JP4469792B2 - Fermented milk for preventing and / or treating oral diseases, and fermented milk for preventing and / or suppressing bad breath - Google Patents

Description

  The present invention relates to the technical field of fermented milk. More specifically, the present invention relates to a fermented milk for preventing and / or treating oral diseases that is inexpensive and has a good flavor, and a fermented milk for preventing and / or suppressing bad breath.

  There are compositions and foods using lactic acid bacteria for the purpose of preventing periodontal disease and bad breath. Most lactic acid bacteria used in these conventional compositions and foods have been isolated from humans, but many are not suitable for foods from the viewpoint of production characteristics and flavor.

  For example, a composition for preventing / treating oral diseases including dental caries and a probiotic product containing a bacterial body belonging to lactic acid bacteria (Lactobacillus salivarius) as an active ingredient (Patent Document 1: WO2002 / 016554) Furthermore, compositions with lactic acid bacteria or their filtrates or treated products and various sugar / plant extracts (Patent Document 2: WO2002 / 080946) have been developed.

  On the other hand, Patent Document 3 (Japanese Patent Laid-Open No. 2003-171292) describes a preventive or therapeutic agent for periodontal disease containing lactic acid bacteria having a periodontal disease preventing effect and saccharides that can be assimilated by these.

  Patent Document 4 (Japanese Patent Application Laid-Open No. 2003-299480) describes that a food containing lactic acid bacteria (reuteri bacteria) having a caries (caries) prevention effect and a yogurt are produced. Reuteri bacteria are known to produce antibiotics (Patent Document 5: US Pat. No. 5,352,586).

WO2002 / 016554 WO2002 / 080946 JP2003-171292 JP2003-299480 U.S. Pat.No. 5,352,586

  Conventionally known lactic acid bacteria, which are said to have a periodontal disease prevention / treatment effect, are mainly used as therapeutic agents or other food forms, and are rarely used as yogurt. This is because, for example, Lactobacillus salivarius described in Patent Document 1 (WO2002 / 016554) is a bacterium present in oral saliva, which is weak under acidic conditions such as lactic acid, and fermented milk using these lactic acid bacteria. This is because the card cannot be formed when trying to manufacture the card.

  Even if it is going to manufacture yogurt using these lactic acid bacteria, an extra manufacturing process is required. For example, when trying to produce fermented milk using lactic acid bacteria that are said to have conventional periodontal disease prevention / treatment effects, (1) culturing lactic acid bacteria alone, and (2) centrifugation according to the situation Or a membrane separation method to concentrate the cultured cells to a predetermined concentration, and (3) a manufacturing process in which the concentrated cells are added to the pre-fermented formula milk together with another lactic acid bacterium forming a card, and mixed and cultured. Necessary. These manufacturing processes are complex to manage and increase the cost and time required for manufacturing. It is also difficult to obtain a flavor with excellent palatability.

  On the other hand, in Patent Document 4 described above, yogurt is produced using Reuteri bacteria as a starter. However, this yogurt is merely a yogurt for preventing caries having a bacteriostatic or bactericidal action against caries (mutans). Not very effective against periodontal disease. Furthermore, the fermentation time of yogurt is extremely long as 10 hours, which is not suitable for production of ordinary commercial yogurt.

  Therefore, the present invention has an object to manufacture fermented milk for treatment and / or prevention of periodontal disease (oral disease) having good taste and inexpensiveness, as well as suppression of bad breath and / or prevention.

  The inventors of the present application have tested in vitro whether fermented milk has effects such as suppression of bad breath and growth of periodontal disease-related bacteria based on a combination of lactic acid strains for preparing fermented milk.

  As a result, oral malodor suppression and gingival swelling are suppressed by administering fermented milk prepared with Bulgarian OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Thermophilus OLS3294 (Streptococcus thermophilus OLS3294) to humans. Was revealed.

  According to the present invention, fermented milk having high palatability and having oral diseases such as periodontal disease prevention / treatment effect and fermented milk having high palatability and prevention of bad breath and / or suppression effect can be provided.

  Furthermore, this invention provides the manufacturing method which can manufacture easily fermented milk with high palatability and a periodontal disease prevention / treatment effect, and fermented milk with high palatability, and a bad breath prevention and / or suppression effect easily.

1. Overview of bacterial species for producing fermented milk Conventionally, fermented milk such as yogurt is made from raw milk (yogurt mix) prepared using milk and dairy products as the main ingredient, and simple lactobacillus helvetics, lactobacillus casei and other ingredients. It is produced by inoculating a starter of one bacterial species, or by inoculating a starter of two or more bacterial species such as Lactobacillus delbruecki subspecies bulgaricus and Streptococcus thermophilus.

  However, it is said that a single fungus cannot produce satisfactory ones for acid generation, flavor generation, and tissue formation. It is necessary for producing yogurt with a good symbiotic effect by using Lactobacillus delbruecki subspecies bulgaricus and Streptococcus thermophilus in combination.

(General fermented milk (yogurt) production overview)
The production process of yogurt is usually
I. (1) Mixing and preparation of raw milk, (2) Homogenization and sterilization, (3) Cooling, (4) Addition of starter, (5) Filling into container, (6) Fermentation, (7) Card And (8) refrigeration or II-1. (1) mixing / preparation of raw milk, (2) homogenization / sterilization, (3) cooling, (4) addition of starter, (5) fermentation, (6) Card formation, (7) Card crushing, (8) Cooling, (9) Filling container, and (10) Refrigeration, or
II-2. (1) mixing and preparation of raw milk, (2) homogenization and sterilization, (3) cooling, (4) addition of starter, (5) fermentation, (6) card formation, (7) It consists of crushing the card, (8) filling the container, (9) cooling, and (10) refrigeration. In the homogenization / sterilization step, homogenization may be performed before sterilization or after sterilization. It is important for commercial production that the number of processes is not as complicated as possible.

2. Periodontal disease Periodontal disease is divided into gingivitis that causes gingival inflammation and periodontitis in which the alveolar bone supporting the teeth is destroyed. So-called alveolar pyorrhea is classified as adult periodontitis. Gingivitis is caused by supragingival plaque, periodontitis is caused by subgingival plaque, and pathogens involved in these include Porphyromonas gingivalis (Pg), Porphyromonas interumedia (Pi), Tannerella forsythensis (Tf), etc. There are several types of bacteria. Many of these are anaerobic bacteria and facultative anaerobic bacteria, and it is said that Porphyromonas gingivalis (Pg) is the main cause.

  By the way, among various enzymes produced by Porphyromonas gingivalis (Pg), trypsin-like cysteine protease (gingipain) has a strong trypsin-like activity as a catalytic activity, causing host protein degradation and host cell damage, It is thought to produce a variety of conditions related to periodontal disease.

  Therefore, inhibiting the action of gingipain protease as a means for preventing or treating periodontal disease is considered to be a very effective means. A protease inhibitor characterized by using a plant extract as an active ingredient has also been reported (Japanese Patent Laid-Open No. 6-25000).

  Moreover, the occurrence of bad breath is one of the symptoms of periodontal disease. It is said that the cause of bad breath is mainly methyl mercaptan produced by anaerobic bacteria such as Fusobacterium nucleatum by methionine metabolism. In order to prevent caries, periodontal disease and bad breath, it is important to inhibit the growth of these bacteria in the lesion. It has been reported that the application of antibacterial substances effective against these bacteria is effective (Japanese Patent Laid-Open No. 9-157144). Something is being explored.

3. Oral diseases of fermented milk, for example, prevention / treatment effect of periodontal disease, search for fermented milk (yogurt) excellent in taste, etc. The present inventors suppress bad breath and suppress the growth of periodontal disease related bacteria. For the purpose of producing fermented milk that suppresses the symptoms of periodontal disease, a comprehensive search was made for combinations of lactic acid strains used in the preparation of fermented milk. In addition, fermented milk is not inoculated as a therapeutic agent, but must have no taste defects that can be ingested as a daily food, and preferably has excellent taste characteristics that can be eaten every day. Since it is desirable to have it, the sensory test of manufactured fermented milk was also performed.

  Specifically, Lactobacillus delbrucky subspecies bulgaricus and Streptococcus thermophilus, which are the main fungi for producing fermented milk, are used in combination, and combinations of these two strains are useful. Selected.

  First, because the most important bacterium for periodontal disease-related bacteria is Porphyromonas (Bacteroides) gingivalis (Pg), search for a combination of lactic acid strains that prepare fermented milk that is highly effective in inhibiting the growth of the bacteria. did. Furthermore, the combination of the strain with high gingipaine inhibitory activity was searched as gingivitis inhibitory activity. And as a bad breath suppression activity, the combination of the strain by which the methyl mercaptan density | concentration which generate | occur | produces at the time of mixed culture with the culture medium which added saliva and fermented milk became low was searched.

  Next, from the searched combinations with high periodontal disease preventive / therapeutic effects, the sensory test was performed to select the ones that are superior in taste. Thereby, it discovered that it was especially preferable to use a specific strain together.

  The fermented milk prepared by using a specific strain in combination thus found is effective in preventing and / or treating oral diseases such as periodontal disease. Furthermore, it confirmed that the said fermented milk was effective also in prevention and / or suppression of bad breath.

4). Fermented milk for prevention and / or treatment of oral diseases such as periodontal disease The present invention relates to (b) Lactobacillus delbrueckii subspecies bulgaricus OLL1255 effective for prevention and / or treatment of oral diseases such as periodontal disease. And fermented milk containing Streptococcus thermophilus OLS3294, (b) the fermented milk containing Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain effective for prevention and / or treatment of oral diseases such as periodontal disease And (c) fermented milk containing Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain, which are labeled as effective for prevention and / or treatment of oral diseases such as periodontal disease .

  Specifically, the selection method of the present invention prepares fermented milk having a high inhibitory effect on (1) periodontal disease-related bacteria, preferably Porphyromonas gingivalis (Pg), from lactic acid bacteria for producing fermented milk. Search for lactic acid strains, (2) Search for strains with high gingipaine inhibitory activity as gingivitis inhibitory activity, and (3) Search for strains with low methyl mercaptan concentration generated during mixed culture with saliva as halitosis suppression activity Is included.

  These fermented milks can also be labeled as being used for the prevention and / or treatment of oral diseases such as periodontal disease.

The Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain has been deposited under the following conditions.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Receipt number: NITE AP-76
(4) Display for identification: Lactobacillus delbrueckii subspecies bulgaricus OLL1255
(5) Date of deposit (date of receipt): February 10, 2005
(6) Accession number: NITE BP-76
The Streptococcus thermophilus OLS3294 strain is deposited under the following conditions.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Receipt number: NITE AP-77
(4) Display for identification: Streptococcus thermophilus OLS3294
(5) Date of deposit (date of receipt): February 10, 2005
(6) Accession number: NITE P-77

5). Fermented milk for prevention and / or suppression of bad breath The present invention relates to (i) fermented milk containing Lactobacillus delbrueckii subspecies bulgaricus OLL1255 and Streptococcus thermophilus OLS3294 for prevention and / or suppression of bad breath, And / or a method for producing the fermented milk containing Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain for suppression, and And fermented milk containing Streptococcus thermophilus OLS3294 strain.

  In addition, these fermented milks can also be labeled with an indication that they are also used for the prevention and / or treatment of oral diseases such as periodontal disease.

6). Production method of fermented milk Conventionally, lactic acid bacteria, which are said to have a periodontal disease prevention / treatment effect as a tablet, need to add another starter lactic acid bacteria in order to produce fermented milk. On the other hand, the fermented milk of the present invention uses Lactobacillus delbruecki subspecies bulgaricus and Streptococcus thermophilus that are used to produce ordinary fermented milk, so other starters are added. It is not necessary to do this, and it can be produced without altering the normal production process of fermented milk.

  Specifically, (1) raw material milk etc. are mixed and prepared, (2) homogenized and sterilized, (3) cooled, and (4) periodontal disease prevention and / or treatment of the present invention, or bad breath Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294 strain) which are effective for prevention and / or suppression are added as 5 starters. ) After filling into a container, (6) fermenting, (7) forming a card, (8) refrigeration can be produced.

  Moreover, (1) raw material milk etc. are mixed and prepared, (2) homogenization and sterilization, (3) after cooling, (4) periodontal disease prevention and / or treatment of this invention, or bad breath prevention and / or Alternatively, Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294 strain), which are effective for suppression, are added as a starter (5) Then, after (6) the card is formed, (7) the card is crushed, (8) filled into a container, and (9) refrigerated. In this case, the start of cooling after fermentation may be performed after the card is formed, after the card is crushed, or after the container is filled. Furthermore, after said (7) card | curd crushing, pulp, vegetables, various sauces, and / or various additives can also be mixed.

  The fermentation time of the present invention is a time that can be used for normal fermented milk production, that is, within 8 hours, preferably within 6 or 5 hours, and more preferably 4 hours.

  Moreover, since the fermented milk of the present invention has a very excellent taste as fermented milk, it can be used regularly and can be taken continuously without difficulty, thereby improving the effect of preventing / treating periodontal disease. Can be increased.

7). About Lactobacillus delbruecki subspecies bulgaricus OLL1255 (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLs3294) The mycological properties of delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS3294) are as follows.

[Example 1] Preparation of fermented milk Test bacteria Lactobacillus delbrucky subspecies bulgaricus and Streptococcus thermophilus strains were used in combination with the following strains, respectively.

(1) Strains for preparing yogurt (L.bulgaricus MEP170801 strain and S.thermophilus MEP170801 strain, L.bulgaricus MEP170802 strain and S.thermophilus MEP170802 strain)
(2) Strains for preparing yogurt (Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294 strain 1 OLS3294 strain or Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain may be abbreviated.)
(3) Standard strains for the preparation of yogurt (JCM 1002T strain (L. bulgaricus) and NCIMB 8510 strain (S. thermophilus))
(4) Strains isolated from plants (L. bulgaricus MEP170803 and S. thermophilus MEP170803, L. bulgaricus MEP170804 and S. thermophilus MEP170803, L. bulgaricus MEP170805 and S. thermophilus MEP170803, L. bulgaricus MEP170804 S.thermophilus MEP170804, L.bulgaricus MEP170805 and S.thermophilus MEP170804)
Moreover, ATCC53608 was used as a Lactobacillus reuteri bacterium.

2. Preparation of fermented milk The composition of the yogurt mix is 857 mL of milk, 25 g of skim milk powder, and 98 mL of purified water.
The yogurt mix was sterilized at a temperature of 95 ° C. for 2 minutes.
The concentration of the bacterial solution is 0.4% (v / v) for Lactobacillus delbrueckii subspecies bulgaricus OLL1255, 1.6% (v / v) for Streptococcus thermophilus, and 0.4% (v / v) for L. reuteri ATCC53608. From these bacterial solutions, a starter was prepared in the above-described combination to produce yogurt. The concentration of the bacterial solution added to the yogurt mix was 3% (w / w).

Fermentation was terminated in 4 hours after adding the test bacteria. Yogurt (Reuteri yogurt) prepared with Reuteri bacteria finished fermentation in 8 hours.
The acidity and pH of the prepared fermented milk are shown in FIG.

[Example 2] Selection by antibacterial activity against periodontal disease-causing bacteria (P. gingivalis, P. intermedia) About fermented milk prepared in Example 1, antibacterial against periodontal disease-causing bacteria (P. gingivalis, P. intermedia) Activity was measured as follows.

  One of the above-mentioned periodontal pathogens was mixed with Brucella HK medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and dispensed in 10 mL portions into a 90 cm diameter petri dish. Furthermore, after overlaying Brucella HK medium, the upper layer portion was aseptically cut out with a cork borer, and 50 mg of fermented milk was added dropwise on an electronic balance. After culturing this medium under anaerobic conditions for 24 hours, the resulting growth inhibition circle diameter (clear zone) was measured. The results are shown in Tables 3 and 4. Tables 3 and 4 are tests performed separately.

[Example 3] Selection by gingipaine inhibitory activity The fermented milk prepared in Example 1 was measured for gingipaine inhibitory activity as follows.
Gindipaine decomposes the substrate Benzoyl-L-Arginine-p-Nitroaniline to produce p-Nitroaniline, whose activity can be investigated by colorimetric determination of p-Nitroaniline. Therefore, it was measured how much the decomposition reaction was inhibited when the fermented milk prepared in Example 1 was added to the reaction solution.

  The degradation reaction was carried out using 400 μL of buffer solution (200 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM Cysteine-HCl, 10 mM CaCl2, 2 mM 2-mercaptoethanol), substrate solution (100 mM Tris-HCl (pH 8.0), 150 mM NaCl). , 100 μL of 10 mM Benzoyl-L-Arg-p-Nitroanilide), 100 μL of gingipain solution (P. gingivalis culture supernatant), and 400 μL of fermented milk diluted 5-fold with physiological saline in a tube, 40 minutes at 40 ° C went. After the reaction, 10 μL of a reaction stop solution (1 mg / mL Leupeptin) was added, the supernatant obtained by centrifugation (15000 rpm, 10 minutes) was filtered, and the absorbance (OD 405 nm) of the permeate was measured. As a positive control, the same treatment was performed by adding 400 μL of physiological saline instead of the diluted solution of fermented milk. The results are shown in FIG. Note that FIG. 2 and Table 5 are tests conducted separately.

[Example 4] Measurement of halitosis suppression activity by methyl mercaptan concentration generated during mixed culture with saliva Todd Hewitt Broth (Difco) containing 0.05% (w / v) methyl mercaptan-generating substrate (L-methionine) 125 mL) was further dispensed in 30 mL, and 0.5 mL of saliva obtained from a healthy adult and 0.012 mL of the fermented milk were added, adjusted to pH 7.8, and cultured at 37 ° C. for 14 hours. After the culture, the methyl mercaptan concentration in the head space of the vial was measured by gas chromatography with a flame photometric detector (manufactured by Shimadzu Corporation). The results are shown in FIG.

[Example 5] Sensory test Method for Producing Fermented Milk The composition of the yogurt mix is 857 mL of milk, 25 g of skim milk powder, and 98 mL of purified water.
The yogurt mix was sterilized at a temperature of 95 ° C. for 2 minutes.
The bacterial solution pre-cultured in the skim milk powder medium was added in the following inoculum amounts.

Bacterial fluid (a) 1.6% (v / v) for Streptococcus thermophilus OLS3294
Bacterial fluid (b) 0.4% (v / v) in Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain
Bacterial fluid (c) 0.4% (v / v) for L. reuteri ATCC53608
The following starters were prepared from the above three types of bacterial solutions to produce yogurt. The concentration of the bacterial solution added to the yogurt mix was 3% (w / w).

Sample (1) Mixed bacterial solution of Streptococcus thermophilus OLS3294 and Lactobacillus delbrueckii subspecies bulgaricus OLL1255 (present invention)
Sample (2) Mixed bacterial solution of Streptococcus thermophilus OLS3294 and L. reuteri ATCC53608 Sample (3) Single bacterial solution of L. reuteri ATCC53608 (Reuteri yogurt)
In consideration of the growth temperature of L. reuteri, all fermentation temperatures were 37 ° C.

2. sensory test
According to the panel of 6 people, (i) taste of acidity (like: dislike), (b) taste of sweetness (like: dislike), (c) balance of acidity / sweetness (good: bad), (d) mouth 6 items of fragrance (good: bad), (e) rich (yes: not), (f) delicious (bad: delicious) were included in the sensory test by the visual analog scale method. Large numbers correspond to “like”, “good”, “present”, “delicious”.

Production characteristics of yogurt Sample (1) formed curd after about 4 hours of fermentation, but samples (2) and (3) did not form curd even after 8 hours of fermentation.

Analysis of sensory test In this test, the yogurt mix was processed at a normal sterilization temperature of 95 ° C for 2 minutes and at a fermentation temperature of 37 ° C. A sample having a fermentation time of 8 hours was used for the sensory test. At this time, as described above, the card was formed in the sample (1), but the card was not formed in the samples (2) and (3), and it was liquid.

  The results of the sensory test are shown in Table 7. As described above, the items with large numerical values correspond to “like”, “good”, “present”, and “delicious”. Compared to samples (2) and (3), sample (1) was remarkably tasty. Statistical processing (Mann-Whitney U test) also showed the superiority of yogurt produced from Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain of the sample (1) of the present invention.

[Example 6] Confirmation of bad breath reduction effect A plain yogurt prepared with Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain was prepared for 24 volunteers (average 36.4 ± 8.6 years old) of 17 men and 7 women. We examined the effects on oral flora, halitosis, and oral findings when ingested for 2 weeks (90g x 2) at any time for 6 weeks. For plain yogurt, homogenize raw material mix (raw milk, skim milk powder, etc.), sterilize and cool, add the above strain, ferment at 43 ° C for about 3 hours, cool at acidity of 0.73. It was prepared with. Brucella HK medium and modified FM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) were used for the bacterial flora analysis, and simple gas chromatography (Oral Chroma, manufactured by Abilit Corporation) was used for the measurement of bad breath. Oral findings were examined by a dentist.

  As a result, about 80% of the subjects who had a bad breath (hydrogen sulfide) above the threshold had a bad breath compared to before intake.

  In addition, saliva pH became more neutral (p <0.01) from pH 7.27 before ingestion to pH 7.08, 6 weeks after ingestion. In oral examination, plaque and gingival swelling were significantly reduced 6 weeks after ingestion compared to before intake (p <0.05). From the above results, it was shown that the administration of yogurt used in this test reduces bad breath and harmful oral bacteria. This result is based on the Wilcoxon test.

[Example 7] Effect of periodontal disease-causing bacteria Porphyromonas gingivalis (PG) on the adherence in oral epithelial cells by prepared fermented milk Four types of fermented milk were prepared. That is, Lactobacillus bulgaricus (L. bulgaricus) OLL1255 and Streptococcus thermophilus (S. thermophilus) OLS3294 (OLL1255-OLS3294 yogurt starter), L. bulgaricus MEP170801 and S. thermophilus MEP170801, L. bulgaricus MEP170802 and 170. .bulgaricus ATCC11842 and S.thermophilus ATCC19258,) were used to ferment a common yogurt mix. PG was cultured at 37 ° C. for 18 hours in GAM broth supplemented with 0.05% hemin and 0.01% vitamin K. While removing the supernatant by centrifugation (3000 rpm, 15 minutes), PG (bacteria) was washed twice with PBS, and then PG was prepared to be about 1 × 10 ⁸ cfu / ml in PBS. At this time, the number of prepared PGs (the number of bacteria) was measured, and this was defined as the “number of bacteria added with PG”.

Subsequently, an oral epithelial cell line (KB cell) was prepared to 1 × 10 ⁵ cells / ml, and 1 ml was seeded per well of a 24-well plate. After culturing them at 37 ° C. for 18 hours, the medium was removed, and 0.8 ml of fresh medium was added. Then, adjusted PG was added in 0.1 ml, and various yogurts diluted 5 times with PBS and yogurt mix were added in 0.1 ml. At this time, the number of lactic acid bacteria in the diluted yogurt was counted, and this was defined as “the number of lactic acid bacteria added bacteria”. After leaving the plate in a CO ₂ incubator for 60 minutes, the medium was removed, and the cells were washed 3 times with PBS. After adding trypsin-EDTA solution at 1 ml and separating PG from the cells, the number of bacteria was counted, and this was designated as “the number of bacteria attached to PG”. The number of adherent bacteria of yogurt lactic acid bacteria was also measured in the same manner, and this was designated as “the number of adherent bacteria of lactic acid bacteria”.

  The results are shown in Table 8 below. The ratio of the number of adherent bacteria to the number of bacteria added to PG was calculated as `` PG adherence rate ''. When PG alone was added to the cell line, it was 9.73 ± 2.57%, while yogurt of OLL1255-OLS3294 was added In this case, it decreased significantly to 1.89 ± 0.63% (P = 0.020).

  On the other hand, when other yogurts or yogurt mixes were added, no significant decrease was observed. In addition, regarding the number of PG-attached bacteria, the PG adhesion inhibition rate was calculated from the ratio of the case where only PG was added and the case where various yogurts were added, and when the yogurt of OLL1255-OLS3294 was added, 80.6 ± 6.5 %, Which is not statistically significant, but higher than other yogurts and yogurt mixes. In the same way, the lactic acid bacteria adhesion rate calculated for the sum of the two types of lactic acid bacteria was significantly higher in the case of yogurt prepared with the standard strain compared to other yogurts and yogurt mixes. (P <0.0001).

  It has already been reported that periodontal inflammation is reduced when the adhesion rate of PG to oral cells decreases. In this experiment, OLL1255-OLS3294, which has an effect of improving the Gingival Index, confirmed the suppression of PG adhesion by yogurt (significant decrease in PG adhesion rate). The results were meaningful for treatment and prevention.

[Example 8]
Effect of conditioned fermented milk on the suppression effect of biofilm formation by S. mutans
Streptococcus mutans (S. mutans) OLS3288 (JCM 5705T) was cultured at 37 ° C. for 24 hours in Brain Heart Infusion (BHI) (237500 Difco, Sparks, MD, USA). S. mutans (bacteria) was recovered by centrifugation (5000 g, 10 min, 4 ° C.). The collected S. mutans was washed twice with a chemically defined medium (CMD) (composition will be described later), and then added to 100 ml of CDM containing 1% (w / v) sucrose.

  On the other hand, yogurt (test fermented milk) using Lactobacillus bulgaricus (L. bulgaricus) OLL1255 and Streptococcus thermophilus (S. thermophilus) OLS3294 (yogurt starter of OLL1255-OLS3294), L. bulgaricus MEP170801 and S. thermphilus MEP170801 0.1 g of yogurt (control fermented milk) and yogurt mix (unfermented milk) was added to 100 ml of CDM containing 1% (w / v) sucrose.

  Then, the above-mentioned CDM containing S. mutans and CDM containing yogurt or yogurt mix were mixed in the same amount of 100 ml each. These mixed solutions were seeded at 100 μl / well in an enzyme immunoassay-radiimmunoassay 96 well plate (9017 Cornig, Cornig, NY) and cultured at 37 ° C. for 48 hours. At this time, 3 wells were used for each sample. After culturing these, the medium was removed, the plate was dried, and the amount of biofilm adhering to each well was measured by the phenol-sulfuric acid method. Specifically, after adding distilled water to a dried plate at 30 μl / well, a 5% phenol solution was further added at 30 μl / well and stirred well. Thereafter, concentrated sulfuric acid was added at 150 μl / well, and immediately after that, the plate was well stirred, cooled to room temperature, and the absorbance at 490 nm was measured.

  The results are shown in FIG. In the case of yogurt (test fermented milk) using L. bulgaricus OLL1255 and S. thermphilus OLS3294, the amount of biofilm by S. mutans was significantly reduced compared to the case of no yogurt added or yogurt mix. However, in the case of yogurt (control fermented milk) using L. bulgaricus MEP170801 and S. thermphilus MEP170801, the amount of biofilm was significantly reduced compared to the case where yogurt was not added. It was equivalent to the yogurt mix.

  From these results, even in the case of yogurt mix, the suppression effect of biofilm formation by S. mutans is recognized, but in the case of yogurt, a higher suppression effect can be expected. And the inhibitory effect of biofilm formation by yogurt was different depending on the type of L. bulgaricus and S. thermophilus, and it was found that these selections are important.

  The fermented milk of the present invention can be used for preventing / treating periodontal disease, and for preventing and / or suppressing bad breath because of its high palatability.

Acidity and pH of the prepared fermented milk. Gindipaine inhibitory activity of the prepared fermented milk. Methyl mercaptan concentration produced during mixed culture in which saliva is mixed with the prepared fermented milk. The influence which adjusted fermented milk has on the suppression effect of biofilm formation by S. mutans.

Claims (11)

  Fermented milk for oral disease prevention and / or treatment containing Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294).   Fermented milk for oral disease prevention and / or treatment prepared using Lactobacillus delbrueckii subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294) .   The fermented milk according to claim 1, wherein the oral disease is periodontal disease.   Fermented milk for bad breath prevention and / or suppression containing Lactobacillus delbrueckii bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294).   Fermented milk for bad breath prevention and / or suppression prepared using Lactobacillus delbrueckii subspecies bulgaricus OLL1255 and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294).   Lactobacillus delbrueckii subspecies bulgaricus OLL1255 and Streptococcus thermophilus OLS3294 (Streptococcus thermophilus OLS 3294) with indications that they are effective for the prevention and / or treatment of periodontal disease ) Fermented milk.   Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain (Lactobacillus delbrueckii subspecies bulgaricus OLL1255) and Streptococcus thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294) with indication that they are effective for prevention and / or suppression of bad breath Fermented milk.   (1) Mixing and preparing raw milk, (2) Homogenizing and sterilizing, (3) After cooling, (4) Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus Lactobacillus characterized by adding thermophilus (Streptococcus thermophilus) as a starter, (5) filling into a container, (6) fermenting, (7) forming curd, (8) refrigerated -Fermented milk for periodontal disease prevention and / or suppression fermented milk or halitosis prevention and / or suppression containing Delbrucky subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) and Streptococcus thermophilus (Streptococcus thermophilus) Production method.   (1) Mixing and preparing raw milk, (2) Homogenizing and sterilizing, (3) After cooling, (4) Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus -Add thermophilus (Streptococcus thermophilus) as a starter, (5) ferment, (6) form curd, (7) crush the card, (8) fill the container, (9) refrigerate Containing Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus characterized in that it prevents fermented milk or halitosis for periodontal disease and / or suppression And / or the manufacturing method of fermented milk for suppression.   (7) Fermented milk for periodontal disease prevention and / or suppression or bad breath prevention and / or suppression according to claim 9, characterized by cooling after crushing the card or (8) filling the container. A method for producing fermented milk.   The Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) and Streptococcus thermophilus (Lactobacillus subspices bulgaricus) OLL1255 strains (Lactobacillus delbuleckis bulgaricus) The method for producing fermented milk according to any one of claims 8, 9 and 10, which is Thermophilus OLS3294 strain (Streptococcus thermophilus OLS3294).

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