CN112759665B - Method for preparing nano-selenium polysaccharide by using tiger milk mushroom hyperbranched polysaccharide - Google Patents

Abstract

The invention relates to a method for preparing nano-selenium polysaccharide by adopting the hyperbranched polysaccharide of tiger milk mushroom. The invention provides a soft template for the synthesis of nano selenium in an acidic solution through the special structure of the hyperbranched polysaccharide, and then obtains a stable hyperbranched polysaccharide nano selenium product through the in-situ reduction of Vc, and the nano selenium content is high, reaching 16.35%, and stable good sex. The method has mild preparation conditions and simple operation, and has potential application value in the field of chemical synthesis of selenium nanomaterials.

Description

Method for preparing nano-selenium polysaccharide by using tiger milk mushroom hyperbranched polysaccharide

technical field

The invention relates to the technical field of nano selenium, in particular to a method for preparing nano selenium polysaccharide by adopting the hyperbranched polysaccharide of tiger milk mushroom.

Background technique

Selenium is an essential trace element for the body. Appropriate selenium supplementation can improve various biological functions such as non-specific immunity, cellular immunity and humoral immunity. It has broad application prospects in food fortification, animal breeding, and crop cultivation. At present, the widely used selenium supplement method in the food and pharmaceutical industries is to add inorganic selenium supplements, such as sodium selenite, sodium hydrogen selenite, sodium selenate, etc. However, inorganic selenium has high toxicity, narrow range of activity and toxicity, relatively small minimum lethal dose, and low utilization rate. According to reports, the use of polysaccharide-selenium complex as a selenium supplement not only has good stability and dissolution, but also the ligand polysaccharide after releasing selenium will not cause toxicity and side effects to the body, and can also promote absorption and utilization.

Tiger milk mushroom hyperbranched polysaccharide is a typical natural hyperbranched polymer with a high degree of branching. It not only has various biological activities, but also contains a large number of terminal active groups, which can be used for modification of natural polysaccharides. In addition, this type of polysaccharide has great potential in drug and small molecule delivery and controlled release systems, and its unique quasi-spherical structure has a good stabilization effect on nano-selenium. Although polysaccharide solution was also used as nano-selenium stabilizer in previous studies, there are still problems such as low selenium content and poor stability.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a method for preparing nano-selenium polysaccharide by adopting the hyperbranched polysaccharide of tiger milk mushroom, through the special structure of the hyperbranched polysaccharide, a soft template is provided for the synthesis of nano-selenium in an acidic solution, and then through the original structure of Vc. The stable hyperbranched polysaccharide nano-selenium product is obtained by in situ reduction. The method has mild preparation conditions and simple operation, and has potential application value in the field of chemical synthesis of selenium nanomaterials.

The first object of the present invention is to provide a method for preparing nano-selenium polysaccharide by adopting tiger milk mushroom hyperbranched polysaccharide, comprising the following steps:

S1, prepare a hyperbranched polysaccharide solution with a concentration of 4-10 mg/mL;

S2, adding nitric acid with a final concentration of 0.5 to 1% to the hyperbranched polysaccharide solution, and mixing until the solution is milky white to obtain an acidified polysaccharide solution;

S3, adding Na ₂ SeO ₃ powder to the acidified polysaccharide solution, and reacting at 60-80° C. for 1-10 hours to obtain a reaction solution;

S4. Under stirring conditions, dropwise add Vc with a concentration of 0.1-0.6M to the reaction solution in step S3. After the color of the solution changes to brick red, continue the reaction at 40-60°C for 1-3 hours, and the reaction solution is dried after dialysis The nano-selenium polysaccharide is obtained.

Further, the hyperbranched polysaccharide is prepared from the sclerotium cell wall of the tiger milk mushroom by alkali extraction and ultrafiltration.

Further, the described alkaline extraction ultrafiltration method specifically comprises the following steps:

(1) grinding the sclerotia of the tiger milk mushroom into powder, successively using water, 0.5-1.5% NaCl solution, and 4-6% NaCl solution for extraction, and retaining the precipitation to obtain the sclerotia cell wall of the tiger milk mushroom;

(2) extracting the sclerotium cell wall of the tiger milk mushroom with water at 90-100° C. to obtain a precipitate, subjecting the precipitate to alkaline extraction with 0.5-2M NaOH, diluting the extract and filtering it with an ultrafiltration membrane to obtain the hyperbranched polysaccharide .

Further, the retention of the ultrafiltration membrane is 50-150 kDa.

Further, in step S1, ultrasonic treatment is used to assist the dissolution of the hyperbranched polysaccharide.

Further, in step S2, the mixing is performed by oscillating ultrasound for 5-10 min.

Further, in step S3, after the reaction is completed, it is cooled to 20-30° C., and the pH is adjusted to 5-6 with anhydrous Na ₂ CO ₃ .

Further, in step S4, after the color of the solution changes to brick red, the reaction is continued in an air bath at 40-60°C.

Further, in step S4, the dialysis is performed in a 3000-4000Da dialysis bag.

The second object of the present invention is to provide the nano-selenium polysaccharide prepared by the method.

By means of the above scheme, the present invention has at least the following advantages:

The invention provides a soft template for the synthesis of nano-selenium in an acidic solution through the special structure of the hyperbranched polysaccharide, and then obtains a stable hyperbranched polysaccharide nano-selenium product through the in-situ reduction of Vc, and the nano-selenium content is high, reaching 16.35%, and stable good sex. The method has mild preparation conditions and simple operation, and has potential application value in the field of chemical synthesis of selenium nanomaterials.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the description, the following description is given with the preferred embodiments of the present invention and the detailed drawings.

Description of drawings

Figure 1 is a comparison diagram of a blank polysaccharide solution with Comparative Examples and Examples.

Figure 2 is a TEM electron microscope image of blank polysaccharide solution and comparative examples and examples.

3 is a field emission TEM image of C and Se in solution particles of Example 1 and Comparative Example 2.

Detailed ways

The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Detection of nano-selenium content: ICP-MS was used to determine the content of selenium in the hyperbranched-nano-selenium solution samples, and the OD334 and selenium content standards of the hyper-branched-nano-selenium solution samples were established.

Sample processing: The synthesized hyperbranched polysaccharide nanoselenium solution was dialyzed to remove residual inorganic selenium, and then freeze-dried.

Preparation of hyperbranched polysaccharide: The hyperbranched polysaccharide of tiger milk mushroom is obtained from the sclerotium cell wall of tiger milk mushroom by alkaline extraction and ultrafiltration method. % NaCl solution and 5% NaCl solution were extracted three times by magnetic stirring at room temperature for 1 h, the extract was removed each time, and the precipitate was retained. Finally, the precipitation was used for 8-10 times with deionized water to obtain the sclerotia cell wall of tiger milk mushroom. Then, extract the water extract three times with 5 times deionized water at 100°C to remove the water extract. The precipitate is stirred and extracted with 5 times the volume of 1M NaOH solution for 6 hours, and the extraction is repeated 8 to 10 times, and the extracts are combined. The extract was diluted 5 times and passed through an ultrafiltration membrane with a cutoff of 100 kDa, the reflux liquid was repeatedly passed through the membrane, washed with deionized water until neutral, and freeze-dried to obtain the hyperbranched polysaccharide of the sclerotium of tiger milk mushroom.

Example 1:

Step 1: Weigh 200 mg of hyperbranched polysaccharide and dissolve it in 20 mL of ultrapure water to prepare a 10 mg/mL polysaccharide solution, and ultrasonically treat it at 100 MHz until it is completely dissolved.

In step 2, 100 μL of concentrated nitric acid (ie, the final concentration of 0.5%) was added to the polysaccharide solution, and the solution was shaken and ultrasonicated for 5 minutes until the solution was milky white.

Step 3. Add 80 mg of Na ₂ SeO ₃ powder to the acidified polysaccharide solution, and heat in a water bath at 70° C. for 6 hours. After the reaction was completed, it was cooled to room temperature, and the pH was adjusted to 5-6 with anhydrous Na ₂ CO ₃ .

Step 4: The sample solution is magnetically stirred and 5 mL of Vc with a concentration of 0.1 M is added dropwise, the color of the solution gradually becomes brick red, and the reaction is continued for 2 hours in an air bath at 50°C. After the reaction, the solution sample was put into a 3500Da dialysis bag, put into a 3L beaker, tap water was continuously dialyzed for 2 days, then changed to deionized water, changed water every 1 hour, magnetically stirred for 3 days, collected intercepted samples, freeze-dried to obtain hyperbranched Polysaccharide nanoselenium sample powder.

After testing, the content of selenium in the obtained hyperbranched polysaccharide nano-selenium was 6.19% (ie, 61.9±2.2 mg/g), and the average particle size was 202.7±6.8 nm. After standing for 96 hours, there is no precipitation at the bottom, and the stability is good.

Example 2:

On the basis of Example 1, the Vc concentration in step 4 was adjusted to 0.4M. After testing, the selenium content in the obtained hyperbranched polysaccharide nano-selenium was 5.40% (ie, 54.0±5.0 mg/g), and the average particle size was 379.2±13.3 nm. After standing for 96 hours, there is no precipitation at the bottom, and the stability is good.

Example 3:

On the basis of Example 1, the Vc concentration in step 4 was adjusted to be 0.6M. After testing, the content of selenium in the obtained hyperbranched polysaccharide nano-selenium was 7.14% (ie, 71.4±6.5 mg/g), and the average particle size was 351.3±15.2 nm. After standing for 96 hours, there is no precipitation at the bottom, and the stability is good.

Example 4:

On the basis of Example 1, the addition amount of concentrated nitric acid in step 2 was adjusted to be 0.75%. After testing, the content of selenium in the obtained hyperbranched polysaccharide nano-selenium was 12.88% (ie, 128.8±6.9 mg/g), and the average particle size was 210.9±19.9 nm. The brick red of the solution was obviously deepened, and after standing for 96 hours, there was no precipitation at the bottom, and the stability was good.

Example 5:

On the basis of Example 1, the addition amount of concentrated nitric acid in step 2 was adjusted to 1%. After testing, the content of selenium in the obtained hyperbranched polysaccharide nano-selenium was 16.35% (ie, 163.5±5.4 mg/g), and the average particle size was 227.7±5.6 nm. The brick red of the solution was obviously deepened, and after standing for 96 hours, there was no precipitation at the bottom, and the stability was good.

Example 6:

On the basis of Example 1, the concentration of hyperbranched polysaccharide in step 1 was adjusted to 4 mg/mL. After testing, the content of selenium in the obtained hyperbranched polysaccharide nano-selenium was 13.75% (ie, 137.5±13.4 mg/g), and the average particle size was 212.0±9.9 nm. The brick red of the solution was obviously deepened, and after standing for 96 hours, there was no precipitation at the bottom, and the stability was good.

Comparative Example 1:

The specific embodiment is the same as in Example 1, except that Vc is not added for the reaction, and the average selenium content and particle size in the sample after the reaction are 1.12% (ie 11.2 mg/g) and 115 nm, respectively, but there is no brick red nano-selenium.

Comparative Example 2:

The specific embodiment is the same as Example 1, the difference is that step 2 is omitted, and step 3 is changed to adding 80mg Na ₂ SeO ₃ to the hyperbranched polysaccharide aqueous solution of the same concentration to react, and finally adding Vc of the same concentration, and the color of the solution gradually changes to Orange red. After testing, the average selenium content and particle size in the samples were 1.55% and 152.6±16.8nm, respectively, which were relatively low, and there was obvious powdery precipitation at the bottom of the solution after standing, presumably because the polysaccharide did not effectively stabilize the nano-selenium. effect.

Figure 1 is a comparison diagram of blank polysaccharide solution with Comparative Example and Example 1. The results showed that: the blank polysaccharide solution and Comparative Example 1 could not synthesize brick red nano-selenium; the content of nano-selenium synthesized in Comparative Example 2 was low and the stability was poor; the solution prepared in Example 2 had the darkest brick red and the largest amount of synthesized nano-selenium , and good stability.

FIG. 2 is the TEM electron microscope images of the blank polysaccharide solution and the comparative example and Example 1. FIG. The results show that the blank polysaccharide molecules are irregular quasi-spherical, while the acidified polysaccharide solution in Comparative Example 1 has a regular spherical shape, but because no nano-selenium is produced, the particle size is smaller; Comparative Example 2 has a relatively regular spherical shape However, due to the low content of the synthesized nano-selenium, the average particle size is small and the particle uniformity is lacking; the sample of Example 1 presents a relatively regular spherical shape, and the average particle size increases due to the high content of nano-selenium contained. , the particle uniformity is significantly improved.

3 is a field emission TEM image of C and Se in solution particles of Example 1 and Comparative Example 2. The results show that both Example 1 and Comparative Example 2 contain nano-selenium, and the nano-selenium is encapsulated in polysaccharide molecules, but the nano-selenium in Example 1 is clearly visible, while the nano-selenium in Comparative Example 2 can only be barely distinguished. The results in the data chart all show that the selenium content in Example 1 is much higher than that in Comparative Example 2.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the technical principles of the present invention. , these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (8)

1. A method for preparing nano selenium polysaccharide by adopting Pleurotus tuber-regium hyperbranched polysaccharide is characterized by comprising the following steps:

s1, preparing a hyperbranched polysaccharide solution with the concentration of 4-10 mg/mL;

s2, adding nitric acid with the final concentration of 0.5-1% into the hyperbranched polysaccharide solution, and mixing until the solution is milky white to obtain an acidified polysaccharide solution;

s3, adding Na into the acidified polysaccharide solution ₂ SeO ₃ Reacting the powder at 60-80 ℃ for 1-10 hours to obtain a reaction solution;

s4, under the stirring condition, dropwise adding Vc with the concentration of 0.1-0.6M into the reaction liquid in the step S3, continuing to react for 1-3 hours at 40-60 ℃ after the color of the solution turns into brick red, dialyzing the reaction liquid, and drying to obtain the nano selenium polysaccharide;

the hyperbranched polysaccharide is prepared from the sclerotium cell wall of the Pleurotus tuber-regium by an alkali extraction ultrafiltration method;

the alkali extraction ultrafiltration method specifically comprises the following steps:

(1) grinding the sclerotium of the Pleurotus tuber-regium into powder, sequentially extracting with water, 0.5-1.5% NaCl solution and 4-6% NaCl solution, and keeping precipitate to obtain the cell wall of the sclerotium of the Pleurotus tuber-regium;

(2) extracting the sclerotium cell walls of the pleurotus tuber-regium with water at 90-100 ℃ to obtain precipitates, carrying out alkali extraction on the precipitates by adopting 0.5-2M NaOH, diluting an extracting solution, and filtering by adopting an ultrafiltration membrane to obtain the hyperbranched polysaccharide.

2. The method according to claim 1, wherein the retention capacity of the ultrafiltration membrane is 50-150 kDa.

3. The method according to claim 1, wherein in step S1, ultrasonic treatment is used to assist the dissolution of the hyperbranched polysaccharide.

4. The method according to claim 1, wherein in step S2, the mixing is performed by shaking ultrasound for 5-10 min.

5. The method according to claim 1, wherein in the step S3, after the reaction is finished, the reaction solution is cooled to 20-30 ℃ and anhydrous Na is adopted ₂ CO ₃ Adjusting pH to 5-6.

6. The method as claimed in claim 1, wherein the reaction is continued in a gas bath at 40-60 ℃ after the color of the solution is changed to brick red in the step of S4.

7. The method according to claim 1, wherein in step S4, the dialysis is performed in a 3000-4000 Da dialysis bag.

8. A nano-selenium polysaccharide prepared by the method of any one of claims 1 to 7.

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