Application of lysine

Abstract

The invention provides application of lysine. The lysine is applied to a disinfectant for inhibiting escherichia coli O157: H7. The lysine is a natural nutrient, the lysine is used for inhibiting and killing escherichia coli O157: H7 in food, the food is free of toxic residues, abuse of preservatives is reduced, in addition, the sensory characteristics of the food can be reserved, and the lysine is particularly suitable for preservation and fresh keeping of uncooked food.

Description

Technical Field

[0001] The present invention relates to the technical field of food sterilization, in particular to a use of lysine. Background Art

[0002] Pathogenic Escherichia coli are widely present in the natural environment, and food may be contaminated by pathogenic E. coli at any of the pathways before entering the digestive environment. Pathogenic E. coli can cause sepsis, meningitis, urinary tract infections, diarrhea, and other diseases associated with the consumption of fecal contaminated food or water, and in the most severe cases, even death. E. coli O157:H7 is a common serotype that is responsible for most diarrheal outbreaks in the world and can cause acute gastroenteritis, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In order to control E. coli O157:H7 in food, the widely used physical sterilization methods include irradiation, pasteurization, pulsed electric field, and high pressure treatment, and the chemical sterilization methods include peracetic acid, chlorine dioxide, sodium hypochlorite, and some organic acids. Although these methods are very effective, they are not suitable for all foods and have inherent disadvantages including changes in sensory properties and toxicity. The limitation of sodium hypochlorite as a disinfectant for washing vegetables and fruits is that it leads to the formation of chlorination byproducts. Benzoate is an effective food and beverage preservative, but benzoate can react with ascorbic acid in beverages to produce benzene, a carcinogen. Aspects of sensory property changes include ultrasonic sterilization, which affects the flavor and nutrition of apple juice and watermelon juice, and heat treatment, which also causes the flavor of melon juice to deteriorate. Therefore, the development of safe and effective alternatives has received increasing attention in recent years. Given the incidence of E. coli O157:H7 infection and the severity of the disease, natural nutrients are becoming increasingly popular as a control option in the food industry to prevent foodborne disease outbreaks, such as shikonin and juglone, which are used to control foodborne Staphylococcus aureus. The main host of E. coli O157:H7 is cattle, where it mainly colonizes at the end of the rectum and is then excreted through feces and irrigated to the land, which has caused some vegetables including lettuce, carrots, tomatoes, cucumbers, etc. to become carriers. Studies have shown that E. coli can survive or grow in a range of minimally processed lettuces, which can have adverse effects on human health after digestion. Therefore, an effective disinfectant is needed to completely inhibit E. coli O157:H7.

[0003] Therefore, it is urgent to choose a natural and harmless disinfectant to inhibit pathogenic Escherichia coli.

[0004] Lysine is an essential amino acid, its chemical name is 2,6-diaminoacetic acid, it can promote brain development, promote fat metabolism and prevent cell degeneration. Lysine can be used as a food flavoring, coloring agent and nutritional enhancer in food processing. The Chinese national standard GB2760-2014 stipulates that when lysine is used as a natural flavoring in the food industry, there is no requirement for lysine in unlimited quantities. So far, no research has reported the inhibitory effect of lysine on E. coli O157:H7. Summary of the invention

[0005] The main purpose of the present invention is to provide a use of lysine to at least solve the problems of residual toxicity and changes in food sensory properties after food sterilization in the prior art.

[0006] In order to achieve the above object, the present invention provides a use of lysine, including the use of lysine in a disinfectant for inhibiting Escherichia coli O157:H7.

[0007] Furthermore, the minimum inhibitory concentration (MIC) of lysine against Escherichia coli O157:H7 is 12.5 mg / mL to 25 mg / mL, and the minimum bactericidal concentration (MBC) is 12.5 mg / mL to 25 mg / mL.

[0008] The technical solution of the present invention provides a use of lysine, which is to apply lysine to a disinfectant for inhibiting E. coli O157:H7. Lysine is a natural nutrient, and the use of lysine to inhibit and kill E. coli O157:H7 on food leaves no toxic residues, reduces the abuse of chemical disinfectants, and retains the sensory properties of food. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0010] Figure 1 This is a graph showing the effect of lysine on the growth curve of EDL933 strain, which can be selected according to an embodiment of the present invention;

[0011] Figure 2 This is a membrane damage diagram of EDL933 strain caused by lysine, which can be selected according to an embodiment of the present invention;

[0012] Figure 3 This is a graph showing the effect of lysine on the cell membrane potential of the EDL933 strain, which can be selected according to an embodiment of the present invention;

[0013] Figure 4 This is a graph showing the effect of lysine on the cell morphology of EDL933 strain under a scanning electron microscope, which can be selected according to an embodiment of the present invention;

[0014] Figure 5 This is a graph showing the effect of lysine on the integrity of the cell membrane of Escherichia coli under a laser confocal microscope (100 times magnification) which can be selected according to an embodiment of the present invention;

[0015] Figure 6 is a graph showing the effect of lysine on AKP leakage in Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0016] Figure 7 is a graph showing the effect of lysine on ATP concentration of Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0017] Figure 8 This is a graph showing the effect of lysine on the ROS content in Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0018] Fig. 9 This is a graph showing the effect of lysine on nucleic acid leakage in Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0019] Fig.10 is a graph showing the effect of lysine on protein leakage in Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0020] Fig.11 is a graph showing the effect of lysine on the biofilm formation ability of Escherichia coli, which can be selected according to an embodiment of the present invention;

[0021] Fig.12 is a graph showing the effect of lysine on the genome of Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0022] Fig.13 It is a lysine that can be selected according to an embodiment of the present invention for the primary structure of Escherichia coli cell protein;

[0023] Fig.14 This is a gel imaging diagram of the effect of lysine on the motility and swarming motility of Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0024] Fig.15 This is a graph showing the effect of lysine on the swimming area of ​​Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0025] Fig.16 is a graph showing the effect of lysine on the surging area of ​​Escherichia coli cells, which can be selected according to an embodiment of the present invention;

[0026] Fig.17 is a graph showing the effect of lysine on the activity of Caco-2 cells, which can be selected according to an embodiment of the present invention;

[0027] Fig.18This is a graph showing the effect of a lysine on the ability of Escherichia coli cells to adhere to Caco-2 cells, which can be selected according to an embodiment of the present invention;

[0028] Fig.19 This is a graph showing the effect of lysine on the ability of Escherichia coli cells to invade Caco-2 cells, which can be selected according to an embodiment of the present invention;

[0029] Fig. 20 This is a graph showing the effect of lysine on the number of EDL933 strain cells on lettuce, which can be selected according to an embodiment of the present invention;

[0030] Fig.21 is a sensory attribute evaluation diagram of lettuce processed in different ways, which can be selected according to an embodiment of the present invention;

[0031] Fig. 22 is an optional experimental feeding time chart according to an embodiment of the present invention;

[0032] Fig.23 is a graph of weight changes of each group of mice during the experiment, which can be selected according to an embodiment of the present invention;

[0033] Fig.24 This is a weight comparison chart of mice in each group at the end of an experiment that can be selected according to an embodiment of the present invention;

[0034] Fig.25 is a comparison diagram of occult blood levels in mouse feces that can be selected according to an embodiment of the present invention;

[0035] Fig.26 This is a comparison chart of myeloperoxidase (MPO) activities of various groups that can be selected according to an embodiment of the present invention;

[0036] Fig. 27 is an optional comparison diagram of colon lengths of various groups according to an embodiment of the present invention;

[0037] Fig.28 This is an optional comparison chart of TNF-α content in each group according to an embodiment of the present invention;

[0038] Fig.29 This is an optional comparison chart of IL-1β content in each group according to an embodiment of the present invention;

[0039] Fig.30 This is an optional comparison chart of IL-6 content in each group according to an embodiment of the present invention;

[0040] Fig.31 This is a colon section comparison diagram that can be selected according to an embodiment of the present invention. DETAILED DESCRIPTION

[0041] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0042] The applicant has found that lysine has an inhibitory effect on E. coli O157:H7 and can be used as the main active ingredient of a disinfectant to inhibit E. coli O157:H7 in food. Lysine is a natural nutrient with no toxicity. By utilizing the inhibitory effect of lysine on E. coli O157:H7, adding the natural nutrient lysine to food can inhibit the growth of bacteria without toxicity and supplement nutrition for the human body, achieving two goals at one stroke.

[0043] This is illustrated by the following examples:

[0044] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art.

[0045] The foodborne pathogen selected in the experiment was Escherichia coli O157:H7EDL933, which was collected from the Food Biomanufacturing and Microbiological Safety Team Laboratory of the School of Food and Engineering, Northwest Agriculture and Forestry University, Yangling, China. Lysine was purchased from Solarbio Biotech Co., Ltd. (SL8330).

[0046] It can only inhibit the growth of E. coli O157:H7, but cannot kill it. After removing lysine, the minimum amount of lysine at which E. coli O157:H7 can resume growth is the minimum inhibitory concentration (MIC), and the minimum amount of lysine that can kill E. coli O157:H7 and still prevent E. coli O157:H7 from growing after lysine removal is the minimum bactericidal concentration (MBC). The MIC and MBC are determined by the microbroth dilution method. In the present application, within the range of the minimum inhibitory concentration and the minimum bactericidal concentration, the solvent used to reflect the inhibitory effect of lysine on E. coli O157:H7 is distilled water.

[0047] After streaking the strain, pick a single colony and continue to culture in lysogeny broth (LB) to obtain a bacterial suspension. Lysine was diluted with distilled water in a 96-well plate and added to the prepared bacterial suspension. The wells with only LB liquid culture medium were used as negative controls. After incubation for 24 hours, the OD value was measured at 600nm. The MIC and MBC of lysine against Escherichia coli from different sources are shown in Table 1.

[0048] Table 1 MIC and MBC of lysine against different pathogenic Escherichia coli strains

[0049]

[0050] Table 1 shows that lysine has an inhibitory effect on all the tested strains. The MIC and MBC of strains EDL933 and D4 were 12.5 mg / mL, and the MIC and MBC values ​​of the other isolated Escherichia coli were both 25 mg / mL. In other words, the minimum inhibitory concentration (MIC) of lysine for Escherichia coli O157:H7 was 12.5 mg / mL to 25 mg / mL, and the minimum bactericidal concentration (MBC) was 12.5 mg / mL to 25 mg / mL.

[0051] The inhibitory effect of lysine on Escherichia coli was further illustrated by the following experiment. In the figure, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0052] 1. Growth Curve and Subinhibitory Concentration (SIC)

[0053] The growth curve describes the growth of bacteria under specific environmental conditions (such as temperature, pH value, nutrient concentration, etc.) over time. By measuring the growth curve, we can understand the growth characteristics of bacteria under different lysine concentrations, including the changes in the lag phase, logarithmic growth phase, stable phase and decay phase. Prepare Escherichia coli suspension so that the concentration of the bacterial suspension is about 10 6 CFU / mL. Prepare different concentrations of lysine and mix the bacterial solution evenly. Use a microbial growth curve analyzer to measure OD every 1 hour 600nm The total operation time was 24 hours. Each group had 3 parallels. The culture time was taken as the horizontal axis, and the OD 600nm The microbial growth curve was plotted with lysine as the ordinate and the lysine concentration that had no effect on the growth of E. coli cells was defined as SIC.

[0054] like Figure 1 As shown in the figure, at 2MIC and MIC concentrations, the growth curve of Escherichia coli almost overlaps with the horizontal axis, indicating that lysine completely inhibits the growth of Escherichia coli. At 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC concentrations, lysine prolongs the lag phase of bacteria. Compared with the control group (Control), lysine at concentrations of 1 / 16MIC, 1 / 32MIC, 1 / 64MIC, and 1 / 128MIC has no significant effect on the growth of the strain. Therefore, 1 / 16MIC, 1 / 32MIC, 1 / 64MIC, and 1 / 128MIC are defined as the SIC of lysine to Escherichia coli. Figure 1Each value is the average of three independent experiments.

[0055] 2. Effect of lysine on Escherichia coli cell membrane

[0056] In the following test methods, the applicant used analysis of variance (ANOVA) to test the significance of the experimental results.

[0057] 2.1. Lysine uptake of N-phenyl-l-naphthylamine (NPN) by Escherichia coli

[0058] E. coli was treated with different concentrations of lysine at 37°C for 30 min. The mixture was centrifuged (8000g, 5 min, 4°C), washed with sterile PBS and resuspended in physiological saline. 200 μL of each sample was added to a 96-well plate, and 1.5 μL of NPN (100 mM) solution was added to each well and mixed by pipetting. The fluorescence value of each sample was immediately measured using a fluorescence spectrophotometer, with an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0059] N-phenyl-l-naphthylamine (NPN) does not fluoresce in aqueous media. When the cell membrane is damaged, NPN will fluoresce after binding to the hydrophobic region of the cell membrane. Cell membrane damage can be determined by NPN uptake and reflected by fluorescence intensity. Figure 2 As shown, compared with the control group (Control), lysine can significantly increase bacterial NPN uptake (p < 0.01), and can cause E. coli cell membrane damage in a concentration-dependent manner. p < 0.01 indicates that the experimental results are highly significant in statistics.

[0060] 2.2 Effect of lysine on membrane potential of Escherichia coli

[0061] The membrane potential change of E. coli is measured using the voltage-sensitive specific fluorescent dye DiBAC4. DiBAC4 is an anionic fluorescent dye that can bind to bacterial cytoplasmic proteins. When the bacteria are stimulated by external stimuli, the dye enters or leaves the cell, which can bring about changes in fluorescence intensity, thereby reflecting changes in cell membrane potential. Although E. coli itself cannot produce any fluorescence, it will emit fluorescence when it binds to depolarized cells or intracellular proteins.

[0062] Add the prepared bacterial solution to the black ELISA plate and add 3 μL of fluorescent dye DiBAC4 to each well, place it in a 37°C incubator for 30 minutes, then add lysine solutions of 2MIC, MIC, 1 / 2MIC, and 1 / 4MIC concentrations to the wells with bacterial solution to allow the fluorescent dye and E. coli to fully react. Use a multifunctional ELISA reader to detect the fluorescence intensity, with an excitation / emission wavelength of 492nm / 515nm, an excitation slit width of 3nm, and an emission slit width of 5nm.

[0063] like Figure 3 As shown in the figure, the fluorescence intensity of E. coli treated with 2MIC, MIC, 1 / 2MIC and 1 / 4MIC lysine was significantly higher than that of the untreated group (p<0.01), and continued to increase with the increase of lysine concentration. The depolarization of membrane potential indicates that the cell membrane of E. coli is damaged. Therefore, the cell membrane of E. coli cells treated with lysine will be damaged.

[0064] 2.3 Scanning electron microscopy observation of Escherichia coli cell morphology

[0065] The morphology of Escherichia coli cells was observed by scanning electron microscopy. Escherichia coli cells were treated with different concentrations of lysine at 37°C for 5 h. The treated samples were washed with physiological saline after centrifugation and then fixed with electron microscope fixative for 12 h. Different concentrations of ethanol were used for gradient dehydration, and the cells were freeze-dried and sprayed with gold. The scanning electron microscope used in this application is a Hitachi cold field emission scanning electron microscope, Regulus 8100, manufactured by Tokyo, Japan.

[0066] like Figure 4 As shown in the figure, the morphology of Escherichia coli cells changed after lysine treatment. Comparing the control group and the experimental group cells without lysine treatment, it was observed that the untreated cells showed a typical Gram-negative bacterial structure, which was rod-shaped, complete in morphology, and had a clear wrinkled structure on the surface. After treatment with different concentrations of lysine, the wrinkled structure changed, the membrane curled and sunken, the cell contents overflowed, and the cells showed obvious shrinkage. With the increase of lysine concentration, the degree of cell damage increased. Therefore, a certain concentration of lysine will destroy the morphology of Escherichia coli cells.

[0067] 2.4. Cell membrane integrity detection

[0068] use BacLightVigor kit L-7007 was used to observe the permeability of lysine to the plasma membrane of Escherichia coli. The Escherichia coli suspension treated with different concentrations of lysine was centrifuged at 8000r / min for 5 minutes, and the supernatant was discarded and suspended in sterile saline. After adding the dye and incubating in the dark for 15 minutes, 5μL was added to the slide, and the fluorescence was observed using a laser confocal microscope and photographed for analysis. The excitation / emission wavelengths of SYTO9 and PI dyes are 485 / 540nm and 535 / 610nm, respectively.

[0069] LIVE / DEAD BacLight TM The staining cell method is used to determine the plasma membrane permeability. It is a double nucleic acid staining method used to assess cell viability based on changes in cell membrane permeability. Low molecular weight (0-10Da) green fluorescent dye (480 / 500nm) can penetrate intact and damaged plasma membranes, while the high molecular weight (668Da) red fluorescent dye PI (490 / 635nm) can only penetrate damaged membranes, replacing green fluorescence with red fluorescence like Figure 5 As shown, the control group is E. coli cells that were not treated with lysine, and cells with intact cell membranes showed The green fluorescence of the dye indicates that the plasma membrane of the control group is intact, while after treatment with 2MIC and MIC concentrations of lysine, all E. coli cells show red fluorescence, and after treatment with 1 / 2MIC concentration of lysine, a small amount of red fluorescence and a large amount of green fluorescence are shown. Green fluorescence and red fluorescence respectively indicate that the cell membrane is intact or damaged. Green fluorescent dye can penetrate the intact bacterial membrane, while red fluorescent dye is hydrophilic and cannot penetrate the intact bacterial membrane. Therefore, as the concentration of lysine increases, the damage of lysine to the E. coli cell membrane increases significantly.

[0070] 3. Effect of lysine on leakage of E. coli cell contents

[0071] The destructive effect of lysine on the E. coli cell membrane is further demonstrated in the following ways.

[0072] 3.1 Effect of lysine on leakage of Escherichia coli alkaline phosphatase (AKP)

[0073] At 37°C, Escherichia coli was treated with different concentrations of lysine for 30 minutes, and a control group without lysine treatment was set up. The mixed solution was centrifuged (8000g, 5min, 4°C), and the supernatant was collected in a pre-cooled centrifuge tube. Alkaline phosphatase (AKP) enzyme activity was determined using an AKP detection kit (Biyuntian), and the absorbance of the sample at 450nm was determined using a UV spectrophotometer.

[0074] AKP is an enzyme that exists between the cell wall and the cell membrane, and its activity is usually undetectable outside the cell. It is usually determined by measuring the AKP activity in the bacterial suspension to confirm whether the cell wall is damaged by antimicrobial substances. Figure 6 As shown, with the increase of lysine concentration in the treated Escherichia coli, the activity of AKP increased, that is, with the increase of lysine concentration, the damage of the Escherichia coli cell wall became more serious.

[0075] 3.2 Effect of lysine on intracellular adenosine triphosphate (ATP) concentration in Escherichia coli

[0076] The bacterial suspension treated with different concentrations of lysine was centrifuged at 12000r / min for 3min, the supernatant was discarded, lysis buffer was added to break the bacteria, the supernatant was centrifuged and the ATP concentration was determined according to the instructions of the ATP detection kit.

[0077] In the ATP bioluminescence method, luciferin is catalyzed to produce fluorescence according to the reaction principle of firefly luciferase and its substrate ATP, and the measured ATP concentration is proportional to the fluorescence intensity. Figure 7 As shown in the figure, the fluorescence intensity of E. coli after 2MIC, MIC and 1 / 2MIC lysine treatment was significantly lower than that of the control group (p<0.01). The ATP level of intact cells is stable, and destroying the stability and integrity of the cells may change the intracellular ATP concentration. Therefore, the experimental results show that lysine reduces the intracellular ATP concentration, that is, lysine damages the cell membrane of E. coli cells.

[0078] 3.3. Reactive Oxygen Species (ROS) Content in Escherichia coli Cells

[0079] The ROS content of bacterial suspensions treated with different concentrations of lysine was determined using an ROS assay kit. The suspended bacteria were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) at a concentration of 5 mol / L and heated at 37°C for 15 minutes to evaluate the production of intracellular ROS. Cell fluorescence was quantified using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0080] like Figure 8 As shown, the ordinate represents the relative fluorescence intensity of ROS content. The ROS content of E. coli treated with lysine at 2MIC and MIC concentrations was significantly higher than that of the control group. The ROS content of 1 / 2MIC and 1 / 4MIC was also significantly different from that of the control group. The change in ROS level can be used as a direct indicator of the degree of damage to E. coli cells by lysine. Therefore, the test results show that lysine causes damage to the cell membrane of E. coli cells, causing the ROS originally controlled in the E. coli cells to leak out.

[0081] 3.4. Protein and nucleic acid release measurements

[0082] The E. coli suspension treated with different concentrations of lysine was centrifuged after 5 h, and the supernatant was collected. The intracellular substance leakage was measured at 260 nm / 280 nm using an ultraviolet spectrophotometer (UV-2600, Shimadzu, Tokyo, Japan). The nucleic acid / protein release content was expressed by the absorbance value at 260 / 280 nm.

[0083] Nucleic acids and proteins dominate the genetic information and cell structure of bacteria and play an important role in bacterial metabolism. Once affected, it will deprive bacteria of their ability to survive. Fig. 9 As shown in Figure 3, nucleic acid leakage of E. coli cells occurred after lysine treatment compared to the control group, while low concentrations of lysine resulted in partial leakage with no significant difference. Fig.10 As shown, after lysine treatment, E. coli cell protein leaked compared with the control group, while low concentrations of lysine caused partial leakage with no significant difference. That is, once the cell membrane is damaged, the integrity of the cell membrane will be unbalanced, leading to the leakage of these cell contents, that is, after a certain concentration of lysine treatment, the cell membrane of E. coli cells is damaged, resulting in the leakage of nucleic acids and proteins of E. coli cells.

[0084] 3.5 Effect of lysine on the biofilm formation ability of Escherichia coli

[0085] Use 96-well plates for crystal violet staining. Inoculate 10 6 CFU / mL of Escherichia coli cells. Different concentrations of Lys were added to the wells of a 96-well plate and cultured at 37°C for 48 hours, then the supernatant was discarded and the wells were rinsed three times with 200 μL PBS (PBS, pH 7.4) and dried at room temperature. The attached bacterial cells were fixed with 200 μL methanol for 15 minutes, and after air drying, 200 μL 1% crystal violet was added to the attached wall for staining for 10 minutes, and then rinsed with PBS. The dye adhered to the cells was dissolved in 150 μL 95% ethanol, and the OD value was measured at a wavelength of 590 nm using a multifunctional microplate reader.

[0086] After treating E. coli EDL933 with different concentrations of lysine for 24 hours, the biofilm formation was observed and quantified using crystal violet staining. Fig.11 As shown, the biofilm of the control strain was intact, while the biofilm formation of the lysine-treated strain was reduced. As the lysine concentration increased, the biofilm formation showed a decreasing trend. Therefore, lysine can significantly inhibit the formation of E. coli biofilm.

[0087] 3.6. Detection of E. coli Cell Genome Integrity

[0088] Bacterial genomic DNA was extracted and purified using a bacterial DNA kit (Tiangen Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. The integrity of the DNA samples was checked by 1% agarose gel electrophoresis. Nucleic acid dyes were added to the gel for UV visualization of DNA. Finally, the gel was photographed using a gel imaging system.

[0089] like Fig.12 As shown, the damage of lysine to bacterial genomic DNA was analyzed by agarose gel electrophoresis. Comparing the gel electrophoresis bands of the genomic DNA of the test group and the control group, it can be seen that the bacterial genomic DNA of the control group has a single, clear electrophoresis band without tailing. After adding lysine, the genomic DNA band of Escherichia coli at a dose of 2MIC almost completely disappeared, and the lysine content at the MIC and 1 / 2MIC doses made the DNA band darker than the control group. There was no significant difference in the DNA under the 1 / 4MIC and 1 / 8MIC doses of lysine compared with the control group. In other words, under the treatment of a certain concentration of lysine, the genomic DNA of Escherichia coli will be damaged.

[0090] 3.7 Analysis of whole-cell proteins by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

[0091] SDS-PAGE was used to analyze the effect of lysine on the primary structure of the protein of Escherichia coli O157:H7. Escherichia coli cells were treated with different concentrations of lysine for 5 hours, then washed and the supernatant discarded. After the Escherichia coli cells were resuspended in buffer, they were heated at 100°C for 15 minutes, and then SDS-PAGE was performed using 5% stacking gel and 12% separation gel. The cells were stained with Coomassie Brilliant Blue and photographed using a gel imager. The photographed images showed that lysine was destructive to the primary structure of bacterial proteins. Membrane proteins play an important role in maintaining the permeability of cell membranes. The destruction of membrane proteins may lead to the destruction of the integrity of the bacterial membrane enzyme system. The protein map of Escherichia coli cells after lysine treatment is shown in Figure 2. Fig.13As shown in the figure, different concentrations of lysine have different effects on the treatment of Escherichia coli. Compared with the control group, the bands after treatment with 2MIC and MIC lysine concentrations are light-colored, while there is no obvious difference in the band changes at 1 / 2MIC and 1 / 4MIC lysine concentrations compared with the control group. Therefore, in the presence of high concentrations of lysine, lysine has a significant destructive effect on the protein structure of cells.

[0092] 3.8. E. coli cell motility and diffusion test

[0093] The swimming ability of E. coli was measured after inoculating E. coli in the center of a plate containing 0.2% agar with different concentrations of lysine. For the clustering test, 2 μL of E. coli was inoculated in the center of an agar plate containing 0.5% agar and 5g / L glucose with different concentrations of lysine and cultured overnight. The movement diameter was measured after 24 hours of incubation. A plate without lysine was used as a control. The gel imager was used to take pictures and record the area of ​​bacterial swimming and the area of ​​the ring formed by the growth around the clustered bacteria.

[0094] Effects of lysine on the motility of Escherichia coli Fig.14 , Fig.15 , Fig.16 As shown in the figure, in the swimming test, the swimming area of ​​E. coli in the control group was significantly different from that in the test group, indicating that lysine can significantly reduce the swimming ability of E. coli. In the clustering test, the area value of the control group and the 1 / 64MIC concentration was not significantly different, and the difference in the clustering area of ​​the control group and 1 / 32MIC and 1 / 16MIC was extremely significant, indicating that a certain concentration of lysine can also inhibit the clustering ability of E. coli.

[0095] 3.9 Effects of lysine on Caco-2 cells

[0096] 3.9.1 Effect of lysine on Caco-2 cell activity

[0097] The effect of lysine on Caco-2 cell toxicity was detected by CCK-8. The cultured Caco-2 cell suspension was inoculated in a 96-well plate and incubated at 37°C with 5% CO 2 The cells were cultured for 24 hours under the conditions of . Subsequently, different concentrations of lysine-DMEM solution were added to the cell sample wells, and the wells with DMEM were used as the control group. The samples were then cultured in a 37°C cell culture incubator for 12 hours, the cell supernatant was discarded, and CCK-8 dye solution was added to each well and cultured in a 37°C cell culture incubator for 2 hours in the dark. Finally, the absorbance of each well of the sample was measured at 450nm.

[0098] Fig.17To investigate the toxic effects of lysine on Caco-2 cells, CCK-8 was used to detect cell viability after co-culturing Caco-2 cells with different concentrations of lysine. It was found that except for the lysine at 2MIC concentration, which had a greater effect on cell viability, the effects of the other concentrations on cell viability were not significantly different from those of the control group. Caco-2 cells are a commonly used intestinal epithelial cell model that can simulate the intestinal environment. By detecting the effects of disinfectant lysine on Caco-2 cells, the applicability of lysine in the intestinal environment can be evaluated. The test results show that a certain concentration of lysine kills Escherichia coli while causing almost no damage to Caco-2 cells, indicating that a certain concentration of lysine can be used safely.

[0099] 3.9.2 Effect of lysine on the ability of E. coli O157:H7 to adhere to and invade Caco-2 cells

[0100] The cultured Caco-2 cells were made into a single cell suspension using trypsin, and the concentration was adjusted with DMEM and inoculated into a 24-well plate and cultured for 24 hours. After washing three times with PBS, the cells were treated with sub-inhibitory concentration of lysine, centrifuged (600g, 5min), and incubated at 37°C, 5% CO 2 Incubate for 2 h at room temperature, discard the liquid, and wash three times with sterile PBS. In the adhesion test, add 0.1% Triton-100 to each well and place it in a 4°C refrigerator for 20 min to lyse the cells; Fig.18 As shown, in the adhesion test, lysine at concentrations of 1 / 16MIC and 1 / 32MIC reduced the adhesion ability of Escherichia coli, and the difference was significant (p<0.05).

[0101] In the invasion assay, the cells were washed once with PBS and then incubated at 37°C with 5% CO in DMEM containing 10% FBS and 100 μg / mL gentamicin. 2 Incubate for 30 minutes at room temperature, discard the liquid, wash three times with sterile PBS, add 0.1% Triton-100 to each well and place in a 4°C refrigerator for 20 minutes to lyse the cells. Dilute the adherent and invaded bacterial samples in PBS in a gradient manner and spread on LB agar medium, and count after incubation at 25°C for 24 hours. Fig.19 As shown, in the invasion test, lysine at concentrations of 1 / 16 MIC and 1 / 32 reduced the invasion ability of Escherichia coli, and the difference was extremely significant (p<0.01).

[0102] 4. Test on the inhibition of Escherichia coli O157:H7 by lysine and evaluation of its sensory properties

[0103] For example, lettuce was purchased from a supermarket in Yangling, China and shipped to the laboratory. The samples were washed with distilled water, sterilized with 75% ethanol to reduce background flora, and dried on a clean bench. The lettuce was then cut into 5 × 5 cm squares using a sterile knife and immersed in bacterial inoculum (10 6 CFU / mL) for 10 minutes and air-dried in a biosafety cabinet for 1 hour. The inoculated sample was divided into six portions, one of which was not washed and used as a benchmark for determining the final bacterial concentration. The remaining five portions were subjected to different washing treatments. The first portion used sterile deionized water, the second portion used 0.1 mg / mL sodium hypochlorite (NaOCl; pH 6.5), the third portion was lysine at a concentration of 2 MIC, the fourth portion was lysine at a concentration of MIC, and the fifth portion was lysine at a concentration of 1 / 2 MIC. The recommended concentration of NaOCl is 0.1 mg / mL. The inoculated samples were placed in a sealed bag and completely immersed in the different washing treatments for 10 minutes with minimal stirring. Subsequently, the lettuce was transferred to 30 mL of sterile saline (0.85% NaCl) and mixed evenly. The sample was then serially diluted 10 times (100 μL) on an LB plate. Incubate at 37°C for 24 hours for colony counting. The initial inoculum level of Escherichia coli was 8.04±0.84logCFU / g, and the detection limit of bacterial flora was 1.0logCFU / g.

[0104] The test results are as follows Fig. 20 As shown, after washing the lettuce with lysine, the number of E. coli colonies on the lettuce was significantly reduced. After washing the lettuce with lysine at concentrations of 2MIC and MIC, a concentration-dependent reduction in E. coli cells was observed. The NaOCl treatment had a similar effect to the 1 / 2MIC treatment. On the other hand, the reducing effect of deionized water on E. coli was negligible compared with the other groups. There was no significant difference in the washing effect between 2MIC and MIC. Therefore, lysine can be used as the main active ingredient of the disinfectant to effectively control the growth of E. coli on agricultural products. The lowercase letters a, b, c, d, and e in the figure indicate that there are statistically significant differences between the means.

[0105] For the quality evaluation of lettuce, a preliminary sensory evaluation method was used. The uninoculated lettuce was soaked in 500 mL of different treatment solutions for 30 minutes and air-dried in a biological safety cabinet for 1 hour. The treatments were deionized water, 0.1 mg / mL NaOCl, and lysine at concentrations of 2 MIC, MIC, and 1 / 2 MIC, respectively. The sensory evaluation panel consisted of five specially trained members. The appearance, smell, taste, texture, and overall acceptance were scored on a 5-point descriptive scale, where "5" represents the best acceptance and "1" represents the worst acceptance. The lower limit of acceptance is "3".

[0106] Sensory attributes such as appearance, smell, taste, texture and overall acceptability Fig.21As shown. The lettuces of the control group and the deionized water group scored the same, and the samples treated with MIC scored the highest among the other four groups because they maintained the original color and smell of the lettuce. NaOCl has a strong pungent taste, and lysine at 2MIC also has a distinct odor, which has the characteristic odor of lysine as a basic amino acid. In other words, lysine at MIC concentration can be used as a natural food disinfectant to provide microbial safety without causing any sensory defects to food.

[0107] 5. Application of lysine in the treatment of Escherichia coli-induced colitis in mice

[0108] Six-week-old male Kunming mice weighing 18–22 g were selected, 8 per cage, and fed with standard laboratory chow at a constant temperature of 23 ± 2 °C with a 12-h light / dark cycle. All mice were housed for at least 1 week before the experiment to acclimate to the environment. The experiment was approved by the Animal Ethics Committee of Northwest Agriculture and Forestry University. The experimental schedule is as follows: Fig. 22 Streptomycin sulfate (5 g / L) was added to the drinking water 3 days before inoculation to reduce the normal flora and promote the colonization of E. coli O157:H7. The normal group was fed regularly throughout the experiment (20 days) and had free access to water. The prevention control group was fed a normal ration from the 4th to the 10th day of the experiment, and 1.0×10 9 The prevention group was gavaged with 20 mg / mL Lys daily from the 4th to the 10th day of the experiment, and then gavaged with E. coli O157:H7 as the prevention control. The treatment control group was gavaged with 1.0×10 9 CFU E. coli O157:H7 and were fed with normal diet and water for the next 7 days. The treatment group consisted of O157:H7-infected mice and were gavaged with 20 mg / mL Lys daily for 7 days.

[0109] Mice infected with E. coli O157:H7 showed clinical symptoms such as weight loss, diarrhea, occult blood in stool, shortened colon length, and increased myeloperoxidase (MPO) activity. Fig.23 and Fig.24 The weight of mice in the normal group was getting heavier. The weight of mice in the prevention group and the treatment group was higher than that of the prevention control group and the treatment control group, and the weight of the prevention group was higher than that of the treatment group, indicating that the weight loss effect of lysine was better than that of the treatment group in the prevention of colitis. The fecal occult blood level of mice was measured using a fecal occult blood kit. Fig.25 The darker the color, the higher the hemoglobin content in the stool. These clinical symptoms and physiological and biochemical indicators are typical clinical manifestations of colitis. Fig.26As shown in Figure 3, the MPO levels in the prevention group and the treatment group were lower than those in the prevention control group and the treatment control group, indicating that Lys can improve the degree of neutrophil MPO-related infiltration. Fig. 27 As shown, the difference in colon length between the prevention group and the normal group was not significant, but the difference between all other groups was significant. Lys reduced the shortening of colon length in mice with E. coli-induced colitis. Proinflammatory cytokines are a series of cytokines that promote inflammation. Typical proinflammatory factors are TNF-α, IL-1β, and IL-6. The levels of TNF-α, IL-1β, and IL-6 in colon tissue were measured, such as Fig.28 , Fig.29 , Fig.30 The H&E-stained colon sections were examined under a microscope. Fig.31 As shown, except for the normal group, all experimental groups showed degeneration and necrosis of mucosal epithelial cells, loss of goblet cells, crypt swelling, and different degrees of inflammatory cell infiltration in the mucosa and submucosa. HE staining of colon tissue showed that the Lys preventive treatment group was more effective than the treatment group. Different lowercase letters in the above experimental figures indicate statistically significant differences between the mean values.

[0110] Therefore, the use of Lys as a nutritional supplement can reduce the severity of colitis induced by E. coli O157:H7 in mice. The symptoms of colitis in the prevention group were alleviated to a greater extent, the upregulation of proinflammatory factors was significantly inhibited, and the intestinal barrier function was protected. Multiple indicators such as colon length, MPO and proinflammatory cytokines in the prevention group returned to normal levels. E. coli O157:H7 can cause severe colitis symptoms, manifested as shortened colon length, increased MPO activity and occult blood in stool. Infection caused by E. coli O157:H7 also affected the expression of immune factors and colon-related protein genes in mice. Nutrients mainly fight diseases by enhancing the body's immunity. The preventive effect of Lys on mice infected with E. coli O157:H7 is stronger than the therapeutic effect. When the body is supplemented with lysine in advance, the body's immunity is enhanced, the colonization of E. coli O157:H7 is reduced, and the enhancement of immunity leads to the inhibition of E. coli O157:H7 colonization. Fundamentally prevented Escherichia coli O157:H7-induced colon lesions in mice.

[0111] All experiments were performed in triplicate and presented as mean ± standard deviation (SD) using OriginPro2021 (Origin Lab, USA). Data were analyzed using SPSS23.0 (SPSS, International Business Machines Corporation, Armonk, New York, USA) for one-way analysis of variance (ANOVA) and Duncan's multiple comparison test. When the p value was < 0.05, the difference was considered statistically significant.

[0112] In general, from the results of field emission scanning electron microscopy, the cell membrane is the main target of lysine. The antibacterial effect of lysine is attributed to the disintegration of the cell membrane, the leakage of cell components, and ultimately the cell lysis. During the research, the applicant found that the level of reactive oxygen in bacterial cells increased, indicating that lysine can also cause lipid peroxidation in bacterial cells, promote cell apoptosis and autophagy. Sub-inhibitory concentrations of lysine can reduce the swimming, clustering, adhesion and invasion of Escherichia coli O157: H7 to Caco-2 cells. The motility of Escherichia coli is controlled by its flagella, which may be due to the fact that sub-inhibitory concentrations of lysine inhibit the flagella of Escherichia coli, resulting in a decrease in the motility of Escherichia coli. Escherichia coli flagella-dependent motility plays a variety of roles in pathogenesis, including its migration to the optimal host site, adhesion and colonization, survival at the infection site, and spread after infection. Flagellar master genes also co-regulate other virulence factors in pathogenic Escherichia coli. Taking lettuce as an example of food to be disinfected, lysine reduces the number of Escherichia coli O157: H7 when washing lettuce. The lysine score at the MIC concentration was also higher than that of other treatment groups during the sensory evaluation, so the role of lysine in disinfecting lettuce is obvious, that is, lysine pretreatment has no effect on the sensory score of lettuce. In addition, lysine also has a certain preventive and mitigating effect on the infection of E. coli O157:H7.

[0113] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of lysine, characterized in that: Application of lysine in disinfectants for inhibiting Escherichia coli O157:H7.

2. The use of lysine according to claim 1, characterized in that: The minimum inhibitory concentration (MIC) of lysine against Escherichia coli O157:H7 is 12.5 mg / mL to 25 mg / mL, and the minimum bactericidal concentration (MBC) is 12.5 mg / mL to 25 mg / mL.

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