Active hawthorn protein for inhibiting bacterial biofilm as well as preparation method and application of active hawthorn protein

Abstract

The invention relates to the field of natural products, in particular to active hawthorn protein for inhibiting a bacterial biofilm and a preparation method and application of the active hawthorn protein for inhibiting the bacterial biofilm, and the active hawthorn protein for inhibiting the bacterial biofilm comprises antibacterial hawthorn protein and a multi-element modification layer compounded with the antibacterial hawthorn protein; the modification layer is obtained by compounding polysaccharide and amine components. The antibacterial protein is extracted from hawthorn fruits, and the surface of the antibacterial protein is subjected to surface modification through polysaccharides and natural amines, so that the purpose of improving the acid resistance of the hawthorn protein is achieved, and the antibacterial protein can stably act on a bacterial biofilm in an acidic microenvironment. Meanwhile, after surface modification, the affinity adhesion capacity of hawthorn protein and a biological membrane can be improved, and the interaction between a composite structure and a bacterial biological membrane structure is enhanced, so that the penetrating effect is achieved, and the antibacterial effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of natural products, and in particular to an active hawthorn protein capable of inhibiting bacterial biofilm, a preparation method and an application thereof. Background Art

[0002] Bacterial infection has become a significant challenge in surgical recovery, with addressing the development and spread of bacterial biofilms being particularly challenging. Bacterial biofilms (BBFs) are membrane-like structures formed by bacteria adsorbed onto inert or active surfaces during growth, adapting to their environment and providing self-protection. These structures consist of bacteria, their own extracellular matrix, and necrotic tissue. Bacterial biofilms are highly common in the body, as seen in chronic wounds, where bacteria adhere to the wound bed and fuse with their own extracellular matrix, as well as in oral biofilm infections. BBF formation creates localized hypoxic conditions beneath the biofilm, providing a strong barrier for bacterial growth. Once mature, the biofilms decompose carbohydrates in the tissue or oral cavity to produce acid, lowering the local pH and leading to various physiological pathologies, such as dental caries and wound ulcers.

[0003] Furthermore, clinical observations indicate that most bacterial infections are closely associated with the formation of bacterial biofilms. Bacteria in biofilms differ in morphology and physiological functions from free bacteria, exhibiting significantly increased tolerance to antibiotics (10-1000 times higher) and exhibiting strong resistance to host immune defenses, leading to the frequent development of drug resistance. When bacteria exist in a biofilm, it is difficult for antimicrobial drugs to penetrate and kill bacteria deep within the biofilm, which can lead to recurrent or persistent infections. More importantly, bacterial biofilms are not simple membranes similar to cell membranes, but rather acidic microecologies composed of numerous components, including tissue fragments, bacteria, and extracellular matrix. This places higher demands on the stability of antibacterial materials in this environment, leading to the development of stable and permeable antibacterial materials targeting this complex structure.

[0004] Natural antibacterial ingredients are a class of materials extracted and isolated from natural products, such as antibacterial proteins and small molecules. Compared to chemically synthesized antibacterial materials, these materials generally offer improved biosafety and are less likely to cause bacterial resistance, leading to their development and use in the current medical community. However, natural antibacterial ingredients often suffer from low acid resistance and instability, limiting their use as general-purpose antibacterial materials. Summary of the Invention

[0005] The present invention aims to overcome the problems of low acid resistance and weak stability of natural antibacterial products in the prior art, and thus provides an active hawthorn protein for inhibiting bacterial biofilms, a preparation method and an application thereof to overcome the above-mentioned shortcomings.

[0006] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions: In the first aspect, the present invention first provides an active hawthorn protein that inhibits bacterial biofilm. The invention comprises an antibacterial hawthorn protein and a multi-layer modified layer compounded with the antibacterial hawthorn protein; The modified layer is obtained by compounding polysaccharide and amine components.

[0007] Hawthorn, a plant in the Rosaceae family, has a long history of health and medicinal use in my country. The Eastern Jin Dynasty's "Zuohou Fang" (Ergou Fang) records that "boiled hawthorn juice can be used to wash lacquer sores," and it has become a current traditional Chinese medicine with both medicinal and edible properties.

[0008] The inventors of this application found during the experiment that a part of the protein components in hawthorn has good antibacterial and antifungal effects, and therefore is expected to be used in medicine as antibacterial materials and drugs. However, hawthorn protein has poor stability in acidic environments, which makes it unstable in the acidic environment of biofilms, thus seriously limiting its application. In addition, hawthorn protein has difficulty penetrating bacterial biofilms, which makes its antibacterial effect unable to directly act on harmful bacteria. Combining the above two major reasons, the current hawthorn protein still cannot meet the standards of antibacterial drugs. After further research, the researchers of this invention accidentally discovered that by compounding and modifying hawthorn protein, it is possible to achieve inhibition of bacterial biofilms.

[0009] The inventors of this application constructed a multi-layered modified hawthorn protein (SHP) based on a complex polysaccharide-natural amine structure and reacted the modified layer with hawthorn protein (HP). This modification has dual functions: 1. It increases the protein's acid resistance, improves protein homeostasis, and enhances its antibacterial effect; 2. It increases the permeability of hawthorn protein. Based on the specific composition and structure of bacterial biofilms, the polysaccharide / amine / protein composite structure can enhance the affinity and adhesion of hawthorn protein to biofilms and strengthen the interaction between the composite structure and bacterial biofilm structure, thereby achieving a penetrating effect and improving the antibacterial effect.

[0010] The results showed that the selected hawthorn antibacterial protein, after multilayer assembly modification, showed significantly improved acid stability and could stabilize bacterial biofilms in acidic microenvironments. In summary, based on the above technology, hawthorn natural protein has stronger antibacterial activity and holds great promise as a natural plant-based antibacterial material, potentially complementing or replacing traditional antibiotics.

[0011] Preferably, the polysaccharide is a combination of any one or more of the acidic polysaccharides inulin polysaccharide, astragalus polysaccharide, and liquorice polysaccharide.

[0012] Preferably, the amine component is any one or more combinations of dopa and its derivatives, arginine and its derivatives, histidine and its derivatives, and guanidine-containing compounds.

[0013] Preferably, the mass ratio of the polysaccharide to the amine component is 10:(1-3).

[0014] Preferably, the molecular weight of the antibacterial hawthorn protein is between 25 and 35 KDa.

[0015] Preferably, the particle size of the active hawthorn protein that inhibits bacterial biofilm is less than or equal to 200 nm.

[0016] In a second aspect, the present invention also provides a method for preparing the active hawthorn protein for inhibiting bacterial biofilm, comprising the following steps: (S.1) removing the core of the hawthorn fruit, washing it, and squeezing out the juice to obtain a hawthorn extract, extracting hawthorn protein from the hawthorn extract, and screening the hawthorn protein to obtain an antibacterial hawthorn protein; (S.2) reacting the polysaccharide with the amine component to assemble a multi-modified layer; (S.3) The antibacterial hawthorn protein is reacted with the multi-modified layer to obtain active hawthorn protein that inhibits bacterial biofilm.

[0017] Preferably, the reaction temperature in step (S.2) is 60-70°C for 1-10 hours, and after the reaction is completed, the reaction is dialyzed at 4-25°C for 8-12 hours, and the dialyzed retentate is taken out to obtain the multi-component modified layer.

[0018] Preferably, in the step (S.3), the antibacterial hawthorn protein and the multi-modified layer are first reacted at a constant temperature of 50-70°C for 0.5-3h, then rapidly heated to 85-95°C for 3-10 min, and rapidly cooled to room temperature to obtain active hawthorn protein that inhibits bacterial biofilm.

[0019] In a third aspect, the present invention also provides the use of the active hawthorn protein for inhibiting bacterial biofilm as described above in natural antibacterial food, antibacterial medicine or antibacterial material.

[0020] Therefore, the present invention has the following beneficial effects: This application extracts a class of antibacterial proteins from hawthorn fruit and modifies their surface with polysaccharides and natural amines, thereby enhancing the acid resistance of hawthorn proteins, thereby stabilizing bacterial biofilms in acidic microenvironments. Furthermore, the surface modification enhances the affinity and adhesion of hawthorn proteins to biofilms, strengthening the interaction between the composite structure and bacterial biofilms, thereby achieving a penetrating effect and improving the antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The antibacterial activity of hawthorn protein separated by graded precipitation with ammonium sulfate of different concentrations.

[0022] Figure 2 It is a hawthorn antibacterial protein obtained by re-dissolving with 50% ammonium sulfate precipitation, and its molecular weight is between 25~35KDa.

[0023] Figure 3 These are the test results of the membrane permeation efficiency of multi-layered hawthorn protein SHP with different side chain ratios.

[0024] Figure 4 These are the test results of acid resistance of hawthorn protein before and after modification.

[0025] Figure 5 These are the test results of the inhibitory effect of hawthorn protein on Candida albicans biofilm before and after modification.

[0026] Figure 6 These are the test results of the inhibitory effect of hawthorn protein on Acinetobacter baumannii biofilm before and after modification. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below generally represent only a portion of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0028] Example 1: Extraction steps of antibacterial hawthorn protein (1) Remove the core of the hawthorn fruit, wash it, squeeze the juice, and filter the residue multiple times to obtain the hawthorn extract. (2) Take the hawthorn extract and adjust the pH to 8.0 with 1 mol / L NaOH solution. Stir at low speed for 2 h at room temperature and centrifuge at 4800 rpm for 20 min. (3) The residue was extracted repeatedly, the supernatant was combined and the pH was adjusted to 4.5 with 1 mol / L HCl. After standing for 30 min, the mixture was centrifuged again and the supernatant was discarded. The precipitate was collected and washed twice with water, and the precipitate was completely dissolved in water. The pH was adjusted to 7.0 with 1 mol / L NaOH to obtain the hawthorn protein solution. (4) Ammonium sulfate was gradually added to the hawthorn protein solution, and the solution was separated by ammonium sulfate precipitation. After the salt concentration reached 10-80%, the solution was placed in a 4°C refrigerator for 4 h, and then the precipitate was collected by centrifugation to form protein samples precipitated with different ammonium sulfate concentrations (10-80%). (5) The eight protein components obtained by precipitation were added to PBS buffer respectively, dialyzed for 24 h, and then used.

[0029] Through the above steps, we can obtain hawthorn protein HP salted out by ammonium sulfate solution of different concentrations. Then we took 2% of different hawthorn protein solutions to conduct antibacterial activity experiments, and selected Candida albicans free solution (10 7 cfu / mL) was used as the observation object, and the antibacterial activity of hawthorn protein salt solutions with different concentrations was compared.

[0030] The results are as follows: Figure 1 As shown, hawthorn protein solutions obtained by ammonium sulfate fractionation precipitation all exhibited a certain degree of antibacterial activity, and hawthorn protein obtained with 50% ammonium sulfate showed the best antibacterial activity compared to hawthorn protein salt solutions at other concentrations, with an antibacterial activity of approximately 74%. The effectiveness of the technology was demonstrated by the ability to isolate natural proteins with antibacterial activity through a combination of alkaline extraction and acid precipitation with metal salts.

[0031] Figure 2 It is a hawthorn antibacterial protein obtained by re-dissolving with 50% ammonium sulfate precipitation, and its molecular weight is between 25~35KDa.

[0032] Example 2: Preparation of Modified Layer and Modified Hawthorn Protein SHP (1) Polysaccharide preheating and mixing: Select one or more of inulin polysaccharide, astragalus polysaccharide, and licorice polysaccharide powders; set up a single polysaccharide group and a composite polysaccharide group (the composite polysaccharide group was mixed at 1:1:1; 2:1:1; 1:2:1; 1:1:2, respectively), and preheat at 65-75°C for 0.5-2 h.

[0033] (2) Preparation of modified layer assembly: Dissolve the polysaccharide mixed powder in (1) in distilled water preheated to 65°C, add a certain amount of natural amine (arginine is selected in this embodiment), stir continuously, and react in a water bath at a constant temperature. The reaction temperature can be selected from 60-70°C (the reaction temperature in this embodiment is 65°C), the reaction time can be selected from 1 to 10 h (the reaction time in this embodiment is 5 h), and the mass ratio of polysaccharide to amine is 10:3.

[0034] (3) The modified layer in (2) is dialyzed. The typical dialysis temperature can be selected from 4 to 25°C (20°C in this embodiment), and the typical dialysis time is 8 to 12 hours (10 hours in this embodiment).

[0035] (4) The dialyzed residue from (3) is removed to form the modified layer.

[0036] (5) Preparation of modified hawthorn protein: The antibacterial hawthorn protein and the modified layer were mixed evenly in a buffer solution with a pH of 7.4 (5:1, w / w), and the mixture was kept at 60°C for 1 h. The mixture was quickly heated to 85-95°C (90°C in this embodiment) and reacted for 0.5-3 min (2 min in this embodiment). The mixture was quickly cooled to room temperature to obtain a multilayer assembled composite modified hawthorn protein (SHP).

[0037] (6) The nanoparticle sizes of different SHPs were measured, and the results are shown in Table 1 below.

[0038] Table 1 Nanostructured properties and reaction index of proteins modified with different polysaccharide side chains Group Nanoscale (nm) Inulin polysaccharide SHP1 689.2±4.5 Astragalus polysaccharide SHP2 559.0±16.7 Licorice polysaccharide SHP3 399.3±2.6 Composite 1:1:1 SHP4 486.7±5.3 Composite 2:1:1 SHP5 379.2±9.3 Composite 1:2:1 SHP6 521.0±6.0 Composite 1:1:2 SHP7 187.5±6.4 The results show that from Table 1 we can see that the combination of different polysaccharide side chain modification layers and proteins formed composite structures of different nanoscales, among which the nanostructure obtained by the composite polysaccharide inulin polysaccharide, astragalus polysaccharide and licorice polysaccharide in a ratio of 1:1:2 was the smallest, which indicates that the overall composite structure was assembled in an orderly manner and the reaction process was controllable.

[0039] It is generally believed that the nanoscale has a great influence on the biomembrane penetration behavior of the complex. After the arginine residues carried by different side chains cause biomembrane disturbance, the complex with appropriate particle size can preferentially enter the biomembrane by perforation or fusion on the biomembrane surface.

[0040] The three types of polysaccharides selected in this patent are all acidic polysaccharides with well-defined molecular weights. The molecular weight order is inulin polysaccharide > astragalus polysaccharide > licorice polysaccharide, with licorice polysaccharide having the highest degree of branching. As shown in the table, inulin polysaccharide SHP has the largest particle size and the highest reaction index, while licorice polysaccharide SHP has the lowest among the three. However, when all licorice polysaccharides are present, the high reaction index prevents the orderly assembly of the modified layer and hawthorn protein. However, a composite polysaccharide ratio of 1:1:2 achieves uniformity between the two indicators. This may be due to the different molecular weights of the side chains, the degree of branching, and the balance of surface hydrophilicity and hydrophobicity.

[0041] Example 3: Membrane permeability of multi-layer hawthorn protein SHP at different side chain ratios The modified layer was assembled using a mixture of polysaccharide and arginine at ratios of 10:1, 10:2, 10:3, 10:5, and 10:7. This was then combined with hawthorn protein (hawthorn protein to modified layer, 5:1, w / w) to construct a SHP. To construct a bacterial biofilm permeability model, 3% sodium alginate, 1% hyaluronic acid, 1% lecithin, and 0.1% calcium ion were dissolved in distilled water and thoroughly dissolved at 75°C with stirring. The hot solution was then dripped onto a 0.45 μm pore size medical microporous membrane (3 cm diameter) and allowed to cool naturally to form a gel layer. This was used to construct a bacterial biofilm under simulated physiological conditions. For the permeability experiment, the sample was slowly dripped into the glass port. Protein concentration was collected at the bottom port over a consistent time period (0.5 h–2 h) and calculated for permeability efficiency.

[0042] Permeation rate (%) = protein concentration permeating under the membrane / protein drop concentration × 100% See the results Figure 3 The results showed that when the ratio of the modified layer of complex polysaccharide: arginine was 10:3, the membrane permeability of the complex protein was the best. The combination of different polysaccharide ratios with amine components such as arginine resulted in different degrees of exposure of the arginine residue chain. At the same time, the further combination of the modified layer and hawthorn protein would also result in different binding sites and different reaction degrees.

[0043] Bacterial biofilms have proven to be more complex than previously imagined, comprising a complex tissue structure composed of bacteria, extracellular matrix, and biological tissue. To address these characteristics, the inventors constructed a hydrogel structure containing extracellular matrix hyaluronic acid and cell membrane phospholipids to simulate bacterial biofilms. Compared to traditional single bacterial membranes, this hydrogel structure more closely resembles the physiological structure of bacterial biofilms, possessing physiological components and rigidity not found in other models. Under optimal conditions, the inventors' SHP achieved a penetration efficiency of 81.7%, approximately 4.4 times higher than HP.

[0044] Example 4: Determination of acid resistance of hawthorn protein before and after modification SHP (complex polysaccharide inulin polysaccharide, astragalus polysaccharide, licorice polysaccharide ratio of 1:1:2, modified layer complex polysaccharide: arginine ratio of 10:3) and HP two samples were placed at pH values ​​of 4, 5, 6, 7, 8, 9, 10, 11, 12 for 120 min, and then adjusted to pH 7.0 and then Escherichia coli (10 7 cfu / mL) inhibition experiment. Figure 4 It shows that SHP still has an inhibition rate of 71% against Escherichia coli in an acidic environment of pH 5.0, which is 1.9 times the antibacterial activity of HP with the same activity. This shows that the acidic environment can inhibit the activity of HP, while SHP can effectively maintain relative stability in the acidic biofilm environment. At the same time, the antibacterial activity of both first increases and then decreases with the increase of pH value. The optimal antibacterial activity of SHP and HP differs by 1 pH unit, indicating that the addition of the modification layer can increase the isoelectric point of the entire structure and improve its stability in the acidic environment.

[0045] Example 5: Construction and Inhibitory Effect of Candida albicans Biofilm In each well of a 24-well culture plate, 1.8 mL of blank culture medium or culture medium containing sample (2% SHP or 2% HP or PA) was added (the ratio of inulin polysaccharide, astragalus polysaccharide and licorice polysaccharide in SHP was 1:1:2, and the ratio of polysaccharide in the modified layer to arginine was 10:3), and 0.2 mL of 10% arginine was added. 7 cfu / mL of bacterial solution (Candida albicans) was cultured for 48 h. After the biofilm matured, the free bacteria were washed with PBS to obtain a Candida albicans biofilm model. The absorbance at a wavelength of 595 nm was measured using the crystal violet method, and the inhibitory effect of the bacterial biofilm was calculated (see Figure 5 ).

[0046] Candida albicans is an opportunistic pathogen in the human oral cavity, capable of causing a variety of oral mucosal diseases under certain conditions. The results showed that SHP exhibited highly effective biofilm inhibition, exceeding 80% against C. albicans biofilms, a remarkable improvement compared to approximately 41% for untreated HP. Furthermore, the modified layer (PA) exhibited some inhibitory effect, albeit less than 25%. Similarly, in experiments comparing free C. albicans, HP exhibited an inhibition rate of approximately 70%, while SHP exhibited an even higher inhibition rate against biofilms. This demonstrates that the construction technology employed in this study effectively enhances the comprehensive antibacterial capacity of hawthorn protein. This improvement is attributed to the modified layer's enhanced acid stability and biofilm penetration ability.

[0047] This indicates that the SHP-modified assembly method has a significant effect on the inhibition of bacterial biofilms, and this strategy can be successfully applied to the field of deep antibacterial inhibition. This is because SHP has a stronger delivery effect than HP and other delivery carriers.

[0048] Example 6: Construction and Inhibitory Effect of Acinetobacter baumannii Biofilm In each well of a 24-well culture plate, 1.8 mL of LB medium was added, and a certain amount of HP, SHP, and a modified layer (LA) were added (wherein the ratio of the complex polysaccharides inulin polysaccharide, astragalus polysaccharide, and licorice polysaccharide in SHP was 1:1:2, and the ratio of the complex polysaccharide in the modified layer to arginine was 10:3), and 0.2 mL of a 10 7 cfu / mL of bacterial solution (Candida albicans) was cultured for 48 h. After the biofilm matured, the free bacteria were washed with PBS to obtain the Acinetobacter baumannii biofilm model. The absorbance at a wavelength of 595 nm was measured using the crystal violet method, and the inhibitory effect on the bacterial biofilm was calculated (see Figure 6 The results showed that SHP's inhibitory effect on Acinetobacter baumannii biofilms was similar to that of the previous study, exceeding 72%. Acinetobacter baumannii is a common Gram-negative opportunistic pathogen in clinical practice, susceptible to infections in various systems, including the respiratory, central nervous, and urinary systems. This bacterium is highly susceptible to developing drug resistance. Therefore, inhibiting infections and biofilms caused by this species is extremely challenging, and SHP has been successfully applied in this area.

[0049] Example 7: Inhibitory effect of amine-modified hawthorn protein on Acinetobacter baumannii biofilm The hawthorn protein used in this example, which is active in inhibiting bacterial biofilms, contains a multi-component modified layer containing amines selected from a mixture of DOPA and histidine (DOPA:Histidine = 1:1). During actual testing, the inventors discovered that histidine can also undergo modification reactions with polysaccharides. However, the imidazole group on histidine is weakly alkaline, resulting in decreased stability in the acidic environment of biofilms. To address this issue, a DOPA-like substance was introduced in this example to enhance overall reaction stability. Other conditions remained consistent with the optimal values ​​from other studies, with the exception of the arginine content, which was replaced with a mixture of DOPA and histidine. Results showed that this modified hawthorn protein achieved a 76% inhibition of Acinetobacter baumannii biofilms.

[0050] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An active hawthorn protein for inhibiting bacterial biofilm, characterized in that: It includes antibacterial hawthorn protein and a multi-modified layer compounded with the antibacterial hawthorn protein; The modified layer is obtained by compounding polysaccharide and amine components.

2. The active hawthorn protein for inhibiting bacterial biofilm according to claim 1, characterized in that: The polysaccharide is any one or more combinations of acidic polysaccharides such as inulin polysaccharide, astragalus polysaccharide and liquorice polysaccharide.

3. The active hawthorn protein for inhibiting bacterial biofilm according to claim 1, characterized in that: The amine component is any one or more combinations of dopa and its derivatives, arginine and its derivatives, histidine and its derivatives, and guanidine-containing compounds.

4. An active hawthorn protein for inhibiting bacterial biofilm according to claim 1, 2 or 3, characterized in that: The mass ratio of the polysaccharide to the amine components is 10:(1-3).

5. The active hawthorn protein for inhibiting bacterial biofilm according to claim 1, characterized in that: The molecular weight of the antibacterial hawthorn protein is between 25 and 35 KDa.

6. The active hawthorn protein for inhibiting bacterial biofilm according to claim 1, characterized in that: The particle size of the active hawthorn protein that inhibits bacterial biofilm is less than or equal to 200 nm.

7. A method for preparing the active hawthorn protein for inhibiting bacterial biofilm as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: (S.1) removing the core of the whole hawthorn fruit, washing it and squeezing out the juice to obtain a hawthorn extract, extracting hawthorn protein from the hawthorn extract, and screening the hawthorn protein to obtain an antibacterial hawthorn protein; (S.2) reacting the polysaccharide with the amine component to assemble a multi-modified layer; (S.3) The antibacterial hawthorn protein is reacted with the multi-modification layer to obtain active hawthorn protein that inhibits bacterial biofilm.

8. The method according to claim 7, characterized in that In the step (S.2), the reaction temperature is 60-70°C for 1-10 hours. After the reaction is completed, the reaction is dialyzed at 4-25°C for 8-12 hours, and the dialyzed retentate is taken out to obtain the multi-component modified layer.

9. The method according to claim 7, characterized in that: In the step (S.3), the antibacterial hawthorn protein and the multi-modified layer are first reacted at a constant temperature of 50-70°C for 0.5-3h, then the temperature is rapidly raised to 85-95°C for reaction for 3-10 min, and then rapidly cooled to room temperature to obtain active hawthorn protein that inhibits bacterial biofilm.

10. Use of the active hawthorn protein for inhibiting bacterial biofilm as claimed in any one of claims 1 to 6 in natural antibacterial food, antibacterial drug or antibacterial material.

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