CN111217934A - Method for removing endotoxin in pectin and pectin for removing endotoxin - Google Patents
Method for removing endotoxin in pectin and pectin for removing endotoxin Download PDFInfo
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- CN111217934A CN111217934A CN201811411715.0A CN201811411715A CN111217934A CN 111217934 A CN111217934 A CN 111217934A CN 201811411715 A CN201811411715 A CN 201811411715A CN 111217934 A CN111217934 A CN 111217934A
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- 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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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Abstract
The invention discloses a method for removing endotoxin in pectin, aiming at providing a method for removing endotoxin, which has simple operation, high product yield and easy industrial production; the technical key points are as follows: (1) dissolving pectin in water to form a solution; (2) microfiltering and separating the pectin solution to remove solid impurities; (3) adding an acid solution into the pectin solution to adjust the pH value to form acid gel, stirring to break the gel and cleaning; after the reaction is finished, carrying out vacuum filtration to remove water, adding an alkali solution to adjust the pH value, and dissolving gel to form a concentrated solution; (4) adding absolute ethyl alcohol into the pectin solution for precipitation; (5) drying the pectin precipitate under normal pressure.
Description
Technical Field
The technology relates to the field of biological pharmacy, in particular to a method for removing endotoxin in pectin.
Background
Pectin is a natural polysaccharide high molecular polymer extracted from plant fruit, is a mixture of oligosaccharide and polysaccharide with common characteristics, and contains D-galacturonic acid as main ingredient, and part of neutral sugar components such as rhamnose, arabinose, galactose, etc. in addition to galacturonic acid. The pectin has molecular weight of 5-30 ten thousand, is white to red brown powder, has no color and odor, no fixed melting point and solubility, is insoluble in organic solvent such as ethanol, is soluble in hot water, and is slightly soluble in cold water. Pectin is a safe and non-toxic natural food additive recommended by the FAO/WHO food additive joint committee, and has no daily addition limit. Pectins are versatile and widely used in the food industry as gelling agents, stabilizers, texturizers, emulsifiers and thickeners; pectin is water-soluble dietary fiber, has the functions of enhancing gastrointestinal motility and promoting nutrient absorption, has better curative effect on preventing and treating diarrhea, intestinal cancer, diabetes, obesity and other diseases, and is an excellent pharmaceutical preparation matrix; pectin is also a good heavy metal adsorbent. With the research work in recent years, the use of pectin is continuously developed, and the pectin has huge development potential and wide market prospect.
Currently, the worldwide annual sales of pectin breaks through 4 million tons, the annual value is nearly $ 50 million, and the annual sales of pectin will increase at a rate of 3% -6% each year. The largest pectin producer worldwide is CP Kelco in the United states, producing 14,000 tons of pectin annually, while the largest pectin producer in China is Andeli pectin, Inc., of Shandong tobacco Tai, producing 4,000 tons of pectin annually. Commercial pectin products sold in the market are all food-grade, have low purity and more impurities, and particularly have high endotoxin content. The main component of endotoxin is lipopolysaccharide substance of gram-negative bacteria cell outer wall layer, and its activity mainly comes from lipoid A in its structure. Endotoxin is a complex with a molecular weight of several thousands to several tens of thousands, capable of forming an association in water with a relative molecular mass of 400,000 to 1,000,000. Endotoxin has remarkable pyrogenicity to mammals, fever can be caused by intravenous injection of a small amount of endotoxin (2ng/kg body weight), blood circulation disorder and endotoxin shock can be caused by a large dose, liver damage and renal function decline are directly or indirectly caused, and endotoxemia can be caused, so that the national pharmacopoeia provides strict limitation on the content of endotoxin in a medicine or a preparation. The high endotoxin content in pectin limits its application in biomedicine and tissue engineering. Endotoxin has strong thermal stability and good heat resistance, is not influenced below 100 ℃, can destroy 98 percent when heated at 120 ℃ for 4 hours, and needs to be heated at 180 ℃ for more than 2 hours for complete inactivation. The endotoxin has strong chemical stability, the activity of the bacterial endotoxin is not influenced by common chemical medicines, and only strong acid, strong alkali or strong oxidizer can destroy the bacterial endotoxin, so the difficulty in removing the endotoxin in the pectin is very high.
Disclosure of Invention
The invention aims to develop a preparation method of high-purity pectin, which is simple to operate, high in product purity and easy for industrial production.
A method for removing endotoxin in pectin is characterized by sequentially comprising the following steps:
(1) pectin dissolution
Weighing pectin, and dissolving in water under stirring to obtain pectin with a mass concentration of 0.5-6%;
(2) microfiltration of pectin solutions
Filtering with microfiltration membrane at 20-80 deg.C after pectin is completely dissolved, wherein the pH value of the solution is 2.5-9; removing particles with the particle size of more than 0.22 mu m;
(3) gel cleaning
Adding acid solution into the ultrafiltered and concentrated pectin solution to adjust pH to 1-2 to form acid gel, stirring to break the gel, stirring and cleaning for 1-4 hr at 20-60 deg.C, and filtering for 1-4 times; after cleaning, carrying out vacuum filtration to remove 70-90% of water, adding an alkali solution to adjust the pH value to 6-7 to dissolve gel to form a concentrated solution;
(4) ethanol precipitation
Precipitating pectin solution with anhydrous ethanol at final volume concentration of 50-90% and precipitation temperature of 2-30 deg.C, preferably 4-10 deg.C;
(5) drying pectin
Drying the pectin precipitate to obtain the product.
In the method for removing endotoxin in pectin, the pectin concentration in the step (1) is 0.5-6%, the stirring speed is 500-1,500rpm, the temperature is 20-80 ℃, and the stirring time is 1-4 h.
Further, the step (2) of the method for removing endotoxin in pectin is microfiltration of the pectin solution, wherein the pectin solution is sequentially filtered by microfiltration membranes with the pore diameters of 0.8, 0.45 and 0.22 mu m, the filtering temperature is 20-80 ℃, the pH value of the solution is 2.5-9, and particles with the particle diameter larger than 0.22 mu m are removed.
Further, the step (3) of the method for removing endotoxin in pectin is gel cleaning to remove endotoxin, acid solution is added into the pectin solution to adjust the pH value to 1-2 to form acid gel, the concentration of the acid solution is 0.5M-5M, and the used acid is one or more than two of hydrochloric acid, acetic acid, citric acid, sulfuric acid and the like; stirring to break gel, stirring and cleaning for 1-4 hr at 20-60 deg.C, and filtering for 1-4 times. And after cleaning, carrying out vacuum filtration under reduced pressure to remove 70-90% of water, adding an alkali solution to adjust the pH to 6-7 to dissolve the gel to form a concentrated solution, wherein the mass concentration of the solution is 5-20%, the concentration of the alkali solution is 0.5-2M, and the alkali is one or more than two of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide and the like.
Further, the step (4) of the method for removing endotoxin in pectin is ethanol precipitation, wherein the mass concentration of the pectin solution is 5-20%, the final volume concentration of ethanol is 50-90%, and the ethanol precipitation temperature is 2-30 DEG C
Further, the step (5) of the method for removing endotoxin in pectin is drying under normal pressure, wherein the drying temperature is 30-80 ℃.
The technical scheme provided by the invention has the following technical advantages:
(1) the technical scheme provided by the invention develops a novel method for removing endotoxin in pectin, and the endotoxin removal rate is high.
(2) The technical scheme provided by the invention is simple to operate, high in product yield and easy for process amplification and industrial production.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not to be construed as limiting the invention in any way. Any modification made by anyone within the scope of the claims of the present invention is still within the scope of the claims of the present invention.
Example 1
The invention provides a method for removing endotoxin in pectin, which sequentially comprises the following steps:
(1) pectin dissolution
20g of pectin is weighed and added into 1L of water to be dissolved under the stirring condition, the mass concentration of the pectin is 2.0 percent, the stirring speed is 1,500rpm, the temperature is 30 ℃, and the stirring time is 2 hours.
(2) Microfiltration of pectin solutions
Filtering the completely dissolved pectin solution with microporous filter membranes with pore diameters of 0.8, 0.45 and 0.22 μm at 70 deg.C and pH of 4 to remove particles with particle diameter of more than 0.22 μm.
(3) Gel cleaning
Adding 1M HCl solution into the filtered pectin solution to adjust pH to 1.0 to form acid gel, stirring to break the gel, stirring and cleaning at 30 deg.C for 2 hr for 3 times. After the washing, the water with the volume of 90% is removed by suction filtration under reduced pressure, and 1M NaOH solution is added to adjust the pH value to 7.0 to dissolve the gel to form a concentrated solution.
(4) Ethanol precipitation
Adding absolute ethanol into the pectin solution for precipitation, wherein the final volume concentration of the ethanol is 70%, and the ethanol precipitation temperature is 10 ℃.
(5) Drying the product
Drying the pectin precipitate at normal pressure at 60 deg.C.
The content of endotoxin in the raw material is 3909.4EU/g, the content of endotoxin in the pectin finished product obtained by the purification process is 36.9EU/g, the removal rate is 99.1 percent, and the yield is 63.9 percent.
Example 2
The invention provides a method for removing endotoxin in pectin, which sequentially comprises the following steps:
(1) pectin dissolution
30g of pectin is weighed and added into 1L of water to be dissolved under the stirring condition, the mass concentration of the pectin is 3.0 percent, the stirring speed is 1,500rpm, the temperature is 30 ℃, and the stirring time is 3 hours.
(2) Microfiltration of pectin solutions
Filtering the completely dissolved pectin solution with microporous filter membranes with pore diameters of 0.8, 0.45 and 0.22 μm at 70 deg.C and pH of 3.5 to remove particles with particle diameter of more than 0.22 μm.
(3) Gel cleaning
Adding 1M HCl solution into the filtered pectin solution to adjust pH to 2.0 to form acid gel, stirring to break the gel, stirring and cleaning at 30 deg.C for 4 hr for 3 times. After the washing, the water with the volume of 90% is removed by suction filtration under reduced pressure, and 1M NaOH solution is added to adjust the pH value to 7.0 to dissolve the gel to form a concentrated solution.
(4) Ethanol precipitation
Adding absolute ethyl alcohol into the pectin solution for precipitation, wherein the final volume concentration of the ethyl alcohol is 60%, and the alcohol precipitation temperature is 10 ℃.
(5) Drying the product
Drying the pectin precipitate at normal pressure at 60 deg.C.
The content of endotoxin in the raw material is 3909.4EU/g, the content of endotoxin in the pectin finished product obtained by the purification process is 41.7E U/g, the removal rate is 98.9 percent, and the yield is 61.4 percent.
Example 3
The invention provides a method for removing endotoxin in pectin, which sequentially comprises the following steps:
(1) pectin dissolution
Weighing 15g of pectin, adding into 1L of dehydrated water under stirring to dissolve, wherein the mass concentration of the pectin is 1.5%, the stirring speed is 1,000rpm, the temperature is 25 ℃, and the stirring time is 1 h.
(2) Microfiltration of pectin solutions
Filtering the completely dissolved pectin solution with microporous filter membranes with pore diameters of 0.8, 0.45 and 0.22 μm at 50 deg.C and pH of 4.5 to remove particles with particle diameter of more than 0.22 μm.
(3) Gel cleaning
Adding 1M HCl solution into the filtered pectin solution to adjust pH to 1.5 to form acid gel, stirring to break the gel, stirring and cleaning at 25 deg.C for 4 hr for 2 times. After the washing, the water with the volume of 80% is removed by suction filtration under reduced pressure, and 1M NaOH solution is added to adjust the pH value to 6.5 to dissolve the gel to form a concentrated solution.
(4) Ethanol precipitation
Adding absolute ethyl alcohol into the pectin solution for precipitation, wherein the final volume concentration of the ethyl alcohol is 80%, and the alcohol precipitation temperature is 20 ℃.
(5) Drying the product
Drying the pectin precipitate at normal pressure at 30 deg.C.
The content of endotoxin in the raw material is 3909.4EU/g, the content of endotoxin in the pectin finished product obtained by the purification process is 39.1EU/g, the removal rate is 90.0 percent, and the yield is 62.2 percent.
Claims (7)
1. A method for removing endotoxin in pectin is characterized by sequentially comprising the following steps:
(1) pectin dissolution
Weighing pectin, and dissolving in water under stirring to obtain pectin with a mass concentration of 0.5-6%;
(2) microfiltration of pectin solutions
Filtering with microfiltration membrane at 20-80 deg.C after pectin is completely dissolved, wherein the pH value of the solution is 2.5-9; removing particles with the particle size of more than 0.22 mu m;
(3) gel cleaning
Adding acid solution into the ultrafiltered and concentrated pectin solution to adjust pH to 1-2 to form acid gel, stirring to break the gel, stirring and cleaning for 1-4 hr at 20-60 deg.C, and filtering for 1-4 times; after cleaning, carrying out vacuum filtration to remove 70-90% of water, adding an alkali solution to adjust the pH value to 6-7 to dissolve gel to form a concentrated solution;
(4) ethanol precipitation
Precipitating pectin solution with anhydrous ethanol at final volume concentration of 50-90% and precipitation temperature of 2-30 deg.C, preferably 4-10 deg.C;
(5) drying pectin
Drying the pectin precipitate to obtain the product.
2. The method for removing endotoxin from pectin according to claim 1, wherein: in the dissolving process in the step (1), the stirring speed is 500-1,500rpm, the temperature is 20-80 ℃, and the stirring time is 1-4 h.
3. The method for removing endotoxin from pectin according to claim 1, wherein: the microfiltration of the pectin solution in the step 2 is sequentially carried out by microfiltration membranes with 3 kinds of pore diameters, wherein the pore diameters are respectively 0.8 mu m, 0.45 mu m and 0.22 mu m.
4. The method for removing endotoxin from pectin according to claim 1, wherein: the concentration of the acid solution in the step 3 is 0.5M-5M, preferably 1M of one or more of hydrochloric acid, acetic acid, citric acid, sulfuric acid and the like; the concentration of the alkali solution is 0.5M-5M, preferably 1M of one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, etc.
5. The method for removing endotoxin from pectin according to claim 1, wherein: the mass concentration of the pectin in the step (4) is 5-20%.
6. The method for removing endotoxin from pectin according to claim 1, wherein: and (5) drying the pectin precipitate at normal pressure, wherein the drying temperature is 30-80 ℃.
7. An endotoxin-removed pectin obtained by the production method as claimed in any one of claims 1 to 6.
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| CN201811411715.0A CN111217934B (en) | 2018-11-25 | 2018-11-25 | Method for removing endotoxin in pectin and pectin for removing endotoxin |
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| CN201811411715.0A CN111217934B (en) | 2018-11-25 | 2018-11-25 | Method for removing endotoxin in pectin and pectin for removing endotoxin |
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| CN111217934B CN111217934B (en) | 2021-11-23 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114573730A (en) * | 2020-12-02 | 2022-06-03 | 中国科学院大连化学物理研究所 | Method for removing endotoxin in alginate and alginate for removing endotoxin |
| CN115386017A (en) * | 2022-10-14 | 2022-11-25 | 大连大学 | Tissue engineering pectin and preparation method and application thereof |
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| CN1184818A (en) * | 1997-12-16 | 1998-06-17 | 张文沂 | Method for purifying sun-flower seed low-ester pectine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114573730A (en) * | 2020-12-02 | 2022-06-03 | 中国科学院大连化学物理研究所 | Method for removing endotoxin in alginate and alginate for removing endotoxin |
| CN114573730B (en) * | 2020-12-02 | 2023-03-28 | 中国科学院大连化学物理研究所 | Method for removing endotoxin in alginate and alginate for removing endotoxin |
| CN115386017A (en) * | 2022-10-14 | 2022-11-25 | 大连大学 | Tissue engineering pectin and preparation method and application thereof |
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| CN111217934B (en) | 2021-11-23 |
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