Method for improving fermentation level of L-histidine through trace element combination

Abstract

The invention belongs to the technical field of microbial fermentation of histidine, and particularly relates to a method for improving the fermentation level of L-histidine through microelement combination. The invention provides a method for improving the fermentation level of L-histidine by combining trace elements. The method comprises a seed solution and a fermentation culture medium, the fermentation culture medium comprises the following components: 20-40 g / L of glucose, 10-20 g / L of ammonium sulfate, 3-6 g / L of monopotassium phosphate, 0.1-0.3 g / L of citric acid, 5-10 g / L of yeast extract powder, 1-3 g / L of magnesium sulfate heptahydrate, 1-2 g / L of ferrous sulfate and trace elements; the trace elements comprise 0.5-2 g / L of zinc chloride, 1-2 g / L of calcium chloride, 0.5-2 g / L of ammonium molybdate, 1-3 g / L of copper sulfate, 1-4 g / L of manganese sulfate and 1-3 g / L of cobalt chloride, and the trace elements are added into the fermentation culture medium according to the proportion of 1-3 ml / L. The trace element composition provided by the invention has a promoting effect on the production of L-histidine, the fermentation unit can be improved by 10-20% and reaches 40-45g / L compared with a contrast, and the sugar-acid conversion rate reaches 21.0%.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation of histidine, and in particular relates to a method for improving the fermentation level of L-histidine by combining trace elements. Background Art

[0002] Histidine was first isolated by German physicist Albrecht Kossel in 1896. For children, histidine is an essential amino acid; for patients with chronic uremia, histidine can alleviate renal anemia and is also an essential amino acid for patients with uremia.

[0003] At present, the process of producing L-histidine includes chemical synthesis, protein hydrolysis and microbial fermentation. Protein hydrolysis is heavily polluted, has poor separation effect, low yield and purity, while fermentation is less polluted, has high yield and high product purity. Patents CN113881726A, CN117660559A and CN1186201971A all introduce methods for preparing L-histidine by fermentation. In the fermentation process control, the flow-added glucose process is generally adopted to reduce the sugar content in the fermentation liquid to less than about 2g / L. Further, in CN117660559A, the intermittent sugar supplement method is adopted to control the sugar content to produce L-histidine. At the same time, CN1186201971A introduces the method of adding adenosine phosphate in the supplement to improve the vitality of the bacteria, thereby increasing the yield of L-histidine.

[0004] At present, the methods for improving the fermentation production of L-histidine are mostly concentrated on the methods of feed control, sugar control, and adding nutrients, but the effect of adding a combination of trace elements on improving L-histidine is not elaborated. Trace elements are activating factors or coenzymes of many biological enzymes and have an important influence on the activity of enzymes. For example, Mg2+ is an activating factor of phosphokinase, etc. The present invention proposes a combination of trace elements, which can effectively improve the fermentation level of L-histidine from practice, and provides a method for improving the fermentation level of L-histidine from another aspect. Summary of the invention

[0005] The purpose of the present invention is to provide a trace element combination to solve the shortcomings of the existing L-histidine fermentation technology.

[0006] Trace elements are essential for bacterial growth and metabolism. Facts show that iron, zinc, magnesium, calcium and other elements play an important role in the activity of biological enzymes. Reasonable combinations of different trace elements are added to the L-histidine fermentation medium to activate the enzyme system in the growth and metabolism process and promote the synthesis of histidine.

[0007] To solve the above technical problems, the technical solution provided by the present invention is a method for improving the fermentation level of L-histidine by using a combination of trace elements, comprising seed liquid and fermentation medium; the fermentation medium components are glucose 20-40g / L, ammonium sulfate 10-20g / L, potassium dihydrogen phosphate 3-6g / L, citric acid 0.1-0.3g / L, yeast extract 5-10g / L, magnesium sulfate heptahydrate 1-3g / L, ferrous sulfate 1-2g / L and trace elements; the trace elements components are zinc chloride 0.5-2g / L, calcium chloride 1-2g / L, ammonium molybdate 0.5-2g / L, copper sulfate 1-3g / L, manganese sulfate 1-4g / L, cobalt chloride 1-3g / L, and the trace elements are added to the fermentation medium at a ratio of 1-3ml / L;

[0008] The preparation method of the L-histidine fermentation broth is:

[0009] Step 1: Inoculate the seed solution into a sterilized fermentation medium, wherein the medium is located in a fermentation tank;

[0010] Step 2, setting the culture temperature of the strain to 35-38°C, and maintaining the content of reducing sugar in the fermentation broth by adding glucose solution;

[0011] Step 3, maintaining the pH in the fermentation tank by adding ammonia water;

[0012] Step 4: The fermentation time is 40-60 hours to obtain L-histidine fermentation liquid.

[0013] In one embodiment, the inoculation amount of the seed solution is 10-15%.

[0014] In one embodiment, the glucose concentration in the glucose solution in step 2 is 400-600 g / L.

[0015] In one embodiment, the concentration of reducing sugars in the fermentation broth in step 2 is ≤ 1 g / L.

[0016] In one embodiment, the fermentation condition in step three is pH 6.7-6.9, and the tank pressure in the fermentation tank is 0.05-0.15 MPa.

[0017] In one embodiment, a defoaming agent needs to be added in step three for defoaming.

[0018] The advantages of the present invention compared with the prior art are:

[0019] The trace element combination provided by the present invention has a promoting effect on the production of L-histidine. Compared with the control, the fermentation unit can be increased by 10% to 20% to 40-45g / L, and the sugar-acid conversion rate reaches 21.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparative table of the effects of a method for improving the fermentation level of L-histidine using a combination of trace elements according to the present invention. DETAILED DESCRIPTION

[0021] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. 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 products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0022] All reagents used in the experiments of the present invention are commonly used reagents.

[0023] Example 1

[0024] Take out the glycerol tube of bacteria from the -80℃ refrigerator, inoculate it into LB liquid medium on the clean bench, and culture it at 37℃ with shaking for 12h. When the OD value is greater than 2, the first-level seed liquid is obtained;

[0025] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 10%, and cultured at 37° C. with shaking until the OD value was about 3, thereby obtaining the second-level seed solution;

[0026] Secondary seed culture medium components: peptone 12g / L, yeast extract powder 8g / L, sodium chloride 5g / L; pH is controlled at 7.0.

[0027] The secondary seed liquid was inoculated into a fermentation tank containing a fermentation medium at a rate of 10%, the culture temperature was 37°C, and the ventilation volume was 1.5 VVM; by adding 500 g / L glucose solution, the reducing sugar content of the fermentation liquid was less than 1 g / L, and the dissolved oxygen content was greater than 25%; 20% ammonia water was added to maintain the pH in the fermentation tank at 6.9; the fermentation time was 56 hours, and L-histidine fermentation liquid was obtained;

[0028] Fermentation medium components: glucose 20g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 3g / L, citric acid 0.1g / L, yeast extract powder 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate 2g / L.

[0029] The components of the trace elements are:

[0030] 2g / L zinc chloride, 2g / L calcium chloride, 2g / L ammonium molybdate, 3g / L copper sulfate, 4g / L manganese sulfate, 3g / L cobalt chloride. Add the above solutions to the fermentation medium at a ratio of 1ml / L.

[0031] Example 2

[0032] Take out the glycerol tube of bacteria from the -80℃ refrigerator, inoculate it into LB liquid medium on the clean bench, and culture it at 37℃ with shaking for 12h. When the OD value is greater than 2, the first-level seed liquid is obtained;

[0033] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 10%, and cultured at 37° C. with shaking until the OD value was about 3, thereby obtaining the second-level seed solution;

[0034] Secondary seed culture medium components: peptone 12g / L, yeast extract powder 8g / L, sodium chloride 5g / L; pH is controlled at 7.0.

[0035] The secondary seed liquid was inoculated into a fermentation tank containing a fermentation medium at a rate of 10%, the culture temperature was 37°C, and the ventilation volume was 1.5 VVM; by adding 500 g / L glucose solution, the reducing sugar content of the fermentation liquid was less than 1 g / L, and the dissolved oxygen content was greater than 25%; 20% ammonia water was added to maintain the pH in the fermentation tank at 6.9; the fermentation time was 54 hours, and L-histidine fermentation liquid was obtained;

[0036] Fermentation medium components: glucose 20g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 3g / L, citric acid 0.1g / L, yeast extract powder 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate 2g / L.

[0037] The components of the trace elements are:

[0038] Zinc chloride 1g / L, calcium chloride 1g / L, ammonium molybdate 1g / L, copper sulfate 1g / L, manganese sulfate 2g / L, cobalt chloride 1g / L. Add the above solutions to the fermentation medium at a ratio of 3ml / L.

[0039] Example 3

[0040] Take out the glycerol tube of bacteria from the -80℃ refrigerator, inoculate it into LB liquid medium on the clean bench, and culture it at 37℃ with shaking for 12h. When the OD value is greater than 2, the first-level seed liquid is obtained;

[0041] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 10%, and cultured at 37° C. with shaking until the OD value was about 3, thereby obtaining the second-level seed solution;

[0042] Secondary seed culture medium components: peptone 12g / L, yeast extract powder 8g / L, sodium chloride 5g / L; pH is controlled at 7.0.

[0043] The secondary seed liquid was inoculated into a fermentation tank containing a fermentation medium at a rate of 10%, the culture temperature was 37°C, and the ventilation volume was 1.5 VVM; by adding 500 g / L glucose solution, the reducing sugar content of the fermentation liquid was less than 1 g / L, and the dissolved oxygen content was greater than 25%; 20% ammonia water was added to maintain the pH in the fermentation tank at 6.9; the fermentation time was 58 hours, and L-histidine fermentation liquid was obtained;

[0044] Fermentation medium components: glucose 20g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 3g / L, citric acid 0.1g / L, yeast extract powder 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate 2g / L.

[0045] The components of the trace elements are:

[0046] 1.5g / L zinc chloride, 1.5g / L calcium chloride, 2g / L ammonium molybdate, 2g / L copper sulfate, 2g / L manganese sulfate, 2g / L cobalt chloride. Add the above solutions to the fermentation medium at a ratio of 2ml / L.

[0047] Comparative Example 1

[0048] Take out the glycerol tube of bacteria from the -80℃ refrigerator, inoculate it into LB liquid medium on the clean bench, and culture it at 37℃ with shaking for 12h. When the OD value is greater than 2, the first-level seed liquid is obtained;

[0049] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 10%, and cultured at 37° C. with shaking until the OD value was about 3, thereby obtaining the second-level seed solution;

[0050] Secondary seed culture medium components: peptone 12g / L, yeast extract powder 8g / L, sodium chloride 5g / L; pH is controlled at 7.0.

[0051] The secondary seed liquid was inoculated into a fermentation tank containing a fermentation medium at a rate of 10%, the culture temperature was 37°C, and the ventilation volume was 1.5 VVM; by adding 500 g / L glucose solution, the reducing sugar content of the fermentation liquid was less than 1 g / L, and the dissolved oxygen content was greater than 25%; 20% ammonia water was added to maintain the pH in the fermentation tank at 6.9; the fermentation time was 56 hours, and L-histidine fermentation liquid was obtained;

[0052] Fermentation medium components: glucose 20g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 3g / L, citric acid 0.1g / L, yeast extract powder 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate 2g / L.

[0053] Comparative Example 2

[0054] Take out the glycerol tube of bacteria from the -80℃ refrigerator, inoculate it into LB liquid medium on the clean bench, and culture it at 37℃ with shaking for 12h. When the OD value is greater than 2, the first-level seed liquid is obtained;

[0055] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 10%, and cultured at 37° C. with shaking until the OD value was about 3, thereby obtaining the second-level seed solution;

[0056] Secondary seed culture medium components: peptone 12g / L, yeast extract powder 8g / L, sodium chloride 5g / L; pH is controlled at 7.0.

[0057] The secondary seed liquid was inoculated into a fermentation tank containing a fermentation medium at a rate of 10%, the culture temperature was 37°C, and the ventilation volume was 1.5 VVM; by adding 500 g / L glucose solution, the reducing sugar content of the fermentation liquid was less than 1 g / L, and the dissolved oxygen content was greater than 25%; 20% ammonia water was added to maintain the pH in the fermentation tank at 6.9; the fermentation time was 54 hours, and L-histidine fermentation liquid was obtained;

[0058] Fermentation medium components: glucose 20g / L, ammonium sulfate 15g / L, potassium dihydrogen phosphate 3g / L, citric acid 0.1g / L, yeast extract powder 5g / L, magnesium sulfate heptahydrate 2g / L, ferrous sulfate 2g / L.

[0059] The present invention and its implementation methods are described above, and such description is not restrictive. What is shown in the specific implementation method is only one of the implementation methods of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in this field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for improving the fermentation level of L-histidine by using a combination of trace elements, characterized in that: It comprises seed liquid and fermentation medium; the fermentation medium comprises 20-40 g / L glucose, 10-20 g / L ammonium sulfate, 3-6 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L citric acid, 5-10 g / L yeast extract, 1-3 g / L magnesium sulfate heptahydrate, 1-2 g / L ferrous sulfate and trace elements; the trace elements comprise 0.5-2 g / L zinc chloride, 1-2 g / L calcium chloride, 0.5-2 g / L ammonium molybdate, 1-3 g / L copper sulfate, 1-4 g / L manganese sulfate and 1-3 g / L cobalt chloride, and the trace elements are added to the fermentation medium at a ratio of 1-3 ml / L; The preparation method of the L-histidine fermentation broth is: Step 1: Inoculate the seed solution into a sterilized fermentation medium, wherein the medium is located in a fermentation tank; Step 2, setting the culture temperature of the strain to 35-38°C, and maintaining the content of reducing sugar in the fermentation broth by adding glucose solution; Step 3, maintaining the pH in the fermentation tank by adding ammonia water; Step 4: The fermentation time is 40-60 hours to obtain L-histidine fermentation liquid.

2. The method for improving the fermentation level of L-histidine by using a combination of trace elements according to claim 1, characterized in that: The inoculation amount of the seed liquid is 10-15%.

3. The method for improving the fermentation level of L-histidine by using a combination of trace elements according to claim 1, characterized in that: The glucose concentration in the glucose solution in step 2 is 400-600 g / L.

4. The method for improving the fermentation level of L-histidine by using a combination of trace elements according to claim 1, characterized in that: In the step 2, the concentration of reducing sugar in the fermentation broth is ≤1 g / L.

5. The method for improving the fermentation level of L-histidine by using a combination of trace elements according to claim 1, characterized in that: The fermentation conditions in step 3 are pH 6.7-6.9, and the tank pressure in the fermentation tank is 0.05-0.15 MPa.

6. The method for improving the fermentation level of L-histidine by using a combination of trace elements according to claim 1, characterized in that: In the step three, a defoaming agent needs to be added for defoaming.

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