CN105039441B - It is conjugated the non-aqueous enzymatic synthesis of alpha linolenic acid isomers - Google Patents
It is conjugated the non-aqueous enzymatic synthesis of alpha linolenic acid isomers Download PDFInfo
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Abstract
The non-aqueous enzymatic synthesis of alpha linolenic acid isomers is conjugated, including：(1) 4mL 50mM, pH6.8 phosphate buffers, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of processing 20min are added in every gram of Lactobacillus casei CGMCC 1.574；Ultrasonic power 200W, often be ultrasonically treated 5s rest 30s, be ultrasonically treated 2 times, centrifuge to obtain permeability thalline；(2) washed with 50mM, pH5.8 phosphate buffer, collect thalline, every gram of permeability wet thallus is resuspended in 20mL50mM, pH5.8 phosphate buffer, add the Tween 80 of bacteria suspension volume 0.5 2.0%, mix, 4 40 DEG C of 2 6h of processing, are collected by centrifugation thalline, thalline must be coated by being freeze-dried；(3) every gram of coating thalline is added in 600mL n-hexanes, stirred, add the alpha linolenic acid of n-hexane volume 0.1 0.3%, 4 40 DEG C of 1 12h of reaction；(4) coating thalline is centrifuged, reclaims n-hexane, product isc9,t11,c15 conjugation alpha linolenic acid isomers.Reaction time of the invention is short, and coating thalline is reusable repeatedly, and yield significantly improves, and non-environmental-pollution, production cost significantly reduces.
Description
Technical field
The invention belongs to biomedicine technical field.
Background technology
Conjugation alpha-linolenic acid is the conjugated isomers of alpha-linolenic acid, is the general designation of one group of CLnA, has more
Kind position isomery and geometric isomer, such as：c9,t11,c15- be conjugated alpha-linolenic acid andt10,c12,c15- conjugation alpha-linolenic acids are different
Structure body etc..Research shows that being conjugated alpha-linolenic acid has the physiological functions such as anticancer, prevention of arterial atherosis, fat-reducing, and its physiology
Activity has isomers specificity, such as：c9,t11,c15- conjugation alpha-linolenic acid isomers has the function that anticancer.In nature
In, conjugation alpha-linolenic acid be primarily present in the milk of ruminant and the fat of meat, mainly byc9,t11,c15- is conjugated α-flax
The isomers such as acid are formed, but its content is generally very low, can not meet the development and application for the purpose of health care and medical treatment.
To realize a large amount of preparations of conjugation alpha-linolenic acid, people are carried out to microbial fermentation synthesis of conjugate alpha-linolenic acid
Some researchs.At present, microbial fermentation is carried out in aqueous, because added fermentation substrate-alpha-linolenic acid is fat-soluble
Material, therefore needed before alpha-linolenic acid addition zymotic fluid through emulsification treatment, and addition is restricted, and is conjugated from zymotic fluid
Alpha-linolenic acid product need to extract through organic solvent, and whole production technology has fermentation period length, production cost is high, it is scarce to yield poorly
Point.Patent of invention CN200410060670.9 reports to be synthesized by Lactobacillus casei CGMCC 1.574 come specific biologicalc9,t11,cThe method that 15- is conjugated alpha-linolenic acid isomers, substrate addition are 1mg/mL, and fermentation time is up to 42h, and substrate turns
Rate is less than 50%, and thalline can not reuse.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, to propose a kind of non-aqueous zymetology for being conjugated alpha-linolenic acid isomers
Synthetic method, significantly shorten generated time, improve yield and conversion ratio, reduce production cost.
The present invention comprises the following steps.
（1）The permeabilized treatment of thalline：Collect the Lactobacillus casei in exponential phase（Lactobacillus casei）The thalline of CGMCC 1.574, through bacteriolyze ferment treatment（The bacteriolyze ferment treatment be added in every gram of wet thallus 4mL 50mM,
PH6.8 phosphate buffers, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of processing 20min）, then through ultrasonic wave at
Reason, ultrasonic power 200W, often be ultrasonically treated 5s rest 30s, be ultrasonically treated 2 times, centrifugation obtain permeability thalline.
（2）Thalline Cotton seeds：After permeability thalline is washed with 50mM, pH5.8 phosphate buffer, bacterium is collected by centrifugation
Body, the phosphate buffer for being resuspended in 20mL50mM, pH5.8 by every gram of permeability wet thallus are fallen into a trap, by permeability wet thallus weight
It is suspended from phosphate buffer, adds bacteria suspension percent by volume 0.5-2.0% Tween-80, is well mixed, at 4-40 DEG C
2-6h is managed, thalline is collected by centrifugation, obtains being coated thalline after thalline is freeze-dried.
（3）Non-aqueous Enzyme catalyzed synthesis：600mL n-hexanes are added to by every gram of coating thalline to fall into a trap, and coating thalline is added
Into n-hexane, stir, add n-hexane percent by volume 0.1-0.3% alpha-linolenic acid, react 1- at 4-40 DEG C
12h。
（4）Collection of products：Coating thalline is centrifuged, hexane solution is evaporated under reduced pressure, reclaims n-hexane, product
Forc9,t11,c15- is conjugated alpha-linolenic acid isomers.
Step of the present invention（2）The addition of middle Tween-80 is preferably the 0.75- of bacteria suspension percent by volume
1.5%。
Step of the present invention（2）Middle treatment temperature is preferably 4-25 DEG C.
Step of the present invention（2）Middle processing time is preferably 3-5h.
Step of the present invention（3）In be preferably added to n-hexane percent by volume 0.125-0.25% alpha-linolenic acid,
2-6h is reacted at 15-30 DEG C.
Step of the present invention（4）Middle coating thalline can be repeated for non-aqueous Enzyme catalyzed synthesisc9,t11,c15- is conjugated
Alpha-linolenic acid isomers.
Lactobacillus casei used in the present invention（Lactobacillus casei）CGMCC 1.574, it is Chinese common micro- life
Thing culture presevation administrative center（CGMCC）Preservation strain, numbering 1.574, the bacterial strain can produce specific linoleic acid isomery
Enzyme, can be by polyunsaturated fatty acidc9,c12 double-bond isomerisms intoc9,t11 conjugated double bonds, by biological isomerization can by α-
Leukotrienes changes intoc9,t11,c15- is conjugated alpha-linolenic acid isomers.At present, definite catalysis has not been separated to from the bacterium also
The isomerase sterling of activity.Therefore, the isomerization reaction is probably to be completed by multi-enzyme system co-catalysis.
With optimal conditions, by the non-aqueous enzymatic synthesis, the n-hexane of the alpha-linolenic acid containing percent by volume 0.15%
It is synthesized after solution reactionc9,t11,cThe content of 15- conjugation alpha-linolenic acid isomers is percent by volume 0.139%, α-flax
The conversion ratio of acid is 92.7%.Step of the present invention（4）Middle coating thalline can be repeated for step（3）Non-aqueous enzymatic
Synthesisc9,t11,c15- is conjugated alpha-linolenic acid isomers, and after coating thalline is reused five times, its catalytic activity is still greater than 90%.
Carried out in aqueous for existing microbial fermentation synthesis of conjugate alpha-linolenic acid, whole production technology has hair
The ferment cycle is grown, and extraction conjugation alpha-linolenic acid product is difficult from zymotic fluid, and thalline can not be reused, and production cost is high, yield
Low shortcomings.The present invention proposes catalyzes and synthesizes conjugation α-Asia by alpha-linolenic acid in organic solvent by microbial cells
The new method of numb acid.This method by lysozyme and ultrasonication Lactobacillus casei CGMCC 1.574, is keeping thalline first
While integrality, somatic cells wall and membrane passage are improved, on the one hand may insure thin in follow-up Cotton seeds
The linoleate isomerase surface energy of intracellular forms complete coatings, on the other hand can improve reaction substrate alpha-linolenic acid and product
The speed of alpha-linolenic acid disengaging thalline is conjugated, high concentration substrate and product is reduced to the inhibitory action of catalytic reaction, greatly shortens
Reaction time.
The present invention is coated from Tween-80 to thalline, an oleic acid moieties is contained in Tween-80 molecule, oleic acid is
The analogue of thalline Linoleic acid isomery zymolyte, when thalline is coated, molecule can be formed in the catalytic active center of enzyme
Trace, make thalline catalytic active center conformation of enzyme after freeze-drying constant, higher catalysis can be kept in organic solvent
Activity.Meanwhile the present invention be incorporated in during thalline Cotton seeds from suitable phosphate buffer, Tween-80 concentration and
Temperature, time are coated, makes gained coating thalline that still there is higher catalytic capability in organic solvent.Enzyme in thalline after coating
Heat endurance improves, and 4h is only needed per the batch reaction time, much smaller than time a couple of days needed for Batch fermentation in traditional aqueous.
Gained coating thalline of the invention has higher catalytic activity in n-hexane, only need to centrifuge coating thalline, will just
Hexane solution is evaporated under reduced pressure, and reclaims n-hexane, you can obtain productc9,t11,c15- is conjugated alpha-linolenic acid.Centrifugation gained
Coating thalline can be repeated several times for non-aqueous Enzyme catalyzed synthesisc9,t11,c15- is conjugated alpha-linolenic acid isomers, significantly shorten
Production time, yield is improved, reduce production cost.In addition, n-hexane is the conventional organic solvent of edible oil and fat industry, this
It also ensure that obtained by the present inventionc9,t11,c15- is conjugated the safety in utilization of alpha-linolenic acid.
The present invention has the following advantages that compared with prior art.
The present invention is directly coated processing to thalline, is used for catalytic reaction as immobilised enzymes, on the one hand reduces enzyme
The cost isolated and purified, avoid the loss of enzyme activity in extraction；On the other hand multi-enzyme system can be wrapped together, made whole
Individual course of reaction is smooth；Furthermore it is coated thalli granule and is more than coating enzyme, is easy to separate from reaction solution, and
Filtration resistance is smaller, and therefore, coating thalline is more suitable for the column reactor for successive reaction.
The present invention carries out catalytic reaction in organic solvent by being coated thalline, avoids in traditional aqueous reaction system
Middle substrate alpha-linolenic acid solubility is low and product conjugation alpha-linolenic acid extracts the problem of difficult.It is coated the heat endurance of enzyme in thalline
Improve, 4h is only needed per the batch reaction time, much smaller than time a couple of days for fermenting required in traditional aqueous.Coating thalline repeats
Using multiple, product yield, and non-environmental-pollution are significantly improved, production cost can be significantly reduced；And traditional aqueous system is sent out
Thalline in zymotic fluid is used only once, and production cost is high, and Wastewater treating is costly, easily pollutes environment.
Embodiment
The present invention is further described by following examples.
Embodiment 1.
By the Lactobacillus casei of activation（Lactobacillus casei）The strains of CGMCC 1.574 are inoculated into MRS culture mediums
In, 20h is cultivated at 30 DEG C, 5000g centrifugations 10min collects thalline, 40mL50mM phosphate is added into 10 grams of wet thallus and is delayed
Fliud flushing（pH6.8）, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking-up,
Through power 200W ultrasonications, often it is ultrasonically treated 5s and rests 30s, be ultrasonically treated 2 times, 5000g centrifugations 10min obtains permeability
Thalline.
Permeability thalline 40mL50mM phosphate buffers（pH5.8）After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 2mL Tween-80s and be well mixed, stirred in 4 DEG C
Mix processing 4h, 6000g centrifugation 10min and collect thalline, thalline is freeze-dried after -80 DEG C of pre-freezes, obtains being coated thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, stirred, 0.9mL alpha-linolenic acids are added, 25
Stirring reaction 4h at DEG C.6000g centrifuge coating thalline, hexane solution is evaporated under reduced pressure at 40 DEG C, recovery just oneself
Alkane, 0.9mL products are obtained, whereinc9,t11,cThe content of 15- conjugation alpha-linolenic acids is 92.7%.The coating thalline that will be collected into
Above-mentioned synthetic reaction is recycled and reused for, when being used continuously five times in productc9,t11,c15- conjugation alpha-linolenic acid content be
90.6%。
Embodiment 2.
By the Lactobacillus casei of activation（Lactobacillus casei）The strains of CGMCC 1.574 are inoculated into MRS culture mediums
In, 18h is cultivated at 30 DEG C, 4000g centrifugations 5min collects thalline, 40mL50mM phosphate-buffereds are added into 10 grams of wet thallus
Liquid（pH6.8）, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking-up, warp
Power 200W ultrasonications, often it is ultrasonically treated 5s and rests 30s, be ultrasonically treated 2 times, 5000g centrifugations 5min obtains permeability bacterium
Body.
Permeability thalline 30mL50mM phosphate buffers（pH5.8）After washing, 5000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 4mL Tween-80s and be well mixed, stirred in 4 DEG C
Mix processing 2h, 5000g centrifugation 10min and collect thalline, thalline is freeze-dried after -80 DEG C of pre-freezes, obtains being coated thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, stirred, 1.8mL alpha-linolenic acids are added, 40
Stirring reaction 12h at DEG C.6000g centrifuge coating thalline, hexane solution is evaporated under reduced pressure at 40 DEG C, recovery just oneself
Alkane, 1.8mL products are obtained, whereinc9,t11,cThe content of 15- conjugation alpha-linolenic acids is 82.7%.The coating thalline that will be collected into
Above-mentioned synthetic reaction is recycled and reused for, when being used continuously five times in productc9,t11,c15- conjugation alpha-linolenic acid content be
72.8%。
Embodiment 3.
By the Lactobacillus casei of activation（Lactobacillus casei）The strains of CGMCC 1.574 are inoculated into MRS culture mediums
In, 18h is cultivated at 30 DEG C, 4000g centrifugations 5min collects thalline, 40mL50mM phosphate-buffereds are added into 10 grams of wet thallus
Liquid（pH6.8）, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking-up, warp
Power 200W ultrasonications, often it is ultrasonically treated 5s and rests 30s, be ultrasonically treated 2 times, 5000g centrifugations 5min obtains permeability bacterium
Body.
Permeability thalline 40mL50mM phosphate buffers（pH5.8）After washing, 5000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 1mL Tween-80s and be well mixed, stirred in 4 DEG C
Mix processing 6h, 5000g centrifugation 10min and collect thalline, thalline is freeze-dried after -80 DEG C of pre-freezes, obtains being coated thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, stirred, 0.6mL alpha-linolenic acids are added, at 4 DEG C
Lower stirring reaction 6h.6000g centrifuges coating thalline, and hexane solution is evaporated under reduced pressure at 40 DEG C, reclaims n-hexane,
0.6mL products are obtained, whereinc9,t11,cThe content of 15- conjugation alpha-linolenic acids is 92.2%.The coating thalline being collected into is repeated
For above-mentioned synthetic reaction, during continuous use five times in productc9,t11,cThe content of 15- conjugation alpha-linolenic acids is 86.9%.
Embodiment 4.
By the Lactobacillus casei of activation（Lactobacillus casei）The strains of CGMCC 1.574 are inoculated into MRS culture mediums
In, 20h is cultivated at 30 DEG C, 5000g centrifugations 10min collects thalline, 40mL50mM phosphate is added into 10 grams of wet thallus and is delayed
Fliud flushing（pH6.8）, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking-up,
Through power 200W ultrasonications, often it is ultrasonically treated 5s and rests 30s, be ultrasonically treated 2 times, 5000g centrifugations 10min obtains permeability
Thalline.
Permeability thalline 40mL50mM phosphate buffers（pH5.8）After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 2mL Tween-80s and be well mixed, stirred in 4 DEG C
Mix processing 4h, 6000g centrifugation 10min and collect thalline, thalline is freeze-dried after -80 DEG C of pre-freezes, obtains being coated thalline.
1 gram of freeze-dried coated thalline is fitted into pillar bioreactor, by 0.9mL alpha-linolenic acids be added to 300mL just oneself
Mixed in alkane, the hexane solution containing alpha-linolenic acid is injected by reactor by pump, flow 3mL/min, collects efflux, will
Efflux is repeatedly injected reactor 2 times, collects efflux, is evaporated under reduced pressure at 40 DEG C, reclaims n-hexane, obtains 0.9mL productions
Thing, whereinc9,t11,cThe content of 15- conjugation alpha-linolenic acids is 91.5%.By the way that several pillar bioreactors are connected on into one
Rise, it is possible to achieve continuous production.
Claims (5)
1. the non-aqueous enzymatic synthesis of alpha-linolenic acid isomers is conjugated, it is characterized in that comprising the following steps：
（1）Collect the Lactobacillus casei in exponential phase（Lactobacillus casei）The thalline of CGMCC 1.574, often
Add 4mL 50mM, pH6.8 phosphate buffers, 5mg/mL lysozymes and 2mM sodium ethylene diamine tetracetates in gram wet thallus, 37 DEG C
Handle 20min, then through ultrasonication, ultrasonic power 200W, be often ultrasonically treated 5s rest 30s, be ultrasonically treated 2 times, centrifuge
To permeability thalline；
（2）After permeability thalline is washed with 50mM, pH5.8 phosphate buffer, thalline is collected by centrifugation, by every gram of permeability dampness elimination
The phosphate buffer that thalline is resuspended in 20mL50mM, pH5.8 is fallen into a trap, and permeability wet thallus is resuspended in into phosphate buffer
In, bacteria suspension percent by volume 0.5-2.0% Tween-80 is added, is well mixed, 2-6h is handled in 4-40 DEG C, bacterium is collected by centrifugation
Body, obtain being coated thalline after thalline is freeze-dried；
（3）600mL n-hexanes are added to by every gram of coating thalline to fall into a trap, and coating thalline is added in n-hexane, stirred,
N-hexane percent by volume 0.1-0.3% alpha-linolenic acid is added, reacts 1-12h at 4-40 DEG C；
（4）Coating thalline is centrifuged, hexane solution is evaporated under reduced pressure, reclaims n-hexane, product isc9,t11,c15-
It is conjugated alpha-linolenic acid isomers.
2. the non-aqueous enzymatic synthesis of conjugation alpha-linolenic acid isomers according to claim 1, it is characterized in that described
Step（2）The addition of middle Tween-80 is the 0.75-1.5% of bacteria suspension percent by volume.
3. the non-aqueous enzymatic synthesis of conjugation alpha-linolenic acid isomers according to claim 1, it is characterized in that described
Step（2）Middle treatment temperature is 4-25 DEG C.
4. the non-aqueous enzymatic synthesis of conjugation alpha-linolenic acid isomers according to claim 1, it is characterized in that described
Step（2）Middle processing time is 3-5h.
5. the non-aqueous enzymatic synthesis of conjugation alpha-linolenic acid isomers according to claim 1, it is characterized in that described
Step（3）The middle alpha-linolenic acid for adding n-hexane percent by volume 0.125-0.25%, reacts 2-6h at 15-30 DEG C.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629302A (en) * | 2004-07-28 | 2005-06-22 | 南昌大学 | Process for preparing conjugated fatty acid |
| CN101058820A (en) * | 2007-05-15 | 2007-10-24 | 吉林大学 | Method of catalytically synthesizing alpha-monolinolenin by using immobilization lipase |
-
2015
- 2015-07-20 CN CN201510423469.0A patent/CN105039441B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629302A (en) * | 2004-07-28 | 2005-06-22 | 南昌大学 | Process for preparing conjugated fatty acid |
| CN101058820A (en) * | 2007-05-15 | 2007-10-24 | 吉林大学 | Method of catalytically synthesizing alpha-monolinolenin by using immobilization lipase |
Non-Patent Citations (3)
| Title |
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| Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species;Lara Gorissen等;《Applied Microbial and Cell Physiology》;20100617;第87卷(第6期);第2257-2266页 * |
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