CN105112462A - Non-aqueous enzymological synthesis method of conjugated linoleic acid isomer - Google Patents
Non-aqueous enzymological synthesis method of conjugated linoleic acid isomer Download PDFInfo
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Abstract
A non-aqueous enzymological synthesis method of a conjugated linoleic acid isomer comprises the following steps: 1, adding 4mL of a phosphate buffer solution with the concentration of 50mM and the pH value of 6.8, lysozyme with the concentration of 5mg/mL and sodium ethylene diamine tetracetate with the concentration of 2mM to every gram of Lactobacillus casei CGMCC 1.574, treating at 37DEG C for 20min, carrying out ultrasonic treatment under 200W for 5s, stopping, carrying out ultrasonic treatment under 200W for 5s 30s later, and centrifuging to obtain permeabilized thalli; 2, washing by using the phosphate buffer solution with the concentration of 50mM and the pH value of 5.8, collecting thalli, re-suspending every gram of the permeabilized wet thalli in 20mL of the phosphate buffer solution with the concentration of 50mM and the pH value of 5.8, adding tween-80 accounting for 0.5-2.0% of the volume of the obtained thallus suspension, uniformly mixing, treating at 4-40DEG C for 2-6h, centrifuging, collecting thalli, and freeze-drying to obtain coated thalli; 3, adding the coated thalli to n-hexane according to a ratio of 1g/600mL, uniformly stirring, adding linolenic acid accounting for 0.1-0.3% of the volume of n-hexane, and reacting at 4-40DEG C for 1-12h; and 4, centrifuging, separating coated thalli, and recovering n-hexane to obtain the product c9,ct11-conjugated gamma-linolenic acid isomer. The method has the advantages of short reaction time, repeated use of the coated thalli, great improvement of the output, no environmental pollution, and great reduction of the production cost.
Description
Technical field
The invention belongs to biomedicine technical field.
Background technology
Conjugated linolic acid (conjugatedlinoleicacid, CLA) is the general designation of one group of CLA, and conjugated double bond has 4 kinds of position isomerisms (8,10; 9,11; 10,12; 11,13) and 4 kinds of rotamerisms (
cis,
cis;
cis,
trans;
trans,
cis;
trans,
trans), total nearly 16 kinds of isomer, as:
c9,
t11-CLA and
t10,
c12-CLA isomer etc.Research finds that CLA has the physiological functions such as anticancer, prevention of arterial is atherosis, enhancing body immunizing power, anti-diabetic, and increasing research shows that its physiologically active has isomer specificity, generally believes now
c9,
t11-CLA isomer has anticancer and that prevention of arterial is atherosis effect.At occurring in nature, CLA is mainly present in the fat in the milk of ruminating animal and meat, primarily of
c9,
t11-CLA is with a small amount of
t9,
t11-CLA,
t10,
cthe isomer such as 11-CLA is formed, but its content is all less than 10mg/g fat usually, cannot meet to keep healthy and Application and Development for the purpose of medical treatment.
For realizing a large amount of preparations of CLA isomer, people prepare CLA to fermentable and have carried out more research.At present, fermentable carries out all in aqueous, because added fermentation substrate-linolic acid is lipid-soluble substance, therefore linolic acid needs through emulsification before adding fermented liquid, and addition is restricted, from fermented liquid, obtain CLA product need through organic solvent extraction, and whole production technique exists the shortcoming that fermentation period is long, production cost is high, yield poorly.Patent of invention CN201110105610 proposes a kind of method of bioconverting conjugated linoleic acid by using Lactobacillus plantarum, and substrate addition is 25mg/mL, and fermentation time reaches 120h, and the output of CLA only has 1.02mg/mL, and the transformation efficiency of substrate only has 4%.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, propose a kind of non-water enzymatic synthesis of conjugated linoleic acid isomers, significantly shorten generated time, improve output and transformation efficiency, reduction production cost.
The present invention includes following steps.
(1) permeabilized treatment of thalline: collect be in logarithmic phase lactobacterium casei (
lactobacilluscasei) CGMCC1.574 thalline, through N,O-Diacetylmuramidase process, (described N,O-Diacetylmuramidase is treated in every gram of wet thallus and adds 4mL50mM, pH6.8 phosphate buffered saline buffer, 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate, 37 DEG C of process 20min), again through ultrasonication, ultrasonication is ultrasonic power 200W, and every supersound process 5s rests 30s, supersound process 2 times, centrifugally obtains permeability thalline.
(2) thalline Cotton seeds: permeability thalline is with after the phosphate buffered saline buffer washing of 50mM, pH5.8, collected by centrifugation thalline, the phosphate buffered saline buffer being resuspended in 20mL50mM, pH5.8 by every gram of permeability wet thallus is fallen into a trap, permeability wet thallus is resuspended in phosphate buffered saline buffer, add the tween-80 of bacteria suspension volume percent 0.5-2.0%, mix, in 4-40 DEG C of process 2-6h, collected by centrifugation thalline, obtains dressing thalline by after thalline lyophilize.
(3) non-water Enzyme catalyzed synthesis: join 600mL normal hexane by every gram of dressing thalline and fall into a trap, dressing thalline is joined in normal hexane, stirs, add the linolic acid of normal hexane volume percent 0.1-0.3%, react 1-12h at 4-40 DEG C.
(4) product collection: centrifugation dressing thalline, carries out underpressure distillation by hexane solution, and reclaim normal hexane, product is
c9,
t11-conjugated linoleic acid isomers.
In step of the present invention (2), the add-on of tween-80 is preferably the 0.75-1.5% of bacteria suspension volume percent.
In step of the present invention (2), treatment temp is preferably 4-25 DEG C.
In step of the present invention (2), the treatment time is preferably 3-5h.
Preferably add the linolic acid of normal hexane volume percent 0.125-0.25% in step of the present invention (3), at 15-30 DEG C, react 2-6h.
In step of the present invention (4), dressing thalline can be recycled and reused for non-water Enzyme catalyzed synthesis
c9,
t11-conjugated linoleic acid isomers.
The present invention's lactobacterium casei used (
lactobacilluscasei) CGMCC1.574, be China General Microbiological culture presevation administrative center (CGMCC) preservation strain, be numbered 1.574, this bacterial strain can produce specific linoleate isomerase, linoleic acid can be become by biological isomerization
c9,
t11-CLA isomer.At present, the linoleate isomerase sterling of definite catalytic activity is not also separated to.Therefore, this isomerization reaction may be completed by multi-enzyme system co-catalysis.
With optimal conditions, by this non-water enzymatic synthesis, synthesized by after the linoleic hexane solution reaction of volume percent 0.15%
c9,
tthe content of 11-CLA isomer is volume percent 0.14%, and linoleic transformation efficiency is 93.3%.In step of the present invention (4), dressing thalline can be recycled and reused for the non-water Enzyme catalyzed synthesis of step (3)
c9,
t11-CLA isomer, after dressing thalline reuses five times, its catalytic activity is still greater than 90%.
Carry out all in aqueous for existing fermentable synthesis CLA, it is long to there is fermentation period in whole production technique, and from fermented liquid, extract CLA product difficult, thalline can not be reused, and production cost is high, and the shortcomings such as yield poorly.The present invention proposes and in organic solvent linolic acid is catalyzed and synthesized by microbial cells
c9,
tthe novel method of 11-CLA.The method is first by N,O-Diacetylmuramidase and ultrasonication lactobacterium casei CGMCC1.574, while maintenance thalline integrity, improve somatic cells wall and membrane passage, can guarantee that intracellular linoleate isomerase surface energy forms complete coatings when follow-up Cotton seeds on the one hand, the speed of reaction substrate linolic acid and product C LA turnover thalline can be improved on the other hand, minimizing high concentration substrate and product are to the restraining effect of catalyzed reaction, greatly Reaction time shorten.
The present invention selects tween-80 to carry out dressing to thalline, containing an oleic acid moieties in tween-80 molecule, oleic acid and linolic acid structural similitude, it is the analog of thalline Linoleic acid isomerase substrate, when thalline dressing, molecular imprinting can be formed at the catalytic active center of enzyme, make thalline catalytic active center conformation of enzyme after lyophilize constant, higher catalytic activity can be kept in organic solvent.Meanwhile, the present invention is combined in thalline Cotton seeds process and selects suitable phosphate buffered saline buffer, tween-80 concentration and dressing temperature, time, makes gained dressing thalline still have higher catalytic capability in organic solvent.After dressing, in thalline, the thermostability of enzyme improves, and every batch of reaction times only needs 4h, time a couple of days in traditional aqueous needed for Batch fermentation.
Gained dressing thalline of the present invention has higher catalytic activity in normal hexane, only needs centrifugation dressing thalline, hexane solution is carried out underpressure distillation, reclaims normal hexane, can obtain product
c9,
t11-CLA.Centrifugal gained dressing thalline repeatedly can be recycled and reused for non-water Enzyme catalyzed synthesis
c9,
t11-CLA isomer, significantly shorten the production time, improves output, reduces production cost.In addition, normal hexane is the organic solvent that food oils industry is commonly used, and this guarantees gained of the present invention
c9,
tthe safety in utilization of 11-CLA.
The present invention compared with prior art tool has the following advantages.
The present invention directly carries out Cotton seeds to thalline, is used for catalyzed reaction as immobilized enzyme, decreases the cost of enzyme purification on the one hand, avoids the loss of enzyme activity when extracting; Can multi-enzyme system be wrapped in together on the other hand, make whole reaction process smooth; Moreover dressing thalli granule is greater than dressing enzyme, be easy to separate from reaction soln, and filtration resistance is less, therefore, dressing thalline is suitable for the column reactor of successive reaction more.
The present invention carries out catalyzed reaction in organic solvent by dressing thalline, avoids the problem that substrate linoleic acid solubleness in traditional aqueous reaction system is low and product C LA extraction is difficult.In dressing thalline, the thermostability of enzyme improves, and every batch of reaction times only needs 4h, time a couple of days of fermenting required in traditional aqueous.Dressing thalline is repeatedly reusable, significantly improves product production, and non-environmental-pollution, significantly can reduce production cost; And the thalline in traditional aqueous system fermented liquid can only use once, production cost is high, and Wastewater treating costly, easy contaminate environment.
Embodiment
The present invention is further described by following examples.
Embodiment 1.
By activation lactobacterium casei (
lactobacilluscasei) CGMCC1.574 strain inoculation is in MRS substratum, 20h is cultivated at 30 DEG C, the centrifugal 10min of 5000g collects thalline, adds 40mL50mM phosphate buffered saline buffer (pH6.8), 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate in 10 grams of wet thallus, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking out, through power 200W ultrasonication, every supersound process 5s rests 30s, supersound process 2 times, the centrifugal 10min of 5000g obtains permeability thalline.
Permeability thalline is with after 40mL50mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 6000g collects thalline, wet thallus is resuspended in 200mL50mM phosphate buffered saline buffer (pH5.8), add 2mL tween-80 and mix, in 4 DEG C of stir process 4h, the centrifugal 10min of 6000g collects thalline, and thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is joined in 600mL normal hexane, stirs, add 0.9mL linolic acid, stirring reaction 4h at 25 DEG C.6000g centrifugation dressing thalline, carries out underpressure distillation by hexane solution at 40 DEG C, reclaims normal hexane, obtains 0.9mL product, wherein
c9,
tthe content of 11-CLA is 93.3%.The dressing thalline collected is recycled and reused for above-mentioned building-up reactions, when using continuously five times in product
c9,
tthe content of 11-CLA is 90.9%.
Embodiment 2.
By activation lactobacterium casei (
lactobacilluscasei) CGMCC1.574 strain inoculation is in MRS substratum, 18h is cultivated at 30 DEG C, the centrifugal 5min of 4000g collects thalline, adds 40mL50mM phosphate buffered saline buffer (pH6.8), 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate in 10 grams of wet thallus, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking out, through power 200W ultrasonication, every supersound process 5s rests 30s, supersound process 2 times, the centrifugal 5min of 5000g obtains permeability thalline.
Permeability thalline is with after 30mL50mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 5000g collects thalline, wet thallus is resuspended in 200mL50mM phosphate buffered saline buffer (pH5.8), add 4mL tween-80 and mix, in 4 DEG C of stir process 2h, the centrifugal 10min of 5000g collects thalline, and thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is joined in 600mL normal hexane, stirs, add 1.8mL linolic acid, stirring reaction 12h at 40 DEG C.6000g centrifugation dressing thalline, carries out underpressure distillation by hexane solution at 40 DEG C, reclaims normal hexane, obtains 1.8mL product, wherein
c9,
tthe content of 11-CLA is 80.8%.The dressing thalline collected is recycled and reused for above-mentioned building-up reactions, when using continuously five times in product
c9,
tthe content of 11-CLA is 70.5%.
Embodiment 3.
By activation lactobacterium casei (
lactobacilluscasei) CGMCC1.574 strain inoculation is in MRS substratum, 18h is cultivated at 30 DEG C, the centrifugal 5min of 4000g collects thalline, adds 40mL50mM phosphate buffered saline buffer (pH6.8), 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate in 10 grams of wet thallus, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking out, through power 200W ultrasonication, every supersound process 5s rests 30s, supersound process 2 times, the centrifugal 5min of 5000g obtains permeability thalline.
Permeability thalline is with after 40mL50mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 5000g collects thalline, wet thallus is resuspended in 200mL50mM phosphate buffered saline buffer (pH5.8), add 1mL tween-80 and mix, in 4 DEG C of stir process 6h, the centrifugal 10min of 5000g collects thalline, and thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is joined in 600mL normal hexane, stirs, add 0.6mL linolic acid, stirring reaction 6h at 4 DEG C.6000g centrifugation dressing thalline, carries out underpressure distillation by hexane solution at 40 DEG C, reclaims normal hexane, obtains 0.6mL product, wherein
c9,
tthe content of 11-CLA is 91.6%.The dressing thalline collected is recycled and reused for above-mentioned building-up reactions, when using continuously five times in product
c9,
tthe content of 11-CLA is 85.8%.
Embodiment 4.
By activation lactobacterium casei (
lactobacilluscasei) CGMCC1.574 strain inoculation is in MRS substratum, 20h is cultivated at 30 DEG C, the centrifugal 10min of 5000g collects thalline, adds 40mL50mM phosphate buffered saline buffer (pH6.8), 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate in 10 grams of wet thallus, 37 DEG C of water-bath oscillation treatment 20min, ice bath after taking out, through power 200W ultrasonication, every supersound process 5s rests 30s, supersound process 2 times, the centrifugal 10min of 5000g obtains permeability thalline.
Permeability thalline is with after 40mL50mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 6000g collects thalline, wet thallus is resuspended in 200mL50mM phosphate buffered saline buffer (pH5.8), add 2mL tween-80 and mix, in 4 DEG C of stir process 4h, the centrifugal 10min of 6000g collects thalline, and thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is loaded in pillar bio-reactor, 0.9mL linolic acid is joined in 300mL normal hexane and mixes, will containing linoleic hexane solution injecting reactor by pump, flow is 3mL/min, collects effluent liquid, effluent liquid is repeated injecting reactor 2 times, collect effluent liquid, carry out underpressure distillation at 40 DEG C, reclaim normal hexane, obtain 0.9mL product, wherein
c9,
tthe content of 11-CLA is 91.6%.By being cascaded by several pillar bio-reactor, continuous seepage can be realized.
Claims (5)
1. the non-water enzymatic synthesis of conjugated linoleic acid isomers, is characterized in that comprising the following steps:
(1) the lactobacterium casei CGMCC1.574 thalline being in logarithmic phase is collected, 4mL50mM, pH6.8 phosphate buffered saline buffer, 5mg/mL N,O-Diacetylmuramidase and 2mM sodium ethylene diamine tetracetate is added in every gram of wet thallus, 37 DEG C of process 20min, again through ultrasonication, ultrasonication is ultrasonic power 200W, every supersound process 5s rests 30s, supersound process 2 times, centrifugally obtains permeability thalline;
(2) permeability thalline is with after the phosphate buffered saline buffer washing of 50mM, pH5.8, collected by centrifugation thalline, the phosphate buffered saline buffer being resuspended in 20mL50mM, pH5.8 by every gram of permeability wet thallus is fallen into a trap, permeability wet thallus is resuspended in phosphate buffered saline buffer, add the tween-80 of bacteria suspension volume percent 0.5-2.0%, mix, in 4-40 DEG C of process 2-6h, collected by centrifugation thalline, obtains dressing thalline by after thalline lyophilize;
(3) join 600mL normal hexane by every gram of dressing thalline to fall into a trap, dressing thalline is joined in normal hexane, stirs, add the linolic acid of normal hexane volume percent 0.1-0.3%, at 4-40 DEG C, react 1-12h;
(4) centrifugation dressing thalline, carries out underpressure distillation by hexane solution, and reclaim normal hexane, product is
c9,
t11-conjugated linoleic acid isomers.
2. the non-water enzymatic synthesis of conjugated linoleic acid isomers according to claim 1, is characterized in that the add-on of tween-80 in described step (2) is the 0.75-1.5% of bacteria suspension volume percent.
3. the non-water enzymatic synthesis of conjugated linoleic acid isomers according to claim 1, is characterized in that in described step (2), treatment temp is 4-25 DEG C.
4. the non-water enzymatic synthesis of conjugated linoleic acid isomers according to claim 1, is characterized in that in described step (2), the treatment time is 3-5h.
5. the non-water enzymatic synthesis of conjugated linoleic acid isomers according to claim 1, is characterized in that the linolic acid adding normal hexane volume percent 0.125-0.25% in described step (3), at 15-30 DEG C, reacts 2-6h.
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Citations (4)
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|---|---|---|---|---|
| CN1928101A (en) * | 2006-09-07 | 2007-03-14 | 河北工业大学 | Method for preparing conjugated linoleic acid |
| CN101565367A (en) * | 2009-05-27 | 2009-10-28 | 浙江大学 | Preparation method of conjugated linoleic acid |
| CN102206687A (en) * | 2011-04-27 | 2011-10-05 | 浙江大学 | Method for bioconverting conjugated linoleic acid by using Lactobacillus plantarum |
| CN104404092A (en) * | 2014-11-04 | 2015-03-11 | 南昌大学 | Conjugated linoleic acid isomer biological enrichment method |
-
2015
- 2015-07-20 CN CN201510424062.XA patent/CN105112462B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928101A (en) * | 2006-09-07 | 2007-03-14 | 河北工业大学 | Method for preparing conjugated linoleic acid |
| CN101565367A (en) * | 2009-05-27 | 2009-10-28 | 浙江大学 | Preparation method of conjugated linoleic acid |
| CN102206687A (en) * | 2011-04-27 | 2011-10-05 | 浙江大学 | Method for bioconverting conjugated linoleic acid by using Lactobacillus plantarum |
| CN104404092A (en) * | 2014-11-04 | 2015-03-11 | 南昌大学 | Conjugated linoleic acid isomer biological enrichment method |
Non-Patent Citations (3)
| Title |
|---|
| SHIGENOBU KISHINO 等: "Linoleic Acid Isomerase in Lactobacillus plantarum AKU1009a Proved to Be a Multi-Component Enzyme System Requiring Oxidoreduction Cofactor", 《BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY》 * |
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