Culture medium suitable for high yield of Indigoidine through fermentation of bacillus licheniformis DW2 / pHY-M4 and application of culture medium

Abstract

The invention belongs to the technical field of fermentation engineering, and discloses a culture medium suitable for high-yield Indigoidine fermentation of bacillus licheniformis DW2 / pHY-M4 and application of the culture medium. The bacillus licheniformis DW2 / pHY-M4 with a specific culture medium formula provided by the invention is used for fermentation verification, and the shake flask yield of the Indigoidine reaches 8.66 / L; and the yield of a 30 L fermentation tank of Indigoidine reaches 33.49 g / L. The process has the advantages of cheap and easily available raw materials, simple culture medium composition, environmental protection, high yield and the like, and has certain application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and particularly relates to a culture medium suitable for high-yield Indigoidine fermentation by Bacillus licheniformis DW2 / pHY-M4 and its application. Background Art

[0002] Indigoidine is a non-toxic, water-soluble natural blue product of microorganisms. It was first discovered and isolated from the plant pathogen Erwinia sp., and later found in other microbial products, such as Arthrobacter and Roseobacter. Indigoidine is a natural blue pigment encoded by a single modular non-ribosomal peptide synthetase (NRPS). Its bright dark blue color makes it an attractive alternative fabric dye. In addition, the biosynthetic pathway of Indigoidine is simple. It is formed by the condensation and oxidation of two molecules of L-glutamine. Compared with other natural blue pigments, it has fewer branched metabolisms and fewer by-products in microbial synthesis, which is conducive to subsequent separation and purification.

[0003] Indigoidine is a natural blue pigment from microorganisms. It is non-toxic and has a bright color similar to indigo. It can be used as a new natural blue pigment and dye in food, medicine, dyeing and finishing industries, and is a strong candidate to replace industrial indigo. Literature reports that with N,N-dimethylformamide as a solvent, Indigoidine has a stronger ability to resist Vc reduction than indigo, and oxidants can reduce the preservation rate of the two pigments to varying degrees; the two pigments have poor thermal stability, and the pigment stability of Indigoidine is better than that of indigo below 75°C. In general, the pigment stability of Indigoidine is significantly better than that of indigo, and it has broad prospects for development and application.

[0004] After preliminary research, we used Bacillus licheniformis DW2 as the starting strain, obtained a genetically engineered bacterium with high yield of Indigoidine, i.e. Bacillus licheniformis DW2 / pHY-M4 (CN118599790A) by metabolic engineering and protein engineering means, and provided a method for fermentation using the fermentation strain. However, the fermentation medium used in the fermentation method is a common fully synthetic medium, which is not conducive to industrial scale-up production. It is mentioned in CN112301070B that the formula of the fermentation medium is a very critical factor in the fermentation industry, which directly affects the output of metabolites and subsequent production costs. Although there are many existing fermentation media and many successful examples, due to the diversity of microbial strains, there is no reference and reference significance for the formula between different strains. In the screening of fermentation media, the components that affect more significantly are carbon source, nitrogen source, inorganic salt and some trace elements. Currently, there are many types of carbon sources, nitrogen sources, inorganic salts, etc. that can be used in industrial production. In particular, carbon sources and nitrogen sources are usually natural components. These components have a great impact on product yield, and also lead to randomness and huge workload when screening formulas.

[0005] Therefore, based on the above problems, the present invention provides a culture medium suitable for high-yield Indigoidine fermentation by Bacillus licheniformis DW2 / pHY-M4, which greatly improves the yield of Indigoidine. Summary of the invention

[0006] The purpose of the present invention is to provide a culture medium suitable for high-yield Indigoidine fermentation by Bacillus licheniformis DW2 / pHY-M4, thereby making up for the deficiencies of the prior art and being suitable for industrial production.

[0007] Another object of the present invention is to provide the use of the above culture medium in the fermentation production of Indigoidine.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The culture medium suitable for the fermentation of Bacillus licheniformis DW2 / pHY-M4 to produce high-yield Indigoidine includes:

[0010] Glucose 30-50g / L, ammonium sulfate 5.02-11.28g / L, nitrogen source 5-40g / L, potassium dihydrogen phosphate 1.0-2.5g / L, magnesium sulfate 1.0-2.5g / L, ferric chloride 0.01-0.03g / L, manganese sulfate 0.01-0.05g / L, calcium chloride 0.01-0.03g / L, pH 6.5-7.5.

[0011] The nitrogen source is soybean meal, corn steep liquor, soy peptone and / or tryptone.

[0012] The culture medium described above, preferably, has a culture medium formula comprising: 30-50 g / L glucose, 6.2-11.28 g / L ammonium sulfate, 5-20 g / L soybean meal, 1.0-2.0 g / L potassium dihydrogen phosphate, 1.0-2.0 g / L magnesium sulfate, 0.01-0.03 g / L ferric chloride, 0.01-0.03 g / L manganese sulfate, 0.01-0.03 g / L calcium chloride, pH 6.5-7.5.

[0013] The above-mentioned culture medium, preferably, the culture medium formula comprises:

[0014] The culture medium formula includes: glucose 50g / L, ammonium sulfate 11.28g / L, soybean meal 20g / L, potassium dihydrogen phosphate 2.0g / L, magnesium sulfate 2.0g / L, ferric chloride 0.02g / L, manganese sulfate 0.02g / L, calcium chloride 0.03g / L, pH 7.0.

[0015] The protection scope of the present invention also includes:

[0016] The culture medium is used in the fermentation production of Indigoidine, and the fermentation strain used in the application is Bacillus licheniformis DW2 / pHY-M4 (CN118599790A).

[0017] The above application preferably comprises the step of inoculating Bacillus licheniformis DW2 / pHY-M4 into the above culture medium and fermenting to produce Indigoidine at 25-30°C.

[0018] In the above application, preferably, the fermentation condition is 30°C.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The production process of the present invention is simple and convenient, and is easy to promote. The combination of specific culture medium components and fermentation conditions can greatly increase the yield of Indigoidine, a secondary metabolite of Bacillus licheniformis. The process of the present invention has the characteristics of high stability and is easy to expand scale production. The fermentation verification of Bacillus licheniformis DW2 / pHY-M4 was carried out using the specific culture medium formula and culture conditions provided by the present invention, and the yield of Indigoidine in a shake flask reached 8.66g / L; the yield of Indigoidine in a 30L fermentation tank can reach 33.49g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Indigoidine liquid phase detection results diagram.

[0022] Figure 2 Schematic diagram of the yield difference of Indigoidine produced using a 30L fermenter. DETAILED DESCRIPTION

[0023] The following will further illustrate a fermentation process for high-yield Indigoigine of Bacillus licheniformis provided by the present invention in conjunction with specific implementation cases. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all purchased from the market unless otherwise specified.

[0024] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0025] Embodiment 1-5:

[0026] Application of the medium suitable for high-yield Indigoidine fermentation by Bacillus licheniformis DW2 / pHY-M4 in the production of Indigoidine:

[0027] 1. Bacteria activation

[0028] Take the working strain Bacillus licheniformis DW2 / pHY-M4 (CN118599790A, prepared in Example 2), thaw at room temperature, draw 0.1 ml of the bacterial suspension and inoculate it onto the LB solid plate, spread it evenly, and place it in a 37°C incubator for 24 hours to obtain an activated strain.

[0029] 2. Seed preparation

[0030] Activate the strain by scraping a loop of colonies with an inoculation loop, then inoculate into a 250ml Erlenmeyer flask containing 50mL LB liquid medium, wrap it up and place it on a shaker at 37°C and 230rpm for 12h. Seed medium composition: 10g / L peptone, 5g / L yeast extract powder, 10g / L NaCl; pH 7.0-7.2;

[0031] 3. Fermentation culture and detection

[0032] The qualified seeds were taken and inoculated into a 250 mL Erlenmeyer flask containing 45 mL of liquid fermentation medium at a ratio of 2% (V / V). The culture temperature was 37° C., the rotation speed was 230 rpm, and the culture was carried out for 48 h.

[0033] Detection and quantification method of Indigoidine: After the fermentation is completed, the fermentation liquid is diluted 10 times with distilled water, and 20 μL is taken and dissolved in 980 μL of DMSO. The yield of Indigoidine is detected by HPLC. HPLC uses Agilent 1200 high performance liquid chromatograph (equipped with quaternary pump, DAD detector and workstation). Chromatographic conditions: Diamonsil C18 Column, 250×4.6mm, 5μm; mobile phase: A (10% methanol), B (100% methanol), 0-9min B 20%-45%; 9-13min 45%-20%; 13-18min B 20%. Flow rate: 1mL min -1 , column temperature 30℃; injection volume 10μL, detection wavelength 600nm. The retention time of pure Indigoidine was used for qualitative analysis and the standard curve method (external standard method) was used for quantitative analysis. The experiment was repeated three times and the results were averaged. The HPLC spectrum of pure Indigoidine is shown in Figure 1 As shown in the figure, it can be seen that the retention time of pure Indigoidine is 10.8min.

[0034] The liquid fermentation medium used in this example and the yield of Indigoidine after fermentation are shown in Table 1;

[0035] The yield of Indigoidine in the fermentation broth was calculated based on the standard curve prepared from the Indigoidine standard.

[0036] From the results in Table 1, it can be seen that the yield of Indigoidine in the culture medium used in Example 1 is the highest, and the carbon source used is glucose at a concentration of 50 g / L.

[0037] In Table 1, glucose and sucrose were sterilized separately and added. Table 1 shows the final concentration of each substance in the culture medium after addition. The pH of the culture medium was 7.0.

[0038] Table 1

[0039]

[0040] Embodiment 6-18:

[0041] Effects of different nitrogen sources on Indigoidine production:

[0042] In this example, the applicant adjusted the type of nitrogen source and the total nitrogen content of the culture medium. The culture medium and Indigoidine yields used in Examples 6-18 are specifically shown in Table 2. Except for the adjustment of the formula of the culture medium used, the fermentation steps and parameters, detection methods, etc. used in this example are the same as those shown in Example 1.

[0043] As can be seen from Table 2, the best fermentation medium is the medium used in Example 9, so this medium was used in the following examples.

[0044] Table 2

[0045]

[0046]

[0047] Furthermore, the content of each component in the formula of Example 9 is adjusted, that is,

[0048] The culture medium formula includes: glucose 30-50g / L, ammonium sulfate 6.2-11.28g / L, soybean meal 5-20g / L, potassium dihydrogen phosphate 1.0-2.0g / L, magnesium sulfate 1.0-2.0g / L, ferric chloride 0.01-0.03g / L, manganese sulfate 0.01-0.03g / L, calcium chloride 0.01-0.03g / L, pH 7.0. The sterilization conditions are 121°C, 30min, and glucose is added after sterilization.

[0049] The fermentation was carried out under the same fermentation conditions as above, and then the content of Indigoidine in the fermentation broth was detected.

[0050] The results are as follows:

[0051]

[0052] Embodiment 22:

[0053] Effects of different culture medium formulations on Indigoidine production:

[0054] Control medium 1

[0055] 1. Bacterial activation is the same as in Example 1

[0056] 2. Seed preparation is the same as in Example 1

[0057] 3. Fermentation and detection Example 1. The components of the liquid fermentation medium used in this example are: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, K2HPO4 18.6g / L, KH2PO4 5.18g / L, manganese chloride 1.81mg / L, zinc sulfate 0.222mg / L, cobalt nitrate 49.4mg / L; glucose 80g / L was sterilized separately and added. The yield of indigoidine was 2.36g / L.

[0058] Control medium 2

[0059] 1. Bacterial activation is the same as in Example 1

[0060] 2. Seed preparation is the same as in Example 1

[0061] 3. Fermentation and detection Example 1. The liquid fermentation culture components used in this example are: 6 g / L tryptone, 12 g / L yeast extract, 12.5 g / L K2HPO4, 2.5 g / L KH2PO4, 10 mg / L manganese sulfate, 2 mg / L zinc sulfate, 4 mg / L cobalt nitrate, 40 mg / L ferrous sulfate, 40 mg / L calcium chloride; 60 g / L glucose was sterilized separately and then added. The yield of indigoidine was 2.58 g / L.

[0062] Control medium 3

[0063] 1. Bacterial activation is the same as in Example 1

[0064] 2. Seed preparation is the same as in Example 1

[0065] 3. Fermentation and Detection Example 1: The liquid fermentation culture components used in this example are: yeast extract 15 g / L, KH2PO4 2 g / L, ammonium sulfate 20 g / L, magnesium sulfate 3 g / L, manganese sulfate 5 mg / L, sodium citrate 1.5 g / L; glucose 80 g / L was sterilized separately and then added. The indigoidine yield was 3.01 g / L.

[0066] Control medium 4

[0067] 1. Bacterial activation is the same as in Example 1

[0068] 2. Seed preparation is the same as in Example 1

[0069] 3. Fermentation and Detection Example 1: The liquid fermentation culture components used in this example are: tryptone 10 g / L, corn steep liquor 10 g / L, NaCl 10 g / L, soy peptone 10 g / L, KH2PO4 3 g / L, yeast extract 15 g / L, ammonium sulfate 6 g / L; glucose 20 g / L was sterilized separately and then added. The indigoidine yield was 1.91 g / L.

[0070] Control medium 5

[0071] 1. Bacterial activation is the same as in Example 1

[0072] 2. Seed preparation is the same as in Example 1

[0073] 3. Fermentation and detection Example 1. The liquid fermentation culture components used in this example are: 30 g / L sodium glutamate, 10 g / L sodium citrate, 10 g / L sodium nitrate, 8 g / L ammonium sulfate, 1 g / L KH2PO4, 1 g / L magnesium sulfate, 1 g / L zinc sulfate, 0.15 g / L manganese sulfate, 1 g / L calcium chloride; 80 g / L glucose is sterilized separately and then added. The yield of indigoidine is 3.18 g / L.

[0074] Embodiment 23:

[0075] Effects of different culture temperatures on Indigoidine production:

[0076] The culture medium used in this example is the culture medium shown in Example 9. The fermentation temperature in the liquid fermentation medium is 25° C. or 30° C., and the rest is the same as in Example 1.

[0077] Finally, at a culture temperature of 25°C, the yield of Indigoidine was 5.65 g / L. At a culture temperature of 30°C, the yield of Indigoidine was 8.66 g / L.

[0078] Therefore, the fermentation temperature suitable for high-yield Indigoidine is 30°C, which is used in the following large-tank fermentation examples.

[0079] Embodiment 24:

[0080] Application of medium suitable for high-yield Indigoidine fermentation by Bacillus licheniformis DW2 / pHY-M4 in large-tank fermentation production of Indigoidine:

[0081] 1. Bacterial activation is the same as in Example 1

[0082] 2. Primary seed preparation

[0083] Take the activated and revived bacterial moss, scrape a ring of colonies with an inoculation loop, and then inoculate it into a 500ml Erlenmeyer flask containing 100ml LB liquid culture medium, wrap it up, and place it on a shaker at 37℃ and 230rpm for 12h.

[0084] 3. Secondary seed preparation

[0085] The first-level seeds that passed the culture were taken and inoculated into a 5L shaking flask containing 500mL of LB liquid culture medium at a ratio of 1% (V / V). The flask was wrapped and placed on a shaking table at 37°C and 230rpm for shaking culture for 12h.

[0086] 4. Fermentation culture

[0087] Take the qualified secondary seeds and inoculate them into a 30L tank containing 22L liquid fermentation medium at a ratio of 20% (V / V). The culture temperature is 30°C, the dissolved oxygen is controlled to be greater than 30%, the air flow rate is 1.0VVM, the initial stirring speed is 500rpm, and the culture is carried out for 60h.

[0088] The liquid fermentation medium formula is composed of: glucose 50g / L, ammonium sulfate 11.28g / L, soybean meal 20g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 2g / L, ferric chloride 0.02g / L, manganese sulfate 0.02g / L, calcium chloride 0.03g / L, pH 7.0. The sterilization conditions are 121℃, 30min, and glucose is added after sterilization.

[0089] The fermentation of LBG medium was used as a control. The LBG medium formula composition is: 10g / L tryptone, 5g / L yeast powder, 10g / L sodium chloride, 50g / L glucose, 14.136g / L K2HPO4, 5.168g / L KH2PO4, 2.86mg / L H3BO3, 1.81mg / L MnCl2·4H2O, 0.222mg / L ZnSO4·7H2O, 0.39mg / L Na2MoO4·2H2O, 79μg / L CuSO4·5H2O, and 49.4μg / L Co(NO3)2·6H2O, pH 7.0-7.2, sterilized at 115℃ for 20min. (CN 110331173 A)

[0090] 5. Fermentation tank control process

[0091] During the culture process, the glucose concentration was controlled to be in the range of 20±5 g / L by adding 500 g / L glucose, and the pH was controlled to be 7.0 by adding 25% ammonia water.

[0092] 6. Experimental results

[0093] After the tank was put into the test, the Indigoidine 30L fermentation tank production reached 33.49g / L, which was 163.9% higher than that of LBG medium (12.69g / L). Figure 2 ).

Claims

1. A culture medium suitable for fermentation of Bacillus licheniformis DW2 / pHY-M4 to produce high yield of Indigoidine, including: Glucose 30-50 g / L, ammonium sulfate 5.02-11.28 g / L, nitrogen source 5-40 g / L, potassium dihydrogen phosphate 1.0-2.5 g / L, magnesium sulfate 1.0-2.5 g / L, ferric chloride 0.01-0.03 g / L, manganese sulfate 0.01-0.05 g / L, calcium chloride 0.01-0.03 g / L, pH 6.5-7.5; The nitrogen source is soybean meal, corn steep liquor, soy peptone and / or tryptone.

2. The culture medium according to claim 1, characterized in that: The culture medium formula includes: glucose 30-50 g / L, ammonium sulfate 6.2-11.28 g / L, soybean meal 5-20 g / L, potassium dihydrogen phosphate 1.0-2.0 g / L, magnesium sulfate 1.0-2.0 g / L, ferric chloride 0.01-0.03 g / L, manganese sulfate 0.01-0.03 g / L, calcium chloride 0.01-0.03 g / L, pH 6.5-7.

5.

3. The culture medium according to claim 2, characterized in that: The culture medium formula includes: the culture medium formula includes: glucose 50 g / L, ammonium sulfate 11.28 g / L, soybean meal 20 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 2.0 g / L, ferric chloride 0.02 g / L, manganese sulfate 0.02 g / L, calcium chloride 0.03 g / L, pH 7.

0.

4. Use of the culture medium according to claim 1 in fermentation production of Indigoidine, wherein the fermentation strain used in the application is Bacillus licheniformis DW2 / pHY-M4.

5. The use according to claim 4, wherein the use process comprises inoculating Bacillus licheniformis DW2 / pHY-M4 into the culture medium according to claim 1, and fermenting to produce Indigoidine at a fermentation condition of 25-30°C.

6. The use according to claim 5, characterized in that: The fermentation condition is 30°C.

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