CN105039442B - The method of biosynthesis conjugation alpha linolenic acid isomers in organic media - Google Patents
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Abstract
The method of biosynthesis conjugation alpha linolenic acid isomers, comprises the following steps in organic media：（1）The thalline of Lactobacillus casei CGMCC 1.574 are resuspended in pulsating medium, after pulse electric field treatment, thalline is collected by centrifugation；（2）After being washed with 50mM, pH5.8 phosphate buffer, thalline is collected by centrifugation, every gram of wet thallus is resuspended in 20mL50mM, pH5.8 phosphate buffer, the Tween 80 for adding bacteria suspension volume 0.5 2.0% is well mixed, 4 40 DEG C of 2 6h of processing, thalline is collected by centrifugation, obtains being coated thalline after freeze-drying；（3）Every gram of coating thalline is added in 600mL n-hexanes, stirred, the alpha linolenic acid of n-hexane volume 0.1 0.3% is added, reacts 1 12h at 4 40 DEG C；（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 is significantly improved, 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 that biosynthesis is conjugated α-flax in a kind of organic media
The method of acid isomer, significantly shorten generated time, improve yield and conversion ratio, reduce production cost.
The present invention comprises the following steps.
（1）The electroporation processing of thalline：Collect the Lactobacillus casei in exponential phase（Lactobacillus casei）The thalline of CGMCC 1.574, after being washed with pulsating medium, thalline is resuspended in pulsating medium（The pulsating medium is
100mM sucrose, 50mM, pH5.8 phosphate buffer, the Tween-80 of percent by volume 0.1%）, cell concentration be 1 ×
109Cfu/mL, ice bath 10min, the impulse electric field processing that through electric-field intensity be 10kV/cm, pulse width is 20 μ s 10 times, every time
Interval time is 2s, and thalline is collected by centrifugation after processing.
（2）Thalline Cotton seeds：After thalline is washed with 50mM, pH5.8 phosphate buffer, thalline is collected by centrifugation, presses
The phosphate buffer that every gram of wet thallus is resuspended in 20mL50mM, pH5.8 is fallen into a trap, and wet thallus is resuspended in into phosphate buffer
In, the Tween-80 for adding bacteria suspension percent by volume 0.5-2.0% is well mixed, and handles 2-6h in 4-40 DEG C, bacterium is collected by centrifugation
Body, obtain being coated thalline after thalline is freeze-dried.
（3）Biosynthesis in organic media：600mL n-hexanes are added to by every gram of coating thalline to fall into a trap, and will be coated thalline
It is added in n-hexane, stirs, adds n-hexane percent by volume 0.1-0.3% alpha-linolenic acid, reacted at 4-40 DEG C
1-12h。
（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 biosynthesis in organic mediac9,t11,c15-
It 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.
Pass through biological synthesis method in the organic media, with optimal conditions, the alpha-linolenic acid containing percent by volume 0.15%
It is contained after hexane solution reactionc9,t11,cThe percent by volume of 15- conjugation alpha-linolenic acid isomers is 0.138%, α-flax
The conversion ratio of acid is 92.0%.Step of the present invention（4）Middle coating thalline can be repeated for step（3）Organic media in
Biosynthesisc9,t11,c15- be conjugated alpha-linolenic acid isomers, coating thalline reuse five times after, its catalytic activity 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 the new method of the biosynthesis conjugation alpha-linolenic acid in organic media.This method is first
Lactobacillus casei CGMCC 1.574 is handled by impulse electric field, while thalline integrality is kept, improve somatic cells wall with
Membrane passage, it on the one hand may insure that the linoleate isomerase surface energy intracellular in follow-up Cotton seeds has been formed
Whole coatings, the speed of reaction substrate alpha-linolenic acid and product conjugation alpha-linolenic acid disengaging thalline on the other hand can be improved, is subtracted
Few high concentration substrate and product greatly shorten the reaction time to the inhibitory action of catalytic reaction.
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, also keep higher catalysis 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 biosynthesis in organic mediac9,t11,c15- is conjugated alpha-linolenic acid isomers, significantly contracting
The short production time, yield is improved, reduces production cost.In addition, n-hexane is conventional organic molten of edible oil and fat industry
Agent, this guarantees 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 biosynthesis reaction by being coated thalline in organic media, avoids and is reacted in traditional aqueous
Substrate alpha-linolenic acid solubility is low in system and product conjugation alpha-linolenic acid extracts the problem of difficult.The heat for being coated enzyme in thalline is steady
Qualitative raising, 4h is only needed per the batch reaction time, much smaller than time a couple of days for fermenting required in traditional aqueous.Being coated thalline can
It is reused many times, significantly improves product yield, and non-environmental-pollution, production cost can be significantly reduced；And traditional aqueous body
It is that thalline in zymotic fluid is used only once, 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, by 10 grams of wet thallus 50mL sucrose containing 100mM, 50mM
Phosphate buffer（pH5.8）, the Tween-80 of percent by volume 0.1% pulsating medium washing, 5000g centrifugation 10min collect bacterium
Body, thalline is resuspended in pulsating medium, adjustment cell concentration is 1 × 109Cfu/mL, ice bath 10min, uses electric-field intensity
10kV/cm, the μ s of pulse width 20 impulse electric field processing, umber of pulse 10 times, each interval time 2s, through pulse electric field treatment
Afterwards, 5000g centrifuges 10min and obtains electroporation thalline.
Electroporation 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 92.0%.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
90.2%。
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, by 10 grams of wet thallus 50mL sucrose containing 100mM, 50mM phosphorus
Phthalate buffer（pH5.8）, the Tween-80 of percent by volume 0.1% pulsating medium washing, 4000g centrifugation 10min collect thalline,
Thalline is resuspended in pulsating medium, adjustment cell concentration is 1 × 109Cfu/mL, ice bath 10min, with electric-field intensity 10kV/
Cm, the μ s of pulse width 20 impulse electric field processing, umber of pulse 10 times, each interval time 2s, after pulse electric field treatment,
5000g centrifugations 5min obtains electroporation thalline.
Electroporation thalline 40mL50mM phosphate buffers（pH5.8）After washing, 5000g centrifugations 5min 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, 6000g centrifugation 5min 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 80.0%.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
68.8%。
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, by 10 grams of wet thallus 50mL sucrose containing 100mM, 50mM phosphorus
Phthalate buffer（pH5.8）, the Tween-80 of percent by volume 0.1% pulsating medium washing, 4000g centrifugation 10min collect thalline,
Thalline is resuspended in pulsating medium, adjustment cell concentration is 1 × 109Cfu/mL, ice bath 10min, with electric-field intensity 10kV/
Cm, the μ s of pulse width 20 impulse electric field processing, umber of pulse 10 times, each interval time 2s, after pulse electric field treatment,
5000g centrifugations 5min obtains electroporation thalline.
Electroporation thalline 40mL50mM phosphate buffers（pH5.8）After washing, 5000g centrifugations 5min 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, 6000g centrifugation 5min 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 90.1%.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 84.4%.
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, by 10 grams of wet thallus 50mL sucrose containing 100mM, 50mM
Phosphate buffer（pH5.8）, the Tween-80 of percent by volume 0.1% pulsating medium washing, 5000g centrifugation 10min collect bacterium
Body, thalline is resuspended in pulsating medium, adjustment cell concentration is 1 × 109Cfu/mL, ice bath 10min, uses electric-field intensity
10kV/cm, the μ s of pulse width 20 impulse electric field processing, umber of pulse 10 times, each interval time 2s, through pulse electric field treatment
Afterwards, 5000g centrifuges 10min and obtains electroporation thalline.
Electroporation 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 89.7%.By the way that several pillar bioreactors are connected on into one
Rise, it is possible to achieve continuous production.
Claims (5)
1. the method for biosynthesis conjugation alpha-linolenic acid isomers in organic media, 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, use
After pulsating medium washing, thalline is resuspended in pulsating medium, cell concentration is 1 × 109Cfu/mL, ice bath 10min, through electric field
The impulse electric field that intensity is 10kV/cm, pulse width is 20 μ s is handled 10 times, and each interval time is 2s, is centrifuged and is received after processing
Collect thalline；
（2）After thalline is washed with 50mM, pH5.8 phosphate buffer, thalline is collected by centrifugation, is resuspended in by every gram of wet thallus
20mL50mM, pH5.8 phosphate buffer are fallen into a trap, and wet thallus is resuspended in phosphate buffer, add bacteria suspension volume
Percentage 0.5-2.0% Tween-80 is well mixed, and handles 2-6h in 4-40 DEG C, thalline is collected by centrifugation, thalline is freeze-dried
After obtain be coated thalline；
（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；
Step（1）Described pulsating medium be 100mM sucrose, 50mM, pH5.8 phosphate buffer, percent by volume
0.1% Tween-80.
2. the method for biosynthesis conjugation alpha-linolenic acid isomers in organic media 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 method for biosynthesis conjugation alpha-linolenic acid isomers in organic media according to claim 1, it is characterized in that
Described step（2）Middle treatment temperature is 4-25 DEG C.
4. the method for biosynthesis conjugation alpha-linolenic acid isomers in organic media according to claim 1, it is characterized in that
Described step（2）Middle processing time is 3-5h.
5. the method for biosynthesis conjugation alpha-linolenic acid isomers in organic media 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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| 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 |
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- 2015-07-20 CN CN201510423650.1A patent/CN105039442B/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 |
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| 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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