Application of vitamin C

Abstract

The invention provides application of vitamin C. The vitamin C is applied to an antibacterial agent for inhibiting staphylococcus aureus. Vitamin C is a natural nutrient, the vitamin C is used for inhibiting and killing staphylococcus aureus on food, the food is free of toxic residues, the original nutrition and sensory characteristics of the food are not affected, abuse of antibiotics is reduced, in addition, nutrition is provided for the human body while the human health is not affected, and the food is healthy. And the preservative is especially suitable for being used as a preservative for milk beverages.

Description

Technical Field

[0001] The present invention relates to the field of sterilization technology, in particular to a use of vitamin C. Background Art

[0002] Staphylococcus aureus is a Gram-positive pathogen that has attracted widespread attention worldwide as a dangerous foodborne pathogen. The enterotoxins and hemolysins produced by Staphylococcus aureus can cause food poisoning with symptoms of vomiting and diarrhea, and can even cause life-threatening infectious diseases such as tissue infection, toxic shock syndrome, and sepsis.

[0003] Due to consumer demand for nutritious and healthy foods, the demand for milk drinks mixed with juice has increased. Conventional heating or pasteurization is a heat treatment commonly used to provide microbial safety and extend the shelf life of beverages and foods. However, high temperatures can have a negative impact on the nutritional, sensory and functional quality of foods. Therefore, existing methods for inhibiting Staphylococcus aureus are not suitable for all foods and have inherent disadvantages including changes in sensory properties and toxicity, so new antimicrobial agents need to be found. Summary of the invention

[0004] The main purpose of the present invention is to provide a use of vitamin C to at least solve the problem in the prior art that the original nutritional and sensory properties of food are affected and the food has toxicity when inhibiting Staphylococcus aureus.

[0005] In order to achieve the above object, the present invention provides a use of vitamin C, including the use of vitamin C in an antibacterial agent for inhibiting Staphylococcus aureus.

[0006] Furthermore, the minimum inhibitory concentration (MIC) of vitamin C against Staphylococcus aureus was 8 mg / mL, and the minimum bactericidal concentration (MBC) was 8 mg / mL.

[0007] The technical solution of the present invention provides a use of vitamin C, which is to apply vitamin C in an antibacterial agent for inhibiting Staphylococcus aureus. Vitamin C is a natural nutrient. Using vitamin C to inhibit and kill Staphylococcus aureus on food leaves no toxic residue in the food, does not affect the original nutritional and sensory properties of the food, reduces the abuse of antibiotics, and provides nutrition to the human body without affecting human health. It is particularly suitable as the main active ingredient of a milk beverage preservative. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] 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:

[0009] Figure 1 is a graph showing the effect of a VC on the growth curve of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0010] Figure 2 is a graph showing the effect of a VC on the leakage of alkaline phosphatase of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0011] Figure 3 This is a graph showing the effect of a VC on the ATP concentration of Staphylococcus aureus cells, which can be selected according to an embodiment of the present invention;

[0012] Figure 4 is a graph showing the effect of a VC on the membrane potential of Staphylococcus aureus, which can be selected according to an embodiment of the present invention;

[0013] Figure 5 is a graph showing the effect of a VC on the cell membrane integrity of Staphylococcus aureus that can be selected according to an embodiment of the present invention;

[0014] Figure 6 This is a graph showing the effect of VC on the cell morphology of Staphylococcus aureus under a scanning electron microscope, which can be selected according to an embodiment of the present invention;

[0015] Figure 7 is a graph showing the effect of a VC on Staphylococcus aureus nucleic acid leakage, which can be selected according to an embodiment of the present invention;

[0016] Figure 8 is a graph showing the effect of a VC on protein leakage of Staphylococcus aureus that can be selected according to an embodiment of the present invention;

[0017] Fig. 9 is a graph showing the effect of a VC on the integrity of the genomic DNA of Staphylococcus aureus that can be selected according to an embodiment of the present invention;

[0018] Fig.10 is a graph showing the effect of a VC on whole cell protein of Staphylococcus aureus that can be selected according to an embodiment of the present invention;

[0019] Fig.11 is a graph showing the effect of a VC on the biofilm formation ability of Staphylococcus aureus that can be selected according to an embodiment of the present invention;

[0020] Fig.12 is a first Staphylococcus aureus supernatant hemolysis rate graph which can be selected according to an embodiment of the present invention;

[0021] Fig.13 is a hemolytic activity graph of a second Staphylococcus aureus supernatant that can be selected according to an embodiment of the present invention;

[0022] Fig.14is a graph showing the effect of optional VC on Hla protein in the supernatant of Staphylococcus aureus according to an embodiment of the present invention;

[0023] Fig.15 is a graph showing the effect of optional VC on SEA protein in the supernatant of Staphylococcus aureus according to an embodiment of the present invention;

[0024] Fig.16 is an SDS-PAGE image showing purified Hla protein, which can be selected according to an embodiment of the present invention;

[0025] Fig.17 This is an SDS-PAGE image showing purified SEA protein, which can be selected according to an embodiment of the present invention;

[0026] Fig.18 This is a graph showing the effect of a VC on purified Hla protein, which can be selected according to an embodiment of the present invention;

[0027] Fig.19 This is a graph showing the effect of a VC on purified SEA protein, which can be selected according to an embodiment of the present invention;

[0028] Fig. 20 is an influence diagram of VC on the quality of milk beverages, which can be selected according to an embodiment of the present invention;

[0029] Fig.21 is a graph showing the effect of VC on the number of microorganisms in a milk beverage, which can be selected according to an embodiment of the present invention;

[0030] Fig. 22 This is an optional graph of the effect of VC on the pH of a milk beverage according to an embodiment of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] This is illustrated by the following examples:

[0033] 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.

[0034] Vitamin C is also called VC. The applicant has found that VC has an inhibitory effect on Staphylococcus aureus and can be used as the main active ingredient of an antibacterial agent to inhibit Staphylococcus aureus in food and the environment. VC is a natural nutrient and is non-toxic. By utilizing the inhibitory effect of VC on Staphylococcus aureus and applying VC in antibacterial agents that inhibit Staphylococcus aureus, it is safer, greener and more efficient. VC is a natural nutrient. Using VC can inhibit and kill Staphylococcus aureus on food, and also has an inhibitory effect on the toxins produced by Staphylococcus aureus. Using VC as an antibacterial agent for food will leave no toxic residue in the food, and will not affect the original nutritional and sensory properties of the food, thus reducing the abuse of antibiotics. In addition, it provides nutrition to the human body without affecting human health, and is particularly suitable as the main active ingredient in a preservative for dairy beverages.

[0035] This is illustrated by the following examples:

[0036] 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.

[0037] The VC used in the experiment of this application was purchased from Solebow Biotechnology Co., Ltd., and Staphylococcus aureus (ATCC29213), Escherichia coli (EDL933), Salmonella (ATCC13311 and CMCC50335), Bacillus cereus (ATCC14579) and Vibrio parahaemolyticus (ATCC17802) were provided by the College of Food Science and Engineering, Northwest Agriculture and Forestry University.

[0038] In the present application, within the range of minimum inhibitory concentration and minimum bactericidal concentration, the solvent used to reflect the inhibitory effect of VC on the above-mentioned bacteria is distilled water.

[0039] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of VC against Staphylococcus aureus were determined by broth microdilution method.

[0040] 10 6 CFU / mL of Staphylococcus aureus suspension was co-cultured with different concentrations of VC 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 TSA after incubation at 37°C for 48 hours was the MBC.

[0041] Table 1 shows the MIC and MBC of VC against common foodborne pathogens. The MIC and MBC of VC against Staphylococcus aureus ATCC29213 are both 8 mg / mL. The test results are shown in Table 1.

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

[0043]

[0044] As shown in Table 1, VC has antibacterial activity against other foodborne pathogens including Escherichia coli, Salmonella, Bacillus cereus and Vibrio parahaemolyticus.

[0045] The inhibitory effect of VC on Staphylococcus aureus is further illustrated by the following test, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. The strain used in 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 VC on the honeycomb plate. OD600 was measured every 1 h using a microbial growth curve analyzer. nm The total operation time was 24h. Each group had 3 parallels. The culture time was taken as the horizontal axis, and the OD600 nm The microbial growth curve is plotted on the vertical axis.

[0048] like Figure 1 As shown in the figure, VC completely inhibited the growth of strain ATCC29213 at 2MIC and MIC concentrations. VC at 1 / 2MIC, 1 / 4MIC and 1 / 8MIC concentrations delayed the growth and proliferation of bacteria.

[0049] 2. Effect of VC on the leakage of alkaline phosphatase (AKP) from Staphylococcus aureus

[0050] Staphylococcus aureus was treated with different concentrations of VC at 37°C for 2 h. The mixture was centrifuged (12000 g, 5 min, 4°C) and the supernatant was collected in a pre-cooled centrifuge tube. AKP enzyme activity was determined using an AKP detection kit (Fancovi), and the absorbance of the sample at 450 nm was determined using a UV spectrophotometer.

[0051] like Figure 2As shown, the content of AKP increases with the increase of VC concentration. The content of AKP after the reaction of VC with 2MIC, 1MIC and 1 / 2MIC concentrations and Staphylococcus aureus is significantly different from that of the control group (p < 0.01). The content of AKP after the reaction of VC with 1 / 4MIC concentration and 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 not detected outside the cell. The present application confirms that the cell wall is destroyed by VC by measuring the AKP activity in the bacterial suspension. As the concentration of VC increases, the concentration of AKP also gradually increases, further proving that VC destroys the cell wall structure of Staphylococcus aureus.

[0052] 3. Effect of VC on ATP content in Staphylococcus aureus cells

[0053] The Staphylococcus aureus suspension treated with different concentrations of VC 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 (Biyuntian S0026).

[0054] The results of the test are as follows Figure 3 As shown in the figure, the intracellular ATP content of Staphylococcus aureus treated with VC 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 the cells may change the intracellular ATP concentration. In this application, it is found and proved that VC can reduce the intracellular ATP concentration. The leakage or rapid depletion of intracellular ATP is due to changes in cell membrane permeability, membrane damage or acceleration of ATP hydrolysis. And as the concentration of VC increases, the intracellular ATP content of Staphylococcus aureus bacteria decreases, further proving that VC destroys the integrity of the cell membrane.

[0055] 4. Effect of VC on membrane potential of Staphylococcus aureus

[0056] Add the prepared bacterial solution to the black ELISA plate and add 3 μL of fluorescent dye DiBAC4 (3) to each well, place in a 37°C incubator for 30 minutes, and then add different concentrations of VC solution to the 96-well plate. After the fluorescent dye and Staphylococcus aureus fully interact, use a multifunctional ELISA reader to detect the fluorescence intensity, with an excitation / emission wavelength of 490 nm / 525 nm, an excitation slit width of 3 nm, and an emission slit width of 5 nm.

[0057] like Figure 4As shown in the figure, the membrane potential of Staphylococcus aureus ATCC29213 cells changed significantly after VC treatment. Compared with the control group, the fluorescence intensity of ATCC29213 cells treated with 2MIC, 1MIC and 1 / 2MIC VC increased significantly (p<0.01), indicating that ATCC29213 was depolarized. The depolarizing effect of VC on ATCC29213 was more obvious at 2MIC, and there was no significant difference from the control group at 1 / 4MIC concentration.

[0058] DiBAC4(3) 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 cause changes in fluorescence intensity, thereby reflecting changes in cell membrane potential. This application discovered and proved that VC can affect the membrane potential of Staphylococcus aureus. As the concentration of VC increases, the fluorescence value of the membrane potential of Staphylococcus aureus gradually increases, indicating cell depolarization.

[0059] 5. Cell membrane integrity detection

[0060] use BacLightVigor kit L-7007, observe the plasma membrane permeability of VC to Staphylococcus aureus. This kit consists of two dyes (green fluorescence) and propidium iodide (PI) (red fluorescence). The bacterial suspension treated with different concentrations of VC was centrifuged at 12000r / min for 5min, the supernatant was discarded and suspended in sterile saline. 3μL of VC was added to each 1mL of bacterial suspension. After incubation in the dark for 15 min, 5 μL of bacterial solution was added to the slide and observed using a laser confocal microscope. The excitation / emission wavelengths of PI and PI dyes are 485 / 540 nm and 535 / 610 nm, respectively.

[0061] LIVE / DEAD BacLight TM The cell staining method is used to measure plasma membrane permeability. It is a double nucleic acid staining method. Cell viability is evaluated based on changes in cell membrane permeability. Figure 5 As shown, cells with intact membranes showed The green fluorescence of the dye indicated that the plasma membrane of the control group was intact, while after treatment with 2MIC and MIC concentrations of VC, all showed red fluorescence, indicating that the cell membrane was damaged. Green fluorescence staining agent and red fluorescence (PI) indicate that the cell membrane is intact or damaged. It can penetrate the intact bacterial membrane, while the red fluorescent dye PI is hydrophilic and cannot penetrate the intact bacterial membrane.

[0062] 6. Observation of Staphylococcus aureus cell morphology using scanning electron microscopy

[0063] Exponential phase Staphylococcus aureus cells were treated with different concentrations of VC at 37°C for 4 h. The treated samples were centrifuged at 12000g for 5 min at 4°C, washed with 0.01M PBS (pH 7.4), and 2.5% glutaraldehyde was used to fix the bacteria for 12 h. 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.

[0064] like Figure 6 As shown in the figure, the morphology of Staphylococcus aureus cells changed after treatment with VC. Comparing the cells in the control group and the test group, it was observed that the untreated cells showed a typical Gram-positive bacterial structure, which was spherical, complete in shape, and smooth on the surface. After treatment with high concentrations of VC, the cell membrane of Staphylococcus aureus ruptured and the contents leaked out. After treatment with low concentrations of VC, wrinkles appeared on the surface of the cell membrane, the cells were significantly swollen, and the shape was less regular compared with the control group. With the increase of VC concentration, the cell morphology changed and the degree of cell damage increased.

[0065] 7. Effect of VC on intracellular nucleic acid and protein leakage of Staphylococcus aureus

[0066] The bacterial suspensions treated with different concentrations of VC were centrifuged at 12,000 r / min for 5 min at 0, 2, 4, 6, 8, and 10 h, and the supernatants were 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.

[0067] like Figure 7 and Figure 8 As shown, compared with the control group, proteins and nucleic acids of Staphylococcus aureus cells leaked rapidly between 2 and 6 hours, and the leakage rate and amount increased with the increase of VC concentration. These results prove that the cell membrane of Staphylococcus aureus was destroyed and the cell integrity was unbalanced, causing the leakage of these cellular substances.

[0068] 8. Effect of VC on the integrity of Staphylococcus aureus genomic DNA

[0069] The genomic DNA of Staphylococcus aureus was extracted using a DNA extraction kit. The genomic DNA (50 μg / mL) was incubated with different concentrations of VC solution at a volume ratio of 1:1 at 37°C for 30 min, and then the DNA binding was detected by electrophoresis on a 1% agarose gel in TAE buffer. The DNA bands were observed using a gel imaging system.

[0070] After VC passes through the cell membrane, it may interact with DNA. Fig. 9 As shown, with the increase of VC concentration, the DNA band gradually faded. Compared with the control group, cells treated with 2MIC and MIC showed significant effects. At the concentration of 2MIC, the total DNA in the reaction solution was significantly reduced, indicating that VC can promote DNA degradation. As the amount of VC increased, the migration of DNA through the gel decreased. VC exhibited DNA binding activity in a dose-dependent manner. When treated with 1 / 2MIC of VC, most of the genomic DNA of Staphylococcus aureus cells could migrate into the gel. When treated with 1MIC of VC, only a small part of the Staphylococcus aureus DNA could migrate into the gel. At 2MIC, the complex of DNA and VC still existed in the band. Therefore, VC can bind to the genomic DNA of Staphylococcus aureus.

[0071] 9. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole-cell proteins

[0072] After incubating different concentrations of VC and Staphylococcus aureus suspension at 37°C for 4 hours, the treated bacterial cells were washed with 0.01M PBS (pH 7.4), centrifuged at 12000g for 5 minutes, and then resuspended with 0.01M PBS (pH 7.4) 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.

[0073] The extracted total protein was subjected to SDS-PAGE gel electrophoresis and then stained with Coomassie Brilliant Blue. Fig.10As shown in the figure, the control group had bright protein bands. After VC treatment of Staphylococcus aureus, 2MIC, MIC and 1 / 2MIC reduced the abundance of intracellular proteins in Staphylococcus aureus. Therefore, VC can cause large molecular proteins to degrade into small molecular proteins or inhibit the synthesis of large molecular proteins. Compared with the control group, there was no significant change in the protein bands after 1 / 4MIC VC treatment. As the VC concentration increased, 2MIC inhibited protein synthesis or degraded proteins.

[0074] 10. Effect of VC on biofilm formation of Staphylococcus aureus

[0075] Staphylococcus aureus cells 10 6 CFU / mL was inoculated in LB liquid medium, and different concentrations of VC 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 adherent cells for staining for 10 minutes and then washed 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.

[0076] After treating Staphylococcus aureus ATCC29213 strain with different concentrations of VC for 24 hours, the formation of biofilm was observed and quantified by crystal violet staining. As shown in 11, the biofilm of the bacterial strain in the control group was intact, while the biofilm formation of the strain after VC treatment was reduced. As the concentration of VC increased, the formation of biofilm showed a downward trend. This shows that VC can significantly inhibit the formation of Staphylococcus aureus biofilm in a dose-dependent manner.

[0077] 11. Hemolytic activity assay

[0078] Staphylococcus aureus cells (10 6 CFU / mL) was cultured in BHI liquid medium at 37°C for 48h, 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 VC were mixed with the minimum hemolytic concentration of the supernatant and an equal volume of 6% rabbit red blood cells was added to mix and the hemolytic activity was determined. Finally, the optical density of the supernatant was measured at 450nm. Positive (0.1% TritonX-100), PBS was negative). The hemolysis percentage was determined based on the ratio of the sample to the positive control.

[0079] Hemolytic ability is an important pathogenic factor of Staphylococcus aureus. Therefore, in order to evaluate whether VC has an effect on the hemolytic activity of Staphylococcus aureus (ATCC29213), 6% fresh rabbit red blood cells were used for hemolytic analysis. First, we used the collected supernatant for hemolytic analysis, such as Fig.12 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 hemolytic effects. We also used this concentration for subsequent experiments. After different concentrations of VC were mixed with the supernatant and incubated for 1 hour, 6% fresh rabbit red blood cells were added. The results after incubation for 4 hours are shown in the figure. Fig.12 and Fig.13 , 2MIC and MIC of VC can completely inhibit the hemolytic ability of the supernatant, 1 / 2MIC concentration of VC reduces the hemolytic ability to 3.67%, 1 / 4MIC concentration of VC reduces the hemolytic ability to 20.33%, and 1 / 8MIC concentration of VC reduces the hemolytic ability to 45.67%.

[0080] 12. Western blot determination of the effect of VC on toxins in the supernatant of Staphylococcus aureus

[0081] The levels of α-hemolysin (Hla) and enterotoxin A (SEA) of Staphylococcus aureus were determined by Western blot.

[0082] After Staphylococcus aureus was cultured in BHI liquid medium for 48 hours, the supernatant was collected and filtered with a 0.22μM filter membrane to obtain the supernatant of Staphylococcus aureus. The supernatant of Staphylococcus aureus was incubated with different concentrations of VC for 2 hours, mixed with sample buffer, heated at 99°C for 10 minutes, separated by electrophoresis using 5% concentrating gel and 15% separating gel, and transferred to a 0.22μM PVDF membrane. The PVDF membrane was then incubated with 5% skim milk at room temperature for 2 hours, the primary antibody (Hla and SEA) 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).

[0083] The results are as follows Fig.14 As shown, VC at 2MIC and MIC concentrations destroyed the Hla protein structure in the supernatant, and there was no significant difference between VC at 1 / 4MIC and the control group, indicating that high concentrations of VC can destroy the structure of Hla to affect its hemolytic activity. Fig.15 It shows that VC also has a very significant inhibitory effect on the SEA activity of Staphylococcus aureus, and both are concentration-dependent.

[0084] 13. Purification of Hla and SEA proteins

[0085] Briefly, Hla and SEA genes were amplified from the wild strain HN37 of Staphylococcus aureus, and then cloned into pCold I plasmid (Takara) and transformed into recipient Escherichia coli BL21 (DE3) cells (Novagen). After induction with 0.5mmol / L isopropyl-1-thiogalactoside (Sigma-Aldrich, St. Louis, MO), the cells were lysed by ultrasound, and then the labeled Hla and SEA recombinant proteins were purified by niNTA spin column (GE Life Science, Westborough, MA). The filtered proteins were then collected on 12% SDS-PAGE and dialyzed into 10mmol / L PBS (pH = 7.4). The results of the purity test of the purified Hla and SEA are shown as follows: Fig.16 and Fig.17 As shown in Figure 2, M represents a protein molecular weight marker. The effect of VC on the purification of Hla protein and SEA protein is shown in Figure 2. Fig.18 and Fig.19 shown.

[0086] 14. Effect of Vitamin C on Milk Beverages

[0087] VC was added to the milk drink so that the final concentration of VC in the milk drink was 0, 4, 8 and 16 mg / mL. Since VC has no color and odor, adding VC will not affect the taste and color of the yogurt drink. 3 CFU / mL of Staphylococcus aureus was inoculated into dairy beverages and incubated at 37°C. The colony count method was used to monitor the viable Staphylococcus aureus. The pH values ​​of the dairy beverage samples were measured using a pH meter.

[0088] like Fig. 20 , Fig.21 and Fig. 22As shown in the figure, after adding different concentrations of VC to milk drinks and inoculating bacteria, it was found that VC with 2MIC and MIC concentrations could completely kill the bacteria in the drinks, and the number of live bacteria in VC with 1 / 2MIC concentration decreased by 0.85 CFU / mL compared with the control group, and the difference was extremely significant. Therefore, it is shown that VC has a bactericidal effect in milk drinks, and low concentrations of VC can also inhibit the growth or reproduction of bacteria. Similarly, the antibacterial activity of VC itself can also play a role in milk drinks. High concentrations of VC can kill live bacteria in milk drinks, and low concentrations of VC can also inhibit the reproduction of bacteria in milk drinks. After adding VC, the pH value of milk drinks, the control group and 2MIC, 1MIC and 1 / 2MIC VC can reduce the pH of the drink, and the difference is extremely significant. After VC is added to milk drinks, the pH of the drink is reduced, but it is not the reduction in pH that affects the growth and reproduction of bacteria, but the antibacterial activity of VC itself can also play a role in milk drinks.

[0089] 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). Significant differences were analyzed using one-way analysis of variance. The significance levels are expressed as follows: *p<0.05, **p<0.01.

[0090] In general, the present application found and confirmed that VC has antibacterial activity against Staphylococcus aureus. VC 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 intracellular contents, thereby showing strong antibacterial activity. VC 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. Moreover, VC can also inhibit the activity of its hemolysin. VC is used in milk beverages as a food antibacterial agent and has a good effect.

[0091] 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 vitamin C, characterized in that, Includes the use of vitamin C in an antimicrobial agent to inhibit Staphylococcus aureus.

2. The use of vitamin C according to claim 1, characterized in that: The minimum inhibitory concentration (MIC) of the vitamin C to Staphylococcus aureus is 8 mg / mL, and the minimum bactericidal concentration (MBC) is 8 mg / mL.

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