Ultrasonic fermentation process of 5-hydroxytryptophan

Abstract

The invention provides a 5-hydroxytryptophan ultrasonic fermentation process, which is characterized in that an ultrasonic-assisted 5-hydroxytryptophan fermentation process is adopted, in the middle and later periods of 5-hydroxytryptophan fermentation, an ultrasonic device is used for ultrasonic treatment, and the ultrasonic conditions are as follows: the ultrasonic power is 560-840W, the ultrasonic frequency is 18-26KHZ, the amplitude is 48-72%, and the ultrasonic time is 16-24S per 500S. According to the present invention, the transfer of extracellular tryptophan to intracellular tryptophan is accelerated, the secretion of 5-hydroxytryptophan is increased, the excessive accumulation problem of L-tryptophan in the middle and later stage of 5-hydroxytryptophan fermentation is effectively solved, the content of the by-product L-tryptophan in the fermentation process is reduced, and the yield and the conversion rate of 5-hydroxytryptophan are effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of 5-hydroxytryptophan fermentation production, in particular to a 5-hydroxytryptophan ultrasonic fermentation process. Background Art

[0002] 5-Hydroxytryptophan (5-HTP), chemically known as 5-hydroxy-3-indolyl-α-alanine, is a natural aromatic amino acid extracted from the seeds of the African leguminous shrub Griffonia suffruticosa. Hence, it is also known as Griffonia suffruticosa seed extract. 5-HTP has important applications in the biopharmaceutical and animal feed industries. In the biopharmaceutical industry, 5-HT is a precursor to the human neurotransmitter serotonin (5-HT). In the animal feed industry, 5-HTP can improve poultry production performance, increase feed utilization, reduce feed intake, enhance animal immunity, and promote growth.

[0003] At present, fermentation is mostly used to produce 5-hydroxytryptophan. In the middle and late stages of fermentation, the 5-hydroxytryptophan precursor L-tryptophan will accumulate excessively, resulting in excessively high extracellular L-tryptophan content and low glucose conversion rate. Therefore, separation and extraction are difficult and the product purity is low. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a 5-hydroxytryptophan ultrasonic fermentation process.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] A 5-hydroxytryptophan ultrasonic fermentation process, wherein ultrasonic treatment is performed in the middle and late stages of fermentation. The specific ultrasonic conditions are: ultrasonic power 560-840W, ultrasonic frequency 18-26KH Z , amplitude 48-72%, ultrasound time 16-24S every 500S.

[0007] Preferably, the above-mentioned 5-hydroxytryptophan ultrasonic fermentation process comprises the following specific steps:

[0008] (1) Slant culture: Take the 5-hydroxytryptophan-producing strain and inoculate it on the slant medium as the first generation slant culture, and incubate it at 36°C for 14 hours; take the colony of the first generation slant culture and inoculate it on a new slant medium as the second generation slant culture, and incubate it at 36°C for 10-12 hours;

[0009] (2) Seed culture: The bacterial moss in the second generation slant was washed with sterile water and transferred to the seed culture medium. The culture temperature was 35°C. The bioreactor was controlled to automatically add ammonia solution to maintain the pH of the seed solution at 7.0±0.2 and the dissolved oxygen value at 40%-50%. When the OD600nm of the seed solution reached 20-30, the next step of fermentation culture was carried out.

[0010] (3) Fermentation culture: The cultured seeds were inoculated into the fermentation medium with an inoculation of 30% and a culture temperature of 36°C. The pH of the seed solution was maintained at 7.0±0.2 and the dissolved oxygen value was maintained at 30%-50% by automatically adding ammonia solution through the bioreactor control. Ultrasonic treatment was performed in the middle and late stages of fermentation under the following ultrasonic conditions: ultrasonic power 560-840W, ultrasonic frequency 18-26kHz Z , amplitude 48-72%, ultrasound time 16-24S every 500S.

[0011] Preferably, the ultrasonic fermentation process of 5-hydroxytryptophan is carried out under the following ultrasonic conditions: ultrasonic power 700W, ultrasonic frequency 22KH Z , amplitude 60%, ultrasound time 20S every 500S.

[0012] Preferably, in the above-mentioned 5-hydroxytryptophan ultrasonic fermentation process, the ultrasonic treatment starts when the L-tryptophan concentration exceeds 3 g / L, and ends when the tryptophan content in the fermentation broth is lower than 2 g / L.

[0013] Preferably, in the above-mentioned 5-hydroxytryptophan ultrasonic fermentation process, the slant culture medium comprises 5 g / L yeast powder, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate, and 20 g / L agar powder.

[0014] Preferably, in the above-mentioned 5-hydroxytryptophan ultrasonic fermentation process, the seed culture medium is 30 g / L glucose, 3 g / L yeast extract powder, 1 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.4 g / L anhydrous magnesium sulfate, 10 mg / L ferrous sulfate heptahydrate, V H 1mg / L, V B1 2mg / L.

[0015] Preferably, in the ultrasonic fermentation process of 5-hydroxytryptophan, the fermentation medium is 10 g / L glucose, 2 g / L yeast extract powder, 1 g / L peptone, 1 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 1 g / L anhydrous magnesium sulfate, 80 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, V H 1 mg / L, V B1 2mg / L, and 700g / L glucose solution was added during the fermentation process.

[0016] Beneficial effects:

[0017] The above-mentioned 5-hydroxytryptophan ultrasonic fermentation process utilizes the cavitation effect caused by ultrasound and adopts an ultrasound-assisted 5-hydroxytryptophan fermentation process to damage the cell membrane lipid bilayer structure and locally rupture the cell membrane, thereby improving the permeability of the bacterial cell membrane, accelerating the transport of extracellular L-tryptophan in the fermentation broth into the cell, and increasing the secretion of 5-hydroxytryptophan. It effectively solves the problem of excessive accumulation of L-tryptophan in the middle and late stages of 5-hydroxytryptophan fermentation, reduces the content of L-tryptophan as a by-product during the fermentation process, and effectively improves the yield and conversion rate of 5-hydroxytryptophan.

[0018] In the middle stage of 5-hydroxytryptophan, the yield of 5-hydroxytryptophan can be as high as 32.2 g / L through intermittent ultrasonic treatment, the by-product tryptophan is reduced to 2.1 g / L, and the sugar-acid conversion rate reaches 13.6%. DETAILED DESCRIPTION

[0019] Example 1

[0020] A 5-hydroxytryptophan ultrasonic fermentation process, comprising the following steps:

[0021] (1) Slant culture: A 5-hydroxytryptophan-producing strain (5-hydroxytryptophan-producing strain E. coli HTP20 in CN118979005B) was inoculated on a slant medium (5 g / L yeast powder, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate, 20 g / L agar powder) as the first-generation slant culture and cultured at 36°C for 14 h; a colony from the first-generation slant culture was inoculated on a new slant medium as the second-generation slant culture and cultured at 36°C for 10-12 h;

[0022] (2) Seed culture: The bacterial moss in the second generation slant was washed with sterile water to a seed culture medium (glucose 30 g / L, yeast extract powder 3 g / L, ammonium sulfate 1 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.4 g / L, ferrous sulfate heptahydrate 10 mg / L, V H 1 mg / L, V B1 2mg / L), the culture temperature is 35°C, and the bioreactor is controlled to automatically add ammonia solution to maintain the seed solution pH at 7.0±0.2 and the dissolved oxygen value at 40%-50%. When the seed solution OD600nm reaches 20-30, the next fermentation culture is carried out;

[0023] (3) Fermentation: The cultured seeds were inoculated into the fermentation medium (glucose 10 g / L, yeast extract powder 2 g / L, peptone 1 g / L, ammonium sulfate 1 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 80 mg / L, manganese sulfate monohydrate 10 mg / L, V H1 mg / L, V B1 2mg / L, 700g / L glucose solution was fed during the fermentation process), the inoculation amount was 30%, the culture temperature was 36°C, and the bioreactor was controlled to automatically feed ammonia solution to maintain the seed solution pH at 7.0±0.2 and the dissolved oxygen value at 30%-50%.

[0024] After 32 h of fermentation, the yield of 5-hydroxytryptophan reached 25.3 g / L, the concentration of by-product tryptophan was 12.0 g / L, and the sugar-acid conversion rate was 10.1%.

[0025] Example 2

[0026] A 5-hydroxytryptophan ultrasonic fermentation process is described. A commercially available fermentation apparatus is used for fermentation. An ultrasonic probe is placed inside a 30L fermentation tank of the fermentation apparatus and penetrates into the lower portion of the fermentation tank. The ultrasonic probe is connected to an ultrasonic controller via a transducer on the fermentation apparatus. The tank is sterilized. The specific steps are as follows:

[0027] (1) Fermentation was carried out using the culture medium and culture conditions described in Example 1, except that ultrasonic treatment was performed in the middle and late stages of fermentation. The ultrasonic conditions were: ultrasonic power 700 W, ultrasonic frequency 22 kHz Z , amplitude 60%, ultrasound time 20S every 500S.

[0028] (2) Real-time monitoring of the concentration of the byproduct L-tryptophan in the fermentation broth. When the L-tryptophan concentration exceeds 3 g / L, ultrasonic treatment is performed to improve the cell permeability of the bacteria. When the tryptophan content in the fermentation broth is lower than 2 g / L, ultrasonic treatment is stopped.

[0029] After 32 hours of fermentation, 5-hydroxytryptophan production reached 32.2 g / L, the byproduct tryptophan concentration dropped to 2.1 g / L, and the sugar-acid conversion rate reached 13.6%. This demonstrates the significant effectiveness of ultrasonic fermentation for 5-hydroxytryptophan fermentation, laying the foundation for the extraction of high-purity 5-hydroxytryptophan.

[0030] Example 3

[0031] A 5-hydroxytryptophan ultrasonic fermentation process, the method is referred to Example 2, except that the ultrasonic conditions are: ultrasonic power 560, ultrasonic frequency 18KH Z , amplitude 48%, ultrasound time 24S every 500S.

[0032] Example 4

[0033] A 5-hydroxytryptophan ultrasonic fermentation process, the method is similar to Example 2, except that the ultrasonic conditions are: ultrasonic power 840W, ultrasonic frequency 26KH Z , amplitude 72%, ultrasound time 16S every 500S.

[0034] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A 5-hydroxytryptophan ultrasonic fermentation process, characterized in that: Ultrasonic treatment was performed in the middle and late stages of fermentation. The specific ultrasonic conditions were: ultrasonic power 560-840W, ultrasonic frequency 18-26KH Z , amplitude 48-72%, ultrasound time 16-24S every 500S.

2. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 1, wherein: The specific steps are as follows: (1) Slant culture: Take the 5-hydroxytryptophan producing strain and inoculate it on the slant medium as the first generation slant culture; take the colony of the first generation slant culture and inoculate it on a new slant medium as the second generation slant culture; (2) Seed culture: The bacterial moss in the second generation slant was washed with sterile water and transferred to the seed culture medium. The bioreactor was controlled to automatically add ammonia solution to maintain the pH of the seed solution at 7.0 ± 0.2 and the dissolved oxygen value at 40%-50%. When the OD600nm of the seed solution reached 20-30, the next step of fermentation culture was carried out. (3) Fermentation: The cultured seeds were inoculated into the fermentation medium, and the ammonia solution was automatically added through the bioreactor to maintain the pH of the seed solution at 7.0±0.2 and the dissolved oxygen value at 30%-50%. Ultrasonic treatment was performed in the middle and late stages of fermentation. The ultrasonic conditions were: ultrasonic power 560-840W, ultrasonic frequency 18-26KH Z , amplitude 48-72%, ultrasound time 16-24S every 500S.

3. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 1 or 2, characterized in that: The ultrasonic conditions are: ultrasonic power 700W, ultrasonic frequency 22KH Z , amplitude 60%, ultrasound time 20S every 500S.

4. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 1 or 2, characterized in that: The ultrasonic treatment starts when the L-tryptophan concentration exceeds 3 g / L, and ends when the tryptophan content in the fermentation broth is lower than 2 g / L.

5. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 2, wherein: The slant culture medium comprises 5 g / L yeast powder, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate, and 20 g / L agar powder.

6. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 2, wherein: The seed culture medium is 30 g / L glucose, 3 g / L yeast extract powder, 1 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.4 g / L anhydrous magnesium sulfate, 10 mg / L ferrous sulfate heptahydrate, V H 1 mg / L, V B1 2mg / L.

7. The 5-hydroxytryptophan ultrasonic fermentation process according to claim 2, wherein: The fermentation medium is 10 g / L glucose, 2 g / L yeast extract powder, 1 g / L peptone, 1 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 1 g / L anhydrous magnesium sulfate, 80 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, V H 1 mg / L, V B1 2mg / L, and 700g / L glucose solution was added during the fermentation process.