KR20040092931A - Lactobacillus confusus inhibiting the growth and the penetration to teeth of Streptococcus Mutans - Google Patents

Abstract

PURPOSE: A lactic acid bacteria composition containing strains of Lactobacillus confusus(Weissella confusa) PL9001, pharmaceutically acceptable excipients or diluents is provided. It has inhibiting effects on the growth of Streptococcus mutans and promotes osteogenesis. Therefore, it can be used in dairy products, fermented food products, various soft drinks, gums or the like. CONSTITUTION: The lactic acid bacteria composition contains strains of Lactobacillus confusus(Weissella confusa) PL9001(KCCM-10245) as lactic acid bacteria, pharmaceutically acceptable excipients or diluents. The concentration of Lactobacillus confusus(Weissella confusa) PL9001 for killing Streptococcus mutans is 1x10¬3-1x10¬9 cfu/ml or cc. The lactic acid bacterial composition can be used as an additive or fermenting agent for lactic acid bacteria formulations, fermented milk, dairy products, soybean milk, gums, cheese, caramel, kimchi, beverages or the like.

Description

Lactobacillus confusus inhibiting the growth and the penetration to teeth of Streptococcus Mutans}

Tooth decay is a disease of the teeth that causes damage to the tooth structure. Tooth decay is the second most common disease after the common cold, which occurs mainly in children and young people. While tooth decay has drastically decreased over the past 30 years, including fluoridation in drinking water, restriction of sugary foods, advances in oral hygiene, and regular dental checks, tooth decay remains a major health problem. In the younger layer, it is the most important cause of tooth damage. Twenty percent of children between the ages of two and four had caries, and 80 percent of young people aged 17 had at least one tooth decay. More than two-thirds of adults between the ages of 35 and 44 lose at least one permanent tooth due to tooth decay, and one-quarter of all ages between 65 and 74 lose all natural teeth ( http://www.ahcpr.gov/clinic/epcsums). /dentsumm.htm .).

Germs always exist normally in the mouth. Bacteria convert all foods, especially sugars and starch into acids. Germs, Acids, Food Leftovers. The needles mix to form a sticky substance that sticks to your teeth. It is most common on the chewing surfaces of large molar teeth, just above the gums and peeling edges of all teeth. Plaque that is not removed from the teeth is mineralized to callus. Plaque and callus irritate the gums, causing gum inflammation and eventually periodontal disease.

The acid in the plaque dissolves the enamel surface of the tooth, creating a hole. The hole is painless until it grows to the inner structure of the tooth and can damage the nerves and blood vessels of the tooth. If left untreated, pus can form. Plaques and bacteria begin to accumulate within 20 minutes after a meal, causing bacterial activity. If plaque and bacteria remain in the teeth, holes are formed and the untreated tooth decays and the inside of the tooth eventually dies.

Sugars and starches in food increase tooth damage. The type, time, and frequency of carbohydrates are more important than the amount consumed. Sticky foods are more harmful than foods that are not, because they remain on the tooth surface. Frequent snacks increase the time the acid contacts the tooth surface. Broken tooth tissue does not regenerate. But caries can stop with treatment. The purpose of treatment is to preserve teeth and prevent complications.

Streptococcus mutants, the main caries, live in the human mouth and grow on the surface of the teeth, especially as they grow and produce acids using sugars in foods to corrode teeth and invade tooth bone cells to destroy toxins. Gum disease caused by various alveolar bone erosion, etc. (Chin-Lo Hahn, Al M. Best, and John G. Tew. 2000. Cytokine induction by Streptococcus mutans and pulpal pathogensis.Infection and Immunity 68: 6785-6789.)

Currently, zircitol is used in the functional gum to prevent tooth decay. However, gyrotol's cavities are prevented from being killed by major caries such as Streptococcus mutans, which are not killed by caries. Therefore, there is no effect of killing Streptococcus mutans or preventing bone cell invasion by xyitol.

Generally, most lactic acid bacteria-containing foods such as yogurt are known to have a strong acidity and thus have a bad effect such as tooth corrosion. However, the acid is formed by the metabolite of lactic acid bacteria, and studies on the effects of lactic acid bacteria control on tooth erosion and bone formation have not been reported separately.

The present invention inhibits the growth of Streptococcus mutants, the main tooth decay fungus, inhibits the invasion of S. mutants into bone cells, and selects lactic acid bacteria that promote bone formation, thereby producing lactic acid bacteria preparations, fermented dairy products, dietary supplements, gum, caramel, cheese It is intended to be used as a fermentation agent or additive to impart the functionality of various products such as food and beverages, including cola.

An object of the present invention is to provide a strain having excellent bone formation promoting ability.

In another aspect, the present invention provides a lactic acid bacterium that inhibits the growth of Streptococcus mutans, which is a representative caries.

In another aspect, the present invention provides a lactic acid bacterium that inhibits the infiltration of bone cells of the representative caries bacteria Streptococcus mutans.

In another aspect, the present invention provides lactic acid bacteria that promote bone cell formation.

Figure 1 Gram Dye Picture of Lactobacillus confusus PL9001 (KCCM-10245)

Figure 2 is an electron micrograph (SEM) of Lactobacillus confusus PL9001 (KCCM-10245).

Figure 3 shows the 16s rRNA sequence of Lactobacillus confusus PL9001 (KCCM-10245)

4th degree Lactobacillus confusus PL9001 (KCCM-10245)

Observation of the effects of bone formation on live bacteria (x200, optical microscope)

5th Degree Lactobacillus confusus PL9001 (KCCM-10245)

Observe the effect of live bacteria on bone formation (von Kossa staining)

Figure 6 shows the effect of Lactobacillus confusus PL9001 (KCCM-10245) on the formation of bone cells (von Kossa staining)

7 shows the effect of Lactobacillus confusus PL9001 (KCCM-10245) on the formation of bones (ALPase color reaction, von Kossa staining)

Figure 8 Identification of the Killing Function of Streptococcusmutants of Lactobacillus confusus PL9001 (KCCM-10245)

Figure 9 Identification of Osteoblast Inhibition Function of Streptococcus mutants of Lactobacillus confusus PL9001 (KCCM-10245)

10 shows the effect of Lactobacillus confusus PL9001 (KCCM-10245) on bone formation (calculated bone area)

In order to achieve the above object, the present invention provides a strain L. confusus PL9001 (KCCM-10245) that promotes bone formation and inhibits the growth and osteoblast invasion of S. mutans, the main caries.

In another aspect, the present invention provides a caries preventive and therapeutic agent comprising the lactic acid bacteria.

The present invention also provides an additive for functional food for the purpose of preventing and treating caries, including lactic acid bacteria and lactic acid bacteria culture filtrate.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

EXAMPLE

Experimental Example 1 Isolation and Identification of L. confusus PL 9001 (KCCM-10245)

Take the feces of the test subjects (Korean infants) and dilute them serially in sterile saline 10 times, MRS medium containing 0.002% Bromophenol (Difco, bacto proteose peptone No.3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g , lactic acid bacteria were isolated by using bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g, dipotassium phosphate 2 g / L). Of these, when colonized with Streptococcus mutans inoculated medium, colonies with inhibitory rings were selected and identified.

The carbohydrate fermentation pattern of the selected strains was determined using API 50 CHL identification kit (bioMerieux Vitex, Inc, France), and strains were analyzed according to the method according to Bergy's manual of systematic bacteriology. Finally, the nucleotide sequence of the 16S rRNA gene was analyzed. The Microseq 16S rRNA gene kit (Perkin Elmer Applied Biosystem) was analyzed and identified. After Gram staining, the bulging form of Lactobacillus confusus, which is typical of Lactobacillus confusus, was confirmed as shown in FIG. 1 (Gram staining picture of PL9001) and FIG. 2 (electron micrograph of PL9001). The sugar fermentation pattern of the isolated Lactobacillus confusus PL9001 is shown in Table 1.

In addition, 16S rRNA of Lactobacillus confusus PL90001 was 99% identical to BISS search results ( http://www.ncbi.nlm.nih.gov/blast ) Weissella confusa (/. Confusus). BankIt AF477495 was given (FIG. 3). The strain was Lactobacillus confusus PL9001 (KCCM-10245) was deposited internationally to the Korea Center for Microbiological Conservation.

[Experimental Example 2] Confirmation of promoting bone formation of L. confusus PL 9001 (KCCM-10245)

To determine whether Lactobacillus confusus PL9001 (KCCM-10245) affects bone formation, a modified test method was used (Stringa E. et al. 1995. Osteoblastic cells from rat long bone: I. Characterization of their differentiation in Bone 16 (6) .663-670; Nair SP et al. 1996. Bacterially induced bone destruction; Mechanism and misconceptions.Infection Immunity 64: 2371-2380). One-day-old rats (ICR) were placed in 70% ethanol, and then separated from the cranial tubes, washed twice with 1 X PBS (phosphate buffered saline, phosphate buffered solution) and culture medium (alpha-MEM, GIBCO, Grand Island, NY, USA). Wash twice with). Alpha-MEM medium containing 0.2% collagenase (Wako pure chemical Industries, Ltd. Japan) and 0.1% dispase (Gibco) was used for the separation of osteoblasts.

The separated cells were plated to a plate cell culture (30 mm diameter) 1 × 10 5 cells / well and cultured in a 5% carbon dioxide incubator for 5 days in alpha-MEM medium containing 10% urea serum. When confluent, 50 ug / ml ascorbic acid (Merck, Darmstadt, Germany) and 10 mM beta-glycerophosphate (Sigma, St. Louis, MI, U.S.A.) were added to all sample groups to aid differentiation. To the experimental group, except for the control group, live bacteria of Lactobacillus confusus PL9001 were added to 1X10 ^ 2 ~ 1X10 ^ 7 cells / well (total culture solution 2 ml), respectively. The medium and lactic acid bacteria were exchanged every 2 days and cultured for 20 days to determine the morphological changes of osteoblasts, whether death, pH of the medium and bone formation. To confirm the bone formation, the bone formed by von Kossa staining, a major enzyme for bone formation, was identified.

Contrary to the general expectation that lactic acid bacteria would form acid and destroy or inhibit the growth of osteocytes, the lactic acid bacteria did not affect the growth and bone formation of osteocytes. After 10 days when the bone nodule began to form, the microorganisms were confirmed to have no effect on bone formation by optical microscopy (FIG. 4), and the results of von Kossa staining (FIG. 5) after 20 days incubation (Vilber Loumat) Image formation system, France) was observed to promote bone formation when comparing the total volume value of the bone formed by analysis (Table 2).

In order to confirm that the bacterium forms lactic acid bacteria also promote bone formation, this time, the activity of ALPase involved in bone formation was measured together. ALPase positive reactions are red and von Kossa stain positive results are brown to black. For staining, discard the culture plate with suction, wash with cold PBS, and use 10% cold Neutral formalin buffer (NFB: 100 ml formalin / formaldehyde, 16 g Na ₂ HPO ₄ , 4 g NaH ₂ PO ₄ .H ₂ O in 1). liter) for 15 minutes. All solutions were discarded, washed with distilled water and left for 15 minutes in fresh distilled water. While waiting, 25 ml of 0.0005 g naphtaol AS MX-PO4, 200 ul DMF (N, N-Dimethylformamide), 25 ml 0.2 M Tris-HCl (pH 8.3), and 0.03 g red violet LB salt were prepared. The culture plate was incubated for 45 minutes at room temperature, washed 3-4 times with distilled water, and left in fresh distilled water for 1 hour. Next, dyeing was performed with 2.5% silver nitrate for 30 minutes. The silver nitrate was removed by suction and washed three times with distilled water. Covered the cells stained with distilled water for a while and the remaining silver nitrate once again removed by washing with distilled water. ( http://stemcell.ibme.utoronto.ca/protocoles -staining-alkalinePhosphatase.htm). As a result, the bone formation promoting phenomenon was observed in the bacterium (FIG. 6) and the same result was observed after staining with ALPase and von Kossa (FIG. 7). This is the first discovery in the world that promotes bone formation by lactic acid bacteria.

Lactobacillus confusus PL9001 (KCCM-11240) is a heterofermenter, and the acid formation is very weak in the case of Lactobacillus confusus PL9001 (KCCM-11240).

Experimental Example 3 Inhibition of Growth of Streptococcus Mutants of L. confusus PL9001 (KCCM-10245) by Overlay Test

In order to confirm the growth inhibition of Streptococcus mutants, L. confusus PL 9001 (KCCM-11240) culture was placed on a blood medium inoculated with Streptococcus mutants and cultured in a carbon dioxide incubator to confirm the production of inhibitory rings. As shown in FIG. 8, growth inhibitory ring of Streptococcus mutants was clearly observed, confirming the killing function of Streptococcus mutants of L. confusus PL9001 (KCCM-10245).

Experimental Example 4 Inhibition of Osteoblastic Invasion of Streptococcus mutants of Lactobacillus confusus PL9001 (KCCM-10245)

Osteoblasts were isolated and cultured for 5 days in the same manner as in Experimental Example 2. For this experiment, Streptococcus mutans (ATCC 27175) isolated from carious dentine were distributed from NCTC and tested according to the reference (Oelschlaeger, TA and BA Tall. 1996. Uptake pathways of clinical isolates of Proteus mirabilis into human epitheial Cell lines.Microb.pathog. 21: 1-16; Marc Jevon. et.al. 1999. Mechanism of internalization of Staphylococcus aureus by cultured human osteoblasts.Infection and Immunity. 67: 2677-2681; Hudson, MC, WK Ramp, NC Nicholson, AS Williams, and MT Nousiaine. 1995. Internalization of Staphylococcus aureus by cultured osteoblasts.Microb. Pathog. 19: 409-419). Streptococcus mutans were cultured in brain-heart infusion (BHI) and suspensions were used to make O.D. 1 at A600. After osteoblasts were confluently cultured, the cells were washed twice with PBS and Streptococcus mutants were added to the medium to 1 × 10 −7 cells / well. After 2 hours of incubation, the cells were washed twice with 1 ml of alpha-MEM. In order to remove non-internalized bacteria, 1 ml of medium containing 100 ug / ml of gentamycin was added and incubated for another 1 hour 30 minutes. After washing twice with PBS, 1 ml of 0.025% TritonX-100 was added to measure the number of internalized bacteria. The cells were destroyed, serially diluted, inoculated into blood agar, and internalized using the Most Probable Method (MPN). The bacteria count was measured. In order to measure the effect of Lactobacillus confusus PL9001 (KCCM-10245), Lactobacillus confusus PL9001 (KCCM-10245), which was suspended in the experimental group to have an OD of 10 in A600, was treated with 1X10 ^ 8 cells / well and incubated for 30 minutes. Subsequently, the Streptococcus mutans were treated to 1 × 10 ^ 7 cells / well and incubated for an additional 1 hour and 30 minutes, followed by serial dilution of the number of internalized Streptococcus mutans in the same manner as the control group. Inoculate to the MPN method to measure the number. As shown in the results of Table 3, S. mutants infiltrated into bone cells by L. confusus PL9001 (KCCM-10245) were reduced to 6%, and when the graph was displayed (Fig. 9), the invasion inhibition effect was more evident. .

As mentioned above, the Lactobacillus confusus PL9001 (KCCM-10245) strain isolated from the infant of the present invention has excellent bone formation promoting effect, killing of main caries Streptococcus mutans, and inhibiting bone cell invasion of Streptococcus mutans. It can be used as a probiotic for prophylactic and therapeutic purposes. It can also be used as a fermentation agent or additive in various foods and beverages and dental health products.

Claims (6)

Functional food and beverage or dental health products for the prevention and treatment of tooth decay using lactic acid bacteria According to claim 1, The lactic acid bacteria Lactobacillus confusus (Weissella confusa) characterized in that the functional food and beverage or dental health products for the purpose of preventing and treating tooth decay The method of claim 2, wherein the lactic acid bacteria functional food and beverage or dental health products for the purpose of preventing and treating caries using Lactobacillus confusus (Weissella confusa) PL9001 (KCCM-10245) A biological net culture and physiologically acceptable excipient or diluent composition of Lactobacillus confusus (Weissella confusa) PL9001 (KCCM-10245) of claim ^{3 at a} cavities killing function concentration ( ¹ × ^{10 3} −1 × ^{10 9} cfu / ml or / cc). Biological net cultures and physiologically acceptable excipients of Lactobacillus confusus (Weissella confusa) PL9001 (KCCM-11240) of claim ^{3 at} a concentration (1x10 ³ -1x10 ⁹ cfu / ml or / cc) that reduces caries bacteria invasion Or diluent composition. 6. Fermented milk according to claim 4, wherein the physiologically acceptable excipient or diluent is. Dairy products such as milk, cheese, soy milk, baby food, fermented foods such as kimchi, salted fish, soy sauce, soybean paste, various soft drinks and gum, caramel, etc., the composition characterized in that the dental health products.