Natural red pigment and fermentation process thereof

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses a natural haematochrome and a fermentation process thereof. According to the present invention, the fermentation process is innovated, the pH value is regulated to induce the engineering bacteria so as to convert the glutamic acid into the N-acetylglutamine, and the N-acetylglutamine is artificially added to inhibit the generation of the N-acetyl-observation blue product and promote the generation of the N, N '-diacetyl-observation blue (natural haematochrome); compared with a natural blue pigment N-acetyl-observation blue, the natural red pigment N, N '-diacetyl-observation blue has the maximum absorption wavelength of 499 + / -2nm, and the colored light of the natural red pigment N, N'-diacetyl-observation blue can be used as one of three primary colors, can be used for dyeing, and also can be matched with other colors of dyes.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of microbial fermentation, and in particular to a natural red pigment and a fermentation process thereof. BACKGROUND

[0002] Red is one of the three primary colors, and it has the lowest frequency and the longest wavelength in the visible light band, so it has the strongest diffraction ability and the strongest ability to penetrate obstacles. Red has a strong impact on vision and can form a strong contrast with many colors, so red pigments are widely used.

[0003] In the early days, ttICHARD KUHN published an article in Archly fiir Mikrobiologie 51, 71-84 (1965) in which indigo was used as a reaction substrate, acetic anhydride was added as an acetylation reagent, and boron trifluoride ether was used as a catalyst to perform acetylation of indigo. Currently, patent publication CN118184576B discloses a natural blue pigment and a biosynthesis method thereof, which synthesizes N-acetyl indigo from one molecule of glutamine and one molecule of N-acetyl glutamine. However, according to the fermentation method, only the natural blue pigment N-acetyl indigo can be produced, and the natural red pigment cannot be obtained by fermentation. Compared with chemical synthesis of natural red pigment, biosynthesis of natural red pigment can greatly reduce the cost and is safe and environmentally friendly. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide a natural red pigment and a fermentation process thereof. Through an innovative fermentation process, the present application regulates the pH to induce the engineered bacteria to convert a large amount of glutamic acid into N-acetyl glutamine, and then artificially adds N-acetyl glutamine to inhibit the production of N-acetyl indigo product and promote the generation of N,N'-diacetyl-indigo (natural red pigment). Compared with the natural blue pigment N-acetyl indigo, the natural red pigment N,N'-diacetyl-indigo has a maximum absorption wavelength of 499±2 nm, and its color light can be used as one of the three primary colors. In addition to being used for dyeing, it can also be used for color mixing with other color dyes.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a natural red pigment, and the structural formula of the natural red pigment is shown as formula (I);

[0007]

[0008] The Chinese name of the above-mentioned natural red pigment is N,N'-diacetyl-indigo.

[0009] The present application also provides a preferred embodiment of the fermentation process of the above-mentioned natural red pigment, which comprises the following steps:

[0010] S1, inoculating the activated recombinant strain seed fermentation liquor into a basic fermentation medium to perform first-stage fermentation culture, maintaining pH at 6.5-7.2, controlling temperature at 25-30°C, and culturing for 0-24 h to obtain a first-stage fermentation liquor;

[0011] S2, maintaining pH of the first-stage fermentation liquor at 7.2-8.0, controlling temperature at 20-25°C, adding a synthetic enzyme inducer and N-acetyl glutamine to perform second-stage fermentation culture, and culturing for 24-30 h to obtain a second-stage fermentation liquor;

[0012] S3, maintaining pH of the second-stage fermentation liquor at 8.0-9.0, controlling temperature at 37-45°C, adding a synthetic enzyme inducer and lactose, and continuously adding N-acetyl glutamine to perform third-stage fermentation culture, and culturing for 30-72 h to obtain a third-stage fermentation liquor, and fermenting until the yield of natural red pigment stops increasing, and ending the fermentation.

[0013] The fermentation production of L-glutamine must be completed in two processes, i.e., a long bacterial period and a production period. The environmental requirements of the long bacterial period and the production period are completely different. After the bacterial amount reaches the optimum concentration, the long bacterial period must be transferred to the production period by regulating pH. That is, the large accumulation of L-glutamine in the L-glutamine fermentation is not a natural process, but a process of artificially forced pH regulation to make the bacteria enter the production period.

[0014] Through a large number of experiments of the present applicant, it is found that the control of pH in the fermentation process directly affects the synthesis efficiency of N,N'-diacetyl-obscurin precursor N-acetyl glutamine. Only under weak alkaline conditions, the corresponding synthetic enzyme activity is high. However, too high fermentation pH will cause slow growth of the strain and inhibit the acid production ability of the strain, thereby reducing the yield. Therefore, a stage pH control strategy is adopted, i.e., the first-stage fermentation is performed for 0-24 h, pH is controlled at 6.5-7.2; the second-stage fermentation is performed for 24-30 h, pH is controlled at 7.2-8.0; and the third-stage fermentation is performed for 30-72 h, pH is controlled at 8.0-9.0. The growth of the recombinant strain, the synthesis of N-acetyl glutamine and N,N'-diacetyl-obscurin are performed under different suitable pH conditions, respectively, so as to promote the yield of N,N'-diacetyl-obscurin to reach the highest.

[0015] Through the fermentation production process of the present application, most of glutamic acid is converted into N-acetyl glutamine, and the biosynthesis of N,N'-diacetyl-obscurin is successfully realized, the content ratio reaches 95%, and the total content of by-products obscurin and N-acetyl obscurin is only 5%, which greatly reduces the production and purification cost, and realizes the biosynthesis of N,N'-diacetyl-obscurin.

[0016] As a preferred embodiment of the fermentation process of the natural red pigment described in the present application, the basic fermentation medium comprises the following components in the following mass concentrations:

[0017] Saccharomyces cervisiae powder P801 5-10 g / L, glucose 70-90 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 0.2-1 g / L, magnesium sulfate heptahydrate 0.2-0.4 g / L, manganese sulfate monohydrate 0.01-0.02 g / L, biotin 2-4 μg / L, VB1 0.2-0.4 mg / L, sodium glutamate 1-2 g / L, water as the solvent, and pH 7.0.

[0018] As a preferred embodiment of the fermentation process of the natural red pigment described in the present application, in the step S1, the conditions of the first-stage fermentation culture further include a dissolved oxygen of 25-35%;

[0019] In the step S2, the conditions of the second-stage fermentation culture further include a dissolved oxygen of 20-25%;

[0020] In the step S3, the conditions of the third-stage fermentation culture further include a dissolved oxygen of 20-30%.

[0021] The first-stage fermentation culture of the present application further includes the above fermentation conditions, which can promote the rapid growth of the strain. The first-stage fermentation culture is carried out under the above fermentation conditions for 24 h, and after the strain enters the late logarithmic growth phase (about OD 600nm = 30), a first-stage fermentation liquor is obtained.

[0022] As a preferred embodiment of the fermentation process of the natural red pigment described in the present application, in the step S2, the second-stage fermentation culture is adjusted to a pH of 7.2-8.0 by supplementing ammonia;

[0023] In the step S3, the third-stage fermentation culture is adjusted to a pH of 8.0-9.0 by supplementing ammonia.

[0024] In the technical solution of the present application, ammonia has two functions in fermentation, i.e., adjusting pH and supplementing NH 4+ The amount of ammonia supplement is based on the pH, and the pH is maintained in a range suitable for the production of N,N'-diacetyl-azorubin precursor and the microgrowth of the strain by supplementing ammonia.

[0025] The second-stage fermentation culture of the present application adopts the above fermentation conditions, and requires the addition of a synthetic enzyme inducer and exogenous N-acetyl glutamine, which can induce the expression of product synthesis enzyme and promote the synthesis of the product, thereby improving the fermentation yield of N,N'-diacetyl-azorubin.

[0026] As a preferred embodiment of the fermentation process of the natural red pigment described in the present application, the synthetic enzyme inducer comprises IPTG.

[0027] The concentration of IPTG in the step S2 is 0.05-0.2 mmol / L.

[0028] The concentration of IPTG in the step S3 is 0.3-0.5 mmol / L, and the concentration of lactose is 5-10 g / L.

[0029] The addition amount of N-acetyl glutamine is 2-5 g / L.

[0030] The N-acetyl glutamine with the above-mentioned addition amount can promote the synthesis of N,N'-diacetyl-obscurine and inhibit the synthesis of N-acetyl obscurine in the application. The N-acetyl glutamine is dissolved in the feed medium and added together with the feed medium.

[0031] As a preferred embodiment of the fermentation process of the natural red pigment, the initial concentration of glucose in the first-stage fermentation culture is 8%.

[0032] The concentration of glucose in the second-stage fermentation culture and the third-stage fermentation culture is 5-10 g / L.

[0033] Different concentrations of glucose are used in the three different fermentation stages in the application, which is beneficial to the growth of the bacteria.

[0034] As a preferred embodiment of the fermentation process of the natural red pigment, the concentration of glucose in the basic medium in the second-stage fermentation culture is lower than 5 g / L, and the feed medium is added.

[0035] As a preferred embodiment of the fermentation process of the natural red pigment, the feed medium comprises: glucose 5-10 g / L, magnesium sulfate heptahydrate 0.2-0.4 g / L, ammonium chloride 4-8 g / L, N-acetyl glutamine 2-5 g / L, and the solvent is water.

[0036] As a preferred embodiment of the fermentation process of the natural red pigment, the recombinant strain contains obscurine synthase encoding gene bpsA, 4'-phosphopantetheine adenylyltransferase encoding gene EntD, glutamine synthetase encoding gene glnA, and N-acetyl glutamate synthetase encoding gene argA.

[0037] In some specific embodiments, the recombinant strain is the recombinant Escherichia coli HG-N-Idg06 of the patent with the publication number CN118360308B (a construction method and application of a metabolic engineering bacterium for biosynthesis of N-acetyl obscurine using glutamic acid as a substrate).

[0038] The application also provides the application of the above-mentioned natural red pigment in fabric dyeing.

[0039] The natural red pigment N,N'-diacetyl-obscurin provided by the application has a maximum absorption wavelength of 499±2nm, and its color light can be used as one of the three primary colors, and can be used for dyeing and color matching with other color dyes.

[0040] Compared with the prior art, the application has the following beneficial effects:

[0041] The application provides a natural red pigment and a fermentation process thereof. The fermentation process comprises a first stage of bacterial proliferation, a second stage of biosynthesis of N-acetyl glutamine, and a third stage of biosynthesis of N,N'-diacetyl-obscurin. Compared with a method for catalyzing glutamic acid to generate N-acetyl obscurin by using resting whole cells, in the fermentation process, glutamic acid is acylated into N-acetyl glutamine by adjusting the pH to 7.2-8.0 in the second stage, and the pH is adjusted to 8.0-9.0 and exogenous N-acetyl glutamine is added in the third stage to promote the engineered bacteria to synthesize N,N'-diacetyl-obscurin by using two molecules of N-acetyl glutamine, while inhibiting the engineered bacteria to synthesize obscurin by using two molecules of glutamine or to synthesize N-acetyl obscurin by using one molecule of glutamine and one molecule of N-acetyl glutamine. The content of the produced N,N'-diacetyl-obscurin can reach 95%, and the total content of by-products obscurin and N-acetyl obscurin is only 5%, which greatly reduces the production and purification cost, and successfully realizes the biosynthesis of N,N'-diacetyl-obscurin. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The biosynthesis route of N,N'-diacetyl-obscurin is shown in the following figure:

[0043] Figure 2 The HPLC spectrum of the product obtained in the example is shown in the following figure:

[0044] Figure 3 The ultraviolet absorption spectrum of the product obtained in the example is shown in the following figure:

[0045] Figure 4 The N,N'-diacetyl-obscurin prepared by the application and the dissolution state in different solvents are shown in the following figure:

[0046] Figure 5 The high-resolution mass spectrum of N,N'-diacetyl-obscurin is shown in the following figure:

[0047] Figure 6 The nuclear magnetic resonance hydrogen spectrum of N,N'-diacetyl-obscurin is shown in the following figure:

[0048] Figure 7 The nuclear magnetic resonance carbon spectrum of N,N'-diacetyl-obscurin is shown in the following figure:

[0049] Figure 8 The two-dimensional nuclear magnetic resonance HSQC spectrum of N,N'-diacetyl-obscurin is shown in the following figure:

[0050] Figure 9 State diagram of N,N'-diacetyl-obscurine in DMSO solution at different temperatures;

[0051] Figure 10 Dyeing cotton fabric diagram of N,N'-diacetyl-obscurine at different concentrations. DETAILED DESCRIPTION

[0052] For the purpose of better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific embodiments.

[0053] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified, and the components used in each parallel experiment are the same.

[0054] The materials used in the following examples were provided by Nanjing Hegot Life Biotechnology Co., Ltd.

[0055] The recombinant strain used in the following examples and comparative examples is: containing obscurine synthase encoding gene bpsA, 4'-phosphopantetheine transferase encoding gene EntD, glutamine synthetase encoding gene glnA and N-acetylglutamate synthetase encoding gene argA.

[0056] The recombinant strain is: the recombinant Escherichia coli HG-N-Idg06 of the patent with publication number CN118360308B (a method for constructing N-acetyl-obscurine biosynthetic metabolic engineering bacteria using glutamic acid as a substrate and application).

[0057] The LB medium includes the following components with mass concentration: sodium chloride 10 g / L, proteose peptone 10 g / L, and yeast extract powder 5 g / L.

[0058] The following examples and comparative examples use a 5L fermenter for fermentation.

[0059] In the following examples, the high performance liquid chromatography detection method of the product is as follows.

[0060] Chromatographic conditions: mobile phase: methanol and pure water gradient elution, gradient table 1 as follows:

[0061] Table 1

[0062] Time min A (pure water) % B (methanol) % 0 70 30 10 40 60 14 70 30 19 70 30

[0063] Wavelength 600 nm, flow rate 1.0 mL / min, sample solution: DMSO, sample volume: 10 μL, column temperature 35 °C, run time 20 min. Column: Galasil EF-C18M 4.6 mm id x 250 mm L (SN B06211801).

[0064] Example 1, a natural red pigment and its fermentation process

[0065] The present embodiment provides a fermentation process of natural red pigment, comprising the following steps:

[0066] (1) The frozen E. coli recombinant strain HG-N-Idg06 bacterial liquid was thawed and then cultured in LB medium containing 50 μg / mL kanamycin and 50 μg / mL streptomycin at 37 °C and 200 rpm for 8 h to obtain an activated bacterial liquid;

[0067] (2) 350 μL of the primary seed liquid obtained in step (1) was inoculated into LB medium at an inoculation amount of 0.1% by flame inoculation, and then cultured at 35 °C and 220 rpm for 10 h to obtain a secondary seed liquid;

[0068] (3) 1.2 L of basic fermentation medium (Saint-Quentin yeast powder P801 8 g / L, glucose 80 g / L, ammonium sulfate 15 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.3 g / L, manganese sulfate monohydrate 0.015 g / L, biotin 3 μg / L, VB1 0.3 mg / L, sodium glutamate 1.5 g / L, solvent water, pH 7.0) was added to a 5 L fermenter, and the secondary seed liquid obtained in step (2) was added to the 5 L fermenter for first-stage fermentation culture, the pH was controlled at 7.0, the temperature was controlled at 27 °C, the rotation speed was controlled at 400 r / min, and the dissolved oxygen was controlled at 30%, and the culture was carried out for 0-24 h, and the bacteria entered the late logarithmic growth phase (about OD600nm=30) to obtain a first-stage fermentation liquid;

[0069] (4) The fermentation conditions of the first-stage fermentation liquid were adjusted for second-stage fermentation culture, when the OD600nm=30 at 24 h, the temperature was lowered to 22 °C, and 0.1 mmol / L IPTG was added for inducible expression of the synthesis enzyme, 3.5 g / L of exogenous N-acetylglutamine was added to inhibit the generation of N-acetyl cyanine, the rotation speed was 300 rpm, and the dissolved oxygen was maintained at 20%. The pH was adjusted to 7.5 by supplementing ammonia. When the glucose concentration in the fermentation liquid was lower than 5 g / L, the feeding medium (including the following components with mass concentration: glucose 7 g / L, magnesium sulfate heptahydrate 3 g / L, ammonium chloride 60 g / L, 3.5 g / L N-acetylglutamine, solvent water) was started to be added, and the glucose concentration was controlled at 5 g / L to obtain a second-stage fermentation liquid.

[0070] (5) fermentation to 30h, temperature to 40℃, pH to 8.5 by adding ammonia and adding IPTG with a concentration of 0.4mmol / L and lactose with a concentration of 8g / L for synthetic enzyme induction expression, continue to feed 3.5g / L of exogenous N-acetyl glutamine to inhibit N-acetyl cyanine generation and promote the generation of N,N'-diacetyl-cyanine, the rotation speed is 350rpm, and the dissolved oxygen is maintained at 25%. When the glucose concentration in the fermentation broth is less than 5g / L, start the flow feeding medium, and prepare the third stage fermentation broth.

[0071] (6) fermentation to 72h, N,N'-diacetyl-cyanine yield stops increasing and has the highest proportion, and fermentation ends. The maximum biomass OD600 during fermentation is 121.3. The final tank fermentation broth has a N,N'-diacetyl-cyanine yield of 13.52g / L, and the proportion is 96.7%.

[0072] Example 2, a natural red pigment and its fermentation process

[0073] The embodiment provides a fermentation process of a natural red pigment, including the following steps:

[0074] (1) After thawing the frozen Escherichia coli recombinant strain HG-N-Idg06 bacterial liquid, the bacterial liquid is cultured in LB medium containing 50μg / mL kanamycin and 50μg / mL streptomycin at 37℃ and 200rpm for 8h to obtain an activated bacterial liquid;

[0075] (2) Take 350μL of the primary seed liquid obtained in step (1) and flame inoculate into LB medium at an inoculation amount of 0.1%, and culture at 35℃ and 220rpm for 10h to obtain a secondary seed liquid;

[0076] (3) Add 1.2L of basic fermentation medium (Saint-Quentin yeast powder P801 8g / L, glucose 80g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.3g / L, manganese sulfate monohydrate 0.015g / L, biotin 3μg / L, VB1 0.3mg / L, sodium glutamate 1.5g / L, and water as a solvent, pH 7.0) to a 5L fermenter, and add the secondary seed liquid obtained in step (2) to the 5L fermenter for first-stage fermentation culture, the pH is controlled to be 7.2, the temperature is controlled to be 30℃, the rotation speed is 400r / min, and the dissolved oxygen is controlled to be 30%, and the culture is carried out for 0-24h, and the bacteria enter the late logarithmic growth phase (about OD600nm=30), and a first-stage fermentation broth is obtained;

[0077] (4) The fermentation conditions of the first-stage fermentation broth are adjusted for second-stage fermentation culture. When the OD600nm of 24h is 30, the temperature is lowered to 25℃ and 0.2mmol / L of IPTG is added for inducing expression of the synthetase, 5g / L of exogenous N-acetylglutamine is added to inhibit the generation of N-acetyltryptophan, the rotation speed is 350rpm, and the dissolved oxygen is maintained at 25%. The second-stage fermentation culture is adjusted to pH 8.0 by supplementing ammonia. When the glucose concentration in the fermentation broth is lower than 5g / L, the feeding medium (including the following components with the mass concentration: glucose 7g / L, magnesium sulfate heptahydrate 3g / L, ammonium chloride 60g / L, 5g / L N-acetylglutamine, and water as the solvent) is started to be added, the glucose concentration is controlled at 5g / L, and the second-stage fermentation broth is prepared.

[0078] (5) When the fermentation is performed to 30h, the temperature is raised to 45℃, the pH is adjusted to 9.0 by supplementing ammonia, 0.5mmol / L of IPTG and 10g / L of lactose are added for inducing expression of the synthetase, 5g / L of exogenous N-acetylglutamine is continuously added to inhibit the generation of N-acetyltryptophan and promote the generation of N,N’-diacetyl-tryptophan, the rotation speed is 400rpm, and the dissolved oxygen is maintained at 30%. When the glucose concentration in the fermentation broth is lower than 5g / L, the feeding medium is started to be added, and the third-stage fermentation broth is prepared.

[0079] (6) When the fermentation is performed to 72h, the yield of N,N’-diacetyl-tryptophan stops increasing and the proportion is the highest, and the fermentation is ended. The maximum biomass OD600 in the fermentation process is 118.4. The yield of N,N’-diacetyl-tryptophan in the final tank fermentation broth is 12.78g / L, and the proportion is 94.5%.

[0080] Example 3, a natural red pigment and its fermentation process

[0081] The embodiment provides a fermentation process of a natural red pigment, which comprises the following steps:

[0082] (1) After the frozen Escherichia coli recombinant strain HG-N-Idg06 bacterial liquid is thawed, the bacterial liquid is cultured in LB medium containing 50μg / mL kanamycin and 50μg / mL streptomycin at 37℃ and 200rpm for 8h, and the activated bacterial liquid is obtained;

[0083] (2) 350μL of the primary seed liquid obtained in step (1) is inoculated into LB medium by flame inoculation at an inoculation amount of 0.1%, and the secondary seed liquid is obtained by culturing at 35℃ and 220rpm for 10h;

[0084] (3) In a 5L fermenter, 1.2L of basic fermentation medium (Saccharomyces cerevisiae powder P801 8g / L, glucose 80g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.3g / L, manganese sulfate monohydrate 0.015g / L, biotin 3μg / L, VB1 0.3mg / L, sodium glutamate 1.5g / L, solvent water, pH 7.0) was added, and the secondary seed liquid obtained in step (2) was added to the 5L fermenter for first-stage fermentation culture. The pH was controlled at 6.5, the temperature was controlled at 25°C, the rotation speed was 350r / min, and the dissolved oxygen was controlled at 25%. After 0-24h of culture, the bacteria entered the late logarithmic growth phase (about OD600nm=30), and the first-stage fermentation liquid was obtained.

[0085] (4) The fermentation conditions of the first-stage fermentation liquid were adjusted for second-stage fermentation culture. When the OD600nm was 30 at 24h, the temperature was lowered to 20°C, and 0.05mmol / L of IPTG was added for inducible expression of the synthetase. 2g / L of exogenous N-acetylglutamine was added to inhibit the generation of N-acetyltryptophan. The rotation speed was 300rpm, and the dissolved oxygen was maintained at 20%. The pH was adjusted to 7.2 by supplementing ammonia. When the glucose concentration in the fermentation liquid was less than 5g / L, the feeding medium (including the following components with mass concentration: glucose 7g / L, magnesium sulfate heptahydrate 3g / L, ammonium chloride 60g / L, 2g / L N-acetylglutamine, solvent water) was started to be added, and the glucose concentration was controlled at 5g / L to obtain the second-stage fermentation liquid.

[0086] (5) At 30h of fermentation, the temperature was raised to 37°C, the pH was adjusted to 8.0 by supplementing ammonia, 0.3mmol / L of IPTG and 6g / L of lactose were added for inducible expression of the synthetase, 2g / L of exogenous N-acetylglutamine was continuously added to inhibit the generation of N-acetyltryptophan and promote the generation of N,N’-diacetyl-tryptophan, the rotation speed was 300rpm, and the dissolved oxygen was maintained at 20%. When the glucose concentration in the fermentation liquid was less than 5g / L, the feeding medium was started to be added to obtain the third-stage fermentation liquid.

[0087] (6) At 72h of fermentation, the yield of N,N’-diacetyl-tryptophan stopped increasing and reached the highest proportion, and the fermentation was ended. The maximum biomass OD600 during fermentation was 119.1. The yield of N,N’-diacetyl-tryptophan in the final tank fermentation liquid was 12.07g / L, and the proportion was 95.1%.

[0088] The embodiments 1-3 are described in detail in terms of culture temperature, rotation speed, dissolved oxygen, and amount of cofactor addition, and the intervals of each condition are determined for different fermentation conditions. The embodiment 1 is the most suitable fermentation condition. The yield of N,N'-diacetyl-obscurine in the final tank fermentation broth of the embodiment 1 is 13.52 g / L, accounting for 96.7%, the yield of N,N'-diacetyl-obscurine in the final tank fermentation broth of the embodiment 2 is 12.78 g / L, accounting for 94.5%, and the yield of N,N'-diacetyl-obscurine in the final tank fermentation broth of the embodiment 3 is 12.07 g / L, accounting for 95.1%. Through the above embodiments, N,N'-diacetyl-obscurine is obtained, and the yield and the proportion under the most suitable condition are the highest, which proves that the above fermentation conditions are used in the second-stage fermentation culture, and the synthetic enzyme inducer and N-acetylglutamine are added to induce the expression of the product synthesis enzyme and promote the synthesis of the product, so that N,N'-diacetyl-obscurine is finally obtained.

[0089] The biosynthetic route of N,N'-diacetyl-obscurine is shown in Figure 1 .

[0090] Example 4, structural identification of the product

[0091] The synthetic product obtained in the embodiment 1 is sent to the Nanjing Normal University Analysis and Testing Center for mass spectrometry and NMR detection.

[0092] The HPLC spectrum of the product obtained in the embodiment 1 is shown in Figure 2 ; and the ultraviolet absorption spectrum of the product obtained in the embodiment 1 is shown in Figure 3 .

[0093] The relative molecular mass of obscurine is 248.19, and the relative molecular mass of acetyl is 43. The high-resolution mass spectrometry detection result is shown in Figure 5 ; ESIMS (m / z, %): 333.0877 ([M+H] - , 100). The relative molecular mass of the sample sent for detection is 332, which is consistent with the theoretical value of N,N'-diacetyl-obscurine.

[0094] As shown in 1 H-NMR, 13 C NMR spectrum Figure 6 , Figure 7 , 1 H NMR (400 MHz, DMSO-d6): δ 11.85 (s, 1H), 9.83 (s, 1H), 9.73 (s, 1H), 2.18 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.36, 164.30, 160.62, 130.70, 130.66, 119.19, 24.79.

[0095] The hydrogen spectrum data was analyzed, and four groups of signals were given in the hydrogen spectrum, wherein the low-field region δ 11.85 (s, 1H) gave a group of amide proton signals, δ 9.83 (s, 1H) a single peak gave an olefin hydrogen proton signal, δ 9.73 (s, 1H) gave a group of imine proton signals, and the high-field region δ 2.18 (s, 3H) with an integral of 3 indicated that there was a group of methyl proton signals in the molecule.

[0096] The carbon spectrum data was analyzed, and the low-field region δ 170.36, 164.30, 160.62 corresponded to the three carbonyl carbon signals in the molecule, δ 119.19 indicated that there was an olefin carbon signal in the molecule, and the low-field region δ 24.79 corresponded to the methyl signal in the hydrogen spectrum.

[0097] In the two-dimensional HSQC spectrum ( Figure 8 ), two groups of carbon-hydrogen related information were given, corresponding to

δ 2.18 (s, 3H), δ 24.79

δ 9.83 (s, 1H), δ 119.19

[0098] The mass spectrometry detection and nuclear magnetic resonance spectroscopy detection results determined that the structure of the product was N,N'-diacetyl-obscurin.

[0099] Comparative Example 1

[0100] Compared with Example 1, the difference lies in that the feed medium of step (4) does not contain N-acetyl glutamine.

[0101] When the fermentation was carried out to 72 h, the fermentation was ended, the maximum biomass OD600 in the fermentation process was 123.1, the N,N'-diacetyl-obscurin in the second stage fermentation broth was 6.19 g / L, the content was 40.5%, and the N-acetyl obscurin content was 55.4%. Compared with Example 1, under the conditions of this comparative example 1, the strain grew normally, and the synthesis of N,N'-diacetyl-obscurin was limited.

[0102] Comparative Example 2

[0103] Compared with Example 1, the difference lies in that the third stage is the same as the second stage, and the rest of the conditions are the same.

[0104] When the fermentation was carried out to 72 h, the fermentation was ended, the maximum biomass OD600 in the fermentation process was 120.2, the N,N'-diacetyl-obscurin in the second stage fermentation broth was 3.21 g / L, the content was 22.6%, and the N-acetyl obscurin content was 73.6%. Compared with Example 1, under the conditions of this comparative example 2, the strain grew normally, the synthesis of N,N'-diacetyl-obscurin was limited, and mainly N-acetyl obscurin was synthesized.

[0105] Comparative Example 3

[0106] The difference compared with Example 1 is that the third stage does not add lactose inducer, and the rest of the conditions are the same.

[0107] The first stage OD600nm is much higher than 30 at 24h fermentation, the maximum biomass OD600 is 118.2 during fermentation, and the N,N'-diacetyl-obscurine content in the fermentation broth is 6.18g / L at 72h fermentation, with a content of 45.8%, and the N-acetyl obscurine content is 51.4%. Compared with Example 1, under the conditions of Example 3, the strain grows normally, and the yield of N,N'-diacetyl-obscurine decreases.

[0108] Comparative Example 4

[0109] The difference compared with Example 1 is that the third stage pH is controlled at 7.0, and the rest of the conditions are the same.

[0110] The maximum biomass OD600 is 119.4 during fermentation, and the N,N'-diacetyl-obscurine content in the fermentation broth is 7.01g / L at 72h fermentation, with a content of 46.2%, and the N-acetyl obscurine content is 52.8%. Compared with Example 1, under the conditions of Example 4, the strain grows normally, and the yield of N,N'-diacetyl-obscurine decreases.

[0111] The results of Examples 1-3 and Comparative Examples 1-4 are shown in Table 2.

[0112] Table 2 N,N'-diacetyl-obscurine yield and purity

[0113]

[0114] Test Example 1, solubility comparison of N,N'-diacetyl-obscurin in different solutions

[0115] 30μg of N,N'-diacetyl-obscurine was weighed and resuspended in 1mL of different solvents, and the color difference was observed. The dissolution state of N,N'-diacetyl-obscurine in different solvents is shown in Table 3. Figure 4 N,N'-diacetyl-obscurine appears bright red in DMSO; a small amount is dissolved in DMF, tetrahydrofuran, and acetic acid, and the color is lighter than DMSO, with a large amount of solid particles at the bottom of the solvent; a small amount is dissolved in isopropanol, and the color is even lighter; no obvious color is observed in ethyl acetate, methanol, ethanol, n-butanol, and water, and a large amount of solid particles are present at the bottom of the solvent. The supernatant of each solvent with dissolved N,N'-diacetyl-obscurine was brought into the standard curve, and the concentration was calculated as shown in Table 3.

[0116] Table 3 Solubility of N,N'-diacetyl-obscurine in different solutions

[0117]

[0118] From Table 2, it can be seen that N,N'-diacetyl-obscurin has the highest solubility in DMSO, reaching 25.6 μg / mL, and has no solubility in ethanol, EA, tert-butanol, water, and isopropanol.

[0119] Test Example 2, detection of N,N'-diacetyl-obscurin acid-base stability

[0120] N,N'-diacetyl-obscurin was dissolved in DMSO, and solutions with pH = 1, pH = 3, pH = 5, pH = 6, pH = 7, pH = 8, pH = 9, pH = 10, and pH = 11 were prepared, respectively. The solutions were left to stand at 30°C for 24 h, after which the absorption at a wavelength of 499 nm was measured by a spectrophotometer, and the preservation rate of N,N'-diacetyl-obscurin was calculated by a standard curve. A standard solution of 20 μg / mL was prepared in advance as a control, and the detection results are shown in Table 4.

[0121] Table 4 N,N'-diacetyl-obscurin acid-base stability

[0122]

[0123] As can be seen from Table 3, N,N'-diacetyl-obscurin is very stable under acidic conditions, and the stronger the acidity, the more stable it is. Under alkaline conditions, it is unstable, and the higher the pH, the greater the damage.

[0124] Test Example 3, detection of N,N'-diacetyl-obscurin temperature stability

[0125] N,N'-diacetyl-obscurin was dissolved in DMSO and stored at -20°C, 4°C, 30°C, 50°C, 80°C, and 100°C, respectively, for 24 h, after which the absorption at a wavelength of 500 nm was measured by a spectrophotometer as shown in Table 5, and the preservation rate of N,N'-diacetyl-obscurin was calculated by a standard curve. A standard solution of 20 μg / mL was prepared in advance as a control, and the detection results are shown in Table 5. Figure 9 Table 5 N,N'-diacetyl-obscurin temperature stability

[0126]

[0127] As can be seen from Table 4, N,N'-diacetyl-obscurin is very stable under conditions of -20°C to 100°C.

[0128]

[0129] Test Example 4, application of N,N'-diacetyl-obscurin in dyeing N,N'-diacetyl-obscurin synthesized in Example 1 above was used to dye cotton textiles according to the following process:

[0130]

[0131] ​N,N'-diacetyl-obsidian blue 2% (o.w.f), 2 times Metol red, citric acid 0.5 mL / L adjusted to pH 5, dyeing at 25℃ for 10 min, bath ratio 1:30.

[0132] After dyeing, the dyed fabric sample was taken out, washed, dried, and the K / S value of the dye was calculated by using the Lambert-Beer law.

[0133] The performances of the dye were tested according to the following standards, and the test results were recorded in Table 6 below.

[0134] The water color fastness was tested according to GB / T 5713-2013; the acid spot color fastness was tested according to GB / T 5715-2013; and the rubbing color fastness was tested according to GB / T 3920-2008.

[0135] Table 6 N,N'-diacetyl-obsidian blue dyeing color fastness

[0136]

[0137]

[0138] N,N'-diacetyl-obsidian blue can dye cotton fabric, with K / S value reaching more than 10, and the washing color fastness, sublimation color fastness and wet rubbing color fastness all reaching level 4-5, and the acid spot color fastness reaching level 5. Figure 10 Cotton fabrics dyed by N,N'-diacetyl-obsidian blue dyeing solution with different concentrations.

[0139] The N,N'-diacetyl-obsidian blue obtained in the application has a maximum absorption wavelength of 499±2 nm, stable color, and is resistant to high temperature and low temperature under acidic conditions, and has a very wide application range and application prospect of industrial production.

[0140] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the application and not to limit the scope of protection of the application. Although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the application.

Claims

1. A natural red colorant, characterized by, The natural red pigment has a structural formula as shown in formula (I); 2. The fermentation process of natural red pigment according to claim 1, characterized in that, The method comprises the following steps: S1, inoculating the activated recombinant strain seed fermentation liquor into a basic fermentation medium to perform first-stage fermentation culture, maintaining the pH at 6.5-7.2, controlling the temperature at 25-30 DEG C, and culturing for 0-24 h to obtain a first-stage fermentation liquor; S2, maintaining the pH of the first-stage fermentation liquor at 7.2-8.0, controlling the temperature at 20-25 DEG C, adding a synthetic enzyme inducer and N-acetyl glutamine to perform second-stage fermentation culture, and culturing for 24-30 h to obtain a second-stage fermentation liquor; S3, maintaining the pH of the second-stage fermentation liquor at 8.0-9.0, controlling the temperature at 37-45 DEG C, adding a synthetic enzyme inducer and lactose, continuously adding N-acetyl glutamine to perform third-stage fermentation culture, and culturing for 30-72 h to obtain a third-stage fermentation liquor, and fermenting until the yield of the natural red pigment stops increasing, and then ending the fermentation.

3. The fermentation process of natural red pigment according to claim 2, characterized in that, The basic fermentation medium comprises the following components in the following mass concentrations: Saccharomyces cervisiae powder P801 5-10 g / L, glucose 70-90 g / L, ammonium sulfate 10-20 g / L, potassium dihydrogen phosphate 0.2-1 g / L, magnesium sulfate heptahydrate 0.2-0.4 g / L, manganese sulfate monohydrate 0.01-0.02 g / L, biotin 2-4 μg / L, VB1 0.2-0.4 mg / L, sodium glutamate 1-2 g / L, and water as the solvent, and the pH is 7.

0.

4. The fermentation process of natural red pigment according to claim 2, characterized in that, In the step S1, the first-stage fermentation culture further comprises a dissolved oxygen of 25-35%; In the step S2, the second-stage fermentation culture further comprises a dissolved oxygen of 20-25%; In the step S3, the third-stage fermentation culture further comprises a dissolved oxygen of 20-30%.

5. The fermentation process of natural red pigment according to claim 2, characterized in that, In the step S2, the second-stage fermentation culture adjusts the pH of the first-stage fermentation liquor to 7.2-8.0 by adding ammonia; In the step S3, the third-stage fermentation culture adjusts the pH of the second-stage fermentation liquor to 8.0-9.0 by adding ammonia.

6. The fermentation process of natural red pigment according to claim 2, characterized in that, The synthetic enzyme inducer comprises IPTG; In the step S2, the concentration of IPTG is 0.05-0.2 mmol / L; In the step S3, the concentration of IPTG is 0.3-0.5 mmol / L, and the concentration of lactose is 5-10 g / L; The addition amount of N-acetyl glutamine is 2-5 g / L.

7. The fermentation process of natural red pigment according to claim 2, characterized in that, The initial concentration of glucose in the first-stage fermentation culture is 8%; The concentration of glucose in the second-stage fermentation culture and the third-stage fermentation culture is 5-10 g / L.

8. The fermentation process of natural red pigment according to claim 2, characterized in that, In the second-stage fermentation culture, the concentration of glucose in the basic medium is lower than 5 g / L, and a feeding medium is added; The feeding medium comprises glucose 5-10 g / L, magnesium sulfate heptahydrate 0.2-0.4 g / L, ammonium chloride 4-8 g / L, N-acetyl glutamine 2-5 g / L, and water as the solvent.

9. The fermentation process of natural red pigment according to claim 2, characterized in that, The recombinant strain contains biosynthetic gene bpsA, 4'-phosphopantetheinyl transferase gene EntD, glutamine synthetase gene glnA and N-acetylglutamate synthetase gene argA.

10. Use of natural red pigment according to claim 1 for dyeing of fabrics.

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