CN108949856B - Preparation method of galacto-oligosaccharide - Google Patents
Preparation method of galacto-oligosaccharide Download PDFInfo
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- CN108949856B CN108949856B CN201810728224.2A CN201810728224A CN108949856B CN 108949856 B CN108949856 B CN 108949856B CN 201810728224 A CN201810728224 A CN 201810728224A CN 108949856 B CN108949856 B CN 108949856B
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- 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
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention belongs to the technical field of functional oligosaccharide preparation, and particularly relates to a preparation method of galactooligosaccharide. The novel organic-inorganic composite material of the polyvinyl chloride-silicon dioxide film used in the invention has good flexibility, and a film enzyme reactor formed after beta-galactosidase is fixed can be used for reaction under a stirring state, and can also be coiled into a column shape to be arranged in a cylindrical container, so that a substrate lactose can be reacted when passing through, and the problems of loss and reaction column blockage in the process of recovering small granular immobilized enzyme can be effectively solved. According to the preparation method, the content of the galactooligosaccharide in the primary conversion syrup is more than 60 percent and is 24 to 57 percent higher than that of the galactooligosaccharide in the primary conversion syrup prepared by a common enzyme method; the preparation method of the invention has the advantages that the lactose content in the primary invert syrup is lower than 15 percent and is far lower than the lactose content of 20 to 35 percent in the primary invert syrup by the common enzyme method.
Description
Technical Field
The invention belongs to the technical field of functional oligosaccharide preparation, and particularly relates to a preparation method of galactooligosaccharide.
Background
Galacto-oligosaccharides (GOS) are functional oligosaccharides and have the characteristics of good solubility, low cariogenic property, low calorific value, low sweetness value and the like. The galactooligosaccharide has the effects of promoting the proliferation of beneficial bacteria in human intestinal tracts, inhibiting the growth of intestinal putrefying bacteria, improving lipid metabolism, reducing the concentration of serum total cholesterol, promoting the absorption of mineral elements, reducing and preventing constipation, reducing the content of toxic substances in colon, promoting the health of the intestinal tracts and the like.
The galacto-oligosaccharide is prepared by catalyzing lactose serving as a raw material by beta-galactosidase (EC3.2.1.23), and is an oligosaccharide mixture of 1-4 galactose molecules connected by beta (1 → 2) bonds or beta (1 → 3) bonds, beta (1 → 4) bonds and beta (1 → 6) bonds on galactose residues of lactose molecules.
The commercial galacto-oligosaccharide is mainly prepared by taking lactose as a substrate through enzymatic synthesis. The yield of the galacto-oligosaccharide synthesized by the enzyme method is 24 to 57 percent at present, and the product purity is not high. In order to further improve the content of the galacto-oligosaccharide in the product, the galacto-oligosaccharide product needs to be further refined by separation and purification technology. The main methods for separating and purifying galactooligosaccharides include chromatographic column separation, membrane separation, microbial fermentation, enzymatic methods, and the like. Although the separation methods can realize the refining of the galactooligosaccharides and improve the purity of the galactooligosaccharides, the separation methods also have some obvious defects, such as high equipment investment of a chromatographic separation method and limitation of the application of the chromatographic separation method; lactose cannot be removed by a membrane separation method, and the purity of the product cannot be guaranteed; non-sugar impurities can be brought in by a microbial fermentation method; the enzymatic method requires addition of various enzymes for separation, requires multiple steps for completion of the test, and some enzymes are expensive, resulting in excessive production costs.
The method for the industrialized production of galacto-oligosaccharide mainly takes lactose as raw material, utilizes beta-galactosidase from microorganism to produce common galacto-oligosaccharide through one-time conversion, and then adopts a microorganism fermentation method to purify the galacto-oligosaccharide. Chinese patent CN102676614A discloses a preparation method of galacto-oligosaccharide, in the method, macroporous weak base styrene resin is taken as a high molecular carrier material, and the carrier material is in a small particle shape and is used for fixing beta-galactosidase; the immobilized enzyme can be suspended in a substrate lactose solution, and the reaction is realized under the stirring state, but the immobilized enzyme is easy to lose after the reaction is finished and the immobilized enzyme is manually recovered; the immobilized enzyme can also be loaded into a reaction column for reaction, but the inlet and the outlet of the enzyme column need to be isolated by gauze with fine aperture, which is easy to block and cause over-high column pressure.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a preparation method of galactooligosaccharide. The novel organic-inorganic composite material of the polyvinyl chloride-silicon dioxide film used in the invention has good flexibility, and a film enzyme reactor formed after beta-galactosidase is fixed can be used for reaction under a stirring state, and can also be coiled into a column shape to be arranged in a cylindrical container, so that the substrate lactose is reacted when passing through, and the problems of loss and reaction column blockage in the process of recovering small granular immobilized enzyme are effectively solved.
The invention is realized by the following technical scheme:
a preparation method of galactooligosaccharide comprises the following steps:
(1) winding a polyvinyl chloride-silicon dioxide film to form a film reactor;
(2) circularly reacting the liquid beta-galactosidase with the membrane reactor at the temperature of 10-30 ℃ for 16-24h to obtain an immobilized beta-galactosidase enzyme membrane reactor;
(3) delivering the lactose solution into an enzyme membrane reactor, and circularly reacting at 45-52 ℃ for 12-24h to obtain galacto-oligosaccharide syrup.
Preferably, the membrane reactor in step (1) is collar-like or cylindrical.
Preferably, the beta-galactosidase in step (2) is subjected to salting-out purification treatment before use.
Preferably, the ratio of the beta-galactosidase to the polyvinyl chloride-silica membrane in the step (2) is 100000-300000u/m2And (3) a membrane.
Preferably, the concentration of the lactose solution in step (3) is 40-52% (w/w), and 6000-20000u of immobilized beta-galactosidase is needed per kg of lactose (on a dry basis) to deliver the lactose solution to the enzyme membrane reactor for recycling reaction.
The preparation method of the galacto-oligosaccharide further comprises the step of nano-filtering treatment of the galacto-oligosaccharide syrup.
Preferably, the membrane used in the nanofiltration treatment is a nanofiltration membrane with a molecular weight cut-off of 200-.
Preferably, the content of the lactose component in the galactooligosaccharide syrup is less than or equal to 15 percent before the galactooligosaccharide syrup is subjected to nanofiltration treatment.
The invention takes the novel organic-inorganic composite material composed of the polyvinyl chloride-silicon dioxide film as the membrane reactor, and the membrane reactor has good flexibility. The membrane enzyme reactor formed by fixing beta-galactosidase on the membrane reactor can be used for reaction under the stirring state, and can also be coiled into a column shape to be arranged in a cylindrical container, so that the substrate lactose can be reacted when passing through, and the problems of loss and reaction column blockage in the process of recovering small granular immobilized enzyme are effectively solved.
The invention has the beneficial effects that:
(1) the invention takes polyvinyl chloride-silicon dioxide membrane material as the immobilized enzyme carrier, and the physical and chemical properties of the immobilized enzyme carrier are stable; the membrane enzyme reactor formed by fixing beta-galactosidase on the polyvinyl chloride-silicon dioxide membrane material can be used for reaction under the stirring state, and can also be coiled into a column shape to be arranged in a cylindrical container, so that the substrate lactose is reacted when passing through, and the problems of loss and reaction column blockage in the recovery process of small granular immobilized enzyme are effectively solved;
(2) according to the preparation method, the content of the galactooligosaccharide in the primary conversion syrup is more than 60 percent and is higher than the content of the galactooligosaccharide in the primary conversion syrup by a common enzyme method by 24 to 57 percent;
(3) the preparation method of the invention has the advantages that the lactose content in the primary invert syrup is lower than 15 percent and is far lower than the lactose content of 20 to 35 percent in the primary invert syrup by the common enzyme method.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A preparation method of galactooligosaccharide comprises the following steps:
(1) mixing polyvinyl chloride-silicon dioxide film 600cm2(about 30g) rolling to form a cylindrical membrane reactor;
(2) 230mL of beta-galactosidase enzyme solution with enzyme activity of 80u/mL after salting-out purification treatment and the membrane reactor obtained in the step (1) are circulated for 20h at the temperature of 20-25 ℃ to obtain an immobilized beta-galactosidase enzyme membrane reactor;
(3) 1800mL of a 52% (w/w) lactose solution was fed to an enzyme membrane reactor and circulated at 45-52 ℃ for 13h to give 1650mL of 55% (w/w) galactooligosaccharide syrup having a monosaccharide content of 24.02%, a lactose content of 14.98% and a galactooligosaccharide content of 61.00%.
Example 2
A preparation method of galactooligosaccharide comprises the following steps:
(1) mixing polyvinyl chloride-silicon dioxide film 750cm2(about 38g) rolling to form a cylindrical membrane reactor;
(2) taking 300mL of beta-galactosidase enzyme solution with enzyme activity of 75u/mL after salting-out purification treatment, and circularly reacting the beta-galactosidase enzyme solution with the membrane reactor obtained in the step (1) at 15-20 ℃ for 18h to obtain an immobilized beta-galactosidase enzyme membrane reactor;
(3) 2300mL of lactose solution with a concentration of 48% (w/w) is delivered to an enzyme membrane reactor, and is circulated for 16h at 45-52 ℃ to obtain 2100mL of galactooligosaccharide syrup with a concentration of 50% (w/w), wherein the content of monosaccharide is 25.88%, the content of lactose is 13.48%, and the content of galactooligosaccharide is 60.64%;
(4) diluting the galactooligosaccharide syrup to 20% (w/w), and performing nanofiltration treatment by using a nanofiltration membrane with the molecular weight cut-off of 200 and 1000 daltons to obtain 3727mL of the galactooligosaccharide syrup, wherein the content of the galactooligosaccharide is 76.47% and the concentration is 22% (w/w).
Example 3
A preparation method of galactooligosaccharide comprises the following steps:
(1) mixing polyvinyl chloride-silicon dioxide film 2.0m2(about 1000g) rolling to form a cylindrical membrane reactor;
(2) taking 9000mL of beta-galactosidase enzyme solution with enzyme activity of 68u/mL after salting-out purification treatment, and circulating the beta-galactosidase enzyme solution with the membrane reactor obtained in the step (1) at 10-15 ℃ for 24h to obtain an immobilized beta-galactosidase enzyme membrane reactor;
(3) 50L of 49% (w/w) lactose solution was fed into an enzyme membrane reactor and circulated at 45-52 ℃ for 24h to obtain 47L of 50% (w/w) galactooligosaccharide syrup having a monosaccharide content of 28.87%, a lactose content of 11.11% and a galactooligosaccharide content of 60.02%.
(4) The galactooligosaccharide syrup was diluted to 20% (w/w), and then subjected to nanofiltration treatment using a nanofiltration membrane having a molecular weight cut-off of 200 and 1000 daltons to obtain 85.6L of galactooligosaccharide syrup, wherein the content of galactooligosaccharide was 78.05% and the concentration was 21% (w/w).
Claims (7)
1. A preparation method of galactooligosaccharide is characterized by comprising the following steps:
(1) winding a polyvinyl chloride-silicon dioxide film to form a film reactor;
(2) circularly reacting the liquid beta-galactosidase with the membrane reactor at the temperature of 10-30 ℃ for 16-24h to obtain an immobilized beta-galactosidase enzyme membrane reactor;
(3) conveying the lactose solution into an enzyme membrane reactor, and carrying out cyclic reaction for 12-24h at the temperature of 45-52 ℃ to obtain galacto-oligosaccharide syrup;
also comprises a step of nanofiltration treatment of the galacto-oligosaccharide syrup.
2. The method for producing galactooligosaccharides according to claim 1, wherein the membrane reactor in step (1) is in a collar shape or a cylinder shape.
3. The method for preparing galactooligosaccharide according to claim 1, wherein the β -galactosidase in step (2) is purified by salting out before use.
4. The method for preparing galactooligosaccharides according to claim 1, wherein the ratio of the β -galactosidase to the polyvinyl chloride-silica membrane in step (2) is 100000-300000u/m2And (3) a membrane.
5. The process for preparing galactooligosaccharides according to claim 1, wherein the concentration of the lactose solution in step (3) is 40-52% w/w, and 6000-.
6. The method for producing galactooligosaccharides according to claim 1, wherein the nanofiltration membrane is a nanofiltration membrane having a molecular weight cut-off of 200 and 1000 daltons.
7. The method of preparing galactooligosaccharide according to claim 1, wherein the content of the lactose component in the galactooligosaccharide syrup is not more than 15% before the galactooligosaccharide syrup is nanofiltered.
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| CN201810728224.2A CN108949856B (en) | 2018-07-05 | 2018-07-05 | Preparation method of galacto-oligosaccharide |
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| CN108949856B true CN108949856B (en) | 2021-12-28 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4251631A (en) * | 1978-02-23 | 1981-02-17 | Research Products Rehovot Ltd. | Cross-linked enzyme membrane |
| CN101182510A (en) * | 2007-11-16 | 2008-05-21 | 清华大学 | Enzyme immobilization method based on excessive expenditure polymer |
| CN102676614A (en) * | 2012-05-29 | 2012-09-19 | 江门量子高科生物股份有限公司 | Method for preparing galacto-oligosaccharide |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69004587T2 (en) * | 1989-02-02 | 1994-05-11 | Abbott Lab | Method and device for quantitative chromatography. |
| CN107828834A (en) * | 2016-12-23 | 2018-03-23 | 南通励成生物工程有限公司 | A kind of preparation method of galactooligosaccharide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4251631A (en) * | 1978-02-23 | 1981-02-17 | Research Products Rehovot Ltd. | Cross-linked enzyme membrane |
| CN101182510A (en) * | 2007-11-16 | 2008-05-21 | 清华大学 | Enzyme immobilization method based on excessive expenditure polymer |
| CN102676614A (en) * | 2012-05-29 | 2012-09-19 | 江门量子高科生物股份有限公司 | Method for preparing galacto-oligosaccharide |
Non-Patent Citations (2)
| Title |
|---|
| Polymer/Silica Nanocomposites: Preparation, Characterization, Properties, and;Hua Zou等;《chemical reviews》;20080823;第108卷(第9期);全文 * |
| 聚氯乙烯/二氧化硅膜复合膜研究;梅硕;《万方学位论文数据库》;20110818;第2、8-9页 * |
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