Bacteriostatic mesoporous starch hemostatic powder and preparation method thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a bacteriostatic mesoporous starch hemostatic powder and a preparation method thereof. The method comprises the following steps: (1) starch gelatinization; (2) enzyme hydrolysis and debranching; (3) regulating self-assembly of the debranching solution; and (4) drying to obtain the bacteriostatic mesoporous starch hemostatic powder. The application regulates the kinetic properties of micro-particle self-assembly of short-chain glucan (SCG) by adding chitosan to the SCG solution produced by hydrolysis of starch by pullulanase, and induces the SCG to form a mesoporous structure. The preparation process is a green and efficient preparation method of the mesoporous starch hemostatic powder, and does not require strong acid and strong base, harmful organic solvents, high temperature and high pressure and other conditions. Meanwhile, the particle size and pore structure of the mesoporous starch can be regulated by the concentration of chitosan, and the prepared starch has stable pore structure, uniform particle size and pore distribution, and has a broad market prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a bacteriostatic mesoporous starch hemostatic powder and a preparation method thereof. BACKGROUND

[0002] It is reported that nearly one-third of traumatic deaths are caused by excessive bleeding, and for life-threatening traumatic bleeding, timely and effective hemostasis is very necessary. So far, a series of hemostatic materials such as hemostatic bandages, hemostatic sponges, hemostatic powders and hemostatic gauze have been researched and developed for rapid and effective control of bleeding. The porous powder hemostatic agent is mainly used for capillary or venous bleeding, is suitable for deep, irregular and unpressable wounds, is used for severe skin surface bleeding in emergency conditions, can be used for large area wounds of external organs in surgical operations, reduces the damage of tissue coagulation to tissue, and can prevent tissue adhesion.

[0003] However, the existing porous powder hemostatic agent has certain defects. For example, a zeolite hemostatic agent of Quikclot is easy to burn the wound in the process of use, has poor biodegradability, and is only suitable for external surface wound bleeding; a starch hemostatic agent of Arista which is of pure plant origin has limited hemostatic way, relies on single physical hemostasis, and has high price; the potato starch hemostatic powder in some patents has a large pore size of the hemostatic powder, so that blood cells are trapped in the pores, affecting the hemostatic effect, and most of the pores are acid-modified, the pore size and particle size cannot be controlled, and the hemostatic powder has potential toxicity.

[0004] From the physical properties of the porous hemostatic powder, it can be known that the factors affecting the hemostatic effect include specific surface area, porosity, pore size, total pore volume and the like. Therefore, a porous hemostatic agent which has adjustable pore structure, good biodegradability, biocompatibility, safety, non-toxicity, low price and strong hemostatic performance is needed to make up for the shortcomings of the prior art.

[0005] Starch is the second most abundant carbohydrate in nature, has good biocompatibility and biodegradability, and is low in price. However, natural starch cannot meet the demand of porous hemostatic powder, and needs to be modified to have the performance of porous materials. The existing modification into pores is mainly acid modification technology. The porous starch prepared by acid pore formation has non-uniform and unstable porous structure and cannot be controlled, the preparation process is complex, the preparation cost is high, and the environment is polluted. Moreover, the hemostatic powder prepared by the prior art is expensive and the preparation process is complex. In addition, the hemostatic performance of the commercially available starch-based hemostatic powder is limited by the original structure characteristics of the original starch. SUMMARY

[0006] The technical problem to be solved by the present application is that the existing acid method for preparing porous starch has uneven and unstable porous structure, cannot be controlled, has complex preparation process, high preparation cost, pollutes the environment, and the hemostatic performance of the commercially available starch-based hemostatic powder is limited by the original structure of the original starch, and the application range is limited.

[0007] To solve the above problems, the present application regulates the kinetic properties of the self-assembly of SCG by adding chitosan to the short-chain glucan (SCG) solution produced by the hydrolysis of starch by pullulanase, and induces the formation of mesoporous structure of the particles. The preparation process is a green and efficient preparation method for mesoporous starch hemostatic powder, which does not require strong acid, strong base, harmful organic solvent and high temperature and pressure conditions; at the same time, the particle size and pore structure of the mesoporous starch can be regulated by the concentration of chitosan, and the prepared starch has stable pore structure, uniform particle size and pore distribution, and has broad market prospect.

[0008] To achieve the above purpose, the present application realizes a preparation method of antibacterial mesoporous starch hemostatic powder, comprising the following steps:

[0009] (1) starch gelatinization;

[0010] (2) enzyme hydrolysis and debranching: after cooling the starch gelatinization liquid, add pullulanase, mix uniformly, and perform water bath enzyme hydrolysis and debranching;

[0011] (3) regulating self-assembly of debranching liquid: centrifuge the debranching liquid obtained in step (2), take out the supernatant to obtain short-chain glucan SCG, immediately add chitosan solution to the SCG solution, and then put it in the refrigerator for self-assembly;

[0012] (4) drying to obtain antibacterial mesoporous starch hemostatic powder.

[0013] Studies have shown that enzyme-hydrolyzed starch can produce short-chain glucan, which can self-assemble into self-assembled starch particles under certain conditions, and regulating the self-assembly of short-chain glucan can produce self-assembled starch with uniform particle size and flower-shaped or spindle-shaped morphology. Chitosan has good biocompatibility, is biodegradable, safe and non-toxic, and has excellent antibacterial properties, and can be used as a hemostatic powder raw material. The present application further utilizes chitosan with steric hindrance effect to regulate the morphology of self-assembled starch, and prepares starch hemostatic powder with porous structure.

[0014] Further, the step (1) is to weigh the amylopectin, add ultrapure water to prepare a 3-4% w / v solution, mix the solution uniformly, and gelatinize uniformly at a high temperature of 100℃ under stirring until the solution boils and becomes transparent. The present application uses amylopectin, and more short-chain glucans can be produced by enzymatic hydrolysis of amylopectin, which is beneficial to subsequent self-assembly. The amylopectin is preferably waxy corn starch or waxy corn starch.

[0015] Further, the step (2) is to cool the starch gelatinized solution to below 60℃, then add pullulanase, and the water bath temperature is 50-60℃. This parameter is the optimal enzymatic hydrolysis temperature of pullulanase. If the temperature exceeds this value, the enzyme may be inactivated. If the temperature is lower than this value, the enzymatic hydrolysis efficiency is low.

[0016] Further, the volume concentration of pullulanase in the step (2) is 0.25-1%, and the enzymatic debranching time is 24-72h. This parameter can produce more short-chain glucans. When the concentration of pullulanase is low, the enzymatic debranching time is long. When the concentration of pullulanase is high, the enzymatic debranching time is short.

[0017] Further, the centrifugation parameter in the step (3) is 3000-4000rpm for 10-15min, and the self-assembly is performed at 2-8℃. The self-assembly efficiency is high and the time is short at this temperature range.

[0018] Further, the final concentration of chitosan in the step (3) is 0.005-0.03% w / v. This parameter can prepare mesoporous starch particles with small particle size and uniform dispersion. If the concentration exceeds this range, the self-assembled starch particles prepared do not meet the requirements of hemostatic agents. The regulation mechanism is that the positive charge of chitosan has a steric hindrance effect, which can make short-chain glucans grow into mesoporous particles during self-assembly. The degree of steric hindrance effect is different when the concentration of chitosan is different, and the pore structure of the prepared mesoporous starch particles is different.

[0019] Further, the step (4) is to wash the self-assembled starch solution obtained in step (3) with ultrapure water and then dry it in a vacuum freeze-drying device.

[0020] A bacteriostatic mesoporous starch hemostatic powder obtained by the above method has a specific surface area of up to 20.54m 2 / g, and a total pore volume of up to 0.131cm 3 / g, and the average pore size is 20-26 nm. The hemostatic powder of the application has good water absorption capacity, and also has excellent antibacterial performance on gram-negative bacteria E. coli and gram-positive bacteria S. aureus due to the addition of chitosan. The hemostatic powder also has good compatibility and coagulation capacity for blood. The good compatibility is due to the fact that the mesoporous starch particles are prepared from starch and chitosan, and the raw material does not cause hemolysis, and the hemolysis rate can be used to explain the coagulation capacity. The coagulation capacity is achieved by the mesoporous structure of the hemostatic agent to quickly absorb the water in the blood, so that the blood cells, platelets and other substances are gathered on the surface of the material to accelerate the coagulation. In addition, the positive charge of chitosan can adsorb the negatively charged blood cells to accelerate the coagulation. The zeolite hemostatic agent generates a large amount of heat after absorbing water during use, which can burn the wound. However, the mesoporous starch hemostatic agent of the application does not generate heat after absorbing water, and is mainly used for capillary and venous hemostasis, and is suitable for irregular, non-pressing and deep wounds, and can be used for hemostasis of external organs during surgery.

[0021] The application has the following advantages:

[0022] (1) The application adjusts the kinetic properties of the self-assembly of short-chain glucan (SCG) particles by adding chitosan to the SCG solution produced by the hydrolysis of pullulanase, and induces the particles to form a mesoporous structure. The preparation method is simple, green and safe, and the cost is low. The particle and pore structure of the hemostatic powder can be controlled, and the problem of uncontrolled pore structure of traditional porous hemostatic powder is solved.

[0023] (2) The chitosan in the hemostatic powder has excellent antibacterial performance, and the positively charged chitosan can electrostatically adsorb negatively charged red blood cells to enhance the aggregation of red blood cells and activate the coagulation pathway, thereby achieving simultaneous hemostasis through multiple pathways.

[0024] (3) The raw materials of the hemostatic powder are starch and chitosan, which are low in cost, abundant in source, biocompatible, degradable, safe and non-toxic, and the preparation process is simple and easy to reproduce. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The preparation flowchart of the hemostatic powder in the application is shown in the figure;

[0026] Figure 2 The scanning electron microscope image of the hemostatic powder in the application is shown in the figure;

[0027] Figure 3 The N2 isothermal adsorption-desorption curve of the hemostatic powder in the application is shown in the figure;

[0028] Figure 4 The pore size distribution graph of the hemostatic powder in the application is shown in the figure;

[0029] Figure 5 Figure 1 is an infrared analysis spectrum of the hemostatic powder of the present application;

[0030] Figure 6 Figure 2 is a water absorption column chart of the hemostatic powder of the present application;

[0031] Figure 7-(1-2) is an antibacterial experiment result chart of the hemostatic powder of the present application;

[0032] Figure 8 Figure 8 is a hemolysis rate result chart of the hemostatic powder of the present application;

[0033] Figure 9 Figure 9 is a coagulation index result chart of the hemostatic powder of the present application. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0035] In the following examples, the amylopectin content of the waxy corn starch used is more than 99%, the chitosan is purchased from Macklin, item number C916461-100g, deacetylation degree 90%, molecular weight 200000, and the pullulanase D2 is purchased from Novozymes. Other materials are commercially available unless otherwise specified.

[0036] Example 1

[0037] A mesoporous starch hemostatic powder with bacteriostatic efficacy, as shown in Figure 1 includes the following preparation steps:

[0038] (1) Prepare a chitosan solution: weigh a certain amount of chitosan powder and prepare a 0.5% w / v chitosan solution with 1% acetic acid. Place it in a 500 rpm shaker to fully dissolve the chitosan powder.

[0039] (2) Gelatinization: add waxy corn starch to 200 mL ultrapure water to a final concentration of 2-4% w / v starch solution, mix well, then place the starch solution in a microwave oven and heat to boiling until it becomes transparent, and cool to below 60°C.

[0040] (3) Enzymatic hydrolysis (debranching): add 0.5% pullulanase to the gelatinized starch and place it in a 60°C water bath for 48 hours of enzymatic hydrolysis.

[0041] (4) Chitosan regulated self-assembly of the debranching solution: immediately centrifuge the debranching solution at 3000 rpm for 10 min, immediately take out the SCG supernatant solution, add chitosan solution to a final concentration of 0.005% w / v, and place the solution in a 4°C refrigerator for self-assembly.

[0042] (5) Drying: wash the self-assembled particles with ultrapure water twice, and then place them in a vacuum freeze dryer for drying, thereby obtaining a starch-based mesoporous hemostatic powder.

[0043] Example 2:

[0044] A mesoporous starch hemostatic powder with antibacterial efficacy includes the following preparation steps:

[0045] (1) Prepare a chitosan solution: weigh a certain amount of chitosan powder, and prepare a 0.5% w / v chitosan solution with 1% acetic acid. The chitosan powder can be fully dissolved in a 500 rpm shaker.

[0046] (2) Gelatinization: add waxy corn starch to ultrapure water to prepare a starch solution with a final concentration of 2-4% w / v. After thoroughly mixing and uniformity, place the starch solution in a microwave oven for high-temperature heating until the solution boils and becomes transparent. Cool to below 60°C.

[0047] (3) Enzymatic hydrolysis (debranching): add 0.5% by volume of pullulanase to the gelatinized starch, and place it in a 60°C water bath for enzymatic hydrolysis for 48 hours.

[0048] (4) Chitosan regulated self-assembly of the debranching solution: immediately centrifuge the debranching solution at 3000 rpm for 10 min, immediately take out the SCG supernatant solution, add chitosan solution to a final concentration of 0.01%-0.05 w / v, and place the solution in a 4°C refrigerator for self-assembly.

[0049] (5) Drying: wash the self-assembled particles with ultrapure water twice, and then place them in a vacuum freeze dryer for drying, thereby obtaining a starch-based mesoporous hemostatic powder.

[0050] Example 3:

[0051] A mesoporous starch hemostatic powder with antibacterial efficacy includes the following preparation steps:

[0052] (1) Prepare a chitosan solution: weigh a certain amount of chitosan powder, and prepare a 0.5% w / v chitosan solution with 1% acetic acid. The chitosan powder can be fully dissolved in a 500 rpm shaker.

[0053] (2) Gelatinization: add waxy corn starch to ultrapure water to prepare a starch solution with a final concentration of 2-4% w / v. After thoroughly mixing and uniformity, place the starch solution in a microwave oven for high-temperature heating until the solution boils and becomes transparent. Cool to below 60°C.

[0054] (3) Enzymatic hydrolysis (debranching): add 0.25% volume concentration of pullulanase to gelatinized starch, and place in a 55°C water bath for 72h of enzymatic hydrolysis.

[0055] (4) Chitosan-regulated self-assembly of debranching solution: immediately centrifuge the debranching solution at 3000 rpm for 10 min, immediately take out the SCG supernatant solution, add chitosan solution to a final concentration of 0.03% w / v, and place the solution in a 4°C refrigerator for self-assembly.

[0056] (5) Drying: wash the self-assembled particles twice with ultrapure water, and then place them in a vacuum freeze dryer for drying, thereby obtaining starch-based mesoporous hemostatic powder.

[0057] Example 4

[0058] A kind of mesoporous starch hemostatic powder with antibacterial efficacy of calcium ion adhesion, comprising the following preparation steps:

[0059] (1) Prepare chitosan solution: weigh a certain amount of chitosan powder, and prepare a 0.5% w / v chitosan solution with 1% acetic acid. The chitosan powder can be fully dissolved in a 500 rpm shaker.

[0060] (2) Gelatinization: add waxy corn starch to ultrapure water to prepare a starch solution with a final concentration of 1% w / v. After mixing evenly, place the starch solution in a microwave oven and heat to boiling until it becomes transparent. Cool to below 60°C.

[0061] (3) Enzymatic hydrolysis (debranching): add 1-3% volume concentration of pullulanase to the gelatinized starch, and place in a 50-60°C water bath for 24-72h of enzymatic hydrolysis.

[0062] (4) Chitosan-regulated self-assembly of debranching solution: immediately centrifuge the debranching solution at 3000 rpm for 10 min, immediately take out the SCG supernatant solution, add chitosan solution to a final concentration of 0.01% w / v, and place the solution in a 4°C refrigerator for self-assembly.

[0063] (5) Calcium ion adhesion: wash the self-assembled particles twice with ultrapure water, add CaCl2 to a final concentration of 50mM, mix the sample and place it for more than 2h. The addition of CaCl2 provides Ca, which promotes coagulation.

[0064] (6) Drying: wash the calcium ion-adhered sample twice with ultrapure water, and then place it in a vacuum freeze dryer for drying, thereby obtaining calcium ion-adhered starch-based mesoporous hemostatic powder.

[0065] Comparative Example 1:

[0066] Dried waxy corn starch (raw starch).

[0067] Comparative Example 2:

[0068] Self-assembly of starch without chitosan regulation includes the following preparation steps:

[0069] (1) Paste: waxy corn starch is added to ultrapure water to prepare a starch solution with a final concentration of 2% w / v. After mixing well, the starch solution is heated to boiling in a microwave oven to become transparent, and then cooled to below 60°C.

[0070] (2) Enzymatic hydrolysis (debranching): 0.5% pullulanase by volume is added to the pasted starch, and the enzyme hydrolysis is carried out in a 60°C water bath for 48h.

[0071] (3) Self-assembly of debranching solution: the debranching solution is immediately centrifuged at 3000 rpm for 10 min, and the supernatant is immediately taken out to obtain the SCG solution. The solution is placed in a 4°C refrigerator for self-assembly.

[0072] (4) Drying: the self-assembled particles are washed twice with ultrapure water and then dried in a vacuum freeze dryer to obtain the starch-based mesoporous hemostatic powder.

[0073] Results verification: the mesoporous starch hemostatic powder with antibacterial efficacy prepared by the present application is proved to have the function of porous hemostatic powder through the following experiments.

[0074] Experiment 1:

[0075] Observation of the surface morphology of the mesoporous starch hemostatic powder with antibacterial efficacy:

[0076] The dried sample is adhered to the sample disc with conductive glue, and after gold spraying treatment, the surface morphology of the sample is observed and photographed by high-resolution scanning electron microscope (MERLIN Carl Zeiss, Oberkochen, Germany) with working distance 3.0mm and accelerating voltage 10kV. The results are shown in Figure 2 As shown in the table, Comparative Example 1 has no pore structure, Comparative Example 2 has a rough surface and no obvious porous structure, and Example 2 has a rough surface, uniform spherical particles, and a porous structure on the surface of the particles, and a large specific surface area of the particles.

[0077] Experiment 2:

[0078] Determination of the specific surface area and pore structure of the mesoporous starch hemostatic powder with antibacterial efficacy:

[0079] The sample was heat treated at 90℃ for 12h, and the N2 isothermal adsorption-desorption curve of the sample was determined by a high-performance multi-channel full-automatic specific surface area and porosity analyzer (TriStar II 3020 3.02, Micromeritics Instrument Corporation), and the specific surface area, average pore size and total pore volume were calculated, the analysis temperature was -195.85℃, the total pore analysis, the vacuum degassing temperature was 50℃, and the time was 24h.

[0080] The pore structure results are shown in Table 1. The BET specific surface area of the comparative example 1 is negative, that is, the nitrogen adsorption is negative, indicating that the original starch has no pore structure, which is consistent with the result of Figure 2 The specific surface area of the comparative example 2 is 1.76m 2 / g, and the average particle size is 3μm, indicating that the particle size of the self-assembled starch is large and the specific surface area is small without chitosan. The specific surface area of the example 2 is up to 20.54m 2 / g, the total pore volume is up to 0.131cm 3 / g, and the average pore size is 25.47nm, which belongs to mesoporous. The starch prepared by the example 1 and the example 3 also belongs to mesoporous starch, but the specific surface area and the total pore volume are smaller than those of the example 2.

[0081] Table 1

[0082]

[0083] The isothermal adsorption-desorption curves of the comparative examples and the examples are shown in Figure 3 The isothermal adsorption-desorption curves of the comparative example 2 and the example 2 are both type V (H3 hysteresis ring) isotherm, the mesoporous nature of the reaction material, the N2 adsorption amount of the example 2 is much higher than that of the comparative example 2, indicating that it has the most pores, and the adsorption and desorption curves are almost coincident, and the pore structure of the reaction material is stable.

[0084] The pore size distribution curves of the comparative examples and the examples are shown in Figure 4 The pore size distribution curve of the comparative example 2 is approximately a straight line, indicating that it has few pores; the pore size distribution of the example 2 is narrow and distributed between mesopores, and the result is consistent with the isothermal adsorption-desorption curve result.

[0085] Experiment three:

[0086] Infrared spectrum analysis of mesoporous starch hemostatic powder with bacteriostatic function:

[0087] The comparative example 2, chitosan, example 2 were vacuum dried at 50℃ for 24h, KBr was baked at 120℃ for more than 4h, the sample and KBr powder were mixed (1:100), ground with a corundum mortar under an infrared baking lamp, and then pressed into a sheet, and then qualitatively detected by a Fourier transform infrared spectrometer (Nicolet iS10, Thermo Fisher Scientific), KBr as background, scanning range 1500-4000cm -1 , scanning resolution 4cm -1 , each sample was scanned 20 times. The infrared spectrum analysis results are shown in Figure 5 , the absorption peaks of comparative example 2 and example 2 in the range of 4000-3300cm -1 are related to O-H and -NH stretching vibration (intermolecular hydrogen bond). The characteristic peak of chitosan at about 1600cm -1 is the -NH2 absorption peak, but there is no same absorption peak in example 2, which may be caused by the electrostatic interaction between the amino group in chitosan and the hydroxyl group on the surface of self-assembled starch. Compared with comparative example 2, the absorption peak in the range of 4000-3000cm -1 in example 2 is wider, indicating that the amino group in chitosan is connected with the hydroxyl group in self-assembled starch through hydrogen bond interaction.

[0088] Experiment four:

[0089] Water absorption of mesoporous starch hemostatic powder with bacteriostatic effect:

[0090] Take 20mg of comparative examples 1, 2 and example 2, add 2mL of PBS (pH 7.2-7.4), and place at 37℃ for 2h, then centrifuge the sample at 15000rpm for 5min, discard the supernatant, weigh the precipitate, weigh as Wmg, and calculate the swelling rate using the following formula.

[0091] Swelling rate (%) = (W-20) ÷ 20 x 100%

[0092] The experimental results are shown in Figure 6 , the water absorption of comparative example 1 is about 100%, the water absorption of comparative example 2 is about 240%, and the water absorption of example 2 is the largest, about 290%. Compared with comparative example 1, the water absorption of example 2 is significantly improved.

[0093] Experiment five:

[0094] Antibacterial experiment of mesoporous starch hemostatic powder with bacteriostatic effect:

[0095] Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria) were selected as bacterial models. Comparative Example 2 and Example 2 were placed in a 121°C high-pressure sterilization pot for sterilization for 20 min. First, 1 mL of bacterial solution (10 4 CFU / mL) was mixed with 4 mL of liquid medium, and then the sample was added to the mixture. No sample was added to the blank group; 5 mg of self-assembled starch was added to Comparative Example 2 to give a final concentration of 1 mg / mL; and 0.5, 2.5, and 5 mg of the hemostatic powder of Example 2 was added to Example 2 to give a final concentration of 0.1, 0.5, and 1 mg / mL, respectively. The bacterial solution with the added sample was placed in a 37°C, 180 rpm constant-temperature shaker, and 100 μL of the suspension was uniformly spread on the surface of an agar plate every 0, 2, 4, and 8 h and incubated at 37°C for 20 h. The agar plate was removed, and the colony-forming units on the agar plate were observed and counted.

[0096] The results of the antibacterial experiment are shown in FIG. 7. From the results, it can be seen that the blank group and Comparative Example 2 had no antibacterial effect on Staphylococcus aureus, and the number of bacteria showed a growth trend as the incubation time was extended. Example 2 had a significant antibacterial effect on Staphylococcus aureus, and the 0.5 and 1 mg / mL concentrations of Example 2 completely inhibited the growth of Staphylococcus aureus after 8 h. From the results, it can be seen that Example 2 had a good antibacterial effect on Staphylococcus aureus. Figure 7-1 The results show that the blank group and Comparative Example 2 had no antibacterial effect on Escherichia coli, and the number of bacteria showed a growth trend as the incubation time was extended. Example 2 had a significant antibacterial effect on Escherichia coli, and the 0.1 mg / mL concentration of Example 2 had no obvious inhibitory effect on Escherichia coli, the 0.5 and 1 mg / mL concentrations of Example 2 had a relatively obvious inhibitory effect on Escherichia coli, and the 1 mg / mL concentration of Example 2 completely inhibited the growth of Escherichia coli after 2 h. From the results, it can be seen that Example 2 had a good antibacterial effect on Escherichia coli. Figure 7-2 The results show that the blank group and Comparative Example 2 had no antibacterial effect on Escherichia coli, and the number of bacteria showed a growth trend as the incubation time was extended. Example 2 had a significant antibacterial effect on Escherichia coli, and the 0.1 mg / mL concentration of Example 2 had no obvious inhibitory effect on Escherichia coli, the 0.5 and 1 mg / mL concentrations of Example 2 had a relatively obvious inhibitory effect on Escherichia coli, and the 1 mg / mL concentration of Example 2 completely inhibited the growth of Escherichia coli after 2 h. From the results, it can be seen that Example 2 had a good antibacterial effect on Escherichia coli.

[0097] Experiment Six:

[0098] Hemolysis rate experiment of mesoporous starch hemostatic powder with bacteriostatic effect:

[0099] The comparative example 1, comparative example 2, example 2, example 4 were dispersed in PBS (pH = 7.4) to make a suspension of 5 mg / mL, the suspension was incubated at 37℃ for 20 min, 1 mL of citrate anticoagulant rabbit blood was mixed with 10 mL of PBS (pH = 7.4), and then centrifuged at 1000 rpm for 10 min to collect red blood cells. 50 μL of red blood cells was added to 500 μL of sample suspension, and the resulting mixture was incubated at 37℃ for 1 h, and then centrifuged at 1000 rpm for 10 min. The supernatant was collected and the absorbance was measured at 540 nm by an enzyme marker. 0.1% Triton X-100 and PBS were positive and negative controls, respectively. The hemolysis rate was calculated by the following formula.

[0100] Hemolysis rate = (Bs-Bp) / (B T -p) x 100%

[0101] Where Bs, Bp and B T are the absorbance at 540 nm of comparative example 1 (comparative example 2, example 2, example 4), PBS and Triton X-100, respectively.

[0102] The results of the hemolysis rate experiment are shown in Figure 8 From the figure, it can be seen that the hemolysis rate of all samples is less than 5%, i.e. it meets the requirements of medical hemostatic agent, indicating that the mesoporous starch hemostatic powder with antibacterial efficacy has good blood compatibility.

[0103] Experiment seven:

[0104] Coagulation index experiment of mesoporous starch hemostatic powder with antibacterial efficacy:

[0105] 10 mg of sample (comparative example 1, comparative example 2, example 2, example 4) was placed in a 5 mL Ep tube and preheated at 37℃ for 5 min, then 200 μL of anticoagulant rabbit blood and 20 μL of CaCl2 were mixed and added to the sample at 37℃ for 5 min. 5 mL of PBS was added to the Ep tube and shaken (30 rpm) for 10 min to wash away the red blood cells that did not aggregate, and the absorbance of the hemoglobin-containing washing solution at 540 nm (A1) was measured by an enzyme marker. The negative control group (A2) was 200 μL of anticoagulant rabbit blood suspended in 5 mL of PBS, and the coagulation index was calculated by the following formula.

[0106] Coagulation index = A1 / A2 x 100%

[0107] The results of the coagulation index experiment are shown in Figure 9 Compared with comparative example 1 and comparative example 2, the coagulation index of example 2 and example 4 is significantly reduced, indicating that it has good coagulation capacity, and the adhesion of calcium ions can increase the coagulation capacity of mesoporous starch hemostatic powder with antibacterial efficacy.

Claims

1. A process for the preparation of a bacteriostatic mesoporous starch hemostatic powder, characterized in that The method comprises the following steps: (1) starch gelatinization; (2) adding enzyme to hydrolyze and depolymerize: after the starch gelatinization solution is cooled, pullulanase is added and mixed uniformly, and the solution is hydrolyzed and depolymerized in a water bath; (3) regulating self-assembly of the depolymerized solution: the depolymerized solution obtained in step (2) is centrifuged at a parameter of 3000-4000 rpm for 10-15 min, and the supernatant is taken out to obtain short-chain glucan (SCG); immediately, chitosan solution is added to the SCG solution, the final concentration of chitosan is 0.005-0.03% w / v, and then the solution is placed in a refrigerator for self-assembly at 2-8°C to prepare antibacterial mesoporous starch hemostatic powder; (4) drying to obtain antibacterial mesoporous starch hemostatic powder. In step (1), amylopectin is weighed, ultrapure water is added, and a 3-4% w / v solution is prepared, the solution is mixed uniformly, and the solution is gelatinized uniformly at 100°C under stirring until the solution is transparent and boiled.

2. The production method according to claim 1, characterized by: In step (2), the starch gelatinization solution is cooled to below 60°C, and then pullulanase is added, and the water bath temperature is 50-60°C.

3. The production method according to claim 1, wherein: In step (2), the volume concentration of pullulanase is 0.25-1%, and the hydrolysis and depolymerization time is 24-72 h.

4. The production method according to claim 1 or 3, characterized by: In step (4), the self-assembled starch solution obtained in step (3) is washed with ultrapure water and then placed in a vacuum freeze-drying device for drying.

5. The production method according to claim 1, wherein: ​ 6. The antimicrobial mesoporous starch hemostatic powder obtained by any of the methods of claims 1-5, characterized in that: The specific surface area is up to 20.54 m 2 The total pore volume is up to 0.131 cm 3 The average pore diameter is 20-26 nm.

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