CN110714040A - Biosynthesis method of dendritic glucosyl nanoparticles - Google Patents
Biosynthesis method of dendritic glucosyl nanoparticles Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
Abstract
The invention discloses a biosynthesis method of dendritic glucosyl nanoparticles, and belongs to the technical field of carbohydrate deep processing. The invention takes cyclodextrin as a raw material, and prepares the dendritic glucosyl nanoparticles by a one-pot synthesis technology based on in vitro polysaccharide enzyme preparation concerted catalysis. The method has the characteristics of simple process, controllable reaction conditions, continuous production, low cost, environmental friendliness and the like, and the diameter size of the obtained dendritic glucosyl nanoparticles is 20-100nm, and the absolute molecular weight is more than 1.0 multiplied by 105g/mol, polydispersion coefficient not more than 1.3, branch density 6-12%, and can be widely used as carrier in the industries of nutritional food, medicine, cosmetics, etc.
Description
Technical Field
The invention belongs to the technical field of carbohydrate deep processing, and particularly relates to a biosynthesis method of dendritic glucosyl nanoparticles.
Background
In recent years, nanoscale delivery systems for entrapping, protecting and delivering lipid-soluble bioactive ingredients have been developed in order to increase the bioavailability of active substances. The nano-transmission system has great potential in the transmission of functional nutrients by virtue of the characteristics of small granularity, large relative surface area, high reaction activity and strong adsorbability, and the manufacturing and property research technology of the nano-transmission system is gradually mature. Of course, the use of nanoscopic transport systems is also required to meet certain requirements. In the face of processing, storage, transportation, heat treatment, drying, illumination, mechanical external force and the like, the nano-transmission system has certain physical and chemical stability and wide applicability. For the delivery of bioactive ingredients, it would be desirable to inhibit their chemical degradation, increase their oral availability, and achieve controlled release of the bioactive ingredient. The existing nano delivery system adopts lipid, protein, surfactant, mineral and other single or combined materials, and the components determine the functional properties of a carrier system, but also have the problems of poor biocompatibility, low hydrophilic drug encapsulation rate, insufficient mechanical strength and durability and the like. A dendrimer is a highly branched polymer, usually linked by chemical bonds from monomers. The molecular structure of dendrimers is not completely symmetrical, their molecular shape is ellipsoidal, and a large number of reactive groups are distributed on the surface of the molecule. The dendritic polymer shows different property characteristics from the linear polymer due to the special structure, can be used as a carrier material, and has important application in the fields of pharmacy, chemical materials, electronics and the like.
At present, most hyperbranched polymers are mainly synthesized by chemical methods, and the main methods comprise the following methods: polycondensation, the most classical method for synthesizing dendritic polymers, generally by polymerization of monomers having carboxyl groups with monomers having amino and hydroxyl groups; addition reactions, generally reactions that occur on unsaturated monomers; ring-opening polymerization, in which a heterocyclic compound is used as a substrate and polymerization is carried out through ring opening of an intermediate. The polymerization degree and the branching degree of the dendritic polymer synthesized by a chemical method are difficult to control, the structure of the dendritic polymer is random, the side reactions of the reaction are more, the product needs to be purified, the preparation cost is higher, and the price is high. However, few studies on glucosyl nanoparticle polymers are reported, and reports related to preparation of dendritic glucosyl nanoparticles by cyclodextrin are not found so far. For the above reasons, the present invention has been made in detail on a method for biosynthesis of a glucosyl dendrimer nanoparticle.
Disclosure of Invention
In order to solve the problems, the invention provides a biosynthesis method for preparing the dendritic glucosyl nanoparticles.
The method for biologically synthesizing the dendritic glucosyl nanoparticles has the characteristics of simple process, environmental protection, low cost, high safety, continuous production and the like, and the application range of the product can relate to a plurality of fields of food, medicine, daily chemicals and the like.
The purpose of the invention is realized by the following technical scheme:
taking cyclodextrin as a raw material, and reacting by a one-pot method under the synergistic catalysis effect of an in vitro polysaccharide enzyme preparation to obtain the dendritic glucosyl nanoparticles; the in vitro polysaccharidase preparation consists of depolymerase and transglycosidase.
In one embodiment of the invention, the ratio of enzymatic activities of the depolymerase and the transglycosidase in the in vitro polysaccharidase preparation is 10:1-1: 10.
In one embodiment of the present invention, the depolymerizing enzyme comprises any one or more of cyclomaltodextrinase, maltoglucosyl enzyme and neopullulanase.
In one embodiment of the invention, the transglycosidase is a 1, 4-alpha-glucan branching enzyme of the hydrolase family 13 and/or the hydrolase family 57.
In one embodiment of the present invention, the cyclodextrin includes any one or more of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and large-ring cyclodextrin in combination.
In one embodiment of the invention, 5-20U of the in vitro polysaccharidase preparation is added per 1g of cyclodextrin to carry out the reaction.
In one embodiment of the invention, the reaction is carried out using a buffer solution as the reaction medium. The buffer solution is phosphate buffer solution with pH 7.0. The addition amount of the reaction medium is 5 to 100 times (mL/g) the mass of the cyclodextrin.
In one embodiment of the invention, the temperature of the reaction is 50-70 ℃.
In one embodiment of the invention, the reaction is carried out at 50-70 ℃ for 30-60min, and then the in vitro polysaccharidase preparation is added for constant temperature reaction for 1-24 h.
In an embodiment of the present invention, the method specifically includes the following steps:
dissolving 10g of cyclodextrin in 50-1000mL of phosphate buffer solution (pH 7.0) every time, placing in a water bath at 50-70 ℃ for heat preservation for 30min, then adding 50-200U of in vitro polysaccharidase preparation for constant temperature reaction for 1-24h, heating to inactivate the enzyme after the reaction is finished, centrifuging, and drying the obtained supernatant in vacuum to obtain the target product.
The invention has the following advantages:
1) the invention fully utilizes bulk cyclodextrin as raw material, develops the product by modern biotechnology, improves the added value of the product, and provides a certain basis for comprehensive development and utilization of the product. The diameter of the obtained glucosyl dendron nanoparticles is 20-100nm, and the absolute molecular weight is more than 1.0 × 105g/mol, polydispersion coefficient not more than 1.2, branch density 6-12%, and can be widely used as carrier in the industries of nutritional food, medicine, cosmetics, etc.
2) The method has the advantages of simple and convenient steps, easy operation, controllable reaction conditions, relatively low cost due to the adoption of a specific enzyme catalysis process, and basically no pollution to the environment due to the adoption of a clean production process.
Drawings
FIG. 1 TEM image of a glucosyl dendron nanoparticle;
FIG. 2 distribution diagram of the diameter of the glucosyl dendron nanoparticles;
FIG. 3 NMR chart of glucosyl dendron nanoparticles.
Detailed Description
The present invention will be further explained with reference to examples, but the present invention is not limited to the examples.
Measurement of Branch Density: by using an AVANCE III-400 MHz nuclear magnetic resonance spectrometer1H spectrum and13c spectral analysis, test temperature 70 ℃, 5mm PABBO-BB probe, zg30 pulse sequence.1The test conditions for the H spectrum were: 8223Hz, relaxation time 1s, recording 16 scans;13the test conditions of the C spectrum are as follows: 24038Hz scan width, relaxation time 2s, 1401 scans were recorded. Meanwhile, panose is taken as a reference. By using1The peak areas at 5.4ppm and 5.0ppm in the H spectrum can be used for quantitatively calculating the alpha-1, 6 glycosidic bond proportion, namely the branch density.
Sources of cyclomaltodextrinase, maltoglucosylase, neopullulanase: commercially available enzyme preparations are available from Novo Nordisk, DuPont, AB Enzymes, Handary, Magazyme, Sigma, etc.
Sources of 1, 4-alpha-glucan branching enzymes of the hydrolase carbohydrase family 13 and/or the hydrolase carbohydrase family 57: one or more of archaea, bacteria, plants and the like, including any one of barley, corn, rice, wheat, sorghum, red algae, chlamydomonas reinhardtii, extreme thermophilic bacteria A. aeolicus, thermophilic fat bacillus B. stearothermophilus, thermophilic archaea T. kodakaraensis and thermophilic Thermus thermophilus, is obtained by extracting endosperm of kernels in a growth period or by activated culture and fermentation to produce enzyme. Can be obtained from the literature reference (microbiological Starch-Converting Enzymes: RecentrationInsights and perspectives. Ming Miao, comprehensive reviews in food science and food safety.2018).
Example 1:
weighing 10g of beta-cyclodextrin, dissolving in 1000mL of phosphate buffer solution (pH 7.0), placing in 70 deg.C water bath, keeping the temperature for 30min, adding 100U of in vitro polysaccharidase preparation (neopullulanase: 1, 4-alpha-glucan branching enzyme of hydrolase family 13 ═ 1:1), reacting at constant temperature for 6h, heating to inactivate enzyme after reaction, centrifuging, and collecting the extractAnd carrying out vacuum drying treatment on the clear liquid to obtain the target product. The average size of the glucosyl dendron nanoparticles is 35nm, and the absolute molecular weight is 4.2 multiplied by 106g/mol, polydispersity 1.0, branching density 7.3%.
Example 2:
weighing 10g of large-ring cyclodextrin, dissolving in 50mL of phosphate buffer solution (pH 7.0), placing in a 70 ℃ water bath, preserving the temperature for 30min, then adding 200U of in vitro polysaccharidase preparation (cyclomaltodextrin enzyme: 1, 4-alpha-glucan branching enzyme of hydrolase family 13 is 10:1), reacting at constant temperature for 24h, heating to inactivate the enzyme after the reaction is finished, centrifuging, and carrying out vacuum drying treatment on the obtained supernatant to obtain the target product. The average size of the glucosyl dendron nanoparticles is 78nm, and the absolute molecular weight is 1.9 multiplied by 107g/mol, polydispersity 1.2, branching density 6.7%.
Example 3:
weighing 10g of gamma-cyclodextrin, dissolving in 250mL of phosphate buffer solution (pH 7.0), placing in a 70 ℃ water bath, preserving heat for 30min, then adding 50U of in vitro polysaccharidase preparation (maltose glucosylase: 1, 4-alpha-glucan branching enzyme of hydrolase 57 family) for reacting for 2h at constant temperature, heating to inactivate enzyme after the reaction is finished, centrifuging, and carrying out vacuum drying treatment on the obtained supernatant to obtain the target product. The average size of the glucosyl dendron nanoparticles is 41nm, and the absolute molecular weight is 8.9 multiplied by 105g/mol, polydispersity 1.1, branching density 6.1%.
Comparative example 1
Referring to example 1, the target product was prepared by replacing the amount distribution of the in vitro polysaccharidase preparation with 30U, and leaving the other conditions unchanged. The results of the obtained product are shown in table 1.
Comparative example 2
Referring to example 1, the target product was prepared by replacing the amount distribution of the in vitro polysaccharidase preparation with 300U, and leaving the other conditions unchanged. The results of the obtained product are shown in table 1.
Comparative example 3
Referring to example 1, the target product was prepared by replacing beta-cyclodextrin with waxy corn starch under otherwise unchanged conditions. The results of the obtained product are shown in table 1.
TABLE 1 Performance results for the products obtained in comparative examples 1-3
| Comparative example | Coefficient of distribution | Branch Density (%) |
| 1 | 1.8 | 5.8 |
| 2 | 2.1 | 5.2 |
| 3 | 2.5 | 4.3 |
The embodiments described herein are merely illustrative of the spirit and some of the experiments performed. Various modifications or additions may be made or substituted in a similar manner to the specific embodiments described herein by those skilled in the art without departing from the spirit of the invention or exceeding the scope thereof as defined in the appended claims.
Claims (10)
1. A method for biologically synthesizing dendritic glucosyl nanoparticles is characterized in that cyclodextrin is used as a raw material, and the dendritic glucosyl nanoparticles are obtained by a one-pot reaction under the catalysis of an in vitro polysaccharase preparation; wherein 50-200U of in vitro polysaccharidase preparation is added into every 10g of cyclodextrin for reaction; the in vitro polysaccharidase preparation comprises depolymerase and transglycosidase.
2. The method of claim 1, wherein the enzymatic activity ratio of depolymerase to transglycosidase in the in vitro polysaccharidase preparation is 10:1 to 1: 10.
3. The method according to claim 1 or 2, wherein the depolymerase comprises any one or more of cyclomaltodextrinase, maltoglucosylase and neopullanase.
4. The method according to any one of claims 1 to 3, wherein the transglycosidase is a 1, 4-alpha-glucan branching enzyme of the hydrolase family 13 and/or the hydrolase family 57.
5. The method of any one of claims 1 to 4, wherein the cyclodextrin comprises any one or more of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, and large-ring cyclodextrin.
6. The method according to any one of claims 1 to 5, wherein the reaction is carried out using a buffer solution having a pH of 4.5 to 6.0 as a reaction medium.
7. The process according to claim 6, characterized in that the reaction medium is added in a volume corresponding to 5-100 times the mass of the cyclodextrin.
8. The process according to any one of claims 1 to 7, wherein the temperature of the reaction is 50 to 70 ℃.
9. The method of any one of claims 1-8, wherein the reacting further comprises incubating the cyclodextrin at 50-70 ℃ for 30-60min prior to the adding of the in vitro polysaccharidase preparation to the reaction.
10. The method of claim 9, wherein the in vitro polysaccharidase preparation is added to the reaction mixture at a temperature in the range of 50-70 ℃ for a reaction time in the range of 1-24 hours.
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