Fermentation method capable of increasing spectinomycin yield

Abstract

The invention relates to the technical field of spectinomycin, in particular to a fermentation method capable of improving the yield of spectinomycin, and solves the problem that although the fermentation process of spectinomycin in the prior art is relatively mature, the yield still needs to be further improved so as to meet the ever-increasing market demand. The invention relates to a fermentation method capable of increasing the yield of spectinomycin. The fermentation method comprises the steps of seed liquid culture, fermentation culture medium preparation, inoculation and fermentation, substance supplementation, fermentation time, fermentation completion and the like. By optimizing fermentation conditions and exploring the possibility of supplementing beneficial substances, the yield of spectinomycin is effectively increased, the market demand is met, and a new way is provided for efficient production of spectinomycin.

Description

Technical Field

[0001] The invention relates to the technical field of spectinomycin, and in particular to a fermentation method capable of increasing the yield of spectinomycin. Background Art

[0002] Spectinomycin, also known as spectinomycin or spectinomycin, is an alkaline water-soluble antibiotic with wide application. It is known for its broad-spectrum antibacterial activity and exhibits strong inhibitory ability against both Gram-positive and Gram-negative bacteria. Spectinomycin has a unique mechanism of action, which effectively curbs the growth of bacteria by irreversibly inhibiting protein synthesis and destroying the integrity of cell membranes. Clinically, spectinomycin is often used to treat urinary tract infections, urethritis, cervicitis, proctitis and other diseases caused by gonococci due to its good in vivo activity. In addition, due to its low toxicity and broad spectrum, spectinomycin is also used as a feed additive to promote the growth of various animals, and is also widely used in the field of pesticides.

[0003] In the production process of spectinomycin, the selection and optimization of fermentation method is very important, which directly affects the yield and quality of spectinomycin. However, in the current fermentation technology, although certain achievements have been made, there are still limitations.

[0004] Specifically, traditional fermentation methods face challenges in increasing spectinomycin production. Although the fermentation process of spectinomycin is relatively mature, the production still needs to be further improved to meet the growing market demand. At the same time, existing fermentation methods may have cumbersome and inefficient problems during operation, which not only increases production costs, but also limits the further expansion of production scale. More importantly, traditional fermentation methods may be difficult to fully tap the potential of spectinomycin, so that its effect in specific applications is limited.

[0005] Citicoline is an important nucleotide derivative, which is composed of choline, ribose and phosphate in chemical structure. It has unique biological activity. It can enhance the fluidity of cell membrane, improve the stability of cell membrane, and maintain the phospholipid ratio of cell membrane by regulating phospholipid metabolism, thereby maintaining the integrity and function of cell membrane. In harmful environments such as oxidative stress, cytiticoline can bind to free radicals to reduce the damage of free radicals to cell membrane. Resveratrol is a natural antioxidant present in plants, which has antioxidant effects such as scavenging or inhibiting the generation of free radicals, inhibiting lipid peroxidation, and regulating the activity of antioxidant-related enzymes. The present invention has found through in-depth research that both cytiticoline and resveratrol can play their unique role in the fermentation process of spectinomycin and improve the fermentation titer of spectinomycin.

[0006] However, the existing fermentation method does not fully utilize this characteristic of citicoline and resveratrol. The parameters such as the method, timing and dosage of adding these two substances in the fermentation process have not been systematically optimized, resulting in limited increase in the yield of spectinomycin. Therefore, in view of these deficiencies in the prior art, we urgently need a fermentation method that can increase the yield of spectinomycin, by scientifically and rationally adding one or both of citicoline and resveratrol, effectively increasing the yield of spectinomycin, and providing strong support for the wide application of spectinomycin. Summary of the invention

[0007] The purpose of the present invention is to provide a fermentation method capable of increasing the yield of spectinomycin, which solves the problem that the fermentation process of spectinomycin in the prior art is relatively mature, but the yield still needs to be further improved to meet the growing market demand.

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

[0009] A fermentation method capable of increasing the yield of spectinomycin, comprising S1, inoculating a cultured seed liquid into a fermentation medium for fermentation;

[0010] S2, adding one or both of cytidine diphosphate choline and resveratrol during the fermentation process;

[0011] S3. After a period of fermentation, the fermentation is stopped to obtain the spectinomycin product.

[0012] Preferably, the inoculation amount is 1%-20% of the volume of the fermentation medium.

[0013] Preferably, the inoculation amount is 5%-10% of the volume of the fermentation medium.

[0014] Preferably, the inoculation amount is 8% of the volume of the fermentation medium.

[0015] Preferably, the composition of the fermentation medium includes: corn starch 40-100g / L, soybean cake powder 0-20g / L, biological nitrogen 5-20g / L, yeast powder 0-20g / L, potassium dihydrogen phosphate 0.5-2g / L, potassium chloride 1-5g / L, ammonium sulfate 1-10g / L, and calcium carbonate 5-30g / L.

[0016] Preferably, the composition of the fermentation medium includes: 60-80 g / L corn starch, 5-15 g / L soybean cake powder, 10-15 g / L biological nitrogen, 5-15 g / L yeast powder, 1-1.5 g / L potassium dihydrogen phosphate, 2-4 g / L potassium chloride, 4-6 g / L ammonium sulfate, and 10-20 g / L calcium carbonate.

[0017] Preferably, the composition of the fermentation medium includes: 70 g / L corn starch, 10 g / L soybean cake powder, 12 g / L biological nitrogen, 10 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, 3 g / L potassium chloride, 5 g / L ammonium sulfate, and 15 g / L calcium carbonate.

[0018] Preferably, the total added amount of citicoline has a mass volume ratio (W / V) of 0.0001-0.05%, and the total added amount of resveratrol has a mass volume ratio (W / V) of 0.0001-0.05%; and the addition cycle is 10-70h during the fermentation process.

[0019] Preferably, the total added amount of citicoline has a mass volume ratio (W / V) of 0.003-0.007%, and the total added amount of resveratrol has a mass volume ratio (W / V) of 0.0001-0.0005%; and the addition cycle is 20-40h during the fermentation process.

[0020] The present invention has at least the following beneficial effects:

[0021] By optimizing the fermentation process and exploring the possibility of adding citicoline and resveratrol, the technical problem of limited yield improvement of traditional fermentation methods was effectively solved, providing a new way to increase the yield of spectinomycin. Secondly, the implementation of this method not only optimized the operation process and improved the fermentation efficiency, but also further explored the production potential of spectinomycin, laying a solid foundation for meeting the growing market demand, and also providing strong support for the wide application of spectinomycin. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Embodiment 1

[0024] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0025] Preparation of fermentation medium: Prepare fermentation medium according to the following composition: corn starch 70g / L, soybean cake powder 10g / L, biological nitrogen 12g / L, yeast powder 10g / L, potassium dihydrogen phosphate 1g / L, potassium chloride 3g / L, ammonium sulfate 5g / L, calcium carbonate 15g / L.

[0026] Inoculation and fermentation: Inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 8% for fermentation.

[0027] Supplemented substances: Citicoline and resveratrol were not supplemented during the fermentation process.

[0028] Fermentation time: The fermentation time is 108h.

[0029] End of fermentation: Stop fermentation and obtain spectinomycin product. After testing, the fermentation titer is 8101u / ml.

[0030] The test results at the end of fermentation are as follows:

[0031] name Fermentation titer (u / ml) Example 1 8101

[0032] The specific workflow is as follows:

[0033] Fermentation medium preparation:

[0034] The fermentation medium was prepared according to the following composition: corn starch 70g / L, soybean cake powder 10g / L, biological nitrogen 12g / L, yeast powder 10g / L, potassium dihydrogen phosphate 1g / L, potassium chloride 3g / L, ammonium sulfate 5g / L, calcium carbonate 15g / L. These ingredients provide the carbon source, nitrogen source, inorganic salt and other nutrients required for the growth of the strain, providing strong support for the growth and metabolism of the strain.

[0035] Inoculation and fermentation:

[0036] The cultured seed liquid is inoculated into the fermentation medium at an inoculation rate of 8% for fermentation. The selection of the inoculation rate is based on a comprehensive consideration of the growth characteristics of the strain and the fermentation efficiency, ensuring that the strain can grow rapidly in the fermentation medium and produce spectinomycin.

[0037] Supplementary materials:

[0038] In the fermentation process of the present invention, although the basic technical solution does not add citicoline and resveratrol, the core of the present invention is to propose the possibility of increasing the yield of spectinomycin by adding these two substances. In practical applications, according to experimental research and optimization results, citicoline and resveratrol can be added at an appropriate time and dosage to give full play to their positive effects on the spectinomycin fermentation process.

[0039] Fermentation time:

[0040] The fermentation time is 108 hours. This time is determined based on the comprehensive consideration of the growth cycle of the strain and the production rate of spectinomycin to ensure that spectinomycin is fully produced during the fermentation process.

[0041] End of fermentation:

[0042] The fermentation was stopped to obtain spectinomycin product. After testing, the fermentation titer under the basic technical solution was 8101u / ml. After adding citicoline and resveratrol, the fermentation titer was expected to increase.

[0043] Beneficial effects:

[0044] The effects of the present invention are mainly reflected in the following aspects:

[0045] Increased yield:

[0046] By scientifically and rationally adding one or both of Citicoline and Resveratrol, the fermentation potency of spectinomycin can be effectively improved, thus meeting the growing market demand. This innovation directly addresses the shortcomings of the traditional fermentation method in terms of limited yield increase.

[0047] Optimize the operation process:

[0048] The fermentation method of the present invention is optimized based on the conventional method, the operation process is optimized, and the fermentation efficiency is improved, which helps to reduce production costs, expand production scale, and improve market competitiveness.

[0049] Unleash the full potential of spectinomycin:

[0050] By adding citicoline and resveratrol, the potential of spectinomycin can be further explored, so that its effect in specific applications can be more fully exerted. This will help expand the application field of spectinomycin and increase its market value.

[0051] Provide strong support:

[0052] The invention provides strong support for the wide application of spectinomycin. By improving the yield and quality of spectinomycin, the needs of more fields can be met and the sustainable development of related industries can be promoted.

[0053] Embodiment 2

[0054] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0055] At 40h of fermentation, citicoline was added, and the total amount of added citicoline was 0.003% by weight to volume ratio (W / V). The other steps were the same as those in Example 1. After testing, the fermentation titer was 11270u / ml.

[0056] The test results at the end of fermentation are as follows:

[0057] name Fermentation titer (u / ml) Example 2 11270

[0058] Embodiment 3

[0059] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0060] At 40h of fermentation, citicoline was added, and the total amount of added citicoline was 0.005% by weight to volume ratio (W / V). The other steps were the same as those in Example 1. After testing, the fermentation titer was 12156u / ml.

[0061] The test results at the end of fermentation are as follows:

[0062] name Fermentation titer (u / ml) Example 3 12156

[0063] Embodiment 4

[0064] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0065] At 40h of fermentation, citicoline was added, and the total amount of added citicoline was 0.007% by weight to volume ratio (W / V). The other steps were the same as those in Example 1. After testing, the fermentation titer was 11459u / ml.

[0066] The test results at the end of fermentation are as follows:

[0067] name Fermentation titer (u / ml) Example 4 11459

[0068] Embodiment 5

[0069] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0070] Resveratrol was added at 40h of fermentation, and the total amount of resveratrol added was 0.0001% by weight to volume ratio (W / V). Other steps were the same as those in Example 1. After testing, the fermentation titer was 8748u / m.

[0071] The test results at the end of fermentation are as follows:

[0072] name Fermentation titer (u / ml) Example 5 8748

[0073] Embodiment 6

[0074] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0075] Resveratrol was added at 40h of fermentation, and the total amount added was 0.0003% by weight to volume ratio (W / V). Other steps were the same as those in Example 1. After testing, the fermentation titer was 9582u / ml.

[0076] The test results at the end of fermentation are as follows:

[0077] name Fermentation titer (u / ml) Example 6 9582

[0078] Embodiment 7

[0079] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0080] Resveratrol was added at 40h of fermentation, and the total amount of resveratrol added was 0.0005% by weight to volume ratio (W / V). The other steps were the same as those in Example 1. After testing, the fermentation titer was 8911u / ml.

[0081] The test results at the end of fermentation are as follows:

[0082] name Fermentation titer (u / ml) Example 7 8911

[0083] Embodiment 8

[0084] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0085] At 40h of fermentation, citicoline and resveratrol were added simultaneously, the total amount of citicoline added was 0.005% by weight and the total amount of resveratrol added was 0.0003% by weight. Other steps were the same as those in Example 1. After testing, the fermentation titer was 13025u / ml.

[0086] The test results at the end of fermentation are as follows:

[0087] name Fermentation titer (u / ml) Example 8 13025

[0088] Embodiment 9

[0089] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0090] The time for adding Citicoline and resveratrol is 30 hours of fermentation. The other steps are the same as those in Example 8. After testing, the fermentation titer is 13886u / ml, which is 71.4% higher than that of conventional technology.

[0091] The test results at the end of fermentation are as follows:

[0092] name Fermentation titer (u / ml) Example 9 13886

[0093] Embodiment 10

[0094] A fermentation method capable of increasing the yield of spectinomycin in this embodiment comprises:

[0095] The time for adding citicoline and resveratrol was 20 hours of fermentation. The other steps were the same as those in Example 8. After testing, the fermentation titer was 12935u / ml.

[0096] The test results at the end of fermentation are as follows:

[0097] name Fermentation titer (u / ml) Example 10 12935

[0098] It can be seen from the above examples that the present invention significantly increases the yield of spectinomycin by supplementing one or both of citicoline and resveratrol during the fermentation process. Among them, the yield of Example 9 is the highest, which is 71.4% higher than that of conventional technology. Therefore, the present invention provides an effective and feasible fermentation method for increasing the yield of spectinomycin.

[0099] A fermentation method for increasing spectinomycin production and its detailed beneficial effects

[0100] Workflow:

[0101] The fermentation method of the present invention aims to increase the yield of spectinomycin, and its workflow includes the following key steps, which are described in detail in each embodiment:

[0102] 1. Preparation of fermentation medium:

[0103] The fermentation medium was prepared, and the composition was: corn starch 70g / L, soybean cake powder 10g / L, biological nitrogen 12g / L, yeast powder 10g / L, potassium dihydrogen phosphate 1g / L, potassium chloride 3g / L, ammonium sulfate 5g / L, calcium carbonate 15g / L. These ingredients provide the carbon source, nitrogen source, inorganic salt and other nutrients required for the growth of the strain.

[0104] 2. Inoculation and fermentation:

[0105] The cultured seed liquid is inoculated into the fermentation medium at an inoculation rate of 8% for fermentation. The selection of the inoculation rate is based on a comprehensive consideration of the growth characteristics of the strain and the fermentation efficiency.

[0106] 3. Added substances (key innovation point):

[0107] Example 1: No citicoline and resveratrol were added as a control.

[0108] Examples 2 to 4: Citicoline was added at 40 h of fermentation, and the mass volume ratio (W / V) of the total amount added was 0.003%, 0.005% and 0.007%, respectively.

[0109] Examples 5 to 7: Resveratrol was added at 40 hours of fermentation, and the total amount added had a mass volume ratio (W / V) of 0.0001%, 0.0003% and 0.0005%, respectively.

[0110] Example 8: Citicoline and resveratrol were added simultaneously at 40 h of fermentation, with the total amount of Citicoline added having a mass volume ratio (W / V) of 0.005% and the total amount of resveratrol added having a mass volume ratio (W / V) of 0.0003%.

[0111] Example 9: The time for adding citicoline and resveratrol is 30 hours of fermentation, and the rest is the same as Example 8.

[0112] Example 10: The time for adding citicoline and resveratrol is 20 hours of fermentation, and the rest is the same as Example 8.

[0113] 4. Fermentation time:

[0114] The fermentation time is 108 h, which is determined based on the growth cycle of the strain and the production rate of spectinomycin.

[0115] 5. End of fermentation:

[0116] The fermentation was stopped to obtain the spectinomycin product. After testing, the fermentation titer of each embodiment was improved.

[0117] Detailed beneficial effects:

[0118] The fermentation method of the present invention significantly increases the yield of spectinomycin by supplementing one or both of citicoline and resveratrol, and the specific beneficial effects are as follows:

[0119] 1. Significantly increase production:

[0120] By comparing Example 1 with the examples of various supplemented substances (Examples 2 to 10), it can be clearly seen that after supplementing with citicoline and resveratrol, the fermentation titer of spectinomycin has been significantly improved. Among them, the yield of Example 9 has increased the most, which is 71.4% higher than that of conventional technology, which fully proves the effectiveness of the present invention.

[0121] 2. Optimize the timing and amount of replenishment:

[0122] By comparing Examples 2 to 4 with Examples 5 to 7, it can be seen that the amount and timing of citicoline and resveratrol supplementation have a significant effect on the fermentation titer. Further explorations in Examples 8 to 10 determined a more optimal supplementation timing (fermentation 30h), which allowed the fermentation titer to reach the highest level (Example 9).

[0123] 3. Optimize the operation process and improve fermentation efficiency:

[0124] The fermentation method of the present invention is optimized based on the conventional method, and the fermentation efficiency is improved by scientifically and rationally adding citicoline and resveratrol, which helps to reduce production costs, expand production scale, and improve market competitiveness.

[0125] 4. Fully tap the potential of spectinomycin:

[0126] By adding citicoline and resveratrol, the potential of spectinomycin can be further explored, so that its effect in specific applications can be more fully exerted. This will help expand the application field of spectinomycin and increase its market value.

[0127] In summary, the fermentation method of the present invention can improve the yield of spectinomycin, which significantly improves the yield of spectinomycin by optimizing the fermentation conditions and supplementing beneficial substances, optimizes the operation process, reduces the production cost, and provides strong support for the wide application of spectinomycin.

[0128] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A fermentation method capable of increasing the yield of spectinomycin, characterized in that: include: S1, inoculating the cultured seed liquid into the fermentation medium for fermentation; S2, adding one or both of citicoline and resveratrol during the fermentation process; S3. After a period of fermentation, the fermentation is stopped to obtain the spectinomycin product.

2. A fermentation method capable of increasing spectinomycin production according to claim 1, characterized in that: The inoculation amount is 1%-20% of the volume of the fermentation medium.

3. A fermentation method capable of increasing spectinomycin production according to claim 2, characterized in that: The inoculation amount is 5%-10% of the volume of the fermentation medium.

4. A fermentation method capable of increasing spectinomycin production according to claim 3, characterized in that: The inoculum size was 8% of the fermentation medium volume.

5. A fermentation method capable of increasing spectinomycin production according to claim 1, characterized in that: The composition of the fermentation medium includes: corn starch 40-100g / L, soybean cake powder 0-20g / L, biological nitrogen 5-20g / L, yeast powder 0-20g / L, potassium dihydrogen phosphate 0.5-2g / L, potassium chloride 1-5g / L, ammonium sulfate 1-10g / L, and calcium carbonate 5-30g / L.

6. A fermentation method capable of increasing spectinomycin production according to claim 5, characterized in that: The composition of the fermentation medium includes: corn starch 60-80g / L, soybean cake powder 5-15g / L, biological nitrogen 10-15g / L, yeast powder 5-15g / L, potassium dihydrogen phosphate 1-1.5g / L, potassium chloride 2-4g / L, ammonium sulfate 4-6g / L, and calcium carbonate 10-20g / L.

7. A fermentation method capable of increasing spectinomycin production according to claim 6, characterized in that: The composition of the fermentation medium includes: corn starch 70g / L, soybean cake powder 10g / L, biological nitrogen 12g / L, yeast powder 10g / L, potassium dihydrogen phosphate 1g / L, potassium chloride 3g / L, ammonium sulfate 5g / L, and calcium carbonate 15g / L.

8. A fermentation method capable of increasing spectinomycin production according to claim 7, characterized in that: The total added amount of citicoline has a mass volume ratio (W / V) of 0.0001-0.05%, and the total added amount of resveratrol has a mass volume ratio (W / V) of 0.0001-0.05%; and the addition cycle is 10-70h during the fermentation process.

9. A fermentation method capable of increasing spectinomycin production according to claim 8, characterized in that: The total added amount of citicoline has a mass volume ratio (W / V) of 0.003-0.007%, and the total added amount of resveratrol has a mass volume ratio (W / V) of 0.0001-0.0005%; and the addition cycle is 20-40h during the fermentation process.