CN105087691A - Method for catalytic synthesis of conjugated linoleic acid isomer by virtue of surfactant-coated bacterium - Google Patents
Method for catalytic synthesis of conjugated linoleic acid isomer by virtue of surfactant-coated bacterium Download PDFInfo
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- CN105087691A CN105087691A CN201510423890.1A CN201510423890A CN105087691A CN 105087691 A CN105087691 A CN 105087691A CN 201510423890 A CN201510423890 A CN 201510423890A CN 105087691 A CN105087691 A CN 105087691A
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- thalline
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Abstract
The invention discloses a method for catalytic synthesis of a conjugated linoleic acid isomer by virtue of a surfactant-coated bacterium, wherein the method comprises the following steps (1) re-suspending each gram of lactobacillus casei CGMCC 1.574 bacterium in 5ml of a permeabilization treatment solution, treating at 37 DEG C for 20min, centrifuging and collecting a permeabilized bacterium; (2) washing the permeabilized bacterium with 60mM of a phosphate buffer solution which is at 5.8 in pH value; centrifuging and collecting the bacterium; re-suspending each gram of the wet bacterium in 20mL of the 60mM of phosphate buffer solution which is at 5.8 in pH value, dropping an acetone solution which contains 1% of dioleyl glutamate ribitol and uniformly mixing at the same time; processing at 4-40 DEG C for 2-6h, centrifuging and collecting the bacterium, and freeze-drying so as to obtain a coated bacterium; (3) adding each gram of the freeze-dried coated bacterium to 500ml of normal hexane, uniformly stirring, adding 0.1-0.3% of linoleic acid in terms of the volume percentage of the normal hexane and reacting at 4-40 DEG C for 1-12h; and (4) centrifuging and isolating the coated bacterium and recovering the normal hexane, so as to obtain a finished product, namely c9,t11-conjugated linoleic acid isomer. The method disclosed by the invention is short in reaction time; the coated bacterium can be recycled for several times, so that yield is significantly improved; and the method is free from environmental pollution and is capable of remarkably reducing 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 method that tensio-active agent dressing thalline catalyzes and synthesizes 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, be resuspended in 5mL permeabilized treatment liquid by every gram of wet thallus to fall into a trap, wet thallus to be resuspended in permeabilized treatment liquid that (described permeabilized treatment liquid is containing the triton x-100 of volume percent 0.5-3.0%, the phosphate buffered saline buffer of 20-200mM, pH5.8), resuspended thalline processes 20min through 37 DEG C, collected by centrifugation permeability thalline.
(2) thalline Cotton seeds: permeability thalline is with after the phosphate buffered saline buffer washing of 60mM, pH5.8, collected by centrifugation thalline, the phosphate buffered saline buffer being resuspended in 20mL60mM, pH5.8 by every gram of wet thallus is fallen into a trap, permeability wet thallus is resuspended in phosphate buffered saline buffer, drip surfactant soln (described surfactant soln is the acetone soln containing the two oleyl alcohol ester ribitol of mass percent 1% L-glutamic acid) more wherein, limit edged mixes, in 4-40 DEG C of process 2-6h, collected by centrifugation thalline, obtains dressing thalline by after thalline lyophilize.
(3) catalyze and synthesize: join 500mL normal hexane by every gram of freeze-dried coated 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%, at 4-40 DEG C, react 1-12h.
(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 (1), permeabilized treatment liquid is preferably the triton x-100 containing volume percent 1.0-2.0%, the phosphate buffered saline buffer of 30-100mM, pH5.8.
The dripping quantity of the middle surfactant soln of step of the present invention (2) is preferably every 50mL bacteria suspension and adds 5-15mL surfactant soln.
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 and catalyze and synthesize
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.
By this dressing thalline process for catalytic synthesis in organic medium, with optimal conditions, synthesized by rear containing the reaction of volume percent 0.15% linoleic hexane solution
c9,
tthe content of 11-CLA isomer is volume percent 0.139%, and linoleic transformation efficiency is 92.7%.In step of the present invention (4), dressing thalline can be recycled and reused for catalyzing and synthesizing 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 medium, linolic acid is catalyzed and synthesized by microbial cells
c9,
tthe novel method of 11-CLA.The method is first by triton x-100 process 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 the two oleyl alcohol ester ribitol of L-glutamic acid to carry out dressing to thalline, containing 2 oleyl alcohol groups in the two oleyl alcohol ester ribitol molecule of L-glutamic acid, oleyl alcohol 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 the two oleyl alcohol ester ribitol concentration of suitable phosphate buffered saline buffer, L-glutamic acid and dressing temperature, time, makes gained dressing thalline still have higher catalytic capability in organic medium.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 can repeatedly be recycled and reused for biosynthesizing in organic medium
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 by dressing thalline in organic medium, 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, 10 grams of wet thallus are resuspended in 50mL containing in the triton x-100 of volume percent 1.5% and the permeabilized treatment liquid of 50mM phosphate buffered saline buffer (pH5.8), 37 DEG C of centrifugal 10min of water bath processing 20min, 5000g obtain permeability thalline.
Permeability thalline is with after 50mL60mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 6000g collects thalline, wet thallus is resuspended in 200mL60mM phosphate buffered saline buffer (pH5.8), slowly drip the two oleyl alcohol ester ribitol (Song Baodong etc. of L-glutamic acid that 40mL mass percent is 1% wherein, petrochemical complex, 2001,30 volume supplementary issue: 376-378) acetone soln, 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 500mL normal hexane, stirs, add 0.75mL 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 0.75mL product, wherein
c9,
tthe content of 11-CLA is 92.7%.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 90.3%.
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, 10 grams of wet thallus are resuspended in 50mL containing in the triton x-100 of volume percent 3.0% and the permeabilized treatment liquid of 200mM phosphate buffered saline buffer (pH5.8), 37 DEG C of centrifugal 10min of water bath processing 20min, 5000g obtain permeability thalline.
Permeability thalline is with after 40mL60mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 5000g collects thalline, wet thallus is resuspended in 200mL60mM phosphate buffered saline buffer (pH5.8), slowly drip the acetone soln that 40mL mass percent is the two oleyl alcohol ester ribitol of L-glutamic acid of 1% wherein, mix, collect thalline in 4 DEG C of centrifugal 5min of stir process 2h, 6000g, thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is joined in 500mL normal hexane, stirs, add 1.5mL 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 1.5mL product, wherein
c9,
tthe content of 11-CLA is 81.7%.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 70.9%.
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, 10 grams of wet thallus are resuspended in 50mL containing in the triton x-100 of volume percent 0.5% and the permeabilized treatment liquid of 20mM phosphate buffered saline buffer (pH5.8), 37 DEG C of centrifugal 10min of water bath processing 20min, 5000g obtain permeability thalline.
Permeability thalline is with after 50mL60mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 4000g collects thalline, wet thallus is resuspended in 200mL60mM phosphate buffered saline buffer (pH5.8), slowly drip the acetone soln that 40mL mass percent is the two oleyl alcohol ester ribitol of L-glutamic acid of 1% wherein, mix, collect thalline in 4 DEG C of centrifugal 5min of stir process 6h, 5000g, thalline carries out lyophilize after-80 DEG C of pre-freezes, obtains dressing thalline.
1 gram of freeze-dried coated thalline is joined in 500mL normal hexane, stirs, add 0.5mL 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 0.5mL product, wherein
c9,
tthe content of 11-CLA is 91.2%.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 84.9%.
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, 10 grams of wet thallus are resuspended in 50mL containing in the triton x-100 of volume percent 1.5% and the permeabilized treatment liquid of 50mM phosphate buffered saline buffer (pH5.8), 37 DEG C of centrifugal 10min of water bath processing 20min, 5000g obtain permeability thalline.
Permeability thalline is with after 50mL60mM phosphate buffered saline buffer (pH5.8) washing, the centrifugal 10min of 6000g collects thalline, wet thallus is resuspended in 200mL60mM phosphate buffered saline buffer (pH5.8), slowly drip the acetone soln that 40mL mass percent is the two oleyl alcohol ester ribitol of L-glutamic acid of 1% wherein, mix, collect thalline in 4 DEG C of centrifugal 10min of stir process 4h, 6000g, 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, 0.75mL linolic acid is joined in 250mL 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 0.75mL product, wherein
c9,
tthe content of 11-CLA is 92.5%.By being cascaded by several pillar bio-reactor, continuous seepage can be realized.
Claims (6)
1. tensio-active agent dressing thalline catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that comprising the following steps:
(1) the lactobacterium casei CGMCC1.574 thalline being in logarithmic phase is collected, be resuspended in 5mL permeabilized treatment liquid by every gram of wet thallus to fall into a trap, be resuspended in by wet thallus in permeabilized treatment liquid, resuspended thalline processes 20min through 37 DEG C, collected by centrifugation permeability thalline;
(2) permeability thalline is with after the phosphate buffered saline buffer washing of 60mM, pH5.8, collected by centrifugation thalline, the phosphate buffered saline buffer being resuspended in 20mL60mM, pH5.8 by every gram of wet thallus is fallen into a trap, permeability wet thallus is resuspended in phosphate buffered saline buffer, drip tensio-active agent wherein again, limit edged mixes, in 4-40 DEG C of process 2-6h, collected by centrifugation thalline, obtains dressing thalline by after thalline lyophilize;
(3) join 500mL normal hexane by every gram of freeze-dried coated 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;
Permeabilized treatment liquid described in step (1) contains the triton x-100 of volume percent 0.5-3.0%, the phosphate buffered saline buffer of 20-200mM, pH5.8;
Tensio-active agent described in step (2) is the acetone soln containing the two oleyl alcohol ester ribitol of mass percent 1% L-glutamic acid.
2. tensio-active agent dressing thalline according to claim 1 catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that in described step (1), permeabilized treatment liquid is the triton x-100 containing volume percent 1.0-2.0%, the phosphate buffered saline buffer of 30-100mM, pH5.8.
3. tensio-active agent dressing thalline according to claim 1 catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that the dripping quantity of tensio-active agent in described step (2) is that every 50mL bacteria suspension adds 5-15mL tensio-active agent.
4. tensio-active agent dressing thalline according to claim 1 catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that in described step (2), treatment temp is 4-25 DEG C.
5. tensio-active agent dressing thalline according to claim 1 catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that in described step (2), the treatment time is 3-5h.
6. tensio-active agent dressing thalline according to claim 1 catalyzes and synthesizes the method for conjugated linoleic acid isomers, it 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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|---|---|---|---|---|
| CN1629302A (en) * | 2004-07-28 | 2005-06-22 | 南昌大学 | Process for preparing conjugated fatty acid |
| 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 |
| CN104404092A (en) * | 2014-11-04 | 2015-03-11 | 南昌大学 | Conjugated linoleic acid isomer biological enrichment method |
-
2015
- 2015-07-20 CN CN201510423890.1A patent/CN105087691B/en not_active Expired - Fee Related
Patent Citations (4)
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| CN1629302A (en) * | 2004-07-28 | 2005-06-22 | 南昌大学 | Process for preparing conjugated fatty acid |
| 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 |
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Non-Patent Citations (3)
| Title |
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| SHIGENOBU KISHINO等: "Linoleic Acid Isomerase in Lactobacillus plantarum AKU1009a Proved to Be a Multi-Component Enzyme System Requiring Oxidoreduction Cofactors", 《BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY》 * |
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