CN113234769B - Biological preparation method of blue pigment - Google Patents

Biological preparation method of blue pigment Download PDF

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CN113234769B
CN113234769B CN202110532132.9A CN202110532132A CN113234769B CN 113234769 B CN113234769 B CN 113234769B CN 202110532132 A CN202110532132 A CN 202110532132A CN 113234769 B CN113234769 B CN 113234769B
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孙磊
李亚
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Nanjing Hegu Life Biotechnology Co ltd
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Abstract

The invention discloses a biological preparation method of blue pigment, which comprises the steps of taking recombinant escherichia coli as a strain, activating and activating the escherichia coli, inoculating the activated escherichia coli into a fermentation culture medium for amplification culture, then placing the escherichia coli into a fermentation tank for continuous fermentation, introducing sterile air during the fermentation process, maintaining the pH value of the fermentation liquid within the range of 6.8-7.2, supplementing materials when the content of residual sugar in the fermentation tank is less than 1g/L, continuing to ferment for a period of time, and separating and purifying to obtain the blue pigment. The biological preparation method of the blue pigment realizes the mass production of the blue pigment, has less pollution, is economic and convenient, and is suitable for the fields of cosmetics, food, health care and the like.

Description

Biological preparation method of blue pigment
Technical Field
The invention relates to a biological preparation method of blue pigment, belonging to the technical field of blue pigment preparation.
Background
The blue pigment is a nontoxic natural microbial blue pigment, has pure color light and high dyeing intensity, and can be applied to food pigments due to natural and nontoxic properties. The molecular formula is C10H8O4N4Relative molecular mass 248, the structural formula is as follows:
Figure 129589DEST_PATH_IMAGE001
. The blue pigment is mainly applied to the dye industry, the dye used in the dye industry at present mainly adopts industrial dye indigo, but the production of the industrial indigo mainly adopts a chemical synthesis method, the produced pollution is serious, the method is one of important sources of industrial pollution, the pollution of the blue pigment produced by microbial fermentation is less, the cost is low, and the method gradually becomes an alternative scheme of the industrial indigo.
The main production mode of the blue pigment at present is to insert the indigo synthetic gene into a proper escherichia coli expression vector (such as pET 28) and transfer a plasmid containing a target gene fragment into an expression host escherichia coli through transformation, and the blue pigment is generally produced in a mode of shaking a flask, but industrialization cannot be realized. Therefore, the invention explores a biological preparation method of the blue pigment, can realize mass production of the blue pigment, is economic and safe, and can be applied in a wide range.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a biological preparation method of blue pigment, which comprises the steps of activating and inoculating recombinant escherichia coli, fermenting in a fermentation tank, performing induced expression, supplementing materials and fermenting to prepare the blue pigment, and realizing mass production of the blue pigment; less pollution, economy, convenience, quality safety and temperature; can be widely applied to the fields of cosmetics, foods, sanitary medical treatment and the like.
In order to achieve the purpose, the invention adopts the following technical scheme: a biological preparation method of blue pigment comprises the steps of taking recombinant Escherichia coli as a strain, activating, inoculating the activated Escherichia coli into a fermentation culture medium for amplification culture, then placing the Escherichia coli into a fermentation tank for continuous fermentation, introducing sterile air during the fermentation process, maintaining the pH value of fermentation liquor within the range of 6.8-7.2, supplementing materials when the content of residual sugar in the fermentation tank is less than 1g/L, continuing the fermentation for a period of time, and separating and purifying to obtain the blue pigment.
The preparation method of the blue pigment comprises the following specific steps:
(1) activating strains: placing the recombinant escherichia coli strain in the conditions of the temperature of 35-38 ℃ and the rotating speed of 170-200 rpm for overnight culture to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated escherichia coli into a fermentation medium for amplification culture at the temperature of 35-38 ℃ until the bacteria density OD600 value is 12-18, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid into a fermentation tank for continuous fermentation at the temperature of 35-38 ℃ and the rotating speed of 200-1200 rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation amount is 2-7 m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L for feeding;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting residual sugar and nitrogen in fermentation tankThe content is less than 1g/L, glucose is manually added to enable the amino nitrogen content to be within the range of 1-2 g/L, continuous fermentation is carried out for 6-10 hours at the temperature of 35-38 ℃ and the rotating speed of 200-1200 rpm, sterile air is introduced during the fermentation process, and the fermentation ventilation volume is 2-7 m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment, removing water to obtain a blue pigment crude product, adding hot water with the temperature of 65-80 ℃ into the blue pigment crude product for pulping for 2 hours, filtering and washing with the hot water until the filtrate is neutral, and drying the trapped substances at a low temperature to obtain the blue pigment product.
Wherein, in the step (5), the content of the blue pigment in the fermentation liquor containing the blue pigment is 1.95-2.20 g/L.
Wherein, in the step (6), the temperature of freeze drying is-60 to-55 ℃; the low-temperature drying temperature is 30-35 ℃.
Wherein, in the step (4), the inducer is IPTG inducer, and the concentration of the IPTG inducer is 200 mM.
More preferably, the temperature in step (1), step (2), step (3) and step (5) is 37 ℃, the density OD600 value in step (2) is 15, and the fermentation aeration rate in step (3) and step (5) is 5m3/h。
In the invention, the activated escherichia coli is inoculated to a fermentation medium according to the inoculation amount of 1-10% (v/v).
In the present invention, the fermentation medium comprises 5.8 g/L tryptone, 3.6 g/L yeast extract, 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerin.
The invention has the beneficial effects that: the method comprises the steps of activating and inoculating recombinant escherichia coli, fermenting in a fermentation tank, performing induced expression, supplementing materials and fermenting to prepare the blue pigment, wherein the fermentation tank replaces a common shake flask in the fermentation process, so that the mass production of the blue pigment is realized; in the preparation method, the yield of the blue pigment in the fermentation liquor reaches about 2g/L, even 2.2g/L, and the yield of the blue pigment is increased; the preparation method has the advantages of less pollution, economy, convenience, safe and stable quality; the prepared blue pigment product can be widely applied to the fields of cosmetics, foods, sanitary medical treatment and the like.
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FIG. 1 is a standard curve of the blue pigment of the present invention in DMSO solution.
Detailed Description
In order to more clearly and completely illustrate the present invention, the following examples are given by way of illustration of the present invention, and are not intended to limit the present invention.
Example 1
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 35 ℃ and the rotating speed of 170rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 1% (v/v), and performing amplification culture at 35 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacteria density OD600 value is 12, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 35 deg.C and 200rpm, introducing sterile air during fermentation with ventilation of 2m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: in the growth period of the strain, adding an IPTG inducer with the concentration of 200mM for inducing and expressing protein;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 6 hours at the temperature of 35 ℃ and the rotating speed of 200rpm, introducing sterile air in the fermentation process,the fermentation aeration is 2m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Example 2
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 38 ℃ and the rotating speed of 200rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculum size of 10% (v/v), and performing amplification culture at 38 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 18, and obtaining an inoculation seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 38 deg.C and 1200rpm, introducing sterile air during fermentation with fermentation ventilation of 7m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 10 hours at the temperature of 38 ℃ and the rotating speed of 1200rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 7m3H, and ammonia water is used for maintaining the pH value of the fermentation liquor6.8-7.2, and obtaining fermentation liquor containing blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at the temperature of-55 ℃, removing water to obtain a crude blue pigment product, adding hot water at the temperature of 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at a low temperature of 30 ℃ to obtain the blue pigment product.
Example 3
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 36 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 8% (v/v), and performing amplification culture at 36 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 16, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 36 deg.C and 800rpm, introducing sterile air during fermentation with ventilation of 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 6 hours at the temperature of 36 ℃ and the rotating speed of 800rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 3.5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at the temperature of-58 ℃, removing water to obtain a crude blue pigment product, adding hot water at the temperature of 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at a low temperature of 33 ℃ to obtain the blue pigment product.
Example 4
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 37 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 6% (v/v), and performing amplification culture at 37 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 15, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 37 deg.C and 500rpm, introducing sterile air during fermentation with ventilation of 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 7 hours at the temperature of 37 ℃ and the rotating speed of 500rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Example 5
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 38 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 6% (v/v), and performing amplification culture at 38 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 15, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 38 deg.C and 500rpm, introducing sterile air during fermentation with ventilation of 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 8 hours at the temperature of 38 ℃ and the rotating speed of 500rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Example 6
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 36 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 6% (v/v), and performing amplification culture at 36 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 15, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 36 deg.C and 500rpm, introducing sterile air during fermentation with ventilation of 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 8 hours at the temperature of 36 ℃ and the rotating speed of 500rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Example 7
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 37 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 6% (v/v), and performing amplification culture at 37 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 16, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 37 deg.C and 500rpm, introducing sterile air during fermentation with ventilation of 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 7 hours at the temperature of 37 ℃ and the rotating speed of 500rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Example 8
A biological preparation method of blue pigment comprises the following specific steps:
(1) activating strains: adding the recombinant escherichia coli strain into a medium shake tube, and placing the medium shake tube on a horizontal shaker for overnight culture at the temperature of 37 ℃ and the rotating speed of 180rpm to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated Escherichia coli into 1L of fermentation medium in conical flask according to inoculation amount of 6% (v/v), and performing amplification culture at 37 deg.C, wherein the fermentation medium comprises tryptone 5.8 g/L, yeast extract 3.6 g/L, and yeast extract 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerol until the bacterial density OD600 value is 15, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid in a fermentation tank for continuous fermentation at 37 deg.C and 500rpm, introducing sterile air during fermentation with fermentation ventilation of 7m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L, and supplementing materials;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 7 hours at the temperature of 37 ℃ and the rotating speed of 500rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation is 5m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment at-60 ℃, removing water to obtain a crude blue pigment product, adding hot water at 65-80 ℃ into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the solid at low temperature of 35 ℃ to obtain the blue pigment product.
Analysis of Experimental results
1. Standard curve of blue pigment
Detecting and analyzing by using a spectrophotometer under the following detection conditions: the wavelength is 602 nm;
adding 1ml DMSO into 1mg blue pigment for ultrasonic dissolution to obtain a blue pigment with a final concentration (C) of 1mg/ml, diluting 1mg/ml blue pigment standard solution to obtain blue pigment with concentrations (C) of 0.01mg/ml, 0.02 mg/ml, 0.06 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.20 mg/ml, 0.25mg/ml and 0.50mg/ml, measuring the corresponding absorbances (A) under the above detection conditions, and fitting to obtain a standard curve with the concentration (C) of the blue pigment as abscissa and the absorbance (A) as ordinate, wherein the result is shown in FIG. 1, and the standard curve of the blue pigment is A = 1.0621C +0.0852, wherein R is R2=0.99。
2. Blue pigment content in fermentation broth
1ml of the fermentation liquid from examples 1 to 8 was added to different centrifuge tubes: centrifuging each centrifuge tube filled with fermentation liquor at the rotation speed of 13000 rpm for 10 min, removing supernatant of the fermentation liquor, adding 1ml of ultrapure water for oscillation suspension, then centrifuging at the rotation speed of 13000 rpm for 10 min, removing supernatant, repeating the process for 3 times, removing water-soluble substances in precipitates, then adding 10ml of DMSO, performing ultrasonic dissolution, centrifuging at 3000 rpm for 5 min in a centrifuge, removing substances insoluble in DMSO to obtain a DMSO solution containing pure blue pigment, then detecting by using a spectrophotometer according to the detection conditions to obtain absorbance (A), calculating the concentration (C) of the blue pigment in the 10ml of DMSO solution according to the standard curve, then calculating the yield of the blue pigment in 1ml of fermentation liquor, and further calculating the content (g/L) of the blue pigment in 1L of fermentation liquor, wherein the results are shown in the following table 2:
example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8
Absorbance (A) 0.2923 0.2938 0.2964 0.3189 0.2987 0.2994 0.3138 0.3072
Concentration of blue pigment in 10ml DMSO solution (C, mg/ml) 0.195 0.196 0.199 0.22 0.201 0.202 0.215 0.209
1L content (g/L) of blue pigment in fermentation broth 1.95 1.96 1.99 2.2 2.01 2.02 2.15 2.09
As can be seen from Table 2 above, the yield of blue pigment in the fermentation broth obtained in the preparation processes of examples 1 to 8 reached about 2g/L, and even in example 4, the temperature was maintained at 37 ℃ and the fermentation aeration rate was 5m3When the density OD600 value of inoculated seed liquid bacteria is 15, the content of the blue pigment in the obtained fermentation liquid reaches 2.2g/L, and the yield of the blue pigment is improved more obviously; the biological preparation method of the blue pigment has the advantages that the fermentation tank replaces a common shake flask in the fermentation process, the mass production of the blue pigment is realized, the preparation method has less pollution, is economical and convenient, has safe and stable quality, and can be widely applied to the fields of cosmetics, food, health care and the like.
Finally, it should be noted that the above embodiments are only used for illustrating and not limiting the technical solutions of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the present invention without departing from the spirit and scope of the present invention, and all modifications or partial substitutions should be covered by the scope of the claims of the present invention.

Claims (7)

1. A biological preparation method of blue pigment is characterized in that recombinant Escherichia coli is used as a strain, activated and activated Escherichia coli is inoculated into a fermentation medium for amplification culture, then the Escherichia coli is placed in a fermentation tank for continuous fermentation, sterile air is introduced during the fermentation process, the pH value of the fermentation liquid is maintained within the range of 6.8-7.2, when the content of residual sugar in the fermentation tank is less than 1g/L, the materials are supplemented, the fermentation is continued for a period of time, and the blue pigment is obtained after separation and purification;
the preparation method comprises the following specific steps:
(1) activating strains: placing the recombinant escherichia coli strain in the conditions of the temperature of 35-38 ℃ and the rotating speed of 170-200 rpm for overnight culture to obtain activated escherichia coli;
(2) and (3) amplification culture: inoculating activated escherichia coli into a fermentation medium for amplification culture at the temperature of 35-38 ℃ until the bacteria density OD600 value is 12-18, and obtaining an inoculated seed solution;
(3) fermentation: placing the inoculated seed liquid into a fermentation tank for continuous fermentation at the temperature of 35-38 ℃ and the rotating speed of 200-1200 rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation amount is 2-7 m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water until the content of residual sugar in the fermentation tank is less than 1g/L for feeding;
(4) inducing expression: adding an inducer to induce and express protein in the growth period of the strain;
(5) feeding and fermenting: detecting the content of residual sugar and nitrogen in a fermentation tank to be less than 1g/L, manually adding glucose to ensure that the content of amino nitrogen is within the range of 1-2 g/L, continuously fermenting for 6-10 hours at the temperature of 35-38 ℃ and the rotating speed of 200-1200 rpm, introducing sterile air in the fermentation process, wherein the fermentation ventilation volume is 2-7 m3Maintaining the pH value of the fermentation liquor to be 6.8-7.2 by using ammonia water to obtain fermentation liquor containing the blue pigment;
(6) separation and purification: freeze-drying the fermentation liquor containing the blue pigment, removing water to obtain a crude blue pigment product, adding hot water into the crude blue pigment product for pulping for 2 hours, filtering, washing with hot water until the filtrate is neutral, and drying the trapped substances at low temperature to obtain the blue pigment product.
2. The method of claim 1, wherein in step (5), the fermentation broth containing the blue pigment has a blue pigment content of 1.95-2.20 g/L.
3. The biological preparation method of a blue pigment according to claim 1, wherein in the step (6), the temperature of freeze drying is-60 to-55 ℃; the low-temperature drying temperature is 30-35 ℃.
4. The process according to claim 1, wherein in step (4), the inducer is IPTG inducer; the concentration of the IPTG inducer is 200 mM.
5. The process according to claim 1, wherein the temperature in step (1), step (2), step (3) and step (5) is 37 ℃, the density OD600 in step (2) is 15, and the aeration rate in step (3) and step (5) is 5m3/h。
6. The method for biologically preparing a blue pigment according to claim 1, wherein the activated Escherichia coli is inoculated into the fermentation medium in an inoculum size of 1% to 10% (v/v).
7. The process of claim 1, wherein the fermentation medium comprises 5.8 g/L tryptone, 3.6 g/L yeast extract, 1.4 g/L K2HPO4、2.2 g/L KH2PO41 ml/L of glycerin.
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