A Streptococcus salivarius strain and its applications

Abstract

The present invention provides a Streptococcus salivarius strain with a preservation number of CGMCC NO. 13308. The present invention also provides the application of Streptococcus salivarius CGMCC NO. 13308 in the preparation of mutans streptococci growth inhibitors and mutans streptococci biofilm formation inhibitors. The present invention discloses for the first time a new Streptococcus salivarius CGMCC NO. 13308. The cells of Streptococcus salivarius CGMCC NO. 13308 have the function of inhibiting the growth of mutans streptococci, and its M17 fermentation supernatant can significantly reduce the formation amount of mutans streptococci biofilm. The present invention broadens the application prospects of Streptococcus salivarius in the prevention and treatment of dental caries and the industrializable resources.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Streptococcus salivarius strain and its application in the preparation of an inhibitor for the growth and biofilm formation of Streptococcus mutans. Background Art

[0002] Dental caries, commonly known as tooth decay or cavities, is a bacterial disease that can secondary pulpitis and periapical periodontitis, and even cause inflammation of the alveolar bone and jawbone. If not treated promptly, the lesion continues to develop, forming a cavity, and eventually the entire crown of the tooth is completely destroyed and disappears. The ultimate result of its development is tooth loss. Dental caries is characterized by a high incidence and wide distribution. This disease is one of the main common diseases in the oral cavity and also one of the most common diseases in humans. The World Health Organization has listed it together with tumors and cardiovascular diseases as the three major key prevention and treatment diseases for humans.

[0003] There are many factors that cause dental caries, such as bacteria, oral environment (food, saliva), host, etc. Among them, bacteria are a necessary condition for the occurrence of dental caries. Generally, there are two types of cariogenic bacteria. One is the acid-producing bacteria genus, mainly Streptococcus mutans (abbreviated as S. mutans), Actinomyces genus, and Lactobacillus, which can decompose carbohydrates to produce acid, resulting in the demineralization of tooth inorganic matter; the other is Gram-positive cocci, which can destroy organic matter and can form cavities in teeth after long-term action. Among the above-mentioned cariogenic bacteria, Streptococcus mutans is recognized by domestic and foreign scholars as the most main and important pathogenic bacterium causing dental caries. Therefore, it is an effective means to control the number of main cariogenic bacteria in the oral cavity and thus prevent and treat dental caries by inhibiting or killing Streptococcus mutans. Currently, there are few control drugs, and silver ammonia nitrate is one of the few. Silver ammonia nitrate is an antiseptic and bactericidal drug with antiseptic, astringent, bactericidal, and corrosive effects, but this drug can stain teeth, so it is not suitable for the treatment of anterior teeth.

[0004] On the other hand, since the biofilm (insoluble polysaccharide) produced by Streptococcus mutans can promote the adsorption and accumulation of cariogenic bacteria on the tooth surface, and then form dental plaque to deteriorate the degree of dental caries, therefore, reducing the production of its biofilm can also achieve the purpose of preventing and treating dental caries. Compared with the research on controlling the number of Streptococcus mutans, the research on preventing and treating dental caries by reducing the production of biofilm is relatively less.

[0005] At present, there are not many methods to directly and specifically inhibit the proliferation of Streptococcus mutans or the production of its biofilm. Clinically, compounds such as silver ammonia nitrate are mostly used, which have a short time effect and strong side effects. However, the situation is different when using microorganisms with the same effect. First of all, microorganisms can colonize on the tooth surface and play a role in inhibiting the growth of bacteria or the formation of biofilm for a long time. Secondly, when using lactic acid bacteria microorganisms that are generally recognized as harmless, there is no need to worry about the risk of side effects. Although some lactic acid bacteria have been reported to have the effect of inhibiting the growth of bacteria or the formation of their biofilm, the number of relevant reports is limited, and the available microbial resources are extremely narrow. Therefore, screening for new lactic acid bacteria strains with the ability to inhibit the growth of Streptococcus mutans or the formation of its biofilm is still an urgent task and a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0006] Based on the above technical problems, the present invention provides a strain of Streptococcus salivarius, and also provides the application of this strain in the preparation of an inhibitor for the growth of Streptococcus mutans and the formation of its biofilm.

[0007] Specifically, on the one hand, the present invention provides a strain of Streptococcus salivarius, and its preservation number is CGMCC NO.13308.

[0008] The Streptococcus salivarius strain BD 3900 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 15, 2016. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101. The preservation number of this strain is: CGMCC No.13308.

[0009] On the second hand, the present invention also provides the application of Streptococcus salivarius CGMCC NO.13308 in the preparation of an inhibitor for the growth of Streptococcus mutans. Streptococcus salivarius CGMCC NO.13308 has an inhibitory effect on the growth of Streptococcus mutans, thus playing a role in preventing and treating dental caries.

[0010] On the third hand, the present invention also provides the application of the Streptococcus salivarius strain in the preparation of an inhibitor for the formation of Streptococcus mutans biofilm. Streptococcus salivarius CGMCC NO.13308 has an inhibitory effect on the formation of Streptococcus mutans biofilm, reducing the number of Streptococcus mutans biofilms and decreasing the adsorption and accumulation of cariogenic bacteria on the tooth surface, thereby inhibiting the formation of dental caries.

[0011] Among them, the fermentation supernatant obtained by fermenting the Streptococcus salivarius strain is used for the preparation of an inhibitor for the formation of Streptococcus mutans biofilm.

[0012] Preferably, the method for preparing the fermentation supernatant is as follows: inoculate the strain CGMCC NO.13308 into the M17 medium containing sucrose, and obtain the supernatant by centrifugation after fermentation culture.

[0013] Preferably, the inoculation amount of the strain CGMCC NO.13308 is 7x10 6 ~3.5x10 7 cfu / mL. When the inoculation amount is too much or too little, the reproduction rate of the strain Streptococcus salivarius CGMCC NO.13308 is slow, resulting in a decrease in the inhibitory activity of the obtained fermentation supernatant on the formation of Streptococcus mutans biofilm.

[0014] Preferably, the content of sucrose in the M17 medium is 0.25-5%. When the content of sucrose is too high or too low, it is not conducive to the reproduction and fermentation of the strain.

[0015] Preferably, the fermentation temperature is 26-42 °C and the time is 8-24 h. When the fermentation temperature is too high or too low, the metabolism of the strain Streptococcus salivarius CGMCC NO.13308 is slow, resulting in a decrease in the inhibitory activity of the fermentation supernatant on the formation of Streptococcus mutans biofilm.

[0016] Preferably, the centrifugation speed is 6000-10000 g and the time is 10-20 min. Within the preferred range, the strain can produce a fermentation supernatant with the activity of inhibiting the formation of Streptococcus mutans biofilm.

[0017] The beneficial effects of the present invention are as follows: The present invention first discloses a new strain of Streptococcus salivarius CGMCC NO.13308. The cells of Streptococcus salivarius CGMCC NO.13308 have the function of inhibiting the growth of Streptococcus mutans, and the supernatant of its M17 fermentation broth can significantly reduce the formation amount of Streptococcus mutans biofilm. The present invention broadens the application prospects of Streptococcus salivarius in the prevention and treatment of dental caries and the industrialization resources. Description of the Drawings

[0018] Figure 1 It shows the antibacterial effect of Streptococcus salivarius CGMCC No.13308 on Streptococcus mutans in Example 2. Detailed Embodiments

[0019] To more clearly illustrate the technical solutions of the present invention, the technical solutions of the present invention will be further described below in combination with specific embodiments:

[0020] In a specific embodiment, the present invention provides a strain of Streptococcus salivarius with the preservation number of CGMCC NO.13308.

[0021] The strain CGMCC NO.13308 was isolated from the oral cavity of a normal human body. The result of 16S rRNA gene sequencing of this strain is shown as SEQ ID NO:1. According to the results of its physiological and biochemical characteristics and 16S rDNA sequence analysis, it was identified as Streptococcus salivarius strain by the China General Microbiological Culture Collection Center (CGMCC). This strain has microbiological characteristics such as Gram-positive staining, a round cell morphology, and no spore formation.

[0022] The Streptococcus salivarius strain has the effect of inhibiting the growth of Streptococcus mutans and the formation of Streptococcus mutans biofilm. Therefore, the present invention also provides the application of Streptococcus salivarius CGMCC NO.13308 in the preparation of an inhibitor for the growth of Streptococcus mutans and in the preparation of an inhibitor for the formation of Streptococcus mutans biofilm.

[0023] Among them, the fermentation supernatant of Streptococcus salivarius CGMCC NO.13308 is used to prepare an inhibitor for the formation of Streptococcus mutans biofilm. The method for preparing the fermentation supernatant of Streptococcus salivarius CGMCC NO.13308 is as follows: inoculate the strain of Streptococcus salivarius CGMCC NO.13308 into the M17 medium containing sucrose, and obtain it by centrifugation after fermentation culture.

[0024] The above technical solution first discloses that the Streptococcus salivarius CGMCC NO.13308 itself has the function of inhibiting the growth of Streptococcus mutans, and the supernatant of its M17 fermentation broth can significantly reduce the formation amount of Streptococcus mutans biofilm. Therefore, the present invention broadens the application prospect and industrialization resources of Streptococcus salivarius in the prevention and treatment of dental caries.

[0025] Preferably, the inoculation amount of the Streptococcus salivarius CGMCC NO.13308 is 7x10 6 ~3.5x10 7 cfu / mL; preferably 1.4x10 7 ~2.8x10 7 cfu / mL, more preferably 2.1x10 7 cfu / mL. When the inoculation amount is too much or too little, the reproduction rate of the Streptococcus salivarius CGMCC NO.13308 strain is slow, resulting in a decrease in the inhibitory activity of the obtained fermentation supernatant on the formation of Streptococcus mutans biofilm.

[0026] Preferably, the sucrose content in the M17 medium is 0.25 - 5% (w / v), preferably 0.5 - 3% (w / v), more preferably 1% (w / v). Sucrose provides a carbon source for the growth of the Streptococcus salivarius CGMCC NO.13308 strain. When the sucrose content is too high or too low, it is not conducive to the reproduction and fermentation of the strain.

[0027] Preferably, the fermentation temperature is 26°C to 42°C; more preferably 30°C to 38°C; most preferably 34°C. The fermentation time is 8 to 24 hours; more preferably 12 to 20 hours; most preferably 16 hours. When the fermentation temperature is too high or too low, the metabolism of the Streptococcus salivarius CGMCC NO. 13308 strain is slow, which will cause the inhibitory activity of the fermentation supernatant on the formation of Streptococcus mutans biofilm to decrease.

[0028] It can also be seen from Comparative Example 1 that when outside the range of the preferred fermentation parameters, the inhibitory effect of the cells or the supernatant of the fermentation broth obtained after centrifugation of the Streptococcus salivarius CGMCC NO. 13308 fermented M17 medium on the growth or biofilm formation of Streptococcus mutans significantly decreases. Within the preferred range, the inoculum amount, sucrose concentration, culture temperature, and fermentation time interact with each other, making the antibacterial and anti-biofilm activities produced by the fermentation of Streptococcus salivarius CGMCC NO. 13308 better.

[0029] Further, in the above steps, the centrifugation speed is 6000 to 10000 g, more preferably 7000 to 9000 g, most preferably 8000 g; the preferred centrifugation time is 10 to 20 minutes, more preferably 13 to 17 minutes, most preferably 15 minutes. Within the preferred range, the strain can produce a fermentation supernatant with the activity of inhibiting the formation of Streptococcus mutans biofilm.

[0030] The following further illustrates the above specific embodiments through examples, but does not limit the present invention to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions. The reagents used in the examples are all analytical pure reagents without further explanation, purchased from the Sinopharm Group. Other test instruments, reagents, and strains, if not specifically stated, can be directly purchased through commercial channels.

[0031] Example 1 Screening, identification, and preservation of Streptococcus salivarius strains

[0032] The Streptococcus salivarius strain in the present invention is obtained through the following steps: Using a sterile pipette, 1 mL of oral saliva from normal people is taken and serially diluted with sterile physiological saline. The diluted solution is evenly spread on a sterile M17 agar medium (purchased from OXOID LTD., UK) by coating. Anaerobic culture is carried out at 37°C for 24 h. Colonies with smooth surfaces and obvious protrusions are selected and transferred to an M17 liquid medium containing 1% (w / v) sucrose. Anaerobic culture is carried out at 37°C for 24 h. The fermentation broth is centrifuged at 9000 rpm for 10 min, and the precipitate part (bacterial cells) and the supernatant part are taken respectively. The precipitate part is washed twice with sterile distilled water, and then a small amount of bacterial cells is picked with an inoculation needle and spot-inoculated on a BHI agar plate (purchased from OXOID LTD., UK) paved with Streptococcus mutans (CGMCC 1.2499, purchased from CGMCC) as the indicator bacteria. After anaerobic culture at 37°C for 24 h, the inhibition of the strain on Streptococcus mutans is observed. The pH of the supernatant part is adjusted to 6.80. 100 μL of the above supernatant and 100 μL of a BHI liquid medium resuspended with Streptococcus mutans (10 5 cfu / mL) containing sucrose (0.5% w / v) are added to a 96-well microplate (purchased from greiner bio~one company, Germany). Anaerobic culture is carried out at 37°C for 24 h, and the amount of biofilm formation is measured. Through the above method, the performance of different strains in inhibiting the growth of Streptococcus mutans or the amount of biofilm formation is measured, and a strain that can both inhibit the growth of Streptococcus mutans and reduce its biofilm production is screened out.

[0033] The result of 16S rRNA gene sequencing of this strain is shown as SEQ ID NO:1 in the sequence listing. According to the results of its physiological and biochemical characteristics and 16S rDNA sequence analysis, it is identified as a Streptococcus salivarius strain by the China General Microbiological Culture Collection Center (CGMCC). This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 15, 2016. The deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101. The deposit number of this strain is: CGMCC No. 13308. The microbiological and physicochemical characteristics of this strain are shown in Table 1:

[0034] Table 1 Physicochemical test results of CGMCC No. 13308 strain

[0035]

[0036] Example 2 Inhibitory effect of Streptococcus salivarius CGMCC NO.13308 on the growth and biofilm formation of Streptococcus mutans

[0037] 1. Materials and Methods

[0038] (1) Preparation of seeds (fermentation strains):

[0039] Preparation of Streptococcus salivarius CGMCC No. 13308 seed solution: Dissolve the freeze-dried powder of Streptococcus salivarius CGMCC No. 13308 with a small amount of sterile distilled water. Use an inoculation loop to pick a loopful and streak it on an M17 solid medium (purchased from OXOID Co., UK). Incubate anaerobically at 37°C for 24 h and take it out. Use an inoculation loop to pick a single colony and put it into 10 mL of M17 liquid medium (purchased from OXOID Co., UK). Use a vortex oscillator to evenly disperse the colony in the liquid medium. Incubate anaerobically at 37°C for 24 h and take it out. Inoculate it into M17 liquid medium (purchased from OXOID Co., UK) at an inoculation amount of 2% (v / v). After anaerobic incubation at 37°C for 24 h, centrifuge the culture at 15000 rpm for 10 min, discard the supernatant, wash the cells twice with sterile distilled water, and then suspend them with sterile distilled water of the original culture volume to obtain the seeds for fermentation. The bacterial concentration of the seed solution is 7x10 8 cfu / mL.

[0040] Preparation of Streptococcus mutans CGMCC 1.2499 seed solution: Dissolve the freeze-dried powder of Streptococcus mutans CGMCC 1.2499 (purchased from CGMCC) with a small amount of sterile distilled water. Use an inoculation loop to pick a loopful and streak it on a BHI solid medium (purchased from OXOID Co., UK). Incubate anaerobically at 37°C for 24 h and take it out. Use an inoculation loop to pick a single colony and put it into 10 mL of BHI liquid medium (purchased from OXOID Co., UK). Use a vortex oscillator to evenly disperse the colony in the liquid medium. Incubate anaerobically at 37°C for 24 h and take it out. Inoculate it into BHI liquid medium (purchased from OXOID Co., UK) at an inoculation amount of 2% (v / v). After anaerobic incubation at 37°C for 24 h, centrifuge the culture at 15000 rpm for 10 min, discard the supernatant, wash the cells twice with sterile distilled water, and then suspend them with sterile distilled water of the original culture volume to obtain the seeds for fermentation. The bacterial concentration of the seed solution is 6x10 8 cfu / mL.

[0041] (2) Detection method for the activity of Streptococcus salivarius cells in inhibiting Streptococcus mutans: Spot inoculation method. Use an inoculation needle to pick a small amount of Streptococcus salivarius cells and spot-inoculate them on a BHI agar plate coated with Streptococcus mutans as the indicator bacterium. Incubate anaerobically at 37°C for 24 h, observe and record the presence and size of the inhibition zone. The larger the diameter of the inhibition zone, the stronger the inhibitory activity.

[0042] (3) Detection method for the inhibition rate of the supernatant of Streptococcus salivarius M17 fermentation broth on the biofilm formation of Streptococcus mutans: Adjust the pH of the supernatant of Streptococcus salivarius M17 fermentation broth to 6.80. Take 100 μL of the above supernatant and 100 μL of BHI liquid medium containing sucrose (0.25% w / v) resuspended with Streptococcus mutans (10 5 cfu / mL) and add them into a 96-well microplate at the same time. Incubate anaerobically at 37 °C for 24 h. Wash the wells involved in the reaction 3 times with deionized water to remove free bacteria. Invert the microplate at room temperature and wait for it to dry naturally. Then add 100 μL of 0.5 g / L crystal violet solution into the wells. After staining at room temperature for 15 min, remove the staining solution, rinse away the unadsorbed crystal violet with a large amount of deionized water, dry naturally, and then add 200 μL of absolute ethanol to each well to dissolve the adsorbed crystal violet. Use a microplate reader to detect the absorbance value OD 样品组 of the sample group at 600 nm. Perform the same operation on the supernatant of the fermentation broth inactivated by boiling water bath and measure the corresponding absorbance value OD 对照组 of the control group at 600 nm. Use the formula "Inhibition rate = (1 - OD 样品组 / OD 对照组 ) × 100%" to calculate the inhibition rate of biofilm formation.

[0043] 2. Inhibitory effect of Streptococcus salivarius CGMCC No. 13308 on the growth and biofilm formation of Streptococcus mutans

[0044] Inoculate the seeds of Streptococcus salivarius CGMCC No. 13308 aseptically at an inoculation amount of 3% (v / v) into M17 liquid medium containing 1% (w / v) sucrose, and incubate anaerobically at 34 °C for 16 h to obtain the fermentation broth.

[0045] Centrifuge the fermentation broth at 8000 g for 15 min, and take the precipitate part (bacterial cells) and the fermentation supernatant part respectively. Wash the precipitate part 2 times with sterile distilled water, and then measure its inhibitory activity against Streptococcus mutans (represented by the diameter of the inhibition zone) by the above spot inoculation method. Measure the inhibition rate of the fermentation supernatant part on the biofilm formation of Streptococcus mutans according to the above method.

[0046] After measurement, the diameter of the inhibition zone of Streptococcus salivarius CGMCC No. 13308 against Streptococcus mutans is 18 mm (as Figure 1 shown), and the inhibition rate of its M17 fermentation broth supernatant on the biofilm formation of Streptococcus mutans is 61%.

[0047] Example 3 Inhibitory effect of Streptococcus salivarius CGMCC No. 13308 on the growth and biofilm formation of Streptococcus mutans

[0048] 1. Materials and methods: The same as in Example 2

[0049] 2. Inhibitory Effect of Streptococcus salivarius CGMCC No. 13308 on the Growth and Biofilm Formation of Streptococcus mutans

[0050] The seeds of Streptococcus salivarius were aseptically inoculated into M17 liquid medium containing 5% (w / v) sucrose at an inoculum size of 1% (v / v) and anaerobically cultured at 42 °C for 24 h to obtain the fermentation broth.

[0051] The fermentation broth was centrifuged at 10,000 g for 10 min, and the precipitate part (bacteria) and the fermentation supernatant part were taken respectively. The precipitate part was washed twice with sterile distilled water, and then its inhibitory activity against Streptococcus mutans was measured by the above-mentioned spot inoculation method (expressed by the diameter of the inhibition zone). The inhibitory rate of the fermentation supernatant part against the biofilm formation of Streptococcus mutans was measured by the above method.

[0052] After measurement, the diameter of the inhibition zone of Streptococcus salivarius CGMCC No. 13308 against Streptococcus mutans was 16 mm, and the inhibitory rate of the supernatant of its M17 fermentation broth against the biofilm formation of Streptococcus mutans was 13%.

[0053] Example 4 Inhibitory Effect of Streptococcus salivarius CGMCC No. 13308 on the Growth and Biofilm Formation of Streptococcus mutans

[0054] 1. Materials and Methods: The same as in Example 2

[0055] 2. Inhibitory Effect of Streptococcus salivarius CGMCC No. 13308 on the Growth and Biofilm Formation of Streptococcus mutans

[0056] The seeds of Streptococcus salivarius CGMCC No. 13308 were aseptically inoculated into M17 liquid medium containing 0.25% (w / v) sucrose at an inoculum size of 5% (v / v) and anaerobically cultured at 26 °C for 8 h to obtain the fermentation broth.

[0057] The fermentation broth was centrifuged at 6,000 g for 20 min, and the precipitate part (bacteria) and the fermentation supernatant part were taken respectively. The precipitate part was washed twice with sterile distilled water, and then its inhibitory activity against Streptococcus mutans was measured by the above-mentioned spot inoculation method (expressed by the diameter of the inhibition zone). The inhibitory rate of the fermentation supernatant part against the biofilm formation of Streptococcus mutans was measured by the above method.

[0058] After measurement, the diameter of the inhibition zone of Streptococcus salivarius CGMCC No. 13308 against Streptococcus mutans was 17 mm, and the inhibitory rate of the supernatant of its M17 fermentation broth against the biofilm formation of Streptococcus mutans was 15%.

[0059] Example 5 Inhibitory Effect of Streptococcus salivarius CGMCC No. 13308 on the Growth and Biofilm Formation of Streptococcus mutans

[0060] 1. Materials and methods: same as Example 2

[0061] 2. Inhibitory effect of Streptococcus salivarius CGMCC No. 13308 on the growth and biofilm formation of Streptococcus mutans

[0062] The seeds of Streptococcus salivarius CGMCC No. 13308 were aseptically inoculated into M17 liquid medium containing 2% (w / v) sucrose at an inoculum size of 2% (v / v) and anaerobically cultured at 38 °C for 12 h to obtain the fermentation broth.

[0063] The fermentation broth was centrifuged at 9000 g for 13 min, and the precipitate part (bacterial cells) and the fermentation supernatant part were taken respectively. The precipitate part was washed twice with sterile distilled water, and then its inhibitory activity against Streptococcus mutans was determined by the above spot inoculation method (represented by the diameter of the inhibition zone). The inhibitory rate of the fermentation supernatant part against the biofilm formation of Streptococcus mutans was determined by the above method.

[0064] After measurement, the diameter of the inhibition zone of Streptococcus salivarius CGMCC No. 13308 against Streptococcus mutans was 16 mm, and the inhibitory rate of the supernatant of its M17 fermentation broth against the biofilm formation of Streptococcus mutans was 30%.

[0065] Example 6 Inhibitory effect of Streptococcus salivarius CGMCC No. 13308 on the growth and biofilm formation of Streptococcus mutans

[0066] 1. Materials and methods: same as Example 2

[0067] 2. Inhibitory effect of Streptococcus salivarius CGMCC No. 13308 on the growth and biofilm formation of Streptococcus mutans

[0068] The seeds of Streptococcus salivarius CGMCC No. 13308 were aseptically inoculated into M17 liquid medium containing 0.5% (w / v) sucrose at an inoculum size of 4% (v / v) and anaerobically cultured at 30 °C for 20 h to obtain the fermentation broth.

[0069] The fermentation broth was centrifuged at 7000 g for 17 min, and the precipitate part (bacterial cells) and the fermentation supernatant part were taken respectively. The precipitate part was washed twice with sterile distilled water, and then its inhibitory activity against Streptococcus mutans was determined by the above spot inoculation method (represented by the diameter of the inhibition zone). The inhibitory rate of the fermentation supernatant part against the biofilm formation of Streptococcus mutans was determined by the above method.

[0070] After measurement, the diameter of the inhibition zone of Streptococcus salivarius CGMCC No. 13308 against Streptococcus mutans was 16 mm, and the inhibitory rate of the supernatant of its M17 fermentation broth against the biofilm formation of Streptococcus mutans was 25%.

[0071] Effect Example 1Hemolytic analysis of Streptococcus salivarius CGMCC No. 13308

[0072] Use an inoculating loop to pick up a small amount of the centrifuged precipitate (bacteria) of the M17 fermentation broth washed with sterile distilled water in Example 2, streak it on an M17 agar plate containing 5% (v / v) horse blood, and incubate anaerobically at 37°C for 48 h. Observe whether there is a hemolytic halo on the blood agar plate.

[0073] The results showed that no hemolytic halo appeared on the blood agar plate for the Streptococcus salivarius CGMCC No. 13308 strain, indicating that the ingestion of Streptococcus salivarius CGMCC No. 13308 is safe and reliable and has no potential harm to the body in terms of hemolysis.

[0074] Effect Example 2 Analysis of the adhesion ability of Streptococcus salivarius CGMCC No. 13308 to teeth

[0075] 1. Hydrophobicity analysis of Streptococcus salivarius CGMCC No. 13308

[0076] Resuspend the centrifuged precipitate (bacteria) of the M17 fermentation broth washed with sterile distilled water in Example 2 with PBS (phosphate buffer) at pH 6.8 to make the absorbance OD of the bacterial suspension at 600 nm 600 be 0.6. Add the hydrophobic solvent xylene (1 mL) to the above bacterial suspension (3 mL). First, let it stand for 5 min, shake it vigorously for 120 s, and then let it stand for 10 min. Take the aqueous phase and measure its OD 600 value. Calculate the adhesion rate to the non-polar solvent xylene according to the following formula.

[0077]

[0078] A0: Initial absorbance value of the bacterial suspension (here it is 0.6);

[0079] A1: Absorbance value of the aqueous phase after standing fully

[0080] After measurement, the adhesion rate of Streptococcus salivarius CGMCC No. 13308 to the non-polar solvent xylene is as high as 95.8%, indicating that this strain has extremely strong surface hydrophobicity.

[0081] 2. Electrostatic interaction analysis of Streptococcus salivarius CGMCC No. 13308

[0082] Resuspend the centrifuged precipitate (bacteria) of the M17 fermentation broth washed with sterile distilled water in Example 2 with PBS (phosphate buffer) at pH 6.8 to make the absorbance OD of the bacterial suspension at 600 nm 600It was 0.6. Ethyl acetate (Lewis base solution) or chloroform (Lewis acid solution) (1 mL) was added to the above-mentioned bacterial suspension (3 mL). After standing still for 5 min, it was shaken vigorously for 120 s and then stood still for 10 min. The aqueous phase was taken to measure its OD 600 value. The adhesion rates to ethyl acetate and chloroform were calculated according to the following formula.

[0083]

[0084] A0: Initial absorbance value of the bacterial suspension (here it is 0.6);

[0085] A1: Absorbance value of the aqueous phase after standing still sufficiently

[0086] It was determined that Streptococcus salivarius CGMCC No. 13308 showed a high degree of adhesion to chloroform and ethyl acetate, and the specific adhesion rates were 88.6% and 99.6% respectively, indicating that a large number of charges capable of generating electrostatic forces were carried on the cell surface.

[0087] 1. Conclusion: In the oral cavity, probiotics bind to the oral mucosa through specific and non-specific adhesion methods. Among them, non-specific adhesion is mainly related to electrostatic, hydrophobic and van der Waals forces. Experiments found that the cell surface of Streptococcus salivarius CGMCC No. 13308 has extremely strong hydrophobicity and carries a large number of charges capable of generating electrostatic forces. This characteristic helps it adhere to the tooth surface and further compete with pathogenic bacteria for binding sites to exert its probiotic characteristics.

[0088] Effect Example 3 Analysis of the survival performance of Streptococcus salivarius CGMCC No. 13308

[0089] 1. Analysis of the tolerance of Streptococcus salivarius CGMCC No. 13308 to antibiotics

[0090] The centrifuged precipitate part (bacterial cells) of the M17 fermentation broth washed with sterile distilled water in Example 2 was resuspended with pH 6.8 PBS (phosphate buffer solution) to make the bacterial concentration of the bacterial suspension 10 7 cfu / mL. 50 μL of the above-mentioned bacterial suspension was added to a microplate containing 150 μL of M17 liquid medium, and antibiotic solutions (spiramycin, gentamicin, chloramphenicol and penicillin) with different concentrations (1, 10, 20, 30, 40, 50, 60 μg / mL) were added thereto. It was anaerobically cultured at 37 °C for 24 h, and the OD 600 absorbance value was measured. The tolerance of the bacterial cells to different antibiotics was judged according to the change of the absorbance value, and the results are shown in Table 2.

[0091] Table 2 Tolerance of Streptococcus salivarius CGMCC No. 13308 to antibiotics

[0092]

[0093] Streptococcus salivarius CGMCC No.13308 is the most sensitive to chloramphenicol, with a tolerance dose < 1 μg / mL. It has a certain tolerance to spiramycin and penicillin, while its tolerance to gentamicin is the strongest, reaching 40 μg / mL. Thus, it can be seen that Streptococcus salivarius CGMCC No.13308 has a certain tolerance to most of the tested antibiotics.

[0094] 2. Tolerance analysis of Streptococcus salivarius CGMCC No.13308 to lysozyme

[0095] The centrifuged precipitate part (bacterial cells) of the M17 fermentation broth washed with sterile distilled water in Example 2 was resuspended with pH 6.8 PBS (phosphate buffer solution). The above bacterial suspension was added to the M17 solid medium at 45°C so that the final viable cell concentration in the medium was 10 6 cfu / mL. Pour the plate. After it cooled and solidified, a sterile Oxford cup was placed on the surface of the solid medium and 100 μL of lysozyme solutions with different concentrations (0.1, 0.2, 0.5, 1, 3, 5 mg / mL) were added thereto. Anaerobic culture was carried out at 37°C for 48 h. The tolerance of the strain to lysozyme was analyzed through the inhibition zone. The results are shown in Table 3.

[0096] Table 3 Tolerance of Streptococcus salivarius CGMCC No.13308 to lysozyme

[0097]

[0098] Note: ~: negative, no inhibition zone; +: positive, with inhibition zone

[0099] Streptococcus salivarius CGMCC No.13308 can tolerate up to 3 mg / mL of lysozyme, and this data is much higher than the lysozyme concentration (1 - 57 μg / mL) in the oral cavity of normal people. Thus, it can be seen that Streptococcus salivarius CGMCC No.13308 will not be inactivated by lysozyme in the oral cavity of normal people.

[0100] 3. Conclusion: Streptococcus salivarius CGMCC No.13308 has tolerance to some antibiotics and a certain concentration of lysozyme. Therefore, this strain will not be inactivated by oral antibiotics and lysozyme in the oral cavity, and has application prospects.

[0101] Comparative Example 1

[0102] The inoculation amount, sucrose concentration, culture temperature, and fermentation time in Example 2 were adjusted one by one, and a group of M17 fermentation broths prepared by different methods were obtained. The inhibitory effects of the fermentation broths obtained in each group on Streptococcus mutans and its biofilm formation were measured according to the method described in Example 2, and the results are shown in Table 4.

[0103] Table 4 Inhibitory effects of M17 fermentation broths prepared by different methods on Streptococcus mutans and its biofilm formation

[0104]

[0105] From the results shown in Table 4, it can be concluded that when the inoculation amount, sucrose concentration, culture temperature, and fermentation time in the preparation method of the M17 fermentation broth are adjusted outside the preferred range, Streptococcus salivarius CGMCC No. 13308 can still inhibit the formation of Streptococcus mutans and its biofilm, but the effect is significantly reduced.

[0106] Comparative Example 2

[0107] Referring to the method described in Example 2, the inhibitory effects of M17 fermentation broths prepared from Streptococcus salivarius CGMCC No. 13308, Lactococcus lactis subsp. lactis CGMCC 1.2472 (purchased from CGMCC), and Streptococcus thermophilus ST-BODY-3 (provided by Chr. Hansen) on the growth of Streptococcus mutans and its biofilm formation were compared. The specific operations are as follows:

[0108] 1. Materials and methods

[0109] (1) Preparation of seeds (fermentation strains):

[0110] The freeze-dried powders of Streptococcus salivarius CGMCC No. 13308, Streptococcus thermophilus ST-BODY-3, and Lactococcus lactis subsp. lactis CGMCC 1.2472 were dissolved in a small amount of sterile distilled water. One loop was taken with an inoculation loop and streaked on M17 solid medium (purchased from Merck Co., Germany). After anaerobic culture at 37 °C for 24 h, it was taken out. A single colony was picked with an inoculation loop and put into 1 mL of M17 liquid (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid medium using a vortex shaker. After anaerobic culture at 40 °C for 24 h, it was taken out. It was inoculated into 50 mL of M17 liquid at an inoculation amount of 2% (v / v) and cultured at 37 °C for 24 h. Then, the culture was centrifuged at 9000 rpm for 10 min, the supernatant was discarded, the cells were washed twice with sterile distilled water, and then suspended in sterile distilled water with the original culture volume to obtain the seeds for fermentation.

[0111] (2) Detection method for the activity of each strain to inhibit Streptococcus mutans: The same as in Example 2.

[0112] (3) Detection method for the inhibition rate of the fermentation supernatant of each strain M17 on the biofilm formation amount of Streptococcus mutans: The same as in Example 2.

[0113] 2. Inhibitory effects of each strain on the growth and biofilm formation of Streptococcus mutans

[0114] Each strain of seeds was aseptically inoculated into M17 liquid medium containing 1% (w / v) sucrose at an inoculation amount of 3% (v / v), and anaerobically cultured at 34 °C for 16 h to obtain fermentation broth.

[0115] The fermentation broth of each group was centrifuged at 8000 g for 15 min, and the precipitate part (bacterial cells) and the fermentation supernatant part were taken respectively. Among them, the precipitate part was washed twice with sterile distilled water, and its inhibitory activity against... was determined by the above spotting method (expressed by the diameter of the inhibition zone). Among them, the inhibitory rate of the fermentation supernatant part on biofilm formation was determined by the above method.

[0116] The inhibition rates of each strain on the growth of Streptococcus mutans and its biofilm formation are shown in Table 5.

[0117] Table 5 Inhibitory effects of M17 fermentation broth prepared from different strains on Streptococcus mutans and its biofilm formation

[0118]

[0119] Note: ~ indicates no inhibition zone

[0120] As can be seen from Table 5, other conventional M17 fermentation strains do not have the ability to ferment M17 to produce substances that inhibit Streptococcus mutans and its biofilm formation, while Streptococcus salivarius CGMCC No. 13308 can ferment M17 medium, and finally obtain bacterial cells that inhibit the growth of Streptococcus mutans, and produce substances that reduce the biofilm yield during metabolism.

[0121] The above has introduced in detail the Streptococcus mutans inhibitor and its preparation method provided by the present invention. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. Sequence Listing <110> Bright Dairy Co., Ltd. <120> A strain of Streptococcus salivarius and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1459 <212> DNA <213> 16S rRNA <400> 1 atacatgcag tagaacgctg aagagaggag cttgctcttc ttggatgagt tgcgaacggg 60 tgagtaacgc gtaggtaacc tgccttgtag cgggggataa ctattggaaa cgatagctaa 120 taccgcataa caatggatga cacatgtcat ttatttgaaa ggggcaattg ctccactaca 180 agatggacct gcgttgtatt agctagtagg tgaggtaacg gctcacctag gcgacgatac 240 atagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300 gggaggcagc agtagggaat cttcggcaat gggggcaacc ctgaccgagc aacgccgcgt 360 gagtgaagaa ggttttcgga tcgtaaagct ctgttgtaag tcaagaacga gtgtgagagt 420 ggaaagttca cactgtgacg gtagcttacc agaaagggac ggctaactac gtgccagcag 480 ccgcggtaat acgtaggtcc cgagcgttgt ccggatttat tgggcgtaaa gcgagcgcag 540 gcggtttgat aagtctgaag ttaaaggctg tggctcaacc atagttcgct ttggaaactg 600 tcaaacttga gtgcagaagg ggagagtgga attccatgtg tagcggtgaa atgcgtagat 660 atatggagga acaccggtgg cgaaagcggc tctctggtct gtaactgacg ctgaggctcg 720 aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg 780 ctaggtgttg gatcctttcc gggattcagt gccgcagcta acgcattaag cactccgcct 840 ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900 gagcatgtgg tttaattcga agcaacgcga acaaccttac caggtcttga catcccgatg 960 ctatttctag agatagaaag ttacttcggt acatcggtga caggtggtgc atggttgtcg 1020 tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctattgttag 1080 ttgccatcat tcagttgggc actctagcga gactgccggt aataaaccgg aggaaggtgg 1140 ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggttg 1200 gtacaacgag ttgcgagtcg gtgacggcaa gctaatctct taaagccaat ctcagttcgg 1260 attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcacg 1320 ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380 acacccgaag tcggtgaggt aaccttttgg agccagccgc ctaaggtggg atagatgatt 1440 ggggtgaagt cgtaacaag 1459

Claims

1. Use of a Streptococcus salivarius strain with deposit number CGMCC NO. 13308 in the preparation of a growth inhibitor for Streptococcus mutans.

2. Use of a Streptococcus salivarius strain with deposit number CGMCC NO. 13308 in the preparation of a biofilm formation inhibitor for Streptococcus mutans. The fermentation supernatant obtained by fermenting the Streptococcus salivarius strain is used in the preparation of the biofilm formation inhibitor for Streptococcus mutans. The preparation method of the fermentation supernatant is as follows: inoculate the CGMCC NO. 13308 strain into an M17 medium containing sucrose, ferment and culture, and then centrifuge to obtain it. The content of sucrose in the M17 medium is 1%; The inoculation amount of the CGMCC NO. 13308 strain is 2.1x10 7 cfu / mL; The fermentation temperature is 34°C and the time is 16 h; The centrifugation speed is 8000g and the time is 15 min.

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