Postbiotics for inhibiting oral pathogenic bacteria as well as preparation method and application thereof

Abstract

The invention discloses a metagen for inhibiting oral pathogenic bacteria as well as a preparation method and application thereof, and belongs to the technical field of microbial fermentation. The metagen provided by the invention is prepared by the following method: inoculating lactobacillus plantarum DSM12028 into a culture medium for activation; inoculating the activated culture of the strain into a culture medium for second-generation culture and fermentation; and after fermentation is finished, performing high-temperature and high-pressure sterilization on the culture solution to obtain the lactobacillus plantarum DSM12028 metagen. The lactobacillus plantarum DSM12028 metaplast has the advantages that the lactobacillus plantarum DSM12028 metaplast has a remarkable inhibition effect on streptococcus mutans, staphylococcus aureus and candida albicans, so that the lactobacillus plantarum DSM12028 metaplast has an important value in the aspect of preventing oral diseases such as decayed teeth and oropharyngeal candidiasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and in particular relates to a postbiotic for inhibiting oral pathogenic bacteria, a preparation method and application thereof. Background Art

[0002] Dental caries and oropharyngeal candidiasis are oral diseases with a very high incidence rate, which are mainly caused by oral pathogens such as Streptococcus mutans, Candida albicans and Staphylococcus aureus.

[0003] Streptococcus mutans can grow and proliferate on the surface of teeth, and form biofilm by secreting extracellular polysaccharides. It is one of the main components of dental plaque, making it difficult to interfere with and destroy it with conventional oral care. In addition, Streptococcus mutans is acid-producing and acid-resistant, and is the main cariogenic bacteria in the oral cavity. It can quickly utilize carbohydrates in the oral cavity and produce a large amount of acid, which reduces the local pH to 4.5-5. The biofilm formed also blocks the buffering effect of saliva, causing the local area of ​​the tooth to be in an acidic environment for a long time and corroded, resulting in the destruction of tooth enamel, the formation of tooth holes and causing caries.

[0004] Staphylococcus aureus is a major human opportunistic pathogen that is associated with a variety of infectious diseases and can cause severe inflammation in many sites, including the oral mucosa, skin, bones, blood, and intestines. In the oral cavity, S. aureus can be isolated from saliva, tongue, mucosal surfaces, gingival surfaces, and periodontal surfaces. Soft tissue infections in the oral and maxillofacial regions and osteomyelitis of the jaws can be caused by this bacterium. In addition, S. aureus can cause dental implant failures. Due to the bacteria's resistance to multiple antibiotics, its infections have been the largest healthcare burden worldwide.

[0005] Candida albicans is a fungus that colonizes the oral mucosal surfaces of 30-70% of healthy individuals. Due to local or systemic immunosuppression, this commensal fungus is able to proliferate, leading to an oral disease known as oropharyngeal candidiasis (OPC). However, in healthy individuals, C. albicans does not cause harm. Commensal fungi, such as C. albicans, are essential for microbiome structure, metabolic function, and immune priming. However, an imbalance in the fungal flora balance, known as fungal dysbiosis, alters the functional composition, structure, and metabolic activity of the host microbiome. Dysfunction may ultimately lead to mucosal fungal infection, causing oropharyngeal candidiasis.

[0006] Therefore, developing a method that can inhibit the above-mentioned oral pathogens is of great value for the prevention of oral diseases such as caries and oropharyngeal candidiasis. Summary of the invention

[0007] In order to overcome the drawbacks of the prior art, the present invention provides a method for preparing a postbiotic for inhibiting oral pathogens, comprising the following steps:

[0008] (1) Strain activation and inoculation:

[0009] Inoculate Lactobacillus plantarum DSM12028 in liquid culture medium for activation, with an inoculation amount of 1-4%;

[0010] (2) Second generation culture and fermentation:

[0011] The activated strain is inoculated into the culture medium for secondary culture and fermentation, with the inoculation amount being 1-4%;

[0012] (3) Sterilization and postbiotic acquisition:

[0013] After the fermentation is completed, the culture solution is sterilized in a high temperature and high pressure sterilizer at 121°C for 30 minutes to obtain Lactobacillus plantarum postbiotics.

[0014] The inoculation amount in step (1) is 2%; the activation condition is anaerobic fermentation at 30-37° C. for 12-36 hours.

[0015] The activation condition of step (1) is anaerobic fermentation at 37° C. for 24 hours.

[0016] The inoculation amount in step (2) is 2%, and the fermentation conditions are anaerobic fermentation at 30-37°C for 12-36h;

[0017] The fermentation condition of step (2) is anaerobic fermentation at 37° C. for 24 hours.

[0018] The liquid culture medium is an MRS liquid culture medium, and the formula of the MRS liquid culture medium is: 20g glucose, 15g whey protein, 20g soy powder, 10g skim milk powder, and 1L distilled water; before inoculation and fermentation, the liquid culture medium is sterilized at a high temperature of 100-115°C for 30min.

[0019] A Lactobacillus plantarum postbiotic is prepared by the method for preparing a postbiotic for inhibiting oral pathogens.

[0020] A preparation for preventing and treating oral pathogens comprises the Lactobacillus plantarum postbiotic.

[0021] Application of a Lactobacillus plantarum postbiotic in the preparation of a preparation for inhibiting Streptococcus mutans, Staphylococcus aureus and / or Candida albicans.

[0022] Application of a Lactobacillus plantarum postbiotic in the preparation of a preparation for preventing and treating dental caries and oropharyngeal candidiasis.

[0023] The beneficial effects of the present invention are:

[0024] The present invention prepares a postbiotic by utilizing Lactobacillus plantarum DSM12028. The Lactobacillus plantarum postbiotic has a significant inhibitory effect on Streptococcus mutans, Staphylococcus aureus and Candida albicans, and thus has important value in preventing oral diseases such as dental caries and oropharyngeal candidiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the effect diagram of the antibacterial effect test;

[0026] Figure 2 This is a picture of the antibacterial effect taken with a scanning electron microscope;

[0027] Figure 3 is the growth curve of Streptococcus mutans;

[0028] Figure 4 is the growth curve of Staphylococcus aureus;

[0029] Figure 5 This is the growth curve of Candida albicans;

[0030] Figure 6 This is a biofilm inhibition test diagram. DETAILED DESCRIPTION

[0031] In the present invention, the Lactobacillus plantarum DSM12028 was purchased from Testol Biotech.

[0032] In the present invention, Streptococcus mutans UA159 was obtained from Shandong University, Staphylococcus aureus ATCC6538 was purchased from Testol Biotechnology, and Candida albicans DAY185 was obtained from Ocean University of China.

[0033] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0034] Example 1

[0035] A method for preparing a postbiotic for inhibiting oral pathogens comprises the following steps:

[0036] (1) Strain activation and inoculation:

[0037] Lactobacillus plantarum DSM12028 was inoculated in a liquid culture medium for activation, the inoculation amount was 1%, and the activation condition was anaerobic fermentation at 30°C for 36h;

[0038] (2) Second generation culture and fermentation:

[0039] The activated strain was inoculated into the culture medium for second generation culture and fermentation, with an inoculation amount of 1%; the fermentation conditions were anaerobic fermentation at 30°C for 36 hours;

[0040] (3) Sterilization and acquisition of postbiotics: After the fermentation is completed, the culture solution is sterilized in a high-temperature and high-pressure sterilizer at 121°C for 30 minutes to obtain Lactobacillus plantarum postbiotics.

[0041] The liquid culture medium is an MRS liquid culture medium, and the formula of the MRS liquid culture medium is: 20g glucose, 15g whey protein, 20g soy powder, 10g skim milk powder, and 1L distilled water; before inoculation and fermentation, the liquid culture medium is subjected to high-temperature sterilization treatment at 115°C for 30min.

[0042] The present invention also relates to a Lactobacillus plantarum postbiotic, which is prepared by the method for preparing a postbiotic for inhibiting oral pathogens.

[0043] The present invention also relates to a preparation for preventing and treating oral pathogenic bacteria, comprising the Lactobacillus plantarum postbiotic.

[0044] The present invention also relates to the use of a plant lactobacillus postbiotic in the preparation of a preparation for inhibiting Streptococcus mutans, Staphylococcus aureus and / or Candida albicans.

[0045] The present invention also relates to the use of a Lactobacillus plantarum postbiotic in the preparation of a preparation for preventing and treating dental caries and oropharyngeal candidiasis.

[0046] Embodiment 2:

[0047] A method for preparing a postbiotic for inhibiting oral pathogens comprises the following steps:

[0048] (1) Strain activation and inoculation: Lactobacillus plantarum DSM12028 was inoculated into a liquid culture medium for activation, with an inoculation amount of 2%; the activation condition was anaerobic fermentation at 37° C. for 24 h;

[0049] (2) Second generation culture and fermentation: The activated strain was inoculated into the culture medium for second generation culture and fermentation, with an inoculation amount of 2%, and anaerobic fermentation was carried out at 37°C for 24 h;

[0050] (3) Sterilization and acquisition of postbiotics: After the fermentation is completed, the culture solution is sterilized in a high-temperature and high-pressure sterilizer at 121°C for 30 min to obtain Lactobacillus plantarum postbiotics.

[0051] The liquid culture medium is an MRS liquid culture medium, and the formula of the MRS liquid culture medium is: 20g glucose, 15g whey protein, 20g soy powder, 10g skim milk powder, and 1L distilled water; before inoculation and fermentation, the liquid culture medium is subjected to high-temperature sterilization treatment at 115°C for 30min.

[0052] The present invention also relates to a Lactobacillus plantarum postbiotic, which is prepared by the method for preparing a postbiotic for inhibiting oral pathogens.

[0053] The present invention also relates to a preparation for preventing and treating oral pathogenic bacteria, comprising the Lactobacillus plantarum postbiotic.

[0054] The present invention also relates to the use of a plant lactobacillus postbiotic in the preparation of a preparation for inhibiting Streptococcus mutans, Staphylococcus aureus and / or Candida albicans.

[0055] The present invention also relates to the use of a Lactobacillus plantarum postbiotic in the preparation of a preparation for preventing and treating dental caries and oropharyngeal candidiasis.

[0056] Example 3: A method for preparing a postbiotic for inhibiting oral pathogens, comprising the following steps:

[0057] (1) Strain activation and inoculation: Lactobacillus plantarum DSM12028 was inoculated into liquid culture medium for activation, with an inoculation amount of 4%; the activation condition was anaerobic fermentation at 37° C. for 24 h;

[0058] (2) Second generation culture and fermentation: The activated strain was inoculated into the culture medium for second generation culture and fermentation, with an inoculation amount of 4%; the fermentation conditions were anaerobic fermentation at 37°C for 24 hours;

[0059] (3) Sterilization and acquisition of postbiotics: After the fermentation is completed, the culture solution is sterilized in a high-temperature and high-pressure sterilizer at 121°C for 30 min to obtain Lactobacillus plantarum postbiotics.

[0060] The liquid culture medium is an MRS liquid culture medium, and the formula of the MRS liquid culture medium is: 20g glucose, 15g whey protein, 20g soy powder, 10g skim milk powder, and 1L distilled water; before inoculation and fermentation, the liquid culture medium is subjected to high-temperature sterilization treatment at 115°C for 30min.

[0061] The present invention also relates to a Lactobacillus plantarum postbiotic, which is prepared by the method for preparing a postbiotic for inhibiting oral pathogens.

[0062] The present invention also relates to a preparation for preventing and treating oral pathogenic bacteria, comprising the Lactobacillus plantarum postbiotic.

[0063] The present invention also relates to the use of a plant lactobacillus postbiotic in the preparation of a preparation for inhibiting Streptococcus mutans, Staphylococcus aureus and / or Candida albicans.

[0064] The present invention also relates to the use of a Lactobacillus plantarum postbiotic in the preparation of a preparation for preventing and treating dental caries and oropharyngeal candidiasis.

[0065] Based on the Lactobacillus plantarum postbiotics prepared in Examples 1 to 3, the specific experiments are as follows:

[0066] 1. Antibacterial test

[0067] 1. Streptococcus mutans

[0068] Add 100 μL of 10% 6 CFU / mL of Streptococcus mutans suspension and 100 μL of Lactobacillus plantarum postbiotics at concentrations of 12.5%, 25%, 50%, and 100%, and an equal amount of water was added to the control group. After culturing in a constant temperature incubator at 37°C for 24 h, the OD was measured using an enzyme marker. 600 value.

[0069] 2. Staphylococcus aureus

[0070] Add 100 μL of 10% 6 CFU / mL of Staphylococcus aureus suspension and 100 μL of Lactobacillus plantarum postbiotics at concentrations of 12.5%, 25%, 50%, and 100%, and an equal amount of water was added to the control group. After culturing in a constant temperature incubator at 37°C for 24 h, the OD was measured using an enzyme marker. 600 value.

[0071] 3. Candida albicans

[0072] Add 100 μL of 10% 6 CFU / mL of Candida albicans suspension and 100 μL of Lactobacillus plantarum postbiotics at concentrations of 12.5%, 25%, 50%, and 100%, and an equal amount of water was added to the control group. After culturing in a constant temperature incubator at 30°C for 24 h, the OD was measured using an enzyme reader. 600 value.

[0073] The test results are as follows Figure 1-Figure 2 As shown:

[0074] The plant lactobacillus postbiotics prepared by the invention have significant inhibitory effects on streptococci, staphylococci aureus and candida albicans.

[0075] 2. Growth Curve

[0076] 1. Streptococcus mutans

[0077] Dilute Streptococcus mutans with saline to 1×10 6 CFU / mL of bacterial solution, 500 μL of bacterial solution was added to a 48-well plate, and then 500 μL of Lactobacillus plantarum postbiotics diluted with water at a concentration of 50% and 100% were added to the wells respectively. The control wells were replaced with an equal amount of water to form a 1 mL culture system. Each sample was repeated 3 times and cultured at 37°C with shaking. The OD was measured every 1 h using a microbial growth analyzer. 600 After 24 hours, all the results were statistically analyzed and plotted.

[0078] 2. Staphylococcus aureus

[0079] Staphylococcus aureus was diluted with saline to 1×10 6 CFU / mL of bacterial solution, 500 μL of bacterial solution was added to a 48-well plate, and then 500 μL of Lactobacillus plantarum postbiotics diluted with water at a concentration of 50% and 100% were added to the wells respectively. The control wells were replaced with an equal amount of water to form a 1 mL culture system. Each sample was repeated 3 times and cultured at 37°C with shaking. The OD was measured every 1 h using a microbial growth analyzer. 600 After 24 hours, all the results were statistically analyzed and plotted.

[0080] 3. Candida albicans

[0081] Candida albicans was diluted with saline to 1×10 6 CFU / mL of bacterial solution, 500 μL of bacterial solution was added to a 48-well plate, and then 500 μL of Lactobacillus plantarum postbiotics diluted with water at a concentration of 50% and 100% were added to the wells respectively. The control wells were replaced with an equal amount of water to form a 1 mL culture system. Each sample was repeated 3 times, shaken and cultured at 30°C, and the OD was measured every 1 hour using a microbial growth analyzer. 600 After 24 hours, all the results were statistically analyzed and plotted.

[0082] The test results are as follows Figure 3 to Figure 5 As shown:

[0083] The growth of microorganisms in the blank control group showed a typical S-shaped growth curve. After adding plant lactobacillus postbiotics, the growth of microorganisms can be significantly inhibited. Plant lactobacillus postbiotics at a concentration of more than 50% can basically completely inhibit the growth of Streptococcus mutans and Staphylococcus aureus, and plant lactobacillus postbiotics at a concentration of 100% can also effectively inhibit the growth of Candida albicans.

[0084] 3. Biofilm inhibition test

[0085] According to the literature [Food Research and Development, 2021, 42(13):180-187], the inhibitory effect of Lactobacillus plantarum postbiotics on the biofilm formation of Streptococcus mutans, Staphylococcus aureus and Candida albicans was determined by crystal violet staining.

[0086] The specific steps are as follows:

[0087] 1. Streptococcus mutans

[0088] Add 100 μL of 10% 6 CFU / mL of Streptococcus mutans suspension and 100 μL of Lactobacillus plantarum postbiotics with concentrations of 12.5%, 25%, 50% and 100%, an equal amount of water was added to the control group, and the cells were cultured in a constant temperature incubator at 37°C for 48 h, the upper culture medium and free bacteria were removed, and the cells were washed three times with 100 μL PBS, and then 100 μL 0.1% crystal violet was added for staining for 15 min, and the crystal violet was absorbed; the cells were washed three times with sterile water to remove the floating color, and after drying, they were decolorized with 30% acetic acid solution, and the OD was measured with an enzyme marker. 570nm Determination of biofilm formation ability The OD value of the blank control group was set as 100% biofilm formation, and the percentage of biofilm formation in the group supplemented with Lactobacillus plantarum was calculated based on this value.

[0089] 2. Staphylococcus aureus

[0090] Add 100 μL of 10% 6 CFU / mL of Staphylococcus aureus suspension and 100 μL of 12.5%, 25%, 50% and 100% Lactobacillus plantarum postbiotics were added. An equal amount of water was added to the control group. After culturing in a constant temperature incubator at 37°C for 48 hours, the upper culture medium and free bacteria were removed, and the cells were washed three times with 100 μL PBS, and then 100 μL of 0.1% crystal violet was added for staining for 15 minutes, and the crystal violet was absorbed; the cells were washed three times with sterile water to remove the floating color, and after drying, they were decolorized with 30% acetic acid solution, and the OD was measured with an enzyme marker. 570nm The OD value of the blank control group was set as 100% biofilm formation, and the percentage of biofilm formation in the group with postbiotics was calculated based on this value.

[0091] 3. Candida albicans

[0092] Add 100 μL of 10% 6CFU / mL of Candida albicans suspension and 100 μL of 12.5%, 25%, 50% and 100% Lactobacillus plantarum postbiotics were added to the control group. An equal amount of water was added to the control group. After culturing in a constant temperature incubator at 30°C for 48 hours, the upper culture medium and free fungi were removed, washed three times with 100 μL PBS, and then 100 μL 0.1% crystal violet was added for staining for 15 minutes, and the crystal violet was absorbed; washed three times with sterile water to wash away the floating color, and after drying, it was decolorized with 30% acetic acid solution, and the OD was measured with an enzyme marker. 570nm The OD value of the blank control group was set as 100% biofilm formation, and the percentage of biofilm formation in the group with postbiotics was calculated based on this value.

[0093] The test results are as follows Figure 6 As shown:

[0094] The plant lactobacillus postbiotics prepared by the present invention have a significant inhibitory effect on the formation of Streptococcus mutans, Staphylococcus aureus and Candida albicans biofilms, and as the concentration increases, the inhibitory effect on the biofilm is stronger, showing typical concentration dependence. And it is reported that the biofilm of microorganisms is closely related to the pathogenicity and virulence of microorganisms, especially related to its antibiotic resistance (Frontiers in Microbiology, 2022). It can be seen from this that the plant lactobacillus postbiotics of the present invention have good anti-Streptococcus mutans, Staphylococcus aureus and Candida albicans effects.

[0095] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a postbiotic for inhibiting oral pathogens, characterized in that: The steps include: (1) Strain activation and inoculation: Inoculate Lactobacillus plantarum DSM12028 in liquid culture medium for activation, with an inoculation amount of 1-4%; (2) Second generation culture and fermentation: The activated strain is inoculated into the culture medium for secondary culture and fermentation, with the inoculation amount being 1-4%; (3) Sterilization and postbiotic acquisition: After the fermentation is completed, the culture solution is sterilized in a high temperature and high pressure sterilizer at 121°C for 30 minutes to obtain Lactobacillus plantarum postbiotics.

2. The method for preparing a postbiotic for inhibiting oral pathogens according to claim 1, characterized in that: The inoculation amount in step (1) is 2%; the activation condition is anaerobic fermentation at 30-37° C. for 12-36 hours.

3. The method for preparing a postbiotic for inhibiting oral pathogens according to claim 2, characterized in that: The activation condition of step (1) is anaerobic fermentation at 37° C. for 24 hours.

4. A method for preparing a postbiotic for inhibiting oral pathogens according to claim 2 or 3, characterized in that: The inoculation amount in step (2) is 2%, and the fermentation conditions are anaerobic fermentation at 30-37° C. for 12-36 hours.

5. The method for preparing a postbiotic for inhibiting oral pathogens according to claim 4, characterized in that: The fermentation condition of step (2) is anaerobic fermentation at 37° C. for 24 hours.

6. The method for preparing a postbiotic for inhibiting oral pathogens according to claim 5, characterized in that: The liquid culture medium is an MRS liquid culture medium, and the formula of the MRS liquid culture medium is: 20g glucose, 15g whey protein, 20g soy powder, 10g skim milk powder, and 1L distilled water; before inoculation and fermentation, the liquid culture medium is sterilized at a high temperature of 100-115°C for 30min.

7. A Lactobacillus plantarum postbiotic, characterized in that The invention discloses a postbiotic for inhibiting oral pathogens according to any one of claims 1 to 6.

8. A preparation for preventing and treating oral pathogens, comprising the Lactobacillus plantarum postbiotic according to claim 7.

9. Use of a Lactobacillus plantarum postbiotic in the preparation of a preparation for inhibiting Streptococcus mutans, Staphylococcus aureus and / or Candida albicans.

10. Use of a Lactobacillus plantarum postbiotic in the preparation of a preparation for preventing and treating dental caries and oropharyngeal candidiasis.

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