CN113170895B - Preparation method of nano-selenium composite system and selenium supplement agent - Google Patents
Preparation method of nano-selenium composite system and selenium supplement agent Download PDFInfo
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- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 claims abstract description 38
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention discloses a preparation method of a nano-selenium composite system and a selenium supplement agent, wherein the preparation method of the nano-selenium composite system comprises the following steps: adding an ascorbic acid aqueous solution and a selenious acid aqueous solution into an aqueous solution of glucan nanoparticles, and stirring to form a nano-selenium mixed solution; and after the nano-selenium mixed solution is purified, adding rosmarinic acid, and stirring to form a nano-selenium composite system. According to the invention, the glucan nanoparticles and the rosmarinic acid are combined on the surface of the nano-selenium, so that the effects of synergistic dispersion and prevention of nano-selenium aggregation can be achieved, the stability of the composite system is improved, and meanwhile, the glucan and the rosmarinic acid have physiological activities of oxidation resistance, tumor resistance and the like, so that the composite system has the functions of oxidation resistance and tumor resistance, and the glucan and the rosmarinic acid are used as selenium supplement components, so that the effect of the selenium supplement is enriched, and the composite system has a better health care effect.
Description
Technical Field
The invention relates to the technical field of nutritional supplements, in particular to a preparation method of a nano-selenium composite system and a selenium supplement.
Background
Selenium is an important nutritional mineral and has great significance to human health, and in humans, 25 genes encoding selenoproteins have been identified. It is also a cofactor for a number of selenium-dependent enzymes, such as the antioxidant glutathione peroxidase (GSH-Px). Maintenance of cellular redox homeostasis depends largely on the selenoprotein group, which in turn depends indirectly on selenium supplementation.
Diet is a major source of selenium for the general population, but due to the low selenium content in crops and soil, human selenium intake is low in many areas of the world, and governments in some areas have begun various selenium supplementation programs that appear to be the best way to compensate for this problem.
The chemical form, redox state and dosage of selenium are reported to be major factors in determining its bioavailability, toxicity and biological properties. Compared to traditional selenium supplements, elemental (zero-valent) Se nanoparticles have high Se density and unique nanoscale properties (size, shape, surface properties, solubility, and chemical composition), can facilitate efficient drug delivery, and are low in cytotoxicity. It is very unstable and easily converts to an inactive form.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a nano-selenium composite system, aiming at solving the problem of poor stability of element (zero-valent) Se nano-particles.
In order to achieve the above object, the present invention provides a method for preparing a nano-selenium composite system, which comprises the following steps:
adding an ascorbic acid aqueous solution and a selenious acid aqueous solution into an aqueous solution of glucan nanoparticles, and stirring to form a nano-selenium mixed solution;
and after the nano-selenium mixed solution is purified, adding rosmarinic acid, and stirring to form a nano-selenium composite system.
Optionally, the weight ratio of the selenious acid to the glucan nanoparticles to the rosmarinic acid is 16-81: 70-85: 15-20.
Optionally, the concentration of the selenious acid aqueous solution is 12.5-62.5 mmol/L.
Optionally, the amount of the ascorbic acid substance is 2.8-3.2 times of the amount of the selenious acid substance.
Optionally, after the nano-selenium mixed solution is purified, rosmarinic acid is added, and in the step of stirring to form a nano-selenium composite system, a dialysis bag with a molecular weight of 3000-4000 Da is used to perform dialysis purification on the nano-selenium mixed solution.
Optionally, before the step of adding ascorbic acid and sodium selenite into the aqueous solution of glucan nanoparticles and stirring to form the nano-selenium mixed solution, the method further comprises the following steps:
dispersing corn starch into a buffer system with the pH value of 4.4-4.8 to obtain a dispersion liquid;
adding saccharifying enzyme into the dispersion liquid, carrying out enzymolysis treatment, and separating and purifying enzymolysis liquid to obtain particles;
and (3) dissolving the particles in water, and homogenizing under high pressure to enable the particle size of the particles to be 200-300 nm, so as to obtain the aqueous solution of the glucan nanoparticles.
Optionally, 0.05 × 10 per gram of corn starch is added4～0.5×104U saccharifying enzyme.
Optionally, in the step of dispersing the corn starch into a buffer system with a pH value of 4.4-4.8 to obtain the dispersion liquid, the buffer system is a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 4.6.
Optionally, the step of adding saccharifying enzyme into the dispersion, performing enzymolysis treatment, and separating and purifying an enzymolysis solution to obtain particles comprises:
adding saccharifying enzyme into the dispersion liquid, stirring and uniformly mixing at 55-65 ℃, then adjusting the pH to 9.5-10.5 by using a sodium hydroxide solution, and standing for 10-16 hours to obtain an enzymolysis liquid;
centrifuging the enzymolysis liquid at the rotating speed of 5500-6500 rpm;
taking the supernatant, and dialyzing for 22-32 h by using a dialysis bag with the molecular weight not less than 3500 Da;
and (3) freezing the concentrated solution at-18 to-25 ℃ for 10 to 14 hours, and then freeze-drying to obtain particles.
Optionally, in the step of taking the particles to dissolve in water, and homogenizing under high pressure so that the particle size of the particles is 200-300 nm to obtain the aqueous solution of the glucan nanoparticles, homogenizing under high pressure for 25-35 min under 580-620 bar.
In order to achieve the above object, the present invention also provides a selenium supplement, which comprises the nano-selenium composite system prepared by the above preparation method of the nano-selenium composite system.
According to the technical scheme provided by the invention, the glucan nanoparticles and the rosmarinic acid are combined on the surface of the nano-selenium, so that the effects of synergistic dispersion and prevention of aggregation of the nano-selenium can be achieved, the stability of a composite system is improved, and meanwhile, the glucan and the rosmarinic acid have the physiological activities of oxidation resistance, tumor resistance and the like, so that the composite system has the functions of oxidation resistance and tumor resistance, and as a selenium supplement component, the effect of the selenium supplement is enriched, and the selenium supplement has a better health care effect. In addition, the method has simple and mild reaction conditions and is easy to industrialize, and the rosmarinic acid is added after purification, so that the ascorbic acid is prevented from damaging the antioxidant function of the rosmarinic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow chart of an embodiment of a method for preparing a nano-selenium complex system provided by the present invention;
FIG. 2 is a scanning electron microscope image of the nano-selenium composite system prepared in example 1;
FIG. 3 is a perspective electron microscope image of the nano-selenium composite system prepared in example 1;
FIG. 4 is a spectrum diagram of the nano-selenium composite system prepared in example 1;
fig. 5 is an energy spectrum analysis diagram of the nano-selenium composite system prepared in example 1, wherein a is a carbon spectrum, b is an oxygen spectrum, and c is a selenium spectrum;
FIG. 6 is an infrared spectrum of the nano-selenium composite system, rosmarinic acid, and dextran nanoparticles prepared in example 1;
FIG. 7 is a graph of the time stability of the nano-selenium composite system prepared in example 1;
fig. 8 is a graph of the time stability of the dextran nanoparticle-nano selenium composite system prepared in comparative example 1;
FIG. 9 is a pH stability diagram of the nano-selenium composite system prepared in example 1;
fig. 10 is a pH stability graph of the dextran nanoparticle-nano selenium composite system prepared in comparative example 1;
FIG. 11 is a graph of the temperature stability of the nano-selenium composite system prepared in example 1;
fig. 12 is a graph of the temperature stability of the dextran nanoparticle-nano selenium composite system prepared in comparative example 1;
fig. 13 is an equivalent graph of DPPH clearance for the nano-selenium composite system prepared in example 1.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B", including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of a nano-selenium composite system, which constructs the nano-selenium composite system with high stability and antioxidant and antitumor functions. Fig. 1 is a diagram illustrating an embodiment of a method for preparing a nano-selenium composite system according to the present invention.
Referring to fig. 1, the preparation method of the nano-selenium composite system includes the following steps:
step S10, adding ascorbic acid and selenious acid aqueous solution into the aqueous solution of the glucan nano particles, and stirring to form a nano selenium mixed solution.
In this embodiment, an aqueous solution of ascorbic acid and selenious acid is added to an aqueous solution of dextran nanoparticles, tetravalent selenium is reduced to selenium under the reducing action of ascorbic acid, and meanwhile, the dextran nanoparticles are combined with the selenium surface, so that selenium aggregation is prevented, and nano-selenium is stably present in the mixed solution.
Wherein the selenious acid aqueous solution can be obtained by dissolving selenious acid in water or selenium dioxide in water; the aqueous solution of dextran nanoparticles is obtained by dissolving dextran nanoparticles in water, which are either commercially available or prepared by themselves. In one embodiment, the preparation of dextran nanoparticles may be performed as follows:
and S101, dispersing corn starch into a buffer system with the pH value of 4.4-4.8 to obtain a dispersion liquid.
The method takes the corn starch as the raw material, the material is nontoxic and edible, the biocompatibility is good, and the starch resource is reasonably utilized. In the embodiment, the corn starch is dispersed into a buffer system with the pH value of 4.4-4.8, so that a proper pH environment is provided for subsequent enzymolysis. The buffer system of pH4.4 to 4.8 may be a citric acid-disodium hydrogen phosphate buffer solution of pH4.6, and hereinafter, a citric acid-disodium hydrogen phosphate buffer solution of pH4.6 will be described as an example.
And S102, adding saccharifying enzyme into the dispersion liquid, carrying out enzymolysis treatment, and separating and purifying enzymolysis liquid to obtain particles.
The addition amount of the saccharifying enzyme is based on the enzyme activity and the amount of the corn starch, and in the embodiment, 0.1-1mL of saccharifying enzyme with the concentration of 10 ten thousand U/mL is correspondingly added to each gram of corn starch.
Specifically, step S102 may include the steps of:
step S1021, adding saccharifying enzyme into the dispersion, stirring and uniformly mixing at 55-65 ℃, then adjusting the pH to 9.5-10.5 by using a sodium hydroxide solution, and standing for 10-16 hours to obtain an enzymolysis solution;
step S1022, centrifuging the enzymolysis liquid at the rotating speed of 5500-6500 rpm;
step S1023, taking the supernatant, and dialyzing for 22-32 h by using a dialysis bag with the molecular weight not less than 3500 Da;
and step S1024, freezing the concentrated solution at-18 to-25 ℃ for 10 to 14 hours, and then freeze-drying to obtain particles.
And S103, dissolving the particles in water, and homogenizing under high pressure to ensure that the particle size of the particles is 200-300 nm to obtain the aqueous solution of the glucan nanoparticles.
In order to ensure that the glucan nanoparticles and the nano-selenium are well dispersed after being combined, the particle size of the glucan nanoparticles is preferably 200-300 nm, and therefore after the particles are obtained, the particles need to be further processed so that the particle size distribution of the particles meets the requirement, the embodiment adopts a high-pressure homogenization mode to ensure that the particle size of the particles is 200-300 nm, and thus the aqueous solution of the glucan nanoparticles is obtained. In the specific implementation, the high-pressure homogenization is carried out under the conditions of 580-620 bar and 25-35 min.
And step S20, after the nano-selenium mixed solution is purified, adding rosmarinic acid, and stirring to form a nano-selenium composite system.
In this embodiment, the nano-selenium mixed solution prepared in step S10 is purified to remove ascorbic acid, and then rosmarinic acid is added and stirred to react, so as to obtain a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Wherein the weight ratio of the selenious acid to the glucan nanoparticles to the rosmarinic acid is 16-81: 70-85: 15-20; the concentration of the selenious acid aqueous solution is 12.5-62.5 mmol/L; the amount of the ascorbic acid substance is 2.8-3.2 times of that of the selenious acid substance.
In specific implementation, the nano selenium mixed solution can be purified by dialysis, specifically, the nano selenium mixed solution is placed in a dialysis bag with the molecular weight of 3000-4000 Da for dialysis for 24 hours, and substances such as ascorbic acid and the like which are not reacted completely and contained in the nano selenium mixed solution can be removed, so that the situation that the ascorbic acid destroys the antioxidant function of the rosmarinic acid is avoided.
According to the technical scheme provided by the invention, the glucan nanoparticles and the rosmarinic acid are combined on the surface of the nano-selenium, so that the effects of synergistic dispersion and prevention of aggregation of the nano-selenium can be achieved, the stability of a composite system is improved, and meanwhile, the glucan and the rosmarinic acid have the physiological activities of oxidation resistance, tumor resistance and the like, so that the composite system has the functions of oxidation resistance and tumor resistance, and as a selenium supplement component, the effect of the selenium supplement is enriched, and the selenium supplement has a better health care effect. In addition, the method has simple and mild reaction conditions and is easy to industrialize, and the rosmarinic acid is added after purification, so that the ascorbic acid is prevented from damaging the antioxidant function of the rosmarinic acid.
In addition, the invention also provides a selenium supplement, which comprises the nano selenium composite system prepared by the preparation method of the nano selenium composite system.
In this embodiment, the nano-selenium composite system can be used alone as a selenium supplement, and can also be mixed with other conventional auxiliary materials, additives and the like in the field to form the selenium supplement. Because the nano-selenium composite system contains the stably existing nano-selenium, and the raw materials of the nano-selenium composite system are substances with good biocompatibility, such as glucan and the like, the nano-selenium composite system has the advantages of good selenium supplementing effect, high bioavailability, small cytotoxicity and the like when being used as a selenium supplementing agent component; meanwhile, the nano selenium composite system contains glucan nano particles and rosmarinic acid which have synergistic effect, so that the selenium supplement has the functions of oxidation resistance and tumor resistance, and has better health care effect.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And (3) putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 12.5mM of ascorbic acid and substance in an amount of 0.066g, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution for 24h by using a 3500Da dialysis bag, adding 18mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to prepare a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
The results of observing the glucan nanoparticle-rosmarinic acid-nano selenium composite system by an electron microscope are shown in fig. 2 and fig. 3, and as can be seen from fig. 2 and fig. 3, the nano selenium is uniform and spherical and is well dispersed by the glucan nanoparticle and rosmarinic acid, which indicates that the composite system has good dispersibility.
The dextran nanoparticle-rosmarinic acid-nano selenium composite system was scanned by an energy spectrometer to analyze the element types, and the results are shown in fig. 4 and 5. The existence of selenium can be seen from fig. 4, and in combination with fig. 5, oxygen and carbon exist on the surface of the nano-selenium, which indicates that glucan nanoparticles and rosmarinic acid are combined on the surface of the nano-selenium.
An infrared spectrometer is adopted to detect the glucan nanoparticle-rosmarinic acid-nano selenium composite system, the glucan nanoparticle and the rosmarinic acid, and the spectrogram is shown in figure 6, wherein a is the infrared spectrogram of the rosmarinic acid, b is the infrared spectrogram of the glucan nanoparticle, and c is the infrared spectrogram of the glucan nanoparticle-rosmarinic acid-nano selenium composite system. As can be seen from the figure, the rosmarinic acid and glucan nanoparticles are 1080cm in length-1The characteristic peak of C-OH appears weakening phenomenon in a glucan nanoparticle-rosmarinic acid-nano selenium composite system, which shows that the hydroxyl is combined with selenium, and proves that the glucan nanoparticle and the rosmarinic acid are combined with the nano selenium through selenium-oxygen bonds, so that the dispersion effect is achieved.
Example 2
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And (3) putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.132g of ascorbic acid and 25mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution with a 3500Da dialysis bag for 24h, adding 18mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to obtain a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Example 3
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And (3) putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.198g of ascorbic acid and 37.5mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution for 24h by using a 3500Da dialysis bag, adding 18mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to prepare a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Example 4
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.264g of ascorbic acid and 50mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution with 3500Da dialysis bag for 24h, adding 18mg of rosmarinic acid, and magnetically stirring at constant speed of 200rpm for 10min to obtain dextran nanoparticle-rosmarinic acid-nano-selenium composite system.
Example 5
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And (3) putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.33g of ascorbic acid and 37.5mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution for 24h by using a 3500Da dialysis bag, adding 18mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to prepare a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Example 6
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 65 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 9.5, and the mixture was allowed to stand for 10 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 5500rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 32h, freezing at-18 deg.C for 10h, and freeze-drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 580bar under high pressure for 35min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
87mL of the prepared glucan nanoparticle solution was put in a beaker, and 0.066g of ascorbic acid and 10mL of selenious acid aqueous solution with the substance amount concentration of 37.5mM were sequentially added, and magnetic stirring was performed at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution for 24h by using a 4000Da dialysis bag, adding 15mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to prepare a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Example 7
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 55 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10.5, and the mixture was allowed to stand for 16 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6500rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 22 hr, freezing at-25 deg.C for 14 hr, and freeze drying. Dissolving 0.24g of freeze-dried particles into a beaker filled with 300mL of deionized water, and homogenizing at 620bar under high pressure for 25min to obtain a glucan nanoparticle solution (the particle size of the particles is 200-300 nm).
And (3) putting 100mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.211g of ascorbic acid and 62.5mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. Dialyzing the prepared nano-selenium mixed solution for 24h by using a 3000Da dialysis bag, adding 20mg of rosmarinic acid, and magnetically stirring at a constant speed of 200rpm for 10min to prepare a glucan nanoparticle-rosmarinic acid-nano-selenium composite system.
Comparative example 1
First, 20g of corn starch was weighed, added to a beaker containing 100mL of a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6, magnetically stirred at a constant speed until the corn starch was uniformly dispersed, 0.1 to 1mL of 10 ten thousand U/mL of saccharifying enzyme was added, and magnetically stirred at a constant speed of 150rpm at 60 ℃ for 2 hours. Then, 4mol/L sodium hydroxide was added to adjust the pH to 10, and the mixture was allowed to stand for 12 hours to obtain an enzymatic hydrolysate. Centrifuging the enzymolysis solution at 6000rpm for 10 min; transferring the supernatant into 3500Da dialysis bag, dialyzing for 24h, freezing at-20 deg.C for 12h, and freeze-drying. Dissolving 0.24g of the freeze-dried particles in a beaker filled with 300mL of deionized water, and homogenizing at 600bar under high pressure for 30min to obtain the glucan nanoparticle solution.
And (3) putting 90mL of the prepared glucan nanoparticle solution into a beaker, sequentially adding 10mL of selenious acid aqueous solution with the concentration of 0.185g of ascorbic acid and 12.5mM of substance, and magnetically stirring at a constant speed of 300rpm for 10min to form a nano-selenium mixed solution. And dialyzing the prepared nano-selenium mixed solution for 24 hours by using a 3500Da dialysis bag to obtain a glucan nano-particle-nano-selenium composite system.
First, stability survey
(1) Stability over time
The glucan nanoparticle-rosmarinic acid-nano selenium composite system prepared in examples 1 to 7 and the glucan nanoparticle-nano selenium composite system prepared in comparative example 1 were taken, left to stand, and the particle size distribution of the composite system was measured at different time points using a malvern particle sizer. For space saving, the results of comparing the time stability of the nano-selenium composite system prepared in example 1 with that of the dextran nanoparticle-nano-selenium composite system are shown in fig. 7 and 8, and the stability data of other examples are shown in table 1.
TABLE 1 stability over time
As can be seen from table 1 and fig. 7 above, the average particle size of the nano-selenium of each example is stabilized at about 100 nm within 25 days, and has no obvious change, while in fig. 8, the particle size distribution of the glucan nanoparticle-nano-selenium composite system prepared in comparative example 1 fluctuates greatly with time, which indicates that the glucan nanoparticle-rosmarinic acid-nano-selenium composite system prepared by the method of the present invention has good stability with time, and is obviously superior to the glucan nanoparticle-nano-selenium composite system.
(2) Stability of pH
The dextran nanoparticle-rosmarinic acid-nano selenium composite system prepared in examples 1 to 7 and the dextran nanoparticle-nano selenium composite system prepared in comparative example 1 were taken, the pH of the system was adjusted, and the particle size distribution of the composite system was measured at different pH using a malvern particle sizer. For space saving, the results of comparing the pH stability of the nano-selenium composite system prepared in example 1 with that of the dextran nanoparticle-nano-selenium composite system are shown in fig. 9 and 10, and the stability data of other examples are shown in table 2.
TABLE 2 pH stability
As can be seen from table 2 and fig. 9 above, the average particle size stability of the nano-selenium of each example is good in the environment of pH 1.4-9.4, while in fig. 10, the particle size distribution of the glucan nanoparticle-nano-selenium composite system prepared in comparative example 1 fluctuates greatly with the change of pH, which indicates that the glucan nanoparticle-rosmarinic acid-nano-selenium composite system prepared by the method of the present invention has good pH stability and is significantly superior to the glucan nanoparticle-nano-selenium composite system.
(3) Temperature stability
The dextran nanoparticle-rosmarinic acid-nano selenium composite system prepared in examples 1 to 7 and the dextran nanoparticle-nano selenium composite system prepared in comparative example 1 were taken, the temperature of the systems was adjusted, and the particle size distribution of the composite systems was measured at different temperatures using a malvern particle sizer. For economy of space, the results of comparing the temperature stability of the nano-selenium composite system prepared in example 1 with that of the dextran nanoparticle-nano selenium composite system are shown in fig. 11 and 12, and the stability data of other examples are shown in table 3.
TABLE 3 temperature stability
As can be seen from table 3 and fig. 11 above, the average particle size stability of the nano-selenium in each example is good in the environment of 4-60 ℃, while in fig. 12, the particle size of the glucan nanoparticle-nano-selenium composite system prepared in comparative example 1 is obviously changed when the temperature reaches 60 ℃, which indicates that the glucan nanoparticle-rosmarinic acid-nano-selenium composite system prepared by the method of the present invention has good temperature stability and is obviously superior to the glucan nanoparticle-nano-selenium composite system.
Second, synergistic antioxidant effect verification test
The method comprises the following steps: taking a polyphenol or polysaccharide nano selenium system (RA rosmarinic acid, Glucan-Senps Glucan nanoparticle-nano selenium composite system, Glucan-RA-Senps nano selenium composite system) with a certain volume, diluting 4-256 times with deionized water, mixing 1mL of samples with different concentrations with 1mL of DPPH solution (0.2mM dissolved in 95% ethanol), shaking up for 3 minutes, and incubating in a dark box at room temperature for 30 minutes. The absorbance was measured by UV-visible spectrophotometry at a wavelength of 517nm using a 50% ethanol solution as a control. The results are shown in FIG. 13.
As a result: the concentrations (IC50) of Glucan-SeNPs and RA for eliminating 50% DPPH free radicals are 107.5 mug/mL and 6.38 mug/mL respectively, and the clearance rate of Glucan-RA-SeNPs to 50% needs 6.41 mug/mL (calculated by Se), wherein RA is 4.86 mug/mL and is lower than the IC50 concentrations of Glucan-SeNPs and RA, which shows that SeNPs and RA show synergistic antioxidation effect on DPPH free radical elimination.
The above are only preferred embodiments of the present invention, and do not limit the scope of the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (8)
1. The preparation method of the nano-selenium composite system is characterized by comprising the following steps of:
adding an ascorbic acid aqueous solution and a selenious acid aqueous solution into an aqueous solution of glucan nanoparticles, and stirring to form a nano-selenium mixed solution;
after the nano-selenium mixed solution is purified, adding rosmarinic acid, and stirring to form a nano-selenium composite system;
wherein the weight ratio of the selenious acid to the glucan nanoparticles to the rosmarinic acid is 16-81: 70-85: 15-20; the amount of the ascorbic acid substance is 2.8-3.2 times of that of the selenious acid substance.
2. The method for preparing the nano-selenium composite system according to claim 1, wherein the concentration of the selenious acid aqueous solution is 12.5-62.5 mmol/L.
3. The method of claim 1, wherein the step of purifying the nano-selenium mixed solution, adding rosmarinic acid, and stirring to form the nano-selenium composite system comprises dialyzing and purifying the nano-selenium mixed solution with a dialysis bag having a molecular weight of 3000-4000 Da.
4. The method of claim 1, wherein the step of adding ascorbic acid and sodium selenite to the aqueous solution of dextran nanoparticles and stirring to form the nano-selenium mixed solution further comprises:
dispersing corn starch into a buffer system with the pH value of 4.4-4.8 to obtain a dispersion liquid;
adding saccharifying enzyme into the dispersion liquid, carrying out enzymolysis treatment, and separating and purifying enzymolysis liquid to obtain particles;
and (3) dissolving the particles in water, and homogenizing under high pressure to enable the particle size of the particles to be 200-300 nm, so as to obtain the aqueous solution of the glucan nanoparticles.
5. The method of claim 4, wherein the step of dispersing corn starch in a buffer system having a pH of 4.4 to 4.8 to obtain a dispersion comprises adding a citric acid-disodium hydrogen phosphate buffer solution having a pH of 4.6 to the corn starch.
6. The method of claim 4, wherein the step of adding saccharifying enzyme to the dispersion, performing enzymolysis treatment, separating and purifying the enzymolysis solution to obtain particles comprises:
adding saccharifying enzyme into the dispersion liquid, stirring and uniformly mixing at 55-65 ℃, then adjusting the pH to 9.5-10.5 by using a sodium hydroxide solution, and standing for 10-16 hours to obtain an enzymolysis liquid;
centrifuging the enzymolysis liquid at the rotating speed of 5500-6500 rpm;
taking the supernatant, and dialyzing for 22-32 h by using a dialysis bag with the molecular weight not less than 3500 Da;
and (3) freezing the concentrated solution for 10-14 h, and then freeze-drying to obtain particles.
7. The method for preparing the nano-selenium composite system of claim 4, wherein in the step of taking the particles to dissolve in water, and homogenizing under high pressure so that the particle size of the particles is 200-300 nm to obtain the aqueous solution of the glucan nanoparticles, the aqueous solution is homogenized under high pressure for 25-35 min under the condition of 580-620 bar.
8. A selenium supplement comprising the nano-selenium composite system obtained by the method for preparing a nano-selenium composite system according to any one of claims 1 to 7.
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