Method for preparing D-calcium pantothenate from fermentation liquor

Abstract

The invention discloses a method for preparing D-calcium pantothenate from fermentation broth, and relates to the technical field of D-calcium pantothenate production.The fermentation broth of D-calcium pantothenate is subjected to flocculation and filter pressing and then enters an ultrafiltration membrane to be filtered, obtained ultrafiltration membrane clear liquid is extracted, subnatant is taken and enters a nanofiltration membrane to be concentrated, obtained concentrated liquid enters a chromatographic column to be eluted, and D-calcium pantothenate is obtained. And collecting a target component containing D-pantothenic acid, then adding a calcium chloride solution for reaction, collecting a reaction solution, cooling and crystallizing, carrying out suction filtration, washing, carrying out suction filtration again, and drying to obtain a finished product of D-calcium pantothenate. According to the method, production links are simplified, the production period is shortened, and the production efficiency is effectively improved; and the purity and yield of the product are improved, the purity is up to 99.5% or above, and the yield is up to 95% or above.

Description

Technical Field

[0001] The invention relates to the technical field of D-calcium pantothenate production, and in particular to a method for preparing D-calcium pantothenate from fermentation liquid. Background Art

[0002] Pantothenic acid is also called vitamin B5, and its chemical formula is C9H 17 NO5 is named "pantothenic acid" because it is widely found in animals and plants. Its structural formula is as follows:

[0003]

[0004] Pantothenic acid is a precursor of coenzyme A and an essential substance for multiple metabolic processes (including carbohydrates, proteins and lipids). It can participate in the synthesis of substances such as steroids, porphyrins, acetylcholine, and maintain normal epithelial function.

[0005] The most important form of pantothenic acid in commercial products is calcium pantothenate, which is a type of B vitamin and one of the nutrients necessary for the normal growth of organisms. Calcium pantothenate exists in three forms due to its chiral carbon atoms: DL-form (racemic form), D-form (dextrorotatory form) and L-form (levorotatory form), and only D-calcium pantothenate has biological activity. The chemical name of calcium pantothenate is calcium N-(2,4-dihydroxy-3,3-dimethylbutyryl)-β-aminopropionic acid, also known as pantothenic acid calcium, and its appearance is white or slightly yellow needle-shaped crystals or powder. The most important form of pantothenic acid in commercial products is calcium pantothenate, which is a type of B vitamin and one of the nutrients necessary for the normal growth of organisms. Calcium pantothenate exists in three forms due to its chiral carbon atoms: DL-form (racemic form), D-form (dextrorotatory form) and L-form (levorotatory form), and only D-calcium pantothenate has biological activity. The chemical name of calcium pantothenate is N-(2,4-dihydroxy-3,3-dimethylbutyryl)-β-alanine calcium, also known as pantothenic acid calcium. It appears as white or slightly yellow needle-shaped crystals or powder.

[0006] Therefore, D-calcium pantothenate belongs to vitamin drugs, which is an indispensable trace substance for the human body and animals to maintain normal physiological functions. It participates in the metabolism of carbohydrates, fats and proteins in the body. It is mainly used in medicine, food and feed additives. In medicine and food health products, it is used to treat vitamin B deficiency, such as beriberi, riboflavin deficiency, pellagra, peripheral neuritis, postoperative colic, mental depression, neurasthenia, strengthen human physique, and improve human immune function. In feed additives, it is used for the growth and development of poultry, livestock and fish, and improve animal reproduction and emergency adaptability.

[0007] The production technology of D-calcium pantothenate mainly includes chemical synthesis, enzymatic method and biological fermentation method. The current production process is mainly based on the first two preparation methods. However, the traditional chemical synthesis of D-calcium pantothenate is relatively complicated, and the racemate needs to be split to obtain D-calcium pantothenate; the enzymatic method requires highly selective enzymes to obtain D-type calcium pantothenate. The use of microbial biofermentation has become the main development direction of the industrial production of D-calcium pantothenate due to its low production cost, low toxicity and low pollution. How to effectively separate D-calcium pantothenate from the liquid fermentation broth with high yield and high purity is a difficult problem in the production of calcium pantothenate by microbial fermentation.

[0008] Chinese patent CN115745827A discloses an extraction process of calcium pantothenate from a fermentation broth, and the extraction steps include fermentation broth, acidification, filtration, nanofiltration primary concentration, ion exchange, nanofiltration secondary concentration, decolorization, neutralization, evaporation concentration, crystallization, centrifugal drying, etc. These process steps are cumbersome and complicated, resulting in a very low yield of calcium pantothenate. Finally, methanol is required to crystallize the crude calcium pantothenate product, thereby further reducing the yield of calcium pantothenate. Chinese patent CN 117510361 A discloses a method for preparing high-purity D-pantothenate calcium, which comprises the following steps: treating the biological fermentation liquid of D-pantothenate calcium with a combination of anion exchange resin (transformed into hydroxyl type), cation exchange resin (transformed into hydrogen type) and weak polar macroporous adsorption resin to obtain a purified D-pantothenate solution; the D-pantothenate solution is converted into a D-pantothenate calcium solution by passing through a cation exchange resin (transformed into calcium ion type), and then decolorized by activated carbon and concentrated by evaporation to obtain high-purity D-pantothenate calcium. The content of the D-pantothenate calcium product prepared by this method is above 99.5%, which greatly improves the quality of the product. However, the ion exchange step is used many times, and a large amount of acid and alkali are used, so that the above calcium pantothenate extraction process has a large amount of wastewater, a large environmental pressure, and a high cost, which is not suitable for industrial production. Chinese patent CN1946851B discloses a method for preparing calcium D-pantothenate from fermentation broth, the method comprising: eluting D-pantothenate from a strongly basic anion exchange resin column with a weak organic acid, and neutralizing the eluted D-pantothenate with an alkaline calcium salt to obtain calcium D-pantothenate. The content of calcium D-pantothenate prepared by this method is low and does not meet the use grade. Summary of the invention

[0009] The technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for preparing D-calcium pantothenate from a fermentation broth is provided, the purity and yield of the product are high and the method is suitable for industrialization.

[0010] In order to solve the above technical problems, the technical solution of the present invention is:

[0011] A method for preparing D-calcium pantothenate from a fermentation broth comprises the following steps:

[0012] A: Add flocculant I to the fermentation broth of D-calcium pantothenate, and after flocculation, filter with a plate and frame filter press to obtain filtrate and filter residue;

[0013] B: Filter the obtained filtrate through an ultrafiltration membrane and collect the clear liquid;

[0014] C: Add the extractant to the collected clear liquid and extract it 2-3 times. After standing and stratifying, take the lower layer;

[0015] D: Concentrate the collected lower layer liquid through a nanofiltration membrane and collect the concentrated liquid;

[0016] E: passing the collected concentrated solution through a chromatography column for chromatography, using eluent A and eluent B for gradient elution to collect the target component containing D-pantothenic acid, wherein eluent A is methanol and eluent B is ammonia water;

[0017] F: Add 1-2%wt calcium chloride solution to the collected target component containing D-pantothenic acid until the pH value of the solution is 6-8, and the reaction end point is within 30 minutes, and the pH value of the solution has no obvious fluctuation, to obtain a reaction solution;

[0018] G: Cool the collected reaction solution to 5-10°C, filter it, wash it with ethanol, filter it, and dry it to obtain the finished product of D-calcium pantothenate.

[0019] Preferably, in step A, the flocculant I is composed of alginic acid, bentonite and chitosan, wherein the mass ratio of alginic acid, bentonite and chitosan is 1:1-4:2-6, the amount of flocculant I is 4-8 g / L, and the flocculation temperature is 30-50°C;

[0020] The filter residue in step A is used for fertilizer production.

[0021] Preferably, in step B, flocculant II is added to the ultrafiltration membrane concentrate, filtered, and the filtrate is collected, and the filtrate is used as a diluent for diluting methanol and ammonia water;

[0022] The composition of flocculant II is chitosan and bentonite in a mass ratio of 2-4:1-2, the dosage of flocculant II is 2-4g / L, the flocculation temperature is 30-50°C, and the pore size of the ultrafiltration membrane is 5000-10000Da.

[0023] Preferably, the stirring speed during the extraction process in step C is 200-300 rpm, and the standing stratification time is 30-60 min.

[0024] Preferably, the extractant in step C is one or a mixture of two or more of ethyl acetate, butyl acetate, petroleum ether, diethyl ether, and isopropyl ether;

[0025] After standing and stratifying, the upper layer liquid is distilled and the extract is collected for the next extraction.

[0026] Preferably, the pore size of the nanofiltration membrane in step D is 150-300 Da, wherein the nanofiltration clear liquid can be used as a diluent for methanol and ammonia water, and can also be used for the preparation of the culture medium during the fermentation process.

[0027] Preferably, the chromatography column in step E is a 100-200 mesh silica gel column or alumina.

[0028] Preferably, in step E, the concentration of methanol is 30-70%, the concentration of ammonia water is 0.5-2.0%wt, and the elution flow rate is controlled to be 0.3-0.8BV / h;

[0029] The amount of eluent A is 2-3 times the column volume, and the amount of eluent B is 1-2 times the column volume.

[0030] Preferably, in step G, the collected reaction solution is concentrated at a temperature of 50-70° C. and a vacuum degree of less than -0.090 MPa to a solid content of 60-80% w / w.

[0031] Preferably, the concentrated reaction solution is cooled to 5-10°C at a rate of 3-5°C / h.

[0032] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0033] 1. The present invention provides a novel composite flocculant. Compared with a single flocculant, the composite flocculant forms larger flocs after reaction, can clarify and preliminarily decolorize D-calcium pantothenate fermentation broth, remove 50-60% of protein and residual sugar in the fermentation broth, and has no loss of D-calcium pantothenate. The process reduces the processing pressure in the subsequent purification process. The filtrate can directly pass through an ultrafiltration membrane to obtain an ultrafiltrate. The filter residue can be used for fertilizer production, thereby improving the economic value and eliminating the trouble of processing, which is environmentally friendly.

[0034] 2. In the process of purifying and preparing calcium D-pantothenate, the present invention first uses an extractant for extraction, then uses column chromatography, and uses methanol and ammonia water for gradient elution, with good elution effect. At the same time, the eluent can be concentrated and recovered for reuse. The method of the present invention not only simplifies the production process, shortens the production cycle, and effectively improves production efficiency; it also improves the purity and yield of the product, with a purity of more than 99.5% and a yield of more than 95%.

[0035] 3. The separation and purification method involved in the present invention has simple operation, low cost and can be industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the liquid chromatogram of the D-calcium pantothenate product in Example 1 of the present invention.

[0037] Figure 2 It is the liquid chromatogram of the D-calcium pantothenate product in Example 2 of the present invention.

[0038] Figure 3 It is the liquid chromatogram of the D-calcium pantothenate product in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the embodiments.

[0040] Example 1

[0041] A method for preparing D-calcium pantothenate from a fermentation broth comprises the following steps:

[0042] A: Flocculant I was added to 50L of D-calcium pantothenate fermentation broth (the content of D-pantothenic acid was 60g / L). After flocculation, the filtrate was filtered using a plate and frame filter press to obtain 45L of filtrate and 450g of filter residue, which was used for fertilizer production. The flocculant I consisted of alginic acid, bentonite and chitosan, wherein the mass ratio of alginic acid, bentonite and chitosan was 1:1:2, the amount of flocculant I was 4g / L, and the flocculation temperature was 30°C.

[0043] B: The obtained filtrate is filtered through an ultrafiltration membrane, and 57.5 L of clear liquid and 7.5 L of ultrafiltration membrane concentrate are collected, wherein flocculant II is added to the ultrafiltration membrane concentrate, and 8.5 L of filtrate is collected. The filtrate is used as a diluent for the subsequent dilution and preparation of eluents methanol and ammonia water;

[0044] The composition of flocculant II is chitosan and bentonite in a mass ratio of 2:1, the dosage of flocculant II is 2 g / L, the flocculation temperature is 30°C, and the pore size of the ultrafiltration membrane is 5000 Da.

[0045] C: Add 3.0 L of extractant to the collected clear liquid and extract twice, stirring at 200 rpm, then let stand for 30 minutes to separate the layers, and take 58.0 L of the lower layer, wherein the extractant is ethyl acetate;

[0046] After standing and stratifying, the upper layer liquid was distilled to collect 2.9L of extract, which was used as the extractant for the next extraction;

[0047] D: The collected lower layer liquid is concentrated through a nanofiltration membrane, and 18.5 L of concentrated liquid is collected, wherein the pore size of the nanofiltration membrane is 150 Da, and 39.5 L of the nanofiltration clear liquid can be used as a diluent for methanol and ammonia water, and can also be used for the configuration of the culture medium during the fermentation process;

[0048] E: The collected concentrate was passed through a chromatography column for chromatography, wherein the chromatography column was a 100-mesh silica gel column, and gradient elution was performed with eluent A and eluent B, and 42.0 L of the target component containing D-pantothenic acid was collected, wherein the eluent A was methanol, and the eluent B was ammonia water, the concentration of methanol was 30%, the concentration of ammonia water was 0.5%wt, the elution flow rate was controlled at 0.3BV / h, the amount of eluent A was 2 times the column volume, and the amount of eluent B was 1 times the column volume;

[0049] F: Add 1%wt calcium chloride solution to the collected target component containing D-pantothenic acid until the pH value of the solution reaches 6. The reaction endpoint is within 30 minutes, and the pH value of the solution has no obvious fluctuation, and 45.0L of reaction solution is obtained;

[0050] G: In step G, the collected reaction solution is concentrated at a temperature of 50° C. and a vacuum degree of <-0.090 MPa to a solid content of 60% w / w. The concentrated reaction solution is cooled to 5° C. at a rate of 3° C. / h, filtered, washed with ethanol, filtered, and dried to obtain 2865 g of a finished product of calcium D-pantothenate with a purity of 99.6% and a yield of 95.5%.

[0051] Example 2

[0052] A method for preparing D-calcium pantothenate from a fermentation broth comprises the following steps:

[0053] A: Flocculant I was added to 50L of D-calcium pantothenate fermentation broth (the content of D-pantothenic acid was 60g / L). After flocculation, the filtrate was filtered using a plate and frame filter press to obtain 46.0L of filtrate and 550g of filter residue, which was used for fertilizer production. The flocculant I consisted of alginic acid, bentonite and chitosan, wherein the mass ratio of alginic acid, bentonite and chitosan was 1:2:4, the amount of flocculant I was 6g / L, and the flocculation temperature was 40°C.

[0054] B: The obtained filtrate was filtered through an ultrafiltration membrane, and 60.0 L of clear liquid and 6.0 L of ultrafiltration membrane concentrate were collected, wherein flocculant II was added to the ultrafiltration membrane concentrate, and 5.8 L of filtrate was collected. The filtrate was used as a diluent for the subsequent dilution and preparation of eluents methanol and ammonia water;

[0055] The composition of flocculant II is chitosan and bentonite in a mass ratio of 1:1, the dosage of flocculant II is 3 g / L, the flocculation temperature is 40°C, and the pore size of the ultrafiltration membrane is 8000 Da.

[0056] C: Add 3.0 L of extractant to the collected clear liquid and extract for 3 times, with a stirring speed of 300 rpm, then let stand for 50 minutes to separate the layers, and take 60.0 L of the lower layer liquid, wherein the extractant is a mixture of butyl acetate and petroleum ether in a volume ratio of 1:1;

[0057] After standing and stratifying, the upper layer liquid was distilled to collect 2.5L of extract, which was used as the extractant for the next extraction;

[0058] D: The collected lower layer liquid is concentrated through a nanofiltration membrane, and 20.0 L of concentrated liquid is collected, wherein the pore size of the nanofiltration membrane is 200 Da, and 39.5 L of the nanofiltration clear liquid can be used as a diluent for methanol and ammonia water, and can also be used for the configuration of the culture medium during the fermentation process;

[0059] E: The collected concentrated solution was chromatographed on a chromatography column, wherein the chromatography column was 200-mesh aluminum oxide, and gradient elution was performed with eluent A and eluent B, and 72.0 L of the target component containing D-pantothenic acid was collected, wherein eluent A was methanol, and eluent B was ammonia water, the concentration of methanol was 50%, the concentration of ammonia water was 1.0%wt, the elution flow rate was controlled at 0.5BV / h, the amount of eluent A was 3 times the column volume, and the amount of eluent B was 2 times the column volume;

[0060] F: Add 1.5%wt calcium chloride solution to the collected target component containing D-pantothenic acid until the pH value of the solution reaches 7. The reaction endpoint is within 30 minutes, and the pH value of the solution has no obvious fluctuation, and 73.5L of reaction solution is obtained;

[0061] G: In step G, the collected reaction solution is concentrated at a temperature of 60° C. and a vacuum degree of <-0.090 MPa to a solid content of 70% w / w. The concentrated reaction solution is cooled to 8° C. at a rate of 4° C. / h, filtered, washed with ethanol, filtered, and dried to obtain 2955 g of a finished product of calcium D-pantothenate with a purity of 99.8% to 98.5%.

[0062] Example 3

[0063] A method for preparing D-calcium pantothenate from a fermentation broth comprises the following steps:

[0064] A: Flocculant I was added to 50.0L of D-calcium pantothenate fermentation broth (the content of D-pantothenic acid was 60g / L). After flocculation, the filtrate was filtered using a plate and frame filter press to obtain 48.0L of filtrate and 750.0g of filter residue, which was used for fertilizer production. The flocculant I consisted of alginic acid, bentonite and chitosan, wherein the mass ratio of alginic acid, bentonite and chitosan was 1:4:6, the amount of flocculant I was 8g / L, and the flocculation temperature was 50°C.

[0065] B: The obtained filtrate was filtered through an ultrafiltration membrane, and 61.0 L of clear liquid and 7.0 L of ultrafiltration membrane concentrate were collected, wherein flocculant II was added to the ultrafiltration membrane concentrate, and 6.5 L of filtrate was collected. The filtrate was used as a diluent for the subsequent dilution and preparation of eluents methanol and ammonia water;

[0066] The composition of flocculant II is chitosan and bentonite in a mass ratio of 4:1, the dosage of flocculant II is 4 g / L, the flocculation temperature is 50°C, and the pore size of the ultrafiltration membrane is 10000 Da.

[0067] C: Add 3.0 L of extractant to the collected clear liquid and extract twice, stirring at 300 rpm, then let stand for 60 minutes to separate the layers, and take 60.5 L of the lower layer liquid, wherein the extractant is a mixture of diethyl ether and isopropyl ether in a volume ratio of 1:1;

[0068] After standing and stratifying, the upper layer liquid was distilled to collect 2.5L of extract, which was used as the extractant for the next extraction;

[0069] D: The collected lower layer liquid is concentrated through a nanofiltration membrane, and 19.5 L of concentrated liquid is collected, wherein the pore size of the nanofiltration membrane is 300 Da, and 41.0 L of the nanofiltration clear liquid can be used as a diluent for methanol and ammonia water, and can also be used for the configuration of the culture medium during the fermentation process;

[0070] E: The collected concentrated solution is chromatographed on a chromatography column, wherein the chromatography column is a 200-mesh silica gel column or alumina column, and gradient elution is performed with eluent A and eluent B to collect 70.0 L of the target component containing D-pantothenic acid, wherein eluent A is methanol, and eluent B is ammonia water, the concentration of methanol is 70%, the concentration of ammonia water is 2.0%wt, the elution flow rate is controlled at 0.8BV / h, the amount of eluent A is 3 times the column volume, and the amount of eluent B is 2 times the column volume;

[0071] F: Add 2%wt calcium chloride solution to the collected target component containing D-pantothenic acid until the pH value of the solution reaches 8. The reaction endpoint is within 30 minutes, and the pH value of the solution has no obvious fluctuation, and 71.5L of reaction solution is obtained;

[0072] G: In step G, the collected reaction solution is concentrated at a temperature of 70° C. and a vacuum degree of <-0.090 MPa to a solid content of 80% w / w. The concentrated reaction solution is cooled to 10° C. at a rate of 5° C. / h, filtered, washed with ethanol, filtered, and dried to obtain 2895 g of a finished product of calcium D-pantothenate with a purity of 99.7% and a yield of 96.5%.

[0073] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for preparing D-calcium pantothenate from a fermentation broth, characterized in that The following steps are involved: A: Add flocculant I to the fermentation broth of D-calcium pantothenate, and after flocculation, filter with a plate and frame filter press to obtain filtrate and filter residue; B: Filter the obtained filtrate through an ultrafiltration membrane and collect the clear liquid; C: Add the extractant to the collected clear liquid and extract it 2-3 times. After standing and stratifying, take the lower layer; D: Concentrate the collected lower layer liquid through a nanofiltration membrane and collect the concentrated liquid; E: passing the collected concentrated solution through a chromatography column for chromatography, using eluent A and eluent B for gradient elution to collect the target component containing D-pantothenic acid, wherein eluent A is methanol and eluent B is ammonia water; F: Add 1-2%wt calcium chloride solution to the collected target component containing D-pantothenic acid until the pH value of the solution is 6-8, and the reaction end point is within 30 minutes, and the pH value of the solution has no obvious fluctuation, to obtain a reaction solution; G: Cool the collected reaction solution to 5-10°C, filter it, wash it with ethanol, filter it, and dry it to obtain the finished product of D-calcium pantothenate.

2. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step A, the flocculant I is composed of alginic acid, bentonite and chitosan, wherein the mass ratio of alginic acid, bentonite and chitosan is 1:1-4:2-6, the amount of flocculant I is 4-8g / L, and the flocculation temperature is 30-50°C; The filter residue in step A is used for fertilizer production.

3. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step B, the ultrafiltration membrane concentrate is added with flocculant II, filtered, and the filtrate is collected, and the filtrate is used as a diluent for diluting methanol and ammonia water; The composition of flocculant II is chitosan and bentonite in a mass ratio of 2-4:1-2, the dosage of flocculant II is 2-4g / L, the flocculation temperature is 30-50°C, and the pore size of the ultrafiltration membrane is 5000-10000Da.

4. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: The stirring speed during the extraction process in step C is 200-300 rpm, and the standing stratification time is 30-60 min.

5. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step C, the extractant is one or a mixture of two or more of ethyl acetate, butyl acetate, petroleum ether, diethyl ether, and isopropyl ether; After standing and stratifying, the upper layer liquid is distilled and the extract is collected for the next extraction.

6. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: The pore size of the nanofiltration membrane in step D is 150-300Da, and the nanofiltration clear liquid can be used as a diluent for methanol and ammonia water, and can also be used for the preparation of the culture medium during the fermentation process.

7. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step E, the chromatography column is a 100-200 mesh silica gel column or alumina.

8. A method for preparing calcium D-pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step E, the concentration of methanol is 30-70%, the concentration of ammonia water is 0.5-2.0%wt, and the elution flow rate is controlled at 0.3-0.8BV / h; The amount of eluent A is 2-3 times the column volume, and the amount of eluent B is 1-2 times the column volume.

9. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 1, characterized in that: In step G, the collected reaction solution is concentrated at a temperature of 50-70° C. and a vacuum degree of less than -0.090 MPa to a solid content of 60-80% w / w.

10. A method for preparing D-calcium pantothenate from a fermentation broth as claimed in claim 9, characterized in that: The concentrated reaction solution was cooled to 5-10°C at a rate of 3-5°C / h.

Images