Novel bacterium-fungus mixed biological membrane system and preparation method thereof
By improving Mcbain culture medium and specific growth environmental conditions, a mixed biofilm of Streptococcus mutans, Viagra and Candida albicans was successfully constructed, which solved the problem of failure to successfully cultivate this system in the prior art, provided an experimental basis for studying the interaction of caries biofilms, and improved the stability and structural integrity of the biofilm.
Patent Information
- Application Number
- CN202510106059.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art has not successfully cultivated a bacteria-fungal hybrid biofilm system formed by Streptococcus mutans, Viagra and Candida albicans, and lacks the experimental basis for studying the distribution of plaque biofilm and the mechanism of bacteria-fungal interaction in caries biofilms.
Through innovative methods, using modified Mcbain culture medium and specific growth environmental conditions, a mixed biofilm of Streptococcus mutans, Viagra and Candida albicans were successfully constructed. The specific steps include culturing each bacterial species separately, mixing and culturing it in different environments to ensure the growth needs and interactions of each bacterial species.
The successful construction of the mixed biofilm of Streptococcus mutans, Viagra and Candida albicans was achieved, providing an experimental basis for studying the distribution of plaque biofilms and the bacteria-fungal interaction mechanism in caries biofilms, and improving the stability and structural integrity of the biofilm.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel bacteria-fungus mixed biofilm system and a preparation method thereof. Background Art
[0002] The oral cavity is one of the important sites for human microbial colonization. These microorganisms interact with each other and with the host to form a stable dynamic balance. When the changes in local environmental factors exceed the regulatory capacity of the microecology, the dynamic balance is broken, which will not only lead to the occurrence of oral diseases such as dental caries, periodontal disease and mucosal diseases, but also become a factor in the occurrence of systemic diseases such as cardiovascular disease and digestive system diseases.
[0003] Streptococcus mutans is recognized as one of the main cariogenic bacteria. With the development of modern molecular biology such as sequencing technology and metagenomics, Streptococcus and Veillonella have been confirmed to be dominant genera in oral saliva and dental plaque of different types of caries, and are considered to be the core microbial community of caries. There is a synergistic relationship between cariogenic streptococci such as Veillonella parvula (ATCC 10790) and Streptococcus mutans (ATCC 25175), which promotes the transformation of oral microbial state to pathogenic state. Streptococcus mutans is a Gram-positive, facultative anaerobic coccus, and Veillonella mutans is a Gram-negative, anaerobic micrococcus. They can grow and reproduce together in an anaerobic environment, at a growth temperature of 37°C, and in brain heart infusion broth (BHI) medium. The laboratory biofilm culture technology of Streptococcus mutans and Veillonella parvula has been very mature. Candida albicans (ATCC 10231) is a Gram-positive, aerobic, round or oval unicellular fungus. It is the most commonly isolated Candida species in clinical practice, with a detection rate of 30-35% in healthy adult oral cavity. When the individual's immunity is low, Candida infection that settles in the human body can cause conditional disease and is an important factor in maintaining the balance of oral microecology. Candida albicans has a synergistic interaction with Streptococcus mutans, resulting in biofilms with greater microbial carrying and infectivity, and will enhance antimicrobial resistance.
[0004] In the etiology of caries, microorganisms are considered to be essential factors for the occurrence and development of caries. Streptococcus mutans is recognized as one of the main cariogenic bacteria. Veillonella parvula is an early colonizer of dental plaque biofilm, distributed in high abundance in the oral cavity, and plays an important role in a variety of oral and systemic diseases. Teng et al. used 20 bacterial groups including Streptococcus mutans and Veillonella parvula to diagnose early childhood caries, with a diagnostic accuracy of 70%. Studies have shown that the abundance of Candida albicans in the saliva of infants and young children with ECC is significantly increased, and there is a high degree of consistency with the increase in the abundance of Streptococcus mutans. Veillonella parvula is widely distributed in the oral microecology, and Candida albicans is one of the resident microorganisms in the oral cavity and an important factor in maintaining the balance of the oral microecology. Streptococcus mutans, Veillonella parvula, and Candida albicans are all related to the occurrence of caries to varying degrees.
[0005] Up to now, the cultivation of bacterial-fungal mixed biofilm systems formed by Streptococcus mutans, Veillonella parvum and Candida albicans is still blank. However, the bacterial-fungal mixed biofilm systems formed by Streptococcus mutans, Veillonella parvum and Candida albicans play an important role in oral health, disease development, drug resistance, colonization ability and metabolic activity of biofilms. The development of bacterial-fungal mixed biofilm systems formed by Streptococcus mutans, Veillonella parvum and Candida albicans provides an experimental basis for further studying the distribution of plaque biofilm flora and exploring the mechanism of bacterial-fungal interaction in caries biofilms. Summary of the invention
[0006] Purpose of the invention: The purpose of the present invention is to address the deficiencies of the prior art and provide a novel bacterial-fungal mixed biofilm system and a preparation method. A bacterial-fungal mixed biofilm system formed by Streptococcus mutans, Veillonella parvula, and Candida albicans is cultivated through an innovative method, which provides an experimental basis for further studying the distribution of plaque biofilm flora and exploring the bacterial-fungal interaction mechanism in caries biofilms.
[0007] Technical solution: The present invention discloses a novel bacteria-fungus mixed biofilm system, wherein the bacteria are Streptococcus mutans and Veillonella parvum, and the fungus is Candida albicans.
[0008] The strain of mutans Streptococcus was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on November 13, 2020, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Xinyang City, Henan Province, and west of Xingsheng Road; the preservation number is BNCC 337082; the strain of Veillonella parvum was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on April 01, 2022, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Yang City, Henan Province, and west of Xingsheng Road; the preservation number is BNCC 294853; the strain of Candida albicans was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on December 24, 2024, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Yang City, Henan Province, and west of Xingsheng Road; the preservation number is BNCC 186382.
[0009] The present invention also provides a method for preparing a novel bacteria-fungus mixed biofilm system, comprising the following steps:
[0010] Step 1, separately culturing the suspensions of Streptococcus mutans, Veillonella parvula and Candida albicans;
[0011] Step 2, preparing a modified McBain medium;
[0012] Step 3, collect cells from the culture medium of Streptococcus mutans and Veillonella parvum, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to prepare a standard bacterial suspension with a concentration of 1×106 cells / ml;
[0013] Step 4: Mix the standard bacterial suspension of Streptococcus mutans and Veillonella parvum in a ratio of 1:1, take 10 ml and inoculate it into the modified McBain medium in step 2, and culture it at 37° C. under anaerobic environment for 10 hours to form a two-species biofilm culture system;
[0014] Step 5: Collect the cells in the culture medium of Candida albicans, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to a concentration of 2×10 5 / ml standard bacterial suspension;
[0015] Step 6: inoculate the standard suspension of Candida albicans into the two-species biofilm culture system, and culture at 37° C. and 5% CO 2 for 48 hours to form a three-species mixed biofilm of Streptococcus mutans, Veillonella parvum and Candida albicans.
[0016] Furthermore, the culture of mutans streptococci in step 1 is specifically as follows: the mutans streptococci are cultured at 37°C, 5% CO2 environment to the logarithmic growth phase, the cells are collected by centrifugation at 2100×g for 10 min at room temperature, washed three times with sterile PBS, and then resuspended in BHI culture medium to prepare a concentration of 1×10 6 10 ml of standard bacterial suspension of Streptococcus mutans was inoculated into a culture dish and cultured at 37°C with 5% CO2.
[0017] Furthermore, the culturing of Veillonella parvum in step 1 is specifically as follows: culturing Veillonella parvum to the logarithmic growth phase at 37°C under anaerobic conditions, collecting the cells by centrifugation at 2100×g for 10 min at room temperature, washing them three times with sterile PBS, and then resuspending them in BHI culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Veillonella parvula and inoculate it into a culture dish, and culture it at 37°C under anaerobic environment.
[0018] Furthermore, the cultivation of Candida albicans in step 1 is specifically as follows: in a shaker at 200 rpm, 37°C, 5% CO2 environment, the Candida albicans is cultured to the logarithmic growth phase, the cells are collected by centrifugation at 2100×g for 10 min at room temperature, washed three times with sterile PBS, and then resuspended in YPD culture medium to prepare a concentration of 1×10 6 10 ml of the standard bacterial suspension of Candida albicans was inoculated into a culture dish and cultured at 37°C and 5% CO2.
[0019] Further, the components of the modified McBain medium in step 2 are: mucin 2.5 g / L, bacterial peptone 2.0 g / L, tryptone 2.0 g / L, yeast extract 1.0 g / L, sodium chloride 0.35 g / L, potassium chloride 0.2 g / L, calcium chloride 0.2 g / L, cysteine hydrochloride 0.1 g / L, hammine 0.001 g / L, vitamin K 0.0001 g / L, PIPES buffer 15 g / L, sodium hydroxide 2.5 g / L, and sucrose 2.0 g / L.
[0020] Beneficial effects: Compared with the prior art, the advantages of the present invention are:
[0021] (1) Based on the successful cultivation of three strains of Streptococcus mutans, Veillonella parvula, and Candida albicans, the present invention successfully discovered that the modified McBain medium can be used as a mixed biofilm culture substrate to culture mixed biofilms;
[0022] (2) Based on the different growth environment requirements of Streptococcus mutans, which is a facultative anaerobe, Veillonella parvum, which is an anaerobic bacterium, and Candida albicans, which is an aerobic fungus, and by utilizing the growth characteristics of Streptococcus mutans as a facultative anaerobe, it was successfully discovered that an early biofilm of Streptococcus mutans and Veillonella parvum could be first cultured in an anaerobic environment, and then Candida albicans could be added to grow together in an aerobic environment, thereby successfully constructing a mixed biofilm of three species of Streptococcus mutans, Veillonella parvum, and Candida albicans;
[0023] (3) The present invention utilizes Streptococcus mutans to synthesize extracellular polysaccharide (EPS) by metabolizing sucrose. EPS can help bacteria adhere to the tooth surface. A high content of EPS is also beneficial to improving the stability and structural integrity of the biofilm. Therefore, Streptococcus mutans and Veillonella parvum are first cultured so that the extracellular polysaccharide synthesized by Streptococcus mutans covers Veillonella parvum, thereby providing an anaerobic environment for Veillonella parvum, thereby adding Candida albicans for aerobic culture without affecting the culture of Veillonella parvum.
[0024] (4) The scanning electron microscope (SEM) image of the three-species mixed biofilm of Streptococcus mutans, Veillonella parvula, and Candida albicans standard strains successfully constructed by the present invention proves the morphology: Streptococcus mutans, Veillonella parvula, and Candida albicans can be seen in the three-species mixed biofilm, confirming the successful construction of the mixed biofilm culture. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a scanning electron micrograph of a mixed biofilm of Streptococcus mutans, Veillonella parvum, and Candida albicans standard strains;
[0026] Figure 2 This is a scanning electron micrograph of a mixed biofilm of Streptococcus mutans and Veillonella parvum;
[0027] Figure 3 is a graph of the test results of Test Example 1;
[0028] Figure 4 is a graph of the test results of Test Example 2;
[0029] Figure 5 is a graph of the test results of Test Example 3;
[0030] Figure 6 It is the culture medium with different ratios of complex nitrogen sources in Experimental Example 3. DETAILED DESCRIPTION
[0031] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.
[0032] Example 1 A novel bacterial-fungal hybrid biofilm system
[0033] like Figure 1 A novel bacterial-fungal mixed biofilm system is shown, wherein the bacteria are Streptococcus mutans and Veillonella parvum, and the fungus is Candida albicans.
[0034] The strain of Streptococcus mutans used in this example was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on November 13, 2020, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Xinyang City, Henan Province, and west of Xingsheng Road; the collection number is BNCC 337082; the strain of Streptococcus parvum was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on April 01, 2022, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Yangyang City, Henan Province, and west of Xingsheng Road; the collection number is BNCC 294853; the strain of Candida albicans was purchased from the Henan Industrial Microbiological Strain Engineering Technology Research Center on December 24, 2024, with an address of No. 12, north of the eastern section of Jingangtai Avenue, Chengguan Town, Shangcheng County, Yangyang City, Henan Province, and west of Xingsheng Road; the collection number is BNCC 186382.
[0035] Figure 1 This is a scanning electron microscope (SEM) image of a mixed biofilm of Streptococcus mutans, Veillonella parvum, and Candida albicans standard strains. The presence of Streptococcus mutans, Veillonella parvum, and Candida albicans can be seen in the image, confirming the successful construction of mixed biofilm culture. Compared with the biofilms of single species of Streptococcus mutans, Veillonella parvum, and Candida albicans, the structure of the mixed biofilm of the three species is more compact, with more extracellular polysaccharides, and the bacteria are richly wrapped. Candida albicans and Veillonella parvum cluster together and adhere tightly to the chain-like Streptococcus mutans; Figure 2 This is a scanning electron microscope image of a mixed biofilm of Streptococcus mutans and Veillonella parvum, showing that Veillonella parvum cannot form a strong biofilm, the biofilm is loose and not dense, the bacterial bodies are far apart, and there are many gaps. Compared with the biofilm of single-species Streptococcus mutans, the dual-species biofilm of Veillonella parvum and Streptococcus mutans has a denser structure, more extracellular polysaccharides, and the bacteria are richly coated. Veillonella parvum clusters and adheres to the chain-like Streptococcus mutans.
[0036] Example 2 Preparation method of a novel bacterial-fungal mixed biofilm system
[0037] Step 1, separately culturing the suspensions of Streptococcus mutans, Veillonella parvula and Candida albicans;
[0038] The specific steps of culturing mutans streptococci were as follows: the mutans streptococci were cultured at 37°C and 5% CO2 until the logarithmic growth phase, the cells were collected by centrifugation at 2100×g for 10 min at room temperature, washed three times with sterile PBS, and then resuspended in BHI culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Streptococcus mutans and inoculate it into a culture dish. Culture and grow it at 37°C and 5% CO2.
[0039] The specific steps of culturing Veillonella parvum were as follows: culturing Veillonella parvum at 37°C in an anaerobic environment until the logarithmic growth phase, centrifuging at 2100×g for 10 min at room temperature to collect the cells, washing them three times with sterile PBS, and resuspending them in BHI culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Veillonella parvula and inoculate it into a culture dish, and culture it at 37°C under anaerobic environment.
[0040] The specific steps of culturing Candida albicans were as follows: the Candida albicans was cultured in a shaking incubator at 200 rpm, 37°C, and 5% CO2 until the logarithmic growth phase, the cells were collected by centrifugation at 2100 × g for 10 min at room temperature, washed three times with sterile PBS, and then resuspended in YPD culture medium to a concentration of 1 × 10 6 10 ml of the standard bacterial suspension of Candida albicans was inoculated into a culture dish and cultured at 37°C and 5% CO2.
[0041] If Streptococcus mutans, Veillonella parvum and Candida albicans need to be revived and subcultured, proceed as follows:
[0042] Mutans Streptococcus: Inoculate mutans Streptococcus preserved at -80℃ into blood agar culture dishes for recovery, and invert and culture at 37℃ for 48h. Take a monoclonal mutans Streptococcus strain in 10ml BHI culture medium, and grow to the logarithmic growth phase at 37℃ and 5% CO2. Blood Agar solid culture medium: Add 1.8% agar to blood agar culture medium, autoclave at 121℃ for 15 minutes, cool to about 50℃ in a clean bench, and dispense into sterile disposable bacterial culture dishes. After cooling and solidification, seal and invert in a 4℃ refrigerator for later use.
[0043] Veillonella parvum: Inoculate the -80℃ preserved Veillonella parvum into a BHIL culture dish for recovery, and invert and culture at 37℃ for 48h. Take a monoclonal Veillonella parvum strain in 10ml BHIL culture medium and grow it to the logarithmic growth phase under anaerobic environment at 37℃. BHIL solid culture medium: Add 1.8% agar to the BHIL culture medium, sterilize it at 121℃ for 15 minutes, cool it to about 50℃ in a clean bench, divide it into portions, pour it into a sterile disposable bacterial culture dish, seal it after cooling and solidifying, and invert it in a 4℃ refrigerator for later use.
[0044] Candida albicans: Inoculate the -80℃ preserved Candida albicans into SDA culture dishes for recovery, and invert and culture at 37℃ for 48h. Take the monoclonal Candida albicans strain in 10ml YPD culture medium, and grow it to the logarithmic growth phase at 37°C, 5% CO2, and 200rpm shaking overnight. Sabouraud Dextrose Agar (SDA) solid culture medium: Add 1.8% agar to SDA culture medium, autoclave at 121℃ for 15 minutes, cool to about 50℃ in the clean bench, dispense, pour into sterile disposable bacterial culture dishes, seal after cooling and solidification, and invert in a 4℃ refrigerator for use. Yeast Extra ct Peptone Dextrose Medium (YPD) culture medium, autoclave at 121℃ for 15 minutes, cool and seal for use.
[0045] Step 2, prepare a modified McBain medium; the components of the modified McBain medium are: 2.5 g / L mucin, 2.0 g / L bacterial peptone, 2.0 g / L tryptone, 1.0 g / L yeast extract, 0.35 g / L sodium chloride, 0.2 g / L potassium chloride, 0.2 g / L calcium chloride, 0.1 g / L cysteine hydrochloride, 0.001 g / L hamming, 0.0001 g / L vitamin K, 15 g / L PIPES buffer, 2.5 g / L sodium hydroxide, and 2.0 g / L sucrose.
[0046] Step 3, collect cells from the culture medium of Streptococcus mutans and Veillonella parvum, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to prepare a standard bacterial suspension with a concentration of 1×106 cells / ml;
[0047] Step 4: Mix the standard bacterial suspension of Streptococcus mutans and Veillonella parvum in a ratio of 1:1, take 10 ml and inoculate it into the modified McBain medium in step 2, and culture it at 37° C. under anaerobic environment for 10 hours to form a two-species biofilm culture system;
[0048] Step 5: Collect the cells in the culture medium of Candida albicans, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to a concentration of 2×10 5 / ml standard bacterial suspension;
[0049] Step 6: inoculate the standard suspension of Candida albicans into the two-species biofilm culture system, and culture at 37° C. and 5% CO 2 for 48 hours to form a three-species mixed biofilm of Streptococcus mutans, Veillonella parvum and Candida albicans.
[0050] Test Example 1
[0051] The culture order of this test example is to culture Streptococcus mutans and Candida albicans first, and then add Veillonella parvula. Other test conditions are consistent with those of Example 2. Figure 3 As shown, the result is that the biofilm system of the three bacteria mixture was not cultivated.
[0052] Test Example 2
[0053] The culture order of this test example is to culture Veillonella parvula and Candida albicans first, and then add Streptococcus mutans. Other test conditions are consistent with those in Example 2, such as Figure 4 As shown, the result is that the biofilm system in which the three bacteria are mixed was not cultivated.
[0054] Test Example 3
[0055] The components of the conventional McBain medium are: 1.0 g / L bacterial peptone, 2.0 g / L tryptone, 2.5 g / L mucin, 1.0 g / L yeast extract, 0.35 g / L sodium chloride, 0.2 g / L potassium chloride, 0.2 g / L calcium chloride, 0.1 g / L cysteine hydrochloride, 0.001 g / L ham, 0.0001 g / L vitamin K, 15 g / L PIPES buffer, 2.5 g / L sodium hydroxide, and 2 g / L sucrose. The modified McBain medium in step 2 of Example 2 is replaced with the conventional McBain medium, such as Figure 5 As shown, the test results are: biofilm formation can be seen, but the combination is not tight and there are many gaps between bacteria.
[0056] Compared with conventional McLaurel's medium, the modified McLaurel's medium in Example 2 has a higher proportion of peptone. It can be seen that the modified McLaurel's medium is more suitable for the culture of a mixed biofilm of Streptococcus mutans, Veillonella parvum and Candida albicans.
[0057] Peptone is rich in organic nitrogen compounds, vitamins and sugars, and is the main raw material for microbial culture media. It can provide nutrients such as nitrogen source, carbon source or growth factor for microorganisms. Peptone and yeast extract are both sources of nitrogen sources. Compared with culture media containing only peptone or only yeast extract, composite nitrogen sources (peptone + yeast extract) have a promoting effect on bacterial growth. Figure 6 As shown, this experiment screened the ratio of complex nitrogen source (peptone + yeast extract) in the culture medium at a ratio of 1:2, 1:1, 2:1, and 3:1 (peptone: yeast extract), and found that a ratio of 2:1 could better meet the nutritional needs of bacterial growth and metabolism.
[0058] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims
1. A novel bacterial-fungal hybrid biofilm system, characterized in that: The bacteria are Streptococcus mutans and Veillonella parvum, and the fungus is Candida albicans.
2. A method for preparing a novel bacterial-fungal mixed biofilm system, characterized in that The steps include: Step 1, separately culturing the suspensions of Streptococcus mutans, Veillonella parvula and Candida albicans; Step 2, preparing a modified McBain medium; Step 3, collect cells from the culture medium of Streptococcus mutans and Veillonella parvum, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to prepare a standard bacterial suspension with a concentration of 1×106 cells / ml; Step 4: Mix the standard bacterial suspension of Streptococcus mutans and Veillonella parvum in a ratio of 1:1, take 10 ml and inoculate it into the modified McBain medium in step 2, and culture it at 37° C. under anaerobic environment for 10 hours to form a two-species biofilm culture system; Step 5: Collect the cells in the culture medium of Candida albicans, centrifuge at 2100×g for 10 min at room temperature, wash three times with sterile PBS, and resuspend in modified McBain culture medium to a concentration of 2×10 5 / ml standard bacterial suspension; Step 6: inoculate the standard suspension of Candida albicans into the two-species biofilm culture system, and culture at 37° C. and 5% CO 2 for 48 hours to form a three-species mixed biofilm of Streptococcus mutans, Veillonella parvum and Candida albicans.
3. The method for preparing a novel bacteria-fungus mixed biofilm system according to claim 2, characterized in that: The specific steps of culturing mutans streptococci in step 1 are as follows: culturing mutans streptococci at 37° C. and 5% CO2 to the logarithmic growth phase, collecting the cells by centrifugation at 2100×g for 10 min at room temperature, washing them three times with sterile PBS, and then resuspending them in BHI culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Streptococcus mutans and inoculate it into a culture dish. Culture and grow it at 37°C and 5% CO2.
4. The method for preparing a novel bacteria-fungus mixed biofilm system according to claim 2, characterized in that: The specific steps of culturing Veillonella parvum in step 1 are as follows: culturing Veillonella parvum to the logarithmic growth phase at 37° C. in an anaerobic environment, collecting the cells by centrifugation at a rate of 2100×g for 10 min at room temperature, washing them three times with sterile PBS, and then resuspending them in BHI culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Veillonella parvula and inoculate it into a culture dish, and culture it at 37°C under anaerobic environment.
5. The method for preparing a novel bacteria-fungus mixed biofilm system according to claim 2, characterized in that: The culturing of Candida albicans in step 1 is specifically as follows: culturing Candida albicans in a shaker at 200 rpm, 37° C., 5% CO2 environment to the logarithmic growth phase, centrifuging at 2100×g for 10 min at room temperature to collect the cells, washing with sterile PBS three times, and then resuspending in YPD culture medium to prepare a concentration of 1×10 6 Take 10 ml of the standard bacterial suspension of Candida albicans and inoculate it into a culture dish. Cultivate and grow it at 37°C and 5% CO2.
6. The method for preparing a novel bacteria-fungus mixed biofilm system according to claim 2, characterized in that: The components of the modified McBain medium in step 2 are: 2.5 g / L mucin, 2.0 g / L bacterial peptone, 2.0 g / L tryptone, 1.0 g / L yeast extract, 0.35 g / L sodium chloride, 0.2 g / L potassium chloride, 0.2 g / L calcium chloride, 0.1 g / L cysteine hydrochloride, 0.001 g / L ham, 0.0001 g / L vitamin K, 15 g / L PIPES buffer, 2.5 g / L sodium hydroxide, and 2.0 g / L sucrose.