A kind of enzymatic-process preparation method of phosphatide type polyunsaturated fatty acid
Technical field
The present invention relates to polyunsaturated fatty acids, more particularly to a kind of a kind of more insatiable hungers of phosphatide type with healthy functions
With the enzymatic-process preparation method of aliphatic acid.
Background technology
It is 18~22 carbon originals that polyunsaturated fatty acid (PUFA), which refers to containing two or more double bonds and carbon chain lengths,
The straight chain fatty acid of son.It is generally divided into omega-3 and omega-6, in PUFA molecules, the double bond away from carboxyl distalmost end is being fallen
On the 3rd carbon atom of number be known as omega-3 aliphatic acid, as eicosapentaenoic acid (EPA), clupanodonic acid (DPA) and
Docosahexaenoic acid (DHA) etc.;On the 6th carbon atom, then referred to as omega-6 aliphatic acid, such as arachidonic acid.
PUFA usually has significant physiological function, is the indispensable nutritional ingredient of human body.
It is several that PUFA products are generally divided into ethyl ester type, triglyceride type and phosphatide type.Wherein triglyceride type and phosphatide type
Belong to natural structure, it is, naturally occurring structure in vivo, and ethyl ester type belongs to non-natural structural, why deposits
In ethyl ester type, it is separation, concentration process because in polyunsaturated fatty acid, PUFA is needed to be converted into monomer, fatty-acid ethyl ester
It is a kind of structure easy to produce and process.
For the PUFA products of different structure, there is a concern which kind of structure is better.Early in 1988, Tarry
Et al. D. the fish oil product for just comparing ethyl ester type, glycerine ester type and fatty acid type, shows that fatty acid type is easiest to absorb, secondly
It is triglyceride type, worst is ethyl ester type, and research later also further demonstrates the conclusion.Contain a large amount of phosphatide in krill oil
The PUFA of type is many to declare that krill oil has higher bioavilability.For phosphatide type and triglyceride type PUFA
Bioavilability, people study, the results showed that, the bioavilability of the two is about the same, shows that phosphatide type PUFA is also
A kind of outstanding structure.Both in addition, phosphatide inherently has healthy functions, phosphatide and PUFA are combined, can obtain
Compound action, so phosphatide type PUFA is a kind of important product form.Natural phosphatide type PUFA is general derived from south
Pole krill oil.Since krill oil is of high cost, just become another acquiring way by artificial mode synthetic phospholipid type PUFA.
The Fatty acid compositions for changing phosphatide, can generally select ester exchange reaction or acidolysis reaction.Ester exchange reaction is
The aliphatic acid on the ester type compound and phosphatide of purpose aliphatic acid is selected to swap;Acidolysis reaction be using free fatty and
Aliphatic acid on phosphatide swaps.Due to phosphatide have very high polarity, usually to select the substrate close with its polarity with
It is reacted, and reaction efficiency just can higher.Studies have shown that phosphatide and the PUFA reaction efficiencies of almost all of esterified form are all very
It is low.When acidolysis reaction, since the polarity and phosphatide of aliphatic acid are more nearly, reaction efficiency is greatly improved.PUFA donors
Form for react it is critically important, it is existing report be mostly free-fat acid type;Also there is the PUFA using esterified form, but react
It needs to add water in the process, thus it is speculated that real reaction mechanism PUFA still occurs mainly in the form of free fatty with phosphatide anti-
It answers.Donor using free fatty as PUFA, has the following disadvantages:First, the preparation of the PUFA of fatty acid type is typically
By the way of chemical hydrolysis, manufacturing cost is high, and the rear separation of reaction product is more complicated;Second, the phosphatide type PUFA face of preparation
Color is deeper.
Invention content
The present invention is directed to disadvantage of the existing technology, provides a kind of substrate and is easy to get, and reaction efficiency is high, the phosphorus of preparation
The enzymatic-process preparation method of the shallower phosphatide type polyunsaturated fatty acid of epoxy-type PUFA colors.
The present invention provides a kind of novel reaction system, develops one kind and being suitable for enzyme process Efficient Conversion, while can obtain again
The reaction system for obtaining high quality product overcomes traditional free-fat acid type PUFA and prepares lacking for phosphatide type PUFA as donor
Point.
A kind of enzymatic-process preparation method of phosphatide type polyunsaturated fatty acid:Use lipase and/or phosphatidase for catalyst,
Catalyst is the enzyme preparation with lipase activity, and enzyme preparation is selected immobilization way, is divided into using five kinds of groups of A, B, C, D and E
The catalyst system and catalyzing of substrate, carries out catalysis reaction, and controlling reaction temperature is 30~80 DEG C;Reaction time is 6~48hr;Control catalysis
The addition of agent is the 1-5% of substrate quality;
A:Free-fat acid type PUFA;
B:Glycerine ester type PUFA;
C:Ethyl ester type PUFA;
D:Phosphatide;
E:Water
Wherein, A:B:C:E:The mass ratio of D is 10~30:50~300:10~30:1~5:100;
After the completion of reaction, immobilised enzymes is filtered off, washing obtains phosphatide type polyunsaturated fatty acid.
The phosphatide selects the mixture of one or both of soybean lecithin and yolk phospholipid.
To further realize the object of the invention, it is preferable that the source of the lipase and/or phosphatidase be rhizopus,
Aspergillus, mucor, bacterium, saccharomycete or animal pancreas.
The addition of the catalyst is the 2-4% of substrate quality.
Aliphatic acid of the present invention may be used any mode and obtain free fatty.Aliphatic acid is sent out in the modification reaction of phosphatide
Important function is waved, the reaction substrate in the present invention includes necessarily a certain amount of free fatty, moreover, the free fatty is
It there is in reaction zero moment.Why requirement reaction zero moment will include a certain amount of free fatty, and main cause exists
It can inhibit the excessive hydrolysis of phosphatide in free fatty.The required free fatty of the present invention, may be used chemical hydrolysis
Method obtains, and enzymatic hydrolysis method can also be used to obtain, and is preferably obtained using enzyme solution using glycerine ester type PUFA as substrate.
A certain amount of ethyl ester type polyunsaturated fatty acid is contained in reaction system of the present invention.Ethyl ester type polyunsaturated fat
Acid and phosphatide have good compatibility, and phosphatide viscosity can be greatly lowered, and the mass transfer for improving reaction system has weight
It acts on.By the variation for monitoring ethyl ester type polyunsaturated fatty acid ingredient in reaction process.Ethyl ester type polyunsaturated fatty acid
Variation to fall behind and fatty acid type PUFA, thus it is speculated that ethyl ester type polyunsaturated fatty acid is preferential to be handed over fatty acid type PUFA
It changes, then fatty acid type PUFA reacts with phosphatide again.
The displacement of lipoid fatty acid is reacted in the participation that water is needed in reaction system of the present invention, and the reaction mechanism mechanism of reaction seems
It is different with glyceride, the aliphatic acid in glyceride is changed using enzyme process, may be used almost water free reaction system, and phosphatide fat
The change of fat acid needs first to generate hemolytic phosphatide or glycerophosphoryl compound, then the binding purpose in a manner of esterification again
Aliphatic acid.General enzyme preparation itself includes moisture, and the amount of moisture often influences reaction efficiency, the amount of moisture in General reactions system
Control is 1~5% (w/w) of phosphatide.
Due to there is the participation of water in reaction, water is preferential and hydrolysis occurs for phosphatide, and phosphatide can lose one or 2 fat
Fat acid generates hemolytic phosphatide or glycerophosphoryl compound, these products are again and fatty acid response generates phosphatide and water, together
When, aliphatic acid exchange reaction constantly is occurring between glyceride, ethyl ester and free fatty in system, in this manner so that anti-
Answer in system that aliphatic acid constantly exchanges in each substrate.
The addition of catalysts of the present invention can take the 1-5% of substrate quality;Reaction temperature is 30~80 DEG C;
Reaction time is 6~48hr.
In reaction system of the present invention, phosphatide can select the mixture of one or both of soybean lecithin, yolk phospholipid.
Krill oil accounts for the 2~5% of the weight in wet base of South Pole phosphinylidyne, and main ingredient includes lecithin 33-36%, phosphatidyl-ethanolamine 5-6%,
Neutral glycerine ester 33-40%, free fatty 8-16%.In phosphatide in krill oil the content of EPA and DHA generally 35% with
It is interior, content can be further increased by enzymatic conversion method obtains the other functional PUFA of addition.Yolk phospholipid and soybean lecithin
In do not include a large amount of functionality PUFA usually, abundance, cost is relatively low, is the important carrier of PUFA.
In reaction system of the present invention, catalyst is the enzyme preparation with lipase activity, and enzyme preparation can select immobilization
Mode, resolvase can also be selected, however, it is preferred to immobilised enzymes.Immobilized enzyme reactor may be used any type of anti-
Device is answered, a reactor is preferably filled with.Enzyme preparation with lipase activity includes lipase or phospholipase A1/A2, they are common
Source is rhizopus, aspergillus, mucor, bacterium, saccharomycete or animal pancreas.In general, lipase and phospholipase A1/A2Boundary
Limit is not very stringent, and lipase also has hydrolysing activity, phospholipase A to phospholipid substrate1/A2Also there is water to triglycerides substrate
Solution activity.
Compared with the existing technology, the invention has the advantages that:
1) substrate is easy to get.PUFA donors of the present invention are mainly glycerine ester type PUFA and ethyl ester type PUFA,
Both substances are the commodity of mass manufacture, are easy buying materials, and free-fat acid type PUFA does not have because it is easy oxidation
There is the raw material supply.Moreover, free-fat acid type PUFA is needed to be prepared by precursor of first two substance, preparation process is to antioxygen
Change has strict demand.
2) there is good reaction efficiency.In general, when producing phosphatide type PUFA, made using the PUFA of free-fat acid type
Reaction efficiency highest is carried out for substrate.Studies have shown that using reaction system of the present invention, can equally obtain and free-fat acid type
PUFA similar reaction efficiencies when being substrate, it is believed that reaction system of the present invention has good reaction efficiency.
3) the phosphatide type PUFA of light color can be obtained.Studies have shown that the color and free-fat acid type PUFA of reaction product
Dosage have a correlation, the mechanism that color generates is unclear.Reaction system of the present invention is due to using less free fatty
Type PUFA can obtain lighter phosphatide type PUFA products.
Specific implementation mode
To more fully understand the present invention, with reference to embodiment, the present invention is further illustrated, but the reality of the present invention
It is unlimited so to apply mode.
Main raw material(s) in following example:
PUFA glyceride, by percentage to the quality, including EPA60%, DPA6%, DHA10%, Zhoushan Xin Nuojia biology works
Journey Co., Ltd produces.
PUFA ethyl esters, by percentage to the quality, including EPA60%, DPA6%, DHA10%, Zhoushan Xin Nuojia bioengineering
Co., Ltd produces.
Fatty acid type PUFA, by percentage to the quality, including EPA60%, DPA6%, DHA10%, Zhoushan Xin Nuojia biologies
Engineering Co., Ltd produces.
Soybean lecithin, ADM companies of the U.S. produce.
Yolk phospholipid, Shaanxi Jiade bioengineering Co., Ltd produce.
Immobilized phospholipase is made by oneself, using a kind of Lecitase Ultra (phospholipase As of Novozymes companies1Product),
With Purolite ECR1030M adsorption of immobilization, by percentage to the quality, the carrying capacity of enzyme is the 12.1% of resin.
Fatty acid compositional analysis:It is more for the relevant free-fat acid type polyunsaturated fatty acid of the present invention, glycerine ester type
The fatty acid compositional analysis of the components such as unsaturated fatty acid, ethyl ester type polyunsaturated fatty acid and phosphatide is all made of following method:
Analysans about 30mg is taken, is added in 50ml round-bottomed flasks, the KOH/methanol solution 2ml of 0.5M is added, is placed in 70 DEG C of water-baths
The saponification that flows back about 10 minutes, reaction process vibrates frequently, until lipid solubilization, appropriate 2 minutes cooling, 3mlBF3 is then added
Methanol solution, is placed in 70 DEG C of water-bath back flow reactions 5 minutes, keeps esterification complete.Then it cools down, 2ml n-hexanes is added, gently shake
Swing the dissolving to promote methyl esters in n-hexane.Then saturated salt solution, which is added, makes n-hexane rise to bottleneck, waits about 1 point
Clock draws upper layer n-hexane mutually in equipped with a small amount of anhydrous Na2SO4Sample bottle carry out gas chromatographic analysis.With reference to " GB/T
The gas chromatographic analysis of 17377-2008 animal and plant fat Fatty acid methyl esters " is analyzed.
Embodiment 1-6
According to catalyst system and catalyzing hybrid reaction substrate shown in table 1, a certain amount of immobilised enzymes is then added and (uses resin
Lecitase Ultra, the Lecitase Ultra of Purolite ECR1030M adsorption of immobilization are a kind of while having typical case
The enzyme preparation of lipase and phospholipase A1 vigor), magnetic agitation reaction, reaction temperature, reaction time and enzyme preparation dosage such as table 2
It is shown.After the completion of reaction, response sample is taken, filters off immobilised enzymes, 200ml acetone, stirring is added, it is seen that precipitation is collected by centrifugation
Acetone insoluble matter precipitates, and adds 30ml acetone and washs 3 times, you can obtains purer acetone insoluble matter.Phosphatide type is mostly not
Saturated fatty acid is a kind of general matter, be easy to show as a variety of method qualitative analyses such as infrared spectrum, TLC and NMR obtained by
The acetone insoluble matter arrived is phosphatide type polyunsaturated fatty acid, is further shown wherein without bright by HPLC-ELSD quantitative analyses
The reactants such as aobvious free fatty, fatty-acid ethyl ester and triglycerides remain.It evaluates the color of the phospholipid fraction and measures anti-
The aliphatic acid composition of phospholipid fraction, the results are shown in Table 3 after answering.
1 substrate of table matches
2 reaction condition of table
3 phospholipid products of table are evaluated
Embodiment 7~8
According to catalyst system and catalyzing hybrid reaction substrate shown in table 4, a certain amount of immobilised enzymes lipozyme TL are then added
IM (the immobilized lipase enzyme product of Novozyme), magnetic agitation reaction, reaction temperature and reaction time are as shown in table 5.Reaction
After the completion, response sample is taken, immobilised enzymes is filtered off, 200ml acetone, stirring is added, it is seen that precipitation is collected by centrifugation precipitation, then adds
Enter 30ml acetone to wash 3 times, you can obtain acetone insoluble matter phosphatide.It evaluates the color of the phospholipid fraction and measures phospholipid fraction
Aliphatic acid forms, and the results are shown in Table 6.
4 substrate of table matches
5 reaction condition of table
|
Embodiment 7 |
Embodiment 8 |
Reaction time (hr) |
48 |
48 |
Reaction temperature (DEG C) |
50 |
50 |
Enzyme preparation dosage (%) |
3 |
3 |
6 phospholipid products of table are evaluated
The present invention, Examples 1 to 5 and embodiment 7 are the embodiment using reaction system of the present invention, table 3 the result shows that, institute
EPA and DHA are combined after being reacted in phospholipid products, i.e., can obtain phosphatide type PUFA products, and color is shallower.It is real
The PUFA donors for applying example 6 and embodiment 8 are free-fat acid type PUFA, and the phosphatide type PUFA coloured products of acquisition are relative to other
Embodiment is deeper.The color generting machanism of lipid material is more complicated, and some is derived from pigment, and some becomes derived from the position of double bond
Change, some is derived from oxidation product, currently, being still difficult to directly research the material base of color generation comprehensively.The embodiment of the present invention
Show when substrate uses more free fatties, at this point, reaction system acid value is higher, obtained coloured product is also relatively deep.
PUFA donors of the present invention are mainly glycerine ester type PUFA and ethyl ester type PUFA, both substances are batch
Quantify the commodity of manufacture, is easy buying materials, and free-fat acid type PUFA is easy oxidation, the not raw material supply because of it.And
And free-fat acid type PUFA is needed to be prepared by precursor of first two substance, preparation process has strict demand to anti-oxidant.
In general, when producing phosphatide type PUFA, reaction effect is carried out as substrate using the PUFA of free-fat acid type
Rate highest.Using reaction system of the present invention, similar reaction when being substrate can be equally obtained with the PUFA of free-fat acid type
Efficiency, it is believed that reaction system of the present invention has good reaction efficiency.
Reaction system of the present invention can obtain lighter phosphatide type due to using less free-fat acid type PUFA
PUFA products.