Method for preparing tetrahydromethylpyrimidine carboxylic acid through fermentation method

Abstract

The invention discloses a method for preparing tetrahydromethylpyrimidine carboxylic acid through a fermentation method. The method comprises the following steps: a) a preparation stage; b) activating the strain; c) seed culture; step d) fermentation treatment; e) extracting and purifying; f) drying treatment; and g) packaging treatment. The step a) preparation stage comprises the following steps: firstly preparing an inclined plane formula. According to the tetrahydromethylpyrimidine carboxylic acid disclosed by the invention, a traditional induction culture mode is changed, and a fermentation preparation mode is adopted, so that the preparation process is not too complicated, the cost is not relatively high, the product yield is not very low, and the preparation quality is not influenced; the tetrahydromethylpyrimidine carboxylic acid is subjected to fermentation preparation through a preparation stage, strain activation, seed culture, fermentation treatment, extraction and purification, drying treatment and packaging treatment, and meanwhile, cleaning treatment is carried out between the steps, so that the tetrahydromethylpyrimidine carboxylic acid is more environment-friendly, and the effect of efficient preparation is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of ectoine carboxylic acid, in particular to a method for preparing ectoine carboxylic acid by fermentation. Background Art

[0002] According to the patents published on the China Patent Network, the patent name is: A method for adding an inducer to an ectoine carboxylic acid fermentation process, and the patent number is: 202310191372.6. The present invention relates to the technical field of amino acid derivative production, and provides a method for adding an inducer to an ectoine carboxylic acid fermentation process. The ectoine carboxylic acid fermentation process comprises slant culture, seed bottle culture, seed tank culture, and fermentation tank culture. The specific steps are as follows: first prepare a slant formula; then culture the seed culture medium in a seed bottle; transfer the seed culture medium to a seed tank for culture, and take samples to measure the OD value and p value during the culture in the seed tank. H value, residual sugar value; finally, the culture in the seed tank is transferred to the fermentation tank for cultivation, the OD content in the fermentation tank is measured, and when the OD content in the fermentation tank reaches 50 or above, an inducer is added for induction, and while the inducer is inducing, the temperature in the fermentation tank is adjusted to 26-30°C for induction for 4 hours, and the inducer is added to the culture tank for induction at 8 hours, 12 hours, and 16 hours, respectively, and the fermentation content in the fermentation tank is measured, and finally the optimal induction time is obtained; and the above-mentioned tetrahydromethylpyrimidine carboxylic acid is prepared by fermentation method. The process is too complicated, the cost is high, and the product yield is also low, which affects the quality of the preparation.

[0003] Therefore, it is necessary to design and modify tetrahydromethylpyrimidine carboxylic acid to effectively prevent the phenomenon that the preparation process is too complicated, the cost is high, and the product yield is low. Summary of the invention

[0004] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a method for preparing tetrahydromethylpyrimidine carboxylic acid by fermentation, which has the advantage of efficient preparation and solves the problems of overly complicated preparation process, high cost and low product yield.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing ectoine by fermentation, comprising step a) preparation stage; step b) activating bacteria; step c) seed culture; step d) fermentation treatment; step e) extraction and purification; step f) drying treatment; step g) packaging treatment; Step a) Preparation stage: first prepare the slant formula, seed culture medium and fermentation medium. The seed culture medium generally includes glucose, yeast peptone, yeast extract, sodium chloride, etc., for culturing in a seed tank, and the fermentation medium includes glucose, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium chloride, magnesium sulfate heptahydrate, yeast peptone, yeast extract and trace element mother solution, etc.; Step b) Activating the bacteria: then inoculating the E. coli onto a solid plate culture medium to obtain activated bacteria; Step c) Seed culture: inoculate the activated bacteria into the seed culture medium and culture it in a seed tank to obtain a seed culture solution. The formula and conditions (such as temperature, pH value, etc.) of the seed culture medium need to be carefully selected and optimized to ensure good growth and metabolism of the bacteria. Step d) fermentation treatment: inoculating the seed culture solution into the fermentation medium and culturing in a fermenter. During the fermentation process, an inducer and a feed medium need to be added to promote the synthesis of ectoine. At the same time, the temperature, pH value, stirring speed and other conditions in the fermenter also need to be strictly controlled to ensure the smooth progress of the fermentation process. Step e) extraction and purification: After a period of fermentation, a certain amount of ectoine will accumulate in the fermentation broth. At this time, the fermentation broth needs to be extracted and purified to obtain a high-purity ectoine product. The specific extraction and purification methods can be selected and optimized according to actual needs and conditions; Step f) Drying: Drying the purified ectoine to remove moisture and impurities; Step g) Packaging: The dried product is then packaged and stored for subsequent use or sale.

[0006] As a preferred embodiment of the present invention, the preparation stage needs to be sorted in a sterile environment, and each culture medium needs to be prepared according to a ratio.

[0007] As a preferred embodiment of the present invention, when activating the bacterial strains, the staff needs to wear protective clothing and goggles and the process is carried out in a sterile environment.

[0008] As a preferred embodiment of the present invention, the seed culture solution obtained during the seed culture needs to be stored in a glass container to avoid contact with air.

[0009] As a preferred embodiment of the present invention, the fermentation process is carried out under appropriate conditions of temperature, pH, ventilation, etc., and various parameters in the fermentation process are controlled to promote the production of THMPC.

[0010] As a preferred embodiment of the present invention, the extraction and purification is performed by steps such as crystallization, solvent washing, column chromatography, etc.

[0011] As a preferred embodiment of the present invention, the drying process needs to be repeated for at least three times, and the drying temperature needs to be adjusted in real time according to the ambient temperature.

[0012] As a preferred embodiment of the present invention, the packaging process uses corrosion-resistant and high-strength containers for packaging, thereby ensuring the quality of the product.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The tetrahydromethylpyrimidine carboxylic acid of the present invention changes the traditional induction culture method and adopts the fermentation method for preparation. The preparation process will not be too complicated, the cost will not be high, the product yield will not be very low, and the quality of the preparation will not be affected. The tetrahydromethylpyrimidine carboxylic acid is fermented and prepared through the preparation stage, activation of bacteria, seed culture, fermentation treatment, extraction and purification, drying treatment and packaging treatment. At the same time, cleaning treatment is performed between each step, making it more environmentally friendly, thereby having the effect of efficient preparation. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] The present invention provides a method for preparing ectoine by fermentation, comprising step a) preparation stage; step b) activating bacteria; step c) seed culture; step d) fermentation treatment; step e) extraction and purification; step f) drying treatment; step g) packaging treatment; Step a) Preparation stage: first prepare the slant formula, seed culture medium and fermentation medium. The seed culture medium generally includes glucose, yeast peptone, yeast extract, sodium chloride, etc., for culturing in a seed tank, and the fermentation medium includes glucose, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium chloride, magnesium sulfate heptahydrate, yeast peptone, yeast extract and trace element mother solution, etc.; Step b) Activating the bacteria: then inoculating the E. coli onto a solid plate culture medium to obtain activated bacteria; Step c) Seed culture: inoculate the activated bacteria into the seed culture medium and culture it in a seed tank to obtain a seed culture solution. The formula and conditions (such as temperature, pH value, etc.) of the seed culture medium need to be carefully selected and optimized to ensure good growth and metabolism of the bacteria. Step d) Fermentation treatment: The seed culture solution is inoculated into the fermentation medium and cultured in a fermenter. During the fermentation process, an inducer and a feed medium need to be added to promote the synthesis of ectoine. At the same time, the temperature, pH value, stirring speed and other conditions in the fermenter also need to be strictly controlled to ensure the smooth progress of the fermentation process; Step e) extraction and purification: After a period of fermentation, a certain amount of ectoine will accumulate in the fermentation broth. At this time, the fermentation broth needs to be extracted and purified to obtain a high-purity ectoine product. The specific extraction and purification methods can be selected and optimized according to actual needs and conditions; Step f) Drying: Drying the purified ectoine to remove moisture and impurities; Step g) Packaging: The dried product is then packaged and stored for subsequent use or sale.

[0016] The present invention is further configured such that, during the preparation stage, sorting needs to be performed in a sterile environment, and each culture medium needs to be prepared in proportion.

[0017] The present invention is further configured such that when activating the bacterial strains, the staff need to wear protective clothing and goggles and perform the activation in a sterile environment.

[0018] The present invention is further configured such that the seed culture solution obtained during seed culture needs to be stored in a glass container to avoid contact with air.

[0019] The present invention is further configured to carry out fermentation under appropriate conditions of temperature, pH, ventilation, etc. during fermentation treatment, and control various parameters in the fermentation process to promote the production of THMPC.

[0020] The present invention is further configured to use crystallization, solvent washing, column chromatography and other steps for operation and treatment during extraction and purification.

[0021] The present invention is further configured such that the drying process needs to be repeated at least three times, and the drying temperature needs to be adjusted in real time according to the ambient temperature.

[0022] The present invention is further configured to use corrosion-resistant and high-strength containers for packaging, thereby ensuring the quality of the product.

[0023] The working principle and use process of the present invention are as follows: first, a slant formula, a seed culture medium and a fermentation culture medium are prepared. The seed culture medium generally includes glucose, yeast peptone, yeast extract, sodium chloride, etc., and is used for culturing in a seed tank. The fermentation culture medium includes glucose, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium chloride, magnesium sulfate heptahydrate, yeast peptone, yeast extract and trace element mother solution, etc., and then Escherichia coli is inoculated on a solid plate culture medium to obtain activated bacteria, and then the activated bacteria is inoculated into the seed culture medium, and cultured in a seed tank to obtain a seed culture solution. The formula and conditions of the seed culture medium (such as temperature, pH value, etc.) need to be carefully selected and optimized to ensure good growth and metabolism of the strain, and then the seed culture solution is inoculated into the fermentation culture medium and cultured in the fermentation tank. During the fermentation process, inducers and feed medium need to be added to promote the synthesis of ectoine. At the same time, the temperature, pH value, stirring speed and other conditions in the fermentation tank also need to be strictly controlled to ensure the smooth progress of the fermentation process. After a period of fermentation, a certain amount of ectoine will accumulate in the fermentation broth. At this time, the fermentation broth needs to be extracted and purified to obtain a high-purity ectoine. The specific extraction and purification methods can be selected and optimized according to actual needs and conditions. The purified ectoine is dried to remove moisture and impurities, and the dried product is packaged and stored for subsequent use or sale, so as to achieve the effect of efficient preparation.

[0024] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0025] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing ectoine by fermentation, characterized in that: The method comprises the steps of a) preparation stage; b) activation of bacterial strains; c) seed culture; d) fermentation treatment; e) extraction and purification; f) drying treatment; and g) packaging treatment. Step a) Preparation stage: first prepare the slant formula, seed culture medium and fermentation medium. The seed culture medium generally includes glucose, yeast peptone, yeast extract, sodium chloride, etc., for culturing in a seed tank, and the fermentation medium includes glucose, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium chloride, magnesium sulfate heptahydrate, yeast peptone, yeast extract and trace element mother solution, etc.; Step b) Activating the bacteria: then inoculating the E. coli onto a solid plate culture medium to obtain activated bacteria; Step c) Seed culture: inoculate the activated bacteria into the seed culture medium and culture it in a seed tank to obtain a seed culture solution. The formula and conditions (such as temperature, pH value, etc.) of the seed culture medium need to be carefully selected and optimized to ensure good growth and metabolism of the bacteria. Step d) fermentation treatment: inoculating the seed culture solution into the fermentation medium and culturing in a fermenter. During the fermentation process, an inducer and a feed medium need to be added to promote the synthesis of ectoine. At the same time, the temperature, pH value, stirring speed and other conditions in the fermenter also need to be strictly controlled to ensure the smooth progress of the fermentation process. Step e) extraction and purification: After a period of fermentation, a certain amount of ectoine will accumulate in the fermentation broth. At this time, the fermentation broth needs to be extracted and purified to obtain a high-purity ectoine product. The specific extraction and purification methods can be selected and optimized according to actual needs and conditions; Step f) Drying: Drying the purified ectoine to remove moisture and impurities; Step g) Packaging: The dried product is then packaged and stored for subsequent use or sale.

2. The method for preparing ectoine by fermentation according to claim 1, characterized in that: During the preparation stage, sorting needs to be performed in a sterile environment, and each culture medium needs to be prepared according to proportion.

3. The method for preparing ectoine by fermentation according to claim 1, characterized in that: When activating the strains, the staff needs to wear protective clothing and goggles and perform the process in a sterile environment.

4. The method for preparing ectoine by fermentation according to claim 1, characterized in that: The seed culture solution obtained during the seed culture needs to be stored in a glass container to avoid contact with air.

5. The method for preparing ectoine by fermentation according to claim 1, characterized in that: During the fermentation process, the fermentation is carried out under appropriate conditions of temperature, pH, ventilation, etc., and various parameters in the fermentation process are controlled to promote the production of THMPC.

6. The method for preparing ectoine by fermentation according to claim 1, characterized in that: The extraction and purification process is performed by using steps such as crystallization, solvent washing, column chromatography, etc.

7. The method for preparing ectoine by fermentation according to claim 1, characterized in that: The drying process needs to be repeated at least three times, and the drying temperature needs to be adjusted in real time according to the ambient temperature.

8. The method for preparing ectoine by fermentation according to claim 1, characterized in that: The packaging process uses corrosion-resistant and high-strength containers to ensure product quality.