Application of Lactobacillus sanfrancisco in preparing products for resisting oral pathogen infection

Abstract

The present invention relates to the use of Lactobacillus sanfrancisco in the preparation of a product for resisting oral pathogen infection. The product for resisting oral pathogen infection is a product for inhibiting Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. The content of Lactobacillus sanfrancisco in the product for resisting oral pathogen infection is 10 6 ~10 10 cfu / mL. The present invention discloses for the first time that Lactobacillus sanfrancisco has the effect of inhibiting oral pathogen infection. Experiments have found that Lactobacillus sanfrancisco has good antibacterial properties against a variety of common oral pathogens such as Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, and can play a significant role in preventing, resisting or clearing common pathogens in the oral cavity. It has the function of inhibiting the growth of oral pathogens and inhibiting the formation of biofilms, and can effectively solve a series of oral problems such as bad breath, oral ulcers, chronic pharyngitis, periodontitis, etc.

Description

Technical Field

[0001] The invention relates to application of Lactobacillus sanfrancisco in preparing products for resisting oral pathogen infection, belonging to the technical field of microorganisms. Background Art

[0002] There are a large number of complex microbial communities in the oral cavity, most of which adhere to the hard or soft tissues of the oral cavity in the form of biofilms. The microbial community may change with changes in the oral environment such as individual growth and tooth growth, and may also vary due to individual or family dietary habits, but generally contains pathogenic bacteria. Oral pathogen infection can easily cause a series of oral problems such as bad breath, oral ulcers, chronic pharyngitis, and periodontitis. There are many types of oral pathogens, including Helicobacter pylori, Prevotella intermedia, Porphyromonas endodontis, Streptococcus mutans, etc. Different pathogens may cause different oral problems, and the same oral problem may also have different pathogens.

[0003] Probiotics can be used as microbial preparations, and when ingested in sufficient amounts, they can play a beneficial role in human health. Currently, common oral probiotics are mainly Lactobacillus and Bifidobacterium, and different types of probiotics may have different antibacterial effects. In view of the certain specificity of the antibacterial effect of probiotics, existing microbial preparations with probiotics as active ingredients are usually used to fight against a certain or a certain class of special pathogens. For example, studies have shown that Streptococcus sanguinis, Streptococcus mitis and Streptococcus salivarius have an inhibitory effect on the colonization of epithelial cells with Actinobacillus actinomycetemcomitans in vitro (W. Teughels et al., J Dent Res 86 (7), 611-617, 2007), and can secrete antibacterial substances to make pathogens weaker or even kill pathogens, thereby reducing the number of oral pathogens and maintaining oral health. However, to date, only a few strains that have been found to have the ability to maintain oral health have been confirmed through experimental processes.

[0004] Lactobacillus sanfrancisco is an obligate heterofermentative lactic acid bacterium. The prior art only discloses the following applications: first, using Lactobacillus sanfrancisco as a fermentation agent to prepare flour products; second, using Lactobacillus sanfrancisco to prepare products such as ethyl acetate. At present, there is no report on the application of Lactobacillus sanfrancisco in the fight against oral pathogens. It is particularly important to explore the functions of bacterial resources and screen out new probiotic strains with specific functional properties and suitable for the physiological characteristics of the Chinese population to promote the development of probiotic products. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of Lactobacillus sanfrancisco in the preparation of products for resisting oral pathogen infection.

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

[0007] Application of Lactobacillus sanfrancisco in the preparation of products for resisting oral pathogen infection.

[0008] Preferably according to the present invention, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco Ls-1001, Lactobacillus sanfrancisco Bj2 or Lactobacillus sanfrancisco Hw; the deposit number of the Lactobacillus sanfrancisco Ls-1001 is CGMCC No.14232; the deposit number of the Lactobacillus sanfrancisco Bj2 is CCTCC NO:M2022514; the deposit number of the Lactobacillus sanfrancisco Hw is CCTCC NO:M2022513.

[0009] More preferably, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco Ls-1001, with the deposit number being CGMCC No.14232.

[0010] Preferably according to the present invention, the oral pathogen is Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia or Aggregatibacter actinomycetemcomitans.

[0011] Preferably according to the present invention, the product for resisting oral pathogen infection contains Lactobacillus sanfrancisco as the main active ingredient.

[0012] According to a preferred embodiment of the present invention, the content of Lactobacillus sanfrancisco in the product for resisting oral pathogen infection is 10 6 ~10 10 cfu / mL.

[0013] Further preferably, the content of Lactobacillus sanfrancisco in the product for resisting oral pathogen infection is 10 10 cfu / mL.

[0014] Preferably according to the present invention, the preparation for resisting oral pathogen infection further comprises at least one carrier.

[0015] Preferably according to the present invention, the product for resisting oral pathogen infection is a medical preparation.

[0016] Further preferably, the medical preparation is an oral spray, a fluoride coating agent, a denture cleaner, an oral effervescent tablet, a chewable tablet or an oral dressing.

[0017] Beneficial effects of the present invention:

[0018] The present invention discloses for the first time that Lactobacillus sanfrancisco has the function of inhibiting oral pathogen infection. The inventor of the present application has found through experiments that Lactobacillus sanfrancisco has good antibacterial properties against a variety of common oral pathogens such as Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, and can play a significant role in preventing, resisting or clearing common pathogens in the oral cavity, and has the function of inhibiting the growth of oral pathogens and inhibiting the formation of biofilms, and can effectively solve a series of oral problems such as halitosis, oral ulcers, chronic pharyngitis, periodontitis, etc. DETAILED DESCRIPTION

[0019] To make the purpose, technical scheme and advantages of the present application clearer, the following will be described in detail with reference to specific embodiments. The embodiments described herein are only a part of the embodiments of the present application and should not be construed as limiting the scope of protection of the present application. The experimental techniques and experimental methods used in the present embodiment are conventional technical methods unless otherwise specified. The materials, reagents, etc. used in the present embodiment, unless otherwise specified, can all be obtained through regular commercial channels.

[0020] The deposit number of the Lactobacillus sanfrancisco Ls-1001 of the present invention is CGMCC No.14232, which has been disclosed in Chinese patent document CN108102974A and does not involve strain preservation. The deposit number of the Lactobacillus sanfrancisco Bj2 is CCTCC NO:M2022514, which has been disclosed in Chinese patent document CN115500454A and does not involve strain preservation. The deposit number of the Lactobacillus sanfrancisco Hw is CCTCC NO:M2022513, which has been disclosed in Chinese patent document CN115500454A and does not involve strain preservation. The Lactobacillus sanfrancisco is available from Ningbo Mingzhou Biotechnology Co., Ltd., and the product number is B336078.

[0021] Example 1: Inhibition of the activity of Lactobacillus sanfrancisco against oral pathogens

[0022] 1. Inoculate frozen Lactobacillus sanfrancisco Ls-1001, Lactobacillus sanfrancisco Bj2 and Lactobacillus sanfrancisco Hw into mMRS liquid culture medium respectively, and culture them in a 30°C constant temperature anaerobic incubator (N2:CO2:O2=79:20:1) for 48 hours. Activate them 2-3 times for standby use. Inoculate the activated bacterial liquid into mMRS culture medium at a volume fraction of 1%, and culture them at 30°C for 24 hours to obtain a fermented liquid. Centrifuge the fermented liquid at 5000r / min for 5 minutes, discard the supernatant, collect the precipitate, and obtain Lactobacillus sanfrancisco cells. Respin the cells with sterilized deionized water and prepare them into 10% Lactobacillus sanfrancisco content. 6 cfu / mL, 10 8 cfu / mL and 10 10cfu / mL of Lactobacillus sanfrancisco suspension to obtain the sample to be tested.

[0023] The components of mMRS medium: peptone 10g / L, yeast extract powder 5g / L, beef extract powder 10g / L, maltose 20g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, ammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1mL / L, pH 5.4.

[0024] 2. Determination of the inhibitory activity of Lactobacillus sanfrancisco against oral pathogens

[0025] The antibacterial activity was determined using the filter paper method: the filter paper was soaked in the San Francisco Lactobacillus suspension for 1 hour, and then placed in a sterile environment to dry for use. The oral pathogens (Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans) cultured to the logarithmic growth phase were diluted with BHI broth to an absorbance of 0.2 at 655nm, and then 0.1mL of the prepared oral pathogen suspension was added to the solid BHI culture medium, and evenly spread with a coating rod, and the dried filter paper was placed in the corresponding position. At the same time, the filter paper soaked in sterile water was used as a blank control. Cultured at 36±1℃ for 48h. The size of the inhibition zone was determined, and the average value of the parallel experiments was taken. The results are shown in Table 1.

[0026] Table 1

[0027]

[0028]

[0029] As shown in Table 1, Lactobacillus sanfrancisco Ls-1001, Lactobacillus sanfrancisco Bj2 and Lactobacillus sanfrancisco Hw all have significant inhibitory effects on oral pathogens Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. Among them, Lactobacillus sanfrancisco Ls-1001 has the strongest inhibitory ability against various oral pathogens. The optimal concentration of Lactobacillus sanfrancisco Ls-1001 is 10 10 cfu / mL.

[0030] Example 2: Inhibition of oral pathogen biofilm formation by Lactobacillus sanfrancisco

[0031] 1. Prepare Lactobacillus sanfrancisco suspension according to the method described in Example 1.

[0032] 2. Determination of the inhibition of oral pathogen biofilm formation by Lactobacillus sanfrancisco

[0033] Oral pathogens (Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans) cultured to the logarithmic growth phase were diluted with BHI broth to an absorbance of 0.2 at 655 nm. Then, 100 μL of Lactobacillus sanfrancisco suspension and 100 μL of oral pathogen suspension were added to the sterilized 96-well cell culture plate and cultured at 37°C for 48 h. The culture medium and suspended bacteria were removed by suction, leaving only the formed biofilm, which was then sterilized with a sterile steam. The biofilm in the wells was rinsed 3 times with distilled water, and 0.4wt% crystal violet was added to the wells to stain the biofilm for 15 minutes; the stained biofilm in the wells was rinsed 4 times with sterile water to remove unbound crystal violet, and dried at 37°C for 2 hours; 100 μL of 95% ethanol solution was added to each well, and the plate was shaken for 10 minutes to release the pigment in the biofilm into ethanol, and the absorbance was tested; the absorbance of the control group with only sterile water was taken as 100%, and the test results are shown in Table 2 below.

[0034] Table 2

[0035]

[0036] As shown in Table 2, Lactobacillus sanfrancisco Ls-1001, Lactobacillus sanfrancisco Bj2 and Lactobacillus sanfrancisco Hw all had significant inhibitory effects on the biofilm formation of oral pathogens Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, and the inhibitory effect on the biofilm formation of oral pathogens became more obvious with the increase of Lactobacillus sanfrancisco concentration. 10 cfu / mL of Lactobacillus sanfrancisco Hw had the best effect in inhibiting the biofilm formation of oral pathogens.

[0037] Example 3: Removal of oral pathogen biofilm by Lactobacillus sanfrancisco

[0038] 1. Prepare Lactobacillus sanfrancisco suspension according to the method described in Example 1.

[0039] 2. Determination of the effect of Lactobacillus sanfrancisco in removing oral pathogen biofilm

[0040] Oral pathogens (Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans) cultured to the logarithmic growth phase were diluted with BHI broth to an absorbance of 0.2 at 655 nm, and then 100 μL of the oral pathogen suspension was added to the wells of a 96-well cell culture plate and cultured at 37°C for 48 h until biofilm was formed. Then, 100 μL of Lactobacillus sanfrancisco suspension was added to each well; after culture at 37°C for 20 min, the culture medium and suspended bacteria were removed and the wells were plated. Only the biofilm is left in the well, and the biofilm in the well is rinsed 3 times with sterile distilled water, and 0.4wt% crystal violet is added to the well to stain the biofilm for 15 minutes; the stained biofilm in the well is rinsed 4 times with sterile water to remove unbound crystal violet, and dried at 37°C for 2 hours; 100μL of 95% ethanol solution is added to each well, and the plate is shaken for 10 minutes to release the pigment in the biofilm into ethanol, and the absorbance is tested; the absorbance of the control group with only sterile water is taken as 100%, and the test results are shown in Table 3 below.

[0041] Table 3

[0042]

[0043] As shown in Table 3, Lactobacillus sanfrancisco Ls-1001, Lactobacillus sanfrancisco Bj2 and Lactobacillus sanfrancisco Hw can remove the biofilm formed by oral pathogens Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, and the ability to remove biofilm is more obvious with the increase of Lactobacillus sanfrancisco concentration. 10 cfu / mL of Lactobacillus sanfrancisco Ls-1001 has the best effect in removing oral pathogen biofilm.

Claims

1. The use of Lactobacillus sanfrancisco in the preparation of products for resisting oral pathogen infection, characterized in that: The Lactobacillus sanfrancisco is Lactobacillus sanfrancisco Ls-1001; the deposit number of the Lactobacillus sanfrancisco Ls-1001 is CGMCC No.14232; The content of Lactobacillus sanfrancisco in the product for resisting oral pathogen infection is 10 6 ~10 10 cfu / mL; The oral pathogens are Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.

2. The use according to claim 1, characterized in that The product for resisting oral pathogen infection uses Lactobacillus sanfrancisco as the main active ingredient.

3. The use according to claim 1, characterized in that The content of Lactobacillus sanfrancisco in the product for resisting oral pathogen infection is 10 10 cfu / mL.

4. The use according to claim 1, characterized in that The product for resisting oral pathogen infection further comprises at least one carrier.

5. The use according to claim 1, characterized in that The product for resisting oral pathogen infection is a medical preparation.

6. The use according to claim 5, characterized in that The medical preparation is an oral spray, a fluoride coating agent, a denture cleaner, an oral effervescent tablet, a chewable tablet or an oral dressing.

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