CN104073457B - Carotenoid high-yield strain and application thereof - Google Patents
Carotenoid high-yield strain and application thereof Download PDFInfo
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Abstract
The invention provides a carotenoid high-yield strain and application thereof, and relates to the field of microbial fermentation and metabolites thereof. Said carotenoidThe high-yield strain is deinococcus mukurossi (R) ((R))<i>Deinococcus</i><i>wulumuqiensis</i>) R12M with the preservation registration number of CCTCC No. M2014099. The invention also provides application of the strain in preparing carotenoid. The invention relates to deinococcus mukurossi (A)<i>Deinococcus</i><i>wulumuqiensis</i>) R12M has a specific strain<i>Deinococcus</i><i>wulumuqiensis</i>sp, nov. and<i>Deinococcus</i><i>radiodurans</i>DSM 20539Thas stronger radiation resistance and carotenoid production performance, and can be used for producing natural pigment and natural antioxidant.
Description
Technical field
The present invention relates to fermentable and metabolite field thereof, particularly to carotenoid superior strain and application thereof。
Background technology
Free radical refers to containing the atom of non-sharing electron, molecule or group, owing to containing unpaired electron in free radical, has the tendency of pairing, has significantly high activity。Free radical is paired to the electronics that stably must make oneself to contiguous atom or molecule abstracts electronics, but also hence in so that atom of being taken by force of electronics or molecule become new free radical, causes chain reaction, and this process is exactly " oxidation "。If free radical produces too much or removed slow, it is by attacking life macromolecule material and various cell, body can be caused in the various damages of molecular level, cellular level and histoorgan level, accelerate the senescence process of body and bring out various disease。The diseases such as common cancer, arteriosclerosis, diabetes, cataract, cardiovascular diseases, senile dementia, arthritis are considered to too much relevant to free radical。
Antioxidant refers to scavenging free radicals maybe can block the material of the oxidation reaction that free radical participates in。Antioxidant of a great variety, can be divided into enzyme scavenger and the big class of non-enzyme scavenger two。Enzyme antioxidant is generally antioxidase。Non-enzyme antioxidant generally comprises the materials such as flavonoid, polysaccharide, vitamin C, vitamin E, carotenoid。They are to help catch and neutralize free radical, thus dispelling a class material of radical pair human body infringement。With advancing age, the ability producing free radical scavenger in body is gradually reduced, thus reducing the defence capability to free radical damage, making body tissue organ easily impaired, accelerating the aging of body, causing a series of disease。In order to prevent the generation of this type of phenomenon, food, health product, medicine, cosmetics can add antioxidant artificially, thus reaching the purpose of defence disease, slow down aging。At present, the antioxidant that major part enterprise uses is synthetic mostly, and synthetic technology is complicated, in the product of synthesis, a small amount of composition is poisonous, causes chromosomal aberration effect, the problem that the Natural antioxidant is then absent from this respect。The mainly extraction from animals and plants due to the current Natural antioxidant, by the restriction extracting raw material, natural anti-reflecting oxide content is relatively low, and extraction process is complicated, so the separation of the Natural antioxidant, screening and a large amount of production have had become as the emphasis of basic research and development, it is of very high actual application value。
Carotenoid (Carotenoids) is the general name of the important natural pigment of a class, it is common to be present among the yellow in animal, higher plant, fungus, algae, orange red or red pigment。Carotenoid has antioxidation, mutation inhibiting, reduction nucleic acid damaging, reduces the plurality of health care functions such as cardiovascular disease and prophylaxis of cancer。Microorganism currently with fermentative Production natural carotenoid has mycete, antibacterial and yeast etc.。But, adopt existing microorganism to prepare natural carotenoid, yield poorly, relatively costly。
Summary of the invention
It is an object of the invention to provide carotenoid superior strain, this bacterial strain has very strong radiation resistance, it is possible to High Yield of Carotenoid。
It is a further object of the present invention to provide above-mentioned bacterial strains application in preparing carotenoid。
The present invention provides carotenoid superior strain, for the unusual coccus in Urumchi (Deinococcuswulumuqiensis) R12M, preservation registration number is CCTCCNo:M2014099。
Carotenoid superior strain R12M strain of the present invention is by bacterial strainDeinococcuswulumuqiensisSp.nov. carrying out atmospheric pressure at room plasma ARTP method mutagenic breeding gained, Classification And Nomenclature isDeinococcuswulumuqiensis。This bacterial strain was preserved in China typical culture collection center (CCTCC) before the applying date。Address: China. Wuhan. Wuhan University, preservation date is on March 23rd, 2014, and preserving number is CCTCCNo:M2014099。
The bacterial strain R12M of High Yield of Carotenoid of the present invention is accredited as unusual coccus (Deinococcus) through microbiology and belongs to。This strain growth, in TGY media surface, was cultivated through 30 DEG C, 14 days, and bacterium colony is orange red, little, circular, protruding, neat in edge, smooth surface, opaque；Cell is circular, in bigeminy or tetrad shape。R12M bacterial strain is carried out morphology and physiological and biochemical test by reference " uncle's Jie Shi Bacteria Identification handbook "。Obtained the 16SrDNA sequence of R12M bacterial strain by PCR, through sequencing and BLAST homology alignment and evolutionary analysis, result show R12M withDeinococcuswulumuqiensisSp.nov.16SrDNA homology is 100%, withDeinococcusradioduransDSM20539T16SrDNA homology be 97%, it is determined that R12M be the unusual coccus in Urumchi (Deinococcuswulumuqiensis)。
Carotenoid superior strain R12M of the present invention, shows have and compare original strain through gamma-rays and UV radiation detectionDeinococcuswulumuqiensisSp.nov. andDeinococcusradioduransDSM20539THigher radiation resistance。
The present invention also provides for the application in preparing carotenoid of the described bacterial strain。By the Urumchi unusual coccus of fermenting (Deinococcuswulumuqiensis) R12MCCTCCNo:M2014099, obtain carotenoid。By unusual for Urumchi coccus (Deinococcuswulumuqiensis) R12MCCTCCNo:M2014099 is inoculated in TGY fluid medium, 28-32 DEG C, shaking speed 180-200rpm when shaken cultivation 36-72h, obtain fermentation liquid；Extract from described fermentation liquid and obtain carotenoid。Described extracting method is: separating thallus from fermentation liquid, by somatic cells breaking cellular wall, obtains cellular lysate liquid；Described lysate centrifuging and taking is precipitated, with organic solvent extraction, it is thus achieved that carotenoid。The method of described somatic cells breaking cellular wall is: with thalline described in soak with hydrochloric acid, put in boiling water bath, then is placed in ice-water bath and processes。
The yield of the carotenoid of experimental result display bacterial strain R12M is significantly higher than bacterial strainDeinococcuswulumuqiensisSp.nov. andDeinococcusradioduransDSM20539T。
Beneficial effect: the present invention is to bacterial strainDeinococcuswulumuqiensisSp.nov. carry out atmospheric pressure at room plasma (ARTP) method and carry out mutagenic breeding, it is thus achieved that mutant strain R12M, the unusual coccus in called after Urumchi (Deinococcuswulumuqiensis) R12M。The unusual coccus in Urumchi (Deinococcuswulumuqiensis) R12M has and compare bacterial strainDeinococcuswulumuqiensisSp.nov. andDeinococcusradioduransDSM20539TThe product carotenoid performance of higher radiation resistance and Geng Gao, can be used for the production of natural pigment and the Natural antioxidant。
Accompanying drawing explanation
Fig. 1 is the R12M bacterial strain homology evolutionary analysis result based on 16SrDNA sequence；Abscissa 0.01 represents evolutionary distance；Numeral on branch node represents the credibility of this branch, and what represent in bracket is No. ID in GENBANK data base。
Fig. 2 be gamma-rays irradiate lower bacterial strain R12M,DeinococcuswulumuqiensisSp.nov. andEscherichiacoliDH5αSurvival curve；Abscissa is gamma Rays dosage；Vertical coordinate is the survival rate of bacterial strain。
Fig. 3 be bacterial strain R12M under UV radiation irradiation,DeinococcuswulumuqiensisSp.nov. andEscherichiacoliDH5αSurvival curve。Abscissa is UV radiation dose；Vertical coordinate is the survival rate of bacterial strain。
Detailed description of the invention
Name embodiment and the present invention is described, but the present invention is not limited to following embodiment。It addition, in the following description, if no special instructions, " % " refers to mass percent。
TGY solid medium: 10g tryptone, 1g glucose, 5g yeast extract, 15g agar are dissolved in water, are settled to 1L。
TGY fluid medium: by 10g tryptone, 1g glucose, 5g yeast extract, be settled to 1L。
Normal saline: 0.85%(w/v) NaCl solution。
Embodiment one: bacterial strainDeinococcuswulumuqiensisSp.nov. mutation
Inoculate from the frozen bacterium of-80 DEG CDeinococcuswulumuqiensisSp.nov.(WangW, MaoJ, ZhangZ, TangQ, XieY, ZhuJ, ZhangL, LiuZ, ShiY, GoodfellowM.2010.Deinococcuswulumuqiensissp.nov.,andDeinococcusxibeiensisSp.nov., isolatedfromradiation-pollutedsoil.Int.J.Syst.Evol.Micro biol.60:2006 2010.) to TGY solid medium, cultivating after 3-4 days for 30 DEG C, picking list colony inoculation, to TGY fluid medium, is cultivated 3-4 days in the 150r/min shaking table of 30 DEG C。
With the absorbance (OD value) of spectrophotometer measurement bacterium solution, adjust bacterium solution OD value to 1 with normal saline。Being coated on small iron plate by the bacterium solution after dilution, each small iron plate is coated with 10 μ l。By small iron plate atmospheric pressure at room plasma (ARTP) machine of coated bacterium solution difference mutation 0s, 60s, 90s, 120s, 150s, 180s when 100W。
After mutation terminates, small iron plate is put in centrifuge tube, carry out gradient dilution with normal saline。Take 10-6With 10-7The bacterium solution of concentration is coated on TGY solid medium, is put in the incubator of 30 DEG C and cultivates 2-3 days。Therebetween, observing the colonial morphology on flat board, and add up total plate count, select compared to the saturate bacterial strain of starting strain, finally select bacterial strain R12M, its bacterium colony is Chinese red, and indication can High Yield of Carotenoid。
Embodiment two: the qualification of bacterial strain R12M
Identifying through microbiology, bacterial strain R12M is that unusual coccus (Deinococcus) belongs to bacterium。
With reference to " uncle's Jie Shi Bacteria Identification handbook " etc., R12M bacterial strain being carried out morphology and physiological and biochemical test, major physiological results of biochemical is as shown in table 1。This strain growth, in TGY solid culture primary surface, was cultivated through 30 DEG C, 14 days, and bacterium colony is Chinese red, little, circular, protruding, neat in edge, smooth surface, opaque；Cell is circular, in tetrad shape。
The physiological and biochemical property that table 1R12M is main
Note: in table 1 "+" for positive reaction；"-" is negative reaction；" ND " be not for having data；" W " is weak positive reaction。
Obtained the 16SrDNA sequence (SEQIDNO:1) of R12M bacterial strain by PCR, through sequencing and BLAST homology alignment and evolutionary analysis, it was shown that R12M withDeinococcuswulumuqiensisSp.nov.16SrDNA homology is 100%, withDeinococcusradioduransDSM20539T(Y11332) 16SrDNA homology is 97%, it is determined that for the unusual coccus in Urumchi (Deinococcuswulumuqiensis)。Comparison analyzes result referring to Fig. 1。Therefore, by the unusual coccus in bacterial strain R12M called after Urumchi (Deinococcuswulumuqiensis) R12M。This bacterial strain was preserved in China typical culture collection center (CCTCC) before the applying date。Address: Luo Jia Shan, wuchang, wuhan, Wuhan University, preservation date is on March 23rd, 2014, and preserving number is CCTCCNo:M2014099。
Embodiment three: gamma-rays measures bacterial strain R12M radiation resistance
With bacterial strain R12M for test strain, arrangeDeinococcuswulumuqiensisSp.nov. it is positive control, arrangesEscherichiacoliDH5αFor negative control。Three strain bacterium are cultivated and grow into exponential phase in TGY fluid medium, and with normal saline flushing, with the resuspended thalline of normal saline after centrifugal at 4 DEG C, respectively obtaining concentration is 1 × 107-1×108The bacterium solution of cfu/mL。Three kinds of bacterium solution are respectively classified into the aliquot of 2mL, in the environment of 25 DEG C, are exposed to 0.167kGy/min dosage60Under the radioactive source of Co, in amount of radiation 0 to 20.0kGy scope, carry out gamma-radiation process with 2.0kGy for gradient。Every 10min, taking the radiation bacterium solution of 0.1mL, carry out gradient dilution with normal saline, each dilution gradient takes 100 microlitre coating TGY flat boards, is placed in 30 DEG C of incubators and cultivates。Count the colony-forming units in TGY culture medium every day, count 15 days。Calculating with the three strain bacterium not bacterium solution via radiation survival rate under same culture conditions, result is as shown in Figure 2。From figure 2 it can be seen thatEscherichiacoliDH5αUnder Low emissivity amount, reached 100% fatality rate, and R12M withDeinococcuswulumuqiensisSp.nov. under the amount of radiation of 20kGy, still have certain survival rate, and under identical amount of radiation, the survival rate of R12M is significantly higher thanDeinococcuswulumuqiensisSp.nov., illustrate that R12M has higher gamma ray resistant radianting capacity than other two strains bacterium, point out this bacterium to have higher product carotenoid ability。
Embodiment four: UV radiation detection bacterial strain radiation resistance
With bacterial strain R12M for test strain, arrangeDeinococcuswulumuqiensisSp.nov. it is positive control, arrangesEscherichiacoliDH5αFor negative control。Three strain bacterium are cultivated and grow into exponential phase in TGY fluid medium, and with normal saline flushing, with the resuspended thalline of normal saline after centrifugal at 4 DEG C, respectively obtaining three kinds of concentration is 1 × 107-1×108The bacterium solution of cfu/mL。Three kinds of bacterium solution are respectively classified into the aliquot of 2mL, irradiate under the UV lamp of 254nm, investigate the survival rate of each bacterium under different exposure dose。Exposure dose is controlled by VLX-30 radiometer。Every 10min, take the irradiation bacterium solution of 0.1mL, carry out gradient dilution with normal saline, each dilution gradient three pieces of TGY flat boards of coating, it is placed in 30 DEG C of incubators and cultivates。Count the colony-forming units in TGY culture medium every day, count 15 days。Calculating with the three strain bacterium not bacterium solution via radiation survival rate under same culture conditions, result is as shown in Figure 3。It is 200J/m in exposure dose as can be seen from Figure 32Under condition,EscherichiacoliDH5αSurvival rate has been 0, but R12M withDeinococcuswulumuqiensisSp.nov. certain survival rate it is respectively provided with, and at 200-400J/m2Between, R12M has higher survival rate, illustrates that R12M has higher tolerance UV radianting capacity, points out this bacterium to have higher product carotenoid ability。
Embodiment five: the mensuration of bacterial strain R12M carotenoid output
By bacterial strain R12M, bacterial strainDeinococcuswulumuqiensisAnd bacterial strain sp.nov.DeinococcusradioduransDSM20539T(DSM20539TBeing Germany's Culture Collection bacterial strain deposit number, T represents it is reference culture) it is inoculated in TGY solid culture primary surface with plate streak, at 30 DEG C of constant temperature culture 60h。By the bacterial strain picking list colony inoculation bred on solid medium in the 250ml shaking flask containing 50mlTGY fluid medium, 30 DEG C, shaking speed 190rpm when shaken cultivation 36h obtain seed liquor。According still further to 1% inoculum concentration, seed liquor is seeded in the 250ml shaking flask containing the fresh TGY fluid medium of 50ml, 30 DEG C, shaking speed 190rpm when shaken cultivation 48h。
Taking 10mL fermentation liquid and proceed in 50ml centrifuge tube, under 3500r/min, centrifugal 15min, abandons supernatant。To precipitate resuspended after, centrifugal 15min under 3500r/min, collect thalline, dry to constant weight for 105 DEG C, obtain dry mycelium, weigh quality the record of dry mycelium。
Weighing 0.1g dry mycelium, add 4mL, concentration is the hydrochloric acid of 3mol/L, at 28 DEG C, 1h is soaked in vibration, is subsequently placed in boiling water bath maintenance 4min, then is positioned in ice-water bath 2min and makes thalline cool down rapidly, obtains cellular lysate liquid。By cellular lysate liquid in the centrifugal 15min of 3500r/min, abandoning supernatant, precipitate and add 4mL acetone with after distilled water wash 3 times, vibrate at 28 DEG C lixiviate 2h, and then the centrifugal 15min of 3500r/min takes supernatant, is carotenoid lixiviating solution。
Carotenoid crude extract is measured in 475nm place absorbance, is calculated as follows the content of carotenoid: carotenoid content (μ g/g)=A475* D*V/0.16*W, wherein, A475-for carotenoid absorbance under 475nm absorbing wavelength；Acetone cumulative volume (mL) used by D-lixiviate；The extension rate of V-carotenoid lixiviating solution；W-dry mycelium quality (g)；The extinction coefficient of 0.16-carotenoid。
The content * Biomass of yield (the mg/L)=carotenoid of carotenoid, wherein Biomass is dry mycelium quality in fermentation liquid。
Obtain bacterial strain R12M, bacterial strain respectivelyDeinococcuswulumuqiensisAnd bacterial strain sp.nov.DeinococcusradioduransDSM20539TIt is 754mg/L fermentation liquid, 304mg/L fermentation liquid and 280mg/L fermentation liquid that the yield of carotenoid is produced in fermentation。The above results illustrates, the bacterial strain R12M after mutation, it is possible to High Yield of Carotenoid。
Embodiment six: extract carotenoid from bacterial strain R12M
Bacterial strain R12M is inoculated in TGY solid medium with plate streak, 30 DEG C of constant temperature culture 60h。From TGY solid medium by the bacterial strain R12M picking list colony inoculation of enrichment culture in the 250ml shaking flask containing 50mlTGY fluid medium, 30 DEG C, shaking speed 190rpm when shaken cultivation 36h after, obtain seed liquor。According still further to 1% inoculum concentration, seed liquor is seeded in the 250ml shaking flask containing the fresh TGY fluid medium of 50ml, 30 DEG C, shaking speed 190rpm when shaken cultivation 48h。The yield of carotenoid is 754mg/L fermentation liquid。
Bacterial strain R12M is inoculated in TGY solid medium with plate streak, 28 DEG C of constant temperature culture 72h。From TGY solid medium by the bacterial strain R12M picking list colony inoculation of enrichment culture in the 250ml shaking flask containing 50mlTGY fluid medium in, 28 DEG C, 180rpm when shaken cultivation 48h, obtain seed liquor。According still further to 1% inoculum concentration, seed liquor is seeded in the 250ml shaking flask containing the fresh TGY fluid medium of 40ml, 28 DEG C, shaking speed 180rpm when shaken cultivation 60h。The yield of carotenoid is 730mg/L fermentation liquid。
Bacterial strain R12M is inoculated in TGY solid medium with plate streak, 32 DEG C of constant temperature culture 48h。From TGY solid medium by the bacterial strain R12M picking list colony inoculation of enrichment culture in the 250ml shaking flask containing 50mlTGY fluid medium, 32 DEG C, 200rpm when shaken cultivation 24h, obtain seed liquor。According still further to 1% inoculum concentration, seed liquor is seeded in the 250ml shaking flask containing the fresh TGY fluid medium of 60ml, 32 DEG C, shaking speed 180rpm when shaken cultivation 36h。The yield of carotenoid is 635mg/L fermentation liquid。
SEQUENCELISTING
<110>Nanjing University of Technology
<120>carotenoid superior strain and application thereof
<130>20140721
<160>1
<170>PatentInversion3.3
<210>1
<211>1459
<212>DNA
<213>the unusual coccus in Urumchi (Deinococcuswulumuqiensis) R12M
<400>1
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cggtcttcggaccgagtggcgcacgggtgagtaacacgtaactgacctacccagaagtca120
tgaataactggccgaaaggtcagctaatacgtgatgtggtgatttgccgtggcaaatcac180
taaagatttatcgcttctggatggggttgcgttccatcagctggttggtggggtaaaggc240
ctaccaaggcgacgacggatagccggcctgagagggtggccggccacaggggcactgaga300
cacgggtcccactcctacgggaggcagcagttaggaatcttccacaatgggcgcaagcct360
gatggagcgacgccgcgtgagggatgaaggttttcggatcgtaaacctctgaatctggga420
cgaaagagccttcgggcagatgacggtaccagagtaatagcaccggctaactccgtgcca480
gcagccgcggtaatacggagggtgcaagcgttacccggaatcactgggcgtaaagggcgt540
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tactggatgtcttgacctctggagaggtaactggaattcctggtgtagcggtggaatgcg660
tagataccaggaggaacaccaatggcgaaggcaagttactggacagaaggtgacgctgag720
gcgcgaaagtgtggggagcaaaccggattagatacccgggtagtccacaccctaaacgat780
gtacgttggctaagcgcaggatgctgtgcttggcgaagctaacgcgataaacgtaccgcc840
tgggaagtacggccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggt900
ggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatgctagg960
aaggcgctggagacagcgccgtgcccttcggggaacctagacacaggtgctgcatggctg1020
tcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgcctt1080
tagttgtcagcattcagttggacactctagagggactgcctgtgaaagcaggaggaaggc1140
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taccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtaga1380
ttgcagttgaaaccgccgggagcctcacggcaggcgtctagactgtggtttatgactggg1440
gtgaagtcgtaacaaggta1459
Claims (6)
1. carotenoid superior strain, be the unusual coccus in Urumchi (Deinococcuswulumuqiensis) R12M, preservation registration number is CCTCCNo:M2014099。
2. the application in preparing carotenoid of the bacterial strain described in claim 1, it is characterised in that by the Urumchi unusual coccus of fermenting (Deinococcuswulumuqiensis) R12MCCTCCNo:M2014099, obtain carotenoid。
3. bacterial strain application in preparing carotenoid according to claim 2, it is characterised in that by unusual for Urumchi coccus (Deinococcuswulumuqiensis) R12MCCTCCNo:M2014099 is inoculated in TGY fluid medium, 28-32 DEG C, 180-200rpm when shaken cultivation 36-72h, obtain fermentation liquid；Extract from described fermentation liquid and obtain carotenoid。
4. bacterial strain application in preparing carotenoid according to claim 3, it is characterised in that described extracting method is: separating thallus from fermentation liquid, by somatic cells breaking cellular wall, obtains cellular lysate liquid；Described lysate centrifuging and taking is precipitated, with organic solvent extraction, it is thus achieved that carotenoid。
5. bacterial strain application in preparing carotenoid according to claim 4, it is characterised in that described organic solvent is acetone。
6. bacterial strain application in preparing carotenoid according to claim 5, it is characterised in that the method for described somatic cells breaking cellular wall is: with thalline described in soak with hydrochloric acid, put in boiling water bath, be subsequently placed in ice-water bath。
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| Deinococcus wulumuqiensis sp. nov.,and Deinococcus;Wang W等;《IJSEM》;20100930;第60卷;2006-2010 * |
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