Salivaria lactobacillus ss12 and application thereof

Abstract

This invention provides a strain of *Lactobacillus salivarius* SS12 and its applications, belonging to the field of microbial technology. This strain was deposited at the China General Microbiological Culture Collection Center on December 1, 2023, with the accession number CGMCC No. 29195. This strain was obtained by screening *Lactobacillus salivarius* SS1 as the starting strain through ultraviolet mutagenesis and gradient high-temperature acclimatization. Compared with the starting strain, *Lactobacillus salivarius* SS12 can grow stably at a high temperature of 50℃ and has better tolerance to salt stress, bile salt stress, oxygen stress, and low temperature stress.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus salivarius SS12 and its applications. Background Technology

[0002] *Ligilactobacillus salivarius*, belonging to the genus *Lactobacillus*, is a non-spore-forming, rod-shaped, Gram-positive bacterium capable of fermenting sugars to produce lactic acid. *Ligilactobacillus salivarius* is part of the normal flora in the human oral cavity and possesses good immunomodulatory and antibacterial effects. For example, patent application CN1639317A (application number: CN02818580.3) discloses a *Ligilactobacillus salivarius* that can exert immunomodulatory effects by regulating cytokine levels or by antagonizing pro-inflammatory microorganisms and clearing them from the gastrointestinal tract. Patent application CN1338940A (application number: CN00802829.X) discloses a *Ligilactobacillus salivarius* that can be administered prophylactically or as a treatment method, alone or in combination with other probiotics, to treat inflammation or functional disorders, particularly intestinal inflammation and intestinal dysfunction.

[0003] Because of its good immune and antibacterial effects, Lactobacillus salivarius is often used to prepare oral care sprays or make bacterial powders to treat inflammatory or immune diseases. However, the preparation or transportation of the above-mentioned products are often affected by environmental factors such as temperature. For example, when preparing oral care sprays, the dry and high-temperature environment will cause a reduction in the number of live Lactobacillus salivarius, and this effect is irreversible.

[0004] To improve the tolerance of strains to environmental stresses such as high salt, high oxygen, high temperature, and low temperature, methods such as artificial mutagenesis, gene editing, or the addition of exogenous protective agents are often used to modify or protect strains. However, these methods have disadvantages such as uncertain safety and high cost. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a strain of Lactobacillus salivarius SS12 and its applications. This strain can grow stably at a high temperature of 50°C and has better tolerance to salt stress, bile salt stress, oxygen stress, and low temperature stress.

[0006] The technical solution of this invention is as follows:

[0007] A strain of *Ligilactobacillus salivarius* SS12 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29195.

[0008] The method for culturing Lactobacillus salivarius SS12 includes the following steps: picking a single colony of Lactobacillus salivarius SS12 and inoculating it into MRS medium, allowing it to be cultured statically to obtain Lactobacillus salivarius SS12 bacterial suspension.

[0009] Preferably, the temperature for static incubation is 37–50°C.

[0010] Preferably, the MRS culture medium comprises the following components: glucose 20 g / L; peptone 10 g / L; yeast extract 5 g / L; triammonium citrate 2 g / L; potassium acetate 2.25 g / L; anhydrous sodium acetate 1.88 g / L; disodium hydrogen phosphate 1.63 g / L; magnesium sulfate heptahydrate 0.58 g / L; manganese sulfate monohydrate 0.25 g / L; beef extract 10 g / L; and Tween 80 1 mL / L.

[0011] The application of *Lactobacillus salivarius* SS12 in responding to high temperature stress, salt stress, bile salt stress, oxygen stress, or low temperature stress environments. Specifically:

[0012] This strain can grow stably at 50℃;

[0013] This strain exhibits good tolerance to environments with salt concentrations of 0–10%, with a survival rate of approximately 70% in environments with a salt concentration of 2% and approximately 30% in environments with a salt concentration of 10%.

[0014] This strain exhibits good tolerance in environments with bile salt concentrations of 0–0.10%, with a survival rate of approximately 90% in environments with bile salt concentrations of 0.05% and approximately 20% in environments with bile salt concentrations of 0.08%.

[0015] This strain exhibits good tolerance to environments with H2O2 concentrations of 0–15 mM, with a survival rate of approximately 95% in an environment with an H2O2 concentration of 5 mM and approximately 25% in an environment with an H2O2 concentration of 15 mM.

[0016] This strain has good tolerance to low temperature stress. After treatment at 4°C for 24 hours, the survival rate is about 72%, and after treatment for 48 hours, the survival rate is about 55%.

[0017] Preferably, the Lactobacillus salivarius SS12 can be used to prepare antibacterial products under high temperature stress, salt stress, bile salt stress, oxygen stress, or low temperature stress.

[0018] More preferably, the antibacterial product is a product for treating diseases caused by Staphylococcus aureus infection.

[0019] Preferably, the Lactobacillus salivarius SS12 can be used to prepare anti-inflammatory products under high temperature stress, salt stress, bile salt stress, oxygen stress, or low temperature stress.

[0020] Preferably, the Lactobacillus salivarius SS12 can be used to prepare products that enhance immunity under high temperature stress, salt stress, bile salt stress, oxygen stress, or low temperature stress.

[0021] Beneficial effects: This invention provides a strain of Lactobacillus salivarius SS12 that can grow stably at 50°C. This strain was obtained by screening Lactobacillus salivarius SS1 as the starting strain through ultraviolet mutagenesis and gradient high temperature acclimatization. Compared with the starting strain, Lactobacillus salivarius SS12 has better tolerance to high temperature stress, salt stress, bile salt stress, oxygen stress and low temperature stress. Attached Figure Description

[0022] Figure 1 The tolerance of Lactobacillus salivarius SS12 to high temperature stress; where (■) represents Lactobacillus salivarius SS1 and (●) represents Lactobacillus salivarius SS12.

[0023] Figure 2 The tolerance of Lactobacillus salivarius SS12 to salt stress is represented by (■), where (●) represents Lactobacillus salivarius SS1 and (●) represents Lactobacillus salivarius SS12.

[0024] Figure 3 The tolerance of Lactobacillus salivarius SS12 to bile salt stress is represented by (■), where (●) represents Lactobacillus salivarius SS1 and (●) represents Lactobacillus salivarius SS12.

[0025] Figure 4 The tolerance of Lactobacillus salivarius SS12 to oxygen stress is represented by (■), where (●) represents Lactobacillus salivarius SS1 and (●) represents Lactobacillus salivarius SS12.

[0026] Figure 5 The tolerance of Lactobacillus salivarius SS12 to low temperature stress; where (■) represents Lactobacillus salivarius SS1 and (●) represents Lactobacillus salivarius SS12. Detailed Implementation

[0027] The following description is based on specific embodiments:

[0028] Source of experimental materials:

[0029] Lactobacillus salivarius SS1: This strain was isolated from the feces of a healthy 6-month-old infant in our laboratory. After 16S rDNA sequencing and comparison of the sequencing results with the database in NCBI Gene Bank, the results showed that the sequence information of this strain was closest to that of Lactobacillus salivarius, and it was named "Lactobacillus salivarius SS1".

[0030] Example 1:

[0031] Obtaining Lactobacillus salivarius SS12

[0032] (1) Ultraviolet mutagenesis treatment: turn on the ultraviolet lamp and preheat for 25 min; take the SS1 bacterial culture of Lactobacillus saliva into a culture dish, place the culture dish under the ultraviolet lamp 25 cm away after preheating and irradiate for 2 min; take the bacterial culture after irradiation and inoculate it into fresh MRS medium at an inoculation amount of 2% by volume, and incubate at 37℃ for 24 h to obtain the bacterial culture after ultraviolet mutagenesis.

[0033] (2) Gradient high temperature acclimatization: The bacterial culture after UV mutagenesis in step (1) was inoculated into fresh MRS medium at an inoculation rate of 2% by volume. It was then cultured statically at 37°C until the logarithmic growth phase. The culture was then transferred to 42°C and cultured statically for 24 hours. The culture was then subcultured repeatedly at 42°C for 7 to 10 times. During this period, the survival rate of the strain was calculated by plate colony counting. After the survival rate stabilized, the culture temperature was increased to 43°C. The survival rate of the strain was calculated using the same method as above. After the survival rate stabilized, the culture temperature was increased again. The strain was subjected to gradient high temperature acclimatization in this way.

[0034] The components of the MRS culture medium in steps (1) and (2) are as follows: glucose 20 g / L; peptone 10 g / L; yeast extract 5 g / L; triammonium citrate 2 g / L; potassium acetate 2.25 g / L; anhydrous sodium acetate 1.88 g / L; disodium hydrogen phosphate 1.63 g / L; magnesium sulfate heptahydrate 0.58 g / L; manganese sulfate monohydrate 0.25 g / L; beef extract 10 g / L; Tween 80 1 mL / L.

[0035] Through the above-mentioned ultraviolet mutagenesis combined with gradient high temperature domestication technology, we finally screened and obtained a mutant strain that can grow stably at 50℃, and named it "Lactobacillus salivarius SS12".

[0036] Ligilactobacillus salivarius SS12 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29195.

[0037] Example 2:

[0038] Culture of Lactobacillus salivarius SS12

[0039] Single colonies of Lactobacillus saliva-associated SS12 were picked and inoculated into MRS medium and incubated at 45°C for 24 hours to obtain Lactobacillus saliva-associated SS12 bacterial suspension.

[0040] Experimental Example 1:

[0041] Tolerance of Lactobacillus salivarius SS12 to high temperature stress

[0042] (1) Single colonies of *Lactobacillus salivarius* SS1 and SS12 were picked and inoculated into MRS medium, and incubated statically at 50°C for 24 h. The OD of the culture medium was then measured within 24 h. 600 The growth curve of the strain was plotted based on the changes in the values, and the number of viable bacteria in the culture medium was detected at 12h and 24h. The results are as follows: Figure 1 As shown in Figures A and B;

[0043] As shown in Figure A, under 50℃ culture conditions, *Lactobacillus salivarius* SS1 has virtually no survival ability, while *Lactobacillus salivarius* SS12 rapidly multiplies after 6 hours, reaches a stationary phase after 14 hours, and ultimately reaches an OD value of [missing value]. 600 The value is approximately 3.5; as shown in Figure B, under 50℃ culture conditions, the viable count of *Lactobacillus salivarius* SS1 was 0 at both 12h and 24h, while the viable count of *Lactobacillus salivarius* SS12 reached 3.25 × 10⁻⁶ at 12h. 8 The CFU / mL concentration resulted in a viable bacterial count of 3.5 × 10⁻⁶ at 24 hours. 8 CFU / mL; The above results indicate that Lactobacillus salivarius SS1 cannot grow at 50℃, but Lactobacillus salivarius SS12 can grow stably at 50℃.

[0044] (2) Single colonies of *Lactobacillus salivarius* SS1 and SS12 were picked and inoculated into MRS medium, and incubated statically at 80°C for 60 seconds. The change in viable bacterial count in the culture medium within 60 seconds was detected, and the survival rate of the two strains at 80°C was calculated. The results are as follows: Figure 1As shown in Figure C, it can be seen from Figure C that neither Lactobacillus salivarius SS1 nor Lactobacillus salivarius SS12 can grow stably at 80℃. The survival rate of Lactobacillus salivarius SS1 drops to 0 after 60s of culture at 80℃, while the survival rate of Lactobacillus salivarius SS12 is about 20% after 60s of culture at 80℃.

[0045] Experimental Example 2:

[0046] Tolerance of Lactobacillus saliva-associated SS12 to salt stress

[0047] Lactobacillus salivarius SS1 was picked and inoculated into MRS medium, and incubated statically at 37°C for 24 h to obtain Lactobacillus salivarius SS1 bacterial suspension. Single colonies of Lactobacillus salivarius SS12 were picked and inoculated into MRS medium, and incubated statically at 45°C for 24 h to obtain Lactobacillus salivarius SS12 bacterial suspension. The Lactobacillus salivarius SS1 and SS12 bacterial suspensions were inoculated separately into PBS buffer with a sodium chloride concentration of 0–22% (22% = 220 g / L) at a volume percentage of 2%, and incubated statically at 37°C for 2 h. The viable counts of Lactobacillus salivarius SS1 and SS12 in the PBS buffer were measured, and the survival rates of the two strains at different salt concentrations were calculated. The results are as follows: Figure 2 As shown;

[0048] The PBS buffer consists of the following components: NaCl 8 g / L; KCl 0.2 g / L; Na2HPO4 1.44 g / L; KH2PO4 0.24 g / L; pH 7.4.

[0049] Depend on Figure 2 It was found that in an environment with a salt concentration of 2%, the survival rate of *Lactobacillus salivarius* SS1 rapidly decreased to about 20%; while the survival rate of *Lactobacillus salivarius* SS12 remained at about 70% in an environment with a salt concentration of 2%, and increased to about 80% in an environment with a salt concentration of 4%, indicating that *Lactobacillus salivarius* SS12 could still continue to reproduce in an environment with a salt concentration of 2%. In an environment with a salt concentration of 10%, the survival rate of *Lactobacillus salivarius* SS12 was about 30%, while the survival rate of *Lactobacillus salivarius* SS1 was only about 13%. In an environment with a salt concentration of 12%, the survival rate of both strains decreased to 0. The above results indicate that *Lactobacillus salivarius* SS12 has a higher tolerance to salt stress than *Lactobacillus salivarius* SS1.

[0050] Experimental Example 3:

[0051] Tolerance of Lactobacillus salivarius SS12 to bile salt stress

[0052] The same method as in Experimental Example 2 was used to obtain *Lactobacillus salivarius* SS1 and SS12 bacterial suspensions. The SS1 and SS12 suspensions were inoculated at a volume percentage of 2% into PBS buffer containing 0–0.14% (0.14% = 1.4 g / L) bovine bile salts (bile acid content ≥75%) and incubated at 37°C for 2 h. The viable counts of *Lactobacillus salivarius* SS1 and SS12 in the PBS buffer were measured, and the survival rates of the two strains at different bile salt concentrations were calculated. The results are as follows: Figure 3 As shown;

[0053] Depend on Figure 3 It was found that the survival rate of *Lactobacillus salivarius* SS1 decreased rapidly in an environment containing bile salts, and it basically lost its ability to survive when the bile salt concentration was 0.05%. In contrast, the survival rate of *Lactobacillus salivarius* SS12 was about 90% at a bile salt concentration of 0.05%, and about 20% at a bile salt concentration of 0.08%. Both strains lost their ability to survive when the bile salt concentration was 0.10%. These results indicate that *Lactobacillus salivarius* SS12 has a higher tolerance to bile salt stress than *Lactobacillus salivarius* SS1.

[0054] Example 4:

[0055] Tolerance of Lactobacillus saliva-associated SS12 to oxygen stress

[0056] The same method as in Experimental Example 2 was used to obtain *Lactobacillus salivarius* SS1 and SS12 bacterial suspensions. The SS1 and SS12 suspensions were inoculated into PBS buffer (0–25 mM H2O2) at a volume percentage of 2%, and incubated at 37°C for 2 hours. The viable counts of *Lactobacillus salivarius* SS1 and SS12 in the PBS buffer were measured, and the survival rate was calculated. The results are shown below. Figure 4 As shown;

[0057] Depend on Figure 4 It was found that *Lactobacillus salivarius* SS1 completely lost its ability to survive after being treated in an environment with a H2O2 concentration of 5 mM for 2 hours, while *Lactobacillus salivarius* SS12 showed good tolerance in environments with H2O2 concentrations of 0–15 mM, with a survival rate of approximately 95% in an environment with a H2O2 concentration of 5 mM and approximately 25% in an environment with a H2O2 concentration of 15 mM. These results indicate that *Lactobacillus salivarius* SS12 has a higher tolerance to oxygen stress than *Lactobacillus salivarius* SS1.

[0058] Example 5:

[0059] Tolerance of Lactobacillus salivarius SS12 to low temperature stress

[0060] The same method as in Experimental Example 2 was used to obtain *Lactobacillus salivarius* SS1 and SS12 bacterial suspensions. The SS1 and SS12 suspensions were inoculated into PBS buffer at a volume percentage of 2%, and cultured statically at 4°C for 24 h and 48 h, respectively. The viable counts of *Lactobacillus salivarius* SS1 and SS12 in the PBS buffer were measured, and the survival rate was calculated. The results are shown below. Figure 5 As shown;

[0061] Depend on Figure 5 The results showed that after treatment at 4℃ for 24 hours, the survival rate of *Lactobacillus salivarius* SS1 was approximately 62%, and the survival rate of *Lactobacillus salivarius* SS12 was approximately 72%. After treatment at 4℃ for 48 hours, the survival rate of *Lactobacillus salivarius* SS1 was approximately 44%, and the survival rate of *Lactobacillus salivarius* SS12 was approximately 55%. These results indicate that *Lactobacillus salivarius* SS12 has a higher tolerance to 4℃ low-temperature stress than *Lactobacillus salivarius* SS1.

[0062] Experimental Example 6:

[0063] Using Staphylococcus aureus as an indicator bacterium, the antibacterial effect of Lactobacillus salivarius SS12 was verified.

[0064] The specific steps are as follows:

[0065] (1) Pick a single colony of Staphylococcus aureus and inoculate it into LB liquid medium. Incubate at 37°C with shaking until OD. 600 The value was adjusted to 0.5 to obtain the bacterial solution. 100 μL of the bacterial solution was added to 100 mL of LB solid medium that had been melted and cooled to 50 °C. After mixing thoroughly, the solution was poured into plates (20 mL / plate). The plates were allowed to stand horizontally until solidified. After solidification, Oxford cups (8 mm in diameter) were placed on the LB solid medium.

[0066] (2) Obtain Lactobacillus salivarius SS1 and Lactobacillus salivarius SS12 bacterial suspensions using the same method as in Experiment Example 2; centrifuge Lactobacillus salivarius SS1 and Lactobacillus salivarius SS12 bacterial suspensions at 6000 rpm for 5 min, collect the supernatant, and add 100 μL of the supernatant to the Oxford cups described in step (1). Incubate the plates containing the Oxford cups at 37℃ for 24 h. An inhibition zone appears on the plate. Measure the diameter of the inhibition zone. The results are shown in Table 1.

[0067] Table 1. Antagonistic effect of Lactobacillus saliva-associated with Staphylococcus aureus

[0068] Diameter of the inhibition zone (cm) 2.95±0.05 2.85±0.05

[0069] As shown in Table 1, there was no significant difference in the size of the inhibition zone produced by *Lactobacillus salivarius* SS1 and SS12 against *Staphylococcus aureus*, indicating that the mutagenized *Lactobacillus salivarius* SS12 also has an antibacterial effect. Therefore, *Lactobacillus salivarius* SS12 can be used in the preparation of anti-inflammatory and antibacterial products.

[0070] In addition, Lactobacillus salivarius SS12 belongs to the Lactobacillus genus of lactic acid bacteria. As an important probiotic, Lactobacillus is often used in the preparation of intestinal microecological preparations. It has the effects of effectively promoting the health of the gastrointestinal digestive system, improving immunity, preventing respiratory infections, and reducing the chance of catching a cold. Therefore, Lactobacillus salivarius SS12 can be used as a probiotic in the preparation of products that improve immunity.

Claims

1. A strain of *Lactobacillus salivarius* ( Ligilactobacillus salivarius SS12 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29195.

2. The method for culturing *Lactobacillus salivarius* SS12 according to claim 1, characterized in that, The procedure includes the following steps: picking a single colony of Lactobacillus salivae SS12 and inoculating it into MRS medium, then incubating it statically to obtain Lactobacillus salivae SS12 bacterial suspension; wherein the static incubation temperature is 37~50℃.

3. The application of Lactobacillus salivarius SS12 as described in claim 1 in response to high temperature stress, salt stress, bile salt stress, oxygen stress and low temperature stress environments.

4. The use of the Lactobacillus SS12 of claim 1 in the preparation of an antibacterial product that inhibits Staphylococcus aureus.

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