CN118026894A - Separation and purification method of citrulline - Google Patents
Separation and purification method of citrulline Download PDFInfo
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- CN118026894A CN118026894A CN202211418016.5A CN202211418016A CN118026894A CN 118026894 A CN118026894 A CN 118026894A CN 202211418016 A CN202211418016 A CN 202211418016A CN 118026894 A CN118026894 A CN 118026894A
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- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 title claims abstract description 269
- 229960002173 citrulline Drugs 0.000 title claims abstract description 259
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 title claims abstract description 247
- 235000013477 citrulline Nutrition 0.000 title claims abstract description 247
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000000926 separation method Methods 0.000 title claims description 31
- 238000000746 purification Methods 0.000 title claims description 29
- 239000012452 mother liquor Substances 0.000 claims abstract description 113
- 238000002425 crystallisation Methods 0.000 claims abstract description 40
- 230000008025 crystallization Effects 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 39
- 230000007935 neutral effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 79
- 239000012043 crude product Substances 0.000 claims description 59
- 239000000047 product Substances 0.000 claims description 58
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 238000011033 desalting Methods 0.000 claims description 16
- 239000010413 mother solution Substances 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 239000011575 calcium Substances 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 9
- BRPQOXSCLDDYGP-UHFFFAOYSA-N calcium oxide Chemical compound [O-2].[Ca+2] BRPQOXSCLDDYGP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000292 calcium oxide Substances 0.000 claims description 8
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 7
- 238000002834 transmittance Methods 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 6
- 238000004064 recycling Methods 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 2
- 239000000920 calcium hydroxide Substances 0.000 claims description 2
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 2
- 125000004122 cyclic group Chemical group 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000002351 wastewater Substances 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 19
- 150000003863 ammonium salts Chemical class 0.000 description 16
- 238000001816 cooling Methods 0.000 description 16
- 238000002386 leaching Methods 0.000 description 12
- 238000001035 drying Methods 0.000 description 11
- 238000000909 electrodialysis Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 7
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- 108010082340 Arginine deiminase Proteins 0.000 description 5
- 230000001276 controlling effect Effects 0.000 description 5
- 238000004042 decolorization Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 235000009697 arginine Nutrition 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 238000010612 desalination reaction Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 239000000498 cooling water Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
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- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000219109 Citrullus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- RHGKLRLOHDJJDR-SCSAIBSYSA-N D-citrulline Chemical compound OC(=O)[C@H](N)CCCNC(N)=O RHGKLRLOHDJJDR-SCSAIBSYSA-N 0.000 description 1
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- -1 ammonium ions Chemical class 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for separating and purifying citrulline, which comprises the steps of 1) adding Ca 2+ into citrulline crystallization mother liquor to make the pH value of the solution neutral; 2) Decoloring, concentrating, crystallizing and separating the obtained feed liquid to obtain citrulline; and the mother liquor after the citrulline is separated in the step 2) is applied to the step 1), and the mother liquor is recycled, so that the citrulline product with high yield is obtained. The invention can simply and effectively purify and recycle the mother liquor, improves the yield of citrulline, reduces the waste water amount, lightens the environmental pollution and saves the production cost.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and relates to a separation and purification method of citrulline.
Background
L-citrulline is an alpha-amino acid, which was first obtained from watermelon. L-citrulline participates in various metabolic processes in the body, such as arginine-citrulline circulation, and has the functions of increasing vasodilation, improving immune system function, relaxing blood vessels of people, maintaining healthy lung function and the like; citrulline also has anti-hydroxyl protection effect on DNA and enzyme, and can be used as antioxidant, thus being widely applied to the fields of medicine, food, health care products and the like.
At present, the preparation method of the L-citrulline mainly comprises a chemical method, a fermentation method and an enzyme method. The chemical method is to hydrolyze L-arginine under alkaline condition to obtain L-citrulline, which is the only method for industrial production of L-citrulline at present in China, but the production process is difficult to control, the product contains optical enantiomer D-citrulline, which affects the quality of the L-citrulline product, and a large amount of waste water is produced in the production process, thus polluting the environment. The fermentation method is characterized in that the microorganism strain is utilized to directly ferment and metabolize L-citrulline, the yield is high, the purity of the product is high, no toxic substances are generated in the production process of the product, and the like, and the method provides convenience for post-processing of the product, but has the defects of low yield (only 1.7g/L at maximum), high cost for extracting the L-citrulline from fermentation broth, and the like. The enzymatic method is that under the action of Arginine Deiminase (ADI), L-arginine is catalyzed and converted into L-citrulline, and has the advantages of mild production conditions, no toxic substances, less impurities in a conversion system, simple extraction process, less pollution and the like.
In the practical production of the enzymatic method, a large amount of citrulline mother liquor is generated, a large amount of sulfate and ammonium salt are added in the mother liquor besides citrulline, and the mother liquor is directly discarded, so that the problem of environmental pollution is solved, and the whole citrulline production yield is low. Directly concentrating the mother solution to extract citrulline, wherein the obtained citrulline contains a large amount of sulfate, and the product is unqualified; the mother liquor is concentrated and extracted after being treated by adding ion exchange, electrodialysis and the like, so that the preparation cost is increased, and a large amount of wastewater is generated to be treated. Therefore, the citrulline is extracted by adopting a proper citrulline mother liquor treatment mode, the total yield is improved, the mother liquor treatment cost is reduced, and the method is particularly important for actual production.
Disclosure of Invention
The invention aims to provide a separation and purification method of citrulline, which effectively improves the yield and purity of citrulline by combining the citrulline mother liquor impurity removal technology with the recycling technology.
In a first aspect, the invention provides a method for separating and purifying citrulline, which comprises the following steps:
1) Adding Ca 2+ into citrulline crystallization mother liquor, and enabling the pH value of the solution to be neutral;
2) And decoloring, concentrating, crystallizing and separating the obtained feed liquid to obtain citrulline.
In the above separation and purification method, in step 1), ca 2+ is added into citrulline crystallization mother liquor, and then the temperature is raised and stirred until the pH of the solution is neutral;
preferably, the citrulline crystallization mother liquor is citrulline crude mother liquor and/or citrulline crystallization mother liquor.
In the separation and purification method, the material liquid obtained after the crystallization and the citrulline separation in the step 2) can be recycled to the step 1).
In a second aspect, the invention provides a method for separating and purifying citrulline, which comprises the following steps:
S1, carrying out impurity removal pretreatment, concentration, crystallization and separation on an L-citrulline conversion solution to obtain a citrulline crude product and a citrulline crude product mother solution;
s2, recrystallizing the citrulline crude product to obtain citrulline fine product and citrulline crystallization mother liquor;
s3, mixing the citrulline crude mother liquor and the citrulline crystallization mother liquor to obtain a mother liquor mixture, adding Ca 2+ into the mother liquor mixture, heating and stirring until the pH value of the feed liquor is neutral, and then carrying out solid-liquid separation to obtain citrulline desalting mother liquor;
s4, decoloring the citrulline desalting mother solution, concentrating the decolored solution, crystallizing and separating to obtain a citrulline crude product and a citrulline crude product mother solution;
s5, applying the citrulline crude product obtained in the step S5 and the citrulline crude product mother liquor to the steps S2 and S3 respectively, and circularly applying the mother liquor to obtain high-yield citrulline fine products.
In the above separation and purification method, in step S1, the mass content of citrulline in the citrulline crude product is 90-99%, and the citrulline concentration in the citrulline crude product mother liquor is 50-200 g/L.
In the above separation and purification method, in step S1, the impurity removal pretreatment includes the following steps:
1) Performing ceramic membrane filtration on the citrulline conversion solution after sterilization treatment, and collecting Tao Qingye;
2) Carrying out ultrafiltration treatment on Tao Qingye to obtain a supernatant, and decoloring to obtain a first decolored solution;
3) Concentrating, crystallizing and separating the first decolorized solution to obtain a citrulline crude product and a citrulline crude product mother solution.
In the above separation and purification method, in step 1), the L-citrulline conversion solution refers to a conversion solution in which arginine is converted into citrulline under the action of arginine deiminase, and can be converted for 3 to 5 hours under the condition that the pH value is 5.5 to 6.5.
In the separation and purification method, in the step 1), tao Qingye is diluted until the citrulline concentration is less than or equal to 5g/L;
in the step 2), the pressure of ultrafiltration treatment is 0.35-0.45 MPa, and the temperature of feed liquid is controlled to be less than or equal to 40 ℃;
Decolorizing the ultra-clear liquid with active carbon at 45-65 ℃ for 25-40 min until the light transmittance reaches more than 98%; the addition amount of the activated carbon is 0.5-1.5% of the volume of the super clear liquid;
in the step 3), the first decolorized solution is concentrated until the citrulline concentration is 400-450 g/L.
In the separation and purification method, in the step S2, the crude citrulline is redissolved until the citrulline concentration is 140-160g/L, and the redissolution temperature is 55-60 ℃;
further, adding activated carbon with the volume of 0.5% -1% into the heavy solution for decolorization to obtain a second decolorized solution, wherein the decolorization condition is 45-65 ℃ for decolorization for 25-40 min, so that the light transmittance reaches more than 98%, and the second decolorized solution is obtained;
Concentrating the second decolorized solution until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, and crystallizing and separating the second concentrated solution to obtain citrulline refined product and citrulline crystallization mother liquor.
In the above separation and purification method, in step S3, the citrulline crude mother liquor and the citrulline crystallization mother liquor both contain separation (centrifugation) aqueous washing solutions.
In the above separation and purification method, in step S3, the Ca 2+ is at least one of calcium carbonate, calcium oxide, and calcium hydroxide;
The addition amount of Ca 2+ is 0.42-0.5 times, preferably 0.42-0.45 times, of the sulfate radical content in the mother liquor mixture.
In the above separation and purification method, in step S3, the temperature is raised to 50-60 ℃ after adding the Ca 2+, and the mixture is stirred and reacted until the pH of the feed liquid returns to 6.5-7.5.
In the above separation and purification method, in step S4, activated carbon with a volume of 0.5% -1% is added into the citrulline desalination mother liquor, and the mixture is decolorized at 45-65 ℃ for 25-40 min, so that the light transmittance reaches more than 98%.
In the separation and purification method, in the step S4, the decolorized solution is concentrated until the concentration of citrulline is 400-450 g/L;
The temperature of the concentration is 45-60 ℃.
In the separation and purification method, the times of recycling the citrulline crude product obtained in the step S4 to the step S2 are 1-3 times;
the cyclic application frequency of the citrulline crude mother solution obtained in the step S4 to the step S3 is 0-2 times.
The mother liquor is repeatedly used, so that the yield of citrulline is improved, and the production cost is reduced.
The invention has the following beneficial technical effects:
(1) According to the invention, the sulfate in the citrulline mother liquor is converted into calcium sulfate by adding Ca 2+, ammonium ions are removed by heating, the sulfate removal rate is improved to the greatest extent, the technical problem that qualified citrulline cannot be extracted due to a large amount of sulfate and ammonium salts in the citrulline mother liquor is solved, the citrulline is simply and effectively extracted from the mother liquor, and the total yield of the separation and extraction of the citrulline by an enzyme method is remarkably improved by recycling the mother liquor, and compared with an electrodialysis method, the citrulline yield is improved by 1-2%.
(2) According to the invention, ca 2+ is added and the heating treatment is carried out to remove sulfate and ammonium salt in the mother solution, so that the desalting process of the citrulline mother solution is simplified, the citrulline mother solution is changed into valuable, the citrulline mother solution is recycled, the production cost is saved, the wastewater amount is reduced by about 80% compared with an electrodialysis method, and the environmental pollution is reduced.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The method comprises the following steps of performing impurity removal pretreatment on citrulline mother liquor to obtain citrulline crude product 1 and citrulline crude product mother liquor 1:
1. Ceramic membrane filtration and ultrafiltration: performing high-temperature sterilization on citrulline conversion solution at 65 ℃, filtering by using a ceramic membrane, wherein the pore diameter of the ceramic membrane is 100nm, the pressure is 0.4MPa, and collecting Tao Qingye L; adding pure water into Tao Qingye to dilute until the citrulline concentration in the ceramic clear liquid is less than or equal to 5g/L; adding the diluted Tao Qingye L into an ultrafiltration system for ultrafiltration, collecting 150L of supernatant, controlling the pressure to be 0.4MPa and the temperature of the feed liquid to be not more than 40 ℃ to obtain citrulline supernatant.
Wherein, the citrulline conversion solution is enzymatic conversion solution and can be obtained by the following steps: adding arginine deiminase solution into substrate solution containing arginine, wherein the concentration of arginine in the substrate solution is 150g/L, and the volume ratio of the substrate solution to the arginine deiminase solution is 10:1, adjusting the pH value to 6.0 during conversion, and converting for 3 hours at the temperature of 37 ℃ to obtain 5L of citrulline conversion solution.
2. Primary decolorization, concentration and centrifugation: adding 1% of activated carbon (namely, the addition amount of the activated carbon is 1% of the volume of the super clear liquid) into the citrulline super clear liquid, and decoloring for 30min at 60 ℃ to obtain a first decolored liquid; concentrating 150L of the first decolorized solution until the citrulline concentration is 400-450 g/L to obtain a first concentrated solution; centrifuging the first concentrated solution, and leaching the citrulline crude product by using pure water with the weight of 0.5 times of wet product to obtain citrulline crude product 1 and citrulline crude product mother liquor 1, wherein the mass content of citrulline in the citrulline crude product 1 is 97%, and the concentration of citrulline in the citrulline crude product mother liquor 1 is 78g/L.
Based on the citrulline crude product 1 and citrulline crude mother liquor 1 obtained as described above, citrulline purification and re-extraction of examples 1 to 5 and comparative example 1 below were performed.
Example 1
1. Redissolving, secondary decoloring, concentrating and centrifuging: and redissolving the citrulline crude product 1 to 160g/L of citrulline content by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution, wherein the light transmittance reaches 99.4%. Concentrating the second decolorized solution at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the weight of 0.5 times of wet product to obtain citrulline refined product and citrulline crystallization mother liquor.
2. Drying and detecting: drying the citrulline fine product in an oven at 60 ℃, weighing to calculate the total yield, detecting the purity of the citrulline fine product and the sulfate and ammonium salt, and generating about 15-20L/kg of waste water in the whole process.
Example 2
1. Redissolving, secondary decoloring, concentrating, cooling, crystallizing and centrifuging: and redissolving the citrulline crude product 1 to the citrulline content of 150g/L by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution after the light transmission reaches 99.6%. Concentrating the secondary decolorized clear liquid at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the weight of 0.5 times of wet product to obtain citrulline fine product 1 and citrulline crystallization mother liquor.
2. Desalting: mixing the citrulline crude mother liquor 1 and citrulline crystallization mother liquor in a storage tank with stirring and heating functions, and adding calcium oxide according to 0.59 times of the sulfate radical content in the mixed mother liquor, wherein the calcium content in the calcium oxide is equal to 0.421 times of the sulfate radical content in the mother liquor; then heating the feed liquid in the storage tank to 60 ℃, and stirring for reaction until the pH value of the feed liquid returns to 7.2, so as to obtain the treated feed liquid; centrifuging the treated feed liquid to obtain citrulline desalting mother liquor.
3. Decoloring, concentrating, centrifuging and washing: adding active carbon into citrulline desalting mother liquor for decoloring to obtain third decoloring clear liquid; concentrating the third decolorized clear solution until the citrulline concentration is 400-450 g/L to obtain a third concentrated solution; and cooling, crystallizing and centrifuging the third concentrated solution, and leaching the crude citrulline product by using pure water with the volume weight of 0.5 times of the wet product to obtain a citrulline crude product 2 and a citrulline crude product mother liquor 2.
4. And returning the citrulline crude product 2 to the step 1, and mechanically applying the citrulline crude product mother liquor and the crystallization mother liquor for 1 time to obtain citrulline refined product 2.
5. Drying and detecting: drying all citrulline essence (citrulline essence 1 and citrulline essence 2) in oven at 60deg.C, weighing to calculate total yield, and detecting purity and sulfate and ammonium salt content of mixed citrulline essence to obtain citrulline with wastewater volume of 6-9L/kg.
Example 3
1. Redissolving, secondary decoloring, concentrating, cooling, crystallizing and centrifuging: and redissolving the citrulline crude product 1 to the citrulline content of 150g/L by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution after light transmittance reaches 99.8%. Concentrating the secondary decolorized clear liquid at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the weight of 0.5 times of wet product to obtain citrulline fine product 1 and citrulline crystallization mother liquor.
2. Desalting: mixing the citrulline crude mother liquor 1 and citrulline crystallization mother liquor in a storage tank with stirring and heating functions, and adding calcium oxide according to 0.59 times of the sulfate radical content in the mixed mother liquor, wherein the calcium content in the calcium oxide is equal to 0.421 times of the sulfate radical content in the mother liquor; then heating the feed liquid in the storage tank to 60 ℃, and stirring for reaction until the pH value of the feed liquid returns to 7.2, so as to obtain the treated feed liquid; centrifuging the treated feed liquid to obtain citrulline desalting mother liquor.
3. Decoloring, concentrating, centrifuging and washing: adding active carbon into citrulline desalting mother liquor for decoloring to obtain third decoloring clear liquid; concentrating the third decolorized clear solution until the citrulline concentration is 400-450 g/L to obtain a third concentrated solution; and cooling, crystallizing and centrifuging the third concentrated solution, and leaching the citrulline crude product by using pure water with the weight of 0.5 times of the wet product to obtain a citrulline crude product 2 and a citrulline crude product mother liquor 3.
4. And (3) returning the citrulline crude product 2 to the step (1) to obtain a citrulline fine product 2, returning the citrulline crude product mother liquor 2 to the step (2) to continuously carry out mother liquor treatment, and circularly recycling the citrulline crude product mother liquor and the crystallization mother liquor for 2 times to obtain a citrulline fine product 3.
5. Drying and detecting: all the citrulline essence (citrulline essence 1, 2 and 3) is dried in an oven at 60 ℃, the total yield is calculated by weighing, the purity and the sulfate and ammonium salt content of the mixed citrulline essence are detected, and the waste water amount generated in the whole process is 5-7L/kg citrulline.
Example 4
1. Redissolving, secondary decoloring, concentrating, cooling, crystallizing and centrifuging: and redissolving the citrulline crude product 1 to 160g/L of citrulline content by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution after light transmittance reaches 99.3%. Concentrating the secondary decolorized clear liquid at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the weight of 0.5 times of wet product to obtain citrulline refined product 1 and citrulline crystallization mother liquor.
2. Desalting: mixing the citrulline crude mother liquor 1 and citrulline crystallization mother liquor in a storage tank with stirring and heating functions, and adding calcium oxide according to 0.61 times of the sulfate radical content in the mother liquor, wherein the calcium content in the calcium oxide is equal to 0.436 times of the sulfate radical content in the mother liquor; and then heating the feed liquid in the storage tank to 60 ℃, and stirring for reaction until the pH value of the feed liquid returns to 7.4, thus obtaining the treated feed liquid.
3. Centrifuging the treated feed liquid to obtain citrulline desalting mother liquor, and adding active carbon into the citrulline desalting mother liquor to decolorize to obtain third decolorized clear liquid; concentrating the third decolorized clear solution until the citrulline concentration is 400-450 g/L to obtain a third concentrated solution; and cooling, crystallizing and centrifuging the third concentrated solution, and leaching the crude citrulline product by using pure water with the volume weight of 0.5 times of the wet product to obtain a citrulline crude product 2 and a citrulline crude product mother liquor 2.
4. And returning the citrulline crude product 2 to the step 1, returning the citrulline crude product mother liquor 2 to the step 2, and continuing to perform mother liquor treatment, wherein the citrulline crude product mother liquor and the crystallization mother liquor are used for 3 times in total, so as to obtain citrulline refined products 1, 2, 3 and 4.
5. Drying and detecting: drying all the citrulline fine products in an oven at 60 ℃, weighing to calculate the total yield, and detecting the purity and sulfate and ammonium salt content of the mixed citrulline fine products, wherein the total process generates 4-6L/kg of citrulline as waste water.
Comparative example 1
1. Redissolving, secondary decoloring, concentrating, cooling, crystallizing and centrifuging: and redissolving the citrulline crude product 1 to the citrulline content of 150g/L by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution after the light transmission reaches 99.6%. Concentrating the secondary decolorized clear liquid at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the volume of 0.5 times of wet product volume to obtain citrulline refined product 1 and citrulline crystallization mother liquor 1.
2. Pumping the citrulline crude mother liquor 1 and the crystallization mother liquor 1 into a rotary steaming bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 70 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450 g/L to obtain a third concentrated solution; and cooling, crystallizing and centrifuging the third concentrated solution, and leaching the crude citrulline product by using pure water with the volume weight of 0.5 times of the wet product to obtain a citrulline crude product 2 and a citrulline crude product mother liquor 2.
3. And (3) returning the citrulline crude product 2 to the step (1) to prepare citrulline fine product 2 and citrulline crystallization mother liquor 2.
4. Drying and detecting: drying all the citrulline fine products in an oven at 60 ℃, weighing to calculate the total yield, detecting the purity of the mixed citrulline fine products and the content of sulfate and ammonium salt, and generating the citrulline with the wastewater amount of 12-16L/kg in the whole process.
Comparative example 2
1. Redissolving, secondary decoloring, concentrating, cooling, crystallizing and centrifuging: and redissolving the citrulline crude product 1 to the citrulline content of 150g/L by pure water, wherein the redissolving temperature is 60 ℃. Adding 1% active carbon into the heavy solution, decoloring for 30min at 60 ℃, and obtaining a second decoloring solution after the light transmission reaches 99.6%. Concentrating the secondary decolorized clear liquid at 70 ℃ until the concentration of citrulline is 400-450 g/L to obtain a second concentrated solution, slowly stirring the second concentrated solution, cooling to normal temperature for crystallization, centrifuging, and leaching the crystals with pure water with the volume of 0.5 times of wet product volume to obtain citrulline refined product 1 and citrulline crystallization mother liquor.
2. Electrodialysis desalination: pouring the citrulline crude mother liquor 1 and crystallization mother liquor into electrodialysis equipment, regulating the pH value of the mixed mother liquor to 5.92 by dilute sulfuric acid or ammonia water, starting electrodialysis, controlling the pH value to 5.92 and controlling the temperature below 40 ℃; when the conductance of the desalting chamber is less than or equal to 1000 mu s/cm, the electrodialysis is closed, and the desalted water is discharged.
3. Pumping the desalted water into a rotary steaming bottle, then starting a water bath kettle, controlling the temperature of the water bath kettle to be 70 ℃, starting cooling water for condensation, and concentrating until the concentration of citrulline is 400-450 g/L to obtain a third concentrated solution; and cooling, crystallizing and centrifuging the third concentrated solution, and leaching the crude citrulline product by using pure water with the volume weight of 0.5 times of the wet product to obtain a citrulline crude product 2 and a citrulline crude product mother liquor 2.
4. And (3) returning the citrulline crude product 2 to the step (1) to prepare citrulline refined product 2, and returning the citrulline crude product mother liquor 2 to the step (2) to continuously carry out electrodialysis desalination on the mother liquor, wherein the citrulline crude product mother liquor and crystallization mother liquor are used for 3 times in total.
5. Drying and detecting: drying all the citrulline fine products in an oven at 60 ℃, weighing to calculate the total yield, and detecting the purity of the mixed citrulline fine products and the content of sulfate and ammonium salt, wherein the total process generates about 30L/kg of waste water.
The total yield and purity of the citrulline fine products obtained in examples 1 to 5 and comparative example 1, sulfate and ammonium salt were measured and calculated.
The yield is that the concentration of citrulline in the conversion liquid is directly measured, the quantity of citrulline to be obtained is calculated, then extraction and purification are carried out, and after the citrulline refined product is dried, the quantity of the citrulline refined product is checked; the yield was calculated according to the following formula:
yield= (amount of citrulline essence/amount of citrulline to be obtained) ×100%.
The purity is the percentage of citrulline in the product to the product, and the citrulline is detected by using a high-efficiency liquid-phase external standard method.
The sulfate detection method is carried out according to the rule 0802 of the Chinese pharmacopoeia 2015.
The ammonium salt detection method is carried out according to the rule 0808 of the Chinese pharmacopoeia 2015.
The total yield and purity of citrulline refined products, sulfate and ammonium salt results obtained by purification of examples 1-4 and comparative example 1 were as follows:
TABLE 1 Total yield and purity of citrulline essence, sulfate and ammonium salt content
As can be seen from table 1, examples 1 and 2-4 show that the overall yield is significantly lower, and the amount of wastewater produced per unit of citrulline produced is also higher, indicating that the mother liquor is not recycled and the yield is severely affected and the amount of wastewater produced is also greater; examples 2-4 and comparative example 1 show that the mother liquor is directly concentrated and then is partially refined, and the yield is improved, but the purity of the refined product is too low, both sulfate and ammonium salt exceed 5000ppm, the product is unqualified, and the amount of the generated wastewater is increased; examples 2-4 and comparative example 2 show that the mother liquor is recycled by electrodialysis treatment, and the purity and index of sulfate and ammonium salt of the product are basically the same as those of the mother liquor recycled by the method, but the total yield is reduced by 1-2%, the amount of wastewater is increased by 2-3 times, the concentration time is increased by 4-5 times, and the energy consumption is greatly improved, so that the sulfate and ammonium salt in the mother liquor can be effectively removed by adding Ca 2+ and performing heat treatment, and the high yield and the high purity are ensured, and compared with the electrodialysis method, the mother liquor treatment process and the wastewater yield are greatly simplified, and the energy conservation and the environmental protection are improved. And as can be seen from examples 2-4, the citrulline mother liquor can be recycled after the purification treatment, so that the yield and purity of citrulline can be obviously improved.
Claims (10)
1. The method for separating and purifying the citrulline is characterized by comprising the following steps of:
1) Adding Ca 2+ into citrulline crystallization mother liquor, and enabling the pH value of the solution to be neutral;
2) And decoloring, concentrating, crystallizing and separating the obtained feed liquid to obtain citrulline.
2. The separation and purification method according to claim 1, wherein: in the step 1), ca 2+ is added into citrulline crystallization mother liquor, and then the temperature is raised and stirred until the pH value of the solution is neutral;
preferably, the citrulline crystallization mother liquor is citrulline crude mother liquor and/or citrulline crystallization mother liquor.
3. The separation and purification method according to claim 1 or 2, characterized in that: and 2) crystallizing and separating citrulline, and recycling the obtained feed liquid to the step 1).
4. A method for separating and purifying citrulline comprises the following steps:
s1, removing impurities from citrulline conversion liquid, pre-treating, concentrating, crystallizing and separating to obtain a citrulline crude product and a citrulline crude product mother liquor;
s2, recrystallizing the citrulline crude product to obtain citrulline fine product and citrulline crystallization mother liquor;
s3, mixing the citrulline crude mother liquor and the citrulline crystallization mother liquor to obtain a mother liquor mixture, adding Ca 2+ into the mother liquor mixture, heating and stirring until the pH value of the feed liquor is neutral, and then carrying out solid-liquid separation to obtain citrulline desalting mother liquor;
s4, decoloring the citrulline desalting mother solution, concentrating the decolored solution, crystallizing and separating to obtain a citrulline crude product and a citrulline crude product mother solution;
S5, applying the citrulline crude product obtained in the step S4 and the citrulline crude product mother liquor to the steps S2 and S3 respectively, and recycling the mother liquor to obtain high-yield citrulline fine products.
5. The separation and purification method according to claim 4, wherein: in step S1, the impurity removal pretreatment includes the following steps:
1) Sterilizing citrulline conversion solution, filtering with ceramic membrane, and collecting Tao Qingye;
2) Performing ultrafiltration treatment on Tao Qingye to obtain a supernatant, and decoloring to obtain a first decolored solution;
3) Concentrating, crystallizing and separating the first decolorized solution to obtain a citrulline crude product and a citrulline crude product mother solution;
preferably, in the step 1), tao Qingye is diluted to the citrulline concentration of less than or equal to 5g/L.
6. The separation and purification method according to any one of claims 4 to 5, wherein: in the step S2, the crude citrulline is redissolved until the citrulline concentration is 140-160g/L;
Preferably, the redissolution temperature is 55-60 ℃.
7. The separation and purification method according to any one of claims 4 to 6, wherein: in the step S2, adding 0.5-1% of activated carbon by volume into the heavy solution, and decoloring for 25-40 min at 45-65 ℃ to ensure that the light transmittance reaches more than 98% to obtain a second decolored solution;
Concentrating, crystallizing and separating the second decolorized solution to obtain citrulline refined product and citrulline crystallization mother liquor;
preferably, the second decolorized solution is concentrated to a citrulline concentration of 400-450 g/L.
8. The separation and purification method according to any one of claims 4 to 7, wherein: in step S3, the Ca 2+ is at least one of calcium carbonate, calcium oxide, and calcium hydroxide;
preferably, the addition amount of the Ca 2+ is 0.42 to 0.5 times of the sulfate radical content in the mother liquor mixture;
More preferably, ca 2+ is added, then the temperature is raised to 50-60 ℃ and the mixture is stirred for reaction, so that the pH of the feed liquid is returned to 6.5-7.5.
9. The separation and purification method according to any one of claims 4 to 8, wherein: in the step S4, adding 0.5-1% of active carbon by volume into the citrulline desalting mother liquor, and decoloring for 25-40 min at 45-65 ℃ to ensure that the light transmission reaches more than 98%;
preferably, the decolorized solution is concentrated to a citrulline concentration of 400-450 g/L.
10. The separation and purification method according to any one of claims 4 to 9, wherein:
the times of circularly applying the citrulline crude product obtained in the step S4 to the step S2 are 1-3 times;
the cyclic application frequency of the citrulline crude mother solution obtained in the step S4 to the step S3 is 0-2 times.
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