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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
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  • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
  • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
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  • A23G9/363—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C15/00—Butter; Butter preparations; Making thereof
  • A23C15/12—Butter preparations
  • A23C15/123—Addition of microorganisms or cultured milk products; Addition of enzymes; Addition of starter cultures other than destillates
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C19/00—Cheese; Cheese preparations; Making thereof
  • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
  • A23C19/061—Addition of, or treatment with, microorganisms
  • A23C19/062—Addition of, or treatment with, microorganisms using only lactic acid bacteria, e.g. pediococcus, leconostoc or bifidus sp., or propionic acid bacteria; Treatment with non-specified acidifying bacterial cultures
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
• • A—HUMAN NECESSITIES
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  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
  • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
  • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
  • A23D7/0056—Spread compositions
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
  • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
  • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
  • A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
  • A23G9/366—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing vitamins, antibiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

• the present invention relates to some novel lactic acid bacteria which inhibit the production of dental plaque in human mouths. More specifically, the production of water- insoluble glucan (mutan) , a major component of dental plaque, which is produced by bacteria normally inhibiting in human mouths, can be inhibited by the novel bacteria. Oral anaerobic bacteria causing gingivitis, periodontitis, and accompanied halitosis (malodor) can be inhibited by the novel bacteria, too. These lactic acid bacteria belong to Enterococcus spp., Lactobacillus spp., Lactococcus spp., and Stretococcus spp.
• Lactic acid bacteria generally ferment carbohydrates to lactic acid. Lactic acid bacteria live in the oral cavities and the alimentary tracts of men and animals and are utilized for the manufacture of fermentative foods, such as yogurt, cheese, etc. In addition, they are used for the production of biologically active materials, such as medicines. Representatives of these lactic acid-producing bacteria are Streptococcus thermophilus, Enterococcus faecalis, Enterococcus durans, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus plantarum. As inhabitants in the entrails of men and animals, these Gram- positive lactic acid bacteria are known to play an important role in maintaining the entrails healthy by the production of lactic acid and antibacterial materials which inhibit the growth of pathogenic bacteria.
• Glucan is either water-soluble glucan, dextran having 1,6- linkage as a predominant linkage, or water- insoluble glucan (mutan) having 1,3- ⁇ linkage as a predominant linkage.
• water-insoluble glucan (mutan) serves as a main matrix of dental plaque.
• Dextranase ⁇ -1,6 glucan hydrolase which digests dextran, was tested as to its ability to prevent dental plaque.
• European patent publication number 0-524-732- A2 disclosed the use of Streptococcus salivarius which was capable of extracellular production of dextranase. But its effect on preventing dental plaque is questionable because dextranase can not digest mutan, the main matrix of dental plaque. Streptococcus salivarius is not used as the starter bacteria fermenting milk.
• Glucan is either water-soluble or water-insoluble (mutan) , each being synthesized from sucrose by the glucosyltransferase secreted from Streptococcus mutans, the most important bacteria among dental plaque-forming bacteria. However, only the water-insoluble glucan, mutan, is the main matrix of dental plaque.
• Enterococcus spp. 1357 Lactobacillus spp. V20, and H 2 0 2 -producing Streptococci such as Streptococcus oralis, Streptococcus mitior, Streptococcus mitis, and Streptococcus sanguis (Bergey's Manual of Systematic Bacteriology vol. 2: Williams & Wilkins, Baltimore, London, Los Angeles, Sydney, 1986) have a growth-inhibitory activity on Streptococcus mutans. When Streptococcus mutans was cultured with Enterococcus spp. 1357, Lactobacillus spp.
• Streptococcus oralis (ATCC 35037) as a representative of H 2 0 2 -producing Streptococci in the broth
• the colony-forming number of Streptococcus mutans was decreased to about one hundredth compared with that of Streptococcus mutans alone.
• the production of water-insoluble glucan or dental plaque was also suppressed significantly due to inhibition of Streptococcus mutans.
• dental plaque adherent to the surface of teeth, provides a suitable habitat at which Streptococcus mutans as well as other bacteria proliferate and causes the dental caries formed, and it was an object of the present study to find novel bacteria which could inhibit the production of water-insoluble glucan or dental plaque by Streptococcus mutans in the mouth.
• Anaerobic bacteria occur in high proportions in periodontitis as well as gingivitis of oral cavities. The proportions of anaerobic bacteria increase significantly (above 90% of microflora in periodontitis lesion) with increasing severity of the diseases.
• Predominant anaerobic bacteria causing gingivitis are Prevotella intermedia and Fusobacterium nucleatum.
• Predominant anaerobic bacteria causing periodontitis include Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum (Contemporary Oral Microbiology and Immunology, Mosby-Year Book, St. Louis, 1992). These anaerobic bacteria produce malodorous components such as volatile sulfur compounds in mouth. The predominant volatile sulfur compounds are hydrogen sulfide from L- cysteine and methyl mercaptan from L-methionine (Persson, S., Oral Microbiol Immunol, 7:378, 1992). Lactobacillus spp.
• V20 and H 2 0 2 -producing Streptococci such as Streptococcus oralis, Streptococcus mitior, Streptococcus mitis, and Streptococcus sanguis produce hydrogen peroxide inhibiting the growth of anaerobic bacteria causing gingivitis and periodontitis, so improving and preventing the lesions, and decreasing the accompanied halitosis.
• the hydrogen peroxide is an oxidizing agent inactivating enzymes by converting functional -SH groups to the oxidized S-S form and is used as a disinfectant against bacteria, especially anaerobics. Another object of this research was to provide foods or beverages employing the lactic acid bacteria.
• the lactic acid bacteria When the foods containing the lactic acid bacteria capable of directly inhibiting the production of water-insoluble glucan or having the growth-inhibitory activity on the microorganisms which contribute to the formation of dental plaque are eaten, the lactic acid bacteria naturally suppress the formation of dental plaque and consequently prevent dental caries formation. And when the foods contain the lactic acid bacteria capable of inhibiting the growth of anaerobic bacteria causing gingivitis and periodontitis, the lactic acid bacteria naturally improve and prevent gingivititis, periodontitis, and accompanied halitosis.
• Lactic acid bacteria were taken from human bodies, streaked on Brain Heart Infusion agar, and cultured at 37°C. Thereafter, separated bacterial colonies were tested whether they could suppress the production of the water-insoluble glucan that Streptococcus mutans (Ingbritt strain) produced.
• Streptococcus mutans Ingbritt strain
• 3 mL of a Brain Heart Infusion medium supplemented with 0.5% yeast extract and 5% sucrose was inoculated with 0.1 mL of Streptococcus mutans and 0.1 L of a culture broth of the separated bacteria.
• Streptococcus mutans was inoculated alone in a Brain Heart Infusion medium containing yeast extract and sucrose.
• the cuvette was placed at an angle of 30° to the horizontal plane of an incubator and incubated for 1 day at 37 °C, in order for the Streptococcus mutans to produce water- insoluble glucan. After removing the culture broth, the cuvette was washed with 4 mL of distilled water and then, filled with 3 mL of distilled water. Its absorbance (OD) at 550 nm was measured by a spectrophotometer . Because the OD value was proportional to the water-insoluble glucan produced, the bacteria which brought about a significantly lower OD value compared with the control, were isolated as dental plaque-inhibitory strains.
• the isolated strains were streaked on the Brain Heart Infusion agar containing 0.25mg/mL 3, 3', 5,5'- tetramethylbenzidine dihydrochloride and O.Olmg/mL horseradish peroxidase and incubated in anaerobic incubator for 48 hours. Lactobacillus spp. V20 formed the blue colonies, indicating that Lactobacillus spp. V20 had the capability to produce hydrogen peroxide.
• microbiological properties of the isolated strains such as morphological and physiological properties (Table 1) , and sugar catabolytic ability (Table 1) were investigated.
• novel lactic acid bacteria being able to inhibit the production of dental plaque were isolated and assayed in comparison with the control in vitro. Further, the novel properties were tested in vivo, that is, the isolated novel bacteria were applied to the human oral cavity.
• M17 medium An equal amount of M17 medium was mixed with MRS medium and supplemented with 0.5% yeast extract, 5% sucrose and 0.1M TES (pH 8.0). Three milliliters of the constituted medium were transferred to a disposable cuvette which was, then, inoculated with 75 ⁇ l of Streptococcus mutans overnight culture. The cuvette was placed at an angle of 30° to the horizontal plane in an incubator and cultured at 30°C for 1 day. The content was removed and then, the cuvette was washed with 3mL of distilled water. Thereafter, the cuvette was filled with 4mL of distilled water and the absorbency at 550nm was measured by a spectrophotometer . This measurement was repeated three times and the average value was obtained (Table 3) . TABLE 3 Inhibitory Effect of Lactic Acid Bacteria on the Production of Water-Insoluble Glucan
• M17 medium An equal amount of M17 medium was mixed with MRS medium and supplemented with 0.5% yeast extract, 5% sucrose,- and 0.1M TES (pH 8.0).
• MRS medium An equal amount of M17 medium was mixed with MRS medium and supplemented with 0.5% yeast extract, 5% sucrose,- and 0.1M TES (pH 8.0).
• One hundred and fifty milliliters of the constituted medium were poured into beaker.
• 0.016 inch stainless steel orthodontic wires (45mg) were immersed in the medium.
• Streptococcus mutans was inoculated at the concentration of 2.5X10 6 per mL of the medium. Thereafter, the lactic acid bacterial strains were inoculated in the medium at the concentration of 5 times higher than that of Streptococcus mutans and incubated in a C0 2 incubator at 37°C for 6.5 hours with shaking.
• Fig. 1(A) is a photograph of the culture of Streptococcus mutans alone while Fig. 1(B) is that of the co-culture of Streptococcus mutans and Lactococcus spp. 1370.
• Plaque scores were performed by Quigley and Hein Plaque Index after disclosing all plaque except third molars.
• the volunteers were randomly assigned to two groups (each nineteen persons), group I mouthrinsing with Lactococcus spp. 1370 while group II mouthrinsing with Lactobacillus spp. V20.
• Test bacterial suspensions were prepared by incubating either Lactococcus spp. 1370 or Lactobacillus spp. V20 in milk for 24 hours. Volunteers rinsed immediately once after oral prophylaxis and twice after meals with 20 mL of
• Lactobacillus spp. V20 was cultured in MRS media for 24 hours.
• Prophyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were cultured in the anaerobic bacteria culture broth containing Brain Heart Infusion media 18.5 g, yeast extract 5.0 g, hemin solution 10 mL (dissolved 50 mg hemin in 1 N sodium hydroxy solution 1 mL and added with distilled water 100 L) , and vitamin K solution 0.2 mL (vitamin K solution 0.15 mL mixed with 95% ethanol 30 mL) per liter in anaerobic incubator for 36 hours.
• Actinobacillus actinomycetemcomitans was cultured in the TSBV media containing Tryptic soy broth 30 g, yeast extract 1.0 g, horse serum 100 mL, bacitracin 75 mg, and vancomycin 5 mg per liter in anaerobic incubator for 36 hours.
• Culture suspension 0.1 mL of Lactobacillus spp. V20 and each anaerobic bacterium at the concentration of 1.4X10 8 per mL were inoculated singly or in combination in the media containing anaerobic bacteria culture broth or TSBV media 3.7 mL mixed with MRS broth 0.3 mL, and cultured in anaerobic incubator for 36 hours.
• the culture suspension was diluted and inoculated on MRS agar, anaerobic bacteria culture agar containing 3% sheep blood or TSBV agar. At 72 hours after culturing, the number of colonies was counted.
• the colony-forming units of Lactobacillus spp. V20 and each anaerobic bacterium were increased after being cultured singly, whereas the colony of each anaerobic bacterium was not found after being cultured in combination with Lactobacillus spp. V20.
• the colony-forming units were not decreased significantly (Table 5) .
• Streptococcus oralis (ATCC 35037) as a representative of H 2 0 2 -producing Streptococci was cultured with each anaerobic bacterium by the above mentioned method, anaerobic bacteria did not form a colony on the media.
• a broth culture containing the novel lactic acid bacterial strains was added at an amount of 0.1 vol. percent to the food just before fermentation and subjected to fermentation along with the existing bacteria to produce yogurt foods.
• the resulting yogurt foods were tasted by 10 panelists. They noted no different flavor between the test samples and the commercially available foods (controls) .
• the lactic acid bacterial strains were added at an amount of 0.2 vol. percent. A response that these test samples thus obtained were not different from the control foods in taste was drawn from 10 panelists who took part in the tasting tests.
• butter foods which were manufactured by a typical procedure were added with 0.2 wt. percent of the freeze-dried lactic acid bacterial strains. These butter foods thus obtained were given as taste samples .
• novel lactic acid bacteria were cultured and freeze-dried (lyophilized) . These freeze-dried bacteria in capsule, tablet, and small package could be taken singly or with other bacteria or materials. These freeze-dried products thus obtained were given taste samples.
• the lactic acid bacterial strains were applied for various foods, including gum, shortening, ice cream, margarine, kimchi, etc.
• novel lactic acid strains have a potent and lasting inhibitory effect on the production of water-insoluble glucan or dental plaque in human mouth, or on the growth of anaerobic bacteria causing gingivitis, periodontitis, and accompanied halitosis .
• novel strains were found to be able to be applied for various foods as well as directly to the teeth.

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• 1998
  • 1998-01-07 KR KR19980000213A patent/KR100266752B1/ko not_active IP Right Cessation
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