Aradicatrat lactobacillus plantarum TG004, application of Aradicatrat lactobacillus plantarum TG004 in preparation of cariogenic bacterium inhibitor or 5-hydroxytryptophan, and gastric cancer-related microbial marker

Abstract

The invention belongs to the technical field of biology, and discloses a phytobacterium atrati TG004, an application of the phytobacterium atrati TG004 in preparation of cariogenic bacterium inhibitors or 5-hydroxytryptophan, and a microbial marker related to gastric cancer. According to the present invention, with the application of the Argentlat lactobacillus TG004, the activity and the biological membrane formation process of cariogenic bacteria such as streptococcus mutans, streptococcus mutans and porphyromonas gingivalis can be well inhibited, and the Argentlat lactobacillus TG004 has excellent potential of being used as the cariogenic bacteria inhibitor, and has important significance on the prevention and treatment of caries.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to Lactobacillus argenteus TG004 and application thereof in preparing a cariogenic bacteria inhibitor or 5-hydroxytryptophan, and microbial markers related to gastric cancer. Background Art

[0002] Dental caries, commonly known as tooth decay, is a common bacterial oral disease that can cause pulpitis and apical periodontitis, and can even cause inflammation of the alveolar bone and jawbone. If not treated in time, the lesions continue to develop, forming cavities, and eventually the crown of the tooth is completely destroyed and disappears. Bacteria are a necessary condition for the formation of dental caries. Streptococcus mutans is the most harmful of these cariogenic bacteria. It achieves initial adhesion through sucrose-independent adhesion such as hydrogen bonding and hydrophobic effects, and then through sucrose-dependent adhesion, it uses carbohydrates such as sucrose in food as a carbon source to polymerize and produce highly viscous glucans, which adhere to the surface of the teeth to form a biofilm, and damage the teeth and surrounding tissues by producing acid or other metabolites, causing oral diseases such as caries.

[0003] At present, the main treatment methods for dental caries include mechanical removal and antibiotics. Mechanical removal is to remove the carious parts of the teeth by physical means and perform tooth restoration, while antibiotics are to kill or inhibit caries-causing bacteria with drugs to achieve a therapeutic effect. However, although mechanical removal is effective, the therapeutic effect is difficult to maintain for a long time. Long-term use of antibiotics will also increase bacterial resistance. In addition, both mechanical removal and antibiotics can cause dysbiosis in the oral environment, thereby causing other problems, and have great limitations.

[0004] Similar to the intestinal environment, the oral environment is also a complex ecosystem, inhabited by hundreds of millions of microorganisms, which are interdependent and mutually restrictive, and together maintain the microecological balance of the oral cavity. Therefore, the introduction of intestinal flora probiotics to re-establish a good oral flora structure to inhibit the growth of cariogenic bacteria such as Streptococcus mutans or destroy the biofilm formed by them is of great significance for the prevention and treatment of dental caries. Summary of the invention

[0005] The first object of the present invention is to provide a Lactobacillus argentulatus TG004. First, the Lactobacillus argentulatus TG004 can well inhibit the activity of cariogenic bacteria such as Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis and their biofilm formation process, and has excellent potential for application as a cariogenic bacteria inhibitor, which is of great significance for the prevention and treatment of dental caries; secondly, the Lactobacillus argentulatus TG004 also has a certain 5-hydroxytryptophan synthesis ability, and can ingest and utilize L-tryptophan to synthesize 5-hydroxytryptophan; furthermore, the Lactobacillus argentulatus TG004 and other strains of the same species but different subspecies thereof have a reduced biological abundance in the intestine and are highly correlated with the occurrence of gastric cancer, and can be used as a microbial marker related to gastric cancer.

[0006] The second object of the present invention is to provide the use of the above-mentioned Lactobacillus argenteus TG004 in the preparation of caries-causing bacteria inhibitors.

[0007] The third object of the present invention is to provide an oral cavity improvement product.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned Lactobacillus argenteus TG004 in the preparation of 5-hydroxytryptophan.

[0009] A fifth object of the present invention is to provide a method for preparing 5-hydroxytryptophan.

[0010] The sixth object of the present invention is to provide a microbial marker associated with gastric cancer.

[0011] Specifically, the Lactobacillus argenteus TG004 provided by the present invention has a deposit number of CCTCC M2025145.

[0012] The present invention provides the use of the Lactobacillus argenteus TG004 in the preparation of a caries-causing bacteria inhibitor.

[0013] Furthermore, the cariogenic bacteria include one or more of Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis.

[0014] The oral cavity improvement product provided by the present invention comprises the above-mentioned Lactobacillus argenteus TG004.

[0015] Furthermore, in the oral care product, the concentration of Lactobacillus argenteus TG004 is 10 5 ~10 8 cell / mL.

[0016] The present invention provides the use of the Lactobacillus argenteus TG004 in the preparation of 5-hydroxytryptophan.

[0017] The present invention provides a method for preparing 5-hydroxytryptophan, which specifically comprises: taking the above-mentioned Lactobacillus argenteus TG004 for anaerobic fermentation culture to obtain the 5-hydroxytryptophan.

[0018] Furthermore, the culture medium for the anaerobic fermentation culture includes L-tryptophan, and the concentration of the L-tryptophan is 0.01 to 2 mg / mL.

[0019] Furthermore, the culture medium for the anaerobic fermentation culture includes hydrogen peroxide, and the concentration of the hydrogen peroxide is 0.2-0.6 mM.

[0020] Furthermore, the anaerobic fermentation culture temperature is 35-40°C and the time is 12-48 hours.

[0021] A gastric cancer-related microbial marker provided by the present invention is Lactobacillus argenteus TG004.

[0022] Biological Deposit Description

[0023] Biological materials: TG004 strain, classified as Lactiplantibacillus argentora tensis, deposited in the China Center for Type Culture Collection on January 15, 2025, the address of the collection center is: Wuhan University, Wuhan, China; the collection number is CCTCC M 2025145. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the Gram staining results of Lactobacillus argenteus TG004 provided in Example 1 of the present invention (scale is 100 μm);

[0025] Figure 2 This is a colony morphology diagram of Lactobacillus argenteus TG004 provided in Example 1 of the present invention;

[0026] Figure 3 This is a graph showing the experimental results of the hydrogen peroxide tolerance test of Lactobacillus argenteus TG004 provided in Example 2 of the present invention;

[0027] Figure 4 This is a graph showing the experimental results of the lysozyme tolerance test of Lactobacillus argenteus TG004 provided in Example 2 of the present invention;

[0028] Figure 5 This is a graph showing the experimental results of the acid production ability test of Lactobacillus argenteus TG004 provided in Example 2 of the present invention;

[0029] Figure 6This is a graph showing the experimental results of the self-aggregation ability test of Lactobacillus argenteus TG004 provided in Example 2 of the present invention;

[0030] Figure 7 This is a graph showing the experimental results of the 5-hydroxytryptophan capacity test of Lactobacillus argenteus TG004 provided in Example 3 of the present invention;

[0031] Figure 8 This is a graph showing the experimental results of optimizing the fermentation conditions for producing 5-hydroxytryptophan by Lactobacillus argenteus TG004 provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The inventor of the present invention takes obtaining a strain with good cariogenic bacteria inhibition activity as a starting point, takes a large number of domestic healthy human fecal samples as research objects, and finally obtains a strain of Lactobacillus argentulatus TG004 with weak acid production ability, good ability to colonize in the oral environment, and excellent inhibitory effect on cariogenic bacteria through screening after a large number of statistics and analyses. The deposit number of Lactobacillus argentulatus TG004 is CCTCC M 2025145; and the inventor further studies it and creatively finds that Lactobacillus argentulatus TG004 also has a certain 5-hydroxytryptophan synthesis ability, can absorb and utilize L-tryptophan to achieve the biosynthesis of 5-hydroxytryptophan, and the reduced biological abundance of Lactobacillus argentulatus TG004 and other strains of the same species but different subspecies in the intestine is highly correlated with the occurrence of gastric cancer. Based on this, the technical solution of the present invention is obtained.

[0033] In the present invention, the molecular biological characteristics of the Lactobacillus argenteus TG004 at least include: the nucleotide sequence of 16S rDNA of the Lactobacillus argenteus TG004 is shown in SEQ ID NO:3.

[0034] In the present invention, the morphological characteristics of the Lactobacillus argenteus TG004 at least include: the colony edge is smooth, the surface is shiny and convex, and the colony color is milky white.

[0035] Based on the excellent properties of the above Lactobacillus argentulatus TG004, such as weak acid production ability, good ability to colonize in the oral environment, and excellent inhibitory effect on cariogenic bacteria, the present invention provides the use of the above Lactobacillus argentulatus TG004 in the preparation of a cariogenic bacteria inhibitor.

[0036] In the present invention, the ability of the Lactobacillus argentulatus TG004 to colonize well in the oral environment includes: the Lactobacillus argentulatus TG004 has excellent tolerance to oral antibacterial substances. Specifically, the Lactobacillus argentulatus TG004 has a bacterial survival rate of more than 90% at a hydrogen peroxide concentration of 0.01 to 1.2 mM, and a bacterial survival rate of more than 80% at a high lysozyme concentration of not less than 0.8 mg / mL, and the bacterial survival rate increases with the increase of the lysozyme concentration, showing a special trend.

[0037] In the present invention, the ability of the Lactobacillus argentulatus TG004 to colonize well in the oral environment includes: the Lactobacillus argentulatus TG004 has excellent self-aggregation ability. Specifically, the self-aggregation ability of the Lactobacillus argentulatus TG004 at 4 hours of culture is not less than 66.55%, and the self-aggregation ability at 12 hours of culture is not less than 85.36%.

[0038] In the present invention, the inhibitory effect of the Lactobacillus argentulatus TG004 on cariogenic bacteria includes: the Lactobacillus argentulatus TG004 can inhibit the growth and reproduction of one or more cariogenic bacteria such as Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis. Specifically, the diameter of the inhibition zone of the Lactobacillus argentulatus TG004 for the clinical strain 593 of Streptococcus mutans is not less than 23.0 mm, the diameter of the inhibition zone for the standard strain ATCC 25175 of Streptococcus mutans is not less than 20.0 mm, and the diameter of the inhibition zone for Porphyromonas gingivalis ATCC 33277 is not less than 29.8 mm.

[0039] In the present invention, the inhibitory effect of the Lactobacillus argentulatus TG004 on cariogenic bacteria includes: the Lactobacillus argentulatus TG004 can copolymerize with one or more cariogenic bacteria such as Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis. Specifically, the copolymerization ability of the Lactobacillus argentulatus TG004 for the clinical strain 593 of Streptococcus mutans is not less than 76.97%, the copolymerization ability for the standard strain ATCC 25175 of Streptococcus mutans is not higher than 76.06%, and the copolymerization ability for Porphyromonas gingivalis ATCC 33277 is not higher than 68.28%.

[0040] In the present invention, the inhibitory effect of the Lactobacillus argentulatus TG004 on cariogenic bacteria includes: the Lactobacillus argentulatus TG004 can inhibit the formation of biofilms of one or more cariogenic bacteria such as Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis. Specifically, the inhibition rate of biofilm formation of the Lactobacillus argentulatus TG004 for the clinical strain 593 of Streptococcus mutans is not higher than 59.15%, the inhibition rate of biofilm formation for the standard strain ATCC 25175 of Streptococcus mutans is not higher than 72.15%, and the inhibition rate of biofilm formation for Porphyromonas gingivalis ATCC 33277 is not higher than 81.88%.

[0041] Based on the potential of the above-mentioned Lactobacillus argentulatus TG004 to be used as a cariogenic bacteria inhibitor, the present invention provides an oral cavity improvement product, which specifically includes the above-mentioned Lactobacillus argentulatus TG004.

[0042] In the present invention, in the oral care product, the concentration of Lactobacillus argenteus TG004 is preferably 10 5 ~10 8 cell / mL, such as 10 5 cell / mL, 10 6 cell / mL, 10 7 cell / mL, 10 8 cell / mL or any value in between.

[0043] In the present invention, the oral improvement product refers to various products used to improve oral hygiene and health conditions, and specific examples thereof include but are not limited to: one or more of toothpaste, toothbrush, dental floss, mouthwash and oral spray.

[0044] Based on the 5-hydroxytryptophan synthesis ability of the above-mentioned Lactobacillus argenteus TG004, the present invention also provides the use of the above-mentioned Lactobacillus argenteus TG004 in the preparation of 5-hydroxytryptophan.

[0045] Based on the potential of Lactobacillus argenteus TG004 in the preparation of 5-hydroxytryptophan, the present invention also provides a method for preparing 5-hydroxytryptophan.

[0046] In the present invention, the method for preparing 5-hydroxytryptophan specifically comprises: taking the above-mentioned Lactobacillus argenteus TG004 for anaerobic fermentation culture to obtain the 5-hydroxytryptophan.

[0047] In the present invention, the culture medium for anaerobic fermentation culture is a conventional technical means in the art. Those skilled in the art can make adaptive selections from existing known culture media as needed. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of LB culture medium, BHIS culture medium and MAC culture medium.

[0048] In some specific embodiments, the culture medium for anaerobic fermentation culture preferably includes L-tryptophan at a concentration of 0.01 to 2 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.3 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL or any value therebetween.

[0049] In some specific embodiments, the culture medium for anaerobic fermentation culture preferably includes hydrogen peroxide at a concentration of 0.2-0.6 mM, such as 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM or any value therebetween.

[0050] In some specific embodiments, the conditions of the anaerobic fermentation culture specifically include a temperature of preferably 35-40°C, 35°C, 36°C, 37.5°C, 38°C, 39°C, 40°C or any value therebetween; and a time of preferably 12-48h, such as 12h, 14h, 16h, 18h, 24h, 36h, 48h or any value therebetween.

[0051] Based on the high correlation between the above-mentioned Lactobacillus argentulatus TG004 and other strains of the same species but different subspecies and the occurrence of gastric cancer, the present invention also provides a microbial marker related to gastric cancer. The microbial marker is specifically Lactobacillus argentulatus.

[0052] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0053] The reagents and their sources involved in the following examples specifically include:

[0054] BHIS agar plate (Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 028361);

[0055] Bacterial genomic DNA rapid extraction kit (Beijing Qingke Biotechnology Co., Ltd., catalog number TSP701-50);

[0056] BHIS liquid culture medium (Guangdong Huankai Microbiological Technology Co., Ltd., catalog number 024053);

[0057] Mutans Streptococcus clinical strain 593 (isolated from the oral cavity of a patient with high dental caries prevalence by a research group of Fujian Medical University);

[0058] Streptococcus mutans standard strain ATCC 25175 (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number B309689);

[0059] Porphyromonas gingivalis ATCC 33277 (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number B81367).

[0060] Example 1

[0061] This example is used to illustrate the identification and biological preservation of a TG004 strain screened from a healthy human feces sample, specifically including:

[0062] 1. Morphological identification: The TG004 strain was inoculated on a BHIS agar plate for streak activation, cultured in an anaerobic incubator at 37°C until a single colony grew, subcultured three times, and subjected to Gram staining and morphological identification. The Gram staining results of TG004 and the colony morphology on the plate were as follows: Figure 1 and 2 shown.

[0063] Depend on Figure 1 It can be seen that the TG004 strain is a Gram-positive bacterium with a rod-shaped cell morphology, non-motile, and no spores. Figure 2 It can be seen that the colony size of the TG004 strain is medium, with smooth edges, a shiny and convex surface, and a milky white color.

[0064] 2. Molecular biological identification: (1) Dip a single colony on the BHIS agar plate into BHIS liquid culture medium, shake evenly, and culture in an anaerobic incubator at 37°C for 12 h. Use a bacterial genomic DNA rapid extraction kit and refer to the instructions for extraction to obtain the genomic DNA of the TG004 strain.

[0065] (2) PCR amplification of genomic DNA was performed using 16S rDNA universal primers to obtain PCR products, which were verified by agarose gel electrophoresis and then sent for sequencing. The 16S rDNA universal primer pair and the 16S rDNA sequencing results of the TG004 strain are shown in Table 1.

[0066] Table 1.

[0067]

[0068] (3) The 16S rDNA sequencing results were compared by Blast and a phylogenetic tree was constructed using MEGA6.0. The homology between the TG004 strain and Lactiplantibacillus argentoratensis DSM 16365 was more than 99.39%, showing a high degree of similarity, and was named Lactiplantibacillus argentoratensis TG004.

[0069] Example 2

[0070] This example is used to illustrate the potential of Lactobacillus argenteus TG004 provided in Example 1 as an inhibitor of caries-causing bacteria, specifically including:

[0071] 1. Tolerance to different antibacterial substances in the oral environment: (1) Hydrogen peroxide tolerance: BHIS liquid culture medium containing a series of H2O2 concentrations (0.01mM, 0.1mM, 0.2mM, 0.4mM, 0.5mM, 1mM, 1.2mM, 1.5mM and 2mM) was prepared, and Lactobacillus argenteus TG004 was inoculated into the above culture medium at an inoculum volume of 1%, and BHIS liquid culture medium was used as a blank control. The cells were cultured in an anaerobic incubator at 37°C for 24h, and the OD values ​​of each group were measured. 600 The bacterial survival rate was calculated according to the following formula. Figure 3 shown.

[0072] Bacterial survival rate = A1 / A0×100%

[0073] Among them, A1 is the OD of the experimental group 600 A0 is the OD of the blank control group. 600 value.

[0074] Depend on Figure 3 The test results show that the Lactobacillus argenteus TG004 provided in Example 1 of the present invention has a good tolerance to H2O2 in the concentration range of 0.01 to 1.2 mM.

[0075] (2) Lysozyme tolerance: BHIS liquid culture medium containing a series of lysozyme concentrations (0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL and 2.0 mg / mL) was prepared, and Lactobacillus argenteus TG004 was inoculated into the above culture medium at an inoculum amount of 1%, and BHIS liquid culture medium was used as a blank control. The culture was incubated in an anaerobic incubator at 37°C for 24 h, and the OD of each group was measured. 600 The bacterial survival rate was calculated according to the following formula. Figure 4 shown.

[0076] Bacterial survival rate = A1 / A0×100%

[0077] Among them, A1 is the OD of the experimental group 600 A0 is the OD of the blank control group. 600 value.

[0078] Depend on Figure 4 From the test results shown, it can be seen that in the presence of low concentration of lysozyme (0.1-0.4 mg / mL), the growth of Lactobacillus argenteus TG004 provided in Example 1 of the present invention is inhibited, but when the lysozyme concentration is greater than 0.4 mg / mL, the bacterial survival rate of Lactobacillus argenteus TG004 increases with the increase of lysozyme concentration.

[0079] 2. Acid production capacity: Lactobacillus argenteus TG004 was inoculated into BHIS liquid medium at a 1% inoculation amount and cultured in an anaerobic incubator at 37°C for 24 h. The supernatant was aspirated at 0 h, 0.5 h, 1 h, 6 h, 12 h and 24 h of culture, and the pH value of the solution was tested. The results were as follows: Figure 5 shown.

[0080] Depend on Figure 5 The test results show that after culturing for 24 hours, the pH value of the culture solution of Lactobacillus argenteus TG004 provided in Example 1 of the present invention decreased from 7.23 to 4.92, and the acid production ability was weak.

[0081] 3. Antibiotic sensitivity: (1) Inoculate Lactobacillus argenteus TG004 into BHIS liquid culture medium, shake evenly, and culture in an anaerobic incubator at 37°C until the culture medium OD reaches 0. 600 is 1, and the seed solution is obtained;

[0082] (2) 100 μL of seed solution was added to the BHIS agar plate and spread evenly. The corresponding antibiotic susceptibility sheets (antibiotic content was 100 μg) were affixed respectively. The paper without antibiotics was used as a blank control. The plates were anaerobically cultured in an anaerobic incubator at 37°C for 24 h. The diameter of the inhibition zone was measured with a vernier caliper. The results are shown in Table 2.

[0083] Table 2.

[0084]

[0085] From the test results shown in Table 2, it can be seen that the Argentuate Lactobacillus TG004 provided in Example 1 of the present invention is sensitive to 6 antibiotics including ampicillin, cefoperazone, ofloxacin, streptomycin, tobramycin, and piperacillin, moderately sensitive to 3 antibiotics including cefuroxime, ceftriaxone, and midecamycin, and insensitive to 8 antibiotics including aztreonam, cefazolin, cephalothin, ceftriaxone, cefepime, ceftazidime, cefoxitin, and kanamycin.

[0086] 4. Antibacterial activity: (1) Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277 were inoculated into BHIS liquid culture medium at a 1% inoculation rate and cultured in an anaerobic incubator at 37°C until the OD of the culture medium reached 0. 600 The value is 1, and the bacterial solution I of each cariogenic bacteria is obtained; 1 mL of each cariogenic bacterial solution I is respectively taken and evenly coated on the BHIS agar culture dish, and then the sterilized Oxford cup is placed in the test plate, and gently pressed to make it contact with the culture dish without gaps, and the operation is performed according to the following groups:

[0087] Blank control group: add 100 μL of BHIS liquid culture medium into the Oxford cup;

[0088] Experimental group: 100 μL of 10 7 cell / mL of BHIS liquid culture medium of Lactobacillus argenteus TG004;

[0089] Positive control group: add 100 μL of BHIS liquid medium containing 0.1 mg / mL chloramphenicol into the Oxford cup;

[0090] (2) Each BHIS agar culture dish was placed in an anaerobic incubator at 37°C for 24 h, and the diameter of the inhibition zone of each group was measured using a vernier caliper. The results are shown in Table 3.

[0091] Table 3.

[0092]

[0093] From the test results shown in Table 3, it can be seen that the Lactobacillus argenteus TG004 provided in Example 1 of the present invention has a good inhibitory effect on Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277.

[0094] 5. Inhibitory effect on the formation of cariogenic bacterial biofilm: (1) Inoculate 1% of Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277 in BHIS liquid culture medium and culture in an anaerobic incubator at 37°C until the OD of the culture medium reaches 0. 600 The value is 0.3, and each cariogenic bacterial solution II is obtained; 100 μL of each cariogenic bacterial solution II is respectively added to a 96-well plate, and the operation is performed according to the following groups:

[0095] Blank control group: add 100 μL of BHIS liquid culture medium into the 96-well plate;

[0096] Experimental group: 100 μL of 10 7 cell / mL of BHIS liquid culture medium of Lactobacillus argenteus TG004.

[0097] (2) The 96-well plate was placed in an anaerobic incubator at 37°C for 24 h. After the incubation, the supernatant was discarded and sterile PBS was added at a volume of 100 μL / well for washing twice. Then, the plate was stained with crystal violet and the OD of each group was measured. 600 The biofilm formation inhibition rate was calculated according to the following formula:

[0098] Biofilm formation inhibition rate = (1-A1 / A0) × 100%

[0099] Among them, A1 is the OD of the experimental group 600 A0 is the OD of the blank control group. 600 The results are shown in Table 4.

[0100] Table 4.

[0101]

[0102] From the test results shown in Table 4, it can be seen that the Lactobacillus argenteus TG004 provided in Example 1 of the present invention has a good inhibitory effect on the biofilm formation of Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277.

[0103] 5. Self-aggregation ability: Lactobacillus argenteus TG004 was inoculated into BHIS liquid culture medium at a 1% inoculation amount, and the absorbance A0 was measured at 600 nm; then, the culture was incubated in an anaerobic incubator at 37°C for 12 h, and the supernatant was aspirated at 2 h, 4 h, 8 h and 12 h of culture, and the absorbance A0 was measured at 600 nm. t The self-aggregation ability was calculated according to the following formula, and the results were measured three times in parallel and the average value was taken. Figure 6 shown.

[0104] Self-aggregation ability = (1-A t / A0)×100%

[0105] Depend on Figure 6 The test results show that the self-aggregation ability of Lactobacillus rhizogenes TG004 provided in Example 1 of the present invention can reach 66.55% after culturing for 4 hours, and the self-aggregation ability reaches 85.36% after culturing for 12 hours.

[0106] 6. Copolymerization ability with cariogenic bacteria: (1) Lactobacillus argenteus TG004, Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC33277 were inoculated into BHIS liquid culture medium at a 1% inoculation amount, cultured in an anaerobic incubator at 37°C for 16 h, centrifuged to remove the supernatant, and the bacterial sludge precipitates were collected and washed 5 times with an equal volume of PBS. The bacterial sludge precipitates were resuspended in BHIS liquid culture medium and the solution OD was 0. 600 The value is adjusted to 0.5;

[0107] (2) 100 μL of the resuspended Lactobacillus argenteus TG004 was mixed evenly with the bacterial suspension of each cariogenic bacteria, and then cultured in an anaerobic incubator at 37°C for 4 h. The supernatant was aspirated and the absorbance A was measured at 600 nm. The co-aggregation ability was calculated according to the following formula. The results are shown in Table 5.

[0108]

[0109] Among them, A x is the OD of Lactobacillus argenteus TG004 600 Value, A y The OD value of Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 or Porphyromonas gingivalis ATCC 33277 600 Value, A mix OD after mixed culture of Lactobacillus argenteus TG004 and cariogenic bacteria 600 value.

[0110] Table 5.

[0111] Caries-causing bacteria Coaggregation ability (%) Streptococcus mutans clinical strain 593 76.97 Streptococcus mutans standard strain ATCC 25175 76.06 Porphyromonas gingivalis ATCC 33277 68.28

[0112] From the test results shown in Table 5, it can be seen that the Lactobacillus rhizogenes TG004 provided in Example 1 of the present invention has excellent coaggregation ability for Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277, and the highest coaggregation ability can reach 76.97% after 4 hours of mixed culture.

[0113] In summary, the Argentuate Lactobacillus TG004 provided in Example 1 of the present invention has a relatively excellent tolerance for antibacterial substances such as H2O2 and lysozyme in the oral cavity, and has excellent self-polymerization ability, and can be well realized in the colonization in the oral environment; at the same time, the Argentuate Lactobacillus TG004 has excellent copolymerization ability with cariogenic bacteria such as Streptococcus mutans clinical strain 593, Streptococcus mutans standard strain ATCC 25175 and Porphyromonas gingivalis ATCC 33277, and can well inhibit the growth, reproduction and biofilm formation of the above-mentioned cariogenic bacteria; in addition, the acid production ability of the Argentuate Lactobacillus TG004 is weak, and the metabolites produced after its colonization in the oral cavity will not cause too much damage to the teeth and their surrounding tissues. Therefore, the Argentuate Lactobacillus TG004 is a highly potential cariogenic bacteria inhibitor, which can be well applied to the reconstruction of a good flora structure in the oral cavity, and is of great significance for the prevention and treatment of caries.

[0114] Example 3

[0115] This example is used to illustrate the 5-hydroxytryptophan (5-HTP) production capacity of Lactobacillus argenteus TG004 provided in Example 1, specifically including:

[0116] 1. 5-HTP production capacity: (1) Lactobacillus argenteus TG004 was inoculated into BHIS liquid culture medium, cultured in an anaerobic incubator at 37°C for 24 h, and the OD of the culture medium was adjusted to 600 is 1, and the seed solution is obtained;

[0117] (2) taking the seed liquid at an inoculum volume of 1% and inoculating it into a BHIS liquid medium containing 0.1 mg / mL of L-tryptophan, culturing it in an anaerobic incubator at 37° C. for 48 h, collecting the supernatant by centrifugation, and freeze-drying the supernatant to obtain a sample to be tested;

[0118] (3) Weigh 20 mg of 5-HTP to prepare a mother solution with a concentration of 1000 μg / mL, and dilute it with methanol to prepare standard solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, and test it by high performance liquid chromatography to draw a standard curve;

[0119] (4) The sample obtained in step (3) was dissolved in 2 mL of deionized water and mixed with 2 mL of ethyl acetate for extraction. The upper organic phase was collected and weighed. 1 mL of the organic phase was dried and dissolved in 1 mL of methanol. The organic phase was filtered through an organic filter membrane and tested by high performance liquid chromatography. The yield of 5-HTP was calculated based on the standard curve. The result is as follows: Figure 7 shown.

[0120] Among them, the conditions of high performance liquid chromatography include: the chromatographic column is a Waters chromatographic column (250mm, 4.6mm, 5.0mm); the mobile phase is methanol + deionized water (5:95), the flow rate is 1.0m / min; the detection time is 15min; the detection wavelength is 276nm; the column temperature is 35℃; and the injection volume is 10μL.

[0121] Depend on Figure 7 The test results shown show that the 5-HTP production of Lactobacillus argenteus TG004 provided in Example 1 of the present invention is 15.852 μg / mL.

[0122] 2. Optimization of fermentation conditions: (1) Lactobacillus argenteus TG004 was inoculated into BHIS liquid culture medium and cultured in an anaerobic incubator at 37°C for 24 h, and the OD value of the culture medium was adjusted. 600 is 1, and the seed solution is obtained;

[0123] (2) The seed solution was inoculated into BHIS liquid medium (containing 0.1 mg / mL L-tryptophan) containing a series of H2O2 concentrations (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM) at an inoculum volume of 1%, and cultured in an anaerobic incubator at 37°C for 48 h. The supernatant was collected by centrifugation and freeze-dried to obtain the sample to be tested;

[0124] (3) The 5-HTP production was tested using the method provided in “1. 5-HTP production capacity”. The results are as follows: Figure 8 shown.

[0125] Depend on Figure 8 The test results shown show that the introduction of hydrogen peroxide in the fermentation process can effectively increase the 5-HTP production of Lactobacillus argenteus TG004, and when the hydrogen peroxide concentration is 0.2-0.6 mM, it has an excellent promoting effect on the synthesis of 5-HTP in Lactobacillus argenteus TG004 cells.

[0126] Example 4

[0127] This example is used to illustrate the correlation between Lactobacillus argenteus TG004 and its different subspecies strains and gastric cancer, specifically including:

[0128] A total of 141 patients who visited a hospital were selected as the research subjects, including a healthy control group without gastrointestinal cancer (HC, N = 36), an early gastric cancer group (EGC, N = 68) and an advanced gastric cancer group (AGC, N = 37). Fecal samples were collected from the patients, intestinal flora was isolated and DNA was extracted, 16S rDNA universal primers were used for PCR amplification, rDNA library was established, sequencing and intestinal flora abundance analysis were performed. The test results showed that the abundance of Lactobacillus argentulatus in the fecal samples of patients with early gastric cancer group and advanced gastric cancer group was statistically significantly different from that in the fecal samples of patients with healthy control group, and the abundance of Lactobacillus argentulatus in the intestinal flora of patients with gastric cancer group was significantly reduced.

[0129] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A Lactobacillus argenteus TG004, characterized in that The deposit number of the Lactobacillus argenteus TG004 is CCTCC M 2025145.

2. Use of Lactobacillus argenteus TG004 according to claim 1 in the preparation of a cariogenic bacteria inhibitor.

3. The use of Lactobacillus argenteus TG004 according to claim 2 in the preparation of a cariogenic bacteria inhibitor, characterized in that: The cariogenic bacteria include one or more of Streptococcus mutans, Streptococcus mitis and Porphyromonas gingivalis.

4. An oral improvement product, characterized in that: The oral improvement product includes the Lactobacillus argenteus TG004 described in claim 1.

5. The oral cavity improving product according to claim 4, characterized in that: In the oral care product, the concentration of Lactobacillus argentulatus TG004 is 10 5 ~10 8 cell / mL.

6. Use of the Lactobacillus argenteus TG004 according to claim 1 in the preparation of 5-hydroxytryptophan.

7. A method for preparing 5-hydroxytryptophan, characterized in that: The preparation method comprises: carrying out anaerobic fermentation culture on the Lactobacillus argenteus TG004 described in claim 1 to obtain the 5-hydroxytryptophan.

8. The method for preparing 5-hydroxytryptophan according to claim 7, characterized in that: The anaerobic fermentation culture medium includes L-tryptophan, and the concentration of the L-tryptophan is 0.01 to 2 mg / mL; Optionally, the culture medium of the anaerobic fermentation culture includes hydrogen peroxide, and the concentration of the hydrogen peroxide is 0.2-0.6 mM.

9. The method for preparing 5-hydroxytryptophan according to claim 7, characterized in that: The temperature of the anaerobic fermentation culture is 35-40° C. and the time is 12-48 hours.

10. A gastric cancer-related microbial marker, characterized in that: The microbial marker is Lactobacillus argenteus.

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