Application of milk-derived extracellular vesicles in inhibiting Staphylococcus aureus and treating mastitis in dairy cows

Abstract

The application of milk-derived extracellular vesicles (EVs) in inhibiting Staphylococcus aureus and treating cow mastitis belongs to the field of animal husbandry technology. To address the technical problem of the difficulty in curing cow mastitis caused by S. aureus infection, the present invention isolates milk-derived EVs from the milk of healthy cows and cows with mastitis. The inhibitory effect of milk-derived EVs on the growth of S. aureus is studied. It is found that milk-derived EVs from both healthy and mastitis cows can inhibit the growth of S. aureus and the formation of S. aureus biofilms. This method further provides a method for treating cow mastitis caused by S. aureus infection using milk-derived EVs.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to the application of milk-derived extracellular vesicles in inhibiting Staphylococcus aureus and treating cow mastitis. Background Art

[0002] Mastitis, also known as cow mastitis, is one of the most common diseases in the dairy industry worldwide. The disease is primarily caused by bacterial invasion, poor feeding conditions, and mechanical damage during milking, which trigger an inflammatory response in the cow's mammary tissue. Pathogenic infection of the mammary tissue is currently the leading cause of mastitis in dairy cows. After contracting mastitis, infected cows struggle to recover quickly, resulting in decreased milk quality and milk production. This not only causes significant economic losses to the dairy industry but also impacts the health of dairy products. The persistent inflammation leads to long-term pathological damage to the cow's mammary tissue and a decrease in immune defenses.

[0003] Currently, mastitis in dairy cows caused by Staphylococcus aureus (also known as Staphylococcus aureus) infection is difficult to cure. In clinical practice, due to the extensive use of antibiotics, affected cows often show varying degrees of antibiotic resistance. In recent years, with the abuse of antibiotics, the resistance of Staphylococcus aureus has continued to increase. About 90% of Staphylococcus aureus are resistant to multiple antibiotics, making the treatment process more difficult and seriously hindering the period of antibiotic treatment (Khanal A, GCS, Gaire A, Khanal A, Estrada R, Ghimire R, Panthee S. Methicillin-resistant Staphylococcus aureus in Nepal: A systematic review and meta-analysis. Int J Infect Dis. 2021 Feb; 103: 48-55.). Staphylococcus aureus invades mammary tissue damaged by physical factors and colonizes mammary epithelial cells, absorbing nutrients from the cytoplasm for its growth and reproduction. After infection, S. aureus not only releases multiple virulence factors but also exploits its unique immune evasion mechanisms, resulting in persistent infection, a long incubation period, and the development of drug resistance in cow mastitis caused by S. aureus. The detection rate of S. aureus in cow mastitis in my country is as high as 80%. S. aureus evades the host immune system, weakening immunity and stimulating the release of inflammatory factors such as IL-6, IL-1β, and TNF-α, causing local damage to mammary tissue and ultimately affecting milk production. Therefore, it is imperative to find an alternative antibiotic that can effectively inhibit the inflammatory response for the treatment of cow mastitis caused by S. aureus infection.

[0004] Extracellular vesicles (EVs) are small vesicles (MVs) with diameters ranging from 30 to 150 nm that are released extracellularly after fusion with the cell membrane. All cells can secrete EVs, and they are widely distributed in biological fluids such as milk, blood, urine, saliva, and amniotic fluid. The contents of secreted EVs vary depending on the cell type, primarily including DNA, RNA, lipids, metabolites, and proteins. In milk, functional molecules such as proteins and miRNAs are encapsulated within EVs. Protected by a phospholipid bilayer, these functional molecules withstand harsh conditions such as high temperatures, RNase digestion, low pH, and gastrointestinal digestion. Currently, research on EVs has largely focused on the identification and discovery of regulatory factors and their application in cancer diagnosis and treatment. Research on bovine milk-derived EVs has focused on the differences in their functional components and anti-inflammatory mechanisms, but their direct effects on bacteria, particularly Staphylococcus aureus, remain unclear. Summary of the Invention

[0005] In order to solve the technical problem of the difficulty in curing cow mastitis caused by Staphylococcus aureus infection, the present invention isolates milk-derived extracellular vesicles from the milk of healthy cows and cows with mastitis, studies the inhibitory effect of milk-derived extracellular vesicles on the growth of Staphylococcus aureus, and finds that extracellular vesicles from both healthy cows and cows with mastitis can inhibit the growth of Staphylococcus aureus and the formation of Staphylococcus aureus biofilms. This further provides a method for treating cow mastitis caused by Staphylococcus aureus infection using cow milk-derived extracellular vesicles.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide the use of cow milk-derived extracellular vesicles in inhibiting the growth of Staphylococcus aureus.

[0008] In one embodiment of the present invention, the cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of healthy cows or extracellular vesicles derived from milk of cows suffering from mastitis.

[0009] In one embodiment of the present invention, the growth of Staphylococcus aureus is inhibited by increasing the active oxygen content in the body of Staphylococcus aureus, reducing the activity of antioxidant enzymes, and inhibiting energy metabolism.

[0010] In one embodiment of the present invention, the minimum inhibitory concentration of cow milk-derived extracellular vesicles is 500 μg / mL.

[0011] The second object of the present invention is to provide the use of cow milk-derived extracellular vesicles in inhibiting the biofilm formation of Staphylococcus aureus.

[0012] In one embodiment of the present invention, the cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of healthy cows or extracellular vesicles derived from milk of cows suffering from mastitis.

[0013] In one embodiment of the present invention, the concentration of cow milk-derived extracellular vesicles is not less than 125 μg / mL.

[0014] The third object of the present invention is to provide the use of cow milk-derived extracellular vesicles as an effective ingredient in the preparation of a medicine for treating cow mastitis caused by Staphylococcus aureus infection.

[0015] In one embodiment of the present invention, the cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of healthy cows or extracellular vesicles derived from milk of cows suffering from mastitis.

[0016] In one embodiment of the present invention, the method for preparing cow milk-derived extracellular vesicles comprises the following steps:

[0017] (1) Fresh milk was centrifuged at 3000 g for 30 min and then frozen for 5 min, and the middle whey was collected;

[0018] (2) centrifuging the whey obtained in step (1) at 15,000 g for 30 min, and aspirating the middle whey again;

[0019] (3) centrifuging the whey obtained in step (2) at 170,000 g for 90 min, discarding the supernatant, and collecting the adherent material in the lower layer;

[0020] (4) Resuspend in PBS buffer, centrifuge at 12000 g for 10 min, and obtain the supernatant. Filter through a 0.45 μm filter and then a 0.22 μm filter to obtain the filtrate.

[0021] (5) The filtrate was centrifuged at 170,000 g for 90 min, the upper liquid was discarded, and the bottom precipitate was mixed by gentle pipetting with PBS buffer and stored at -80 °C until use.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses ultraspeed differential centrifugation to extract milk-derived extracellular vesicles from the milk of healthy cows and cows with mastitis. Through transmission electron microscopy observation, particle size analysis and protein immunoblotting to detect markers, it is identified that the morphological characteristics, particle diameter size, particle concentration and surface markers of the milk-derived extracellular vesicles are consistent with the typical characteristics of milk-derived exosomes.

[0024] The present invention directly acts on the milk-derived extracellular vesicles to the standard strain of Staphylococcus aureus and Staphylococcus aureus isolated from the milk sample of cows with mastitis. It is found that the extracellular vesicles from healthy milk and the extracellular vesicles from mastitis milk have an inhibitory effect on the growth of Staphylococcus aureus, with a minimum inhibitory concentration of 500 μg / mL; the extracellular vesicles from healthy milk and the extracellular vesicles from mastitis milk also have an inhibitory effect on the formation of Staphylococcus aureus biofilm, with a minimum inhibitory concentration of 125 μg / mL.

[0025] In addition, the present invention found that milk-derived extracellular vesicles can activate the oxidative stress response of Staphylococcus aureus, resulting in a significant concentration-dependent increase in ROS, and antioxidant enzymes including POD, CAT and SOD showed a significant downward trend as the concentration of milk-derived extracellular vesicles increased. Milk-derived extracellular vesicles activate the oxidative stress in Staphylococcus aureus bacteria, leading to the production of ROS, further activating the antioxidant reductase system, reducing the activity of antioxidant enzymes and weakening the ability of bacteria to scavenge free radicals. The accumulation of ROS in the bacteria interferes with its energy metabolism pathway, thereby causing excessive ATP consumption in the bacteria, ultimately inhibiting the logarithmic growth phase of Staphylococcus aureus, inhibiting the formation of biofilm, and then achieving an antibacterial effect. Mastitis milk-derived extracellular vesicles have a more significant inhibitory effect than healthy milk-derived extracellular vesicles.

[0026] The present invention provides a basis for the study of the anti-inflammatory mechanism of milk-derived extracellular vesicles; at the same time, it lays a foundation for the application research of engineered extracellular vesicles for improving inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of identification of extracellular vesicles from healthy milk cells; Figure 1 A in the figure is the morphology of extracellular vesicles from healthy milk cells under transmission electron microscope. Figure 1 B in the figure is the size diagram of extracellular vesicles from healthy milk cells. Figure 1 C in the figure is the particle size distribution of extracellular vesicles from healthy milk cells. Figure 1 D in the figure is a Western blot analysis of the expression of extracellular vesicle surface marker proteins CD9 and TSG101 in healthy milk-derived cells;

[0028] Figure 2 This is the result of identification of extracellular vesicles from mastitis milk; Figure 2 A in the figure is the morphology of extracellular vesicles from mastitis milk under transmission electron microscope. Figure 2 B in the figure is the size diagram of extracellular vesicles from healthy milk cells. Figure 2 C in the figure is the particle size distribution of extracellular vesicles from healthy milk cells. Figure 2D in the figure is a Western blot analysis of the expression of extracellular vesicle surface marker proteins CD9 and TSG101 in mastitis milk-derived cells;

[0029] Figure 3 The figure shows the culture results of pathogens in milk samples of mastitis cows in blood plates; Figure 3 A in the figure is the result of culture of pathogens in milk samples of cows with obvious clinical symptoms of mastitis in blood plates. Figure 3 Figure B shows the results of culture of pathogens in a blood plate from a milk sample of a cow with mastitis whose somatic cell count exceeded 500,000 / mL.

[0030] Figure 4 Figure 2 shows the results of PCR amplification of the nuc gene of 26 isolates; where M is DL2000 DNA marker, lane 1 is the negative control, lane 2 is the Staphylococcus aureus standard strain BNCC186335, and lanes 3 to 28 correspond to isolates SA1, SA2, SA3, SA4, SA5, SA6, SA7, SA8, SA9, SA10, SA11, SA12, SA13, SA14, SA15, SA16, SA17, SA18, SA19, SA20, SA21, SA22, SA23, SA24, SA25, and SA26, respectively;

[0031] Figure 5 This is the phylogenetic tree of 11 Staphylococcus aureus isolates and reference strains;

[0032] Figure 6 The growth curve of Staphylococcus aureus standard strain and isolated strain and the result of colony count determination corresponding to OD value are shown in the figure; Figure 6 A in the figure is the growth curve of the standard strain of Staphylococcus aureus. Figure 6 B in the figure is the growth curve of isolate SADQ1. Figure 6 C in the figure is the growth curve of isolate SADQ2. Figure 6 D in the figure is the result of the colony count determination corresponding to the OD value of the Staphylococcus aureus standard strain. Figure 6 E in the figure is the result of the determination of the OD value of the isolated strain SADQ1 corresponding to the number of colonies. Figure 6 F in the figure is the result of the determination of the colony count corresponding to the OD value of the isolate SADQ2;

[0033] Figure 7 The figure shows the screening results of the inhibitory concentration of extracellular vesicles from milk of different sources on Staphylococcus aureus; Figure 7 A in the figure is the result of screening the inhibitory concentration of extracellular vesicles from healthy milk against Staphylococcus aureus. Figure 7 Figure B shows the results of the screening of the inhibitory concentration of extracellular vesicles derived from mastitis milk against Staphylococcus aureus;

[0034] Figure 8 The growth curve of Staphylococcus aureus standard strain after adding different concentrations of milk-derived extracellular vesicles from the same source; Figure 8 A in the figure is the growth curve of the Staphylococcus aureus standard strain after adding different concentrations of extracellular vesicles from healthy milk. Figure 8 B in the figure is the growth curve of the Staphylococcus aureus standard strain after adding different concentrations of extracellular vesicles from mastitis milk;

[0035] Figure 9 The growth curve of Staphylococcus aureus standard strain after adding the same concentration of extracellular vesicles from milk of different sources; Figure 9 A in the figure is the growth curve of Staphylococcus aureus standard strain after adding extracellular vesicles from milk of different sources at a concentration of 250 μg / mL. Figure 9 Figure B is the growth curve of Staphylococcus aureus standard strain after adding extracellular vesicles from milk of different sources at a concentration of 500 μg / mL. Figure 9 C in the figure is the growth curve of the Staphylococcus aureus standard strain after adding extracellular vesicles from milk of different sources at a concentration of 1000 μg / mL;

[0036] Figure 10 is the growth curve of Staphylococcus aureus isolates after treatment with milk-derived extracellular vesicles; Figure 10 A in the figure is the growth curve of Staphylococcus aureus SADQ1 after treatment with milk-derived extracellular vesicles. Figure 10 B is the growth curve of Staphylococcus aureus SADQ2 after treatment with milk-derived extracellular vesicles;

[0037] Figure 11 This is the result diagram of the effect of extracellular vesicles from milk of different sources on the biofilm formation of Staphylococcus aureus; Figure 11 A in the figure shows the effect of extracellular vesicles from milk of different sources on the biofilm formation of Staphylococcus aureus standard strain. Figure 11 B in the figure shows the effect of extracellular vesicles from milk of different sources on the biofilm formation of Staphylococcus aureus SADQ1. Figure 11 C in the figure shows the effect of extracellular vesicles from milk of different sources on the biofilm formation of Staphylococcus aureus SADQ2;

[0038] Figure 12 This is the result of ROS fluorescence staining of Staphylococcus aureus after treatment with extracellular vesicles from healthy milk;

[0039] Figure 13 This is the result of ROS fluorescence staining of Staphylococcus aureus after treatment with extracellular vesicles from mastitis milk;

[0040] Figure 14 This is the fluorescence quantitative result of ROS in Staphylococcus aureus after treatment with milk-derived extracellular vesicles; Figure 14a, b, c, d, and e indicate significant differences (P < 0.05);

[0041] Figure 15 The statistical results of SOD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations are shown in the figure. Figure 15 A in the figure is the statistical result of SOD activity of Staphylococcus aureus standard strain after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 15 B in the figure is the statistical result of SADQ1 SOD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 15 C in the figure is the statistical result of SADQ2 SOD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations; H is HmEVs, M is MmEVs; **P < 0.01, ***P < 0.001;

[0042] Figure 16 The statistical results of CAT activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations are shown in the figure. Figure 16 A in the figure is the statistical result of CAT activity of Staphylococcus aureus standard strain after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 16 Figure B is the statistical result of SADQ1 CAT activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 16 C in the figure is the statistical result of SADQ2 CAT activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations; H is HmEVs, M is MmEVs; ***P < 0.001;

[0043] Figure 17 The statistical results of POD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations are shown in the figure. Figure 17 A in the figure is the statistical result of POD activity of Staphylococcus aureus standard strain after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 17 Figure B shows the statistical results of SADQ1 POD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 17 C in the figure is the statistical result of SADQ2 POD activity of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations; H is HmEVs, M is MmEVs; ***P < 0.001;

[0044] Figure 18 The statistical results of ATP content of Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations are shown in the figure. Figure 18 A in the figure is the statistical result of ATP content of Staphylococcus aureus standard strain after being treated with milk-derived extracellular vesicles from different sources and concentrations. Figure 18Figure B is the statistical result of SADQ1 ATP content in Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations. Figure 18 Figure C shows the statistical results of SADQ2 ATP content in Staphylococcus aureus after treatment with milk-derived extracellular vesicles from different sources and concentrations; H stands for HmEVs, and M stands for MmEVs; ***P < 0.001. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified and can be obtained from commercial channels by those skilled in the art.

[0047] The data of the present invention were analyzed using SPSS 20.0 software and plotted using GraphPad Prism 9, with P < 0.05 considered statistically significant.

[0048] (I) Isolation and identification of extracellular vesicles from milk from different sources of dairy cows

[0049] (1) Collection of milk from healthy cows and cows with mastitis

[0050] ① In this experiment, milk from healthy cows and cows with mastitis was provided by Yongxing Ranch in Lindian County, Daqing City, Heilongjiang Province. Holstein cows within 30 days after calving were selected as healthy cows. The production performance index (DHI) data of dairy cows from Yongxing Ranch were analyzed, and cows with mastitis and a somatic cell count exceeding 500,000 / mL were selected for sampling.

[0051] ② When collecting milk samples, 6 healthy cows and 6 cows with mastitis were fixed in pens respectively. The area around the mammary glands was wiped three times with alcohol. The milk was squeezed out by hand and the first three handfuls of milk were discarded.

[0052] ③ Collect the collected milk into 50mL sterile centrifuge tubes, label the 12 milk samples separately, and place the total volume of each milk sample at about 2L in a foam insulation box with an ice pack. Bring it back to the laboratory for centrifugation within 24 hours and store it at 4℃.

[0053] (2) Isolation and extraction of milk-derived extracellular vesicles

[0054] ① Transfer fresh milk to a 50 mL sterile plug-top centrifuge tube and centrifuge at 3000 g for 30 min in a low-temperature high-speed centrifuge set at 4°C. After centrifugation, remove the milk and freeze it in a -20°C refrigerator for 5 min. To solidify the upper layer of milk fat, use a syringe to draw out the middle layer of whey and transfer it to a new sterile plug-top centrifuge tube.

[0055] ② To remove excess milk fat and cell debris, place the whey in a low-temperature high-speed centrifuge, set at 4°C, centrifuge at 15,000g for 30 minutes, and then aspirate the middle layer of whey into a new centrifuge tube;

[0056] ③ Take a sterile ultracentrifuge tube filled with whey, balance with sterile PBS buffer, seal it, and ultracentrifuge at 170,000g for 90 minutes at 4°C. Aspirate and discard the supernatant. The adherent material in the lower layer is crude milk-derived extracellular vesicles.

[0057] ④ Add PBS buffer to resuspend the crude milk-derived extracellular vesicles, centrifuge at 12000g for 10 minutes, and collect the supernatant. Filter the supernatant using a 0.45μm filter and then a 0.22μm filter. Transfer the filtrate after two filtrations to an ultracentrifuge tube.

[0058] ⑤ Ultracentrifuge the filtrate at 4°C, 170,000g for 90 min, discard the supernatant, gently pipette with 2 mL of PBS buffer, mix the bottom precipitate, transfer to a sterile EP tube, and store in a -80°C refrigerator until use.

[0059] (3) Identification of milk-derived extracellular vesicles

[0060] ① Transmission electron microscopy observation of the morphology of extracellular vesicles from milk

[0061] 10 μL of milk-derived extracellular vesicles were dropped onto a copper grid and allowed to settle for 5 minutes, after which the floating liquid was removed with filter paper. 10 μL of uranyl acetate was dropped onto the copper grid and incubated for 1 minute, after which the floating liquid was removed with filter paper and allowed to stand at room temperature until dry. The dried copper grid was placed under a transmission electron microscope and examined using 100 kV imaging conditions.

[0062] ②Analysis of milk-derived extracellular vesicle size

[0063] Take 1 μL of milk-derived extracellular vesicles and dilute it 1000 times in 1 mL of PBS buffer to prevent the sample from clogging the injection needle; use standard samples to test the instrument performance, and after passing the test, load the milk-derived extracellular vesicle sample; after the sample is tested, obtain the particle size and concentration information of the milk-derived extracellular vesicles.

[0064] ③ Western blot detection of extracellular vesicle surface marker proteins of milk-derived cells

[0065] A. Take 50 μL of milk-derived extracellular vesicles and add 5× RIPA lysis buffer; mix well and place on ice for 30 minutes, shaking and mixing once every 15 minutes; prepare standard samples for BCA protein concentration determination, take 20 μL of diluted milk-derived extracellular vesicles and add them to the BCA mixture and mix well; incubate at 37°C for 30 minutes, and measure the OD using a microplate reader. 562nm The absorbance value under the absorbance is recorded; the protein concentration of the milk-derived extracellular vesicles to be tested is calculated according to the standard curve;

[0066] B. Prepare a 15% SDS PAGE gel according to the size of the sample protein to be tested. After assembling the electrophoresis apparatus, add an appropriate amount of Running Buffer.

[0067] C. Take out the protein sample, boil it in a constant temperature metal bath for 3-5 minutes, and centrifuge it. Add the protein sample and protein maker into the wells of the electrophoresis gel in the required order using a pipette or loading needle.

[0068] D. Cover the tank, turn on the power supply, and run the gel at 80V until the sample runs out of the stacking gel. Then, run the gel at 100V until the bromophenol blue reaches the bottom of the gel.

[0069] E. After electrophoresis is complete, remove the electrophoresis gel and trim it to size, cutting off the edges and excess material. Mark the lower left corner. Cut a PVDF membrane to the appropriate size based on the trimmed gel size, mark the lower left corner, activate it in methanol for 20 seconds, and then soak it in pre-chilled transfer buffer.

[0070] F. Cut 8 pieces of filter paper into 8 cm x 10 cm pieces and soak them in pre-chilled transfer buffer. Create a transfer "sandwich" by following the order of sponge, filter paper, electrophoresis gel, membrane, filter paper, and sponge.

[0071] G. Add transfer buffer to the tank, assemble the transfer apparatus, add floating ice, bury the apparatus in an ice bath or place it in the refrigerator, turn on the power, and transfer the membrane at 300mA. The transfer time is determined by the size of the protein to be detected. The general rule is 1 kd = 1 min.

[0072] H. After transfer, remove the PVDF membrane and soak it in 5% skim milk (TBST) with the membrane protein side facing up for 1 hour.

[0073] I. Primary Antibody Blocking: After milk blocking, cut the membrane as needed and soak it in the prepared primary antibody solution (antibody dilution ratio is 1:1000) and incubate at 4°C overnight.

[0074] G. Recover the primary antibody and wash the membrane with 20 mL of TBST at room temperature for 10 minutes. Repeat three times.

[0075] K. Select secondary antibody based on the primary antibody. Dissolve the secondary antibody at a dilution of 1:5000 in 5% skim milk in TBST. Soak the membrane in the secondary antibody solution and incubate at room temperature for 1 hour.

[0076] L. Recover the secondary antibody and wash the membrane with 20 mL of TBST at room temperature for 10 minutes. Repeat three times.

[0077] M. Remove the PVDF membrane, drain the water, and lay it flat on plastic wrap with the protein side facing up. Add equal volumes of ECL A / B solution mixture dropwise onto the membrane and react in the dark for 5 minutes. Remove the membrane and transfer it to plastic wrap with the protein side facing up.

[0078] N. Place the membrane in the imager, set the parameters, and start exposure; adjust the brightness and contrast, and save the image.

[0079] The present invention uses ultraspeed differential centrifugation to separate extracellular vesicles from cow milk. The morphological characteristics of the samples obtained are observed using transmission electron microscopy. Under the electron microscope, the extracellular vesicle samples from healthy milk and mastitis milk all show a bowl-shaped structure, most of which have a diameter of 50-100 nm. The morphological characteristics and diameter size are consistent with the typical characteristics of extracellular vesicles (see Figure 1 A and Figure 2 The particle size distribution of the extracted milk-derived extracellular vesicles was analyzed using a NanoFCM particle size analyzer. The results showed that the average particle size of healthy milk-derived extracellular vesicles was 79.2 nm and the concentration was 1.95×10 12 Particles / mL; the average particle size of extracellular vesicles in mastitis milk was 79.3 nm, and the concentration was 4.12×10 10 Particles / mL (see Figure 1 B in Figure 1 C in Figure 2 B and Figure 2 The present invention detected the presence of CD9 and TSG101 in extracellular vesicles from healthy milk and extracellular vesicles from mastitis milk (see Figure 1 D and Figure 2 D). CD9 and TSG101 are surface markers of extracellular vesicles. After the extracellular vesicles are lysed, their proteins are extracted for detection. If CD9 and TSG101 are detected, it can further prove that the sample extracted in the present invention is an extracellular vesicle.

[0080] (II) Isolation, culture and identification of Staphylococcus aureus in the milk of cows with mastitis

[0081] (1) Centrifuge the milk at 4000 rpm for 10 min at 4°C, discard the upper layer of fat and the middle layer of whey, resuspend the precipitate in physiological saline, inoculate it into a blood plate, and incubate it at 37°C for 24 h.

[0082] (2) Single colonies with morphological characteristics consistent with Staphylococcus aureus were selected for isolation and purification.

[0083] (3) The purified strains were subjected to Gram staining to distinguish Gram-positive bacteria from Gram-negative bacteria.

[0084] (4) The colonies initially identified as Gram-positive bacteria were inoculated into LB broth culture medium, and suspected Staphylococcus aureus were screened out by performing hydrogen peroxide catalase test and rabbit plasma coagulase test.

[0085] (5) The strain initially identified as suspected Staphylococcus aureus was inoculated into LB broth medium and incubated overnight at 37°C in a shaking incubator. 100 μL of the bacterial solution was taken out and centrifuged at 4000 rpm for 10 minutes at room temperature. The supernatant was discarded and 100 μL of pure water was added. The mixture was boiled at 150°C for 10 minutes and centrifuged at 4000 rpm for 5 minutes at room temperature. The supernatant was used as the strain DNA template. A 200 μL sterile centrifuge tube was used for PCR reaction (the reaction system was 4 μL of dNTP mixture, 6 μL of strain DNA template, 0.5 μL of Taq enzyme, 5 μL of 10×PCR Buffer, 2 μL of upstream and downstream primers, and 28.5 μL of dH2O). The nuc gene specific for Staphylococcus aureus was amplified and detected by agarose gel electrophoresis. The target band was compared to identify whether it was Staphylococcus aureus. The nucleotide sequences of the primers nuc-F and nuc-R used in the PCR reaction are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0086] SEQ ID NO.1: 5'-GCGATTGATGGTGATACGGTT-3'

[0087] SEQ ID NO.2: 5'-AGCCAAGCCTTGACGAACTAAAGC-3'

[0088] (6) DNA template was extracted from the strain and amplified using universal primers for the 16S rRNA gene. The nucleotide sequences of universal primers 27F and 1541R-new are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The amplification reaction system consisted of 2 μL of strain DNA template, 2 μL of upstream and downstream primers, 19 μL of dH2O, and 25 μL of Mix premix. The PCR product was recovered and purified and sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. Sequence alignment was performed on NCBI, and a phylogenetic tree was constructed using the Newton-Joint Joint method for analysis.

[0089] SEQ ID NO.3: 5'-AGAGTTTGATCMTGGCTCAG-3'

[0090] SEQ ID NO.4: 5'-AAGGAGGTGATCCAGCCGCA-3'

[0091] (7) The bacterial suspension of the strain identified as Staphylococcus aureus was evenly mixed with sterile glycerol at a volume ratio of 1:1 to prepare glycerol bacteria, which were then frozen at -80°C for later use.

[0092] The present invention isolates and cultures the bacteria from the milk samples of mastitis cows, observes the morphological characteristics and growth of the bacteria in the blood plate, and selects 26 single colonies (see Figure 3 ), respectively named SA1, SA2, ..., SA26, and the picked suspected Staphylococcus aureus colonies were purified and cultured. At the same time, Gram staining was used for microscopic morphological observation, and Gram-positive bacteria (appearing blue-purple) were selected and inoculated into LB broth. The isolates were subjected to catalase test and coagulase test. The isolates with bubble generation in the catalase test and the coagulase test that could coagulate rabbit plasma were preliminarily judged to be Staphylococcus aureus. Through the above biochemical identification, 11 isolates (SA1, SA3, SA4, SA5, SA7, SA8, SA9, SA12, SA17, SA18 and SA19) were preliminarily judged to be Staphylococcus aureus.

[0093] The nuc gene was amplified by PCR in 26 isolated strains, with the Staphylococcus aureus standard strain BNCC186335 as a control. The 279bp nuc gene band was amplified in both the Staphylococcus aureus standard strain and the 11 strains that were initially identified as Staphylococcus aureus isolates. Figure 4 .

[0094] The full-length genes of 26 clinical bovine mastitis isolates were amplified using the universal primers 27F and 1541R-new for the 16S rRNA gene. The amplified samples were used to detect the size of the target fragments using agarose gel electrophoresis. The PCR products containing the target fragments were recovered and sent to a biological company for sequencing. The sequencing results were compared by BLAST on the NCBI website. The results showed that the 16S rRNA sequences of the 11 isolates identified as Staphylococcus aureus by biochemical identification and Staphylococcus aureus-specific nuc gene PCR were more than 99% similar to those of known Staphylococcus aureus. The remaining isolates all belonged to other Staphylococcus species. The phylogenetic tree constructed using the NJ method is shown in Figure 2. Figure 5As shown, the 11 S. aureus isolates were classified in the same clade as Staphylococcus aureus CICC 10384 and Staphylococcus aureus ATCC 12600, but not in the same clade as the outgroup reference strains S. epidermidis, S. brunensis, S. succinus, and S. haemolyticus. Based on the phylogenetic tree, the 11 S. aureus isolates were divided into two groups for subsequent experiments and designated SADQ1 (including SA1, SA8, SA9, SA12, SA17, SA18, and SA19) and SADQ2 (including SA3, SA4, SA5, and SA7).

[0095] Example 1: Application of extracellular vesicles from healthy milk and extracellular vesicles from mastitis milk in inhibiting the growth of Staphylococcus aureus

[0096] (1) Growth curves of Staphylococcus aureus standard strains and isolates and corresponding colony count determination

[0097] (1) Preparation of bacterial solution

[0098] The glycerol culture of the Staphylococcus aureus standard strain and the clinical bovine mastitis Staphylococcus aureus isolate frozen in a -80℃ refrigerator was taken out, and a disposable sterile inoculation loop was used to pick the glycerol culture to streak on LB solid medium, and then placed in a 37℃ constant temperature incubator for overnight culture; a single colony was picked and inoculated into LB broth medium, and placed in a 37℃ shaking incubator for overnight culture; cultured until the bacterial solution OD 600nm =0.5, dilute with LB broth at 1:100, mix thoroughly, and use as bacterial solution for later use.

[0099] (2) Growth curve determination

[0100] 24 aliquots of the bacterial suspension of the standard strain of Staphylococcus aureus and the clinical strain of Staphylococcus aureus isolated from dairy cow mastitis were taken, 50 μL each, and inoculated into 24 aliquots of LB broth culture medium respectively. The culture was placed in a shaker at 37°C for 24 h. Every hour, a portion of LB broth culture medium was taken, 2 mL of bacterial suspension was pipetted into a cuvette, and the OD was measured using a UV spectrophotometer. 600nm The absorbance of the bacterial solution was measured and repeated 3 times, and the results were recorded.

[0101] (3) Determination of colony counts corresponding to the OD values ​​of the standard strain of Staphylococcus aureus and the isolated strain of Staphylococcus aureus with clinical bovine mastitis

[0102] The experimental steps are the same as above step (2), and the OD 600nmAt the same time as the value, 1 mL of bacterial solution was aspirated and diluted 10-fold with physiological saline. After dilution to the appropriate multiple, 1 mL of the suspension was aspirated and inoculated into LB solid culture medium. The suspension was evenly spread with a spreading rod until the suspension was fully absorbed by the LB solid culture medium. The experiment was repeated three times; the suspension was placed in a 37°C constant temperature incubator and cultured overnight. The colonies were counted and recorded the next day.

[0103] In fresh sterile LB liquid culture medium, the standard strains of Staphylococcus aureus, SADQ1 and SADQ2 grew well. Figure 6 As shown in Figure 2, the logarithmic growth phase was reached at about 6 hours, and the lag phase was reached at 12 hours. 600nm =0.5, the colony counts of Staphylococcus aureus standard strain, SADQ1 and SADQ2 were all above 10 8 CFU / mL, and the colony count was calculated based on this OD value in subsequent experiments.

[0104] (II) Screening of the inhibitory concentration of extracellular vesicles from milk derived from different sources of dairy cows against Staphylococcus aureus

[0105] (1) Extracellular vesicles (HmEVs) from healthy cow milk and extracellular vesicles (MmEVs) from mastitis cow milk were diluted to 2 mg / mL using sterile LB broth medium, and Staphylococcus aureus suspension was obtained by the same method as described in step (1) above. The following experimental steps were the same for the extracellular vesicles from milk of two different sources.

[0106] (2) Add 100 μL of LB broth medium to wells 1-12 of a 96-well plate, add 100 μL of milk-derived extracellular vesicles to well 1 as a negative control, and add 100 μL of bacterial suspension to well 2 as a positive control.

[0107] (3) Pipette 100 μL of diluted milk-derived extracellular vesicles and inoculate them into the third well, and pipette and aspirate them repeatedly 5 times; Pipette 100 μL of the liquid in the third well and add it to the fourth well, and pipette and aspirate them repeatedly 5 times; Pipette 100 μL of the liquid in the fourth well and add it to the fifth well, until it is diluted to the twelfth well, and then pipette and aspirate 100 μL of the liquid in the twelfth well and discard it; Add 100 μL of the prepared bacterial suspension to wells 3-12 in sequence, and pipette and aspirate again to mix.

[0108] (4) Place the 96-well plate after mixing by pipetting in a 37°C constant temperature incubator and incubate for 24 hours. Make 3 wells for each concentration group.

[0109] (5) Use a microplate reader to measure the OD at 24 h 620nm The absorbance under the condition of 40 nm was measured and the absorbance and inhibition rate curve was drawn.

[0110] According to the method of visual observation, after culturing the standard strain of Staphylococcus aureus for 24 hours, it can be observed that the extracellular vesicles of healthy milk and mastitis milk reduce the bacterial precipitation of the standard strain of Staphylococcus aureus at 500 μg / mL and above, and 500 μg / mL is determined to be the minimum inhibitory concentration. The OD value is measured using a microplate reader. 620nm The absorbance value under the condition of 40 μg / mL was compared with that of the positive control group, and the bacterial survival rate of the positive control group was set as 100%. Figure 7 As shown in the results, the inhibition rates of extracellular vesicles from healthy milk at concentrations of 500 μg / mL and 1000 μg / mL were 37.04% and 44.12%, respectively, while the inhibition rates of extracellular vesicles from mastitis milk at concentrations of 500 μg / mL and 1000 μg / mL were 40.86% and 48.72%, respectively. Figure 7 The results showed that when the initial amount of bacterial liquid was the same, the inhibitory effect of extracellular vesicles from mastitis milk at the same mass concentration was more significant than that of extracellular vesicles from healthy milk.

[0111] (III) Screening of the effect time of extracellular vesicles from milk of different cow sources on Staphylococcus aureus

[0112] (1) Obtain a bacterial suspension using the same method as described in step (1) above, ensuring that the initial inoculum volume is the same.

[0113] (2) Take 7 shake tubes, numbered 1 to 7. Add the diluted bacterial suspension to the first 6 shake tubes, add 2 times the minimum inhibitory concentration, minimum inhibitory concentration, and sub-inhibitory concentration of extracellular vesicles from milk of healthy cows to 1000 μg / mL, 500 μg / mL, and 250 μg / mL, respectively; add the same concentration gradient of extracellular vesicles from milk of mastitis cows to 4 to 6 shake tubes, and add only the diluted bacterial suspension to the seventh shake tube as a control group.

[0114] (3) Pipette and thoroughly mix the 7 shaking tubes, and incubate at 37°C and 180 rpm for 24 h.

[0115] (4) Measure OD using an enzyme-labeled instrument 620nm The absorbance value was measured every hour, and the experiment was repeated three times.

[0116] By plotting the growth curve of the standard strain of Staphylococcus aureus after adding milk-derived extracellular vesicles, it was found that the addition of milk-derived extracellular vesicles inhibited the growth and reproduction of Staphylococcus aureus in the logarithmic growth phase. Figure 8 As shown in the figure, with the increase of the concentration of milk-derived extracellular vesicles, the concentration growth of Staphylococcus aureus liquid was significantly inhibited.

[0117] like Figure 9As shown, when added to the same concentration of milk-derived extracellular vesicles, mastitis milk-derived extracellular vesicles significantly inhibited the growth curve of a standard strain of Staphylococcus aureus compared to healthy milk-derived extracellular vesicles. By affecting the reproduction and division of S. aureus during its logarithmic growth phase, the logarithmic growth phase was slowed, resulting in a corresponding decrease in colony counts. Treatment of the S. aureus suspension with 500 μg / mL milk-derived extracellular vesicles had a more pronounced inhibitory effect, with growth initially suppressed. Treatment with 1000 μg / mL milk-derived extracellular vesicles showed a highly significant inhibitory trend. The results show that as the concentration of milk-derived extracellular vesicles acting on S. aureus increases, the degree of inhibition of S. aureus growth increases. Under the same treatment concentration, due to the different components of the two groups of milk-derived extracellular vesicles, the inhibitory effect of the extracellular vesicles from mastitis milk is significantly higher than that of the extracellular vesicles from healthy milk. However, both groups acted on the logarithmic growth phase of S. aureus. After 12 hours of treatment at a milk-derived extracellular vesicle concentration of 1000 μg / mL and 8 hours in the low and medium concentration treatment groups, growth rates showed a trend of recovery. Therefore, it is inferred that the inhibition time of milk-derived extracellular vesicles on S. aureus is within 8 hours.

[0118] The two milk-derived extracellular vesicles were treated with Staphylococcus aureus isolates SADQ1 and SADQ2 at a concentration of 1000 μg / mL, and the growth curves of the Staphylococcus aureus isolates were determined using the same method. Figure 10 As shown in the results, both showed the same trend as the standard strain of Staphylococcus aureus, indicating that both milk-derived extracellular vesicles had a significant inhibitory effect on the growth and reproduction of Staphylococcus aureus.

[0119] Example 2: Application of extracellular vesicles from healthy milk and extracellular vesicles from mastitis milk in inhibiting the formation of Staphylococcus aureus biofilm

[0120] The present invention studies the effect of extracellular vesicles from milk of different sources on the biofilm formation of Staphylococcus aureus using the following method:

[0121] (1) Obtain a bacterial suspension using the same method as described in step (1) above.

[0122] (2) According to the minimum inhibitory concentrations of extracellular vesicles from healthy milk and extracellular vesicles from mastitis milk against Staphylococcus aureus in previous experiments, different amounts of extracellular vesicles from healthy milk and extracellular vesicles from mastitis milk were added. In this experiment, four concentration gradients were set up, namely 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL as experimental groups, and the control group without adding the two types of extracellular vesicles from milk was set up. Three parallel groups were set up for each group, and the 96-well plate was placed at 37°C for incubation for 12 hours.

[0123] (3) After 12 h of incubation, the 96-well plate was removed, the bacterial suspension was discarded, and the surface planktonic bacteria were rinsed three times with pure water and dried at room temperature.

[0124] (4) Add methanol for fixation, discard the methanol after 15 minutes, and dry at room temperature.

[0125] (5) Add 1% crystal violet solution and stain for 30 minutes. Aspirate and discard the staining solution, rinse with pure water until there is no free dye, and dry at room temperature.

[0126] (6) Prepare 33% glacial acetic acid solution and add 200 μL to each well to dissolve the dye.

[0127] (7) Use an enzyme-labeled instrument to measure OD 570nm The absorbance values ​​were recorded and plotted to compare the effects of different concentrations of homologous milk-derived extracellular vesicles on the biofilm formation of Staphylococcus aureus. The experiment was repeated three times.

[0128] The content of Staphylococcus aureus biofilm after treatment with milk-derived extracellular vesicles was determined by crystal violet staining to explore whether milk-derived extracellular vesicles have an inhibitory effect on the formation of Staphylococcus aureus biofilm. 570nm The absorbance of the biofilm after dissolution with 33% glacial acetic acid was measured. The data of the test group were averaged three times (D). The negative control group without inoculated bacterial suspension was taken as the negative value and averaged (DC). 2 times the negative value was taken as the cutoff value (2DC). When D ≤ 2DC, the formation of Staphylococcus aureus biofilm was inhibited. The results are shown in Figure 2. Figure 11 It was shown that when the concentration of the two milk-derived extracellular vesicles reached 125 μg / mL, they could significantly inhibit the formation of Staphylococcus aureus biofilm.

[0129] Example 3: Study on the antibacterial mechanism of extracellular vesicles from healthy milk and mastitis milk against Staphylococcus aureus

[0130] (1) Preparation of bacterial suspension

[0131] Pick a single colony and place it in LB broth medium, shake it at 37℃ and 180r / min until the logarithmic growth phase, and adjust the bacterial solution to OD using a spectrophotometer. 600nm = 0.5, centrifuge at 4000 rpm for 5 min, discard the supernatant, add fresh sterile LB medium, pipette to mix, and divide the bacterial suspension into three equal parts;

[0132] 1 mL of bacterial suspension was added to each well of a 12-well plate. Different concentrations of healthy milk-derived extracellular vesicles were added and mixed by pipetting to achieve final concentrations of 500 μg / mL and 250 μg / mL, respectively. The bacterial suspension without milk-derived extracellular vesicles was set as the control group and incubated in a 37°C incubator for 6 hours.

[0133] After incubation, the bacterial suspension in the 12-well plate was transferred to a 1.5 mL centrifuge tube and centrifuged at 4000 rpm for 5 minutes to retain the precipitate. The experimental procedure for the mastitis milk-derived extracellular vesicles group was the same as above.

[0134] (2) Detection of Reactive Oxygen Species (ROS) Content in Staphylococcus aureus

[0135] Using the Solebol Reactive Oxygen Species Detection Kit, 1 μL of DCFH-DA (10 mM) was added to each 1 mL of fresh sterile LB broth medium to a final concentration of 10 μmol / L. The bacterial precipitate collected in step (1) was resuspended in the diluted DCFH-DA to a bacterial concentration of approximately 10 μmol / L. 8 The probe was placed in a 37°C incubator and incubated for 20 min, with inversion mixing every 5 min to ensure full contact between the probe and the bacteria. The bacteria were washed three times with fresh sterile LB broth to fully remove the DCFH-DA that had not entered the bacteria. The probe-loaded samples were directly observed using a fluorescence microscope for DCF fluorescence spectroscopy in the dark. The fluorescence intensity was analyzed using Image J.

[0136] like Figure 12 and Figure 13 As shown in the figure, Staphylococcus aureus treated with milk-derived extracellular vesicles was loaded with DCFH-DA probe, which showed green fluorescence under the DCF fluorescence spectrum. The fluorescence intensity was positively correlated with ROS. The green fluorescence intensity emitted by the control group was weak. When the sub-inhibitory concentration of milk-derived extracellular vesicles was 250μg / mL, the green fluorescence gradually increased. At 500μg / mL, the green fluorescence intensity of the bacteria was the brightest and the number of fluorescent cells was the largest. Figure 14 As shown, after 6 hours of exposure to S. aureus at 250 μg / mL and 500 μg / mL of extracellular vesicles from healthy milk, the fluorescence intensity was approximately 1.91 times and 2.37 times that of the control group, respectively. After treatment with 250 μg / mL and 500 μg / mL of extracellular vesicles from mastitis milk, the fluorescence intensity was approximately 1.94 times and 2.56 times that of the control group, respectively. This indicates that both milk-derived extracellular vesicles can promote ROS production in S. aureus.

[0137] (3) Detection of superoxide dismutase (SOD) activity of Staphylococcus aureus

[0138] The bacteria collected in step (1) above were transferred to a 1.5 mL centrifuge tube, 1 mL of SOD extract was added, the bacteria were ultrasonically disrupted, and the tubes were centrifuged at 8000 g for 10 min at 4°C. The supernatant was placed on ice for testing. The SOD activity of Staphylococcus aureus was determined according to the instructions of the superoxide dismutase (SOD) activity detection kit of Solebold Technology Co., Ltd.

[0139] like Figure 15As shown in the results, after 6 hours of treatment with milk-derived extracellular vesicles, the SOD activity of S. aureus in the treated group increased with the increase of treatment concentration, showing a dose-dependent negative correlation compared with the control group. When the standard strain of S. aureus was cultured for 6 hours, the SOD activity of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle-treated groups decreased by 9.94% and 23.80%, respectively, and the SOD activity of the mastitis milk-derived extracellular vesicle-treated group decreased by 12.53% and 26.01%, respectively, compared with the control group; when the S. aureus SADQ1 was cultured for 6 hours, the SOD activity of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle-treated groups decreased by 12.53% and 26.01%, respectively, compared with the control group. The SOD activity of SADQ2 in the 250 μg / mL and 500 μg / mL healthy milk EVs groups decreased by 7.63% and 21.76%, respectively, compared with the control group, and the SOD activity of the mastitis milk EVs group decreased by 10.01% and 20.86%, respectively. These data suggest that mastitis milk EVs significantly inhibit SOD activity in S. aureus compared with healthy milk EVs.

[0140] (4) Detection of catalase (CAT) activity of Staphylococcus aureus

[0141] The bacteria collected in step (1) above were transferred to a 1.5 mL centrifuge tube, 1 mL of CAT extract was added, the bacteria were ultrasonically disrupted, and the tube was centrifuged at 8000 g for 10 min at 4°C. The supernatant was placed on ice for testing. The CAT activity of S. aureus was determined according to the instructions of the catalase (CAT) activity detection kit of Solebold Technology Co., Ltd.

[0142] like Figure 16As shown in the results, after 6 hours of treatment with milk-derived extracellular vesicles, S. aureus had a strong inhibitory effect on the CAT activity in S. aureus. With the increase of milk-derived extracellular vesicle concentration, the CAT activity in S. aureus was inhibited in a dose-dependent manner. When the standard strain of S. aureus was cultured for 6 hours, the CAT activity of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle-treated groups decreased by 22.13% and 34.91%, respectively, compared with the control group, and the CAT activity of the mastitis milk-derived extracellular vesicle-treated group decreased by 42.95% and 55.55%, respectively; when S. aureus SADQ1 was cultured for 6 hours, the CAT activity of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle-treated groups decreased by 1.3% and 2.91%, respectively, compared with the control group. The CAT activity of S. aureus SADQ2 decreased by 26.91% and 35.05% in the 250 μg / mL and 500 μg / mL healthy milk EVs treatment groups, respectively, compared with the control group. The CAT activity of the mastitis milk EVs treatment group decreased by 34.50% and 58.29%, respectively. After 6 hours of culture, the CAT activity of S. aureus SADQ2 decreased by 15.63% and 38.90% in the 250 μg / mL and 500 μg / mL healthy milk EVs treatment groups, respectively, compared with the control group. The CAT activity of the mastitis milk EVs treatment group decreased by 40.49% and 55.02%, respectively. Mastitis milk EVs showed a more significant inhibitory effect on the CAT activity of S. aureus.

[0143] (5) Detection of peroxidase (POD) activity of Staphylococcus aureus

[0144] The bacteria collected in step (1) above were transferred to a 1.5 mL centrifuge tube, 1 mL of POD extract was added, the bacteria were ultrasonically disrupted, and the tube was centrifuged at 8000 g for 10 min at 4°C. The supernatant was placed on ice for testing. The POD activity of Staphylococcus aureus was determined according to the instructions of the peroxidase (POD) activity detection kit of Solebold Technology Co., Ltd.

[0145] like Figure 17As shown in the results, with the addition of milk-derived extracellular vesicles, the POD enzyme activities of Staphylococcus aureus standard strain and Staphylococcus aureus SADQ2 were significantly lower than those of the control group, and showed a downward trend with increasing concentration. When the Staphylococcus aureus standard strain was cultured for 6 hours, the POD activities of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle treatment groups decreased by 29.71% and 57.39%, respectively, compared with the control group, and the POD activities of the mastitis milk-derived extracellular vesicle treatment group decreased by 52.66% and 58.84%, respectively; when Staphylococcus aureus SADQ1 was cultured for 6 hours, the POD activities of the 250μg / mL and 500μg / mL healthy milk-derived extracellular vesicle treatment groups decreased by 1. The POD activity of the S. aureus SADQ2 strain decreased by 17.35% and 20.40%, respectively, in the mastitis milk-derived extracellular vesicle-treated group, and by 20.86% and 30.46%, respectively. After 6 hours of culture, the POD activity of the S. aureus SADQ2 strains treated with 250 μg / mL and 500 μg / mL healthy milk-derived extracellular vesicles decreased by 4.81% and 35.45%, respectively, compared with the control group, and the POD activity of the mastitis milk-derived extracellular vesicle-treated group decreased by 17.29% and 77.85%, respectively. Comparison of POD activity after treatment with the same concentration of milk-derived extracellular vesicles revealed that the mastitis milk-derived extracellular vesicles had a more significant inhibitory effect than the healthy milk-derived extracellular vesicle-treated group.

[0146] (6) Detection of ATPase activity of Staphylococcus aureus

[0147] The detection of ATPase activity refers to the research method of Ning et al. (Ning HouQi NHQ, Li YingQiu LYQ, WangZhaoSheng WZS, et al. The synergistic antibacterial properties of glycininbasic peptide against bacteria via membrane damage and inactivation of enzymes[J]. 2019.). ATP can be decomposed into ADP and inorganic phosphorus by ATPase. The ATP content is determined by measuring the amount of inorganic phosphorus. The operation method refers to the instructions of the ATPase test kit of Solebold Technology Co., Ltd.

[0148] ATPase decomposes ATP to generate ADP and inorganic phosphorus. The ATPase activity is determined by measuring the content of inorganic phosphorus. Figure 18As shown, ATP levels in the treated groups decreased with increasing concentrations of milk-derived extracellular vesicles. After 6 hours of exposure to a standard strain of Staphylococcus aureus, ATP levels in the 250 μg / mL and 500 μg / mL healthy milk-derived extracellular vesicle-treated groups decreased by 17.82% and 47.31%, respectively, compared to the control group. ATP levels in the mastitis milk-derived extracellular vesicle-treated groups decreased by 37.63% and 55.52%, respectively. After 6 hours of incubation, ATP levels in S. aureus SADQ1 treated with 250 μg / mL and 500 μg / mL EVs from healthy milk decreased by 14.78% and 32.26%, respectively, compared to the control group, and decreased by 26.72% and 49.34%, respectively, in the group treated with EVs from mastitis milk. After 6 hours of incubation, ATP levels in S. aureus SADQ2 treated with 250 μg / mL and 500 μg / mL EVs from healthy milk decreased by 26.65% and 42.64%, respectively, compared to the control group, and decreased by 28.71% and 55.10%, respectively, compared to the mastitis milk group. At the same concentration of EVs, EVs from mastitis milk had a more significant inhibitory effect on ATP levels in S. aureus. S. aureus isolates SADQ1 and SADQ2 showed the same inhibitory trend as the standard strain.

[0149] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Extracellular vesicles from dairy cow milk inhibit Staphylococcus aureus ( Staphylococcus aureus ) growth application, characterized in that, The cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of cows suffering from mastitis; The average particle size of the extracellular vesicles from milk of cows with mastitis is 79.3 nm; The extracellular vesicles derived from milk of cows with mastitis contain CD9 and TSG101; The inhibition of Staphylococcus aureus growth is to increase the active oxygen content in Staphylococcus aureus, reduce the activity of antioxidant enzymes, and inhibit energy metabolism; The minimum inhibitory concentration of the cow milk-derived extracellular vesicles is 500 μg / mL.

2. The use of cow milk-derived extracellular vesicles in inhibiting Staphylococcus aureus biofilm formation, characterized in that: The cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of cows suffering from mastitis; The average particle size of the extracellular vesicles from milk of cows with mastitis is 79.3 nm; The extracellular vesicles derived from milk of cows with mastitis contain CD9 and TSG101; The concentration of the cow milk-derived extracellular vesicles is not less than 125 μg / mL.

3. Use of cow milk-derived extracellular vesicles as an active ingredient in the preparation of a medicine for treating cow mastitis caused by Staphylococcus aureus infection, characterized in that: The cow milk-derived extracellular vesicles are extracellular vesicles derived from milk of cows suffering from mastitis; The average particle size of the extracellular vesicles from milk of cows with mastitis is 79.3 nm; The extracellular vesicles derived from milk of the cow suffering from mastitis contain CD9 and TSG101.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of milk-derived extracellular vesicles comprises the following steps: (1) Fresh milk was centrifuged at 3000 g for 30 min and then frozen for 5 min, and the middle whey was aspirated; (2) Centrifuge the whey obtained in step (1) at 15,000 g for 30 min, and aspirate the middle whey again; (3) Centrifuge the whey obtained in step (2) at 170,000 g for 90 min, discard the supernatant, and collect the adherent material in the lower layer; (4) Resuspend in PBS buffer, centrifuge at 12,000 g for 10 min, and obtain the supernatant. Filter through a 0.45 μm filter and then a 0.22 μm filter to obtain the filtrate. (5) The filtrate was centrifuged at 170,000 g for 90 min, the upper liquid was discarded, and the precipitate at the bottom was mixed and stored at -80 °C until use.

Images