CN111995698A - Method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on enzymolysis method - Google Patents
Method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on enzymolysis method Download PDFInfo
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- CN111995698A CN111995698A CN202010789438.8A CN202010789438A CN111995698A CN 111995698 A CN111995698 A CN 111995698A CN 202010789438 A CN202010789438 A CN 202010789438A CN 111995698 A CN111995698 A CN 111995698A
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- C07K1/18—Ion-exchange chromatography
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention relates to a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymolysis method, which comprises the following steps: preparing intestinal mucosa mixed solution; preparing enzymolysis liquid by an enzymolysis method; filtering to obtain clear filtrate and enzymolysis concentrated solution; adsorbing the filtrate with heparin through ion exchange resin column to obtain effluent liquid; taking down the ion exchange resin column which is adsorbed and saturated, firstly washing the resin in the ion exchange resin column by using washing saline, and then carrying out heparin elution on the resin by using desorption saline to obtain heparin sodium eluent; filtering, desalting and concentrating the heparin sodium eluent to obtain a heparin sodium concentrated solution; drying to obtain crude heparin sodium; cooling the effluent, and then desalting and concentrating to obtain a first concentrated solution; vacuum concentrating the first concentrated solution to obtain protein peptide concentrated solution; and drying the protein peptide concentrated solution to obtain the protein peptide. The method has the advantages of high extraction rate of heparin sodium, short production cycle, low cost and little pollution, and can simultaneously extract protein peptide.
Description
Technical Field
The invention relates to a method for extracting heparin sodium, in particular to a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymolysis method.
Background
At present, the heparin extraction in China has two processes of salting out method and enzymolysis method. The salting-out method comprises adding salt to intestinal mucosa, adjusting pH with alkali, heating to about 50 deg.C, maintaining the temperature to dissolve heparin, transferring to water, heating to 95 deg.C to solidify intestinal membrane protein, separating to obtain byproduct intestinal membrane residue (or called intestinal membrane protein), and subjecting the separated clear liquid to resin adsorption, analysis, alcohol precipitation, oven drying to obtain heparin. The enzymolysis method comprises the steps of adding salt to the intestinal mucosa, adding alkali to adjust the pH value, adding protease, heating to about 50 ℃, keeping the temperature to carry out enzymolysis on the intestinal mucosa to obtain enzymolysis liquid, and carrying out enzyme deactivation, resin adsorption, resolution, alcohol precipitation, drying and the like on the enzymolysis liquid to obtain the heparin. Compared with salting-out method, the enzymolysis method has the advantages of high heparin yield, less waste water, low salt consumption and the like, so the method is widely applied.
However, the enzymatic method still has the following problems:
the production period is long: the traditional method has long resin adsorption process time, generally needs 8-10 hours, is easy to cause deterioration and stink of feed liquid, increases the content of amino nitrogen and other components, and causes adverse effects on subsequent protein component extraction and utilization;
the resin utilization rate is low: in the traditional method, resin is put into an adsorption tank, and as the intestinal mucosa tissue in the adsorption tank is more and the resin is easily blocked by grease components in the adsorption tank, the adsorption capacity of the blocked resin is greatly reduced, and the adsorption of the resin is influenced by the problems;
the resin breakage rate is high: the resin is subjected to long-term stirring in the tank in the adsorption and desorption processes, so that the resin breakage rate is high;
large amount of waste water: the traditional industrial production method has large wastewater discharge amount and high salt content, and particularly the enzymolysis process has high organic content of discharged water, high wastewater treatment difficulty and high operation cost;
the use amount of the dangerous organic solvent is large: the traditional production method adopts the modes of ethanol precipitation and standing overnight to collect heparin, has long production period, high workshop grade requirement, strict running management requirements of ethanol recovery, storage and the like, and higher cost.
Disclosure of Invention
In order to solve the problems, the invention provides a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymolysis method, which can improve the extraction efficiency of heparin sodium, increase the yield, reduce the consumption and the breakage rate of adsorption resin, realize the comprehensive utilization of raw materials, reduce the discharge of high-salt and high-organic wastewater, reduce environmental pollution and reduce the cost, and the specific technical scheme is as follows:
a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymolysis method comprises the following steps:
s101, preparing intestinal mucosa mixed liquor;
s102, preparing the mixed solution of the intestinal mucosa into enzymolysis solution by an enzymolysis method;
s103, filtering the enzymolysis liquid to obtain clear filtrate and enzymolysis concentrated liquid;
s104, adsorbing the clear filtrate by heparin by using an ion exchange resin column, and simultaneously obtaining an effluent liquid;
s105, taking down the ion exchange resin column which is adsorbed and saturated, firstly washing the resin in the ion exchange resin column by using washing saline, and then carrying out heparin elution on the resin by using desorption saline to obtain heparin sodium eluent;
s106, filtering, desalting and concentrating the heparin sodium eluent to obtain a second permeate and a heparin sodium concentrated solution;
s107, drying the heparin sodium concentrated solution to obtain a crude product heparin sodium;
s201, cooling the effluent liquid in the step S104;
s202, filtering, desalting and concentrating the cooled effluent to obtain a first permeate and a first concentrate;
s203, carrying out vacuum concentration on the first concentrated solution to obtain a protein peptide concentrated solution;
and S204, drying the protein peptide concentrated solution to obtain the protein peptide.
Further, the concentrated solution of enzymolysis in step S103 is returned to S101 to be extracted again.
Further, an inorganic microfiltration membrane is used for filtering the enzymatic hydrolysate in step S103.
Further, at least two ion exchange resin columns are connected in series for adsorption in step S104.
Further, the concentration of the cleaning brine in the step S105 is 4-8%; the concentration of the desorption brine is 20-30%, and the temperature of the desorption brine is 45-50 ℃.
Further, the effluent in step S201 heats the desorption brine in step S105 through a heat exchanger.
Further, in the step S106, an ultrafiltration membrane is used for filtering the heparin sodium eluate, and when the heparin sodium eluate is concentrated to 5-6 times, purified water is supplemented to the original volume for continuous filtration, and the operation is repeated for 1-3 times.
Further, in the step S202, a nanofiltration membrane is used for desalination and concentration, and when the concentration is 5 to 6 times, purified water is supplemented to the original volume for continuous filtration, and the operation is repeated for 1 to 3 times.
Further, the second permeate in step S106 is used for dissolving the intestinal mucosa in step S101;
the first permeate in step S202 is used for dissolving the intestinal mucosa in step S101;
condensed water is obtained during vacuum concentration in step S203, and the condensed water is used for dissolving intestinal mucosa in step S101.
Further, the spray drying is performed after the excipient is added in step S204.
Further, the washing saline is washed in a forward direction, and the desorption saline is desorbed in a reverse direction.
Compared with the prior art, the invention has the following beneficial effects:
the method for simultaneously extracting the heparin sodium and the intestinal membrane protein peptide based on the enzymolysis method can improve the extraction efficiency of the heparin sodium, increase the yield, reduce the using amount and the breakage rate of the adsorption resin, realize the comprehensive utilization of raw materials, reduce the discharge of high-salt high-organic-matter wastewater, reduce the environmental pollution, reduce the production input cost of enterprises, and simultaneously extract the intestinal membrane protein peptide.
Drawings
FIG. 1 is a flow chart of a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymatic hydrolysis method.
Detailed Description
The invention will now be further described with reference to the accompanying drawings.
Example one
As shown in fig. 1, a method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymatic hydrolysis method comprises the following steps:
s101, preparing intestinal mucosa mixed liquor;
s102, preparing the mixed solution of the intestinal mucosa into enzymolysis solution by an enzymolysis method;
s103, filtering the enzymolysis liquid to obtain clear filtrate and enzymolysis concentrated liquid;
s104, adsorbing the clear filtrate by heparin by using an ion exchange resin column, and simultaneously obtaining an effluent liquid;
s105, taking down the ion exchange resin column which is adsorbed and saturated, firstly washing the resin in the ion exchange resin column by using washing saline, and then carrying out heparin elution on the resin by using desorption saline to obtain heparin sodium eluent;
s106, filtering, desalting and concentrating the heparin sodium eluent to obtain a second permeate and a heparin sodium concentrated solution;
s107, drying the heparin sodium concentrated solution to obtain a crude product heparin sodium;
s201, cooling the effluent liquid in the step S104;
s202, filtering, desalting and concentrating the cooled effluent to obtain a first permeate and a first concentrate;
s203, carrying out vacuum concentration on the first concentrated solution to obtain a protein peptide concentrated solution;
and S204, drying the protein peptide concentrated solution to obtain the protein peptide.
In step S103, an inorganic microfiltration membrane is used for filtering the enzymatic hydrolysate.
The concentration of the cleaning saline water in the step S105 is 4-8%; the concentration of the desorption brine is 20-30%, and the temperature of the desorption brine is 45-50 ℃.
Firstly, removing the adsorbed small molecular protein component in the resin by using low-concentration cleaning saline; then high-concentration desorption saline is used for heparin elution, so that the content of impurities in the heparin sodium eluent is reduced.
And S106, filtering the heparin sodium eluent by using an ultrafiltration membrane, and supplementing purified water to the original volume for continuous filtration when the heparin sodium eluent is concentrated to 5-6 times, and repeating the operation for 1-3 times.
And S202, desalting and concentrating by adopting a nanofiltration membrane, supplementing purified water to the original volume and continuously filtering when the concentration is 5-6 times, and repeating the operation for 1-3 times.
In step S204, the excipient is added and then spray-dried.
In the step S203, the concentration can be carried out until the concentration content of the soluble solid is 40-60%.
The heparin sodium concentrated solution in step S107 may be spray-dried.
Example two
In addition to the first embodiment, the concentrated solution of enzymolysis in step S103 is returned to step S101 for further extraction. The extraction rate is improved.
EXAMPLE III
On the basis of any one of the above embodiments, at step S104, at least two ion exchange resin columns are connected in series for adsorption.
The ion exchange resin columns are arranged and corrected in a ' saturated adsorption leakage-free ' dual-purpose one-standby ' three-branch series connection mode or in a ' one-purpose one-standby ' two-branch series connection mode; the ion exchange resin column with saturated adsorption is separated from the technical process through valve switching, and the rest ion exchange resin columns continue to operate, so that the working efficiency is high. In the traditional process, because the impurity content in the enzymolysis liquid is high, the enzymolysis liquid can only be adsorbed by adopting a mode of scattering into an adsorption tank, but the method adopts filtration and adopts a mode of adsorbing by adopting an ion exchange resin column, so that the adsorption rate and the working efficiency are greatly improved, and the breakage rate of the resin can be effectively reduced. The extraction rate of heparin sodium can be improved by series connection, because the content of heparin sodium in the enzymolysis liquid is lower, the heparin sodium is easily lost by adopting first-stage filtration, and therefore, the extraction rate of the heparin sodium is ensured by adopting the series connection mode to carry out multi-stage filtration after the content of the heparin sodium is determined.
Example four
On the basis of any of the above embodiments, the effluent in step S201 heats the desorption brine in step S105 through a heat exchanger. The utilization rate of energy is improved, and the cost is reduced. And the temperature of the effluent liquid is reduced to 20-30 ℃ of the working temperature of the organic membrane, so that the service life of the organic membrane is ensured.
EXAMPLE five
In any of the above embodiments, the second permeate in step S106 is used for dissolving the intestinal mucosa in step S101; the first permeate in step S202 is used for dissolving the intestinal mucosa in step S101; condensed water is obtained during vacuum concentration in step S203, and the condensed water is used for dissolving intestinal mucosa in step S101.
The first permeating liquid and the second permeating liquid mainly contain salt and water, and can be used as intestinal mucosa dissolving water for recycling, so that the utilization rate is improved.
The condensed water can also be used to dissolve the intestinal mucosa, increasing the water availability.
EXAMPLE six
On the basis of any one of the above embodiments, the washing brine is positively washed, and the desorption brine is reversely desorbed. The forward washing means that washing brine enters from the top of the ion exchange resin column and flows out from the bottom. The reverse desorption means that desorption brine enters from the bottom of the ion exchange resin column and flows out from the top.
Firstly, removing the adsorbed small molecular protein component in the resin by using low-concentration cleaning saline; then high-concentration desorption saline is used for heparin elution, and the counter-current desorption effect is good because the saturation degree of the upper resin adsorption of the ion exchange resin column is higher than that of the lower resin adsorption of the ion exchange resin column.
The production efficiency is improved: the production period is greatly shortened, and the resin adsorption time is shortened to 0.5-1 hour from 8-10 hours of the traditional method; while adsorption, the components of the fully soluble protein peptide are concentrated and collected by the nanofiltration membrane, the time is short, the generation amount of unfavorable components such as amino nitrogen and the like is greatly reduced, and the quality of the protein peptide product is improved.
The utilization rate of the resin is improved, and the breakage rate is reduced: after the extract is clarified and filtered by the inorganic microfiltration membrane, components such as grease, intestinal mucosa tissues and the like are isolated, the blockage of resin adsorption holes is greatly reduced, the heparin adsorption capacity is enhanced, and due to the fixed bed adsorption and desorption, the damage rate of the resin is reduced compared with a stirring mode, and the production cost is saved.
Obviously improves the water quality and the water quantity of the discharged wastewater: the concentrated solution of the inorganic microfiltration membrane returns to the previous procedure for repeated extraction, thereby reducing the emission of organic matters; the main components of the organic membrane permeate liquid are salt and water, and condensed water obtained by evaporation and concentration can be recycled to the intestinal mucosa dissolving procedure, so that the recycling of the saline solution is realized, and the wastewater treatment cost is saved.
Use of non-hazardous organic solvents: the purification and recovery of the heparin component in the desorption solution adopt a mode of combining membrane separation concentration and pure water dialysis, thereby improving the product recovery rate and the heparin titer.
The technical principle of the present invention is described above in connection with specific embodiments. The description is made for the purpose of illustrating the principles of the invention and should not be construed in any way as limiting the scope of the invention. Based on the explanations herein, those skilled in the art will be able to conceive of other embodiments of the present invention without inventive step, which shall fall within the scope of the appended claims.
Claims (10)
1. A method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on an enzymolysis method is characterized by comprising the following steps:
s101, preparing intestinal mucosa mixed liquor;
s102, preparing the mixed solution of the intestinal mucosa into enzymolysis solution by an enzymolysis method;
s103, filtering the enzymolysis liquid to obtain clear filtrate and enzymolysis concentrated liquid;
s104, adsorbing the clear filtrate by heparin by using an ion exchange resin column, and simultaneously obtaining an effluent liquid;
s105, taking down the ion exchange resin column which is adsorbed and saturated, firstly washing the resin in the ion exchange resin column by using washing saline, and then carrying out heparin elution on the resin by using desorption saline to obtain heparin sodium eluent;
s106, filtering, desalting and concentrating the heparin sodium eluent to obtain a second permeate and a heparin sodium concentrated solution;
s107, drying the heparin sodium concentrated solution to obtain a crude product heparin sodium;
s201, cooling the effluent liquid in the step S104;
s202, filtering, desalting and concentrating the cooled effluent to obtain a first permeate and a first concentrate;
s203, carrying out vacuum concentration on the first concentrated solution to obtain a protein peptide concentrated solution;
and S204, drying the protein peptide concentrated solution to obtain the protein peptide.
2. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
the enzymolysis concentrated solution in step S103 is returned to S101 for re-extraction.
3. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
and in the step S103, an inorganic microfiltration membrane is adopted for filtering the enzymolysis liquid.
4. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
and at least two ion exchange resin columns are connected in series for adsorption in the step S104.
5. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
the concentration of the cleaning saline water in the step S105 is 4-8%; the concentration of the desorption brine is 20-30%, and the temperature of the desorption brine is 45-50 ℃.
6. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 5,
the effluent in step S201 heats the desorption brine in step S105 through a heat exchanger.
7. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
and S106, filtering the heparin sodium eluent by using an ultrafiltration membrane, and supplementing purified water to the original volume for continuous filtering when the heparin sodium eluent is concentrated to 5-6 times, and repeating the operation for 1-3 times.
8. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
and in the step S202, a nanofiltration membrane is adopted for desalination and concentration, and when the concentration is 5-6 times, purified water is supplemented to the original volume for continuous filtration, and the operation is repeated for 1-3 times.
9. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
the second permeate in step S106 is used for dissolving the intestinal mucosa in step S101;
the first permeate in step S202 is used for dissolving the intestinal mucosa in step S101;
condensed water is obtained during vacuum concentration in step S203, and the condensed water is used for dissolving intestinal mucosa in step S101.
10. The method for simultaneously extracting heparin sodium and intestinal membrane protein peptide based on the enzymolysis method as claimed in claim 1,
the washing saline water is washed in a forward direction, and the desorption saline water is desorbed in a reverse direction.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112624479A (en) * | 2020-12-31 | 2021-04-09 | 无锡优普克生物科技有限公司 | Resourceful treatment method and device for high-salt and high-alcohol wastewater in heparin extraction |
| CN113999330A (en) * | 2021-12-07 | 2022-02-01 | 潢川县鹏升畜产品有限公司 | Low-salt-concentration heparin sodium and active intestinal protein peptide separation and co-production process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112624479A (en) * | 2020-12-31 | 2021-04-09 | 无锡优普克生物科技有限公司 | Resourceful treatment method and device for high-salt and high-alcohol wastewater in heparin extraction |
| CN113999330A (en) * | 2021-12-07 | 2022-02-01 | 潢川县鹏升畜产品有限公司 | Low-salt-concentration heparin sodium and active intestinal protein peptide separation and co-production process |
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Application publication date: 20201127 |