CN106609286A - Preparation method for phosphatide containing long-chain polyunsaturated fatty acid - Google Patents
Preparation method for phosphatide containing long-chain polyunsaturated fatty acid Download PDFInfo
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Abstract
The invention relates to a preparation method for phosphatide containing long-chain polyunsaturated fatty acid. The preparation method for phosphatide containing long-chain polyunsaturated fatty acid comprises the following steps: (1) contacting raw phosphatide with grease and a solvent and adding an enzyme preparation for a reaction; and (2) removing the solvent and adding an immobilized enzyme for a reaction. The preparation method is characterized in that a step of addition of carbamide is additionally carried out after the step (1). With the method, price cost is greatly lowered, and the added value of the prepared phosphatide is substantially increased.
Description
Technical field
The present invention provides a kind of phospholipid rich in long-chain polyunsaturated fatty acid and preparation method thereof.
Background technology
Long-chain polyunsaturated fatty acid（PUFAs）Refer to containing two or more double bond and carbon chain lengths are the straight chain fatty acid of 18～22 carbon atoms, such as EPA, DHA, DPA and ARA etc. has important physiologically active to human body, with the irreplaceable effect of other fatty acids.Sweet three ester and phospholipid are two kinds of main natural carriers of PUFAs, wherein, sweet three ester type PUFAs is mainly derived from abyssal fishes fat and fermentable, and phosphatide type PUFAs is then mainly extracted from the organ-tissue of Antarctic krill and abyssal fishes.Some researchs recently show, phosphatide type PUFAs has more preferable oxidation stability and bioavailability than sweet three ester type PUFAs, phosphatide type PUFAs major parts can not be hydrolyzed and directly be absorbed by cell, dual physiologically active with phospholipid and PUFAs, therefore have a clear superiority than sweet three ester type PUFAs in terms of human body physiological function is maintained, phosphatide type PUFAs can be widely used for the aspects such as food additive, health product and medicine.
But, limited by source, phosphatide type PUFAs is affected by krill catching season and heavy metal pollution that may be present etc. is threatened, and the soybean phospholipid and egg yolk lecithin of abundance does not then contain PUFAs in its natural acid composition, therefore, progressively launched using the research that enzymatic clarification contains PUFAs structure phospholipid based on common phospholipid.
CN101701229B discloses the method that concentrated phosphatide and consaturated oil mixing are added lipase-catalyzed phospholipid ester exchange preparation quality structure phospholipid and lecithin.The method carries out catalysis transesterification, but the method reaction efficiency is relatively low using phospholipid or concentrated phosphatide and consaturated oil as raw material with lipase, and the response time is up to 60h or so.
CN102181498A applications high-purity seal oil sequestered Ω -3 unsaturated fatty acids and LYSO-PHOSPHATIDYLCHOLINE LYSOPC, under non-aqueous enzymatic catalysis effect, obtain phosphatidylcholine type Ω -3 unsaturated fatty acidss acid products.The patent uses dicyandiamide solution, and the response time is long, while in catalytic reaction process, hydrolytic side reactions occur seriously, cause efficiency of pcr product relatively low.
CN101195637B provides a kind of technique for preparing in subcritical R134a systems and being rich in polyunsaturated fatty acid phospholipid, but the method needs high-pressure sealed reactor and prepares the relevant apparatus of subcritical gas, equipment investment is big, simultaneous reactions medium R134a is a kind of industrial chemicals, is not suitable for the production of food.
CN104531790A discloses a kind of anion/cation surfactant is dissolved in hydrophobic alkanes solvent and prepares micro- reaction system, carries out the method that catalytic reaction obtains phospholipid DHA using phospholipase.
In addition domestic some other prepares the document of phosphatide type PUFAs with regard to enzyme process, is also concentrated mainly on the immobilized-lipase used in dicyandiamide solution or immobilized phospholipase A1Catalysis phospholipid carries out the reaction of acidolysis or ester-ester exchange to carry out, and such as Jinhua is beautiful etc.（The research [J] of lipase-catalyzed Acetylating Modification of Soybean Phosphatide）With novozym435 catalysis soybean phospholipids and EPA-DHA ester exchanges, the response time, the insertion rate of EPA-DHA was 24% or so up to 60h；Sun Zhaomin（The technique that enzyme process prepares n-3 polyunsaturated fatty acid type phospholipid）Immobilized phospholipase A is used in solvent-free system1It is 25% that catalysis soybean phospholipid and ethyl ester type fish oil carry out the insertion rate of ester exchange reaction, EPA and DHA, but phospholipid hydrolysis side reaction generation is very serious.
US2005282033A provides a kind of enzyme catalysiss and prepares the improved method containing PUFAs, and the method is added amine substance during the course of the reaction, improves reaction rate, but the insertion rate and phospholipid final yield of PUFAs are not improved.
WO2005038037A2 carries out esterification and transesterification preparation structure phospholipid using E.C. 3.1.1.32 and A2, and the method response time is long, and insertion rate is not high, and hydrolytic side reactions occur serious.
EP0494881B1 uses phospholipase A2 catalytic esterification in dicyandiamide solution, the shortcomings of the method has active unstable after the limited source and immobilization of enzyme in addition to the drawbacks of there is above method, also.
The content of the invention
It is an object of the present invention to provide the preparation method of the phospholipid containing long-chain polyunsaturated fatty acid, including：
（1）Raw material phospholipid, oils and fatss and solvent are contacted, adds enzyme preparation to be reacted；
（2）After removing solvent, immobilized enzyme is added to be reacted, it is characterised in that in step（1）There is the step of adding phosphoamide afterwards.
Preparation in accordance with the present invention, wherein, the raw material phospholipid does not contain long-chain polyunsaturated fatty acid.
Preparation in accordance with the present invention, wherein, the raw material phospholipid is the phospholipid of the phospholipid or animal origin of plant origin.
Preparation in accordance with the present invention, wherein, the phospholipid of the plant origin is selected from least one in soybean phospholipid, rapeseed phosphatide, sunflower phosphatide, peanut phosphatide, Semen Sesami phospholipid or Semen Maydiss phospholipid.
Preparation in accordance with the present invention, wherein, the phospholipid of the animal origin is egg yolk lecithin.
Preparation in accordance with the present invention, wherein, long-chain polyunsaturated fatty acid of the oils and fatss containing 5～60 weight %.
Preparation in accordance with the present invention, wherein, at least one of the oils and fatss in bathypelagic fish oil and microbial grease.
Preparation in accordance with the present invention, wherein, at least one of the oils and fatss in tunny fish oil, herring oil, salmon fish oil, pilchard oil, docosahexenoic acid algae oil or arachidonic acid oil.
Preparation in accordance with the present invention, wherein, the solvent is ethanol solution.
Preparation in accordance with the present invention, wherein, the solvent is 95% ethanol solution.
Preparation in accordance with the present invention, wherein, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：1～1：15.
Preparation in accordance with the present invention, wherein, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：2～1：10.
Preparation in accordance with the present invention, wherein, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：4～1：8.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：5～1：80.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：8～1：40.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：10～1：30.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：12～1：18.
Preparation in accordance with the present invention, wherein, step（1）Described in enzyme preparation be resolvase form.
Preparation in accordance with the present invention, wherein, step（1）Described in enzyme preparation be selected from E.C. 3.1.1.32, at least one in phospholipase A2 or lipase.
Preparation in accordance with the present invention, wherein, step（1）Described in enzyme preparation derive from aspergillus niger（Aspergillus
niger）, antarctic candida（Candida antarctic）, fold candida（Candida rugosa）, Candida cylindracea（Candida
cylindracea）, thermophilic fungal（Thermomyces lanuginosus）, rhizomucor miehei（Rhizomucor miehei）, rice black wool mould（Mucor
miehei）, Rhizopus delemar（Rhizopus delemar）, Rhizopus oryzae（Rhizopus oryzae）, Pseudomonas（Pseudononas sp. ）In at least one.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 10：1～200：1.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 20：1～100：1.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 40：1～80：1.
Preparation in accordance with the present invention, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 50：1～60：1.
Preparation in accordance with the present invention, wherein, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 100 weight portion～800 weight portions.
Preparation in accordance with the present invention, wherein, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 200 weight portion～600 weight portions.
Preparation in accordance with the present invention, wherein, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 250 weight portion～350 weight portions.
Preparation in accordance with the present invention, wherein, the step（2）It is to carry out at 60～80 DEG C in temperature.
Preparation in accordance with the present invention, wherein, the step（2）Immobilized enzyme be the lipase or phospholipase that are fixed on macroporous adsorbent resin or deacidite carrier at least one.
Preparation in accordance with the present invention, wherein, with the weight ratio of the addition total amount of weight ratio meter, the raw material phospholipid and the oils and fatss and the immobilized enzyme for 10：1～200：1.
Preparation in accordance with the present invention, wherein, with the weight ratio of the addition total amount of weight ratio meter, the raw material phospholipid and the oils and fatss and the immobilized enzyme for 20：1～150：1.
Preparation in accordance with the present invention, wherein, the step（2）Reacted under evacuation or protective gas atmosphere.
Preparation in accordance with the present invention, wherein, the step（2）Protective gas atmosphere be inert gas atmosphere.
Preparation in accordance with the present invention, wherein, the step（2）Middle addition water absorbing agent.
Preparation in accordance with the present invention, wherein, at least one of the water absorbing agent in molecular sieve or hydroscopic high-molecular resin.
Preparation in accordance with the present invention, wherein, at least one of the hydroscopic high-molecular resin in acrylamide and acrylic acid salt crosslinking copolymerization thing and starch grafted acrylate crosslinking copolymerization thing.
Preparation in accordance with the present invention, wherein, the phosphatidylcholine content of the raw material phospholipid is 15～60 weight %.
Preparation in accordance with the present invention, the long-chain polyunsaturated fatty acid is containing two or more double bond and carbon number is 18～22 straight chain fatty acid.
Preparation in accordance with the present invention, the long-chain polyunsaturated fatty acid are at least one in eicosapentaenoic acid, docosahexenoic acid, clupanodonic acid and arachidonic acid.
Preparation in accordance with the present invention, the phosphatidylcholine content of the described phospholipid containing long-chain polyunsaturated fatty acid is 70～85 weight %.
Preparation in accordance with the present invention, during the fatty acid of the described phospholipid containing long-chain polyunsaturated fatty acid is constituted, content of polyunsaturated fatty acid is 15～60 weight %.
Preparation in accordance with the present invention, during the fatty acid of the described phospholipid containing long-chain polyunsaturated fatty acid is constituted, content of polyunsaturated fatty acid is 25～52 weight %.
It is a further object to provide the phospholipid containing long-chain polyunsaturated fatty acid, which is prepared by the preparation method described in any one in claim 100～135.
Phospholipid containing long-chain polyunsaturated fatty acid of the invention, its phosphatidylcholine content are 70～85 weight %.
Phospholipid containing long-chain polyunsaturated fatty acid of the invention, in its fatty acid composition, content of polyunsaturated fatty acid is 15～60 weight %.
Phospholipid containing long-chain polyunsaturated fatty acid of the invention, in its fatty acid composition, content of polyunsaturated fatty acid is 25～52 weight %.
Phospholipid containing long-chain polyunsaturated fatty acid of the invention, the long-chain polyunsaturated fatty acid is containing two or more double bond and carbon number is 18～22 straight chain fatty acid.
Phospholipid containing long-chain polyunsaturated fatty acid of the invention, the long-chain polyunsaturated fatty acid are at least one in eicosapentaenoic acid, docosahexenoic acid, clupanodonic acid and arachidonic acid.
It is a further object to provide the phosphatidylcholine containing long-chain polyunsaturated fatty acid, in its fatty acid composition, content of polyunsaturated fatty acid is 20～80 weight %.
Phosphatidylcholine containing long-chain polyunsaturated fatty acid of the invention, in its fatty acid composition, content of polyunsaturated fatty acid is 30～60 weight %.
Phosphatidylcholine containing long-chain polyunsaturated fatty acid of the invention, the long-chain polyunsaturated fatty acid is containing two or more double bond and carbon number is 18～22 straight chain fatty acid.
Phosphatidylcholine containing long-chain polyunsaturated fatty acid of the invention, the long-chain polyunsaturated fatty acid are at least one in eicosapentaenoic acid, docosahexenoic acid, clupanodonic acid and arachidonic acid.
Invention effect
The present invention mainly has the advantage that：(1) without the need for high purity long chain polyunsaturated fatty acid acry radical donor and highly purified phosphatidylcholine（Or LYSO-PHOSPHATIDYLCHOLINE LYSOPC）Make reaction raw materials, greatly reduce Costco Wholesale.(2) in product, long-chain polyunsaturated fatty acid and phosphatidylcholine content are significantly improved, and are realized the purpose of the access and phosphatidylcholine purification of long-chain polyunsaturated fatty acid in phospholipid, are obviously improved added value of product.
This method need not adopt lecithin in high purity（PC）With highly purified long-chain polyunsaturated fatty acid raw material, process is simple, in product, long-chain polyunsaturated fatty acid insertion rate is high and phosphatidylcholine content brings up to more than 70%, greatly improves value-added content of product.
Specific embodiment
Polyunsaturated fatty acid（PUFA）It is different according to the position for starting the 1st double bond from methyl end, ω -3 and omega 6 polyunsaturated fatty acid can be divided into.In omega-3 unsaturated fatty acid, two kinds of unsaturated fatty acids most important to human body are DHA and EPA.EPA is the english abbreviation of eicosapentaenoic acid, with the rubbish in cleaning blood vessel（Cholesterol and triglyceride）Function, be commonly called as " blood vessel scavenger ".DHA is the english abbreviation of docosahexenoic acid, has functions that vessel softening, nourishing the brain and improving intelligence, improves vision, is commonly called as " NAOHUANGJIN ".
The preparation method of the phospholipid containing long-chain polyunsaturated fatty acid
The preparation method of the phospholipid containing long-chain polyunsaturated fatty acid of the present invention, including：
（1）Raw material phospholipid, oils and fatss and solvent are contacted, adds enzyme preparation to be reacted；
（2）After removing solvent, immobilized enzyme is added to be reacted, it is characterised in that in step（1）There is the step of adding phosphoamide afterwards.
In the present invention, the long-chain polyunsaturated fatty acid is referred to containing two or more double bond and carbon number is 18～22 straight chain fatty acid, such as eicosapentaenoic acid（EPA）, docosahexenoic acid（DHA）, clupanodonic acid（DPA）And arachidonic acid（ARA）Deng.
In the present invention, the raw material phospholipid is the phospholipid of the phospholipid or animal origin of plant origin.At least one of the phospholipid of the plant origin in soybean phospholipid, rapeseed phosphatide, sunflower phosphatide, peanut phosphatide, Semen Sesami phospholipid or Semen Maydiss phospholipid.The phospholipid of the animal origin is egg yolk lecithin.Raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acid.
In the present invention, " without long-chain polyunsaturated fatty acid " to refer to and do not detect long-chain polyunsaturated fatty acid by the assay method described in the present invention.The phospholipid that " the raw material phospholipid " of the present invention is used as raw material in referring to reaction." phospholipid containing long-chain polyunsaturated fatty acid " of the present invention refers to the phospholipid prepared by the preparation method of the above-mentioned phospholipid containing long-chain polyunsaturated fatty acid.In the present invention, the oils and fatss contain 5～60 weight %, preferably 10～55 weight %, the long-chain polyunsaturated fatty acid of more preferably 20～45 weight %.In the specific embodiment of the present invention, long-chain polyunsaturated fatty acid of the oils and fatss containing 16.2 weight %, 22.7 weight %, 27 weight %, 28.6 weight %, 40 weight % or 50 weight %.
At least one of the oils and fatss in bathypelagic fish oil and microbial grease.At least one of the oils and fatss in tunny fish oil, herring oil, salmon fish oil, pilchard oil, docosahexenoic acid algae oil or arachidonic acid oil.
In the present invention, the solvent is ethanol solution, preferably 95% ethanol solution.
In a preferred embodiment of the invention, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：1～1：20, preferably 1：1～1：15, more preferably 1：2～1：10, more preferably 1：4～1：8.
In the specific embodiment of the present invention, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：1、1：2、1：4、1：5、1：8、1：10、1：15.
In a preferred embodiment of the invention, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：2～1：100, preferably 1：5～1：80, more preferably 1：8～1：40, more preferably 1：10～1：30, particularly preferably 1：12～1：20, most preferably 1：12～1：18.
In the specific embodiment of the present invention, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：5、1：8、1：10、1：12、1：18、1：20、1：30、1：40.
In a preferred embodiment of the invention, step（1）Described in enzyme preparation be resolvase form.Step（1）Described in enzyme preparation be selected from E.C. 3.1.1.32, at least one in phospholipase A2 or lipase.
In a preferred embodiment of the invention, step（1）Described in enzyme preparation derive from aspergillus niger（Aspergillus
niger）, antarctic candida（Candida antarctic）, fold candida（Candida rugosa）, Candida cylindracea（Candida
cylindracea）, thermophilic fungal（Thermomyces lanuginosus）, rhizomucor miehei（Rhizomucor miehei）, rice black wool mould（Mucor
miehei）, Rhizopus delemar（Rhizopus delemar）, Rhizopus oryzae（Rhizopus oryzae）, Pseudomonas（Pseudononas sp. ）In at least one.
In a preferred embodiment of the invention, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 10：1～200：1, preferably 20：1～100：1, more preferably 40：1～80：1, it is further 50：1～60：1.
In the specific embodiment of the present invention, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 20：1、30：1、45：1、55：1、60：1、80：1、100：1.
In a preferred embodiment of the invention, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 100 weight portion～800 weight portions, preferably 200 weight portion～600 weight portions, more preferably 250 weight portion～350 weight portions.
In the specific embodiment of the present invention, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 100 weight portions, 200 weight portions, 267 weight portions, 300 weight portions, 350 weight portions, 400 weight portions, 600 weight portions.
In the present invention, the step（1）Reaction condition be not particularly limited, as long as the purpose of the present invention can be realized.For example can carry out at 30～40 DEG C, such as 35 DEG C.Response time is, for example, 1～10 hour, preferably 2～6 hours.
After adding phosphoamide to be reacted, crystallization treatment can be carried out at 0～4 DEG C, sub-argument goes out filtrate.The time of crystallization treatment is, for example, 1～6 hour, preferably 2～4 hours.Subsequently by filtrate by distillation（For example rotate）Remove solvent.
In the present invention, the step（2）It is to carry out at 60～80 DEG C in temperature.Response time is, for example, 2～10 hours, preferably 4～8 hours.The step（2）Reacted under evacuation or protective gas atmosphere.Evacuation is, for example, 0.1～5KPa.The step（2）Protective gas atmosphere be inert gas atmosphere, e.g. nitrogen atmosphere.
In a preferred embodiment of the invention, the step（2）Immobilized enzyme be the lipase or phospholipase that are fixed on macroporous adsorbent resin or deacidite carrier at least one.The lipase or phospholipase are the lipases or phospholipase of the micro-organisms in mucor, Penicillium, aspergillus, Rhizopus, thermophilic fungal category, Pseudomonas and candida mycoderma source.The immobilized enzyme can pass through commercially available, for example, novozyme
435th, Lipozyme RM IM, or by CN201310307403.6 and document Vikbjerg, Huiling Mu,
Xuebing Xu, Synthesis of structured phospholipids by
immobilized phospholipase A2Catalyzed acidolysis, Journal of Biotechnology, 2007,128：Method described in 545～554 grades is obtained, such as immobilized PLA1, immobilized phospholipase A2 etc..
In a preferred embodiment of the invention, with weight ratio meter, the addition total amount of the raw material phospholipid and the oils and fatss is 10 with the weight ratio of the immobilized enzyme：1～200：1, preferably 20：1～150：1, more preferably 40：1～100：1, it is further 50：1～60：1.
In the specific embodiment of the present invention, with the weight ratio of the additions total amount of weight ratio meter, the raw material phospholipid and the oils and fatss and the immobilized enzyme for 25：1、29：1、30：1、50：1、55：1、60：1、67：1、100：1.
In the present invention, the step（2）Middle addition water absorbing agent.The addition of the water absorbing agent suitably can determine, with the weight ratio of the addition total amount of weight ratio meter, the raw material phospholipid and the oils and fatss and the water absorbing agent for 10：1～50：1, preferably 15：1～40：1.For example with weight ratio meter, the addition total amount of the raw material phospholipid and the oils and fatss is 16 with the weight ratio of the water absorbing agent：1、17：1、22：1、30：1.
At least one of the water absorbing agent in molecular sieve or hydroscopic high-molecular resin.The molecular sieve is, for example, 3A molecular sieves.At least one of the hydroscopic high-molecular resin in acrylamide and acrylic acid salt crosslinking copolymerization thing and starch grafted acrylate crosslinking copolymerization thing.
In the present invention, the phosphatidylcholine content of the raw material phospholipid be 15～60 weight %, for example, 16 weight %, 18 weight %, 22 weight %, 60 weight %.
After reaction terminates, immobilized enzyme is filtered to remove by conventional method, adds solvent（Such as acetone etc.）Washed, then by centrifugal treating（For example under 8000～10000r/min）.
Prepared by the preparation method of the phospholipid containing long-chain polyunsaturated fatty acid of the invention described above, the phospholipid containing long-chain polyunsaturated fatty acid can be prepared.
The phospholipid containing long-chain polyunsaturated fatty acid prepared by the preparation method of the phospholipid containing long-chain polyunsaturated fatty acid of the invention described above, its phosphatidylcholine content be 70～85 weight %, for example, 70 weight %, 72 weight %, 73 weight %, 74 weight %, 78 weight %, 81 weight %, 82 weight %, 85 weight %.
The phospholipid containing long-chain polyunsaturated fatty acid prepared by the preparation method of the phospholipid containing long-chain polyunsaturated fatty acid of the invention described above, in its fatty acid composition, content of polyunsaturated fatty acid is 20～80 weight %, preferably 30～60 weight %, for example, 24.7 weight %, 30 weight %, 38 weight %, 41.5 weight %, 44.7 weight %, 45.9 weight %, 63.3 weight %, 76.3 weight %.As the raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acid, therefore the content of polyunsaturated fatty acid of the phospholipid containing long-chain polyunsaturated fatty acid of the present invention can also regard content of polyunsaturated fatty acid increase rate as.
Phospholipid containing long-chain polyunsaturated fatty acid prepared by the preparation method of the phospholipid containing long-chain polyunsaturated fatty acid of the present invention by the invention described above, in its fatty acid composition, it is 38.7 weight % that DPA is 12.9 weight %, DHA；It is 21.6 weight % that EPA is 5.5 weight %, DHA；It is 27.3 weight % that EPA is 3.8 weight %, DHA；ARA is 40.2 weight %；It is 32.4 weight % that EPA is 28.6 weight %, DHA；It is 20.7 weight % that DPA is 8.9 weight %, DHA；It is 30.2 weight % that EPA is 8.6 weight %, DHA；It is 25.2 weight % that EPA is 2.7 weight %, DHA；Or EPA is that 7.6 weight %, DHA are 12.4 weight %.
Phospholipid containing long-chain polyunsaturated fatty acid
The present invention the phospholipid containing long-chain polyunsaturated fatty acid, its phosphatidylcholine content be 70～85 weight %, for example, 70 weight %, 72 weight %, 73 weight %, 74 weight %, 78 weight %, 81 weight %, 82 weight %, 85 weight %.
The phospholipid containing long-chain polyunsaturated fatty acid of the present invention, in its fatty acid composition, content of polyunsaturated fatty acid is 15～60 weight %, preferably 25～52 weight %, for example, 20 weight %, 27.1 weight %, 27.9 weight %, 29.6 weight %, 31.1 weight %, 38.8 weight %, 40.2 weight %, 51.6 weight %, 61 weight %.As the raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acid, therefore the content of polyunsaturated fatty acid of the phospholipid containing long-chain polyunsaturated fatty acid of the present invention can also be regarded as relative to raw material phospholipid, the content of polyunsaturated fatty acid increase rate of product phospholipid.In the present invention, the long-chain polyunsaturated fatty acid is referred to containing two or more double bond and carbon number is 18～22 straight chain fatty acid, such as eicosapentaenoic acid（EPA）, docosahexenoic acid（DHA）, clupanodonic acid（DPA）And arachidonic acid（ARA）Deng.
The phospholipid containing long-chain polyunsaturated fatty acid of the present invention, in its fatty acid composition, it is 38.7 weight % that DPA is 12.9 weight %, DHA；It is 21.6 weight % that EPA is 5.5 weight %, DHA；It is 27.3 weight % that EPA is 3.8 weight %, DHA；ARA is 40.2 weight %；It is 32.4 weight % that EPA is 28.6 weight %, DHA；It is 20.7 weight % that DPA is 8.9 weight %, DHA；It is 30.2 weight % that EPA is 8.6 weight %, DHA；It is 25.2 weight % that EPA is 2.7 weight %, DHA；Or EPA is that 7.6 weight %, DHA are 12.4 weight %.
Phosphatidylcholine containing long-chain polyunsaturated fatty acid
Phosphatidylcholine is separated from the phospholipid containing long-chain polyunsaturated fatty acid of the present invention（Separation method reference literature Reddy,
J. R. C., Vijeeta, T., Karuna, M. S. L., Rao, B. V. S., & Prasad, R. B. N.
(2005), Lipase-Catalyzed Preparation of Palmitic
And Stearic Acid-rich Phosphatidylcholine, Journal
of the American Oil Chemists’ Society,
82(10), 727–730）, isolated phosphatidylcholine（The phosphatidylcholine containing long-chain polyunsaturated fatty acid of phosphatidylcholine otherwise referred to as of the invention or the present invention）, isolated phosphatidylcholine is carried out into pretreatment and determination of fatty acid then, method respectively refers to standard GB/T/T
17376-2008 and GB/T 24894-2008.
In the fatty acid composition of above-mentioned phosphatidylcholine, content of polyunsaturated fatty acid is 20～80 weight %, preferably 30～60 weight %, for example, 24.7 weight %, 30 weight %, 38 weight %, 41.5 weight %, 44.7 weight %, 45.9 weight %, 63.3 weight %, 76.3 weight %.As the raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acid, therefore the content of polyunsaturated fatty acid of the phosphatidylcholine of the present invention can also regard as relative to raw material phospholipid, the content of polyunsaturated fatty acid increase rate of isolated phosphatidylcholine.
The phosphatidylcholine containing long-chain polyunsaturated fatty acid of the present invention, in its fatty acid composition, it is 45.2 weight % that DPA is 18.1 weight %, DHA；It is 30.6 weight % that EPA is 7.4 weight %, DHA；It is 35.6 weight % that EPA is 9.1 weight %, DHA；ARA is 57.1 weight %；It is 40.1 weight % that EPA is 36.2 weight %, DHA；It is 28.7 weight % that DPA is 12.8 weight %, DHA；It is 35.6 weight % that EPA is 10.3 weight %, DHA；It is 26.8 weight % that EPA is 3.2 weight %, DHA；Or EPA is that 8.9 weight %, DHA are 15.8 weight %.
The features described above that the present invention is mentioned, or the feature that embodiment is mentioned can be in any combination.All features disclosed in this case description can be used in combination with any combinations thing form, each feature disclosed in description, can be replaced with any alternative characteristics for providing identical, impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only the general example of impartial or similar features.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise all of percent, ratio, ratio or number be by weight.
The unit in percent weight in volume in the present invention is well-known to those skilled in the art, for example, refer to the weight of the solute in 100 milliliters of solution.
Unless otherwise defined, all specialties used in text and scientific words and same meaning familiar to one skilled in the art institute.Additionally, any similar to described content or impartial method and material are all can be applicable in the inventive method.Preferable implementation described in text is only presented a demonstration with material and is used.
Embodiment
E.C. 3.1.1.32, phospholipase A2, lipase TLL used in following embodiments, lipase Candida
Cylindracea, lipase pseudononas sp., lipase Rhizopus oryzae are respectively the Lecitase of commercialization
Ultra, Lecitase 10L, Lipozyme TL 100L,
Lipase AY“Amano” 30、Lipase from Pseudononas sp.、Lipase
F-AP 15。
Immobilized-lipase Novozyme 435 used in following embodiments is purchased from Novozymes companies, immobilized PLA1 and immobilized phospholipase A2 are obtained in existing publication method, such as CN201310307403.6 and document Vikbjerg, Huiling Mu, Xuebing
Xu, Synthesis of structured phospholipids by
immobilized phospholipase A2Catalyzed acidolysis, Journal of Biotechnology, 2007,128：545～554 etc..
In following embodiments, the raw material sources for using are as follows：Powdered soybean phospholipid and egg yolk lecithin are purchased from Beijing Merya's Lecithin Co., Ltd.；DHA algal oil and ARA oils and fatss are purchased from Xiamen Huisheng Biological Co., Ltd.；Tunny fish oil, herring oil and pilchard oil are purchased from Norsk
Hydro A/S。
Phosphatidylcholine（PC）The assay method of content is determined according to the method in standard GB/T/T 21493-2008；The assay method of lipoid fatty acid composition is determined according to the method in standard GB/T/T 24894-2008, and wherein preprocess method is carried out according to the method for standard GB/T/T 17376-2008；PC is separated from product by PC fatty acid composition measurings first, separation method reference literature Reddy, J. R. C., Vijeeta, T., Karuna, M.
S. L., Rao, B. V. S., ＆ Prasad, R. B. N. (2005), Lipase-Catalyzed Preparation of Palmitic and Stearic
Acid-rich Phosphatidylcholine, Journal of the
American Oil Chemists’ Society,
82 (10), 727-730, pretreatment and determination of fatty acid are then carried out, method respectively refers to standard GB/T/T
17376-2008 and GB/T 24894-2008.
Embodiment 1
Weigh 15g powdered soybean phospholipids（PC contents are 18%）, 30g DHA algal oils（DHA 35%, DPA 15%）With 95% ethanol of 450mL in reaction bulb, after stirring under the conditions of 35 DEG C, add 0.5mL E.C. 3.1.1.32s and 1mL lipase TL L, after reaction 4h, 90g phosphoamides are added, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 2h, filtrate is filtered to isolate, after 70 DEG C of revolvings remove ethanol, 1.5g novozyme is added
435, extracting vacuum reacts 6h under the conditions of 70 DEG C to 5KPa ~ 0.1KPa.After the completion of question response, Novozyme 435 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 10000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 1.
Table 1
。
Comparing embodiment 1
（1）Weigh 15g powdered soybean phospholipids（PC contents are 18%）With 95% ethanol of 150mL in reaction bulb, after stirring under the conditions of 35 DEG C, 0.5mL E.C. 3.1.1.32s are added, after reaction 4h, 70 DEG C of revolvings remove ethanol；（2）Weigh 30g DHA algal oils（DHA 35%, DPA 15%）With 95% ethanol of 300mL in reaction bulb, 1mL lipase TL L are added, after reaction 4h, 90g phosphoamides is added, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 2h, filter to isolate filtrate, 70 DEG C of revolvings remove ethanol；（3）By step（1）And step（2）After products therefrom mixing, 1.5g novozyme are added
Extracting vacuum 5KPa ~ 0.1KPa under the conditions of 435,70 DEG C, reacts 6h.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 10000r/min is centrifuged and obtains acetone insoluble matter, and testing result is as shown in table 2 below.
Table 2
。
Comparing embodiment 2
Weigh 15g powdered soybean phospholipids（PC contents are 18%）, 30g DHA algal oils（DHA 35%, DPA 15%）With 95% ethanol of 450mL in reaction bulb, after stirring under the conditions of 35 DEG C, 0.5mL E.C. 3.1.1.32s and 1mL lipase TL L are added, after reaction 4h, 70 DEG C of revolvings remove ethanol；1.5g novozyme 435 are added, extracting vacuum reacts 6h under the conditions of 70 DEG C to 5KPa ~ 0.1KPa.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 10000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 3.
Table 3
。
Comparing embodiment 3
Weigh 5g soybean lecithins（PC contents are 60%）With 15g DHA concentrated solutions（DPA and DHA total contents are 97.2%）, 2g novozyme 435 are added, extracting vacuum reacts 6h under the conditions of 70 DEG C to 5KPa ~ 0.1KPa.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 10000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 4.
Table 4
。
Embodiment 2
Weigh 10g egg yolk lecithin（PC contents are 60%）, 100g tunny fish oils（DHA 18.2%, EPA 4.5%）With 95% ethanol of 2.2L in reaction bulb, after stirring under the conditions of 35 DEG C, 1mL E.C. 3.1.1.32s and 1mL lipase Candida are added
Cylindracea, after reaction 2h, adds 440g phosphoamides, until completely dissolved, reaction bulb is placed in 4 DEG C of environment and crystallizes 4h, filter to isolate filtrate, after revolving removes ethanol, the immobilized PLA1 and 5g 3A molecular sieves of 2g are added, 60 DEG C of reaction 8h under the conditions of nitrogen charging.After the completion of question response, immobilized PLA1 is filtered to remove（Recyclable recycling）, addition 20mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 5.
Table 5
。
Embodiment 3
Weigh 10g powdered soybean phospholipids（PC contents are 18%）, 80g herring oils（DHA 20.1%, EPA 8.5%）With 95% ethanol of 720mL in reaction bulb, after stirring under the conditions of 35 DEG C, add 0.8mL phospholipase A2s and 1.2mL lipase Pseudononas sp., after reaction 4h, 270g phosphoamides are added, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 4h, filtrate is filtered to isolate, after revolving removes ethanol, 1.8g immobilized phospholipase A2 and 3g hydroscopic high-molecular resins is added（Acrylamide and acrylic acid salt crosslinking copolymerization thing）, 70 DEG C of reaction 8h under the conditions of nitrogen charging.After the completion of question response, immobilized phospholipase A2 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 6.
Table 6
。
Embodiment 4
Weigh 20g powdered soybean phospholipids（PC contents are 16%）, 100g ARA oils and fatss（ARA 40%）With 95% ethanol of 2.16L in reaction bulb, after stirring under the conditions of 35 DEG C, add 0.5mL E.C. 3.1.1.32s and 1mL lipase Rhizopus oryzae, after reaction 3h, 240g phosphoamides are added, until completely dissolved, reaction bulb is placed in 2 DEG C of environment and crystallizes 3h, filtrate is filtered to isolate, after revolving removes ethanol, 2g is added
Novozyme 435, to 5KPa ~ 0.1KPa, 80 DEG C are reacted 4h to extracting vacuum.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 40mL washing with acetones 3 times, 10000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 7.
Table 7
。
Embodiment 5
Weigh 20g powdered soybean phospholipids（PC contents are 22%）, 160g pilchard oils（DHA 16.8%, EPA 10.2%）With 95% ethanol of 900mL in reaction bulb, after stirring under the conditions of 35 DEG C, 1mL E.C. 3.1.1.32s and 2mL lipase TL L are added, after reaction 4h, add 480g phosphoamides, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 4h, filter to isolate filtrate, after revolving removes ethanol, 1.8g Novozyme 435 are added, to 5KPa ~ 0.1KPa, 75 DEG C are reacted 4h to extracting vacuum.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 35mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 8.
Table 8
。
Embodiment 6
Weigh 15g powdered soybean phospholipids（PC contents are 18%）, 15g DHA algal oils（DHA 35%, DPA 15%）With 95% ethanol of 360mL in reaction bulb, after stirring under the conditions of 35 DEG C, add 0.1mL E.C. 3.1.1.32s and 0.2mL lipase TL L, after reaction 6h, 30g phosphoamides are added, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 5h, filtrate is filtered to isolate, after 70 DEG C of revolvings remove ethanol, 0.45g novozyme is added
435, extracting vacuum reacts 8h under the conditions of 70 DEG C to 5KPa ~ 0.1KPa.After the completion of question response, novozyme 435 is filtered to remove（Recyclable recycling）, addition 15mL washing with acetones 3 times, 10000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 9.
Table 9
。
Embodiment 7
Weigh 10g egg yolk lecithin（PC contents are 60%）, 150g tunny fish oils（DHA 18.2%, EPA 4.5%）With 95% ethanol of 4.8L in reaction bulb, after stirring under the conditions of 35 DEG C, 1mL E.C. 3.1.1.32s and 7mL lipase TLL are added, after reaction 2h, 960g phosphoamides is added, until completely dissolved, reaction bulb is placed in 2 DEG C of environment and crystallizes 4h, filter to isolate filtrate, after revolving removes ethanol, the immobilized PLA1 and 10g 3A molecular sieves of 5.6g are added, 60 DEG C of reaction 8h under the conditions of nitrogen charging.After the completion of question response, immobilized PLA1 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 10.
Table 10
。
Embodiment 8
Weigh 10g powdered soybean phospholipids（PC contents are 18%）, 40g herring oils（DHA 20.1%, EPA 8.5%）With 95% ethanol of 2L in reaction bulb, after stirring under the conditions of 35 DEG C, add 0.5mL phospholipase A2s and 0.75mL lipase Pseudononas sp., after reaction 5h, 175g phosphoamides are added, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 4h, filtrate is filtered to isolate, after revolving removes ethanol, 2g immobilized phospholipase A2 and 3g hydroscopic high-molecular resins is added（Starch grafted acrylate crosslinking copolymerization thing）, 65 DEG C of reaction 8h under the conditions of nitrogen charging.After the completion of question response, immobilized phospholipase A2 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 11.
Table 11
。
Embodiment 9
Weigh 10g egg yolk lecithin（PC contents are 60%）, 40g salmon fish oil（DHA9.5%, EPA6.7%）With 95% ethanol of 2L in reaction bulb, after stirring under the conditions of 35 DEG C, 0.5mL phospholipase A2s and 0.75mL lipase Rhizopus oryzae are added, after reaction 3h, add 175g phosphoamides, until completely dissolved, reaction bulb is placed in 0 DEG C of environment and crystallizes 4h, filter to isolate filtrate, after revolving removes ethanol, 2g immobilized phospholipase A2, extracting vacuum is added to react 6h under the conditions of 70 DEG C to 5KPa ~ 0.1KPa.After the completion of question response, immobilized phospholipase A2 is filtered to remove（Recyclable recycling）, addition 30mL washing with acetones 3 times, 8000r/min centrifugations obtain acetone insoluble matter.Testing result is shown in Table 11.
Table 12
。
The foregoing is only presently preferred embodiments of the present invention, it is not limited to the substantial technological context of the present invention, the substantial technological content of the present invention is broadly defined in the right of application, any technology entities that other people complete or method, if identical with defined in the right of application, also or a kind of equivalent change, among being considered to be covered by the right.
Claims (19)
1. a kind of preparation method of the phospholipid containing long-chain polyunsaturated fatty acid, including：
（1）Raw material phospholipid, oils and fatss and solvent are contacted, adds enzyme preparation to be reacted；
（2）After removing solvent, immobilized enzyme is added to be reacted, it is characterised in that in step（1）There is the step of adding phosphoamide afterwards.
2. preparation method according to claim 1, wherein, the raw material phospholipid does not contain long-chain polyunsaturated fatty acid.
3. the preparation method according to Claims 2 or 3, wherein, the raw material phospholipid is the phospholipid of the phospholipid or animal origin of plant origin, it is preferred that at least one of the phospholipid of the plant origin in soybean phospholipid, rapeseed phosphatide, sunflower phosphatide, peanut phosphatide, Semen Sesami phospholipid or Semen Maydiss phospholipid, the phospholipid of the preferably animal origin is egg yolk lecithin.
4. the preparation method according to any one in claims 1 to 3, wherein, long-chain polyunsaturated fatty acid of the oils and fatss containing 5～60 weight %, and/or at least one of the oils and fatss in bathypelagic fish oil and microbial grease, at least one of the preferred oils and fatss in tunny fish oil, herring oil, salmon fish oil, pilchard oil, docosahexenoic acid algae oil or arachidonic acid oil.
5. the preparation method according to any one in Claims 1 to 4, wherein, the solvent is ethanol solution.
6. the preparation method according to any one in Claims 1 to 5, wherein, with weight ratio meter, step（1）Described in raw material phospholipid and the oils and fatss addition ratio be 1：1～1：15, preferably 1：2～1：10, more preferably 1：4～1：8.
7. the preparation method according to any one in claim 1～6, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the solvent ratio be 1：5～1：80, preferably 1：8～1：40, more preferably 1：10～1：30, more preferably 1：12～1：18.
8. the preparation method according to any one in claim 1～7, wherein, step（1）Described in enzyme preparation be resolvase form, and/or described enzyme preparation be selected from E.C. 3.1.1.32, at least one in phospholipase A2 or lipase.
9. the preparation method according to any one in claim 1～8, wherein, with weight/volume（g/mL）Than meter, step（1）Described in raw material phospholipid and the oils and fatss addition total amount and the enzyme preparation ratio be 10：1～200：1, preferably 20：1～100：1, more preferably 40：1～80：1, more preferably 50：1～60：1.
10. the preparation method according to any one in claim 1～9, wherein, relative to 100 weight portion of addition total amount of the raw material phospholipid and the oils and fatss, with weight ratio meter, the addition of the phosphoamide is 100 weight portion～800 weight portions, preferably 200 weight portion～600 weight portions, more preferably 250 weight portion～350 weight portions.
11. preparation methoies according to any one in claim 1～10, wherein, the step（2）Immobilized enzyme be the lipase or phospholipase that are fixed on macroporous adsorbent resin or deacidite carrier at least one.
12. preparation methoies according to any one in claim 1～11, wherein, with the weight ratio of the addition total amount of weight ratio meter, the raw material phospholipid and the oils and fatss and the immobilized enzyme for 10：1～200：1, preferably 20：1～150：1.
13. preparation methoies according to any one in claim 1～12, wherein, the step（2）Reacted under evacuation or protective gas atmosphere, the step（2）Protective gas atmosphere be inert gas atmosphere.
14. preparation methoies according to any one in claim 1～13, wherein, the step（2）At least one of middle addition water absorbing agent, the preferably water absorbing agent in molecular sieve or hydroscopic high-molecular resin, preferably at least one of the hydroscopic high-molecular resin in acrylamide and acrylic acid salt crosslinking copolymerization thing and starch grafted acrylate crosslinking copolymerization thing.
15. preparation methoies according to any one in claim 1～14, wherein, the phosphatidylcholine content of the raw material phospholipid be 15～60 weight %, and/or the phosphatidylcholine content of the described phospholipid containing long-chain polyunsaturated fatty acid be 70～85 weight %.
16. according to the preparation method described in any one in claim 1～15, the long-chain polyunsaturated fatty acid is containing two or more double bond and carbon number is 18～22 straight chain fatty acid, and the preferably long-chain polyunsaturated fatty acid is at least one in eicosapentaenoic acid, docosahexenoic acid, clupanodonic acid and arachidonic acid.
17. according to the preparation method described in any one in claim 1～16, and in the fatty acid composition of the described phospholipid containing long-chain polyunsaturated fatty acid, content of polyunsaturated fatty acid is 15～60 weight %, preferably 25～52 weight %.
A kind of 18. phospholipid containing long-chain polyunsaturated fatty acid, which is prepared by the preparation method described in any one in claim 1～17.
A kind of 19. phosphatidylcholines containing long-chain polyunsaturated fatty acid, in its fatty acid composition, content of polyunsaturated fatty acid is 20～80 weight %, preferably 30～60 weight %, and/or the long-chain polyunsaturated fatty acid is containing two or more double bond and carbon number is 18～22 straight chain fatty acid, the preferably long-chain polyunsaturated fatty acid is at least one in eicosapentaenoic acid, docosahexenoic acid, clupanodonic acid and arachidonic acid.
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