KR100266752B1 - Plaque inhitition by novel human oral strains of lacticacid producing bacteria - Google Patents

Abstract

PURPOSE: Provided are novel lactic acid bacteria which effectively inhibit the formation of glucan and plaque in human oral cavity. CONSTITUTION: The lactic acid bacteria, Enterococcus sp.(1357 KCTC 0360BP), Lactobacillus sp. V20 (KCTC 0361BP), and Lactococcus sp. 1370(KCTC 0415BP) inhibit the formation of glucan and plaque in human oral cavity. They are isolated by: collecting lactic acid bacteria from the human cavity; spreading the lactic acid bacteria on brain heart infusion agar and incubating them at 37 deg.C for 16 hours; inoculating Streptococcus mutans and the isolated lactic acid bacteria in a brain heart infusion medium; fermenting them at 37 deg.C for 3 days; and removing the fermented medium and measuring optical density to isolate lactic acid bacteria inhibiting the production of glucan. The lactic acid bacteria may be added into foods.

Description

Plaque inhitition by novel human oral strains of lactic acid producing bacteria

The present invention relates to a novel bacterium that inhibits the formation of glucan, a major component of plaque in the human oral cavity. More specifically, the present invention relates to novel lactic acid bacteria isolated from microorganisms normally present in the oral cavity of a human. These lactic acid bacteria are known as Enterococcus genus, Lactobacillus genus Lactobacillus, and Lactobacillus which inhibit the activity of glucosyltransferase or antagonize bacteria involved in glucan formation. It relates to the lactic acid bacteria in the genus (Lactococcus).

Lactobacillus refers to bacteria that ferment carbohydrates and produce lactic acid as the final metabolite. Lactobacillus is present in the oral cavity and digestive tract of humans and animals and is commonly used in the manufacturing process of fermented foods such as kimchi or yogurt. In addition, it is used for the production of biologically active substances such as pharmaceuticals. Bacteria belonging to these lactic acid bacteria include Streptococcus, Enterococcus, Lactococcus, and Lactobacillus, Bifidobacterium, and Micrococcus. Among these, lactic acid bacteria used in food are Str. thermophilus, E. faecalis, E. durans, Lactococcus lactis, L. lactis, L.acidophilus, L. bulgaricus, L. thermophilus, L. casei, L. plantarum. These lactic acid bacteria are Gram-positive bacteria, which are known to play an important role in maintaining the health of the intestine by decomposing carbohydrates ingested by humans and animals, producing lactic acid and antibiotics, and inhibiting the growth of harmful intestinal bacteria.

Conventionally, enzymes acting on glucan have been separated to decompose glucan, which is a major component of plaque, to promote oral hygiene. First, dextranase (α-1,6 glucan hydrolase) acting on dextran has been purified and tested in humans and animals (Staat RH Schachtete CF, 1975; Raster, AG Brown, LR, 1983). In addition, studies on the isolation and purification of mutanase (mutanase, endo-α-1,3 glucanase), which break down mutan, an important component of glucan formation, have been reported (Guggenheim B. et al., 1972). Takehara et al., 1981). However, animal experiments on the use of glucan formation inhibitors have not been shown to have much effect on human experiments. The isolated mutanacea has a low plaque decay effect and takes a long time. Because of their time and consumption, their economic and practical use proved to be difficult.

Therefore, the present inventors focus on these facts that lactic acid bacteria can be directly collected and separated from human oral cavity, and several lactic acid bacteria significantly inhibit plaque formation through a number of experiments including glucan inhibitory effect, plaque formation inhibitory effect and actual clinical experiments. Achieved by confirming the fact. Among the lactic acid strains that inhibit plaque formation in the present invention, particularly important strains are Enterococcus spp. 1357, Lactobacillus spp. V20, and Lactococcus spp. 1370 strains, respectively. These strains were deposited on July 30, 1997, and December 11, 1997, respectively, to the Genetic Bank of the Genetic Resource Center, Korea Institute of Science and Technology. Enterococcus 1357 Accession No .: KCTC0360BP, Lactobacillus V20 Accession No .: KCTC 0361BP, Lactococcus 1370 Accession No .: KCTC 0415BP)

The main component of the human oral plaque is glucan, a complex of carbohydrates. Glucan can be classified into water-soluble lanthanum and water-insoluble mutan. Glucan is synthesized from sucrose by glucose transferase secreted by Streptococcus mutans. Mutanes have α-1,3 bonds and are water-insoluble and become the main component of intraoral plaque. In general, plaque is a cause of dental caries and periodontal disease because the growth of other bacteria in addition to Streptococcus mutans on the tooth surface and promote the attachment of various substances such as food waste. Accordingly, another object of the present invention is to prevent lactic acid bacteria from inhibiting glucan formation in the oral cavity, thereby suppressing plaque formation in the teeth, thereby maintaining cleanness in the oral cavity.

Still another object of the present invention is to provide a novel beverage or food using these lactic acid bacteria. That is, by directly inhibiting glucan formation or by directly edible food containing lactic acid bacteria that are antagonistic to the microorganism that forms glucan, it is possible to suppress plaque formation and further prevent tooth caries and periodontal disease.

Hereinafter, the specific configuration and operation of the present invention.

1A-1C are photographs showing the inhibition of water-insoluble glucan formation in disposable cuvettes of lactic acid bacteria of the present invention.

Figure 2a-2c is a photograph showing that the lactic acid bacteria of the present invention inhibits the formation of artificial plaque using a wire for correction.

Figure 3a-3c is a photograph showing the use of inhibiting plaque formation in the human oral cavity of the present invention lactic acid bacteria.

Therefore, the present invention is to collect the lactic acid bacteria samples in the oral cavity of humans in the brain heart infusion agar (Brain heart infusion agar) and then incubated for 16 hours at 37 ℃ in a thermostat generated by the Streptococcus mutans Inhibition of water-insoluble glucan formation was tested and identified. 3 ml of Brain Heart Infusion medium added with 0.5% yeast extract and 5% sucrose was placed in a disposable cuvette and inoculated with 0.1 ml of Streptococcus mutans and 0.1 ml of the bacterial culture. At this time, only the Streptococcus mutans was inoculated into the brain heart infusion agar medium containing yeast extract and sucrose as a control. Disposable cuvettes were placed in the incubator at an angle of 30 ° to the horizontal plane and incubated at 37 ° C. for 3 days to form Streptococcus mutans to form water-insoluble glucan. The culture solution was removed, washed with 4 mL of distilled water, and then 3 mL of distilled water was added to measure absorbance (OD) at a wavelength of 550 nm of a spectrophotometer. When significantly less glucan was formed compared to the amount of water-insoluble glucan produced in the control group, the absorbance in the spectrophotometer was remarkably reduced, and these bacteria were isolated as plaque inhibiting lactic acid bacteria. Several morphological and physiological and microbiological characteristics and glycolytic ability of the isolated lactic acid strains were examined (Table 1, 2).

The microbiological characteristics of isolated plaque formation inhibiting lactic acid bacteria are described as follows.

Table 1 describes the morphological and physiological properties of the isolated lactic acid strains. Table 2 shows the results of investigation of the sugar resolution of the same bacteria.

Morphology and Physiological Properties of Lactic Acid Bacteria Strains of the Invention Characteristics Isolated bacterial strains Enterococcus 1357 Lactobacillus genus V20 Lactococcus 1370 Size Form Gram Staining Spore Formation Catalase Cultivation Temperature 10 ℃ Culture Temperature 45 ℃ pH9.640% Bile Acid6.5% NaClMs Proliferation Acetoin-Producing Hippurate Hydrolysis Pyridyridylarylamidates Pyrrolidonylary amidase) Alpha-Galactosidase Beta-glucuronidase Beta-Glaccuronidase Beta-Galactosidase Alkaline phosphatase Leucine arylamidases Leucine arylamidase Arginine dihydrolase 0.5-1 μm cocci, serial positive-+++++-+++-+-++ 0.6-0.9 × 2-6μmBacterial, Serial Positive --- + --- + 0.5-1 μm, Streptococcus positive-+-+-+ ------ +-

Glycolytic Activity of the Lactic Acid Bacteria Strain of the Invention Types of Carbohydrates (Sugar) Enterococcus 1357 Lactobacillus genus V20 Lactococcus 1370 Arabinos Amygdalin Cellobiose Esculin Fructose Galactose Glucose Lactose Maltose Mannitol Mannose Melannose Melezitose Raffinose Rhamnose Salinine Sorcinol Trebitose Trehalose Inulin Starch Glycogen -++++++++-+ --- +-+-+- -+++++++-+ ----- + -+-+++++++ ----- +-+-

As described above, the present invention comprises a process for isolating and identifying novel lactic acid bacteria for inhibiting plaque formation and an in vitro process and an actual clinical test process by comparing the isolated bacteria with a control group.

Hereinafter, the present invention will be described in detail with reference to Examples and Examples for glucan or novel lactic acid bacteria for plaque formation inhibition.

Example 1 Inhibition of Water-insoluble Glucan Formation in Disposable Cuvettes

M17 medium and MRS medium were mixed in the same amount, 0.5% yeast extract and 5% sucrose were added, and 0.2M TS buffer solution (TES buffer, pH 8.5) was added to the third branch of medium. Then, 3 ml thereof was placed in a disposable cuvette and inoculated with 75 µl of Streptococcus mutans culture solution. This was installed at an angle of 30 ° to the horizontal plane in the incubator and incubated at 37 ° C for 1 day. The contents were removed, washed with 4 ml of distilled water, and photographed (FIGS. 1A, 1B, 1C). Then, 3 ml of distilled water was added to the cuvettes, and the absorbance was measured at 550 nm wavelength of the spectrophotometer. This was repeated three times to find an average (Table 3). Figures 1a (A), 1b (A) and 1c (A) of Cuvette are the control group cultured only Streptococcus mutans, Figure 1a (B) is a culture of Streptococcus mutans and Enterococcus 1357 together. 1a (C) is cultured only 1357 genus Enterococcus. Figure 1b (B ') is cocultured with Streptococcus mutans and Lactobacillus V20 strain and Figure 1b (C') is a culture of Lactobacillus V20 alone. In addition, Fig. 1c (B) is cocultured with 1370 strains of Streptococcus mutans and Lactococcus and Fig. 1c (C) is a culture of only 1370 Lactococcus.

In Table 3a, the absorbance of the control group inoculated with Streptococcus mutans was 2.122, whereas the absorbance of experimental group II inoculated with Enterococcus 1357 dropped to 0.713, and the absorbance of experimental group IV inoculated with Lactobacillus V20 was 1.154. fell. Therefore, Enterococcus 1357 and Lactobacillus V20 inhibit glucan formation by Streptococcus mutans, respectively. In addition, the absorbances of experimental group I inoculated with enterococcus 1357 strain alone and experimental group III inoculated solely with Lactobacillus V20 strain were 0.434 and 0.506, respectively, indicating that the glucan formation inhibitory effect was very large. . In Table 3b, the absorbance of the control group inoculated with Streptococcus mutans was 1.765, whereas the absorbance of the experimental group VI inoculated with Lactococcus 1370 dropped to 0.848 and glucan formation in Experimental group V inoculated with Lactococcus 1370 strain alone. The inhibitory effect was found to have an absorbance of 0.479, indicating that this strain also had an effect of inhibiting plaque formation.

Glucan formation inhibitory effect division Lactobacillus strains inoculated into the medium Absorbance / 550nm Control Streptococcus mutans 2.122 Experiment Group I Enterococcus 1357 0.434 Experimental Group II Enterococcus 1357 + Streptococcus mutans 0.713 Experimental Group III Lactobacillus V20 0.506 Experimental Group IV Lactobacillus V20 + Streptococcus mutans 1.154

Glucan formation inhibitory effect division Lactobacillus strains inoculated into the medium Absorbance / 550nm Control Streptococcus mutans 1.765 Experimental Group V Lactococcus 1370 0.479 Experimental Group VI Lactococcus 1370 + Streptococcus mutans 0.848

Example 2 artificial plaque formation inhibition experiment using orthodontic wire

M17 (M17) medium and MRS (MRS) medium were mixed in the same amount, 0.5% yeast extract and 5% sucrose were added, and 0.2M TS buffer solution (TES buffer, pH 8.5) was added to the third branch of medium. Then 150 ml of it was taken and placed in a beaker. Here, about 45 mg of 0.016 inch stainless steel calibration wire was prepared and suspended in a beaker to be immersed in the culture. The lactic acid bacteria strains of the present invention inoculated with 2.5 × 10 6 Streptococcus mutans per 1 ml of medium and purely isolated were added with 10 times the number of Streptococcus mutans bacteria, and shaken for 6 hours in a 37 ° C. carbon dioxide incubator. Incubated. The wires were transferred to new beakers and photographed (FIGS. 2A-2C). Figure 2a (A), Figure 2b (A) and 2c (A) is a culture of Streptococcus mutans alone and Figure 2a (B) is a co-culture of 1357 strains of Streptococcus mutans and Enterococcus. In addition, Fig. 2b (B) shows the strains of Streptococcus mutans and Lactobacillus V20 strains, and Fig. 2c (B) shows the results of coculture with 1370 strains of Streptococcus mutans and Lactococcus. The weight of the artificial plaque on the wire was measured to extract the weight of the wire, and the weight of the artificial plaque on the wire during the culture was calculated as shown in Table 4. Artificial plaque production was 75.4 mg in the control group inoculated with Streptococcus mutans alone. In experimental group I inoculated with Enterococcus 1357 and experimental group III inoculated with Lactococcus 1370, no artificial plaque was formed. In experimental group II inoculated with the genus V20 strain, artificial plaque production dropped to 30.9 mg. As a result of the experiment, it was found that the formation of artificial plaque of Streptococcus mutans was inhibited by the novel lactic acid bacteria Enterococcus 1357, Lactobacillus V20, and Lactococcus 1370 strains of the present invention, respectively.

Artificial plaque formation effect division Lactobacillus strains inoculated into the medium Artificial plaque production Control Streptococcus mutans 75.4mg Experiment Group I Streptococcus mutans + Enterococcus 1357 0.0mg Experimental Group II Streptococcus mutans + Lactobacillus V20 30.9mg Experimental Group III Streptococcus mutans + Lactococcus 1370 0.0mg

Example 3 Experimental Use of Inhibiting Plaque Formation in Human Oral cavity

In order to test how strongly the novel lactic acid bacteria strain exhibits plaque formation inhibitory effect, the same experiment was conducted at the same time intervals in the same person.

After 6 hours of brushing, the closing solution (disclosing solution), a plaque staining agent, was applied to the front of the tooth, rinsed with water, and photographed (photo 3a). Two days later, the same person was brushed, 50 ml of lactic acid bacteria drink incubated with Enterococcus 1357 in skim milk, and after 6 hours, the disc-logging solution was reapplied and rinsed with water and photographed (photo 3b). Again, the same person was brushed after 13 days and lactobacillus V20 in 50 ml of lactic acid bacteria drink cultured in skim milk, after 6 hours, the disc-roofing solution was applied and rinsed with water and photographed. The result was as in FIG. 3C. As shown in Figure 3a, it can be seen that a large amount of plaque or glucan is produced on the surface of the teeth. It can be seen that is significantly high. Therefore, the continuous use of the lactic acid bacteria of the present invention is expected to increase synergistically inhibiting plaque formation.

Example 4 plaque index reduction experiment

In order to prevent the plaque formation inhibitory effect of the novel lactic acid bacteria strain of the present invention, 38 volunteers were subjected to the same experiment, and plaque scores were determined based on the plaque index of Quigley and Hein. The degree of index reduction was examined.

Scaling was performed for 38 volunteers. After 1 week of scalding, the scalp was washed for 10 minutes, and then the foods were eaten as usual, but not limited to brushing. Then, after 24 hours, the disc-logging solution was applied and rinsed with water. At this time, the degree of plaque formation on buccal side and lingual side of all teeth except the third molars should be determined by the panelist who majors in preventive dentistry based on Quigley and Hein's plaque index. The average plaque score for all teeth except the third molar was averaged by individual, and 38 volunteers were divided into two groups and averaged for each group. Shampoo the same people and rinse your mouth for 2 minutes with 20ml (109 CFU / ml) of lactic acid bacteria drink incubated in skim milk with Lactococcus 1370 in one group of 19 and Lactobacillus V20 in 19 other groups. It was made. This rinsing was carried out three times a day, after shampooing, after breakfast and lunch, to prevent brushing and to eat as usual, and after 24 hours, the disc rinsing solution was applied and rinsed with water. In this case, the same panelist assigns plaque scores based on Quigley and Hein's plaque index to the degree of plaque formation formed on the entire surface of the tooth except the third molar. Averaged by each group and again by each group. The results are as shown in Table 5. The plaque score of the group rinsed with Lactobacillus 1370 cultured Lactobacillus 1370 was 1.20, and the plaque score was 0.97 compared to 2.17 without Lactic Acid Bacteria, and Lactobacillus The plaque score of the group rinsed with lactic acid bacterium cultured with genus V20 was 1.60, and the plaque score decreased by 0.55 compared to 2.15 when the lactic acid bacteria were not added. In addition, both groups using lactic acid bacteria showed statistically significant (p0.05) inhibition of plaque formation, and it was academically proved that the lactic acid bacteria strain of the present invention had high plaque formation inhibitory effect.

Effect of reduction of plaque score by new lactic acid bacteria Lactic acid bacteria Basic plaque score average Average plaque score when using lactic acid bacteria Plaque score difference Lactococcus 1370 2.17 1.20 -0.97 ^* Lactobacillus V20 2.15 1.60 -0.55 ^*

* P <0.01 by paired t test

Hereinafter, the use of the novel lactic acid bacteria strain of the present invention to foods to explain the use of inhibiting the formation of plaque in the human oral cavity.

Use Example 1: Yogurt Product

In the process of manufacturing yogurt prepared by the yogurt manufacturing company, 0.1% by volume of the lactic acid strain culture of the present invention was added immediately before fermentation and mixed and fermented with an existing strain to prepare yogurt and tasting to 10 panelists. ) And no difference in flavor was found. In addition, as a result of tasting 10 panelists of the experimental group sealed by adding 0.2% by volume of the lactic acid strain of the present invention and the control product without adding the lactic acid strain of the present invention before sealing the yogurt manufacturing process, the difference in flavor between both products Answered no.

Use Example 2: Kimchi Product

Five cabbages were cut into 4-5 cm, pickled in brine, washed, and dehydrated. Addition of seasoning ingredients prepared separately such as leek, ginger, garlic, red pepper powder, salted fish, and aged for 3 days at 20 ℃ aging temperature. As a result of tasting 10 kimchi to the panel by adding the group and no addition group (control group) to each of the lactic acid bacteria strain culture of the present invention to the naturally aged kimchi cultured (control group), the respondents did not feel the difference in the flavor of kimchi.

Use Example 3: Butter Products

0.2 wt% of the lactic acid bacteria strain lyophilized product of the present invention was mixed before packaging of the butter product prepared by a conventional method, and then packaged into products and provided for tasting.

Use Example 4: Cheese Products

0.2 wt% of the lactic acid bacteria strain lyophilized product of the present invention was mixed before packaging of the cheese product prepared by a conventional method, and then packaged into a product and used for tasting. The use example of the present invention was applied to any food, such as gum, shortening, ice cream, margarine, in addition to the food described in the specification, and the scope of the present invention is not limited only to these foods.

As a result of the inhibition of glucan formation through the disposable cuvettes and the test of inhibition of artificial plaque formation through orthodontic wires and the purpose of inhibiting the formation of plaque in the oral cavity of the human body, the novel lactic acid bacteria strain of the present invention has a strong and sustained effect of inhibiting glucan or plaque formation. Knowing that the bacteria is a very useful invention in the dentition industry.

In addition, since the novel lactic acid bacteria strain of the present invention can be utilized for food, the present invention is a very useful invention in the food industry and intestinal health medicine.

Claims (5)

Enterococcus sp. 1357 KCTC 0360BP having an activity of inhibiting the formation of glucan or plaque in the human oral cavity. Lactobacillus sp. V20 KCTC 0361BP having activity to inhibit the formation of glucan or plaque in human oral cavity. Lactococcus sp. 1370 KCTC 0415BP having the activity of inhibiting the formation of glucan or plaque in the human oral cavity. Glucan or plaque formation inhibiting food prepared by using the bacteria of any one of claims 1 to 3 alone or in combination. The method of claim 4, wherein the food is selected from the group consisting of kimchi, yogurt, butter, cheese, ice cream, gum, shortening, and margarine.