CN115232178A - Process for extracting sialic acid from fermentation liquor and application of process - Google Patents
Process for extracting sialic acid from fermentation liquor and application of process Download PDFInfo
- Publication number
- CN115232178A CN115232178A CN202210993063.6A CN202210993063A CN115232178A CN 115232178 A CN115232178 A CN 115232178A CN 202210993063 A CN202210993063 A CN 202210993063A CN 115232178 A CN115232178 A CN 115232178A
- Authority
- CN
- China
- Prior art keywords
- acid
- ethanol
- liquid phase
- sialic acid
- ferroferric oxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 53
- 230000004151 fermentation Effects 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 47
- 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 44
- 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 44
- 230000008569 process Effects 0.000 title claims abstract description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 112
- 239000002253 acid Substances 0.000 claims abstract description 61
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 47
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000007791 liquid phase Substances 0.000 claims abstract description 33
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000000413 hydrolysate Substances 0.000 claims abstract description 27
- 239000002244 precipitate Substances 0.000 claims abstract description 25
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 16
- 229940087562 sodium acetate trihydrate Drugs 0.000 claims abstract description 16
- 238000001027 hydrothermal synthesis Methods 0.000 claims abstract description 15
- 239000006247 magnetic powder Substances 0.000 claims abstract description 15
- 238000010438 heat treatment Methods 0.000 claims abstract description 14
- 238000005406 washing Methods 0.000 claims abstract description 12
- 239000013078 crystal Substances 0.000 claims abstract description 11
- 230000000813 microbial effect Effects 0.000 claims abstract description 10
- 230000007062 hydrolysis Effects 0.000 claims abstract description 8
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 7
- 239000002245 particle Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims description 25
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 12
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 12
- 229910001447 ferric ion Inorganic materials 0.000 claims description 12
- 229910001448 ferrous ion Inorganic materials 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 239000012265 solid product Substances 0.000 claims description 6
- 230000000274 adsorptive effect Effects 0.000 claims description 4
- 238000004042 decolorization Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 2
- 229920005989 resin Polymers 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- 239000005909 Kieselgur Substances 0.000 claims 1
- 239000003463 adsorbent Substances 0.000 claims 1
- 239000003712 decolorant Substances 0.000 claims 1
- 230000009471 action Effects 0.000 abstract description 10
- 238000000605 extraction Methods 0.000 abstract description 8
- 239000007787 solid Substances 0.000 abstract description 6
- 238000010899 nucleation Methods 0.000 abstract description 3
- 230000006911 nucleation Effects 0.000 abstract description 3
- 108010009736 Protein Hydrolysates Proteins 0.000 abstract 1
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 238000006386 neutralization reaction Methods 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 description 22
- 230000007935 neutral effect Effects 0.000 description 15
- 238000001914 filtration Methods 0.000 description 9
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 6
- 229960002089 ferrous chloride Drugs 0.000 description 6
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 6
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004251 human milk Anatomy 0.000 description 2
- 235000020256 human milk Nutrition 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004641 brain development Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- 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
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a process for extracting sialic acid from fermentation liquor, which comprises the following steps: s1, performing solid-liquid separation on fermentation liquor containing polysialic acid obtained by a microbial fermentation method, and then adding ferroferric oxide magnetic powder into the obtained liquid phase; adding ethanol to precipitate the crystal, and separating out the precipitate to obtain polysialic acid. And S2, adding hydrochloric acid into the aqueous solution of the polysialic acid for hydrolysis, and adding ferroferric oxide for neutralization after the hydrolysis is finished to obtain hydrolysate. And S3, decoloring the hydrolysate, concentrating, adding ethanol, separating out precipitates, washing and drying to obtain sialic acid. And S4, heating the liquid phase after the precipitate is separated to separate out ethanol, adding sodium acetate trihydrate and ethylene glycol into the residual liquid phase for hydrothermal reaction, and separating out solids in the reaction liquid to obtain ferroferric oxide particles. The process uses ferroferric oxide magnetic powder as an auxiliary extracting agent, and realizes better extraction of polysialic acid in fermentation liquor under the combined action of a micro magnetic field and a heterogeneous nucleation interface provided by the auxiliary extracting agent.
Description
Technical Field
The invention relates to the field of sialic acid preparation by a fermentation method, in particular to a process for extracting sialic acid from fermentation liquor and application of the process.
Background
Sialic acid plays an important role in regulating the physiological function of a human body by participating in the physiological function of the cell surface, is one of components of breast milk, and is beneficial to the brain development of infants in a growth period. Sialic acid is widely distributed in the natural world, and besides the breast milk, the sialic acid content in the cubilose can reach 7-12%, so that the cubilose becomes a heavy-duty raw material for extracting sialic acid, but the yield of the cubilose is very low, and the production requirement of sialic acid cannot be met, so that the sialic acid prepared by a chemical process becomes a feasible choice. Through development for many years, sialic acid can be prepared by a chemical synthesis method, an enzymatic synthesis method, a microbial fermentation method and the like, and compared with other methods, the microbial fermentation method has the advantages of short production period, high yield, simplicity, rich raw material sources, low price, high economic benefit and the like. However, in the process of extracting polysialic acid, the currently widely used alcohol precipitation method has the problem that part of polysialic acid remains in the fermentation broth and cannot be extracted, so that a method with better extraction effect needs to be researched.
Disclosure of Invention
Aiming at the problems, the invention provides a process for extracting sialic acid from fermentation liquor and application of the process. In order to achieve the purpose, the invention discloses the following technical scheme:
in a first aspect of the invention, a process for extracting sialic acid from a fermentation broth is disclosed, comprising:
s1, performing solid-liquid separation on fermentation liquor containing polysialic acid obtained by a microbial fermentation method, adding ferroferric oxide magnetic powder into the obtained liquid phase, and stirring to disperse the powder in the liquid phase. Adding ethanol under stirring, and stirring for a while. And finally, standing the mixture until a crystal is precipitated, and separating the precipitate to obtain the polysialic acid.
S2, dissolving the polysialic acid in water to form a polysialic acid solution, then adding hydrochloric acid into the solution to hydrolyze the polysialic acid, and adding ferroferric oxide to neutralize redundant hydrochloric acid to obtain a hydrolysate.
And S3, adding an adsorptive decolorizing agent into the hydrolysate for decolorizing, concentrating the obtained decolorized solution after the completion, adding ethanol into the obtained concentrated solution, uniformly stirring, standing to precipitate crystals after the completion, separating the precipitate, washing and drying to obtain the sialic acid.
And S4, heating the liquid phase obtained after the precipitate is separated in the step S3 to separate ethanol in the liquid phase, condensing and collecting the ethanol, adding sodium acetate trihydrate and ethylene glycol into the residual liquid phase, performing hydrothermal reaction, and separating a solid product in a reaction liquid after the hydrothermal reaction is completed to obtain ferroferric oxide particles which are used in the liquid phase obtained in the step S1.
Preferably, in the step S1, the addition amount of the ferroferric oxide magnetic powder in the liquid phase is 0.17-0.22 g/L. More preferably, the ferroferric oxide magnetic material is micron-sized powder, and the micron-sized powder is dispersed in a liquid phase containing polysialic acid and is matched with ethanol, so that the extraction effect of the polysialic acid can be effectively improved.
Preferably, in step S1, the volume ratio of ethanol to liquid phase is 1:3.35 to 4.7, and the standing time is 30 to 55min.
Preferably, in step S2, the pH of the polysialic acid solution after the addition of the hydrochloric acid is maintained between 1 and 3.2, the hydrolysis time is 2 to 3 hours, and the hydrolysis temperature is 75 to 85 ℃. The polysialic acid can be hydrolyzed and decomposed into sialic acid under acidic conditions. More preferably, the concentration of hydrochloric acid is 20 to 25wt.%.
Preferably, in step S3, the adsorptive decoloring agent includes any one of adsorption resin, activated carbon, diatomite, and the like. More preferably, the content of the decolorizer in the hydrolysate is 3-5 g/L, the decolorization time is 40-55 min, and the temperature is 50-65 ℃.
Preferably, in step S3, the destaining solution is heated and concentrated to 55% or less, preferably 45 to 50% of the original volume.
Preferably, in step S3, the volume ratio of ethanol to the concentrated solution is 1:3.35 to 4.7, and the standing time is 40 to 55min.
Preferably, in step S3, the precipitate is washed with ethanol and then dried by any one of vacuum drying or freeze drying.
Preferably, in step S4, the heating temperature is 80 to 85 ℃.
Preferably, in step S4, the ratio of the total molar amount of ferric ions and ferrous ions in the liquid phase to the molar amount of sodium acetate trihydrate is 5.8 to 6.3:50 to 54, wherein the molar ratio of the ferric ions to the ferrous ions is 1.75 to 2.1.
Preferably, in step S4, the mass-to-volume ratio of sodium acetate trihydrate to ethylene glycol is 7 to 7.5g:50 to 70ml.
Preferably, in step S4, the temperature of the hydrothermal reaction is 160 to 200 ℃ and the time is 3 to 5 hours.
In the second aspect of the invention, the application of the process for extracting sialic acid from fermentation liquor in the fields of biology, medicine, chemical industry and the like is disclosed.
Compared with the prior art, the beneficial effects obtained by the invention at least comprise the following parts: the process of the invention uses ferroferric oxide magnetic powder as an auxiliary extracting agent, and realizes better extraction of polysialic acid in fermentation liquor under the combined action of a micro magnetic field and a heterogeneous nucleation interface provided by the auxiliary extracting agent. The method comprises the following specific steps:
(1) According to the method, ferroferric oxide magnetic powder is added into fermentation liquor, polysialic acid dissolved in the fermentation liquor is gradually crystallized and precipitated after ethanol is added, and at the initial stage of crystallization and precipitation, the ferroferric oxide magnetic powder dispersed in the fermentation liquor containing the polysialic acid is used as a heterogeneous interface to accelerate nucleation and crystallization of the polysialic acid, reduce the difficulty of separating the polysialic acid from the fermentation liquor and enable the polysialic acid to be more easily separated. Meanwhile, each ferroferric oxide magnetic particle is equivalent to a micro magnetic field, and the generated magnetic force can effectively weaken hydrogen bonds between polysialic acid and water molecules, so that the polysialic acid can be more easily separated from fermentation liquor to be nucleated, crystallized, grown and separated out, and the extraction rate of the polysialic acid is improved.
(2) After the obtained polysialic acid containing ferroferric oxide is further hydrolyzed, the ferroferric oxide is converted into ferric chloride and ferrous chloride under the action of hydrochloric acid, sialic acid in hydrolysate is separated out and precipitated in the further alcohol precipitation process, and the ferric chloride and the ferrous chloride cannot be separated out along with the sialic acid due to the fact that the ferric chloride and the ferrous chloride can be dissolved in water and ethanol, so that the sialic acid is separated from the sialic acid.
(3) The invention also utilizes the characteristic that the molar ratio of ferric ions to ferrous ions obtained by converting the ferroferric oxide into ferric chloride and ferrous chloride under the action of hydrochloric acid is 2, and the ferroferric oxide can be directly converted into the ferroferric oxide again under the action of sodium acetate trihydrate and ethylene glycol without adjustment, thereby realizing the cyclic utilization of the ferroferric oxide.
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 embodiments.
Example 1
A process for extracting sialic acid from a fermentation broth comprising the steps of:
s1, filtering fermentation liquor containing polysialic acid obtained by a microbial fermentation method in a filter, removing solid matters in the fermentation liquor, standing, and taking supernatant. According to the condition that the volume of the supernatant and the mass of ferroferric oxide are 1L: adding micron ferroferric oxide magnetic powder into the supernatant according to the proportion of 0.2g, then stirring at a high speed to disperse the ferroferric oxide magnetic powder into the supernatant, then continuously adding ethanol (the mass fraction is 95%) which is 4 times of the volume of the supernatant under the stirring action, and slowly stirring for 20min after the ethanol is added. Standing for 45min to precipitate, centrifuging (speed 9500r/min for 10 min) to separate out precipitate, washing with ethanol, and vacuum drying at 80 deg.C for 1 hr to obtain polysialic acid.
S2, dissolving the polysialic acid in water to form a polysialic acid solution with the mass fraction of 2%, adding hydrochloric acid with the concentration of 20wt.% into the solution, adjusting the pH of the polysialic acid solution to 2.5, and hydrolyzing the polysialic acid at 80 ℃ for 3 hours. And after the completion, continuously adding ferroferric oxide into the obtained hydrolysate to neutralize redundant hydrochloric acid, thus obtaining the neutral hydrolysate.
And S3, adding activated carbon into the neutral hydrolysate according to the proportion of 4g/L, and keeping the neutral hydrolysate at 60 ℃ for 50min for decolorization. And filtering to remove the active carbon in the neutral hydrolysate to obtain the decolorized solution. And then heating and concentrating the decolored solution to 50% of the original volume to obtain a concentrated solution. And (2) cooling the concentrated solution to room temperature, adding ethanol (the mass fraction is 95%) with the volume of 4.5 times of the concentrated solution, stirring, standing for 50min after the completion to precipitate crystals, then centrifuging (the speed is 9500r/min, the time is 10 min) to separate the precipitates, washing with ethanol, and drying in vacuum at 80 ℃ for 1 hour to obtain sialic acid.
And S4, heating the liquid phase obtained after the precipitate is separated in the step S3 at 80 ℃ to separate out ethanol, condensing and collecting the ethanol for recycling. Then, the weight ratio of 7.2g:55ml of sodium acetate trihydrate is dissolved in ethylene glycol, the solution obtained is added to the liquid phase remaining after the separation of the ethanol, and the ratio of the total molar amount of ferric ions and ferrous ions in the liquid phase remaining to the molar amount of sodium acetate trihydrate is 6:52. after the completion, hydrothermal reaction is carried out in a reaction kettle at 185 ℃ for 4 hours. And centrifuging (the speed is 9500r/min, the time is 10 min) after the completion to separate a black solid product in the hydrothermal reaction liquid, thereby obtaining the ferroferric oxide particles.
Example 2
A process for extracting sialic acid from a fermentation broth comprising the steps of:
s1, filtering fermentation liquor containing polysialic acid obtained by a microbial fermentation method in a filter, removing solid matters in the fermentation liquor, standing and taking supernatant. According to the volume of the supernatant and the mass of ferroferric oxide of 1L: adding micron ferroferric oxide magnetic powder into the supernatant according to the proportion of 0.17g, then stirring at a high speed to disperse the ferroferric oxide magnetic powder into the supernatant, then continuously adding ethanol (the mass fraction is 95%) which is 4.7 times of the volume of the supernatant under the stirring action, and slowly stirring for 25min after the ethanol is added. Standing for 30min to precipitate, centrifuging (speed 9500r/min for 10 min) to separate out precipitate, washing with ethanol, and vacuum drying at 80 deg.C for 1 hr to obtain polysialic acid.
S2, dissolving the polysialic acid in water to form a polysialic acid solution with the mass fraction of 2%, adding hydrochloric acid with the concentration of 20wt.% into the solution, adjusting the pH of the polysialic acid solution to 3.2, and hydrolyzing the polysialic acid at 85 ℃ for 2 hours. And after the completion, continuously adding ferroferric oxide into the obtained hydrolysate to neutralize redundant hydrochloric acid, thus obtaining the neutral hydrolysate.
And S3, adding diatomite into the neutral hydrolysate according to the proportion of 5g/L, and keeping the neutral hydrolysate at the temperature of 50 ℃ for 55min for carrying out decoloration treatment. Filtering to remove diatomite in the neutral hydrolysate to obtain the decolorized solution. And then heating and concentrating the decolored solution to 55 percent of the original volume to obtain a concentrated solution. And (3) cooling the concentrated solution to room temperature, adding ethanol (the mass fraction is 95%) of which the volume is 3.35 times of that of the concentrated solution, stirring, standing for 40min after the concentrated solution is finished to precipitate crystals, then centrifuging (the speed is 9500r/min, the time is 10 min) to separate out the precipitates, washing with ethanol, and drying in vacuum at the temperature of 80 ℃ for 1 hour to obtain the sialic acid.
And S4, heating the liquid phase after the precipitate is separated in the step S3 at 85 ℃ to separate out ethanol in the liquid phase, and condensing and collecting the ethanol for recycling. Then, the weight ratio of 7.0g:50ml of sodium acetate trihydrate is dissolved in ethylene glycol, the solution obtained is added to the liquid phase remaining after the separation of the ethanol, and then the molar ratio of ferric ions to ferrous ions is adjusted to 1.75:1, and the molar amount ratio of the total molar amount of ferric ions and ferrous ions to the molar amount of sodium acetate trihydrate is 5.8:50. after the completion, hydrothermal reaction is carried out in a reaction kettle at the temperature of 160 ℃ for 5 hours. And centrifuging (the speed is 9500r/min, the time is 10 min) after the completion to separate a black solid product in the hydrothermal reaction liquid, thereby obtaining the ferroferric oxide particles.
Example 3
A process for extracting sialic acid from a fermentation broth comprising the steps of:
s1, filtering fermentation liquor containing polysialic acid obtained by a microbial fermentation method in a filter, removing solid matters in the fermentation liquor, standing, and taking supernatant. According to the condition that the volume of the supernatant and the mass of ferroferric oxide are 1L: adding micron ferroferric oxide magnetic powder into the supernatant according to the proportion of 0.22g, then stirring at a high speed to disperse the ferroferric oxide magnetic powder into the supernatant, then continuously adding ethanol (the mass fraction is 95%) which is 3.35 times of the volume of the supernatant under the stirring action, and slowly stirring for 25min after the ethanol is added. Standing for 55min to precipitate crystal, centrifuging (speed 9500r/min for 10 min) to separate out the precipitate, washing with ethanol, and vacuum drying at 80 deg.C for 1 hr to obtain polysialic acid.
S2, dissolving the polysialic acid in water to form a polysialic acid solution with the mass fraction of 2%, then adding hydrochloric acid with the concentration of 25wt.% into the solution, adjusting the pH of the polysialic acid solution to 1, and performing hydrolysis of the polysialic acid at 75 ℃ for 3 hours. And after the completion, continuously adding ferroferric oxide into the obtained hydrolysate to neutralize redundant hydrochloric acid, thus obtaining the neutral hydrolysate.
And S3, adding diatomite into the neutral hydrolysate according to the proportion of 3g/L, and keeping the mixture at 65 ℃ for 40min for carrying out decolorization treatment. Filtering to remove diatomite in the neutral hydrolysate to obtain the decolorized solution. And then heating and concentrating the decolored solution to 45% of the original volume to obtain a concentrated solution. And (3) cooling the concentrated solution to room temperature, adding ethanol (the mass fraction is 95%) of which the volume is 4.7 times of that of the concentrated solution, stirring, standing for 40min after the concentrated solution is finished to precipitate crystals, then centrifuging (the speed is 9500r/min, the time is 10 min) to separate out the precipitates, washing with ethanol, and drying in vacuum at the temperature of 80 ℃ for 1 hour to obtain the sialic acid.
And S4, heating the liquid phase after the precipitate is separated in the step S3 at 85 ℃ to separate out ethanol in the liquid phase, and condensing and collecting the ethanol for recycling. Then, the weight ratio of 7.5g:70ml of sodium acetate trihydrate is dissolved in ethylene glycol, the obtained solution is added to the liquid phase remaining after the separation of the ethanol, and then ferric chloride and ferrous chloride are added to adjust the molar ratio of ferric ions to ferrous ions to 2.1:1, and the molar amount ratio of the total molar amount of ferric ions and ferrous ions to the molar amount of sodium acetate trihydrate is 6.3:54. after the completion, hydrothermal reaction is carried out in a reaction kettle at the temperature of 200 ℃ for 3h. And centrifuging (the speed is 9500r/min, the time is 10 min) after the completion to separate a black solid product in the hydrothermal reaction liquid, so as to obtain the ferroferric oxide particles.
Example 4
A process for extracting polysialic acid from a fermentation broth, comprising the steps of: filtering fermentation liquor containing polysialic acid obtained by microbial fermentation in a filter to remove solid substances in the fermentation liquor, standing and taking supernatant. Then, ethanol (mass fraction: 95%) 4 times of the volume of the supernatant was added under stirring, and the mixture was slowly stirred for 20min after the addition of the ethanol was completed. Standing for 45min to precipitate, centrifuging (speed 9500r/min for 10 min) to separate out precipitate, washing with ethanol, and vacuum drying at 80 deg.C for 1 hr to obtain polysialic acid.
Example 5
A process for extracting sialic acid from a fermentation broth comprising the steps of:
s1, filtering fermentation liquor containing polysialic acid obtained by a microbial fermentation method in a filter, removing solid matters in the fermentation liquor, standing and taking supernatant. According to the volume of the supernatant and the mass of ferroferric oxide of 1L: adding demagnetized micron ferroferric oxide powder into the supernatant according to the proportion of 0.17g, then stirring at a high speed to disperse the ferroferric oxide powder into the supernatant, then continuously adding ethanol (the mass fraction is 95%) with the volume 4.7 times of that of the supernatant under the stirring action, and slowly stirring for 25min after the ethanol is added. Then standing for 30min until the crystal is precipitated, centrifuging (speed 9500r/min, time 10 min) to separate out the precipitate, washing with ethanol, and vacuum drying at 80 deg.C for 1 hr to obtain polysialic acid.
S2, dissolving the polysialic acid in water to form a polysialic acid solution with the mass fraction of 2%, adding 20wt.% hydrochloric acid into the solution, adjusting the pH of the polysialic acid solution to 3.2, and performing hydrolysis of the polysialic acid at 85 ℃ for 2 hours. And after the completion, continuously adding ferroferric oxide into the obtained hydrolysate to neutralize redundant hydrochloric acid, thus obtaining the neutral hydrolysate.
And S3, adding diatomite into the neutral hydrolysate according to the proportion of 5g/L, and keeping the neutral hydrolysate at the temperature of 50 ℃ for 55min for carrying out decoloration treatment. Filtering to remove diatomite in the neutral hydrolysate to obtain the decolorized solution. And then heating and concentrating the decolored solution to 55 percent of the original volume to obtain a concentrated solution. And (3) cooling the concentrated solution to room temperature, adding ethanol (the mass fraction is 95%) of which the volume is 3.35 times of that of the concentrated solution, stirring, standing for 40min after the concentrated solution is finished to precipitate crystals, then centrifuging (the speed is 9500r/min, the time is 10 min) to separate out the precipitates, washing with ethanol, and drying in vacuum at the temperature of 80 ℃ for 1 hour to obtain the sialic acid.
And S4, heating the liquid phase after the precipitate is separated in the step S3 at 85 ℃ to separate out ethanol, condensing and collecting the ethanol for recycling. Then, the weight ratio of 7.0g:50ml of sodium acetate trihydrate is dissolved in ethylene glycol, the solution obtained is added to the liquid phase remaining after the separation of the ethanol, and then the molar ratio of ferric ions to ferrous ions is adjusted to 1.75:1, and the molar amount ratio of the total molar amount of ferric ions and ferrous ions to the molar amount of sodium acetate trihydrate is 5.8:50. after the completion, hydrothermal reaction is carried out in a reaction kettle at the temperature of 160 ℃ for 5 hours. And centrifuging (the speed is 9500r/min, the time is 10 min) after the completion to separate a black solid product in the hydrothermal reaction liquid, so as to obtain the ferroferric oxide particles.
The polysialic acid extraction rate and the purity of sialic acid in examples 1 to 5 were measured, and the results are as follows.
From the test results of examples 1 to 3 and examples 4 to 5, it can be seen that the extraction rates of polysialic acid are significantly balanced when no ferroferric oxide powder is added during the extraction of polysialic acid or when demagnetized ferroferric oxide powder is added. And the obtained sialic acid has high purity, which shows that the process can also effectively realize the separation of ferric chloride and ferrous chloride formed by ferroferric oxide under the action of hydrochloric acid from the sialic acid, and ensures the purity of the sialic acid.
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 process for extracting sialic acid from a fermentation broth, comprising the steps of:
s1, performing solid-liquid separation on fermentation liquor containing polysialic acid obtained by a microbial fermentation method, adding ferroferric oxide magnetic powder into the obtained liquid phase, and stirring to disperse the powder in the liquid phase; adding ethanol under the stirring condition, and continuously stirring for a period of time after the completion; separating the precipitate after the crystal precipitates to obtain polysialic acid;
s2, dissolving the polysialic acid in water to form a polysialic acid solution, then adding hydrochloric acid into the solution to hydrolyze polysialic acid, and adding ferroferric oxide to neutralize redundant hydrochloric acid to obtain a hydrolysate;
s3, adding an adsorptive decoloring agent into the hydrolysate for decoloring, concentrating the obtained decolored solution after the decoloring treatment is finished, adding ethanol into the obtained concentrated solution, uniformly stirring, standing to precipitate crystals after the decoloring treatment is finished, separating the precipitates, washing and drying to obtain sialic acid;
and S4, heating the liquid phase obtained after the precipitate is separated in the step S3 to separate ethanol in the liquid phase, condensing and collecting the ethanol, adding sodium acetate trihydrate and ethylene glycol into the residual liquid phase, performing hydrothermal reaction, and separating a solid product in a reaction liquid after the hydrothermal reaction is completed to obtain ferroferric oxide particles which are used in the liquid phase obtained in the step S1.
2. The process for extracting sialic acid from fermentation broth according to claim 1, wherein in step S1, the amount of ferroferric oxide magnetic powder added to the liquid phase is 0.17-0.22 g/L; preferably, the ferroferric oxide is micron-sized powder.
3. The process for extracting sialic acid from fermentation broth according to claim 1, wherein the volume ratio of ethanol to liquid phase in step S1 is 1:3.35 to 4.7, and the standing time is 30 to 55min.
4. The process for extracting sialic acid from fermentation broth according to claim 1, wherein in step S2, the pH of the polysialic acid solution after adding the hydrochloric acid is maintained between 1 and 3.2, the hydrolysis time is 2-3 h, and the hydrolysis temperature is 75-85 ℃; preferably, the concentration of the hydrochloric acid is 20 to 25wt.%.
5. The process for extracting sialic acid from fermentation broth of claim 1, wherein in step S3, the adsorptive decolorant comprises any one of an adsorbent resin, activated carbon, diatomaceous earth; preferably, the content of the decolorizer in the hydrolysate is 3-5 g/L, the decolorization time is 40-55 min, and the temperature is 50-65 ℃.
6. The process of claim 1, wherein in step S3, the destaining solution is concentrated by heating to 55% or less, preferably 45 to 50%, of the original volume;
preferably, in step S3, the volume ratio of ethanol to the concentrated solution is 1:3.35 to 4.7, wherein the standing time is 40 to 55min;
preferably, in step S3, the precipitate is washed with ethanol and then dried by any one of vacuum drying and freeze drying.
7. The process for extracting sialic acid from fermentation broth according to any of claims 1-6, wherein in step S4, the ratio of the total molar amount of ferric and ferrous ions in the liquid phase to the molar amount of sodium acetate trihydrate is 5.8-6.3: 50 to 54, wherein the molar ratio of the ferric ions to the ferrous ions is 1.75 to 2.1; preferably, in step S4, the heating temperature is 80 to 85 ℃.
8. The process for extracting sialic acid from fermentation broth according to any one of claims 1-6, wherein in step S4, the mass volume ratio of sodium acetate trihydrate to ethylene glycol is 7-7.5 g:50 to 70ml.
9. Process for extracting sialic acid from a fermentation broth according to any of claims 1-6, wherein in step S4 the hydrothermal reaction is carried out at a temperature of 160-200 ℃ for a time of 3-5 h.
10. Use of a process for extracting sialic acid from a fermentation broth according to any of claims 1-9 in the biological, pharmaceutical or chemical field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210993063.6A CN115232178A (en) | 2022-08-18 | 2022-08-18 | Process for extracting sialic acid from fermentation liquor and application of process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210993063.6A CN115232178A (en) | 2022-08-18 | 2022-08-18 | Process for extracting sialic acid from fermentation liquor and application of process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115232178A true CN115232178A (en) | 2022-10-25 |
Family
ID=83678524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210993063.6A Pending CN115232178A (en) | 2022-08-18 | 2022-08-18 | Process for extracting sialic acid from fermentation liquor and application of process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115232178A (en) |
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 |
-
2022
- 2022-08-18 CN CN202210993063.6A patent/CN115232178A/en active Pending
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 |
Non-Patent Citations (1)
| Title |
|---|
| 黄家贤等译: "《现代微生物学实验技术及其应用》", 华中科技大学出版社, pages: 385 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109265498B (en) | Method for preparing N-acetylneuraminic acid by integrated separation and purification of polysialic acid | |
| CN112979482B (en) | High-purity L-valine as well as preparation method and application thereof | |
| CN113004347B (en) | Method for separating and purifying 2' -fucosyllactose | |
| CN113005161B (en) | Preparation method of polysialic acid and polysialic acid product | |
| JPH04235192A (en) | 1-kestose crystal and production thereof | |
| CN115232178A (en) | Process for extracting sialic acid from fermentation liquor and application of process | |
| CN113045610B (en) | Method for extracting glucosamine from N-acetylglucosamine fermentation liquor | |
| CN108299220B (en) | Method for extracting L-4-hydroxyisoleucine from fermentation catalytic liquid | |
| CN114195835B (en) | New process for preparing coenzyme I injection bulk drug | |
| CN115772549A (en) | Preparation method for extracting nicotinamide containing trace nicotinic acid from fermentation liquor | |
| CN114507262B (en) | Hydrolysis process of polysialic acid | |
| CN112813115B (en) | Production process of high-purity L-arginine | |
| CN110606863B (en) | Preparation method of N-acetylneuraminic acid dihydrate | |
| JPS6338B2 (en) | ||
| CN1042703A (en) | Water diacolation-ion exchange method is extracted the levodopa novel process | |
| CN113754704A (en) | Preparation method for efficiently preparing glucose powder by using ionic resin | |
| CN113292613A (en) | Preparation method of D-glucosamine sulfate | |
| CN113045409A (en) | Crystallization method of calcium gluconate for injection | |
| CN113248553A (en) | Preparation method of D-glucosamine hydrochloride | |
| CN112125935A (en) | Preparation method of rhamnose | |
| CN116496330B (en) | Sialic acid extraction method and sialic acid extracted by same | |
| CN115246864A (en) | Method for preparing sialic acid | |
| CN113929615B (en) | Method for purifying nojirimycin | |
| CN112457166B (en) | Separation and purification method of erythritol fermentation liquor | |
| JPS6272646A (en) | Separation and purification of lactic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |