CN103146776A - Method for producing indigo pigment with bacillus subtilis - Google Patents
Method for producing indigo pigment with bacillus subtilis Download PDFInfo
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Abstract
The invention relates to a method for producing natural indigo pigment and particularly discloses a method for producing indigo pigment with the bacillus subtilis. The method includes the following steps: (1) bacterial strain fermental cultivation: inoculating seed solution of China General Microbiological Culture Collection Center (CGMCC) 5698 of the bacillus subtilis producing the indigo pigment into a sterilized liquid fermentation medium, and cultivating the bacillus subtilis at 26-34 DEG C for 46-60h, wherein the rotation speed of a swing bed is 200r/min; and (2) extracting indigo pigment from the bacillus subtilis. The method for producing the indigo pigment with the bacillus subtilis has the advantages that bacterial strains of the bacillus subtilis can synthesize indigo pigment in the liquid fermentation medium efficiently, and the production efficiency is high. The requirements for a carbon source and a nitrogen source by the bacterial strains are low, the genetic stability is good and the operation is simple.
Description
Technical field:
The present invention relates to a kind of a kind of method that produces natural indigo pigment that the present invention relates to.
Background technology:
Indigo (indigo) is a kind of bright-colored and stable blue pigment.Structurally, indigo is a kind of aromatics, is mainly used in the dyeing of cloth, is will to obtain after the textile dyeing processing by indigo as jeans.Except as dyestuff, indigo and Indirubin (a kind of pigment of indigo class) also has important application in pharmaceutical industries, can comprise cancer relative disease, senile dementia, tardy allergy etc. to some diseases, and important result for the treatment of is arranged.In foodstuffs industry, an indigo class additive that can be used as food color adds in food.Traditional indigo production is mainly extracted from plant, contains abundant indigo pigment in the blades such as some plants such as woaded blue, indigo plant, acanthaceous indigo, wild blue or green tree.Indigo safe by plant extract pollutes less, but its output and purity are lower, have limited indigo production.Chemical synthesis output is high, and purity is high, and production technique is relatively simple.At present, indigo mainly by chemical synthesis production.Yet chemosynthesis is indigo, because synthesis material, catalyzer, intermediate product etc. in synthetic all have certain toxicity, not only HUMAN HEALTH is had a strong impact on, and has produced a large amount of by products, as aniline, oil of mirbane and metal-salt etc.The discharging of this class material causes serious environmental pollution.The microorganism catalysis synthesis of natural is indigo is mainly the intracellular enzyme of using microbe, in vivo or the specific reaction of external catalysis, realizes bio-transformation.Gentle because of its catalytic reaction condition, the effect substrate is had highly selective, reduces production costs, reduces the characteristics such as energy consumption, environmentally safe and green safety and caused scientific worker's concern.At present, also there are problems in the microorganism catalysis of reporting in the production application of indigo pigment: the culture cycle as bacterial strain is longer, and the colouring matter secretion ability is low, and enzyme is unstable etc. in reaction medium, therefore further develop Microbial resources, screen indigo superior strain significant.
Summary of the invention:
The object of the present invention is to provide a kind of bacillus subtilis strain of producing indigo pigment.
A kind of indigo bacterial strain JWDL that produces of the present invention, it is characterized in that described bacterial strain is Bacillus Subtilis, be subtilis, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on January 9th, 2012, preserving number is: CGMCC 5698.
Bacterial strain of the present invention is 37 ℃ of cultivation 24h on the LB solid medium, and it is black-and-blue that bacterium colony is, and circle is opaque, and surface folding is rough, the irregular expansion in edge.Utilize transmission electron microscope observing, peritrichous without pod membrane, has gemma, and the thalline size is (0.7～0.9) μ m * (3～3) μ m.Physiological and biochemical property sees Table 1.
The physiological and biochemical property of table 1 bacterial strain JWDL
Annotate "+" expression biochemical reaction positive, "-" expression biochemical reaction is negative
This bacterial strain 16sRNA gene is checked order.Resulting sequence is carried out homology relatively with existing 16SrDNA sequence in Blast software and Genbank, utilize software MEGA4.0 to build evolutionary tree, result shows that it is one that bacterial strain of the present invention and Bacillus Subtilis (EU543578) gather, sequence similarity is 99%, the results are shown in Figure 1.Morphological feature, physiological and biochemical property and the 16S rDNA nucleotide sequence homology analytical results of comprehensive JWDL bacterial strain are accredited as subtilis (Bacillus Subtilis) with bacterial strain JWDL.
Bacillus Subtilis (EU543578) can not produce indigo pigment through fermentation test.
A kind of method of producing indigo pigment with bacillus subtilis strain, step is as follows:
(1) the dull and stereotyped cultivation: with the bacterial classification of subtilis CGMCC5698 on the LB solid medium in 30-37 ℃ of constant temperature culture;
(2) preparation of seed liquor: single bacterium colony of step (1) is accessed 30-34 ℃ of lower constant temperature culture of seed liquor substratum 18 hours, and rotating speed is 180r/min;
(3) fermentation culture: in the sterilized liquid fermentation medium of the cultured seed liquor access of step (2), constant temperature culture 48-60h, shaking speed 200r/min;
(4) extract the indigo pigment of subtilis: after fermentation ends, with fermented liquid 12000r/min under room temperature, centrifugal 15min collects navy blue bacterial sediment thing; Supernatant liquor adds isopyknic acetone, and after the concussion extraction, the centrifugal 10min of 12000r/min collects the acetone layer that contains indigo pigment repeatedly; The bacterial sediment thing of collecting suspends with the phosphate buffer solution concussion piping and druming of 60ml20mmol/L pH7.5, then 12000r/min, centrifugal 10min; As above repetitive scrubbing is 3 times, and non-thalline impurity is washed off, and the thalline after centrifugal is with the acetone of the 40ml thalline that fully suspends, then adopts the broken somatic cells of ultrasonic method under the ice bath state; Ultrasonic power 600-800W, working hour 18s, 20s intermittently, work times 25-35 time; The centrifugal 10min of suspension 10000r/min after ultrasonication, obtain navy blue supernatant liquor and dark cell debris precipitation for the first time; Again add acetone 10ml in the cell debris precipitation, repeat above ultrasonication cell step 1-2 time; Fermented supernatant fluid with the blue supernatant liquor that obtains after each ultrasonication and acetone extract merges at last; The employing Rotary Evaporators is concentrated, and then vacuum lyophilization is removed remaining acetone, obtains indigo pigment.
Preferred 32 ℃ of fermentation culture temperature.
During the preferred 46h of fermentation time; Select peptone and ammonium sulfate as nitrogenous source.Preferred 0.5% glucose+0.018% indoles is compounded carbons in compounded carbons, the time indigo output the highest;
The composition of the described LB solid medium of step (1), in percent weight in volume, unit is g/100ml, wherein peptone 1, yeast powder 0.5, NaCl1, the pH value is 7.2.
The composition of the described seed liquor substratum of step (2), in percent weight in volume, the g/100ml of unit, wherein peptone 1, glucose 0.5, yeast powder 0.5, NaCl0.2, (NH
4)
2SO
40.2, NH
4NO
30.1, K
2HPO
40.01, MgSO
47H
2O0.01, pH7.5.
The composition of the described fermention medium of step (3), in percent weight in volume, the g/100ml of unit, wherein peptone 0.5, glucose 0.5, yeast powder 0.5, (NH
4)
25O
40.25, MgSO
47H
2O0.01, FeSO
47H
2O0.04, NH
4NO
30.1 pH is 7.0-9.0; Add the indoles ethanolic soln after thalline fermentation culture 18h, make that in fermented liquid, the indoles final concentration reaches 180mg/L.
Beneficial effect:
Subtilis provided by the present invention has the following advantages when being used for indigo pigment production:
1) bacillus subtilis strain that uses of the present invention, can be in the liquid state fermentation substratum efficient synthesizing indigo, production efficiency is higher.Bacterial strain of the present invention in the shaking flask of 1000ml, fermentation period 48-720h, indigo pigment production can reach 271mg/L.
2) bacterial strain of the present invention is lower to the Carbon and nitrogen sources requirement, and carbon source is mixed carbon source, and nitrogenous source is also mixed nitrogen.
3) this bacterial strain genetic stability is good.This bacterial strain is simple to operate, and more than 10 generations of continuous passage, the synthesis capability of indigo pigment is substantially constant.
Description of drawings:
The phyletic evolution of the indigo pigment formation bacterium of Fig. 1 JWDL is grown tree
The thin layer chromatography analysis of Fig. 2 cyanine sample
The Broad Spectrum Analysis of Infinitesimal of Fig. 3 cyanine sample
Embodiment:
The Screening and Identification that embodiment 1 produces indigo pigment bacterial strain
1, sample collecting
Gather soil sample near Xuzhou City, Jiangsu sewage work mud.
2, the separation screening of bacterial strain gathers soil sample on September 12nd, 2011 from the mud of Xuzhou City of Jiangsu Province Kui river, and soil sample gathers people Gao Zhao and builds.
Preparation solid primary dcreening operation substratum (g/100ml of unit): yeast extract paste 0.2, NaCl0.2, (NH
4)
2SO
40.2, NH
4NO
30.1, K
2HPO
40.01, MgSO
47H
2O0.01, FeSO
47H
2O0.004, agar powder 1.5, pH7.5.The cultivation for preparing is based on 121 ℃, and sterilization 15min is down flat plate.Indoles is made into ethanol the indoles solution that final concentration is 0.5g/100ml, and the substratum of the bacterium of having gone out is cooled to 50 ℃ of left and right, adds indoles solution 3ml according to every 100ml substratum, is down flat plate after mixing again.
Preparation enrichment medium (g/100ml of unit): NaCl0.2, (NH
4)
25O
40.2, NH
4NO
30.1, MgSO
47H
2O0.01, FeSO
47H
2O0.004, NaH
2PO
40.02, KH
2PO
40.02, indoles, pH7.5.Note the indoles add-on with solid primary dcreening operation substratum, medium sterilization and cooling after, under aseptic condition, indoles solution is added.
The soil sample that collects is got 2g, is added in the 250ml triangular flask that the 60ml enrichment medium is housed shaking flask concussion enrichment culture 3d under 32 ℃, the condition of 160r/min.The suspension liquid of then therefrom getting the 1ml enrichment culture is added in the test tube that the 9ml sterilized water is housed, and then is diluted to successively 10
-3, 10
-4, 10
-5, 10
-6, from the test tube of suitable dilution, get 200 μ l diluents and coat in solid primary dcreening operation substratum, after fully drying, cultivate 48-72h in 30 ℃ of incubators.Observe bacterium colony size and bacterium colony color and luster, select the thalline line purifying that produces cyanine, the bacterial strain preservation after purifying.
Obtaining liq sieves substratum (g/100ml of unit) again: peptone 0.5, yeast extract paste 0.2, NaCl0.2, (NH
4)
25O
40.2, NH
4NO
30.1, K
2HPO
40.01, MgSO
47H
2O0.01, FeSO
47H
2O0.004, pH7.5.The cultivation for preparing is based on 121 ℃, and 15min sterilizes.After cooling, every 100ml substratum adds indoles solution 3ml.The single bacterium colony of primary dcreening operation inoculation after purifying is in the LB liquid nutrient medium, and 18h is cultivated in 32 ℃ of concussions, receives above liquid according to 6% inoculum size and sieves again in substratum, 250ml triangular flask liquid amount 50ml, 30 ℃, 180r/min concussion cultivation 4860h.Select the darker corresponding bacterial strain JWDL preservation of triangular flask fermented liquid cyanine color.
3, the evaluation of bacterial strain
The bacterial strain Physiology and biochemistry is identified: according to " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) method is carried out the tests such as utilization of carbon source test, catalase test, hydrogen sulfide production test, indole test, nitrate and nitrite reduction test; Morphological Identification: dibbling is on the beef-protein medium solid plate respectively with bacterial strain to be identified, and 37 ℃ of cultivation 2d describe and record bacterium colony cultural characteristic and morphological features; The extraction of molecular biology identification: bacterial genomes DNA is with reference to the method for " molecular cloning experiment guide " (the 3rd edition).According to the most conservative primers in bacterial 16 S rDNA.Primers F: 5 '-AGA GTT TGATCM TGGCTC AG-3 '; Primer R:5 '-AAG GAG GTG WTCCARCC-3 ' is synthetic by Shanghai living work biotechnology company limited.The PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 40s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min eventually.Pcr amplification product is delivered to Beijing three rich polygala root Bioisystech Co., Ltd and is checked order.After sequencing result carries out two-way splicing, the 16S rDNA sequence that obtains is submitted to carries out the BLAST on-line analysis in the NCBI nucleic acid database.Choose at random afterwards the part genus bacillus 16S rDNA sequence in report from the GenBank nucleic acid database of NCBI, use Clustal X1.81 and Phylodraw082 software building systematic evolution tree and analyze, phylogenetic tree as shown in Figure 1.
The fermentation culture of embodiment 2 bacterial strains and the extraction of indigo pigment crude product
1, the cultivation of bacterial strain
Preparation seed liquor substratum (g/100ml of unit): peptone 1, glucose 0.5, yeast powder 0.5, NaCl0.2, (NH
4)
25O
40.2, NH
4NO
30.1, K
2HPO
40.01, MgSO
47H
2O0.01, pH7.5.The cultivation for preparing is based on 121 ℃, and 15min sterilizes.The single bacterium colony of inoculating strain JWDL in above substratum, 250ml triangular flask liquid amount 40ml, 32 ℃, 180r/min concussion are cultivated 20h.
Preparation fermented liquid substratum (g/L): peptone 0.5, glucose 0.5, yeast powder 0.5, (NH
4)
25O
40.25, MgSO
47H
2O0.01, FeSO
47H
2O0.04, NH
4NO
30.1, pH7.5.The cultivation for preparing is based on 121 ℃, and 15min sterilizes.The seed liquor of cultivating is inoculated in the fermentation of having sterilized according to 4% inoculum size supports in base, 1000ml triangular flask liquid amount 150ml, 32 ℃, 180r/min shake cultivation.Add indoles solution 3.6ml (indoles is made into ethanol the indoles solution that final concentration is 0.5g/100ml) during fermentation 18h.When cultivating 24h, three bottles darken, and thalline begins chromogenesis, and fermentation is to the 48h left and right, and it is maximum that amount of pigment reaches, and continues to cultivate, and pigment production changes little, stops fermentation during 48h.
2, indigo pigment extracts
After fermentation ends, with 12000r/min under the fermented liquid room temperature of 150ml, centrifugal 15min collects navy blue bacterial sediment thing supernatant.Supernatant liquor adds isopyknic acetone, and after the concussion extraction, the centrifugal 10min of 12000r/min collects the acetone layer that contains indigo pigment repeatedly.Thalline after centrifugal suspends with the phosphate buffer solution concussion piping and druming of 60ml20mmol/L pH7.5, then 12000r/min, centrifugal 10min; As above repetitive scrubbing is 3 times, and non-thalline impurity is washed off.Thalline after centrifugal is with the acetone of the 40ml thalline that fully suspends, then adopts the broken somatic cells of ultrasonic method under the ice bath state.Ultrasonic power: 600-800W, working hour 18s, 20s intermittently, work times 25-35 time.The centrifugal 10min of suspension 10000r/min after ultrasonication, obtain navy blue supernatant liquor and dark cell debris precipitation for the first time.Again go up in cell debris precipitation and add acetone 10ml, repeat above ultrasonication cell step 1-2 time.The blue supernatant liquor that will at every turn obtain at last and the fermented supernatant fluid of acetone extract merge.The employing Rotary Evaporators is concentrated, and then vacuum lyophilization is removed remaining acetone, obtains indigo pigment.
The Structural Identification of embodiment 3 pigments
1, thin-layer chromatography (TLC) is analyzed: the indigo crude product sample dissolution for preparing is in DMF (DMF).The same standard model of this sample is distinguished point sample together in the thin utmost point of TLC.Developping agent adopts methanol-acetone/1: 1 (v/v) solvent systems.After fully launching, blower dries up.Result as shown in Figure 2, identical position display blue spot on chromatoplate.
2, UV, visible light wide spectrum scanning analysis: be dissolved in the indigo sample of DMF (DMF) with the indigo sample of standard, carry out the scanning of ultraviolet-visible (250nm-700nm) wide spectrum.Result as shown in Figure 3, obtained sample is basically identical with standard model scanning wide spectrum absorption curve, all at 285nm, 600nm place demonstration absorption peak.Prove thus, the blue pigment that bacterial strain JWDL fermentation obtains is indigo.
Claims (7)
1. a method of producing indigo pigment, is characterized in that, comprises the steps:
(1) strain fermentation is cultivated: in the sterilized liquid fermentation medium of seed liquor access with subtilis (Bacillus Subtilis) CGMCC5698, in 26-34 ℃ of constant temperature culture 46-60h, shaking speed 200r/min;
(2) extract the indigo pigment of subtilis.
2. a kind of method of producing indigo pigment as claimed in claim 1, is characterized in that, described strain fermentation adds the indoles ethanolic soln after cultivating 18h, makes that in fermented liquid, indoles concentration reaches 180mg/L.
3. a kind of method of producing indigo pigment as claimed in claim 1, is characterized in that, described fermention medium forms, in percent weight in volume, and the g/100ml of unit, wherein peptone 0.5, glucose 0.5, yeast powder 0.5, (NH
4)
2SO
40.25, MgSO
47H
2O0.01, FeSO
47H
2O0.04, NH
4NO
30.1 pH is 7.0-9.0.
4. a kind of method of producing indigo pigment as claimed in claim 1, it is characterized in that, the preparation method of the seed liquor of described subtilis CGMCC5698 is as follows: the single bacterium colony access seed liquor that flat board is cultivated gained is cultivated based on 30-34 ℃ of lower constant temperature culture 18 hours, and shaking speed is 180r/min; The composition of described seed liquor substratum, in percent weight in volume, the g/100ml of unit, wherein peptone 1, glucose 0.5, yeast powder 0.5, NaCl0.2, (NH
4)
2SO
40.2, NH
4NO
30.1, K
2HPO
40.01, MgSO
47H
2O0.01, pH7.5.
5. a kind of as claimed in claim 1 or 2 or 3 or 4 method of producing indigo pigment, it is characterized in that, described subtilis CGMCC5698 also cultivates through dull and stereotyped before inoculation, on the LB solid medium in 30-37 ℃ of constant temperature culture, described LB solid medium forms, and in percent weight in volume, unit is g/100ml, wherein peptone 1, yeast powder 0.5, NaCl1, the pH value is 7.2.
6. a kind of method of producing indigo pigment as claimed in claim 1, is characterized in that, described fermentation culture temperature is 32 ℃.
7. a kind of method of producing indigo pigment as claimed in claim 1, it is characterized in that, the process of extracting the indigo pigment of subtilis in described step (2) is: after fermentation ends, with fermented liquid 12000r/min under room temperature, centrifugal 15min collects navy blue bacterial sediment thing; Supernatant liquor adds isopyknic acetone, and after the concussion extraction, the centrifugal 10min of 12000r/min collects the acetone layer that contains indigo pigment repeatedly; The bacterial sediment thing of collecting suspends with the phosphate buffer solution concussion piping and druming of 60ml20mmol/L pH7.5, then 12000r/min, centrifugal 10min; As above repetitive scrubbing is 3 times, and non-thalline impurity is washed off, and the thalline after centrifugal is with the acetone of the 40ml thalline that fully suspends, then adopts the broken somatic cells of ultrasonic method under the ice bath state; Ultrasonic power 600-800W, working hour 18s, 20s intermittently, work times 25-35 time; The centrifugal 10min of suspension 10000r/min after ultrasonication, obtain blue supernatant liquor and dark cell debris precipitation for the first time; Again add acetone 10ml in the cell debris precipitation, repeat above ultrasonication cell step 1-2 time; Fermented supernatant fluid with the blue supernatant liquor that obtains after each ultrasonication and acetone extract merges at last; The employing Rotary Evaporators is concentrated, and then vacuum lyophilization is removed remaining acetone, obtains indigo pigment.
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Cited By (4)
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| CN106497980A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of extracting method of natural blue pigment and colouring method |
| CN111518718A (en) * | 2020-04-29 | 2020-08-11 | 天津科技大学 | Recombinant escherichia coli indinoid production culture medium and method for fermenting and preparing crude pigment |
| US10975243B2 (en) | 2018-12-28 | 2021-04-13 | Industrial Technology Research Institute | Genetically modified microorganism and method for producing indigo dye |
| CN113234769A (en) * | 2021-05-17 | 2021-08-10 | 南京合谷生命生物科技有限公司 | Biological preparation method of blue pigment |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497980A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of extracting method of natural blue pigment and colouring method |
| US10975243B2 (en) | 2018-12-28 | 2021-04-13 | Industrial Technology Research Institute | Genetically modified microorganism and method for producing indigo dye |
| CN111518718A (en) * | 2020-04-29 | 2020-08-11 | 天津科技大学 | Recombinant escherichia coli indinoid production culture medium and method for fermenting and preparing crude pigment |
| CN113234769A (en) * | 2021-05-17 | 2021-08-10 | 南京合谷生命生物科技有限公司 | Biological preparation method of blue pigment |
| CN113234769B (en) * | 2021-05-17 | 2022-03-11 | 南京合谷生命生物科技有限公司 | Biological preparation method of blue pigment |
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| CN103146776B (en) | 2014-08-27 |
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