Lactobacillus delbrueckii subsp. Lactis TG001 and application thereof in improving oral health

Abstract

The invention discloses a lactobacillus delbrueckii subsp. Lactis TG001 and application thereof in improving oral health, and belongs to the technical field of probiotic application, the preservation number of the lactobacillus delbrueckii subsp. Lactis TG001 is CCTCC NO: M 20241941, the preservation place is China Center for Type Culture Collection in Wuhan, and the preservation date is September 9, 2024. The lactobacillus delbrueckii subsp. Lactis TG001 has a good inhibition effect on common oral pathogens such as streptococcus mutans and porphyromonas gingivalis, particularly can inhibit the growth of cariogenic bacteria streptococcus mutans by generating hydrogen peroxide and remove biofilm formation, and has good tolerance to antibacterial substances in an oral environment. Therefore, the oral cavity diseases such as dental caries, periodontitis and ozostomia can be prevented, the micro-ecological environment of the oral cavity is effectively improved, meanwhile, the peculiar smell of the oral cavity is relieved, and good application prospects are achieved in preparation of products for preventing and / or treating the oral cavity diseases such as dental caries and periodontitis.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic application, and particularly relates to a strain of Lactobacillus delbrueckii lactis subspecies TG001 and an application thereof in improving oral health. Background Art

[0002] As the entrance to the human digestive tract, the oral cavity has the second most complex microbial community after the intestines. It is known that there are more than 700 different bacterial species in the oral cavity. These microorganisms form a unique oral microecological system in different parts of the oral cavity, such as the lips, cheeks, tongue, hard palate, soft palate, teeth, gums, alveolar bone, gingival sulcus and saliva. In a healthy state, a dynamic balance is maintained between the oral microbial communities and between the microorganisms and the host. Once this balance is broken, it may lead to a series of oral diseases, such as caries, periodontal disease and halitosis, and may even cause oral cancer.

[0003] Common oral pathogens include Porphyromonas gingivalis ( Porphyromona sgingivalis )、Prevotella intermedia( Prevotellainterme-Dia ), Streptococcus gordonii ( Streptococcus sgordonii )、Streptococcus mutans( Streptococcus smutans ) etc. Streptococcus mutans is considered to be one of the main causes of dental caries and is also one of the bacteria that forms biofilms. It can cause tooth enamel loss, gingival swelling and inflammation, and affect the beauty and appearance of teeth, causing many inconveniences in patients' lives.

[0004] Traditional methods of oral disease prevention and treatment mainly include the use of antibacterial agents (such as chlorhexidine, fluoride, etc.) to kill pathogenic bacteria in the oral cavity. However, this method has obvious limitations and may destroy the balance of the oral microecological system and aggravate the problem of bacterial resistance. Given the importance and irreplaceability of microorganisms to the human body, as well as their complexity among different individuals, it is not feasible to directly eliminate pathogenic microorganisms.

[0005] In recent years, probiotics have been widely used in the treatment of gastrointestinal diseases, urinary tract infections, respiratory tract infections and other diseases. There are also more and more clinical studies on probiotics for oral diseases. Probiotics can inhibit oral pathogens through mechanisms such as adhesion, copolymerization, nutritional competition, and production of antibacterial substances. They can also maintain oral health by restoring the ecological balance of oral microbial communities.

[0006] Probiotic therapy for dental caries is a current research hotspot, but there are still relatively few probiotics in China that can prevent dental caries. We still need to tap into excellent local microbial resources and develop probiotics for dental caries prevention with independent intellectual property rights. As a new type of "bacteria-to-bacteria" biological therapy, the safety of probiotic therapy has been recognized by European and American food safety organizations and the National Health Commission of China (NHC). Summary of the invention

[0007] The present invention obtains a strain of Lactobacillus delbrueckii subspecies TG001 by isolating it from the feces of healthy people. The strain has a good inhibitory effect on common oral pathogens such as Streptococcus mutans and Porphyromonas gingivalis, and can especially inhibit the growth of Streptococcus mutans, a cariogenic bacterium, by producing hydrogen peroxide, and remove biofilm formation. Therefore, the occurrence of oral diseases such as caries, periodontitis, and halitosis can be prevented, the oral microecological environment can be effectively improved, and oral odor can be reduced at the same time. The strain has a good application prospect in the preparation of products for preventing and or treating oral diseases such as caries and periodontitis.

[0008] The present invention provides a strain of Lactobacillus delbrueckii subspecies lactis TG001 ( Lactobacillus delbrueckii subsp. lactis TG001), the Lactobacillus delbrueckii subspecies lactis TG001 has a preservation number of CCTCC NO: M 20241941, a preservation location of Wuhan China Type Culture Collection Center, and a preservation date of September 9, 2024.

[0009] Furthermore, the 16S rRNA sequence of Lactobacillus delbrueckii subsp. lactis TG001 is shown in SEQ ID NO:1.

[0010] The present invention also provides the use of the Lactobacillus delbrueckii subspecies lactis TG001 in inhibiting Streptococcus mutans or Porphyromonas gingivalis.

[0011] The present invention also provides the use of the Lactobacillus delbrueckii subspecies lactis TG001 in coaggregating mutans streptococci or Porphyromonas gingivalis.

[0012] The present application provides a probiotic preparation comprising the above-mentioned Lactobacillus delbrueckii subsp. lactis TG001 or at least one of its inactivated product, fermentation broth, and metabolites.

[0013] Furthermore, the probiotic preparation is an oral probiotic preparation that inhibits oral pathogenic bacteria.

[0014] Furthermore, the dosage form of the probiotic preparation includes tablets, lyophilized powders, capsules, granules, pills, pastes, liquid preparations, gels or solid beverages.

[0015] The present application also provides the use of the above-mentioned probiotic preparation in the preparation of products for preventing or treating dental caries, periodontitis and halitosis.

[0016] The beneficial effects of the present invention are: 1. The present invention provides a Lactobacillus delbrueckii subsp. lactis TG001 strain that can simultaneously inhibit oral pathogenic bacteria Streptococcus mutans and Porphyromonas gingivalis, which can effectively eliminate pathogenic bacteria in the oral cavity and improve oral health.

[0017] 2. The Lactobacillus delbrueckii subsp. lactis TG001 strain provided by the present invention can effectively eliminate the biofilm formation of oral pathogenic bacteria Streptococcus mutans clinical strain 593 and standard strain ATCC 25175.

[0018] 3. The Lactobacillus delbrueckii subsp. lactis TG001 strain provided by the present invention has good self-aggregation ability, and has a certain co-aggregation ability with Streptococcus mutans and Porphyromonas gingivalis, which is beneficial to the adhesion and colonization of the strain.

[0019] 4. Lactobacillus delbrueckii subspecies lactis TG001 has good tolerance, can survive in a low pH, high salt environment, and has a certain tolerance to hydrogen peroxide and lysozyme, indicating that it can exert its probiotic properties in the oral cavity.

[0020] 5. The Lactobacillus delbrueckii subsp. lactis TG001 strain of the present invention is sensitive to five antibiotics including macrolides and cephalosporins, and is moderately sensitive to five antibiotics including streptomycin, and is a safe and reliable probiotic. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology observation result of Lactobacillus delbrueckii subspecies lactis TG001.

[0022] Figure 2 This is the phylogenetic tree of Lactobacillus delbrueckii subsp. lactis TG001.

[0023] Figure 3 This is a graph showing the tolerance of Lactobacillus delbrueckii subspecies TG001 to lysozyme.

[0024] Figure 4 This is a graph showing the tolerance of Lactobacillus delbrueckii subspecies lactis TG001 to hydrogen peroxide.

[0025] Figure 5 This is a graph showing the acid tolerance results of Lactobacillus delbrueckii subspecies TG001.

[0026] Figure 6 This is a graph showing the salt tolerance results of Lactobacillus delbrueckii subspecies TG001.

[0027] Figure 7 This is the result of the hemolytic experiment of Lactobacillus delbrueckii subspecies TG001.

[0028] Figure 8 These are the results of the hydrogen peroxide production experiment with Lactobacillus delbrueckii subsp. lactis TG001. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0031] In view of the above problems, the present invention attempts to use an in vitro verified screening method to use 40 strains isolated from the intestines of healthy humans as an experimental group, and uses the Oxford cup punch method antibacterial experiment to screen out probiotics that have inhibitory effects on oral pathogens Streptococcus mutans and Porphyromonas gingivalis, and observes the colony morphology of the strains and detects the physiological and biochemical characteristics, and combines 16S rDNA analysis to determine the strain species, and analyzes the aggregation ability, tolerance and antibiotic sensitivity of probiotics through in vitro experiments, and uses crystal violet staining to determine the effect of probiotics on the biofilm formation of Streptococcus mutans and Porphyromonas gingivalis. In addition, the hydrogen peroxide production experiment proves that the probiotics mediate the inhibitory effect on Streptococcus mutans by producing hydrogen peroxide.

[0032] Example 1 Screening of probiotics The strains were screened using brain heart infusion (BHI) medium (g / L): peptone 10.0, dehydrated calf brain extract powder 12.5, dehydrated calf heart extract powder 5.0, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, and agar 15.0. The strains were derived from the feces of healthy adults in Xiamen, Fujian. Single colonies were inoculated on BHI agar plates and cultured at 37 °C and 200 rpm in a shaking incubator for 24-48 h. The Oxford cup punch method was then used to screen out probiotic strains that inhibit oral pathogens Streptococcus mutans and Porphyromonas gingivalis. BHI medium was used for Streptococcus mutans, and TSB medium was used for Porphyromonas gingivalis. A sterilized Oxford cup was placed in a disposable sterile culture dish. The solid culture medium was cooled to about 55 °C. The pathogenic bacteria suspension was added to the culture medium at a 1% inoculum rate. After being fully mixed with the culture medium, it was quickly poured into the plate with the Oxford cup placed in advance. After the culture medium was cooled and solidified, the Oxford cup was removed and 100 μL of probiotic suspension (1.0×10 8 CFU / mL). The positive control for Streptococcus mutans was 100 μg / mL chloramphenicol, and the negative control for Porphyromonas gingivalis was 200 μg / mL gentamicin. The negative control used an equal amount of BHI medium and cultured at 37 °C for 48 h. The strains with obvious antibacterial transparent zones were selected, and the diameter of the antibacterial zone was measured with a vernier caliper and recorded.

[0033] The Oxford cup punch method was used to screen the antibacterial effects of the 40 probiotic strains isolated, and the strains with inhibitory effects on Streptococcus mutans were screened according to the diameter of the inhibition zone. The results showed that among the 40 probiotic strains initially isolated, one strain had a significant inhibitory effect on Streptococcus mutans and could also inhibit Porphyromonas gingivalis. The diameter of the inhibition zone is shown in Table 2.

[0034] Table 2 Antibacterial effect of Lactobacillus delbrueckii subsp. lactis TG001 on Streptococcus mutans and Porphyromonas gingivalis

[0035] Example 2 Morphological observation and biological identification of strains The morphological identification of the selected probiotics with the ability to simultaneously inhibit Streptococcus mutans and Porphyromonas gingivalis was performed, and the results were as follows Figure 1 (A: Colony morphology of Lactobacillus delbrueckii subsp. lactis TG001 on BHI medium, B: Gram staining microscopic image of Lactobacillus delbrueckii subsp. lactis TG001) and Table 3. The colony size of the bacteria is medium, the colony edge is smooth, the colony surface is shiny, the colony morphology is convex, and the colony color is milky white. Gram staining is positive. The probiotic strain morphology observed under an optical microscope is curved rod-shaped, non-motile, and has no spores.

[0036] Table 3 Colony morphology and bacterial characteristics of Lactobacillus delbrueckii subsp. lactis TG001

[0037] The genomic DNA was extracted using a bacterial genomic DNA rapid extraction kit, and the extracted genomic DNA was used as a template for PCR amplification. The PCR product obtained by amplification was sent for sequencing. The sequence obtained by sequencing was compared with BLAST on the NCBI website. The results showed that the bacteria was a strain of Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp. lactis ), whose 16S rDNA sequence is shown in SEQ ID NO: 1.

[0038] SEQ ID NO: 1 The strain was compared with the known Lactobacillus delbrueckii subsp. lactis by BLAST comparison on the NCBI website. Lactobacillus delbrueckii subsp. lactis (Sequence ID: CP031023.1) has a similarity of 100%. In order to further understand the position of this strain in the phylogenetic relationship, the phylogenetic tree was constructed by combining the existing sequences of Lactobacillus delbrueckii subsp. lactis using the neighbor-joining method (NJ), and Aerococcus hominis (CCUG 42038B) was used as an external strain. Figure 2 The phylogenetic tree shows that the strain belongs to Lactobacillus delbrueckii subspecies lactis and is named Lactobacillus delbrueckii subspecies lactis TG001 ( Lactobacillus delbrueckii subsp. Lactis TG001).

[0039] Example 3 Determination of the self-aggregation ability of Lactobacillus delbrueckii subsp. lactis TG001 and its co-aggregation ability with pathogenic bacteria Self-aggregation ability The culture liquid of Lactobacillus delbrueckii subsp. lactis TG001 after 16 h of culture was centrifuged, the precipitated bacterial sludge was collected and washed three times with PBS, resuspended and the optical density value A at 600 nm was adjusted. 0 The optical density of the supernatant was 0.6±0.05 and incubated at 37°C for 4 h. t Repeat the test three times to calculate the strain self-aggregation. The self-aggregation calculation formula is:

[0040] Among them A 0 Representative initial OD of Lactobacillus delbrueckii subsp. lactis TG001 600 Value, A t Represents the OD measured after standing 600 value.

[0041] Coaggregation ability The culture of Lactobacillus delbrueckii subsp. lactis TG001 for 16 h was centrifuged, the supernatant culture was discarded, the bacterial sludge precipitate was collected, and washed with PBS for 3-5 times to remove the residual culture solution. After the activation of Streptococcus mutans and Porphyromonas gingivalis, the cells were collected by centrifugation, washed with PBS buffer twice, and resuspended to adjust the OD 600 0.5±0.05. Take 100 μL of Lactobacillus delbrueckii subspecies TG001 suspension and mix thoroughly with pathogenic bacteria, then shake (200 r / min for 5 min) and let stand at 37 °C for 4 h. The optical density of the bacterial suspension at 600 nm was measured by the supernatant, and the determination was repeated three times to calculate the copolymerization rate of Lactobacillus delbrueckii subspecies TG001 and pathogenic bacteria. The formula for calculating the cross-agglutination rate is:

[0042] Where: A x is the initial OD of probiotics 600 Value, A y is the initial OD of Streptococcus mutans 600 , A mix OD after 4 h of mixed culture of probiotics and Streptococcus mutans 600 value.

[0043] In the oral environment, for some pathogenic microorganisms that cause oral diseases, a higher self-aggregation ability helps them exist in the oral cavity in the form of biofilms, thereby leading to the occurrence of oral diseases such as dental caries. This example measured the self-aggregation ability of Lactobacillus delbrueckii subspecies TG001 at 37°C for 4 h. As shown in Table 4, Lactobacillus delbrueckii subspecies TG001 showed a strong self-aggregation ability, and the self-aggregation ability of 4 h reached 71.53%, proving that it is easier to survive in the oral cavity and has the advantage of rapid reproduction.

[0044] Coaggregation refers to the aggregation ability between different strains, which is expressed by the cross-aggregation rate. After the probiotics cross-aggregate with the planktonic mutans streptococci, there are two possible results. One result is that the aggregates do not adhere to the teeth and are discharged from the mouth with swallowing, cleaning and other actions; the other result is that the aggregates adhere to the teeth, accelerating the formation of plaque biofilm. As can be seen from Table 4, Lactobacillus delbrueckii subsp. lactis TG001 has a certain cross-aggregation ability with the planktonic mutans streptococcus clinical strain 593 and the standard strain ATCC25175, which are 63.34% and 58.46% respectively at 4 h. And the coaggregation ability with the periodontitis pathogen Porphyromonas gingivalis ATCC 33277 at 4 h is 58.90%, which proves that Lactobacillus delbrueckii subsp. lactis TG001 has a strong coaggregation ability, which can inhibit the colonization of mutans streptococci and Porphyromonas gingivalis in the oral cavity, balance the oral flora, and improve the oral microecology.

[0045] Table 4 Self-aggregation of Lactobacillus delbrueckii subsp. lactis TG001 and coaggregation with pathogenic bacteria

[0046] Example 4 Inhibitory ability of Lactobacillus delbrueckii subsp. lactis TG001 on biofilm formation of Streptococcus mutans and Porphyromonas gingivalis Crystal violet staining was used to semi-quantitatively detect the formation of mutans Streptococcus biofilm. After the pathogenic bacteria were activated by streaking on a BHI solid plate, a single colony was picked and placed in a BHI liquid medium and cultured at 37 °C for 24 h. The OD value of the pathogenic bacteria suspension was then adjusted. 600About 0.5. Take 25 μL of pathogenic bacteria suspension and add it to each well of a 96-well microplate containing 175 μL of BHI liquid medium supplemented with 20 g / L sucrose. Incubate at 37 °C for 24 h and stain the pathogenic bacteria biofilm with 0.1% crystal violet. Rinse the microplate with PBS buffer. Then add 200 μL of DMSO to dissolve the biofilm stain and measure the OD value at 570 nm.

[0047] After Lactobacillus delbrueckii subsp. lactis TG001 was streaked and activated on a BHI solid plate, a single colony was picked and placed in a BHI liquid medium and cultured at 37 °C for 24 h. Then the OD value of the Lactobacillus delbrueckii subsp. lactis TG001 suspension was adjusted. 600 About 0.5. Take 25 μL of probiotic and pathogenic bacterial suspensions, mix them and add them to a 96-well microplate containing 150 μL of BHI liquid culture medium with 20 g / L sucrose in each well, and culture them anaerobically at 37 °C for 24 h. After culture, aspirate the bacterial suspension in the well, wash it three times with phosphate buffer to remove planktonic bacteria, dry it at room temperature for 30 min, add 0.1% crystal violet to stain for 20 min, aspirate the dye, wash it three times with phosphate buffer to remove residual dye, dry it at room temperature for 30 min, and add 200 μL DMSO to each well. After fully dissolved, read the absorbance value at 570 nm on the microplate reader. The biofilm inhibition rate is calculated as follows:

[0048] The semi-quantitative analysis results of biofilm crystal violet staining showed that Lactobacillus delbrueckii subsp. lactis TG001 could inhibit the biofilm formation of Streptococcus mutans clinical strain 593 by 47.09% (Table 5), and the inhibition rate of biofilm formation of Porphyromonas gingivalis ATCC 33277 was 71.88%. The inhibitory effect on the biofilm of Streptococcus mutans standard strain ATCC 25175 was weaker, and it could inhibit the biofilm formation by 34.63%. The results showed that Lactobacillus delbrueckii subsp. lactis TG001 could inhibit the biofilm formation of Streptococcus mutans and Porphyromonas gingivalis to varying degrees, reduce the amount of biofilm formation, and can be used as a product for preventing and treating dental caries in food, medicine, and daily chemical products.

[0049] Table 5 Effect of Lactobacillus delbrueckii subsp. lactis TG001 on biofilm formation of pathogenic bacteria

[0050] Example 5 Determination of the tolerance of Lactobacillus delbrueckii subsp. lactis TG001 to lysozyme The tolerance of Lactobacillus delbrueckii subsp. lactis TG001 to lysozyme was investigated by spectrophotometry. BHI medium was added to a 96-well culture plate, and then different concentrations of lysozyme solution were added to make the final concentrations 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 mg / mL, respectively. The culture of Lactobacillus delbrueckii subsp. lactis TG001 was inoculated into a 96-well culture plate at a volume fraction of 5%, cultured at 37 °C for 24 h, and the OD was measured. 600 The value was used to judge the tolerance of Lactobacillus delbrueckii subsp. lactis TG001 to lysozyme. 8 CFU / mL of Lactobacillus delbrueckii subsp. lactis TG001 culture, the amount of lysozyme solution was replaced with an equal volume of sterile water, and the survival rate was calculated: survival rate = number of viable bacteria containing lysozyme / number of viable bacteria without lysozyme × 100%. The experimental results are shown in Figure 3 .

[0051] Lysozyme at a concentration of 0-1.5 mg / mL did not affect the growth of Lactobacillus delbrueckii subsp. lactis TG001. When the lysozyme concentration was 0.4 mg / mL, the survival rate was 94.58%; when the lysozyme concentration increased to 0.6 mg / mL, the survival rate increased to 101.29%; and as the lysozyme concentration continued to increase, the survival rate of Lactobacillus delbrueckii subsp. lactis TG001 continued to increase, and all were higher than 100%. It proved that Lactobacillus delbrueckii subsp. lactis TG001 had high tolerance to lysozyme. The experimental results showed that within the lysozyme concentration of 1.5 mg / mL, Lactobacillus delbrueckii subsp. lactis TG001 had good tolerance to lysozyme, which was much higher than the lysozyme concentration in human oral saliva (0-57 μg / mL), indicating that Lactobacillus delbrueckii subsp. lactis TG001 could show high survival ability in the extreme environment of the oral cavity.

[0052] Example 6 Determination of the tolerance of Lactobacillus delbrueckii subsp. lactis TG001 to hydrogen peroxide The tolerance of Lactobacillus delbrueckii subsp. lactis TG001 to hydrogen peroxide was investigated by spectrophotometry. BHI medium was added to a 96-well culture plate, and hydrogen peroxide of different molar concentrations was added to make the final concentrations 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mM, respectively. The culture of Lactobacillus delbrueckii subsp. lactis TG001 was inoculated into a 96-well culture plate at a volume fraction of 5%, cultured at 37 °C for 24 h, and the OD 600 The control group only added an equal volume of 1.0×10 8CFU / mL of Lactobacillus delbrueckii subsp. lactis TG001 culture, the amount of hydrogen peroxide solution was replaced with an equal volume of sterile water. And the survival rate was calculated: survival rate = number of viable bacteria with hydrogen peroxide / number of viable bacteria without hydrogen peroxide × 100%.

[0053] The experimental results are shown in Figure 4 After 24 h of hydrogen peroxide treatment, hydrogen peroxide at a concentration of 0-1.2 mM had little effect on the growth of Lactobacillus delbrueckii subsp. lactis TG001. When the hydrogen peroxide concentration was as high as 1.2 mM, the survival rate was 96.43%, indicating that Lactobacillus delbrueckii subsp. lactis TG001 had a high tolerance to hydrogen peroxide.

[0054] Example 7 Determination of acid tolerance of Lactobacillus delbrueckii subsp. lactis TG001 In order to detect the acid resistance of probiotics, probiotics were inoculated in 96-well plates at a 5% inoculum in BHI medium with pH values ​​of 3, 4, 5, and 6, and the control group was inoculated in BHI medium with pH value of 7. The cells were cultured at 37 °C for 24 h, and the OD values ​​were measured. 600 The acid tolerance of probiotics was determined based on their growth. The survival rate was calculated as follows: Survival rate = OD of treatment group 600 / control group OD 600 ×100%.

[0055] The experimental results showed that after culturing Lactobacillus delbrueckii subsp. lactis TG001 in a medium with a pH of 4 for 24 h, the survival rate of the strain was 96.58%, indicating that Lactobacillus delbrueckii subsp. lactis TG001 had strong survival ability in a slightly acidic environment. Figure 5 .

[0056] Example 8 Determination of salt tolerance of Lactobacillus delbrueckii subsp. lactis TG001 In a 96-well microtiter plate, the salt tolerance of Lactobacillus delbrueckii subsp. lactis TG001 was evaluated in BHI medium supplemented with 0.5%, 1.0%, 2.0%, 4.0% and 8.0% NaCl at a 5% inoculum. The untreated BHI medium represented the control group. After culturing at 37 °C for 24 h, the OD 600 The survival rate was calculated as follows: survival rate = OD of treatment group 600 / control group OD 600 ×100%.

[0057] Results Figure 6 The survival rate of Lactobacillus delbrueckii subsp. lactis TG001 after 24 h of culture in 8% NaCl medium was 68.96%, and the survival rate under 4% NaCl was 99.92%. The results showed that Lactobacillus delbrueckii subsp. lactis TG001 had good tolerance to high salt environment.

[0058] Example 9 Determination of antibiotic sensitivity of Lactobacillus delbrueckii subsp. lactis TG001 The standardized drug sensitivity method (Kirby-Bauer, KB) was used to test the antibiotic sensitivity of probiotics. 75 μL of 1.0×10 8 CFU / mL probiotic suspension was evenly spread on the surface of BHI culture medium and allowed to stand for 15-20 minutes until there was no obvious liquid on the agar surface. Two drug-sensitive paper sheets were pasted on each culture medium and allowed to stand for 10 minutes until the drug-sensitive paper sheets were tightly attached. The culture was anaerobically cultured at 37 °C for 48 hours, and the diameter of the inhibition zone around each drug-sensitive paper sheet was measured and recorded. The sensitivity judgment criteria are shown in Table 6.

[0059] The KB method was used to investigate the sensitivity of Lactobacillus delbrueckii subsp. lactis TG001 to 17 antibiotics. The results showed that Lactobacillus delbrueckii subsp. lactis was sensitive to five antibiotics including macrolides and cephalosporins, and moderately sensitive to five antibiotics including streptomycin. It is a safe and reliable probiotic.

[0060] Table 6 Antibiotic sensitivity results of Lactobacillus delbrueckii subsp. lactis TG001

[0061] Note: S-susceptible, sensitive; I-intermediate, moderately sensitive; R-resistance, resistant.

[0062] Example 10 Hemolytic Test Prepare BHI solid culture medium, sterilize at 121℃, cool to about 55℃ in a clean bench, add defatted sheep blood at a ratio of 5%, mix well and pour onto a plate. After the plate is cooled, spread the probiotic solution onto the plate by the plate streak method, and after culturing at 37℃ for 24 hours, observe whether a transparent hemolytic ring appears on the plate.

[0063] The growth of Lactobacillus delbrueckii subsp. lactis TG001 on the blood agar plate for 24 h was observed. No transparent hemolytic ring appeared after 24 h of culture, proving that Lactobacillus delbrueckii subsp. lactis TG001 was non-hemolytic. Figure 7 .

[0064] Example 11 Experiment on hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis TG001 To detect H produced by probiotic isolates 2 O 2 Prepare tetramethylbenzidine (0.25 mg / mL) and horseradish peroxidase (HRP, 0.01 mg / mL) agar plates for qualitative determination. Probiotics were inoculated onto TMB agar plates and cultured in an anaerobic chamber at 37°C. After 48 h, the plates were exposed to ambient air. If the probiotics produced H2 O 2 , it reacts with horseradish peroxidase in the culture medium, which oxidizes TMB and causes the colonies to turn blue. In addition, in order to prove whether hydrogen peroxide plays an antibacterial role, in the pathogenic bacteria inhibition experiment, 0.166 mg / mL of catalase was added to the probiotic culture medium, and then the probiotics were added to the Oxford cup wells and incubated at 37°C under microaerobic conditions for 24 hours. If the inhibition zone disappears, it means that hydrogen peroxide mediates the inhibition of the growth of Streptococcus mutans.

[0065] Preparation of tetramethylbenzidine (0.25 mg / mL) and horseradish peroxidase (HRP, 0.01 mg / mL) agar plates for qualitative determination of H produced by probiotic isolates 2 O 2 The results showed that on the TMB-HRP plate, the colonies of Lactobacillus delbrueckii subsp. lactis TG001 turned blue, proving that Lactobacillus delbrueckii subsp. lactis TG001 produced hydrogen peroxide. In order to prove whether hydrogen peroxide played an antibacterial role, in the pathogenic bacteria inhibition experiment, 0.166 mg / mL of catalase was added to the probiotic culture medium, and then Lactobacillus delbrueckii subsp. lactis TG001 was added to the Oxford cup wells and incubated at 37°C for 24 h. The disappearance of the inhibition zone was observed, indicating that hydrogen peroxide mediated the inhibition of the growth of Streptococcus mutans, see Figure 8 (A: Lactobacillus delbrueckii subsp. lactis TG001 produces blue colonies on TMB plates, B: The inhibition zone of Lactobacillus delbrueckii subsp. lactis TG001 disappears after adding catalase).

[0066] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus delbrueckii subspecies lactis TG001, characterized in that: The preservation number of the Lactobacillus delbrueckii subspecies lactis TG001 is CCTCC NO: M 20241941, the preservation location is Wuhan China Type Culture Collection Center, and the preservation date is September 9, 2024.

2. The Lactobacillus delbrueckii subspecies lactis TG001 according to claim 1, characterized in that Its 16S rRNA sequence is shown in SEQ ID NO:

1.

3. Use of Lactobacillus delbrueckii subspecies lactis TG001 described in claim 1 in inhibiting Streptococcus mutans or Porphyromonas gingivalis.

4. Use of Lactobacillus delbrueckii subspecies lactis TG001 described in claim 1 in coaggregating Streptococcus mutans or Porphyromonas gingivalis.

5. A probiotic preparation comprising at least one of the Lactobacillus delbrueckii lactis subsp. lactis TG001 according to any one of claims 1 to 4 or an inactivated product, fermentation broth, or metabolite thereof.

6. The probiotic preparation according to claim 5, characterized in that The probiotic preparation is an oral probiotic preparation that inhibits oral pathogenic bacteria.

7. The probiotic preparation according to claim 5, characterized in that The dosage form of the probiotic preparation includes tablets, freeze-dried powder, capsules, granules, pills, pastes, liquid preparations, gels or solid beverages.

8. Use of the probiotic preparation according to claim 5 in the preparation of products for preventing or treating dental caries, periodontitis and halitosis.

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