Composition capable of effectively inhibiting growth of staphylococcus aureus biofilm

Abstract

The invention discloses a composition capable of effectively inhibiting growth of a staphylococcus aureus biofilm. According to the method, firstly, a nitrogen source in a culture medium of the staphylococcus epidermidis CCSM0287 strain is optimized, and the growth difference of the staphylococcus epidermidis under different culture media and culture conditions and the inhibition effect of fermentation supernatant on a staphylococcus aureus biological membrane are fully explored; and then compounding a staphylococcus epidermidis CCSM0287 fermentation supernatant obtained from an optimized culture medium (tryptone, soy peptone and fresh bovine bone collagen peptide powder are adopted as a nitrogen source) with a lactobacillus paracasei S20 fermentation supernatant, so that the growth of a staphylococcus aureus biological membrane can be obviously inhibited, the surface structure of the staphylococcus aureus biological membrane can be obviously damaged, and the staphylococcus epidermidis CCSM0287 fermentation supernatant can be applied to antibacterial products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus, is a representative of Gram-positive bacteria, and is one of the main pathogens causing skin and soft tissue infections. Its pathogenic substances include biofilm, efflux pumps, toxins and plasmids of Staphylococcus aureus. At present, the best method for treating Staphylococcus aureus infection is mainly antibiotic treatment, but the frequent use of antibiotics has enhanced the drug resistance of Staphylococcus aureus, leading to its spread around the world, seriously affecting social public health. How to treat infections caused by Staphylococcus aureus with increasing drug resistance is a hot topic in current research. Biofilm is a viscous extracellular matrix with a unique three-dimensional structure formed by Staphylococcus aureus attaching, proliferating, and secreting extracellular polymer molecules on a certain plane. It attaches to the bacteria to form a protective barrier, preventing adverse external factors from contacting the bacteria. That is, the formation of Staphylococcus aureus biofilm is the main reason for protecting Staphylococcus aureus from escaping immunity and developing drug resistance. Therefore, destroying biofilm synthesis and degradation is one of the effective methods to prevent Staphylococcus aureus colonization and infection.

[0003] As the dominant flora on the skin, Staphylococcus epidermidis plays an important role in maintaining the health of the skin. As part of the skin ecosystem, Staphylococcus epidermidis can not only communicate with skin cells, but also interact with different microorganisms on the skin. The antimicrobial peptides and other substances it produces have certain defensive functions, which can protect the host from pathogens and assist the host in shaping the immune system to defend against the colonization of pathogenic microorganisms. Staphylococcus epidermidis can also inhibit the growth and pathogenicity of Staphylococcus aureus through the quorum sensing system. This inhibitory effect is achieved through certain substances secreted by Staphylococcus epidermidis, which can inhibit the secretion of toxins and proteases of Staphylococcus aureus, thereby reducing damage to the skin.

[0004] CN115637235B provides a strain of Staphylococcus epidermidis CCSM0287, which was deposited in the China Center for Type Culture Collection (CCTCC) on June 1, 2022, with a deposit number of CCTCC M 2022779. Experiments have confirmed that the strain of Staphylococcus epidermidis CCSM0287 is a potential skin probiotic with good antioxidant and anti-inflammatory effects. It can be seen that the CCSM0287 strain is a strain with development potential. The formation of strain biofilm is inseparable from the existence of quorum sensing. The fermentation metabolites of Staphylococcus epidermidis contain a variety of active molecules that can effectively inhibit the formation of Staphylococcus aureus biofilm, but different culture conditions and the selection of culture medium have a certain effect on the fermentation metabolites of Staphylococcus epidermidis, thereby affecting its efficacy. However, the prior art lacks the selection of Staphylococcus epidermidis culture medium and a composition that can effectively inhibit the growth of Staphylococcus aureus biofilm. Summary of the invention

[0005] In view of the above problems, the present invention provides a composition that can effectively inhibit the growth of Staphylococcus aureus biofilm. The present invention first optimizes the nitrogen source in the culture medium of Staphylococcus epidermidis CCSM0287 strain, fully explores the growth difference of Staphylococcus epidermidis under different culture media and culture conditions and the inhibitory effect of its fermentation supernatant on Staphylococcus aureus biofilm, and then compounding the Staphylococcus epidermidis CCSM0287 fermentation supernatant obtained by optimizing the culture medium with the Lactobacillus paracasei S20 fermentation supernatant, which can significantly inhibit the growth of Staphylococcus aureus biofilm and destroy its surface structure, and can be used in antibacterial products.

[0006] In order to achieve the above technical purpose, the present invention first provides a method for preparing the fermentation supernatant of Staphylococcus epidermidis CCSM0287, which is characterized in that the seed liquid of CCSM0287 strain is inoculated into a liquid culture medium according to an inoculation amount of 1-3%, the rotation speed is 80-240r / min, and the culture is shaken and fermented for 6-16h to obtain a fermentation liquid, and the fermentation liquid is frozen and centrifuged to take the supernatant, which is the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain. The liquid culture medium is: 2-3g / L glucose, 4-6g / L sodium chloride, 2-3g / L dipotassium hydrogen phosphate, 16-18g / L tryptone, 2-4g / L soy peptone, and 1-3g / L fresh bovine collagen peptide powder.

[0007] The preparation method of the seed liquid culture medium is as follows: Staphylococcus epidermidis CCSM0287 cultured on a plate is inoculated into TSB liquid culture medium, and cultured on a shaking table at 35-40°C for 16-20h, and the OD is adjusted. 600 When the concentration reaches 0.55-0.56, it will be used as seed solution.

[0008] Preferably, the method comprises the following steps: inoculating the seed liquid of CCSM0287 strain into a liquid culture medium at an inoculum volume of 2%, shaking and culturing at a rotation speed of 160 r / min, and fermenting for 6 hours to obtain a fermentation liquid. The liquid culture medium comprises: 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 17 g / L tryptone, 3 g / L soy peptone, and 2 g / L fresh bovine collagen peptide powder.

[0009] The second object of the present invention is to provide a composition that can effectively inhibit the growth of Staphylococcus aureus biofilm, wherein the composition is compounded by fermentation supernatant of Staphylococcus epidermidis CCSM0287 and fermentation supernatant of Lactobacillus paracasei S20 in a volume ratio of 1:0.5 to 2.0 (preferably 1:1). The strain deposit number of Lactobacillus paracasei S20 is CGMCCNO.17120 (CN 110205266 B).

[0010] Preferably, the method for obtaining the fermentation supernatant of Lactobacillus paracasei S20 is: using MRS medium for anaerobic culture for 14 to 24 hours, preferably 19 hours, freezing and centrifuging the fermentation liquid, and taking the supernatant, which is the fermentation supernatant of Lactobacillus paracasei S20.

[0011] The technical effects of the present invention are:

[0012] 1. The present invention first optimizes the nitrogen source in the culture medium of Staphylococcus epidermidis CCSM0287 strain, and fully explores the growth differences of Staphylococcus epidermidis under different culture media and culture conditions and the inhibitory effect of its fermentation supernatant on Staphylococcus aureus biofilm; the results show that when the nitrogen source is trypsin + soy peptone + fresh bovine collagen peptide powder, and under optimized fermentation conditions (liquid volume 120ml, rotation speed 160r / min, fermentation 6h), the fermentation supernatant has the best inhibition rate of biofilm formation of Staphylococcus aureus, reaching 33%.

[0013] 2. The fermentation supernatant of Lactobacillus paracasei S20 cultured anaerobically in MRS medium for 19 hours had the best inhibition rate on biofilm formation of Staphylococcus aureus, reaching 38%.

[0014] 3. The fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain and the fermentation supernatant of Lactobacillus paracasei S20 strain synergistically enhance the efficacy. When combined in a volume ratio of 1:1, their inhibition rate on biofilm reached 82%, and they had a good effect on reducing the adhesion of Staphylococcus aureus biofilm growth process.

[0015] 4. After 24 hours of culture, Staphylococcus aureus cultured without adding the composition grew densely and formed a dense film on the surface. However, after adding the composition, the number of bacteria was significantly reduced, grew in a dispersed state, and had a smooth surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Effects of different nitrogen sources on the growth of Staphylococcus epidermidis CCSM0287;

[0017] Figure 2 :The biofilm inhibition rate of Staphylococcus epidermidis CCSM0287 against Staphylococcus aureus under different fermentation processes;

[0018] Figure 3 : The biofilm inhibition rate of Lactobacillus paracasei S20 against Staphylococcus aureus at different fermentation times;

[0019] Figure 4 : The biofilm inhibition rate of compositions with different ratios on Staphylococcus aureus;

[0020] Figure 5 :Effects of different proportions of compositions on the adhesion of Staphylococcus aureus;

[0021] Figure 6 : Effects of compositions on Staphylococcus aureus biofilm surfaces. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is further described below in conjunction with specific embodiments. The raw materials involved in the embodiments are all common commercially available products unless otherwise specified. The experimental methods without specific conditions specified in the embodiments are selected according to conventional methods and conditions, or according to the product instructions.

[0023] The materials and bacterial culture media used in the examples are as follows: crystal violet (Haibo Biotechnology); sodium chloride (Exploration Platform); Staphylococcus aureus (purchased from Beina Biotechnology); Staphylococcus epidermidis CCSM0287 (isolated from a healthy human body); Lactobacillus paracasei S20 (isolated from the fermented food "milk pimples"); TSA solid culture medium; MRS solid culture medium; MRS liquid culture medium.

[0024] The preparation method of seed liquid in the present invention is as follows:

[0025] (1) Bacterial Activation

[0026] The Staphylococcus epidermidis CCSM0287 and Staphylococcus aureus strains stored at -80°C were inoculated onto TSA solid plates using the three-zone streak method using a disposable inoculation loop, placed in a constant temperature incubator at 37°C for incubation for 16 hours, then taken out, and single colonies were picked and activated for two generations for use; Lactobacillus paracasei S20 was inoculated onto MRS solid plates using the three-zone streak method using a disposable inoculation loop, placed in a constant temperature incubator at 37°C for anaerobic incubation for 48 hours, then taken out, and single colonies were picked and activated for two generations for use.

[0027] (2) Seed solution preparation

[0028] The Staphylococcus epidermidis CCSM0287 and Staphylococcus aureus strains cultured on the plate were inoculated into TSB liquid culture medium, respectively, and cultured in a shaking incubator at 37°C for 16-20 hours. The fermentation broth was then taken out and the OD 600 , adjust OD 600 The Lactobacillus paracasei S20 cultured on the plate was inoculated into the MRS liquid medium, placed in a 37°C constant temperature incubator for anaerobic culture for 24 to 48 hours, and then taken out to measure the OD of the fermentation liquid. 600 , adjust OD 600 When the concentration reaches 0.8-1.0, it will be used as seed solution.

[0029] Example 1: Effects of culture media with different nitrogen source compositions on the growth of Staphylococcus epidermidis CCSM0287

[0030] The optimization of the culture medium is an important part of the strain culture process. Different culture medium compositions have a great influence on the growth of the strain and its products. The liquid culture medium used to prepare the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain includes carbon source, nitrogen source, inorganic salt and growth factor, among which the carbon source and growth factor are glucose, the inorganic salt is dipotassium hydrogen phosphate and sodium chloride, and the nitrogen source includes: beef extract, yeast extract, tryptone, soy peptone, fish meal peptone, millet collagen peptide powder, fresh bovine bone collagen peptide powder, walnut collagen peptide powder. The composition and content of the other nutrients in the culture medium except the nitrogen source are: glucose 2.5g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, and the nitrogen source in the culture medium is combined. The specific combination is shown in Table 1.

[0031] Table 1 Screening of nitrogen sources in culture medium

[0032] Nitrogen source composition (g / L) Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Beef extract - 8 - - 10 Yeast extract - - 15 - - Tryptone 17 - - 10 - Soybean Peptone 3 2 - - 2 Fishmeal Peptone - - 2 2 - Millet collagen peptide powder - - - - - Fresh Beef Bone Collagen Peptide Powder 2 - - 2 - Walnut collagen peptide powder - - 2 - 2

[0033] To verify the effect of different nitrogen sources on the growth of Staphylococcus epidermidis CCSM0287, the culture medium was prepared according to Table 1. The seed liquid of CCSM0287 strain was inoculated into 120 mL liquid culture medium at a 2% inoculation rate. The culture was shaken at 170 r / min. Samples were taken every two hours to measure the OD 600 The growth of Staphylococcus epidermidis CCSM0287 under the influence of different nitrogen sources was plotted. The results are as follows Figure 1 As shown in the results, Example 1 (nitrogen source using tryptone + soy peptone + fresh bovine collagen peptide powder) has the best effect.

[0034] Example 2: Effect of the fermentation process of Staphylococcus epidermidis CCSM0287 strain on Staphylococcus aureus biofilm

[0035] The growth mode of Staphylococcus epidermidis belongs to aerobic and facultative anaerobic type, so the oxygen content in the fermentation process has a great influence on the active substances in the fermentation broth. In order to explore the influence of fermentation supernatant under different fermentation processes on biofilm and find the best fermentation process, the culture medium in the above Example 1 was used for experiments, and the fermentation process was optimized from three aspects: the amount of liquid in the conical flask, the speed of the shaking table and the fermentation time. The specific group settings are shown in Table 2.

[0036] Table 2 Fermentation process optimization group settings

[0037] Group Liquid volume (mL) Speed ​​(r / min) Time (h) S1 80 80 6 S2 80 160 10 S3 80 240 16 S4 100 80 10 S5 100 160 16 S6 100 240 6 S7 120 80 16 S8 120 160 6 S9 120 240 10

[0038] The above fermentation culture used 250mL conical flasks and was inoculated into liquid culture medium at a 2% inoculum. After the culture was completed, a refrigerated centrifuge was used to centrifuge at 4°C for 10 minutes at 10,000 r / min. The supernatant was taken as the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain, and the supernatant was passed through a 0.22 μm membrane to remove excess bacteria. 136 ml TSB + 12 ml Staphylococcus aureus suspension (OD 600 =0.55-0.56) + 52 ml sterile saline, add 136 ml TSB + 12 ml Staphylococcus aureus suspension (OD 600 =0.55-0.56) + 52 ml of fermentation supernatant of CCSM0287 strains of different groups, 4 duplicate wells are set for each group. The well plate is placed in a 37°C constant temperature incubator for 24 hours to form a mature biofilm, then taken out and the liquid therein is poured out.

[0039] Well plate crystal violet staining method: Wash the well plate three times with 0.9% saline, dry it, add 0.01% crystal violet solution to the completely dried well plate, let it stand for 15 minutes to determine the remaining total biofilm, then wash it twice with sterile deionized water to wash away the excess crystal violet solution, dry it in a drying oven, and then use 95% ethanol to release the fixed crystal violet solution, and measure the absorbance at 595nm. According to OD 595 The biofilm inhibition rate was calculated based on the value of , where the inhibition rate was calculated as follows:

[0040] Inhibition rate (%) = (OD 595 - Experimental group OD 595 ) / control group OD 595

[0041] The results of the inhibition rate of fermentation supernatant on biofilm at different culture times are shown in Figure 2From the results, it can be seen that the fermentation supernatant of the S8 group (liquid volume 120ml, rotation speed 160r / min, fermentation 6h) has the best inhibition rate on the biofilm formation of Staphylococcus aureus, reaching 33%.

[0042] Example 3: Effect of Lactobacillus paracasei S20 fermentation supernatant on Staphylococcus aureus biofilm

[0043] CN 110205266 B mentions that Lactobacillus paracasei S20 is reported to have a strong inhibitory effect on Staphylococcus aureus, so it is speculated that its fermentation supernatant also has a good inhibitory effect on the formation of Staphylococcus aureus biofilm. Lactobacillus paracasei S20 is an anaerobic cultured microorganism. The fermentation supernatant obtained at different fermentation times contains different active molecules. In order to explore the effect of the supernatant cultured at different fermentation times on the Staphylococcus aureus biofilm, 4 parallel experiments were set up. The seed liquid of Lactobacillus paracasei S20 was inoculated in MRS liquid culture medium at an inoculum amount of 2%. The bacterial suspension was taken out after culturing for 14h, 19h, 24h and 29h respectively. A refrigerated centrifuge was used to centrifuge at 10000r / min for 10min at 4°C. The supernatant was taken as the fermentation supernatant of Lactobacillus paracasei S20, and passed through a 0.22μm membrane to remove excess bacteria. 136ml TSB+12ml Staphylococcus aureus suspension (OD 600 =0.55-0.56) + 52 ml sterile saline, add 136 ml TSB + 12 ml Staphylococcus aureus suspension (OD 600 =0.55~0.56)+52ml S20 strain fermentation supernatant cultured for different time periods, 4 duplicate wells were set in each group, the well plate was placed in a 37°C constant temperature incubator and cultured for 24 hours to form a mature biofilm, then taken out, the liquid therein was discarded, the well plate crystal violet staining method experimental steps in Example 2 were repeated, and the inhibition rate of Lactobacillus paracasei S20 on Staphylococcus aureus biofilm was calculated.

[0044] The results of the inhibition rate of the fermentation supernatant of Lactobacillus paracasei S20 at different culture times on biofilm are shown in Figure 3 From the results, it can be seen that the 19-hour fermentation supernatant has the best inhibition rate on the biofilm formation of Staphylococcus aureus, reaching 38%.

[0045] Example 4: Effect of the composition on the growth and adhesion of Staphylococcus aureus biofilm

[0046] By screening the nitrogen source in the liquid culture medium for culturing Staphylococcus epidermidis, the liquid culture medium with the best growth effect of Staphylococcus epidermidis under the same conditions was selected. Based on this liquid culture medium, the fermentation process of Staphylococcus epidermidis was optimized. It was found that the liquid culture medium containing nitrogen sources as tryptone, soy peptone and fresh bovine collagen peptide powder, the fermentation process was: liquid volume 120mL, speed 160r / min, time 6h, the best effect of inhibiting Staphylococcus aureus biofilm. The fermentation supernatant obtained by anaerobic culture of Lactobacillus paracasei S20 for 19h also has a good effect of inhibiting Staphylococcus aureus biofilm. The fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain and the fermentation supernatant of Lactobacillus paracasei S20 strain were combined into mixtures according to the volume ratio of 1:2, 1:1 and 2:1, and the crystal violet staining method was used to determine the inhibition rate of Staphylococcus aureus biofilm and the adhesion effect of different proportions of the composition during the growth of the biofilm.

[0047] The determination method of biofilm inhibition rate is the same as in Examples 2 and 3, and the results are shown in Figure 4 It can be found that when the ratio of Staphylococcus epidermidis fermentation filtrate to Lactobacillus paracasei fermentation filtrate is 1:1, the inhibition rate of biofilm is 82%, which can effectively inhibit the growth of Staphylococcus aureus biofilm. When determining the adhesion effect, the grouping scheme is the same as in Examples 2 and 3. Seven 96-well plates are set up, and each group is set up with 4 duplicate wells. After culturing for 4, 8, 10, 12, 16, 20, and 24 hours, a plate is taken out, the liquid therein is poured out, and the experimental steps of the well plate crystal violet staining method in Example 2 are repeated. The results are shown in FIG. Figure 5 From the adhesion effect point of view, the composition has a good effect of reducing the adhesion of Staphylococcus aureus biofilm growth process, among which the adhesion reduction effect is best when the ratio of Staphylococcus epidermidis fermentation filtrate to Lactobacillus paracasei fermentation filtrate is 1:1.

[0048] Example 5: Effect of the composition on the surface of Staphylococcus aureus biofilm

[0049] The surface morphology of the Staphylococcus aureus biofilm after and without the addition of the composition was observed using a scanning electron microscope. Two groups of experiments were set up using a 24-well plate. A sterilized PVC plate of 0.8 cm × 0.8 cm × 0.08 cm was added to the well to allow Staphylococcus aureus to attach and grow thereon. The experimental group was added with: PVC plate + 50 μL of Staphylococcus aureus suspension + 200 μL of 1:1 mixed 287 strain and S20 strain fermentation supernatant + 500 μL of TSB medium, and the control group was added with: PVC plate + Staphylococcus aureus suspension (OD 600=0.55~0.56) 50μL + 200μL physiological saline + 500μL TSB culture medium, set 3 duplicate wells for each group, place the 24-well plate in a 37℃ constant temperature incubator for 24 hours, take it out, wash it twice with sterilized PBS, take out the PVC material plate, dry it, mark the front side of the PVC material (the side with biofilm) with a marker, spray a layer of metal foil on the front side of the PVC material to increase conductivity, put it under a scanning electron microscope for observation and filming with a magnification of 8000 times, and the results are shown in Figure 6 It can be clearly observed that after culturing Staphylococcus aureus for 24 hours without adding the composition, the bacteria grew densely and formed a dense film on the surface, while after adding the composition, the number of bacteria was significantly reduced, grew in a dispersed state, and the surface was smooth.

Claims

1. A method for preparing the fermentation supernatant of Staphylococcus epidermidis CCSM0287, characterized in that: The seed liquid of CCSM0287 strain is inoculated into a liquid culture medium at an inoculation rate of 1-3%, and the culture is shaken at a rotation speed of 80-240 r / min and fermented for 6-16 hours to obtain a fermentation liquid. The fermentation liquid is freeze-centrifuged and the supernatant is taken, which is the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain; the liquid culture medium comprises: 2-3 g / L glucose, 4-6 g / L sodium chloride, 2-3 g / L dipotassium hydrogen phosphate, 16-18 g / L tryptone, 2-4 g / L soy peptone and 1-3 g / L fresh bovine collagen peptide powder.

2. The preparation method according to claim 1, characterized in that: The preparation method of the seed liquid culture medium is as follows: Staphylococcus epidermidis CCSM0287 cultured on a plate is inoculated into TSB liquid culture medium, and cultured on a shaking table at 35-40° C. for 16-20 hours, and the OD is adjusted. 600 When the concentration reaches 0.55-0.56, it will be used as seed solution.

3. The preparation method according to claim 1, characterized in that: The seed liquid of CCSM0287 strain was inoculated into the liquid culture medium at an inoculum rate of 2%, and the culture was shaken at a rotation speed of 160 r / min and fermented for 6 hours to obtain a fermentation liquid.

4. The preparation method according to claim 1, characterized in that: The liquid culture medium comprises: 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 17 g / L tryptone, 3 g / L soy peptone, and 2 g / L fresh bovine collagen peptide powder.

5. The fermentation supernatant of Staphylococcus epidermidis CCSM0287 prepared by the method according to any one of claims 1 to 4.

6. A composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm, the composition being prepared by compounding the fermentation supernatant of Staphylococcus epidermidis CCSM0287 and the fermentation supernatant of Lactobacillus paracasei S20 according to claim 5 in a volume ratio of 1:0.5 to 2.

0.

7. The composition according to claim 6, characterized in that The fermentation supernatant of Staphylococcus epidermidis CCSM0287 and the fermentation supernatant of Lactobacillus paracasei S20 are compounded in a volume ratio of 1:

1.

8. The composition according to claim 6, characterized in that The method for obtaining the fermentation supernatant of Lactobacillus paracasei S20 is as follows: using MRS medium for anaerobic culture for 14 to 24 hours, freezing and centrifuging the fermentation liquid, and taking the supernatant, which is the fermentation supernatant of Lactobacillus paracasei S20.

9. The composition according to claim 8, characterized in that The cells were cultured anaerobically using MRS medium for 19 h.

10. Use of the composition according to any one of claims 6 to 9 in inhibiting the growth of Staphylococcus aureus biofilm.

Images