pressure 0.03mbar of system, knifing spinner velocity 500rpm;

C) second step separated: get step heavy ends sample introduction again, and 70 ℃ of preheatings, feed rate 1800ml/h, 120 ℃ of distillations, the absolute pressure 0.03mbar of system, knifing spinner velocity 500rpm collects light component and promptly gets pure ursine fat.

The content of three kinds of ω-3 polyenoic fatty acids is respectively in the prepared pure ursine fat product: the content of EPA is 20.4%, and the content of DHA is 29.98%, and the content of DPA is 19.96%, acid number is 0.5, iodine number is 298, and peroxide value is 0.15, and total recovery is 59.65%.

Experimental example 1

This experimental example is the pre-treatment of Adeps Phocae vitulinae sample.

1. the esterification of Adeps Phocae vitulinae

Fatty acid in the natural Adeps Phocae vitulinae all exists with the form of mixing triglyceride, the very high difficult vaporization of its boiling point, and Adeps Phocae vitulinae complicated component, 1,2 of triglyceride with 3 on may be respectively saturation and the great different fatty acids of carbon chain lengths difference.Therefore for purification PUFA wherein, must earlier it be disintegrated down from triglyceride.Can reach this purpose by transesterificationization, and generate boiling point lower, be easy to vaporize and more stable monoester form.

2. Adeps Phocae vitulinae ethyl ester completeness is investigated

Get Adeps Phocae vitulinae and derivatization sample spot on silica gel g thin-layer plate (110 ℃ of activation 1h), petroleum ether (30～60 ℃ of boiling ranges)-ether (9: 1) launches, and spray 0.02%Rhodamin6G ethanol liquid is observed (see figure 2) down in uviol lamp (365nm).The R of fatty acid methyl ester _fValue is greater than triglyceride, and the disappearance of triglyceride point illustrates that derivatization is complete in the back sample of deriving.

1,4 were esterification sample as stated above during Fig. 2 esterification completeness was investigated, and 2,5 is the no esterification sample, and 3 is complete esterification sample, and by this figure as seen, triglyceride speckle complete obiteration in 1,4 proves that said method can be with the complete ethyl esterization of sample.

Experimental example 2

This experimental example is the assay of three kinds of polyenoic fatty acids in the pure ursine fat.

1. instrument, reagent and sample

Instrument: Tianjin, island (Shimadzu) 201OA high performance liquid chromatograph, ultraviolet double-wavelength detector, Class-VP chromatographic work station; Waters515 pump, 996 type diode array detector, Millennium32 chromatographic work station; MiIlipore ultra-pure water draft machine.

Reagent: 2,6 one ditertbutylparacresols (BHT), sodium metal, metallic potassium, glacial acetic acid, absolute ether, normal hexane, anhydrous sodium sulfate, potassium hydroxide, ethanol, phenolphthalein, hydrochloric acid, acetone, triethylamine are homemade analytical pure; High pure nitrogen; Methanol, acetonitrile chromatographically pure, Fisher company product, trifluoroacetic acid, chromatographically pure, Merck company product; Alpha-brominated 1-Phenylethanone., chromatographically pure, sample: Adeps Phocae vitulinae, Canadian TerraNova fishery company limited.

Raw material: Adeps Phocae vitulinae ethyl ester sample.

2. sample pre-treatments

For the content to three kinds of polyenoic fatty acids in the Adeps Phocae vitulinae before and after the purification compares, its content is measured with the HPLC method.

3. chromatographic condition

Chromatographic column: anti-phase carbon 18 posts,

Mobile phase: acetonitrile: methanol: water=68: 12: 20, constant gradient eluting;

Flow velocity: 1.5ml/min; Column temperature: 45 ℃; Detect wavelength: 210nm; Sample size: 20 μ l.

4. quantitative approach

Get EPAM (EPA methyl ester), DHAM (DHA methyl ester), DPAM (DPA methyl ester) sample introduction gets the reference substance chromatogram,, with external standard method with the content of three kinds of polyenoic fatty acids of each sample of calculated by peak area.

Because what directly record is the content of Adeps Phocae vitulinae fatty acid methyl ester, try to achieve corresponding content of fatty acid, still needs and carries out following conversion:

EPA content=EPAM content * 302.450/316.478=EPAM content * 0.95567

DHA content=DHAM content * 328.487/342.515=DHAM content * 0.95904

DPA content=DPAM content * 330.530/344.531=DPAM content * 0.95936;

Experimental example 3

This experimental example is to study the distil process condition in the molecular distillation pure ursine fat.

1. experimental raw and segregation apparatus

Raw material: Adeps Phocae vitulinae, Canadian TerraNova fishery company limited is produced

Segregation apparatus: the German VTA VKL70 of company type wiped-film short-distance distillation equipment, see Fig. 1.

2. technology path determines

Raw material enters short course distillation device from feeder, under the effect of blade applicator, is uniformly distributed on the heating evaporation face, and evaporating surface is by heat-conducting oil heating.When raw material is heated on evaporating surface, under high vacuum condition, volatile component flies to interconderser straight and is condensed into liquid, flows into the light component receiving flask along condenser.Unevaporated non-volatilization component flows into the heavy ends receiving flask.Cold-trap is set on the pipeline, adds liquid nitrogen in the cold-trap as the deep cooling agent, doing like this has three benefits: prevent that 1. volatile matter from entering vacuum pump, prolong the service life of vacuum pump; 2. reduce vacuum effectively, improve the work efficiency of vacuum pump; 3. reduce of the pollution of pump oil to separating mixture.Most of fishy smell material in the raw material promptly enters cold-trap.

Because the dual condensation of cold-trap that vacuum system has intermediate condensation pipe and liquid nitrogen to add has guaranteed the equilibrium of whole system operating pressure.The knifing system can guarantee the uniformity of liquid film on the whole evaporating surface, and the liquid film surface constantly is updated, thereby has guaranteed to be heated enough weak points of raw material time of staying on evaporating surface.

At first raw material is carried out separated under higher feed rate and knifing rotor speed in test, the weight component sampling that at every turn obtains is analyzed.

Because the volatility difference, when molecular distillation, saturated and monounsaturated fatty acid ester is steamed earlier, esters of polyunsaturated fatty acids secondly, glycerol is the most difficult to be steamed (the glycerol boiling point is 290.9 ℃ under the normal pressure).Therefore technology path of the present invention is to carry out for two steps to separate: the first step, remove light component, and promptly look for an appropraite condition that saturated and monounsaturated fatty acid ester are steamed earlier, make it enter light component, purpose component PUFA ethyl ester staying in the heavy ends as much as possible; Second step, remove heavy ends, promptly look for an appropriate condition that purpose component PUFA ethyl ester is steamed as far as possible, enter light component, glycerol, BHT, sodium acetate etc. are then stayed in the heavy ends, finish purification.Second step separation simultaneously has decolorization concurrently.In addition, in each step separation, separate if be necessary all can carry out repeatedly multistage distillation.

3. vapo(u)rizing temperature is to the influence of EPAE, DHAE, DPAE content

In molecular distillation, vapo(u)rizing temperature is the factor that separation is had the greatest impact.Shown in Fig. 3-8 be molecular distillation when operating pressure is 0.4～0.06mbar, temperature is to the influence curve of EPAE, DHAE, DPAE content in light, the heavy ends:

From Fig. 3-8 as can be known, in vapo(u)rizing temperature was 80～120 ℃ scope, the content of PUFAE raise with temperature earlier in the heavy ends, descended with the temperature rising again after reaching a maximum, and is then opposite in the light component.This is because when vapo(u)rizing temperature was low, saturated and monounsaturated fatty acid was steamed earlier, and the PUFAE overwhelming majority is stayed in the heavy ends; After temperature was elevated to a certain degree, PUFAE is also steamed gradually entered light component.

Can be found out also that by Fig. 3-8 the molecular distillation behavior of EPAE is different with DHAE and DPAE, it is maximum that the concentration of EPAE in heavy ends reaches when 85 ℃ of left and right sides, and it is maximum that DHAE and DPAE reach about 100 ℃.Therefore can utilize this characteristic that EPAE and other PUFAE are carried out fine separation, also point out our first step to separate and should carry out under between 80 ℃～100 ℃ simultaneously, second step separated and should carry out under higher temperature.

4. vapo(u)rizing temperature is to the influence of yield

Not only the lay down hard and fast rule content of PUFAE of molecular distillation process operation temperature, and the yield of PUFAE also had considerable influence.Fig. 9-14 is a temperature to the influence curve of PUFAE yield in light, the heavy ends:

Raise with temperature by rate and descend gradually of PUFAE in heavy ends as can be seen from Fig. 9-14, then opposite in the light component, temperature should be low as far as possible when this pointed out our first step to separate, and the second step separation temperature should be high as far as possible.Certain temperature can not be selected too high, not only can destroy PUFAE stability because temperature is too high, and also can cause other impurity to steam with PUFAE, influences separating effect.Take all factors into consideration temperature to the double influence of content and yield and we requirement to product, our first step is separated and is adopted 80 ℃ and 85 ℃ of two-step distillations; Second step was separated 120 ℃ of employings.Result of the test confirms can reach the expection purification effect like this.

5. the influence of feed rate

Molecular distillation process material feeding speed is very big to the yield influence of product.Because feeding temperature is lower than evaporating temperature, when feed rate was big, some effective evaporating surface was used to preheated feed, thereby caused the actual effectively minimizing of disengagement area, also obviously reduced so the amount of steaming accounts for the ratio of total feed; But feed rate is also unfavorable to separating too slowly.Find in experiment: if feed rate is too fast, the esterification Adeps Phocae vitulinae also is not heated evaporation and just flows into the heavy ends catcher in the raw material, and PUFAE content improves less; If feed rate is too slow, because raw material increased in the time of staying of evaporating surface, the probability that heavy ends escape into cryosurface also increases, and makes its colour changed into yellow of product, and yield descends simultaneously.

6. the influence of blade applicator rotor speed

The blade applicator rotor speed also has to a certain degree influence to still-process, and slewing rate is too slow, is difficult to form uniform liquid film on the heating wall, and the speed of liquid film Surface Renewal is slow excessively simultaneously, is unfavorable for mass-and heat-transfer; Rotor rotation rate is too fast, can cause part material directly to be thrown on the interconderser by blade applicator, rather than from evaporating surface through pervaporation condensation on cryosurface then, therefore can cause the reduction of separation efficiency.

7. the influence of feeding temperature

Test finds that along with the increase of feeding preheating temperature, the content of PUFAE improves in the product, and this is that actual effectively disengagement area increases, the separation efficiency raising owing to the disengagement area that is used for the material preheating after the feeding temperature raising is less.But the influence degree of relative and other factor feeding temperatures is less.This is that though the part evaporating surface is used to preheated feed, feed rate used in the experiment is also slower because when feeding temperature is low, and it is less that the evaporating surface area that is used for the material preheating accounts for the ratio of vaporizer evapo tranpiration area.Too high feeding temperature can quicken the decomposition of material simultaneously.

After measured, the product E PA content that makes with this technology can reach 15.5%, and DHA is 26.94%, and DPA is 18.59%, and total recovery is 57.98%.

Experimental example 4

This experimental example is the attributional analysis of the refining front and back of Adeps Phocae vitulinae.

1. acid number

Measure by two appendix VII of " The People's Republic of China's pharmacopeia " version in 2000 H method: get refining preceding Adeps Phocae vitulinae 5g or refining back Adeps Phocae vitulinae 10g, the accurate title in the fixed rearmounted 250ml conical flask, adding alcohol-ether (1: 1) mixed liquor (faces with before adding instructions phenolphthalein solution 1.0ml, transfer to little existing pink with 0.1mol/L sodium hydroxid volumetric solution) 50ml, jolting makes dissolving fully, with the titration of 0.1mol/L sodium hydroxid volumetric solution, continued for 30 seconds to pink and do not take off.Volume (ml) with consumption 0.1mol/L sodium hydroxid volumetric solution is A, and example weight (g) is G, calculates acid number according to following formula: sample acid number=(A * 5.61)/G

2. iodine number

Measure by two appendix VII of " The People's Republic of China's pharmacopeia " version in 2000 H method: sample thief 0.1g, the accurate title, decide, and puts in the dry iodine flask of 250ml, add chloroform 10ml, accurate IBr solution 25ml, the close plug of adding in dissolving back, shake up, in the dark placed 30 minutes.The potassium iodide test solution 10ml and the water 100ml that add new system, shake up,, note shake well during titration with the remaining iodine of sodium thiosulfate volumetric solution (0.1mol/L) titration, it is faint yellow that liquid to be mixed brown becomes, and adds starch indicator solution 1ml and continue titration and disappear to blue; Do blank experiment simultaneously.Molten long-pending (ml) with sample consumptive use sodium thiosulfate volumetric solution is A, and the volume (ml) that blank assay consumes is B, and example weight (g) is G, calculates iodine number according to following formula: sample iodine number=((B-A) * 1.269)/G

3. peroxide value

Measure by " The People's Republic of China's pharmacopeia " version in 2000 two text kinds soybean oil (injection) peroxide assay method: sample thief 10.0g, put in the 250ml iodine flask, add glacial acetic acid one chloroform (6: 4) mixed liquor 30ml immediately, jolting makes dissolving, the saturated potassium iodide 0.5ml of accurate adding, close plug, jolting 1 minute, add water 30ml, with sodium thiosulfate volumetric solution (0.01mol/L) titration, add starch indicator solution 0.5ml during to nearly terminal point, continue titration and disappear, and titration results is proofreaied and correct with blank experiment to blue.The volume (ml) of waiting until the sodium sulfate volumetric solution with sample consumption is A, and the volume of blank assay (ml) is B, and example weight (ml) is G, calculates peroxide value according to following formula: sample peroxide value=((A-B) * 0.01 * 1000)/G

4. moisture and volatile matter

Measure by two appendix VII of " The People's Republic of China's pharmacopeia " version in 2000 H method: the about 5g of sample thief puts in the flat weighing bottle that is dried to constant weight, accurately claims surely, takes out in 40 minutes 105 ℃ of dryings, puts to put coldly in the exsiccator, and weight decided in title; Again 105 ℃ of dryings 20 minutes, put coldly, claim decide weight, be no more than 0.001g to the difference of double dry weighing afterwards.The weight that subtracts mistake is the weight that contains moisture and volatile matter in the sample.

5. result and discussion

Attributional analysis table before and after table 1 Adeps Phocae vitulinae is refining

Project	Raw material	Esterification feed	Refining back product
				Abnormal smells from the patient	Stronger fishlike smell	Intensive fishlike smell	Very light fishlike smell
Color and luster	Light yellow	Buff	Very light yellow
				Acid number	0.6	0.4	0.3
Iodine number	146	139	286
				Peroxide value	0.26	0.58	0.12
Moisture and volatile matter (%)	0.2	0.2	0.01
				EPA content (w/w)	7.028％	6.95％	15.5％
DHA content (w/w)	9.75％	9.71％	26.94％
				DPA content (w/w)	4.256％	4.05％	18.59％

Acid number means the weight (mg) of the required potassium hydroxide of free fatty that contains among neutralization fat, fatty oil or other similar substance 1g.Back spoiled by rancid oil or fat produces a large amount of free fatties, and acid number increases, and therefore available acid number comes the measure oil degree of becoming sour.Iodine number means fat, fatty oil or other similar substance 100g, required iodine amount (g) when abundant halogenation.It reflects greasy degree of unsaturation.Peroxide value reflection sample contains the mole of peroxide, can weigh the degree of oxidation of sample to a certain extent, judges the oils and fats quality.

As seen from the above table, after the raw material esterification, quality has bigger variation, this be since during esterification the part Adeps Phocae vitulinae oxidized, so peroxide value raises, color and luster is deepened, fishy smell increases the weight of.And follow esterification the two keys of part also to be arranged by saturated, so iodine number descends to some extent.But every index all has bigger improvement after refining, illustrates that refining back Adeps Phocae vitulinae quality greatly improves.

Claims (10)

1. a pure ursine fat is characterized in that, the content of three kinds of ω-3 polyenoic fatty acids is respectively in the Adeps Phocae vitulinae: EPA 10-20%, DHA 25-30%, DPA 15-20%.

2. a kind of pure ursine fat according to claim 1 is characterized in that, the content of three kinds of ω-3 polyenoic fatty acids is respectively in the Adeps Phocae vitulinae: EPA 14-16%, DHA 26-28%, DPA 17-19%.

3. a kind of pure ursine fat according to claim 1 is characterized in that, the content of three kinds of ω-3 polyenoic fatty acids is respectively in the Adeps Phocae vitulinae: the content of EPA is 15-16%, and the content of DHA is 26-27%, and the content of DPA is 18-19%.

4. the preparation method of a pure ursine fat is characterized in that, may further comprise the steps:

1) esterification treatment of Adeps Phocae vitulinae

Get the raw material Adeps Phocae vitulinae, mix mutually with the alkali metal salt equal-volume of the alcohol of 0.2-1.0mol/L, adjust pH is to 6.5-7.5;

2) molecular distillation purification

A) degassing: will enter molecular distillation apparatus after the preheating of esterification Adeps Phocae vitulinae, vapo(u)rizing temperature 75-85 ℃, get heavy ends, repeat to distill 1-5 time;

B) first step is separated: get the distillation of step heavy ends sample introduction, vapo(u)rizing temperature 60-110 ℃, repeat to distill 1-5 time;

C) second step separated: get step heavy ends sample introduction again, 100-120 ℃ of distillation collected light component and promptly got pure ursine fat.

5. the preparation method of a kind of pure ursine fat according to claim 4 is characterized in that, may further comprise the steps:

1) esterification treatment of dog oil

Get the raw material Adeps Phocae vitulinae, mix mutually with the alkali metal salt equal-volume of the alcohol of 0.2-1.0mol/L, adjust pH is to 6.5-7.5;

2) molecular distillation purification

A) degassing: the esterification Adeps Phocae vitulinae is entered molecular distillation apparatus after 50-70 ℃ of preheating, feed rate 650-750ml/h, vapo(u)rizing temperature 75-85 ℃, the absolute pressure 0.04-0.06mbar of system, knifing spinner velocity 110-170rpm, polyene fatty acid ethylester enters heavy ends, repeats to distill 1-5 time;

B) first step is separated: get the distillation of step heavy ends sample introduction, and 60-110 ℃ of preheating, feed rate 90-1800ml/h, vapo(u)rizing temperature 60-110 ℃, the absolute pressure 0.001-0.03mbar of system, knifing spinner velocity 100-500rpm repeats to distill 1-5 time;

C) second step separated: get step heavy ends sample introduction again, and 15-70 ℃ of preheating, feed rate 90-1800ml/h, 100-120 ℃ of distillation, the absolute pressure 0.001-0.03mbar of system, knifing spinner velocity 100-500rpm collects light component and promptly gets pure ursine fat.

6. the preparation method of a kind of pure ursine fat according to claim 5, it is characterized in that, the alkali metal salt of the alcohol in the described esterification treatment is alcoholic acid sodium salt or the potassium salt of 0.5-0.8mol/L, also can contain the 0.001%-0.005% antioxidant in the alkali metal salt of alcohol, preferred content is 0.003%-0.005%, described antioxidant is a 2,6 ditertiary butyl p cresol or to hydroxyl tert-butyl group methoxybenzene; The adjusting pH value is 6.8-7.2;

The preheat temperature that outgases in the described distillation is 55-65 ℃, and feed rate is 680-720ml/h, and vapo(u)rizing temperature 78-82 ℃, system's absolute pressure is 0.04-0.05mbar, and knifing spinner velocity 130-150rpm repeats to distill 1-3 time.Antioxidant is in the alkali metal salt of alcohol.

7. the preparation method of a kind of pure ursine fat according to claim 4, it is characterized in that, the isolating preheat temperature of the first step is 60-70 ℃ in the described distillation, feed rate is 200-300ml/h, vapo(u)rizing temperature 70-90 ℃, system's absolute pressure is 0.001-0.01mbar, and knifing spinner velocity 100-200rpm repeats to distill 1-3 time.

8. the preparation method of a kind of pure ursine fat according to claim 4, it is characterized in that, in the described distillation second the step isolating preheat temperature be 50-70 ℃, feed rate is 100-200ml/h, vapo(u)rizing temperature is 110-120 ℃, the absolute pressure 0.001-0.02mbar of system, knifing spinner velocity 100-200rpm.

9. the preparation method of a kind of pure ursine fat according to claim 4 is characterized in that, the alkali metal salt of the alcohol that esterification is used is a Sodium ethylate, and its molar concentration is 0.5mol/L, and the pH value of esterification is 7.1.

10. the preparation method of a kind of pure ursine fat according to claim 4 is characterized in that, may further comprise the steps:

1) esterification treatment of Adeps Phocae vitulinae:

Get the raw material Adeps Phocae vitulinae, mix mutually with isopyknic Sodium ethylate of the 0.5mol/L that contains 0.005% 2,6 ditertiary butyl p cresol, fill nitrogen under the room temperature and stir 10 minutes adding glacial acetic acid adjusting pH value to 7.1, the decompression rotary evaporation is removed ethanol, removes the bottom small amount of solid and promptly gets the ethyl ester Adeps Phocae vitulinae;

2) molecular distillation purification

A) degassing: material is entered molecular distillation apparatus after 60 ℃ of preheatings, feed rate 720ml/h, 80 ℃ of vapo(u)rizing temperatures, the absolute pressure 0.040mbar of system, knifing spinner velocity 140rpm, polyene fatty acid ethylester enters heavy ends;

B) first step is separated: get step heavy ends sample introduction, 60 ℃ of preheatings, feed rate 210ml/h, 80 ℃ of distillations, the absolute pressure 0.005mbar of system, knifing spinner velocity 140rpm; Get heavy ends sample introduction again, vapo(u)rizing temperature changes 85 ℃ into, feed rate 90ml/h, and other are the same;

C) second step separated: get step heavy ends sample introduction again, and 60 ℃ of preheatings, 120 ℃ of distillations, the absolute pressure 0.012mbar of system, knifing spinner velocity 140rpm, feed rate 150ml/h collects light component and promptly gets pure ursine fat.

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