A method for preparing selenized dextran using pulsed electric field

Abstract

The invention discloses a method for preparing selenized glucan by utilizing a pulse electric field. The method comprises the following steps: by introducing a pulse electric field treatment technology in an acidic environment to gradually activate binding sites such as hydroxyl groups of glucan, promoting the selenization process, improving the selenization rate of polysaccharide, shortening the selenization time, efficiently obtaining selenized glucan with high yield and high activity, saving a large amount of production and preparation costs, and realizing high-value utilization of polysaccharide. The extracted selenized glucan has a stronger scavenging rate for hydroxyl groups and superoxide radicals, exerts better antioxidant activity, can be used for supplementing the trace element selenium in human body, can be used for developing functional foods such as nutrition enhancers, and is applied to the fields of medicine, health care, cosmetics, etc., and has huge market development potential.

Description

Technical field:

[0001] The invention relates to a method for preparing selenized dextran by utilizing a pulse electric field. Background technology:

[0002] Glucan is the most common hexose in nature. The homopolysaccharide formed by the polymerization of multiple glucoses via glycosidic bonds is called glucan. Existing studies have shown that glucan has multiple biological activities such as scavenging free radicals, improving immunity, anti-virus, and improving intestinal function, and is widely used in the fields of medicine, health care food, and cosmetics. According to the results published by the World Health Organization, selenium and iron, zinc, iodine and other elements are listed as 14 essential trace elements for the human body. Selenium is a component of the glutathione peroxidase system and has a good antioxidant effect. It can prevent vision loss and cardiovascular and cerebrovascular diseases. Selenium is also known as the "anti-liver necrosis protection factor" and the "guardian of human health and life." The latest report shows that due to the changes in the physical and chemical structure, the selenized glucan has significantly enhanced biological activity and is easier to be digested and absorbed by the human body.

[0003] At present, the sources of selenized glucan are often divided into three categories: soil selenium enrichment, microbial selenium enrichment, chemical modification, etc., but all of them have certain defects. Soil selenium enrichment is to enrich inorganic selenium elements by planting crops on selenium-rich soil and obtain selenium-containing plant polysaccharides through biotransformation. Selenium-rich crops can be processed into selenium-rich feed and then fed to harvest selenium-rich pork, eggs and other livestock products. However, according to surveys, since 72% of my country's land belongs to selenium-poor soil or selenium-deficient soil, this method has practical difficulties such as high cost, long cycle, low efficiency, and high difficulty in industrialization. Microbial selenium enrichment represented by selenium-enriched yeast, by adding inorganic selenium such as sodium selenite to the yeast culture medium, converting it into an organic selenium form and obtaining a variety of microbial fermentation products through enrichment, this method has the defects of low product purity and inability to directional regulation. Water extraction or organic solvent extraction destroys its activity at high temperature and has low efficiency. Supercritical fluid extraction is expensive and not suitable for industrial production. Chemical modification is a method of generating organic selenium compounds by introducing selenium into organic compounds such as polysaccharides and polyphenols. Common reaction modes include selenization reaction, selenium substitution reaction, selenium oxidation reaction, selenium coupling reaction, etc. For example, CN114957500A discloses a selenization modification method for improving the immunological activity of yam polysaccharides and its application. However, since nitric acid is generally required to create an acidic reaction environment, and long-term heating and stirring operations are required, there are disadvantages such as large environmental pollution, high industrial cost, long selenization time, and low selenization rate. Summary of the invention:

[0004] The purpose of the present invention is to provide a method for preparing selenized dextran using a pulsed electric field, which solves the problems of large environmental pollution, high industrial cost, long selenization time and low selenization rate in the prior art of preparing selenized dextran using chemical modification.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing selenized dextran using a pulsed electric field, the method comprising the following steps:

[0007] (1) adding dextran and dilute nitric acid at a material-liquid ratio of 1:50 to 1:150 g / mL, stirring until fully dissolved; adding sodium selenite and barium chloride, the mass ratio of sodium selenite and barium chloride to the dextran in step (1) is (0.4-2.0):1, mixing thoroughly, placing in a pulse electric field environment for pulse treatment, using a peristaltic pump to maintain stable flow of the solution during the treatment process, the flow rate is 5-20 L / h; the pulse treatment conditions are: field strength 1-10 kV / cm, frequency 30-100 Hz, pulse width 10-50 μs, treatment time 10-60 min;

[0008] (2) After step (1), sodium sulfate was added and the precipitate was removed by centrifugation until no turbidity was observed after further addition of sodium sulfate;

[0009] (3) adding an appropriate amount of supersaturated sodium carbonate solution (>300 mg / mL at room temperature) to adjust the pH to 7-9; taking the supernatant and dialyzing it for 48-72 hours, changing the water every 12 hours until no free selenium can be detected by the ascorbic acid method; and freeze-drying the treated solution to obtain selenized glucan.

[0010] Preferably, the concentration of the dilute nitric acid in step (1) is 0.1-0.5 wt %.

[0011] Preferably, in step (1), the field strength is 4.4-10 kV / cm, the frequency is 30-50 Hz, the pulse width is 10-30 μs, and the treatment time is 30-60 min.

[0012] More preferably, the field strength in step (1) is 4.4-7.8 kV / cm, further preferably 6.2 kV / cm-7.8 kV / cm.

[0013] The mass ratio of sodium sulfate in step (2) to barium chloride in step (1) is (1.0-3.0):1.

[0014] In step (2), the centrifugal speed is 8000-10000 r / min, and the centrifugal time is 10-30 min.

[0015] Preferably, the specific parameters of the dialysis in step (3) are: the molecular weight cut-off of the dialysis bag is 3000-5000Da, and the diameter of the dialysis bag is 34-77mm.

[0016] The invention utilizes pulse electric field to strengthen the preparation process of selenized dextran. Under the environment of 0.1wt%-0.5wt% dilute nitric acid, the pH value of the reaction system is 3-6. Under the action of pulse electric field and free radicals and the catalysis of barium chloride, the dextran molecules absorb the electric field energy, and the originally stable structure becomes unstable, the chemical activity of the molecules gradually increases, and the activation sites such as hydroxyl groups are gradually exposed, and are combined with inorganic selenium in sodium selenite to obtain selenized dextran. The method gradually activates the hydroxyl and other binding sites of polysaccharides such as glucan by introducing pulse electric field treatment technology in an acidic environment, promotes the selenization process under the catalysis of barium chloride, improves the selenization rate of polysaccharides, shortens the selenization time, and efficiently obtains high-yield and high-activity selenized glucan, saving a large amount of production and preparation costs, and realizing the high-value utilization of polysaccharides. The extracted selenized glucan has a stronger scavenging rate for hydroxyl and superoxide free radicals than the selenized yam polysaccharide, and exerts better antioxidant activity. It can be used to supplement the trace element selenium in the human body, can be used to develop functional foods such as nutritional enhancers, and can also be used in the fields of medicine, health care, cosmetics, etc., and has huge market development potential.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1) The present invention effectively improves the efficiency of selenized dextran by introducing pulsed electric field treatment under acidic conditions and combining it with barium chloride catalysis, thereby saving production costs and promoting the industrial production process of selenized dextran;

[0019] 2) The present invention adopts a low-acid environment with a pH of 3-6, which further reduces the impact of the inorganic acid used in the selenization process on the environment and improves the safety of the pulse treatment process. At the same time, the inventors found that the pulse electric field has a step-by-step activation effect, and further selected the most efficient pulse electric field treatment conditions, which is of great significance to maintaining economic benefits and natural benefits;

[0020] 3) The selenized glucan prepared by the present invention has a stronger scavenging rate for hydroxyl and superoxide free radicals, plays a better role in promoting blood circulation and promoting yang function, can be used to supplement the trace element selenium in the human body, and can also be explored for the development of functional foods and cosmetics.

[0021] In summary, the present invention gradually activates the hydroxyl and other binding sites of glucan by introducing pulse electric field treatment technology in an acidic environment, combines with barium chloride catalysis, promotes the selenization process, improves the selenization rate of polysaccharides, shortens the selenization time, and efficiently obtains high-yield and high-activity selenized glucan, saves a lot of production and preparation costs, and realizes the high-value utilization of polysaccharides. The extracted selenized glucan has a stronger scavenging rate for hydroxyl and superoxide free radicals than the selenized yam polysaccharide, and exerts better antioxidant activity. It can be used to supplement the trace element selenium in the human body, can be used to develop functional foods such as nutritional enhancers, and is applied to the fields of medicine, health care, cosmetics, etc., and has huge market development potential. Description of the drawings:

[0022] Figure 1 The selenium content in selenized dextran prepared under different field strengths. As the field strength increases, the selenium content increases in a step-like manner.

[0023] Figure 2 The XPS graphs are from left to right and from top to bottom, which are the XPS graphs corresponding to conventional heat treatment (Comparative Example 2) / pulsed electric field treatment (Example 1) in a 0.3% dilute nitric acid environment, and conventional heat treatment (Comparative Example 4) / pulsed electric field treatment (Example 6) in a 0.5% dilute nitric acid environment. Under different acid environments, compared with conventional heat treatment, the CO bond ratio of pulse-treated selenized dextran decreased, indicating that the hydrogen atoms on the hydroxyl groups of dextran were replaced.

[0024] Figure 3 It is a photoelectron energy spectrum diagram; from left to right and from top to bottom are the photoelectron energy spectrum diagrams corresponding to conventional heat treatment in a 0.3% dilute nitric acid environment (referring to comparative example 2) / pulse electric field treatment (Example 1), conventional heat treatment in a 0.5% dilute nitric acid environment (Comparative example 4) / pulse electric field treatment (Example 6). Among them, the results show that on the selenized dextran chain, selenium element exists in the form of +4 valence, but not -2 valence, which indicates that selenium has been successfully grafted onto dextran, and is carried out in a way of replacing the hydroxyl group on carbon No. 6. Specific implementation method:

[0025] The following is a further description of the present invention, rather than a limitation of the present invention.

[0026] Embodiment 1:

[0027] Dextran and dilute nitric acid were added at a material-liquid ratio of 1:100 g / mL, and stirred until fully dissolved; the concentration of the dilute nitric acid was 0.3%, sodium selenite and barium chloride (both at a mass ratio of 1:2 to dextran) were added, and stirred until fully mixed, and placed in a pulsed electric field environment for pulse treatment. A peristaltic pump was used to maintain a stable flow of the solution during the treatment process, and the flow rate was 18 L / h. The pulsed electric field treatment conditions were: field strength 1.2 kV / cm, frequency 30 Hz, pulse width 10 μs, and treatment time 60 min; end Then add enough sodium sulfate, centrifuge (speed 10000r / min, time 15min) to remove the precipitate, repeat the precipitation and centrifugation until there is no turbidity when sodium sulfate is added again, add an appropriate amount of supersaturated sodium carbonate solution to adjust the pH to about 7, take the supernatant and dialyze for 72h, change the water every 12h, until no free sodium selenate can be detected by ascorbic acid method, freeze-dry the treated solution to obtain selenized glucan, and the selenium content in the product is 11.4μg / g measured by ICP-MS, see Figure 1 The hydroxyl radical scavenging rate of the selenized glucan was 90.28%, and the superoxide anion radical scavenging rate was 8.97%. The hydroxyl radical scavenging rate of the glucan that was not selenized was 87.36%, and the superoxide anion radical scavenging rate was 6.35%. The results showed that the selenized glucan obtained in this example had a stronger scavenging rate for hydroxyl and superoxide radicals, and exerted better antioxidant activity.

[0028] Comparative Example 1:

[0029] Refer to Example 1, except that no barium chloride was added.

[0030] Dextran and dilute nitric acid were added at a material-liquid ratio of 1:100 g / mL, and stirred until fully dissolved; the concentration of the dilute nitric acid was 0.3%, sodium selenite (with a mass ratio of 1:2 to dextran) was added, stirred until fully mixed, and placed in a pulsed electric field environment for pulse treatment. A peristaltic pump was used to maintain the solution flowing stably during the treatment process, and the flow rate was 18 L / h. The pulsed electric field treatment conditions were: field strength 1.2 kV / cm, frequency 30 Hz, pulse width 10 μs, and treatment time 60 min; after the end, sufficient sodium sulfate was added, centrifuged (speed 10000 r / min, time 15 min) to remove the precipitate, and the precipitation and centrifugation were repeated until there was no turbidity when sodium sulfate was added again, and an appropriate amount of supersaturated sodium carbonate solution was added to adjust the pH to about 7, and the supernatant was dialyzed for 72 h, and the water was changed every 12 h until no free sodium selenate could be detected by the ascorbic acid method. The treated solution was freeze-dried to obtain selenized dextran, and the selenium content in the product was measured by ICP-MS to be 3.7 μg / g.

[0031] Comparative Example 2:

[0032] Refer to Example 1, except that the pulse electric field treatment was not performed.

[0033] Dextran and dilute nitric acid were added at a material-liquid ratio of 1:100 g / mL, and stirred until fully dissolved; the concentration of the dilute nitric acid was 0.3%, sodium selenite and barium chloride (both in a mass ratio of 1:2 to dextran) were added, stirred until fully mixed, and placed in a water bath heating environment for selenization treatment. During the treatment process, magnetic stirring was used to keep the solution stirred stably, the speed was 300 r / min, and the water bath heating treatment conditions were: temperature 70°C, treatment time 5 h; after the end, sufficient sodium sulfate was added, centrifuged (speed 10000 r / min, time 15 min) to remove the precipitate, and the precipitation and centrifugation were repeated until there was no turbidity when sodium sulfate was added again, and an appropriate amount of supersaturated sodium carbonate solution was added to adjust the pH to about 7, the supernatant was dialyzed for 72 h, and the water was changed every 12 h until no free sodium selenate could be detected by the ascorbic acid method, and the treated solution was freeze-dried to obtain selenized dextran, and the selenium content in the product was measured by ICP-MS to be 6.2 μg / g.

[0034] Comparison of Example 1 with Comparative Examples 1-2 shows that the pulsed electric field treatment of the present application and the barium chloride catalyst have a synergistic effect, thereby improving the selenization rate of polysaccharides.

[0035] Comparative Example 3:

[0036] Refer to Example 1, except that the glucan is replaced by yam polysaccharide.

[0037] Add yam polysaccharide and dilute nitric acid at a solid-liquid ratio of 1:100 g / mL, and stir until fully dissolved; the dilute nitric acid concentration is 0.3%, add sodium selenite and barium chloride (the mass ratio to yam polysaccharide is 1:2), stir until fully mixed, and place in a pulse electric field environment for pulse treatment. During the treatment process, a peristaltic pump is used to maintain a stable flow of the solution, the flow rate is 18 L / h, and the pulse electric field treatment conditions are: field strength 1.2 kV / cm, frequency 30 Hz, pulse width 10 μs, treatment time 60 min; After the end, add enough sodium sulfate, centrifuge (speed 10000r / min, time 15min) to remove the precipitate, repeat the precipitation centrifugation until there is no turbidity when adding sodium sulfate again, add an appropriate amount of supersaturated sodium carbonate solution to adjust the pH to about 7, take the supernatant and dialyze for 72h, change the water every 12h, until the ascorbic acid method can no longer detect free sodium selenate, freeze-dry the treated solution to obtain selenized yam polysaccharide, and the selenium content in the product is 3.6μg / g measured by ICP-MS. The hydroxyl radical scavenging rate of the selenized yam polysaccharide is 88.47%, and the superoxide anion radical scavenging rate is 6.56%.

[0038] From the comparison between Example 1 and Comparative Example 3, it can be seen that the selenized glucan extracted by the present invention has a stronger scavenging rate for hydroxyl and superoxide free radicals than the selenized yam polysaccharide, and exerts better antioxidant activity.

[0039] Example 2

[0040] Refer to Example 1, except that: the pulse electric field treatment conditions are: field intensity 4.4 kV / cm, frequency 30 Hz, pulse width 10 μs, and treatment time 60 min.

[0041] Dextran and dilute nitric acid were added at a material-liquid ratio of 1:100 g / mL, and stirred until fully dissolved; the concentration of the dilute nitric acid was 0.3%, sodium selenite and barium chloride (both at a mass ratio of 1:2 to dextran) were added, and stirred until fully mixed, and placed in a pulse electric field environment for pulse treatment. A peristaltic pump was used to maintain a stable flow of the solution during the treatment process, and the flow rate was 18 L / h. The pulse electric field treatment conditions were: field strength 4.4 kV / cm, frequency 30 Hz, pulse width 10 μs, and treatment time 60 min; end Then add enough sodium sulfate, centrifuge (speed 10000r / min, time 15min) to remove the precipitate, repeat the precipitation and centrifugation until there is no turbidity when sodium sulfate is added again, add an appropriate amount of supersaturated sodium carbonate solution to adjust the pH to about 7, take the supernatant and dialyze for 72h, change the water every 12h, until no free sodium selenate can be detected by ascorbic acid method, freeze-dry the treated solution to obtain selenized glucan, and the selenium content in the product is 32.9μg / g measured by ICP-MS (see Figure 1 ).

[0042] Example 3

[0043] With reference to Example 1, the difference is that: the pulse electric field treatment conditions are: field strength 7.8 kV / cm, frequency 30 Hz, pulse width 10 μs, treatment time 60 min, to obtain selenized dextran, and the selenium content in the product measured by ICP-MS is 82.7 μg / g (see Figure 1 ).

[0044] Embodiment 4:

[0045] Referring to Example 1, the difference is that the pulse electric field is 9.6 kV / cm, and the selenium content in the selenized glucan is 72.3 μg / g (see Figure 1 ).

[0046] Embodiment 5:

[0047] Reference Example 1, except that the pulse electric field treatment conditions are: field strength 6.2 kV / cm, frequency 30 Hz, pulse width 10 μs, treatment time 60 min; the selenium content in the product measured by ICP-MS is 33.2 μg / g (see Figure 1 ).

[0048] It is known from the comparison of Example 1 to Example 5 (see Figure 1), as the field strength increases, the selenium content rises in a step-by-step manner, and the pulsed electric field has a step-by-step activation effect. Between 1.2kV / cm-4.4kV / cm, the selenium content rises rapidly, and between 4.4-6.2kV / cm, the selenium content continues to rise steadily, although the rising speed slows down, with a good effect. The selenium content rises rapidly at 6.2kV / cm-7.8kV / cm, and the rising speed is faster than that at 1.2kV / cm-4.4kV / cm, reaching the highest value at 7.8kV / cm, and then falling back. Therefore, the field strength is preferably 4.4-7.8kV / cm, and more preferably 6.2kV / cm-7.8kV / cm.

[0049] Embodiment 6:

[0050] Refer to Example 1, except that the concentration of the dilute nitric acid is 0.5%, and the selenium content in the obtained selenized glucan is 11.8 μg / g.

[0051] Comparative Example 4:

[0052] Referring to Comparative Example 2, the difference is that the concentration of the dilute nitric acid is 0.5%, and the selenium content in the selenized glucan is 4.9 μg / g.

[0053] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing selenized dextran using a pulsed electric field, characterized in that: The method comprises the following steps: (1) adding dextran and dilute nitric acid at a material-liquid ratio of 1:50 to 1:150 g / mL, stirring until fully dissolved; adding sodium selenite and barium chloride, the mass ratio of sodium selenite and barium chloride to the dextran in step (1) is (0.4-2.0):1, fully mixing, placing in a pulse electric field environment for pulse treatment, using a peristaltic pump to maintain stable flow of the solution during the treatment process, the flow rate is 5-20 L / h; the pulse treatment conditions are: field strength 1-10 kV / cm, frequency 30-100 Hz, pulse width 10-50 μs, treatment time 10-60 min; (2) After step (1), sodium sulfate was added and the precipitate was removed by centrifugation until no turbidity was observed after further addition of sodium sulfate; (3) adding a supersaturated sodium carbonate solution to adjust the pH to 7-9; dialyzing the supernatant for 48-72 hours, changing the water every 12 hours, until no free selenium can be detected by the ascorbic acid method; and freeze-drying the treated solution to obtain selenized glucan.

2. The method according to claim 1, characterized in that The concentration of the dilute nitric acid in step (1) is 0.1-0.5wt%.

3. The method according to claim 1, characterized in that: In step (1), the field strength is 4.4-10 kV / cm, the frequency is 30-50 Hz, the pulse width is 10-30 μs, and the treatment time is 30-60 min.

4. The method according to claim 1, characterized in that: The field strength in step (1) is 4.4-7.8 kV / cm.

5. The method according to claim 1, characterized in that The field strength in step (1) is 6.2 kV / cm-7.8 kV / cm.

6. The method according to claim 1, characterized in that The mass ratio of sodium sulfate in step (2) to barium chloride in step (1) is (1.0-3.0):

1.

7. The method according to claim 1, characterized in that In step (2), the centrifugal speed is 8000-10000 r / min, and the centrifugal time is 10-30 min.

8. The method according to claim 1, characterized in that: The specific parameters of the dialysis in step (3) are: the molecular weight cut-off of the dialysis bag is 3000-5000Da, and the diameter of the dialysis bag is 34-77mm.

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