CN107955826B - Preparation method and application of high-purity manno-mannan - Google Patents
Preparation method and application of high-purity manno-mannan Download PDFInfo
- Publication number
- CN107955826B CN107955826B CN201711341567.5A CN201711341567A CN107955826B CN 107955826 B CN107955826 B CN 107955826B CN 201711341567 A CN201711341567 A CN 201711341567A CN 107955826 B CN107955826 B CN 107955826B
- Authority
- CN
- China
- Prior art keywords
- enzymolysis
- powder
- molecular weight
- mannan
- mannonous
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- 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
Abstract
The invention discloses high-purity mannosidase which is generated by β -mannase controllable enzymolysis of konjac fine powder, and a polysaccharide system with the weight-average molecular weight of 60000-65000Da is obtained through purification.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a preparation method of high-purity mannosidase and application thereof in the field of medicines.
Background
In recent years, functional polysaccharides have been actively studied at home and abroad, and many developed countries have defined various polysaccharide products as specific health foods for preventing and treating obesity, hyperglycemia, hyperlipidemia, arteriosclerosis, coronary heart disease and other diseases.
Konjac Glucomannan (KGM) is polysaccharide formed by combining D-glucose and D-mannose through β -1, 4 glycosidic bonds, the average polymerization degree of the KGM is between 1000-10000.
KGM can be degraded to generate glucomannan with different polymerization degrees, wherein, the mannan oligosaccharide is a kind of saccharide with lower polymerization degree, the polymerization degree of saccharide molecules is 2-10 saccharides, and the KGM is widely applied to the fields of food, chemical industry, medicine and the like.
Chinese patent (application number is 201310022943. X) discloses a method for synergistically preparing konjac glucomannan with medium polymerization degree, and provides KGM with medium polymerization degree, the polymerization degree of the KGM is between 100-900, the sugar reduces the viscosity of KGM hydrosol on the premise of keeping the original characteristics of KGM, and effectively increases the KGM solubility, but the method adopts irradiation to degrade the glucomannan, has long irradiation time, and has small treatment capacity in each test, and is not beneficial to large-scale industrial production.
Chinese patent (application No. 201110309468.5) discloses a method for preparing konjac glucomannan and glucomannan with different molecular weights, proposes to degrade glucomannan into zymolytes with different molecular weights, but does not determine the polymerization degree of the zymolytes, does not define the functional characteristics of the zymolytes with different molecular segments, has simple process, and only carries out preliminary alcohol precipitation separation, and the purity of the product is lower.
Chinese patent (application No. 201210331680.6) discloses a konjak dietary fiber food with high bioavailability and a preparation method thereof, wherein the konjak dietary fiber food is formed by mixing all degradation products with molecular weight less than 52650 after konjak powder is degraded, oligosaccharide and glucomannan with polymerization degree of 2-65 are contained, and the molecular weights of all components of the mixture are in gradient, and the preparation method comprises the following steps: the microbial preparation is prepared by mixing a degradation product with a larger molecular weight and a degradation product with a smaller molecular weight, wherein the molecular weight is defined as the larger molecular weight between 5265 and 52650, and the molecular weight is defined as the smaller molecular weight less than 5265, and the degradation product with the larger molecular weight is prepared by acid degradation.
Disclosure of Invention
The invention aims to solve the technical problem of providing high-purity mannosidase which is a polysaccharide system with the weight-average molecular weight of 60000-65000Da and is prepared by performing controllable and accurate enzymolysis on konjac powder through β -mannase to obtain a polymer formed by connecting 300-325 monosaccharides through β -1, 4 glycosidic bonds and purifying.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of high-purity mannonous sugar powder sequentially comprises the following steps:
(1) adding water into an enzymolysis tank according to a production ratio, controlling the temperature to be 30-50 ℃, adding neutral β -mannase into the enzymolysis tank, then weighing konjac fine powder and uniformly putting the konjac fine powder into the enzymolysis tank, controlling the pH of materials in the enzymolysis tank to be 6.0-7.5, rapidly reducing the viscosity of enzymolysis liquid along with the advance of enzymolysis time, and immediately inactivating enzymes when a real-time viscosity detection device arranged on the enzymolysis tank detects that the viscosity of the enzymolysis liquid reaches 1100-1200mpa.s, wherein the enzyme inactivation time is 20-30min, and cooling;
(2) performing microfiltration and impurity removal, and performing microfiltration treatment by using a ceramic membrane to obtain filtrate, wherein the aperture of the ceramic membrane is 0.2 mu m, and the working pressure is as follows: 0.01-0.2 MPa;
(3) performing primary ultrafiltration, namely performing ultrafiltration on the filtrate by using a ceramic ultrafiltration membrane with the molecular weight cutoff of 65000Da, wherein the working conditions are as follows: the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 400L-450L/h, and a filtrate 1 and a trapped fluid 2 are respectively obtained;
(4) performing secondary ultrafiltration, namely selecting a ceramic ultrafiltration membrane with the molecular weight cutoff of 60000Da to perform ultrafiltration on the filtrate 1 in the step (3), wherein the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, and the material flow rate is 450L-500L/h, so as to respectively obtain a filtrate 3 and a trapped liquid 4;
(5) pumping the trapped liquid 2 in the step (3) into an enzymolysis tank, and continuing enzymolysis in a continuous feeding manner;
(6) desalting, decoloring and deodorizing the trapped liquid 4 obtained in the step (4) to obtain a refined mannan liquid;
(7) and (3) vacuum concentration: concentrating the refined mannan liquid prepared in the step (6), wherein the concentration of a solid matter after concentration is 45-55%;
(8) spray drying: and (4) carrying out spray drying on the concentrate concentrated in the step (7) to obtain the high-purity mannonous sugar powder.
In the step (1), the viscosity of the enzymolysis end point is controlled at 1100-.
In a further technical scheme, in the step (1), the input amount of the neutral β -mannase is calculated according to the mass ratio of enzyme materials of 1: 10-30.
In a further technical scheme, the total sugar content of the high-purity mannosylidene powder prepared in the step (8) is more than 95%, wherein the content of the mannosylidene with the molecular weight of 60000-65000Da is more than 90%.
In a further technical scheme, the invention also provides a method for applying the polysaccharide system to an anti-tumor product, and the polysaccharide system has an obvious inhibition effect on tumors.
In a further technical scheme, the polysaccharide system is subjected to derivatization treatment to prepare a corresponding product or be applied to an antiviral product for use, and the polysaccharide system has a good broad-spectrum antiviral effect.
Because a large number of hydroxyl groups exist in the molecule of the konjac mannan, a series of chemical modifications can be carried out to prepare various derivatives, thereby greatly enriching the research and development and application of the mannan. The original characteristics of the mannan are improved through chemical modification, so that the mannan has better action effect, and particularly, a new derivative can be prepared, and a more attractive new black-technology product is developed.
The enzymolysis tank is provided with a device for quickly and automatically detecting the viscosity of the material, and the specific structure can refer to the structural arrangement of a device for quickly and automatically detecting the viscosity of the material (Chinese patent 201620963523.0).
Compared with the prior art, the invention has the following advantages:
(1) the invention judges the enzymolysis end point by controlling the viscosity of the enzymolysis liquid, and can accurately and conveniently obtain products in the required molecular weight range in industrial production. The enzymolysis end point viscosity is controlled at 1100-1200mpa.s for enzyme deactivation, the molecular weight distribution measured after microfiltration is that when the viscosity value is 1100-1200mpa.s, the percentage of the zymolyte with the molecular weight range of 60000-65000Da is the highest, and therefore, the precision enzyme deactivation is carried out at the position of the viscosity range.
(2) The invention integrates the controllable enzymolysis, the ion exchange technology and the ultrafiltration purification technology to prepare the high-purity mannosidase, optimizes the preparation process, and ensures that the purity of the mannosidase reaches over 90 percent and the molecular weight is intensively distributed between 60000Da and 65000 Da.
(3) The product adopts a mature controllable enzymolysis technology, can realize large-scale production, has simple operation process, good purification effect and high effective components, and has important economic and production values.
(4) The mannan prepared by the invention is applied to anti-tumor products, has obvious anti-tumor effect, and is beneficial to a large number of tumor patients.
(5) The application of the prepared mannan in antiviral products, which is disclosed by the invention, is to perform sulfation modification treatment on the mannan to obtain a sulfated derivative of the mannan with good water solubility, wherein the sulfated derivative has a good broad-spectrum antiviral effect.
Noun interpretation
The mannan is a polymer formed by connecting β -1, 4 glycosidic bonds of 300-plus 325 monosaccharides obtained by β -mannase through controllable enzymolysis by using konjac fine powder as a raw material, has the molecular weight of 60000-plus 65000Da, has low heat, stability, safety and no toxicity, has obvious antitumor effect when being applied to an antitumor product, and has good broad-spectrum antiviral effect after being subjected to derivatization treatment.
Drawings
FIG. 1 is a process flow diagram of the present invention for preparing mannonous powder.
Detailed Description
Example 1A method for preparing mannosylidene polysaccharide, as shown in FIG. 1, comprises the following steps:
(1) adding 200kg of production water into an enzymolysis tank, controlling the temperature in the enzymolysis tank to be 30-50 ℃, adding neutral β -mannase into the enzymolysis tank (the adding amount of the enzyme is calculated according to the mass ratio of the enzyme to the konjac fine powder being 1: 10-30), weighing 10kg of konjac fine powder, uniformly putting the konjac fine powder into the enzymolysis tank with a device for quickly and automatically detecting the viscosity of the material, controlling the pH of the material in the enzymolysis tank to be 6.0-7.5, quickly reducing the viscosity of the enzymolysis liquid along with the advancing of enzymolysis time, immediately inactivating the enzyme when a real-time viscosity detection device arranged on the enzymolysis tank detects that the viscosity of the enzymolysis liquid reaches 1100-enriched 1200mpa.s, and cooling the product;
(2) performing microfiltration and impurity removal, and performing microfiltration treatment by using a ceramic membrane to obtain filtrate, wherein the pore diameter of the ceramic membrane is 0.2 mu m, and the working pressure is as follows: 0.01-0.2 MPa; the molecular weight distribution was then determined and is shown in Table 1.
TABLE 1
| Molecular weight distribution Range Da | Weight average molecular weight Da | Percentage of peak area% |
| ＞1000000 | 2256355 | 0.33 |
| 1000000~200000 | 556109 | 3.20 |
| 200000~65000 | 106563 | 9.98 |
| 65000~60000 | 62989 | 58.64 |
| 60000~10000 | 29858 | 11.65 |
| 10000~2000 | 6568 | 8.69 |
| ＜2000 | 989 | 7.51 |
Table 1 shows the measured molecular weight distribution after microfiltration when the enzymolysis end point viscosity is controlled at 1100-1200mpa.s for enzyme deactivation. When the viscosity value is 1100-.
(3) Performing primary ultrafiltration, namely performing ultrafiltration on the filtrate by using a ceramic ultrafiltration membrane with the molecular weight cutoff of 65000Da, wherein the working conditions are as follows: the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 250L-300L/h, filtrate 1 (the molecular weight range is less than 65000 Da) and trapped fluid 2 (the molecular weight range is more than or equal to 65000 Da) are respectively obtained, and the washing temperature is 55-65 ℃;
(4) performing secondary ultrafiltration, namely selecting a ceramic ultrafiltration membrane with the molecular weight cutoff of 60000Da to perform ultrafiltration on the filtrate 1 in the step (4), wherein the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 600L-650L/h, and obtaining a filtrate 3 (the molecular weight range is less than 60000 Da) and a retentate 4 (the molecular weight range is less than 60000Da and less than 65000 Da) respectively, and the washing temperature is 55-65 ℃;
(5) pumping the trapped liquid 2 in the step (4) into an enzymolysis tank, and continuing enzymolysis in a continuous feeding manner;
(6) desalting, decoloring and deodorizing the trapped liquid 4 in the step (5) by using D301-G macroporous weak base anion exchange resin, 001 × 7 strong acid cation exchange resin and D296R type anion exchange resin to obtain mannonous glucose solution;
(7) and (3) vacuum concentration: concentrating mannan liquid by adopting a VEZJM-20 falling film evaporator, wherein the concentration of the material before concentration is about 10-15%, the concentration of the solid matter after concentration can reach 45-55%, the concentration temperature is 55-60 ℃, the vacuum degree is-0.085 Mpa, the flow meter rotor is positioned at 7-9L/min, and the discharge temperature is less than 50 ℃.
(8) Spray drying: and (3) spray-drying the concentrated mannosylidene solution obtained in the step (8) by using an LPG-3 centrifugal spray-drying machine, wherein the rotating speed of an atomizer is 260-400r/min, the air inlet temperature is 150-200 ℃, and the air outlet temperature is 80-100 ℃ to obtain the high-purity mannosylidene powder.
(9) The liquid chromatography and mass spectrometry are used for detecting the mannan, the total sugar content is more than 95%, and the mannan functional component (the molecular weight is 60000-65000) is more than 90%.
Comparative example 1 preparation of mannan zymolyte I with molecular weight range of not less than 65000Da
(1) Injecting 200kg of production water into an enzymolysis tank, controlling the temperature in the enzymolysis tank to be 30-50 ℃, adding neutral β -mannase into the enzymolysis tank (the adding amount of the enzyme is calculated according to the mass ratio of the enzyme to the materials being 1: 10-30), weighing 10kg of konjac fine powder, uniformly adding the konjac fine powder into the enzymolysis tank with a device for quickly and automatically detecting the viscosity of the materials, controlling the pH of the materials in the enzymolysis tank to be 6.0-7.5, rapidly reducing the viscosity of the enzymolysis liquid along with the advancing of enzymolysis time, immediately inactivating the enzyme when a real-time viscosity detection device arranged on the enzymolysis tank detects that the viscosity of the enzymolysis liquid reaches 1100 slab-1200 mpa.s, and cooling the tank;
(2) performing microfiltration and impurity removal, and performing microfiltration treatment by using a ceramic membrane to obtain filtrate, wherein the pore diameter of the ceramic membrane is 0.2 mu m, and the working pressure is as follows: 0.01-0.2 MPa;
(3) performing primary ultrafiltration, namely performing ultrafiltration on the filtrate by using a ceramic ultrafiltration membrane with the molecular weight cutoff of 65000Da, wherein the working conditions are as follows: the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 250L-300L/h, filtrate 1 (the molecular weight range is less than 65000 Da) and trapped fluid 2 (the molecular weight range is more than or equal to 65000 Da) are respectively obtained, and the washing temperature is 55-65 ℃;
(4) collecting the trapped liquid 2 in the step (3), and preparing the mannan zymolyte I with the molecular weight range of more than or equal to 65000Da according to the following steps of desalting, decoloring and deodorizing the trapped liquid 2 in the step (3) by adopting D301-G macroporous weak base anion exchange resin, 001 × 7 strong acid cation exchange resin and D296R type anion exchange resin to obtain the mannan zymolyte I;
(5) and (4) concentrating in vacuum.
(6) Spray drying to obtain mannan zymolyte I with molecular weight not less than 65000 Da.
Comparative example 2 preparation of mannan zymolyte II with molecular weight Range < 60000Da
(1) Injecting 200kg of production water into an enzymolysis tank, controlling the temperature in the enzymolysis tank to be 30-50 ℃, adding neutral β -mannase into the enzymolysis tank (the adding amount of the enzyme is calculated according to the mass ratio of the enzyme to the materials being 1: 10-30), weighing 10kg of konjac fine powder, uniformly adding the konjac fine powder into the enzymolysis tank with a device for quickly and automatically detecting the viscosity of the materials, controlling the pH of the materials in the enzymolysis tank to be 6.0-7.5, rapidly reducing the viscosity of the enzymolysis liquid along with the advancing of enzymolysis time, immediately inactivating the enzyme when a real-time viscosity detection device arranged on the enzymolysis tank detects that the viscosity of the enzymolysis liquid reaches 1100 slab-1200 mpa.s, and cooling the tank;
(2) performing microfiltration and impurity removal, and performing microfiltration treatment by using a ceramic membrane to obtain filtrate, wherein the pore diameter of the ceramic membrane is 0.2 mu m, and the working pressure is as follows: 0.01-0.2 MPa;
(3) performing primary ultrafiltration, namely performing ultrafiltration on the filtrate by using a ceramic ultrafiltration membrane with the molecular weight cutoff of 65000Da, wherein the working conditions are as follows: the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 250L-300L/h, filtrate 1 (the molecular weight range is less than 65000 Da) and trapped fluid 2 (the molecular weight range is more than or equal to 65000 Da) are respectively obtained, and the washing temperature is 55-65 ℃;
(4) performing secondary ultrafiltration, namely selecting a ceramic ultrafiltration membrane with the molecular weight cutoff of 60000Da to perform ultrafiltration on the filtrate 1 in the step (4), wherein the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 600L-650L/h, and obtaining a filtrate 3 (the molecular weight range is less than 60000 Da) and a retentate 4 (the molecular weight range is less than 60000Da and less than 65000 Da) respectively, and the washing temperature is 55-65 ℃;
(5) collecting the filtrate 3 of the step (4), and preparing the mannan zymolyte II with the molecular weight range less than 60000Da according to the following steps of desalting, decoloring and deodorizing the trapped liquid 2 in the step (4) by adopting D301-G macroporous weak base anion exchange resin, 001 × 7 strong acid cation exchange resin and D296R type anion exchange resin to obtain the mannan zymolyte II;
(6) and (4) concentrating in vacuum.
(7) Spray drying to obtain mannan zymolyte II with molecular weight less than 60000 Da.
Effect experiment embodiment 1: mannan for antitumor test
The application of the produced mannan in resisting tumors is to prepare the mannan into corresponding products or apply the mannan into anti-tumor products, has obvious inhibition effect on tumors, and the ratio of the amount of the mannan to the body weight is 100-300 mg/kg.
The anti-tumor test scheme includes that a mouse inoculated with a tumor for 10 days is selected, ascites of the mouse is aseptically extracted, the mouse is diluted by 3 times of 0.9% sodium chloride injection, the number of tumor cells is adjusted to be 1 × 108/ml, 0.2ml is subcutaneously inoculated to each mouse armpit, the mouse is randomly grouped, administration is started after 24 hours after inoculation, a control group is physiological saline with the same amount and is 1 time a day, a 5-fluorouracil group is 10mg/kg, subcutaneous injection is adopted and is 1 time a week, the products of example 1, the products of comparative example 1 and the products of comparative example 2 are 200mg/kg respectively, gastric perfusion is adopted, tumor-bearing mice are killed 1 time a day and 7 days continuously after neck breakage, tumor masses are stripped, tumor weights are weighed, and tumor inhibition rates are calculated, and specific experimental results are shown in the following table 2:
TABLE 2 inhibitory Effect of mannosidasan on mouse S-180 sarcoma
| Group of | Sample(s) | Dosage (mg/kg) | Mean tumor weight. + -. SD | Tumor inhibition Rate (%) |
| Control group (salt water) | 10 | 0 | 2.33±0.39 | — |
| 5-FU group | 10 | 10 | 0.51±0.33 | 77.8%** |
| EXAMPLE 1 product group | 10 | 200 | 0.88±0.50 | 58.1%** |
| Comparative example 1 product group | 10 | 200 | 1.52±0.24 | 35.2%* |
| Comparative example 2 product group | 10 | 200 | 1.98±0.25 | 30.7%* |
P < 0.01 compared to control; p < 0.05 compared to control.
The experimental results show that: the products of comparative example 1 and comparative example 2 have obvious inhibition effect (P < 0.05) on mouse transplanted S-180 sarcoma, while the product of mannosylidene example 1 has very obvious inhibition effect (P < 0.01), which is closer to the inhibition effect of 5-fluorouracil.
Effect experiment embodiment 2: mannan sulfated derivatives antiviral assays
The mannan is modified by sulfation to prepare corresponding products or applied to antiviral products, so that the sulfated mannan has good broad-spectrum antiviral effect when the concentration is more than 200 mu g/ml.
The sulfated polysaccharide is natural and semi-synthetic acidic poly-vinyl containing sulfate groups, is a polyanion compound, and has active research on the antiviral aspect since the discovery that sulfated glucan has HIV (human immunodeficiency Virus) inhibiting activity in 1987, and can possibly become another HIV-1 inhibitor of a secondary virus reverse transcriptase activity inhibitor and a protease inhibitor. The sulfated mannan is sulfated and modified by chlorosulfonic acid-pyridine method, and the sulfated modification can significantly improve the biological activity, especially the antiviral activity, of the mannan.
The antiviral experimental protocol: the existing method for determining the antiviral activity in vitro cell test mainly comprises a cytopathic effect inhibition method, a pH value conversion method, a staining method and a plaque reduction method, wherein the cytopathic effect inhibition method, the staining method and the plaque reduction method are adopted in the test.
(1) Inhibition of cytopathic effects of sulfated derivatives
The sulfated derivatives were diluted in a non-cytotoxic concentration range and observed for inhibition of the cytopathic effect caused by the herpes simplex virus HSV-2 by staining and microscopy, as shown in Table 3 below:
TABLE 3 Effect of sulfated derivatives on HSV-2 induced cytopathic effects
Note: the symbols in table 3 represent: and (2) preparing: (ii) cell-free lesions; + -: cytopathic effect is less than 5%; + is as follows: cytopathic effect is 5% -25%; + of: 25% -50% of cytopathic effect; + to the utmost extent: cytopathic effect is 50% -75%; + + of: cytopathy is 75% -100%.
The results in Table 3 show that the product of example 1, i.e. the sulfated derivative of mannosylidene polysaccharide, has a significantly greater inhibitory effect (less than 5% of cytopathic effect) on the cytopathic effect induced by the herpes simplex virus HSV-2 than the sulfated derivative of the product of comparative example 1 and the product of comparative example 2 (25% to 50% of cytopathic effect).
(2) Experiment of sulfated derivative on inhibition of herpes simplex virus HSV-2 plaque
The sulfated derivative of the product of example 1 already produced a significant inhibition of viral plaque formation at a concentration of 200. mu.g/ml. The virus formation can be completely inhibited at a concentration of 400. mu.g/ml, and the specific experimental results are shown in the following table 4:
TABLE 4 inhibition of herpes simplex virus HSV-2 plaque by sulfated derivatives
Note: the symbols in table 4 represent: and (2) preparing: (ii) cell-free lesions; + -: cytopathic effect is less than 5%; + is as follows: cytopathic effect is 5% -25%; + of: 25% -50% of cytopathic effect; + to the utmost extent: cytopathic effect is 50% -75%; + + of: cytopathy is 75% -100%.
The results in Table 4 show that the inhibition of the HSV-2 plaque by the sulfated derivative of the product of example 1, i.e. the mannosylidene polysaccharide (cytopathic effect less than 5%), is significantly greater than the inhibition of the sulfated derivative of the product of comparative example 1 and the product of comparative example 2 (cytopathic effect 5% -25%).
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (5)
1. The preparation method of the high-purity mannonous sugar powder is characterized in that the high-purity mannonous sugar powder is produced by controllably performing enzymolysis on konjac refined powder through neutral β -mannase, and a polysaccharide system with the weight-average molecular weight of 60000-65000Da is obtained through purification, and the preparation method sequentially comprises the following steps:
(1) adding water into an enzymolysis tank according to a production ratio, controlling the temperature to be 30-50 ℃, adding neutral β -mannase into the enzymolysis tank, wherein the adding amount of the neutral β -mannase is calculated according to the mass ratio of enzyme to material of 1: 10-30, then weighing konjac powder, uniformly adding the konjac powder into the enzymolysis tank, controlling the pH of the material in the enzymolysis tank to be 6.0-7.5, rapidly reducing the viscosity of the enzymolysis liquid along with the advancing of enzymolysis time, and immediately deactivating enzyme when a real-time viscosity detection device arranged on the enzymolysis tank detects that the viscosity of the enzymolysis liquid reaches 1100-;
(2) performing microfiltration and impurity removal, and performing microfiltration treatment by using a ceramic membrane to obtain filtrate, wherein the aperture of the ceramic membrane is 0.2 mu m, and the working pressure is as follows: 0.01-0.2 MPa;
(3) performing primary ultrafiltration, namely performing ultrafiltration on the filtrate obtained in the step (2) by using a ceramic ultrafiltration membrane with the molecular weight cutoff of 65000Da, wherein the working conditions are as follows: the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, the material flow rate is 250L-300L/h, and a filtrate 1 and a trapped fluid 2 are respectively obtained;
(4) performing secondary ultrafiltration, namely selecting a ceramic ultrafiltration membrane with the molecular weight cutoff of 60000Da to perform ultrafiltration on the filtrate 1 in the step (3), wherein the temperature is 20-30 ℃, the pressure is 0.1-0.12MPa, and the material flow rate is 600L-650L/h to respectively obtain a filtrate 3 and a trapped fluid 4;
(5) pumping the trapped liquid 2 in the step (3) into an enzymolysis tank, and continuing enzymolysis in a continuous feeding manner;
(6) desalting, decoloring and deodorizing the trapped liquid 4 obtained in the step (4) to obtain a refined mannan liquid;
(7) and (3) vacuum concentration: concentrating the refined mannan liquid prepared in the step (6), wherein the concentration of a solid matter after concentration is 45-55%;
(8) spray drying: and (4) carrying out spray drying on the concentrate concentrated in the step (7) to obtain the high-purity mannonous sugar powder.
2. A high purity mannonous powder prepared by the process of claim 1.
3. The highly pure mannonous powder according to claim 2 wherein the highly pure mannonous powder has a total sugar content of more than 95% and a mannonous content of more than 90% with a molecular weight of 60000-65000 Da.
4. Use of the high purity mannosidase powder of claim 3 for the preparation of anti-tumor products.
5. The use of the high purity mannosidase powder of claim 3 for preparing antiviral products wherein: and carrying out sulfation derivatization treatment on the mannan powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711341567.5A CN107955826B (en) | 2017-12-14 | 2017-12-14 | Preparation method and application of high-purity manno-mannan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711341567.5A CN107955826B (en) | 2017-12-14 | 2017-12-14 | Preparation method and application of high-purity manno-mannan |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107955826A CN107955826A (en) | 2018-04-24 |
| CN107955826B true CN107955826B (en) | 2020-07-07 |
Family
ID=61958964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711341567.5A Active CN107955826B (en) | 2017-12-14 | 2017-12-14 | Preparation method and application of high-purity manno-mannan |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107955826B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008024A (en) * | 2007-01-30 | 2007-08-01 | 武汉东方天琪生物工程有限公司 | Process for producing high-purity kanjak mannan-oligosaccharides |
| CN103102430A (en) * | 2013-01-22 | 2013-05-15 | 广州城市职业学院 | Method for synergistically preparing konjac glucomannan with medium-polymerization degree |
| CN103382493A (en) * | 2012-05-03 | 2013-11-06 | 桂林微邦生物技术有限公司 | Method for fast producing high-purity mannan-oligosaccharide |
| CN103468766A (en) * | 2013-09-18 | 2013-12-25 | 恩施天天佳生物科技有限公司 | Preparation method of high-purity mannan oligosaccharide |
| CN205990407U (en) * | 2016-08-29 | 2017-03-01 | 宁波拜尔玛生物科技有限公司 | A kind of device of fast automatic detecting material viscosity |
| CN107213972A (en) * | 2017-05-17 | 2017-09-29 | 宁波拜尔玛生物科技有限公司 | The mannosan and its anti-tumor virus applications prepared based on nanometer technology |
-
2017
- 2017-12-14 CN CN201711341567.5A patent/CN107955826B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008024A (en) * | 2007-01-30 | 2007-08-01 | 武汉东方天琪生物工程有限公司 | Process for producing high-purity kanjak mannan-oligosaccharides |
| CN103382493A (en) * | 2012-05-03 | 2013-11-06 | 桂林微邦生物技术有限公司 | Method for fast producing high-purity mannan-oligosaccharide |
| CN103102430A (en) * | 2013-01-22 | 2013-05-15 | 广州城市职业学院 | Method for synergistically preparing konjac glucomannan with medium-polymerization degree |
| CN103468766A (en) * | 2013-09-18 | 2013-12-25 | 恩施天天佳生物科技有限公司 | Preparation method of high-purity mannan oligosaccharide |
| CN205990407U (en) * | 2016-08-29 | 2017-03-01 | 宁波拜尔玛生物科技有限公司 | A kind of device of fast automatic detecting material viscosity |
| CN107213972A (en) * | 2017-05-17 | 2017-09-29 | 宁波拜尔玛生物科技有限公司 | The mannosan and its anti-tumor virus applications prepared based on nanometer technology |
Non-Patent Citations (3)
| Title |
|---|
| Molecular weight distribution and fermentation of mechanically pre-treated konjac enzymatic hydrolysates;Jun Yang等;《Carbohydrate Polymers》;20161206;第159卷;全文 * |
| Synergetic degradation of konjac glucomannan by γ-ray irradiation and hydrogen peroxide;Tingtiao Pan等;《Carbohydrate Polymers》;20130402;第93卷(第2期);摘要，第762页左栏第3段-第763页右栏第2段，图1，图3 * |
| 酶催化魔芋葡甘聚糖的可控降解;祁黎等;《高分子学报》;20031031(第5期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107955826A (en) | 2018-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107760740B (en) | High-purity konjac mannose and preparation method and application thereof | |
| Rao et al. | Extraction, purification, bioactivities and prospect of lentinan: A review | |
| CN110128562B (en) | An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs | |
| CN110551230B (en) | Preparation method of astragalus polysaccharide | |
| CN105001352A (en) | Beta-1,3/1,6-glucan, preparation method therefor, and application thereof in preparing immune enhancement and anti-tumor medicine and functional food | |
| CN101654485B (en) | Spreading hedvotis herb polysaccharide for curing malignant tumor and preparation method thereof | |
| CN111803515A (en) | Application of algal polysaccharide and derivatives thereof in preparation of medicine for preventing and/or treating novel coronavirus infection | |
| CN112321737B (en) | Oyster mushroom polysaccharide selenoside-II, preparation method thereof and application thereof in preparation of medicines for specifically killing non-small lung adenocarcinoma | |
| CN104861085B (en) | Glucans of Chinese chestnut kernel α 1,6 and preparation method thereof and the application in antineoplastic | |
| CN107955826B (en) | Preparation method and application of high-purity manno-mannan | |
| CN111217935A (en) | Micromolecule citrus pectin and preparation method thereof | |
| CN111363060B (en) | Polysaccharide with anti-tumor activity and application and preparation method thereof | |
| US4614733A (en) | Polysaccharides pharmaceutical compositions and the use thereof | |
| CN110218262B (en) | Application of low-sulfated heteroglycan rich in glucuronic acid and derived from brown algae in preparation of medicines for treating type 2 diabetes | |
| CN107936135B (en) | Preparation method and application of high-purity mannan | |
| CN116003645A (en) | Fucoidan, and preparation and purification method thereof | |
| CN115161361A (en) | Method for preparing unbranched linear galactan by enzyme molecular machine technology and application | |
| EP1411956B1 (en) | Use of oversulfated polysaccharides as inhibitors of hiv | |
| CN112143769B (en) | A method for preparing radix Puerariae polypeptide extract from radix Puerariae residue and radix Puerariae polypeptide extract prepared thereby | |
| CN110878129B (en) | Glucosamine heparin salt and application thereof | |
| CN110982862B (en) | Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide | |
| CN109232756B (en) | Suaeda salsa polysaccharide extract and preparation method and application thereof | |
| CN112321751A (en) | Refining method of high-quality enoxaparin sodium | |
| CN106084085B (en) | A kind of preparation method and application of low-molecular-weight algal polysaccharide sulfate | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |