A fermentation medium for the ophiophagus hannah antimicrobial peptide OH-CATH30

Abstract

The present invention belongs to the technical field of microbial fermentation, and discloses a fermentation medium for the antimicrobial peptide OH-CATH30 of the king cobra. The main components of its formula are as follows: 50 g / L of glucose, 0.186 - 0.372 g / L of calcium sulfate dihydrate, 3.64 - 7.28 g / L of potassium sulfate, 2.98 - 5.96 g / L of magnesium sulfate, 0.826 - 1.652 g / L of potassium hydroxide, 5.34 - 8.01 ml / L of 85% phosphoric acid, 14 ml / L of PTM14; the pH value of the optimized fermentation medium is 4.5 - 6.0; the culture time is 24 - 48 h; the inoculation amount is 20 - 40%; the culture method is aerobic fermentation tank culture; the fermentation culture temperature is 30 °C. The optimized medium of the present invention has a lower salt content, optimizes the subsequent separation and purification process, saves costs at the same time, and is conducive to large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation, and more specifically relates to a fermentation medium for the antimicrobial peptide OH-CATH30 of the king cobra. Background Art

[0002] Cathelicidins are cationic host defense peptides that play an important role in the innate immune system. The antimicrobial peptide OH-CATH30 of the king cobra is a truncated peptide of cathelicidins, consisting of 30 amino acid sequences. Currently, studies have found that it has good antibacterial effects against various Gram-negative and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterobacter aerogenes.

[0003] Currently, the fermentation medium used for the antimicrobial peptide OH-CATH30 of the king cobra is the BSM medium. The BSM medium is an inorganic salt medium with simple components and convenient preparation. Experiments have found that at the end of a fermentation cycle, the content of unutilized components in the BSM medium is relatively high. On the one hand, it is easy to cause waste of raw materials during production. On the other hand, due to the large salt content, the subsequent separation and purification process is relatively cumbersome, which is not conducive to large-scale production. Summary of the Invention

[0004] The present invention provides a fermentation medium for the antimicrobial peptide OH-CATH30 of the king cobra, and the formula of this medium saves costs, has a low salt content, and optimizes the subsequent treatment process.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In the present invention, the fermentation medium is composed of the following components:

[0007] 1) Glucose 50 g / L

[0008] 2) Calcium sulfate dihydrate 0.186 - 0.372 g / L

[0009] 3) Potassium sulfate 3.64 - 7.28 g / L

[0010] 4) Magnesium sulfate 2.98 - 5.96 g / L

[0011] 5) Potassium hydroxide 0.826 - 1.652 g / L

[0012] 6) 85% Phosphoric acid 5.34 - 8.01 ml / L

[0013] 7) PTM1 4 ml / L

[0014] 8) The pH value of the optimized fermentation medium is 4.5 - 6.0; the culture time of the optimized fermentation medium is 24 - 48 h; the inoculation amount of the optimized fermentation medium is 20 - 40%; the optimized fermentation culture method is aerobic fermentation tank culture; the fermentation culture temperature is 30 °C.

[0015] Preferably, the calcium sulfate dihydrate is 0.186 g / L.

[0016] Preferably, the potassium sulfate is 3.64 g / L.

[0017] Preferably, the magnesium sulfate is 2.98 g / L.

[0018] Preferably, the potassium hydroxide is 0.826 g / L.

[0019] Preferably, the 85% phosphoric acid is 5.34 ml / L.

[0020] Preferably, the components of the PTM1 solution are as follows: 6 g / L copper sulfate pentahydrate, 0.08 g / L potassium iodide, 3 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride hexahydrate, 20 g / L zinc chloride, 65 g / L ferric sulfate heptahydrate, 0.2 g / L biotin, 5 mL / L concentrated sulfuric acid, and the balance is pure water.

[0021] Preferably, the pH value of the optimized fermentation medium is 6.0; the fermentation culture time is 24 h; the inoculation amount is 20%; the culture method is aerobic fermentation tank culture; the fermentation culture temperature is 30 °C.

[0022] In the present invention, the medium for dissolving the optimized fermentation medium is conventional in the art, preferably water; the water is conventional in the art, such as distilled water, double-distilled water, etc.

[0023] In the present invention, the preparation method of the optimized fermentation medium is conventional in the art, and only the components contained therein need to be simply mixed and then prepared according to the conventional preparation conditions of microbial media in the art; the conventional preparation conditions are 121 °C, high-pressure moist heat sterilization for 15 min.

[0024] The reagents and raw materials used in the present invention are all commercially available.

[0025] The positive and progressive effects of the fermentation medium for the antimicrobial peptide OH-CATH30 of Ophiophagus hannah in the present invention are as follows: the optimized fermentation medium of the present invention has a low salt content, optimizes the subsequent separation and purification process, saves costs at the same time, and is conducive to large-scale production. Detailed implementation manners

[0026] The present invention will be further described below by way of experimental examples, but the present invention is not limited to the scope of the examples described herein.

[0027] Example 1: The preparation method of the culture medium of the present invention is as follows: Take each raw material, glucose 50 g / L,

[0028] , calcium sulfate dihydrate 0.186 g / L, potassium sulfate 3.64 g / L, magnesium sulfate 2.98 g / L, potassium hydroxide 0.826 g / L, 85% phosphoric acid 5.34 ml / L, PTM1 4 ml / L. After stirring each raw material evenly, stir and preheat to 80 °C, adjust the pH to 6.0, sterilize at 115 °C for 30 min, cool with cold air for 10 min, turn on the stirrer, and cool the equipment to room temperature to obtain the fermentation medium. The preparation method of the above PTM1 solution is as follows: Weigh 6 g of copper sulfate pentahydrate, 0.08 g of potassium iodide, 3 g of manganese sulfate monohydrate, 0.2 g of sodium molybdate dihydrate, 0.02 g of boric acid, 0.5 g of cobalt chloride hexahydrate, 20 g of zinc chloride, 65 g of ferric sulfate heptahydrate, 0.2 g of biotin, 5 mL of concentrated sulfuric acid, add 800 mL of pure water to dissolve, and make up the volume to 1000 mL to obtain the trace salt solution PTM1.

[0029] Adopt the common fermentation process: Prepare the first-stage seed liquid and the second-stage seed liquid for standby, sterilize the fermentation tank and the fermentation medium, calibrate the dissolved oxygen and calibrate the pH electrode. After the dissolved oxygen calibration and the pH electrode calibration are completed, inoculate the prepared second-stage seed liquid into the fermentation tank through the fire ring for fermentation. The fermentation process is divided into growth culture and induction culture. The growth culture is the feeding culture stage. Control the fermentation temperature at 30 °C, set the pH at 6, maintain the dissolved oxygen value between 35% - 45%, ferment and culture for 24 h. During the culture process, sample and detect the residual glucose in the fermentation broth. When the glucose is detected to be consumed completely, start to flow in a 30% glucose solution at a rate of 50 ml / h. The feeding time depends on the bacterial concentration at the start of induction. When the bacterial concentration reaches the bacterial concentration condition at the start of induction, carry out starvation culture for 60 - 90 min. The induction culture is the expression culture stage. After the starvation culture ends, start methanol induction. In the induction culture stage, control the fermentation temperature at 30 °C, set the pH at 6, ferment and culture for 48 h. Initially adjust the ventilation volume to 1 L / min. As the methanol consumption increases, appropriately adjust the ventilation volume. Start to flow in 1% of the fermentation broth volume of 100% methanol at one time. When the methanol is consumed completely, start to flow in methanol at a rate of 2 ml / h / L. After the bacteria are completely adapted to methanol, adjust the methanol flow rate to 5 ml / h / L, and maintain the dissolved oxygen value between 20% - 40% until the fermentation ends.

[0030] Treatment of the fermentation broth: After the fermentation is completed, the fermentation broth is centrifuged by a tubular centrifuge at a speed of 10,000 rpm to remove the yeast strain, and the supernatant is taken. Then, a membrane filtration device is used to sequentially pass through 10,000D, 5,000D ultrafiltration membranes and 600D nanofiltration membranes to remove macromolecular impurities and small molecular inorganic salts. Finally, a freeze dryer is used to prepare the crude product of cobra antimicrobial peptide OH-CATH30.

[0031] Example 2

[0032] The preparation method of the medium is as follows: Take each raw material, glucose 50 g / L, calcium sulfate dihydrate 0.372 g / L, potassium sulfate 7.28 g / L, magnesium sulfate 5.96 g / L, potassium hydroxide 1.652 g / L, 85% phosphoric acid 10.68 ml / L, PTM1 4 ml / L. After stirring each raw material evenly, stir and preheat to 80 °C, adjust the pH to 6.0, sterilize at 115 °C for 30 min, cool with cold air for 10 min, turn on the stirrer, and cool the equipment to room temperature to obtain the fermentation medium.

[0033] The remaining steps are repeated according to the fermentation process of Example 1 for experiments.

[0034] Example 3

[0035] The preparation method of the medium is as follows: Take each raw material, glucose 50 g / L,

[0036] , calcium sulfate dihydrate 0.465 g / L, potassium sulfate 9.1 g / L, magnesium sulfate 7.45 g / L, potassium hydroxide 4.13 g / L, 85% phosphoric acid 26.7 ml / L, PTM1 4 ml / L. After stirring each raw material evenly, stir and preheat to 80 °C, adjust the pH to 6.0, sterilize at 115 °C for 30 min, cool with cold air for 10 min, turn on the stirrer, and cool the equipment to room temperature to obtain the fermentation medium.

[0037] The remaining steps are repeated according to the fermentation process of Example 1 for experiments.

[0038] Example 4

[0039] The preparation method of the medium is as follows: Take each raw material, glucose 50 g / L,

[0040] , calcium sulfate dihydrate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate 14.9 g / L, potassium hydroxide 4.13 g / L, 85% phosphoric acid 26.7 ml / L, PTM1 4 ml / L. After stirring each raw material evenly, stir and preheat to 80 °C, adjust the pH to 6.0, sterilize at 115 °C for 30 min, cool with cold air for 10 min, turn on the stirrer, and cool the equipment to room temperature to obtain the fermentation medium.

[0041] The remaining experiments were carried out by repeating the fermentation process steps of Example 1.

[0042] The results showed that: according to the experimental results of Examples 1-4, during the fermentation culture, the OD600 value of the fermentation broth, the wet weight of the bacteria, and the content of the crude antibacterial peptide were measured, and the results are shown in the following table.

[0043] Table 1: Experimental results of BSM medium with different contents

[0044]

[0045] It can be seen from the above table that the highest OD600 value and the highest wet weight in the fermentation of the engineering bacteria of Ophiophagus hannah antibacterial peptide OH-CATH30 with the 20% BSM medium formula and the 100% BSM medium formula are the same, and the content of the crude Ophiophagus hannah antibacterial peptide OH-CATH30 prepared is the same. That is, the growth of Ophiophagus hannah antibacterial peptide OH-CATH30 fermented with the 20% BSM optimized medium is consistent with that fermented with the 100% BSM medium, and it does not affect the expression of Ophiophagus hannah antibacterial peptide OH-CATH30. The 20% BSM medium optimized formula has a lower salt content. Using the optimized 20% BSM medium for fermentation can optimize the subsequent separation and purification process, save costs at the same time, and contribute to large-scale production.

Claims

1. A fermentation medium for the ophiophagus hannah antimicrobial peptide OH-CATH30, characterized in that: The fermentation medium consists of the following components: 1) Glucose 50 g / L; 2) Calcium sulfate dihydrate 0.186 - 0.372 g / L; 3) Potassium sulfate 3.64 - 7.28 g / L; 4) Magnesium sulfate 2.98 - 5.96 g / L; 5) Potassium hydroxide 0.826 - 1.652 g / L; 6) 85% Phosphoric acid 5.34 - 8.01 ml / L; 7) PTM1 4 ml / L; Among them, the pH value of the fermentation medium is 4.5 - 6.

0.

2. The fermentation medium for the ophiophagus hannah antimicrobial peptide OH-CATH30 according to claim 1, characterized in that, The composition components of the fermentation medium are: Glucose 50 g / L; Calcium sulfate dihydrate 0.186 g / L; Potassium sulfate 3.64 g / L; Magnesium sulfate 2.98 g / L; Potassium hydroxide 0.826 g / L; 85% Phosphoric acid 5.34 ml / L; PTM1 4 ml / L.

3. According to the fermentation medium for cobra antimicrobial peptide OH-CATH30 described in claim 1, the components of the PTM1 solution are as follows: Copper sulfate pentahydrate 6 g / L, Potassium iodide 0.08 g / L, Manganese sulfate monohydrate 3 g / L, Sodium molybdate dihydrate 0.2 g / L, Boric acid 0.02 g / L, Cobalt chloride hexahydrate 0.5 g / L, Zinc chloride 20 g / L, Ferric sulfate heptahydrate 65 g / L, Biotin 0.2 g / L, Concentrated sulfuric acid 5 mL / L, and the balance is pure water.

4. According to the fermentation medium for cobra antimicrobial peptide OH-CATH30 described in claim 1, the pH value of the fermentation medium is 6.0.

Images