CN104651422A - Method of extracting DHA and EPA in type of triglyceride from deep-sea fish - Google Patents
Method of extracting DHA and EPA in type of triglyceride from deep-sea fish Download PDFInfo
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Abstract
The invention belongs to the technical field of biopharmacy and provides a method of extracting DHA and EPA in the type of triglyceride from deep-sea fish. The method includes steps of: preparing crude fish oil with enzymolysis technology, preparing free fatty acid, separating poly-unsaturated fatty acids containing the EPA and the DHA through a urea-embedding method, and preparing the fatty acid triglyceride of the DHA and the EPA. The method can effectively extract and enrich the DNA and the EPA from the deep-sea fish. The obtained crude fish oil is low in acidic value and is high in hydrolysis degree of esterified fatty acids. The finally products of the DHA and the EPA are in the type of triglyceride with the contents of the DHA and the EPA are in the type of triglyceride being high.
Description
Technical field
The invention belongs to biological pharmacy technical field, particularly relate to a kind of method extracting triglyceride type DHA and EPA from bathypelagic fish.
Background technology
Bathypelagic fish, if tuna etc. is a kind of large-scale ocean property important goods food fish.Because bathypelagic fish must keep travelling fast often, could maintain the supply of health, it is only movable in depths, marine site to add, the therefore tender deliciousness of meat, and not by environmental pollution, is the healthy food that modern is rare.The processing industry of bathypelagic fish becomes an important industry, but bathypelagic fish exists quality in the course of processing is difficult to a series of significant problems such as controlling, scrap stock producing level is low, production energy consumption is high, significant loss is serious, and business economic loss is larger.Therefore operation revenue is increased to the tankage that the bathypelagic fish course of processing produces, realize economic worth.The application of fish oil widely, can be applicable to the field such as health care, medicine, food, chemical industry and feed.By being a kind of extraordinary mode realizing economic benefit by the offal of bathypelagic fish for the preparation of fish oil.
In fish oil, n-3 polyunsaturated fatty acid (n-3PUFA) has important biological significance in human body, the physiologically active of its characteristic fatty acid timnodonic acid (EPA) and docosahexenoic acid (DHA) is clear and definite, EPA has prevention coronary heart disease, hypotensive, Ginseng Extract, prevention of arterial are atherosis and cerebral thrombosis, the physiologically active such as anticancer, DHA can promote the intelligent growth of baby significantly, improve cerebral function, improve memory, EPA and DHA is the good base-material producing preventing cardiovascular disease and baby's intelligence-improving food.
Polyene fatty acid in fish oil, comprise EPA and DHA, belong to heat and chemically unstable material, be isolated and purify and difficulty, the technology of existing conventional separation and purification has rectifying separation technology, molecular distillation technique, chromatographic separation technology, cryogenic freezing technology, metal salt precipitate technology and fish oil lactide, but all there is respective deficiency:
Wherein, the technical process of traditional purification techniques comprise come unstuck, depickling, decolouring, deodorization etc., this process for refining is loaded down with trivial details, and product color is undesirable, and heating required in this leaching process easily causes grease oxidation rancid.Rectifying separation technology, it is current most popular industrial separation technology, its principle utilizes each component volatilization in mixed fatty acid different, be separated according to the character of different saturated vapor pressure at the same temperature, but the method service temperature is higher, time is longer, causes unsaturated fatty acids generation thermosensitive response, affects quality product and yield.Molecular distillation technique, under high vacuum condition, be heated can overflow from the page according to fluid molecule, the mean free path of molecular motion is different and be separated in the gas phase, this kind of technological operation temperature is low, separated material is not easily separated or is polymerized, and molecular distillation apparatus one-time investment is comparatively large, and maintenance cost is higher.Supercritical fluid technology facility investment is large, extraction pressure is high, and conventional supercritical fluid extraction plant is mainly made up of single autoclave and separating still, only single separating reaction can be carried out, in addition, supercritical extraction cannot realize saturated or lower concentration unsaturated fatty acids close with other molecular weight to EPA, DHA to be separated.Chromatographic separation technology, a kind of modern separation, analysing and detecting method, the chromatographic process that can be used for fatty acid separation purifying and detection has gas-chromatography, thin-layer chromatography, high performance liquid chromatography etc., in most cases be separated with easy silica gel chromatography, this method can be close by polarity, the unsaturated fatty acids of structural similitude is separated, and main drawback is that power consumption is large, and running cost is high.Cryogenic freezing technology is the object reaching separation according to fatty acid mixt crossing the difference of solubleness in cold organic solvent.The length of saturated fatty acid solubleness in organic solvent and carbochain is inversely proportional to, and the lipid acid of same degree of unsaturation, carbochain is longer, and solubleness is lower, and to the unsaturated fatty acids of same carbochain number, with the increase of double bond, solubleness increases.This kind of technology main drawback to reclaim a large amount of solvent, and separation efficiency is not high, and need the cooling apparatus of pole low temperature, cost is higher.Metal salt precipitate technology, be utilize saturated fatty acid, the difference of metal-salt solubleness in certain organic solvent of low unsaturated fatty acids and EPA, DHA is separated, conventional is silver nitrate legitimate network, the method is simple to operate, mild condition, the cycle is short, yield is high, but Silver Nitrate is expensive, reclaim difficulty, production cost is high.Fish oil hands over esterification techniques, refers to when basic catalyst exists, by the glycerine in ethanol replacement grease, produces ethyl esterified high unsaturated fatty acid (EPA and DHA).Ethyl ester type ω-3PUFA absorption and digestion in human body is more difficult, and may there is potential safety hazard.
In prior art, often EPA, DHA of extraction are processed into ethyl ester type fish oil, quality product is caused to be affected, and the process need of ethyl ester type fish oil replaces quantitative ethanol in esterification process, cause the imbalance of lipid acid, the metabolic adsorption rate of ethyl ester type fish oil in human body is low, bioavailability is low, poor stability; And ethyl ester type fish oil easily decomposites ethanol in gi tract, cause easily causing untoward reaction as allergy to the crowd of ethyl ester tolerance difference, therefore, its security is low.In fish oil, the native state of EPA, DHA is triglyceride type, and the lipase with specificity hydrolyzing triglyceride of human body pancreas and liver, the readily digested absorption of EPA, DHA under native state, and its stable in properties, can effectively ensure its nutritive ingredient, be convenient to absorption of human body utilize, glyceride type fish oil specific absorption is about three times of ethyl ester type fish oil.
In view of this, in above-mentioned steps S04, in order to obtain high triglyceride type EPA and DHA of gamma value, need the polyunsaturated fatty acid containing EPA, DHA after to above-mentioned concentrating to carry out esterification.Compare with traditional catalyzer, lipase-catalyzed reaction has good specificity and high efficiency, but due to the limitation of enzyme industrial development, enzymatic reaction is difficult to control, and causes esterification yield on the low side.And the participation of subcritical fluids, the reversed reaction of enzymatic hydrolysis reaction can be carried out, as esterification, lactonize, the synthesis etc. of transesterify and peptide; And minimizing substrate or product, to the suppression of enzyme, the thermostability increasing enzyme, minimizing byproduct of reaction, can solve the problems referred to above existed when being used alone enzymatic reaction effectively simultaneously.In order to increase contact area and the contact frequency of substrate and enzyme, improve the efficiency of enzymatic reaction further, described subcritical enzymatic reaction can be selected to carry out under hyperacoustic effect.
Particularly, in above-mentioned supercritical extraction reactor, also containing normal hexane, glycerine, as preferred embodiment, described normal hexane, glycerine, lipase Novozym435, be normal hexane containing the amount ratio of the polyunsaturated fatty acid of free EPA, DHA: glycerine: lipase Novozym435: the polyunsaturated fatty acid containing free EPA, DHA is (8-12) ml:(2-6) g:(0.01-0.05) g:1g.
In the embodiment of the present invention, the condition of described subcritical enzymatic reaction is: the pressure of described reactor is 4-8MPa, and ultrasonic oscillation magnetic field is 2-10Hz, and temperature of reaction is 30 DEG C-60 DEG C, and the reaction times is 1h-12h.
Described subcritical enzyme reaction schematic diagram is as shown in Figure 2: join in supercritical extraction reactor form mixture, at CO by containing the polyunsaturated fatty acid of EPA, DHA, normal hexane, glycerine, lipase Novozym435
2subcritical enzymatic reaction is carried out under the acting in conjunction in subcritical fluids and ultrasonic wave magnetic field.
Through the polyunsaturated fatty acid containing EPA, DHA of above-mentioned process, gamma value can reach 80%-90%.
In above-mentioned steps S04, in order to reduce the impact on triglyceride type EPA and DHA quality, and realize the efficient extraction of triglyceride type EPA and DHA simultaneously and refine, adopting the fatty acid triglycercide of method to above-mentioned EPA, DHA of supercritical extraction to process.In supercritical extraction process, because supercutical fluid is at Near The Critical Point, the subtle change of temperature and pressure can cause the larger change of supercritical fluid densities, can regulate the dissolving power to material thus.Therefore, the percentage extraction of supercritical extraction is high, being separated of triglyceride type EPA with DHA and solvent is easily realized in extraction process, and exist simultaneously organic solvent-free residual, to advantages such as heat-sensitive substance are survivable, effectively can remove free fatty acids, fat-soluble pigment, volatility and nonvolatile aldehydes possible remaining in above-mentioned subcritical enzymatic reaction, ketone, alcohols stink substance, thus reach the object of depickling, decolouring, deodorization.
In the step of the fatty acid triglycercide of preparation described EPA, DHA, described supercritical extraction is divided into extraction and fractionation two step.Particularly, after described subcritical enzymatic reaction terminates, using the reaction product after above-mentioned esterification as substrate, supercritical extraction being reacted setting model is: extracting pressure is 20MPa-30(MPa, and extraction temperature is 35 DEG C-40 DEG C, CO
2flow is 4-6L/h, completes extraction process with this understanding.After extraction terminates, be separated by extracting substance, described separation is divided into two-stage to be separated, and wherein, the pressure of flash trapping stage is 6MPa-10MPa, and temperature is 45 DEG C-50 DEG C; The pressure of the second-order separation is 4MPa-6MPa, and temperature is 55 DEG C-60 DEG C.
In embodiment of the present invention S04 step, in order to enhance productivity, increase the productive rate of the fatty acid triglycercide of EPA, DHA simultaneously, and then carry out rectification process after carrying out supercritical extraction process, liquid extracting and separating obtained is introduced in rectifying column and is carried out purifies and separates again.Concrete, the level Four rectifying successively of the reaction product after supercritical extraction, wherein, the temperature of one-level rectifying is 60 DEG C-65 DEG C, and the temperature of two-stage rectification is 65 DEG C-70 DEG C, and the temperature of three grades of rectifying is 70 DEG C-75 DEG C, and the temperature of level Four rectifying is 75 DEG C-80 DEG C.
After above-mentioned rectifying column, collect the cut obtained is based on the close high purity polyunsaturated fatty acid triglycercide of the density of the unsaturated fatty acids triglyceride level of EPA and DHA, wherein, the mass percentage of triglyceride type EPA is 14%-15%, the mass percentage 71%-75% of triglyceride type DHA.
Particularly, as shown in Figure 3, CO
2cO in steel cylinder 1
2successively after the process of strainer 2 and cold 3, and carry agent 11 and mix in mixing tank 5, after the thermal pretreatment of preheater 6, CO
2become supercritical state fluid, mix with the reaction product after subcritical enzymatic reaction in reactor 7 and carry out supercritical extraction, extraction product enters rectifying column 10 successively and carries out level Four rectification process after flash trapping stage still 8, the second-order separation still 9, finally obtain purifying, concentrated triglyceride type DHA and EPA, product flows out through flow totalizer 12.
The method extracting triglyceride type DHA and EPA from bathypelagic fish that the embodiment of the present invention provides, enzymolysis process is adopted to extract fish oil, the crude fish oil DHA obtained and EPA content is high, acid number is low, can reach 1.5 (mg KOH)/g in an inert atmosphere, not easily to become sour qualitative change, directly can carry out enzymolysis glycerine reaction, save the treatment step reducing acid number, simplify and extract flow process; Carry out enzyme digestion reaction by subcritical state, obtain triglyceride type lipid acid, esterifying efficiency obviously increases, and esterification yield can reach 90%; The fatty acid triglycercide of EPA and DHA is concentrated by supercritical fluid extraction, concentrated and refining object can be reached simultaneously, save handling procedure, production efficiency is high, cost is low, in the polyunsaturated triglyceride finally obtained, the productive rate of triglyceride level EPA and DHA is high, wherein, triglyceride type EPA can up to 15%, and triglyceride type DHA can up to 75%.
Be further described below in conjunction with specific embodiment.
Summary of the invention
The object of the present invention is to provide a kind of method extracting triglyceride type DHA and EPA from bathypelagic fish, be intended to solve that DHA and the EPA purity extracted from fish oil of prior art is low, acid number is high, color needs to decolour deeply and obtain the mixture such as direactive glyceride, Witepsol W-S 55 that DHA and EPA is DHA and EPA or ethyl ester class fish oil and the high problem of cost.
The present invention is achieved in that a kind of method extracting triglyceride type DHA and EPA from bathypelagic fish, comprises the following steps:
Bathypelagic fish is rubbed into meat slurry, after carrying out heat treated under anaerobic after adding water, after using trypsinase to be carried out the first enzyme digestion reaction, carry out solid-liquid separation, collect filtrate, obtain crude fish oil;
Utilize lipase 1LVK-F100 that described crude fish oil is carried out the second enzyme digestion reaction, obtain free fatty acids;
Under water bath condition, mixture solution is obtained after the methyl alcohol of urea or alcohol saturated solution being mixed with described free fatty acids, described mixture solution is carried out cooling process to temperature is 4 DEG C-10 DEG C, after insulation leaves standstill process 48h-72h, carry out filtration treatment and obtain filtrate, described filtrate is carried out extraction treatment and collected organic layer, obtains the polyunsaturated fatty acid containing EPA, DHA concentrated;
With the described polyunsaturated fatty acid containing free EPA, DHA for substrate, put it in the supercritical extraction reactor containing lipase Novozym435, subcritical enzymatic reaction is carried out in ultrasonic wave magnetic field, reaction product is carried out supercritical extraction, rectifying, obtains the fatty acid triglycercide of EPA, DHA.
The method extracting triglyceride type DHA and EPA from bathypelagic fish provided by the invention, effectively can extract DHA and EPA from bathypelagic fish or even as the bathypelagic fish head of scrap stock, its production cost is low.The crude fish oil acid number obtained in preparation process is low, can reach 1.5 (mg KOH)/g; In crude fish oil, esterified fatty acid hydrolysis degree is high, can reach 50%; DHA and EPA finally obtained is triglyceride type DHA and EPA, and the content of glycerine three type EPA and DHA is high, and wherein, triglyceride type EPA can up to 15%, and triglyceride type DHA can up to 75%.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet extracting glycerine ester type DHA and EPA from bathypelagic fish that the embodiment of the present invention provides.
Fig. 2 is the subcritical enzymatic reaction of the fatty acid triglycercide for the preparation of EPA, DHA schematic diagram that the embodiment of the present invention provides.
Fig. 3 is the schematic flow sheet of the fatty acid triglycercide for the preparation of EPA, DHA that the embodiment of the present invention provides; Wherein, in Fig. 3, each numbering is respectively: 1.CO
2steel cylinder, 2. strainer, 3. cold, 4. high-pressure metering pump, 5. mixing tank, 6. preheater, 7. reactor, 8. flash trapping stage still, 9. the second-order separation still, 10. rectifying column (10-1 to 10-4 is respectively and rectifying column, two-stage rectification post, three grades of rectifying columns, level Four rectifying columns), 11. carry agent, 12. flow totalizers.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiments provide a kind of method that bathypelagic fish extracts triglyceride type DHA and EPA from bathypelagic fish, comprise the following steps, as described in Figure 1:
S01. adopt zymolysis technique to prepare crude fish oil: bathypelagic fish to be rubbed into meat slurry, after carrying out heat treated under anaerobic after adding water, after using trypsinase to be carried out the first enzyme digestion reaction, carry out solid-liquid separation, collect filtrate, obtain crude fish oil;
S02. free fatty acids is prepared: utilize lipase 1LVK-F100 that described crude fish oil is carried out the second enzyme digestion reaction, obtain free fatty acids;
S03. urea entraing method is adopted to be separated the polyunsaturated fatty acid containing EPA, DHA: under water bath condition, mixture solution is obtained after the methyl alcohol of urea or alcohol saturated solution being mixed with described free fatty acids, described mixture solution is carried out cooling process to temperature is 4 DEG C-10 DEG C, after insulation leaves standstill process 48h-72h, carry out filtration treatment and obtain filtrate, described filtrate is carried out extraction treatment and collected organic layer, obtains the polyunsaturated fatty acid containing EPA, DHA concentrated;
S04. the fatty acid triglycercide of EPA, DHA is prepared: with the described polyunsaturated fatty acid containing free EPA, DHA for substrate, put it in the supercritical extraction reactor containing lipase Novozym435, subcritical enzymatic reaction is carried out in ultrasonic wave magnetic field, reaction product is carried out supercritical extraction, rectifying, obtains the fatty acid triglycercide of EPA, DHA.
Particularly, in above-mentioned steps S01, the method for bathypelagic fish being rubbed into meat slurry is unrestricted.As preferred embodiment, in order to reach even, efficient crushing effect, bathypelagic fish can be pulverized through bone cutter and form rotten shape, bone cutter power can be set to about 4KW, and the meat slurry size obtained is advisable with 5mm-80mm.Wherein, the kind of bathypelagic fish can select tuna, cod, sardines, salmon, shark etc.In order to reduce Financial cost, the tankage after preferably processing with bathypelagic fish are that raw material extracts.
Taking meat obtained above slurry, is (0.5-1) in the mass ratio of meat slurry and water: the ratio of (1-1.5) adds water-lute.Because the grease in fish oil, particularly tuna oil can decompose generation under aerobic environment, there is volatile aldehydes, ketone and alcohol, oxycarbide, epoxide and acid and so on lower-molecular substance, thus cause the instability of acid number, even acid number obviously to raise, cause fish oil to become sour rotten.Therefore, for avoiding the oxidative rancidity of polyunsaturated fatty acid, carry out heat treated under anaerobic, described oxygen free condition can be inert gas atmosphere, vacuum environment and other common oxygen free conditions, as nitrogen atmosphere etc.As preferred embodiment, in order to obtain uniform mixture solution, the temperature of described heat treated is 85 DEG C ~ 90 DEG C, and heat-up time is 0.5h-1h.Through above-mentioned process, avoid described crude fish oil generation oxidative rancidity, meanwhile, eliminate the step that the needs caused due to acid number increase carry out reducing acid number process.
In above-mentioned steps S01, in order to can effectively by the fish oil constituents extraction in bathypelagic fish out, and reach the object that extraction efficiency is high and impurity is few simultaneously, described first enzyme digestion reaction adopts following method to carry out: regulate the pH value of mixture solution to 7.0-8.5, add the trypsinase that massfraction is 1.5%-3.0%, enzyme digestion reaction 15h-19h under lucifuge condition, temperature of reaction is 20 DEG C-30 DEG C.
After the first enzyme digestion reaction, zymolyte can be carried out centrifugal treating and realize solid-liquid separation, get upper strata enzymolysis solution, namely obtain crude fish oil.Wherein, the condition of centrifugal treating can be set to normal condition.In the present embodiment, in order to improve purity and the centrifugal efficiency of crude fish oil, adopt the centrifugation 15min-30min of 4500r/min-6000r/min.
In above-mentioned steps S02, due in above-mentioned crude fish oil, the lipid acid such as EPA, DHA all exist with the form of fish oil glyceryl ester, are directly separated concentrated EPA and DHA difficulty large and be not easy to remove the saturated or monounsaturated lipid acid existed with impurity from crude fish oil.Therefore, the embodiment of the present invention needs to carry out the second enzyme digestion reaction to crude fish oil, use the tri-glyceride in Lipase catalyzed hydrolysis fish oil, obtain the free fatty acid form of DHA and EPA, saturated or monounsaturated free fatty acids, fish oil glycerine ester hydrolysis is shown below; Remove desat or monounsaturated lipid acid by urea clathration mode again, thus reach the object of concentrated EPA and DHA.By above-mentioned process, be convenient to the free fatty acids obtaining being easy to purification process.
As preferred embodiment, in order to improve the hydrolysis efficiency of fish oil glyceryl ester in crude fish oil, it is that the lipase 1LVK-F100 of 1.5%-3.0% is as catalyzer that the embodiment of the present invention adds massfraction in described crude fish oil, at the temperature of 45 DEG C-55 DEG C, enzyme digestion reaction 20h-28h, obtains free fatty acids.
After above-mentioned process, in crude fish oil, the percent hydrolysis of esterified fatty acid is up to 30%-50%.
In above-mentioned steps S03, due to the complicated component in the above-mentioned free fatty acids that obtains, containing multiple monounsaturated fatty acids and polyunsaturated fatty acid composition, cause the content of EPA, DHA and purity all lower, therefore, need to carry out separating treatment to described free fatty acids.To the separation of above-mentioned free fatty acids, isolation technique conventional in this area can be adopted, as rectifying separation technology, molecular distillation technique, chromatographic separation technology etc.But the service temperature of rectifying separation technology is higher and the time is longer, easily causes EPA, DHA that thermosensitive response occurs, thus affect its quality; Molecular distillation technique is on the contrary, because service temperature is low, separated material is not easily separated, and expends height; Although chromatographic separation technology can obtain the high product of purity, often productive rate is low, cost large, and other conventional isolation techniques are also difficult to meet the requirement of EPA and DHA for purity, productive rate, quality and cost simultaneously.In above-mentioned free fatty acids, because EPA and DHA is polyunsaturated fatty acid, containing multiple unsaturated link(age), cause its carbon chain molecules to form certain sterie configuration, be not easy by urea clathration; And the higher lipid acid of saturation ratio or monounsaturated fatty acids higher due to its degree of unsaturation, often easily by the urea molecule inclusion of crystallization, and separate out with stable crystal saturates form.In view of this, as preferred embodiment, urea clathration technology can be adopted, under the wash-out of suitable elutriant, it is separated with other free fatty acidies.
In the embodiment of the present invention, in order to obtain the mixed solution mixed, as preferred embodiment, described bath temperature is 60 DEG C-65 DEG C.In order to do not make as carrier urea excess load, simultaneously can reach good separating effect again, repeatedly study discovery through contriver: the allocation ratio of described free fatty acids and urea methyl alcohol saturated solution or urea alcohol saturated solution is 1g:(5-10) ml.
In above-mentioned steps S03, the methyl alcohol of described urea or alcohol saturated solution can adopt conventional hybrid mode with mixing of above-mentioned free fatty acids, as preferred embodiment, in order to mix and not destroy the structure of each composition, described mixing adopts the form stirred, and churning time is with until mixture solution is even, transparent is advisable.
In order to urea molecule the lipid acid higher with saturation ratio or monounsaturated fatty acids can form more stable crystal inclusion compound in crystallisation process, cooling process need be carried out to said mixture solution.So-called specific embodiment, the method for described cooling process is: after making the temperature of mixed solution solution be down to 4 DEG C-10 DEG C with the rate of temperature fall of 5 DEG C/10min-10 DEG C/10min, it is placed 48-72h under the condition of 4 DEG C-5 DEG C.
Sample after cooling process is carried out filtration treatment, and described filter type is unrestricted.As preferred embodiment, in order to improve filtration efficiency, described filter type selects the mode of suction filtration to carry out.After suction filtration, use corresponding methyl alcohol, ethanolic soln wash crystallization body, collect and merging filtrate.
Above-mentioned filtrate is carried out extraction treatment, and the extraction system that described extraction treatment adopts is filtrate, normal hexane, distilled water system, wherein, filtrate: normal hexane: the amount ratio of distilled water is 10ml:(1-2) g:(2-3) g.Mixing solutions is shaken up rear standing separation and collected organic layer, organic layer solution is carried out underpressure distillation, namely obtain EPA, the much higher unsaturated free fatty acids concentrated solution of DHA content.
Embodiment 1
S11. the preparation of crude fish oil
Zymolysis technique is adopted to prepare bathypelagic fish crude fish oil.Bathypelagic fish is pulverized through bone cutter and forms rotten shape, takes 600g meat slurry, in meat: water=0.5:1(m:m) ratio add water-lute, under the condition of darkroom, logical nitrogen, in 85 DEG C of boiling 1h.Use NaOH adjust ph to 8.0.Add the trypsinase that massfraction is 2%, (25 DEG C) enzymolysis 17h under darkroom, room temperature condition.After the centrifugal 20min of 5000r/min, get upper strata enzymolysis solution, be crude fish oil.
S12. the preparation of free fatty acids
Adopt zymolysis technique that the fish oil glyceride form of crude fish oil is hydrolyzed, obtain free fatty acids.Utilize lipase 1LVK-F100 for catalyzer, enzyme dosage is 2%, and temperature of reaction is 50 DEG C, and the reaction times is 24h, obtains free fatty acids.
S13. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Urea entraing method is adopted to concentrate the how unsaturated free fatty acids such as EPA, DHA.Under the water bath of 65 DEG C, prepare the alcohol saturated solution of urea, then add free fatty acids, wherein, configuration proportion is as follows, free fatty acids: urea alcohol saturated solution=1:10(w:v).Stir to transparent, after making the temperature of mixed solution be down to 4 DEG C with the cooling rate of 5 DEG C/10min, place 4 DEG C of refrigerator 72h, then carry out suction filtration, after using the ethanolic soln wash crystallization body of 100%, collect filtrate.Filtrate is added normal hexane, distilled water, wherein, configuration proportion is as follows, filtrate: normal hexane: distilled water=10:2:2(v:m:m).Mixing solutions is shaken up rear standing separation organic phase, finally by upper strata organic solution underpressure distillation, namely obtain the how unsaturated free fatty acids concentrated solution such as EPA, DHA.
The preparation of the fatty acid triglycercide of S14.EPA and DHA
With above-mentioned free unsaturated fatty acid concentrated solution for substrate puts into supercritical extraction reactor, under the booster action in ultrasonic wave magnetic field, carry out the optionally enzyme digestion reaction under subcritical state.Add reactant to have: normal hexane, glycerine, lipase Novozym435, to add in following ratio: normal hexane: free fatty acids is 10:1(v:m), glycerine: free fatty acids is 4:1 (w:w), lipase Novozym435: free fatty acids is 0.02:1(w:w).Arranging reactor pressure is 4MPa, when pressure keeps stablizing within 10min, opens ultrasonic wave magnetic field, oscillation frequency 2.5Hz, and setting temperature of reaction kettle 40 DEG C, reacts 3h with this understanding, the main fatty acid triglycercide generating many carbon.
Using the triglyceride level after above-mentioned esterification as reaction substrate, supercritical extraction reaction setting model is: extracting pressure is 25MPa, and extraction temperature is 40 DEG C, CO
2flow is 4L/h, and the pressure of separation reactor I is 10MPa, and the temperature of separation reactor I is 50 DEG C, to be separated the pressure of II be the temperature of 6MPa, separation reactor I I is 60 DEG C, and the temperature of rectifying column I is 65 DEG C, the temperature of rectifying column II is 70 DEG C, and the temperature of rectifying column III is 75 DEG C, and the temperature of rectifying column IV is 80 DEG C.The high purity polyunsaturated fatty acid triglycercide that to be the unsaturated fatty acids triglyceride level of EPA and DHA be main density that rectifying column collects the material obtained is close.
Embodiment 2
S21. the preparation of crude fish oil
Zymolysis technique is adopted to prepare bathypelagic fish crude fish oil.Bathypelagic fish is pulverized through bone cutter and forms rotten shape, takes 600g meat slurry, in meat: water=0.5:1(w:w) ratio add water-lute, under the condition of darkroom, logical nitrogen, in 85 DEG C of boiling 0.5h.Use NaOH adjust ph to 8.0.Add the trypsinase that massfraction is 1.5%, (20 DEG C) enzymolysis 15h under darkroom, room temperature condition.After the centrifugal 20min of 4500r/min, get upper strata enzymolysis solution, be crude fish oil.
S22. the preparation of free fatty acids
Adopt zymolysis technique that the fish oil glyceride form of crude fish oil is hydrolyzed, obtain free fatty acids.Utilize lipase 1LVK-F100 for catalyzer, enzyme dosage is 1.5%, and temperature of reaction is 45 DEG C, and the reaction times is 20h, obtains free fatty acids.
S23. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Urea entraing method is adopted to concentrate the how unsaturated free fatty acids such as EPA, DHA.Under the water bath of 60 DEG C, prepare the alcohol saturated solution of urea, then add free fatty acids, wherein, configuration proportion is as follows, free fatty acids: urea alcohol saturated solution=5:10(w:v).Stir to transparent, after making the temperature of mixed solution be down to 10 DEG C with the cooling rate of 10 DEG C/10min, place 4 DEG C of refrigerator 48h, then carry out suction filtration, after using the ethanolic soln wash crystallization body of 100%, collect filtrate.Filtrate is added normal hexane, distilled water, wherein, configuration proportion is as follows, filtrate: normal hexane: distilled water=10:1:2(v:m:m).Mixing solutions is shaken up rear standing separation organic phase, finally by upper strata organic solution underpressure distillation, namely obtain the how unsaturated free fatty acids concentrated solution such as EPA, DHA.
The preparation of the fatty acid triglycercide of S24.EPA and DHA
With above-mentioned free unsaturated fatty acid concentrated solution for substrate puts into supercritical extraction reactor, under the booster action in ultrasonic wave magnetic field, carry out the optionally enzyme digestion reaction under subcritical state.Add reactant to have: normal hexane, glycerine, lipase Novozym435, to add in following ratio: normal hexane: free fatty acids is 8:1(v:m), glycerine: free fatty acids is 2:1 (w:w), lipase Novozym435: free fatty acids is 0.01:1(w:w).Arranging reactor pressure is 6MPa, when pressure keeps stablizing within 10min, opens ultrasonic wave magnetic field, oscillation frequency 6Hz, and setting temperature of reaction kettle 30 DEG C, reacts 5h with this understanding, the main fatty acid triglycercide generating many carbon.
Loaded by triglyceride level after above-mentioned esterification as reaction substrate in reactor, supercritical extraction reaction setting model is: extracting pressure is 20MPa, and extraction temperature is 30 DEG C, CO
2flow is 4L/h, and the pressure of separation reactor I is 6MPa, and the temperature of separation reactor I is 45 DEG C, to be separated the pressure of II be the temperature of 4MPa, separation reactor I I is 55 DEG C, and the temperature of rectifying column I is 60 DEG C, the temperature of rectifying column II is 65 DEG C, and the temperature of rectifying column III is 70 DEG C, and the temperature of rectifying column IV is 75 DEG C.Rectifying column collects the triglyceride level that the material obtained is the unsaturated fatty acids of EPA and DHA.It is the close high purity polyunsaturated fatty acid triglycercide of main density.
Embodiment 3
S31. the preparation of crude fish oil
Zymolysis technique is adopted to prepare bathypelagic fish crude fish oil.Bathypelagic fish is pulverized through bone cutter and forms rotten shape, takes 600g meat slurry, in meat: water=0.5:1.5(m:m) ratio add water-lute, under the condition of darkroom, logical nitrogen, in 90 DEG C of boiling 1h.Use NaOH adjust ph to 8.0.Add the trypsinase that massfraction is 3%, enzymolysis 15h under darkroom, the condition of 30 DEG C.After the centrifugal 20min of 6000r/min, get upper strata enzymolysis solution, be crude fish oil.Crude fish oil detects according to standard GB/T/T17377-2008 " gas chromatographic analysis of animal-plant oil fatty acid methyl ester ".
S32. the preparation of free fatty acids
Adopt zymolysis technique that the fish oil glyceride form of crude fish oil is hydrolyzed, obtain free fatty acids.Utilize lipase 1LVK-F100 for catalyzer, enzyme dosage is 3.0%, and temperature of reaction is 55 DEG C, and the reaction times is 28h, obtains free fatty acids.
S33. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Urea entraing method is adopted to concentrate the how unsaturated free fatty acids such as EPA, DHA.Under the water bath of 65 DEG C, prepare the methyl alcohol saturated solution of urea, then add free fatty acids, wherein, configuration proportion is as follows, free fatty acids: urea methyl alcohol saturated solution=1:10(w:v).Stir to transparent, after making the temperature of mixed solution be down to 10 DEG C with the cooling rate of 5 DEG C/10min, place 4 DEG C of refrigerator 72h, then carry out suction filtration, after using the methanol solution wash crystallization body of 100%, collect filtrate.Filtrate is added normal hexane, distilled water, wherein, configuration proportion is as follows, filtrate: normal hexane: distilled water=10:2:3(v:m:m).Mixing solutions is shaken up rear standing separation organic phase, finally by upper strata organic solution underpressure distillation, namely obtain the how unsaturated free fatty acids concentrated solution such as EPA, DHA.
The preparation of the fatty acid triglycercide of S34.EPA and DHA
With above-mentioned free unsaturated fatty acid concentrated solution for substrate puts into supercritical extraction reactor, under the booster action in ultrasonic wave magnetic field, carry out the optionally enzyme digestion reaction under subcritical state.Add reactant to have: normal hexane, glycerine, lipase Novozym435, to add in following ratio: normal hexane: free fatty acids is 12:1(v:m), glycerine: free fatty acids is 6:1 (w:w), lipase Novozym435: free fatty acids is 0.05:1(w:w).Arranging reactor pressure is 8MPa, when pressure keeps stablizing within 10min, opens ultrasonic wave magnetic field, oscillation frequency 10Hz, and setting temperature of reaction kettle 60 DEG C, reacts 12h with this understanding, the main fatty acid triglycercide generating many carbon.
Loaded by triglyceride level after above-mentioned esterification as reaction substrate in reactor, supercritical extraction reaction setting model is: extracting pressure is 30MPa, and extraction temperature is 40 DEG C, CO
2flow is 6L/h, and the pressure of separation reactor I is 10MPa, and the temperature of separation reactor I is 50 DEG C, to be separated the pressure of II be the temperature of 6MPa, separation reactor I I is 60 DEG C, and the temperature of rectifying column I is 65 DEG C, the temperature of rectifying column II is 70 DEG C, and the temperature of rectifying column III is 75 DEG C, and the temperature of rectifying column IV is 80 DEG C.The high purity polyunsaturated fatty acid triglycercide that to be the unsaturated fatty acids triglyceride level of EPA and DHA be main density that rectifying column collects the material obtained is close.
Comparative example 1
1. the preparation of crude fish oil
Zymolysis technique is adopted to prepare bathypelagic fish crude fish oil.Bathypelagic fish is pulverized through bone cutter and forms rotten shape, takes 600g meat slurry, in meat: water=0.5:1(m:m) ratio add water-lute, under the condition of darkroom, logical nitrogen, in 85 DEG C of boiling 1h.Use NaOH adjust ph to 8.0.Add the trypsinase that massfraction is 2%, (25 DEG C) enzymolysis 17h under darkroom, room temperature condition.After the centrifugal 20min of 5000r/min, get upper strata enzymolysis solution, be crude fish oil.
2. supercritical extraction concentrates the triglyceride level of EPA/DHA
Loaded by above-mentioned crude fish oil as reaction substrate in reactor, supercritical extraction reaction setting model is: extracting pressure is 25MPa, and extraction temperature is 40 DEG C, CO
2flow is 4L/h, and the pressure of separation reactor I is 10MPa, and the temperature of separation reactor I is 50 DEG C, and to be separated the pressure of II be the temperature of 6MPa, separation reactor I I is 60 DEG C, collects the material being separated and obtaining.
In above-described embodiment, reagent source is:
Trypsinase: food grade, Zhengzhou Hong Cheng Chemicals company limited;
LVK-F100 lipase: Shenzhen Leveking Biology Engineering Co., Ltd;
Novozym435 lipase: Novozymes Company.
Test the mass percentage of glycerine ester type EPA and DHA in the content of EPA and DHA in crude fish oil in above-described embodiment 1-3, the degree of hydrolysis of free fatty acids, the gamma value of lipid acid and the finished product, testing method is as follows:
(1) first DHA/EPA detection method of content in fish oil: detect according to GB/T17377-2008 " analysis of animal-plant oil fatty acid methyl ester chromatography of gases ".
(2) detection method (i.e. determination of acid value) of free fatty acid content: detect according to GB/T5530-2005 " mensuration of animal-plant oil acid number and acidity ".
(3) mensuration of fatty acid ester rate: according to the acid number of GB/T5530-2005 method assaying reaction system, then according to company's calculating below: esterification yield (%)=[(acid number before the reacted acid number/reaction of 1-)] × 100%.
(4) mensuration of glycerine ester type DHA/DHA purity: detect according to GB/T17377-2008 " analysis of animal-plant oil fatty acid methyl ester chromatography of gases " " measure.
Test result is as shown in table 1 below:
Table 1
As seen from the above table, the bathypelagic fish that the embodiment of the present invention provides extracts the method for triglyceride type DHA and EPA from bathypelagic fish, and in crude fish oil, the content of DHA/EPA is high, can reach 28.94% and 4.93% respectively; The degree of hydrolysis of free fatty acids is high, can reach more than 50%; Fatty acid esterification degree is up to 90%; In the finished product obtained, glycerine ester type DHA mass percentage is high, can reach 15.02% and 74.98% respectively.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. from bathypelagic fish, extract a method of triglyceride type DHA and EPA, comprise the following steps:
Bathypelagic fish is rubbed into meat slurry, after carrying out heat treated under anaerobic after adding water, after using trypsinase to be carried out the first enzyme digestion reaction, carry out solid-liquid separation, collect filtrate, obtain crude fish oil;
Utilize lipase 1LVK-F100 that described crude fish oil is carried out the second enzyme digestion reaction, obtain free fatty acids;
Under water bath condition, mixture solution is obtained after the methyl alcohol of urea or alcohol saturated solution being mixed with described free fatty acids, described mixture solution is carried out cooling process to temperature is 4 DEG C-10 DEG C, after insulation leaves standstill process 48h-72h, carry out filtration treatment and obtain filtrate, described filtrate is carried out extraction treatment and collected organic layer, obtains the polyunsaturated fatty acid containing EPA, DHA concentrated;
With the described polyunsaturated fatty acid containing free EPA, DHA for substrate, put it in the supercritical extraction reactor containing lipase Novozym435, subcritical enzymatic reaction is carried out in ultrasonic wave magnetic field, reaction product is carried out supercritical extraction, rectification process, obtains the fatty acid triglycercide of EPA, DHA.
2. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claim 1, is characterized in that: in the step of the fatty acid triglycercide of preparation described EPA, DHA, and described supercritical extraction is divided into extraction and fractionation two step, wherein,
The condition of described extraction is: extracting pressure is 20MPa-30MPa, and extraction temperature is 35 DEG C-40 DEG C, CO
2flow be 4-6L/h;
Described separation is divided into two-stage to be separated, and wherein, the pressure of flash trapping stage is 6MPa-10MPa, and temperature is 45 DEG C-50 DEG C; The pressure of the second-order separation is 4MPa-6MPa, and temperature is 55 DEG C-60 DEG C.
3. from bathypelagic fish, extract the method for triglyceride type DHA and EPA as claimed in claim 1, it is characterized in that: in the step of the fatty acid triglycercide of preparation described EPA, DHA, described rectification process is divided into level Four rectifying, wherein, the temperature of one-level rectifying is 60 DEG C-65 DEG C, the temperature of two-stage rectification is 65 DEG C-70 DEG C, and the temperature of three grades of rectifying is 70 DEG C-75 DEG C, and the temperature of level Four rectifying is 75 DEG C-80 DEG C.
4. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the fatty acid triglycercide of preparation described EPA, DHA, the condition of described subcritical enzymatic reaction is: the pressure of described reactor is 4-8MPa, ultrasonic oscillation magnetic field is 2-10Hz, temperature of reaction is 30 DEG C-60 DEG C, and the reaction times is 1h-12h.
5. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the fatty acid triglycercide of preparation described EPA, DHA, in supercritical extraction reactor, also containing normal hexane, glycerine, and described normal hexane, glycerine, lipase Novozym435, be normal hexane containing the amount ratio of the polyunsaturated fatty acid of free EPA, DHA: glycerine: lipase Novozym435: the polyunsaturated fatty acid containing free EPA, DHA is (8-12) ml:(2-6) g:(0.01-0.05) g:1g.
6. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the described polyunsaturated fatty acid containing EPA, DHA of preparation, the extraction system that described extraction treatment adopts is filtrate, normal hexane, distilled water system, wherein, filtrate: normal hexane: the amount ratio of distilled water is 10ml:(1-2) g:(2-3) g.
7. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the described polyunsaturated fatty acid containing EPA, DHA of preparation, the methyl alcohol of described free fatty acids and urea or the amount ratio of alcohol saturated solution are 1g:(5-10) ml.
8. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the described polyunsaturated fatty acid containing EPA, DHA of preparation, the method for described cooling process is after making the temperature of mixed solution solution be down to 4 DEG C-10 DEG C with the rate of temperature fall of 5 DEG C/10min-10 DEG C/10min; And/or
The temperature leaving standstill process is 4 DEG C-5 DEG C.
9. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the described free fatty acids of preparation, the method of described second enzyme digestion reaction is: in described crude fish oil, add the lipase 1LVK-F100 that massfraction is 1.5%-3.0%, enzyme digestion reaction 20h-28h at the temperature of 45 DEG C-55 DEG C.
10. the method extracting triglyceride type DHA and EPA from bathypelagic fish as described in as arbitrary in claims 1 to 3, it is characterized in that: in the step of the described crude fish oil of preparation, the method of described first enzyme digestion reaction is: after regulating the pH value of described mixture solution to 7.0-8.5, add the trypsinase that massfraction is 1.5%-3.0%, enzyme digestion reaction 15h-19h under lucifuge condition, temperature of reaction is 20 DEG C-30 DEG C.
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