A microalgae protein antibacterial hydrogel applicable to the gastrointestinal environment, preparation method, and application

Abstract

The present invention belongs to the technical field of bacteriostatic agents, and specifically relates to a microalgal protein bacteriostatic hydrogel applicable to the gastrointestinal environment, a preparation method, and an application. The microalgal protein bacteriostatic hydrogel uses a microalgal protein hydrogel as a carrier for the bacteriostatic agent. The carrier is prepared by using a crosslinking agent with microalgal protein and sodium organic acid as the basic raw materials; the microalgae are Chlorella vulgaris; the mixing ratio of microalgal protein to sodium organic acid is 1-10:1-15, and the mass concentration after mixing is 1%-30%. The prepared microalgal protein hydrogel carrier has good loading performance and is excellent in loading bacteriostatic agents; the microalgal protein hydrogel carrier has a wide pH application range, with a pH of 1-8, and can be used under extremely acidic and neutral conditions; the microalgal protein hydrogel carrier loaded with a bacteriostatic agent can be used for the slow release of the bacteriostatic agent in a harsh gastrointestinal environment. In the gastrointestinal environment, it can not only effectively retain the active ingredients of the bacteriostatic agent but also achieve the long-term slow release effect of the bacteriostatic agent in an extreme environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacteriostatic agents, and particularly relates to a microalgae protein bacteriostatic hydrogel applicable to the gastrointestinal environment; in particular, it also relates to its preparation method and application. Background Art

[0002] At present, gastrointestinal inflammation has become the most common disease globally with a high incidence rate. Research shows that the main reason for this phenomenon is the imbalance of the intestinal flora caused by the human body's contact with food and water contaminated by pathogenic microorganisms, which ultimately leads to the large-scale reproduction of pathogenic bacteria in the gastrointestinal tract. Based on this situation, inhibiting the reproduction of pathogenic bacteria in the gastrointestinal environment has become the preferred solution for alleviating gastrointestinal inflammation, and bacteriostatic agents with biological activity are currently the best choice for inhibiting the reproduction of pathogenic bacteria. How to ensure that the bacteriostatic agent can retain its bacteriostatic activity for a long time in a harsh pH environment is an urgent problem to be solved in inhibiting the reproduction of gastrointestinal pathogenic bacteria, and the core key to solving this problem is to find a bacteriostatic agent carrier applicable to the gastrointestinal environment.

[0003] Most of the currently commercially available bacteriostatic agent carriers are prepared by chemical condensation reactions using chemical substances such as high molecular polymers and nanomaterials, and problems such as harmful substance residues caused by the use of toxic raw materials and catalysts are likely to occur.

[0004] In the prior art, there are also bacteriostatic agent carriers synthesized using chemical substances such as polyethyleneimine - polyacrylic acid and isosorbide mononitrate as raw materials. However, since this bacteriostatic agent carrier contains a large amount of chemical substances that cannot be digested and absorbed by the human body, it is likely to cause a large amount of chemical substance residues in the body, causing certain harm to the human body. In addition, the applicable pH range of the bacteriostatic agent carrier in the prior art is relatively narrow, and it is only applicable under neutral conditions, while the bacteriostatic carrier applicable to the gastrointestinal environment has higher requirements for the pH range of use, and it cannot meet the requirements for protecting the biological activity of the bacteriostatic agent and long-term slow release in a harsh pH environment.

[0005] In view of the above situation, there is an urgent need to prepare a bacteriostatic agent carrier with a wide applicable pH range, which can not only effectively retain the active ingredients of the bacteriostatic agent in the gastrointestinal environment, but also achieve the long-term slow release of the bacteriostatic agent in the harsh gastrointestinal environment. Summary of the Invention

[0006] To solve the above problems, the present invention prepares a microalgae protein antibacterial hydrogel applicable to the gastrointestinal environment. The microalgae protein antibacterial hydrogel of the present invention uses a microalgae protein hydrogel as an antibacterial agent carrier; the microalgae protein and sodium organic acid used are all biological substances extracted from natural products, avoiding the harm caused by chemical substances, having the advantages of good biocompatibility and no residue, and ensuring the safety of the antibacterial carrier. The antibacterial agent carrier prepared by the present invention has a wide applicable pH range and has a certain antibacterial sustained-release effect within the range of pH 1-8. The cumulative release rate of the antibacterial agent carrier in intestinal fluid can reach 99.51%, which is significantly higher than the release rate of general carriers.

[0007] The specific technical solution of the present invention is as follows:

[0008] A microalgae protein antibacterial hydrogel applicable to the gastrointestinal environment, the microalgae protein antibacterial hydrogel uses a microalgae protein hydrogel as an antibacterial agent carrier, and the carrier is prepared by using microalgae protein and sodium organic acid as basic raw materials and using a cross-linking agent.

[0009] The antibacterial agent carrier can also enable the antibacterial agent to retain antibacterial activity for a long time in a harsh pH environment; the applicable pH range of the antibacterial agent carrier is 1-8.

[0010] Preferably, the microalgae is Chlorella vulgaris.

[0011] Preferably, the microalgae is Chlorella pyrenoidosa.

[0012] Preferably, the cross-linking agent is at least one of 2,5-dimethyl-2,5-di-tert-butylperoxyhexane (bis-25), EDC / NHS, acetic anhydride, and modified carbodiimide.

[0013] Preferably, the sodium organic acid is at least one of sodium polyacrylate, sodium alginate, sodium citrate, sodium tartrate, and sodium carboxymethylcellulose.

[0014] Preferably, the antibacterial agent is at least one of polylysine, curcumin, and berberine.

[0015] The present invention also provides a preparation method of the microalgae protein antibacterial hydrogel applicable to the gastrointestinal environment, which specifically includes the following steps:

[0016] (1) Preparation of antibacterial agent carrier

[0017] Select microalgae protein and sodium organic acid as basic materials for mixing, adjust the pH to 0-7, then add a cross-linking agent, stir at room temperature and then refrigerate to obtain a microalgae protein hydrogel, that is, an antibacterial agent carrier;

[0018] (2) Loading and sustained release of antibacterial agent

[0019] Load and slowly release the bacteriostatic agent from the bacteriostatic agent carrier in phosphate buffer in step (1). After the loading is completed, wipe the surface moisture dry and then carry out the slow release to complete the loading and slow release of the bacteriostatic agent.

[0020] (3) Select Staphylococcus aureus and Escherichia coli as pathogenic bacteria, and use the Oxford cup antibacterial circle method to verify the antibacterial effect of the hydrogel; After activating the test bacteria for 3 - 18 h, coat them on the culture dish. Put 10 - 200 μL of the slow release solution into the Oxford cup in the culture dish, and culture it in a constant temperature incubator for 5 - 72 h, and observe the diameter of the antibacterial circle.

[0021] Preferably, in step (1), the mixing ratio of microalgae protein to sodium organic acid is 1 - 10:1 - 15 (w / w), and the mass concentration of the mixed dispersion system after mixing is 1% - 30% (proportion of the total reaction system).

[0022] Preferably, in step (1), the mass of the cross-linking agent accounts for 1% - 50% of the total mass of the mixed substances.

[0023] Preferably, in step (1), the stirring speed is 50 r / min - 500 r / min, and the stirring time is 0.5 - 4 h; the refrigeration temperature is -18 - 18 °C, and the time is 12 - 72 h.

[0024] Preferably, in step (2), the loading temperature is -4 - 40 °C, the pH range is 0 - 12, and the time is 1 - 70 h; the slow release temperature is 10 - 60 °C, the pH range is 0 - 12, and the time is 1 - 200 h.

[0025] The protection scope of the present invention also includes the application of the microalgae protein antibacterial hydrogel, specifically the application of the microalgae protein antibacterial hydrogel in the preparation of products with the efficacy of inhibiting the reproduction of gastrointestinal pathogenic bacteria.

[0026] The beneficial effects of the present invention are as follows:

[0027] (1) The bacteriostatic agent carrier is safe, stable, efficient, and residue-free

[0028] In the development process of this product of the present invention, the hydrogel carrier formed by the biocrosslinking reaction of protein and organic acid, the used microalgae protein and sodium organic acid are both biological substances extracted from natural products, and no toxic components participate in the reaction, avoiding the harm brought by chemical substances, having the advantages of good biocompatibility and no residue, ensuring the safety of the bacteriostatic carrier; and the formed structure is stable and can be applied to commercial production;

[0029] (2) The gel of the present invention has excellent antibacterial performance

[0030] The bacteriostatic agent was introduced into the hydrogel carrier through a method of sustained release of the load. The results showed that the microalgae protein hydrogel had excellent loading and sustained release effects on the biological bacteriostatic agent. The reason was that both loading and sustained release depended on the concentration gradient difference. As long as there was a concentration gradient difference, the bacteriostatic agent could slowly release from the porous structure of the hydrogel and exert its bacteriostatic performance. In the present invention, the bacteriostatic diameter of the bacteriostatic agent carrier was as high as 4.55 mm, far superior to the bacteriostatic effect of commercially available carriers synthesized by chemical substances;

[0031] (3) Wide pH application range

[0032] The bacteriostatic agent carrier of the present invention can not only effectively retain the active ingredients of the bacteriostatic agent, but also maintain its stable structure within a wide pH range (pH 1 - 8), enabling the bacteriostatic agent carrier to achieve a long-term sustained release effect in the extreme pH environment of the gastrointestinal tract; and the cumulative release rate of the bacteriostatic agent carrier in intestinal fluid can reach 99.51%;

[0033] (4) Great potential for industrialization of the bacteriostatic carrier

[0034] The microalgae protein used in the present invention is derived from marine microalgae, which has the advantages of wide source, large yield, and low raw material cost. The prepared carrier has a low cost, and the preparation method is simple and efficient, with great application potential in industrial scale-up. Brief description of the drawings

[0035] Figure 1 is the loading rate of each example and comparative example at different pH values;

[0036] Figure 2 is the sustained release rate of each example and comparative example at different pH values;

[0037] Figure 3 is the sustained release rate of each example and comparative example in gastric juice at different times;

[0038] Figure 4 The sustained release rate of each example in the intestine at different times;

[0039] Figure 5 is the bacteriostatic circle diameter of the gastric juice sustained release solution of each example; among them, Example X - S represents the bacteriostatic circle diameter of Example X against Staphylococcus aureus; Example X - E represents the bacteriostatic circle diameter of Example X against Escherichia coli;

[0040] Figure 6 is the bacteriostatic circle diameter of the intestinal fluid sustained release solution of each example; among them, Example X - S represents the bacteriostatic circle diameter of Example X against Staphylococcus aureus; Example X - E represents the bacteriostatic circle diameter of Example X against Escherichia coli. Detailed implementation manners

[0041] To enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0042] Example 1

[0043] Prepare a microalgae protein antibacterial hydrogel suitable for the gastrointestinal environment

[0044] (1) Preparation of antibacterial agent carrier

[0045] Select microalgae protein and sodium alginate as the base materials for mixing (w:w = 1:3). The mass concentration of this mixed dispersion system is 20% (the proportion in the total reaction system, the same below). After adjusting the pH of the mixed system to 4.0, add the cross-linking agent EDC / NHS with a mass ratio of 10% (the proportion in the total mixture); control the rotation speed at 150 r / min, stir at room temperature for 1 h, and then refrigerate at 4 °C for 24 h to obtain a microalgae protein hydrogel, that is, the antibacterial agent carrier;

[0046] The microalgae described in this example is Chlorella pyrenoidosa, the same below;

[0047] (2) Loading and slow release of antibacterial agent

[0048] Load and slowly release polylysine from the antibacterial agent carrier in step (1) in phosphate buffer. The loading temperature is 37 °C, the pH is 1 - 8, and the loading time is 48 h; after the loading is completed, dry the surface moisture and perform slow release. The slow release temperature is 37 °C, the pH is 1 - 8, and the slow release time is 150 h to complete the loading and slow release of the antibacterial agent;

[0049] (3) Verification of antibacterial effect of hydrogel

[0050] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, verify the antibacterial effect of the hydrogel by the Oxford cup antibacterial circle method; activate the test bacteria for 10 h, spread the activated test bacteria on the culture dish, and put 150 μL of the slow release solution in the Oxford cup in the culture dish, and culture in a constant temperature incubator for 12 h, and observe the diameter of the antibacterial circle.

[0051] Example 2

[0052] Prepare a microalgae protein antibacterial hydrogel suitable for the gastrointestinal environment

[0053] (1) Preparation of antibacterial agent carrier

[0054] Select microalgae protein and sodium citrate as the base materials for mixing (w:w = 1:1). The mass concentration of this mixed dispersion system is 10%. After adjusting the pH of the mixed system to 5.0, add the cross-linking agent acetic anhydride with a mass ratio of 10% (proportion of the total mixture); control the rotation speed at 200 r / min, stir at room temperature for 1 h, and then refrigerate at 4°C for 24 h to obtain the microalgae protein hydrogel, which is the antibacterial agent carrier;

[0055] (2) Loading and slow release of antibacterial agent

[0056] Load and slowly release curcumin from the antibacterial agent carrier in step (1) in phosphate buffer. The loading temperature is 37°C, the pH is 1 - 8, and the loading time is 48 h; after the loading is completed, dry the surface moisture and then carry out slow release. The slow release temperature is 37°C, the pH is 1 - 8, and the slow release time is 150 h to complete the loading and slow release of the antibacterial agent;

[0057] (3) Verification of antibacterial effect of hydrogel

[0058] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, verify the antibacterial effect of the hydrogel by the Oxford cup antibacterial circle method; activate the test bacteria for 10 h, spread the activated test bacteria on the culture dish, and put 150 μL of the slow release solution into the Oxford cup in the culture dish, and culture in a constant temperature incubator for 12 h, and observe the diameter of the antibacterial circle.

[0059] Example 3

[0060] Prepare a microalgae protein antibacterial hydrogel suitable for the gastrointestinal environment

[0061] (1) Preparation of antibacterial agent carrier

[0062] Select microalgae protein and sodium polyacrylate as the base materials for mixing (w:w = 1:2). The mass concentration of this mixed dispersion system is 10%. After adjusting the pH of the mixed system to 4.8, add the cross-linking agent bis(2,4,6-trichlorophenyl) carbonate with a mass ratio of 2% (proportion of the total mixture); control the rotation speed at 200 r / min, stir at room temperature for 1 h, and then refrigerate at 4°C for 24 h to obtain the microalgae protein hydrogel, which is the antibacterial agent carrier;

[0063] (2) Loading and slow release of antibacterial agent

[0064] Load and slowly release berberine from the antibacterial agent carrier in step (1) in phosphate buffer. The loading temperature is 37°C, the pH is 1 - 8, and the loading time is 48 h; after the loading is completed, dry the surface moisture and then carry out slow release. The slow release temperature is 37°C, the pH is 1 - 8, and the slow release time is 150 h to complete the loading and slow release of the antibacterial agent;

[0065] (3) Verification of antibacterial effect of hydrogel

[0066] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, the antibacterial effect of the hydrogel was verified by the Oxford cup antibacterial circle method; the test bacteria were activated for 10 h, the activated test bacteria were spread on the culture dish, and 150 μL of the sustained-release solution was placed in the Oxford cup in the culture dish, and it was cultured in a constant temperature incubator for 12 h, and the diameter of the antibacterial circle was observed.

[0067] Example 4

[0068] Prepare a microalgae protein antibacterial hydrogel suitable for the gastrointestinal environment

[0069] (1) Preparation of antibacterial agent carrier

[0070] Select microalgae protein and sodium carboxymethylcellulose as the base materials for mixing (w:w = 1:1), the mass concentration of this mixed dispersion system is 15%, after adjusting the pH of the mixed system to 5.2, add 10% (by proportion of the total mixture) of the cross-linking agent modified carbodiimide; control the rotation speed at 200 r / min, stir at room temperature for 1 h, and then refrigerate at 4 °C for 24 h to obtain the microalgae protein hydrogel, that is, the antibacterial agent carrier;

[0071] (2) Loading and sustained release of antibacterial agent

[0072] Load and sustain the release of the mixture of polylysine and curcumin on the antibacterial agent carrier in step (1) in phosphate buffer, the loading temperature is 37 °C, the pH is 1 - 8, and the loading time is 48 h; after the loading is completed, dry the surface moisture for sustained release, the sustained release temperature is 37 °C, the pH is 1 - 8, and the sustained release time is 150 h, to complete the loading and sustained release of the antibacterial agent;

[0073] (3) Verification of the antibacterial effect of the hydrogel

[0074] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, the antibacterial effect of the hydrogel was verified by the Oxford cup antibacterial circle method; the test bacteria were activated for 10 h, the activated test bacteria were spread on the culture dish, and 150 μL of the sustained-release solution was placed in the Oxford cup in the culture dish, and it was cultured in a constant temperature incubator for 12 h, and the diameter of the antibacterial circle was observed.

[0075] Example 5

[0076] Prepare a microalgae protein antibacterial hydrogel suitable for the gastrointestinal environment

[0077] (1) Preparation of antibacterial agent carrier

[0078] Microalgae protein and sodium tartrate were selected as the base materials for mixing (w:w = 1:1). The mass concentration of this mixed dispersion system was 15%. After adjusting the pH of the mixed system to 5.2, a cross-linking agent, carbodiimide, with a mass ratio of 10% (proportion of the total mixture) was added. The rotation speed was controlled at 200 r / min, and after stirring at room temperature for 1 h, it was refrigerated at 4°C for 24 h to obtain a microalgae protein hydrogel, which was the antibacterial agent carrier.

[0079] (2) Loading and slow release of antibacterial agent

[0080] The antibacterial agent carrier in step (1) was used for the loading and slow release of the mixture of curcumin and berberine in phosphate buffer. The loading temperature was 37°C, the pH was 1 - 8, and the loading time was 48 h. After the loading was completed, the surface moisture was wiped dry for slow release. The slow release temperature was 37°C, the pH was 1 - 8, and the slow release time was 150 h, thus completing the loading and slow release of the antibacterial agent.

[0081] (3) Verification of the antibacterial effect of the hydrogel

[0082] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, the antibacterial effect of the hydrogel was verified by the Oxford cup antibacterial circle method. The test bacteria were activated for 10 h, and the activated test bacteria were spread on the culture dish. 150 μL of the slow release solution was placed in the Oxford cup in the culture dish and cultured in a constant temperature incubator for 12 h, and the diameter of the antibacterial circle was observed.

[0083] Control Example 1

[0084] (1) Preparation of antibacterial agent carrier

[0085] Pea protein and sodium citrate were selected as the base materials for mixing (w:w = 1:1). The mass concentration of this mixed dispersion system was 15%. After adjusting the pH of the mixed system to 5.2, a cross-linking agent, acetic anhydride, with a mass ratio of 10% (proportion of the total mixture) was added. The rotation speed was controlled at 200 r / min, and after stirring at room temperature for 1 h, it was refrigerated at 4°C for 24 h to obtain a pea protein hydrogel, which was the antibacterial agent carrier.

[0086] (2) Loading and slow release of antibacterial agent

[0087] The antibacterial agent carrier in step (1) was used for the loading and slow release of curcumin in phosphate buffer. The loading temperature was 37°C, the pH was 1 - 8, and the loading time was 48 h. After the loading was completed, the surface moisture was wiped dry for slow release. The slow release temperature was 37°C, the pH was 1 - 8, and the slow release time was 150 h, thus completing the loading and slow release of the antibacterial agent.

[0088] (3) Verification of the antibacterial effect of the hydrogel

[0089] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, the antibacterial effect of the hydrogel was verified by the Oxford cup antibacterial circle method; the test bacteria were activated for 10 h, and the activated test bacteria were spread on a culture dish. 150 μL of the sustained-release solution was placed in the Oxford cup in the culture dish, and it was cultured in a constant-temperature incubator for 12 h, and the diameter of the antibacterial circle was observed.

[0090] Comparative Example 2

[0091] (1) Preparation of antibacterial agent carrier

[0092] Microalgae protein and sodium carboxymethylcellulose were selected as the base materials for mixing (w:w = 1:1), and the mass concentration of the mixed dispersion system was 15%. After adjusting the pH of the mixed system to 5.2, glyoxal with a mass ratio of 5% (proportion of the total mixture) was added; the rotation speed was controlled at 200 r / min, stirred at room temperature for 1 h, and then refrigerated at 4 °C for 24 h to obtain microalgae protein hydrogel, that is, the antibacterial agent carrier;

[0093] (2) Loading and sustained release of antibacterial agent

[0094] The antibacterial agent carrier in step (1) was used for the loading and sustained release of curcumin in phosphate buffer. The loading temperature was 37 °C, the pH was 1 - 8, and the loading time was 48 h; after the loading was completed, the surface moisture was wiped dry for sustained release. The sustained release temperature was 37 °C, the pH was 1 - 8, and the sustained release time was 150 h, completing the loading and sustained release of the antibacterial agent;

[0095] (3) Verification of antibacterial effect of hydrogel

[0096] Using Staphylococcus aureus and Escherichia coli as pathogenic bacteria samples, the antibacterial effect of the hydrogel was verified by the Oxford cup antibacterial circle method; the test bacteria were activated for 10 h, and the activated test bacteria were spread on a culture dish. 150 μL of the sustained-release solution was placed in the Oxford cup in the culture dish, and it was cultured in a constant-temperature incubator for 12 h, and the diameter of the antibacterial circle was observed.

[0097] Under the in vitro simulated gastrointestinal environment, the antibacterial degree, loading rate, and sustained release rate of the microalgae protein antibacterial hydrogel in different examples were different.

[0098] The loading rate, sustained release rate, and antibacterial circle of each example were measured and compared with the comparative example, and the results are shown in the following table and drawings. Figure 5-6 Among them, S represents Staphylococcus aureus, and E represents Escherichia coli; Example X - S represents the diameter of the antibacterial circle of Example X against Staphylococcus aureus, and Example X - E represents the diameter of the antibacterial circle of Example X against Escherichia coli, and "X" is Examples 1 - 5.

[0099] Table 1 Loading rate % of each example and comparative example under different pH environments

[0100] Item pH = 1.2 pH = 2 pH = 3 pH = 4 pH = 5 pH = 6 pH = 7.4 pH = 8 Example 1 12.3 18.6 24.9 26.9 30.8 36.8 46.8 49.5 Example 2 11.5 15.2 21.6 24.8 29.7 33.2 40.5 46.9 Example 3 12.9 16.7 23.8 25.2 28.3 31.9 39.1 45.2 Example 4 10.5 17.8 25.8 27.9 31.9 35.9 42.9 44.9 Example 5 13 19.5 20.8 28.6 32.8 34.7 44.8 46.9 Comparative Example 1 8.54 10.98 12.68 16.85 20.64 25.68 27.92 30.56 Comparative Example 2 6.95 8.75 10.86 12.65 15.98 20.37 23.65 25.98

[0101] Table 2 Sustained release rate % of each example and comparative example under different pH environments

[0102]

[0103]

[0104] Table 3 Sustained release rate % of each example and comparative example in gastric juice within different time periods

[0105] Item 12h 24h 36h 48h 96h 120h 132h 150h Example 1 8.24 10.58 14.28 17.86 2.68 27.62 28.49 28.64 Example 2 7.52 11.98 15.35 19.56 22.85 24.13 25.06 25.21 Example 3 8.46 12.68 16.48 19.85 22.03 24.92 26.34 26.72 Example 4 9.56 12.56 15.85 20.56 23.51 25.98 27.46 27.81 Example 5 8.56 12.67 15.96 20.56 23.68 25.96 27.24 27.53 Comparative Example 1 6.95 8.56 9.85 10.67 16.98 18.92 20.43 20.68 Comparative Example 2 4.85 6.98 7.56 8.85 10.95 13.56 15.76 15.84

[0106] Table 4 Cumulative sustained release rate % of each example in the intestine within different time periods

[0107] Item 12h 24h 36h 48h 96h 120h 132h 150h Example 1 18.64 34.87 49.92 70.85 88.79 95.02 98.23 99.52 Example 2 15.21 24.62 34.85 52.73 70.21 75.52 76.60 76.91 Example 3 16.72 30.85 40.26 56.37 74.98 80.12 80.74 81.26 Example 4 17.81 20.86 44.91 62.92 79.98 85.93 86.31 86.92 Example 5 19.50 28.83 38.67 68.82 84.76 90.86 90.92 91.25 Comparative Example 1 10.64 17.67 28.95 32.52 40.65 49.12 49.87 50.98 Comparative Example 2 7.96 12.38 18.96 25.64 30.24 38.95 39.99 40.85

[0108] Table 5 Inhibition zone diameter mm of gastric juice sustained release solution of each example

[0109]

[0110]

[0111] Table 6 Inhibition zone diameter mm of intestinal sustained release solution of each example

[0112] Item 1 day 2 days 3 days 4 days 5 days Example 1-S 4.55 3.5 2.1 1.5 1.0 Example 2-S 3.3 2.2 1.4 0.6 0.3 Example 3-S 3.8 2.4 1.6 0.8 0.4 Example 4-S 3.1 2.8 1.9 1.0 0.6 Example 5-S 3.5 3.0 2.0 1.2 0.8 Commercially available carrier 1.5 1.0 0.6 0.3 0.0 Example 1-E 3.5 2.6 1.1 0.8 0.1 Example 2-E 2.3 1.2 0.4 0.2 0.0 Example 3-E 2.5 1.4 0.6 0.3 0.0 Example 4-E 2.8 1.8 0.9 0.4 0.0 Example 5-E 3.0 2.0 1.0 0.6 0.05 Commercially available carrier 1.0 0.7 0.4 0.1 0.0

[0113] According to the above table and the attached drawings, it can be seen that compared with the comparative example, under the same pH conditions, the loading rate of the microalgae protein antibacterial hydrogel in the example can be about 15% higher at most, and the sustained release rate can be about 40% higher at most; it shows that the microalgae protein hydrogel carrier of the present invention has good loading performance. At 150 h, the sustained release rates in gastric juice and intestinal juice can still reach about 0.2% and 0.6% respectively, and the cumulative sustained release rates can respectively reach about 25% and 90%; it can still be applicable under the conditions of pH 1 - 8, and the pH application range is wide, which fully proves that the microalgae protein hydrogel carrier loaded with antibacterial agent of the present invention can be used for slow release in a harsh gastrointestinal environment.

[0114] According to the in vitro antibacterial analysis, the microalgae protein antibacterial hydrogel of the present invention has good antibacterial effects on Escherichia coli and Staphylococcus aureus, helps to balance the intestinal flora, is more conducive to inhibiting the growth of harmful bacteria and promoting the growth of beneficial bacteria; the antibacterial diameter against Staphylococcus aureus in intestinal juice can reach 4.55 mm. In the gastrointestinal environment, it can not only effectively retain the active ingredients of the antibacterial agent, but also achieve the long-term slow release effect of the antibacterial agent in an extreme environment.

Claims

1. A microalgae protein antibacterial hydrogel applicable to the gastrointestinal environment, characterized in that, The described microalgae protein antibacterial hydrogel is prepared by using a microalgae protein hydrogel as a carrier for the antibacterial agent and utilizing a crosslinking agent; The preparation steps of the described microalgae protein antibacterial hydrogel are as follows: (1) Preparation of the antibacterial agent carrier: Select microalgae protein and sodium alginate as the basic materials for mixing to obtain a mixed dispersion system. The mass concentration of this mixed dispersion system in the total reaction system is 20%. After adjusting the pH of this mixed dispersion system to 4.0, add 10% of the crosslinking agent EDC / NHS based on the mass of this mixed dispersion system. Control the rotation speed at 150 r / min, stir at room temperature for 1 h, and then refrigerate at 4°C for 24 h to obtain a microalgae protein hydrogel, that is, the antibacterial agent carrier. The described microalgae protein is Chlorella pyrenoidosa, and the mass ratio of microalgae protein to sodium alginate is 1:3; (2) Loading and slow release of the antibacterial agent: Load and slowly release polylysine from the antibacterial agent carrier in (1) in a phosphate buffer solution. The loading temperature is 37°C, the pH is 1.0 - 8.0, and the loading time is 48 h. After the loading is completed, dry the surface moisture and then carry out slow release. The slow release temperature is 37°C, the pH is 1.0 - 8.0, and the slow release time is 150 h to complete the loading and slow release of the antibacterial agent.

2. Use of the microalgae protein antibacterial hydrogel according to claim 1 in the preparation of a product with the efficacy of inhibiting the reproduction of gastrointestinal pathogenic bacteria.

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