KR20060043665A - Lactic acid bacteria inhibiting halitosis - Google Patents

Abstract

The present invention relates to a novel lactic acid bacterium that inhibits halitosis (oral malodor), and combines with anaerobic bacteria that produce volatile sulfur compounds to produce hydrogen peroxide in aerobic and anaerobic conditions, thus producing a novel genus Weissra (Lactobacillus genus Weissella ), the novel bacteria are isolated and identified among the naturally occurring lactic acid bacteria in the oral Weissella cibaria CMU (Accession No .: KCTC 10650BP), Weissella sibaria CMS- 1 (Accession No .: KCTC 10678BP), Weiquila Sibaria CMS-2 (Accession No .: KCTC 10679BP), Weiquila Sibaria CMS-3 (Accession No .: KCTC 10680BP) and were deposited. Through the strains of genus Weylla genus Sibaria it is possible to provide lactic acid bacteria and foods using the same that can effectively inhibit bad breath-producing anaerobic bacteria without causing corrosion on teeth.

Bad Breath, Lactic Acid Bacteria, Anaerobic Bacteria, Volatile Sulfur Compounds, Weispilla Sibaria

Description

Lactic acid bacteria inhibiting halitosis

The present invention relates to novel bacteria that inhibit bad breath from the human oral cavity. More specifically, the present invention relates to a novel lactic acid bacteria isolated from microorganisms normally present in the oral cavity of a person. These lactic acid bacteria are related to a novel genus Weissella lactic acid bacterium that binds with representative anaerobic bacteria producing volatile sulfur compounds and produces hydrogen peroxide in aerobic and anaerobic conditions to inhibit anaerobic bacteria.

Bad breath refers to odors derived from the oral cavity and neighboring organs, and many people have an inconvenience in daily life due to bad breath. 85-90% of bad breath comes from the oral cavity, especially from the back of the tongue. The main component of bad breath is volatile sulfur compounds, of which 90% of the total amount of volatile sulfur compounds is hydrogen sulfide made from cysteine and methyl mercaptan made from methionine. These components are produced by anaerobic bacteria, and the back of the tongue is the most important habitat. This area is poorly cleaned by saliva and has many small depressions, making it a place where bacteria live continuously. Production of volatile sulfur compounds by anaerobic bacteria is the most important cause of bad breath, but is also caused by oral diseases such as caries and periodontitis. The bacteria that make hydrogen sulfide from cysteine are Peptostreptococcus genus, Eubacterium genus, Selenomonas genus, Centipeda genus, Bacteroides genus, and Fusobacterium spp. ( Fusobacterium ), bacteria that make methylmercaptan from methionine include the genus Fusobacterium , bacteroids, Porphyromonas (genus genus), Eubacterium genus. In particular, the bacteria found in the mouth of bad breath patients are Fusobacterium nucleatum , Prevotella intermedia , Porphyromonas gingivalis . Previously, the genus Bacteroids and Popiromonas, together with the genus Prevotella , are similarly similar bacteria. Although many types of anaerobic bacteria are involved in the development of bad breath, the most common bacterial causes of bad breath are Fusobacterium nucreatum and Popiromonas gingivalis.

In order to suppress bad breath, it is most effective to suppress the proliferation of anaerobic bacteria that produce volatile sulfur compounds, which are the main components of bad breath, and conventionally, tongue tongue was used to remove the growth substrate of bacteria from the tongue. In order to remove or to inhibit the growth of anaerobic bacteria or the generation of volatile sulfur compounds by anaerobic bacteria, metal salts such as zinc chloride or disinfectants such as alcohol or chlorhexidine were used. However, these metal salts and disinfectants suppress not only anaerobic bacteria that cause bad breath, but also other microorganisms in the oral cavity, and because these substances do not swallow into the esophagus, they do not reach the back of the tongue, which is an important place for bad breath, and is mainly in front of the oral cavity. Gargled and spit out of mouth Therefore, there is a problem that can not be sterilized until the back of the tongue, which is the main growth site of bad breath-producing anaerobic bacteria. In addition, such a substance is diluted by saliva and swallowed with the saliva into the esophagus, the effect in the oral cavity is only about 20 minutes to 2 hours, after which the microorganisms multiply again in the oral cavity to cause bad breath. Therefore, the metal salt or disinfectant used to reduce bad breath has only a short effect.

Therefore, in recent years, there has been an attempt to suppress the proliferation of anaerobic S. bacteria in vitro using lactic acid bacteria. Lactic acid bacteria refers to bacteria that ferment carbohydrates to produce lactic acid as a final metabolite. Lactic acid bacteria are present in the oral cavity and digestive tract of humans and animals, and are commonly used in the manufacturing process of fermented foods such as kimchi or yogurt. The lactic acid bacteria to be used in food products and the like Enterococcus (Enterococcus) genus, Lactobacillus put up Russ (Lactobacillus) genus Lactococcus (Lactococcus), A flow Pocono stock (Leuconostoc), A Streptococcus (Streptococcus) in, Wei Cell rasok . However, such lactic acid bacteria have a problem that when the lactic acid bacteria are administered to the oral cavity, they are diluted with saliva, such as metal salts or disinfectants, and swallowed directly into the esophagus, making it difficult to remain in the oral cavity. In addition, lactic acid bacteria such as Lactobacillus acidophilus , Lactobacillus casei , and Lactobacillus salivarius can inhibit the growth of anaerobic bacteria in vitro. In the oral cavity of normal people, the strong acid produced by lactic acid bacteria is neutralized by the buffering action of saliva, making it difficult to inhibit the growth of other bacteria. In addition, strong acid causes dental caries, so there is a problem that long-term administration of such bacteria is bad for oral hygiene.

The present inventors attempted to settle in the oral cavity to lactic acid bacteria that inhibit anaerobic bacteria that cause bad breath to solve the above problems. The present invention was intended to produce lactic acid bacteria to produce hydrogen peroxide by combining with anaerobic bacteria that produce volatile sulfides in aerobic and anaerobic conditions.

Until now, the role of lactic acid bacteria in the oral cavity to produce lactic acid to corrode teeth because the role was considered negative. However, lactic acid bacteria according to the present invention produce less lactic acid, so that the acidity is not high, so that bad breath can be suppressed by preventing anaerobic bacteria that cause bad breath without causing corrosion to the teeth, and therefore, bad breath is completely different from metal salts and disinfectants. The purpose is to suppress anaerobic bacteria that occur. In addition, the lactic acid bacteria according to the present invention is prepared by the food lactic acid bacteria preparation to drink or eat, because it passes through the back of the oral cavity to settle on the back of the tongue, while proliferating the development of anaerobic bacteria that cause bad breath. .

The present invention has been made in order to achieve the above object, the lactic acid bacteria according to the present invention is catalase production negative, grows at 25 ^o C to 42 ^o C, combined with anaerobic bacteria that produce volatile sulfides It relates to the Weissella cibaria bacteria that produce hydrogen peroxide in aerobic and anaerobic conditions.

In detail, the Weyquila shibaria bacterium according to the present invention is a Weyquila sibaria lactic acid bacterium, characterized in that it combines with bad breath-producing anaerobic bacteria without producing a low lactic acid so that the acidity is not high and causing corrosion to the teeth. In addition, the present invention is characterized in that it relates to the genus Weylla cibaria CMU KCTC 10650BP which binds to the anaerobic bacteria causing bad breath and generates hydrogen peroxide in aerobic and anaerobic conditions. In addition, other Weiquila sibaria lactic acid bacteria according to the present invention relates to Weiquila sibaria CMS-1 KCTC 10678BP or to Weiquila sibaria CMS-2 KCTC 10679BP or Weiquila sibaria CMS-3 KCTC 10680BP.

Another feature of the present invention may be in the form of drinking or eaten prepared by the lactic acid bacterium agent to suppress anaerobic bacteria that produce volatile coalitions by using singly or in combination with the genus Weissra sibaria lactic acid bacteria. Since it passes through the back of the oral cavity, it is a food prepared to suppress the proliferation of bad breath-producing anaerobic bacteria while settling and proliferating on the back of the tongue.

In addition, the present invention is characterized by a method for separating and identifying the lactic acid bacteria that inhibits the wetting of the genus Weylla genus Sibaria lactic acid bacteria and anaerobic bacteria and the generation of hydrogen peroxide, which inhibits bad breath. In this method, the lactic acid bacteria sample was cultured in MRS medium for 1 day and centrifuged (6,000 rpm, 10 min, 4 ^o C), washed once in 0.85% saline, and then suspended. Add each of the mixture for 10 seconds, stir at 37 ^° C. and 110 rpm for 30 minutes, and leave it at room temperature for 3 minutes, selecting a lactic acid bacterium from which the supernatant is transparent from the test tube, and generating a strain of hydrogen peroxide in the selected lactic acid bacteria. For isolation, 3 μl of lactic acid bacteria culture medium was added to the medium prepared by adding 0.25 mg / ml of 3,3 ', 5,5'-tetramethylbenzidine and 0.01 mg / ml of peroxidase to the MS solid medium. Inoculating each step to examine the formation of blue colonies, separating the lactic acid bacteria that produce hydrogen peroxide in anaerobic and aerobic conditions, and identifying the isolated lactic acid strains by 16S rRNA gene analysis As to a method for separating, identifying the Weissella cibaria lactic acid value.

The present inventors collected and separated the lactic acid bacteria naturally present in the human oral cavity, and studied through the inhibitory effect experiments and the actual clinical experiments on anaerobic bacteria, it was confirmed that the Weirella sibaria lactic acid bacteria significantly inhibited bad breath. . Strains that inhibit the occurrence of bad breath in the present invention was named Weyquila sybaria CMU, Weyquira sybaria CMS-1, Weyquira sybaria CMS-2, Weyquira sybaria CMS-3, and these strains were named June 4, 2004. On August 6th, it was deposited with the Korea Institute of Bioscience and Biotechnology, Biological Resource Center (Waquila Sibaria CMU Accession No .: KCTC 10650BP, Waquia Sibaria CMS-1 Accession No .: KCTC 10678BP, Weijura Sibaria CMS-2 Accession No. : KCTC 10679BP, Weiquila Sibaria CMS-3 Accession No .: KCTC 10680BP).

Since the lactic acid bacteria according to the present invention are immediately combined with bacteria such as the representative bad breath-producing anaerobic bacteria, such as Fusobacterium nucleatum and Popiromonas jinjivalis, when the lactic acid bacterium of the present invention is administered orally, Fusobacterium nucrea Combination with bacteria such as Tomb and Popiromonas jinjivalis can produce hydrogen peroxide in both anaerobic and anaerobic conditions, thereby inhibiting the combined anaerobic bacteria. In particular, anaerobic bacteria proliferate in the anaerobic state and metabolic activity occurs, so hydrogen peroxide production in the anaerobic state is important in suppressing these anaerobic bacteria.

When describing the form and physiological characteristics of the strain of lactic acid bacteria according to the present invention. According to the present invention, Lactobacillus strains that inhibit the occurrence of bad breath, Weissula sibaria CMU, Weissella sibaria CMS-1, Weissella sibaria CMS-2 and Weissella sibaria CMS-3 belong to the genus Weissura, It is grown in MS medium and is Gram-positive bacilli. Catalase production negative, grows at 25 ^o C, 35 ^o C, 42 ^o C and combines with major anaerobic bacteria to produce volatile sulfides to form hydrogen peroxide in aerobic and anaerobic conditions to form blue colonies . In addition, lactic acid bacteria according to the present invention (L-arabinose), amigdalin (amygdalin), cellobiose (cellobiose), esculin (esculin), fructose (fructose), galactose (gentiobiose), gentiobiose, Gluconate, N-acetyl-glucosamine, Glucose, Maltose, Mannose, Salicin, Sucrose, and D-xylose It has the ability to decompose carbohydrates such as).

As described above, the present invention consists of a process for isolating and identifying novel lactic acid bacteria for inhibiting bad breath incidence and the actual clinical experimental process of the separated bacteria. Hereinafter, the novel lactic acid bacteria for inhibiting bad breath of the present invention will be described in detail with reference to Examples and Examples. The invention can be better understood by the following examples, which are intended to illustrate the invention and are not intended to limit the scope of protection defined by the appended claims.

Example 1 Isolation and Identification of Lactic Acid Bacteria

First, the present inventors collected 3,000 strains of lactic acid bacteria samples from the oral cavity of humans to see the degree of binding to the anaerobic bacteria Fusobacterium nucreateum, lactic acid bacteria in MRS medium and Fusobacte Leeum Nucreatum ATCC 10953 was incubated in a brain heart infusion medium supplemented with 1% yeast extract, 5 μg / ml hemin, and 1 μg / ml vitamin K ₁ for 1 day and centrifuged ( 6,000 rpm, 10 min, 4 ^° C), 0.85% saline was washed once and then suspended. Then, 1 ml each of the suspension was added to the test tube, mixed for 10 seconds, stirred at 37 ^° C. and 110 rpm for 30 minutes, and left to stand at room temperature for 3 minutes. Among the lactic acid bacteria examined in this way, the supernatant was selected as a lactic acid bacterium transparent. To isolate the hydrogen peroxide-producing strain among the selected lactic acid bacteria, 0.25 mg / ml of 3,3 ', 5,5'-tetramethylbenzidine and 0.01 mg / ml of peroxidase were added to MS solid medium. 3 μl of lactic acid bacteria culture medium was inoculated into the prepared medium to examine the formation of blue colonies. Subsequently, four strains of lactic acid bacteria, which immediately bind to Fusobacterium nucleatum and generate hydrogen peroxide in the anaerobic and aerobic states, were isolated. Identification of the 4 strains of isolated lactic acid bacteria was carried out by 16S rRNA gene analysis, all 4 strains of the lactic acid bacteria were identified as Weisilla Sibaria.

Example 2 Cross-linking and Hydrogen Peroxide Production Experiment of Anaerobic Bacteria Producing Volatile Sulfur Compounds and Lactic Acid Bacteria and Standard Lactic Acid Bacteria According to the Present Invention

Standard lactic acid bacteria Enterococcus faecalis ATCC 29212, Lactobacillus casei ATCC 334, Lactobacillus acidophilus ATCC 4356, Lactobacillus fermentum ATCC 14931 ), Lactobacillus salivarius ATCC 11742 , Streptococcus oralis ATCC 35037, Streptococcus salivarius, Weissella confusa ATCC 10881, Weissella kimchi CHJ3, Weissella cibaria CCUG41967 and the main anaerobic bacteria Fusobacterium nucreatum and Popiromonas, which produce the four strains of lactic acid bacteria and volatile sulfur compounds isolated and identified in Example 1 according to the present invention. Experiments were conducted to determine the degree of binding to the zygovalis ( Porphyromonas gingivalis A7A1-28). First, Fusobacterium nucleatum was incubated in Brain Heart Infusion medium supplemented with 1% yeast extract, 5 μg / ml hemin, and 1 μg / ml vitamin K ₁ for 1 day, and Pyromonas ginjivalis was 0.5% yeast. The extract, 5 μg / ml hemin, 1 μg / ml vitamin K ₁ was added to the brain heart infusion medium added for 2 days, lactic acid bacteria were incubated for 1 day in MS medium or brain heart infusion medium, centrifuged (6,000 rpm, 10 min, 4 ^° C.), once washed and then suspended in a similar saliva (0.021 M Na ₂ HPO ₄ / NaH ₂ PO ₄ , 36 mM NaCl, 0.96 mM CaCl ₂ , pH 7.3). Then, lactic acid bacteria and anaerobic bacteria suspensions were added to the test tube alone or in combination, respectively, for 10 seconds, and stirred at 37 ^° C. and 110 rpm for 30 minutes, and then allowed to stand at room temperature for 3 minutes.

Here, 0.5 ml of the supernatant was stirred before and after 30 minutes of stirring, and the absorbance (optical density) of the medium was measured at a wavelength of 660 nm of a spectrophotometer. To determine whether hydrogen peroxide was produced in anaerobic and aerobic conditions, 3 μl of lactic acid bacteria culture was inoculated into a medium prepared by adding 3,3 ', 5,5'-tetramethylbenzidine and peroxidase to examine blue colony formation. .

Table 1 below shows the results of the cross-linking of the standard lactic acid bacteria and the present invention lactic acid bacteria and the main volatile sulfur compound-producing anaerobic bacteria and hydrogen peroxide production experiments.

      Lactobacillus Absorbance when combined with FN ^* ____________________ 0 min 30 min Absorbance when combined with PG ^** ____________________ 0 min 30 min   Hydrogen peroxide production __________________ Anaerobic state Enterococcus picalis    1.238 0.707    0.963 0.485     -- Lactobacillus casei    1.883 1.103    1.857 0.681     -- Lactobacillus ashdophyllus    1.829 1.485    1.713 1.368     -- Lactobacillus Fermentum    1.717 1.608    1.565 1.233     -- Lactobacillus salivarius    1.974 0.547    2.028 1.430     -+ Streptococcus Oralis    1.340 0.489    1.441 1.176     -+ Streptococcus salivarius    1.531 0.192    1.608 0.238     -- Weiqilla Confusa    1.824 1.017    1.532 1.338     -- Weiqilla Kimchi    1.817 1.652    1.689 1.434     -+ Weiquila Sibaria CCUG 41967    1.790 1.517    1.680 1.368     + + Weiquila Sibaria CMU    1.854 0.363    1.783 0.067     + + Weispilla Sibaria CMS-1    1.861 0.382    1.731 0.808     + + Weiqilla Sibaria CMS-2    1.864 0.308    1.781 0.154     + + Weiquila Sibaria CMS-3    1.871 0.418    1.707 0.725     + +

^* FN: Fusobacterium New Creatum

^** PG: Popiromonas Jinjivalis

As shown in Table 1 above, the lactic acid bacteria and the anaerobic bacteria and the hydrogen peroxide production experiment, both standard lactic acid bacteria in the anaerobic state did not produce hydrogen peroxide, but only in Wetilla sibaria to produce hydrogen peroxide in the anaerobic and aerobic state Confirmed. However, compared to the Lactobacillus strain of the present invention, the strains of the strains of the strains of the strains of the strains, such as the strains of the strains of the strains of the strains, were strains of the supernatant after 1.5 minutes of stirring. It was as high as 1.368. The supernatant absorbance of the lactic acid bacteria and Fusobacterium nucleatum according to the present invention was reduced to 0.363, 0.382, 0.308, 0.418 after stirring for 30 minutes at 1.854, 1.861, 1.864, 1.871, and lactic acid bacteria and foreskin according to the present invention. The supernatant absorbance at the time of incorporation of Monas Genjivalis was reduced to 0.067, 0.808, 0.154, 0.725 after stirring for 30 minutes at 1.783, 1.731, 1.781, 1.707, and the Weiquila sibaria CCUG 41967, which is the Weiquila sibarian standard strain Lactobacillus showed a difference with 4 weeks. Only four weeks of lactic acid bacteria according to the present invention can produce hydrogen peroxide in the anaerobic and aerobic state and can be seen that the interaction with the major volatile sulfur compound-producing anaerobic bacteria.

Example 3: Lactic acid bacteria and standard lactic acid bacteria in accordance with the present invention bad breath suppression experiment

In order to confirm the clinical effect of the standard lactic acid bacteria and the lactic acid bacteria of the present invention to inhibit the bad breath, the concentration of volatile sulfur compounds, especially hydrogen sulfide, methyl mercaptan and dimethyl sulfide, in the oral cavity was measured in the human body. Was measured using Oralchroma ^(R) (Tagasa, Japan). In each individual, the volatile sulfur compound concentrations in the oral cavity vary greatly depending on whether they brush their teeth, when they are eating, when they are, and when they are stressed. First, they wake up in the morning as usual and do not eat or drink or brush their teeth. Oral chromatographic measurement of oral volatile sulfur compounds is repeated for 3 days and averaged. To test the degree of inhibition of lactic acid bacteria on the production of oral volatile sulfur compounds, after brushing after lunch and dinner, 30 minutes later, 10 ⁹ lactic acid strains suspended in 15 ml of distilled water were put in the mouth, and gargled for 1 minute. I had a safe and swallowed it. The next morning, the oral chromatographic concentration of oral volatile sulfur compounds was measured three times without ingesting or brushing food or drink. Again, after lunch and dinner, brushing teeth after the same day, 30 minutes later, 15 ml of distilled water and 10 ⁹ isolated strains were put in the mouth, gargled for 1 minute, and swallowed for 1 minute. The next morning, oral chromatographic determination of oral volatile sulfur compounds was repeated three times without eating or drinking or brushing teeth.

Table 2 below shows the bad breath inhibitory effect upon oral administration of the lactic acid bacteria according to the present invention.

 Administration strain  Bad breath inhibition rate (deaf person / subject) _________________________________________________ Total dose on the first day of administration Weiquila Sibaria CMU      52.9% (37/70) 17.1% (12/70) 70.0% (49/70) Weispilla Sibaria CMS-1      54.5% (6/11) 18.2% (2/11) 72.7% (8/11) Weiqilla Sibaria CMS-2      50.0% (5/10) 10.0% (1/10) 60.0% (6/10) Weiquila Sibaria CMS-3      40.0% (4/10) 30.0% (3/10) 70.0% (7/10)

As shown in Table 2, 37 (52.9%) of the 70 subjects who consumed the Wyquilla sibaria CMU decreased volatile sulfur compounds concentration by more than 50% in 1 day, and more than 50% in 2 days. The total number of patients was 12 (17.1%), a total of 49 (70.0%), and 21 (30.0%) were not. Eight out of 11 subjects who consumed the Weissula Sibaria CMS-1 (72.7%), 6 out of 10 (60.0%) who consumed the Weissula Sibaria CMS-2, Seven out of 10 drinking subjects (70.0%) had a reduction of more than 50% in control values. In particular, eight patients had a more than 50% reduction in 1 day after eating Weissula Sibaria CMU in 11 people who had bad breath with concentration of volatile sulfur compounds more than 1000 ppb as measured by oral chroma. The bad breath was reduced to two people in a total of 10 people (90.9%) showed that the bad breath was reduced, the lactic acid bacteria strain of the present invention was more effective in people with severe bad breath. However, when the standard lactic acid bacteria were tested in each of three subjects, all standard lactic acid bacteria showed no inhibitory effect on bad breath. Therefore, only four strains of lactic acid bacteria according to the present invention had a clinically bad breath inhibitory effect.

Hereinafter, the use of the novel lactic acid bacteria strain according to the present invention to the food and the bad breath development inhibitor.

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 can be found. In addition, there was no difference in flavor between the two products as a result of tasting 10 control panel products of the experimental group which added 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. Answered.

Use Example 2: Kimchi Product

Five cabbages were cut into 4-5 cm, pickled in brine, washed, and dehydrated. After adding the seasoning, it was aged for 3 days at 20 ^o C. Naturally aged kimchi 0.2% by weight of the lactic acid bacteria culture of the present invention and the addition group (control group) was provided to each of the panelists to tasting 10 panelists as a result of responding that the kimchi flavor does not feel the difference.

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. After tasting 10 panelists, they answered that there was no difference in the flavor of butter products.

Use Case 4: Gum Products

0.2 wt% of the lactic acid bacteria strain lyophilized product of the present invention was mixed before packing of the chewing gum product prepared by a conventional method, and then packaged into products and provided for tasting. The results were presented to 10 panelists, who answered that they did not notice any difference in the flavor of the gum products.

In this way, by directly edible new beverages or foods using the lactic acid bacteria according to the present invention to the development of hydrogen peroxide to the anaerobic bacteria to suppress the generation of volatile sulfides and further prevent the occurrence of bad breath.

The use example of the present invention was applied to any food such as cheese, shortening, ice cream, margarine, in addition to the foods described in the specification, and the scope of the present invention is not limited to these foods.

In addition, stability against the bad breath-inhibiting lactic acid bacteria Weiquila sibaria CMU KCTC 10650BP, Weiquila sibaria CMS-1 KCTC 10678BP, Weiquila sibaria CMS-2 KCTC 10679BP and Weiquila sibaria CMS-3 KCTC 10680BP according to the present invention. The inspection was carried out and will be described below.

Stability test of bad breath inhibiting lactic acid strain according to the present invention

Safety test was performed for the bad breath inhibiting lactic acid strain according to the present invention, first, inoculated with the isolated strain in blood agar (blood agar) and incubated for 24 hours at 37 ^o C, all isolates did not show β-hemolysis . Gelatin was not liquefied even after inoculating the isolate with the gelatin media and incubating for 6 weeks at 35 ^o C.

Next, it was examined whether or not the production of harmful metabolites of lactic acid bacteria according to the present invention, first inoculated with the isolate strain in urea agar medium (urea agar) to see whether ammonia production and incubated for 12 hours at 37 ^o C all isolates The culture medium was negative with no change in color, inoculated with isolate strain in tryptophan media for 18 hours at 37 ^o C, and 5 drops of Kovac's reagent were added to the isolate to determine whether indole was produced. It was negative with no color change, and inoculated with phenylalanine agar medium inoculated with phenylalanine agar medium to see if phenylpyruvic acid was formed by deamined from phenylalanine and incubated at 37 ^o C for 24 hours. When 5 drops of ferric chloride were added, all isolates were negative with no change in medium color.

Subsequently, it was tested for the generation of noxious enzymes from isolated bacteria. First, ρ-nitrophenyl was added to 0.1 M sodium phosphate buffer (pH 6.0) to see whether β-glucuronidase was produced. 0.2% of ρ-nitrophenyl-β-D-glucuronide was mixed with the same amount of the isolated bacterial suspension and reacted at 37 ^o C for 16 hours. It was not a voice. To see if 7α-dehydroxylase was produced, dissolved 0.016% of cholic acid and chenodeoxycholic acid in 0.1 M phosphate buffer solution were added to the isolated bacterial suspension. The reaction was stopped at 37 ^o C for 30 minutes, 3 drops of 6 N hydrochloric acid was added to stop the enzymatic reaction. The mixture was extracted three times with ethyl acetate, dried over nitrogen gas, and then thin-layer chromatography. , TLC). Thin layer chromatography was placed on a TLC plate under 20 μl per sample and dried, then developed with a developing solution, dried at 40 ^o C for 10 minutes, and then treated with 50% sulfuric acid and treated at 180 ^o C for 2 minutes. The cholic acid was not converted to deoxycholic acid and the kenodicholic acid to lithocholic acid. To see if nitroreductase was formed, 4-nitrobenzoic acid and trichloroacetic acid were added to the pulverized bacterial supernatant and reacted at 37 ^o C for one hour. The voice did not turn red. To determine platelet aggregation, the degree of platelet aggregation was measured by adding isolated bacteria and ADP, a platelet aggregation trigger, to the platelet-rich plasma.

As described above, the bad breath inhibiting lactic acid bacterium according to the present invention was confirmed to be safe in all of the safety tests, such as test for the production of harmful metabolites and test for the generation of harmful enzymes of isolated bacteria.

As described above, the novel lactic acid bacteria strain according to the present invention confirmed by mutual binding experiment with volatile sulfur compound-producing anaerobic bacteria and bad breath control experiment in human oral cavity has a bad breath inhibitory effect, which is very useful in the dental industry. According to the present invention, lactic acid bacteria which produce hydrogen peroxide were combined with anaerobic bacteria producing volatile sulfides even in aerobic and anaerobic conditions, thereby allowing lactic acid bacteria to inhibit bad breath-producing anaerobic bacteria in the oral cavity. .

The lactic acid bacteria according to the present invention is to produce less lactic acid, so that the acidity is not high, it is possible to suppress bad breath by inhibiting anaerobic bacteria generated bad breath without causing corrosion on teeth. In addition, by using the novel lactic acid bacteria strain according to the present invention to drink or eat by food, passing through the back of the oral cavity, because it can settle on the back of the tongue and proliferate while inhibiting the proliferation of bad breath-producing anaerobic bacteria, food industry and health It is a very useful invention in medicine.

Claims (5)

Weissella cibaria , catalase-producing negative, growing from 25 ^o C to 42 ^o C, combined with anaerobic bacteria producing volatile sulfides to produce hydrogen peroxide in aerobic and anaerobic conditions. The method of claim 1, wherein the Weyquila sibaria bacteria produce less lactic acid, the acidity is not high, so as not to cause erosion of the teeth, Weiquila sibaria lactic acid bacteria, characterized in that combined with the bad breath-producing anaerobic bacteria. The method according to claim 1 or claim 2, wherein the Weylla shivaria lactic acid bacterium is called Weylla sibaria CMU KCTC 10650BP, Weysha sibaria CMS-1 KCTC 10678BP, Weyshilla sibaria CMS-2 KCTC 10679BP or 3 KCTC 10680BP, Weiweila Sibarian lactic acid bacteria characterized in that. By preparing the Wetilla civaria lactic acid bacterium according to claim 3 as a food lactic acid bacterium for drinking or eating, so that the lactic acid bacteria pass through the back of the mouth and settles on the back of the tongue to proliferate and prevent the growth of anaerobic bacteria that cause bad breath. Manufactured food. The lactic acid bacteria sample was cultured in MRS medium for 1 day and centrifuged (6,000 rpm, 10 min, 4 ^o C), washed once in 0.85% saline, and then suspended. 1 ml each of the suspension was added to the test tube, mixed for 10 seconds, stirred at 37 ^° C. and 110 rpm for 30 minutes, and then allowed to stand at room temperature for 3 minutes, Selecting a lactic acid bacterium from which the supernatant is transparent among the test tubes left untreated, In order to isolate hydrogen peroxide-producing strains from the selected lactic acid bacteria, 0.25 mg / ml of 3,3 ', 5,5'-tetramethylbenzidine and 0.01 mg / ml of peroxidase in MS solid medium. Inoculating 3 μl of the lactic acid bacteria culture medium into the medium prepared by adding the same to examine the formation of blue colonies. Isolating lactic acid bacteria producing hydrogen peroxide in anaerobic and aerobic conditions; and Identifying the isolated lactic acid strain by 16S rRNA gene analysis, Method for isolating and identifying the Weissella sibarian lactic acid bacteria according to claim 3.