CN114106087A - Starch polymer with core-shell-crown structure and preparation method thereof - Google Patents
Starch polymer with core-shell-crown structure and preparation method thereof Download PDFInfo
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- CN114106087A CN114106087A CN202111421752.1A CN202111421752A CN114106087A CN 114106087 A CN114106087 A CN 114106087A CN 202111421752 A CN202111421752 A CN 202111421752A CN 114106087 A CN114106087 A CN 114106087A
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The invention discloses a starch polymer with a core-shell-crown structure and a preparation method thereof, belonging to the technical field of deep processing of starch. The invention takes linear starch dextrin as raw material, and prepares the starch polymer with core-shell-crown structure by multienzyme coupling catalysis and temperature-controlled crystallization technology, and concretely comprises the following steps: dissolving linear amylodextrin in a buffer salt solution system with the pH value of 5.0-7.0 to obtain a uniform solution with the mass concentration of 10-30%, adding multifunctional amylase which is from a hydrolase family 13 or 57 and has sugar chain branching activity/depolymerization activity of less than 30 to perform a constant temperature reaction, then placing the obtained starch enzymolysis product in a water bath at the temperature of 95 ℃ to heat for 30-60min, and continuously adding glycosyltransferase to the starch enzymolysis product to perform a constant temperature reaction; after the reaction is finished, heating to inactivate enzyme, adjusting the temperature of the system to 0-20 ℃, storing, centrifuging, taking the precipitate, and drying to obtain the starch polymer with the core-shell-crown structure. The method has simple and convenient operation, controllable reaction conditions and continuous green production.
Description
Technical Field
The invention belongs to the technical field of starch deep processing, and particularly relates to a starch polymer with a core-shell-crown structure and a preparation method thereof.
Background
Starch is an important renewable resource next to cellulose as an important energy storage substance in plants. China has abundant starch resources, the annual total yield exceeds 3000 million tons, but more than 90 percent of the starch resources are used for producing primary products such as starch sugar, sugar alcohol, fermentation products and the like, have a larger gap with developed countries in Europe and America, and mainly reflect low utilization rate of raw materials, imperfect production technology and especially lack of high value-added products. Bulk starch is modified properly to change its original molecular structure, so that the physical and chemical properties of starch, such as gelatinization, water solubility, gelatinization, aging resistance, viscosity, film forming property, etc. are changed, and the starch can be widely applied in the fields of food, paper making, textile, fine chemical industry, medicine, etc. For example, the physical modification process is simple and easy to operate, but the physical modification has low modification degree on starch, and is often required to be combined with other modification means; the chemical modified starch is the largest amount used in the starch industry at present, but the modification cost is high, and the generated waste causes pollution to the environment; the enzyme modification reaction condition is mild, the reaction efficiency is high, the substrate specificity is strong, and the method is green and environment-friendly; the composite modification is to combine multiple modification methods to obtain a modified derivative with combined characteristics. At present, international multinational companies such as Yiranan, Jiaji, Rogaite and the like utilize green clean production technology to develop nano starch, intelligent hydrogel, drug delivery carriers, biodegradable materials and the like and form large-scale production and sale; domestic enterprises mainly produce and sell traditional chemically modified products such as acid hydrolysis starch, oxidized starch, acetate starch and the like, and need to enrich and upgrade the types of the existing products and improve the added value of the products. For the above reasons, the present invention has designed and prepared a novel starch polymer having a core-shell-crown structure.
Disclosure of Invention
The invention aims to provide a method for synthesizing a starch polymer with a core-shell-crown structure, which has the characteristics of simple and convenient method operation, controllable reaction conditions, continuous green production and the like.
The purpose of the invention is realized by the following technical scheme: the preparation method comprises the following steps of preparing a starch polymer with a core-shell-crown structure by taking linear starch dextrin as a raw material through multi-enzyme coupling catalysis and a temperature-controlled crystallization process, wherein the preparation method sequentially comprises the following steps:
(1) dissolving linear amylodextrin in a buffer salt solution system with the pH value of 5.0-7.0 to obtain a linear amylodextrin solution with the mass concentration of 10-30%, adding 50-600U/g of multifunctional amylase, and reacting at the constant temperature of 35-70 ℃ for 6-48 h;
wherein the multifunctional amylase is derived from the family 13 or 57 of hydrolytic carbohydrases, having a sugar chain branching activity/depolymerizing activity (enzymatic catalytic polymerization forming 1,6 and 1,4 bond ratio) < 30;
(2) after the reaction in the step (1) is finished, heating to inactivate enzyme, adding 30-200U/g glycosyltransferase into the mixture, and reacting for 2-12h at the constant temperature of 35-50 ℃;
(3) after the reaction in the step (2) is finished, heating to inactivate enzyme, adjusting the temperature of the system to 0-20 ℃, storing for 6-24h, centrifuging, taking the precipitate, and drying to obtain the target product, namely the starch polymer with the core-shell-crown structure.
As an embodiment of the present invention, the sugar chain branching activity of the multifunctional amylase refers to activity of reducing the absorbance of the linear starch-iodine complex at 660nm, and is based on the ability of the multifunctional amylase to cleave an α -1,4 glycosidic bond and transfer to another glucose residue to form a cyclic chain structure to reduce linear starch fragments, the branching activity (U/mL) × 100/10 × 20 [ (absorbance of the linear starch-iodine complex at 660 nm-absorbance of the linear starch-iodine complex at 660nm with addition of an enzyme preparation)/(absorbance of the linear starch-iodine complex at 660 nm) ]. The sugar chain depolymerization activity means: the activity of reducing the molecular weight of the starch is the amount of enzyme required for reducing the molecular weight of the starch to 500000Da when the multifunctional amylase acts on 1g of starch substrate and reacts for 8 hours under the condition of the optimal temperature and the pH for catalyzing the carbohydrase. The specific test method comprises the following steps: molecular weight reduction activity (U/mL) ═ 1/[ (amount of enzyme required to reduce starch molecular weight to 500000 Da/1000) × (1000 mg/sample mass) ]. Wherein the activities are all measured at 70 ℃ and pH 7.0.
In one embodiment of the present invention, the multifunctional amylase comprises: preparing multifunctional amylase by activating and culturing hydrolase family 13 or 57 in archaea and bacteria, fermenting to produce enzyme, collecting thallus, and pulverizing lyophilized powder; the ratio of the sugar chain branching activity to the depolymerization activity of the multifunctional amylase is less than 30.
In one embodiment of the present invention, the specific steps of the preparation method of the multifunctional amylase refer to the chinese patent with application number 2019106991744.
In one embodiment of the invention, the microbial source comprises: bacillus stearothermophilus ATCC 7953, Thermus thermophilus Caldicoloyamamurae UTM801 CGMCC 6185, Thermus thermophilus Streptococcus thermophilus ATCC 14485, Thermus thermophilus ATCC33923, Thermus archaebacterium thermophilus Aeropyrumpernix K1 and the like are purchased from biological resource centers such as American ATCC and JCM Japan.
In one embodiment of the present invention, the glycosyltransferase is at least one of amylosucrase, dextran sucrase, maltose glucosyltransferase, dextran branching enzyme, and dextran phosphorylase.
As an embodiment of the present invention, the multifunctional amylase is used in an amount of 60U/g.
In one embodiment of the present invention, the glycosyltransferase is used in an amount of 100U/g.
As an embodiment of the present invention, the linear starch dextrin is a degradation product of starch of plant origin, and the molecular weight of the linear starch dextrin is (1.0-20.0). times.104g/mol。
As an embodiment of the present invention, the plant-derived starch includes at least one of cereal starch, potato starch, and bean starch.
As an embodiment of the invention, the buffered salt solution system is at least one of phosphate, citrate, acetate, Tris-hydrochloride and barbiturate.
As an embodiment of the present invention, the heating and inactivating conditions in step (2) are specifically: heating in water bath at 95 deg.C for 30-60 min.
The second purpose of the invention is to provide the starch derivative prepared by the method, the microstructure of the starch derivative is of a core-shell-crown structure, and the molecular weight of the starch derivative is (0.1-5.0) × 108g/mol, particle size of 200-2000nm, crystallinity of 10-35%, transverse grain size of 3-15nm, and indigestible nutrient fragment ratio>45％。
The third purpose of the invention is to provide the application of the starch derivative in the fields of special medical food, host-guest encapsulated carriers, biomedical materials and the like.
Has the advantages that:
1) the method adopts a multienzyme coupling catalysis and temperature-controlled crystallization process, and specifically comprises the following steps: firstly, linear amylodextrin is polymerized to form an inner core with a multi-branch sugar chain structure under the enzymatic catalysis of multifunctional amylase, then partial chain extension is carried out on the surface of the inner core under the enzymatic catalysis of glycosyltransferase, finally, parameters such as processing temperature, time and the like are regulated and controlled to enable sugar chains to be self-assembled and aggregated on the surface of the inner core to form a shell-crown structure, and finally, the starch derivative with the core-shell-crown structure is formed.
2) The method has simple and convenient steps, easy operation, controllable reaction conditions and relatively low cost; the clean production process is adopted, so that the environment is basically not polluted; the obtained starch derivative has core-shell-crown structure in microscopic morphology, and molecular weight of 0.1-5.0 x 108g/mol, particle size of 200-2000nm, crystallinity of 10-35%, transverse grain size of 3-15nm, and indigestible nutrient fragment ratio>45％。
3) The starch polymer with a core-shell-crown structure prepared by the invention forms a sugar chain double-helix crystal structure on the surface of an inner core, and the product can be applied to the fields of special medical food, host-object encapsulated carriers, biomedical materials and the like.
4) The invention fully utilizes the starch with abundant resources in China to design the processing method of the starch polymer with the core-shell-crown structure, creates products with different application performances, increases the additional value of the starch, expands the application field of the starch, and meets the requirements of the application industry on the structure and the performance of the starch. The product prepared by the invention can be applied to a plurality of fields such as food, medicine, daily chemicals and the like, has good market prospect and wide economic benefit.
Drawings
FIG. 1 XRD patterns of linear corn starch dextrin and core-shell-crown structured starch polymer prepared in example 1.
FIG. 2 is a schematic representation of the structure of the starch polymers of the present invention having a core-shell-crown structure.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
The test method comprises the following steps:
determination of absolute molecular weight:a combined system of high performance liquid phase size exclusion chromatography, a multi-angle laser light scattering detector and a refractive index detector is adopted, a Shodx Ohpak SB-805HQ gel chromatographic column is selected, 0.1mol/L sodium nitrate solution is used as a mobile phase, the flow rate is set to be 0.7mL/min, and the refractive index is set to be dn/dc which is 0.138.
Particle size determination:the particle size is measured by a laser particle size analyzer, a fixed laser with the wavelength of 658nm is used as a light source, the angle of scattered light is 90 degrees, and the operating temperature is 25 ℃.
Crystallinity and crystal size determination:and analyzing by using an X-ray diffractometer, setting power of 1600W, copper target Cu Ka (lambda is 1.5406nm), scanning speed of 4 DEG/min and scanning range of 3-40 DEG, and measuring and calculating crystallinity I and crystal granularity Dc of the sample by using MDI jade software. The specific formula is as follows:
in the formula: i isC-crystalline diffraction peak diffraction intensity; i isP-amorphous peak diffraction intensity; λ -radiation wavelength (nm); b-half-peak width of the crystallization peak; theta-diffraction angle.
Determination of indigestibility:the content of indigestible starch nutritive fragments was determined by the Englyst method using the whole starch analysis kit from Megazyme of ireland.
Sources of enzymes:
the preparation method of the multifunctional amylase comprises the following steps:
the microbial transglycosidase preparation is derived from archaea and hydrolase family 13 or 57 in bacteria, has sugar chain branching activity/depolymerization activity of less than 30, is prepared by screening archaea or bacteria from nature, performing activation culture, fermenting to produce enzyme, collecting thalli, freezing, drying, crushing and the like, and has the specific method steps of the preparation method of the microbial transglycosidase preparation in Chinese patent with the application number of 2019106991744.
Sources of glycosyltransferases:commercially available amylosucrase, dextran sucrase, maltose glucosyltransferase, dextran branching enzyme, dextran phosphorylase, and the like.
Example 1:
the molecular weight is 2.7X 104Dissolving g/mol linear corn starch dextrin in a Tris-hydrochloride buffer solution system with the pH value of 7.0 to prepare a starch dextrin uniform solution with the mass concentration of 10%, and then adding 60U/g substrate of hydrolase family 13 multifunctional amylase (the sugar chain branching activity/depolymerization activity is 26.2) into the solution to perform constant temperature reaction for 10 hours at 35 ℃; heating in 95 deg.C water bath for 30min, adding 100U/g substrate dextran sucrase, and reacting at 40 deg.C for 5 hr; heating to inactivate enzyme after reaction, adjusting system temperature to 4 deg.C, storing for 10 hr, centrifuging, precipitating, and drying to obtain target productStarch polymers having a core-shell-crown structure.
The molecular weight of the starch polymer having a core-shell-crown structure obtained in example 1 was determined to be 1.3X 108g/mol, particle size of 283nm, crystallinity of 24 percent, transverse grain size of 5.1nm, and the proportion of indigestible nutrition fragments of 65 percent.
From the comparison of fig. 1, it can be seen that linear corn amylodextrin forms a specific structure of inner amorphous-outer crystalline through multi-enzyme coupled catalysis and temperature controlled crystallization process, demonstrating the successful preparation of starch polymer of core-shell-crown structure (see fig. 2).
Example 2:
weighing molecular weight of 1.2 × 105Dissolving g/mol linear potato amylodextrin in an acetate buffer salt solution system with the pH value of 5.0 to prepare an amylodextrin uniform solution with the mass concentration of 15%, and then adding 200U/g substrate of hydrolysis carbohydrase family 57 multifunctional amylase (the sugar chain branching activity/depolymerization activity is 18.4) into the solution to perform thermostatic reaction at 40 ℃ for 12 hours; heating in water bath at 95 deg.C for 40min, adding amylosucrase 100U/g substrate, and reacting at constant temperature of 35 deg.C for 3 hr; heating to inactivate enzyme after reaction, adjusting system temperature to 20 deg.C, storing for 6h, centrifuging, precipitating, and drying to obtain the final product, i.e. starch polymer with core-shell-crown structure.
The molecular weight of the starch polymer having a core-shell-crown structure obtained in example 2 was determined to be 3.5X 108g/mol, particle size of 1080nm, crystallinity of 31 percent, transverse grain size of 9.5nm, and the proportion of indigestible nutrient fragments of 57 percent.
Example 3:
weighing molecular weight of 5.6 × 104Dissolving g/mol linear pea starch dextrin in a phosphate buffer salt solution system with the pH value of 6.0 to prepare a starch dextrin uniform solution with the mass concentration of 30%, and then adding 500U/g substrate of hydrolase family 13 multifunctional amylase (the sugar chain branching activity/depolymerization activity is 10.5) to the solution to perform constant temperature reaction at 60 ℃ for 36 hours; heating in 95 deg.C water bath for 60min, adding 200U/g substrate dextran sucrase, and reacting at 50 deg.C for 12 hr; heating to inactivate enzyme after reaction, and adjusting system temperature to 10 deg.CAfter 24 hours of storage, centrifugal treatment and precipitation drying are carried out to obtain the target product, namely the starch polymer with a core-shell-crown structure.
The molecular weight of the starch polymer having a core-shell-crown structure obtained in example 3 was determined to be 2.7X 108g/mol, particle size of 793nm, crystallinity of 25%, transverse grain size of 7.0nm, and indigestible nutrient fragment ratio as high as 62%.
Comparative example 1:
referring to example 1, the only difference is that the step of adding 60U/g substrate of hydrolase family 13 multifunctional amylase (sugar chain branching activity/depolymerization activity is 26.2) is omitted, and starch derivatives obtained when other conditions are not changed, namely:
weighing the molecular weight of 2.7X 104Dissolving the linear corn starch dextrin in a Tris-hydrochloride buffer solution system with the pH value of 7.0 in g/mol, preparing a starch dextrin uniform solution with the mass concentration of 10%, and then carrying out constant temperature treatment at 35 ℃ for 10 hours; heating in 95 deg.C water bath for 30min, adding 100U/g substrate dextran sucrase, and reacting at 40 deg.C for 5 hr; heating to inactivate enzyme after the reaction is finished, adjusting the temperature of the system to 4 ℃, storing for 10h, centrifuging, precipitating and drying to obtain the target product.
The molecular weight of the starch derivative obtained in comparative example 1 was determined to be 7.5X 104g/mol, particle size of 38nm, crystallinity of 4%, transverse grain size of 0.6nm, and indigestible nutrient fraction of only 26%.
Comparative example 2:
referring to example 1, the only difference is that the multifunctional amylase is derived from the hydrolase family 13, the sugar chain branching activity/depolymerization activity is adjusted to 53.6, and the starch derivative obtained when other conditions are not changed, namely:
weighing the molecular weight of 2.7X 104Dissolving g/mol linear corn amylodextrin in a Tris-hydrochloride buffer solution system with the pH value of 7.0 to prepare an amylodextrin uniform solution with the mass concentration of 10%, and then adding 60U/g substrate of hydrolase family 13 multifunctional amylase (the sugar chain branching activity/depolymerization activity is 53.6) into the solution to perform constant temperature reaction at 35 ℃ for 10 hours; heating in 95 deg.C water bath for 30min, and adding 100U/g substrate dextran sucrase 4Reacting for 5 hours at constant temperature of 0 ℃; heating to inactivate enzyme after the reaction is finished, adjusting the temperature of the system to 4 ℃, storing for 10h, centrifuging, precipitating and drying to obtain the target product.
The molecular weight of the starch derivative prepared in comparative example 2 was determined to be 2.6X 106g/mol, 54nm of particle size, 7 percent of crystallinity, 1.3nm of transverse grain size and 36 percent of indigestible nutrient fragment.
Comparative example 3:
with reference to example 1, the only difference is that the "dextran sucrase added 100U/g substrate" step is omitted, while the starch derivative obtained when the other conditions are unchanged, namely:
weighing the molecular weight of 2.7X 104Dissolving g/mol linear corn starch dextrin in a Tris-hydrochloride buffer solution system with the pH value of 7.0 to prepare a starch dextrin uniform solution with the mass concentration of 10%, and then adding 60U/g substrate of hydrolase family 13 multifunctional amylase (the sugar chain branching activity/depolymerization activity is 26.2) into the solution to perform constant temperature reaction for 10 hours at 35 ℃; heating in 95 deg.C water bath for 30min, and reacting at 40 deg.C for 5 hr; heating to inactivate enzyme after the reaction is finished, adjusting the temperature of the system to 4 ℃, storing for 10h, centrifuging, precipitating and drying to obtain the target product.
The molecular weight of the starch derivative obtained in comparative example 2 was determined to be 5.2X 107g/mol, particle size 71nm, crystallinity 0%, and indigestible fraction of 12%.
Comparative example 4:
referring to example 1, except that the amount of the multifunctional amylase used was changed from 60U/g substrate to 25U/g and 1000U/g, respectively, the corresponding starch derivative was prepared. The results of the properties of the starch derivative product obtained are shown in table 1.
TABLE 1 results of starch derivatives obtained with different amounts of multifunctional Amylase
Comparative example 5:
with reference to example 1, the only difference is that the corresponding starch derivatives were prepared by replacing the substrate with 100U/g of dextran sucrase by 10U/g and 500U/g, respectively. The results of the properties of the starch derivative product obtained are shown in table 2.
TABLE 2 results of starch derivatives obtained with different amounts of dextran sucrase
Example 4:
according to the special requirements of diabetics on carbohydrates, a special product for diabetics is designed and created by taking the starch polymer with the core-shell-crown structure prepared in the embodiment 1 of the invention as a main carbohydrate nutritional component, and the product is rich in indigestible starch nutritional fragments (> 65%) and has a postprandial glycemic index GI < 55.
Example 5:
aiming at the problems of low solubility, easy oxidation degradation and the like of the current fat-soluble plant compounds such as beta-carotene, curcumin and the like, the starch compound of phytochemicals is carried by evaluating, screening and combining the starch polymer with a specific core-shell-crown structure obtained in the embodiments 1 to 3 through the raw materials science, so that the content, the oxidation stability, the solubility and the bioavailability of the natural fat-soluble plant compounds are improved, and the bad flavor of the fat-soluble plant compounds can be improved.
The specific 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. The preparation method of the starch derivative is characterized by sequentially comprising the following steps of:
(1) dissolving linear amylodextrin in a buffer salt solution system with the pH value of 5.0-7.0 to obtain a linear amylodextrin solution with the mass concentration of 10-30%, adding 50-600U/g of multifunctional amylase, and reacting at the constant temperature of 35-70 ℃ for 6-48 h;
wherein the multifunctional amylase is derived from the family 13 or 57 of hydrolytic carbohydrases, having a sugar chain branching activity/depolymerizing activity < 30;
(2) after the reaction in the step (1) is finished, heating to inactivate enzyme, adding 30-200U/g glycosyltransferase into the mixture, and reacting for 2-12h at the constant temperature of 35-50 ℃;
(3) after the reaction in the step (2) is finished, heating to inactivate enzyme, adjusting the temperature of the system to 0-20 ℃, storing for 6-24h, centrifuging, taking the precipitate, and drying to obtain the target product, namely the starch polymer with the core-shell-crown structure.
2. The method for producing a starch derivative according to claim 1, wherein the glycosyltransferase is at least one of amylosucrase, dextran sucrase, maltotransglucosylase, dextran branching enzyme, and dextran phosphorylase.
3. The method for preparing a starch derivative according to claim 1, wherein the multifunctional amylase is used in an amount of 60U/g.
4. The method for producing a starch derivative according to claim 1 or 2, wherein the glycosyltransferase is used in an amount of 100U/g.
5. The method for producing a starch derivative according to claim 1, wherein the linear starch dextrin is a degradation product of a plant-derived starch, and the molecular weight of the linear starch dextrin is (1.0-20.0) x 104g/mol。
6. The method for preparing a starch derivative according to claim 5, wherein the plant-derived starch comprises at least one of cereal starch, potato starch and legume starch.
7. The method for preparing starch derivatives according to claim 1, wherein the buffered salt solution system is at least one of phosphate, citrate, acetate, Tris-hydrochloride, barbiturate.
8. The method for preparing a starch derivative according to claim 1, wherein the conditions for inactivating the enzyme in the step (2) by heating are as follows: heating in water bath at 95 deg.C for 30-60 min.
9. The starch derivative prepared by the method of any one of claims 1 to 8, wherein the starch derivative has a core-shell-crown structure in a micro-morphology and a molecular weight of (0.1-5.0) x 108g/mol, particle size of 200-2000nm, crystallinity of 10-35%, transverse grain size of 3-15nm, and indigestible nutrient fragment ratio>45％。
10. Use of starch derivatives according to claim 9 for the preparation of specialist foods, host-guest encapsulated carriers and biomedical materials.
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