CN110317744A - A method of producing Marseille bacterium and its production basket purpurin of Indigo pigment - Google Patents
A method of producing Marseille bacterium and its production basket purpurin of Indigo pigment Download PDFInfo
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Abstract
The invention discloses one plant of Marseille bacterium and its applications, Marseille bacterium (Massilia sp.SOD) provided by the invention, from the Hefei City, Anhui Province garden Nong Cui experimental plot soil, and it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC No.1.13687.The condition of bacterial strain production Indigo pigment is: the strain of activation culture being inoculated in 1% tryptone fluid nutrient medium, 28 DEG C, stands ferment at constant temperature culture 3-4 days；Thallus in bacterium solution is collected by centrifugation, using dehydrated alcohol as extractant, extract is concentrated through rotary evaporation and obtains.The Indigo pigment has preferable thermal stability and ph stability.The present invention provides the novel bacterials of production purpurin, the purpurin of bacterial strain production is natural pigment, highly-safe, there is good bioactivity, new way also is provided for the natural purpurin of industrialized production simultaneously, is had good prospects in industrial applications such as food, dyeing and weaving and medicine.
Description
Technical field
The invention belongs to pigment production technical fields, and in particular to a kind of Marseille bacterium for producing Indigo pigment and its production basket
The method of purpurin.
Background technique
Pigment can be divided into synthetic food color and natural pigment according to source, in the row such as food, textile printing and dyeing and cosmetics
Industry has important application.Wherein, synthetic food color mainly extracts from coal tar or passes through by raw material of arene compounds
Chemically synthesized organic pigment, strong coloring force is cheap, but partially synthetic pigment is to human health, or even induction cancer
Disease, therefore its application is subject to certain restrictions.Natural pigment is widely present in a variety of organisms, and many animals and plants and microorganism are
Main source.It is separated into pure physical process from the extraction of animals and plants and microorganism, it is highly-safe, therefore benefit is developed to natural pigment
Research becomes the research hotspot of foods and cosmetics industry.But animals and plants material is by the system of many factors such as resource, environment
About, cause to underproduce, is expensive, limiting its application development.And microbial resources is utilized to produce natural pigment, it overcomes
The shortcomings of natural pigment are produced using animals and plants as raw material, and are easy to industrialize.Therefore, using micro-organisms natural colour
Element is not limited by resource, environment and space, is easier to realize industrialization, is increasingly becoming the mainstream in natural pigment source.
Currently, the cyanine to circulate in the market is mainly artificial chemistry synthetic dyestuff, natural blue pigment is less, and comes blue more
Alum mine, indigo plant, acanthaceous indigo and cape jasmine etc., few Indigo pigments from microbial source.Wherein, gardenia blue pigment is with cape jasmine fruit
For raw material, processes, complex process and the higher cost such as extracted, evaporation and concentration and purification, raw material is limited, raw in large-scale industry
There is certain difficulties in production.The microorganism for being able to produce cyanine for having disclosed report at present is seldom, mainly has sky blue
Color streptomycete (Streptomyces coelicolor) A3, pseudomonad, Du roll Salmonella (Duganella sp.) T2013, shut out
Roll Salmonella B2, Chromobacterium (Chromobacterium), Jansen bacillus (Janthinobacterium lividum) and fluorescence
Pseudomonad (Pseudomonasfluorescens) B1 etc..The yield of the types of these bacterial strains, quantity and bacterial strain pigment is also remote
It is not able to satisfy the needs that people research and develop natural blue.
Marseille Pseudomonas (Massilia) bacterial strain is under the jurisdiction of bacterium domain (Bacteria) Proteobacteria (Proteobacteria) β
Deform Gammaproteobacteria (Betaproteobacteria) bulkholderia cepasea mesh (Burkholderiales) Oxalobacter section
(Oxalobacteraceae), 43 effectively descriptions kind are had now been found that.Marseille Pseudomonas member shows a variety of physiological activity, example
Such as, Massilia tieshanensis TS3TIt is resistant to As3+, Cu2+, Zn2+, Ni2+And Cd2+Etc. various heavies；Stench horse
Match bacterium (M.putida6NM-7T) dimethyl disulfide can be generated；Massilia sp.WG5 can degrade phenanthrene；
M.chloroacetimidivorans TA-C7eTCan degrade chloroacetamide；Massilia sp.UMI-21 can synthesize poly-
Hydroxyalkanoate (Polyhydroxyalkanoates, PHA) etc., however the research of cyanine Marseille bacterium can be generated and be rarely reported.
Summary of the invention
The object of the present invention is to provide a kind of Massilia for generating Indigo pigment to belong to new strains.
Marseille Pseudomonas (Massilia) bacterial strain SOD provided by the present invention belongs to Oxalobacteraceae section, in
In September, 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: the micro- life of the Chinese Academy of Sciences
Object research institute, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), preservation registration number: CGMCC No.1.13687.In 2019 03
The moon 18 issues the biomaterial preservations for being used for proprietary program, and registers on the books.
Non- gemma type aerobic bacteria of the bacterial strain for rod-short, Gram-negative, whole body amphitrichous can move.Growth temperature
8-30 DEG C of range；NaCl tolerance is less than or equal to 1%；PH range 5-9.Colony colour is atropurpureus.Bacterial strain to nitrous acid also
Original is positive；Hydrolyzable aesculin, gelatin, polysorbas20 and starch；To oxidizing ferment, catalase reacting positive；Indoles is generated, urea
Enzyme, hydrogen sulfide generation are negative.Bacterial strain is in N-acetylglucosamine, the assimilation of capric acid, adipic acid, citric acid and phenylacetic acid
It is negative.Bacterial strain can utilize aesculin ferrum citricum, D- cellobiose, D-Maltose, the sea D- bath sugar, starch, glycerol and rough gentian
Disaccharides produces sour (API50 CH).In API ZYM test, acid phosphatase, esterase (C4), the lipoid esterase (C8), figured silk fabrics of bacterial strain
Propylhomoserin virtue amidase, leucine virtue amidase, trypsase, acid phosphatase, naphthols-As-BI- phosphohydrolase, α-grape
Glycosidase and activity of beta-glucosidase are positive.In GenBank, with its highest mould of 16Sr DNA gene order similarity
Formula bacterial strain is Massilia violaceinigra B2T.G+C content is 65.4mol% in DNA.
Another purpose of the application is to provide a kind of method for producing Indigo pigment, and this method is fermentation Marseille Pseudomonas
(Massilia) bacterial strain SOD, production obtain Indigo pigment.
As preferred means, Marseille Pseudomonas (Massilia) the bacterial strain SOD fermentation culture conditions are to issue at 8-28 DEG C
Ferment culture 3-4 days.
As further preferred means, Marseille Pseudomonas (Massilia) the bacterial strain SOD fermentation culture conditions are further
Fermented and cultured 4 days at preferably 25 DEG C.
As further preferred means, Marseille Pseudomonas (Massilia) the bacterial strain SOD fermentation culture conditions are: fermentation
3-4 days, liquid amount 50-100mL/500mL, inoculum concentration 1-5%, pH7.0-7.4,25-28 DEG C of temperature, static gas wave refrigerator.
As further preferred means, every liter in the solid medium of Marseille Pseudomonas (Massilia) the bacterial strain SOD
Contain: yeast extract powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, leachable starch 0.5g, phosphorus
Sour hydrogen dipotassium 0.3g, anhydrous magnesium sulfate 0.024g, Sodium Pyruvate 0.3g, agar 20.0g.1000mL is added water to, pH value is transferred to
7.0-7.4 range.Multiplying culture and miscellaneous bacteria are carried out in solid culture primary surface with plate streak inoculation Massilia sp.SOD
Detection.
As further preferred means, the liquid tryptone of Marseille Pseudomonas (Massilia) the bacterial strain SOD ferments
Contain for every liter in culture medium: tryptone 5-15g.PH is adjusted to the bottled 200ml fermentation liquid of 7.2,500ml triangle, fermented and cultured 3-4
It.
As further preferred means, after the fermented and cultured of Marseille Pseudomonas (Massilia) the bacterial strain SOD by from
The heart collects thallus, extracts Indigo pigment with organic solvent, extract is concentrated and dried through rotary evaporation and obtains Indigo pigment crude extract.
As further preferred means, the organic solvent is elected to be dehydrated alcohol or methanol as extractant.
The invention has the advantages that:
1, Marseille bacterium SOD of the present invention can generate a large amount of violaceins in static gas wave refrigerator, deposit with reported bacterial strain
Significantly different, compared with shaken cultivation, more saving electric power energy and equipment.
2, Marseille bacterium SOD of the present invention is Marseille Pseudomonas one newfound new bacterium, and existing bacterial strain is significantly different with belonging to.
3, the chromogenic plain thermal stability of Marseille bacterium SOD of the present invention is good, 100 DEG C of processing 4-26h of methanol solution, methanol solution
Light absorption value is basically unchanged at 570nm, and pigment stability is apparently higher than other reported bacterial strain produced pigments, in processing 4
Hour pigment loss late is 1.56%；As 100 DEG C of processing 4h of Pseudomonas fluorescens B1 CGMCC cyanine that No.1194 is produced are
Loss late is up to 60.57%；Pigment loss late reaches when 100 DEG C of processing 2h of the produced cyanine of Xanthomonas campestris B29 CGMCC No.3699
18.8%, so the pigment loss late at 4 hours is greater than 18.8%.
4, the Indigo pigment yield of Marseille bacterium SOD of the present invention reaches 0.57-2.42g/L, domestic about the Marseille for producing purpurin
The document of bacterium is rarely reported.
5, Indigo pigment prepared by the present invention has better ph stability.
Detailed description of the invention
Fig. 1 is the systematic evolution tree constructed based on bacterial strain SOD and its reference strains 16S rRNA gene order.
Fig. 2 is bacterial strain SOD transmission electron microscope photo.
Fig. 3 is that Indigo pigment crude extract thin-layer chromatography is detected in the upper figure of silica gel plate.
Fig. 4 is Indigo pigment crude extract liquid chromatographic detection result figure.
Fig. 5 is dissolution characteristics result figure of the Indigo pigment in different solvents.
Fig. 6 is the influence of Indigo pigment semifinished product (methanol solution) stability under natural light irradiation.
Fig. 7 is the influence of dark lower Indigo pigment semifinished product (methanol solution, pH2-13) 72 hours rear stabilities.
Fig. 8 is influence of the processing time to Indigo pigment semifinished product stability under different pH；
Fig. 9 is influence of the metal ion to Indigo pigment semifinished product.
Specific embodiment
Present invention is further described in detail with reference to the accompanying drawing:
A kind of method producing Indigo pigment of the invention, the related experiment for preparing Indigo pigment are as follows:
One, the separation, screening and identification of bacterial strain
1, sample acquires:
Soil picks up from AnHui Agriculture University, HeFei City, AnHui Province agriculture kingfisher garden mould sample.
2, the separation and screening of bacterial strain
Soil extract liquid culture medium: weighing 500 grams of soil, be added in 2L aqua sterilisa, stir and evenly mix, and stands 10-
20min takes supernatant, dilutes 3 times respectively, 10 times, adds 2% agar, pH7.0-7.2 matches soil extract liquid culture medium, inverted plate.
It is separated using dilution method, weighs 10.0g soil sample and pour into the triangular flask containing 100mL sterile water and shake up, according to
Subgradient dilution, is made soil supension；100 μ L soil supensions are taken to be coated on above-mentioned isolation medium plate, 28 DEG C of culture 2-3
It, selects atropurpureus single colonie, purifying of being crossed repeatedly, and vigorous atropurpureus bacterium colony can be gone out in R2A cultured on solid medium；
By sediments microscope inspection, until microscopy is pure bacterial strain (cellular morphology is consistent), it is SOD, that is, Marseille bacterium by this Strain Designation
Belong to (Massilia sp.) SOD.Short-term preservation can be placed in 4 DEG C of refrigerators, need to be activated before experiment.Can also in R2A and
In 40% glycerol mixed liquor, long-term preservation at -80 DEG C.
Preparation solid R2A culture medium activated strains: yeast extract powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g,
Glucose 0.5g, leachable starch 0.5g, dipotassium hydrogen phosphate 0.3g, anhydrous magnesium sulfate 0.024g, Sodium Pyruvate 0.3g, agar
20.0g adds water 1000ml, adjusts final pH 7.0-7.4.
3, the identification of bacterial strain
28 DEG C are taken, the bacterial strain sample after R2A culture medium culture 3d, in optical microscopy and observed under electron microscope bacterial strain
The morphological feature and flagellum movement mode of cell.Gram staining experiment is carried out with standard Gram's stain.Analyze bacterial strain SOD
It is different with culture medium in different temperatures (8,20,28,30,33 DEG C), different NaCl concentrations (0,0.3,0.5,0.7,1,1.5,2%)
Cultural characteristic under the conditions of initial pH (5.0,6.0,7.0,8.0,9.0,10.0).3% (v/v) H2O2Solution is to fresh Growth
Cell carries out catalase activity measurement, detects the work of oxidizing ferment with oxidase reagent bioM é rieux according to standard step
Property.Indoles, hydrogen sulfide experiment are carried out referring to Tindall experimental method.It is studied by API20 NE, 50CH and API ZYM test paper
It is utilized to different carbon source and enzyme activity.
The result shows that: Massilia sp.SOD has whip in rod-short, non-gemma type aerobic bacteria, Gram-negative all over the body
Hair, can move, 8-33 DEG C of growth temperature range, and NaCl tolerance is less than or equal to 1%, pH range 5.0-9.0, and colony colour is
Atropurpureus.
16s rDNA sequencing is carried out to Massilia sp.SOD, sequence is in Genbank accession number
MH551481；16S rDNA based on type strain and reference strains nearest with its affiliation in bacterial strain SOD and GenBank
Gene order has carried out the auxology analysis of system.Using MEGA6 software with adjacent method, maximum likelihood method and maximum parsimony method structure
Build systematic evolution tree, such as Fig. 1.And the draft genome of the bacterial strain is determined, the results show that the strain gene group sketch contains
735558bp is spliced into 81 contigs.G+C content is 65.4mol%.
1 bacterial strain SOD of table and Marseille Pseudomonas have type strain Intraspecific differences in physiological
Note: "+" positive reaction, "-" negative reaction.Bacterial strain: 1, SOD；2, M.violaceinigra B2T；3,
M.glaciei B448-2T；4, M.eurypsychrophila CGMCC1.12828T；5, Rugamonas rubra
CCM3730T.ND, without document report.
Two, basket purpurin is prepared using Massilia sp.SOD
1, bacterial strain activates
Prepare solid R2A culture medium.Configured culture medium sterilizes 20-30min at 121 DEG C, inverted plate, and cooling is followed by
Enter Massilia sp.SOD, cultivates 2-3d at 28 DEG C.
Liquid tryptone culture medium is prepared to strain fermentation culture: tryptone 10g adds water 1000ml, adjusts final
pH7.2.Configured culture medium sterilizes 20-30min at 121 DEG C, it is cooling after the appropriate single colonie of picking be inoculated in Liquid Culture
In base, stationary culture 3-4d at 28 DEG C.
2, the extraction of purpurin
The bacterium solution 10000r/min of fermented and cultured is centrifuged 10min, abandoning supernatant can be obtained thallus.It is added into thallus suitable
Dehydrated alcohol is measured, vortex concussion is mixed, and then (UC-6200, the Tianjin Tyke Mei Rui science and technology are limited in ultrasonic cleaner
Company) in maximum power vibrate 0.5-1h, will oscillation liquid 10000r/min be centrifuged 10min, obtain the ethanol solution of pigment.If still
There is pigment to remain in cell residue, aforesaid operations step is repeated, until no longer extracting pigment.Gained supernatant warp
I.e. obtaining purpurin slightly proposes component after vacuum drying or 70 DEG C of Rotary Evaporators evaporation dryings.
3, the constituent analysis of purpurin runic object
1) thin-layer chromatography inspection is known:
As shown in figure 3, using 50 × 100G silica gel plate, solvent ethyl acetate: methanol (80:1), as a result such as Fig. 3 institute
Show, cyanine is containing there are two components, respectively blue and light violet magenta.Mobility of cyanine component I, II on silica gel plate
It (Rf) is respectively 0.28 and 0.56.
2) cyanine crude extract HPLC is analyzed:
As shown in figure 4, high performance liquid chromatography: using Agilent-1100 high performance liquid chromatograph, chromatographic column Agilent
Eclipse XDB-C18,150mm × 4mm, 5 μm.Column temperature is 30 DEG C, and eluant, eluent is methanol: water (70: 30)；Flow velocity is
1.0mL·min-1, Detection wavelength 570nm.
Three, the physicochemical characteristics detection of basket purpurin
1, dissolution characteristics: 5 milligrams of basket purpurin crude extracts are weighed, are dissolved in 1ml water, acetonitrile, methanol, ethyl alcohol, isopropyl respectively
Alcohol, acetone, tetrahydrofuran, ethyl acetate, chloroform, n-hexane, in 11 kinds of solvents of petroleum ether (60-90) (mentioned reagent sequence with
Sequence consensus in Fig. 5), oscillation dissolution 1h, stands at room temperature, dissolution situation of the pigment in each solvent is observed, as a result such as Fig. 5
Shown, which can be dissolved in methanol, ethyl alcohol, acetone, be slightly soluble in tetrahydrofuran, ethyl acetate and n-hexane, be insoluble in
Water, acetonitrile, chloroform, petroleum ether etc., wherein methanol, the solubility of ethyl alcohol are maximum, are shown as navy blue, and ethyl alcohol is nontoxic, therefore select
It is best pigment Extraction solvent with ethyl alcohol.
2, the photostability of pigment
Take be added in the above-mentioned pigment for preparing basket purpurin and the dissolution of undried methanol of 3mL translucency preferable 15 ×
In 150mm teat glass, as irradiating under outdoor natural light, pigment loss late is as shown in Figure 7 after a certain period of time.
3, the ph stability of pigment
Such as Fig. 7 and 8, HCl or NaOH is added in the undried pigment dissolved with methanol into 10mL, adjusts pH points
Not Wei 2,3,4,5,6,7,8,9,10,11,12,12.5,13 set dark place placement, measured its suction at 24,48,72,96 hours respectively
Light value, and take pictures.The pigment is very stable within the scope of pH6-10 as the result is shown, more stable within the scope of pH4-5, is lower than in pH
3 or more than 11 when it is unstable, the color of solution quickly becomes yellow, OD570Light absorption value at nm also declines very serious.
4, the thermal stability of pigment
The obtained Indigo pigment extract of the present invention is configured to the methanol solution of concentration 3mg/mL, be respectively placed in 50 DEG C,
60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, handle in 100 DEG C of water-baths in 4-26h, measure light absorption value under 570nm, calculate pigment residue
Amount.Pigment high-temperature process 4 hours as the result is shown, except 100 DEG C of loss lates be 1.56% in addition to, other be lower than 1%, even across
100 DEG C of processing 26h, loss late are also only 5.48%, illustrate that pigment of the invention has stronger temperature capacity.
The loss late of 2 pigment of table at different temperatures
5, influence of the metal ion to the semifinished product of Indigo pigment
The obtained Indigo pigment extract of the present invention is configured to 3mg/mL methanol solution, take respectively the above-mentioned solution of 5mL with
The Fe of 0.05mol/L3+、Fe2+、K+、Na+、Mg2+、Pb2+Solion is uniformly mixed in equal volume, is placed 48 hours at room temperature dark
Afterwards, with survey its light absorption value under 570nm, calculate pigment residue amount, the results are shown in attached figure 9, and ion is followed successively by CK, K from left to right+、Fe3 +、Na+、Fe2+、Mg2+、Pb2+.CK is pigment methanol solution, does not add the control of ion.
Fe is wherein added3+Pigment solution turns yellow rapidly, other are without significant change.After placing for 24 hours, it can be seen from attached drawing 9
Except K+Less outer, Fe is influenced on cyanine of the present invention3+、Fe2+、Na+、Mg2+、Pb2+It is affected Deng to cyanine of the present invention.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
The present invention is not limited to above to the description of embodiment, the content that those skilled in the art disclose according to the present invention,
The improvement and modification that need not be carried out by creative work on the basis of the present invention, all should protection scope of the present invention it
It is interior.
SEQUENCE LISTING
<110>Agricultural University Of Anhui
<120>a kind of Marseille bacterium for producing Indigo pigment and its method for producing basket purpurin
<130> 2019/04/04
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1490
<212> DNA
<213>Marseille Pseudomonas (Massilia sp.)
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcatgc cttacacatg caagtcgaac 60
ggcagcgcgg ggcaacctgg cggcgagtgg cgaacgggtg agtaatatat cggaacgtac 120
cctggagtgg gggataacgt agcgaaagtt acgctaatac cgcatacgat ctaaggatga 180
aagtggggga tcgcaagacc tcatgctcct ggagcggccg atatctgatt agctagttgg 240
tggggtaaag gcctaccaag gcatcgatca gtagctggtc tgagaggacg accagccaca 300
ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat tttggacaat 360
gggcgaaagc ctgatccagc aatgccgcgt gagtgaagaa ggccttcggg ttgtaaagct 420
cttttgtcag ggaagaaacg gtggaggcta atatcctctg ctaatgacgg tacctgaaga 480
ataagcaccg gctaactacg tgccagcagc cgcggtaata cgtagggtgc aagcgttaat 540
cggaattact gggcgtaaag cgtgcgcagg cggttttgta agtctgtcgt gaaatccccg 600
ggctcaacct gggaattgcg atggagactg caaggctaga atctggcaga ggggggtaga 660
attccacgtg tagcagtgaa atgcgtagag atgtggagga acaccgatgg cgaaggcagc 720
cccctgggtc aagattgacg ctcatgcacg aaagcgtggg gagcaaacag gattagatac 780
cctggtagtc cacgccctaa acgatgtcta ctagttgtcg ggttttaatt aacttggtaa 840
cgcagctaac gcgtgaagta gaccgcctgg ggagtacggt cgcaagatta aaactcaaag 900
gaattgacgg ggacccgcac aagcggtgga tgatgtggat taattcgatg caacgcgaaa 960
aaccttacct acccttgaca tgtacggaag ccacgagaga tcgaggcgtg ctcgaaagag 1020
agccgtaaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080
tcccgcaacg agcgcaaccc ttgtcattag ttgctacgaa agggcactct aatgagactg 1140
ccggtgacaa accggaggaa ggtggggatg acgtcaagtc ctcatggccc ttatgggtag 1200
ggcttcacac gtcatacaat ggtacataca gagggccgcc aacccgcgag ggggagctaa 1260
tcccagaaag tgtatcgtag tccggattgt agtctgcaac tcgactacat gaagttggaa 1320
tcgctagtaa tcgcggatca gcatgtcgcg gtgaatacgt tcccgggtct tgtacacacc 1380
gcccgtcaca ccatgggagc gggttttacc agaagtaggt agcttaaccg caaggggggc 1440
gcttaccacg gtaggattcg tgactggggt gaagtcgtaa caaggtaacc 1490
Claims (10)
1. a kind of Marseille bacterium for producing Indigo pigment, Marseille Pseudomonas (Massilia) bacterial strain SOD, deposit number are as follows: CGMCC
No.1.13687。
2. a kind of method for producing Indigo pigment, it is characterised in that: culture Marseille Pseudomonas (Massilia) bacterial strain SOD is produced
To Indigo pigment.
3. a kind of method for producing Indigo pigment according to claim 2, it is characterised in that: the Marseille Pseudomonas
(Massilia) bacterial strain SOD fermentation culture conditions are fermented and cultured 3-4 days at 8-28 DEG C.
4. a kind of method for producing Indigo pigment according to claim 3, it is characterised in that: the Marseille Pseudomonas
(Massilia) bacterial strain SOD fermentation culture conditions are fermented and cultured 4 days at 25 DEG C.
5. a kind of method for producing Indigo pigment according to claim 2, it is characterised in that: the Marseille Pseudomonas
(Massilia) bacterial strain SOD fermentation culture conditions are: fermentation 3-4 days, liquid amount 50-100mL/500mL, inoculum concentration 1-5%,
PH7.0-7.4,25-28 DEG C of temperature, static gas wave refrigerator.
6. a kind of method for producing Indigo pigment according to claim 5, it is characterised in that: the Marseille Pseudomonas
(Massilia) in the solid medium of bacterial strain SOD every liter contain: yeast extract powder 0.5g, peptone 0.5g, casein hydrolysis
Object 0.5g, glucose 0.5g, leachable starch 0.5g, dipotassium hydrogen phosphate 0.3g, anhydrous magnesium sulfate 0.024g, Sodium Pyruvate
0.3g, agar 20.0g, remaining is settled to 1000mL with water, and pH is adjusted to 7.0-7.4.
7. a kind of method for producing Indigo pigment according to claim 1, it is characterised in that: the Marseille Pseudomonas
(Massilia) in the liquid tryptone fermentation medium of bacterial strain SOD every liter contain: tryptone 5-15g.
8. according to a kind of described in any item methods for producing Indigo pigment of claim 2-7, it is characterised in that: the production is blue
The method of purpurin further includes the steps that the extraction purification Indigo pigment from Marseille Pseudomonas (Massilia) bacterial strain SOD.
9. a kind of method for producing Indigo pigment according to claim 8, it is characterised in that: the Marseille Pseudomonas
(Massilia) by thalline were collected by centrifugation after the fermented and cultured of bacterial strain SOD, Indigo pigment, extract warp are extracted with organic solvent
Rotary evaporation, which is concentrated and dried, obtains Indigo pigment crude extract.
10. a kind of method for producing Indigo pigment according to claim 9, it is characterised in that: the organic solvent is elected to be
Dehydrated alcohol or methanol are as extractant.
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