CN103146628B - Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof - Google Patents
Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof Download PDFInfo
- Publication number
- CN103146628B CN103146628B CN201310066107.1A CN201310066107A CN103146628B CN 103146628 B CN103146628 B CN 103146628B CN 201310066107 A CN201310066107 A CN 201310066107A CN 103146628 B CN103146628 B CN 103146628B
- Authority
- CN
- China
- Prior art keywords
- pai
- lactococcus lactis
- lactis
- lactococcal
- cla
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as a construction method and an application thereof. The method for constructing the recombined lactococcal lactis comprises the step of introducing an encoding gene of a protein PAI (Plasminogen Activator Inhibitor) into lactococcal lactis serving as host bacteria to obtain the recombined lactococcal lactis, the conversion rate of the trans-10, cis-12 conjugated linoleic acid of which is higher than that of the host bacteria, wherein the protain PAI is a protein (a) composed of amino acid sequences shown in SEQ IN No. 2 or a protein (b) which is formed by replacing the amino acid sequences shown in SEQ IN No. 2 with one or several amino acid residues and/or deleting and/or adding the amino acid sequences shown in SEQ IN No. 2 and has the activity of linoleate isomerase, or is derived from the protein (1) and has the activity of linoleate isomerase. The food-grade recombined lactococcal lactis disclosed by the invention can produce t10c12-CLA, and the content of the t10c12-CLA accounts for 15.19-23.91% of the total quantity of thallus fatty acids.
Description
Technical field
The present invention relates to produce food-grade recombination lactic acid galactococcus and construction process and the application of conjugated linolic acid.
Background technology
Conjugated linolic acid (CLA) belongs to octadecadienoic acid, is the isomer of linolic acid (LA), comprises that more than 20 plant component.Wherein to cis-9, anti-form-1 1 conjugated linolic acid and anti-form-1 0, cis-12 conjugated linolic acid (trans-10, cis-12; T10c12-CLA) biologic activity of isomer is more clearly.Concerning t10c12-CLA, it can promote the metabolism of health, suppresses the synthetic of fat, can reduce the probability of occurrence of obesity.2008, U.S. FDA was issued food license licensed licenser licence, agreed to CLA to use as foodstuff additive.At present, CLA is produced and sells as healthcare products by numerous food product and pharmacy corporation.
Occurring in nature, some rumen microorganisms in ox, sheep body have the ability of synthetic CLA, and these CLA can enter in milk, and the CLA total amount of one liter of milk is 1.4 grams of left and right, and wherein the proportion of t10c12-CLA is less than 10%.Industrial, to after linolic acid alkali isomerization, generate CLA, product is the form of mixture, except above-mentioned two kinds of CLA components, also contain multiple isomer impurities, the content of impurity even can reach more than 30%, if these impurity enter human body, easily derives the multiple conjugate class isomer that has negative biological effect by arachidonic acid metabolic pathway.Can find out, if the CLA adding in food does not contain impurity, composition is single, more be conducive to human consumer's health.
Under the support of biotechnology, utilize microorganism to produce in a large number the one-component of CLA, be a more satisfactory approach.Research is found, more natural lower bacteria classes have the ability that self synthesizes t10c12-CLA component, for example, the linoleate isomerase PAI(Propionibacterium acnes isoenzyme of propionibacterium acnes (Propionibacterium acnes)) just linolic acid LA isomery can be become to t10c12-CLA.Codon optimized pai gene, can be in cells of mamma animals heterogenous expression, also linolic acid can be changed into t10c12-CLA composition (Gou Kemian, Li Shili, the dual purpose of linoleate isomerase aspect dehydrogenation and isomery, number of patent application: 201110112685.5).But also there is not the report of the reconstituted food level microbe that t10c12-CLA transformation efficiency is high at present.
Summary of the invention
A technical problem to be solved by this invention is to provide Recombinant Lactococcus lactis and construction process and the application that t10c12-CLA transformation efficiency is high.
The high Recombinant Lactococcus lactis of a strain t10c12-CLA transformation efficiency provided by the present invention, be Lactococcus lactis (Lactococcus lactis) PAI-75, it is numbered CGMCC No.7148 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The method of the high Recombinant Lactococcus lactis of transformation efficiency of structure t10c12-CLA provided by the present invention, comprise the encoding gene of protein PAI is imported and in the Lactococcus lactis of Host Strains, obtains anti-form-1 0, the transformation efficiency of cis-12 conjugated linolic acid is higher than the step of the Recombinant Lactococcus lactis of described Host Strains;
Described protein PAI is following protein a) or b):
A) protein being formed by the aminoacid sequence shown in SEQ ID No.2;
B) aminoacid sequence of SEQ ID No.2 passed through to replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has linoleate isomerase activity, or thering is linoleate isomerase activity by a) derivative protein.
Wherein, described protein PAI has linoleate isomerase activity.SEQ ID No.2 is made up of 424 amino-acid residues.
In aforesaid method, the transformation efficiency of described t10c12-CLA refers to that described Host Strains or Recombinant Lactococcus lactis are taking linolic acid as substrate, anti-form-1 0 in the thalline that cultivation obtains, anti-form-1 0 in the quality of cis-12 conjugated linolic acid and thalline, the ratio of cis-12 conjugated linolic acid and linolic acid quality sum.
The encoding gene of described protein PAI specifically can be following 1) or 2) or 3) DNA molecular:
1) its encoding sequence is the DNA molecular of SEQ ID No.1;
2) under stringent condition with 1) DNA molecule hybridize that limits and the DNA molecular of code for said proteins PAI;
3) with 1) DNA molecular that limits has more than 90% consistence and the DNA molecular of code for said proteins PAI.
Wherein, SEQ ID No.1 is made up of 1275 Nucleotide, and its encoding sequence is 1-1275 position.
In aforesaid method, described Host Strains is food grade Lactococcus lactis and/or its derivative strain.
The encoding gene of described protein PAI imports in described Host Strains by reconstituted food grade expression vector.
Wherein, described food grade Lactococcus lactis refers to edible Lactococcus lactis, can be Lactococcus lactis NZ3900 or its derivative strain and/or Lactococcus lactis MG1363 or its derivative bacterium and/or Lactococcus lactis LM0230 or its derivative bacterium and/or Lactococcus lactis IL1403 or its derivative bacterium etc.Described reconstituted food grade expression vector is that selection markers is the expression vector of food grade selection markers, and described food grade selection markers specifically can be the lacF gene of Lactococcus lactis (Lactococcus lactis).
In aforesaid method, described food grade Lactococcus lactis can be Lactococcus lactis NZ3900 or its derivative strain and/or Lactococcus lactis MG1363 or its derivative bacterium and/or Lactococcus lactis LM0230 or its derivative bacterium and/or Lactococcus lactis IL1403 or its derivative bacterium etc., and described reconstituted food grade expression vector can be the reconstituted food grade expression vector of the encoding gene of expressing described protein PAI.
Further, described reconstituted food grade expression vector specifically can be the recombinant expression vector that the multiple clone site of the encoding gene insertion pNZ8149 of described protein PAI is obtained.
The anti-form-1 0 that above-mentioned arbitrary described method builds, the transformation efficiency of cis-12 conjugated linolic acid also belongs to protection scope of the present invention higher than the Recombinant Lactococcus lactis of described Host Strains.
The present invention also provides the reconstituted food grade expression vector of the encoding gene of expressing described protein PAI.
Wherein, described reconstituted food grade expression vector specifically can be the recombinant expression vector that the multiple clone site of the encoding gene insertion pNZ8149 of described protein PAI is obtained.
The Recombinant Lactococcus lactis that t10c12-CLA transformation efficiency provided by the present invention is high specifically can be food grade Lactococcus lactis, be preferably the recombinant type Lactococcus lactis that gene engineering method is produced, described Recombinant Lactococcus lactis has been integrated the linoleate isomerase PAI gene of external source, this bacterial strain can be expressed linoleate isomerase, this enzyme can become linolic acid biocatalysis anti-form-1 0, cis-12 conjugated linolic acid.
Of the present invention experimental results show that, patent of invention (Gou Kemian will be applied for, Li Shili, the dual purpose of linoleate isomerase aspect dehydrogenation and isomery, number of patent application: the 201110112685.5) encoding gene of described PAI, insert in food grade expression vector pNZ8149, then transform Lactococcus lactis (Lactococcus lactis) NZ3900, and then the food-grade recombination lactic acid galactococcus of acquisition stably express PAI gene, can effectively produce t10c12-CLA, the content of product t10c12-CLA accounts for the 15.19-23.91% of thalline fatty acid total amount; Wherein, in the best recombinant bacterial strain of effect, t10c12-CLA content has exceeded 23.91% of thalline fatty acid total amount, and substrate linolic acid isomery becomes the transformation efficiency of product t10c12-CLA to exceed 37.6%; The present invention provides method and bacterial strain for utilizing Recombinant Lactococcus lactis to produce the related application of t10c12-CLA.
Biomaterial information
Classification And Nomenclature: Lactococcus lactis (Lactococcus lactis)
Strain number: PAI-75
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 01 18th, 2013
The preservation center numbering of registering on the books: CGMCC No.7148
Describe the present invention in detail below in conjunction with specific embodiment, these embodiment are for understanding instead of restriction the present invention.
Brief description of the drawings
Fig. 1 is linolic acid and anti-form-1 0 in the thalline of each bacterial strain, and cis-12 conjugated linolic acid accounts for the percentage composition of thalline total fatty acids.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Lactococcus lactis (Lactococcus lactis) NZ3900(in following embodiment is purchased from German Mo Bi Tec company), be food grade strain, be lactose deficient strain, can not utilize lactose, can only be growing containing in the substratum of glucose.
PNZ8149(is purchased from German Mo Bi Tec company), be food grade expression vector, the gene that contains metabolising lactose.
Embodiment 1, the high Recombinant Lactococcus lactis of structure t10c12-CLA transformation efficiency
The present embodiment, using Lactococcus lactis (Lactococcus lactis) NZ3900 as Host Strains, taking pNZ8149-PAI as reconstituted food grade expression vector as example, is illustrated the method that builds the Recombinant Lactococcus lactis that t10c12-CLA transformation efficiency is high.The method proceeds to pNZ8149-PAI after Lactococcus lactis (Lactococcus lactis) NZ3900 bacterium, utilize lactose medium screening recombinant bacterial strain, the restructuring thalline that can grow just may carry the pai gene of external source, and the PAI isomerase of this genetic expression can change into t10c12-CLA by linolic acid.This expression system has been avoided antibiotic use, does not also produce intracellular toxin, is a kind of food grade vector expression system efficiently.At this, by above-mentioned experimental principle and method, pai gene is imported in food grade Lactococcus lactis by food grade expression vector, obtain the food grade engineering strain that can produce t10c12-CLA.Specific as follows:
1, the reconstituted food level of the encoding gene of marking protein PAI is expressed the structure that carries pNZ8149-PAI
For the optimization pai gene shown in SEQ ID No.1 is inserted to pNZ8149 plasmid, design PCR primer (sense:5 '-aaa act gca gcc atg agc att agt aag gac agc a-3 ', anti-sense:5 '-cgg ggt acc tta gac aaa gaa ccg ggt cac ca-3 '), Pst I and Acc651 restriction enzyme site between upstream and downstream primer, are introduced respectively, taking the optimization pai gene shown in SEQ ID No.1 as template, with the optimization pai gene shown in high-fidelity pfu enzyme (Tian Gen biochemical technology company limited) amplification SEQ ID No.1, PCR condition is: 95 DEG C, 5min, 94 DEG C, 30sec, 60 DEG C, 30sec, 72 DEG C, 150sec, totally 32 circulations, 72 DEG C, 10min.The PCR product obtaining is with after Pst I and Acc651 double digestion, electrophoresis reclaims fragment and is connected on pNZ8149 plasmid (purchased from German MoBiTec company) by Pst I and Acc651 double enzyme site, be built into pNZ8149-PAI plasmid, enzyme is cut qualification and DNA sequencing proves, the nucleotide sequence of the optimization pai gene in pNZ8149-PAI is SEQ ID No.1.The albumen PAI of optimization pai genes encoding SEQ ID No.2 shown in SEQ ID No.1.
Conventional phenol/chloroform/primary isoamyl alcohol extraction process purifying pNZ8149-PAI plasmid, for transforming food grade Lactococcus lactis (Lactococcus lactis) NZ3900.
2, the preparation of the Lactococcus lactis common cultivation of (Lactococcus lactis) NZ3900 and competence bacterium
The German MoBiTec of reference company product description
(
http:// www.mobitec.com/download/handbook/VS-NICE_Handbook.pdf) operation.Particularly, the Lactococcus lactis of-80 DEG C of refrigerator defrostings (Lactococcus lactis) NZ3900, is seeded to by 1% dilution in the basic culture solution (M17 meat soup+0.5M sucrose+2.5% glycine+0.5% glucose) of 5 milliliters, and 30 DEG C leave standstill and cultivate; This 5 ml soln was diluted with 50 milliliters of above-mentioned basic culture solutions again in second day, 30 DEG C leave standstill cultivation; Above-mentioned 25 milliliters of nutrient solutions were diluted in 200 milliliters of fresh basic culture solutions in the 3rd day, continue to cultivate, as the OD of bacterium liquid
600when value reaches 0.2-0.3, collect thalline, after 4 DEG C centrifugal (6000 × g, 20min), with the resuspended precipitation thalline of 0.5M sucrose+10% glycerine solution of 200 milliliters, continue centrifugal (6000 × g, 20min); Use again the resuspended thalline of 0.5M sucrose+10% glycerine+50mM EDTA solution of 100 milliliters, ice bath 15min; 4 DEG C are continued centrifugal (8000 × g, 20min), with the thalline of 50 milliliters of 0.5M sucrose+10% glycerine solutions washing precipitation, 4 DEG C of centrifugal (6000 × g, 20min), with 2 milliliters of resuspended thalline of 0.5M sucrose+10% glycerine solution, 40 microlitres divide and in the micro centrifugal pipe of the 1.5-mL that installs to precooling, are placed in stand-byly on ice, or after liquid nitrogen flash freezer, are stored in-80 DEG C of refrigerators.
3, the qualification of the conversion of the electricity of Lactococcus lactis (Lactococcus lactis) NZ3900 and restructuring bacterium colony
In 40 microlitre Lactococcus lactis (Lactococcus lactis) the NZ3900 competent cell that above-mentioned steps 2 is prepared, the 5 microlitre pNZ8149-PAI plasmid purifications that add step 1 to prepare, after mixing gently, transfer in the electric revolving cup (Bio-Rad company of the U.S.) of 2-mm, after ice bath 5 minutes, use Gene pulser X-cell type electroporation (Bio-Rad company of the U.S.) to carry out electricity and transform, electricity turns parameter and is: 2000V, 200 Ω, 25 μ F; Subsequently, add immediately 1 ml soln (basic culture solution+20mM MgCl described in step 2
2+ 2mM CaCl
2) in, ice bath 5 minutes; Then, 30 DEG C leave standstill cultivation approximately 1 hour; Draw the bacterium liquid of appropriate recovery, coat Elliker solid medium (containing 0.004% Bromocresol purple and 0.5% lactose) upper, 30 DEG C of constant temperature culture 36-48 hour, screening mono-clonal transforms bacterium colony, and wherein, it is yellow that positive periphery of bacterial colonies substratum is.
Above-mentioned positive monoclonal bacterium colony is chosen to 1 ml soln (M17 meat soup+0.5% lactose) to 30 DEG C of incubated overnight; Second day, draw 200 microlitre solution centrifugals wherein, use again 50 microlitre lysates (4M Guanidinium hydrochloride+50mM EDTA+50mM Tris-HCl+10mM DTT) resuspended, boiling water bath boils 15 minutes, centrifugal and with twice of distilled water wash, finally use 20 microlitre distilled water resuspended, get the template of 1 microlitre as thalline pcr amplification pai gene.The inner a pair of Auele Specific Primer of design pai gene (sense:5 '-caggattccacgattacac-3 ', anti-sense:5 '-cagtcaggaccagcacat-3 ').PCR condition is: 95 DEG C, and 10min; 94 DEG C, 30sec, 60 DEG C, 30sec, 72 DEG C, 1min, totally 35 circulations; 72 DEG C, 10min.PCR product carries out DNA gel electrophoretic analysis and takes pictures.The recombinant bacterial strain of the PCR positive, extract plasmid according to ordinary method, carry out enzyme and cut qualification, and order-checking, the step 4 that the bacterial strain (called after Recombinant Lactococcus lactis (Lactococcus lactis) PAI-22, PAI-45, PAI-75, PAI-76, PAI-80, PAI-91) to the Recombinant Lactococcus lactis (Lactococcus lactis) that contains the optimization pai gene shown in SEQ ID No.1 continues below operates.
Meanwhile, according to the method described above pNZ8149 is imported to Lactococcus lactis (Lactococcus lactis) NZ3900 and obtain recombinant bacterial strain as empty carrier control strain (called after Recombinant Lactococcus lactis (Lactococcus lactis) P8149).
4, Lactococcus lactis abduction delivering, fatty acid content is measured
Respectively from Lactococcus lactis (Lactococcus lactis) NZ3900, Recombinant Lactococcus lactis (Lactococcus lactis) P8149, PAI-22, PAI-45, PAI-75, PAI-76, PAI-80 and the PAI-91 bacterium liquid of 5 milliliters of incubated overnight, getting wherein 40 microlitres is inoculated in 10 milliliters of M17 meat soup solution, cultivate 2-3 hour, work as OD for 30 DEG C
600reach at 0.4 o'clock, in bacterium liquid, add Sigma company of the inductor nisin nisin(U.S. simultaneously) and substrate linolic acid (Sigma, L1012), require the final concentration of two kinds of reagent to reach respectively every milliliter of 50 nanograms and 0.5 milligram, continue to cultivate 48 hours, centrifugal collection thalline, reference standard method is carried out methyl esterification of fatty acid processing, measures thalline and comprises t10c12-CLA and the linolic acid percentage composition at interior various lipid acid.Concrete steps are: thalline is transferred to the glass of tetrafluoroethylene bottle cap and is methylated in pipe, every part adds 4 milliliters of chloracetyl methyl alcohol (chloracetyl: methyl alcohol=1:10) and 2 ml n-hexanes, covers tightly pipe lid, 80 DEG C of water-baths 2 hours, taking-up is cooled to after room temperature, then adds 7% K
2cO
35 milliliters of solution, vibration mixes latter standing 10 minutes, centrifugal (1000rpm, supernatant direct injection 5min) separating is to HP-88 type chromatographic column (Agilent company of the U.S.), use HP7890 full-automatic gas chromatography (Agilent) to analyze, concrete gas-chromatography program is: 120 DEG C of initial temperatures, keep 1min; Be warming up to 175 DEG C with the speed of 10 DEG C/min, keep 10min; Be warming up to 210 DEG C with the speed of 5 DEG C/min again, keep 5min; Be warming up to 240 DEG C with the speed of 5 DEG C/min again, keep finishing after 10min.Carrier gas is helium, and splitting ratio is 10:1.Adopt external standard method (calibration curve method) to carry out quantitatively lipid acid.With reference to lipid acid standard substance (Sigma, 47885-U and O5632) peak area, the signal that instrument is detected, with GC ChemStation Software(Agilent) software processes, calculate relative content, the experiment of cell cultures and determination of fatty acid repeats 4 times continuously, and the significance analysis of experimental data adopts t inspection.Under above-mentioned condition, the t10c12-CLA in standard substance, linolic acid retention time are respectively 31.166 minutes, 28.239 minutes.In Recombinant Lactococcus lactis (Lactococcus lactis) PAI-22, PAI-45, PAI-75, PAI-76, PAI-80 and PAI-91 thalline, occur that retention time is respectively the chromatographic peak of the t10c12-CLA of 31.176 minutes, 31.180 minutes, 31.181 minutes, 31.182 minutes, 31.179 minutes, 31.175 minutes; In Lactococcus lactis (Lactococcus lactis) NZ3900, Recombinant Lactococcus lactis (Lactococcus lactis) P8149, PAI-22, PAI-45, PAI-75, PAI-76, PAI-80 and PAI-91 thalline, occur that retention time is respectively the linolic acid chromatographic peak of 28.241 minutes, 28.262 minutes, 28.261 minutes, 28.260 minutes, 28.281 minutes, 28.267 minutes, 28.265 minutes, 28.278 minutes.
Result shows Lactococcus lactis (Lactococcus lactis) NZ3900(Host Strains), Recombinant Lactococcus lactis (Lactococcus lactis) p8149(empty carrier control strain) linoleic content accounts for 64% left and right of thalline fatty acid total amount, there is not isomerization process, in thalline, can't detect product t10c12-CLA; On the contrary, transform all bacterial strains of pNZ8149-PAI plasmid, comprise the bacterial strains such as Recombinant Lactococcus lactis (Lactococcus lactis) PAI-22, PAI-45, PAI-75, PAI-76, PAI-80 and PAI-91, can both stably express pai gene, the isomerase PAI of translation can effectively produce t10c12-CLA, and the content of product t10c12-CLA accounts for the 15.19-23.91%(quality percentage composition of thalline fatty acid total amount).Also can find out from Fig. 1, between different strains, there are significantly different (in Fig. 1, in same component, when the different letter of mark, representing to have significant difference, i.e. p<0.05 between bacterial strain) in the content of product and substrate.Wherein, in Recombinant Lactococcus lactis (Lactococcus lactis) PAI-75 thalline, the content of t10c12-CLA is the highest, account for 23.91% of thalline total fatty acid content, significantly be better than Recombinant Lactococcus lactis (Lactococcus lactis) PAI-22, PAI-45, PAI-76, PAI-80, other recombinant bacterial strain such as PAI-90 (p<0.05), its the t10c12-CLA transformation efficiency (anti-form-1 0 in thalline, anti-form-1 0 in the quality of cis-12 conjugated linolic acid and thalline, the ratio of cis-12 conjugated linolic acid and linolic acid quality sum), be t10c12-CLA and (t10c12-CLA+LA) ratio of quality, reach 37.62%.These presentation of results, PAI recombinant lactic acid bacteria can effectively become t10c12-CLA product by linolic acid substrate isomery, wherein Recombinant Lactococcus lactis (Lactococcus lactis) PAI-75 effect is best, for the exploitation of the PAI recombinant lactic acid bacteria of food grade provides foundation and bacterial strain.
Wherein, Recombinant Lactococcus lactis (Lactococcus lactis) PAI-75 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 18th, 2013, the preservation center numbering of registering on the books: CGMCC No.7148.
Claims (2)
1. Recombinant Lactococcus lactis, it is characterized in that: described Recombinant Lactococcus lactis is Lactococcus lactis (Lactococcus lactis) PAI-75, and it is numbered CGMCC No.7148 registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. Recombinant Lactococcus lactis claimed in claim 1 is being produced anti-form-1 0, the application in the conjugated linolic acid of cis-12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310066107.1A CN103146628B (en) | 2013-03-01 | 2013-03-01 | Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310066107.1A CN103146628B (en) | 2013-03-01 | 2013-03-01 | Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103146628A CN103146628A (en) | 2013-06-12 |
| CN103146628B true CN103146628B (en) | 2014-11-05 |
Family
ID=48544951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310066107.1A Expired - Fee Related CN103146628B (en) | 2013-03-01 | 2013-03-01 | Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103146628B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039385B (en) * | 2015-05-22 | 2019-04-19 | 天津科技大学 | T10, c12- conjugated linoleic acid engineered strain and its recombinant expression plasmid and construction method and application |
| EP3675642A1 (en) * | 2017-08-30 | 2020-07-08 | Chr. Hansen A/S | Process for producing a mesophilic fermented milk product |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250864A (en) * | 2011-05-03 | 2011-11-23 | 中国农业大学 | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism |
-
2013
- 2013-03-01 CN CN201310066107.1A patent/CN103146628B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250864A (en) * | 2011-05-03 | 2011-11-23 | 中国农业大学 | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism |
Non-Patent Citations (2)
| Title |
|---|
| 王腊梅 等.硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达.《生物工程学报》.2012,第28卷(第9期),摘要，正文1107页右栏，1116页左栏. * |
| 硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达;王腊梅 等;《生物工程学报》;20120925;第28卷(第9期);摘要，正文1107页右栏，1116页左栏 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103146628A (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ye et al. | Bacterial community dynamics and product distribution during pH‐adjusted fermentation of vegetable wastes | |
| Bourgoin et al. | Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci? | |
| CN104974944B (en) | A kind of schizochytrium limacinum genetic engineering bacterium for producing DHA and its construction method and application | |
| EP2768966B1 (en) | Bacteria and method for synthesizing fatty acids | |
| Wada et al. | Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli | |
| CN103146628B (en) | Food-grade recombined lactococcal lactis capable of producing conjugated linoleic acid as well as construction method and application thereof | |
| Wang et al. | RNA-Seq reveals transcriptomic interactions of Bacillus subtilis natto and Bifidobacterium animalis subsp. lactis in whole soybean solid-state co-fermentation | |
| Xiong et al. | Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression | |
| Soriano-Perez et al. | Production of lactic acid from cheese whey by batch cultures of Lactobacillus helveticus | |
| CN105602880B (en) | The method of one plant of Corynebacterium glutamicum and its excessive synthetic phospholipid acyl serine | |
| Kobayashi et al. | Comparison of biomass and lipid production under ambient carbon dioxide vigorous aeration and 3% carbon dioxide condition among the lead candidate Chlorella strains screened by various photobioreactor scales | |
| Deptula et al. | Red-brown pigmentation of Acidipropionibacterium jensenii is tied to haemolytic activity and cyl-like gene cluster | |
| Liu et al. | Lactic acid bacteria: microbial metabolism and expanding applications | |
| CN104152483A (en) | Application of argJ gene in fermentation production of L-citrulline | |
| CN102382779B (en) | Manufacture method for set type additive-free yogurt and lactobacillus casei used therein | |
| CN113061542B (en) | Pichia pastoris engineering bacteria capable of producing conjugated linoleic acid and application thereof | |
| Luo et al. | Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli | |
| CN102250864B (en) | Dual purposes of linoleate isomerase in aspects of dehydrogenation and isomerism | |
| Honda et al. | Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323T | |
| CN103060219A (en) | Manufacturing method of fermented milk beverage and Lactobacillus casei used by same | |
| CN104630079A (en) | Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof | |
| AU2015211754A1 (en) | Modified cyanobacteria | |
| Zhang et al. | Lactobacillus delbrueckii subsp. bulgaricus proteinase: purification by ion-exchange and hydrophobic interaction chromatography | |
| CN113061541B (en) | Recombinant saccharomyces cerevisiae and application thereof in production of conjugated linoleic acid | |
| Chen et al. | Screening and identification of lactic acid bacteria strains with high acid-producing from traditional fermented yak yogurt |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: CHINA AGRICULTURAL UNIVERSITY Free format text: FORMER OWNER: GOU KEMIAN Effective date: 20141218 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20141218 Address after: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee after: China Agricultural University Address before: 100193 Beijing City, Haidian District Feng Lian villa No. 19 Building 2 unit 102 room Patentee before: Gou Kemian |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141105 Termination date: 20170301 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |