CN112063675A - High-hydrolysis-degree polypeptide nano-selenium particle and preparation method and application thereof - Google Patents
High-hydrolysis-degree polypeptide nano-selenium particle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses polypeptide nano selenium particles with high hydrolysis degree and a preparation method and application thereof. The particle size of the polypeptide nano selenium particles with high hydrolysis degree is 255-448nm, and the hydrolysis degree is 15-30%. Compared with the conventional polypeptide nano selenium particles, the egg white polypeptide with high reducibility and hydrolysis degree, which can reduce sodium selenite into elemental selenium, is prepared by utilizing the egg white powder through ultrasonic and enzymolysis limitation, so that the problems of completeness and absorptivity of the egg white polypeptide are solved, and the polypeptide nano selenium particles with small particle size are obtained, and have good oxidation resistance, cell growth promotion effect and bacterial growth inhibition effect.
Description
Technical Field
The invention relates to the field of nano biological materials, in particular to high-hydrolysis-degree polypeptide nano selenium particles and a preparation method and application thereof.
Background
Selenium is one of the essential trace elements for humans and animals. Selenium has an important relationship with the anticancer, antiviral and immunological functions of the body. A great deal of research and clinical trials show that selenium deficiency can cause dysfunction of some important organs in human body, thereby causing various diseases, such as tumor, cardiovascular disease, keshan disease and the like. At present, the selenium element is supplemented mainly by directly taking sodium selenite (Na)2SeO3) Or sodium selenate (Na)2SeO4) Inorganic salts, however, are not easily controlled by the body's absorption of inorganic salts and have potential residual toxicity, thereby risking selenium poisoning.
Nanoparticle materials, also known as ultrafine particle materials, are composed of nanoparticles. The nanoparticles generally mean particles having a size of 1 to 100 nm. The nanometer technology can make the production process of the medicine more and more precise, and the medicine with specific functions can be manufactured by directly utilizing the arrangement of atoms and molecules on the scale of nanometer materials. The nanometer material particles can facilitate the transmission of medicine in human body, and intelligent medicine coated with several layers of nanometer particles can search and attack cancer cell or repair damaged tissue after entering human body. The new diagnostic devices and related technologies using nanotechnology undoubtedly open new directions for drug development.
At present, elemental selenium prepared by inorganic selenium reduction in the traditional process is easy to aggregate to form grey or black insoluble aggregated elemental selenium, and the aggregated elemental selenium has no biological activity and is not beneficial to absorption and utilization of cells. The nano selenium particles prepared by using macromolecules such as protein or chitosan and the like as the dispersing agent or embedding agent of the elemental selenium can prevent the elemental selenium from aggregating, but the particle size of the formed macromolecular nano selenium particles is relatively large, so that the macromolecular nano selenium particles are not beneficial to absorption and utilization of organisms, and have single function.
Therefore, aiming at the defects of the existing research technology of nano-selenium materials, the invention provides a novel nano-selenium material preparation scheme, and the prepared egg white polypeptide nano-selenium particles not only can be used as an edible selenium supplement, but also can promote cell growth and be used as an antibacterial material, and have extremely high practical value.
Disclosure of Invention
The invention aims to provide a high-hydrolysis polypeptide nano selenium particle;
the invention also aims to provide a preparation method of the polypeptide nano selenium particles with high hydrolysis degree;
the invention also aims to provide the application of the polypeptide nano selenium particles with high hydrolysis degree in inhibiting the growth of bacteria;
the invention also aims to provide the application of the polypeptide nano selenium particles with high hydrolysis degree in antioxidation;
the invention also aims to provide the application of the polypeptide nano selenium particles with high hydrolysis degree in promoting cell growth.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
the high-hydrolysis-degree polypeptide nano selenium particle has the particle size of 255-448nm and the hydrolysis degree of 15-30%.
Preferably, the particle size of the polypeptide nano selenium particles with high hydrolysis degree is less than 260 nm.
Compared with inorganic selenium, the nano selenium with smaller particle size has safer and more efficient absorption and utilization rate. The inventor finds that in the polypeptide nano selenium with high hydrolysis degree and 30% of hydrolysis degree, the proportion of the polypeptide of 0-500Da reaches more than 65%, and the molecular weight of more than 1500Da is almost not existed; the polypeptide with 0-500Da in the polypeptide nano-selenium with high hydrolysis degree of 15% has only 50%, and the molecular weight distribution is concentrated in 0-1000 Da. From the analysis, the polypeptide with higher hydrolysis Degree (DH) has smaller molecular weight and higher absorptivity of the polypeptide with high hydrolysis degree nano-selenium.
The spectrum of the high-hydrolysis-degree polypeptide nano selenium shows a negative peak in a 201nm ultraviolet region, which shows that in the formation process of the high-hydrolysis-degree polypeptide nano selenium, an alpha-helical structure is increased, and the hydrogen bonding effect is enhanced, so that the stability between egg white polypeptide (EWP, a main component of egg white powder) and the nano selenium is enhanced.
In a second aspect of the present invention, there is provided:
the preparation method of the polypeptide nano selenium particles with high hydrolysis degree comprises the following steps:
(1) ultrasonically treating protein raw materials, adjusting pH, adding Alcalase enzyme for enzymolysis, adding alkaline solution for adjusting pH, inactivating enzyme, and centrifuging to obtain polypeptide supernatant;
(2) adding Na2SeO3Heating to obtain polypeptide nano selenium particles with high hydrolysis degree;
wherein the ultrasonic frequency is 35-40kHz, and the ultrasonic time is 10-15 min;
the pH value of the solution after the protein raw material is added into the alkaline solution is equal to the pH value of the solution before enzymolysis.
The inventor finds that the ultrasonic treatment can destroy the disulfide bond conformation of the egg white protein, reduce the viscosity of the solution and reduce the total sulfydryl number, thereby greatly increasing the contact area of enzyme and substrate and leading the hydrolysis degree to be relatively increased. The egg white hydrolysis degree after ultrasonic pretreatment is improved, and the protein hydrolysis is more sufficient. In the comparison of the degree of hydrolysis, the degree of hydrolysis (ultrasonic pretreatment) > the degree of hydrolysis (heat pretreatment) > the degree of hydrolysis (untreated), and the degree of hydrolysis of the egg white hardly changed after reaching 30%.
The egg white polypeptide with small hydrolysis degree has low hydrolysis degree and incomplete hydrolysis, which can cause large molecular weight and uneven distribution of the polypeptide, thereby influencing the action effect of the polypeptide.
Further, the amount of the alkaline solution added in the above step (1) is 2.1 to 4.1 ml.
The amount of alkaline solution added (or consumed) indicates the degree of hydrolysis of the egg white polypeptide.
The alkaline solution includes sodium hydroxide.
Further, the protein raw material in the step (1) is egg white powder. The main component of the egg white powder is egg white polypeptide.
Of course, other protein polypeptide raw materials can be reasonably replaced according to actual requirements.
Further, the heating temperature in the step (2) is 70-80 ℃, and the heating time is 3-3.5 h.
Further, the preparation method further comprises the following steps: after the protein raw material is treated by ultrasonic wave, the protein raw material is subjected to heat preservation treatment.
Furthermore, the temperature of the heat preservation treatment is 50-55 ℃, and the heat preservation time is 10-15 min.
In a third aspect of the present invention, there is provided:
the application of the polypeptide nano selenium particles with high hydrolysis degree in inhibiting the growth of bacteria.
Such bacteria include Staphylococcus aureus (gram positive) and Escherichia coli (gram negative).
In a fourth aspect of the present invention, there is provided:
the application of the polypeptide nano selenium particles with high hydrolysis degree in preparing an antioxidant preparation.
In a fifth aspect of the present invention, there is provided:
the application of the polypeptide nano selenium particles with high hydrolysis degree in promoting cell growth.
The inventor finds that the polypeptide nano selenium particles with high hydrolysis degree are applied to cells, and the nano selenium particles not only improve the survival rate of the cells, but also have the effect of promoting the growth of the cells at an extremely low concentration.
The invention has the beneficial effects that:
1. the method prepares the reductive polypeptide capable of reducing sodium selenite into elemental selenium by using the egg white powder, is safe and environment-friendly, and cannot cause damage to human bodies;
2. according to the invention, egg white polypeptide with different degrees of hydrolysis is obtained by pretreating egg white protein, polypeptide nano selenium particles with different properties and particle sizes are prepared, and the application is wide;
3. the polypeptide nano selenium particles with high hydrolysis degree have good oxidation resistance, have the function of promoting cell growth at low concentration, and can inhibit bacterial growth.
Drawings
FIG. 1 is a graph showing the effect of different pretreatment methods on the degree of hydrolysis of egg white polypeptide;
FIG. 2 is a liquid phase diagram (A) of egg white polypeptide obtained by different degrees of hydrolysis and a molecular weight ratio diagram (B) thereof;
FIG. 3 is a comparison of the reducing power of egg white polypeptide and high-hydrolysis polypeptide nano-selenium particles with 15% and 30% hydrolysis degree
FIG. 4 is a comparison of the lipid peroxidation resistance of egg white polypeptide and high-hydrolysis polypeptide nano selenium particles with 15% and 30% hydrolysis degrees;
FIG. 5 is an appearance diagram (A) and a particle size diagram (B) of polypeptide nano selenium particles with high hydrolysis degrees of 15% and 30%;
FIG. 6 is a circular dichroism spectrum (A) of egg white polypeptide and high-hydrolysis polypeptide nano selenium particles with 30% hydrolysis degree and a secondary structure diagram (B) of the circular dichroism spectrum;
FIG. 7 is a schematic diagram of the formation and structure of high-hydrolysis polypeptide nano-selenium particles;
FIG. 8 is a picture of the growth inhibition zone of high-hydrolysis polypeptide nano-selenium particles with 15% and 30% hydrolysis degree on pathogen inhibition around the inner hole of the culture medium;
FIG. 9 is a morphological diagram of cells cultured in the sample for 24 hours ((a) is Na)2SeO3Polypeptide nano selenium with 15% hydrolysis degree, (c) polypeptide nano selenium with 30% hydrolysis degree, and control is normal growth cells);
FIG. 10 shows Na2SeO3And the effect of high-hydrolysis polypeptide nano selenium particles with 15% and 30% of hydrolysis degree on cell viability.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Example 1
The example provides a preparation method of polypeptide nano selenium particles with 15% polypeptide hydrolysis degree:
weighing 1g of egg white powder, adding the egg white powder into 100mL of distilled water, uniformly oscillating, and then carrying out ultrasonic (40kHz) pretreatment for 15 min.
② preserving the heat of the egg white liquid after the ultrasonic treatment for 10min at 55 ℃, then adjusting the pH to the optimum pH of 8.0 by 0.1M HCl, adding 200uL of Alcalase enzyme, and carrying out enzymolysis in water bath at 55 ℃. Titrate to 8.0 with 0.5M NaOH every 30min (consuming 2.1mL NaOH solution) to give a polypeptide solution with 15% hydrolysis. After enzyme deactivation at 90 ℃, the mixture is centrifuged to separate polypeptide supernatant.
③ regulating the concentration of the polypeptide solution to be 35mg/mL, adding Na with the concentration of 6mM2SeO3The reaction temperature is 80 ℃, the reaction time is 3.5 hours, and the polypeptide nano selenium particles with high hydrolysis degree are prepared.
The polypeptide nano selenium particles with high hydrolysis degree prepared by the method are 448 nm.
The calculation formula of the enzymolysis degree in the invention is as follows:
wherein B is the consumption volume of NaOH, mL;
Nbthe molar concentration of NaOH is mol/L;
MPthe mass of the egg white powder is g;
htotis egg white protein htot(gram equivalent of peptide bond per gram of protein) value, h of egg white proteintotThe value was 7.67 mmol/g;
α represents the average degree of dissociation of α amino acids (about 0.9091 when the pH is 8.0 at 55 ℃).
Example 2
The example provides a preparation method of polypeptide nano selenium particles with 30% polypeptide hydrolysis degree:
weighing 1g of egg white powder, adding the egg white powder into 100mL of distilled water, uniformly oscillating, and then carrying out ultrasonic (40kHz) pretreatment for 15 min.
② preserving the heat of the egg white liquid after the ultrasonic treatment for 10min at 55 ℃, then adjusting the pH to the optimum pH of 8.0 by 0.1M HCl, adding 200uL of Alcalase enzyme, and carrying out enzymolysis in water bath at 55 ℃. Titrate to 8.0 with 0.5M NaOH every 30min (consuming 4.1mL NaOH solution) to give a polypeptide solution with 30% hydrolysis. After enzyme deactivation at 90 ℃, the mixture is centrifuged to separate polypeptide supernatant.
③ regulating the concentration of the polypeptide solution to be 35mg/mL, adding Na with the concentration of 6mM2SeO3The reaction temperature is 80 ℃, the reaction time is 3.5 hours, and the polypeptide nano selenium particles with high hydrolysis degree are prepared.
The polypeptide nano selenium particles with high hydrolysis degree prepared by the method are 255 nm.
Example 3
Weighing 1g of egg white powder, adding the egg white powder into 100mL of distilled water, uniformly oscillating, and then carrying out ultrasonic (40kHz) pretreatment for 15 min.
② preserving the heat of the egg white liquid after the ultrasonic treatment for 10min at 55 ℃, then adjusting the pH to the optimum pH of 8.0 by 0.1M HCl, adding 200uL of Alcalase enzyme, and carrying out enzymolysis in water bath at 55 ℃. Titrate to 8.0 with 0.5M NaOH every 30min (consuming 3.2mL NaOH solution) to give a polypeptide solution with 23% hydrolysis. After enzyme deactivation at 90 ℃, the mixture is centrifuged to separate polypeptide supernatant.
③ regulating the concentration of the polypeptide solution to be 35mg/mL, adding Na with the concentration of 6mM2SeO3The reaction temperature is 80 ℃, the reaction time is 3.5 hours, and the polypeptide nano selenium particles with high hydrolysis degree are prepared.
The polypeptide nano-selenium particles with high hydrolysis degree prepared by the method are 265 nm.
The effect detection of the polypeptide nano selenium particles with high hydrolysis degree
The antioxidant activity of the high-hydrolysis polypeptide nano-selenium particles (obtained by using the high-hydrolysis polypeptide nano-selenium particles obtained in examples 1 and 2) of the present invention is indicated by reducing power and lipid peroxidation resistance; the bacteriostatic effect takes the size of a bacteriostatic zone of a culture medium for staphylococcus aureus (gram positive) and escherichia coli (gram negative) as an index; cytotoxicity is detected by using liver cancer cell activity as an index.
Reduction force measurement
Taking egg white as a reference, respectively taking 10mL of high-hydrolysis-degree polypeptide nano selenium solution with different concentrations, then adding 2.5mL of 0.2M phosphate buffer (pH6.6) and 2.5mL of 1% potassium ferricyanide solution, placing in a water bath at 50 ℃ for constant temperature reaction for 20min, and then rapidly cooling. Immediately adding 2.5mL of 10% trichloroacetic acid solution, centrifuging (3000Xg,10min,20 ℃), collecting 2.5mL of supernatant, adding 2.5mL of distilled water and 0.5mL of 0.1% ferric trichloride solution, shaking, mixing, standing for 10min, and measuring light absorption value at 700 nm.
Determination of anti-lipid peroxidation Capacity
Taking egg white as a reference, mixing 7.2mL of soybean lecithin with the concentration of 0.4% (w/v) and 0.8mL of polypeptide nano selenium solutions with different concentrations and high hydrolysis degrees, and then adding 0.8mL of FeSO4 solution with the concentration of 10mmol/L for catalytic oxidation reaction. After mixing, the sample was incubated in a 37 ℃ water bath for 15min, then 2mL of 20% (w/v) trichloroacetic acid solution was added and centrifuged at 5000r/min for 10 min. 4mL of the supernatant was added with 2mL of 0.8% (w/v) thiobarbituric acid, shaken well and then placed in a boiling water bath for 15 min. Measuring absorbance A at 532nm, and measuring absorbance A with distilled water as blank control instead of hydrolysate0The lipid peroxidation resistance is calculated according to the following formula:
determination of bacteriostatic Effect
The cultured staphylococcus aureus and escherichia coli were inoculated into a specific agar culture dish, two holes were punched in each culture dish in sequence using a 5mm punch, 0.5mL of high-hydrolysis polypeptide nano-selenium having a selenium content of 20mg/mL was added to the holes, and the other was added with physiological saline as a blank. After incubating the dishes in a growth chamber at 37 ℃ for 24 hours, the size of the zone of inhibition was measured.
Determination of cell viability
The high-hydrolysis polypeptide nano-selenium particles of the invention and Na were evaluated by the CCK-8 assay according to the instructions of the assay kit manufacturer2SeO3The effect on cell viability.
The test cells used in the present invention were HepG-2 cells.
Cells were seeded at a density of 50000 cells/well in 96-well plates and then incubated in 100uL of medium. And after 24 hours of culture, changing the culture solution, culturing by using the nano selenium particles, preparing culture media containing the polypeptide nano selenium particles with different concentrations and high hydrolysis degrees, and culturing the cells in the pore plates for 24 hours. Finally, 10uL of CCK-8 reagent was added to each well and 5% CO at 37 deg.C2And further incubated at 95% humidity for 0.5 hour. Measuring absorbance at 450nm with enzyme reader to quantify cell survival rate, wherein the control group uses Na containing equal amount of selenium according to the mass ratio of selenium element in high-hydrolysis degree polypeptide nano selenium particles2SeO3. Cell viability was calculated using the following formula:
wherein, As is the light absorption value measured under the condition that nano selenium particles exist;
ac is the absorbance value measured under the condition without nano selenium particles;
ab is the absorbance measured with medium and CCK-8 only.
Measurement results
Influence of pretreatment mode on hydrolysis degree of polypeptide nano-selenium with high hydrolysis degree
As can be seen from FIG. 1, the hydrolysis degree of the egg white polypeptide increases with the increase of time, and the hydrolysis degree value tends to be flat when the enzymolysis time reaches 300min by using protease, and hardly changes after the hydrolysis degree reaches 30%.
With different pretreatment modes, it was further found that the degree of hydrolysis (ultrasonic pretreatment) > the degree of hydrolysis (thermal pretreatment) > the degree of hydrolysis (untreated). The ultrasonic treatment is proved to destroy the disulfide bond conformation of the egg white protein, reduce the viscosity of the solution and reduce the total sulfydryl number, thereby greatly increasing the contact area of the enzyme and the substrate and leading the hydrolysis degree to be relatively increased. The egg white hydrolysis degree after ultrasonic pretreatment is improved, and the protein hydrolysis is more sufficient.
As can be seen from FIG. 2, the egg white polypeptide with low hydrolysis degree is found by LC-MS analysis of the polypeptide nano-selenium with high hydrolysis degree, and the egg white polypeptide with low hydrolysis degree is low in hydrolysis degree, incomplete in hydrolysis, large in molecular weight and uneven in distribution. In the high-hydrolysis-degree polypeptide nano selenium with the hydrolysis degree of 30%, the polypeptide proportion of 0-500Da reaches more than 65%, and almost no molecular weight is larger than 1500 Da; the polypeptide with 0-500Da in the polypeptide nano-selenium with high hydrolysis degree of 15% has only 50%, and the molecular weight distribution is concentrated in 0-1000 Da. From the above analysis, it can be concluded that the larger the DH, the smaller the molecular weight of the polypeptide.
As can be seen from fig. 3, the reducing power of the nano-selenium particles with different polypeptide hydrolysis degrees is stronger than that of the egg white polypeptide, and the reducing power of the nano-selenium particles with the polypeptide hydrolysis degree of 30% is higher than that of the nano-selenium particles with the polypeptide hydrolysis degree of 15%.
As can be seen from FIG. 4, the lipid peroxidation resistance of the nano-selenium particles with different polypeptide hydrolysis degrees is stronger than that of egg white polypeptide. The anti-lipid peroxidation capacity of the nano selenium particles with the polypeptide hydrolysis degree of 30% is obviously higher than that of the nano selenium particles with the polypeptide hydrolysis degree of 15%. The higher the hydrolysis degree of the polypeptide for preparing the nano selenium particles is, the better the oxidation resistance of the prepared polypeptide nano selenium particles is.
As can be seen from fig. 5, the egg white polypeptide nano-selenium with the hydrolysis degree of 30% is clearer in appearance and darker in color than the egg white polypeptide nano-selenium with the hydrolysis degree of 15% compared with the egg white polypeptide nano-selenium with the hydrolysis degree of 30%. The molecular weight of the egg white polypeptide with larger hydrolysis degree is smaller, and the prepared polypeptide nano selenium with high hydrolysis degree also shows the rule. The high-hydrolysis polypeptide nano-selenium with the hydrolysis degree of 30 percent has the grain diameter of about 255nm, and the high-hydrolysis polypeptide nano-selenium with the hydrolysis degree of 15 percent has the grain diameter of about 448nm with the difference of about 200 nm. After the polypeptide nano selenium with high hydrolysis degree is formed by the polypeptide and the nano selenium, the particle size of the polypeptide nano selenium with high hydrolysis degree prepared by the polypeptide with small hydrolysis degree is smaller, and the required stabilizing force is relatively smaller.
As can be seen from FIG. 6, the spectra of the egg white polypeptide and the high-hydrolysis polypeptide nano-selenium respectively show two negative peaks in the ultraviolet regions of 198nm and 201nm, which are the characteristics of the alpha-helical structure, and illustrate that the alpha-helical structure changes after the egg white polypeptide nano-selenium is formed. From the secondary structure analysis of the egg white polypeptide to the polypeptide nano-selenium with high degree of hydrolysis, it can be seen that the alpha-helix structure is increased, which corresponds to the helix structure transition of the previous reaction. The above results show that in the process of forming the egg white polypeptide nano selenium, the alpha-helical structure is increased, and the hydrogen bonding effect is enhanced. Therefore, the secondary structure is changed due to the interaction between the egg white polypeptide and the polypeptide nano selenium with high hydrolysis degree, and the stability of the egg white polypeptide to the nano selenium can be increased due to the increase of hydrogen bonds.
As can be seen from fig. 7, in a Transmission Electron Microscope (TEM). The high-hydrolysis polypeptide nano-selenium has different particle sizes, including solid and hollow, but still spherical structures, and the particle size of the high-hydrolysis polypeptide nano-selenium egg white polypeptide nano-selenium is not much different from that of the high-hydrolysis polypeptide nano-selenium in a solution, and the internal structure is probably hollow. After aggregation, the nano-selenium increases in particle size and consists of many small spheres, with incompletely formed spheres on the sides. The forming structure diagram and the microscopic structure diagram of the polypeptide nano selenium with high hydrolysis degree can be obtained. From the above, the egg white polypeptide nano-selenium has a tendency of aggregating to spheres, and a hollow sphere mode can exist inside, which is similar to the self-assembly process.
As can be seen from fig. 8, the inhibitory effect of the nano-selenium particles having a polypeptide hydrolysis degree of 15% was not as good as that of the nano-selenium particles having a polypeptide hydrolysis degree of 30% for escherichia coli; the inhibiting effect of the nano selenium particles with the polypeptide hydrolysis degree of 15% on staphylococcus aureus is better than that of the nano selenium particles with the polypeptide hydrolysis degree of 30%, but the nano selenium particles with the polypeptide hydrolysis degrees of 15% and 30% have the capacity of inhibiting escherichia coli and staphylococcus aureus.
FIG. 9 shows that Na is used2SeO3And independently culturing HepG-2 cells by egg white polypeptide nano selenium with different degrees of hydrolysis. The cells showed a significant dose dependence of the cell number, with the increase in sample concentration the contact between the cells was lost, forming cytocontractile and apoptotic bodies. Control cells that grew normally adhered to each other, were intact in shape, and grew well. Under the condition of the same dose of Se, the number of the treated cells is the minimum when the cells are subjected to microscopic examination, which indicates that Na2SeO3Severely limits the growth of cells, and the number of polypeptide nano-selenium cultured cell strains with 15 percent of hydrolysis degree and 30 percent of hydrolysis degree is obviously more than that of Na2SeO3And (4) grouping. This also shows that the toxicity of the egg white polypeptide nano-selenium is less than that of Na under the same selenium dose2SeO3。
In fig. 10, 30% hydrolysis polypeptide nano-selenium with smaller particle size can promote cell growth at very low concentration, and can inhibit cell growth only when the concentration is increased to 6.25ug/ml, and the inhibition effect on cell growth is stronger and stronger with the increase of the concentration. And the polypeptide nano-selenium with 15 percent of hydrolysis degree and larger granularity also shows the inhibition effect on the cell growth when the concentration is extremely low. The cytotoxicity may have a certain relationship with the size of the polypeptide nano-selenium, and the smaller the particle size of the polypeptide nano-selenium is, the smaller the toxic effect on cells is.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The polypeptide nano selenium particle with high hydrolysis degree is characterized in that the particle size of the polypeptide nano selenium particle with high hydrolysis degree is 255-448nm, and the hydrolysis degree is 15% -30%.
2. The method for preparing the high-hydrolysis-degree polypeptide nano selenium particles as claimed in claim 1, which comprises the following steps:
(1) ultrasonically treating protein raw materials, adjusting pH, adding Alcalase enzyme for enzymolysis, adding alkaline solution for adjusting pH, inactivating enzyme, and centrifuging to obtain polypeptide supernatant;
(2) adding Na2SeO3Heating to obtain polypeptide nano selenium particles with high hydrolysis degree;
wherein the ultrasonic frequency is 35-40kHz, and the ultrasonic time is 10-15 min;
the pH value of the solution of the protein raw material added with the alkaline solution is equal to the pH value of the solution before enzymolysis.
3. The method according to claim 2, wherein the amount of the basic solution added in step (1) is 2.1 to 4.1 mL.
4. The method according to claim 2, wherein the protein material in step (1) is egg white powder.
5. The preparation method according to claim 2, wherein the heating temperature in the step (2) is 70-80 ℃ and the heating time is 3-3.5 h.
6. The method of manufacturing according to claim 2, further comprising: after the protein raw material is treated by ultrasonic wave, the protein raw material is subjected to heat preservation treatment.
7. The preparation method according to claim 7, wherein the temperature of the heat preservation treatment is 50-55 ℃, and the heat preservation time is 10-15 min.
8. The use of the high-hydrolysis polypeptide nano-selenium particle of claim 1 for inhibiting bacterial growth.
9. The use of the high-hydrolysis polypeptide nano-selenium particles of claim 1 in the preparation of an antioxidant preparation.
10. The use of the high-hydrolysis polypeptide nano-selenium particle of claim 1 for promoting cell growth.
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