CN105087691B - The method that surfactant coating thalline catalyzes and synthesizes conjugated linoleic acid isomers - Google Patents
The method that surfactant coating thalline catalyzes and synthesizes conjugated linoleic acid isomers Download PDFInfo
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Abstract
The method that surfactant coating thalline catalyzes and synthesizes conjugated linoleic acid isomers, comprises 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 60mM, pH5.8, thalline is collected by centrifugation, every gram of wet thallus is resuspended in the phosphate buffer of 20mL60mM, pH5.8, the acetone soln of double oleyl alcohol ester ribitol containing 1% glutamic acid is added dropwise again, side edged mixes, 4 40 DEG C of 2 6h of processing, are collected by centrifugation thalline, thalline must be coated after freeze-drying;(3)Every gram of freeze-dried coated thalline is added in 500mL n-hexanes, is stirred evenly, the linoleic 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, recycles n-hexane, product isc9,t11 conjugated linoleic acid 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
Conjugated linoleic acid (conjugated linoleic acid, CLA) is the system of one group of Octadecane Conjugated Dienoic Acid
Claim, conjugated double bond there are 4 kinds of position isomeries(8,10; 9,11; 10,12; 11,13)With 4 kinds of geometrical isomerisms(cis,cis; cis,trans; trans,cis; trans,trans), up to 16 kinds of isomers are shared, such as:c9,t11-CLA andt10,c12-CLA
Isomers etc..Research finds that CLA has the physiology work(such as anticancer, prevention of arterial atherosis, enhancing immunity of organisms, anti-diabetic
Can, and more and more researchs show that its physiological activity has isomers specificity, generally believe nowc9,t11-CLA isomeries
Body has the function that anticancer and prevention of arterial atherosis.In nature, CLA is primarily present in the milk and meat of ruminant
Fat in, mainly byc9,t11-CLA and a small amount oft9,t11-CLA,t10,cThe isomers such as 11-CLA are formed, but its content is led to
Often both less than 10mg/g is fatty, can not meet the development and application for the purpose of health care and medical treatment.
To realize prepared by a large amount of of CLA isomers, people prepare CLA to microbial fermentation and have carried out more research.Mesh
Before, microbial fermentation carries out in aqueous, since added fermentation substrate-linoleic acid is liposoluble substance, sub- oil
Acid is needed through emulsification treatment before adding zymotic fluid, and additive amount is restricted, and CLA products are obtained from zymotic fluid need to be through organic solvent
Extraction, there are the shortcomings that fermentation period is long, production cost is high, low output for whole production technology.Patent of invention
CN201110105610 proposes a kind of method of bioconverting conjugated linoleic acid by using Lactobacillus plantarum, and substrate additive amount is 25mg/
ML, fermentation time are up to 120h, and the yield of CLA only has 1.02mg/mL, and the conversion ratio of substrate only has 4%.
The content of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, propose that a kind of surfactant coating thalline catalyzes and synthesizes altogether
The method of conjugated linoleic acid isomers, significantly shortens generated time, improves yield and conversion ratio, reduces 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 triton x-100 of permeabilized treatment liquid 0.5-3.0% containing percent by volume, 20-200mM,
The phosphate buffer of pH5.8), thalline is resuspended through 37 DEG C of processing 20min, permeability thalline is collected by centrifugation.
(2)Thalline Cotton seeds:After permeability thalline is washed with the phosphate buffer of 60mM, pH5.8, bacterium is collected by centrifugation
Body, the phosphate buffer that 20mL60mM, pH5.8 are resuspended in by every gram of wet thallus are fallen into a trap, and permeability wet thallus is resuspended in phosphorus
In phthalate buffer, then surfactant solution is added dropwise thereto(The surfactant solution is containing mass percent 1%
The acetone soln of the double oleyl alcohol ester ribitol of glutamic acid), side edged is uniformly mixed, and handles 2-6h in 4-40 DEG C, thalline is collected by centrifugation,
Coating thalline is obtained after thalline is freeze-dried.
(3)Catalyze and synthesize:500mL n-hexanes are added to by every gram of freeze-dried coated thalline to fall into a trap, and coating thalline is added to
In n-hexane, stir evenly, add the linoleic acid of n-hexane percent by volume 0.1-0.3%, react 1-12h at 4-40 DEG C.
(4)Collection of products:Coating thalline is centrifuged, hexane solution is evaporated under reduced pressure, recycles n-hexane, product
Forc9,t11- conjugated linoleic acid isomers.
Step of the present invention(1)Middle permeabilized treatment liquid is preferably that the Qula of the 1.0-2.0% containing percent by volume leads to X-
100,30-100mM, the phosphate buffer of pH5.8.
Step of the present invention(2)The dripping quantity of middle surfactant solution is preferably to add 5- per 50mL bacteria suspensions
15mL surfactant solutions.
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 linoleic 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 catalyzing and synthesizingc9,t11- conjugated linoleic acid isomeries
Body.
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, by biological isomerization can by linoleic acid intoc9,t11-CLA isomers.At present, definite catalysis has not been separated to also
The linoleate isomerase sterling of activity.Therefore, which is probably to be completed by multi-enzyme system co-catalysis.
By coating thalline process for catalytic synthesis in organic media, with optimal conditions, containing percent by volume 0.15%
It is synthesized after linoleic hexane solution reactionc9,tThe content of 11-CLA isomers is percent by volume 0.139%, linoleic acid
Conversion ratio be 92.7%.Step of the present invention(4)Middle coating thalline can be repeated for step(3)Catalyze and synthesizec9,t11-CLA isomers, after coating thalline is reused five times, its catalytic activity is still greater than 90%.
Carried out in aqueous for existing microbial fermentation synthesis CLA, whole production technology there are fermentation period length,
CLA products difficulty is extracted from zymotic fluid, thalline cannot be reused, and production cost is high, and yield is low to wait shortcomings.The present invention
Propose and catalyzed and synthesized linoleic acid in organic media by microbial cellsc9,tThe new method of 11-CLA.This method is first
Lactobacillus casei CGMCC 1.574 is handled by triton x-100, while thalline integrality is kept, improves somatic cells
Wall and membrane passage, on the one hand may insure the linoleate isomerase surface energy shape intracellular in follow-up Cotton seeds
Into complete coatings, the speed of reaction substrate linoleic acid and product CLA disengaging thalline on the other hand can be improved, reduces high concentration
Substrate and product greatly shorten the reaction time to the inhibitory action of catalytic reaction.
The present invention selects the double oleyl alcohol ester ribitol of glutamic acid to be coated thalline, the double oleyl alcohol ester ribitol molecules of glutamic acid
In contain 2 oleyl alcohol groups, oleyl alcohol is similar with linoleic acid structure, is the analogue of thalline Linoleic acid isomery zymolyte,
When thalline is coated, molecular engram can be formed in the catalytic active center of enzyme, make the catalytic activity of thalline enzyme after freeze-drying
Center conformation is constant, can keep higher catalytic activity in organic solvent.Meanwhile the present invention is incorporated in thalline Cotton seeds mistake
The double oleyl alcohol ester ribose determining alcohols of suitable phosphate buffer, glutamic acid and coating temperature, time are selected in journey, is coated gained
Thalline still has higher catalytic capability in organic media.The heat endurance of enzyme improves in thalline after coating, every batch of secondary response
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 productc9,t11-CLA.Centrifugation gained coating thalline can be multiple
It is recycled and reused for biosynthesis in organic mediac9,t11-CLA isomers, significantly shorten the production time, improve yield, reduce
Production cost.In addition, n-hexane is the common organic solvent of edible oil and fat industry, this guarantees obtained by the present inventionc9,tThe safety in utilization of 11-CLA.
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 linoleic acid solubility is low and product CLA extracts the problem of difficult.The heat endurance for being coated enzyme in thalline improves, every batch of
Reaction time only needs 4h, much smaller than time a couple of days for fermenting required in traditional aqueous.It is reusable repeatedly to be coated thalline, shows
Write and improve product yield, and non-environmental-pollution, production cost can be significantly reduced;And the thalline in traditional aqueous system zymotic fluid
It is used only once, 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 and 50mM phosphate buffers(pH5.8)Permeabilized treatment liquid in, 37 DEG C of water bath processing 20min,
5000g centrifugations 10min obtains permeability thalline.
Permeability thalline 50mL60mM phosphate buffers(pH5.8)After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL60mM phosphate buffers(pH5.8)In, 40mL mass percents are slowly added dropwise thereto as 1%
The double oleyl alcohol ester ribitol of glutamic acid(Song Baodong etc., petrochemical industry, the supplementary issues of volume 2001,30:376-378)Acetone soln, mix
Closing uniformly, thalline are collected in 4 DEG C of stir process 4h, 6000g centrifugation 10min, thalline is freeze-dried after -80 DEG C of pre-freezes,
Obtain coating thalline.
1 gram of freeze-dried coated thalline is added in 500mL n-hexanes, is stirred evenly, 0.75mL linoleic acid is added, at 25 DEG C
Lower stirring reaction 4h.6000g centrifuges coating thalline, and hexane solution is evaporated under reduced pressure at 40 DEG C, recycles n-hexane,
0.75mL products are obtained, whereinc9,tThe content of 11-CLA is 92.7%.The coating thalline being collected into is recycled and reused for above-mentioned synthesis
Reaction, when being used continuously five times in productc9,tThe content of 11-CLA is 90.3%.
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 and 200mM phosphate buffers(pH5.8)Permeabilized treatment liquid in, 37 DEG C of water bath processings
20min, 5000g centrifugation 10min obtain permeability thalline.
Permeability thalline 40mL60mM phosphate buffers(pH5.8)After washing, 5000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL60mM phosphate buffers(pH5.8)In, 40mL mass percents are slowly added dropwise thereto as 1%
The double oleyl alcohol ester ribitol of glutamic acid acetone soln, be uniformly mixed, bacterium collected in 4 DEG C of stir process 2h, 6000g centrifugation 5min
Body, thalline are freeze-dried after -80 DEG C of pre-freezes, obtain coating thalline.
1 gram of freeze-dried coated thalline is added in 500mL n-hexanes, is stirred evenly, 1.5mL linoleic acid is added, at 40 DEG C
Lower stirring reaction 12h.6000g centrifuge coating thalline, hexane solution is evaporated under reduced pressure at 40 DEG C, recycling just oneself
Alkane, obtains 1.5mL products, whereinc9,tThe content of 11-CLA is 81.7%.The coating thalline being collected into is recycled and reused for above-mentioned conjunction
Into reaction, during continuous use five times in productc9,tThe content of 11-CLA is 70.9%.
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 and 20mM phosphate buffers(pH5.8)Permeabilized treatment liquid in, 37 DEG C of water bath processing 20min,
5000g centrifugations 10min obtains permeability thalline.
Permeability thalline 50mL60mM phosphate buffers(pH5.8)After washing, 4000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL60mM phosphate buffers(pH5.8)In, 40mL mass percents are slowly added dropwise thereto as 1%
The double oleyl alcohol ester ribitol of glutamic acid acetone soln, be uniformly mixed, bacterium collected in 4 DEG C of stir process 6h, 5000g centrifugation 5min
Body, thalline are freeze-dried after -80 DEG C of pre-freezes, obtain coating thalline.
1 gram of freeze-dried coated thalline is added in 500mL n-hexanes, is stirred evenly, 0.5mL linoleic acid is added, at 4 DEG C
Stirring reaction 6h.6000g centrifuges coating thalline, and hexane solution is evaporated under reduced pressure at 40 DEG C, recycles n-hexane, obtains
To 0.5mL products, whereinc9,tThe content of 11-CLA is 91.2%.It is anti-that the coating thalline being collected into is recycled and reused for above-mentioned synthesis
Should, when being used continuously five times in productc9,tThe content of 11-CLA is 84.9%.
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 and 50mM phosphate buffers(pH5.8)Permeabilized treatment liquid in, 37 DEG C of water bath processing 20min,
5000g centrifugations 10min obtains permeability thalline.
Permeability thalline 50mL60mM phosphate buffers(pH5.8)After washing, 6000g centrifugations 10min collects thalline,
Wet thallus is resuspended in 200mL60mM phosphate buffers(pH5.8)In, 40mL mass percents are slowly added dropwise thereto as 1%
The double oleyl alcohol ester ribitol of glutamic acid acetone soln, be uniformly mixed, collected in 4 DEG C of stir process 4h, 6000g centrifugation 10min
Thalline, thalline are freeze-dried after -80 DEG C of pre-freezes, obtain coating thalline.
1 gram of freeze-dried coated thalline is fitted into pillar bioreactor, 0.75mL linoleic acid is added to 250mL n-hexanes
Middle mixing, will contain linoleic hexane solution by pump and inject reactor, flow 3mL/min, collects efflux, will flow out
Liquid is repeatedly injected reactor 2 times, collects efflux, is evaporated under reduced pressure at 40 DEG C, recycles n-hexane, obtains 0.75mL products,
Whereinc9,tThe content of 11-CLA is 92.5%.By the way that several pillar bioreactors are cascaded, it is possible to achieve continuous raw
Production.
Claims (6)
1. surfactant coating thalline catalyzes and synthesizes the method for conjugated linoleic acid isomers, it is characterized in that comprising the following steps:
(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 60mM, pH5.8, thalline is collected by centrifugation, by every gram of wet thallus weight
The phosphate buffer for being suspended from 20mL60mM, pH5.8 is fallen into a trap, and permeability wet thallus is resuspended in phosphate buffer, then to
Surfactant is wherein added dropwise, side edged is uniformly mixed, and handles 2-6h in 4-40 DEG C, thalline is collected by centrifugation, and thalline is freezed dry
Coating thalline is obtained after dry;
(3)500mL 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 linoleic 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 isc9,t11- is conjugated
Linoleic acid isomers;
Step(1)The triton x-100 of permeabilized treatment liquid 0.5-3.0% containing percent by volume, 20-200mM, pH5.8
Phosphate buffer;
Step(2)The surfactant is the acetone soln of double oleyl alcohol ester ribitol containing 1% glutamic acid of mass percent.
2. the method that surfactant coating thalline according to claim 1 catalyzes and synthesizes conjugated linoleic acid isomers, its
It is characterized in the step(1)Middle permeabilized treatment liquid be the 1.0-2.0% containing percent by volume triton x-100,30-
The phosphate buffer of 100mM, pH5.8.
3. the method that surfactant coating thalline according to claim 1 catalyzes and synthesizes conjugated linoleic acid isomers, its
It is characterized in the step(2)The dripping quantity of middle surfactant is to add 5-15mL surfactants per 50mL bacteria suspensions.
4. the method that surfactant coating thalline according to claim 1 catalyzes and synthesizes conjugated linoleic acid isomers, its
It is characterized in the step(2)Middle treatment temperature is 4-25 DEG C.
5. the method that surfactant coating thalline according to claim 1 catalyzes and synthesizes conjugated linoleic acid isomers, its
It is characterized in the step(2)Middle processing time is 3-5h.
6. the method that surfactant coating thalline according to claim 1 catalyzes and synthesizes conjugated linoleic acid isomers, its
It is characterized in the step(3)The middle linoleic acid for adding n-hexane percent by volume 0.125-0.25%, reacts at 15-30 DEG C
2-6h。
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