CN106434416B - It is a kind of produce eicosapentaenoic acid bacterial strain and its application - Google Patents
It is a kind of produce eicosapentaenoic acid bacterial strain and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention discloses a kind of bacterial strain for producing eicosapentaenoic acid and its applications, belong to microorganism field.The bacterial strain includes Shewanella baltica.The bacterial strain is producing the application in eicosapentaenoic acid, and the application is the following steps are included: activate the bacterial strain, activation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；It ferments to the bacterial strain after activation, fermentation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm.The bacterial strain of this four plants production eicosapentaenoic acids is wild-type strain, can satisfy human body to the needs of EPA, these four wild type superior strains enrich the resources bank of Microbe synthesis EPA, provide potential strain resource and genetic resources to commercially produce EPA.In addition, the application method can further improve the ability that bacterial strain produces eicosapentaenoic acid, to obtain more eicosapentaenoic acids by the bacterial strain.
Description
Technical field
The present invention relates to microorganism field, in particular to a kind of bacterial strain for producing eicosapentaenoic acid and its application.
Background technique
EPA (Eicosapentaenoic Acid, eicosapentaenoic acid) is of great significance for human health, research
Show the physiological function that it has prevention cardiovascular disease, inhibits breast cancer and colon cancer.In addition, before being converted into human body
The eicosanoids active material such as column parathyrine, thromboxane and leukotriene.The commercial source of EPA is mainly deep sea fish oil at present, but
The production of fish oil is limited by fish, season and place, and processing cost is high and has the dirt of fishlike smell, the especially marine eco-environment
The increasingly scarcity of dye and fishery resources, makes fish oil be difficult meet the needs of people are to EPA.
Microorganism has the advantages such as the speed of growth is fast, nutritional need is low, metabolic regulation is simple.Many microbial strains can be with
Synthesize the metabolite of other high added values, such as squalene, phytosterol and astaxanthin.Wherein, the metabolism of high added value produces
Object EPA and DHA (Docosahexaenoic Acid, docosahexaenoic acid) belong to the serial polyunsaturated fatty acid of ω -3.
Currently, the microorganism of DHA is rich in, as thraustochytriale (Thraustochytrium) and schizochytrium limacinum (Schizochytrium) are equal
Have been used for commercialization large-scale production DHA, the horse Tyke (Martek) and the good of China for representing such as U.S., company must be excellent
(Cabio).However, it has not been found that can be used for commercially producing the wild type superior strain of EPA.
In the implementation of the present invention, the inventor finds that the existing technology has at least the following problems:
EPA is the indispensable important nutrient of human body.Although can be converted after human body intake essential fatty acid linolenic acid
For EPA or DHA, but this speed of reaction in human body is very slow and conversion ratio is very low, is far from satisfying human body to the need of EPA
It asks, especially infant and the middle-aged and the old.Since there is presently no the wild type Producing Strains that discovery can be used for commercially producing EPA
Strain, therefore the wild type superior strain that acquisition meets the production EPA of business demand is extremely urgent.
Summary of the invention
In order to solve the problems, such as not can be used for commercially producing the wild type superior strain of EPA, this hair in the prior art
Bright embodiment provide it is a kind of produce eicosapentaenoic acid bacterial strain and its application.The technical solution is as follows:
On the one hand, the embodiment of the invention provides a kind of bacterial strain for producing eicosapentaenoic acid, the bacterial strain is
Shewanella baltica, Shewanella baltica was preserved in China typical culture collection on June 6th, 2016
The heart, deposit number are CCTCC NO:M 2016312.
Specifically, the deposit number is in the bacterial strain 16Sr DNA sequence dna such as sequence table of CCTCC NO:M 2016312
Shown in SEQ IN NO:4.
On the other hand, the embodiment of the invention provides a kind of above-mentioned bacterial strains to produce the application in eicosapentaenoic acid, described
Using the following steps are included:
The bacterial strain is activated, activation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；After activation
The bacterial strain ferments, fermentation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；
The activation and fermentation of the bacterial strain are carried out using fluid nutrient medium, the fluid nutrient medium includes the light blue of 3g/mL
Rhzomorph.
Specifically, the fluid nutrient medium include: yeast extract, it is peptone, casein hydrolysate, glucose, solvable
Property starch, Sodium Pyruvate, dipotassium hydrogen phosphate, epsom salt and water, wherein yeast extract, peptone, casein hydrolysis
The mass ratio of object, glucose and soluble starch is 1:1:1:1:1, yeast extract, Sodium Pyruvate, dipotassium hydrogen phosphate, seven water
The mass ratio of magnesium sulfate and water is 5:3:3:180, and the mass ratio of yeast extract and water is 1:2000, the fluid nutrient medium
PH value is 7.3 ± 0.2.
Specifically, the bacterial strain after taking activation ferments according to the inoculum concentration that volume ratio is 1%.
Further, the fluid nutrient medium passes through 121 DEG C of sterilizing 20min.
Specifically, the activation condition of the bacterial strain are as follows: 10 DEG C.
Specifically, the fermentation condition of the bacterial strain are as follows: 10 DEG C.
Specifically, shaking speed is 180rpm when the bacterial strain is activated and fermented.
Technical solution provided in an embodiment of the present invention, which has the benefit that, produces two the embodiment of the invention provides a kind of
The bacterial strain of the bacterial strain of ten carbon 5 alkene acids and its application, this four plants production eicosapentaenoic acids is wild-type strain, can satisfy people
For body to the needs of EPA, these four wild type superior strains enrich the resources bank of Microbe synthesis EPA, and can be used for being commercialized
Production, while the research for PKS approach provides material, lays the foundation for the building of high yield ω -3VLCPUFAs engineered strain, this
Outside, which can further improve the ability that bacterial strain produces eicosapentaenoic acid, to obtain more two by the bacterial strain
Ten carbon 5 alkene acids.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is the degree and EPA methyl esters mark product of the fatty acid component of bacterial strain 6-42 provided in an embodiment of the present invention
Comparison diagram；
Fig. 2 is the gas chromatogram of bacterial strain 6-42 provided in an embodiment of the present invention；
Fig. 3 is the gas chromatogram of EPA methyl esters mark product provided in an embodiment of the present invention；
Fig. 4 is agarose gel electrophoresis figure provided in an embodiment of the present invention.
Bacterial strain provided in an embodiment of the present invention was sent to China typical culture collection center progress on June 6th, 2016
Preservation, preservation address: Luo Jia Shan Wuhan University, Wuhan City, Hubei China province, strain classification name are respectively as follows: Compostimona
Ssuwonensis, Flavobacteriu momnivorum and Shewanella baltica；Deposit number is respectively as follows: CCTCC
NO:M 2016311, CCTCC NO:M 2016310 and CCTCC NO:M 2016312.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.
Embodiment
The embodiment of the invention provides a kind of bacterial strains to produce the application in eicosapentaenoic acid, which includes following step
It is rapid: activation and fermentation, the activation condition of bacterial strain are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；The fermentation condition of bacterial strain are as follows:
0-15 DEG C, shaking speed is 180 ± 10rpm.
The present embodiment chooses 6 batches of bacterial strains, and 6 batches of bacterial strains are by China typical culture collection center (China center
For type culture collection, CCTCC) it provides, 6 batches of bacterial strains pick up from Norway's Svalbard Ny-Alesund
Regional (Ny-Alesund, Svalbard Archipelago, Norway).Wherein, first and second batch pick up from this area
Glacial snout soil, geographical coordinate be respectively (78 ° of 54 ' N, 12 ° of 06 ' E&78 ° 53 ' N, 12 ° of 10 ' E)；Third batch picks up from the ground
The tundra tundra soil in area is located at (78 ° of 53 ' N, 12 ° of 09 ' E)；4th batch and the 5th batch is picked up from this area
Stuphallet steep cliff tomography is located at (78 ° of 57.575 ' N, 11 ° of 36.288 ' E&78 ° 57.521 ' N, 11 ° of 36.447 ' E)；
The 6th batch of MidtreLovenbreen glacial snout stripping zone for picking up from this area, be located at (78 ° of 53.704 ' N, 12 ° 5.262 '
E)。
Activation: it will live on strain inoculated of the culture on slant medium to fluid nutrient medium (for activated strains)
Change, obtains original strain, the soil that the bacterium source on slant medium is extracted in Norway, Ny-Alesund area, Svalbard
In earth sample, specifically, the strain inoculated saved on picking slant medium extremely contains the sterile of 3ml fresh fluid nutrient medium
In tool plug test tube, it is placed on the shaking table that 0-15 DEG C of revolving speed is 180 ± 10rpm and is activated.In the present embodiment, the culture of activation
Temperature can be 10 DEG C, and revolving speed can be 180rpm.
Wherein, the activation and fermentation of bacterial strain are carried out using fluid nutrient medium, fluid nutrient medium includes: yeast extract, egg
White peptone, casein hydrolysate, glucose, soluble starch, Sodium Pyruvate, dipotassium hydrogen phosphate, epsom salt and water, wherein
Yeast extract, peptone, casein hydrolysate, glucose and soluble starch mass ratio be 1:1:1:1:1, yeast extracts
Object, Sodium Pyruvate, dipotassium hydrogen phosphate, epsom salt and water mass ratio be 5:3:3:180, the quality of yeast extract and water
Than being 7.3 ± 0.2 for the pH value of 1:2000, fluid nutrient medium.Specifically, in the present embodiment, fluid nutrient medium specifically includes:
Yeast extract 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, Sodium Pyruvate
0.3g, dipotassium hydrogen phosphate 0.3g, epsom salt 0.049g, water 1L, pH value are 7.3 ± 0.2,121 DEG C of sterilizing 20min.
In addition, fluid nutrient medium (can also be mentioned including the cerulenin of 3 μ g/mL by Sigma-Aldrich
For).The cerulenin of 3 μ g/mL is obtained by the way that cerulenin to be dissolved in dehydrated alcohol, is placed in -20 DEG C of preservations.
Fermentation: original strain is seeded on fluid nutrient medium and cultivates (for fermenting) to strain growth to logarithmic growth
Phase obtains bacterium solution.Specifically, activated bacterium solution (original strain) is forwarded to according to 1% (V/V) inoculum concentration new containing 100mL
In the 250mL shaking flask of fresh fluid nutrient medium.Cultivation temperature is 0-15 DEG C, and revolving speed is 180 ± 10rpm.In the present embodiment, it ferments
Cultivation temperature can be 10 DEG C, revolving speed can be 180rpm.
Bacterial strain screening
Bacterium solution is measured by fatty acid component, obtains the bacterial strain for producing eicosapentaenoic acid.Specific method include: with rouge
In the identical situation of fat acid constituents determination condition, with EPA (Eicosapentaenoic Acid, eicosapentaenoic acid) methyl esters mark
Product (purchasing in Larodan company, Sweden) are as control, according to appearance time of the EPA methyl esters mark product in fatty acid gas-chromatography
The bacterial strain that may produce EPA is picked out, then gas chromatography-mass spectrography analysis is carried out to the bacterial strain that may produce EPA, according to EPA first
The mass spectrogram of ester mark product judges whether the thallus in the bacterium solution produces EPA, when the mass spectrogram of EPA methyl esters mark product and the mass spectrogram of bacterium solution
When identical, then judge that the bacterial strain in the bacterium solution produces EPA.
Fatty acid component measurement:
Thalli dry powder is made in grinding after bacterium solution is dried in vacuo, and thalli dry powder is placed in vial, adds into vial
Enter methanol hydrochloride solution and heneicosanoic acid and carry out vortex oscillation, sealed glass jars are placed in 80 DEG C of water-bath 2h, are cooled to room
Sodium-chloride water solution and n-hexane is added in warm backward vial and carries out vortex oscillation, supernatant is taken after standing, by supernatant
Centrifugation obtains the supernatant containing esterification fatty acid, and the supernatant containing esterification fatty acid is added with after being dried with nitrogen
Silylating reagent, the volume of silylating reagent is equal with the volume of the supernatant containing esterification fatty acid, in 90 DEG C of reaction 1h,
It can be directly used for gas chromatographic analysis after being cooled to room temperature.
The present embodiment uses Agilent 7890A gas chromatograph, hydrogen ion flame detector (Flame Ionization
Detector, FID)；HP-FFAP (30m × 0.25mm × 0.25m) chromatographic column；Carrier gas is high pure nitrogen, and inlet pressure is
25psi, sample volume 1L, split ratio 30:1；Temperature program: then 150 DEG C of holding 1min of initial temperature are warming up to 5 DEG C/min
230 DEG C and 8min is kept, injection port and detector temperature are kept at 250 DEG C and 280 DEG C.Rouge is calculated using area normalization method
The percentage contents of fat acid.GC-MS (gas chromatography-mass spectrography) is using identical instrument, splitter and separation condition, matter
Compose model Agilent 5975C.
The present embodiment obtains the bacterial strain of four plants of production EPA using the above method altogether, as shown in table 1, four plants of bacterial strain difference
Number is 1-1,2-37,2-42 and 6-42, wherein four plants of bacterial strain 16Sr DNA sequence dnas respectively as SEQ IN NO:1 in sequence table,
SEQ IN NO:2 in sequence table, in sequence table in SEQ IN NO:3 and sequence table shown in SEQ IN NO:4.Wherein, bacterial strain 6-
The degree of 42 fatty acid component as shown in Figure 1, the gas chromatogram of bacterial strain 6-42 as shown in Fig. 2, EPA methyl esters mark product
Gas chromatogram as indicated at 3, due to the appearance time of the gas-chromatography of the gas-chromatography and EPA methyl esters mark product of bacterial strain 6-42,
Then think that bacterial strain 6-42 produces EPA, other three plants similarly judge.
Table 1 is four plants of bacterial strains for producing EPA
As shown in Table 1, the EPA of four plants of bacterial strains 1-1,2-37,2-42 and 6-42 measurement accounts for total fatty acids percentage and is followed successively by
1.1%, 2.3%, 0.9% and 4.9%.
The classification position of bacterial strain
The present embodiment analyzes the classification position to identify screened bacterial strain using 16S rDNA sequence.
Identification: extracting the total DNA of original strain, and specifically, the bacterial strain seed liquor (original strain) after taking activation is seeded to
In fresh fluid nutrient medium, it is placed in 10 DEG C of shaking table 180rpm cultures.According to bacterial genomes extracts kit (Minibest
Bacterial Genomic DNA Extraction Kit, Japanese Takara company) operation instruction extracts the total of bacterium solution
DNA.The total DNA for taking 3 μ L to extract carries out agarose gel electrophoresis detection, and remaining total DNA sample is placed in -20 DEG C of preservations.
Pcr amplification reaction is carried out by template of total DNA, obtains amplified production；Specifically, reaction system is 50 μ L, specifically
Reaction system and response procedures it is as shown in table 2:
Table 2 is pcr amplification reaction system
Wherein, forward primer sequence is as shown in table SEQ IN NO:5, and reverse primer sequences are as shown in table SEQ IN NO:6.
Table 3 is pcr amplification reaction program
Response procedures are referring to Phusion High-Fidelity DNA Polymerase operation instructions.3 μ L are taken to expand
Product carries out agarose gel electrophoresis detection according to pcr amplification reaction program shown in table 3.
Amplified production is recycled, 16S rDNA segment is obtained；Agarose gel electrophoresis to pcr amplification product detects
After correct, using purification and recovery kit, (Minibest DNA Fragment purification Kit, Japanese Takara is public
Department) pcr amplification product is recycled, concrete operation step is referred to specification.
16S rDNA segment is connected on plasmid, connection product is obtained；Using the flat end Cloning Kit (Zero of PCR
Blunt PCR Cloning Kit, Life company, the U.S.) by the 16S rDNA segment of recycling be connected to 0 plasmid of Top (purchase in
Life company, the U.S.) on.Reaction system is 6 μ L, specific as shown in table 4:
Table 4 is the ingredient of linked system
Configured linked system is placed in and connects 10min at room temperature, reaction solution can be directly used for transformation experiment after connection.
Connection product is transferred in competent escherichia coli cell, part supernatant is removed after centrifugation and is resuspended, is obtained
To re-suspension liquid, re-suspension liquid is coated on kanamycins solid medium；The specific method is as follows:
(1) the DH5 α competent cell that -70 DEG C save is placed in and is dissolved on ice, after dissolution is added in 6 μ L connection reaction solutions
Competent cell in, 30min is stood after mixing gently on ice, obtains mix products；
(2) mix products addition centrifuge tube is built in thermal shock 60s in 42 DEG C of water-baths, pays attention to acting in operating process light
It is soft；
(3) the fresh kanamycins solid culture of 500 μ L is then added after the completion by tube stand in 2min on ice in thermal shock
Base (LB solid medium), shaken cultivation 1h in 37 DEG C of constant-temperature tables；Wherein, kanamycins solid medium includes: that yeast mentions
Take object, peptone, sodium chloride and agar, wherein yeast extract, peptone, sodium chloride and agar mass ratio be 5:10:
10:18, the pH value of kanamycins solid medium are 7.0.In the present embodiment, yeast extract 5g, peptone 10g, chlorination
Sodium 10g, agar 18g, 7.0,121 DEG C of sterilizing 20min of pH value, the culture for Escherichia coli.
(4) centrifuge tube is taken out and is centrifuged 1min in 4000rpm, be resuspended after removing part supernatant, obtain re-suspension liquid,
Re-suspension liquid is coated on the LB solid plate culture medium of 50 μ g/mL, 37 DEG C of constant incubators is subsequently placed at and is incubated overnight.Pass through
Competent cell is concentrated in the mode of resuspension.
Single colonie on picking LB solid plate culture medium (its component is identical as LB solid medium component) carries out bacterium colony
PCR detection, using M13F/M13R as primer (sequence of M13F as shown in table SEQ IN NO:7, the sequence of M13R such as table SEQ IN
Shown in NO:8), PCR reaction, reaction system and condition are carried out referring to specification using Taq enzyme.Then take the PCR reaction solution of 3 μ L
For agarose gel electrophoresis, whether testing goal stripe size is correct, as a result as shown in figure 4, in Fig. 4, M DNA
Maker, 1,2,3,4 respectively represent bacterial strain 1-1,2-37,2-42 and 6-42, wherein the 16S rDNA amplified production of four bacterial strains
Size is each about 1.5Kb, it is seen that the stripe size of four kinds of bacterial strains provided in this embodiment is correct.
Picking positive colony is sequenced and (holds up Kechuang neoformation Science and Technology Ltd. in Wuhan), determines the consistent of sequencing result
After property by the 16S rDNA sequence of acquisition NCBI (National Center of Biotechnology Information,
National Center for Biotechnology Information) carry out on database BLAST (Basic Local Alignment Search Tool,
Basic Local Alignment Search Tool) sequence analysis, four bacterial strains respectively with Nocardioidesplantarum,
Compostimonassuwonensis, Flavobacteriumomnivorum and Shewanellabaltica homology are higher,
To assert four plants of bacterial strains be belonging respectively to Nocardioidesplantarum, Compostimonassuwonensis,
Flavobacteriumomnivorum and Shewanellabaltica, and then the classification position of four plants of bacterial strains has been determined.
Temperature is to bacterial strain
Influence the embodiment of the invention provides temperature to bacterial strain 6-42 by the temperature of activation and is cultivated to logarithmic growth phase
Temperature simultaneously be set as 0 DEG C, 5 DEG C, 10 DEG C, 15 DEG C and 20 DEG C, other condition of culture are identical, by 20 light dydrocarbons of production obtained
The bacterial strain of olefin(e) acid carries out fatty acid component measurement respectively, and measurement result is as shown in table 5:
Table 5 is the influence that temperature produces EPA content to bacterial strain 6-42
Note: data are indicated with mean ± SD (n=3) in table 5；Similarly hereinafter.
As shown in Table 5,3.4% when accounting for the percentage of total fatty acids by 20 DEG C with the reduction EPA of temperature is stepped up
8.8% when to 0 DEG C, corresponding dry cell weight is raised to 6.2mg/g by 2.2mg/g.Simultaneously because its biomass of the reduction of temperature
Also 0.3g/L is dropped to by initial 0.5g/L therewith, it is seen that yield (unit volume culture medium content) highest at 10 DEG C of EPA
Up to 2.2mg/L, therefore, the temperature of the temperature of activation and culture to logarithmic growth phase is disposed as 10 DEG C by the present embodiment, thus
Improve the ability that four kinds of thallus produce EPA.
The variation of its fatty acid each component is further analyzed, as shown in table 6.
Table 6 is influence of the temperature to the fatty acid component of bacterial strain 6-42
As shown in Table 6, as reduction linear saturated fatty acids Short-Chain Fatty Acids C12:0 and the C14:0 content of temperature increases
Add, middle long chain fatty acids C16:0, C17:0 and C18:0 content is reduced, and linear saturated fatty acids total amount first increases to be subtracted afterwards.Each branch is full
With fatty acid as the reduction changing rule of temperature is different, and amplitude of variation is little, and branch chain saturated fatty acid total amount first subtracts to be increased afterwards.
The changes of contents of comprehensive straight chain and branch chain saturated fatty acid, saturated fatty acid total amount subtracts with the reduction of temperature as the result is shown
It is few.Monounsaturated fatty acids C16:1 content with temperature reduce and increase and C18:1 variation tendency on the contrary, total amount variation tendency
It is unobvious；Growth trend is presented with the reduction of temperature in polyunsaturated fatty acid EPA content.On the whole, with the drop of temperature
Low, unsaturated fatty acid total amount increases and saturated fatty acid total amount is reduced.
Influence of the cerulenin to bacterial strain
Fluid nutrient medium in the embodiment of the present invention may include cerulenin, and the additional amount of cerulenin is respectively set to
0,1,2,3,4,5 each concentration of μ g/mL carry out shake flask fermentation at 10 DEG C.The yield of EPA is as shown in table 7 under each concentration.
Table 7 is influence of the cerulenin to EPA
As shown in Table 7, the amount that bacterial strain produces EPA after cerulenin processing increases, and wherein cerulenin concentration is 3 μ
Reach maximum when g/mL, EPA account for total fatty acids percentage, dry cell weight and unit volume culture medium content respectively reach 7.6%,
4.5mg/g and 3.6mg/L.Two kinds of condition of culture are stood and vibrated when comparing 10 DEG C, it can be seen that EPA accounts for total fatty acids when standing
Percentage and dry cell weight are higher, but shaken cultivation biomass is higher therefore EPA unit volume culture medium yield is higher than standing training
It supports, therefore, the cerulenin in fluid nutrient medium in the present embodiment is 3 μ g/mL.
Further variation of the analysis cerulenin to fatty acid each component, as shown in table 8.
Table 8 is influence of the cerulenin to the fatty acid component of bacterial strain 6-42
As can be seen from Table 8 after cerulenin is added, carbon chain lengths are in 15 carbon and its linear saturated fatty acids below
Content increases and the above content of 15 carbon is reduced, and C13:0 content increases in branch chain saturated fatty acid and C15:0 and C17:0 content subtracts
Less, straight chain and branch chain saturated fatty acid total amount increase but the latter's variation is smaller.Hydroxy fatty acid overall variation is little, and list is not
Saturated fatty acid content is reduced, and opposite EPA content first increases to be subtracted afterwards.Generally, cerulenin makes the short chain rouge of bacterial strain 6-42
Fat acid content increases, and monounsaturated fatty acids content is reduced, and hydroxy aliphatic acid content is constant and EPA content increases.
Meanwhile combination temperature and cerulenin carry out Comprehensive Experiment to bacterial strain 6-42, are separately added into 3 μ at 0 DEG C and 10 DEG C
The cerulenin of g/mL cultivates thallus 6-42.The results are shown in Table 9 for it.
Table 9 is the influence that temperature and cerulenin produce EPA content to bacterial strain 6-42
Note: * indicates shaken cultivation in table 9, and other is stationary culture.
Be added the cerulenin stationary culture of 3 μ g/mL at 0 DEG C as can be seen from Table 9, EPA account for total fatty acids percentage and
Dry cell weight content reaches maximum, and respectively 11.5% and 6.7mg/g compares 10 DEG C of shaken cultivations (not adding cerulenin)
Under the conditions of be respectively increased to 2.3 and 2.2 times；The cerulenin shaken cultivation of 3 μ g/mL, unit volume culture are added at 10 DEG C
EPA content highest in base, reaches 3.6mg/L, and compared to 0 DEG C stationary culture (not adding cerulenin) of EPA yield increases to
1.8 again.
The embodiment of the invention provides a kind of bacterium bacterium that eicosapentaenoic acid is produced from extreme environment screening, identification, optimization
The bacterial strain of strain and its application, this four plants production eicosapentaenoic acids is wild type producing bacterial strain, wherein two plants are to find energy for the first time
Enough synthesize EPA.These four wild type producing bacterial strains enrich bacterial strain and the genetic resources library of Microbe synthesis EPA, and potential
For commercially producing, while the research for PKS approach provides material, establishes for the building of high yield ω -3VLCPUFAs engineered strain
Fixed basis, in addition, the application method can further improve the ability that bacterial strain produces eicosapentaenoic acid, to be obtained by the bacterial strain
More eicosapentaenoic acids.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of bacterial strain for producing eicosapentaenoic acid, which is characterized in that the bacterial strain is Shewanella baltica,
Shewanella baltica is preserved in China typical culture collection center, deposit number CCTCC on June 6th, 2016
NO:M 2016312.
2. bacterial strain according to claim 1, which is characterized in that the deposit number is the bacterium of CCTCC NO:M 2016312
Strain 16Sr DNA sequence dna is as shown in SEQ IN NO:4 in sequence table.
3. a kind of bacterial strain as claimed in claim 1 or 2 is producing the application in eicosapentaenoic acid, which is characterized in that described to answer
With the following steps are included:
The bacterial strain is activated, activation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；
It ferments to the bacterial strain after activation, fermentation condition are as follows: 0-15 DEG C, shaking speed is 180 ± 10rpm；
The activation and fermentation of the bacterial strain are carried out using fluid nutrient medium, the fluid nutrient medium includes the cerulenin of 3g/mL.
4. application according to claim 3, which is characterized in that the bacterial strain after taking activation is 1% according to volume ratio
Inoculum concentration is fermented.
5. application according to claim 3, which is characterized in that the fluid nutrient medium passes through 121 DEG C of sterilizing 20min.
6. application according to claim 3, which is characterized in that the activation condition of the bacterial strain are as follows: 10 DEG C.
7. application according to claim 3, which is characterized in that the fermentation condition of the bacterial strain are as follows: 10 DEG C.
8. application according to claim 3, which is characterized in that shaking speed is when the bacterial strain activation and fermentation
180rpm。
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