CN1586130A - Method for producing blue pigment and its special strain - Google Patents
Method for producing blue pigment and its special strain Download PDFInfo
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- CN1586130A CN1586130A CN 200410074325 CN200410074325A CN1586130A CN 1586130 A CN1586130 A CN 1586130A CN 200410074325 CN200410074325 CN 200410074325 CN 200410074325 A CN200410074325 A CN 200410074325A CN 1586130 A CN1586130 A CN 1586130A
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- cyanine
- pseudomonas fluorescens
- blue pigment
- pigment
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- 239000001055 blue pigment Substances 0.000 title abstract description 10
- 238000004519 manufacturing process Methods 0.000 title description 10
- 238000000034 method Methods 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 241000589540 Pseudomonas fluorescens Species 0.000 claims abstract description 14
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 12
- 239000012137 tryptone Substances 0.000 claims abstract description 12
- 239000012138 yeast extract Substances 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 52
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 20
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 13
- 239000012530 fluid Substances 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
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- 238000007747 plating Methods 0.000 claims description 7
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- 239000000047 product Substances 0.000 description 9
- 238000004040 coloring Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
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- 239000000287 crude extract Substances 0.000 description 6
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- NNMALANKTSRILL-ZUTFDUMMSA-N 3-[(2z,5z)-2-[[3-(2-carboxyethyl)-5-[(z)-[(3z,4r)-3-ethylidene-4-methyl-5-oxopyrrolidin-2-ylidene]methyl]-4-methyl-1h-pyrrol-2-yl]methylidene]-5-[(4-ethyl-3-methyl-5-oxopyrrol-2-yl)methylidene]-4-methylpyrrol-3-yl]propanoic acid Chemical compound O=C1C(CC)=C(C)C(\C=C/2C(=C(CCC(O)=O)C(=C/C3=C(C(C)=C(\C=C/4\C(\[C@@H](C)C(=O)N\4)=C/C)N3)CCC(O)=O)/N\2)C)=N1 NNMALANKTSRILL-ZUTFDUMMSA-N 0.000 description 2
- MQBFFYQCZCKSBX-UHFFFAOYSA-N 5-hydroxy-6,7,8-trimethoxy-2-(3,4,5-trimethoxyphenyl)chromen-4-one Chemical compound COC1=C(OC)C(OC)=CC(C=2OC3=C(OC)C(OC)=C(OC)C(O)=C3C(=O)C=2)=C1 MQBFFYQCZCKSBX-UHFFFAOYSA-N 0.000 description 2
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- INPDFIMLLXXDOQ-UHFFFAOYSA-N Phycocyanobilin Natural products CCC1=C(C)C(=CC2=NC(=C/c3[nH]c(C=C/4C(C(C(N4)=O)C)=CC)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O INPDFIMLLXXDOQ-UHFFFAOYSA-N 0.000 description 2
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides blue pigment producing pseudomonas fluorescens strain B1 CGMCC No. 1194, and the blue pigment producing process therewith. The blue pigment producing process includes the following steps: culturing blue pigment producing pseudomonas fluorescens strain B1 CGMCC No. 1194 through fermentation at 4-30 deg.c for 24-240 hr to obtain thallus; and crushing the thallus cells to obtain blue pigment. The liquid culture medium includes: tryptone 8-12 g, yeast extract 4-6 g, glucose 0.8-1.2 g, dipotassium hydrogen phosphate 2-4 g and water 1000 ml. The blue pigment thus produced is natural product and has no latent harm and no side effect on animal body, so that it may be used widely in food industry.
Description
Technical field
The present invention relates to the production method of natural colouring matter and produce bacterial strain, particularly relate to a kind of method and special-purpose bacterial strain of cultivating thereof of producing cyanine.
Background technology
Pigment generally is divided into synthetic food color and natural colouring matter, and both all have been widely used in aspects such as food color, daily cosmetics and feed addictive.Synthetic food color is meant with chemical synthesis process prepared organic pigment, mainly is to extract from coal tar or is that raw material is synthetic with arene compounds such as benzene, toluene, naphthalene, aniline.After synthetic food color occurs, accepted by the user rapidly with advantages such as its lovely luster, strong coloring force, good stability, color matching convenience, low prices, but along with subject development such as modern medicine, food hygienes, it is found that synthetic dyestuff exists toxicological issues, some is harmful to health, even brings out cancer etc.Therefore, more and more countries has been stopped using the bigger kind of some toxicity, country that has even the use of having forbidden synthetic dyestuff.Because the natural colouring matter major part is the edible part from plant, microorganism, animal, extracting process is pure physical process, can not change on the structure.Therefore, natural colouring matter is safe and have the extensive attention that certain nutrient value causes people mostly because of it, and its Application and Development research becomes the focus in Phytochemistry and the Food Science field.
Cyanine has very high added value as one of 3 basic pigments, can be deployed into multiple color tones with natural colouring matter red, yellow strain, makes natural colouring matter abundanter.Natural blue at present commonly used have two kinds of mast cyanine and phycocyanobilins.Mast cyanine is to be raw material with the plant cape jasmine fruit, obtain cyanine through preliminary treatment, active ingredient extracting, enzymatic reaction, evaporation and concentration and process such as refining, but this preparation method's complicated process of preparation, the cost height, raw materials for production are subjected to the cape jasmine fruit production restriction, give the large-scale industrial production cyanine bring certain difficulty (Liu Chenglun, Xu Longjun. the gardenin progress of research. research and development of natural products, 1996,8 (2): 69～72).Phycocyanobilin is with spirulina (Zhang Kun, Fang Yanxiong. the research of spirulina blue color. food and machinery, 1995, (4): 21～23.) and beads algae (Zhao Liangzhong, Duan Lindong. the extraction and the property research thereof of beads algae blue pigment. food industry science and technology, 1998, (2): 24～26) be raw material, through lixiviate, centrifugation, concentrate, purifying and getting, but the shortcoming of phycocyanobilin be heat-resisting, light resistance is poor.
From microorganism, extract pigment, be not subjected to the restriction in resource, environment and space, have plant pigment and animal pigment incomparable superiority, but the microorganism that can produce cyanine at present seldom, the streptomyces that is of report is disclosed, as streptomyces coelicolor (Streptomyces coelicolor) A3 (2) (Leonid V.Bystrykh, Miguel
A.Fern á ndez-Moreno, Jan K.Herrema, Francisco Malpartida, David A.Hopwood, LubbertDijkhuizen.Production of Actinorhodin-Related " Blue Pigments " by Streptomycescoelicolor A3 (2) .Journal of Bacteriology, 1996,178 (8): 2238-2244), Streptomycescoelicolor 100 (opens and the spring, Ji Wenming, Wang Wu. streptomyces coelicolor produces the industrialization medium optimization of cyanine. Wuxi Light Industry Univ.'s journal, 2000,19 (2): 138～141), (Lu Lingsun prolongs great waves to Streptomyces sp LS-1, Tang Yong, the blue a kind of actinomycetes of Qin Huai send out the research microbiology circular .2001 that alcohol produces natural blue pigment, 28 (4): 50～54) etc.These strain growth cycles are long, produce the temperature range narrow (26 ℃-30 ℃) of pigment, and its cyanine production ratio is lower, generally at 0.2g pigment crude extract/g cell.
Summary of the invention
The purpose of this invention is to provide a kind of bacterial strain that can secrete, produce cyanine.
Product cyanine pseudomonas fluorescens provided by the present invention (Pseudomonas fluorescens) B1 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 07 21st, 2004, preserving number is CGMCC № 1194.
It is oval shaft-like that this bacterial strain is, no brood cell, pod membrane, Gram-negative.On LB medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L) plate, bacterium colony is rounded, and surface wettability is smooth, neat in edge.Before producing pigment, the bacterial clump size is 1～2mm, and bacterium colony is yellow.At the cultivation initial stage, pigment produces at the bacterium colony center, after extend to around, final colony diameter can reach 2～3mm, the center blueness is darker, blueness is more shallow all around.The oxidase of this bacterium, cytochrome oxidase, the two water Jie enzyme reactions of arginine are positive, indoles, acidifying, urase, glucosaccharase, protease and galactoside enzyme reaction are negative, assimilation glucose, arabinose, mannose, mannitol, N-acetyl-gucosamine, gluconate, capric acid, malic acid and citric acid.(BioM é rieux Biomerieux SA) identifies this bacterial classification with API 20 NE kits, qualification result be encoded to 1147555, identify that percentage is 99.9%, mode frequency T=0.93, bacterium is called pseudomonas fluorescens (Pseudomonasfluorescens).This bacterium all can grow under the scope of 4 ℃-30 ℃ cultivation temperature and pH6-7.6.
Second purpose of the present invention provides the method that bacterial strain of the present invention is produced cyanine of using.
The method of production cyanine provided by the present invention comprises the steps: 1) strain culturing: will produce cyanine pseudomonas fluorescens (Pseudomonas fluorescens) B1 CGMCC № 1194 and obtain thalline in 24-240 hour 4 ℃ of-30 ℃ of condition bottom fermentations cultivations; 2) broken somatic cells obtains cyanine.
The medium of described fermented and cultured contains following substances: tryptone 8-12g, and yeast extract 4-6g, glucose 0.8-1.2g, dipotassium hydrogen phosphate 2-4g adds water to 1000ml.Wherein preferably: tryptone 10g, yeast extract 5g, glucose 1g, dipotassium hydrogen phosphate 3g adds water to 1000ml, and pH is 6-7.6.
In order to make the better effects if of fermentation, before carrying out liquid culture, frozen bacterial classification also activates through dull and stereotyped the cultivation, and plating medium commonly used contains following substances: tryptone 5g, and yeast extract 2.5g, NaCl 2.5g, agar 10g adds water to 500ml; Described plating medium pH is 7.2-7.6.
In process of production, the described thalline that obtains generally can adopt centrifugation, as obtaining mycelium in that 6000rpm is centrifugal.The process that described broken somatic cells obtains cyanine is that adding concentration is the ethanol of 5-100%, the isopropyl alcohol of 5-100% or the oxolane of 5-100% in thalline, obtains the ethanolic solution of cyanine behind the sonic oscillation, obtains cyanine after the drying.Solvent is the ethanol of 5-100% preferably, with the alcohol extract cyanine time, amount of alcohol added can be controlled in every 100ml zymotic fluid usually and adds 20-300ml, and the ethanolic solution drying mode commonly used that has pigment can adopt vacuum drying, promptly can obtain the thick product of cyanine after the drying.
Strain growth speed of the present invention is fast, and the cultivation temperature wide ranges all can be produced pigment at 4 ℃ of-30 ℃ of condition bottom fermentations, is convenient to suitability for industrialized production; The productive rate of bacterial strain production cyanine of the present invention is higher, generally can reach the thick product of the above cyanine of 5g/L zymotic fluid, the cyanine that obtains belongs to all-natural product, there is not the potential harm of artificial color, experimental results show that, can not produce any side effect to animal body, can be widely used in each field such as food industry.
Embodiment
The separation screening of embodiment 1, product cyanine bacterium B1
1, sample collection
Soil specimen is gathered in No. 1 glacier from Xinjiang.
2, the separation of bacterial strain and screening
The preparation plating medium: beef extract 3g, peptone 10g, NaCl 5g, agar 20g adds water to 1000ml, pH7.4-7.6.With above-mentioned medium with 1.05kg/cm
2, 121 ℃, high pressure steam sterilization 20 minutes falls dull and stereotyped.
Soil specimen is made 0.2% suspension, and being diluted to suspension concentration by 10 times is 2 * 10
-8, get each dilution factor sample liquid 0.2ml and evenly coat respectively on the culture medium flat plate, cultivated 24-240 hour for 4 ℃-30 ℃, observe and select the bacterium colony that produces cyanine, a strain bacterium called after B1 wherein, preservation behind the purifying.
The extraction of the cultivation of embodiment 2, bacterial strain and cyanine crude product
1, strain culturing
The preparation plating medium: tryptone 5g, yeast extract 2.5g, NaCL 2.5g, agar 10g adds water to 500ml, regulates pH7.2-7.6.Plating medium is at 121 ℃ of 15min that sterilize down, dull and stereotyped, and cooling back access B1 bacterial strain was cultivated 48 hours under 30 ℃.
The obtaining liq medium: tryptone 10g, yeast extract 5g, glucose 1g, dipotassium hydrogen phosphate 3g adds water to 1000ml, regulates pH6-7.The 100ml medium packed into sterilize in the 500ml blake bottle, the colony inoculation of the single product pigment of picking is cultivated under 30 ℃ of temperature in aseptic liquid nutrient medium from flat board, and speed of agitator is 150rpm-250rpm.Cultivate that thalline is in logarithmic growth mid-term and begins to produce pigment after 14 hours, pigment produced and then is in mid-term stage when thalline entered deadtime after 50 hours, and pigment produces and enters deadtime after thalline enters 40 hours deadtimes, can stop fermentation.
2, the extraction of cyanine
With zymotic fluid centrifugal 10min under 6000rpm, abandon supernatant, in sediment, add absolute ethyl alcohol (every 100ml zymotic fluid adds 50ml ethanol), with eddy mixer with its mixing, in the 200W ultrasonic cleaner, vibrated 1 hour then, the centrifugal 5min of vibration liquid 8000rpm is obtained the ethanolic solution of pigment.If still have pigment to remain in the cell residue, can repeat the aforesaid operations step, till no longer pigment can being extracted.
With resulting ethanolic solution vacuum drying, promptly obtain the crude extract of cyanine, productive rate is the thick product of 5.3g cyanine/L zymotic fluid.
The extraction of the cultivation of embodiment 3, bacterial strain and cyanine crude product
1, strain culturing
Incubation is identical with embodiment 2, and difference is that used medium is: tryptone 8g, and yeast extract 6g, glucose 0.8g, dipotassium hydrogen phosphate 4g adds water to 1000ml; Cultivation temperature is 4 ℃, and the incubation speed of agitator is 150rpm-250rpm, cultivates promptly can stop after 240 hours cultivating.
2, cyanine extracts
The centrifugal 10min of zymotic fluid 6000rpm is obtained thalline, in thalline, add 5% aqueous isopropanol (every 100ml zymotic fluid adds the 300ml aqueous isopropanol), the sonic oscillation smudge cells obtains the aqueous isopropanol of cyanine, vacuum drying promptly obtains the crude extract of cyanine, and productive rate is the thick product of 5.1g cyanine/L zymotic fluid.
The extraction of the cultivation of embodiment 4, bacterial strain and cyanine crude product
1, strain culturing
Incubation is identical with embodiment 2, and difference is that used medium is: tryptone 12g, and yeast extract 4g, glucose 1.2g, dipotassium hydrogen phosphate 2g adds water to 1000ml; Cultivation temperature is 30 ℃, and the incubation speed of agitator is 150rpm-250rpm, cultivates promptly can stop after 100 hours cultivating.
2, cyanine extracts
The centrifugal 10min of zymotic fluid 6000rpm is obtained thalline, in thalline, add 50% tetrahydrofuran solution (every 100ml zymotic fluid adds the 100ml tetrahydrofuran solution), the sonic oscillation smudge cells obtains the aqueous isopropanol of cyanine, vacuum drying promptly obtains the crude extract of cyanine, and productive rate is the thick product of 5g cyanine/L zymotic fluid.
The physicochemical characteristics of embodiment 5, cyanine of the present invention detects
1, photostability
The pigment ethanolic solution of undried among the 2ml embodiment 2 is added to light transmission preferably in the 10ml teat glass, in case the ethanol volatilization places outdoor natural daylight under and shines, the percent of loss of pigment is as shown in table 1 behind the illumination certain hour with sealing the sealing of film and paraffin.
The percent of loss of table 1. pigment under natural light irradiation
| The strong natural light irradiation time (h) | B1 bacterium cyanine | ||
| Pigment concentration (g/L) | Percent of loss (%) | Pigmentary colours | |
| ??0 | ???0.587 | ??0 | Dark blue |
| ??12 | ???0.130 | ??77.9 | Pale yellow |
| ??24 | ???0.042 | ??92.8 | Pale yellow |
By the data in the table as can be seen, the photostability of pigment in ethanolic solution is relatively poor, and after 24 hours, the pigment loss surpasses 90%, and color also changes, and becomes light yellow by original navy blue.
2, heat endurance
With 1ml concentration is that pigment ethanol (concentration of alcohol is 100%) solution and the concentration of 0.5g/L is that the 1.0g/L aqueous solution places the 1.5ml polypropylene tube to carry out Temperature Treatment respectively, qualitatively judges the heat endurance of pigment according to the percent of loss of pigment.Take three kinds of Temperature Treatment modes: airbath is heated 4h respectively for 60 ℃, 100 ℃, 121 ℃ of heating of high steam pot 15min, and heat endurance result is as shown in table 2.The result shows, when with ethanol during as solvent, the pigment absorbance is loss not.When with water during as solvent, the pigment loss when operative temperature is 100 ℃ is greater than 60 ℃.
The percent of loss of table 2. pigment under different temperatures
| Solvent | Temperature | B1 bacterium cyanine | |
| Absorbance (576nm) | Percent of loss (%) | ||
| Ethanol | Original | ?0.853 | ????0 |
| ?60℃??4h | ?0.955 | ????-11.96 | |
| ?100℃?4h | ?1.174 | ????-60.57 | |
| ?121℃?15min | ?0.905 | ????-6.10 | |
| Water | Original | ?0.952 | ????0 |
| ?60℃??4h | ?0.892 | ????6.30 | |
| ?100℃?4h | ?0.619 | ????34.98 | |
| ?121℃?15min | ?0.684 | ????28.15 | |
The pigment extract of undried among the embodiment 2 is preserved under 4 ℃ in refrigerator with the masking foil shading, and in 30 days, pigmentary colours do not change substantially at least.
3, the ph stability of pigment
The pigment ethanolic solution of undried adds HCl in 5ml embodiment 2, and when Solution H Cl concentration was 2.4mol/L, solution colour became green by original navy blue; Add NaOH, when solution NaOH concentration was 0.24mol/L, solution colour became yellow by original navy blue.
4, dissolubility and the dissolving color of pigment in different solvents
Gained cyanine shown color in different solvents has a great difference.With water, benzinum is solvent, is shown as dusty blue; As solvent, be shown as blueness with methyl alcohol, ethanol, isopropyl alcohol, 50% methyl alcohol, 50% ethanol, 50% isopropyl alcohol, 50% acetone, 50% oxolane; As solvent, be shown as purple with acetone, ethyl acetate, oxolane.
Can observe directly a certain amount of pigment crude extract by range estimation can be dissolved in methyl alcohol very soon, and dissolubility is best; Oxolane and ethanol take second place; Water and benzinum are the poorest to the dissolubility of pigment crude extract.
Claims (10)
1, produces cyanine pseudomonas fluorescens (Pseudomonas fluorescens) B1 CGMCC № 1194.
2, a kind of method of producing cyanine comprises the steps: 1) strain culturing: will produce cyanine pseudomonas fluorescens (Pseudomonas fluorescens) B1 CGMCC № 1194 and obtain thalline in 24-240 hour 4 ℃ of-30 ℃ of condition bottom fermentations cultivations; 2) broken somatic cells obtains cyanine.
3, method according to claim 2 is characterized in that: the medium of described fermented and cultured contains following substances: tryptone 8-12g, and yeast extract 4-6g, glucose 0.8-1.2g, dipotassium hydrogen phosphate 2-4g adds water to 1000ml.
4, method according to claim 3 is characterized in that: described medium contains following substances: tryptone 10g, and yeast extract 5g, glucose 1g, dipotassium hydrogen phosphate 3g adds water to 1000ml, and pH is 6-7.6.
5, according to claim 2 or 3 or 4 described methods, it is characterized in that: described product cyanine pseudomonas fluorescens (Pseudomonas fluorescens) B1 CGMCC № 1194 also cultivates through dull and stereotyped before inoculation, described plating medium contains following substances: tryptone 5g, yeast extract 2.5g, NaCl 2.5g, agar 10g adds water to 500ml; Described plating medium pH is 7.2-7.6.
6, according to claim 2 or 3 or 4 described methods, it is characterized in that: described thalline obtains by centrifugal in the culture fluid that ferments.
7, according to claim 2 or 3 or 4 described methods, it is characterized in that: the process that described broken somatic cells obtains cyanine is to add the oxolane of the isopropyl alcohol of ethanol that concentration is 5-100%, 5-100% or 5-100% as solvent in thalline, obtain the solution of cyanine behind the sonic oscillation, obtain cyanine after the drying.
8, method according to claim 7 is characterized in that: described solvent is that concentration is the ethanol of 5-100%.
9, method according to claim 8 is characterized in that: described amount of alcohol added is that every 100ml zymotic fluid adds 20-300ml ethanol.
10, method according to claim 7 is characterized in that: the described dry vacuum drying of adopting.
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