CN114085297A - Cyclo beta-1, 2-glucan and curcumin clathrate compound and preparation method thereof - Google Patents
Cyclo beta-1, 2-glucan and curcumin clathrate compound and preparation method thereof Download PDFInfo
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- CN114085297A CN114085297A CN202111514741.8A CN202111514741A CN114085297A CN 114085297 A CN114085297 A CN 114085297A CN 202111514741 A CN202111514741 A CN 202111514741A CN 114085297 A CN114085297 A CN 114085297A
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- glucan
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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
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
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- A—HUMAN NECESSITIES
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
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- 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
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Abstract
The invention discloses a cyclobeta-1, 2-glucan and curcumin clathrate compound and a preparation method thereof, belonging to the technical field of biology. The invention provides a method for preparing and characterizing cyclic beta-1, 2-glucan by a fermentation method and a method for preparing curcumin by the fermentation method, and structural analysis, and belongs to the technical field of biology. The invention is synthesized by fermentation in a 7-L fermentation tank (two-stage pH fermentation), and the yield reaches 2.79 g/L. The pure neutral oligosaccharide is obtained by purifying methods such as ethanol grading and alcohol precipitation, SPE solid phase extraction, DEAE-Sepharose FF ion exchange column chromatography and the like, and is identified as the ring beta-1, 2-glucan which consists of glucose monomers, has the polymerization degree range of 17-23 and mainly has the polymerization degree of 19. The solubility of the curcumin can be obviously improved and the bioavailability can be improved by successfully forming the inclusion compound by the cyclo beta-1, 2-glucan and the curcumin.
Description
Technical Field
The invention relates to a clathrate compound of cyclic beta-1, 2-glucan and curcumin and a preparation method thereof, belonging to the technical field of biology.
Background
The Cyclic beta-1, 2-glucan (Cyclic beta-1, 2-glucans) is a Cyclic polysaccharide which is composed of glucose monomers and is connected by beta-1, 2 glycosidic bonds, the distribution of the degree of polymerization is 17-25, and the degree of polymerization can reach 40 in some strains. Cyclo-beta-1, 2-glucan is classified into unbranched and branched structures, and substituents having branched structures include phosphoglyceryl, methylmalonyl, and succinyl. And the degree of polymerization and substituents vary greatly among different bacterial species. Because the structure of the beta-1, 2-glucan and cyclodextrin is very similar, the beta-glucan and cyclodextrin are both cyclic structures consisting of glucose monomers.
Curcumin (curcumin) with the following structural formula:
is a natural polyphenol compound extracted from rhizome of some plant of Curcuma of Zingiberaceae, and is orange yellow crystalline powder, insoluble in water, and soluble in organic solvent such as methanol, ethanol, acetone, etc. Has multiple physiological and biochemical activities such as antioxidant action, anti-inflammatory action, anti-tumor action and the like, and is increasingly concerned by scholars at home and abroad.
Curcumin has the effects of resisting oxidation, tumors and blood fat, but has the defects of poor solubility, low oral bioavailability and the like, so that the application of curcumin in the fields of medical and health care products or the cosmetic industry is limited. At present, turmeric root powder and health products containing curcumin which are available in the market are mostly imported products. The product has rich varieties, has curcumin capsules, tablets, tea and functional beverages, and has the main effects of resisting oxidation, protecting liver and the like. But the turmeric root powder and the curcumin have the characteristics of poor water solubility, easy decomposition and the like, so that the application of the curcumin in functional foods and health-care products is greatly hindered. Meanwhile, China has entered the aging society, and the improvement of life quality and health care become more and more requirements of people on health care products and functional foods. Curcumin is a natural compound currently recognized as effective in preventing and treating senile dementia. Therefore, the promotion of the health care products and the functional foods is certainly popular in the market.
But curcumin has the characteristics of low water solubility, low bioavailability, easy degradation, poor stability and the like, and limits the wide clinical application of curcumin. Therefore, it is important to increase the water solubility of curcumin and thus improve its bioavailability.
Disclosure of Invention
In order to obtain a method for increasing the water solubility of curcumin, the invention adopts the beta-1, 2-glucan and curcumin to carry out inclusion, and the beta-1, 2-glucan has stronger water solubility (the solubility of the beta-1, 2-glucan is 250 g/L; the cyclodextrin is 18g/L), and the polymerization degree is larger than that of the cyclodextrin (the polymerization degree of the beta-1, 2-glucan is 17-25, the maximum is 40; the polymerization degree of the cyclodextrin is 6-12), the diameter of a circular cavity is larger (the diameter of the beta-1, 2-glucan cavity is 0.88-1.30 nm; and the diameter of the cyclodextrin is 0.85 nm). The cyclic beta-1, 2-glucan per unit volume can include more substances, carry larger molecules.
The invention firstly provides a preparation method of cyclic beta-1, 2-glucan, which comprises the following steps:
(1) inoculating a seed solution of Rhizobium radiobacter (Rhizobium radiobacter) ATCC1333 serving as a fermentation strain into a fermentation culture medium, and fermenting to prepare a fermentation supernatant;
(2) purifying the fermentation supernatant to prepare the cyclo-beta-1, 2-glucan.
In one embodiment of the present invention, step (1) is: inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 10-15% by volume, wherein the conversion temperature is 30-33 ℃.
In one embodiment of the present invention, in the step (1), Rhizobium radiobacter (Rhizobium radiobacter) ATCC1333 is activated in a seed culture medium to obtain a seed solution; inoculating the seed liquid into a 7-L fermentation tank according to the inoculation amount of 10-15% by volume ratio, and fermenting to obtain fermentation liquid; and centrifuging and purifying the fermentation liquor to obtain the cyclic beta-1, 2-glucan.
In one embodiment of the present invention, step (1) is: activating Rhizobium radiobacter (ATCC 1333) in a seed culture medium to obtain a seed solution; inoculating the seed liquid into a 7-L fermentation tank in an inoculation amount of 10-15% by volume ratio, controlling the pH value in two stages at a conversion temperature of 30 ℃, setting an initial aeration ratio of 1vvm and an initial rotation speed of 400r/min as a 100% DO value, and fermenting to obtain a fermentation liquid; and centrifuging the fermentation liquor to obtain fermentation supernatant.
In one embodiment of the present invention, the reaction conditions of the fermentation are: culturing for 24 hours at the temperature of 30-33 ℃ and under the condition of 200 rpm.
In one embodiment of the invention, the two stages are controlled to have a pH value of 7.0 in the growth period (fermentation time is 0-24 h) of the strain at the early stage of fermentation and a pH value of 7.0 in the sugar production period after fermentation for 24 h.
In one embodiment of the invention, the seed medium comprises (g/L): peptone 10, yeast extract 2, MgSO4·7H2O1, and adjusting the pH value to 7.0.
In one embodiment of the invention, the fermentation medium comprises: 10-30 g/L mannitol, 1.0-5.0 g/L glutamic acid, 0.2-0.6 g/L NaCl, K2HPO4 1.0～3.0g/L，MgSO4·7H2O 0.2～0.6g/L，CaCl2·2H20.02-0.06 g/L of O and 0.1L/L of trace elements;
and (3) a microelement mother solution: FeCl3·6H2O 2.5g/L，MnCl2·4H2O 1g/L，Na2MoO4·2H2O 0.01g/L，ZnSO4·7H2O 0.01g/L，CuSO4·5H2O 0.01g/L，H3BO3 0.01g/L，CoCl2·6H2O0.01 g/L, biotin 0.01g/L, thiamine 0.01 g/L.
In one embodiment of the invention, the fermentation medium is: 20g/L of mannitol and 1.0g/L of glutamic acid; NaCl 0.2g/L, K2HPO4 1.0g/L，MgSO4·7H2O 0.2g/L，CaCl2·2H20.04g/L of O and 0.1L/L of trace elements;
and (3) a microelement mother solution: FeCl3·6H2O 2.5g/L，MnCl2·4H2O 1g/L，Na2MoO4·2H2O 0.01g/L，ZnSO4·7H2O 0.01g/L，CuSO4·5H2O 0.01g/L，H3BO3 0.01g/L，CoCl2·6H2O0.01 g/L, biotin 0.01g/L, thiamine 0.01 g/L.
In an embodiment of the present invention, the purification method in step (2) specifically comprises:
step 1: performing rotary evaporation and concentration on the fermentation supernatant prepared in the step (1), taking the supernatant, adding absolute ethyl alcohol for incubation, taking the supernatant, and concentrating to obtain a concentrated solution; adding absolute ethyl alcohol into the concentrated solution, carrying out alcohol precipitation, taking supernate, and concentrating to 1/5-1/10 of the original volume to obtain concentrated solution;
step 2: adding anhydrous ethanol with ten times of volume into the concentrated solution obtained in the step 1 to obtain a mixture, carrying out alcohol precipitation on the mixture, taking a precipitate, removing an organic reagent, and freeze-drying to obtain crude oligosaccharide powder;
and step 3: adding water into the crude oligosaccharide powder obtained in the step 2 to prepare a solution, removing protein, activating the graphitized carbon solid phase extraction column by using the graphitized carbon solid phase extraction column, eluting by using water, loading the sample, eluting the sample by using acetonitrile with different concentrations, and taking 10-20% acetonitrile eluent as the sample for solid phase extraction and purification;
and 4, step 4: collecting the sample purified by the solid phase extraction in the step 3, removing acetonitrile by rotary evaporation concentration, and carrying out freeze drying concentration to obtain a pretreated sample;
and 5: dissolving the sample obtained in the step 4 in water to prepare an oligosaccharide ultrapure water solution, injecting the solution into a DEAE-Sepharose FF ion exchange column chromatography column for purification to obtain neutral oligosaccharide;
step 6: and (4) dialyzing the neutral oligosaccharide obtained in the step (5) by a 2000D dialysis bag to remove salt ions, and freeze-drying in a vacuum freeze-drying machine to obtain a pure neutral oligosaccharide product.
In an embodiment of the present invention, the purification method in step (2) specifically comprises:
step 1: concentrating the fermentation supernatant obtained in example 1 by rotary evaporation, centrifuging at 10000rpm for 10min, collecting the supernatant, adding 3 times of anhydrous ethanol into the supernatant, incubating at 4 deg.C for 12h, centrifuging at 10000rpm for 10min, and collecting the supernatant;
concentrating the supernatant to 1/10 to obtain a concentrated solution; adding 3 times volume of anhydrous ethanol into the concentrated solution to make the final concentration of ethanol reach 75% (v/v), standing at 4 deg.C for 12h, centrifuging at 10000rpm for 10min, collecting supernatant, and concentrating the supernatant to 1/5 of original volume to obtain concentrated solution.
Step 2: adding ten times of volume of absolute ethanol into the concentrated solution in the step 1 to enable the final concentration of the ethanol to reach 91 percent to obtain a mixture, carrying out ethanol precipitation on the mixture at 4 ℃ for 12 hours, centrifuging at 10000rpm for 10min, taking the precipitate, removing the organic reagent by nitrogen blowing, and freeze-drying to obtain crude oligosaccharide powder.
And step 3: preparing a solution from the crude oligosaccharide powder in the step 2, removing protein by using a Sevag reagent, purifying by using a graphitized carbon solid phase extraction column, activating the graphitized carbon solid phase extraction column by using acetonitrile, and eluting by using water and then loading the sample; eluting the sample by acetonitrile (5%, 10%, 15%, 20%, 25%, 30%) with different concentrations in turn, and collecting the eluent after the acetonitrile with different concentrations is eluted to the sample;
separating and detecting the target sugar by TLC thin-layer chromatography of the eluent respectively; the detection method of TLC thin layer chromatography comprises the following steps:
a sample (2 mu L) is separated by taking a silica gel-60 plate (20cm multiplied by 10cm) as a fixed stage and optimized n-butyl alcohol-ethanol-water (5:5:9v/v/v) as a mobile phase, and after a color developing agent (3,5 dihydroxytoluene 900mg + absolute ethanol 375mL + distilled water 25mL + concentrated sulfuric acid 50mL) soaks a thin-layer plate, the thin-layer plate is immediately placed in a drying oven at 105 ℃ for color development for 5min, and dark red spots appear.
TLC results showed: the target sugar was eluted at 15% and 20% acetonitrile concentration.
Collecting and combining the eluent containing the target sugar: and combining the eluates with the concentration of 15% and 20% acetonitrile to obtain a purified sample.
And 4, step 4: and (3) carrying out rotary evaporation and concentration on the purified sample obtained in the step (3) to remove acetonitrile, pre-freezing at the temperature of-40 ℃, carrying out freeze drying and concentration, and then, filtering the sample through a 0.22 mu m filter membrane to obtain a pretreated sample.
And 5: dissolving the sample obtained in step 4 in ultrapure water to prepare an oligosaccharide ultrapure water solution (final concentration of 20g/L), and injecting the solution into a DEAE-Sepharose FF ion exchange column chromatography (1.9 cm. times.20 cm). Eluting with ultrapure water and 0.1mol/L, 0.2mol/L and 0.3mol/L gradient KCL aqueous solution at the flow rate of 1.0mL/min, collecting the eluate every 5min, detecting whether the collected eluate contains the target product by anthrone sulfuric acid method and TLC thin-layer chromatography, combining the eluates containing the target product, and concentrating to obtain the target neutral oligosaccharide.
The results show that: the sugar content of the target neutral oligosaccharide was 92.9%.
And detecting the protein concentration of the target neutral sugar by adopting Coomassie brilliant blue, wherein the protein concentration is 0 g/L.
Step 6: and (3) dialyzing the neutral oligosaccharide obtained in the step (5) by a 2000D dialysis bag to remove salt ions, and finally freeze-drying in a vacuum freeze-drying machine to obtain a purified product of the cyclo-beta-1, 2-glucan.
The invention also provides a cyclic beta-1, 2-glucan prepared by the method, wherein the structural formula of the cyclic beta-1, 2-glucan is shown as follows (shown in figure 7):
the invention also provides application of the cyclo beta-1, 2-glucan in preparation of curcumin dissolution accelerator.
The invention also provides a curcumin dissolution promoter, which contains the cyclo beta-1, 2-glucan.
The invention also provides an inclusion compound which contains the beta-1, 2-glucan and curcumin.
In one embodiment of the present invention, the inclusion is prepared according to the following method:
mixing the curcumin solution and the cyclo beta-1, 2-glucan solution according to the molar ratio of (10: 1) - (5: 2), stirring for 48-72 hours to obtain a mixture, filtering the mixture, and freeze-drying to obtain the clathrate compound.
In one embodiment of the invention, the curcumin solution and the cyclo β -1,2-glucan solution are mixed in a molar ratio of 1: 1.
The invention also provides application of the clathrate compound in preparation of curcumin-containing products in medical health products and food beverages.
The invention also provides structural analysis of the cyclic beta-1, 2-glucan.
In one embodiment of the invention, the molecular weight of the cyclo-beta-1, 2-glucan and its degree of polymerization are analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).
In one embodiment of the invention, the monosaccharide composition of the cyclo- β -1,2-glucan is analyzed by ion chromatography.
In one embodiment of the invention, the functional group composition of the cyclo-beta-1, 2-glucan is analyzed by Fourier infrared spectroscopy.
The invention also provides the establishment and the structural characterization of the embedded system of the cyclo beta-1, 2-glucan and the curcumin.
In one embodiment of the invention, the construction and structural characterization of the embedded system of the cyclic beta-1, 2-glucan and curcumin are carried out.
In one embodiment of the invention, the cycloβ -1,2-glucan is analyzed by DSC with curcumin clathrate.
In one embodiment of the invention, the cyclobeta-1, 2-glucan is analyzed by FI-TR for inclusion complexes with curcumin.
In one embodiment of the invention, by1H NMR analysis of the inclusion complex of cyclobeta-1, 2-glucan with curcumin.
In one embodiment of the invention, the inclusion complex of cyclobeta-1, 2-glucan with curcumin is analyzed by SEM.
In one embodiment of the invention, the polymerization degree is in the range of 17-23, mainly 19 polymerization degree, and only glucose monomer composed of dextran; has typical absorption peak of polysaccharide, and is pyranose configuration; at 891.20cm-1The absorption peak at (A) is a characteristic absorption peak of a β -glycosidic bond.
The invention also provides application of the cyclo beta-1, 2-glucan and curcumin in preparation of bioactive products.
Advantageous effects
(1) According to the invention, the yield of the ring beta-1, 2-glucan is improved from 1.9g/L to 2.79g/L and is improved by 46.8% through the tank-feeding fermentation of the 7-L bioreactor of the radioactive rhizobium.
(2) The cyclo beta-1, 2-glucan provided by the invention has good water solubility (250g/L), can wrap more curcumin and is a good embedding material. The curcumin/cyclo beta-1, 2-glucan clathrate compound prepared by the invention is prepared by DSC, FT-IR,1HNMR and SEM identification prove that the curcumin/cyclodextrin inclusion compound has better stability than the traditional curcumin/cyclodextrin inclusion compound.
(3) The invention can provide a new inclusion material for curcumin after curcumin drug molecules are prepared into the inclusion compound of cyclo-beta-1, 2-glucan. Not only prevents the oxidative decomposition of the effective components of the curcumin and ensures the original efficacy of the curcumin, but also obviously improves the physicochemical properties of the medicine and solves the problems of low bioavailability, difficult preparation and the like. The clathrate has the advantages of simple preparation process, no adverse side effect, edible safety, long-term administration, and remarkable health promotion effect.
Drawings
FIG. 1: the fermentation process curve of the ring beta-1, 2-glucan under the condition of pH control (7.0 in the growth phase and 5.5 in the sugar production phase).
FIG. 2: phase solubility curves for curcumin, cyclo-beta-1, 2-glucan, cyclodextrin and analogs thereof.
FIG. 3: differential scanning calorimetry plots of curcumin, cyclo-beta-1, 2-glucan, a control mixture of the two, and clathrates; a: curcumin; b: cyclo β -1, 2-glucan; c: a cyclo-beta-1, 2-glucan and curcumin inclusion; d: cyclo β -1,2-glucan with curcumin control mix.
FIG. 4: infrared spectra of curcumin, cyclo-beta-1, 2-glucan, a control mixture of the two, and inclusion complex; a: curcumin; b: cyclo β -1, 2-glucan; c: a cyclic beta-1, 2-glucan and curcumin control mixture; d: cyclo beta-1, 2-glucan and curcumin embedded material.
FIG. 5: hydrogen spectra of curcumin, cyclo-beta-1, 2-glucan and inclusion complex; a: curcumin; b: cyclo β -1, 2-glucan; c: cyclo beta-1, 2-glucan and curcumin embedded material.
FIG. 6: scanning electron microscope spectra of curcumin, cyclo-beta-1, 2-glucan, control mixtures of the two, and inclusion complex; a: curcumin; b: cyclo β -1, 2-glucan; c: a cyclic beta-1, 2-glucan and curcumin control mixture; d: cyclo beta-1, 2-glucan and curcumin embedded material.
FIG. 7: the structural formula of the ring beta-1, 2-glucan.
FIG. 8: cyclo beta-1, 2-glucan NMR spectra (a: 1H; b: 13C).
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, but the present invention is not limited to these specific examples. The media, reagents, etc. described in the examples below are all commercially available to one of ordinary skill in the art.
Rhizobium radiobacter ATCC1333 referred to in the following examples originated from the sugar Bio manufacturing and bioreactor laboratory of the university of south Jiangnan. Curcumin, alpha-cyclodextrin, beta-cyclodextrin, 2 hydroxypropyl-beta-cyclodextrin referred to in the following examples were purchased from the national pharmaceutical group.
The detection methods referred to in the following examples are as follows:
determination of extracellular neutral oligosaccharide yield:
exopolysaccharide content-total sugar content-acidic sugar content.
The total sugar content is measured by adopting an anthrone-sulfuric acid colorimetric method;
the acidic sugar content was determined by the resorcinol method, and the remaining sugar content was about that of the extracellular neutral oligosaccharide sugar.
TLC (thin layer chromatography) for identifying cyclo-beta-1, 2-glucan
Cyclo beta-1, 2-glucan can be separated under the action of a developing agent (n-butyl alcohol: ethanol: water: 5:9), and a sugar reaction color developing agent (3, 5-dihydroxytoluene 900mg, absolute ethanol 375mL, distilled water 25mL and concentrated sulfuric acid 50mL) is immediately placed in a drying oven at 105 ℃ for color development for 3-5min until dark red spots appear.
The solubility detection method comprises the following steps: the content of curcumin in the clathrate is detected by high performance liquid chromatography, and the chromatographic column is ZORBAX SB-C18(4.6 × 150mm, Agilent). Mobile phase: 0.1 percent phosphoric acid water solution (A) -acetonitrile (B), and gradient elution (0-10 min, 30-60 percent B, 10-12 min, 60-65 percent B, 12-15 min, 65-70 percent B, 15-20 min, 70-100 percent B). Detection wavelength: 280 nm. Sample introduction amount: 15 mu L, flow rate of 1mL/min, detection temperature: at 30 ℃. The regression equation for curcumin was found to be: y18229 x-2.8; correlation coefficient 0.9994; the retention time is 11.215 min; the range (mM/L) is 0.1-5. Plotting the main concentration of the clathrate compound to the curcumin concentration in the clathrate compound to obtain a phase solubility diagram
Calculation of stability constants:
wherein S0The curcumin concentration is about 1.65 × 10 without host molecules-2mM/L. The slope in the formula is the slope of the phase solubility plot.
The media involved in the following examples are as follows:
slant culture Medium (C)g/L): peptone 10, yeast extract 2, MgSO4·7H2O1 and agar powder 15, and adjusting the pH value to 7.0.
Seed medium (g/L): peptone 10, yeast extract 2, MgSO4·7H2O1, and adjusting the pH value to 7.0.
Fermentation medium (g/L): mannitol 20; 1.0 of glutamic acid; 0.2 parts of NaCl; k2HPO4 1.0；MgSO4·7H2O 0.2；CaCl2·2H2O0.04; 0.1L/L of trace elements;
microelement mother liquor (g/L): FeCl3·6H2O 2.5；MnCl2·4H2O 1；Na2MoO4·2H2O 0.01；ZnSO4·7H2O0.01；CuSO4·5H2O 0.01；H3BO3 0.01；CoCl2·6H2O0.01; 0.01 parts of biotin; thiamine 0.01.
Example 1: preparation of cyclic beta-1, 2-glucans
The method comprises the following specific steps:
1. production of extracellular neutral oligosaccharide by Shake flask fermentation of Rhizobium radiobacter ATCC1333
(1) Inoculating Rhizobium radiobacter ATCC1333 to a slant culture medium, performing inverted culture at 30 ℃, culturing for 48-72h, selecting 2-3 rings of single colonies on an ultra-clean workbench, inoculating to a seed culture medium containing 50mL, and culturing for 24h under the conditions of 30 ℃ and 200rpm to obtain activated seed liquid;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium with the inoculation amount of 10% (v/v), performing fermentation culture at 30 ℃ and 200rpm for 144h to prepare a fermentation liquid, and detecting extracellular neutral oligosaccharide (the content of acid sugar subtracted from the total sugar content in the fermentation liquid, roughly estimated as the content of neutral sugar) in the fermentation liquid, wherein the content of the extracellular neutral oligosaccharide is 1.9 g/L.
2. Preparation of extracellular neutral oligosaccharide in fermentation tank
(1) Inoculating Rhizobium radiobacter ATCC1333 into a slant culture medium, performing inverted culture at 30 ℃ for 48-72h, then selecting 2-3 rings of single colonies on an ultra-clean workbench, inoculating into a seed culture medium containing 50mL, and performing culture at 30 ℃ and 200rpm for 24h to obtain an activated seed solution.
(2)7-L fermentation tank horizontal system:
inoculating the seed liquid obtained in the step (1) into a fermentation medium with the total liquid loading of the fermentation tank of 3.5L at an inoculation amount of 10% (v/v), and supplementing glutamic acid exogenously every 4h during the cell growth period until the total concentration of the glutamic acid is 5 g/L;
the carbon source is fed in batches, and when the fermentation time is 36h, mannitol is added to a final concentration of 20 g/L.
The incubation temperature was 30 ℃ and the initial pH was 7.0. By supplementing 2mol/L hydrochloric acid and 20% NH3·2H2Controlling the pH of the O aqueous solution to be 7.0 within 0-24 h, controlling the pH at the later stage (after 24h) to be 5.5 (shown in figure 1), obtaining fermentation liquor after the fermentation is finished within 144h, and centrifuging the fermentation liquor to obtain fermentation supernatant; the aeration ratio is set to be 1vvm, and the rotating speed of 400r/min is 100 percent DO value.
The detection result of the extracellular neutral oligosaccharide in the fermentation liquor shows that the content of the extracellular neutral oligosaccharide produced by the strain reaches 2.79g/L at most. The content of extracellular neutral oligosaccharide in the fermentation liquor obtained by shake flask fermentation is 1.9g/L, which is 48% lower than that in horizontal fermentation in the upper tank.
Example 2: purification of cyclic beta-1, 2-glucans
The method comprises the following specific steps:
step 1: concentrating the fermentation supernatant obtained in example 1 by rotary evaporation, centrifuging at 10000rpm for 10min, collecting the supernatant, adding 3 times of anhydrous ethanol into the supernatant, incubating at 4 deg.C for 12h, centrifuging at 10000rpm for 10min, and collecting the supernatant;
concentrating the supernatant to 1/10 to obtain a concentrated solution; adding 3 times volume of anhydrous ethanol into the concentrated solution to make ethanol final concentration reach 75% (v/v) to obtain mixture, standing at 4 deg.C for 12 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and concentrating the supernatant to 1/5 of original volume to obtain concentrated solution.
Step 2: and (2) continuously adding ten times of volume of absolute ethyl alcohol into the concentrated solution in the step (1) to ensure that the final concentration reaches 91 percent of ethyl alcohol, carrying out alcohol precipitation on the mixture at 4 ℃ for 12h, centrifuging at 10000rpm for 10min, taking the precipitate, removing the organic reagent by nitrogen blowing, and freeze-drying to obtain crude oligosaccharide powder.
And step 3: adding water into the crude oligosaccharide powder obtained in the step 2 to prepare a solution, removing protein by using a Sevag reagent, purifying by using a graphitized carbon solid phase extraction column, activating the graphitized carbon solid phase extraction column by using acetonitrile, and washing by using water and then loading the sample; eluting the sample by adopting acetonitrile (5%, 10%, 15%, 20%, 25% and 30%) with different volume fractions in sequence, and collecting the eluent after the acetonitrile with different volume fractions is used for eluting the sample;
detecting whether the eluent contains target products or not by TLC thin-layer chromatography; the detection method of TLC thin layer chromatography comprises the following steps: a sample (2 mu L) is separated by taking a silica gel-60 plate (20cm multiplied by 10cm) as a fixed stage and optimized n-butyl alcohol-ethanol-water (5:5:9v/v/v) as a mobile phase, and a color developing agent (3,5 dihydroxytoluene 900mg, absolute ethanol 375mL, distilled water 25mL and concentrated sulfuric acid 50mL) is placed in a drying oven at 105 ℃ immediately after being soaked in a thin-layer plate, and is developed for 5min, so that dark red spots appear.
TLC results showed: the target sugar was eluted at 15% and 20% acetonitrile concentration. The combined eluates containing the target saccharide (eluates eluted at 15% and 20% acetonitrile) were collected.
And 4, step 4: and (3) carrying out rotary evaporation and concentration on the eluent obtained in the step (3) to remove acetonitrile, pre-freezing at the temperature of-40 ℃, carrying out freeze drying and concentration, and then, filtering the obtained product through a 0.22 mu m filter membrane to obtain a pretreated sample.
And 5: dissolving the sample obtained in step 4 in ultrapure water to prepare an oligosaccharide ultrapure water solution (final concentration of 20g/L), and injecting the solution into a DEAE-Sepharose FF ion exchange column chromatography (1.9 cm. times.20 cm). Eluting with ultrapure water and 0.1mol/L, 0.2mol/L and 0.3mol/L gradient KCL aqueous solution at the flow rate of 1.0mL/min, collecting the eluate every 5min, detecting whether the collected eluate contains the target product by anthrone sulfuric acid method and TLC thin-layer chromatography, combining the eluates containing the target product, and concentrating to obtain the target neutral oligosaccharide.
Wherein: anthrone sulfuric acid method:
anthrone reagent: 0.1g of anthrone is accurately weighed and dissolved in 100mL of dilute sulfuric acid (the dilute sulfuric acid solution is diluted to 100mL by 76mL of concentrated sulfuric acid (1.84)), and the anthrone reagent is required to be prepared in situ.
Glucose standard solution: weighing 100mg of glucose dried to constant weight, adding water for dissolving, and diluting to 500mL with a constant volume of 0.2 mg/mL-1。
The solutions were added in the order according to Table 1, immediately shaken and mixed, boiled in a boiling water bath for 10min, taken out and cooled at room temperature for 10min, and the absorbance value was measured at 620nm after shaking. The standard curve is obtained as y-7.2858 x-0.0004, where R2＝0.9994。
TABLE 1 determination of glucose Standard concentration Curve
TLC thin layer chromatography: a silica gel-60 plate (20cm multiplied by 10 cm; inclusive) is used as a fixed stage, optimized n-butanol-ethanol-water (5:5:9v/v/v) is used as a mobile phase for separation, and a color developing agent (3,5 dihydroxytoluene 900mg, absolute ethanol 375mL, distilled water 25mL and concentrated sulfuric acid 50mL) is immediately placed in a drying oven at 105 ℃ after being soaked in a thin-layer plate, and is developed for 5min, so that dark red spots appear.
The results show that: the sugar content of the target neutral oligosaccharide was 92.9%.
And detecting the protein concentration of the target neutral sugar by adopting Coomassie brilliant blue, wherein the protein concentration is 0 g/L.
Step 6: dialyzing the neutral oligosaccharide obtained in the step 5 by a 2000D dialysis bag to remove salt ions, and finally freeze-drying in a vacuum freeze-drying machine to obtain pure target neutral oligosaccharide, wherein the purity is detected as follows: 92.9 percent.
Comparative example 1:
the specific implementation steps are the same as those in embodiment 2, except that the adjustment step 1 is as follows:
performing rotary evaporation and concentration on the fermentation supernatant obtained in the example 1, centrifuging for 10min at 10000rpm, taking the supernatant, adding 3 times, 5 times and 7 times of volume of absolute ethyl alcohol into the supernatant respectively, incubating for 12h at 4 ℃, centrifuging for 10min at 10000rpm, and taking 1-3 supernatants respectively;
respectively concentrating the supernatant liquid 1-3 to 1/10 of the original volume to obtain concentrated solution 1-3; respectively adding 3 times of volume of absolute ethyl alcohol into the concentrated solutions 1-3 to enable the final concentration to respectively reach 75% of ethyl alcohol, standing at 4 ℃ for 12 hours, centrifuging at 10000rpm for 10min, taking supernate, and concentrating the supernate to 1/5 of the original volume to respectively obtain concentrated solutions 1-3;
the results show that: the purity of the target neutral oligosaccharide obtained by respectively adding 3 times, 5 times and 7 times of ethanol is respectively as follows: 2.5%, 31.3% and 65.4%.
Comparative example 2:
the specific example is the same as example 2, except that the graphitized carbon solid phase extraction column in step 2 is adjusted to be an ion exchange column, and the result shows that the purity of the target neutral oligosaccharide obtained by using the ion exchange column is as follows: 73.8 percent.
Comparative example 3:
the difference between the specific embodiment and embodiment 2 is that the 2000D dialysis bag of step 6 was adjusted to: 500D, 1000D, 3500D, the results show that the purity of the target neutral oligosaccharide obtained by respectively adopting 500D, 1000D, 3500D dialysis bags is respectively: 81.5%, 86.4% and 12.3%.
Comparative example 4:
the specific implementation manner is the same as that of example 2, except that the detection method of TLC thin layer chromatography in step 3 is adjusted to: using a silica gel-60 plate (20 cm. times.10 cm) as a fixing stage, separation was carried out using n-butanol-acetic acid-water (2:1:1v/v/v) as a mobile phase, and a color developing agent (3, 5-dihydroxytoluene 900mg + absolute ethanol 375mL + distilled water 25mL + concentrated sulfuric acid 50 mL).
The results show that: the target sugar cannot be separated from other components efficiently, and the presence of the target sugar cannot be detected.
Example 3: structural analysis of Cyclic beta-1, 2-glucan
The neutral oligosaccharides obtained in example 2 were subjected to structural analysis.
(1)MALDI-TOF-MS
And (3) testing conditions are as follows: the molecular weight of the purified neutral oligosaccharides was analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The specific method comprises the following steps: mu.L of oligosaccharide sample (1g/L) was spotted on the target and dried under negative pressure in a desiccator. At the same site, 1. mu.L of DHB matrix solution was applied and dried in a desiccator under reduced pressure. The sample is mixed with the matrix and crystallized for measurement. The analysis was performed in the mass range of m/z 2000-. The result shows that the polymerization degree of the neutral oligosaccharide is 17-23.
(2) Monosaccharide composition
And (3) testing conditions are as follows: the obtained neutral oligosaccharide is completely hydrolyzed at 100 ℃ for 10h, and then monosaccharide composition is detected by ion chromatography. The specific method comprises the following steps: accurately weighing 5mg of a sample, adding 300 mu L of trifluoroacetic acid with the concentration of 2mol/L, carrying out acidolysis at the constant temperature of 100 ℃ for 12h, blowing the sample to a blowing instrument after the acidolysis is finished, adding methanol for three times for removing the trifluoroacetic acid, dissolving a hydrolysate with ultrapure water, sucking out the dissolved hydrolysate, fixing the volume to 5mL, and carrying out chromatographic analysis by an ICS-5000 ion chromatograph (pulse amperometric detector) of the American Minian company. The results using a CarboPac PA20 chromatography column showed that the neutral oligosaccharides obtained in example 2 were glucans composed of only glucose monomers.
(3)FI-TR
And (3) testing conditions are as follows: fourier Infrared Spectroscopy (FI-TR) is a mass spectrometric means of determining the structure of functional groups of a substance. Taking 4-5mg of sample, mixing with a proper amount of KBr crystal, grinding fully, flaking, and carrying out infrared wavelength (400-4000 cm) on the sample to be measured by a Fourier infrared spectrometer-1) And (6) scanning. The result shows that the absorption peak of the polysaccharide is typical of the pyranose configuration. At 891.20cm-1The absorption peak at (A) is a characteristic absorption peak of a β -glycosidic bond.
(4) Nuclear magnetic resonance identification
And (3) testing conditions are as follows: 1D NMR of sample (1H and13c NMR) spectra were determined at 25 ℃ using an Avance III 400MHz spectrometer (Bruker, switzerland). Approximately 50mg of the sample was dissolved in D2O (99.9%). The results show (as shown in fig. 8): the chemical shift of the anomeric proton H-1 is less than 5.0ppm, which indicates that the purified product only contains beta-pyranose. 104.30ppm in the target glucan corresponds to non-reducing C-1-terminal, chemical shift now occurs at 83ppm representing the C-2 absorption peak of the β -1, 2-glycosidic linkage polysaccharide and indicates that the sample contains no non-reducing residues and exhibits a cyclic structure. In addition, the chemical shift values of C-1, C-2, C-3 and C-4 indicate that the sample is a polymer with different polymerization degrees.
Example 4: establishment of embedded system of cyclo beta-1, 2-glucan and curcumin
The method comprises the following specific steps:
step 1: preparing a 5mM curcumin solution: preparing the purchased curcumin solution into the ultrapure water of the curcumin solution with the final concentration of 5mM and the volume of 2mL (containing 200 muL of absolute ethyl alcohol);
meanwhile, after the purified product of Cyclo-beta-1, 2-glucan prepared in example 2 was dissolved in water, 2mL volumes of Cyclo-beta-1, 2-glucan solutions (0.0mM, 0.5mM, 1.0mM, 1.5mM, 2.0mM) were prepared at different concentrations.
The prepared curcumin solutions were mixed with the cyclic β -1,2-glucan solutions at concentrations of 0.0mM, 0.5mM, 1.0mM, 1.5mM, and 2.0mM, respectively, to prepare mixtures, respectively.
Step 2: respectively placing the mixture prepared in the step 1 in a black centrifuge tube, stirring for 48h on a magnetic stirrer at the temperature of 30 +/-2 ℃, balancing the reaction, respectively centrifuging the mixture for 20min at 10000g, filtering by using a 0.45-micrometer filter membrane to remove the non-embedded curcumin, and freezing and freeze-drying to obtain the inclusion compound.
Namely: clathrating cyclobeta-1, 2-glucan with curcumin at concentrations of 0.0mM, 0.5mM, 1.0mM, 1.5mM and 2.0mM respectively to obtain clathrates 1-5.
Comparative example 5:
the specific method is the same as the step 1-2, and is characterized in that the beta-cyclodextrin, the 2 hydroxypropyl-beta-cyclodextrin and the curcumin are replaced by cyclic beta-1, 2-glucan, and the clathrate compound is prepared by mixing the alpha-cyclodextrin, the beta-cyclodextrin, the 2 hydroxypropyl-beta-cyclodextrin and the curcumin respectively; the method specifically comprises the following steps:
alpha-cyclodextrin, beta-cyclodextrin and 2 hydroxypropyl-beta-cyclodextrin with different concentrations (0.0mM, 0.5mM, 1.0mM, 1.5mM and 2.0mM) are mixed with the curcumin solution in the step 1 to respectively prepare an inclusion compound of the alpha-cyclodextrin and the curcumin (inclusion compound 6-10), an inclusion compound of the beta-cyclodextrin and the curcumin (inclusion compound 11-15) and an inclusion compound of the 2 hydroxypropyl-beta-cyclodextrin and the curcumin (inclusion compound 16-20).
The inclusion compounds 1 to 20 were subjected to phase solubility detection, and the solubility results of the inclusion compounds 1 to 20 are shown in table 2 and fig. 2.
TABLE 2 solubility results for the inclusion complex
By combining Higuchi and Connors theories, plotting the concentration of guest molecules versus host molecules, a curve, i.e., a phase solubility map, can be generated (fig. 2). If the phase solubility curve has a good linear relationship, the stability constant of the clathrate can be obtained from the slope of the straight line. The phase solubility curve of the cyclo beta-1, 2-glucan and the cyclodextrin and the derivatives thereof belongs to ALType, namely, a 1:1 inclusion compound is formed in the system. At the same time, the stability constant can be calculated from fig. 2. The larger the stability constant, the more stable the clathrate.
The stability constants of the cyclodextrin beta-1, 2-glucan, the alpha-cyclodextrin, the beta-cyclodextrin and the 2 hydroxypropyl-beta-cyclodextrin are calculated to be 930M, 121M, 444M and 358M respectively-1I.e. the cyclodextrin is 7.68 times as much as the alpha-cyclodextrin and 2.09 times as much as the beta-cyclodextrin.
The results show that the entrapment effect of the cyclo beta-1, 2-glucan is significantly higher than that of the cyclodextrin and its derivatives because the cavity size and solubility of the cyclo beta-1, 2-glucan are larger than those of the cyclodextrin and its derivatives, and therefore, the cyclo beta-1, 2-glucan is selected next to be included with curcumin.
Control mixture:
preparation of control mixture:
curcumin powder and cyclo-beta-1, 2-glucan powder (obtained after freeze-drying the cyclo-beta-1, 2-glucan solution prepared in example 2) were mixed in a molar ratio of 1:1 to form a physical mixture of curcumin and cyclo-beta-1, 2-glucan, which was placed in a ceramic crucible and ground for 15min to obtain a homogeneous blend, i.e., a control mixture was prepared.
Example 5: structure identification of inclusion compound
The inclusion compound obtained in example 4 (curcumin-cyclo β -1, 2-glucan) and the corresponding control (curcumin, cyclo β -1,2-glucan and a control mixture of curcumin and cyclo β -1, 2-glucan) were subjected to structural characterization.
The detailed scheme is as follows:
(1)DSC
and (3) testing conditions are as follows: sample (4-5mg) was heated at 50 ℃ to 250 ℃ in an aluminum crucible at a heating rate of 10 ℃/min under flowing N2Measurements were made at a flow rate of 50mL/min in gas using DSC Q200(TA Instruments, USA). Appropriate amounts of samples (curcumin, cyclo β -1,2-glucan, a control mixture of the two (1: 1 molar ratio) and inclusion) were placed in an aluminum pan and tested under simultaneous thermal analyzer under the above conditions, and a blank pan was used as a reference.
The results of the experiment are shown in FIG. 3.
As can be seen from FIG. 3, the melting point of curcumin has a sharp phase transition endothermic peak at 171 ℃, which is the melting point of curcumin; the cyclic beta-1, 2-glucan has a broad endothermic peak at 130-150 ℃; the DSC curve of the control mixture showed a sharp peak; in the inclusion compound, a shallow and wide endothermic peak exists, and the phase transition endothermic peak of the curcumin disappears, which shows that a new phase is formed after the curcumin and the cyclic beta-1, 2-glucan are included.
(2)FI-TR
And (3) testing conditions are as follows: a plurality of samples (curcumin, cyclo-beta-1, 2-glucan, a control mixture (molar ratio is 1:1) of the curcumin and the cyclo-beta-1, 2-glucan and an inclusion compound) are mixed with a proper amount of KBr crystal, are fully ground, are prepared into sheets, and are subjected to infrared wavelength (400-4000 cm) by a Fourier infrared spectrometer-1) And (6) scanning.
The results of the experiment are shown in FIG. 4.
A and b in fig. 4 are the infrared chromatograms of curcumin and cyclo β -1,2-glucan, respectively, with their typical characteristic absorption peaks; in the control mixture, it is clearly seen that it is curcumin and cyclo β -1,2-glucanSimple superposition of sugar infrared spectra shows that weak or no interaction exists in the physical mixing process; the inclusion complex showed almost complete coverage of 800cm of curcumin, similar to cyclo-beta-1, 2-glucan species-1To 1600cm-1Typical characteristic peaks in the range indicate the formation of clathrates.
(3)1H NMR
And (3) testing conditions are as follows: of samples1The H NMR spectrum was measured at 25 ℃ using an Avance III 400MHz spectrometer. Solubilisation of Cyclo-beta-1, 2-glucan in D2O is in; curcumin was dissolved in DMSO-D6, and the inclusion compound was 80% D2Dissolving in O.
The results of the experiment are shown in FIGS. 5a to 5 c.
As can be seen from FIG. 5, the inclusion compound was obtained by complexation with cyclo-. beta.1, 2-glucan1The H NMR spectrum shows that the chemical shifts (delta) of H-1, H-2, H-3 and H-6 of curcumin are respectively changed significantly and are respectively 0.033, 0.041, 0.018 and 0.073 ppm. At the same time, downward chemical shifts of H-1(0.1172ppm) and H-2(0.1254ppm) of cyclic β -1,2-glucan were detected, which directly determined the formation of inclusion complex.
(4)SEM
And (3) testing conditions are as follows: the surface morphology of curcumin, cyclo beta-1, 2-glucan and inclusion compound was studied using Scanning Electron Microscopy (SEM). Fixing the sample powder with double-sided adhesive tape carbon tape, spraying gold, and making it conductive under low vacuum condition to obtain SEM picture.
The results of the experiment are shown in FIGS. 6a to 6 d.
As can be seen from fig. 6a, curcumin appears in the form of repeating rod-like crystals; cyclo β -1,2-glucan (fig. 6b) consists of fairly amorphous planar sheets; the control mixture (fig. 6c) is in a mixed shape, with rod-shaped crystals of curcumin clearly adsorbed on the surface of cyclo β -1, 2-glucan; after inclusion, the inclusion complex (fig. 6d) exhibited an amorphous structure with no original morphology of curcumin and cyclic β -1, 2-glucan.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing cyclic beta-1, 2-glucan, comprising the steps of:
(1) inoculating a seed solution of Rhizobium radiobacter (Rhizobium radiobacter) ATCC1333 serving as a fermentation strain into a fermentation culture medium, and fermenting to prepare a fermentation supernatant;
(2) purifying the fermentation supernatant to prepare the cyclo-beta-1, 2-glucan.
2. The method according to claim 1, wherein the method of step (1) is embodied as: inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 10-15% by volume, wherein the conversion temperature is 30-33 ℃.
3. The method according to claim 1 or 2, wherein the purification method of step (2) is in particular:
step 1: performing rotary evaporation and concentration on the fermentation supernatant prepared in the step (1), taking the supernatant, adding absolute ethyl alcohol for incubation, taking the supernatant, and concentrating to obtain a concentrated solution; adding absolute ethyl alcohol into the concentrated solution, precipitating with ethanol, taking the supernatant, and concentrating to 1/5-1/10 of the original volume to obtain a concentrated solution;
step 2: adding anhydrous ethanol with ten times of volume into the concentrated solution obtained in the step 1 to obtain a mixture, carrying out alcohol precipitation on the mixture, taking the precipitate, removing an organic reagent, and freeze-drying to obtain crude oligosaccharide powder;
and step 3: adding water into the crude oligosaccharide powder obtained in the step 2 to prepare a solution, and after protein removal, performing solid-phase extraction on the solution by using graphitized carbon to purify the solution; activating a graphitized carbon solid-phase extraction column by adopting acetonitrile, carrying out elution on a sample by using water, eluting the sample by adopting acetonitrile with different concentrations, and taking 10-20% of acetonitrile eluent as a sample for solid-phase extraction and purification;
and 4, step 4: collecting the sample purified by the solid phase extraction in the step 3, removing acetonitrile by rotary evaporation concentration, and carrying out freeze drying concentration to obtain a pretreated sample;
and 5: dissolving the sample obtained in the step 4 in water to prepare an oligosaccharide ultrapure water solution, injecting the solution into a DEAE-Sepharose FF ion exchange column chromatography column for purification to obtain neutral oligosaccharide;
step 6: and (3) dialyzing the neutral oligosaccharide obtained in the step (5) by a 2000D dialysis bag to remove salt ions, and freeze-drying in a vacuum freeze-drying machine to obtain a purified product of the cyclo-beta-1, 2-glucan.
4. The cyclic beta-1, 2-glucan prepared by the method of any one of claims 1 to 3, wherein the structural formula of the cyclic beta-1, 2-glucan is shown as follows:
5. use of the cyclic beta-1, 2-glucan of claim 4 in the preparation of a curcumin solubilizing agent.
6. A curcumin solubilizing agent comprising the cyclic β -1,2-glucan according to claim 4.
7. A clathrate compound comprising the cyclic β -1,2-glucan according to claim 4 and curcumin.
8. The clathrate of claim 7, wherein the inclusion is made by:
mixing the curcumin solution and the cyclo beta-1, 2-glucan solution according to the molar ratio of (10: 1) - (5: 2), stirring for 48-72 hours to obtain a mixture, filtering the mixture, and freeze-drying to obtain the clathrate compound.
9. The clathrate compound according to claim 7 or 8, characterized in that curcumin solution and cyclo β -1,2-glucan solution are mixed in a molar ratio of 1: 1.
10. The use of the inclusion compound of any one of claims 7 to 9 in the preparation of curcumin-containing products in medical and health products and food and beverage products.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103386136A (en) * | 2013-07-31 | 2013-11-13 | 西南大学 | Application of hydrophobic glucan in preparation of curcumin solubilizing agent and utilization method of hydrophobic glucan |
| CN108440683A (en) * | 2018-03-30 | 2018-08-24 | 福建师范大学 | A kind of glucan curcumin inclusion compound and preparation method thereof |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103386136A (en) * | 2013-07-31 | 2013-11-13 | 西南大学 | Application of hydrophobic glucan in preparation of curcumin solubilizing agent and utilization method of hydrophobic glucan |
| CN108440683A (en) * | 2018-03-30 | 2018-08-24 | 福建师范大学 | A kind of glucan curcumin inclusion compound and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHUAN-CHAO WU 等: ""Enhanced solubility of curcumin by complexation with fermented cyclic β-1,2-glucans "", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 》 * |
| GEETHA VENKATACHALAM 等: ""Characterization and applications of cyclic b-(1,2)- glucan produced from R. meliloti"", 《RSC ADVANCES》 * |
| 曲娟娟: ""放射型根瘤菌产环β-1,2-葡聚糖的合成过程优化"", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 欧阳可宣等: "姜黄素与β-葡聚糖的结合研究", 《福建师范大学学报(自然科学版)》 * |
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