CN115246864A - Method for preparing sialic acid - Google Patents
Method for preparing sialic acid Download PDFInfo
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- CN115246864A CN115246864A CN202210828998.9A CN202210828998A CN115246864A CN 115246864 A CN115246864 A CN 115246864A CN 202210828998 A CN202210828998 A CN 202210828998A CN 115246864 A CN115246864 A CN 115246864A
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- sialic acid
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 56
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 239000002253 acid Substances 0.000 claims abstract description 68
- 239000000706 filtrate Substances 0.000 claims abstract description 51
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 50
- 239000000413 hydrolysate Substances 0.000 claims abstract description 46
- 239000012265 solid product Substances 0.000 claims abstract description 32
- 230000007935 neutral effect Effects 0.000 claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000010438 heat treatment Methods 0.000 claims abstract description 27
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 25
- 239000000843 powder Substances 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 13
- 230000007062 hydrolysis Effects 0.000 claims abstract description 11
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 11
- 108010009736 Protein Hydrolysates Proteins 0.000 claims abstract description 10
- 230000000813 microbial effect Effects 0.000 claims abstract description 10
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 9
- 239000007787 solid Substances 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 8
- 239000007791 liquid phase Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000010907 mechanical stirring Methods 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 5
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 claims description 4
- 229910000020 calcium bicarbonate Inorganic materials 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 239000000049 pigment Substances 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 20
- 239000000047 product Substances 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 7
- 238000001035 drying Methods 0.000 abstract description 6
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 abstract description 5
- 229910001424 calcium ion Inorganic materials 0.000 abstract description 5
- 239000013589 supplement Substances 0.000 abstract description 4
- 239000008346 aqueous phase Substances 0.000 abstract description 2
- 238000001816 cooling Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 15
- 238000000926 separation method Methods 0.000 description 11
- 238000004042 decolorization Methods 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 230000006911 nucleation Effects 0.000 description 6
- 238000010899 nucleation Methods 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 238000006386 neutralization reaction Methods 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000000225 synapse Anatomy 0.000 description 2
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical group CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
- C07H7/02—Acyclic radicals
- C07H7/027—Keto-aldonic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for preparing sialic acid, which comprises the following steps: (1) Filtering polysialic acid fermentation liquor obtained by a microbial fermentation method, collecting filtrate, adding calcium carbonate powder and ethanol into the filtrate, standing, and separating a solid product to obtain polysialic acid. (2) And dissolving the polysialic acid in hydrochloric acid for hydrolysis to obtain hydrolysate. (3) Neutralizing the hydrolysate, decoloring the obtained neutral hydrolysate, concentrating the obtained decolored solution, cooling the obtained concentrated solution in an ice bath, adding ethanol, standing, separating a solid product in an aqueous phase, washing the solid product, and drying under a heating condition in vacuum to obtain the sialic acid solid. The process can improve the extraction rate of the polysialic acid in the fermentation liquor and the extraction rate of the sialic acid in the hydrolysate, and the obtained sialic acid product can supplement calcium ions.
Description
Technical Field
The invention relates to the technical field of sialic acid preparation, in particular to a method for preparing sialic acid.
Background
The sialic acid is also called sialic acid, and the chemical name is N-acetylneuraminic acid, which is a derivative of 9-carbon monosaccharide. Sialic acid can act on brain cell membrane and synapse to improve response speed of brain nerve cell synapse, thereby promoting memory and intelligence development, and has antiviral effect. Currently, sialic acid production methods include natural extraction, chemical synthesis, polymer decomposition, and microbial fermentation. Compared with other centralized methods, the microbial fermentation method can produce polysialic acid in large quantity, and the polysialic acid can be further hydrolyzed and then separated and purified to obtain the sialic acid. The above process includes two steps of extracting polysialic acid dissolved in a fermentation liquid and extracting sialic acid dissolved in a hydrolysate. However, the traditional alcohol precipitation process has the problem of unsatisfactory extraction rate, the extraction rate influences the yield of the target product, and further directly influences the overall practical prospect of the process, for example, if the extraction rate is too low, more target products remain in a liquid phase and cannot be extracted, the product waste is caused, and the cost of the finished product is increased.
Disclosure of Invention
Aiming at the problems, the invention provides a method for preparing sialic acid, which can not only simultaneously improve the extraction rate of polysialic acid in fermentation liquor and the extraction rate of sialic acid in hydrolysate, but also supplement calcium ions to the obtained sialic acid product. In order to achieve the purpose, the invention discloses the following technical scheme:
a method of preparing sialic acid comprising the steps of:
(1) Filtering polysialic acid fermentation liquor obtained by a microbial fermentation method, collecting filtrate, and placing the filtrate in an ice bath so as to keep the filtrate at a low temperature. And then adding nano calcium carbonate powder into the filtrate, performing ultrasonic oscillation, adding ethanol, performing mechanical stirring, standing, and separating a solid product in an aqueous phase to obtain polysialic acid for later use.
(2) And dissolving the polysialic acid in water to form a polysialic acid solution, and then adding hydrochloric acid into the polysialic acid solution to hydrolyze the polysialic acid to obtain a hydrolysate for later use.
(3) And adding a neutralizing agent into the hydrolysate to treat the hydrolysate to be neutral, thereby obtaining neutral hydrolysate. And then carrying out decoloring treatment on the neutral hydrolysate, concentrating the obtained decolored solution after the decoloring treatment is finished, then placing the obtained concentrated solution in an ice bath to cool the concentrated solution to be stable, adding ethanol into the concentrated solution to mechanically stir, then standing the concentrated solution, separating a solid product in a liquid phase after the standing is finished, washing the solid product, and then carrying out vacuum drying under a heating condition to obtain the sialic acid solid.
In a typical embodiment, in the step (1), the content of the calcium carbonate powder added to the filtrate is 0.12 to 1.15g/L. The calcium carbonate powder is dispersed in the filtrate, which is more conducive to nucleation when the polysialic acid is precipitated under the action of ethanol, the difficulty in precipitating the polysialic acid is reduced, and the extraction rate of the polysialic acid is improved.
In a typical embodiment, in the step (1), the volume of the added ethanol is 3.5 to 5 times of the volume of the filtrate, and the standing time is 40 to 60min.
In a preferred embodiment, in step (1), the filtrate is concentrated and then placed in an ice bath for subsequent treatment. Optionally, the filtrate is concentrated to 50-60% of the original volume by heating.
In a typical embodiment, in step (2), the added hydrochloric acid adjusts the polysialic acid solution to a pH value of between 1 and 3.5 to facilitate hydrolysis of the polysialic acid. Optionally, the mass concentration of the hydrochloric acid solution is between 15% and 23%.
In a typical embodiment, in the step (2), the hydrolysis time is 2 to 3 hours, and the hydrolysis is performed under heating at 80 to 85 ℃.
In a typical embodiment, in step (3), the neutralizing agent comprises calcium carbonate and/or calcium bicarbonate, which not only consumes the excess acid solution in the hydrolysate, but also facilitates the conversion to ethanol-insoluble crystalline alcohol in the later process to promote the precipitation of sialic acid in the hydrolysate.
In a typical embodiment, in step (3), a decolorizing agent such as adsorbent resin, activated carbon, diatomite and the like is added into the neutral hydrolysate to adsorb pigments in the hydrolysate, so as to improve the purity and appearance quality of the obtained sialic acid product.
In a typical embodiment, in the step (3), the content of the decoloring agent added into the neutral hydrolysate is 3-5 g/L, the decoloring time is 45-60 min, and the temperature is 50-65 ℃.
In a typical embodiment, in the step (3), the decolorized solution is concentrated by heating to 50-60% of the original volume.
In a typical embodiment, in the step (3), the volume of the ethanol added is 3.5 to 5 times of the volume of the concentrated solution, and the standing time is 35 to 50min.
In a typical embodiment, in the step (3), the heating temperature is 70 to 75 ℃ and the heating time is 1 to 1.5 hours.
Compared with the prior art, the invention has the following beneficial effects: the process of the invention can not only simultaneously improve the extraction rate of the polysialic acid in the fermentation liquor and the extraction rate of the sialic acid in the hydrolysate, but also supplement calcium ions to the obtained sialic acid product. The reason for this is that: firstly, calcium carbonate powder and ethanol are added into filtered polysialic acid fermentation liquor, water in the filtrate is mutually dissolved with the ethanol under the action of the ethanol, so that the concentration of the polysialic acid in the filtrate begins to increase, the polysialic acid begins to be separated out after the maximum solubility is exceeded, and the calcium carbonate powder provides a large number of heterogeneous sites for nucleation of the polysialic acid to accelerate the separation of the polysialic acid. Meanwhile, the existence of the heterogeneous sites can obviously reduce the nucleation energy of the polysialic acid, so that the polysialic acid is easier to crystallize and precipitate, and the extraction rate of the polysialic acid in the filtrate is promoted. When the polysialic acid is further acidolyzed, the calcium carbonate in the polysialic acid is converted into calcium chloride under the action of hydrochloric acid, the calcium chloride reacts with subsequently added ethanol to form insoluble crystalline alcohol which can provide a large number of heterogeneous sites for nucleation of the sialic acid, so that the sialic acid precipitation is accelerated, and the sialic acid extraction rate is improved. The crystalline alcohol decomposes during subsequent further heating to release the ethanol bound therein and is converted there to calcium chloride which provides calcium ion supplementation to the resulting sialic acid product.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. The invention will now be further illustrated by specific examples.
Example 1
A method of making sialic acid comprising the steps of:
(1) Preparing fermentation liquor containing polysialic acid by using a microbial fermentation method, filtering the fermentation liquor in a filter to perform solid-liquid separation, and collecting filtrate for later use.
(2) And (2) putting the container containing the filtrate in an ice bath to reduce the temperature of the filtrate, adding nano calcium carbonate powder (the content of calcium carbonate in the filtrate is 0.13 g/L) into the filtrate after the temperature is reduced to be stable, then carrying out ultrasonic oscillation for 5min to uniformly disperse the added calcium carbonate powder in the filtrate, and then adding ethanol with the mass concentration of 95% and the volume of 4.3 times of the volume of the filtrate under the action of mechanical stirring. Standing for 50min after the completion, and centrifuging the liquid phase at the speed of 9500r/min for 10min after the completion of standing to perform solid-liquid separation to obtain a solid product, namely polysialic acid.
(3) And (3) adding the solid product polysialic acid obtained in the last step into water for dissolving to obtain a polysialic acid solution with the mass concentration of 2.5%. Then, 20% by mass hydrochloric acid was added thereto, and the pH of the polysialic acid solution was adjusted to 3. Then heating in water bath to 85 ℃ for hydrolysis for 2.5 hours to obtain hydrolysate.
(4) And adding calcium carbonate into the hydrolysate for neutralization reaction, and neutralizing the hydrolysate to be neutral to obtain neutral hydrolysate. And then adding diatomite into the neutral hydrolysate for decolorization, wherein the content of the diatomite added into the neutral hydrolysate is 4g/L, the decolorization time is 55min, and the temperature is 60 ℃. Filtering to separate diatomite from liquid phase, and collecting filtrate as decolorizing solution.
(5) Heating and concentrating the obtained decolorized solution to 60% of the original volume, then placing the obtained concentrated solution in an ice bath to cool to be stable, adding ethanol with 4 times of the volume of the concentrated solution while stirring, and standing for 45min after the completion. And after standing, centrifuging the liquid phase at the speed of 9500r/min for 10min to separate a solid product in the liquid phase, washing the solid product with 95% ethanol, and drying the solid product at 75 ℃ for 1.5 hours in vacuum to obtain the sialic acid solid.
Example 2
A method of making sialic acid comprising the steps of:
(1) Preparing fermentation liquor containing polysialic acid by using a microbial fermentation method, filtering the fermentation liquor in a filter to perform solid-liquid separation, collecting filtrate, and heating and concentrating the filtrate to 60% of the original volume to obtain concentrated solution.
(2) And (2) placing a container containing the concentrated solution in an ice bath to reduce the temperature of the concentrated solution, adding nano calcium carbonate powder (the content of calcium carbonate in the filtrate is 0.15 g/L) into the concentrated solution after the concentrated solution is cooled to be stable, then carrying out ultrasonic oscillation for 5min to uniformly disperse the added calcium carbonate powder into the filtrate, and then adding ethanol with the mass concentration of 95% which is 5 times of the volume of the filtrate under the action of mechanical stirring. Standing for 60min after completion, and centrifuging the liquid phase at the speed of 9500r/min for 10min after the standing for solid-liquid separation to obtain a solid product, namely polysialic acid.
(3) And (3) adding the solid product polysialic acid obtained in the last step into water for dissolving to obtain a polysialic acid solution with the mass concentration of 2.5%. Then, hydrochloric acid having a mass concentration of 15% was added thereto, and the pH of the polysialic acid solution was adjusted to 3.5. Then heating in water bath to 85 ℃ for hydrolysis for 2 hours to obtain hydrolysate.
(4) And adding calcium bicarbonate into the hydrolysate for neutralization reaction, and neutralizing the hydrolysate to be neutral to obtain neutral hydrolysate. And then adding active carbon into the neutral hydrolysate for decolorization, wherein the content of the active carbon added into the neutral hydrolysate is 3g/L, the decolorization time is 45min, and the temperature is 65 ℃. Filtering to separate the active carbon in the liquid phase, and collecting the filtrate as decolorized liquid.
(5) Heating and concentrating the obtained decolorized solution to 55% of the original volume, then placing the obtained concentrated solution in an ice bath to cool to be stable, adding ethanol with the volume 3.5 times of the volume of the concentrated solution while stirring, and standing for 35min after the completion. And after standing, centrifuging the liquid phase at the speed of 9500r/min for 10min to separate a solid product in the liquid phase, washing the solid product with 95% ethanol, and drying at 70 ℃ for 1 hour in vacuum to obtain the sialic acid solid.
Example 3
A method of making sialic acid comprising the steps of:
(1) Preparing fermentation liquor containing polysialic acid by using a microbial fermentation method, filtering the fermentation liquor in a filter to perform solid-liquid separation, collecting filtrate, and heating and concentrating the filtrate to 50% of the original volume to obtain concentrated solution.
(2) And (2) placing a container containing the concentrated solution in an ice bath to reduce the temperature of the concentrated solution, adding nano calcium carbonate powder (the content of calcium carbonate in the filtrate is 0.12 g/L) into the concentrated solution after the concentrated solution is cooled to be stable, then carrying out ultrasonic oscillation for 5min to uniformly disperse the added calcium carbonate powder into the filtrate, and then adding ethanol with the mass concentration of 95% and the volume of 3.5 times of the volume of the filtrate under the action of mechanical stirring. Standing for 40min after the completion, and centrifuging the liquid phase at the speed of 9500r/min for 10min after the completion of standing to perform solid-liquid separation to obtain a solid product, namely polysialic acid.
(3) And (3) adding the solid product polysialic acid obtained in the last step into water for dissolving to obtain a polysialic acid solution with the mass concentration of 2.5%. Then, 23% by mass hydrochloric acid was added thereto, and the pH of the polysialic acid solution was adjusted to 1. Then heating to 80 ℃ in water bath for hydrolysis for 3 hours to obtain hydrolysate.
(4) And adding calcium carbonate into the hydrolysate for neutralization reaction, and neutralizing the hydrolysate to be neutral to obtain neutral hydrolysate. And then adding adsorption resin into the neutral hydrolysate for decolorization, wherein the content of the adsorption resin added into the neutral hydrolysate is 5g/L, the decolorization time is 60min, and the temperature is 50 ℃. Filtering to separate the active carbon in the liquid phase, and collecting the filtrate as decolorized liquid.
(5) Heating and concentrating the obtained decolorized solution to 50% of the original volume, then placing the obtained concentrated solution in an ice bath to cool to be stable, adding ethanol with 5 times of the volume of the concentrated solution while stirring, and standing for 50min after the completion. And after standing, centrifuging the liquid phase at the speed of 9500r/min for 10min to separate a solid product in the liquid phase, washing the solid product with 95% ethanol, and drying the solid product at 75 ℃ for 1 hour in vacuum to obtain the sialic acid solid.
Example 4
A method of making sialic acid comprising the steps of:
(1) Preparing fermentation liquor containing polysialic acid by using a microbial fermentation method, filtering the fermentation liquor in a filter to perform solid-liquid separation, and collecting filtrate for later use.
(2) And (3) putting the container containing the filtrate in an ice bath to reduce the temperature of the filtrate, and adding ethanol with the mass concentration of 95% and the volume of 4.3 times that of the filtrate while stirring into the filtrate after the temperature is reduced to be stable. Standing for 50min after the completion, and centrifuging the liquid phase at the speed of 9500r/min for 10min after the completion of standing to perform solid-liquid separation to obtain a solid product, namely polysialic acid.
(3) And (3) adding the solid product polysialic acid obtained in the last step into water for dissolving to obtain a polysialic acid solution with the mass concentration of 2.5%. Then, 20% by mass hydrochloric acid was added thereto, and the pH of the polysialic acid solution was adjusted to 3. Then heating in water bath to 85 ℃ for hydrolysis for 2.5 hours to obtain hydrolysate.
(4) And adding calcium carbonate into the hydrolysate for neutralization reaction, and neutralizing the hydrolysate to be neutral to obtain neutral hydrolysate. And then adding diatomite into the neutral hydrolysate for decolorization, wherein the content of the diatomite added into the neutral hydrolysate is 4g/L, the decolorization time is 55min, and the temperature is 60 ℃. Filtering to separate diatomite from liquid phase, and collecting filtrate as decolorizing liquid.
(5) Heating and concentrating the obtained decolorized solution to 60% of the original volume, then placing the obtained concentrated solution in an ice bath to cool to be stable, adding ethanol with 4 times of the volume of the concentrated solution while stirring, and standing for 45min after the completion. And after standing, centrifuging the liquid phase at the speed of 9500r/min for 10min to separate a solid product in the liquid phase, washing the solid product with 95% ethanol, and drying the solid product at 75 ℃ for 1.5 hours in vacuum to obtain the sialic acid solid.
Example 5
A method of making sialic acid comprising the steps of:
(1) Preparing fermentation liquor containing polysialic acid by using a microbial fermentation method, filtering the fermentation liquor in a filter to perform solid-liquid separation, collecting filtrate, and heating and concentrating the filtrate to 60% of the original volume to obtain concentrated solution.
(2) And (3) putting the container containing the concentrated solution in an ice bath to reduce the temperature of the concentrated solution, and adding ethanol with the mass concentration of 95% and the volume 5 times that of the filtrate into the concentrated solution while stirring after the temperature is reduced to be stable. Standing for 60min after completion, and centrifuging the liquid phase at the speed of 9500r/min for 10min after the standing for solid-liquid separation to obtain a solid product, namely polysialic acid.
(3) And (3) adding the solid product polysialic acid obtained in the last step into water for dissolving to obtain a polysialic acid solution with the mass concentration of 2.5%. Hydrochloric acid at a mass concentration of 15% was then added thereto, and the pH of the polysialic acid solution was adjusted to 3.5. Then heating in water bath to 85 ℃ for hydrolysis for 2 hours to obtain hydrolysate.
(4) And adding calcium bicarbonate into the hydrolysate for neutralization reaction, and neutralizing the hydrolysate to be neutral to obtain neutral hydrolysate. And then adding active carbon into the neutral hydrolysate for decolorization, wherein the content of the active carbon added into the neutral hydrolysate is 3g/L, the decolorization time is 45min, and the temperature is 65 ℃. Filtering to separate the active carbon in the liquid phase, and collecting the filtrate as decolorized liquid.
(5) Heating and concentrating the obtained decolorized solution to 55% of the original volume, then placing the obtained concentrated solution in an ice bath to cool to be stable, adding ethanol with the volume 3.5 times of the volume of the concentrated solution while stirring, and standing for 35min after the completion. And after standing, centrifuging the liquid phase at the speed of 9500r/min for 10min to separate a solid product in the liquid phase, washing the solid product with 95% ethanol, and drying at 70 ℃ for 1 hour in vacuum to obtain the sialic acid solid.
Performance testing
The extraction rate of polysialic acid and the extraction rate of sialic acid in examples 1 to 5 were measured, and the results are shown below.
Polysialic acid, sialic acid extraction rate test result
From the above test results, it can be seen that the processes of examples 1 to 3 can simultaneously improve the extraction rate of polysialic acid in the fermentation broth and the extraction rate of sialic acid in the hydrolysate, and the obtained sialic acid product can also supplement calcium ions, compared to examples 4 and 5. The calcium carbonate powder is added to provide a large number of heterogeneous sites for nucleation of the polysialic acid, so that the precipitation of the polysialic acid is accelerated, and the extraction rate of the polysialic acid is improved. The calcium carbonate is converted into calcium chloride under the action of hydrochloric acid, the calcium chloride reacts with subsequently added ethanol to form insoluble crystalline alcohol which can be precipitated, and the crystalline alcohol can provide a large number of heterogeneous sites for nucleation of sialic acid, so that the precipitation of the sialic acid is accelerated, and the extraction rate of the sialic acid is improved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of preparing sialic acid comprising the steps of:
(1) Filtering polysialic acid fermentation liquor obtained by a microbial fermentation method, collecting filtrate, and placing the filtrate in an ice bath; then adding nano-scale calcium carbonate powder into the filtrate, performing ultrasonic oscillation, adding ethanol, performing mechanical stirring, standing, and separating a solid product in a liquid phase to obtain polysialic acid;
(2) Dissolving the polysialic acid in water to form a polysialic acid solution, and then adding hydrochloric acid into the polysialic acid solution to hydrolyze polysialic acid to obtain a hydrolysate;
(3) Adding a neutralizer into the hydrolysate to treat the hydrolysate to be neutral, thereby obtaining a neutral hydrolysate; and then carrying out decoloring treatment on the neutral hydrolysate, concentrating the obtained decolored solution after the decoloring treatment is finished, then placing the obtained concentrated solution in an ice bath to cool the concentrated solution to be stable, adding ethanol into the concentrated solution to mechanically stir, then standing the concentrated solution, separating a solid product in a liquid phase after the standing is finished, washing the solid product, and then carrying out vacuum drying under a heating condition to obtain the sialic acid solid.
2. The method for preparing sialic acid according to claim 1, wherein the calcium carbonate powder is added to the filtrate in an amount of 0.12 to 1.15g/L in step (1).
3. The method for preparing sialic acid according to claim 1, wherein the ethanol is added in an amount of 3.5 to 5 times the volume of the filtrate in the step (1), and the standing time is 40 to 60min.
4. The method for preparing sialic acid according to claim 1, wherein, in the step (2), the poly-sialic acid solution is adjusted to a pH value of 1-3.5 by the added hydrochloric acid;
preferably, the mass concentration of the hydrochloric acid solution is between 15 and 23 percent;
preferably, in the step (2), the hydrolysis time is 2 to 3 hours, and the hydrolysis is performed under heating at 80 to 85 ℃.
5. The method for preparing sialic acid according to claim 1, wherein, in the step (3), the neutralizing agent comprises calcium carbonate and/or calcium bicarbonate.
6. The method for preparing sialic acid according to claim 1, wherein in the step (3), any one of adsorption resin, activated carbon and diatomite is added into the neutral hydrolysate to adsorb pigment in the hydrolysate;
preferably, in the step (3), the content of the decoloring agent added into the neutral hydrolysate is 3-5 g/L, the decoloring time is 45-60 min, and the temperature is 50-65 ℃.
7. The method for preparing sialic acid according to claim 1, wherein in step (3), the destaining solution is concentrated by heating to 50-60% of the original volume.
8. The method for preparing sialic acid according to claim 1, wherein the ethanol is added in a volume of 3.5 to 5 times the volume of the concentrated solution in the step (3), and the standing time is 35 to 50min.
9. The method for producing sialic acid according to claim 1, wherein the heating temperature is 70 to 75 ℃ and the heating time is 1 to 1.5 hours in step (3).
10. The method for preparing sialic acid according to any one of claims 1 to 9, wherein in step (1), the filtrate is concentrated and then placed in an ice bath for subsequent treatment; preferably, the filtrate is concentrated by heating to 50-60% of the original volume.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046671A (en) * | 2014-06-24 | 2014-09-17 | 东北农业大学 | Fermentation production method of sialic acid |
| CN111386350A (en) * | 2017-11-21 | 2020-07-07 | 詹尼温生物技术有限责任公司 | Method for purifying sialic acid from fermentation broth |
| CN114539333A (en) * | 2022-03-01 | 2022-05-27 | 德元堂(上海)健康科技发展有限公司 | Cyclic production process of sialic acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046671A (en) * | 2014-06-24 | 2014-09-17 | 东北农业大学 | Fermentation production method of sialic acid |
| CN111386350A (en) * | 2017-11-21 | 2020-07-07 | 詹尼温生物技术有限责任公司 | Method for purifying sialic acid from fermentation broth |
| CN114539333A (en) * | 2022-03-01 | 2022-05-27 | 德元堂(上海)健康科技发展有限公司 | Cyclic production process of sialic acid |
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| 黄家贤等译: "现代微生物学实验技术及其应用", vol. 1, 华中科技大学出版社, pages: 385 * |
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Application publication date: 20221028 |