KR20240003144A - Novel oral lactic acid bacteria, Lactobacillus reuteri TSB-R7 with remarkable propolis resistance - Google Patents

Abstract

The present invention relates to Lactobacillus reuteri TSB-R7, an oral lactic acid bacterium. Unlike known oral beneficial bacteria that cannot grow in high concentration propolis, the Lactobacillus reuteri TSB-R7 strain of the present invention has excellent propolis tolerance. As a result, it survives in the oral cavity even when ingested at the same time as propolis, and when applied to food together with propolis, it has the advantage of effectively demonstrating antibacterial effect against oral harmful bacteria through its synergistic effect.

Description

Novel oral lactic acid bacteria, Lactobacillus reuteri TSB-R7 with remarkable propolis resistance}

The present invention relates to Lactobacillus reuteri TSB-R7, a novel oral lactic acid bacterium, and more specifically, to Lactobacillus reuteri TSB-R7, a novel oral lactic acid bacterium with excellent propolis tolerance.

Streptococcus mutans is a mesophilic bacterium that grows at 18-40°C and inhabits not only the oral cavity, but also the pharynx and intestines. Additionally, it plays an important role in the formation of cavities due to its ability to adhere to enamel and produce acidic by-products. This breaks down sugar to obtain energy, which creates lactic acid and creates an acidic environment. This removes salts from the surface structure, dissolving calcium and creating holes in the tooth.

Accordingly, the market for oral lactic acid bacteria, which are probiotics that help with oral immunity by suppressing harmful bacteria in the mouth and proliferating beneficial bacteria, is growing rapidly. As the intake of lactic acid bacteria to increase intestinal immunity has become common for the reason that oral lactic acid bacteria have been attracting attention for a short period of time, the oral cavity, which has the largest number of bacteria after the intestines, is attracting attention, and there are concerns about bad breath and tooth decay due to the use of masks that have become routine due to the spread of COVID-19. I believe this is because the number of consumers is increasing.

About 700 species and 10 billion bacteria in the mouth are divided into beneficial and harmful bacteria and maintain coexistence and balance. When this balance is broken and harmful bacteria multiply excessively, various oral diseases occur, so intake of oral lactic acid bacteria is helpful for oral health. Consuming oral lactic acid bacteria can naturally maintain the bacterial balance in the oral cavity through the proliferation of beneficial bacteria, thereby correcting the fundamental problem of oral disease.

Accordingly, research on oral lactic acid bacteria has been actively conducted worldwide since the early 2000s, and currently, about 100 products are sold in over 30 countries, including the United States, Europe, and Japan, and are becoming popular.

Meanwhile, propolis refers to a product made by collecting tree secretions from bees and mixing them with bee saliva and beeswax. It is mainly dark brown, but comes in various colors such as red, black, and green. Propolis has been used as a folk medicine for a long time. It has been used as a treatment for inflammation in Egypt since 5,000 years ago, and was also used as a preservative when making mummies.

In Korea, propolis began to be produced and sold as a health supplement in 1996. Currently, the main known effects of propolis include antioxidant, anti-inflammatory, antibacterial, and blood circulation improvement.

In particular, propolis is known to have antibacterial properties against Streptococcus mutans, a cavity-causing bacteria, and is used in mouthwash and toothpaste.

The daily intake of propolis extract recognized by the Ministry of Food and Drug Safety is 16-17 mg of total flavonoids for antioxidant activity, and the daily intake does not apply for oral antibacterial activity. This is because it is a food that has been consumed safely for a long time and there have been no reports of serious toxicity, and the flavonoid ingredient comes in direct contact with the mouth and throat to suppress inflammation and bacteria in the oral cavity.

However, according to previous research, high-concentration propolis treatment such that the total flavonoid content is 8.5 mg affects the growth of some beneficial bacteria, and long-term use may cause bacterial imbalance in the oral cavity.

Accordingly, the present inventor designed the present invention to select and provide strains with excellent survival even in a high concentration propolis environment in order to simultaneously provide the antibacterial activity of Streptococcus mutans and improve the oral environment.

Korean Patent No. 10-1966772 (2019.04.02) discloses an oral pathogen inhibitory composition containing Lactobacillus reuteri CS 132 (KCTC 11452BP) or a culture thereof. Korean Patent Publication No. 10-2007-0018985 (2007.02.14) discloses the use of lactic acid bacteria to reduce gingival bleeding and alleviate oral inflammation.

One object of the present invention is to provide a novel Lactobacillus reuteri TSB-R7 (Accession number: KCTC19008P) that has excellent propolis resistance and can be used as an oral probiotic material.

Another object of the present invention is to provide a food composition for preventing or improving oral diseases containing Lactobacillus reuteri TSB-R7 (Accession Number: KCTC19008P) as an active ingredient.

The present invention provides Lactobacillus reuteri TSB-R7 (Accession number: KCTC19008P).

Meanwhile, in the present invention, the Lactobacillus reuteri TSB-R7 has excellent propolis resistance and antibacterial activity against Streptococcus mutans .

Meanwhile, the present invention provides a food composition for preventing or improving oral diseases containing Lactobacillus reuteri TSB-R7 (Accession Number: KCTC19008P) as an active ingredient.

The food composition of the present invention preferably further contains propolis.

The Lactobacillus reuteri TSB-R7 strain of the present invention, unlike known oral beneficial bacteria that cannot grow in high-concentration propolis, has excellent propolis tolerance and survives in the oral cavity even when ingested simultaneously with propolis, making it applicable to food along with propolis. In this case, the synergy effect has the advantage of effectively demonstrating antibacterial effect against oral harmful bacteria.

Figure 1 shows the results of analyzing the 23S rRNA gene base sequence of the strain selected in the 7th selection experiment.
Figure 2 shows the results of confirming the viability of Lactobacillus reuteri TSB-R7, test strain, and commercial strain according to high concentration propolis treatment time.
Figure 3 shows the results of confirming the antibacterial activity of Lactobacillus reuteri TSB-R7, the test strain and the commercial strain against cavity-causing bacteria.
Figure 4 shows the results of confirming the antibacterial synergy effect of propolis and Lactobacillus reuteri TSB-R7, test strains and commercial strains, respectively, through the inhibition rate of harmful bacteria.

Propolis is known to help with antibacterial effects in the oral cavity.

However, in high-concentration propolis with a total flavonoid content of 8.5 mg, some beneficial bacteria die or cannot grow, so there is a problem that the balance of the oral environment may be destroyed when high-concentration propolis products are used.

Accordingly, the present inventor completed the present invention by selecting new lactic acid bacteria that grow even at high concentrations of propolis and confirming the synergistic effect between the strain and propolis.

In the present invention, the above-mentioned strain was named ' Lactobacillus reuteri TSB-R7', and was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center and granted accession number KCTC19008P on June 15, 2022. The purpose is to

In addition, products using the strain of the present invention preferably further include propolis. At this time, the propolis preferably has a total flavonoid content of 8.5 to 17 mg.

According to one embodiment of the present invention, Lactobacillus reuteri KCCM40717, Lactobacillus reuteri A, Lactobacillus reuteri B, and Weicella sibaria, known as oral lactic acid bacteria. cibaria ) A, unlike the strain of the present invention, it was confirmed that the growth ability was reduced in a high-concentration propolis environment with 17 mg of total flavonoids.

In addition, it was confirmed that the above-mentioned publicly available and commercial strains had no combination effect at all or showed a very low level of harmful bacteria inhibition compared to the strain of the present invention when combined with propolis.

In other words, Lactobacillus reuteri TSB-R7, a strain discovered in the present invention, has excellent propolis resistance and, when administered alone or in combination with propolis, has excellent antibacterial activity against Streptococcus mutans , an oral harmful bacteria. It has been confirmed that it is effective.

Accordingly, the present invention provides a food composition for preventing or improving oral diseases containing Lactobacillus reuteri TSB-R7 (Accession Number: KCTC19008P) as an active ingredient. At this time, the oral disease may preferably be cavities or periodontitis, and may further include dry mouth, efflorescence, etc. caused by an imbalance of beneficial bacteria in the oral cavity.

The term “prevention” used in the present invention refers to all actions that suppress or delay the onset of oral disease by administering the pharmaceutical composition according to the present invention.

The term "improvement" in the present invention refers to any action in which an oral disease is improved or beneficially changed by administration of the composition of the present invention.

In the food composition of the present invention, the Lactobacillus reuteri TSB-R7 (Accession number: KCTC19008P) is preferably included in an amount of 0.00001 to 50% by weight compared to the food composition for improving oral diseases. If it is less than 0.00001% by weight, the effect is insignificant, and if it exceeds 50% by weight, the increase in effect compared to the amount used is minimal, making it uneconomical.

Food compositions of the present invention include, for example, noodles, gums, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, and vitamin complexes. and other health supplements, but is not necessarily limited thereto.

When the food composition of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples or experimental examples, and includes modifications of the technical idea equivalent thereto.

[ Example 1: Selection and identification of new oral lactic acid bacteria]

1. Isolation of new oral lactic acid bacteria surviving in high concentration propolis

Saliva was collected from 27 adults who had excellent oral health and had not recently consumed lactic acid bacteria products, each was placed in 10 ml MRS liquid medium, and cultured at 37°C for 18 hours.

Afterwards, 2 ml of culture medium was taken, 1 ml of water-soluble propolis liquid extract (Green propolis extract, Apis-Flora, Brazil) with a total flavonoid content of 17 mg per 1 ml was added, and treated at 37°C for 20 minutes. Afterwards, it was diluted 100 times with PBS, plated on MRS solid medium, and cultured at 37°C for 48 hours.

The resulting colonies were inoculated into MRS liquid medium and grown by culturing at 37°C for 24 hours, and 1 ml was added to the freezing solution containing MRS liquid medium: Glycerol in a 4:1 ratio. Afterwards, it was placed in a cryo-tube and stored frozen at -70°C before being used in the following experiment.

2. Identification of isolated strains

To identify the strain isolated above, genomic DNA was extracted using the Accuprep genome extraction kit (Bioneer, Korea). The extracted DNA was synthesized with universal primers 1F, 16R, 27F, and 1492R to amplify the gene fragment. After requesting Cosmogenetech to analyze the base sequence, the results were reported to NCBI (National Center for Biotechnology Information, Bethesda, MD, USA). Homology was confirmed using the BLAST program.

Through a total of 8 screening experiments, 8 strains with propolis resistance were purely isolated from the oral saliva of 27 adults, and are shown in Table 1 below.

Classification (number of saliva samples) produce colony Identification result 1st (2) Candida albicans 2nd (3) Lactobacillus plantarum , Lactococcus lactis 3rd (4) Streptococcus salivarius , staphylococcus capitis 4th (3) Bacillus subtilis , Rummeliibacillus stabekisii 5th (4) Weissella confusa 6th (5) Lactobacillus plantarum , paenibacillus polymyxa 7th (4) Weissella confusa , Lactobacillus reuteri 8th (2) Candida albicans

Among the above strains, Lactobacillus reuteri is an oral lactic acid bacterium that has already been widely used as its oral efficacy has been proven, and the strain selected in the 7th screening experiment was named 'Lactobacillus reuteri TSB-R7' (Figure 1). Figure 1 shows the results of analyzing the 23S rRNA gene base sequence of the strain selected in the 7th selection experiment, confirming that it has more than 99.9% homology with Lactobacillus reuteri .

[ Example 2: Lactobacillus Lutheri TSB -Propolis resistance evaluation of R7 strain]

In order to evaluate the survival ability according to the high concentration propolis treatment time, the Lactobacillus reuteri TSB-R7 strain selected above was placed in 10 ml of MRS liquid medium and cultured at 37°C for 18 hours.

Afterwards, mix 2 ml of culture medium with the number of viable cells adjusted to 10 ⁸ CFU/ml and 1 ml of water-soluble propolis liquid extract (Green propolis extract, Apis-Flora, Brazil) with a total flavonoid content of 17 mg per 1 ml, and then incubate at 37°C. were treated for 20 minutes, 40 minutes, and 60 minutes, respectively.

Afterwards, Lactobacillus reuteri TSB-R7 strain was diluted 100-fold with PBS, spread on MRS solid medium, and cultured at 37°C for 48 hours to observe colony formation depending on the treatment time of high-concentration propolis.

Comparative experiments were performed in the same manner as above for the test strain Lactobacillus reuteri KCCM40717 and the commercial strain Lactobacillus reuteri A, Lactobacillus reuteri B, and Weicella cibaria A.

As a result of the experiment, the strain described above and oral lactic acid bacteria from other companies showed a similar pattern of surviving only up to 20 minutes after treatment with high concentration propolis. On the other hand, Lactobacillus reuteri TSB-R7 was the only one that survived up to 60 minutes after treatment with high-concentration propolis, and it was confirmed that it showed the best propolis resistance (Figure 2). Figure 2 shows the results of confirming the viability of Lactobacillus reuteri TSB-R7, test strain, and commercial strain according to high concentration propolis treatment time.

As shown in the above results, in the case of Lactobacillus reuteri TSB-R7, which has excellent propolis tolerance, its growth was least affected by propolis, so it has a high probability of surviving and establishing itself in the oral cavity when used in oral care products using propolis. Therefore, it was expected that the various effects of lactic acid bacteria could be effectively demonstrated, and the following experiment was additionally performed.

[ Example 3: Lactobacillus Lutheri TSB -Analysis of antibacterial activity against cavity-causing bacteria of R7 strain]

To analyze the antibacterial activity of Streptococcus mutans, a cavity-causing bacterium, the Streptococcus mutans KCTC3065 strain, a cavity-causing bacterium used to measure antibacterial activity, was pre-cultured in BHI liquid medium and then grown to a viable cell count of 1×10 ⁶ CFU. It was suspended and plated on BHI solid medium.

Lactobacillus reuteri TSB-R7 strain was placed in MRS liquid medium and cultured at 37°C. The culture was inoculated at 1% in 50 ml of MRS medium and cultured at 37°C for 24 hours. Afterwards, the culture was centrifuged (5,000 g, 10 minutes, 4°C), and a sterile supernatant was obtained using an injection filter (0.22㎛, Sartorius, Germany).

Afterwards, a sterilized disk was placed on the culture medium to absorb 10 ㎕ each, cultured at 37°C for 48 hours, and then the size of the growth-inhibiting rings in the culture medium was checked.

Comparative experiments were performed in the same manner as above for the test strain Lactobacillus reuteri KCCM40717 and the commercial strain Lactobacillus reuteri A, Lactobacillus reuteri B, and Weicella cibaria A.

As a result of the experiment, the Lactobacillus reuteri TSB-R7 strain produces various antibiotic substances such as lactic acid, hydrogen peroxide, and bacteriocins, and is similar to commercial strains for which excellent antibiotic activity against periodontal disease bacteria has already been reported. When compared, it was confirmed that it exhibited a more significant antibacterial effect, confirming the possibility of utilizing the Lactobacillus reuteri TSB-R7 strain as an oral lactic acid bacterium (Figure 3). Figure 3 shows the results of confirming the antibacterial activity of Lactobacillus reuteri TSB-R7, the test strain and the commercial strain against cavity-causing bacteria.

[ Example 4: Lactobacillus Lutheri TSB -R7 strain and [Evaluation of antibacterial synergy efficacy of propolis]

To evaluate the effect of combining propolis and lactic acid bacteria through mixed culture, Lactobacillus reuteri TSB-R7 strain and ^{Streptococcus} mutans were each suspended to a final concentration of 1 Each was inoculated into a mixed medium (MRS liquid medium and BHI liquid medium mixed 1:1 (v/v)). As a control, an equal volume of PBS was used instead of Lactobacillus reuteri TSB-R7 strain.

Afterwards, the cells were cultured for 6 hours at 37°C and then plated on MSB solid medium to confirm the viable count of Streptococcus mutans. The inhibition rate of Streptococcus mutans was expressed as a ratio of {(Streptococcus mutans CFU of untreated control - CFU of Streptococcus mutans after sample treatment)/CFU of untreated control Streptococcus mutans} x 100.

Comparative experiments were performed in the same manner as above for the test strain Lactobacillus reuteri KCCM40717 and the commercial strain Lactobacillus reuteri A, Lactobacillus reuteri B, and Weicella cibaria A.

As a result of the experiment, the inhibition rate of Streptococcus mutans was significantly increased when Lactobacillus reuteri TSB-R7 and propolis were combined compared to when propolis was treated alone.

On the other hand, in the case of combined treatment with test strains and commercial strains and propolis, there was no combination effect at all, or in the case of combined treatment with Lactobacillus reuteri TSB-R7 and propolis, the inhibition rate of harmful bacteria was less than that and did not show a significant combination effect (Figure 4). Figure 4 shows the results of confirming the antibacterial synergy effect of propolis and Lactobacillus reuteri TSB-R7, test strains and commercial strains, respectively, through the harmful bacteria inhibition rate.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC19008P 20220615

<110> TS CORPORATION <120> Novel oral lactic acid bacteria, Lactobacillus reuteri TSB-R7 with remarkable propolis resistance <130> YP-22-068 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 2540 <212> DNA <213> Lactobacillus reuteri <220> <221>rRNA <222> (1)..(2540) <223> 23S rRNA of Lactobacillus reuteri TSB-R7 <400> 1 agctagcaag aggtagacgc agtgaactga aacatcttag tagctgcagg aagagaaaga 60 aacatcgatt ccctgagtag cggcgagcga aaagggaaga gcccaaacca acaagcttgc 120 ttgttggggt tgtaggactg aacattagag ttaccaaagt gcgacgtagt cgcaacagtt 180 gggaagctgt gccatagagg gtgaaagccc cgtagacgaa acgtcacact ctctgttcag 240 gatcctgagt acggcgggac acgtgaaacc ccgtcggaac ccgcgaggac catctcgcaa 300 ggctaaatac tccctagtga ccgatagtga accagtaccg tgagggaaag gtgaaaagca 360 ccccggaagg ggagtgaaat agttcctgaa accatgtgcc tacaagctgt cggagcccgt 420 taatgggtga cggcgtgcct cttgcagaat gaaccggcga gttacgattg catgcaaggt 480 taaggtggaa aaaccggagc cgtagcgaaa gcgagtctta aatgggcgta agaagtatgt 540 agttgtagac ccgaaaccag gtgacctacc catgtccagg ttgaaggtgc ggtaaagcgc 600 actggaggac cgaacccgtg tcagttgaaa atggctggga tgaggtgtgg gtagcggtga 660 aattccaaac gaacttggag atagctggtt ctctccgaaa tctctttagg gggagccttg 720 aggtaaagaa tcgtggaggt agagctactg tttggacaag gggcccgtca tgggttacca 780 acttcagata aactccgaat gccatcgatt tatactcagg agtcagacga tgagtgataa 840 gatccaccgt cgaaaggggga acagcccaga tcaccagtta aggtccctaa atatatgcta 900 agtggaaaag gatgtggagt tgcatagaca actaggatgt tggcttagaa gcagccacca 960 tttaaagagt gcgtaatagc tcactagtcg agtgatcctg cgccgaaaat gtaccggggc 1020 taagcatatt accgaaactg tggatgtgca ctatgtgcac gtgataggag agcgttctaa 1080 gggcggcgaa gtcagaccgt gaggactggt ggagcgctta gaagtgagaa tgccggtatg 1140 agtagcgaaa gacaggtgag aatcctgtcc accgaatgac taaggtttcc tggggaaggc 1200 tcgtcctccc agggtaagtc gggacctaag ccgaggccga gaggcgtagg cgatggataa 1260 caggttgaga ttcctgtacc agttaactgc gtttgacgat ggagggacgc aggaggctaa 1320 gcaaaccgta cgactggaag agtacggcca agcagtaagt caggatgtga gtcaaatgtt 1380 tacatccgcg ttgacaagct gtgatgggga gcgaaattaa agtagcgaag ttgccgatgt 1440 cacactgccg agaaaagctt ctaaggagta gttaactgcc cgtaccgcaa accgacacag 1500 gtagtcgagg agagaatcct aaggtgagcg agagaactct cgttaaggaa ctcggcaaaa 1560 tgaccccgta acttcgggag aaggggtgct gcccgcaagg ccagccgcag tgaataggcc 1620 caggcgactg tttatcaaaa acacaggttt ctgcaaaatc gtaagatgaa gtatagaggc 1680 tgacgcctgc ccggtgctgg aaggttaaaa ggatgcgtta gcttcggcga agctcagaat 1740 tgaagcccca gtaaacggcg gccgtaacta taacggtcct aaggtagcga aattccttgt 1800 cgggtaagtt ccgacccgca cgaaaggcgt aacgatctgg gcgctgtctc aacgagagac 1860 tcggtgaaat tgaaatccct gtgaagatgc agggtacccg cgacaggacg gaaagacccc 1920 atggagcttt actgtagctt gatattgagt gtttgtactg cttgtacagg ataggtagga 1980 gccgtagaag tcgggacgct agtttcgaca gaggcgctgg tgggatacta cccttgcaat 2040 atgaccactc taacccgcag cactgaacgt gctgggagac agtgtcaggt gggcagtttg 2100 actggggcgg tcgcctccta aaaggtaacg gaggcgccca aaggttcgct cagtatggtt 2160 ggaaatcatg cgcagagtgt aaaggcacaa gcgagcttga ctgcgagaca gacaggtcga 2220 gcagggacga aagtcgggct tagtgatccg gtggttccgt atggaagggc catcgctcaa 2280 cggataaaag ctaccctggg gataacaggc ttatctcccc caagagtcca catcgacggg 2340 gaggtttggc acctcgatgt cggctcatcg catcctgggg ctgtagtcgg tcccaagggt 2400 tgggctgttc gcccattaaa gcggtacgcg agctgggttc agaacgtcgt gagacagttc 2460 ggtccctatc cgtcgcgggc gtaggaaatt tgagaggacc tgtccttagt acgagaggac 2520 cgggatggac ataccgctgg 2540

Claims (4)

Lactobacillus reuteri TSB-R7 (Accession number: KCTC19008P).
According to paragraph 1,
The Lactobacillus reuteri TSB-R7,
Lactobacillus reuteri TSB-R7, characterized by excellent propolis resistance and antibacterial activity against Streptococcus mutans .
A food composition for preventing or improving oral diseases containing Lactobacillus reuteri TSB-R7 (Accession Number: KCTC19008P) as an active ingredient.
According to paragraph 3,
The food composition is,
A food composition for preventing or improving oral disease, further comprising propolis.