A foodborne pathogenic bacteria biofilm inhibitor and its preparation method and application

Abstract

The present invention discloses an inhibitor of foodborne pathogenic bacteria biofilm, a preparation method and application thereof, wherein the inhibitor comprises a nanoparticle core, the outer surface of the nanoparticle core is coupled with a dextrorotatory amino acid to form a dextrorotatory amino acid modified silicon nanoparticle that can be phagocytosed by bacteria; the inhibitor inhibits the growth and decomposition of the foodborne pathogenic bacteria biofilm after 72 hours of formation. In the present invention, the dextrorotatory amino acid is coupled to the outer surface of the silicon nanoparticle to form a core-shell structure, which can be phagocytosed by bacteria to decompose and inhibit the growth of the foodborne pathogenic bacteria biofilm, especially 72 hours when the growth of the foodborne pathogenic bacteria biofilm reaches the maximum, and the growth and decomposition can be well inhibited, and the inhibition rate of the foodborne pathogenic bacteria biofilm reaches up to 81%.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety, and specifically relates to an inhibitor of foodborne pathogenic bacteria biofilm and a preparation method and application thereof. Background Art

[0002] Biofilms formed by foodborne pathogens can not only cause cross-contamination and post-processing contamination of food, but may also cause food poisoning and the spread of disease, posing a huge hidden danger to food safety and public safety. Biofilm is a type of bacterial self-protective growth and coordinated multicellular group behavior. Its surface growth can induce bacteria to express unique phenotypes that are different from those of planktonic bacteria. Pathogenic bacteria can form biofilms on biological surfaces and non-biological surfaces, including liquid-solid interfaces, liquid-gas interfaces, interfaces between immiscible liquids, and solid-gas interfaces; they can form on the hydrophobic cuticle of fruits and vegetables and surfaces rich in protein such as meat, as well as on surfaces such as stainless steel, glass, and wooden cutting boards.

[0003] At present, the research mainly explores the formation process of biofilm at the liquid-solid interface. First, the substances in the fluid (such as proteins in meat, etc.) are quickly adsorbed to the surface of the medium to form a conditional layer. The bacteria complete the early colonization under the action of active movement, diffusion, gravity or fluid shear force. This process usually takes 5-30 seconds. During this period, it is mainly maintained by van der Waals force, electrostatic force, hydrophobic force, and London dispersion force. The adhesion is not very tight. With a slightly stronger scraping force, it is very easy to detach from the surface and return to a floating growth mode. Subsequently, under the mediation of its polymer appendages (flagellum, pili, etc.) and adhesion factors, it further enters the irreversible adsorption stage, and the contact time usually takes several minutes to several hours. Studies have shown that type IV cilia participate in the formation of microcolonies through twitching produced by contraction. Bacteria at this stage are more difficult to be destroyed by external forces, detergents and surfactants. At this time, a large number of floating cells are constantly attached to the fixed bacteria to further proliferate. While forming microcolonies, they proliferate and continue to secrete extracellular polymers. The structure of mature biofilms has spatial distribution heterogeneity. The inner gaps formed by water channels and extracellular polymers are intertwined to form a highly organized three-dimensional network structure. The water channels are conducive to the transportation of nutrients and metabolic waste. Under different environments, mature biofilms with loose structures and different thicknesses are formed. For example, in still water, they are "mushroom" structures, while in torrents, they are often flat. The physiological states of bacteria at different levels of biofilms are not the same. This is because the pH, oxygen concentration and nutrient concentration of their microenvironment affect the growth rate and metabolic activity of the flora. Due to the severe supply limitation of nutrients and oxygen, the cells at the bottom of the biofilm are in a living non-culturable dormant state, which responds slowly to adverse external stress and activates their own related tolerance genes. In addition, the permeability barrier function of the biofilm matrix makes it difficult for antimicrobial agents to act on them. After stopping, the remaining bacteria multiply rapidly, resulting in repeated infections that cannot be eradicated.

[0004] Once formed, biofilm is difficult to eradicate, so preventing and inhibiting its formation is the key to controlling the formation of Cronobacter biofilm. Generally, two aspects are considered. One is the change in the characteristics of the contact surface. The surface free energy, wetting properties and interfacial energy roughness of the medium (aluminum alloy, stainless steel, plastic and glass products, etc.) directly affect the adhesion of bacteria. Studies have found that the adhesion and growth of pathogens can be inhibited by using antibacterial coatings; the second is to add antibacterial active ingredients, such as allicin, black fungus extract, nisin, pediocin and antimicrobial peptides, which have a good inhibitory effect on the formation of biofilm. However, the preparation process of the above extracts is complicated, the price is high, and it is sensitive to temperature, so it cannot be applied to food processing in food factories. Summary of the invention

[0005] In view of the problems existing in the above-mentioned background technology, the present invention provides an inhibitor of foodborne pathogenic bacteria biofilm and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] Disclosed is an inhibitor of foodborne pathogenic bacteria biofilm, comprising a nanoparticle core, the outer surface of which is coupled with dextrorotatory amino acids to form dextrorotatory amino acid-modified silicon nanoparticles that can be phagocytosed by bacteria; the inhibitor can decompose and inhibit the growth of foodborne pathogenic bacteria biofilm 72 hours after it is formed.

[0008] In a further embodiment, the nanoparticle core is a silicon nanoparticle, and the dextrorotatory amino acid includes dextrorotatory tryptophan.

[0009] In a further embodiment, the foodborne pathogens in the foodborne pathogenic bacteria biofilm include Listeria monocytogenes, Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa.

[0010] The second invention object of the present invention is to provide an application of the above-mentioned inhibitor of foodborne pathogenic bacteria biofilm, wherein the inhibitor decomposes and inhibits the growth of foodborne pathogenic bacteria biofilm, specifically: the inhibitor is added to the foodborne pathogenic bacteria biofilm and statically cultured for 1-2.5 hours, the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1: (5-8), and the maximum concentration of foodborne pathogens in the foodborne pathogenic bacteria biofilm is 3.9*10 6 CFU / mL.

[0011] In a further embodiment, when the foodborne pathogenic bacteria biofilm is a Listeria monocytogenes biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:6;

[0012] When the foodborne pathogenic bacteria biofilm is a Staphylococcus aureus biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:8;

[0013] When the foodborne pathogenic bacteria biofilm is a Salmonella biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:7;

[0014] When the foodborne pathogenic bacteria biofilm is an Escherichia coli biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1 hour, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:5;

[0015] When the foodborne pathogenic bacteria biofilm is a Cronobacter sakazakii biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:5;

[0016] When the foodborne pathogenic bacteria biofilm is a Pseudomonas aeruginosa biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:7.

[0017] The third invention object of the present invention is to provide a method for preparing the above-mentioned inhibitor of foodborne pathogenic bacteria biofilm, which comprises the following steps:

[0018] (1) Preparation of activation mixture

[0019] Mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution with an N-hydroxysuccinimide solution to prepare an activated mixed solution;

[0020] (2) Preparation of silicon nanoparticle solution

[0021] Mixing the sodium ascorbate solution with 3-aminopropyltrimethoxysilane, and adding ultrapure water eight times the volume of the mixture, stirring and mixing; and obtaining a silicon nanoparticle solution after dialysis;

[0022] (3) Preparation of right-handed amino acid modified silicon nanoparticle solution

[0023] The silicon nanoparticle solution and the activation mixture are mixed and incubated at room temperature; then the dextrorotatory amino acid solution is added and incubated on a shaking table to obtain a dextrorotatory amino acid-modified silicon nanoparticle solution, which is the inhibitor of the present application.

[0024] In a further embodiment, in step (1), the volume ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution to the N-hydroxysuccinimide aqueous solution is 1:1, the concentration of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is 10 mM, and the concentration of the N-hydroxysuccinimide aqueous solution is 5 mM.

[0025] In a further embodiment, the reducing agent in step (2) is sodium ascorbate, and the silane is 3-aminopropyltrimethoxysilane;

[0026] The volume ratio of the reducing agent to silane is 1:1.

[0027] The dialysis is performed using a 500Da dialysis bag for 15-20 hours, and the retentate obtained by dialysis is diluted 12-18 times to obtain a silicon nanoparticle solution.

[0028] In a further embodiment, the volume ratio of the silicon nanoparticle solution to the activation mixture in step (3) is 2:1, and the incubation time at room temperature is 20-40 minutes;

[0029] The amount of the dextrorotatory amino acid solution added is 0.08-0.12 times the volume of the silicon nanoparticle solution;

[0030] The shaking incubation is carried out at room temperature at a rotation speed of 150-250 rpm for 2-4 hours.

[0031] The preparation of the food-borne pathogenic bacteria biofilm in the present invention is as follows:

[0032] The foodborne pathogens were inoculated into LB broth at a volume ratio of 1:100, and the final concentration of the foodborne pathogens was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate, and cultured at 37°C for 72 hours to obtain a foodborne pathogenic bacteria biofilm; the inhibitor was added to the above-mentioned 96-well cell culture plate at a volume ratio of 1: (5-8), and cultured at room temperature 37°C for 1 hour to 2.5 hours.

[0033] The beneficial technical effects of the present invention are embodied in the following aspects:

[0034] The present invention couples right-handed amino acids to the outer surface of silicon nanoparticles to form a core-shell structure, which can be phagocytosed by bacteria to inhibit the growth and decomposition of foodborne pathogenic bacteria biofilms, especially 72 hours when the growth of foodborne pathogenic bacteria biofilms reaches the maximum, and can also achieve good inhibition and decomposition, with an inhibition rate of 69-81% on foodborne pathogenic bacteria biofilms.

[0035] The right-handed amino acid modified silicon nanoparticles prepared by the present invention have a good inhibitory effect on the biofilm formed by common foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa.

[0036] Although the prior art has disclosed the inhibitory effect of right-handed amino acids on bacterial biofilms, they are usually added for inhibition in the early stages of biofilm formation. The growth of foodborne pathogens reaches its maximum at 72 hours after the formation of the foodborne pathogenic bacteria biofilm, but the inhibitory effect of right-handed amino acids on biofilms formed at 72 hours is poor. This is because the cells of the biofilm enter a living, non-culturable dormant state, which slows down the response to adverse external stress and activates their own related tolerance genes. In addition, the permeability barrier effect of the biofilm matrix makes it difficult to effectively remove them.

[0037] Since silicon nanoparticles (whose freeze-dried powder is a crystal structure) may have a certain aggregation effect, when the right-handed amino acids are covalently coupled with the silicon nanoparticles, due to the bacterial intake mechanism of amino acids, the right-handed amino acids carry the silicon nanoparticles into the bacteria. On the one hand, a large number of silicon nanoparticles modified with right-handed amino acids are stored inside the bacteria and cannot be used; on the other hand, the action of some hydrolases causes the right-handed amino acids to be separated, and the bacteria cannot use the right-handed amino acids for normal physiological metabolism. In general, the prior art does not disclose the combined use of right-handed amino acids and silicon nanoparticles.

[0038] The present invention creatively utilizes the bacterial intake mechanism of amino acids and combines it with the aggregation effect of silicon nanoparticles to inhibit the growth and reproduction of foodborne pathogenic bacteria biofilms. It is very suitable for the cleaning of biofilms in the food industry and can be applied to the treatment of stubborn biofilms in the future.

[0039] The inhibitor of the present invention works at room temperature and is therefore suitable for food processing.

[0040] The inhibitor of the present invention can still inhibit the growth and reproduction of foodborne pathogenic bacteria biofilm 72 hours after it is formed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a graph of relative ultraviolet absorption intensity before and after covalent coupling of dextrorotatory tryptophan and silicon nanoparticles in the present invention;

[0042] Figure 2 This is the ultraviolet absorption graph of the inhibition of Listeria monocytogenes biofilm by Application Example 1 of the present invention;

[0043] Figure 3 This is the ultraviolet absorption graph of the inhibition of Staphylococcus aureus biofilm by Application Example 2 of the present invention;

[0044] Figure 4 This is the ultraviolet absorption graph of the inhibition of Salmonella biofilm by Application Example 3 of the present invention;

[0045] Figure 5 This is the UV absorption graph of the inhibition of Escherichia coli biofilm by Application Example 4 of the present invention;

[0046] Figure 6 This is the ultraviolet absorption graph of the inhibition of Cronobacter sakazakii biofilm by Application Example 5 of the present invention;

[0047] Figure 7 This is the ultraviolet absorption graph of the inhibition of Pseudomonas aeruginosa biofilm by Application Example 6 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with specific embodiments.

[0049] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0051] In the following examples, the aptamers used were purchased from Shanghai Bioengineering Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, guanidine hydrochloride, vancomycin, sodium ascorbate and 3-aminopropyltrimethoxysilane used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the 500Da dialysis bag used was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; unless otherwise specified, other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] The phosphate buffered saline solution used in the present application is a sterile phosphate buffered saline solution with a pH value of 7.4 and a 0.1 M concentration.

[0053] All strains used in this application are purchased from the American Type Culture Collection (ATCC), where Listeria monocytogenes is numbered ATCC 43251, Staphylococcus aureus is numbered ATCC 29213, Salmonella is numbered ATCC 14028, Escherichia coli is numbered ATCC 25922, Cronobacter sakazakii is numbered ATCC29544, and Pseudomonas aeruginosa is numbered ATCC 15442. In addition, the above strains are purchased through overseas agents of Hefei Xiyue Biotechnology Co., Ltd.

[0054] The instruments, equipment, raw materials, reagents or method steps used in this application are all ensured to be processed under sterile conditions. The instruments, equipment, raw materials, reagents or method steps not mentioned in this application are conventional or well-known technical methods for those skilled in the art and are not described in detail in this application.

[0055] Preparation Example 1

[0056] Preparation of Dextrorotatory Amino Acid Modified Silica Nanoparticle Solution

[0057] (1) Preparation of activation mixture

[0058] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to prepare an activation mixture;

[0059] (3) Preparation of silicon nanoparticle solution

[0060] Mix a 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane in a volume ratio of 1:1, add eight times the volume of ultrapure water, and stir to mix;

[0061] The solution was dialyzed for 18 h using a 500 Da dialysis bag to obtain the retentate and diluted fifteen times;

[0062] (3) Preparation of right-handed amino acid modified silicon nanoparticle solution

[0063] The diluted retentate solution and the activated mixed solution were mixed at a volume ratio of 2:1 and incubated at room temperature for 30 minutes; a 100uM dextrorotatory tryptophan solution was added, and the amount of dextrorotatory tryptophan solution added was 0.1 times the volume of the retentate solution; the mixture was incubated on a shaking table at a speed of 200rpm and room temperature for 3 hours to obtain a dextrorotatory amino acid modified silicon nanoparticle solution.

[0064] like Figure 1 As shown, when the ultraviolet absorption wavelength of the silicon nanoparticles shifts from 346 nm (curve A) to 354 nm (curve B), it indicates that the dextrorotatory amino acids in the dextrorotatory amino acid-modified silicon nanoparticle solution are successfully modified onto the silicon nanoparticles.

[0065] Preparation Example 2

[0066] (1) Preparation of activation mixture

[0067] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to prepare an activation mixture;

[0068] (3) Preparation of silicon nanoparticle solution

[0069] Mix a 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane in a volume ratio of 1:1, add eight times the volume of ultrapure water, and stir to mix;

[0070] The retentate was dialyzed for 20 h using a 500 Da dialysis bag and diluted 12 times;

[0071] (3) Preparation of right-handed amino acid modified silicon nanoparticle solution

[0072] The diluted retentate solution and the activated mixed solution were mixed at a volume ratio of 2:1 and incubated at room temperature for 40 minutes; a 100uM dextrorotatory tryptophan solution was added, and the amount of dextrorotatory tryptophan solution added was 0.12 times the volume of the retentate solution; the mixture was incubated on a shaking table at a speed of 180rpm and room temperature for 4 hours to obtain a dextrorotatory amino acid modified silicon nanoparticle solution.

[0073] Application Example 1 (Inhibition of Listeria monocytogenes biofilm)

[0074] The method for inhibiting Listeria monocytogenes biofilm by right-handed amino acid-modified silicon nanoparticles is as follows:

[0075] Listeria monocytogenes was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of Listeria monocytogenes was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare a Listeria monocytogenes biofilm, one of which served as a blank control;

[0076] Then, the dextrorotatory amino acid modified silicon nanoparticle solution prepared in Preparation Example 1 was added to the above 96-well cell culture plate at a volume ratio of 1:6, and cultured at room temperature 37° C. for 2 h, which served as the treatment group.

[0077] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0078] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0079] like Figure 2 As shown, compared with the blank control, positive control 1 (inhibition rate 13.6%) and positive control 2 (inhibition rate 26.8%), the treatment group showed that the combination of right-handed amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Listeria monocytogenes biofilm (inhibition rate 80.4%). Inhibition rate = UV absorption value of the blank group - UV absorption value of the experimental group (values ​​taken from Figure 2 UV absorption graph).

[0080] In addition, it is also proved that right-handed amino acids also have a certain inhibition rate on Listeria monocytogenes biofilm, but it is significantly lower than the inhibition rate of the inhibitors of the present application; and silicon nanoparticles have very low toxicity to bacteria outside the bacteria and need to be delivered into the bacteria to have a strong bactericidal effect.

[0081] Application Example 2 (Inhibition of Staphylococcus aureus biofilm)

[0082] The method for inhibiting Staphylococcus aureus biofilm by right-handed amino acid-modified silicon nanoparticles is as follows:

[0083] Staphylococcus aureus was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of Staphylococcus aureus was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare a Staphylococcus aureus biofilm, one of which served as a blank control;

[0084] Then, the dextrorotatory amino acid-modified silicon nanoparticle solution prepared in Preparation Example 1 was added to a 96-well cell culture plate at a volume ratio of 1:8, and cultured at room temperature 37° C. for 2.5 h as treatment.

[0085] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0086] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0087] like Figure 3 As shown, compared with the blank control, positive control 1 (inhibition rate 14.8%) and positive control 2 (inhibition rate 22.6%), the treatment group demonstrated that the combination of right-handed amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Staphylococcus aureus biofilm (inhibition rate 74%).

[0088] Application Example 3 (Inhibition of Salmonella biofilm)

[0089] The method for inhibiting Salmonella biofilm by silicon nanoparticles modified with right-handed amino acids is as follows:

[0090] Salmonella was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of Salmonella was adjusted to 3.9*106 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare a Salmonella biofilm, one of which served as a blank control;

[0091] Then, the dextrorotatory amino acid-modified silicon nanoparticle solution prepared in Preparation Example 1 was added to a 96-well cell culture plate at a volume ratio of 1:7, and cultured at room temperature 37° C. for 1.5 h as treatment.

[0092] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0093] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0094] like Figure 4 As shown, compared with the blank control, positive control 1 (inhibition rate 14.2%) and positive control 2 (inhibition rate 17.4%), the treatment group demonstrated that the combination of right-handed amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Salmonella biofilm (inhibition rate 69.4%).

[0095] Application Example 4 (Inhibition of Escherichia coli biofilm)

[0096] The method for inhibiting Escherichia coli biofilm by right-handed amino acid-modified silicon nanoparticles is as follows:

[0097] E. coli was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of E. coli was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare an E. coli biofilm, one of which served as a blank control;

[0098] Then, the dextrorotatory amino acid-modified silicon nanoparticle solution prepared in Preparation Example 1 was added to a 96-well cell culture plate at a volume ratio of 1:5, and cultured at room temperature 37° C. for 1 hour as treatment.

[0099] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0100] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0101] like Figure 5 As shown, compared with the blank control, positive control 1 (inhibition rate 13.4%) and positive control 2 (inhibition rate 20%), the treatment group demonstrated that the combination of right-handed amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Escherichia coli biofilm (inhibition rate 71.2%).

[0102] Application Example 5 (Inhibition of Cronobacter sakazakii biofilm)

[0103] The method for inhibiting Cronobacter sakazakii biofilm by right-handed amino acid-modified silicon nanoparticles is as follows:

[0104] Cronobacter sakazakii was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of Cronobacter sakazakii was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare Cronobacter sakazakii biofilm, one of which was used as a blank control;

[0105] Then, the dextrorotatory amino acid-modified silicon nanoparticle solution prepared in Preparation Example 1 was added to a 96-well cell culture plate at a volume ratio of 1:5, and cultured at room temperature 37° C. for 1.5 h as treatment.

[0106] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0107] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0108] like Figure 6As shown, compared with the blank control, positive control 1 (inhibition rate 11.8%) and positive control 2 (inhibition rate 30.2%), the treatment group demonstrated that the combination of right-handed amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Cronobacter sakazakii biofilm (inhibition rate 79.4%).

[0109] Application Example 6 (Inhibition of Pseudomonas aeruginosa biofilm)

[0110] The method for inhibiting the biofilm of Pseudomonas aeruginosa by silicon nanoparticles modified with right-handed amino acids is as follows:

[0111] Pseudomonas aeruginosa was inoculated into LB broth at a volume ratio of 1:100, and the final concentration of Pseudomonas aeruginosa was adjusted to 3.9*10 6 CFU / mL, and transferred to a 96-well cell culture plate and cultured at 37°C for 72 h to prepare a Pseudomonas aeruginosa biofilm, one of which served as a blank control;

[0112] Then, the dextrorotatory amino acid-modified silicon nanoparticle solution prepared in Preparation Example 1 was added to a 96-well cell culture plate at a volume ratio of 1:7, and cultured at room temperature 37° C. for 2 h as treatment.

[0113] Under the same conditions, single D-amino acid and silicon nanoparticles of unmodified D-amino acid were used as positive controls 1 and 2, respectively.

[0114] The method for determining the biofilm content is as follows: discard the culture medium from the 96-well cell culture plate, wash it repeatedly with sterile saline for 3 times until the bacteria that are not attached to the wall are washed away. After fixing it at 60°C for 1 hour, add 200 μL of 0.1% crystal violet dye solution to each well for 30 minutes, then wash it with sterile saline for 3 times (try to wash away the unbound dye), air-dry it at room temperature, dissolve it in 33% acetic acid, shake it gently, and use an enzyme reader to measure the absorbance value at a wavelength of 590 nm.

[0115] like Figure 7 As shown, compared with the blank control, positive control 1 (inhibition rate 11.6%) and positive control 2 (inhibition rate 19.2%), the treatment group demonstrated that the combination of dextrorotatory amino acids and silicon nanoparticles had a better inhibitory and disintegrating effect on Pseudomonas aeruginosa biofilm (inhibition rate 71.4%).

[0116] It will be easily understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An inhibitor of foodborne pathogenic bacteria biofilm, characterized in that: The inhibitor comprises a silicon nanoparticle core, and the outer surface of the silicon nanoparticle core is coupled with a dextrorotatory amino acid to form a dextrorotatory amino acid-modified silicon nanoparticle that can be phagocytosed by bacteria; the inhibitor decomposes and inhibits the growth of foodborne pathogenic bacteria biofilm 72 hours after it is formed; The preparation method comprises the following steps: (1) Preparation of activation mixture Mixing an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with an aqueous solution of N-hydroxysuccinimide to prepare an activated mixed solution; (2) Preparation of silicon nanoparticle solution The sodium ascorbate solution and 3-aminopropyltrimethoxysilane are mixed in a volume ratio of 1:1, and ultrapure water in a volume eight times that of the mixed solution is added and stirred to mix well; after dialysis, a silicon nanoparticle solution is obtained; The dialysis is performed using a 500Da dialysis bag for 15-20 hours, and the retentate obtained by dialysis is diluted 12-18 times; (3) Preparation of right-handed amino acid modified silicon nanoparticle solution The silicon nanoparticle solution and the activation mixed solution are mixed at a volume ratio of 2:1 and incubated at room temperature; then a dextrorotatory amino acid solution is added and incubated on a shaking table to obtain a dextrorotatory amino acid-modified silicon nanoparticle solution; the amount of the dextrorotatory amino acid solution added is 0.08-0.12 times the volume of the silicon nanoparticle solution.

2. The inhibitor according to claim 1, characterized in that: The dextrorotatory amino acids include dextrorotatory tryptophan.

3. The inhibitor according to claim 1, characterized in that: The food-borne pathogenic bacteria in the food-borne pathogenic bacteria biofilm include Listeria monocytogenes, Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa.

4. The use of an inhibitor of foodborne pathogenic bacteria biofilm as claimed in claim 1, characterized in that: The inhibitor is used for decomposing and inhibiting the growth of foodborne pathogenic bacteria biofilm, specifically: adding the inhibitor to the foodborne pathogenic bacteria biofilm and culturing it statically for 1-2.5 hours, the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:(5-8), and the maximum concentration of foodborne pathogenic bacteria in the foodborne pathogenic bacteria biofilm is 3.9×10 6 CFU / mL.

5. The use according to claim 4, characterized in that: When the foodborne pathogenic bacteria biofilm is a Listeria monocytogenes biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:6; When the foodborne pathogenic bacteria biofilm is a Staphylococcus aureus biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:8; When the foodborne pathogenic bacteria biofilm is a Salmonella biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:7; When the foodborne pathogenic bacteria biofilm is an Escherichia coli biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1 hour, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:5; When the foodborne pathogenic bacteria biofilm is a Cronobacter sakazakii biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 1.5 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:5; When the foodborne pathogenic bacteria biofilm is a Pseudomonas aeruginosa biofilm, the inhibitor is added to the foodborne pathogenic bacteria biofilm and cultured at room temperature for 2 hours, and the volume ratio of the inhibitor to the foodborne pathogenic bacteria biofilm is 1:

7.

6. The inhibitor according to claim 1, characterized in that: In step (1), the volume ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution to the N-hydroxysuccinimide aqueous solution is 1:1, the concentration of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution is 10 mM, and the concentration of the N-hydroxysuccinimide aqueous solution is 5 mM.

7. The inhibitor according to claim 1, characterized in that: The incubation time at room temperature in step (3) is 20-40 minutes.

8. The inhibitor according to claim 1, characterized in that: In step (3), the shaking incubation is carried out at room temperature at a rotation speed of 150-250 rpm for 2-4 hours.

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