A composition effective to inhibit staphylococcus aureus biofilm growth

Abstract

The application discloses a composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm. The application firstly 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 inhibition effect of the fermentation supernatant on Staphylococcus aureus biofilm, then compounds the fermentation supernatant of Staphylococcus epidermidis CCSM0287 obtained by using the optimized culture medium (the nitrogen source adopts tryptone + soybean peptone + fresh beef bone collagen peptide powder) with the fermentation supernatant of Lactobacillus paracasei S20, can obviously inhibit the growth of Staphylococcus aureus biofilm and destroy the surface structure of the Staphylococcus aureus biofilm, and can be applied in bacteriostatic products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm. BACKGROUND

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of gram-positive bacteria, and is one of the main pathogenic bacteria causing skin and soft tissue infections. The related substances causing pathogenicity of Staphylococcus aureus include Staphylococcus aureus biofilm, efflux pump, toxin and plasmid, etc. At present, the best method for treating Staphylococcus aureus infection is mainly antibiotic treatment, but the frequent use of antibiotics enhances the drug resistance of Staphylococcus aureus, leading to its spread worldwide, seriously affecting social public health. How to treat the infection caused by Staphylococcus aureus with gradually enhanced drug resistance is a research hotspot at present. Biofilm is a thick and unique three-dimensional structure of extracellular matrix formed by the adhesion, proliferation and secretion of extracellular polymer molecules of Staphylococcus aureus on a certain plane. It forms a protective barrier around the bacteria, which hinders the contact of external adverse factors with the bacteria. Therefore, the generation of Staphylococcus aureus biofilm is the main reason for Staphylococcus aureus to escape immunity and produce drug resistance, and thus destroying the synthesis and degradation of biofilm is one of the effective methods to prevent the colonization and infection of Staphylococcus aureus.

[0003] Staphylococcus epidermidis, as the dominant flora on the skin, plays an important role in maintaining the healthy state of the skin. As a part of the skin ecosystem, Staphylococcus epidermidis not only can communicate with skin cells, but also can interact with different microorganisms on the skin. The substances such as antibacterial peptides produced by Staphylococcus epidermidis have certain defense functions, which can protect the host from pathogen invasion and help the host shape the immune system to prevent 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 realized by some substances secreted by Staphylococcus epidermidis, which can inhibit the secretion of toxins and proteases of Staphylococcus aureus, thereby reducing the damage to the skin.

[0004] CN115637235B provides a strain of Staphylococcus epidermidis CCSM0287, which is preserved in China Center for Type Culture Collection (CCTCC) on June 1, 2022, and the preservation number is CCTCC M 2022779. Experiments have proved that the Staphylococcus epidermidis CCSM0287 strain is a potential probiotic strain with good antioxidant and anti-inflammatory effects. The CCSM0287 strain is a strain with development potential. The formation of the bacterial biofilm cannot be separated from the existence of the quorum sensing phenomenon. The fermentation metabolites of Staphylococcus epidermidis contain various active molecules that can effectively inhibit the formation of Staphylococcus aureus biofilm. However, the selection of different culture conditions and culture media has a certain influence on the fermentation metabolites of Staphylococcus epidermidis, and thus affects its efficacy. However, the prior art lacks the selection of Staphylococcus epidermidis culture medium and compositions that can effectively inhibit the growth of Staphylococcus aureus biofilm. SUMMARY

[0005] To solve the above problems, the present application provides a composition that can effectively inhibit the growth of Staphylococcus aureus biofilm. The present application first optimizes the nitrogen source in the culture medium of Staphylococcus epidermidis CCSM0287 strain, fully explores the growth differences of Staphylococcus epidermidis under different culture media and culture conditions, and the inhibition of the fermentation supernatant of Staphylococcus epidermidis on Staphylococcus aureus biofilm, and then the fermentation supernatant of Staphylococcus epidermidis CCSM0287 obtained by optimizing the culture medium is compounded with the fermentation supernatant of Lactobacillus paracasei S20, which can obviously inhibit the growth of Staphylococcus aureus biofilm and destroy its surface structure, and can be applied in bacteriostatic products.

[0006] In order to achieve the above technical purpose, the present application first provides a preparation method of fermentation supernatant of Staphylococcus epidermidis CCSM0287, characterized in that the seed liquid of CCSM0287 strain is inoculated in liquid culture medium at an inoculation amount of 1-3%, and is shaken and cultured at a speed of 80-240 r / min, and is fermented for 6-16 h to obtain fermentation liquid. The fermentation liquid is frozen and centrifuged, and the upper clear liquid is taken as the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain. The liquid culture medium is: glucose 2-3 g / L, sodium chloride 4-6 g / L, potassium phosphate dibasic 2-3 g / L, tryptone 16-18 g / L, soybean peptone 2-4 g / L, and fresh bovine collagen peptide powder 1-3 g / L.

[0007] The preparation method of the above seed liquid culture medium is: Staphylococcus epidermidis CCSM0287 cultured on a plate is inoculated into TSB liquid culture medium, and shaken and cultured at 35-40℃ for 16-20 h, and the OD value is adjusted to 0.55-0.56 as seed liquid for standby. 600

[0008] ​Preferably, it is specifically: the seed liquid of the CCSM0287 strain is inoculated in a liquid culture medium at an inoculation amount of 2%, and is cultured by shaking at a speed of 160 r / min, and is fermented for 6 h to obtain a fermentation liquor. The liquid culture medium is: glucose 2.5 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, tryptone 17 g / L, soybean peptone 3 g / L, and fresh beef collagen peptide powder 2 g / L.

[0009] A second object of the present application is to provide a composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm, which is prepared by compounding the fermentation supernatant of Staphylococcus epidermidis CCSM0287 and the fermentation supernatant of Lactobacillus paracasei S20 at a volume ratio of 1:0.5-2.0 (preferably 1:1). The strain preservation number of Lactobacillus paracasei S20 is CGMCC NO.17120 (CN 110205266 B).

[0010] Preferably, the fermentation supernatant of Lactobacillus paracasei S20 is obtained by anaerobic culture using MRS medium for 14-24 h, preferably 19 h. The fermentation liquor is frozen and centrifuged, and the upper clear liquid is taken as the fermentation supernatant of Lactobacillus paracasei S20.

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

[0012] 1. The present application first optimizes the nitrogen source in the culture medium of Staphylococcus epidermidis CCSM0287 strain, fully explores the growth differences of Staphylococcus epidermidis under different culture media and culture conditions, and the inhibition effect of the fermentation supernatant on the biofilm of Staphylococcus aureus; it is found that the fermentation supernatant under the optimized fermentation conditions (liquid volume 120 ml, rotation speed 160 r / min, fermentation time 6 h) has the best inhibition rate on the biofilm formation of Staphylococcus aureus, reaching 33%.

[0013] 2. The fermentation supernatant of Lactobacillus paracasei S20 anaerobically cultured for 19 hours using MRS medium has the best inhibition rate on the 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 have synergistic effect, and the inhibition rate on the biofilm reaches 82% when they are combined at a volume ratio of 1:1, and they have good effect on reducing adhesion during the growth process of Staphylococcus aureus biofilm.

[0015] 4. The Staphylococcus aureus cultured without adding the composition grows densely after 24 hours, and a dense film is formed on the surface, while the number of bacteria is significantly reduced after adding the composition, and the bacteria grow in a dispersed manner, and the surface is smooth. BRIEF DESCRIPTION OF DRAWINGS

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

[0017] Figure 2 Inhibition rate of Staphylococcus epidermidis CCSM0287 on Staphylococcus aureus biofilm under different fermentation processes;

[0018] Figure 3 Inhibition rate of Lactobacillus paracasei S20 on Staphylococcus aureus biofilm under different fermentation time;

[0019] Figure 4 Inhibition rate of different proportion compositions on Staphylococcus aureus biofilm;

[0020] Figure 5 Effect of different proportion compositions on Staphylococcus aureus adhesion;

[0021] Figure 6 Effect of compositions on Staphylococcus aureus biofilm surface. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be further described below in combination with specific examples. The raw materials involved in the examples are all ordinary commercially available products, and the experimental methods not specified in the examples are selected according to the conventional methods and conditions or the product instructions.

[0023] The materials and bacterial culture media used in the examples are as follows: crystal violet (Hibio); sodium chloride (Exploration Platform); Staphylococcus aureus (purchased from Bei Na Biological); Staphylococcus epidermidis CCSM0287 (isolated from healthy human body); Lactobacillus paracasei S20 (isolated from fermented food "nai ge da"); TSA solid culture medium; MRS solid culture medium; MRS liquid culture medium.

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

[0025] (1) Strain activation

[0026] The Staphylococcus epidermidis CCSM0287 and Staphylococcus aureus strains preserved at -80℃ were inoculated on TSA solid plates using a disposable inoculation loop with a three-zone streaking method, and then taken out after being cultured in a 37℃ constant temperature incubator for 16h. Single colonies were picked and activated for two generations for standby use. The Lactobacillus paracasei S20 was inoculated on MRS solid plates using a disposable inoculation loop with a three-zone streaking method, and then taken out after being cultured in a 37℃ constant temperature incubator for 48h. Single colonies were picked and activated for two generations for standby use.

[0027] (2) Seed liquid preparation

[0028] Staphylococcus epidermidis strain CCSM0287 and Staphylococcus aureus strain, respectively, were inoculated into TSB liquid medium and cultured at 37°C with shaking for 16–20 h. The OD values ​​of the fermentation broth were then measured. 600 Adjust OD 600 The culture medium was prepared as a seed culture at a concentration of 0.55–0.56. Lactobacillus paracasei S20 cultured on plates was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24–48 hours. The OD value of the fermentation broth was then measured. 600 Adjust OD 600 Prepare a seed solution at a concentration of 0.8–1.0.

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

[0030] Optimization of the culture medium is a crucial step in the bacterial strain cultivation process, as different medium compositions significantly impact strain growth and its products. The liquid culture medium used to prepare the fermentation supernatant of *Staphylococcus epidermidis* strain CCSM0287 consisted of a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source and growth factor were glucose, the inorganic salts were dipotassium hydrogen phosphate and sodium chloride, and the nitrogen sources included several of the following: beef extract, yeast extract, tryptone, soybean peptone, fish meal peptone, millet collagen peptide powder, fresh bovine bone collagen peptide powder, and walnut collagen peptide powder. The composition and content of the remaining nutrients in the culture medium, excluding the nitrogen source, were: glucose 2.5 g / L, sodium chloride 5 g / L, and dipotassium hydrogen phosphate 2.5 g / L. The specific combinations of nitrogen sources in the culture medium are shown in Table 1.

[0031] Table 1. Screening of nitrogen sources in culture media

[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 Fish meal 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 effects of different nitrogen sources on the growth of Staphylococcus epidermidis CCSM0287, the culture medium was prepared according to Table 1. The seed culture of CCSM0287 strain was inoculated into 120 mL of liquid culture medium at a 2% inoculation rate. The medium was then shaken at 170 rpm and cultured. Samples were taken every two hours to measure the OD. 600 Values ​​were used to plot the growth of Staphylococcus epidermidis CCSM0287 under different nitrogen sources. The results are as follows: Figure 1 As shown, Example 1 (using tryptone + soybean peptone + fresh bovine bone collagen peptide powder as the nitrogen source) showed the best results.

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

[0035] The growth mode of Staphylococcus epidermidis belongs to aerobic facultative anaerobic type, and therefore 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 Example 1 above was used for experiment, and the fermentation process was optimized from three aspects of conical flask liquid volume, shaking speed and fermentation time by orthogonal optimization experiment method. The specific group setting is shown in Table 2.

[0036] Table 2 Fermentation process optimization group setting

[0037] Group Liquid volume (mL) Rotation 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 flask, inoculated in liquid culture medium according to 2% inoculation amount, after culture, used refrigerated centrifuge at 4℃, 10000r / min centrifugation for 10min, took the upper clear liquid as the fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain, passed through 0.22μm membrane to remove excess bacteria, took 96-well plate, added 136ml TSB+12ml Staphylococcus aureus bacterial suspension (OD 600 =0.55-0.56) + 52ml sterilized physiological saline in control group holes, added 136ml TSB+12ml Staphylococcus aureus bacterial suspension (OD 600 =0.55-0.56) + 52ml different groups of CCSM0287 strain fermentation supernatant in experimental group holes, set 4 duplicate holes in each group. After placing the hole plate in 37℃ constant temperature incubator for 24 hours to form mature biofilm, it was taken out and the liquid was poured out.

[0039] Crystal violet staining method of hole plate: washed the hole plate with 0.9% physiological saline for three times, dried, added 0.01% crystal violet solution to the completely dried hole plate, stood for 15min for staining to determine the remaining total biofilm amount, then washed twice with sterile deionized water to wash away the excess crystal violet solution, placed in a drying oven for drying, then released the fixed crystal violet solution with 95% ethanol, measured the absorbance at 595nm. According to the value of OD 595 , the biofilm inhibition rate was calculated, wherein the calculation method of inhibition rate was:

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

[0041] The inhibition rate results of biofilm by fermentation supernatant under different culture time are shown in Table 3. Figure 2From the results, it can be seen that the fermentation supernatant of the S8 group (liquid volume 120 ml, rotation speed 160 r / min, fermentation time 6 h) has the best inhibitory effect on the biofilm formation of S. aureus, reaching 33%.

[0042] Example 3: Effect of L. paracasei S20 fermentation supernatant on S. aureus biofilm

[0043] It is mentioned in CN 110205266 B that L. paracasei S20 has a very strong inhibitory effect on S. aureus, and it is therefore presumed that the fermentation supernatant of L. paracasei S20 has a good inhibitory effect on the formation of S. aureus biofilm. L. paracasei S20 belongs to anaerobic microorganisms, and the fermentation supernatant obtained at different fermentation times contains different active molecules. In order to explore the effect of the supernatant obtained at different fermentation times on S. aureus biofilm, four parallel experiments were set up. The seed liquid of L. paracasei S20 was inoculated into MRS liquid medium at an inoculation amount of 2%, and the bacterial suspension was taken out after 14 h, 19 h, 24 h and 29 h of culture, respectively. The bacterial suspension was centrifuged at 10,000 r / min for 10 min at 4°C using a refrigerated centrifuge, and the supernatant was taken as the fermentation supernatant of L. paracasei S20. The supernatant was filtered through a 0.22 μm membrane to remove excess bacteria. A 96-well plate was prepared by adding 136 ml of TSB + 12 ml of S. aureus bacterial suspension (OD 600 = 0.55-0.56) + 52 ml of sterilized physiological saline to the control group, and adding 136 ml of TSB + 12 ml of S. aureus bacterial suspension (OD 600 = 0.55-0.56) + 52 ml of fermentation supernatant of S20 strain cultured for different times to the experimental group. Four duplicate wells were set in each group. After the mature biofilm was formed by culturing the plate in a 37°C constant temperature incubator for 24 hours, the liquid was poured out, and the hole plate crystal violet staining method experiment steps in Example 2 were repeated. The inhibitory rate of L. paracasei S20 on S. aureus biofilm was calculated.

[0044] The inhibitory rate of L. paracasei S20 fermentation supernatant on biofilm at different culture times is shown in Table 3. Figure 3 From the results, it can be seen that the fermentation supernatant obtained at 19 hours has the best inhibitory effect on the biofilm formation of S. aureus, reaching 38%.

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

[0046] The nitrogen source in the liquid culture medium for Staphylococcus epidermidis was screened, and the liquid culture medium with the best growth effect of Staphylococcus epidermidis under the same conditions was selected. Based on the liquid culture medium, the fermentation process of Staphylococcus epidermidis was optimized, and it was found that the liquid culture medium containing nitrogen source of tryptone, soybean peptone and fresh bovine bone collagen peptide powder had the best inhibitory effect on Staphylococcus aureus biofilm when the fermentation process was as follows: liquid volume 120 mL, rotation speed 160 r / min, time 6 h. The fermentation supernatant of Lactobacillus paracasei S20 obtained after anaerobic culture for 19 h also had a good inhibitory effect on Staphylococcus aureus biofilm. The fermentation supernatant of Staphylococcus epidermidis CCSM0287 strain and the fermentation supernatant of Lactobacillus paracasei S20 strain were combined into a mixture in the volume ratio of 1:2, 1:1 and 2:1 respectively, and the inhibitory rate of the mixture with different proportions on Staphylococcus aureus biofilm and the adhesion effect in the growth process of biofilm were determined by crystal violet staining method.

[0047] The determination method of biofilm inhibition rate was the same as in examples 2 and 3, and the results were as follows: 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 on biofilm reaches 82%, which can effectively inhibit the growth of Staphylococcus aureus biofilm. The grouping scheme for determining the adhesion effect is the same as in examples 2 and 3, and 7 96-well plates are set, 4 duplicate wells are set in each group, and one plate is taken out after 4, 8, 10, 12, 16, 20 and 24 hours of culture respectively, and the liquid in the plate is poured out. The well plate crystal violet staining experiment steps in example 2 are repeated, and the results are as follows: Figure 5 From the adhesion effect, the composition has a good effect on reducing the adhesion of Staphylococcus aureus biofilm in the growth process, and the adhesion effect is best when the ratio of Staphylococcus epidermidis fermentation filtrate to Lactobacillus paracasei fermentation filtrate is 1:1.

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

[0049] The surface morphology of Staphylococcus aureus biofilm after adding and not adding the composition was observed by scanning electron microscope. Two groups of experiments were set using 24-well plates, and sterilized PVC plates with a size of 0.8 cm x 0.8 cm x 0.08 cm were added in the wells to make Staphylococcus aureus adhere to them and grow. In the experimental group, the PVC plate + Staphylococcus aureus bacterial suspension 50 μL + 200 μL 1:1 mixed 287 strain and S20 strain fermentation supernatant + TSB culture medium 500 μL were added, and in the control group, the PVC plate + Staphylococcus aureus bacterial suspension (OD 600= 0.55~0.56) 50 μL + 200 μL physiological saline + TSB medium 500 μL, 3 wells were set in each group, the 24-well plate was taken out after being cultured in a 37°C constant temperature incubator for 24 hours, was gently washed twice using sterilized PBS, the PVC material plate was taken out, dried, marked on the front side (the side with the biofilm) using a marker, a layer of metal foil was sprayed on the front side of the PVC material to increase the conductivity, was placed under a scanning electron microscope for observation at 8000 times magnification, and the results are shown in Figure 6 It can be obviously observed that the Staphylococcus aureus does not add the composition and is cultured for 24 hours, the bacterial bodies grow densely, and a dense film is formed on the surface; and after the composition is added, the number of bacterial bodies is obviously reduced, the bacterial bodies grow in a scattered manner, and the surface is smooth.

Claims

1. A composition capable of effectively inhibiting the growth of Staphylococcus aureus biofilm, the composition being compounded by Staphylococcus epidermidis CCSM0287 fermentation supernatant and Lactobacillus paracasei S20 fermentation supernatant at a volume ratio of 1:1; the accession number of the Staphylococcus epidermidis CCSM0287 is CCTCC M 2022779; the accession number of the Lactobacillus paracasei S20 is CGMCC NO.17120; The preparation method of the fermentation supernatant of the Staphylococcus epidermidis CCSM0287 is as follows: the seed liquid of the CCSM0287 strain is inoculated into a liquid medium at an inoculation amount of 1-3%, and is shaken and cultured at a speed of 80-160 r / min for 6 h to obtain a fermentation liquid; the fermentation liquid is frozen and centrifuged, and the upper clear liquid is taken as the fermentation supernatant of the Staphylococcus epidermidis CCSM0287 strain; the liquid medium is as follows: glucose 2-3 g / L, sodium chloride 4-6 g / L, potassium phosphate dibasic 2-3 g / L, tryptone 16-18 g / L, soybean peptone 2-4 g / L, and fresh bovine bone collagen peptide powder 1-3 g / L. The Lactobacillus paracasei S20 fermentation supernatant is obtained by anaerobic culture of the MRS medium for 19-24 h, freezing and centrifuging the fermentation liquid, and taking the upper clear liquid as the Lactobacillus paracasei S20 fermentation supernatant.

2. The composition of claim 1, wherein The Lactobacillus paracasei S20 fermentation supernatant is obtained by anaerobic culture of the MRS medium for 19-24 h, freezing and centrifuging the fermentation liquid, and taking the upper clear liquid as the Lactobacillus paracasei S20 fermentation supernatant.

3. The composition of claim 1, wherein The preparation method of the seed liquid medium of the CCSM0287 strain is that Staphylococcus epidermidis CCSM0287 cultured on a plate is inoculated into a TSB liquid medium, and is shaken and cultured at 35-40°C for 16-20 h, and the OD is adjusted to 0.55-0.56 as a seed liquid for standby. 600 to 0.55-0.56 as a seed liquid for standby.

4. The composition of claim 1, wherein The seed liquid of the CCSM0287 strain is inoculated into a liquid medium at an inoculation amount of 2%, and is shaken and cultured at a speed of 160 r / min for 6 h to obtain a fermentation liquid.

5. The composition of claim 1, wherein The liquid medium is as follows: glucose 2.5 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, tryptone 17 g / L, soybean peptone 3 g / L, and fresh bovine bone collagen peptide powder 2 g / L.

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