CN105112461B - It is coated the method for thalline biosynthesis conjugation acid and gamma-linolenic isomers in organic media - Google Patents
It is coated the method for thalline biosynthesis conjugation acid and gamma-linolenic isomers in organic media Download PDFInfo
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Abstract
The method of thalline biosynthesis conjugation acid and gamma-linolenic isomers in organic media is coated, is comprised the following steps：（1）Every gram of 1.574 thalline of Lactobacillus casei CGMCC is resuspended in 5mL permeabilized treatment liquid, 37 DEG C of processing 20min, are collected by centrifugation permeability thalline；（2）After being washed with the phosphate buffer of 50mM, pH5.8, thalline is collected by centrifugation, every gram of permeability wet thallus is resuspended in the phosphate buffer of 20mL50mM, pH5.8, the polysorbas20 for adding bacteria suspension volume 0.5 2.0% is uniformly mixed, 4 40 DEG C of 2 6h of processing, thalline is collected by centrifugation, coating thalline is obtained after freeze-drying；（3）Every gram of coating thalline is added in 600mL n-hexanes, is stirred evenly, the acid and gamma-linolenic of n-hexane volume 0.1 0.3% is added, reacts 1 12h at 4 40 DEG C；（4）Coating thalline is centrifuged, recycles n-hexane, product isc6,c9,t11 conjugation acid and gamma-linolenic isomers.Reaction time of the invention is short, and coating thalline is reusable repeatedly, and yield is significantly improved, and non-environmental-pollution, significantly reduces production cost.
Description
Technical field
The invention belongs to biomedicine technical field.
Background technology
Conjugation gamma-Linolenic acid is the conjugated isomers of gamma-Linolenic acid, is the general designation of one group of eighteen carbon conjugated trienoic acid, has
A variety of position isomeries and geometric isomer, such as：c6,c9,t11- be conjugated gamma-Linolenic acid andc6,t10,c12- is conjugated gamma-Linolenic acid
Isomers etc..Research shows that being conjugated gamma-Linolenic acid has the physiological functions such as anticancer, prevention of arterial atherosis, weight-reducing, and it is given birth to
Reason activity has isomers specificity, such as：c6,c9,t11- conjugation gamma-Linolenic acid isomers has the function that anticancer.In nature
In boundary, conjugation gamma-Linolenic acid be primarily present in the milk of ruminant and the fat of meat, mainly byc6,c9,t11- conjugation γ-
The isomers such as leukotrienes are formed, but its content is usually very low, can not meet the development and application for the purpose of health care and medical treatment.
There is presently no conjugation gamma-Linolenic acid preparation method report, people simply to microbial fermentation synthesis of conjugate α-
Leukotrienes has carried out some researchs.At present, microbial fermentation carries out in aqueous, due to fermentation substrate-gamma-Linolenic acid
It is liposoluble substance, therefore is needed before gamma-Linolenic acid addition zymotic fluid through emulsification treatment, and additive amount is restricted, and will be from hair
Conjugation gamma-Linolenic acid product is obtained in zymotic fluid to be extracted through organic solvent, whole production technology there are fermentation period it is long, be produced into
The shortcomings that this height, low output.
The content of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, propose a kind of coating thalline biosynthesis in organic media
The method of gamma-Linolenic acid isomers is conjugated, significantly shortens generated time, raising 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）1.574 thalline of CGMCC, are resuspended in 5mL permeabilized treatment liquid by every gram of wet thallus and fall into a trap, wet thallus is resuspended in
In property treatment fluid（The permeabilized treatment liquid is the triton x-100 of percent by volume 0.5-3.0%, 5-50 μM of γ-
Leukotrienes, the phosphate buffer of 20-200mM, pH5.8）, thalline is resuspended through 37 DEG C of processing 20min, permeability bacterium is collected by centrifugation
Body.
（2）Thalline Cotton seeds：After permeability thalline is washed with the phosphate buffer of 50mM, pH5.8, 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, the Tween-20 for adding bacteria suspension percent by volume 0.5-2.0% is uniformly mixed, in 4-40 DEG C of processing
2-6h, is collected by centrifugation thalline, and coating thalline is obtained after thalline is freeze-dried.
（3）Biosynthesis in organic media：600mL n-hexanes are added to by every gram of freeze-dried coated thalline to fall into a trap, and will be coated
Thalline is added in n-hexane, is stirred evenly, and the gamma-Linolenic acid of n-hexane percent by volume 0.1-0.3% is added, at 4-40 DEG C
Lower reaction 1-12h.
（4）Collection of products：Coating thalline is centrifuged, hexane solution is evaporated under reduced pressure, recycles n-hexane, product
Forc6,c9,t11- is conjugated gamma-Linolenic acid isomers.
Step of the present invention（1）Middle permeabilized treatment liquid is preferably that the Qula of percent by volume 1.0-2.0% leads to X-
100,15-35 μM of gamma-Linolenic acid, the phosphate buffer of 30-100mM, pH5.8.
Step of the present invention（2）The addition of middle Tween-20 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 the gamma-Linolenic acid of n-hexane percent by volume 0.125-0.25%,
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 mediac6,c9,t11- is total to
Yoke gamma-Linolenic acid isomers.
Lactobacillus casei used in the present invention（Lactobacillus casei）CGMCC 1.574, 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 γ-
Leukotrienes（c6,c9,c12-18:3）Changing at 268nm has the novel substance of characteristic absorption peak, and absworption peak is altogether at 268nm
The characteristic peak of yoke triolefin, shows that the bacterium can change into gamma-Linolenic acidc6,c9,t11- is conjugated gamma-Linolenic acid isomers.At present,
The isomerase sterling of definite catalytic activity is not separated to from the bacterium also.Therefore, which is probably by multienzyme
System co-catalysis is completed.
By coating thalline biological synthesis method in organic media, with optimal conditions, containing percent by volume 0.15%
It is synthesized after the hexane solution reaction of gamma-Linolenic acidc6,c9,tThe content of 11- conjugation gamma-Linolenic acid isomers is volume hundred
Divide ratio 0.1388%, the conversion ratio of gamma-Linolenic acid is 92.5%.Step of the present invention（4）Middle coating thalline can be repeated for
Step（3）Organic media in biosynthesisc6,c9,t11- is conjugated gamma-Linolenic acid isomers, and coating thalline is reused five times
Afterwards, its catalytic activity is still greater than 90%.
Carried out in aqueous for existing microbial fermentation synthesis of conjugate gamma-Linolenic acid, whole production technology exists
Fermentation period is grown, and extraction conjugation gamma-Linolenic acid product is difficult from zymotic fluid, and thalline cannot be reused, and production cost is high, production
Measure low shortcomings.The present invention is proposed catalyzes and synthesizes conjugation in organic media by microbial cells by gamma-Linolenic acid
The new method of gamma-Linolenic acid.This method handles Lactobacillus casei CGMCC 1.574 by triton x-100 first, is keeping bacterium
While body integrality, somatic cells wall and membrane passage are improved, on the one hand may insure in follow-up Cotton seeds
Intracellular isomerase surface energy forms complete coatings, on the other hand can improve reaction substrate gamma-Linolenic acid and product is total to
Yoke gamma-Linolenic acid passes in and out the speed of thalline, reduces the inhibitory action of high concentration substrate and product to catalytic reaction, greatly shortens anti-
Between seasonable.
The present invention adds 5-50 μM of gamma-Linolenic acid in permeabilized treatment thalline, gamma-Linolenic acid can with thalline
Linoleate isomerase catalytic active center combines, and in thalline Cotton seeds, can form molecule print in the catalytic active center of enzyme
Mark, makes thalline catalytic active center conformation of enzyme after freeze-drying constant, higher catalysis can be kept to live in organic media
Property.Meanwhile the present invention be incorporated in thalline Cotton seeds during select suitable phosphate buffer, Tween-20 concentration and bag
Clothing temperature, time, make gained coating thalline still have higher catalytic capability in organic media.After coating in thalline enzyme heat
Stability improves, and the every batch of reaction time only needs 4h, 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 recycles n-hexane, you can obtain productc6,c9,t11- is conjugated gamma-Linolenic acid.Centrifugation gained
Coating thalline can be repeated several times for biosynthesis in organic mediac6,c9,t11- is conjugated gamma-Linolenic acid isomers, significantly contracting
The short production time, yield is improved, reduces production cost.In addition, n-hexane is that edible oil and fat industry is common organic molten
Agent, this guarantees obtained by the present inventionc6,c9,t11- is conjugated the safety in utilization of gamma-Linolenic acid.
The present invention has the following advantages that compared with prior art.
The present invention is directly coated thalline processing, is used for catalytic reaction as immobilised enzymes, on the one hand reduces enzyme
The cost isolated and purified, avoids the loss of enzyme activity in extraction；On the other hand multi-enzyme system can be wrapped together, made whole
A reaction process 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 by being coated thalline in organic media, avoids in traditional aqueous reaction system
Middle substrate gamma-Linolenic acid solubility is low and product conjugation gamma-Linolenic acid extracts the problem of difficult.It is coated the thermostabilization of enzyme in thalline
Property improve, the every batch of reaction time only needs 4h, much smaller than time a couple of days for fermenting required in traditional aqueous.Coating thalline can weigh
It is multiple to use repeatedly, product yield, and non-environmental-pollution are significantly improved, production cost can be significantly reduced；And traditional aqueous system
Thalline in zymotic fluid is used only once, and production cost is high, and Wastewater treating is costly, environment easy to pollute.
Embodiment
The present invention is further described by following embodiments.
Embodiment 1.
By the Lactobacillus casei of activation（Lactobacillus casei）1.574 strains of CGMCC are inoculated into MRS culture mediums
In, 20h is cultivated at 30 DEG C, 5000g centrifugations 10min collects thalline, 10 grams of wet thallus are resuspended in 50mL and contain percent by volume
1.5% triton x-100,25 μM of gamma-Linolenic acid and 50mM phosphate buffers（pH5.8）Permeabilized treatment liquid in, 37
DEG C water bath processing 20min, 5000g centrifugation 10min obtains permeability thalline.
Permeability thalline 50mL50mM phosphate buffers（pH5.8）After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 2 mL Tween-20s and be uniformly mixed, in 4 DEG C
Stir process 4h, 6000g centrifugation 10min collects thalline, and thalline is freeze-dried after -80 DEG C of pre-freezes, obtains coating thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, is stirred evenly, 0.9mL gamma-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, recycling just oneself
Alkane, obtains 0.9mL products, whereinc6,c9,tThe content of 11- conjugation gamma-Linolenic acids is 92.5%.The coating thalline that will be collected into
Above-mentioned synthetic reaction is recycled and reused for, when being used continuously five times in productc6,c9,t11- conjugation gamma-Linolenic acid content be
90.5%。
Embodiment 2.
By the Lactobacillus casei of activation（Lactobacillus casei）1.574 strains of CGMCC are inoculated into MRS culture mediums
In, 18h is cultivated at 30 DEG C, 4000g centrifugations 5min collects thalline, 10 grams of wet thallus are resuspended in 50mL and contain percent by volume
3.0% triton x-100,5 μM of gamma-Linolenic acid and 200mM phosphate buffers（pH5.8）Permeabilized treatment liquid in, 37
DEG C water bath processing 20min, 4000g centrifugation 10min obtains permeability thalline.
Permeability thalline 50mL50mM 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-20s and be uniformly 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 coating thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, is stirred evenly, 1.8mL gamma-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, recycling just oneself
Alkane, obtains 1.8mL products, whereinc6,c9,tThe content of 11- conjugation gamma-Linolenic acids is 79.2%.The coating thalline that will be collected into
Above-mentioned synthetic reaction is recycled and reused for, when being used continuously five times in productc6,c9,t11- conjugation gamma-Linolenic acid content be
68.9%。
Embodiment 3.
By the Lactobacillus casei of activation（Lactobacillus casei）1.574 strains of CGMCC are inoculated into MRS culture mediums
In, 18h is cultivated at 30 DEG C, 4000g centrifugations 5min collects thalline, 10 grams of wet thallus are resuspended in 50mL and contain percent by volume
0.5% triton x-100,50 μM of gamma-Linolenic acid and 20mM phosphate buffers（pH5.8）Permeabilized treatment liquid in, 37
DEG C water bath processing 20min, 4000g centrifugation 10min obtains permeability thalline.
Permeability thalline 50mL50mM 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-20s and be uniformly 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 coating thalline.
1 gram of freeze-dried coated thalline is added in 600mL n-hexanes, is stirred evenly, adds 0.6mL gamma-Linolenic acids, at 4 DEG C
Lower stirring reaction 6h.6000g centrifuges coating thalline, and hexane solution is evaporated under reduced pressure at 40 DEG C, recycles n-hexane,
0.6mL products are obtained, whereinc6,c9,tThe content of 11- conjugation gamma-Linolenic acids is 88.3%.The coating thalline being collected into is repeated
For above-mentioned synthetic reaction, during continuous use five times in productc6,c9,tThe content of 11- conjugation gamma-Linolenic acids is 80.1%.
Embodiment 4.
By the Lactobacillus casei of activation（Lactobacillus casei）1.574 strains of CGMCC are inoculated into MRS culture mediums
In, 20h is cultivated at 30 DEG C, 5000g centrifugations 10min collects thalline, 10 grams of wet thallus are resuspended in 50mL and contain percent by volume
1.5% triton x-100,25 μM of gamma-Linolenic acid and 50mM phosphate buffers（pH5.8）Permeabilized treatment liquid in, 37
DEG C water bath processing 20min, 5000g centrifugation 10min obtains permeability thalline.
Permeability thalline 50mL50mM phosphate buffers（pH5.8）After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL50mM phosphate buffers（pH5.8）In, add 2 mL Tween-20s and be uniformly mixed, in 4 DEG C
Stir process 4h, 6000g centrifugation 10min collects thalline, and thalline is freeze-dried after -80 DEG C of pre-freezes, obtains coating thalline.
1 gram of freeze-dried coated thalline is fitted into pillar bioreactor, by 0.9mL gamma-Linolenic acids be added to 300mL just oneself
Being mixed in alkane, the hexane solution containing gamma-Linolenic acid is injected into reactor by pumping, flow 3mL/min, collects efflux,
Efflux is repeatedly injected reactor 2 times, efflux is collected, is evaporated under reduced pressure at 40 DEG C, n-hexane is recycled, obtains 0.9mL
Product, whereinc6,c9,tThe content of 11- conjugation gamma-Linolenic acids is 90.9%.By the way that several pillar bioreactors are connected on
Together, it is possible to achieve continuous production.
Claims (6)
1. the method for thalline biosynthesis conjugation gamma-Linolenic acid isomers in organic media is coated, it is characterized in that including following
Step：
（1）Collect the Lactobacillus casei in exponential phase（Lactobacillus casei）1.574 thalline of CGMCC, are pressed
Every gram of wet thallus is resuspended in 5mL permeabilized treatment liquid and falls into a trap, and wet thallus is resuspended in permeabilized treatment liquid, and thalline is resuspended through 37
DEG C processing 20min, permeability thalline is collected by centrifugation；
（2）After permeability thalline is washed with the phosphate buffer of 50mM, pH5.8, 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 phosphate buffer
In, the Tween-20 for adding bacteria suspension percent by volume 0.5-2.0% is uniformly mixed, and handles 2-6h in 4-40 DEG C, bacterium is collected by centrifugation
Body, obtains coating thalline after thalline is freeze-dried；
（3）600mL n-hexanes are added to by every gram of freeze-dried coated thalline to fall into a trap, and coating thalline is added in n-hexane, is stirred
Uniformly, the gamma-Linolenic acid of n-hexane percent by volume 0.1-0.3% is added, reacts 1-12h at 4-40 DEG C；
（4）Coating thalline is centrifuged, hexane solution is evaporated under reduced pressure, recycles n-hexane, product isc6,c9,t11-
It is conjugated gamma-Linolenic acid isomers；
Step（1）The permeabilized treatment liquid be percent by volume 0.5-3.0% triton x-100,5-50 μM of γ-Asia
Numb acid, the phosphate buffer of 20-200mM, pH5.8.
2. the side of coating thalline according to claim 1 biosynthesis conjugation gamma-Linolenic acid isomers in organic media
Method, it is characterized in that the step（1）Middle permeabilized treatment liquid be percent by volume 1.0-2.0% triton x-100,15-35
μM gamma-Linolenic acid, the phosphate buffer of 30-100mM, pH5.8.
3. the side of coating thalline according to claim 1 biosynthesis conjugation gamma-Linolenic acid isomers in organic media
Method, it is characterized in that the step（2）The addition of middle Tween-20 is the 0.75-1.5% of bacteria suspension percent by volume.
4. the side of coating thalline according to claim 1 biosynthesis conjugation gamma-Linolenic acid isomers in organic media
Method, it is characterized in that the step（2）Middle treatment temperature is 4-25 DEG C.
5. the side of coating thalline according to claim 1 biosynthesis conjugation gamma-Linolenic acid isomers in organic media
Method, it is characterized in that the step（2）Middle processing time is 3-5h.
6. the side of coating thalline according to claim 1 biosynthesis conjugation gamma-Linolenic acid isomers in organic media
Method, it is characterized in that the step（3）The middle gamma-Linolenic acid for adding n-hexane percent by volume 0.125-0.25%, in 15-30
2-6h is reacted at DEG C.
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| CN1629302A (en) * | 2004-07-28 | 2005-06-22 | 南昌大学 | Process for preparing conjugated fatty acid |
| CN104404092A (en) * | 2014-11-04 | 2015-03-11 | 南昌大学 | Conjugated linoleic acid isomer biological enrichment method |
| CN104480150A (en) * | 2014-11-04 | 2015-04-01 | 南昌大学 | Biological enrichment method of conjugated linolenic acid isomer |
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