Method for extracting and preparing D-calcium pantothenate from pantothenic acid-containing fermentation liquor

Abstract

The invention belongs to the technical field of biopharmacy, and relates to a method for extracting and preparing D-calcium pantothenate from pantothenic acid-containing fermentation broth, which comprises the following steps: acidifying the pantothenic acid-containing fermentation broth, removing solids in the acidified fermentation broth, extracting the obtained clear fermentation broth by using an extracting agent, and concentrating to obtain D-calcium pantothenate. The extraction agent contains tri-n-octylamine, di (2-ethylhexyl) phosphate and a modifier, the modifier is a reaction product obtained by reaction of choline chloride and glucose, an organic phase obtained by extraction is washed with water and then is subjected to back extraction and calcification, and D-calcium pantothenate is obtained. The method provided by the invention can improve the purity and yield of D-calcium pantothenate, is easy to operate, convenient to control and simple in step, and has the advantages of low discharge capacity, low energy consumption, low cost, low solvent residue, good repeatability, suitability for industrial large-scale production and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for extracting and preparing D-pantothenate calcium from a pantothenic acid-containing fermentation broth. Background Art

[0002] D-calcium pantothenate, chemically named N-(α,γ-dihydroxy-β,β-dimethylbutyryl)-β-alanine calcium, is a water-soluble B vitamin and one of the nutrients necessary for normal growth of organisms. D-calcium pantothenate can release pantothenic acid and calcium into the body, and pantothenic acid, aminoethanol and adenosine diphosphate constitute the coenzyme A of acyltransferase in humans and animals, and then participate in the metabolism of proteins, fats and sugars, playing an important role in material metabolism. At present, D-calcium pantothenate is widely used in feed, medicine, food and other industries with its unique biochemical functions.

[0003] The methods for industrial production of D-calcium pantothenate mainly include chemical synthesis and microbial fermentation. The chemical synthesis method first requires the preparation of two important intermediates, namely β-aminopropionic acid and DL-pantothenate lactone. There are two methods for preparing D-calcium pantothenate from the above intermediates: one is to first chemically synthesize DL-calcium pantothenate, and then obtain D-calcium pantothenate by splitting DL-calcium pantothenate; the other is to split the key intermediate DL-pantothenate lactone by chemical or biological methods to obtain D-pantothenate lactone, and then synthesize D-calcium pantothenate by reacting D-pantothenate lactone with β-aminopropionic acid. At the same time, butyrolactone, hydrocyanic acid and isobutyraldehyde are the main raw materials for the production of DL-pantothenate lactone, acrylonitrile is the main raw material for the production of β-aminopropionic acid, and formaldehyde is the main auxiliary material for the production of β-aminopropionic acid. It can be seen that the initial raw materials for the industrial production of D-calcium pantothenate are all chemical products, and the cost of raw materials accounts for about 60% of the production cost of calcium pantothenate, and the process is complicated and difficult to control. Compared with chemical synthesis methods, microbial fermentation is more efficient and environmentally friendly to produce D-calcium pantothenate. However, a large amount of nutrients and basic substances need to be added during the fermentation process, and a large amount of impurities such as microbial cells, pigments, proteins, and organic matter similar to the product structure will be produced, thereby increasing the cost of purification and waste treatment as well as energy consumption.

[0004] CN115745827A discloses an extraction process for producing calcium pantothenate by fermentation, the main process of which includes fermentation liquid acidification, filtration, nanofiltration primary concentration, ion exchange, nanofiltration secondary concentration, decolorization, neutralization, evaporation concentration, crystallization, centrifugal drying; or fermentation liquid acidification, filtration, nanofiltration primary concentration, decolorization, ion exchange, nanofiltration secondary concentration, neutralization, evaporation concentration, crystallization, centrifugal drying. However, the purity and yield of calcium pantothenate obtained by this method still need to be further improved, and the method has complicated steps, uses activated carbon for decolorization, and has a large industrial production amount, does not involve the recycling of activated carbon, has a lot of solid waste, and uses centrifugal drying, which has a long drying cycle and high energy consumption, and is not suitable for large-scale production.

[0005] CN118084708A discloses a method for preparing calcium D-pantothenate, which comprises performing solid-liquid separation on a fermentation liquid containing D-pantothenic acid to obtain a first liquid phase and a first solid phase; adjusting the pH value of the first liquid phase to 4-5 using a pH regulator to obtain a second liquid phase; performing electrodialysis on the second liquid phase and water to obtain a D-pantothenic acid solution; performing a cyclic decolorization on the D-pantothenic acid solution and a calcium compound using activated carbon fiber cotton after a neutralization reaction to obtain a calcium D-pantothenate solution; and removing water from the calcium D-pantothenate solution to obtain calcium D-pantothenate. However, the purity and yield of calcium D-pantothenate obtained by this method still need to be further improved. Furthermore, the number of cycles for cyclic decolorization using activated carbon fiber cotton is 5-10 times, the decolorization period is relatively long, the amount of activated carbon fiber membrane used is large, and the number of decolorization times will inevitably reduce the decolorization efficiency of the activated carbon fiber cotton, resulting in the final solid calcium D-pantothenate having an unqualified color. In addition, this method finally utilizes a spray dryer to remove water. Although it avoids the use of organic solvents, D-calcium pantothenate has strong hygroscopicity, the dehydration time is 3 to 10 seconds, the moisture content can reach 3.5%, the product stability is poor, and it is not easy to mass produce.

[0006] In summary, among the methods for preparing D-calcium pantothenate in the prior art, on the one hand, the chemical direct synthesis method is complex and difficult to control, the raw material cost is high, and all of them are chemical products with serious pollution; on the other hand, the microbial fermentation method is difficult, has low purity, low yield, and high energy consumption, and it is difficult to achieve industrial-scale production. Summary of the invention

[0007] The object of the present invention is to provide a method for extracting and preparing D-calcium pantothenate from a fermentation broth containing pantothenic acid, which can improve the purity and yield of D-calcium pantothenate.

[0008] Specifically, the method for extracting and preparing D-pantothenate calcium from pantothenic acid-containing fermentation broth provided by the present invention comprises acidifying the pantothenic acid-containing fermentation broth, removing solids from the obtained acidified fermentation broth, and then extracting the obtained fermentation clear liquid with an extractant, wherein the extractant contains tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier, wherein the modifier is a reaction product obtained by reacting choline chloride and glucose, and then washing the organic phase obtained by extraction with water, performing back extraction and calcification to obtain D-pantothenate calcium.

[0009] The key to the present invention is that after the pantothenic acid-containing fermentation broth is acidified and solids are removed, a specific extractant is used for extraction, and then the organic phase obtained by extraction is washed with water, stripped and calcified to convert D-pantothenic acid into D-calcium pantothenate. Among them, a complex containing tri-n-octylamine, di(2-ethylhexyl) phosphate and a reaction product obtained by the reaction of choline chloride and glucose is used as an extractant, which can effectively extract D-pantothenic acid in the fermentation broth and effectively remove impurities, thereby improving the purity and yield of the obtained D-calcium pantothenate.

[0010] The method for preparing calcium D-pantothenate provided by the present invention uses fermentation broth as a raw material, and compared with the existing chemical synthesis method, can avoid the use of chemical raw materials from the source, and does not need to first obtain pantothenic acid lactone and then obtain calcium pantothenate. D-pantothenic acid is directly extracted from the fermentation broth to prepare calcium D-pantothenate. The method has simple steps, is easy to operate, and is easy to control. It has the advantages of low pollution discharge, low energy consumption, low cost, low solvent residue, good repeatability, and is suitable for industrial-scale production, etc. It complies with the principle of green environmental protection and is conducive to the realization of industrialized scale-up production.

[0011] In a preferred embodiment, the method for extracting and preparing D-pantothenate calcium from pantothenic acid-containing fermentation broth further includes drying the activated carbon after decolorization and then regenerating and recycling it using microwave radiation. At this time, the utilization rate of the activated carbon can be improved and the amount of solid waste processed can be reduced.

[0012] In a preferred embodiment, the concentrated solution is dissolved in methanol and dried after adding seed crystals for crystallization, and the drying method is selected as boiling drying, which can not only perfectly solve the problems of D-calcium pantothenate being easily dissolved and bonded by moisture in the early stage of drying, having strong thermal sensitivity, being difficult to dry, and having high power consumption, but also shorten the drying cycle, improve the production capacity of a single machine, reduce the drying energy consumption and be suitable for large-scale production, and at the same time solve the problem of high residual solvent in the crystallization of methanol aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention provides a specific process flow chart for extracting and preparing D-calcium pantothenate from pantothenic acid-containing fermentation broth. DETAILED DESCRIPTION

[0014] The method for extracting and preparing D-pantothenate calcium from pantothenic acid-containing fermentation broth provided by the present invention comprises acidifying the pantothenic acid-containing fermentation broth, removing solids in the obtained acidified fermentation broth, and then extracting the obtained fermentation clear liquid with an extractant, wherein the extractant contains tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier, and the modifier is a reaction product obtained by reacting choline chloride and glucose, and then washing the organic phase obtained by extraction with water, performing back extraction and calcification to obtain D-pantothenate calcium.

[0015] The present invention has no particular limitation on the source of the pantothenic acid-containing fermentation broth, which can be obtained by commercial purchase, donation, etc., or prepared according to various existing methods.

[0016] In the present invention, the purpose of the acidification is mainly to denature and inactivate the impurities in the fermentation broth so as to facilitate its removal. The method of the acidification can be a conventional choice in the art without special limitation. In a preferred embodiment, the method of acidifying the pantothenic acid fermentation broth to obtain an acidified fermentation broth comprises adjusting the pH value of the pantothenic acid fermentation broth to 1.5-2.5, then keeping it warm at a temperature of 90°C-95°C for 5min-30min, and cooling it to 20°C-30°C after the end of the insulation, so as to obtain the acidified fermentation broth. Among them, the reagent used to adjust the pH value can be various existing acid solutions, such as at least one of sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, etc. The amount of the acid solution is based on adjusting the pH value of the pantothenic acid fermentation broth to 1.5-2.5, for example, it can be 1.5, 1.8, 2, 2.2, 2.5 or any value therebetween. After the pH value is adjusted, the temperature of the heat preservation reaction can be, for example, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C or any value therebetween, and the heat preservation reaction time can be, for example, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value therebetween. After the heat preservation is completed, the temperature needs to be lowered to, for example, 20°C, 23°C, 25°C, 28°C, 30°C or any value therebetween.

[0017] In the present invention, the method for removing solids from the acidified fermentation broth may be, for example, filter pressing, centrifugation, plate and frame filtration, membrane filtration, etc., preferably using a ceramic membrane for filtration, and the permeate obtained is the fermentation clear liquid. The molecular weight cutoff of the ceramic membrane is preferably 1000 to 10000 Da, such as 1000 Da, 2000 Da, 4000 Da, 6000 Da, 8000 Da, 10000 Da or any value therebetween.

[0018] In the present invention, the extractant contains tri-n-octylamine, di(2-ethylhexyl)phosphate and a modifier. The concentrations of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant are preferably independently 5 g / L to 50 g / L, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 g / L or any value therebetween. When the concentrations of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant are lower than 5 g / L or higher than 50 g / L, the enhancement effect of the synergistic coefficient of the two solvents will slowly weaken. If the concentration of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant is lower than 5g / L, the concentration of tri-n-octylamine in the extract phase cannot offset the negative effect of the increased concentration of di(2-ethylhexyl)phosphate on the synergistic effect; if the concentration of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant is higher than 50g / L, the two extractants will react in the extract phase, thereby affecting the extraction efficiency. The ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant to the volume of the modifier is preferably (2-4):(6-8), such as 2:8, 2.2:7.8, 2.5:7.5, 2.8:7.2, 3:7, 3.2:6.8, 3.4:6.6, 3.6:6.4, 3.8:6.2, 4:6 or any value therebetween. When the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant to the volume of the modifier is lower than 2:8 (i.e., the total content of tri-n-octylamine and di(2-ethylhexyl)phosphate is too low), a stable third phase will be formed, which will affect the extraction effect; when the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant to the volume of the modifier is higher than 4:6 (i.e., the total content of tri-n-octylamine and di(2-ethylhexyl)phosphate is too high), the modifier provides fewer active groups, and the solubility of the active complex in the extractant is reduced, thereby affecting the extraction efficiency. The modifier is a reaction product obtained by reacting choline chloride and glucose. The process of reacting choline chloride and glucose preferably includes stirring choline chloride and glucose at a temperature of 60°C to 80°C (such as 60°C, 62°C, 65°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C, 80°C or any value therebetween) until a transparent liquid is naturally cooled to room temperature. The molar ratio of choline chloride and glucose used in the preparation process of the modifier is preferably (1-3):1, such as 1:1, 1.3:1, 1.5:1, 1.8:1, 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1 or any value therebetween.

[0019] In the present invention, the extraction method is preferably cross-current extraction, that is, multi-stage continuous extraction is adopted, and the extraction phase of the previous stage continues to be used as the phase to be extracted for the next stage extraction. The number of extraction stages is preferably 10 to 15 stages, such as 10, 11, 12, 13, 14 or 15. The conditions of the extraction are not particularly limited, as long as it is conducive to effectively separating the target product and impurities in the phase to be extracted. In a preferred embodiment, in the cross-current extraction process, each stage of extraction independently includes stirring and mixing the phase to be extracted and the extractant at 20 to 30°C for 0.5 to 2 hours, and then standing for extraction for 10 to 30 hours. At this time, it is more conducive to the separation of the target product and impurities, and the purity of the product can be further improved. Wherein, the temperature for stirring and mixing the phase to be extracted with the extractant can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C or any value therebetween, and the time for stirring and mixing can be 0.5h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h or any value therebetween. The time for standing extraction after stirring and mixing the phase to be extracted with the extractant can be, for example, 10h, 13h, 15h, 18h, 20h, 22h, 25h, 28h, 30h or any value therebetween. In each stage of extraction, the volume ratio of the phase to be extracted to the extractant is preferably independently (0.5-1.5):1, such as 0.5:1, 0.8:1, 1:1, 1.2:1, 1.5:1 or any value therebetween.

[0020] In the present invention, the conditions of the stripping are not particularly limited, as long as they are conducive to effectively separating the target product and impurities in the phase to be stripped. In a preferred embodiment, the stripping method includes stirring and mixing the washed organic phase with the stripping agent at 65-75°C for 1-3 hours, and then standing for stripping for 5-10 hours, which is more conducive to the separation of the target product and impurities, and can further improve the purity of the product. Among them, the temperature for stirring and mixing the washed organic phase with the stripping agent can be 65°C, 68°C, 70°C, 72°C, 75°C or any value therebetween, and the stirring and mixing time can be 1h, 1.2h, 1.5h, 1.8h, 2, 2.2h, 2.4h, 2.6h, 2.8h, 3h or any value therebetween. The time for standing and stripping after the organic phase after washing with the stripping agent is stirred and mixed can be, for example, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h or any value therebetween. The stripping agent is preferably an aqueous solution of n-heptane. Wherein, the volume ratio of water to n-heptane in the stripping agent is preferably 1:(1-2), such as 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2 or any value therebetween. When the volume ratio of water to n-heptane in the stripping agent is lower than 1:1 (that is, the content of n-heptane is too low), the extraction phase cannot be fully recovered; when the volume ratio of water to n-heptane in the stripping agent is higher than 1:2 (that is, the content of n-heptane is too high), the product stripping is insufficient. The volume ratio of the organic phase to the stripping agent is preferably 1:(3-8), such as 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8 or any value therebetween. In addition, the organic phase obtained by stripping is preferably recycled as an extractant.

[0021] In the present invention, the calcification agent is preferably calcium hydroxide and / or calcium oxide. The mass ratio of the calcification agent to the pure mass of D-pantothenic acid in the aqueous phase obtained by back extraction is preferably 1:(5-10), such as 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10 or any value therebetween.

[0022] In a preferred embodiment, the method for extracting and preparing D-pantothenate calcium from a pantothenic acid-containing fermentation broth provided by the present invention further includes decolorizing with activated carbon after calcification, concentrating with a reverse osmosis membrane, dissolving the resulting concentrated solution with methanol, adding seed crystals for crystallization, and then drying to obtain D-pantothenate calcium crystals.

[0023] In the present invention, the amount of the activated carbon is preferably 10% to 20% of the mass of calcium D-pantothenate, such as 10%, 12%, 14%, 16%, 18%, 20% or any value therebetween. The activated carbon is preferably powdered activated carbon.

[0024] In the present invention, the decolorization conditions preferably include a temperature of 75°C to 85°C, such as 75°C, 78°C, 80°C, 82°C, 84°C, 85°C or any value therebetween; and a time of 1h to 5h, such as 1h, 2h, 3h, 4h, 5h or any value therebetween.

[0025] In a preferred embodiment, the method for extracting and preparing D-pantothenate calcium from pantothenic acid fermentation broth also includes drying the activated carbon after decolorization and regenerating and recycling it by microwave radiation. Wherein, the microwave radiation conditions preferably include a microwave frequency of 20Hz to 25Hz, such as 20Hz, 21Hz, 22Hz, 23Hz, 24Hz, 25Hz or any value therebetween; the radiation time is 10min to 30min, such as 10min, 15min, 20min, 25min, 30min or any value therebetween. The number of times the activated carbon is regenerated is preferably 1 to 10 times, such as 1 time, 2 times, 4 times, 6 times, 8 times, 10 times, etc. The activated carbon can be all regenerated activated carbon or all fresh activated carbon. Preferably, 60% to 70% of the regenerated activated carbon and 30% to 40% of the new activated carbon are selected for use in a single batch decolorization process. At this time, not only a good decolorization effect can be achieved, but also the regenerated activated carbon can be fully utilized.

[0026] In the present invention, the concentration temperature is preferably 20°C to 30°C, such as 20°C, 22°C, 24°C, 26°C, 28°C, 30°C or any value therebetween. The concentration conditions are preferably such that the solid content of the concentrate is increased to 80 g / L or more, more preferably concentrated to 80 to 100 g / L, such as 80, 85, 90, 95, 100 g / L or any value therebetween.

[0027] In the present invention, the volume ratio of the methanol to the concentrated solution is preferably (2 to 6): 1, such as 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1 or any value therebetween. In a preferred embodiment, the crystal seeds are added after dissolving in methanol, kept for 3h to 6h, and then cooled to below -10°C. Wherein, the crystal seeds are D-calcium pantothenate crystals. In order to be more conducive to improving the purity of the final product, the purity of the D-calcium pantothenate crystals as crystal seeds is preferably 99% or more. The crystal seeds preferably account for 0.5‰ to 1.5‰ of the pure mass of D-calcium pantothenate in the concentrated solution, such as 0.5‰, 0.8‰, 1‰, 1.2‰, 1.5‰ or any value therebetween.

[0028] In the present invention, the drying method is preferably boiling drying. The drying conditions preferably include a temperature of 80°C to 150°C, such as 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C or any value therebetween; a time of 5min to 30min, such as 5min, 10min, 15min, 20min, 25min, 30min or any value therebetween.

[0029] like Figure 1 As shown, in a preferred embodiment, the method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth comprises the following steps:

[0030] S1. Acidification: The pH value of the pantothenic acid fermentation broth was adjusted to 1.5 to 2.5, and then kept at a temperature of 90°C to 95°C for 5min to 30min. After the end of the insulation, the temperature was lowered to 20°C to 30°C to obtain an acidified fermentation broth;

[0031] S2. Filtration pretreatment: The acidified fermentation broth is filtered using a ceramic membrane to remove solids therein to obtain a fermentation clear liquid;

[0032] S3. Extraction: The fermentation liquid is subjected to cross-flow extraction using an extractant. In the cross-flow extraction process, each stage of extraction independently comprises stirring and mixing the phase to be extracted with the extractant at 20 to 30° C. for 0.5 to 2 h, and then standing for extraction for 10 to 30 h to obtain an organic phase;

[0033] S4. Stripping: Wash the organic phase obtained by extraction with water, and then stir and mix the washed organic phase with the stripping agent at 65-75° C. for 1-3 hours, and then stand for stripping for 5-10 hours to obtain an organic phase (extraction agent, which can be recycled) and an aqueous phase;

[0034] S5. Calcification and decolorization: adding a calcification reagent to the aqueous phase obtained by stripping and mixing evenly, and then decolorizing the obtained D-calcium pantothenate solution using activated carbon to obtain a decolorized D-calcium pantothenate solution;

[0035] S6. Concentration, crystallization and drying: The decolorized D-calcium pantothenate solution is concentrated to a solid content of more than 80 g / L using a reverse osmosis membrane. The resulting D-calcium pantothenate concentrate is then dissolved in methanol and seed crystals are added for crystallization. The resulting wet crystals are then dried by boiling to obtain D-calcium pantothenate crystals.

[0036] The present invention will be described in detail below through examples.

[0037] In the following examples and comparative examples, the pantothenic acid fermentation broth is the pantothenic acid fermentation broth disclosed in Example 1 of CN117327747A.

[0038] In the following examples and comparative examples, the purity of D-calcium pantothenate crystals was calculated by area normalization method after measuring the absorbance with a spectrophotometer; the content of D-calcium pantothenate crystals was calculated with reference to D-calcium pantothenate standard.

[0039] Example 1

[0040] S1. Acidification: The pH value of the pantothenic acid-containing fermentation liquid is adjusted to 2.0 with concentrated sulfuric acid. After acidification, the fermentation liquid enters the heater, the temperature is raised to 90°C and kept warm for 10 minutes, and then the temperature is lowered to 20°C after the insulation is completed to obtain the acidified fermentation liquid.

[0041] S2. Filtration pretreatment: The acidified fermentation broth is filtered through a ceramic membrane with a molecular weight cutoff of 10,000 Da to obtain a fermentation clear liquid.

[0042] S3. Extraction: The fermentation clear liquid is used as the feed phase, and a mixture of tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier is used as the extractant for cross-current extraction, and the cross-current extraction level is 13. In each stage of extraction, the volume ratio of the feed phase and the extractant is 1:1, and the phase to be extracted and the extractant need to be stirred at 25°C for 1 hour, and then left to extract for 20 hours. After the extraction is completed, the organic phase is washed with pure water, and the washing liquid is circulated for cleaning the ceramic membrane. The concentration of tri-n-octylamine in the extractant is 5g / L, the concentration of di(2-ethylhexyl) phosphate is 50g / L, the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl) phosphate to the volume of the modifier is 3:7, and the modifier is obtained by stirring choline chloride and glucose at a molar ratio of 2:1 at 70°C until a transparent liquid and then naturally cooling to room temperature.

[0043] S4. Back extraction: The organic phase obtained from the above extraction is washed with pure water, then mixed with a back extraction agent, heated to 69.5°C and stirred for 2 hours, then allowed to stand for extraction for 7.5 hours, and the extraction phase and the aqueous phase (D-pantothenic acid solution) are separated. The extraction phase is regenerated and recycled. The back extraction agent is composed of n-heptane and pure water in a volume ratio of 2:3. During back extraction, the volume ratio of the organic phase to the back extraction agent is 1:5.

[0044] S5. Calcification decolorization: Add calcium oxide to the aqueous phase obtained after back extraction at a mass ratio of 7.8:1 of D-pantothenic acid to calcium oxide, stir evenly, and obtain D-pantothenate calcium solution, and then decolorize it through an activated carbon column. The mass of the activated carbon column is 20% of the mass of D-pantothenate calcium, the decolorization temperature is 80°C, and the decolorization time is 2h. The activated carbon is selected as powdered activated carbon. After drying the activated carbon, regenerate it at a microwave frequency of 22Hz for 20min. For the next decolorization, select a mixture of 67% regenerated activated carbon and 33% new activated carbon.

[0045] S6. Concentration, crystallization and drying: The D-pantothenate calcium solution passed through the activated carbon column is concentrated by reverse osmosis membrane to obtain D-pantothenate calcium concentrate; the reverse osmosis membrane temperature is 20°C, and the concentration is carried out twice, and the secondary concentration is carried out at 20L / h / m 2 The final concentration is 90 g / L. After adding 2 times the volume of methanol to the obtained D-pantothenate calcium concentrate to dissolve it completely, add D-pantothenate calcium crystals with a purity of 99% as seeds, the seeds account for 1‰ of the pure mass of D-pantothenate calcium in the concentrate, keep the temperature down to -10°C for 3 hours, crystallize for 24 hours, filter, and dry the obtained wet crystals in a boiling drying equipment at 100°C for 10 minutes to obtain D-pantothenate calcium crystals.

[0046] After testing, the purity of the D-calcium pantothenate crystals was 99.85%, the content was 99.10%, the methanol residue was 0.013%, and the total yield was 99.45%.

[0047] Example 2

[0048] S1. Acidification: The pantothenic acid-containing fermentation liquid is adjusted to pH 1.5 with concentrated sulfuric acid. After acidification, the fermentation liquid enters the heater, the temperature is raised to 95°C and kept for 5 minutes. After the insulation, the temperature is lowered to 30°C to obtain the acidified fermentation liquid.

[0049] S2. Filtration pretreatment: The acidified fermentation broth is filtered through a ceramic membrane with a molecular weight cutoff of 1000 Da to obtain a fermentation clear liquid.

[0050] S3. Extraction: The fermentation clear liquid is used as the feed phase, and a mixture of tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier is used as the extractant for cross-current extraction, and the cross-current extraction stage is 10. In each stage of extraction, the volume ratio of the feed phase and the extractant is 1.5:1, and the phase to be extracted and the extractant need to be stirred at 30°C for 0.5h, and then left to extract for 30h. After the extraction is completed, the organic phase is washed with pure water, and the washing liquid is circulated for cleaning the ceramic membrane. The concentration of tri-n-octylamine in the extractant is 50g / L, the concentration of di(2-ethylhexyl) phosphate is 5g / L, the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl) phosphate to the volume of the modifier is 2:8, and the modifier is obtained by stirring choline chloride and glucose at a molar ratio of 1:1 at 60°C until a transparent liquid and then naturally cooling to room temperature.

[0051] S4. Back extraction: The organic phase obtained from the above extraction is washed with pure water, then mixed with a back extraction agent, heated to 70.5°C, stirred for 1 hour, and then allowed to stand for extraction for 5 hours, and the extraction phase and the aqueous phase (D-pantothenic acid solution) are separated. The extraction phase is regenerated and recycled. The back extraction agent is composed of n-heptane and pure water in a volume ratio of 1:1. During back extraction, the volume ratio of the organic phase to the back extraction agent is 1:4.

[0052] S5. Calcification decolorization: Add calcium hydroxide to the aqueous phase obtained after back extraction at a mass ratio of 5:1 of D-pantothenic acid to calcium hydroxide and stir evenly to obtain D-pantothenate calcium solution, and then decolorize it through an activated carbon column. The mass of the activated carbon column is 10% of the mass of D-pantothenate calcium. The decolorization temperature is 75°C and the decolorization time is 5h. Powdered activated carbon is selected. After drying the activated carbon, regenerate it at a microwave frequency of 22Hz for 20min. For the next decolorization, a mixture of 70% regenerated activated carbon and 30% new activated carbon is selected.

[0053] S6. Concentration, crystallization and drying: The D-pantothenate calcium solution passed through the activated carbon column is concentrated by reverse osmosis membrane to obtain D-pantothenate calcium concentrate; the reverse osmosis membrane temperature is 25°C, and the concentration is carried out twice, and the secondary concentration is carried out at 20L / h / m 2 The final concentration is 90 g / L. After adding 4 times the volume of methanol to the obtained D-pantothenate calcium concentrate to dissolve it completely, add D-pantothenate calcium crystals with a purity of 99% as seeds, the seeds account for 1‰ of the pure mass of D-pantothenate calcium in the concentrate, keep the temperature down to -10°C for 3 hours, crystallize for 24 hours, filter, and dry the obtained wet crystals at 150°C for 5 minutes in a boiling drying equipment to obtain D-pantothenate calcium crystals.

[0054] After testing, the purity of the D-calcium pantothenate crystals was 99.54%, the content was 99.14%, the methanol residue was 0.014%, and the total yield was 99.42%.

[0055] Example 3

[0056] S1. Acidification: The pH value of the pantothenic acid-containing fermentation liquid is adjusted to 2.5 with concentrated sulfuric acid. After acidification, the fermentation liquid enters the heater, the temperature is raised to 92°C and kept warm for 30 minutes, and then the temperature is lowered to 22°C to obtain the acidified fermentation liquid.

[0057] S2. Filtration pretreatment: The acidified fermentation broth is filtered through a ceramic membrane with a molecular weight cutoff of 20,000 Da to obtain a fermentation clear liquid.

[0058] S3. Extraction: The fermentation clear liquid is used as the feed phase, and a mixture of tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier is used as the extractant for cross-current extraction, and the cross-current extraction stage is 15. In each stage of extraction, the volume ratio of the feed phase to the extractant is 0.5:1, and the phase to be extracted and the extractant need to be stirred at 20°C for 2h, and then left to extract for 10h. After the extraction is completed, the organic phase is washed with pure water, and the washing liquid is circulated for cleaning the ceramic membrane. The concentration of tri-n-octylamine in the extractant is 25g / L, the concentration of di(2-ethylhexyl) phosphate is 25g / L, the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl) phosphate to the volume of the modifier is 4:6, and the modifier is obtained by stirring choline chloride and glucose at a molar ratio of 3:1 at 80°C until a transparent liquid and then naturally cooling to room temperature.

[0059] S4. Back extraction: The organic phase obtained from the above extraction is washed with pure water, then mixed with a back extraction agent, heated to 69.5°C and stirred for 3 hours, then allowed to stand for extraction for 10 hours, and the extraction phase and the aqueous phase (D-pantothenic acid solution) are separated. The extraction phase is regenerated and recycled. The back extraction agent is composed of n-heptane and pure water in a volume ratio of 1:2. During back extraction, the volume ratio of the organic phase to the back extraction agent is 1:6.

[0060] S5. Calcification decolorization: Add calcium oxide to the aqueous phase obtained after back extraction at a mass ratio of D-pantothenic acid to calcium oxide of 10:1 and stir evenly to obtain a D-pantothenate calcium solution, and then decolorize it through an activated carbon column. The mass of the activated carbon column is 30% of the mass of D-pantothenate calcium. The decolorization temperature is 85°C and the decolorization time is 1h. Powdered activated carbon is selected. After drying the activated carbon, regenerate it at a microwave frequency of 30Hz for 20min. For the next decolorization, a mixture of 60% regenerated activated carbon and 40% new activated carbon is selected.

[0061] S6. Concentration, crystallization and drying: The D-pantothenate calcium solution passed through the activated carbon column is concentrated by reverse osmosis membrane to obtain D-pantothenate calcium concentrate; the reverse osmosis membrane temperature is 30°C, and the concentration is carried out twice, and the secondary concentration is carried out at 20L / h / m 2 6 times the volume of methanol was added to the obtained D-pantothenate calcium concentrate to dissolve it completely, and then D-pantothenate calcium crystals with a purity of 99% were added as seeds, and the seeds accounted for 1‰ of the pure mass of D-pantothenate calcium in the concentrate. The temperature was kept at -10°C for 3 hours and crystallized for 24 hours, filtered, and the wet crystals were dried at 80°C for 30 minutes in a boiling drying equipment to obtain D-pantothenate calcium crystals.

[0062] After testing, the purity of the D-calcium pantothenate crystals was 99.82%, the content was 99.28%, the methanol residue was 0.014%, and the total yield was 99.08%.

[0063] Example 4

[0064] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the concentration of tri-n-octylamine in the extractant was 50 g / L, the concentration of di(2-ethylhexyl) phosphate was 50 g / L, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0065] After testing, the purity of the D-calcium pantothenate crystals was 99.84%, the content was 99.75%, the methanol residue was 0.012%, and the total yield was 99.67%.

[0066] Example 5

[0067] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the volume ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl) phosphate in the extractant to the volume of the modifier was controlled at 5:5, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0068] After testing, the purity of the D-calcium pantothenate crystals was 86.05%, the content was 90.56%, the methanol residue was 0.013%, and the total yield was 86.32%.

[0069] Example 6

[0070] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the concentrations of tri-n-octylamine and di(2-ethylhexyl) phosphate in the extractant were both 1 g / L, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0071] After testing, the purity of the D-calcium pantothenate crystals was 87.28%, the content was 82.86%, the methanol residue was 0.012%, and the total yield was 91.29%.

[0072] Example 7

[0073] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the concentrations of tri-n-octylamine and di(2-ethylhexyl) phosphate in the extractant were both 80 g / L, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0074] After testing, the purity of the D-calcium pantothenate crystals was 90.63%, the content was 87.88%, the methanol residue was 0.013%, and the total yield was 88.05%.

[0075] Example 8

[0076] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the molar ratio of choline chloride and glucose used in the preparation of the modifier was 5:1, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0077] After testing, the purity of the D-calcium pantothenate crystals was 83.98%, the content was 85.64%, the methanol residue was 0.012%, and the total yield was 86.21%.

[0078] Example 9

[0079] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the molar ratio of choline chloride and glucose used in the preparation of the modifier was 0.5:1, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0080] After testing, the purity of the D-calcium pantothenate crystals was 82.08%, the content was 88.97%, the methanol residue was 0.012%, and the total yield was 86.26%.

[0081] Example 10

[0082] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the back-extraction step S4, the volume ratio of water to n-heptane in the back-extraction agent used was 1:5, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0083] After testing, the purity of the D-calcium pantothenate crystals was 97.97%, the content was 90.54%, the methanol residue was 0.013%, and the total yield was 96.05%.

[0084] Embodiment 11

[0085] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the temperature at which the phase to be extracted and the extractant were stirred was controlled at 40° C. The other conditions were the same as those of Example 1, and D-calcium pantothenate crystals were obtained.

[0086] After testing, the purity of the D-calcium pantothenate crystals was 92.68%, the content was 96.71%, the methanol residue was 0.013%, and the total yield was 88.77%.

[0087] Example 12

[0088] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the stripping step S4, the temperature at which the washed organic phase and the stripping agent were stirred was controlled at 50° C. The other conditions were the same as those of Example 1, and D-calcium pantothenate crystals were obtained.

[0089] After testing, the purity of the D-calcium pantothenate crystals was 93.01%, the content was 94.84%, the methanol residue was 0.012%, and the total yield was 90.11%.

[0090] Comparative Example 1

[0091] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the di(2-ethylhexyl) phosphate in the extractant was replaced by the same weight portion of tri-n-octylamine, and the other conditions were the same as in Example 1, and D-calcium pantothenate crystals were obtained.

[0092] After testing, the purity of the D-calcium pantothenate crystals was 78.56%, the content was 81.24%, the methanol residue was 0.015%, and the total yield was 66.98%.

[0093] Comparative Example 2

[0094] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, the tri-n-octylamine in the extractant was replaced by the same weight portion of di(2-ethylhexyl) phosphate, and the other conditions were the same as those of Example 1, and D-calcium pantothenate crystals were obtained.

[0095] After testing, the purity of the D-calcium pantothenate crystals was 75.85%, the content was 67.56%, the methanol residue was 0.013%, and the total yield was 52.24%.

[0096] Comparative Example 3

[0097] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 1, except that in the extraction step S3, no modifier was added to the extractant. The other conditions were the same as those of Example 1, and D-calcium pantothenate crystals were obtained.

[0098] After testing, the purity of the D-calcium pantothenate crystals was 74.38%, the content was 60.64%, the methanol residue was 0.014%, and the total yield was 65.27%.

[0099] Comparative Example 4

[0100] D-calcium pantothenate was extracted and prepared from the pantothenic acid-containing fermentation broth according to the method of Example 4, except that in the extraction step S3, no modifier was added to the extractant, and the other conditions were the same as those of Example 4, and D-calcium pantothenate crystals were obtained.

[0101] After testing, the purity of the D-calcium pantothenate crystals was 70.52%, the content was 62.03%, the methanol residue was 0.013%, and the total yield was 79.68%.

[0102] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth, characterized in that: The method comprises acidifying a pantothenic acid-containing fermentation broth, removing solids from the obtained acidified fermentation broth, and then extracting the obtained fermentation clear liquid with an extractant, wherein the extractant contains tri-n-octylamine, di(2-ethylhexyl) phosphate and a modifier, wherein the modifier is a reaction product obtained by reacting choline chloride and glucose, and then washing the organic phase obtained by the extraction with water, performing back extraction and calcification to obtain D-calcium pantothenate.

2. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 1, characterized in that: The method for acidifying pantothenic acid-containing fermentation broth to obtain acidified fermentation broth comprises adjusting the pH value of the pantothenic acid-containing fermentation broth to 1.5-2.5, then keeping the broth at 90°C-95°C for 5-30 minutes, and cooling the broth to 20°C-30°C after the keeping, to obtain the acidified fermentation broth.

3. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 1, characterized in that: The method for removing solids from the acidified fermentation broth comprises filtering the acidified fermentation broth using a ceramic membrane, and the obtained permeate is the fermentation clear liquid; preferably, the molecular weight cutoff of the ceramic membrane is 1000 to 10000 Da.

4. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 1, characterized in that: The concentrations of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant are independently 5 g / L to 50 g / L; Preferably, the ratio of the total volume of tri-n-octylamine and di(2-ethylhexyl)phosphate in the extractant to the volume of the modifier is (2-4):(6-8); Preferably, the process of the reaction of choline chloride and glucose comprises stirring choline chloride and glucose at a temperature of 60° C. to 80° C. until they become a transparent liquid and then naturally cooling to room temperature; preferably, the molar ratio of choline chloride to glucose is (1 to 3):1; Preferably, the extraction method is cross-current extraction, and the number of extraction stages is 10 to 15; Preferably, in the cross-current extraction process, each stage of extraction independently comprises stirring and mixing the phase to be extracted with the extractant at 20-30° C. for 0.5-2 h, and then standing for extraction for 10-30 h; Preferably, the volume ratio of the phase to be extracted to the extractant is (0.5-1.5):

1.

5. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 1, characterized in that: The stripping method comprises stirring and mixing the washed organic phase with a stripping agent at 65-75° C. for 1-3 hours, and then standing for stripping for 5-10 hours; preferably, the stripping agent is an aqueous solution of n-heptane, and the volume ratio of water to n-heptane in the stripping agent is 1:(1-2); Preferably, the volume ratio of the organic phase to the stripping agent is 1:(3-8); Preferably, the method further comprises recycling the organic phase obtained by back extraction as an extractant.

6. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 1, characterized in that: The calcification reagent is calcium hydroxide and / or calcium oxide; the mass ratio of the calcification reagent to the pure mass of D-pantothenic acid in the aqueous phase obtained by back extraction is 1:(5-10).

7. The method for extracting and preparing calcium D-pantothenate from a pantothenic acid-containing fermentation broth according to any one of claims 1 to 6, characterized in that: The method also includes decolorizing with activated carbon after calcification, concentrating with a reverse osmosis membrane, dissolving the obtained concentrated solution with methanol, adding crystal seeds for crystallization, and then drying to obtain D-calcium pantothenate crystals.

8. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 7, characterized in that: The amount of activated carbon used is 10% to 20% of the mass of D-calcium pantothenate; Preferably, the decolorization conditions include a temperature of 75°C to 85°C and a time of 1h to 5h; Preferably, the method for extracting and preparing calcium D-pantothenate from pantothenic acid-containing fermentation broth further comprises drying the activated carbon after decolorization and then regenerating and recycling it by microwave radiation.

9. The method for extracting and preparing D-calcium pantothenate from a pantothenic acid-containing fermentation broth according to claim 7, characterized in that: The concentration temperature is 20°C to 30°C; Preferably, the concentration condition is based on increasing the solid content of the concentrated solution to above 80 g / L; Preferably, the volume ratio of the methanol to the concentrated liquid is (2-6):1; Preferably, the drying method is boiling drying; Preferably, the drying conditions include a temperature of 80° C. to 150° C. and a time of 5 min to 30 min.

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