Application of nicotinic acid

Abstract

The invention provides application of nicotinic acid. The nicotinic acid is applied to a disinfectant for inhibiting staphylococcus aureus. Nicotinic acid is a natural nutrient and is used for inhibiting and killing staphylococcus aureus on food, the food is free of toxic residues, abuse of other chemical disinfectants is reduced, and in addition, nutrients are provided for the human body while human health is not affected.

Description

Technical Field

[0001] The invention relates to the technical field of sterilization, and in particular to a use of nicotinic acid. Background Art

[0002] Staphylococcus aureus is a common foodborne pathogen that is widely found in foods such as meat, milk and related products. Staphylococcus aureus can produce exotoxins and endotoxins. Exotoxins mainly include hemolysins that can cause hemolysis, while endotoxins are mainly staphylococcal enterotoxins, which can cause symptoms such as nausea, vomiting, and abdominal cramps. They can cause host diseases such as gastroenteritis and purulent skin infections. In severe cases, life-threatening complications such as bacteremia and endocarditis may occur. Although the use of antibiotics can effectively prevent and inhibit diseases caused by Staphylococcus aureus, the overuse of antibiotics leads to excessive antibiotic residues in food and the environment, thereby enhancing the resistance of pathogenic bacteria to antibiotics, resulting in the production of "super-resistant" oxacillin-resistant Staphylococcus aureus strains, threatening human health. Currently, alternatives to antibiotics include enzyme preparations, probiotics, and plant extracts, but they all have certain defects. For example, enzymes have complex biochemical structures and are easily inactivated or degraded quickly. Maintaining the stability of enzymes is considered the biggest challenge in practical applications. It is not clear what the ideal dosage of enzymes should be, and what potential side effects may occur when using them. The mechanism of action of probiotics is not fully understood, and safety is also a problem for probiotics. Long-term use of a single plant substance or its extracts may also cause some microorganisms to produce resistance genes.

[0003] Therefore, it is urgent to develop safe, green and efficient antibacterial agents.

[0004] Vitamins are a type of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions. They play an important role in human growth, metabolism, and development. Insufficient vitamin intake will cause the body's metabolism to lose balance, lead to decreased immunity, and may lead to malnutrition, making it susceptible to various diseases. Niacin is an organic compound, a white crystalline powder, mainly found in animal viscera and muscle tissue, and also in trace amounts in fruits and egg yolks. It is one of the 13 essential vitamins for the human body. Niacin is also mainly used as a feed additive to improve the utilization rate of feed protein, increase the milk production of dairy cows, and increase the yield and quality of fish, chicken, duck, cattle, sheep and other livestock meat.

[0005] So far, no studies have reported the inhibitory effect of niacin on Staphylococcus aureus. Summary of the invention

[0006] The main purpose of the present invention is to provide a use of nicotinic acid to at least solve the problem that the methods used in the prior art to prevent and inhibit diseases caused by Staphylococcus aureus threaten human health.

[0007] In order to achieve the above object, the present invention provides a use of nicotinic acid, including the use of nicotinic acid in an antibacterial agent for inhibiting Staphylococcus aureus.

[0008] Furthermore, the minimum inhibitory concentration (MIC) of niacin against Staphylococcus aureus was 4 mg / mL, and the minimum bactericidal concentration (MBC) was 4 mg / mL.

[0009] The technical solution of the present invention provides a use of nicotinic acid, which is used in a disinfectant for inhibiting Staphylococcus aureus. Nicotinic acid is a natural nutrient. Nicotinic acid is used to inhibit and kill Staphylococcus aureus on food, leaving no toxic residue in the food, reducing the abuse of other chemical disinfectants, and providing nutrients to the human body without affecting human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] 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:

[0011] Figure 1 is an influence diagram of a nicotinic acid Staphylococcus aureus growth curve that can be selected according to an embodiment of the present invention;

[0012] Figure 2 is a graph showing the effect of niacin on AKP leakage of Staphylococcus aureus cells, which can be selected according to an embodiment of the present invention;

[0013] Figure 3 This is a graph showing the effect of nicotinic acid on the cell membrane potential of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0014] Figure 4 is a graph of membrane damage of Staphylococcus aureus caused by nicotinic acid, which can be selected according to an embodiment of the present invention;

[0015] Figure 5 This is a graph showing the effect of nicotinic acid on the cell membrane integrity of Staphylococcus aureus under a laser confocal microscope (100 times magnification) which can be selected according to an embodiment of the present invention;

[0016] Figure 6 This is a graph showing the effect of nicotinic acid on the cell morphology of Staphylococcus aureus under a scanning electron microscope, which can be selected according to an embodiment of the present invention;

[0017] Figure 7is a graph showing the effect of nicotinic acid on ATP concentration of Staphylococcus aureus cells, which can be selected according to an embodiment of the present invention;

[0018] Figure 8 This is a graph showing the effect of nicotinic acid on the intracellular ROS content of Staphylococcus aureus cells, which can be selected according to an embodiment of the present invention;

[0019] Fig. 9 is a graph showing the effect of nicotinic acid on nucleic acid and protein leakage in Staphylococcus aureus cells, which can be selected according to an embodiment of the present invention;

[0020] Fig.10 is a graph showing the effect of nicotinic acid on the integrity of the genomic DNA of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0021] Fig.11 is a graph showing the effect of nicotinic acid on whole cell protein of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0022] Fig.12 is a graph showing the effect of nicotinic acid on the biofilm formation ability of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0023] Fig.13 is a first Staphylococcus aureus hemolytic activity graph that can be selected according to an embodiment of the present invention;

[0024] Fig.14 is a second Staphylococcus aureus hemolytic activity graph which can be selected according to an embodiment of the present invention;

[0025] Fig.15 is a graph showing the effect of the first nicotinic acid on the hemolytic activity of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0026] Fig.16 is a graph showing the effect of a second nicotinic acid on the hemolytic activity of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0027] Fig.17 This is a graph showing the effect of niacin on Caco-2 cell activity, which can be selected according to an embodiment of the present invention;

[0028] Fig.18 is the toxicity of a Staphylococcus aureus supernatant to Caco-2 cells that can be selected according to an embodiment of the present invention;

[0029] Fig.19 is a graph showing the effect of nicotinic acid on the toxicity of Staphylococcus aureus supernatant on Caco-2 cells, which can be selected according to an embodiment of the present invention;

[0030] Fig. 20 is an optional effect of nicotinic acid on the expression of Hla and SEA proteins in the supernatant of Staphylococcus aureus according to an embodiment of the present invention;

[0031] Fig.21 This is a graph showing the influence of nicotinic acid on the grayscale value of Hla protein expression in the supernatant of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0032] Fig. 22 is an optional grayscale value effect diagram of nicotinic acid on SEA protein expression in the supernatant of Staphylococcus aureus according to an embodiment of the present invention;

[0033] Fig.23 is a diagram of wound states of each back of a mouse that can be selected according to an embodiment of the present invention;

[0034] Fig.24 This is a staining diagram of a mouse subcutaneous tissue section that can be selected according to an embodiment of the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] The applicant has found that nicotinic acid, also known as VB3, has an inhibitory effect on Staphylococcus aureus and can be used as the main active ingredient of a disinfectant to inhibit Staphylococcus aureus in food and the environment. Nicotinic acid is a natural nutrient and is non-toxic. By utilizing the inhibitory effect of nicotinic acid on Staphylococcus aureus and applying nicotinic acid in disinfectants that inhibit Staphylococcus aureus, it is safer, greener and more efficient. Nicotinic acid is a natural nutrient. Using nicotinic acid to inhibit and kill Staphylococcus aureus on food leaves no toxic residue in the food, reduces the abuse of antibiotics, and provides nutrients to the human body without affecting human health.

[0037] This is illustrated by the following examples:

[0038] 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.

[0039] In the experiments of the present application, niacin was purchased from Solebow Biotech Co., Ltd., and foodborne pathogens such as Staphylococcus aureus (ATCC29213), Escherichia coli O157:H7 (EDL933), Salmonella (ATCC1311 and CMCC50335), Bacillus cereus (ATCC14579) and Vibrio parahaemolyticus (ATCC17802) were preserved in the laboratory of Food Biomanufacturing and Microbiological Safety Team of the College of Food Science and Engineering, Northwest Agriculture and Forestry University. Within the range of minimum inhibitory concentration and minimum bactericidal concentration, the solvent used to reflect the inhibitory effect of niacin on the above-mentioned foodborne pathogens is distilled water.

[0040] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of VB3 against Staphylococcus aureus were determined by broth microdilution method.

[0041] 10 6 CFU / mL of Staphylococcus aureus suspension was co-cultured with different concentrations of nicotinic acid on a 96-well microplate. After incubation at 37°C for 24 hours, the lowest concentration at which no bacterial growth was observed in the test group was the MIC. The concentration at which no bacterial growth was observed in 100 μL of the suspension on LB agar after incubation at 37°C for 48 hours was the MBC. Using the same method, the MIC and MBC of VB3 against Staphylococcus aureus (ATCC29213), Escherichia coli O157:H7 (EDL933), Salmonella (ATCC1311 and CMCC50335), Bacillus cereus (ATCC14579) and Vibrio parahaemolyticus (ATCC17802) were determined. The results are shown in Table 1.

[0042] Table 1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of VB3 against foodborne pathogens

[0043]

[0044] As shown in Table 1, the MIC and MBC of niacin against Staphylococcus aureus ATCC29213 are both 4 mg / mL, and it has antibacterial activity against other foodborne pathogens including Escherichia coli, Salmonella, Bacillus cereus and Vibrio parahaemolyticus.

[0045] The inhibitory effect of nicotinic acid on Staphylococcus aureus is further illustrated by the following test, in which * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. The strain selected for the following test is Staphylococcus aureus ATCC29213.

[0046] 1. Growth curve

[0047] The final concentration was 10 6 CFU / mL of Staphylococcus aureus was treated with different concentrations of nicotinic acid on honeycomb plates. OD was measured every 1 h using a microbial growth curve analyzer. 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 is plotted on the vertical axis.

[0048] like Figure 1As shown in the growth curve of Staphylococcus aureus, niacin completely inhibited the growth of strain ATCC29213 at 2MIC and MIC concentrations. Nicotinic acid at concentrations of 1 / 2MIC, 1 / 4MIC and 1 / 8MIC delayed the growth and proliferation of bacteria.

[0049] 2. Effect of niacin on the cell wall of Staphylococcus aureus

[0050] At 37°C, Staphylococcus aureus was treated with nicotinic acid at concentrations of 2MIC, 1MIC, 1 / 2MIC, and 1 / 4MIC for 2 hours. The mixture was centrifuged (12000g, 5min, 4°C), and the supernatant was collected in a pre-cooled centrifuge tube. Alkaline phosphatase (AKP) activity was determined using an AKP detection kit (FANKEW), and the absorbance of the sample at 450nm was determined using a UV spectrophotometer. Figure 2 As shown in the figure, the concentration of AKP increases with the increase of nicotinic acid concentration. The AKP content after the reaction of nicotinic acid with 2MIC, 1MIC and 1 / 2MIC concentrations with Staphylococcus aureus is significantly different from that of the control group (p < 0.01). The AKP content after the reaction of nicotinic acid with 1 / 4MIC concentration with Staphylococcus aureus is not significantly different from that of the control group (p > 0.05). AKP, as an enzyme, is distributed between the cell wall and the cell membrane, and its activity is usually undetectable outside the cell. The AKP activity in the bacterial suspension is measured to confirm whether the cell wall is destroyed by antibacterial substances. As the concentration of nicotinic acid increases, the concentration of AKP also gradually increases, indicating that nicotinic acid can destroy the cell wall structure of Staphylococcus aureus.

[0051] 3. Effect of niacin on the cell membrane of Staphylococcus aureus

[0052] 3.1 Effect of niacin on membrane potential of Staphylococcus aureus

[0053] Add the prepared bacterial solution to the black ELISA plate and add 3 μL of fluorescent dye DiBAC4 to each well, place in a 37°C incubator and culture for 30 minutes, then add nicotinic acid solution to the 96-well plate to make the concentrations 2MIC, 1MIC, 1 / 2MIC and 1 / 4MIC respectively. After the fluorescent dye and Staphylococcus aureus have fully reacted, use a multifunctional ELISA instrument to detect the fluorescence intensity, the excitation / emission wavelength is 492nm / 515nm, the excitation slit width is 3nm, and the emission slit width is 5nm. The membrane potential of Staphylococcus aureus cells changed significantly after nicotinic acid treatment. Figure 3As shown, compared with the control group, the intracellular fluorescence intensity of cells treated with 2MIC, 1MIC and 1 / 2MIC niacin increased significantly (p<0.01), indicating that the strain was depolarized. The depolarizing effect of niacin on the strain was more obvious at 2MIC, and there was no significant difference from the control group at a concentration of 1 / 4MIC. The membrane potential change was measured using the voltage-sensitive specific fluorescent dye DiBAC4. DiBAC4 is an anionic fluorescent dye that can bind to bacterial cytoplasmic proteins. After 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. In this study, niacin can also affect the membrane potential of Staphylococcus aureus. As the concentration of niacin increases, the membrane potential fluorescence value of Staphylococcus aureus gradually decreases, indicating cell depolarization.

[0054] 3.2 Effect of nicotinic acid on N-phenyl-l-naphthylamine (NPN) uptake by Staphylococcus aureus

[0055] After treating Staphylococcus aureus with different concentrations of nicotinic acid for 30 minutes, the mixture was centrifuged (8000g, 5min, 4°C), washed with sterile saline and resuspended. 200μL of each sample was added to a 96-well plate, and 1.5μL of NPN (100mM) solution was added to each well and mixed by pipetting. The fluorescence value of each group of samples was immediately measured using a fluorescence spectrophotometer with an excitation wavelength of 350nm and an emission wavelength of 420nm.

[0056] 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 4 As shown, compared with the control group, niacin can significantly increase bacterial NPN uptake (p < 0.01), and can cause damage to the cell membrane of Staphylococcus aureus in a concentration-dependent manner. p < 0.01 indicates that the experimental results are highly significant in statistics.

[0057] 3.3. Cell membrane integrity detection

[0058] use BacLightVigor kit L-7007 was used to observe the plasma membrane permeability of niacin on Staphylococcus aureus. The bacterial suspension treated with different concentrations of niacin was centrifuged at 12000r / min for 5min, and the supernatant was discarded and suspended in sterile saline.

[0059] Add 3 μL of bacterial suspension 9 / PI dye mixture, incubate in the dark for 15 min, drop 5 μL on a glass slide, and observe using a laser confocal microscope. The excitation / emission wavelengths of 9 and PI dyes are 485 / 540 nm and 535 / 610 nm, respectively.

[0060] like Figure 5 As shown, cells with intact membranes showed The green fluorescence of the 9 dye indicated that the plasma membrane of the control group was intact, while after treatment with 2MIC and MIC concentrations of niacin, all showed red fluorescence, indicating that the cell membrane was damaged. 9) and red fluorescence (PI) show that the cell membrane is intact or damaged, respectively. Green fluorescent stain 9 can penetrate the complete bacterial membrane, while the red fluorescent dye PI is hydrophilic and cannot penetrate the complete bacterial membrane. The 9 dye can penetrate the intact plasma membrane, while the high molecular weight (668Da) red fluorescent dye PI (490 / 635nm) can only penetrate the damaged membrane. Therefore, it can be further explained that nicotinic acid is destructive to the cell membrane of Staphylococcus aureus.

[0061] 3.4 Scanning electron microscopy observation of Staphylococcus aureus cell morphology

[0062] It was observed using a scanning electron microscope (SEM). Exponential phase Staphylococcus aureus cells were treated with nicotinic acid at concentrations of 2MIC, 1MIC and 0 at 37°C for 4h. The treated samples were centrifuged at 12000g for 5min at 4°C, washed with 0.01M PBS (pH7.4), and fixed with 2.5% glutaraldehyde overnight. Different concentrations of ethanol (30%, 50%, 70%, 80%, 90% and 100%) were used for dehydration. The cells were then freeze-dried and coated with a layer of gold. The microstructure of the bacteria was observed using a Hitachi cold field emission scanning electron microscope (Regulus 8100, Tokyo, Japan).

[0063] like Figure 6 As shown in the figure, the cell morphology of Staphylococcus aureus changed after treatment with niacin. Comparing the cells in the control group and the experimental group, it was observed that the cells without niacin treatment showed a typical Gram-positive bacterial structure, which was spherical, complete in morphology, and smooth on the surface. Slight wrinkles appeared on the surface of the cell membrane after treatment with low concentrations of niacin. As the concentration of niacin increased, the degree of cell damage increased and the wrinkles became more severe. Therefore, a certain concentration of niacin will destroy the cell morphology of Staphylococcus aureus.

[0064] 4. Effects of niacin on intracellular substances of Staphylococcus aureus

[0065] The destructive effect of niacin on the cell membrane of Staphylococcus aureus is further demonstrated in the following ways.

[0066] 4.1 Effect of niacin on intracellular adenosine triphosphate (ATP) concentration in Staphylococcus aureus

[0067] The Staphylococcus aureus suspension treated with different concentrations of nicotinic acid was centrifuged at 12000r / min for 5min. The supernatant was discarded and washed three times with PBS. Then, cell lysis buffer was added to break the bacteria. The supernatant was taken by centrifugation and the intracellular ATP concentration was determined according to the ATP detection kit.

[0068] like Figure 7 As shown in the figure, the intracellular ATP content of Staphylococcus aureus treated with nicotinic acid at concentrations of 2MIC, 1MIC, 1 / 2MIC and 1 / 4MIC was significantly reduced compared with that of untreated cells, and the difference was extremely significant (p<0.01). The ATP level of intact cells is stable, and destroying the stability and integrity of cells may change the intracellular ATP concentration. The experimental results show that nicotinic acid reduces the intracellular ATP concentration, and the leakage or rapid depletion of intracellular ATP is due to changes in cell membrane permeability, membrane damage or accelerated ATP hydrolysis. As the concentration of nicotinic acid increases, the intracellular ATP content of Staphylococcus aureus bacteria decreases, further indicating that nicotinic acid destroys the integrity of the cell membrane. Scanning electron microscopy results also show that nicotinic acid can destroy the cell membrane of Staphylococcus aureus, leading to the leakage of contents.

[0069] 4.2. Reactive Oxygen Species (ROS) Content in Staphylococcus aureus Cells

[0070] Fluorescent molecule dichlorodihydrofluorescein was obtained from China Institute of Biotechnology. The intracellular ROS level of Staphylococcus aureus in nicotinic acid treated samples was determined using fluorescent molecule dichlorodihydrofluorescein (DCFH-DA). Each treated bacterial suspension was incubated with DCFH-DA (5 μmol / L) at 37 °C for 10 min. Samples were centrifuged (12000 g, 10 min) and a multimode microplate reader platform was used with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0071] like Figure 8 As shown in the figure, the ROS content of Staphylococcus aureus treated with nicotinic acid at a concentration of 2MIC was significantly higher than that of the control group. 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.

[0072] 5. Effect of niacin on the release of intracellular components of Staphylococcus aureus

[0073] The Staphylococcus aureus suspension treated with different concentrations of nicotinic acid for 6 hours was centrifuged at 12000 r / min for 5 minutes, and the supernatant was collected. The intracellular substance leakage was measured at 260nm / 280nm using an ultraviolet spectrophotometer (UV-2600, Shimadzu, Tokyo, Japan). The nucleic acid / protein release content was expressed by the absorbance value at 260 / 280nm.

[0074] like Fig. 9 As shown, nucleic acids and proteins leaked from the treated S. aureus cells. These results demonstrate that once the cell membrane is damaged, an imbalance in cell membrane integrity occurs, leading to leakage of these cell contents.

[0075] 6. Effect of niacin on the integrity of Staphylococcus aureus genomic DNA

[0076] The genomic DNA of Staphylococcus aureus was extracted according to the instructions of the DNA extraction kit. The genomic DNA (50 μg / mL) was incubated with different concentrations of nicotinic acid solution (0, 1 / 2MIC, 1MIC, 2MIC) at a volume ratio of 1:1 at 37°C for 30 minutes, and then the DNA binding was detected by electrophoresis on 1% agarose gel in TAE buffer. The DNA bands were observed using a gel imaging system.

[0077] Nicotinic acid can interact with the extracted genomic DNA. Fig.10 As shown, as the concentration of niacin increases, the DNA band gradually fades. Compared with the control group, cells treated with 2MIC and MIC showed significant effects. As the amount of niacin increases, the migration of DNA through the gel decreases. Niacin exhibits DNA binding activity in a dose-dependent manner. When treated with 1 / 2MIC of niacin, most of the genomic DNA of Staphylococcus aureus cells can migrate into the gel. When treated with 1MIC of niacin, only a small part of the S. aureus DNA can migrate into the gel. Therefore, niacin can bind to the genomic DNA of S. aureus.

[0078] 7. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole-cell proteins

[0079] After incubating different concentrations of nicotinic acid and Staphylococcus aureus suspension at 37°C for 6 hours, the treated bacterial cells were washed with 0.01M PBS (pH7.4), centrifuged at 12000g for 5 minutes, and then resuspended with 0.01M PBS (pH7.4) and mixed with SDS-PAGE loading buffer (supernatant: buffer = 4:1). The sample was heated at 99°C for 10 minutes and analyzed by SDS-PAGE using 5% concentrating gel and 15% separating gel. The protein bands in the gel were stained with Coomassie Brilliant Blue R-250 and observed using a gel imager.

[0080] Proteins are also the most important biomacromolecules that support the structure and vitality of living cells, therefore, the effects of niacin and the whole protein of S. aureus were studied by SDS-PAGE, such as Fig.11 As shown, the control group had bright protein bands. After treating Staphylococcus aureus with nicotinic acid, 2MIC, MIC and 1 / 2MIC reduced the abundance of intracellular proteins in Staphylococcus aureus. Compared with the control group, there was no significant change in the protein bands after treatment with 1 / 4MIC nicotinic acid. However, with the increase in nicotinic acid concentration, 2MIC inhibited protein synthesis or degraded proteins. Therefore, nicotinic acid can cause the degradation of large molecular proteins into small molecular proteins or inhibit the synthesis of large molecular proteins.

[0081] 8. Effect of niacin on biofilm formation of Staphylococcus aureus

[0082] Crystal violet staining was performed using a 96-well plate. 6 CFU / mL was inoculated in LB liquid medium, and different concentrations of nicotinic acid were added to the wells of the 96-well plate. After culturing at 37°C for 48 hours, the supernatant was discarded, and the wells were rinsed 3 times with 200μL PBS (PBS, pH 7.4) and dried at room temperature. The attached bacterial cells were fixed with 200μL methanol for 15 minutes, and after drying, 200μL 1% crystal violet was added to the wells for staining for 10 minutes and then washed twice with PBS. The dye adhered to the cells was dissolved in 150μL 95% ethanol, and the OD value was measured at 590nm using a multifunctional microplate reader.

[0083] After 24 h of treatment with different concentrations of nicotinic acid, the biofilm formation of Staphylococcus aureus was observed and quantified by crystal violet staining. Fig.12As shown, the biofilm of the bacterial strain in the control group was intact, while the biofilm formation of the strain after niacin treatment was reduced. As the concentration of niacin increased, the formation of biofilm showed a downward trend. Nicotinic acid can significantly inhibit the formation of Staphylococcus aureus biofilm in a dose-dependent manner. Biofilm provides a relatively stable living environment for the survival, proliferation and recolonization of bacteria, and therefore can be used as an important target for drugs. Biofilm is also one of the key virulence factors of Staphylococcus aureus. Therefore, niacin can significantly inhibit the formation of biofilm.

[0084] 9. Effect of niacin on the toxicity of Staphylococcus aureus fermentation supernatant

[0085] 9.1 Hemolytic activity

[0086] Staphylococcus aureus ATCC29213 cells (106 CFU / mL) were cultured in brain heart infusion broth (BHI) liquid medium at 37°C for 48 hours, and the supernatant was collected and filtered with a 0.22μM filter membrane. Staphylococcus aureus supernatant was added to a 96-well plate and an equal volume of 6% rabbit red blood cells was added to calculate the minimum hemolytic concentration of the supernatant. Then, different concentrations of nicotinic acid were mixed with the minimum hemolytic concentration of the supernatant and an equal volume of 6% rabbit red blood cells were added to mix and the hemolytic activity was determined. Finally, the optical density of the supernatant was measured at 405nm. Positive (0.1% TritonX-100), PBS was negative). The hemolysis percentage was determined based on the ratio of the sample to the positive control.

[0087] Hemolytic ability is an important pathogenic factor of Staphylococcus aureus. Therefore, in order to evaluate whether niacin has an effect on the hemolytic activity of Staphylococcus aureus, 6% fresh rabbit red blood cells were used for hemolytic analysis. First, we used the collected supernatant for hemolytic analysis, such as Fig.13 and Fig.14 As shown in the figure, after the supernatant was diluted in multiples, it was found that the minimum hemolytic concentration of the supernatant was 1 / 64. All supernatant concentrations above 1 / 64 could produce a strong hemolytic effect. We also used this concentration for subsequent experiments. Different concentrations of nicotinic acid were mixed with the supernatant and incubated for 2 hours. Then 6% fresh rabbit red blood cells were added. The results after incubation for 4 hours are shown in the figure. Fig.15 and Fig.16 As shown, 2MIC nicotinic acid can reduce the hemolytic ability of the supernatant to 19.51%, and 1MIC concentration of nicotinic acid reduces the hemolytic ability to 31.83%. Hla is an important virulence factor of Staphylococcus aureus. High concentrations of nicotinic acid reduce the hemolytic effect of the bacterial supernatant, which indicates that nicotinic acid inhibits the activity of Hla in the bacterial supernatant.

[0088] 9.2 Cell Viability

[0089] In a 96-well plate, Caco-2 cells were injected. When the cells grew to about 70%, the culture medium was removed and washed twice with PBS in the wells. Culture medium containing 10 μg / mL BHI and different concentrations of nicotinic acid was added and incubated for 6 hours, and PBS was washed twice and discarded. 10% CCK-8 (AbMole) solution was added to each well and incubated in an incubator for 0.5 to 2 hours. The absorbance value obtained by measuring the absorbance at 450 nm was used to evaluate cell viability.

[0090] Caco-2 cells are a human clonal colon adenocarcinoma cell line that is structurally and functionally similar to differentiated intestinal epithelial cells and can be used to evaluate the cytotoxicity of toxic proteins. Fig.17 , Fig.18 ,and Fig.19 As shown in the figure, nicotinic acid at a concentration of 2MIC can reduce the activity of cells. After the supernatant was incubated with cells, a supernatant at a concentration of 1 / 64 was selected for cell testing. When nicotinic acid was incubated with the supernatant and then incubated with Caco-2 cells, the toxicity of the supernatant incubated with nicotinic acid at the MIC concentration disappeared, and the activity was the same as that of the control group Caco-2 cells, and the difference was not significant. The toxicity of nicotinic acid at a concentration of 1 / 2MIC and 1 / 4MIC was also reduced after incubation with the supernatant. This shows that nicotinic acid at a certain concentration can also reduce the toxic effect of the supernatant on Caco-2 cells, and it is concentration-dependent.

[0091] 9.3. Western blot analysis of the effect of nicotinic acid on toxins in the supernatant of Staphylococcus aureus

[0092] Human Leucocyte Antigen (HLA) and Staphylococcal Enterotoxin A (SEA) were determined by Western blot. After Staphylococcus aureus was cultured in BHI liquid medium for 48 hours, the supernatant was collected and filtered with a 0.22 μM filter membrane to obtain the supernatant of Staphylococcus aureus. The supernatant of Staphylococcus aureus was incubated with different concentrations of nicotinic acid for 2 hours, mixed with the loading buffer, and the sample was heated at 99°C for 10 minutes, separated by electrophoresis using 5% concentrating gel and 15% separating gel, and transferred to a 0.22 μM PPVDF membrane. The PVDF membrane was then incubated with 5% skim milk at room temperature for 2 hours, the primary antibody (Hla) was incubated at 4°C overnight, and the secondary antibody was incubated at room temperature for 2 hours. The chemiluminescence enhancement solution was evenly dropped on the PVDF membrane, and then exposed and imaged using a chemiluminescence imager (Tanon, Shanghai, China).

[0093] Western blot test results Fig. 20 , Fig.21 and Fig. 22 As shown, niacin destroys the structure of Hla and SEA proteins in the supernatant, and some natural substances inhibit the activity of toxin proteins. High concentrations of niacin can kill Staphylococcus aureus and inhibit the toxin proteins it produces.

[0094] 10. Back skin infection of suckling mice

[0095] Lactating Kunming mice (6-8 days old) were selected, regardless of gender, and randomly divided into 7 groups, 5 mice in each group. After the mice were anesthetized with CO2, 50 μL of a mixture of different doses of niacin and supernatant after incubation, a negative control (PBS), a drug control, and a supernatant control were injected subcutaneously on the back. At the 10-h time point, the status of the lactating mice was photographed and recorded, and then the mice were euthanized. Staphylococcus aureus infection mainly affects the skin and soft tissues, which is illustrated by establishing a suckling mouse back skin infection model. Fig.23 The back wounds of newborn mice were recorded at 0 and 10 hours after subcutaneous injection of a mixture of Staphylococcus aureus supernatant and nicotinamide (VB3). Ten hours after the injection of different treatments on the back skin of suckling mice, the backs of suckling mice in the BHI group, BHI+1 / 2MIC group and BHI+1 / 4MIC group showed a lot of redness and swelling, and some died. The suckling mice in the BHI group and BHI+1 / 4MIC group all died; the mortality rate of suckling mice in the BHI+1 / 2MIC group was 60%, and the skin of the mice that did not die also showed redness and swelling, and the mental state was poor, and they could only show slight activity; while the BHI+2MIC group and BHI+MIC group did not die, and the mental state was good and the activity was strong.

[0096] Then, the dorsal skin of the euthanized mice was taken for tissue fixation, fixed in 4% formalin (Sangon) at 4°C for 24 h, embedded in paraffin, separated, stained with hematoxylin and eosin (HE), and photographed under a 100x light microscope. Fig.24 As shown, HE staining also revealed that compared with the PBS group, a large number of inflammatory cells such as neutrophils and lymphocytes infiltrated the epidermis, dermis and hair follicle cells in the BHI, BHI+1 / 2MIC and BHI+1 / 4MIC groups, and fibroblast proliferation was observed around the blood vessels.

[0097] All experiments in this application were repeated three times or more, and the data shown are mean ± standard deviation. All experimental data were analyzed using SPSS20.0 (SPSS Inc., Chicago, IL, USA). The Student's t test was used to analyze the significant differences between two groups of data. The significant differences of three or more groups of data were analyzed by one-way analysis of variance (ANOVA).

[0098] In general, nicotinic acid destroys the integrity of the cell wall and cell membrane of Staphylococcus aureus, depolarizes the cell membrane, reduces the intracellular ATP concentration, changes the cell morphology, and causes leakage of some contents in the cell, thereby showing a strong antibacterial activity. Nicotinic acid also has a certain destructive effect on the genomic DNA and protein structure of Staphylococcus aureus, and can also inhibit the formation of biofilm of Staphylococcus aureus. In addition, the present application further illustrates the effect of nicotinic acid on the toxicity reduction of Staphylococcus aureus supernatant through hemolysis, cytotoxicity and lactating mouse wound models.

[0099] 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 niacin, characterized in that: Use of niacin in disinfectants against Staphylococcus aureus.

2. The use of nicotinic acid according to claim 1, characterized in that: The minimum inhibitory concentration (MIC) of niacin against Staphylococcus aureus is 4 mg / mL, and the minimum bactericidal concentration (MBC) is 4 mg / mL.

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