CN103602615A - Streptomyces Iividans and application thereof - Google Patents
Streptomyces Iividans and application thereof Download PDFInfo
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- CN103602615A CN103602615A CN201310562683.5A CN201310562683A CN103602615A CN 103602615 A CN103602615 A CN 103602615A CN 201310562683 A CN201310562683 A CN 201310562683A CN 103602615 A CN103602615 A CN 103602615A
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Abstract
The invention discloses Streptomyces Iividans and application thereof. The preservation number of Streptomyces Iividans is CGMCC (China General Microbiological Culture Collection Center) No.8083. The application refers to the application of Streptomyces Iividans in producing blue pigments, and is specifically implemented through the steps of inoculating Streptomyces Iividans to a seed culture solution to carry out multiplication culture, thereby obtaining a seed solution; transferring the seed solution to a fermented culture solution to carry out fermented culture, thereby obtaining fermentation broth; separating and drying the fermentation broth so as to obtain a coarse blue pigment preparation. Compared with the prior art, the invention discloses Streptomyces Iividans capable of producing blue pigments, which enriches the germplasm resources of blue pigment producing bacteria; a blue pigment produced by Streptomyces Iividans is stable in performance and has certain bacteriostasis, and maximum OD585 is 15.4.
Description
Technical field
The present invention relates to technical field of bioengineering, be specifically related to the shallow Streptomyces glaucoviolaceus of a strain and application thereof.
Background technology
Pigment refers to and can make, by the painted material of mordant dyeing thing, in productive life and scientific research, to have a wide range of applications.Natural pigment typically refers to the pigment that utilizes the material of nature existence or the secondary metabolites of cultural method production to carry out certain processing and make.Due to its have safe and reliable, have no side effect, tone nature and the advantage such as multi-functional, be accompanied by the development of foodstuffs industry, medicine industry, daily chemical industry and aquaculture etc. and be widely used.
The starting material wide material sources (coming from animals and plants, microorganism, mineral substance etc.) of processing natural pigment, of a great variety, on the booksly by 2004 just have an appointment 600 kinds.But that the natural pigment that existing processing makes mainly be take is red, yellow pigment is as main, the very rare ， of cyanine China GB2760-2007 < < foodstuff additive are used in 56 kinds of pigments listing in hygienic standard > > cyanine only to have two kinds of gardenin and phycocyanins.Because blueness is one of three primary colors, cyanine can be used for the allotment of multiple color tones.But because cyanine is rare, make to develop natural blue pigment and in pigment association area, there is great realistic meaning.
The natural source of natural blue pigment is very limited.In prior art, inorganic natural blue pigment is mainly derived from the blue navajoite of nature, and organic natural blue pigment is mainly derived from plant and microbial material, as the leaf of the anils such as indigo plant, tea indigo plant, acanthaceous indigo, Wu Lan, woaded blue can be for the preparation of beautiful blue dyestuff.Although most natural blue pigment product is still and take that animals and plants material is waste, this class material obtain the impact such as the factor that is subject to season, weather, the place of production, make the output of cyanine very limited.And utilize Microbial resources to produce natural pigment, not limited by these, and, utilize that microorganism fermentative production is not only produced, extracting method is all comparatively simple, and cost is relatively low, is comparatively desirable cyanine production method.
But the bacterial strain that can produce cyanine in microorganism is also very rare, mainly concentrates on actinomycetes.That has reported has: marine streptomyces (Streptommycete sp.) M259(Deng Xiang unit etc., the research of marine streptomyces M259 high yield cyanine physico-chemical property, Food science, 2006,7:35-39.), streptomyces coelicolor (Streptomyces coelicolor) 100 bacterial strains (and spring etc., cyanine Study on Extraction Method in streptomyces coelicolor born of the same parents, biotechnology, 2000,3:19-21.); Streptomycete ZLT bacterial strain (Lee one reed etc., the research of cyanine Biological Characteristics of Strain and pigment essential property is produced in a strain, biology magazine, 2007,1:41-43.); The Chinese patent literature that is also CN 101864380B just like publication number discloses a kind of crude preparation by using and the method for blue pigment producing bacteria and preparation thereof, this blue pigment producing bacteria is a kind of Flavobacterium (Flavobacterium sp.) B29, and deposit number is CGMCC No.3699.
The kind of the microorganism strains of the product cyanine of existing report and quantity also far can not meet the needs of every profession and trade to natural blue pigment research and development, therefore, filter out the cyanine that can produce in a large number the novel microorganism bacterial classification of cyanine and there is different qualities and there is important scientific research and using value.
Summary of the invention
The invention provides the shallow Streptomyces glaucoviolaceus (Streptomyces lividans) that a strain can produce cyanine, the cyanine that utilizes this bacterial strain to produce is comparatively stable, and intestinal bacteria and streptococcus aureus are had to certain bacteriostatic action.
The shallow Streptomyces glaucoviolaceus of one strain (Streptomyces lividans), Classification And Nomenclature is shallow Streptomyces glaucoviolaceus (Streptomyces lividans), strain number is jx-02, on August 28th, 2013, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center that is positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.8083.
The present invention also provides the application of described shallow Streptomyces glaucoviolaceus in preparing cyanine.Specifically comprise:
(1) described shallow Streptomyces glaucoviolaceus is inoculated in seed culture fluid and carries out multiplication culture, obtain seed liquor;
The shallow Streptomyces glaucoviolaceus of freezing preservation need first carry out actication of culture, and activation condition is: temperature is 37 ℃, and the time is 48h.Single colony inoculation on picking activation flat board, in seed culture fluid, carries out multiplication culture.As preferably, the condition of described multiplication culture is: temperature is 25～30 ℃, and the time is 10～14h; As further preferred, the condition of described multiplication culture is: temperature is 28 ℃, and the time is 12h.More preferably, described multiplication culture is that shaking table is cultivated, and rotating speed is 150～200r/min.
(2) described seed liquor is transferred in fermentation culture and carries out fermentation culture, obtain fermented liquid;
The inoculum size of described seed liquor is preferably 5～10%, and more preferably 8%.The condition optimization of described fermentation culture is: pH is 7, and temperature is 25～30 ℃, and the time is 6～8 days; More preferably pH is 7, and temperature is 28 ℃, and the time is 7 days.
For described shallow Streptomyces glaucoviolaceus, the present invention is optimized fermention medium on the basis of Gause I substratum, and in 1L, described fermentation culture comprises: 18～22g carbon source, 0.8～1.2g nitrogenous source, 0.4～0.6g inorganic salt; Preferably, described fermentation culture comprises: 20g carbon source, 1g nitrogenous source, 0.5g inorganic salt; More preferably, described carbon source is glycerine, and described nitrogenous source is saltpetre.Fermentation culture is also cultivated for shaking table, and rotating speed is 150～200r/min.Under the fermentation condition of setting in the present invention, it is maximum that the cyanine output of described shallow Streptomyces glaucoviolaceus reaches, OD
585can reach 15.4.
(3) separated from described fermented liquid, dryly obtain described cyanine crude preparation by using; Be about to fermented liquid pH and be adjusted to the centrifugal 10min of 12,8000r/min, get supernatant its pH is adjusted to l～2,10000r/min is centrifugal, and 10min abandons supernatant, and throw out vacuum-drying to constant weight obtains cyanine crude preparation by using.
The present invention also provides the cyanine that utilizes described shallow Streptomyces glaucoviolaceus fermented extracted to obtain rough.The maximum absorption wavelength of this cyanine is 585nm left and right, has very large conjugated system in molecular structure, may have the conjugated structure of more conjugated double bond, phenyl ring or similar phenyl ring, or containing more heteroatomic heterocycles, concrete structure need further analysis.
Research shows, outdoor directing light, high temperature (being greater than 60 ℃), oxygenant H
2o
2, metal cations Fe
3+and Cu
2+this blueness is have to obvious destruction, and outdoor scattered light, UV-light, reductive agent Na
2sO
3, part metals ion is (as Na
+, K
+and Ca
2+) and foodstuff additive salt, sucrose, citric acid this cyanine is had no significant effect.The applied research that this result of study can be the fields such as weaving, food provides basic data.
This cyanine also has certain restraining effect to intestinal bacteria and streptococcus aureus.
Compared with prior art, beneficial effect of the present invention is:
The invention discloses a strain and can produce the shallow Streptomyces glaucoviolaceus of cyanine, enriched the germ plasm resource of blue pigment producing bacteria; The not only stable performance of cyanine that this shallow Streptomyces glaucoviolaceus produces, has certain bacteriostatic action, and OD
585reach as high as 15.4.
Accompanying drawing explanation
Fig. 1 is the flat board growth figure of the shallow Streptomyces glaucoviolaceus of the present invention;
Fig. 2 is the observation by light microscope figure of the shallow Streptomyces glaucoviolaceus of the present invention;
Fig. 3 is the electron microscope observation figure of the shallow Streptomyces glaucoviolaceus of the present invention;
Fig. 4 is the genome agarose gel electrophoresis figure of the shallow Streptomyces glaucoviolaceus of the present invention;
The 16S rRNA agarose gel electrophoresis figure that Fig. 5 is;
Fig. 6 is the phyletic evolution site plan of the shallow Streptomyces glaucoviolaceus of the present invention;
Fig. 7 is the carbon source optimizing figure of fermention medium;
Fig. 8 is the nitrogenous source optimization figure of fermention medium;
Fig. 9 is the carbon source concentration optimization figure of fermention medium;
Figure 10 is the carbon-nitrogen ratio optimization figure of fermention medium;
Figure 11 is the inorganic salt concentration optimization figure of fermention medium;
Figure 12 is the ferrous ion concentration optimization figure of fermention medium;
Figure 13 is the affect figures of different pH on cyanine output;
Figure 14 is the affect figure of differing temps on cyanine output;
Figure 15 is the affect figures of different seed liquor inoculum sizes on cyanine output;
Figure 16 is the ultraviolet-visible spectrogram of cyanine of the present invention;
Figure 17 is the affect figure of pH on cyanine stability of the present invention;
Figure 18 is outdoor directing light and the affect figure of room scattering light on cyanine stability of the present invention;
Figure 19 is the affect figure of UV-irradiation on cyanine stability of the present invention;
Figure 20 is the affect figure of temperature on cyanine stability of the present invention;
Figure 21 is oxygenant H
2o
2with reductive agent Na
2sO
3the figure that affects on cyanine stability of the present invention;
Figure 22 a is that the cyanine solution of different concns is to 10
-1the inhibition zone test pattern of dilution intestinal bacteria spread plate;
Figure 22 b is that the cyanine solution of different concns is to 10
-2the inhibition zone test pattern of dilution intestinal bacteria spread plate;
Figure 23 a is that the cyanine solution of different concns is to 10
-1the inhibition zone test pattern of dilution streptococcus aureus spread plate;
Figure 23 b is that the cyanine solution of different concns is to 10
-2the inhibition zone test pattern of dilution streptococcus aureus spread plate;
The cyanine solution of Figure 24 a different concns is to 10
-1the inhibition zone test pattern of dilution yeast saccharomyces cerevisiae spread plate;
The cyanine solution of Figure 24 b different concns is to 10
-2the inhibition zone test pattern of dilution yeast saccharomyces cerevisiae spread plate.
Embodiment
(1) Morphological Identification
From the pedotheque of park, Ningbo, screening and separating obtains the bacterial strain that cyanine is produced in a strain, through further screening and purifying, inoculation is cultivated to plate.Flat board is cultivated after 3d, observes and produces cyanine colony characteristics, finds that the cultural characteristic of bacterial strain on Gause I solid medium is: thalli growth is slower than bacterium, after cultivation 36-48h, starts to occur bacterium colony, can observe cyanine and produce after 60-72h.Bacterium colony surface is canescence, and aerial hyphae is more luxuriant, is fine hair shape, and it is blue that substrate mycelium is, and can produce soluble blue pigment (see figure 1).
Through further opticmicroscope and electron microscope microscopy, observe, we tentatively judge that this bacterial strain is actinomycetes, Gram-negative bacteria (seeing Fig. 2 and Fig. 3).As seen from Figure 3, the fibrillae of spores of this bacterial strain is longer, has 3-5 circle spiral, and spore is ellipse or Long Circle.
(2) Molecular Identification
According to document (Orsini M, Romano-Spica V.A microwave-based method for nucleic acid isolation from environmental samples[J] .Let Appl Microbiol, 2001, experimental procedure 33:17-20.) is extracted the genome that produces cyanine bacterial strain, and the genome extracting is carried out to agarose gel electrophoresis, result is as shown in Figure 4.
The genomic dna extracting of take is template, utilizes the 16S rRNA sequence of following primer pair bacterial strain to carry out pcr amplification, and primer sequence is as follows:
Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAGAACGAAC-3 ';
Downstream primer: 5 '-TACGGTTACCTTGTTACGACTTCACCCC-3 '.
Amplified production carries out agarose gel electrophoresis, and as shown in Figure 5, product length is approximately 1500bp to result; And entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out gene sequencing, base sequence is as shown in SEQ ID No.1.To in sequencing result NCBI, register, it is for No. GenBank: KC898819.
Online Blast software (http://www.ncbi.nlm.nih.gov/blast) for the gene sequencing result obtaining is carried out to homology analysis, result finds significantly similar gene order of a plurality of streptomyces in GenBank, and its homology is up to 98%.By Blast instrument, in NCBI protein sequence database, carry out homology search, and utilize Clustal W program to carry out multiple sequence comparison.Utilizing MEGA4.0 software, adopt contiguous concatenation method (Neighbor Joining Method), obtain this protein system evolutionary tree (Fig. 6), is shallow Streptomyces glaucoviolaceus by this dientification of bacteria.
This bacterial strain is delivered to the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms on August 28th, 2013, and deposit number is CGMCC No.8083.
From the test tube of preservation bacterial classification, picking colony carries out actication of culture, at 37 ℃, cultivates 48h; The formula of activation medium is: 2% Zulkovsky starch, 0.05%NaCl, 0.05%K
2hPO
4, 0.05%MgSO
4, 0.001%FeSO
4, 2% agar.
Then the single bacterium colony on picking activation flat board is transferred in seed culture fluid and carries out multiplication culture, cultivates 12h, rotating speed 200r/min at 28 ℃; The formula of seed culture fluid is: 2% Zulkovsky starch, 0.2% extractum carnis, 0.05%NaCl, 0.05%K
2hPO
4, 0.05%MgSO
4, all the other are water, 0.001%FeSO
47H
2o.
Finally seed liquor is inoculated in fermentation culture, carries out fermentation culture.For obtaining the cyanine of maximum production, fermentation culture based component, fermentation condition have been carried out to series of optimum.
(1) medium component optimization
1) carbon source optimizing
With Gause I substratum in contrast, with glycerine, N.F,USP MANNITOL, glucose, sucrose, as carbon source, replace the Zulkovsky starch in control medium, the activated good bacterial strain of picking is cultivated and is concentrated to 250mL, cultivate centrifugal to fermented liquid after 7 days, get supernatant liquor and measure absorbancy under wavelength 585nm, relatively OD
585select optimum carbon source, the results are shown in Figure 7.As seen from Figure 7, when take glycerine during as carbon source, OD
585the highest, be 7.01, therefore selecting glycerine is carbon source.
2) optimization of nitrogenous source
Determining on the basis of carbon source, with extractum carnis, peptone, yeast extract paste, (NH
4)
2sO
4as nitrogenous source, replace the saltpetre in control medium, the activated good bacterial strain of picking is cultivated and is concentrated to 250mL, cultivates after 7 days centrifugally to fermented liquid, gets supernatant liquor and measure absorbancy under wavelength 585nm, comparison OD
585select optimum nitrogenous source, the results are shown in Figure 8.As seen from Figure 8, except ammonium sulfate, other four kinds of nitrogenous sources can grow cyanine bacterial strain, and saltpetre has the cyanine of production peak during as nitrogenous source, and saltpetre is also the common inorganic nitrogen in laboratory, so select saltpetre as optimum nitrogen source.
3) carbon nitrogen concentration is determined and optimum carbon nitrogen ratio
Determine after best carbon nitrogen source, with 4%, 6%, 8% carbon source concentration replaces in control medium 2% carbon source concentration, and the activated good bacterial strain of picking is cultivated and concentrated to 250mL, cultivates after 7 days centrifugal to fermented liquid, get supernatant liquor and measure absorbancy under wavelength 580nm, relatively OD
585select optimum carbon source concentration, the results are shown in Figure 9.As seen from Figure 9, along with the increase of glycerol concentration, cyanine output reduces gradually, may be that the carbon source of high density has suppressed the growth of cyanine, so select 2% as optimum carbon source concentration.
According to optimum carbon source concentration, configure different carbon-nitrogen ratios: 30:1,25:1,20:1,15:1,10:1, the activated good bacterial strain of picking cultivate to be concentrated to 250mL, cultivates after 7 days centrifugally to fermented liquid, gets supernatant liquor and measure absorbancy under wavelength 585nm, relatively OD
585select optimum carbon-nitrogen ratio, the results are shown in Figure 10.As seen from Figure 10, when carbon-nitrogen ratio is 20:1, cyanine output reaches maximum, so selection optimum carbon nitrogen ratio is 20:1.
Optimum carbon source concentration is 2%, and optimum carbon nitrogen ratio is 20:1, can guestimate optimum nitrogen source concentration be 0.1% thus.
4) optimization of the inorganic salt concentration such as sodium-chlor, dipotassium hydrogen phosphate and magnesium sulfate
According to the ratio of these three composition 1:1:1 on Gause I substratum, configure respectively 0.025%, 0.05%, the substratum of 0.075%, 0.1%, 0.15% concentration, the activated good bacterial strain of picking is cultivated and is concentrated to 250mL, cultivate centrifugally to fermented liquid after 7 days, get supernatant liquor and measure absorbancy under wavelength 585nm, relatively OD
585under absorbancy select optimum concn, the results are shown in Figure 11.As seen from Figure 11, it is maximum that inorganic salt concentration cyanine output in 0.05% reaches, so select inorganic salt concentration, is 0.05%.
5) optimization of ferrous ion
On the basis of optimal conditions, choose 0.0005%, 0.001%, the concentration of 0.0015%, 0.002% pair of ferrous ion is optimized, and the activated good bacterial strain of picking is cultivated and concentrated to 250mL, cultivate centrifugally to fermented liquid after 7 days, get supernatant liquor and measure absorbancy under wavelength 585nm, relatively OD
585select best ferrous ion concentration, the results are shown in Figure 12.As seen from Figure 12, ferrous ion concentration between 0.001% and 0.0015% time output reach the highest, select 0.001% as best ferrous ion concentration.
(2) optimization of fermentation condition
1) optimization of pH
At carbon nitrogen source, concentration of carbon and nitrogen sources and carbon-nitrogen ratio are carried out the optimization of pH after determining to substratum.Initial pH is: 5,6,7,8,9.Then the activated good bacterial strain of picking cultivate to be concentrated to 250mL, cultivates after 7 days centrifugally to fermented liquid, gets supernatant liquor and measure absorbancy under wavelength 585nm, relatively OD
585select optimum pH, the results are shown in Figure 13.As seen from Figure 13, when pH is 7, OD
580value is maximum, shows that cyanine output is maximum.
2) optimization of temperature
Picking bacterial strain, in the substratum of having optimized, is placed in respectively 25 ℃, 28 ℃, 30 ℃, 33 ℃, 37 ℃ bottom fermentations and cultivates, and cultivates after 7 days centrifugally to fermented liquid, gets supernatant liquor and under wavelength 580nm, measures absorbancy (OD
585), compare OD
585select optimum fermentation temp, the results are shown in Figure 14.As seen from Figure 14, OD when 28 ℃ and 30 ℃
585value is maximum, shows that cyanine output is maximum; Consider laboratory equipment requirement, select 28 ℃ for optimum temps.
3) optimization of inoculum size
By bacterial strain seed liquor, with 2%, 4%, 6%, 8%, 10% inoculum size is inoculated on the substratum of having optimized carries out fermentation culture, cultivates after 7 days centrifugally to fermented liquid, gets supernatant liquor and under wavelength 585nm, measures absorbancy (OD
585), compare OD
585select optimum inoculation amount, the results are shown in Figure 15.As seen from Figure 15, when inoculum size is 8%, OD
585value is maximum, shows that cyanine output is maximum.
(3) orthogonal test
According to above-mentioned optimal conditions, to choose the larger factor of cyanine yield effect is carried out to orthogonal experiment, orthogonal, in Table 1, arranges glycerol concentration, carbon-nitrogen ratio, inorganic salt concentration, four factors of inoculum size altogether, and each factor is established three levels.
Table 1 orthogonal
According to the setting of table 1, carry out fermentation culture, cultivate after 7 days centrifugally to fermented liquid, get supernatant liquor and under wavelength 585nm, measure absorbancy (OD
585), according to spectrophotometric analysis Orthogonal experiment results (in Table 2).
Table 2 orthogonal experiments
From table 2, optimum fermentation scheme is: glycerine 2%, and carbon-nitrogen ratio 20:1, inorganic salt concentration 0.05%, inoculum size 8%, this orthogonal experiments is consistent with optimum result before this.
(4) utilize bacterial strain to carry out cyanine fermentation
According to the condition of having optimized, utilize the shallow Streptomyces glaucoviolaceus of embodiment 1 to carry out cyanine fermentation, specifically comprise:
1) from the test tube of preservation bacterial classification, picking colony carries out actication of culture, at 37 ℃, cultivates 48h;
The formula of activation medium is: 2% Zulkovsky starch, 0.05%NaCl, 0.05%K
2hPO
4, 0.05%MgSO
4, 0.001%FeSO
4, 2% agar;
2) the single bacterium colony on picking activation flat board is transferred in seed culture fluid and carries out multiplication culture, at 28 ℃, cultivates 12h, and rotating speed 200r/min, obtains seed liquor;
The formula of seed culture fluid is: 2% solubility is formed sediment, 0.2% extractum carnis, 0.05%NaCl, 0.05%K
2hPO
4, 0.05%MgSO
4, 0.001%FeSO
47H
2o, all the other are water;
3) seed liquor is inoculated in fermentation culture and carries out fermentation culture with 8%, cultivate 7 days at 28 ℃, rotating speed 200r/min, obtains fermented liquid;
The formula of fermentation culture is: 2% glycerine, and 0.1% saltpetre, 0.05% inorganic salt, 0.001%FeSO47H2O, all the other are water;
4) fermented liquid pH is adjusted to the centrifugal 10min of 12,8000r/min, gets supernatant its pH is adjusted to l～2,10000r/min is centrifugal, and 10min abandons supernatant, and throw out vacuum-drying is to constant weight.
The cyanine crude preparation by using that embodiment 2 is prepared carries out a series of test, to obtain the basic data of this cyanine.
(1) solvability
Get equivalent cyanine crude preparation by using and be dissolved in respectively 10mL dehydrated alcohol, distilled water, methyl alcohol, acetone, methyl-sulphoxide, in ethyl acetate, sherwood oil, after static 2h, observe solvability and colour-change.
(2) uv-vis spectra
Cyanine crude preparation by using is soluble in water, cyanine pH value of water solution is adjusted to respectively to 3,5,7,9,11,13, with UV-2550 type ultraviolet-visible pectrophotometer, within the scope of 200-800nm, the cyanine aqueous solution under different pH is scanned, obtain the uv-vis spectra of cyanine.
From test (1) and (2), this cyanine is soluble in alkaline aqueous solution, dissolve in the acidic solution of pH > 3, the aqueous solution that is insoluble to pH < 3, dissolves in methyl-sulphoxide, is insoluble to acetone, sherwood oil, methyl alcohol, dehydrated alcohol, the organic solvents such as ethyl acetate.Visible ray spectrogram is shown in Figure 16.As shown in Figure 16, the maximum absorption wavelength of this pigment is 585nm left and right, the molecular structure that this pigment is described has very large conjugated system, the conjugated structure that may have more conjugated double bond, phenyl ring or similar phenyl ring, or containing more heteroatomic heterocycles, the concrete structure of this pigment needs further to be resolved.
(3) stability
1) impact of pH on cyanine stability
Regulate cyanine pH value of water solution to 3,5,7,9,11,13, every 1h, measure light absorption value (seeing Figure 17), and observe its colour-change (in Table 3).
The impact of table 3 pH on cyanine stability
Under acidity or alkaline condition, natural pigment easily fades or variable color, and the tolerance that pigment changes pH value, is determining its range of application.From Figure 17 and table 3, this cyanine is all comparatively stable under different pH, and under acidic conditions, this cyanine takes on a red color, and during pH value 3, maximum absorption band appears at 520nm left and right; Under neutrallty condition, be lavender, maximum absorption band is in 560nm left and right; Under alkaline condition, present blueness, during pH value 9, maximum absorption band is in 585nm left and right, and during pH value 13, maximum absorption band is in 620nm left and right.The possible cause that occurs this result is that under different pH, the conjugated system in this cyanine molecular structure changes, thereby shows different maximum absorption wavelengths and colour-change.
2) impact of light on cyanine stability
Regulate cyanine pH value of water solution to 5,7,9,11, different pH value pigment aqueous solutions, respectively with outdoor direct projection and room scattering rayed, are measured to light absorption value every 1h, the above-mentioned cyanine aqueous solution is placed under ultraviolet lamp and is irradiated simultaneously, every 10min, measure light absorption value, and observe colour-change.The results are shown in Figure 18 and Figure 19.
From Figure 18 and Figure 19, this cyanine is under room scattering light and UV-irradiation, and light absorption value changes little, and stability is better; But under outdoor directing light irradiates, this cyanine is received remarkable destruction, and after 8h, survival rate is only 40% left and right.Show should avoid high light direct projection in preserving and using the process of this cyanine.
3) impact of temperature on cyanine stability
Regulate cyanine pH value of water solution to 9, the cyanine aqueous solution is placed in to 20 ℃, 40 ℃, 60 ℃, 80 ℃, constant temperature is placed at 100 ℃, measures its absorbancy, and observe colour-change every 1.5h.The results are shown in Figure 20.
As seen from Figure 20, this pigment solution is preserved after 9h at the temperature lower than 60 ℃, and light absorption value changes little, and the survival rate of pigment is higher than 80%.Under more than 80 ℃ condition, this pigment light absorption value significantly declines, survival rate less than 30% after 9h.Illustrate that this pigment solution is more stable at the temperature lower than 60 ℃, higher temperature has larger destruction to it.
4) oxygenant H
2o
2with reductive agent Na
2sO
3impact on cyanine stability
Regulate cyanine pH value of water solution to 9, add respectively 0.1%, 0.2%, 0.3%, 0.4%, 0.5% different concns H
2o
2and Na
2sO
3, every 1h sampling and measuring absorbancy, and observe colour-change.The results are shown in Figure 21.
As seen from Figure 21, oxygenant H
2o
2this blueness is have to significant destruction, and concentration is larger, affects more obviously, work as H
2o
2when concentration reaches 0.5%, pigment survival rate only 19.4%, illustrates that this cyanine is easily oxidized, should avoid sneaking into or adding appropriate antioxidant to extend preservation period of oxygenant when preserving.
Reductive agent Na
2sO
3stability influence to this cyanine is less, and when concentration is 0.10%, the survival rate of pigment is about 81.3%, but with the increase of reductant concentration, does not reduce.Its possible cause is Na
2sO
3partial double bond reduction in this pigment molecular, has destroyed conjugated structure, causes color and maximum absorption band to change.
5) impact of metal ion on cyanine stability
Regulate cyanine pH value of water solution to 9, add respectively 0.0025mol/L, 0.005mol/L, the Na of 0.01mol/L different concns
+, K
+, Ca
2+, Mg
2+, Cu
2+, Fe
3+deng metal ion, standing 2h measures its absorbancy, observes the affect situation of each metal ion on pigment, the results are shown in Table 4.
The impact of table 4 metal ion on cyanine stability
From table 4, within the scope of finite concentration, Na
+, K
+and Ca
2+this cyanine is not had to impact substantially, and Mg
2+its absorbancy is increased, therefore can utilize containing Mg in actual applications
2+reagent reaches hyperchromic effect.Add Fe
3+after this pigment aqueous solution by blue flavescence, light absorption value significantly reduces, and produces yellow mercury oxide, adds Cu
2+produce blue flocks.So Fe
3+and Cu
2+larger on this pigment impact, in using and preserving, should note avoiding using irony, copper container, and contact with the chemical reagent containing iron ion and cupric ion.
6) impact of foodstuff additive on cyanine stability
Regulate cyanine pH value of water solution to 9, add respectively 1%, 2%, 3%, 4%, 5% different mass mark sucrose and salt, survey absorbancy, and observe colour-change.The results are shown in Table 5.
The impact of table 5 foodstuff additive on cyanine stability
From table 5, within the scope of finite concentration, salt and sucrose on this cyanine substantially without impact; Because citric acid is acid, add the rear cyanine aqueous solution to be reddened by indigo plant, light absorption value has small size decline, but little on this cyanine impact on the whole.
By investigating different factors, grey violet streptomycete is produced to the impact of cyanine stability, find outdoor directing light, high temperature (being greater than 60 ℃), oxygenant H
2o
2, metal cations Fe
3+and Cu
2+this pigment aqueous solution is had to obvious destruction, and outdoor scattered light, UV-light, reductive agent Na
2sO
3, part metals ion is (as Na
+, K
+and Ca
2+) and foodstuff additive salt, sucrose, citric acid this cyanine is had no significant effect.The applied research that this result of study can be the fields such as weaving, food provides basic data.
(4) anti-microbial activity
Take appropriate cyanine crude preparation by using and be dissolved in the sodium hydroxide solution of 0.05mol/L, make certain density liquid, detect the anti-microbial activity of this cyanine to intestinal bacteria, streptococcus aureus, yeast saccharomyces cerevisiae.
1) indicator preparation
By the intestinal bacteria bacteria suspension being kept in subzero 70 ℃ of refrigerators, after dissolving at normal temperatures, streak culture on LB solid medium flat board, at 37 ℃, be inverted and cultivate after 18～24h, with transfering loop picking list bacterium colony, transfer in the Erlenmeyer flask of LB liquid nutrient medium is housed, 37 ℃ of isothermal vibrations are cultivated 12-18h, stand-by as indicator.
To be kept at the streptococcus aureus bacteria suspension in subzero 70 ℃ of refrigerators, after dissolving at normal temperatures, streak culture on LB solid medium flat board, at 37 ℃, be inverted and cultivate after 18～24h, with transfering loop picking list bacterium colony, transfer in the Erlenmeyer flask of LB liquid nutrient medium is housed, 37 ℃ of isothermal vibrations are cultivated 12-18h, stand-by as indicator.
Take 2g glucose and 1g dry yeast is put into shaking flask, add and be distilled to 100mL, in the electric heating constant-temperature blowing drying box of 38 ℃, place 60min, stand-by as indicator.
2) inhibition zone is measured
Adopt K-B disk diffusion method to carry out.
First original bacterium liquid (intestinal bacteria, streptococcus aureus and yeast saccharomyces cerevisiae) is done to 10 with sterilized water
-1, 10
-2doubly dilution, draws the bacteria suspension of each concentration of 0.1mL on corresponding nutrition flat board, with spreading rod by the coating of bacterium liquid evenly.
Dry susceptibility filter paper is lain against on clean aseptic plate with tweezers, drawing respectively 20 μ L concentration is 2g/L, the cyanine of 1g/L is on dry susceptibility filter paper (diameter 5mm), then plate is uncapped to put into after thermostatic drying chamber (37 ℃) is dried and be attached to containing on bacterium flat board, with the sodium hydroxide solution of 0.02mol/L, compare, every kind of bacterium is cooked three repetitions.To be placed in 37 ℃ of (28 ℃ of fungies) thermostat containers containing bacterium flat-plate inverted and cultivate 24h(fungi 48h), adopt right-angled intersection method to measure the size of pastille filter paper antibacterial circle diameter, average, relatively fungistatic effect.
The calculation formula of antibacterial circle diameter is:
Antibacterial circle diameter (mm)=measurement colony diameter mean value-5.0; The results are shown in Figure 22a, Figure 22 b, Figure 23 a, Figure 23 b, Figure 24 a, Figure 24 b and table 6, table 7.
Table 6 different concns cyanine is to colibacillary antibacterial circle diameter (mm)
| Intestinal |
10 -1 | 10 -2 |
| 2g/L cyanine | 11 | 4 |
| 1g/ |
6 | 4 |
| 0.02mol/ |
0 | 0 |
Table 7 different concns cyanine is to the antibacterial circle diameter of streptococcus aureus (mm)
| Gold Portugal |
10 -1 | 10 -2 |
| 2g/ |
9 | 3 |
| 1g/ |
7 | 3 |
| 0.02mol/ |
0 | 0 |
From Figure 22 a, Figure 22 b and table 6, this cyanine has obvious bacteriostatic activity to Gram-negative bacteria intestinal bacteria; From Figure 23 a, Figure 23 b and table 7, this cyanine also has obvious bacteriostatic activity to streptococcus aureus.From Figure 24 a, Figure 24 b, this cyanine to yeast saccharomyces cerevisiae without any bacteriostatic activity.
Claims (9)
1. the shallow Streptomyces glaucoviolaceus of a strain (Streptomyces lividans), is characterized in that, the shallow Streptomyces glaucoviolaceus of called after (Streptomyces lividans) jx-02, and deposit number is CGMCC No.8083.
2. the application of shallow Streptomyces glaucoviolaceus as claimed in claim 1 in producing cyanine.
3. application as claimed in claim 2, is characterized in that, comprising:
(1) shallow Streptomyces glaucoviolaceus as claimed in claim 1 is inoculated in seed culture fluid and carries out multiplication culture, obtain seed liquor;
(2) described seed liquor is transferred in fermentation culture and carries out fermentation culture, obtain fermented liquid;
(3) separated from described fermented liquid, the dry cyanine crude preparation by using that obtains.
4. application as claimed in claim 3, is characterized in that, the condition of described multiplication culture is: temperature is 25～30 ℃, and the time is 10～14h.
5. application as claimed in claim 3, is characterized in that, the inoculum size of described seed liquor is 5～10%.
6. application as claimed in claim 3, is characterized in that, the condition of described fermentation culture is: pH is 7, and temperature is 25～30 ℃, and the time is 6～10 days.
7. application as claimed in claim 3, is characterized in that, in 1L, described fermentation culture comprises: 18～22g carbon source, 0.8～1.2g nitrogenous source, 0.4～0.6g inorganic salt.
8. application as claimed in claim 7, is characterized in that, described carbon source is glycerine, and described nitrogenous source is saltpetre.
9. the cyanine crude preparation by using being obtained by shallow Streptomyces glaucoviolaceus fermented extracted described in claim 1.
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|---|---|---|---|---|
| CN105176873A (en) * | 2015-09-30 | 2015-12-23 | 菏泽学院 | Streptomyces sp. strain for producing environment-friendly blue pigment |
| CN106497980A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of extracting method of natural blue pigment and colouring method |
| CN106498767A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of method of natural blue pigment to textile dyeing |
| CN107118588A (en) * | 2017-05-04 | 2017-09-01 | 山东科技大学 | A kind of method and apparatus for extracting marine streptomyces natural blue pigment |
| CN111471316A (en) * | 2020-03-19 | 2020-07-31 | 当阳市鸿阳新材料科技有限公司 | Extraction method of microbial pigment and method for dyeing lyocell fibers by using microbial pigment |
| CN114933980A (en) * | 2022-02-21 | 2022-08-23 | 中国科学院西双版纳热带植物园 | Streptomyces lividans HJB-XTBG45 for preventing and treating polygonatum sibiricum root rot and application thereof |
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-
2013
- 2013-11-13 CN CN201310562683.5A patent/CN103602615B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 谢勇 等: "浅青紫链霉菌蓝色苏的稳定性研究", 《嘉兴学院院报》 * |
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| CN105176873A (en) * | 2015-09-30 | 2015-12-23 | 菏泽学院 | Streptomyces sp. strain for producing environment-friendly blue pigment |
| CN105176873B (en) * | 2015-09-30 | 2018-07-24 | 菏泽学院 | Produce the streptomycete bacterial strain of environment protecting blue pigment |
| CN106497980A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of extracting method of natural blue pigment and colouring method |
| CN106498767A (en) * | 2016-10-11 | 2017-03-15 | 嘉兴学院 | A kind of method of natural blue pigment to textile dyeing |
| CN106498767B (en) * | 2016-10-11 | 2019-02-05 | 嘉兴学院 | A kind of method of natural blue pigment to textile dyeing |
| CN107118588A (en) * | 2017-05-04 | 2017-09-01 | 山东科技大学 | A kind of method and apparatus for extracting marine streptomyces natural blue pigment |
| CN107118588B (en) * | 2017-05-04 | 2019-05-24 | 山东科技大学 | A kind of method and apparatus for extracting marine streptomyces natural blue pigment |
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| CN114933980A (en) * | 2022-02-21 | 2022-08-23 | 中国科学院西双版纳热带植物园 | Streptomyces lividans HJB-XTBG45 for preventing and treating polygonatum sibiricum root rot and application thereof |
| CN114933980B (en) * | 2022-02-21 | 2024-02-20 | 中国科学院西双版纳热带植物园 | Streptomyces shallowus HJB-XTBG45 for preventing and treating rhizoma polygonati root rot and application thereof |
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