CN105176873B - Produce the streptomycete bacterial strain of environment protecting blue pigment - Google Patents
Produce the streptomycete bacterial strain of environment protecting blue pigment Download PDFInfo
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Abstract
The invention discloses a kind of streptomycete bacterial strains of production environment protecting blue pigment, belong to microorganism field.The present invention produces the streptomycete of environment protecting blue pigment（Streptomyces sp.）YLA0 bacterial strains, the bacterial strain have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No：11335, the preservation time is：On September 8th, 2015.The streptomycete of the production environment protecting blue pigment（Streptomyces sp.）YLA0 bacterial strains are used for the production of blue pigment.
Description
Technical field
The present invention relates to microorganism field, more particularly to a kind of streptomycete bacterial strain of production environment protecting blue pigment.
Background technology
With cosmetics, the development of food service industry, people want its safety while food coloring demand increases
It asks and is also being continuously improved.
As pigment used in food additives, there are two main classes:A kind of artificial chemistry synthetic dyestuff, one kind are biology
Pigment；How toxic chemical synthesis pigment is, and to human body, there are larger threats, therefore is now allowed to the chemical pigment used
Decline year by year；Biochrome is applied widely gradually to get the favour of people because its is safe.
Biochrome can be divided into three classes according to its source:Plant extract pigment, animal extraction pigment and microorganisms pigments.
Low to environmental requirement when generally microbial fermentation is chromogenic plain, production amount of pigment is stablized, and has compared with plant and animal pigment apparent superior
Property.As the blue pigment of one of three big key colours, colourful color can be deployed into the natural pigment of red, yellow strain
It adjusts.Multiple-microorganism can ferment chromogenesis in nature, but the bacterial strain that can produce cyanine is relatively fewer.
The main source of natural blue pigment is to be extracted from tropical plants such as cape jasmine, but obtained from plant in the prior art
The stability of the pigment arrived is not high, is also frequently subjected to type, geography, region and the influence in season of plant, to supply
It cannot be guaranteed.
Currently, domestic and international market is in blue pigment the state that supply falls short of demand.Therefore, isolating can big volume production indigo plant
The microorganism fungus kind of color pigment probes into its optimal conditions of fermentation and extraction process, and the exploitation of natural edible blue pigment is answered
With being extremely important.The generally first centrifugal solid-liquid separation of the extraction of cyanine, then uses the side such as alcohol precipitation, spray drying
Method, often albumen and pigment are difficult to efficiently separate, and the organic solvents consumption such as ethyl alcohol is big, and cost recovery is higher；Spray drying
Though quickly, high energy consumption, and due to containing protein, tend to adhesion bottle wall, be not readily separated.
Invention content
In order to make up for the deficiencies of the prior art, it efficiently producing environment protecting blue pigment the present invention provides one kind and produces blue
Pigment is easily by the streptomycete bacterial strain of the production environment protecting blue pigment of separating-purifying.
The technical scheme is that：
A kind of streptomycete (Streptomyces sp.) YLA0 bacterial strains of production environment protecting blue pigment, the bacterial strain have been preserved in
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC No：11335, preservation
Time is：On September 8th, 2015.
Streptomycete (Streptomyces sp.) YLA0 bacterial strains of the production environment protecting blue pigment, the part of 16rDNA
Gene order is：
Streptomycete (Streptomyces sp.) YLA0 bacterial strains of the production environment protecting blue pigment, obtaining step are：
1) tree peony Rhizosphere sampling is taken, is placed in the triangular flask equipped with sterile water, concussion is uniform, and upper layer suspension is taken after standing；
2) suspension is diluted 5-20 times, and the suspension after 0.05-0.5mL is diluted is coated on improvement Gause I conjunction
At agar medium tablet, cultivated 5-7 days at 28-30 DEG C；
3) after there is single bacterium colony, the bacterium colony for generating water colo(u)r is picked out；
4) continuous several times are crossed, and are isolated and purified, that is, obtain the starting strain of production blue pigment, that is, streptomycete
(Streptomyces sp.) YLA11 bacterial strains；
5) YLA11 inoculations are in seed culture medium for the streptomycete (Streptomyces sp.) of production blue pigment, through luring
Become, screening, obtains high streptomycete (Streptomyces sp.) the YLA0 bacterial strains of stable hereditary property, blue pigment yield.
Preferably, the improvement Gause I synthetic agar culture medium is carried by soluble starch 15-25g, yeast
Take object 0.6-1.5g, potassium nitrate 0.6-1.5g, sodium chloride 0.3-0.8g, dipotassium hydrogen phosphate 0.3-0.8g, magnesium sulfate 0.3-0.8g,
Ferrous sulfate 0.008-0.012g, agar 15-25g and distilled water 1000mL compositions, the improvement Gause I synthetic agar training
The pH value for supporting base is 7.2-7.4.
Preferably, in step 5), the conidium of starting strain, it is 1 × 10 to make spore concentration8-109cfu/
The spore suspension of ml, mutagenesis under the action of nitrosoguanidine treatment fluid carry out complex mutation in conjunction with ultra violet lamp, and interval picking is not
It with the sample of processing time, carries out plate screening and fermentation is screened, the bacterial strain for screening of fermenting carries out fermentation secondary screening, obtains production blue
The mutant strain of pigment, that is, streptomycete (Streptomyces sp.) YLA0 bacterial strains.
Streptomycete (Streptomyces sp.) YLA0 bacterial strains of the production environment protecting blue pigment are for blue pigment
Production.
Using the blue color of streptomycete (Streptomyces sp.) YLA0 bacterial strains production of the production environment protecting blue pigment
The method of element, it is characterised in that：Bacterial strain is placed in fermentation medium, and is fermented 2-9 days at 35-37 DEG C；The fermented and cultured
Base is made of corn, potassium nitrate, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate and water, the fermented and cultured
The pH of base is 7.2-7.4.
Preferably, the fermentation medium is by corn 20.0g, potassium nitrate 1.0g, sodium acetate 0.5g, phosphoric acid hydrogen two
Potassium 0.5g, magnesium sulfate 0.5g, manganese sulfate 0.01g, ferrous sulfate 0.01g and water 1000mL compositions.
Beneficial effects of the present invention are：
Starch and corn flour Efficient Conversion can be blue pigment by bacterial strain of the present invention, while fermentation byproduct is less,
Separation is simple, and environmental pollution is few, and water consumption is few or does not have to water, can be used as work after simple acid extraction or ethanol precipitation
Industry raw material is the blue pigment production bacterial strain of one plant of great research and development value.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without having to pay creative labor, may be used also for those of ordinary skill in the art
With obtain other attached drawings according to these attached drawings.
Fig. 1 is the separation of three ride of tablet, purifying figure；
Fig. 2 is flat-plate bacterial colony aspect graph；
Fig. 3 is 3 bacterium colonies of 4*10 light microscope OLYMPUS DP72 micro imaging systems (48h)；
Fig. 4 is 10*10 light microscope OLYMPUS DP72 micro imaging systems (48h)；
Fig. 5 is 4*10 light microscope OLYMPUS DP72 micro imaging systems (60h)；
Fig. 6 is 4*10 light microscope OLYMPUS DP72 micro imaging systems (72h)；
Fig. 7 is 4*10 light microscope OLYMPUS DP72 micro imaging system colonial morphology figures (4d=96h)；
Fig. 8 is that 4*10 light microscope OLYMPUS DP72 micro imaging systems (7d) show that blue pigment is secreted；
Fig. 9 is that 10*10 light microscope OLYMPUS DP72 micro imaging systems (7d) show that blue pigment is secreted；
Figure 10 is that 10*10 light microscope OLYMPUS DP72 micro imaging systems (7d) show edge aerial hyphae；
Figure 11 is YLA0 strain culturing 4d inserted sheets, spore filament shapes microexamination figure (60*10)；
Figure 12 is that YLA0 cultivates 7d printingout microexamination figures (100*10)；
Figure 13 is the systematic evolution tree of YLA0 bacterial strains；
Figure 14 is the accumulation of fermentation process cyanine；
Figure 15 is single day yield of fermentation process pigment；
Figure 16 is enhancing effect figure of the metal salt to pigment；
Figure 17 is influence of the temperature to pigment stability；
Figure 18 is HPLC high-efficient liquid phase chromatogram of the pigment at different pH；
The high performance liquid chromatography of blue pigment after Figure 19 purifications.
Streptomycete (Streptomyces sp.) YLA0 bacterial strains, the bacterial strain have been preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number are
CGMCC No：11335, the preservation time is：On September 8th, 2015.
Specific implementation mode
Embodiment 1 produces the acquisition of streptomycete (Streptomyces sp.) YLA0 bacterial strains of environment protecting blue pigment
The tree peony rhizosphere 3-30cm soil samples of Heze City, Shandong Province Mudan District field production, take 5g pedotheques be put into equipped with 45mL without
In the triangular flask of bacterium water (bead), shaken well takes upper layer suspension after standing 5-10min, dilutes 10 times, takes 0.1mL with sterile
Glass applies stick and is uniformly coated on improvement No. 1 synthetic agar culture medium flat plate of Gao Shi, is inverted in 28 DEG C of incubators and cultivates 5-7d,
After there is single bacterium colony, the bacterium colony for generating blue water colo(u)r is picked out, continuous 3 scribing line isolate and purify (Fig. 1 and Fig. 2), can
Obtain the starting strain of production blue pigment, that is, streptococcus (Streptomyces sp.) YLA11 bacterial strains.
Starting strain is inoculated in seed culture medium, is collected with physiological saline and the conidium for the starting strain that suspends, is done
It is 1 × 10 at spore concentration8-109The spore suspension of cfu/ml, mutagenesis under the action of nitrosoguanidine treatment fluid are shone in conjunction with ultraviolet lamp
Row complex mutation is injected, the sample of picking different disposal time is spaced, plate screening is carried out and fermentation is screened, the bacterium for screening of fermenting
Strain carries out fermentation secondary screening, obtains the mutant strain of the high production blue pigment of stable hereditary property, blue pigment yield, that is, strepto-
Bacterium (Streptomyces sp.) YLA0 bacterial strains, it is commonly micro- which has been preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, deposit number are CGMCC No：11335, the preservation time is：On September 8th, 2015.
Improve Gause I synthetic agar culture medium：Soluble starch 20g, yeast extract 1g, potassium nitrate 1g, sodium chloride
0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, ferrous sulfate 0.01g, agar 20g and distilled water 1000mL compositions, pH value are
7.2-7.4。
Seed culture medium：Soluble starch 20.0g, casein 0.5g, potassium nitrate (KNO3) 1.0g, sodium chloride (NaCl)
0.5g, dipotassium hydrogen phosphate (K2HPO4) 0.5g, magnesium sulfate (MgSO4.7H2O) 0.5g, zinc sulfate (ZnSO4) 0.01g, ferrous sulfate
(FeSO4.7H2O) 0.01g, distilled water 1000ml, pH7.2-7.4
Streptomycete (Streptomyces sp.) YLA0 bacterial strains of the production environment protecting blue pigment are for blue pigment
Production.
Embodiment 2 produces the blue color of streptomycete (Streptomyces sp.) YLA0 strain fermentations production of environment protecting blue pigment
Element
Using the blue color of streptomycete (Streptomyces sp.) YLA0 bacterial strains production of the production environment protecting blue pigment
The method of element, is placed in fermentation medium by bacterial strain, and ferment 2-9 days at 35-37 DEG C, obtains zymotic fluid；The fermentation medium
By corn 20.0g, potassium nitrate 1.0g, sodium acetate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, manganese sulfate 0.01g, sulfuric acid
Ferrous 0.01g and water 1000mL compositions, the pH of the fermentation medium is 7.2-7.4；
Manganese sulfate is added in fermentation medium, plays stimulation, more blue pigments can be produced.
Embodiment 3 produces the biological property of streptomycete (Streptomyces sp.) YLA0 bacterial strains of environment protecting blue pigment
Analysis
Gause I culture medium：Soluble starch 20.0g, casein 0.5g, potassium nitrate (KNO3) 1.0g, sodium chloride
(NaCl) 0.5g, dipotassium hydrogen phosphate (K2HPO4) 0.5g, magnesium sulfate (MgSO4.7H2O) 0.5g, zinc sulfate (ZnSO4) 0.01g, sulphur
Sour ferrous iron (FeSO4.7H2O) 0.01g, distilled water 1000ml, pH7.2-7.4.
1. morphological feature
On Gause I culture medium, scribing line can form fluffy colony, early stage visible formation under the microscope when detaching
Sparse shallow white fluffy bacterium colony.48h bacterium colonies as shown in Figure 3 and Figure 4.Gradually become dense afterwards, white, but go out without blue pigment
It is existing.Fig. 5 (culture 60h), Fig. 6 (culture 72h), Fig. 7 (culture 96h), culture can see and begins with blue formation for 96 hours.The
It can see within 6 days that periphery of bacterial colonies has apparent blue pigment to occur, a large amount of shapes of blue pigment can be clearly visible on tablet by the 7th day
At, and thering are pigment secretion of droplets to form (Fig. 8, Fig. 9), aerial hyphae is more flourishing (such as Figure 10).
Visible aerial hyphae and spiral helicine fibrillae of spores in the micro- sem observation of inserted sheet and printingout, it can be seen that the bacterium
Strain spore is more, spore chain 3-4 circle loose-screws (Figure 11, Figure 12).
2. carbon source, nitrogen source generate the bacterial strain influence situation of blue pigment
On the basis of Gause I culture medium, use respectively the glucose of equivalent, mannose, xylose, lactose, maltose,
Sucrose, mannitol, xylan, sodium carboxymethylcellulose, hemicellulose, lignin, pectin, malt flour substituted starch carry out carbon source
Utilization power is tested, as a result such as table 1：
1 utilization of carbon source situation of table
Compared with good utilisation：+++, it utilizes：++, utilization is poor：+, it does not utilize：—
As can be seen from Table 1, the carbon source material that cannot function as blue pigment secretion has：Xylose, sucrose and carboxymethyl cellulose
Plain sodium, pectin；The carbon source material that a small amount of pigment can be secreted is：Glucose, mannose, lactose, maltose, mannitol；Be conducive to
The carbon source material of colouring matter secretion is：Lignin is best, starch takes second place, hemicellulose and xylan third, malt flour takes second place again.
On the basis of Gause I culture medium, ammonium nitrate, ammonium chloride, potassium nitrate, ammonium sulfate, the phosphoric acid of equivalent are used respectively
Ammonium, ammonium dihydrogen phosphate, ammonium oxalate, ammonium tartrate, urea, peptone, beef extract, casein, tryptone, yeast extract,
Soy meal replaces potassium nitrate to carry out nitrogen source utilization power experiment, and the results are shown in Table 2：
2 nitrogen source utilization power of table
Compared with good utilisation：+++, it utilizes：++, utilization is poor：+, it does not utilize：-
As can be seen from Table 2, which cannot utilize ammonium phosphate, ammonium dihydrogen phosphate, beef extract, yeast extract, casein
Nitrogen source chromogenesis is done, but in addition to ammonium chloride and ammonium dihydrogen phosphate utilization are weaker, others, which are substantially all, can utilize growth；Production
The less nitrogen source of element that adds lustre to has ammonium sulfate, ammonium chloride, ammonium oxalate, and so ammonium salt is unfavorable for the generation of pigment.Beef
Cream, peptone, tryptone and yeast extract, which do nitrogen source, cannot generate cyanine；It is main to generate the preferable nitrogen source of cyanine
Have corn flour is best, potassium nitrate takes second place, urea third；For biomass, casein is apparently higher than other nitrogen sources, is carrying out
Addition is contemplated that when seed culture.
3. the influence that initial pH generates pigment
On the basis of Gause I culture medium, the first of culture medium is adjusted with 10% hydrochloric acid and sodium hydroxide solution respectively
Beginning pH value is to be inoculated with 10% inoculum concentration after 3,4,5,6,7,8,9,10,11 conventional sterilants cool down, and observes initial pH to pigment
The influence of generation, the results are shown in Table 3；
The influence that 3 initial pH of table generates pigment
As can be seen from Table 3, it is most suitable for the secretion of pigment and mycelial growth when initial pH is 7 or so.
As shown in figure 18, the HPLC high-efficient liquid phase chromatograms under different pH, the as seen from Figure 18 pigment under difference pH
Appearance time does not change substantially, illustrates that its structure of matter is essentially identical.
4. the influence that difference C/N generates pigment
Based on Gause I culture medium, the constant (KNO of amount of fixed nitrogen source3For 1g/L), 100mL cultures are weighed respectively
Different amounts of starch in base (3.2,2.88,2.56,2.24,1.92,1.6,1.28,0.96,0.64 and original formulation in 2.0g
It is that C/N ratios 12.5) is made to meet following table requirement to make C/N.115 DEG C, high pressure sterilization 15Min, 26-28 DEG C of culture after inoculation.As a result such as
Shown in table 4：
The influence that 4 difference C/N of table generates pigment
As shown in Table 4,14 C/N：When 1, the amount of chromogenesis is maximum, and biomass is also larger.
5. the influence that inorganic ion-pair pigment generates
Based on Gause I culture medium, the amount of fixed other materials is constant, weighs respectively different in 100mL culture mediums
Inorganic salts 0.05g, instead of the NaCl in culture medium.115 DEG C, high pressure sterilization 15Min, 30 DEG C of cultures after inoculation.As a result such as table 5
It is shown：
The influence that 5 inorganic ion-pair pigment of table generates
As shown in Table 5, SO4 2-、Cl-、NO3 -, wait influence of the inorganic anions to colouring matter secretion smaller, and acetate (AC-)
The secretion of pigment can be obviously promoted；Ca, Na, K, plasma influence certain promotion to the secretion of pigment, Fe, Cu, Ag, Hg,
Co is unfavorable for the secretion of pigment, and Mn ions can be obviously promoted the secretion of pigment, and can promote the fast-growth of biomass.It is contemplated that
A small amount of MnSO is added in seed culture medium and fermentation medium4, it is used to be inoculated with to generate more seeds, and in fermentation
Generate more pigments.
6. influence of the incubation time to colouring matter secretion
Since the 24th hour of fermented and cultured, took 10ml bacterium solutions in two centrifuge tubes in fermentation flask every 24 hours
In.10min first is centrifuged through 5000rpm, uses its spectral curve of measurement of ultraviolet-visible spectrophotometer after abandoning precipitation, then pass through
12000rpm centrifuges 10min, uses its spectral curve of measurement of ultraviolet-visible spectrophotometer after equally abandoning precipitation, and take in collection of illustrative plates
Wavelength is the numerical value at 596nm, is OD value of the pigment at 596nm.According to the variation of pigment collection of illustrative plates observation pigment yield, directly
No longer change to pigment concentration.Obtain data make Figure 14 pigments accumulation and Figure 15 pigments in yield in different time periods.
The accumulation of pigment is as shown in figure 14, and single day yield of pigment is as shown in figure 15.
By the analysis to the above pigment OD value line charts, it is found that streptomycete YLA0 in incubation, starts in 2d
Chromogenesis, but yield is smaller, 4d starts largely to generate, and single day yield of pigment reaches most in 6d, pigment when 8d
Cumulant it is most, hereafter pigment total amount is reduced.Thalline pigment yield significantly reduces when reaching 9d between when fermenting.
It can stop fermenting in 7d when therefore carrying out fermenting and producing to pigment, carry out the extraction of pigment, optimum point of production can be obtained at this time.
Streptomycete (Streptomyces sp.) YLA0 bacterial strains of the production environment protecting blue pigment, the part of 16rDNA
Gene order is：
The above gene sequencing result is obtained by Hua Da genetic test.
Forward primer 27F：GAGAGTTTGATCCTGGCTCAG；
Reverse primer 1541R：AAGGAGGTGATCCAGCCGCA.
By obtained gene sequencing result with online Blast softwares (http：//www.ncbi.nlm.nih.gov/
Blast homology analysis) is carried out, the significantly similar gene order of multiple streptomyces is as a result found in GenBank, it is homologous
Property is up to 98%.Homology search is carried out in NCBI protein sequence databases by Blast tools, and utilizes Clustal
W programs carry out Multiple sequence alignments.Using MEGA4.0 softwares, using contiguous concatenation method (Neighbor Joining
Method), which is streptomycete by the systematic evolution tree (Figure 13) for obtaining the bacterial strain, and the bacterial strain and common bacterial strain
There are certain features different, it may be possible to a novel species of blue pigment can be generated.
The bacterial strain is sent to China Committee for Culture Collection of Microorganisms's common micro-organisms center in September in 2015 on the 8th
Preservation, deposit number are CGMCC No：11335.
The property and stability study of pigment
1. the dissolubility of pigment：
To stopping fermentation after production cyanine streptomycete shaking table culture 7d, collects zymotic fluid and carry out 5000rpm centrifugations first
10min takes supernatant averagely to assign in two beakers, be constantly added dropwise into supernatant respectively 1mol/L sodium hydroxide solutions and
1mol/L hydrochloric acid solutions find that pigment dissolves in aqueous slkali and dissolves in acid solution again, and to get over low solubility smaller by pH, as pH ＜
1h is stood after being shaken up when 4 and will produce brick-red precipitation, and when being adjusted to alkalinity, solution restores blue again.By pigment of the acid after heavy
After drying, water imbibition is very poor, but is soluble in aqueous slkali, and property is constant, it is possible to the pigment long period preservation that acid is heavy.
5 portions of equivalent supernatants are equally taken, it is organic molten with the polarity of concentration or nonpolarity that equivalent is added into supernatant respectively
Agent, as a result, it has been found that the cyanine generates fine granularity precipitation in the stronger organic solvent methanol of polarity and ethyl alcohol, it can in acetone
Filamentous precipitation is generated, insoluble in nonpolar organic solvents such as chloroform, ether and ethyl acetate.
Experimental result：According to the principle that the above experimental result, speculates that the molecular structure of the pigment is pole in conjunction with similar compatibility
Property, there is certain help to probing into for pigment molecular structure.
2.pH changes the influence to pigment colour generation
In above-mentioned pigmentolysis it was found that, acid is constantly added dropwise into pigment or when alkali, pigmentary colours with pH change
Change and change, specific change procedure is as follows：
Experimental result：The transition interval of pigment is allowed to preferably distinguish weak acid or weak as indicator between pH6-7
The acid-base property of aqueous slkali can be used as a kind of preferable acid-base indicator.
HPLC high-efficient liquid phase chromatograms under different pH, as seen from the figure the pigment appearance time under difference pH do not have substantially
Great changes have taken place, illustrates that its structure of matter is essentially identical.
3. influence of the different ions to pigment stability
Prepare the following salting liquid (FeSO of 0.05mol/L4、ZnSO4、MgSO4、CaSO4、CuSO4、MnSO4、BaSO4、Pb
(NO3)2、AgNO3, according to the preparation method that redox is concentrated, prepare the solution for 25% zymotic fluid that salinity is 1mmol/L
Stand a period of time.It chooses and the solution of complex reaction does not occur surveys OD values at its 596nm, it is found that the absorbance of pigment solution has
Enhanced.The salt ion of complex reaction does not occur with pigment, it is as shown in figure 16 to the enhancing effect of pigment solution absorbance：
Experimental result：The Fe in conjunction with known to the above experiment3+、Gu2+、Pb+Can with cyanine occur complex reaction and generate it is heavy
It forms sediment.Fe3+What is generated is precipitated as dark brown, and supernatant is yellow.Gu2+、Pb+What is generated is precipitated as blue, supernatant be it is light blue or
It is colourless.Remaining ion can enhance the stability of pigment.
4. influence of the temperature to pigment stability
It takes the zymotic fluid of appropriate four times of dilution and pH is adjusted to 9, take 10mL to be respectively charged into 11 test tubes every time, jump a queue.
Retain one and be in room temperature, remaining 10 are put into 100 DEG C of water-bath and keep the temperature, and take out one every 10min and are put into cold water
Influence of the temperature to absorbance at pigment 596nm is probed into middle cooling.Influence of 100 DEG C of the temperature to absorbance at pigment 596nm
As shown in figure 17：
Understand that 100 DEG C of temperature can shine the absorbance at pigment 596nm influences at certain, make the reduction of its OD value.Upper
The minimum temperature further probed on the basis of testing and influence absorbance is stated, 11 test tubes is taken to be separately added into a concentration of of 10mL
The zymotic fluid of 25%pH=9.Same to retain 1 in room temperature, remaining 9 are respectively placed in 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C
Water-bath in keep the temperature, after 2h take out survey its 596nm at absorbance.
Experimental result：According to the above experiment it is found that the absorbance when temperature is 100 DEG C at pigment 596nm at any time
Increase and reduce, therefore high temperature is larger to the stability influence of pigment.Due to time restriction, fail to carry out 100 DEG C or more of temperature
Degree influences to test on pigment stability, is speculated according to 50-100 DEG C of influence result trend, and 100 DEG C or more of high temperature is steady to pigment
Qualitative effect bigger.Prolonged high temperature process should be avoided in use.
5. influence of the illumination to pigment stability
The pH of appropriate a concentration of 25% zymotic fluid is adjusted to 9,10mL is taken to be placed in 10 test tubes respectively, then 10 are tried
Pipe is respectively placed in varying environment：1, light-exposed outside Room 2, it is protected from light outside Room 3,4, it is light-exposed in Room 5,6, it is protected from light in Room 7,8,9,10 is ultraviolet
Illumination.Every surveying the absorbance at its 596nm for 24 hours, continuously survey five days.
Experimental result：By analyzing obvious effect of the illumination known to line chart to pigment, outdoor strong light is to pigment
Influence becomes apparent, so outdoor become apparent from than indoor pigment change.Interior is protected from light, ultraviolet light and refrigerator preserve three conditions
Under influence to pigment be not particularly evident, one can be selected as storage conditions.
Embodiment 4
The extraction process of environment protecting blue pigment, including step：
1) 2 gained zymotic fluid of embodiment is centrifuged；Specifically, it is centrifuged twice；For the first time centrifuge with
Rotating speed 4000-5000rpm is centrifuged described zymotic fluid 5-10 minutes；Second centrifuges with ten thousand rpm of rotating speed 1.0-1.2 centrifugations the
It is primary to centrifuge gained supernatant 10-15 minutes；It is supernatant one that second, which centrifuges gained supernatant, twice centrifugation point
Summarize for precipitation one from gained precipitation；
2) use the ultra-filtration centrifuge tube of 1-10Kda with rotating speed 4000-5000rpm ultra-filtration and separations supernatant one 5-15 minutes,
It obtains ultrafiltration permeate one and liquid one is concentrated by ultrafiltration, enriching hydrochloric acid in liquid one is concentrated by ultrafiltration, carry out sour heavy processing, the addition of concentrated hydrochloric acid
Amount meets 1mL concentrated hydrochloric acids/300mL zymotic fluids；Heavy be disposed of acid centrifuges to obtain supernatant two and precipitation two；Into precipitation two
Add the sodium hydrate aqueous solution of 20%-40%, adjust pH to 7-8, two dissolving of precipitation obtains clear liquid A；
3) be added into clear liquid A and account for the ethyl alcohol of clear liquid A total volumes 40-60%, carry out alcohol precipitation processing, alcohol precipitation be disposed from
The heart detaches to obtain supernatant four and precipitation four, drying precipitated four, obtains solid blue pigment.
Gained dry product adds 1mol/L lye after acid is heavy, the pigment after super filter tube is crossed, in Agilent Technogies HPLC
Chromatographic column, Eclipse XDB-C18,4.6*250mm, 5um, mobile phase 10-40% methanol aqueous solutions can, flow velocity 0.5-
1.0mL/min, Detection wavelength 587-596nm.As a result as shown in figure 19, the pigment is overall purer as seen from the figure,
His content of material is seldom.
Embodiment 5
The extraction process of environment protecting blue pigment, including step：
1) 2 gained zymotic fluid of embodiment is centrifuged；Specifically, it is centrifuged twice；For the first time centrifuge with
Rotating speed 4000-5000rpm is centrifuged described zymotic fluid 5-10 minutes；Second centrifuges with ten thousand rpm of rotating speed 1.0-1.2 centrifugations the
It is primary to centrifuge gained supernatant 10-15 minutes；It is supernatant one that second, which centrifuges gained supernatant, twice centrifugation point
Summarize for precipitation one from gained precipitation；
2) the direct enriching hydrochloric acid into supernatant one, carries out the heavy processing of acid, the addition of concentrated hydrochloric acid meet 1mL concentrated hydrochloric acids/
300mL zymotic fluids；Heavy be disposed of acid centrifuges to obtain supernatant three and precipitation three；Add the hydrogen of 20%-40% into precipitation three
Aqueous solution of sodium oxide, adjusts pH to 7-8, and three dissolving of precipitation obtains clear liquid B；Use the ultra-filtration centrifuge tube of 1-10Kda with rotating speed
4000-5000rpm ultra-filtration and separation clear liquids B5-15 minutes obtain ultrafiltration permeate two and liquid two are concentrated by ultrafiltration, it is standby that liquid two is concentrated by ultrafiltration
With；
3) it is added into ultrafiltration concentration liquid two and accounts for the ethyl alcohol that two total volume 40-60% of liquid is concentrated by ultrafiltration, carry out alcohol precipitation processing,
Alcohol precipitation, which is disposed, centrifuges to obtain supernatant four and precipitation four, drying precipitated four, obtains solid blue pigment.
Certainly, also directly clear liquid A and ultrafiltration concentration liquid two can be spray-dried, obtains solid blue pigment and is protected
It deposits；If higher to blue pigment purity requirement, before use, carrying out alcohol precipitation processing.
The high performance liquid chromatography of blue pigment after purification is as shown in figure 19, has Figure 19 it is found that present invention gained pigment is pure
Spend very high, impurity content is few.
Claims (5)
1. a kind of streptomycete of production environment protecting blue pigment（Streptomyces sp.）YLA0 bacterial strains, during which has been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No：11335, when preservation
Between be：On September 8th, 2015.
2. the streptomycete of production environment protecting blue pigment as described in claim 1（Streptomyces sp.）YLA0 bacterial strains, it is special
Sign is that the partial gene sequence of 16rDNA is：
cgtcccatcg ccagtcccac cttcgacagc tccctcccac aaggggttgg gccaccggct 60
tcgggtgtta ccgactttcg tgacgtgacg ggcggtgtgt acaaggcccg ggaacgtatt 120
caccgcagca atgctgatct gcgattacta gcgactccga cttcatgggg tcgagttgca 180
gaccccaatc cgaactgaga ccggcttttt gagattcgct ccaccttgcg gtatcgcagc 240
tcattgtacc ggccattgta gcacgtgtgc agcccaagac ataaggggca tgatgacttg 300
acgtcgtccc caccttcctc cgagttgacc ccggcggtct cccgtgagtc cccaacaccc 360
cgaagggctt gctggcaaca cgggacaagg gttgcgctcg ttgcgggact taacccaaca 420
tctcacgaca cgagctgacg acagccatgc accacctgta caccgaccac aaggggggca 480
ccatctctga tgctttccgg tgtatgtcaa gccttggtaa ggttcttcgc gttgcgtcga 540
attaagccac atgctccgcc gcttgtgcgg gcccccgtca attcctttga gttttagcct 600
tgcggccgta ctccccaggc ggggcactta atgcgttagc tgcggcacgg acaacgtgga 660
atgttgccca cacctagtgc ccaccgttta cggcgtggac taccagggta tctaatcctg 720
ttcgctcccc acgctttcgc tcctcagcgt cagtatcggc ccagagatcc gccttcgcca 780
ccggtgttcc tcctgatatc tgcgcatttc accgctacac caggaattcc gatctcccct 840
accgaactct agcctgcccg tatcgactgc agacccgggg ttaagccccg ggctttcaca 900
accgacgtga caagccgcct acgagctctt tacgcccaat aattccggac aacgcttgcg 960
ccctacgtat taccgcggct gctggcacgt agttagccgg cgcttcttct gcaggtaccg 1020
tcactttcgc ttcttccctg ctgaaagagg tttacaaccc gaaggccgtc atccctcacg 1080
cggcgtcgct gcatcaggct ttcgcccatt gtgcaatatt ccccactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggccggtcgc cctctcaggc cggctacccg 1200
tcgtcgcctt ggtgagccat tacctcacca acaagctgat aggccgcggg ctcatccttc 1260
accgccggag ctttcgaacc tcgcagatgc ctgcgagggt cagtatccgg tattagaccc 1320
cgtttccagg gcttgtccca gagtgaaggg cagattgccc acgtgttact cacccgttcg 1380
ccactaatcc ccaccgaagt ggttcatcgt tcgactgcat gtgta 1425。
3. the streptomycete of production environment protecting blue pigment as described in claim 1（Streptomyces sp.）YLA0 bacterial strains are for indigo plant
The production of color pigment.
4. using the streptomycete of production environment protecting blue pigment as described in claim 1（Streptomyces sp.）YLA0 bacterial strains are given birth to
The method for producing blue pigment, it is characterised in that：Bacterial strain is placed in fermentation medium, and is fermented 2-9 days at 35-37 DEG C；It is described
Fermentation medium is made of corn, potassium nitrate, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate and water, described
The pH of fermentation medium is 7.2-7.4.
5. as claimed in claim 4 using the streptomycete of production environment protecting blue pigment（Streptomyces sp.）YLA0 bacterial strains are given birth to
The method for producing blue pigment, it is characterised in that：The fermentation medium by corn 20.0g, potassium nitrate 1.0g, sodium acetate 0.5g,
Dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, manganese sulfate 0.01g, ferrous sulfate 0.01g and water 1000mL compositions.
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| CN101525586A (en) * | 2009-01-22 | 2009-09-09 | 广东省微生物研究所 | Streptomyces caeruleatus GIMN4.002 and application thereof |
| CN103602615A (en) * | 2013-11-13 | 2014-02-26 | 嘉兴学院 | Streptomyces Iividans and application thereof |
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| JP2003304890A (en) * | 2002-04-15 | 2003-10-28 | Ajinomoto Co Inc | Method for producing erasable dye with microorganism, erasable dye produced thereby and erasable ink containing erasable dye |
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| CN103602615A (en) * | 2013-11-13 | 2014-02-26 | 嘉兴学院 | Streptomyces Iividans and application thereof |
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