CN104651422B - A kind of method that triglyceride type DHA and EPA are extracted from deep-sea fish - Google Patents
A kind of method that triglyceride type DHA and EPA are extracted from deep-sea fish Download PDFInfo
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Abstract
The present invention is applied to biopharmaceutical technology, there is provided a kind of method that deep-sea fish extracts triglyceride type DHA and EPA from deep-sea fish, comprises the following steps：Crude fish oil is prepared using zymolysis technique, free fatty is prepared, the polyunsaturated fatty acid containing EPA, DHA is separated using urea entraing method, prepares EPA, DHA fatty acid triglycercide.This method can effectively be extracted from deep-sea fish, be enriched with DHA and EPA；The crude fish oil acid number that is obtained in preparation process is low, esterified fatty acid hydrolysis degree is high in crude fish oil, and the DHA and EPA finally given be glycerine ester type DHA and EPA, and glycerine ester type EPA and DHA content height.
Description
Technical field
The invention belongs to biopharmaceutical technology, more particularly to one kind extracted from deep-sea fish triglyceride type DHA and
EPA method.
Background technology
Deep-sea fish, such as tuna are a kind of large-scale pelagic important goods food fish.Because deep-sea fish must protect often
Hold quick travelling, could maintain the supply of body, plus only movable in marine site depths, therefore meat is tender delicious, and not by ring
Border is polluted, and is the rare healthy food of modern.The processing industry of deep-sea fish turns into an important industry, but deep-sea fish
There have that quality is unmanageable, leftover pieces producing level is low, energy consumption is high, significant loss is serious in process etc. to be a series of
Significant problem, business economic loss are larger.Therefore operating income is increased to leftover bits and pieces caused by deep-sea fish process, realizes warp
Ji value.The application of fish oil is very extensive, can be applied to the fields such as health care, medicine, food, chemical industry and feed.By by deeply
It is a kind of extraordinary mode for realizing economic benefit that the offal of ocean fish, which is used to prepare fish oil,.
N-3 polyunsaturated fatty acids in fish oil（n-3PUFA）There is important biological significance, its feature fat in human body
Fat acid eicosapentaenoic acid（EPA）And docosahexaenoic acid（DHA）Physiologically active it is clear and definite, EPA have prevention coronary disease
Physiologically active, the DHA such as disease, hypotensive, dispelling fatigue, prevention of arterial atherosis and cerebral thrombus, anticancer can be obviously promoted baby
The intelligence development of youngster, improve cerebral function, improve memory, EPA and DHA are production prevention of cardiovascular disease and baby's intelligence development food
The good base-material of product.
Polyenoic fatty acid in fish oil, including EPA and DHA, be category heat and chemically unstable material, be isolated purification and
It is difficult, and the existing conventional technology isolated and purified has rectifying isolation technics, molecular distillation technique, chromatographic separation technology, low temperature
Refrigeration Technique, metal salt precipitate technology and fish oil lactide, but respective deficiency all be present：
Wherein, the technical process of traditional purification techniques includes degumming, depickling, decolouring, deodorization etc., and the process for refining is numerous
Trivial, product color is undesirable, and the heating required in this extraction process easily causes grease oxidation rancid.Rectifying separates
Technology, is current most popular industrial separation technology, and its principle is to utilize in fatty acid mixed each component volatility not
Together, separated at the same temperature according to the property of different saturated vapor pressures, but this method operation temperature is higher, the time compared with
It is long, cause unrighted acid that thermosensitive response occurs, influence product quality and yield.Molecular distillation technique, in high vacuum
Under the conditions of, escaped according to the heated meeting of fluid molecule from the page, the mean free path of molecular motion in the gas phase is different and is divided
From, this kind of technical operation temperature is low, and separated material is not readily separated or polymerize, and molecular distillation apparatus one-time investment compared with
Greatly, and maintenance cost is higher.Supercritical fluid technique equipment investment is big, and extraction pressure is high, and conventional supercritical fluid extraction equipment master
If being made up of single autoclave and separating still, it is only capable of carrying out single separation reaction, in addition, supercritical extract can not
Realization separates saturation EPA, DHA close with other molecular weight or low concentration unrighted acid.Chromatographic separation technology, it is one
The modern separation of kind, analyzing detecting method, the chromatographic process for purifying and detecting available for Separation of Fatty Acids have gas-chromatography, thin layer color
Spectrum, high performance liquid chromatography etc., separated in most cases with easy silica gel chromatograph, the method can be close by polarity, structure phase
As unrighted acid separate, major defect is that power consumption is big, and running cost is high.Cryogenic freezing technology, mixed according to aliphatic acid
Compound in organic solvent is subcooled solubility difference and reach the purpose of separation.The dissolving of saturated fatty acid in organic solvent
Spend and be inversely proportional with the length of carbochain, and the aliphatic acid of same degree of unsaturation, carbochain are longer, solubility is lower, to same carbochain number
Purpose unrighted acid, with the increase of double bond, solubility increase.This kind of technology major defect is need to reclaim a large amount of solvents, point
From inefficient, it is necessary to which the cooling device of extremely low temperature, cost are higher.Metal salt precipitate technology, be using saturated fatty acid, it is low not
Saturated fatty acid and EPA, DHA metal salt difference of solubility in certain organic solvent are separated, conventional for nitre
The silver-colored complexometry of acid, this method is simple to operate, mild condition, cycle are short, high income, but silver nitrate is expensive, recovery is difficult, produces
Cost is high.Fish oil hands over esterification techniques, refers in the presence of base catalyst, by the glycerine in ethanol replacement grease, produces second
The high unsaturated fatty acid of esterification（EPA and DHA）.Ethyl ester type ω -3PUFA digest and absorb in human body it is relatively difficult, and
It there may be potential safety hazard.
The content of the invention
It is an object of the invention to provide a kind of method that triglyceride type DHA and EPA are extracted from deep-sea fish, it is intended to solves
DHA and EPA purity that certainly prior art is extracted from fish oil is low, acid number is high, color needs to decolourize deeply and obtains DHA and EPA is
The problem of mixture or ethyl ester class fish oil and the costs such as DHA and EPA monoglyceride, glyceryl ester are high.
The present invention is achieved in that a kind of method that triglyceride type DHA and EPA is extracted from deep-sea fish, including with
Lower step：
Deep-sea fish is rubbed into meat and starched, is added after being heated under anaerobic after water, using trypsase by its
After carrying out the first enzyme digestion reaction, separation of solid and liquid is carried out, filtrate is collected, obtains crude fish oil；
The crude fish oil is subjected to the second enzyme digestion reaction using lipase 1LVK-F100, obtains free fatty；
Under water bath condition, obtained after the methanol of urea or alcohol saturated solution are mixed with the free fatty
Mixture solution, the mixture solution is subjected to cooling processing to temperature and is 4 DEG C -10 DEG C, is incubated stewing process 48h-72h
Afterwards, carry out filtration treatment and obtain filtrate, the filtrate is subjected to extraction processing and collected organic layer, concentrated containing EPA,
DHA polyunsaturated fatty acid；
Using the polyunsaturated fatty acid containing free EPA, DHA as substrate, put it into containing lipase
In Novozym435 supercritical extract reactor, subcritical enzymatic reaction is carried out in ultrasonic wave magnetic field, reaction product is entered
Row supercritical extract, rectifying, obtain EPA, DHA fatty acid triglycercide.
The method provided by the invention that triglyceride type DHA and EPA is extracted from deep-sea fish, can effectively from deep-sea fish,
It is even low as extraction DHA and EPA, its production cost in the deep-sea fish head of leftover pieces.The crude fish oil obtained in preparation process
Acid number is low, can reach 1.5 (mg KOH)/g；Esterified fatty acid hydrolysis degree is high in crude fish oil, can reach 50%；Finally give
DHA and EPA is triglyceride type DHA and EPA, and glycerine three type EPA and DHA content are high, wherein, triglyceride type EPA can
Up to 15%, triglyceride type DHA may be up to 75%.
Brief description of the drawings
Fig. 1 is the schematic flow sheet provided in an embodiment of the present invention that glycerine ester type DHA and EPA are extracted from deep-sea fish.
Fig. 2 is the fatty acid triglycercide subcritical enzymatic reaction provided in an embodiment of the present invention for being used to prepare EPA, DHA
Schematic diagram.
Fig. 3 is the schematic flow sheet provided in an embodiment of the present invention for being used to prepare EPA, DHA fatty acid triglycercide；Its
In, each numbering is respectively in Fig. 3：1.CO2Steel cylinder, 2. filters, 3. cold, 4. high-pressure metering pumps, 5. blenders, 6. preheatings
Device, 7. reactors, 8. one-level separating stills, 9. the second-order separation kettles, 10. rectifying columns（10-1 to 10-4 be respectively and rectifying column, two
Level rectifying column, three-level rectifying column, level Four rectifying column）, 11. carry agent, 12. flow totalizers.
Embodiment
In order that technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Drawings and Examples, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
A kind of method for extracting triglyceride type DHA and EPA from deep-sea fish the embodiments of the invention provide deep-sea fish, bag
Following steps are included, as described in Figure 1：
S01. crude fish oil is prepared using zymolysis technique：Deep-sea fish is rubbed into meat and starched, is carried out under anaerobic after adding water
After heating, after being carried out the first enzyme digestion reaction using trypsase, separation of solid and liquid is carried out, filtrate is collected, obtains thick fish
Oil；
S02. free fatty is prepared：The crude fish oil is subjected to the second enzyme digestion reaction using lipase 1LVK-F100, obtained
To free fatty；
S03. using polyunsaturated fatty acid of the urea entraing method separation containing EPA, DHA：Under water bath condition, by urea
Methanol or alcohol saturated solution obtain mixture solution after being mixed with the free fatty, and the mixture solution is entered
Row cooling processing to temperature is 4 DEG C -10 DEG C, after insulation stewing process 48h-72h, carries out filtration treatment and obtains filtrate, will described in
Filtrate carries out extraction processing and collected organic layer, the polyunsaturated fatty acid containing EPA, DHA concentrated；
S04. EPA, DHA fatty acid triglycercide are prepared：Using the polyunsaturated fatty acid containing free EPA, DHA as
Substrate, put it into the supercritical extract reactor containing lipase Novozym435, Asia is carried out in ultrasonic wave magnetic field and is faced
Boundary's enzymatic reaction, reaction product is subjected to supercritical extract, rectifying, obtains EPA, DHA fatty acid triglycercide.
Specifically, it is in above-mentioned steps S01, deep-sea fish rubbing is unrestricted into the method for meat slurry.As the presently preferred embodiments,
In order to reach uniform, efficient crushing effect, deep-sea fish can be pulverized to form rotten shape by bone cutter, bone cutter power can be set
For 4KW or so, obtained meat slurry size is advisable with 5mm-80mm.Wherein, the species of deep-sea fish can select tuna, cod,
Sardine, salmon, shark etc..In order to reduce financial cost, the leftover bits and pieces after preferably being processed with deep-sea fish is raw material extraction.
Meat slurry obtained above is weighed, the mass ratio by meat slurry and water is (0.5-1):The ratio of (1-1.5) adds water-stop.
Because the grease in deep sea fish oil, particularly tunny fish oil can decompose generation with volatile aldehydes, ketone under aerobic environment
Class and alcohol, oxycarbide, epoxides and acid etc lower-molecular substance, so as to cause the unstable of acid number, even acid number obvious
Rise, causes fish oil to become sour rotten.Therefore, to avoid the oxidative rancidity of polyunsaturated fatty acid, added under anaerobic
Heat treatment, the oxygen free condition can be inert gas atmosphere, vacuum environment and other common oxygen free conditions, such as nitrogen atmosphere
Deng.As the presently preferred embodiments, in order to obtain uniform mixture solution, the temperature of the heating is 85 DEG C~90 DEG C, is added
The hot time is 0.5h-1h.By above-mentioned processing, avoid the crude fish oil and oxidative rancidity occurs, meanwhile, eliminate due to acid number
The step of needing to carry out reducing acid number processing caused by increase.
In above-mentioned steps S01, in order to effectively come out the fish oil constituents extraction in deep-sea fish, and reach extraction simultaneously
Efficiency high and the few purpose of impurity, first enzyme digestion reaction are carried out using following methods：Adjust mixture solution pH value to
After 7.0-8.5, the trypsase that mass fraction is 1.5%-3.0%, the enzyme digestion reaction 15h-19h under the conditions of lucifuge, reaction are added
Temperature is 20 DEG C -30 DEG C.
After the first enzyme digestion reaction, zymolyte can be subjected to centrifugal treating and realize separation of solid and liquid, take upper strata enzymolysis liquid, i.e.,
Obtain crude fish oil.Wherein, the condition of centrifugal treating may be configured as normal condition.In the present embodiment, in order to improve the pure of crude fish oil
Degree and centrifugal efficiency, using 4500r/min-6000r/min centrifugation 15min-30min.
In above-mentioned steps S02, because in above-mentioned crude fish oil, the aliphatic acid such as EPA, DHA is deposited in the form of fish oil glyceride
Concentration EPA and DHA difficulty is being directly separated from crude fish oil greatly and is being not easy to remove with saturation existing for impurity or list not
The aliphatic acid of saturation.Therefore, the embodiment of the present invention needs to carry out the second enzyme digestion reaction to crude fish oil, uses Lipase catalyzed hydrolysis
Triglyceride in fish oil, obtain DHA and EPA free fatty acid form, saturation or monounsaturated free fatty,
Fish oil glycerine ester hydrolysis is shown below；Desaturation or monounsaturated aliphatic acid is removed by urea clathrate mode again, from
And reach concentration EPA and DHA purpose.By above-mentioned processing, it is easy to obtain the free fatty for being easy to purification process.
As the presently preferred embodiments, in order to improve the hydrolysis efficiency of fish oil glyceride in crude fish oil, the embodiment of the present invention is in institute
State and lipase 1LVK-F100 of the mass fraction for 1.5%-3.0% is added in crude fish oil as catalyst, in 45 DEG C -55 DEG C of temperature
The lower enzyme digestion reaction 20h-28h of degree, obtains free fatty.
After above-mentioned processing, the percent hydrolysis of esterified fatty acid is up to 30%-50% in crude fish oil.
In above-mentioned steps S03, due to the complicated component in obtained above-mentioned free fatty, contain a variety of single unsaturated lipids
Fat acid and polyunsaturated fatty acid composition, cause EPA, DHA content and purity relatively low, therefore, it is necessary to the free fat
Fat acid carries out separating treatment.Separation to above-mentioned free fatty, conventional isolation technics in the art can be used, such as rectifying point
From technology, molecular distillation technique, chromatographic separation technology etc..But the operation temperature of rectifying isolation technics is higher and the time is longer,
Easily cause EPA, DHA that thermosensitive response occurs, so as to influence its quality；Molecular distillation technique is on the contrary, because operation temperature
Low, separated material is not readily separated, and is expended high；Although chromatographic separation technology can obtain the high product of purity, often yield
It is low, cost is big, and other conventional isolation techniques are also difficult to meet EPA and DHA for purity, yield, quality and cost simultaneously
It is required that.In above-mentioned free fatty, because EPA and DHA is polyunsaturated fatty acid, containing multiple unsaturated bonds, cause it
Carbon chain molecules can form certain steric configuration, it is not easy to by urea clathrate；And the aliphatic acid or single unsaturated that saturation degree is higher
Aliphatic acid tends to the urea molecule inclusion being crystallized because its degree of unsaturation is higher, and with stable crystal saturate shape
Formula separates out.In view of this, as the presently preferred embodiments, can use urea clathrate technology, under the elution of suitable eluent by it
Separated with other free fatties.
In the embodiment of the present invention, in order to obtain well mixed mixed liquor, as the presently preferred embodiments, the bath temperature is
60℃-65℃.It is anti-through inventor in order to not make urea excess load while and can as carrier reach preferable separating effect
Multiple research is found：The allocation ratio of free fatty and urea the methanol saturated solution or urea alcohol saturated solution is 1g：
(5-10)ml。
In above-mentioned steps S03, the methanol or alcohol saturated solution of the urea can be adopted with the mixing of above-mentioned free fatty
With the hybrid mode of routine, as the presently preferred embodiments, in order to be well mixed and not destroy the structure of each composition, the mixing is adopted
With the form of stirring, mixing time is with until mixture solution is uniform, transparent is advisable.
In order to urea molecule can be higher with saturation degree in crystallization process aliphatic acid or monounsaturated fatty acids formed compared with
Stable crystal inclusion compound, need to carry out cooling processing to said mixture solution.So-called specific embodiment, the cooling processing
Method is：After the temperature of mixing liquor is down to 4 DEG C -10 DEG C with 5 DEG C/10min-10 DEG C/10min rate of temperature fall, by it
48-72h is placed under conditions of 4 DEG C -5 DEG C.
Sample after cooling is handled is subjected to filtration treatment, the filter type is unrestricted.As being preferable to carry out
Example, in order to improve filter efficiency, the filter type is carried out from the mode filtered.After suction filtration, corresponding methanol, ethanol are used
Solution wash crystallization body, collect simultaneously merging filtrate.
Above-mentioned filtrate is subjected to extraction processing, the extraction system that the extraction processing uses is filtrate, n-hexane, distilled water
System, wherein, filtrate：N-hexane：The amount ratio of distilled water is 10ml：(1-2)g：(2-3)g.Stood after mixed solution is shaken up
Simultaneously collected organic layer is separated, organic layer solution is evaporated under reduced pressure, that is, obtains the free fat of the high how unsaturated of EPA, DHA content
Fat acid concentrate.
In the prior art, EPA, DHA of extraction are often processed into ethyl ester type fish oil, cause product quality to be affected,
And the process of ethyl ester type fish oil needs to replace quantitative ethanol in esterification process, causes the imbalance of aliphatic acid, ethyl ester type
Metabolic adsorption rate of the fish oil in human body is low, bioavailability is low, stability is poor；And ethyl ester type fish oil easily divides in intestines and stomach
Ethanol is solved, causes easily to cause the crowd of ethyl ester poor resistance adverse reaction such as allergy, therefore, its security is low.Fish
EPA, DHA native state are triglyceride type in oil, and human body pancreas and liver has selectivity hydrolyzing triglyceride
Lipase, EPA, DHA under native state are easily digested and assimilated, and its property is stable, can effectively ensure that its nutritional ingredient, just
Utilized in absorption of human body, glyceride type fish oil absorptivity is three times of ethyl ester type fish oil or so.
In view of this, in above-mentioned steps S04, in order to obtain the high triglyceride type EPA and DHA of esterification degree, it is necessary to upper
State the polyunsaturated fatty acid containing EPA, DHA after concentration and carry out esterification.It is lipase-catalyzed compared with traditional catalyst
Reaction has good selectivity and high efficiency, and still, due to the limitation of enzyme industrial development, enzymatic reaction is difficult to control, and is caused
Esterification yield is relatively low.And the participation of subcritical fluids, the back reaction of enzymatic hydrolysis reaction can be carried out, be such as esterified, lactonize, ester exchange and
Synthesis of peptide etc.；And the suppression to enzyme of substrate or product is reduced simultaneously, increases the heat endurance of enzyme, reduce byproduct of reaction, energy
Efficiently solve existing above mentioned problem when enzymatic reaction is used alone.In order to increase the contact area of substrate and enzyme and contact frequency
Rate, further improves the efficiency of enzymatic reaction, and the subcritical enzymatic reaction may be selected to carry out in the presence of ultrasonic wave.
Specifically, in above-mentioned supercritical extract reactor, also containing n-hexane, glycerine, as the presently preferred embodiments, it is described just
Hexane, glycerine, lipase Novozym435, the amount ratio of polyunsaturated fatty acid containing free EPA, DHA are n-hexane：It is sweet
Oil：Lipase Novozym435：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 the subcritical enzymatic reaction is：The pressure of the reactor is 4-8MPa, is surpassed
Acoustic vibration magnetic field is 2-10Hz, and reaction temperature is 30 DEG C -60 DEG C, reaction time 1h-12h.
The subcritical enzyme reaction schematic diagram is as shown in Figure 2：By the polyunsaturated fatty acid containing EPA, DHA, n-hexane,
Glycerine, lipase Novozym435, which are added in supercritical extract reactor, forms mixed material, in CO2Subcritical fluids and super
Subcritical enzymatic reaction is carried out under the collective effect in sound wave magnetic field.
By the polyunsaturated fatty acid containing EPA, DHA of above-mentioned processing, esterification degree is up to 80%-90%.
In above-mentioned steps S04, in order to reduce the influence to triglyceride type EPA and DHA mass, and glycerine three is realized simultaneously
Ester type EPA and DHA efficient extraction and refined, using the method for supercritical extract to above-mentioned EPA, DHA fatty acid glycerine three
Ester is handled.During supercritical extract, because supercritical fluid is in Near The Critical Point, the minor variations meeting of temperature and pressure
Cause the large change of supercritical fluid densities, it is possible thereby to adjust the solvability to material.Therefore, the extraction of supercritical extract
Take rate high, separating for triglyceride type EPA and DHA and solvent is easily realized in extraction process, and organic solvent-free simultaneously be present
The advantages that remaining, be survivable to heat-sensitive substance, it can effectively remove the trip of possible remaining in above-mentioned subcritical enzymatic reaction
From aliphatic acid, fat-soluble pigment, volatility and non-volatile aldehydes, ketone, alcohols stink substance, so as to reach depickling, take off
Color, the purpose of deodorization.
In the step of preparing described EPA, DHA fatty acid triglycercide, the supercritical extract is divided into extraction and divided
From two steps.Specifically, after the subcritical enzymatic reaction terminates, using the reaction product after above-mentioned esterification as substrate, face super
Boundary extractive reaction sets the condition to be：Extracting pressure is 20MPa-30（MPa, extraction temperature are 35 DEG C -40 DEG C, CO2Flow is 4-
6L/h, extraction process is completed on this condition.After extraction terminates, extracting substance is separated, it is described to be divided into two fractions
From, wherein, the pressure of one-level separation is 6MPa-10MPa, and temperature is 45 DEG C -50 DEG C；The pressure of the second-order separation is 4MPa-6MPa,
Temperature is 55 DEG C -60 DEG C.
In S04 steps of the embodiment of the present invention, in order to improve production efficiency while increase EPA, DHA fatty acid glycerine three
The yield of ester, rectification process is and then carried out after carrying out supercritical extract processing, the liquid that extract and separate is obtained introduces rectifying
Purifies and separates again are carried out in post.Specifically, the reaction product level Four rectifying successively after supercritical extract, wherein, one-level
The temperature of 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-level rectifying is 70 DEG C -75 DEG C, four
The temperature of level rectifying is 75 DEG C -80 DEG C.
Collected after above-mentioned rectifying column obtained cut be using EPA and DHA unsaturated fat acid glycerol three ester as
The close high-purity polyunsaturated fatty acid triglycercide of main density, wherein, triglyceride type EPA weight/mass percentage composition is
14%-15%, triglyceride type DHA weight/mass percentage composition 71%-75%.
Specifically, as shown in Figure 3, CO2CO in steel cylinder 12Successively after the processing by filter 2 and cold 3, with taking
Band agent 11 mixes in blender 5, after the pre-heat treatment of preheated device 6, CO2Become supercriticality fluid, in reactor 7
Mixed with the reaction product after subcritical enzymatic reaction carry out supercritical extract, extraction product successively through one-level separating still 8,
After the second-order separation kettle 9 enter rectifying column 10 carry out level Four rectification process, finally give purifying, concentrate triglyceride type DHA and
EPA, product flow out through flow totalizer 12.
The method provided in an embodiment of the present invention that triglyceride type DHA and EPA are extracted from deep-sea fish, in an inert atmosphere
Using enzymatic isolation method extraction fish oil, obtained crude fish oil DHA and EPA content are high, acid number is low, can reach 1.5 (mg KOH)/g, are not easy
Become sour qualitative change, can directly carry out enzymolysis glycerine reaction, save the processing step for reducing acid number, simplify extraction flow；Faced by Asia
Boundary's state carries out enzyme digestion reaction, obtains triglyceride type aliphatic acid, esterifying efficiency substantially increases, and esterification yield can reach 90%；Pass through
Supercritical fluid extraction concentrates EPA and DHA fatty acid triglycercide, can reach concentration and refined purpose simultaneously, saves
Processing routine, production efficiency is high, cost is low, in the polyunsaturated triglyceride finally given, triglycerides EPA and DHA production
Rate is high, wherein, triglyceride type EPA may be up to 15%, and triglyceride type DHA may be up to 75%.
It is further described with reference to specific embodiment.
Embodiment 1
S11. the preparation of crude fish oil
Deep-sea fish crude fish oil is prepared using zymolysis technique.Deep-sea fish pulverizes to form rotten shape by bone cutter, weighs 600g meat
Slurry, by meat：Water=0.5:1（m：m）Ratio add water-stop, under conditions of darkroom, logical nitrogen, in 85 DEG C of boiling 1h.Use
NaOH adjusts pH value to 8.0.The trypsase that mass fraction is 2% is added, under darkroom, room temperature condition（25℃）Digest 17h.
After centrifuging 20min by 5000r/min, upper strata enzymolysis liquid, as crude fish oil are taken.
S12. the preparation of free fatty
The fish oil glyceride form for making crude fish oil using zymolysis technique is hydrolyzed, and obtains free fatty.Utilize fat
Enzyme 1LVK-F100 is catalyst, and enzyme dosage 2%, reaction temperature is 50 DEG C, reaction time 24h, obtains free fatty.
S13. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Using how unsaturated free fatties such as the concentration of urea entraing method EPA, DHA.Under 65 DEG C of water bath, prepare
The alcohol saturated solution of urea, adds free fatty, wherein, configuration proportion is as follows, free fatty：Urea ethanol saturation
Solution=1:10（w：v）.It is evenly stirred until transparent, after the temperature of mixed liquor is down to 4 DEG C with 5 DEG C/10min cooling rate, puts
4 DEG C of refrigerator 72h are put, then are filtered, using collecting filtrate after 100% ethanol solution wash crystallization body.By filtrate add just oneself
Alkane, distilled water, wherein, configuration proportion is as follows, filtrate：N-hexane：Distilled water=10：2：2（v:m:m）.After mixed solution is shaken up
Upper strata organic solution, is finally evaporated under reduced pressure by standing separation organic phase, that is, it is dense to obtain the how unsaturated free fatty such as EPA, DHA
Contracting liquid.
The preparation of S14.EPA and DHA fatty acid triglycercide
It is put into using above-mentioned free unsaturated fatty acid concentrate as substrate in supercritical extract reactor, in ultrasonic wave magnetic field
Booster action under carry out subcritical state under selectivity enzyme digestion reaction.Addition reactant has：N-hexane, glycerine, fat
Enzyme Novozym435, added in following ratio：N-hexane：Free fatty is 10:1（v:m）, glycerine：Free fatty is 4:1
(w:W), lipase Novozym435：Free fatty is 0.02：1（w:w）.Setting reactor pressure is 4MPa, when pressure exists
When keeping stable within 10min, ultrasonic wave magnetic field is opened, frequency of oscillation 2.5Hz, 40 DEG C of temperature of reaction kettle is set, in this condition
Lower reaction 3h, mainly generate the fatty acid triglycercide of more carbon.
It is using the triglycerides after above-mentioned esterification as reaction substrate, supercritical extract reaction setting condition：Extracting pressure
For 25MPa, extraction temperature is 40 DEG C, CO2Flow is 4L/h, and the pressure of separation reactor I is 10MPa, and the temperature of separation reactor I is 50
DEG C, the pressure for separating II is 6MPa, and separation reactor I I temperature is 60 DEG C, and rectifying column I temperature is 65 DEG C, rectifying column II temperature
For 70 DEG C, rectifying column III temperature is 75 DEG C, and rectifying column IV temperature is 80 DEG C.The material that rectifying column is collected to obtain be EPA and
The close high-purity polyunsaturated fatty acid triglycercide of density based on DHA unsaturated fat acid glycerol three ester.
Embodiment 2
S21. the preparation of crude fish oil
Deep-sea fish crude fish oil is prepared using zymolysis technique.Deep-sea fish pulverizes to form rotten shape by bone cutter, weighs 600g meat
Slurry, by meat：Water=0.5:1（w：w）Ratio add water-stop, under conditions of darkroom, logical nitrogen, in 85 DEG C of boiling 0.5h.Use
NaOH adjusts pH value to 8.0.The trypsase that mass fraction is 1.5% is added, under darkroom, room temperature condition（20℃）Enzymolysis
15h.After centrifuging 20min by 4500r/min, upper strata enzymolysis liquid, as crude fish oil are taken.
S22. the preparation of free fatty
The fish oil glyceride form for making crude fish oil using zymolysis technique is hydrolyzed, and obtains free fatty.Utilize fat
Enzyme 1LVK-F100 is catalyst, and enzyme dosage 1.5%, reaction temperature is 45 DEG C, reaction time 20h, obtains free fatty.
S23. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Using how unsaturated free fatties such as the concentration of urea entraing method EPA, DHA.Under 60 DEG C of water bath, prepare
The alcohol saturated solution of urea, adds free fatty, wherein, configuration proportion is as follows, free fatty：Urea ethanol saturation
Solution=5:10（w：v）.Be evenly stirred until it is transparent, after the temperature of mixed liquor is down to 10 DEG C with 10 DEG C/10min cooling rate,
4 DEG C of refrigerator 48h are placed, then are filtered, using collecting filtrate after 100% ethanol solution wash crystallization body.Filtrate is added just
Hexane, distilled water, wherein, configuration proportion is as follows, filtrate：N-hexane：Distilled water=10：1：2（v:m:m）.Mixed solution is shaken up
Upper strata organic solution, is finally evaporated under reduced pressure by standing separation organic phase afterwards, that is, obtains the how unsaturated free fatty such as EPA, DHA
Concentrate.
The preparation of S24.EPA and DHA fatty acid triglycercide
It is put into using above-mentioned free unsaturated fatty acid concentrate as substrate in supercritical extract reactor, in ultrasonic wave magnetic field
Booster action under carry out subcritical state under selectivity enzyme digestion reaction.Addition reactant has：N-hexane, glycerine, fat
Enzyme Novozym435, added in following ratio：N-hexane：Free fatty is 8:1（v:m）, glycerine：Free fatty is 2:1
(w:W), lipase Novozym435：Free fatty is 0.01：1（w:w）.Setting reactor pressure is 6MPa, when pressure exists
When keeping stable within 10min, ultrasonic wave magnetic field is opened, frequency of oscillation 6Hz, sets 30 DEG C of temperature of reaction kettle, on this condition
5h is reacted, mainly generates the fatty acid triglycercide of more carbon.
Triglycerides after above-mentioned esterification is loaded in reactor as reaction substrate, supercritical extract reaction setting condition
For：Extracting pressure is 20MPa, and extraction temperature is 30 DEG C, CO2Flow is 4L/h, and the pressure of separation reactor I is 6MPa, separation reactor I
Temperature is 45 DEG C, and the pressure for separating II is 4MPa, and separation reactor I I temperature is 55 DEG C, and rectifying column I temperature is 60 DEG C, rectifying column
II temperature is 65 DEG C, and rectifying column III temperature is 70 DEG C, and rectifying column IV temperature is 75 DEG C.Rectifying column collects obtained thing
Matter is the triglycerides of EPA and DHA unrighted acid.Based on the close high-purity polyunsaturated fatty acid glycerine of density
Three esters.
Embodiment 3
S31. the preparation of crude fish oil
Deep-sea fish crude fish oil is prepared using zymolysis technique.Deep-sea fish pulverizes to form rotten shape by bone cutter, weighs 600g meat
Slurry, by meat：Water=0.5:1.5（m：m）Ratio add water-stop, under conditions of darkroom, logical nitrogen, in 90 DEG C of boiling 1h.Use
NaOH adjusts pH value to 8.0.The trypsase that mass fraction is 3% is added, 15h is digested under conditions of darkroom, 30 DEG C.Pass through
After 6000r/min centrifugations 20min, upper strata enzymolysis liquid, as crude fish oil are taken.Crude fish oil is according to standard GB/T/T17377-2008
《Animal and plant fat fatty acid methyl ester gas chromatographic analysis》Detected.
S32. the preparation of free fatty
The fish oil glyceride form for making crude fish oil using zymolysis technique is hydrolyzed, and obtains free fatty.Utilize fat
Enzyme 1LVK-F100 is catalyst, and enzyme dosage 3.0%, reaction temperature is 55 DEG C, reaction time 28h, obtains free fatty.
S33. the free polyunsaturated fatty acids such as EPA, DHA are concentrated
Using how unsaturated free fatties such as the concentration of urea entraing method EPA, DHA.Under 65 DEG C of water bath, prepare
The methanol saturated solution of urea, adds free fatty, wherein, configuration proportion is as follows, free fatty：Urea methanol saturation
Solution=1:10（w：v）.Be evenly stirred until it is transparent, after the temperature of mixed liquor is down to 10 DEG C with 5 DEG C/10min cooling rate,
4 DEG C of refrigerator 72h are placed, then are filtered, using collecting filtrate after 100% methanol solution wash crystallization body.Filtrate is added just
Hexane, distilled water, wherein, configuration proportion is as follows, filtrate：N-hexane：Distilled water=10：2：3（v:m:m）.Mixed solution is shaken up
Upper strata organic solution, is finally evaporated under reduced pressure by standing separation organic phase afterwards, that is, obtains the how unsaturated free fatty such as EPA, DHA
Concentrate.
The preparation of S34.EPA and DHA fatty acid triglycercide
It is put into using above-mentioned free unsaturated fatty acid concentrate as substrate in supercritical extract reactor, in ultrasonic wave magnetic field
Booster action under carry out subcritical state under selectivity enzyme digestion reaction.Addition reactant has：N-hexane, glycerine, fat
Enzyme Novozym435, added in following ratio：N-hexane：Free fatty is 12:1（v:m）, glycerine：Free fatty is 6:1
(w:W), lipase Novozym435：Free fatty is 0.05：1（w:w）.Setting reactor pressure is 8MPa, when pressure exists
When keeping stable within 10min, ultrasonic wave magnetic field is opened, frequency of oscillation 10Hz, sets 60 DEG C of temperature of reaction kettle, on this condition
12h is reacted, mainly generates the fatty acid triglycercide of more carbon.
Triglycerides after above-mentioned esterification is loaded in reactor as reaction substrate, supercritical extract reaction setting condition
For：Extracting pressure is 30MPa, and extraction temperature is 40 DEG C, CO2Flow is 6L/h, and the pressure of separation reactor I is 10MPa, separation reactor I
Temperature be 50 DEG C, the pressure for separating II be 6MPa, and separation reactor I I temperature is 60 DEG C, and rectifying column I temperature is 65 DEG C, rectifying
Post II temperature is 70 DEG C, and rectifying column III temperature is 75 DEG C, and rectifying column IV temperature is 80 DEG C.Rectifying column collects what is obtained
Material is the close high-purity polyunsaturated fatty acid glycerine of the density based on EPA and DHA unsaturated fat acid glycerol three ester
Three esters.
Comparative example 1
1. the preparation of crude fish oil
Deep-sea fish crude fish oil is prepared using zymolysis technique.Deep-sea fish pulverizes to form rotten shape by bone cutter, weighs 600g meat
Slurry, by meat：Water=0.5:1（m：m）Ratio add water-stop, under conditions of darkroom, logical nitrogen, in 85 DEG C of boiling 1h.Use
NaOH adjusts pH value to 8.0.The trypsase that mass fraction is 2% is added, under darkroom, room temperature condition（25℃）Digest 17h.
After centrifuging 20min by 5000r/min, upper strata enzymolysis liquid, as crude fish oil are taken.
2. supercritical extract concentrates EPA/DHA triglycerides
Above-mentioned crude fish oil is loaded in reactor and is as reaction substrate, supercritical extract reaction setting condition：Extraction
Pressure is 25MPa, and extraction temperature is 40 DEG C, CO2Flow is 4L/h, and the pressure of separation reactor I is 10MPa, and the temperature of separation reactor I is
50 DEG C, the pressure for separating II is 6MPa, and separation reactor I I temperature is 60 DEG C, collects isolated material.
In above-described embodiment, reagent source is：
Trypsase：Food-grade, Zhengzhou Hong Cheng chemical products Co., Ltd；
LVK-F100 lipase：Shenzhen Leveking Biology Engineering Co., Ltd；
Novozym435 lipase：Novozymes Company.
To EPA and DHA content in crude fish oil in above-described embodiment 1-3, the degree of hydrolysis of free fatty, aliphatic acid ester
Glycerine ester type EPA and DHA weight/mass percentage composition is tested in change degree and final products, and method of testing is as follows：
(1) first DHA/EPA detection method of content in fish oil:According to GB/T17377-2008《Animal and plant fat fatty acid methyl
Ester chromatography of gases is analyzed》Detection.
(2) detection method of free fatty acid content（That is acid value measuring）：According to GB/T5530-2005《Animal and plant fat
The measure of acid number and acidity》Detection.
(3) measure of fatty acid ester rate：According to the acid number of GB/T5530-2005 methods measure reaction system, then press
Calculated according to following company：Esterification yield（%）=[（Acid number before the reacted acid number/reactions of 1-）]×100%.
(4) measure of glycerine ester type DHA/DHA purity：According to GB/T17377-2008《Animal and plant fat fatty acid methyl ester
Chromatography of gases is analyzed》Detection " determines.
Test result is as shown in table 1 below:
Table 1
As seen from the above table, deep-sea fish provided in an embodiment of the present invention extracts triglyceride type DHA and EPA from deep-sea fish
Method, DHA/EPA content is high in crude fish oil, respectively reaches 28.94% and 4.93%；The degree of hydrolysis of free fatty is high, can
Reach more than 50%；Aliphatic acid esterification degree is up to 90%；In obtained final products, glycerine ester type DHA weight/mass percentage compositions are high, can
Respectively reach 15.02% and 74.98%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (9)
1. a kind of method that triglyceride type DHA and EPA are extracted from deep-sea fish, comprises the following steps：
Deep-sea fish is rubbed into meat and starched, adds after being heated under anaerobic after water, is carried out using trypsase
After first enzyme digestion reaction, separation of solid and liquid is carried out, filtrate is collected, obtains crude fish oil；Wherein, the heating-up temperature of the heating is
85 DEG C~90 DEG C, heat time 0.5h-1h；
The crude fish oil is subjected to the second enzyme digestion reaction using lipase 1LVK-F100, obtains free fatty；
Under water bath condition, mixed after the methanol of urea or alcohol saturated solution are mixed with the free fatty
Thing solution, it is 4 DEG C -10 DEG C, after insulation stewing process 48h-72h that the mixture solution is carried out into cooling processing to temperature, is entered
Row filtration treatment obtains filtrate, and the filtrate is carried out into extraction processing and collected organic layer, concentrated containing the more of EPA, DHA
Unrighted acid；
Using the polyunsaturated fatty acid containing free EPA, DHA as substrate, put it into containing lipase Novozym435's
In supercritical extract reactor, subcritical enzymatic reaction is carried out in ultrasonic wave magnetic field, by reaction product carry out supercritical extract,
Rectification process, obtain EPA, DHA fatty acid triglycercide；Wherein, the condition of the subcritical enzymatic reaction is：The reaction
The pressure of kettle is 4-8MPa, and ultrasonic oscillation magnetic field 2-10Hz, reaction temperature is 30 DEG C -60 DEG C, reaction time 1h-12h.
2. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claim 1 is any, it is characterised in that：
In the step of preparing described EPA, DHA fatty acid triglycercide, the supercritical extract is divided into two steps of extraction and separation, its
In,
The condition of the extraction is：Extracting pressure is 20MPa-30MPa, and extraction temperature is 35 DEG C -40 DEG C, CO2Flow be 4-
6L/h；
It is described to be divided into two-stage separation, wherein, the pressure of one-level separation is 6MPa-10MPa, and temperature is 45 DEG C -50 DEG C；Two level
The pressure of separation is 4MPa-6MPa, and temperature is 55 DEG C -60 DEG C.
3. triglyceride type DHA and EPA method are extracted from deep-sea fish as claimed in claim 1, it is characterised in that：Making
In the step of standby described EPA, DHA fatty acid triglycercide, the rectification process is divided into level Four rectifying, wherein, one-level rectifying
Temperature be 60 DEG C -65 DEG C, the temperature of two-stage rectification is 65 DEG C -70 DEG C, and the temperature of three-level rectifying is 70 DEG C -75 DEG C, level Four essence
The temperature evaporated is 75 DEG C -80 DEG C.
4. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing described EPA, DHA fatty acid triglycercide, in supercritical extract reactor, also containing n-hexane,
Glycerine, and the amount ratio of the n-hexane, glycerine, lipase Novozym435, polyunsaturated fatty acid containing free EPA, DHA
For n-hexane：Glycerine：Lipase Novozym435：Polyunsaturated fatty acid containing free EPA, DHA is (8-12) ml:(2-6)
g:(0.01-0.05)g:1g。
5. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing the polyunsaturated fatty acid containing EPA, DHA, extraction system that the extraction processing uses is filters
Liquid, n-hexane, distillation aqueous systems, wherein, filtrate：N-hexane：The amount ratio of distilled water is 10ml：(1-2)g：(2-3)g.
6. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing the polyunsaturated fatty acid containing EPA, DHA, the methanol or second of the free fatty and urea
The amount ratio of alcohol saturated solution is 1g：(5-10)ml.
7. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing the polyunsaturated fatty acid containing EPA, DHA, the method for the cooling processing be with 5 DEG C/
After 10min-10 DEG C/10min rate of temperature fall makes the temperature of mixing liquor be down to 4 DEG C -10 DEG C；And/or
The temperature of stewing process is 4 DEG C -5 DEG C.
8. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing the free fatty, the method for second enzyme digestion reaction is：Matter is added in the crude fish oil
Measure the lipase 1LVK-F100, the enzyme digestion reaction 20h-28h at a temperature of 45 DEG C -55 DEG C that fraction is 1.5%-3.0%.
9. the method that triglyceride type DHA and EPA are extracted from deep-sea fish as described in claims 1 to 3 is any, its feature exist
In：In the step of preparing the crude fish oil, the method for first enzyme digestion reaction is：Adjust the pH value of the mixture solution
To 7.0-8.5, addition mass fraction is 1.5%-3.0% trypsase, the enzyme digestion reaction 15h-19h under the conditions of lucifuge,
Reaction temperature is 20 DEG C -30 DEG C.
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| CN111647633B (en) * | 2020-05-22 | 2022-02-08 | 西华大学 | Method for enriching EPA and DHA in deep sea fish oil |
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