CN115089605A - Preparation method of auricularia polytricha polysaccharide nano-selenium compound - Google Patents
Preparation method of auricularia polytricha polysaccharide nano-selenium compound Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering, and discloses a preparation method of auricularia polytricha polysaccharide nano-selenium compound, which comprises the following steps: s1, fully and uniformly mixing the auricularia polytricha polysaccharide solution and the sodium selenite solution, adding ascorbic acid solution, and carrying out water bath reaction to obtain reaction liquid; s2, dialyzing the reaction solution obtained in the step 1 to obtain a auricularia polytricha polysaccharide nano selenium compound solution; s3, freeze-drying the auricularia polytricha polysaccharide nano-selenium compound solution obtained in the step 2 into powder solid to obtain the auricularia polytricha polysaccharide nano-selenium compound. The auricularia polytricha polysaccharide nano-selenium compound prepared by the process has the advantages of small particle size, low toxicity, good stability and stronger oxidation resistance.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a preparation method of auricularia polytricha polysaccharide nano-selenium compound.
Background
Selenium is one of the essential trace elements and is also a constituent element and active center of various enzymes in the human body, such as selenoprotein and selenase. Selenium can play an important role in the synthesis of certain enzymes that scavenge free radicals and can act as an antioxidant to protect cells and tissues from damage. The biological active dose of selenium is extremely narrow, excessive selenium can cause selenium poisoning of a human body, and the lack of selenium in the human body can cause various diseases such as cardiovascular diseases, keshan disease, osteopathy, diabetes and the like. Nano-selenium (SeNPs) has the characteristics of high activity, high safety, high bioavailability and the like, and is called selenium supplement with minimum toxicity. However, nano-selenium is generally unstable and tends to aggregate into black or gray elemental selenium, which also results in a reduction in its bioavailability. Therefore, the template with biological activity is added to be used as a stabilizer to prepare the selenium supplement with small particle size, low toxicity and high biological activity, and the selenium supplement is a breakthrough for developing a high-efficiency and safe selenium supplement.
The auricularia polytricha polysaccharide is a main active ingredient in auricularia polytricha. Has anticoagulant, antiinflammatory, antioxidant, antibacterial, radioprotective, mutation resisting, blood sugar lowering, and immunity enhancing effects. The auricularia polytricha polysaccharide is a non-toxic natural product with various high biological activities, so that the related process conditions for preparing the nano selenium by using the auricularia polytricha polysaccharide as a template regulator need to be researched.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a preparation method of a auricularia polytricha polysaccharide nano-selenium compound, which has the advantages that the preparation process is simple, fast and strong in operability, the prepared nano-selenium has small particle size, the average particle size can reach 66nm, the particle size does not change obviously and does not generate precipitates after being stored for 28 days at 4 ℃, the oxidation resistance of the auricularia polytricha polysaccharide nano-selenium compound is greatly improved compared with the auricularia polytricha polysaccharide, and the problem that the nano-selenium is generally unstable and is easy to aggregate into black or gray element selenium, so that the bioavailability of the auricularia polytricha polysaccharide nano-selenium compound is reduced is solved.
(II) technical scheme
In order to realize the simple, fast and strong operability of the preparation process, the prepared nano selenium has small particle size, the average particle size can reach 66nm, the particle size does not change obviously and does not generate precipitate after being stored for 28 days at the temperature of 4 ℃, and the antioxidant capacity of the auricularia polytricha polysaccharide nano selenium compound is greatly improved compared with the auricularia polytricha polysaccharide.
The invention provides the following technical scheme:
a preparation method of an auricularia polytricha polysaccharide nano-selenium compound comprises the following steps:
s1 preparation of reaction liquid
Fully and uniformly mixing the auricularia polytricha polysaccharide solution and the sodium selenite solution, further adding an ascorbic acid solution, and carrying out water bath reaction to obtain a reaction liquid;
s2, preparing auricularia polytricha polysaccharide nano selenium compound solution
Dialyzing the reaction solution obtained in the step 1 to obtain a auricularia polytricha polysaccharide nano selenium compound solution;
s3, obtaining the auricularia polytricha polysaccharide nano selenium compound
And (3) freeze-drying the auricularia polytricha polysaccharide nano selenium compound solution obtained in the step (2) into powdery solid to obtain the auricularia polytricha polysaccharide nano selenium compound.
Preferably, in the step 1, the concentration of the auricularia polytricha polysaccharide is 50-150 mg/L, the concentration of the sodium selenite solution is 0.25-1 mmol/L, and the concentration of the ascorbic acid solution is 2-3 mmol/L.
Preferably, in the step 1, the reaction temperature is 25-50 ℃ and the reaction time is 0.5-1.5 h.
Preferably, in the step 2, the dialysis process includes transferring the reaction solution into a 3000Da dialysis bag, and continuously dialyzing for 48-50 h at 4 ℃ in a dark environment.
Preferably, in step S1, auricularia polytricha polysaccharide is used as a stabilizer, ascorbic acid is used as a reducing agent, and sodium selenite is used as a selenium source.
Preferably, in step S3, the grain size of the auricularia polytricha polysaccharide nano-selenium complex is characterized by a dual-wavelength colorimetry.
Preferably, the characterization is carried out by measuring absorbance of the auricularia polytricha polysaccharide nano-selenium complex solution at the wavelength of 410nm and 490nm through an ultraviolet spectrophotometer according to the change of the colloid particle size: the larger the ratio of A410/A490 is, the smaller the colloid particle size is, so as to judge the particle size of the auricularia polytricha polysaccharide nano-selenium.
Preferably, in the step S3, the auricularia polytricha polysaccharide nano-selenium complex solution is placed in a freezing tank, and then liquid nitrogen is injected into the freezing tank, so that freeze drying can be performed.
(III) advantageous effects
Compared with the prior art, the preparation method of the auricularia polytricha polysaccharide nano selenium compound provided by the invention has the following beneficial effects:
1. according to the preparation method of the auricularia polytricha polysaccharide nano-selenium compound, ascorbic acid is used as a reducing agent, auricularia polytricha polysaccharide is used as a stabilizing agent, the preparation process is simple, fast and strong in operability, the prepared nano-selenium has small particle size, the average particle size can reach 66nm, the particle size does not change obviously and does not generate precipitates after being stored for 28 days at 4 ℃, and the bioavailability is effectively improved.
2. According to the preparation method of the auricularia polytricha polysaccharide nano-selenium compound, compared with auricularia polytricha polysaccharide, the antioxidative capacity of the auricularia polytricha polysaccharide nano-selenium compound is greatly improved, and the overall effect is further improved.
Drawings
FIG. 1 is a graph showing the effect of Auricularia polytricha polysaccharide concentration on the particle size of nanometer selenium in Auricularia polytricha polysaccharide in example 1;
FIG. 2 is a graph showing the effect of sodium selenite and ascorbic acid on the size of nano-selenium particles in Auricularia polytricha in example 1;
FIG. 3 is a graph showing the effect of reaction temperature on the particle size of nano-selenium in Auricularia polytricha in example 1;
FIG. 4 is a graph showing the effect of reaction time on the particle size of nano-selenium in Auricularia polytricha in example 1;
FIG. 5 is a TEM image of the nano-selenium complex in example 2
FIG. 6 is a schematic diagram showing the particle size change of Auricularia polytricha polysaccharide nano-selenium complex for 28 days at 4 ℃ in example 2
FIG. 7 is a comparison of Auricularia polytricha polysaccharide nano-selenium complex and nano-selenium (without Auricularia polytricha polysaccharide) stored for 28 days in example 2.
FIG. 8 is a graph comparing the ability of Auricularia polytricha polysaccharide nano-selenium complexes to scavenge DPPH free radicals in example 3;
FIG. 9 is a graph comparing the ABTS free radical scavenging ability of Auricularia polytricha polysaccharide nano-selenium complex in example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
A preparation method of auricularia polytricha polysaccharide nano-selenium compound comprises the following steps:
s1 preparation of reaction liquid
Fully and uniformly mixing the auricularia polytricha polysaccharide solution and the sodium selenite solution, further adding an ascorbic acid solution, and carrying out water bath reaction to obtain a reaction liquid;
s2, preparing auricularia polytricha polysaccharide nano selenium compound solution
Dialyzing the reaction solution obtained in the step 1 to obtain a auricularia polytricha polysaccharide nano selenium compound solution;
s3, obtaining the auricularia polytricha polysaccharide nano selenium compound
And (3) freeze-drying the auricularia polytricha polysaccharide nano-selenium compound solution obtained in the step (2) into powder solid to obtain the auricularia polytricha polysaccharide nano-selenium compound, specifically, placing the auricularia polytricha polysaccharide nano-selenium compound solution in a freezing tank, and then filling liquid nitrogen into the tank to carry out freeze-drying.
The first embodiment is as follows:
the preparation method of the auricularia polytricha polysaccharide nano selenium compound provided by the embodiment specifically comprises the following steps:
adding ascorbic acid and sodium selenite into the auricularia polytricha polysaccharide solution, obtaining the auricularia polytricha polysaccharide nano-selenium compound through water bath reaction, and respectively exploring a plurality of different proportions and process conditions for preparing the auricularia polytricha polysaccharide nano-selenium compound as follows:
(1) influence of auricularia polytricha polysaccharide concentration on nano-selenium: the nanometer selenium complex of auricularia polytricha polysaccharide is prepared by different auricularia polytricha polysaccharide concentrations (25, 50, 100, 200, 300, 400, 500mg/L), and the particle size of the nanometer selenium complex is characterized by a double-wavelength colorimetry, and the particle size of the nanometer selenium complex is the smallest when the auricularia polytricha polysaccharide concentration is 50mg/L as shown in figure 1.
(2) Effect of molar ratio of sodium selenite and ascorbic acid on nano-selenium: the auricularia polytricha polysaccharide nano selenium complex is prepared by adjusting the molar ratio of ascorbic acid to sodium selenite (1:1, 1:2, 1:3, 1:4 and 1:5), and the particle size of the auricularia polytricha polysaccharide nano selenium complex is characterized by a dual-wavelength colorimetry method, and the results shown in figure 2 show that the particle size of the nano selenium complex is the smallest when the molar ratio of sodium selenite to ascorbic acid is 1: 6.
(3) Influence of reaction temperature on nano-selenium: the auricularia polytricha polysaccharide nano selenium compound is prepared by changing the reaction temperature (20, 30, 40, 60 and 80 ℃), and the particle size of the auricularia polytricha polysaccharide nano selenium compound is characterized by a double-wavelength colorimetry, and the particle size of the nano selenium compound is the smallest when the reaction temperature is 60 ℃ as shown in figure 3.
(4) Influence of reaction time on the nano-selenium: the Auricularia polytricha polysaccharide nano selenium complex is prepared by changing the reaction time (0.25, 0.5, 1, 3, 5, 7h), and the particle size of the Auricularia polytricha polysaccharide nano selenium complex is characterized by a dual wavelength colorimetry, and the particle size of the nano selenium complex is the smallest when the reaction time is 1h, as shown in figure 4.
(5) The orthogonal experiment was performed based on the one-factor results, the orthogonal design and results are shown in table 1. As can be seen from Table 1, the optimal conditions for preparing the Auricularia polytricha polysaccharide nano-selenium complex are that the Auricularia polytricha polysaccharide concentration is 100mg/L, the molar ratio of sodium selenite to ascorbic acid is 1:5, the reaction temperature is 40 ℃, and the reaction time is 1 h. The results of the orthogonal experiments were verified by repeating the experiments three times, and the results showed that the A410/A490 ratio was approximately 1.98. The values obtained by the orthogonal experiment, and therefore the conditions optimized by the orthogonal experiment are valid.
TABLE 1 analysis of Auricularia polytricha polysaccharide nanocomplex orthogonal experimental design results
Example two:
the auricularia polytricha polysaccharide nano selenium compound is prepared by utilizing the conditions and is characterized by means such as a transmission electron microscope. As shown in FIG. 6, the Auricularia polytricha polysaccharide nano-selenium compound is observed under a transmission electron microscope, and is a sphere with a smooth surface, the particle size is 20-82 nm, and the average particle size is 66 nm. The prepared auricularia polytricha polysaccharide nano selenium compound is placed in an environment with the temperature of 4 ℃ for storage, the grain diameter of the compound is represented by a double-wavelength colorimetry every 7 days, and as can be seen from figure 7, the ratio of A410 to A490 does not change obviously. Fig. 8 is a comparison graph of 0 day and 28 days of the auricularia polytricha nano selenium complex stored at 4 ℃, and it can be seen from fig. 8 that the auricularia polytricha polysaccharide nano selenium complex solution is stable and transparent, while the nano selenium (without auricularia polytricha polysaccharide) of the control group is completely precipitated at the bottom after 28 days, and the result shows that the auricularia polytricha polysaccharide plays a crucial role in the stability of nano selenium.
EXAMPLE III
Determination of antioxidant activity of different samples:
(1) determination of DPPH radical scavenging: the experiment should be operated in a dark room, 2mL of nano-selenium solution with different concentration gradients and 2X 10 solution are respectively added into a test tube -4 mol L -1 Shaking 2ml DPPH-ethanol solution, reacting in 37 deg.C water bath for 30min, measuring A at 517nm wavelength S (ii) a Adding 2mL of anhydrous ethanol instead of DPPH solution into the sample solution, reacting in the above water bath for 30min, and measuring A at a wavelength of 517nm X (ii) a 2mL of distilled water was added to 2mL of the DPPH solution in place of the sample solution, and after 30min of the water bath reaction, A was measured at a wavelength of 517nm 0 Vc and polysaccharide were used as controls. Clearance was calculated according to the following formula:
DPPH radical clearance:
in the formula A 0 Is the absorbance of the blank control group; a. the S Is the absorbance of the sample solution; a. the x Is the absorbance of the sample control.
FIG. 8 is a graph comparing the DPPH radical scavenging rate for samples of different concentrations. As shown, DPPH of the auricularia polytricha polysaccharide nano-selenium complex and the auricularia polytricha polysaccharide increased with increasing sample concentration. When the concentration of the auricularia polytricha polysaccharide nano selenium compound reaches 1mg/mL, the clearance rate can reach 76 percent, which shows that the auricularia polytricha polysaccharide nano selenium compound has strong clearance capability on DPPH free radicals and is far higher than the auricularia polytricha polysaccharide with the same concentration.
(2) Determination of elimination of ABTS free radicals: reacting 2,2' -dinitrogen bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (7mM) with K 2 S2O 8 Equal volumes (2.45mM) were mixed and then incubated in the dark for 14-16 hours to obtain ABTS working solution. Before measurement, the ABTS working solution was diluted with absolute ethanol so that its absorbance at 734nm was 0.70. + -. 0.02. Mixing 0.2mL of sample with 5.8mL of ABTS working solution, vortexing, reacting at room temperature for 6min, determining absorbance of 734nm, replacing ABTS with 5.8mL of absolute ethanol solution in control group, replacing sample with 0.2mL of deionized water in blank group, and using V C As a positive control. In the three methods described above, each sample was measured in triplicate and the mean clearance was calculated. The clearance rate was calculated as follows:
in the formula A 0 Is the absorbance of the blank control; a. the S Is the absorbance of the sample solution; a. the x Is the absorbance of the sample control.
As shown in fig. 9, the auricularia polytricha polysaccharide nano-selenium complex has a certain dosage relationship for eliminating ABTS free radicals, and the ABTS free radical eliminating capability of the auricularia polytricha polysaccharide nano-selenium is much higher than that of the auricularia polytricha polysaccharide.
It should be noted that, within the scope of the components, ratios and process parameters described in the present invention, the technical effects described in the present invention can be achieved by specifically selecting other components, ratios or values, and therefore, they are not listed one by one.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The preparation method of the auricularia polytricha polysaccharide nano-selenium compound is characterized by comprising the following steps:
s1 preparation of reaction liquid
Fully and uniformly mixing the auricularia polytricha polysaccharide solution and the sodium selenite solution, further adding an ascorbic acid solution, and carrying out water bath reaction to obtain a reaction liquid;
s2, preparing auricularia polytricha polysaccharide nano selenium compound solution
Dialyzing the reaction solution obtained in the step 1 to obtain a auricularia polytricha polysaccharide nano selenium compound solution;
s3, obtaining the auricularia polytricha polysaccharide nano selenium compound
And (3) freeze-drying the auricularia polytricha polysaccharide nano selenium compound solution obtained in the step (2) into powdery solid to obtain the auricularia polytricha polysaccharide nano selenium compound.
2. The method for preparing auricularia polytricha polysaccharide nano-selenium compound according to claim 1, wherein in the step 1, the concentration of auricularia polytricha polysaccharide is 50-150 mg/L, the concentration of sodium selenite solution is 0.25-1 mmol/L, and the concentration of ascorbic acid solution is 2-3 mmol/L.
3. The method for preparing auricularia polytricha polysaccharide nano selenium compound according to claim 1, wherein in the step 1, the reaction temperature is 25-50 ℃ and the reaction time is 0.5-1.5 h.
4. The method for preparing auricularia polytricha polysaccharide nano selenium compound according to claim 1, wherein in the step 2, the dialysis process comprises transferring the reaction solution into a 3000Da dialysis bag, and the dialysis temperature is controlled at 4 ℃ and the dialysis is continued for 48-50 h in a dark environment.
5. The method of claim 1, wherein in step S1, the Auricularia polytricha polysaccharide is used as a stabilizer, the ascorbic acid is used as a reducing agent, and the sodium selenite is used as a selenium source.
6. The method of claim 1, wherein in step S3, the particle size of the Auricularia polytricha polysaccharide nano-selenium complex is characterized by dual wavelength colorimetry.
7. The method for preparing Auricularia polytricha polysaccharide nano-selenium compound according to claim 6, wherein the characterization comprises measuring absorbance of Auricularia polytricha polysaccharide nano-selenium compound solution at wavelengths of 410nm and 490nm by ultraviolet spectrophotometry, according to the change of colloid particle size: the larger the ratio of A410 to A490 is, the smaller the colloid particle size is, and the particle size of the auricularia polytricha polysaccharide nano-selenium is judged.
8. The method of claim 1, wherein the auricularia polytricha polysaccharide nano-selenium complex is freeze-dried by placing the auricularia polytricha polysaccharide nano-selenium complex solution in a freezing tank and then injecting liquid nitrogen into the freezing tank in step S3.
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| CN112370465A (en) * | 2020-10-29 | 2021-02-19 | 华南理工大学 | Sargassum fusiforme polysaccharide functionalized nano-selenium and preparation method and application thereof |
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