Method for refining D-pantothenic acid from fermentation liquor

Abstract

The invention discloses a method for refining D-pantothenic acid from fermentation liquor, and belongs to the technical field of D-pantothenic acid extraction. Comprising the following steps: (1) D-pantothenic acid expression, (2) centrifugal treatment, (3) decoloration, (4) concentration, (5) crystallization, (6) filtration or centrifugation, (7) dissolution, (8) ultrasonic-assisted recrystallization, (9) filtration, and (10) washing and drying. The D-pantothenic acid crystal with a fine crystal form is obtained through ultrasonic-assisted crystallization, the surface area of the crystal is increased through the fine crystal form, impurity removal in the washing stage is facilitated, and therefore the D-pantothenic acid crystal with the higher purity is prepared. The D-pantothenic acid crystals are washed by using a detergent and acetone in sequence, so that the formation of small crystals is further promoted while impurities are fully washed away. The D-pantothenic acid prepared by the invention is fine in crystal form, the bioavailability and the dispersibility of a product containing the D-pantothenic acid are improved, and in addition, the fine crystal form can also reduce the bitter taste of the D-pantothenic acid and improve the taste.

Description

Technical Field

[0001] The invention relates to the technical field of D-pantothenic acid extraction, and in particular to a method for refining D-pantothenic acid from fermentation liquid. Background Art

[0002] D-Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin that is essential for maintaining human health. Pantothenic acid plays many roles in biology and is involved in important biochemical processes such as fatty acid metabolism, energy production, cholesterol synthesis and hormone synthesis. The human body cannot synthesize pantothenic acid on its own, so it must be obtained through diet. Pantothenic acid is widely present in many foods, such as meat, eggs, milk and vegetables. A lack of pantothenic acid may cause symptoms such as skin problems, fatigue and indigestion.

[0003] The process of preparing D-pantothenic acid by chemical synthesis is highly polluting, costly and complicated. When D-pantothenic acid is prepared by microbial fermentation, the D-pantothenic acid extracted from the fermentation broth has low purity and large crystal form, which is not conducive to digestion and absorption by organisms. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for purifying D-pantothenic acid from a fermentation broth, so as to overcome the problem that the D-pantothenic acid extracted from the fermentation broth in the prior art has low purity and large crystal form.

[0005] In a first aspect, the present invention provides a method for refining D-pantothenic acid from a fermentation broth, comprising the following steps:

[0006] (1) D-pantothenic acid expression: fermenting recombinant E. coli containing the D-pantothenic acid gene to obtain an E. coli fermentation broth;

[0007] (2) Centrifugation: centrifuging the E. coli fermentation broth to obtain a supernatant containing D-pantothenic acid;

[0008] (3) Decolorization: Add 50-100 g of activated carbon per liter of supernatant, stir for 0.5-2 h, filter, and collect the filtrate;

[0009] (4) Concentration: heating the filtrate to 50° C. to 60° C. and concentrating the filtrate under vacuum conditions to 1 / 5 to 1 / 3 of the original volume of the filtrate to obtain a concentrated solution;

[0010] (5) Crystallization: stirring the concentrated solution and lowering the temperature to 2° C. to 4° C. to crystallize the D-pantothenic acid in the concentrated solution;

[0011] (6) Filtration or centrifugation: The solid-liquid mixture obtained after crystallization is filtered or centrifuged at low temperature, and the solid is collected and dried to obtain crude D-pantothenic acid;

[0012] (7) Dissolving: using an organic solvent to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol;

[0013] (8) Recrystallization: Under stirring, add an anti-solvent in an amount of 1 to 1.5 times the volume of the organic solvent to the dissolved solution, wherein the anti-solvent is a mixed solution of chloroform and dichloromethane, and control the temperature to be 15° C. to 22° C. to perform recrystallization;

[0014] The recrystallization is assisted by ultrasound, and the ultrasonic power is 20-30 W / cm 2 , the ultrasonic frequency is 20 to 40 kHz, the ultrasound works for 2 to 4 seconds and rests for 2 to 4 seconds;

[0015] (9) Filtration: Filter the solid-liquid mixture after recrystallization to collect the solid;

[0016] (10) Washing and drying: The solid obtained after filtration is washed with a detergent and acetone in sequence, and then dried to obtain D-pantothenic acid. The detergent is any one or both of chloroform and dichloromethane.

[0017] Compared with the prior art, the present invention cultured the recombinant Escherichia coli in a suitable culture medium to promote its growth and produce D-pantothenic acid, and the crude D-pantothenic acid can be obtained by centrifugation, decolorization, concentration and crystallization, filtration or centrifugation under the above-mentioned specific conditions. Further, the present invention uses a mixed solution of methanol and ethanol to dissolve the obtained crude D-pantothenic acid to improve the solubility of the crude D-pantothenic acid therein, promote the full dissolution of the crude D-pantothenic acid, and then add an anti-solvent to the above-mentioned dissolving solution, which can reduce the solubility of D-pantothenic acid in the dissolving solution, so that D-pantothenic acid reaches an oversaturated state, thereby forming crystals to precipitate, and the anti-solvent is selected as a mixed solution of chloroform and dichloromethane, which can more effectively remove impurities during the recrystallization process and improve the purity of the D-pantothenic acid crystals. When combined with ultrasound-assisted crystallization during recrystallization, the above synergistic effect can recrystallize to obtain fine D-pantothenic acid crystals, and the fine crystal increases the surface area of ​​the crystals, which is conducive to the removal of impurities in the washing stage, thereby preparing D-pantothenic acid crystals with higher purity. In addition, when washing the D-pantothenic acid crystals obtained by recrystallization, the present application sequentially uses detergent and acetone for washing, and the two washings can fully wash away impurities, and the use of acetone for washing is also conducive to the formation of small crystals. The D-pantothenic acid crystals prepared by the present invention are fine, and the fine crystals make D-pantothenic acid more easily absorbed through the intestinal mucosa, thereby improving bioavailability, and the fine crystals of D-pantothenic acid have better dispersibility in food or feed. In addition, the fine crystals can also reduce the bitterness of D-pantothenic acid, thereby improving the taste.

[0018] Furthermore, the centrifugation in step (2) is: centrifugation at a speed of 6000 to 10000 rpm for 10 to 30 minutes.

[0019] The above technical solution further limits the centrifugal speed and centrifugal time. Under the centrifugal speed and centrifugal time, the supernatant and Escherichia coli can be fully separated to obtain the supernatant containing D-pantothenic acid.

[0020] Furthermore, the stirring rate during decolorization in step (3) is 50-100 rpm; and the filter pore size is 0.65-10 um.

[0021] The above technical solution further limits the stirring rate when using activated carbon for decolorization, as well as the filtration pore size, so that the activated carbon can be filtered out after decolorization using the activated carbon.

[0022] Furthermore, the vacuum degree during concentration in step (4) is -0.06 to -0.08 MPa.

[0023] The above technical solution further limits the vacuum degree during concentration. Concentrating the filtrate under the above vacuum degree can ensure that the concentration process is carried out at a lower temperature, thereby avoiding high-temperature decomposition of D-pantothenic acid.

[0024] Furthermore, the stirring rate during crystallization in step (5) is 30 to 60 rpm, and the cooling rate is 0.5 to 1° C. / min.

[0025] The above technical solution further limits the stirring rate and cooling rate during crystallization. By limiting the above specific stirring rate and cooling rate, it can ensure that the D-pantothenic acid in the concentrated solution is fully crystallized, and the crystal morphology of the crystals can be controlled, which is convenient for subsequent further processing.

[0026] Furthermore, in step (6), the temperature of low-temperature filtration or low-temperature centrifugation is 2-8°C, the filtration pore size is 10-50 um, and the centrifugation is performed at a rotation speed of 5000-6000 rpm for 10-20 min.

[0027] The above technical scheme further limits the separation conditions of the solid-liquid mixture after crystallization. By limiting the temperature of low-temperature filtration or low-temperature centrifugation to 2 to 8°C, the loss during the separation of crude D-pantothenic acid can be further reduced, thereby ensuring the output rate of crude D-pantothenic acid obtained during filtration.

[0028] Furthermore, during the dissolution in step (7), 4 to 5 L of organic solvent is added for each kg of crude D-pantothenic acid to dissolve; the volume ratio of methanol to ethanol in the organic solvent is 1:(0.2 to 1).

[0029] The above technical scheme further limits the amount of organic solvent used during dissolution, as well as the volume ratio of methanol to ethanol in the organic solvent, which can fully dissolve the crude D-pantothenic acid. Using a mixed solution of methanol and ethanol in the above volume ratio is conducive to obtaining fine crystals of D-pantothenic acid during recrystallization.

[0030] Furthermore, the stirring rate during recrystallization in step (8) is 80 to 120 rpm, and a vertical stirring blade is used during stirring;

[0031] The volume ratio of chloroform to dichloromethane in the anti-solvent is 1:(0.5-2);

[0032] The method of adding the anti-solvent includes: first adding 1 / 5 volume of the anti-solvent to the dissolving solution, and then adding the remaining 4 / 5 volume of the anti-solvent to the dissolving solution at a flow rate of 20 to 50 ml / min.

[0033] The above technical solution uses a vertical stirring blade and controls the stirring speed to 80-120 rpm, which is conducive to the crystal form obtained by recrystallization being finer; further, the above technical solution also defines the volume ratio of the anti-solvent when chloroform and dichloromethane are used, and the synergistic effect of the above technical solution can better promote the precipitation of D-pantothenic acid crystals. Further controlling the anti-solvent to be added twice, especially with a specific droplet speed during the second addition, can better control the crystallization process, thereby forming more uniform and controllable fine crystals.

[0034] Furthermore, in step (8), before adding the anti-solvent, an organic acid or an organic amine in an amount of 0.1 to 0.3 times the volume of the dissolving solution may be added, wherein the organic acid is any one or both of citric acid and acetic acid, and the organic amine is ethanolamine.

[0035] In the above technical solution, before adding the anti-solvent, an organic acid or an organic amine is added, which can further promote the formation of small crystals by adjusting the pH value of the solution.

[0036] Furthermore, the pore size during filtration in step (9) is 2 to 10 μm;

[0037] The washing in step (10) comprises: washing the filtered solid with a detergent having an equal volume to the anti-solvent, stirring for 10 to 30 minutes, filtering to remove the detergent at a pore size of 2 to 10 um, and then washing the filtered solid with acetone having a volume of 2 to 3 times that of the anti-solvent, stirring for 10 to 30 minutes, and filtering to remove the acetone at a pore size of 2 to 10 um to obtain D-pantothenic acid crystals.

[0038] The above technical solution further defines a specific method of washing the solid obtained after recrystallization with a detergent and acetone in sequence, which can fully remove the impurities adsorbed on the D-pantothenic acid crystals and further improve the purity of the D-pantothenic acid crystals. In addition, the final washing with acetone is also conducive to the further formation of small D-pantothenic acid crystals. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific 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.

[0040] In a first aspect, an embodiment of the present invention provides a method for refining D-pantothenic acid from a fermentation broth, comprising the following steps:

[0041] S1. Expression of D-pantothenic acid: fermenting the recombinant Escherichia coli containing the D-pantothenic acid gene to obtain an Escherichia coli fermentation broth.

[0042] For example, the D-pantothenic acid gene and the promoter pTrc can be connected to the expression vector pBAD to obtain a recombinant expression vector, which is then transformed into Escherichia coli to obtain a recombinant Escherichia coli containing the D-pantothenic acid gene, and the Escherichia coli fermentation broth is obtained using high-density cell fermentation technology. The OD of the Escherichia coli fermentation broth is 600 The value is 100 to 150.

[0043] The specific high-density cell fermentation technology can be referred to as follows:

[0044] The recombinant E. coli working seeds were inoculated into a seed shake flask containing LB liquid medium and cultured at a temperature of 35-37°C and a rotation speed of 200-220 rpm for 10-18 h to obtain the OD 600 The invention discloses a seed shake flask liquid with a pH value of 1 to 2; the seed shake flask liquid is added to a fermentation tank at a volume ratio of 5 to 10% to perform high-density fermentation, and the fermentation parameters are as follows: temperature of 35 to 37°C, pH of 6.8 to 7.3 controlled by ammonia water, and DO value of 10 to 40%; feeding is started after the DO value rebounds, feed medium A: glucose solution with a mass percentage of 50 to 60%, feed medium B: yeast powder peptone solution with a mass percentage of 20 to 30% (the mass ratio of yeast powder to peptone is 1:1), and the feeding rate is 10 to 30 g / L / h (the mass ratio of feed medium A to feed medium B is 1:(0.5 to 1)); OD 600 When the OD value exceeds 60, IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2-0.4 mmol / L was added to start induction. 600After stabilization, the fermentation is terminated to obtain a fermentation liquid containing D-pantothenic acid.

[0045] The culture medium in the fermentation tank is: anhydrous potassium hydrogen phosphate (K 2 HPO 4 ): 3g / L, anhydrous potassium dihydrogen phosphate (KH 2 PO 4 ): 1.5g / L, sodium chloride (NaCl): 0.5g / L, ammonium chloride (NH 4 Cl): 1g / L, trisodium citrate (Na 3 C 6 H 5 O 7 ): 1g / L, yeast extract: 10g / L, glucose: 30g / L, peptone: 20g / L.

[0046] S2. Centrifugation: The E. coli fermentation broth is centrifuged to obtain a supernatant containing D-pantothenic acid.

[0047] The centrifugation in the above step is: centrifugation at a speed of 6000-10000 rpm for 10-30 min. For example, the centrifugation speed can be 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm; the centrifugation time can be 10 min, 20 min or 30 min. After the fermentation liquid is centrifuged, a supernatant containing D-pantothenic acid and an Escherichia coli precipitate are obtained, and the supernatant is retained and the precipitate is discarded.

[0048] S3, decolorization: add 50-100g activated carbon per liter of supernatant, stir for 0.5-2h, filter and collect the filtrate. For example, 50g, 60g, 70g, 80g, 90g or 100g activated carbon can be added per liter of supernatant, preferably 60-80g activated carbon. The particle size of the activated carbon particles is 1mm-3mm.

[0049] In the above steps, the stirring rate is 50-100 rpm, and the filter pore size is 0.65-10 um. For example, the stirring rate can be 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm or 100 rpm, preferably 60-90 rpm; the filter pore size can be 0.65 um, 2 um, 4 um, 6 um, 8 um or 10 um, preferably 2-8 um. Activated carbon is an efficient adsorbent that can effectively remove small molecule pigments in the supernatant. After decolorization is completed, the activated carbon and other small molecule solid impurities in the supernatant are removed by filtration.

[0050] S4, concentration: the filtrate is heated to 50°C to 60°C, and the filtrate is concentrated under vacuum to 1 / 5 to 1 / 3 of the original volume of the filtrate to obtain a concentrated solution. For example, the filtrate can be heated to 50°C, 55°C or 60°C.

[0051] The vacuum degree during concentration in the above steps is -0.06 to -0.08 MPa, and for example, the vacuum degree can be -0.06 MPa, -0.07 MPa or -0.08 MPa. Concentrating under vacuum conditions can reduce the temperature during concentration, so that the concentration can be carried out at a lower temperature to avoid high-temperature degradation of D-pantothenic acid. After concentration is completed, the D-pantothenic acid content in the concentrate can reach 30 to 50 g / L.

[0052] S5, crystallization: stirring the above concentrated solution, and lowering the temperature to 2°C to 4°C to crystallize the D-pantothenic acid in the concentrated solution. For example, the temperature can be lowered to 2°C, 3°C or 4°C.

[0053] In the above steps, the stirring rate is 30-60 rpm, and the cooling rate is 0.5-1°C / min. For example, the stirring rate can be 30 rpm, 40 rpm, 50 rpm or 60 rpm, preferably 40-50 rpm; the cooling rate can be 0.5°C / min, 0.6°C / min, 0.7°C / min, 0.8°C / min, 0.9°C / min or 1°C / min, preferably 0.6-0.8°C / min. The above concentrated solution is stirred and cooled for crystallization, so that the D-pantothenic acid in the concentrated solution can be fully crystallized.

[0054] S6. Filtration or centrifugation: The solid-liquid mixture obtained after crystallization is subjected to low-temperature filtration or low-temperature centrifugation, and the solid is collected and dried to obtain a crude D-pantothenic acid product.

[0055] After the crystallization in step S5 is completed, the D-pantothenic acid crystals obtained by crystallization can be separated by low temperature filtration or low temperature centrifugation. The temperature during low temperature filtration or low temperature centrifugation is controlled at 2 to 8°C, for example, the temperature can be controlled at 2°C, 4°C, 6°C or 8°C.

[0056] If low temperature filtration is selected, the filtration pore size is 10-50 um, and the solid obtained by filtration is D-pantothenic acid crystals. The filtration pore size can be 10 um, 20 um, 30 um, 40 um or 50 um, preferably 20-40 um.

[0057] If low temperature centrifugation is selected, the centrifugation can be performed at a speed of 5000-6000 rpm for 10-20 min, and the resulting precipitate is D-pantothenic acid crystals. For example, the centrifugation speed can be 5000 rpm, 5500 rpm or 6000 rpm; the centrifugation time can be 10 min, 15 min or 20 min.

[0058] When the collected solid D-pantothenic acid crystals are dried, they can be dried for 2 to 3 hours at a vacuum degree of -0.05 to 0.08 MPa and a temperature of 50 to 70°C.

[0059] S7. Dissolving: using an organic solvent to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol.

[0060] During the dissolution, 4 to 5 L of organic solvent is added for each kg of crude D-pantothenic acid to dissolve. The volume ratio of methanol to ethanol in the organic solvent is 1:(0.2 to 1), and for example, the volume ratio of methanol to ethanol can be 1:0.2, 1:0.4, 1:0.6, 1:0.8, or 1:1, preferably 1:(0.4 to 0.8).

[0061] The above step uses a mixed solution of methanol and ethanol in a volume ratio of 1: (0.2-1) to fully dissolve the dried crude D-pantothenic acid, and under the joint action of the anti-solvent, it is also beneficial to obtain finer D-pantothenic acid crystals in the subsequent recrystallization step.

[0062] S8, recrystallization: under stirring, add an anti-solvent of 1 to 1.5 times the volume of the organic solvent to the above-mentioned solution, the anti-solvent is a mixed solution of chloroform and dichloromethane, and control the temperature to 15° C. to 22° C. for recrystallization. For example, the temperature can be controlled to 15° C., 17° C., 20° C. or 22° C.

[0063] The stirring rate in the recrystallization step is 80 to 120 rpm, and a vertical stirring blade is used during stirring. For example, the stirring rate may be 80 rpm, 100 rpm or 120 rpm. By using a vertical stirring blade and controlling the stirring rate, D-pantothenic acid crystals with finer crystal forms are obtained.

[0064] Furthermore, ultrasound is used to assist the recrystallization process, and the ultrasound power is 20 to 30 W / cm 2 The ultrasonic frequency is 20-40 kHz, and the ultrasound works for 2-4 seconds and rests for 2-4 seconds. For example, the ultrasonic power can be 20 W / cm 2 , 22W / cm 2 , 24W / cm 2 、26W / cm 2 、28W / cm 2 or 30W / cm 2 , preferably 24 to 28 W / cm 2 ; The ultrasonic frequency can be 20kHz, 25kHz, 30kHz, 35kHz or 40kHz, preferably 20-30kHz.

[0065] The cavitation effect of ultrasound and the strong shear force generated when ultrasound propagates in liquid can interrupt the growth process of crystals, destroy the large crystals that have been formed, break them into smaller particles, and generate a large number of nucleation centers to promote the formation of fine crystals. In addition, the intermittent ultrasonic mode can reduce energy consumption, save energy, reduce equipment wear and extend the service life of equipment while ensuring the crystal treatment effect.

[0066] In the above step, a mixed solution of chloroform and dichloromethane is used as an anti-solvent for recrystallization, which can better promote the precipitation of D-pantothenic acid crystals and is also conducive to the formation of fine crystals. The volume ratio of chloroform to dichloromethane is 1:(0.5-2), and for example, the volume ratio can be 1:0.5, 1:1, 1:1.5 or 1:2.

[0067] Furthermore, the anti-solvent is added in the above step by first adding 1 / 5 volume of the anti-solvent to the dissolving solution, and then adding the remaining 4 / 5 volume of the anti-solvent to the dissolving solution at a flow rate of 20 to 50 ml / min. For example, the flow rate may be 20 ml / min, 30 ml / min, 40 ml / min or 50 ml / min, preferably 20 to 40 ml / min.

[0068] Adding 1 / 5 volume of anti-solvent first can ensure that the anti-solvent is evenly distributed in the solution, reduce the solubility of the solution, and make D-pantothenic acid begin to nucleate, forming a large number of nucleation centers, providing a basis for subsequent crystal growth, and avoiding adding all the anti-solvent at once, resulting in local oversaturation, forming large crystals or uneven crystals. Adding the remaining anti-solvent at a rate of 20-50 ml / min can continuously maintain the supersaturation of the solution, which on the one hand helps to form more nucleation centers, and on the other hand can avoid the formation of large crystals due to too fast a crystallization rate, thereby forming small and uniform D-pantothenic acid crystals.

[0069] Furthermore, before adding the anti-solvent, an organic acid or an organic amine in an amount of 0.1 to 0.3 times the volume of the dissolving solution may be added, wherein the organic acid is any one or both of citric acid and acetic acid, and the organic amine is ethanolamine. By adding an organic acid or an organic amine in an amount of 0.1 to 0.3 times the volume of the dissolving solution before adding the anti-solvent, the pH value of the solution may be adjusted, and the nucleation and growth process of the D-pantothenic acid crystals may be effectively controlled, thereby obtaining fine and uniform crystals.

[0070] S9, filtration: filtering the solid-liquid mixture after recrystallization to collect the solid. The filtration pore size is 2-10 um, for example, the filtration pore size can be 2 um, 4 um, 6 um, 8 um or 10 um, preferably 4-8 um.

[0071] S10, washing and drying: washing the solid obtained after filtering with a detergent and acetone in sequence, drying after washing to obtain D-pantothenic acid, wherein the detergent is any one or both of chloroform and dichloromethane. Washing twice with a detergent and acetone in sequence can fully remove impurities on the D-pantothenic acid crystals and improve the purity of the D-pantothenic acid. In addition, washing with acetone not only helps to remove impurities on the crystal surface, but also helps to control the crystal form to obtain fine D-pantothenic acid crystals.

[0072] Furthermore, the washing includes: washing the filtered solid with a detergent of the same volume as the anti-solvent, stirring for 10 to 30 minutes, filtering to remove the detergent at a pore size of 2 to 10 um, and then washing the filtered solid with acetone of 2 to 3 times the volume of the anti-solvent, stirring for 10 to 30 minutes, and filtering to remove the acetone at a pore size of 2 to 10 um to obtain D-pantothenic acid crystals. The stirring rate is 60 to 120 rpm.

[0073] After washing is completed, the washed D-pantothenic acid crystals can be dried using a blower dryer, the drying temperature can be set to 60-80° C., and the drying time can be 4-8 hours.

[0074] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0075] Example 1

[0076] This embodiment provides a method for refining D-pantothenic acid from a fermentation broth, comprising the following steps:

[0077] S1. D-pantothenic acid expression: The D-pantothenic acid gene and promoter pTrc were connected to the expression vector pBAD to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Escherichia coli to obtain a recombinant Escherichia coli containing the D-pantothenic acid gene, and the Escherichia coli fermentation liquid was obtained by high-density cell fermentation technology. The OD value of the Escherichia coli fermentation liquid was 120.

[0078] The specific fermentation steps are as follows:

[0079] The recombinant E. coli working seeds were inoculated into a seed shake flask containing LB liquid medium and cultured at 37°C and 200 rpm for 14 h to obtain the OD 600The seed shake flask liquid has an OD value of 1.5; the seed shake flask liquid is connected to a fermentation tank at a volume ratio of 7% for high-density fermentation, and the fermentation parameters are: temperature 37°C, pH 7 controlled by ammonia water, and DO value 20%; when the DO value rebounds, feeding begins, feed medium A: 55% by mass glucose solution, feed medium B: 25% by mass yeast powder peptone (the mass ratio of yeast powder to peptone is 1:1) solution, and the feeding rate is: 20g / L / h (the mass ratio of feed medium A to feed medium B is 1:(0.7)); OD 600 When the OD value exceeded 60, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.3 mmol / L to start induction. 600 After stabilization, the fermentation is terminated to obtain a fermentation liquid containing D-pantothenic acid.

[0080] The culture medium in the fermentation tank is: anhydrous potassium hydrogen phosphate (K 2 HPO 4 ): 3g / L, anhydrous potassium dihydrogen phosphate (KH 2 PO 4 ): 1.5g / L, sodium chloride (NaCl): 0.5g / L, ammonium chloride (NH 4 Cl): 1g / L, trisodium citrate (Na 3 C 6 H 5 O 7 ): 1g / L, yeast extract: 10g / L, glucose: 30g / L, peptone: 20g / L.

[0081] S2. Centrifugation: Centrifuge the E. coli fermentation broth at 7000 rpm for 20 min to obtain a supernatant containing D-pantothenic acid.

[0082] S3. Decolorization: Add 70 g of activated carbon with a particle size of 1 mm to each liter of supernatant, stir at a rate of 80 rpm for 1 hour, filter with a pore size of 4 um, and collect the filtrate.

[0083] S4. Concentration: The filtrate is heated to 55° C. and concentrated under a vacuum degree of -0.07 MPa to 1 / 4 of the original volume of the filtrate to obtain a concentrated solution.

[0084] S5. Crystallization: The concentrated solution was stirred at a rate of 40 rpm, and the temperature was reduced to 3° C. at a rate of 0.5° C. / min to crystallize the D-pantothenic acid in the concentrated solution.

[0085] S6. Filtration: The solid-liquid mixture obtained after crystallization was filtered at low temperature at 4°C with a pore size of 30 um, the solid was collected, and dried at 55°C under -0.07 MPa for 2 h to obtain crude D-pantothenic acid.

[0086] S7. Dissolution: For every kilogram of crude D-pantothenic acid, 4 L of organic solvent is added to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol, wherein the volume ratio of methanol to ethanol is 1:0.4.

[0087] S8. Recrystallization: Use an upright stirring blade to stir the above-mentioned solution at a rate of 100 rpm. Under stirring, add 0.2 times the volume of the solution of citric acid to the solution, and then add 1.5 times the volume of the organic solvent of the anti-solvent, the anti-solvent is a mixed solution of chloroform and dichloromethane, wherein the volume ratio of chloroform to dichloromethane is 1:1, control the temperature to 20°C, and perform recrystallization.

[0088] The anti-solvent is added as follows: first, 1 / 5 volume of the anti-solvent is added to the dissolving solution, and then the remaining 4 / 5 volume of the anti-solvent is added to the dissolving solution at a rate of 30 ml / min.

[0089] Ultrasonic assistance was used in the above recrystallization process, and the ultrasonic power was 24 W / cm 2 The ultrasonic frequency is 20kHz, and the ultrasound works for 2 seconds and rests for 2 seconds.

[0090] S9, filtration: filter the solid-liquid mixture after recrystallization with a pore size of 4 um to collect the solid.

[0091] S10, washing and drying: the filtered solid is washed with chloroform in an equal volume to the anti-solvent, stirred at 80 rpm for 20 min, and then filtered to remove chloroform at a pore size of 4 μm, and then the filtered solid is washed with acetone in an amount of 2 times the anti-solvent volume, stirred at 80 rpm for 20 min, and then filtered to remove acetone at a pore size of 4 μm. After washing, the washed D-pantothenic acid crystals are dried using a blower dryer, the drying temperature is set to 60° C., and dried for 4 h to obtain D-pantothenic acid crystals.

[0092] Example 2

[0093] This embodiment provides a method for refining D-pantothenic acid from a fermentation broth, comprising the following steps:

[0094] S1. D-pantothenic acid expression: D-pantothenic acid gene, promoter pTrc and expression vector pBAD were connected to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Escherichia coli to obtain a recombinant Escherichia coli containing the D-pantothenic acid gene, and the Escherichia coli fermentation liquid was obtained by high-density cell fermentation technology. The OD value of the Escherichia coli fermentation liquid was 120. For specific fermentation steps, refer to Example 1.

[0095] S2. Centrifugation: The E. coli fermentation broth was centrifuged at 6000 rpm for 30 min to obtain a supernatant containing D-pantothenic acid.

[0096] S3. Decolorization: Add 50 g of activated carbon with a particle size of 2 mm to each liter of supernatant, stir at a rate of 50 rpm for 0.5 h, filter with a pore size of 0.65 um, and collect the filtrate.

[0097] S4. Concentration: The filtrate is heated to 50° C. and concentrated under a vacuum degree of -0.06 MPa to 1 / 5 of the original volume of the filtrate to obtain a concentrated solution.

[0098] S5. Crystallization: The concentrated solution was stirred at a rate of 30 rpm, and the temperature was reduced to 2° C. at a rate of 0.5° C. / min to crystallize the D-pantothenic acid in the concentrated solution.

[0099] S6. Filtration: The solid-liquid mixture obtained after crystallization was filtered at low temperature at 2°C with a pore size of 10 um, the solid was collected, and dried at 50°C under -0.06 MPa for 2 h to obtain crude D-pantothenic acid.

[0100] S7. Dissolution: For every kilogram of crude D-pantothenic acid, 5 L of organic solvent is added to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol, wherein the volume ratio of methanol to ethanol is 1:0.2.

[0101] S8. Recrystallization: Use an upright stirring blade to stir the above-mentioned solution at a rate of 80 rpm. Under stirring, add 0.1 times the volume of ethanolamine to the solution, and then add an anti-solvent with an equal volume to the organic solvent, wherein the anti-solvent is a mixed solution of chloroform and dichloromethane, wherein the volume ratio of chloroform to dichloromethane is 1:0.5, and control the temperature to 15°C for recrystallization.

[0102] The anti-solvent is added as follows: first, 1 / 5 volume of the anti-solvent is added to the dissolving solution, and then the remaining 4 / 5 volume of the anti-solvent is added to the dissolving solution at a rate of 20 ml / min.

[0103] Ultrasonic assistance was used in the above recrystallization process, and the ultrasonic power was 20 W / cm 2 The ultrasonic frequency is 30kHz, and the ultrasound works for 3 seconds and rests for 3 seconds.

[0104] S9, filtration: filter the solid-liquid mixture after recrystallization with a pore size of 2 um to collect the solid.

[0105] S10, washing and drying: The filtered solid was washed with dichloromethane of the same volume as the anti-solvent, stirred at 80 rpm for 10 min, and then filtered to remove dichloromethane at a pore size of 2 um, and then the filtered solid was washed with acetone of 3 times the volume of the anti-solvent, stirred at 80 rpm for 10 min, and then filtered to remove acetone at a pore size of 2 um. After washing, the washed D-pantothenic acid crystals were dried using a blast dryer, the drying temperature was set to 80° C., and dried for 8 hours to obtain D-pantothenic acid crystals.

[0106] Example 3

[0107] This embodiment provides a method for refining D-pantothenic acid from a fermentation broth, comprising the following steps:

[0108] S1. D-pantothenic acid expression: D-pantothenic acid gene, promoter pTrc and expression vector pBAD were connected to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Escherichia coli to obtain a recombinant Escherichia coli containing the D-pantothenic acid gene, and the Escherichia coli fermentation liquid was obtained by high-density cell fermentation technology. The OD value of the Escherichia coli fermentation liquid was 120. For specific fermentation steps, refer to Example 1.

[0109] S2. Centrifugation: centrifuge the E. coli fermentation broth at 10,000 rpm for 10 min to obtain a supernatant containing D-pantothenic acid.

[0110] S3. Decolorization: Add 100 g of activated carbon with a particle size of 3 mm per liter of supernatant, stir at a rate of 100 rpm for 2 hours, filter with a pore size of 10 um, and collect the filtrate.

[0111] S4. Concentration: The filtrate is heated to 60° C. and concentrated under a vacuum degree of -0.08 MPa to 1 / 3 of the original volume of the filtrate to obtain a concentrated solution.

[0112] S5. Crystallization: The concentrated solution was stirred at a rate of 60 rpm, and the temperature was reduced to 4°C at a rate of 1°C / min to crystallize the D-pantothenic acid in the concentrated solution.

[0113] S6. Centrifugation: The solid-liquid mixture obtained after crystallization was centrifuged at 4°C and 6000 rpm for 15 min, the supernatant was discarded, the solid precipitate was collected, and dried at 50°C under -0.06 MPa for 2 h to obtain crude D-pantothenic acid.

[0114] S7. Dissolution: For every kilogram of crude D-pantothenic acid, 4 L of organic solvent is added to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol, wherein the volume ratio of methanol to ethanol is 1:1.

[0115] S8. Recrystallization: Use an upright stirring blade to stir the above-mentioned solution at a rate of 120 rpm. Under stirring, add an anti-solvent with a volume of 1.2 times that of the organic solvent to the solution, wherein the anti-solvent is a mixed solution of chloroform and dichloromethane, wherein the volume ratio of chloroform to dichloromethane is 1:2, and the temperature is controlled at 22°C for recrystallization.

[0116] The anti-solvent is added as follows: first, 1 / 5 volume of the anti-solvent is added to the dissolving solution, and then the remaining 4 / 5 volume of the anti-solvent is added to the dissolving solution at a rate of 50 ml / min.

[0117] Ultrasonic assistance was used in the above recrystallization process, and the ultrasonic power was 30 W / cm 2 The ultrasonic frequency is 40kHz, and the ultrasound works for 4 seconds and rests for 4 seconds.

[0118] S9, filtration: filter the solid-liquid mixture after recrystallization with a pore size of 10 um to collect the solid.

[0119] S10, washing and drying: The filtered solid was washed with chloroform in an equal volume to the anti-solvent, stirred at 80 rpm for 30 min, and then filtered to remove chloroform at a pore size of 10 μm, and then the filtered solid was washed with acetone in an amount of 3 times the volume of the anti-solvent, stirred at 80 rpm for 30 min, and then filtered to remove acetone at a pore size of 10 μm. After washing, the washed D-pantothenic acid crystals were dried using a blast dryer, the drying temperature was set to 70° C., and dried for 6 h to obtain D-pantothenic acid crystals.

[0120] Comparative Example 1

[0121] D-pantothenic acid was purified from the fermentation broth according to the method of Example 1, except that ultrasound assistance was not used in the recrystallization process in step S8.

[0122] Comparative Example 2

[0123] D-pantothenic acid was purified from the fermentation broth according to the method of Example 1, except that the acetone used in the second washing in step S10 was replaced by dichloromethane in an equal amount.

[0124] Comparative Example 3

[0125] D-pantothenic acid was purified from the fermentation broth according to the method of Example 1, except that the anti-solvent was added in step S8 by directly adding the entire volume of the anti-solvent to the dissolved solution at a rate of 30 ml / min.

[0126] Comparative Example 4

[0127] D-pantothenic acid was purified from the fermentation broth according to the method of Example 1, except that the anti-solvent in step S8 was not added in a flowing manner, but 1 / 5 volume of the anti-solvent was first added to the dissolving solution, and then the remaining 4 / 5 volume of the anti-solvent was added to the dissolving solution.

[0128] Test Case

[0129] The purity of the D-pantothenic acid crystals prepared in Examples 1 to 3 and Comparative Examples 1 to 4 was tested, and the results are shown in Table 1.

[0130] Table 1

[0131] Purity of D-pantothenic acid crystals Crystal size Example 1 98.8％ 40um Example 2 99.2％ 43um Example 3 98.9％ 42um Comparative Example 1 98.1％ 52um Comparative Example 2 97.9％ 51um Comparative Example 3 98.2％ 49um Comparative Example 4 96.9％ 60um

[0132] From the above results, it can be seen that in comparative example 1, the purity of the obtained D-pantothenic acid crystals is reduced and the crystal size is enlarged without using ultrasound assistance during the recrystallization process in step S8, indicating that the use of ultrasound assistance during the recrystallization process can improve the purity of the D-pantothenic acid crystals and reduce the crystal size of the D-pantothenic acid. In comparative example 2, after replacing the acetone used in the second washing in step S10 with dichloromethane, the purity of the obtained D-pantothenic acid crystals is reduced and the crystal size is enlarged, indicating that the use of acetone washing can further improve the purity of the D-pantothenic acid crystals and is conducive to obtaining fine crystalline D-pantothenic acid crystals. After the anti-solvent addition method was changed in Comparative Examples 3 and 4, the purity of the obtained D-pantothenic acid crystals decreased and the crystal size became larger, indicating that the specific addition method of the present invention of "first adding 1 / 5 volume of the anti-solvent to the dissolving solution, and then adding the remaining 4 / 5 volume of the anti-solvent to the dissolving solution at a rate of 20 to 50 ml / min" can improve the purity of the D-pantothenic acid crystals obtained by recrystallization and reduce the crystal size of the D-pantothenic acid obtained by recrystallization.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for purifying D-pantothenic acid from a fermentation broth, characterized in that: The following steps are involved: (1) D-pantothenic acid expression: fermenting recombinant E. coli containing the D-pantothenic acid gene to obtain an E. coli fermentation broth; (2) Centrifugation: centrifuging the E. coli fermentation broth to obtain a supernatant containing D-pantothenic acid; (3) Decolorization: Add 50-100 g of activated carbon per liter of supernatant, stir for 0.5-2 h, filter, and collect the filtrate; (4) Concentration: heating the filtrate to 50° C. to 60° C. and concentrating the filtrate under vacuum conditions to 1 / 5 to 1 / 3 of the original volume of the filtrate to obtain a concentrated solution; (5) Crystallization: stirring the concentrated solution and lowering the temperature to 2° C. to 4° C. to crystallize the D-pantothenic acid in the concentrated solution; (6) Filtration or centrifugation: The solid-liquid mixture obtained after crystallization is filtered or centrifuged at low temperature, and the solid is collected and dried to obtain crude D-pantothenic acid; (7) Dissolving: using an organic solvent to dissolve the dried crude D-pantothenic acid to obtain a solution, wherein the organic solvent is a mixed solution of methanol and ethanol; (8) Recrystallization: Under stirring, add an anti-solvent in an amount of 1 to 1.5 times the volume of the organic solvent to the dissolved solution, wherein the anti-solvent is a mixed solution of chloroform and dichloromethane, and control the temperature to be 15° C. to 22° C. to perform recrystallization; The recrystallization is assisted by ultrasound, and the ultrasonic power is 20-30 W / cm 2 , the ultrasonic frequency is 20 to 40 kHz, the ultrasound works for 2 to 4 seconds and rests for 2 to 4 seconds; (9) Filtration: Filter the solid-liquid mixture after recrystallization to collect the solid; (10) Washing and drying: The solid obtained after filtration is washed with a detergent and acetone in sequence, and then dried to obtain D-pantothenic acid. The detergent is any one or both of chloroform and dichloromethane.

2. The method according to claim 1, characterized in that The centrifugal treatment in step (2) is: centrifuging at a speed of 6000 to 10000 rpm for 10 to 30 minutes.

3. The method according to claim 1, characterized in that The stirring rate during decolorization in step (3) is 50-100 rpm; and the filtration pore size is 0.65-10 um.

4. The method according to claim 1, characterized in that The vacuum degree during concentration in step (4) is -0.06 to -0.08 MPa.

5. The method according to claim 1, characterized in that The stirring rate during crystallization in step (5) is 30-60 rpm, and the cooling rate is 0.5-1°C / min.

6. The method according to claim 1, characterized in that The temperature of the low-temperature filtration or low-temperature centrifugation in step (6) is 2-8°C, the filtration pore size is 10-50 um, and the centrifugation is performed at a speed of 5000-6000 rpm for 10-20 min.

7. The method according to claim 1, characterized in that During the dissolution in step (7), 4 to 5 L of organic solvent is added for each kg of crude D-pantothenic acid to dissolve the crude product; the volume ratio of methanol to ethanol in the organic solvent is 1:(0.2 to 1).

8. The method according to claim 1, characterized in that The stirring rate during recrystallization in step (8) is 80 to 120 rpm, and a vertical stirring blade is used during stirring; The volume ratio of chloroform to dichloromethane in the anti-solvent is 1:(0.5-2); The method of adding the anti-solvent includes: first adding 1 / 5 volume of the anti-solvent to the dissolving solution, and then adding the remaining 4 / 5 volume of the anti-solvent to the dissolving solution at a flow rate of 20 to 50 ml / min.

9. The method according to claim 1, characterized in that: In step (8), before adding the anti-solvent, an organic acid or an organic amine in an amount of 0.1 to 0.3 times the volume of the dissolving solution may be added. The organic acid is any one or both of citric acid and acetic acid, and the organic amine is ethanolamine.

10. The method according to claim 1, characterized in that The pore size during filtration in step (9) is 2 to 10 μm; The washing in step (10) comprises: washing the filtered solid with a detergent of an equal volume to the anti-solvent, stirring for 10 to 30 minutes, filtering to remove the detergent at a pore size of 2 to 10 um, and then washing the filtered solid with acetone of 2 to 3 times the volume of the anti-solvent, stirring for 10 to 30 minutes, and filtering to remove the acetone at a pore size of 2 to 10 um to obtain D-pantothenic acid crystals.