CN108330094A - A kind of the sequestered recombinant Mycobacterium smegmatis and its construction method of production niacin - Google Patents
A kind of the sequestered recombinant Mycobacterium smegmatis and its construction method of production niacin Download PDFInfo
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Abstract
The invention discloses a kind of the sequestered recombinant Mycobacterium smegmatis and its construction method of production niacin.A kind of recombinant Mycobacterium smegmatis of claimed production niacin, the recombinant Mycobacterium smegmatis contains recombination episomal plasmids, wherein recombination episomal plasmids containnudCGene,nudCGene nucleotide series such as SEQ ID NO：Shown in 1.The engineering bacteria being prepared through the invention can directly obtain niacin with a step, and independent of adding 3 cyanopyridines, not only avoid the various disadvantages during existing niacin chemical synthesis, and working condition is simple, and it is pollution-free, it is simple and easy to do, easily amplification, it is at low cost, it is suitable for large-scale industrial production application, there is the value of prodigious popularization and application.
Description
Technical field
The present invention relates to the production technical fields of niacin, more particularly, to a kind of sequestered recombined smegmatis of production niacin
Mycobacteria and its construction method.
Background technology
Niacin, that is, acidum nicotinicum is also referred to as used as vitamin B3, belongs to vitamin B complex, be a kind of water soluble vitamin, belongs to
It is one of 13 kinds of vitamin needed by human in vitamin B complex.Niacin is hydrogen carrier and antipellagra important in body tissue
The factor, have and maintain skin and neurological health, promote the effect of digestion.If it lacks, pellagra can be generated, skin is shown as
The symptoms such as inflammation, glossitis, oropharynx, diarrhea and agitation, insomnia cacesthesia.It is referred to as nicotinic acid with niacinamide, is used for antipellagra,
Also it can be used as blood expansion medicine.As medicine intermediate, can stop for isoniazid, niacinamide, Buddhist nun and the production of hexanicit etc..
Existing niacin is also used for cereal product, and meat additive and feed addictive are to prevent pellagra.Especially niacin is added in feed
The disease resistance of livestock and poultry can be improved afterwards, accelerate the speed of growth, improve the utilization rate of feed, can largely save feed, reduce raising
Cost.The nursing milk cow of the feed with addition niacin is once fed in the U.S., and compared with the control group, 15%-20% can be improved in the output of milk.
In addition niacin is alternatively arranged as forming the biochemical hormone of activated sludge, the deodorant of air and waste gas.Niacin is in dye industry, sense
Luminescent material industry, hair dyeing auxiliary agent, detergent etc. also have certain application.
What the production that niacin niacin is mainly produced with chemical synthesis industrial at present was industrially applied at present predominantly changes
Method, synthetic method mainly have liquid phase method（Potassium permanganate oxidation method and nitric acid oxidation method）And vapor phase method（Ozonation, ammonia oxygen
Change method and air oxidation process）, it is raw materials used predominantly 3- picolines, 2- methyl -5- ethylpyridines, nicotinonitrile and
Beautiful jade etc..Current most common method is to synthesize niacin by raw material air oxidation process of 3- picolines.By 3- picolines, sky
Gas and ammonia are passed through in fluidized-bed reactor in proportion, the reaction generation nicotinic acid nitrile under 290~360 DEG C, V2O5 catalysis；Then at hydrogen-oxygen
Highly pressured hydrolysis generates naotin in change sodium water solution, at 160 DEG C, is finally acidified to obtain niacin with hydrochloric acid.In this building-up process
It needs by this intermediate product of nicotinic acid nitrile, nicotinic acid nitrile is hydrolyzed to niacin by chemical reaction generation and needs high temperature and pressure, strong acid strong
The condition of alkali has certain requirement and meeting etching apparatus to equipment, and also unfriendly to environment.Company of Qing Hua Ziguang
Patent CN114288A, which reports to be catalyzed with one step of 3- picolines, generates niacin, and process is that 3- picolines and sulfuric acid is anti-
Picoline sulfate should be generated, then oxidation generates niacin sulfate under the action of oxidant nitric acid, and niacin sulfate adds
It can be 90% or so with obtained niacin, raw material yield to enter in aqueous slkali.The strong acid such as sulfuric acid, nitric acid, aqueous slkali are needed during this
Strong base substance, it is seriously polluted.
During niacin chemical synthesis, it is necessary to have specific high temperature and high pressure environment or using strong acid, highly basic or change
Chemical catalyst processing, reaction selectivity is not high, and by-product is more, and the yield of product is not high, and environmental pollution is big.In contrast, biological
Method, which prepares niacin, has the characteristics that substrate selective height, high catalytic efficiency, reaction condition is mild, environmental pollution is small.In addition, biological
Method prepares easily amplification, at low cost, is suitable for large-scale industrial production application.It has been reported that at present with microorganism Bacillus gemma bar
Bacterium, rhodococcus rhodochrous, Nocard's bacillus, Fusarinm solani and pseudomonas putida fermentation carry out living things catalysis nicotinonitrile system
Standby niacin.But biological catalysis prepares niacin and depends primarily on microbial fermentation production nitrilase, is catalyzed by this enzyme
Nicotinonitrile is converted into niacin, it is still desirable to add raw material nicotinonitrile.
Invention content
The technical problem to be solved by the present invention is in order to overcome the shortcomings of above-mentioned existing niacin technology of preparing, provide one kind
The engineering bacteria of niacin can be directly produced, which is that can expressnudCThe engineering bacteria of gene.The engineering bacteria be bynudCBase
It obtains, is being overexpressed because ORF sequences are connected to episomal plasmids and are transformed into mycobacterium smegmatisnudCThe shame dirt of gene point
Niacin can be largely obtained in the bacterium solution of branch bacillus.
The first purpose of the invention is to provide a kind of recombinant Mycobacterium smegmatis producing niacin.
Second object of the present invention is to provide a kind of method for the recombinant Mycobacterium smegmatis preparing production niacin.
Third object of the present invention is to provide the recombinant Mycobacterium smegmatis of the production niacin of the method structure.
Fourth object of the present invention is to provide application of the recombinant Mycobacterium smegmatis in producing niacin.
Fifth object of the present invention is to provide a kind of methods producing niacin.
To achieve the goals above, the present invention is achieved by the following technical programs：
A kind of recombinant Mycobacterium smegmatis of production niacin, the recombinant Mycobacterium smegmatis contain recombination episomal plasmids, wherein
Recombination episomal plasmids containnudCGene,nudCGene nucleotide series such as SEQ ID NO：Shown in 1.
The construction method of the recombinant Mycobacterium smegmatis will containnudCThe recombination episomal plasmids of gene order convert
Enter mycobacterium smegmatis.
Preferably, the episomal plasmids are pMV261 plasmids.
Preferably, the mycobacterium smegmatis is mycobacterium smegmatis mc2155。
Preferably, it is converted using electric shocking method, shock parameters are：2.5 kV of voltage, resistance 1000,25 μ of capacitance
F。
Preferably, the construction method of recombinant Mycobacterium smegmatis is divided into following steps：
S1. mycobacterium smegmatis mc is prepared2155 competent cells；
S2. conversion mycobacterium smegmatis mc2155；
S3. positive restructuring bacterium is screened.
It is highly preferred that the construction method of recombinant Mycobacterium smegmatis is divided into following steps：
S1. the recombinant Mycobacterium smegmatis is prepared：
By mycobacterium smegmatis mc2155 single bacterium colonies are inoculated in 7H9 fluid nutrient mediums, 37 DEG C of 200rpm shaken cultivations to logarithm
Growth period (OD600It is 0.5~1.0)；Culture is with 1:（80~120）Ratio be inoculated in fresh 7H9 fluid nutrient mediums,
37 DEG C are incubated overnight to OD600To 0.6 or so, in 4 DEG C, 5000 rpm centrifuge 10 min and collect thalline, by thalline precooling
10% sterile glycerol washs, and is eventually adding 10% glycerine of 10 ml precoolings, and after thalline is resuspended, -80 DEG C of separating device freezes spare.
S2. the recombinant Mycobacterium smegmatis is cultivated：
Take positive plasmid that the mycobacterium smegmatis mc of 200 μ l is added2155 electricity turn in competent cell, are incubated 8~12 on ice
Then min is transferred in 2 mm BTX electricity revolving cups, cleans the water on electric revolving cup outer wall, then use BTX ECM630 electrotransformation instrument
Electric shock, shock parameters are：2.5 kV of voltage, resistance 1000,25 μ F of capacitance.After having shocked by electricity, 1 ml 7H9 liquid is added immediately
Body culture medium, 37 DEG C of incubators take appropriate culture solution coating 7H10 solid plates after being incubated overnight（Containing 40~60 μ g/ml sulfuric acid
Kanamycins）, tablet sets 37 DEG C of incubator cultures 3~5 days.
S3. the niacin in purifying culture solution is collected：
Monoclonal to grow on picking tablet is inoculated into 7H9 fluid nutrient mediums and cultivates 2~3 days to exponential phase, from
The heart collects thalline and with sterile water washing, after thalline is resuspended with sterile water again, boiling water boiling 10~20 minutes, then 14000~
16000rpm takes DNA profiling of the supernatant as PCR, PCR to be using primer pair after centrifuging 15 minutes：5'-
GTGGCAGCGAGGACAACTTG-3', 5'-GATGCCTGGCAGTCGATCGTAC-3', PCR verification plasmid are transferred to shame dirt branch
In bacillus.
The recombinant Mycobacterium smegmatis of the production niacin of the method structure, also belongs to protection scope of the present invention.
Application of the recombinant Mycobacterium smegmatis of the production niacin in producing niacin, also belongs to the protection model of the present invention
It encloses.
A method of niacin is produced, is included the following steps：
S1. the recombinant Mycobacterium smegmatis is prepared；
S2. the recombinant Mycobacterium smegmatis is cultivated；
S3. the niacin in purifying culture solution is collected.
Preferably, it in the step S2, is cultivated using 7H9 fluid nutrient mediums.
Preferably, in the step S3, the specific practice for collecting the niacin in culture solution is：It is filtered and is trained using sterile filters
Nutrient solution simultaneously collects filtrate, with the protein and other macromolecular substances that are concentrated by ultrafiltration in pipe removal filtrate and collects filtrate, carries out
Efficient liquid phase chromatographic analysis detaches niacin.
Preferably, the parameter of efficient liquid phase chromatographic analysis is chromatographic column：Thermo Fisher, Hypersil Gold
AQ, 150 × 4.6 mm, 3 μm；Column temperature：30℃；Mobile phase：Water phase is the deionized water containing 0.1% formic acid, and organic phase is first
Alcohol；Gradient elution：When 0 min ,+5% organic phase of 95% water phase, when 30 min ,+95% organic phase of 5% water phase；Flow velocity：0.3
mL/min；Sample size：10 μL；Wavelength：284 nm.
Compared with prior art, the present invention has the advantages that：
The engineering bacteria being prepared through the invention can directly obtain niacin with a step, and independent of addition nicotinonitrile,
Existing various disadvantages during niacin chemical synthesis are not only avoided, and working condition is simple, it is pollution-free, it is easy to be easy
Row easily amplifies, at low cost, is suitable for large-scale industrial production application.
Description of the drawings
Fig. 1 is pMV261-NudCmsPlasmid nucleic acid gel electrophoresis, M after III digestion with restriction enzyme of BamH I and Hind：
DNA marker；1-3：Positive plasmid.
Fig. 2 is pMV261-NudCmsPlasmid schematic diagram.
Fig. 3 is pcr amplification product nucleic acid gel electrophoresis；M：DNA marker；1~3：Positive clone molecule.
Fig. 4 is control strain and is overexpressed strain cultured solution filtrate and niacin standard items through HPLC separation chromatograms；A is pair
According to bacterial strain mycobacterium smegmatis mc2155 culture solution filtrates；B：It is overexpressed the mycobacterium smegmatis mc of NudC genes2155-
pMV261-NudCmsCulture solution filtrate；C：Niacin standard items.
Specific implementation mode
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1nudCGene magnification and structure plasmid pMV261-NudCms
1、nudCGene magnification
With mycobacterium smegmatis mc2155 genomic DNAs are template, use primer pair 5'-ATCGGGATCCATGAGCGAACACCG
CACGT-3'/5'-TGCAAAGCTTTCAGTCGAGTGCGGCCCAGG-3', PCR amplificationnudCGene ORF sequences.PCR product
Target DNA fragment is separated by electrophoresis through nucleic acid gel, returns kit using Omega companies glue and recycles target DNA fragment, by sequencing
After obtainnudCGene ORF sequences such as SEQ ID NO：Shown in 1, the amino acid sequence such as SEQ ID NO of the protein of coding：
2.DNA after recycling uses BamH I and III digestion with restriction enzyme of Hind and is recycled by Omega companies Cycle-pure
The DNA that kit recycles after digestion is spare.
2, digestion
LB cultivates the e.colistraindh5α of the plasmid containing pMV261, and plasmid is extracted using Omega companies plasmid extraction kit,
The pMV261 plasmids of acquisition are using progress nucleic acid gel electrophoresis after III digestion with restriction enzyme of BamH I and Hind and use
The plasmid of Omega companies plastic recovery kit recycling linearisation.
3, it connects
The DNA fragmentation of digestion recycling and the linearization plasmid pMV261 recycled through same digestion react for 16 DEG C through T4 DNA ligases
Overnight, connection product converts bacillus coli DH 5 alpha competent cell and is coated with LB solid plates（Containing 100 μ g/ml sulfuric acid cards, that is mould
Element）, tablet 37 DEG C of constant incubator overnight incubations of placement.
4, the screening of positive plasmid
The monoclonal colonies inoculation LB liquid medium grown on picking tablet, 37 DEG C of shaking table 200rpm concussions are incubated overnight.So
Omega companies plasmid extraction kit is used to extract plasmid afterwards, the plasmid of acquisition uses III restriction enzyme of BamH I and Hind
It is verified through nucleic acid gel electrophoresis detection after digestion,nudCGene ORF sequences are connected on pMV261 carriers（Such as Fig. 1）.It tests simultaneously
Demonstrate,prove correct plasmid pMV261-NudCmsThrough Qing Ke companies sequence verification sequence without mutation, successful pMV261-NudC is builtms
Plasmid map such as Fig. 2.
Embodiment 2 builds NudC and is overexpressed mycobacterium smegmatis bacterial strain
1, mycobacterium smegmatis mc is prepared2155 competent cells
The fresh mycobacterium smegmatis mc of picking2155 single bacterium colonies are inoculated in 5 ml 7H9 fluid nutrient mediums, 37 DEG C of 200rpm
Shaken cultivation is to exponential phase (OD0.5~1.0)；Culture is with 1:100 ratio is inoculated in 100 fresh ml 7H9 liquid
In body culture medium, 37 DEG C are incubated overnight to OD600To 0.6 or so, in 4 DEG C, 5000 rpm centrifuge 10 min and collect thalline, by bacterium
10% sterile glycerol of body precooling at least washes twice, and is eventually adding 10 ml（In right amount）Thalline is resuspended in 10% glycerine of precooling
Afterwards, -80 DEG C of separating device freezes spare.
2, conversion mycobacterium smegmatis mc2155
Take the correct positive plasmid pMV261-NudC of structuremsDNA be added 200 μ l mycobacterium smegmatis mc2155 electricity turn sense
In by state cell, it is incubated 10 min on ice, is then transferred in 2 mm BTX electricity revolving cups, cleans the water on electric revolving cup outer wall, then
It is shocked by electricity using BTX ECM630 electrotransformation instrument, shock parameters are：2.5 kV of voltage, resistance 1000,25 μ F of capacitance.It has shocked by electricity
Afterwards, 1 ml 7H9 fluid nutrient mediums are added immediately, 37 DEG C of incubators take appropriate culture solution coating 7H10 solids flat after being incubated overnight
Plate（Containing 50 μ g/ml kanamycin sulfates）, tablet sets 37 DEG C of incubator cultures 3~5 days.
3, positive restructuring bacterium is screened
Monoclonal to grow on picking tablet, is inoculated into 5mL 7H9 fluid nutrient mediums and cultivates 2-3 days to logarithmic growth
Phase, thalline were collected by centrifugation and with sterile water washing 2 times, after thalline is resuspended with sterile water again, boiling water boiling 15 minutes, then
15000rpm takes DNA profiling of the supernatant as PCR, PCR to use primer pair 5'-GTGGCAGCGAGGACAACT after centrifuging 15 minutes
TG-3', 5'-GATGCCTGGCAGTCGATCGTAC-3', using the genomic DNA of extraction as template, PCR verifies pMV261-
NudCmsPlasmid is transferred in mycobacterium smegmatis（PCR results such as Fig. 3）.Correct NudC will be verifiedmsThe Strain Designation of overexpression
For mycobacterium smegmatis mc2155-pMV261-NudCms。
3 recombinant Mycobacterium smegmatis mc of embodiment2155-pMV261-NudCmsSecrete niacin
1, mycobacterium smegmatis mc2155-pMV261-NudCmsIt is inoculated with 7H9 fluid nutrient mediums, 37 DEG C of cultures to exponential phase
Afterwards, using 0.22 μm of sterile filters broth filtrate of Millipore and the filtrate of filtering is collected, then uses Millipore
The macromoleculars such as the protein that 3-kDa is concentrated by ultrafiltration in pipe removal filtrate simultaneously collect filtrate after filtering.The filtrate of acquisition uses efficient
Liquid chromatograph（HPLC）It detaches and analyzes niacin.
The specific conditions of HPLC：
Unit type：Thermo Fisher Scientific UltiMate 3000 ultrahigh-performance
liquid chromatography
Chromatographic column：Thermo Fisher, Hypersil Gold aQ, 150 × 4.6 mm, 3 μm, column temperature：30℃；
Mobile phase：Water phase：Deionized water（Containing 0.1% formic acid）；Organic phase：Methanol；
Gradient elution：When 0 min ,+5% organic phase of 95% water phase；
When 30 min ,+95% organic phase of 5% water phase；
Flow velocity：0.3 mL/min；
Sample size：10 μL；
Wavelength：284 nm.
2, HPLC is the results show that mycobacterium smegmatis mc2155-pMV261-NudCmsContain niacin in culture solution（Such as figure
4）.
Sequence table
<110>The Shanghai bio tech ltd Jing Nuo
<120>A kind of the sequestered recombinant Mycobacterium smegmatis and its construction method of production niacin
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 936
<212> DNA
<213>Mycobacterium smegmatis (Mycobacterium smegmatis)
<400> 1
atgagcgaac accgcacgtt cgggctccgt aacgtcccgc tgctgtcccg ggtcggcgcc 60
gatcgcgccg ataccttgcg caccgacgtc gacgccgccc tggcgggctg gcccgacgcg 120
ctggtgctac gcgtggaccg ccgcaaccag gtgctcatcg ccaacggtca ggtggtgctc 180
ggtgaggccg gcgcactcgg agaccggccg cccgagcacg cggtgttcct gggacgtctg 240
caggacggca ggcacgtatg gggtatccgg gcggatctgg aggcgcccga ggatgccgac 300
ctggggaccg aggtgctcga cctgcgccgg gccgggcaga tcttcgacga caccagcgcc 360
cagttggtgg cgaccgccac ggcgctgctc aactggcatg acaacgcgcg gcacagcgcg 420
atcgacgggg cgccgacccg gcccgccaag ggcggctggt cgcgcgtcaa cccgctgacc 480
ggccacgagg agttcccgcg gatcgacccc gccatcatct gcctggtgca cgacgggcat 540
gaccgggcgg tgctggcccg tcagacgctg tggccggagc ggttgttctc gatcctggcc 600
ggcttcgtcg aggccggcga gtcgttcgag acatgcgtgc agcgcgagat cgccgaggag 660
atcgggctca cggtcaccga cgtgcagtac ctcggcagtc agccgtggcc gttcccgcgc 720
tcgctcatgg tgggattcca cgcgatcggc gacccggagc agccgttctc ctacaacgac 780
ggcgagatcg ccgaggccgc gtggttcacc cgcgatgaga tccgcgcggc actcgatcag 840
ggggactgga acagcgactc gccgtcacgg ctcctgctgc caggctccat ctcgatcgcc 900
cgcgagatca tcgaatcctg ggccgcactc gactga 936
<210> 2
<211> 311
<212> PRT
<213>Mycobacterium smegmatis (Mycobacterium smegmatis)
<400> 2
Met Ser Glu His Arg Thr Phe Gly Leu Arg Asn Val Pro Leu Leu Ser
1 5 10 15
Arg Val Gly Ala Asp Arg Ala Asp Thr Leu Arg Thr Asp Val Asp Ala
20 25 30
Ala Leu Ala Gly Trp Pro Asp Ala Leu Val Leu Arg Val Asp Arg Arg
35 40 45
Asn Gln Val Leu Ile Ala Asn Gly Gln Val Val Leu Gly Glu Ala Gly
50 55 60
Ala Leu Gly Asp Arg Pro Pro Glu His Ala Val Phe Leu Gly Arg Leu
65 70 75 80
Gln Asp Gly Arg His Val Trp Gly Ile Arg Ala Asp Leu Glu Ala Pro
85 90 95
Glu Asp Ala Asp Leu Gly Thr Glu Val Leu Asp Leu Arg Arg Ala Gly
100 105 110
Gln Ile Phe Asp Asp Thr Ser Ala Gln Leu Val Ala Thr Ala Thr Ala
115 120 125
Leu Leu Asn Trp His Asp Asn Ala Arg His Ser Ala Ile Asp Gly Ala
130 135 140
Pro Thr Arg Pro Ala Lys Gly Gly Trp Ser Arg Val Asn Pro Leu Thr
145 150 155 160
Gly His Glu Glu Phe Pro Arg Ile Asp Pro Ala Ile Ile Cys Leu Val
165 170 175
His Asp Gly His Asp Arg Ala Val Leu Ala Arg Gln Thr Leu Trp Pro
180 185 190
Glu Arg Leu Phe Ser Ile Leu Ala Gly Phe Val Glu Ala Gly Glu Ser
195 200 205
Phe Glu Thr Cys Val Gln Arg Glu Ile Ala Glu Glu Ile Gly Leu Thr
210 215 220
Val Thr Asp Val Gln Tyr Leu Gly Ser Gln Pro Trp Pro Phe Pro Arg
225 230 235 240
Ser Leu Met Val Gly Phe His Ala Ile Gly Asp Pro Glu Gln Pro Phe
245 250 255
Ser Tyr Asn Asp Gly Glu Ile Ala Glu Ala Ala Trp Phe Thr Arg Asp
260 265 270
Glu Ile Arg Ala Ala Leu Asp Gln Gly Asp Trp Asn Ser Asp Ser Pro
275 280 285
Ser Arg Leu Leu Leu Pro Gly Ser Ile Ser Ile Ala Arg Glu Ile Ile
290 295 300
Glu Ser Trp Ala Ala Leu Asp
305 310
Claims (10)
1. a kind of recombinant Mycobacterium smegmatis of production niacin, which is characterized in that the recombinant Mycobacterium smegmatis contains recombination trip
Release plasmid, wherein recombination episomal plasmids containnudCGene,nudCGene nucleotide series such as SEQ ID NO：Shown in 1.
2. the construction method of recombinant Mycobacterium smegmatis described in claim 1, which is characterized in that will containnudCGene order
Recombination episomal plasmids are transformed into mycobacterium smegmatis.
3. according to the method described in claim 2, it is characterized in that, the episomal plasmids are pMV261 plasmids.
4. according to the method described in claim 2, it is characterized in that, being converted using electric shocking method, shock parameters are：Voltage
2.5 kV, resistance 1000,25 μ F of capacitance.
5. the recombinant Mycobacterium smegmatis of the production niacin of claim 2 the method structure.
6. application of the recombinant Mycobacterium smegmatis of claim 1 or 5 in producing niacin.
7. a kind of method producing niacin, which is characterized in that include the following steps,
S1. the recombinant Mycobacterium smegmatis described in claim 1 or 5 is prepared；
S2. the recombinant Mycobacterium smegmatis is cultivated；
S3. the niacin in purifying culture solution is collected.
8. the method according to the description of claim 7 is characterized in that in the step S2, trained using 7H9 fluid nutrient mediums
It supports.
9. the method according to the description of claim 7 is characterized in that in the step S3, the tool of the niacin in culture solution is collected
Body way is：Using sterile filters broth filtrate and collect filtrate, be concentrated by ultrafiltration pipe removal filtrate in protein and its
His macromolecular substances simultaneously collect filtrate, carry out efficient liquid phase chromatographic analysis, detach niacin.
10. according to the method described in claim 9, it is characterized in that, the parameter of efficient liquid phase chromatographic analysis is chromatographic column：
Thermo Fisher, Hypersil Gold aQ, 150 × 4.6 mm, 3 μm；Column temperature：30℃；Mobile phase：Water phase is containing 0.1%
The deionized water of formic acid, organic phase are methanol；Gradient elution：When 0 min ,+5% organic phase of 95% water phase, when 30 min, 5%
+ 95% organic phase of water phase；Flow velocity：0.3 mL/min；Sample size：10 μL；Wavelength：284 nm.
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