Culture medium for fermentation of phenazino-1-carboxylic acid and fermentation method

Abstract

The invention relates to the technical field of biology, in particular to a culture medium for fermentation of phenazino-1-carboxylic acid and a fermentation method. According to the invention, an active component A (organic acid) and an active component B (phenazino-1-carboxylic acid) are added into a basic culture medium for fermentation, and the fermentation culture medium and the method capable of shortening the fermentation time of the phenazino-1-carboxylic acid and increasing the yield of the phenazino-1-carboxylic acid are obtained by screening the active component A and optimizing the component proportion of the active component A, the active component B and the basic culture medium; the fermentation medium and the method are used for fermentation of phenazino-1-carboxylic acid, are short in fermentation time and low in fermentation cost, and have wide industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a culture medium and a fermentation method for the fermentation of phenazine-1-carboxylic acid Background Art

[0002] Phenazine-1-carboxylic acid is a highly effective, low-toxic, broad-spectrum, and environmentally compatible biological antibacterial agent. It is the main antibacterial active substance extracted and purified from the fermentation broth of Pseudomonas fluorescens strain M18, and is a secondary metabolite obtained by microbial fermentation. Its main active ingredient is phenazine-1-carboxylic acid (abbreviated as PCA).

[0003] At present, the longer the fermentation cycle of phenazine-1-carboxylic acid fermentation, the higher the corresponding costs of equipment power, labor, etc. As a result, the overall production cost of phenazine-1-carboxylic acid is high, which restricts the market promotion and wide application of phenazine-1-carboxylic acid. Therefore, it is particularly necessary to develop a phenazine-1-carboxylic acid with a short fermentation time and low cost. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a culture medium and a fermentation method for the fermentation of phenazine-1-carboxylic acid.

[0005] The present invention provides a culture medium, which comprises a basal medium, active ingredient A and active ingredient B;

[0006] The basal medium comprises: glucose, corn steep liquor, bone peptone, dipotassium hydrogen phosphate, magnesium sulfate and / or ammonium sulfate;

[0007] The active ingredient A is an organic acid; the organic acid is selected from citric acid, formic acid or acetic acid;

[0008] The active ingredient B is phenazine-1-carboxylic acid.

[0009] Furthermore, in the culture medium, the basal medium comprises: 1 wt% - 10 wt% glucose, 1 wt% - 10 wt% corn steep liquor, 0.1 wt% - 5 wt% bone peptone, 0.1 wt% - 0.3 wt% dipotassium hydrogen phosphate, 0.1 wt% - 0.3 wt% magnesium sulfate and 0.1 wt% - 0.3 wt% ammonium sulfate.

[0010] Furthermore, the basal medium comprises: 1 wt% - 2 wt% glucose, 1 wt% - 2 wt% corn steep liquor, 0.2 wt% - 0.5 wt% bone peptone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate and 0.2 wt% ammonium sulfate; pH 6.5 - 7.0.

[0011] In a specific embodiment of the present invention, the basal medium can be basal medium 1: 2 wt% glucose, 2 wt% corn steep liquor, 0.2 wt% peptone from bone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, and 0.25 wt% ammonium sulfate; pH 6.5 - 7.0;

[0012] In a specific embodiment of the present invention, the basal medium can be basal medium 2: 1 wt% glucose, 1 wt% corn steep liquor, 0.5 wt% peptone from bone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, and 0.25 wt% ammonium sulfate; pH 6.5 - 7.0;

[0013] In some other embodiments of the present invention, the basal medium can be basal medium 3: 1.5 wt% glucose, 1.5 wt% corn steep liquor, 0.4 wt% peptone from bone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, and 0.25 wt% ammonium sulfate; pH 6.5 - 7.0;

[0014] In a specific embodiment of the present invention, the basal medium can be basal medium 1, and active ingredients A and B are added thereto. When A is 0.2 wt% citric acid and B is 0.5 wt% fermented phenazine - 1 - carboxylic acid, the fermentation time of phenazine - 1 - carboxylic acid is the shortest and the yield is the highest.

[0015] In a specific embodiment of the present invention, the screening of active ingredient A was first carried out. The test results showed that among formic acid, acetic acid, and / or citric acid, acetic acid and / or citric acid as active ingredient A was superior to formic acid, and citric acid was the best. Then, through the optimization of the addition ratios of the basal medium, active ingredient A, and active ingredient B, the highest yield of phenazine - 1 - carboxylic acid in the short - time fermentation process at 48 h was obtained, and the results are as shown in Example 4.

[0016] In a specific embodiment of the present invention,

[0017] The addition amount of citric acid is 0.1 wt% - 0.5 wt% of the basal medium;

[0018] The addition amount of phenazine - 1 - carboxylic acid is 0.45 wt% - 0.6 wt% of the basal medium.

[0019] In the present invention, the addition amounts of active ingredients A and B were optimized. The test results showed that when the basal medium was basal medium 3, the organic acid was citric acid with an addition amount of 0.2 wt%, and the addition amount of active ingredient B, phenazine - 1 - carboxylic acid, was 0.5 wt%, the fermentation time was the shortest and the yield was the highest.

[0020] By directly adding active ingredients to the fermented basal medium instead of feeding during the fermentation process, the present invention simplifies the fermentation process and also avoids the risk of contamination that may be brought about by feeding. Secondly, through the screening of active ingredients and the optimization of the addition amount, the present invention obtains active ingredients and the optimal addition amount that can significantly shorten the fermentation time on the basis of a considerable yield of phenazine-1-carboxylic acid; on this basis, through the optimization of the basal medium, the fermentation time is further shortened; at the same time, the addition of active ingredient B further reduces the risk of contamination during the fermentation process. In the fermentation method of the present invention, the fermentation cycle of phenazine-1-carboxylic acid is shortened to 36-48 h, which can effectively reduce the production cost of phenazine-1-carboxylic acid, thereby promoting the popularization and application of the biopesticide phenazine-1-carboxylic acid. Moreover, the added active ingredient A of the present invention is economical and practical, and has a wide source, and active ingredient B is the target product, so both are convenient and easy to obtain, so this method is conducive to popularization and implementation.

[0021] The present invention provides a fermentation method of phenazine-1-carboxylic acid, which is fermented with the medium described in the present invention.

[0022] The strain for fermentation is Pseudomonas fluorescens strain M18.

[0023] Furthermore, the fermentation method includes the following steps:

[0024] Step 1: Cultivate Pseudomonas fluorescens strain M18 to obtain a seed solution;

[0025] Step 2: Inoculate the seed solution into the medium according to any one of claims 1-5 at 1 vt% for fermentation to obtain phenazine-1-carboxylic acid.

[0026] The temperature of the fermentation is 30 °C.

[0027] The rotation speed of the fermentation is 180 rpm - 260 rpm.

[0028] In a specific embodiment of the present invention, the active ingredient A needs to be pre-prepared into a mother liquor, and then filtered and sterilized through a microporous filter membrane, and added to the basal medium before inoculation so that the final concentration of the active ingredient A is 0.2 wt% - 0.5 wt%; Active ingredient B: Weigh the technical material and dissolve it in sterile water, and make it homogeneous by ultrasonic treatment, and add it to the fermentation medium before inoculation, and the final concentration of active ingredient B is 0.45 wt% - 0.6 wt%.

[0029] The present invention adds active ingredient A (organic acid) and active ingredient B (shenqinmycin) to a basic fermentation medium, and obtains a fermentation medium and method capable of shortening the fermentation time of shenqinmycin and increasing the yield of shenqinmycin by screening the active ingredient A and optimizing the proportion of active ingredients A, B and basic medium components. The fermentation medium and method are used for the fermentation of shenqinmycin, with short fermentation time and low fermentation cost, and have broad industrial application prospects. DETAILED DESCRIPTION

[0030] The present invention provides a culture medium and a fermentation method for shinazomib fermentation. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0031] The fermented strain used in the present invention is Pseudomonas fluorescens M18, and the seed liquid of Pseudomonas fluorescens M18 is prepared by the following steps: activating the strain on a KB plate, inoculating one inoculation loop into a KB liquid culture medium, for 16 to 18 hours, at 30° C. and 200 rpm.

[0032] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0033] Example 1

[0034] The basic fermentation formula is as follows: 2wt% glucose, 2wt% corn steep liquor, 0.2wt% bone peptone, 0.2wt% dipotassium hydrogen phosphate, 0.2wt% magnesium sulfate, 0.25wt% ammonium sulfate, and the balance is water, with a pH of 7.0, followed by sterilization at 115°C for 20min.

[0035] The active ingredient A is selected from citric acid, formic acid or acetic acid, and the addition amount is 0.1wt%~0.5wt%, and the addition amount of the active ingredient B is 0.5wt%.

[0036] Fermentation conditions: 30°C, 250 rpm;

[0037] Fermentation time: 48 hours.

[0038] The HPLC test results are shown in Table 1:

[0039] Table 1. HPLC test results without or with different active ingredients A added

[0040]

[0041] From the comparison results of the improvement of fermentation titer by adding formic acid, acetic acid, and citric acid, the improvement effects of acetic acid and citric acid on fermentation titer are better than that of formic acid, and citric acid is the best.

[0042] Example 2

[0043] Fermentation basic formula: 1 wt% glucose, 1 wt% corn steep liquor, 0.5 wt% bone peptone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, 0.25 wt% ammonium sulfate, the balance is water, pH is 7.0, then sterilized at 115 °C for 20 min;

[0044] The active ingredient A is citric acid, and the addition amount is 0.5 wt%, and the addition amount of the active ingredient B is 0.6 wt%.

[0045] Fermentation conditions: 30 °C, 250 rpm;

[0046] Fermentation time: 48 hours.

[0047] The HPLC detection results are shown in Table 2:

[0048] Table 2. HPLC detection results 1

[0049]

[0050] Example 3

[0051] Fermentation basic formula: 1.5 wt% glucose, 1.5 wt% corn steep liquor, 0.4 wt% bone peptone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, 0.25 wt% ammonium sulfate, the balance is water, pH is 7.0, then sterilized at 115 °C for 20 min;

[0052] The active ingredient A is citric acid, and the addition amount is 0.4 wt%, and the addition amount of the active ingredient B is 0.45 wt%.

[0053] Fermentation conditions: 30 °C, 250 rpm;

[0054] Fermentation time: 48 hours.

[0055] The HPLC detection results are shown in Table 3:

[0056] Table 3. HPLC detection results 1

[0057]

[0058] Example 4

[0059] Fermentation basic formula: 2 wt% glucose, 2 wt% corn steep liquor, 0.2 wt% bone peptone, 0.2 wt% dipotassium hydrogen phosphate, 0.2 wt% magnesium sulfate, 0.25 wt% ammonium sulfate, the balance is water, pH is 7.0, then sterilize at 115 °C for 20 min;

[0060] The active ingredient A is selected as citric acid, and the addition amount is 0.2 wt%, and the addition amount of the active ingredient B is 0.5 wt%.

[0061] Fermentation conditions: 30 °C, 250 rpm;

[0062] Fermentation time: 48 hours.

[0063] The HPLC detection results are shown in Table 4:

[0064] Table 4. HPLC detection results 3

[0065]

[0066] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Culture medium, characterized in that, It includes a basal medium, active ingredient A and active ingredient B; The basal medium includes: glucose, corn steep liquor, peptone from bone, dipotassium hydrogen phosphate, magnesium sulfate and / or ammonium sulfate; The active ingredient A is an organic acid; The active ingredient B is phenazine - 1 - carboxylic acid; 2. The culture medium according to claim 1, wherein The basal medium includes: glucose 1wt% - 10wt%, corn steep liquor 1wt% - 10wt%, peptone from bone 0.1wt% - 5wt%, dipotassium hydrogen phosphate 0.1wt% - 0.3wt%, magnesium sulfate 0.1wt% - 0.3wt% and ammonium sulfate 0.1wt% - 0.3wt%.

3. The culture medium according to claim 2, wherein The basal medium includes: glucose 1wt% - 2wt%, corn steep liquor 1wt% - 2wt%, peptone from bone 0.2wt% - 0.5wt%, dipotassium hydrogen phosphate 0.2wt%, magnesium sulfate 0.2wt% and ammonium sulfate 0.2wt%; pH 6.5 - 7.

0.

4. The culture medium according to claim 1, characterized in that The organic acid includes citric acid.

5. The culture medium according to claim 1 or 4, characterized in that, The addition amount of the citric acid is 0.1wt% - 0.5wt% of the basal medium; The addition amount of the phenazine - 1 - carboxylic acid is 0.45wt% - 0.6wt% of the basal medium.

6. The fermentation method of phenazine methosulfate, characterized in that, It is for fermentation using the culture medium according to any one of claims 1 - 5.

7. The fermentation method according to claim 6, wherein The strain for the fermentation is Pseudomonas fluorescens strain M18.

8. The fermentation method according to claim 6 or 7, characterized in that, It includes the following steps: Step 1, cultivate Pseudomonas fluorescens strain M18 to obtain a seed solution; Step 2, inoculate the seed solution into the culture medium according to any one of claims 1 - 5 at 1vt% for fermentation to obtain phenazine - 1 - carboxylic acid.

9. The fermentation method according to claim 8, wherein The temperature of the fermentation is 30°C.

10. The fermentation method according to claim 9, characterized in that, The rotation speed of the fermentation is 200rpm - 250rpm.

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