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    • 专利摘要:
    PURPOSE: A probiotic strain isolated from Korean feces, having improved gastric juice- and bile acid-resistance and the use thereof are provided, thereby effectively decreasing lactose intolerance and inhibiting the infection of Listeria and Escherichia coli. CONSTITUTION: A probiotic strain Lactobacillus fermentum HY701 (KCCM 10351) is isolated from Korean feces, has improved gastric juice- and bile acid-resistance, produces beta-galactosidase and has the antimicrobial activity to Listeria and Escherichia coli. A composition for the treatment of lactose intolerance contains the probiotic strain Lactobacillus fermentum HY701 (KCCM 10351) as an effective component. A functional health food composition contains the probiotic strain Lactobacillus fermentum HY701 (KCCM 10351) as an effective component. A composition for inhibiting the infection of Listeria and Escherichia coli contains the probiotic strain Lactobacillus fermentum HY701 (KCCM 10351) as an effective component.
    公开号:KR20030064030A
    申请号:KR1020020004436
    申请日:2002-01-25
    公开日:2003-07-31
    发明作者:백현동；정황영
    申请人:주식회사 푸코；
    IPC主号:
    专利说明:

    Novel probiotic strain isolated from Korean feces having gastric juice-resistance, bile acid-resistance and its use} with high intestinal viability with high resistance to gastric juice and bile acids
    [4] The present invention relates to a novel useful probiotic probiotic and its use with high intestinal viability with high resistance to gastric juice and bile acids, and more particularly, isolated from human feces of Koreans, and high resistance to gastric juice and bile acids. It is related to Lactobacillus fermentum HY701, which is a probiotic probiotic having a superior intestinal viability and superior to existing strains.
    [5] Probiotic probiotics are products that are produced for the purpose of improving the body function through the intestinal diseases as well as normalizing the intestinal flora by enzymes, organic acids, vitamins and non-toxic antibacterial substances secreted by microorganisms by ingesting live microbial cells (Fuller , R. 1989. Probiotics in man and animals.J. Appl. Bacteriol. 66: 365-378). These probiotic probiotics are associated with stabilizing the intestinal flora, reducing rot production and preventing disease, preventing immunity, and activating immunity, indirect anticancer activity, lowering cholesterol, reducing lactose intolerance, constipation suppression and prevention. Functionality is recognized (Goldin, BR and Borbach, SL 1977. Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 40: 2421-2426; Fuller, R. 1991. Probiotics in human medicine. Gut. 32: 439-442; Shah, N. 1994.Lactobacillus acidophilusand lactose intolerance, a review. ASEAN Food J. 9: 47-54; Takiguchi et al. 1997.Lactobacillus acidophilusSBT 2062 andBifidobacterium longumSBT 2928 on harmful intestinal bacteria. J. Int. Microbiol. 11: 11-17). Bacteria widely used as probiotic probiotics are Lactobacillus (Lactobacillus)genus,Streptococcus(Streptococcus)Genus, Bifidobacterium (Bifidobacterium)Genus and diffuser lactobacillus Bacillus (Bacillus)Genus and so on. On the other hand, as a probiotic probiotic, the rationale for bacteria should be based on safety, functional aspects (survivability, fixability, formatting, antimicrobial production ability, immune promoting ability, antigenotoxic activity, inhibitory ability of pathogenic bacteria) and technical aspects (functional characteristics). , Safety, bacteriophage resistance, and viability during manufacturing), and GRAS (Generally Recognized As Safe) microorganisms require high intestinal viability.
    [6] In view of the above, the present inventors select a strain having excellent resistance to gastric juice and bile acid among commercially available probiotic probiotics among useful lactic acid bacteria isolated from feces of Koreans, and analyze the characteristics of the strain. Was completed.
    [7] Accordingly, an object of the present invention is to provide useful human probiotic probiotic resistant to gastric juice and bile acid.
    [8] Still another object of the present invention is to provide a use of the probiotic probiotic for functional health foods and medicines.
    [9] The object of the present invention is to isolate and isolate the Lactobacillus fermentum (HY701) strain from human feces of the human, and to confirm the resistance to artificial gastric juice and artificial bile solution of the isolated strain, enzyme activity, antibiotic Achieved by measuring the resistance to the material and the antimicrobial activity.
    [1] Figure 1 is a graph showing the resistance to artificial gastric juice of the probiotic live bacteria of the present invention.
    [2] Figure 2 is a graph showing the resistance to artificial bile acids of the probiotic live bacteria of the present invention.
    [3] Figure 3 shows the results of the growth inhibition of Listeria monocytogenes ATCC 19111 (A) and E. coli JM109 (B) of the probiotic live bacteria of the present invention.
    [10] The present invention comprises the steps of separating the Lactobacillus fermentum HY701 from the human feces of the human body; Measuring the resistance of the strain of the present invention to artificial gastric juice and artificial bile acid according to the method of Kobayashi et al .; Performing microbiological identification of the strains of the invention by examining the availability of 49 carbon sources by Gram staining and API CHL (bioMereux co., France); Measuring enzymatic activity of the strain using an API ZYM kit; Investigating antibiotic resistance according to the paper disc method and measuring the antimicrobial activity against Listeria monocytogenes ATCC 19111 and E. coli JM109.
    [11] In the step of measuring the resistance to artificial gastric juice, the resistance to artificial bile acid, the total number of bacteria was diluted 10 times serially with 0.1% peptone water, and then plated by plating 0.1 ml each plate, After incubation at each optimum temperature, the colony forming unit was measured to calculate the total bacterial count.
    [12] Commercial probiotic strains DH65, KM332, and PA212 used in the above steps were used in Korea's representative probiotic probiotics (Cellbiotech Co., Ltd.), Listeria monocytogenes ATCC 19111 used to measure the antimicrobial activity was purchased from ATCC Was used.
    [13] The probiotic probiotic of the present invention was named as Lactobacillus fermentum HY701 and was deposited on January 16, 2001 to the Korea Bacterial Association Microbial Conservation Center, an international depository institution (Accession Number: KCCM-10351).
    [14] The present invention provides a functional food, health supplement foods and pharmaceuticals comprising the strain as an active ingredient.
    [15] The composition of the present invention may be administered to the subject individual at least once daily, and the dosage unit for oral administration of an adult is preferably administered at 1 μg to 200 mg per kg of body weight. However, dosage may vary depending on body weight, age, health condition, method of administration, drug mixture and severity of disease, and may be in unit dosage forms such as tablets, capsules, granules, suspensions, emulsions or several times by conventional methods. It may be formulated or encapsulated in a dosage form for use.
    [16] When the composition of the present invention is taken periodically, the microorganisms of the present invention in the intestine form a bacterium and produce β-galactosidase to alleviate lactose intolerance, and can inhibit intestinal infections such as Listeria and E. coli. have.
    [17] In addition, the term 'composition' of the present specification does not necessarily mean that the drug is approved, it is a concept that includes a conventional functional food or health supplements.
    [18] Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.
    [19] Example 1 Isolation and Culture of Probiotic Probiotics of the Invention
    [20] In order to isolate probiotic live bacteria, about 100 bacteria were isolated from human feces of Koreans and 28 strains of strains having similar morphological characteristics as those of Lactobacillus sp. Were selected. Using these strains, anaerobic Gram-positive bacillus producing lactic acid was selected, and the enzyme activity was measured in these strains, and the β-galactosidase activity was strong, and β-glucuronidase (β- Strains without glucuronidase) activity were selected and abbreviated as HY701 strains. The isolated lactic acid bacteria were cultured using lactobacilli MRS medium (Difco Laboratories, USA) as a culture medium.
    [21] Lactic acid bacteria were purely isolated and preserved on a MRS agar plate, inoculated with platinum in MRS broth of a 10 ml test tube, and cultured at 32 ° C. for 12 hours.
    [22] Example 2 Resistance of Probiotic Probiotics of the Present Invention to Artificial Gastric Fluids
    [23] There are a number of characteristics that must be present as probiotic probiotics, one of the most important of which is the high viability (Fuller, R. 1989. Probiotics in amn and animals.J. Appl. Bacteriol. 66: 365 -378). Bacteria ingested through the oral cavity survive through the stomach and duodenum and reach the final target site of the intestine, so they must pass strong acidic gastric juice to function as a probiotic probiotic. Pure gastric juice has a pH of 1.4-2.0, and most of the microbes die from it. However, due to the buffering effect of food intake, the pH is slightly increased, and the killing rate of microorganisms is somewhat reduced (Conway et al. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.J. Dairy Sci. 70: 1-12). For acid resistance experiments, methods for confirming survival rate in vivo and indirect measurement using gastric juice are known. It has been reported that the killing of probiotic probiotics is mainly due to low pH and that the results of experiments in vitro and in vivo are almost identical (Conway et al. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to . intestinal cells J. Dairy Sci 70: .. 1-12; Giannella et al 1972. Gastric acid barrier to ingested microorganisms in man: Studies in vivo and in vitro Gut 13:.. 251-256; Berrada et al 1991. Bifidobacterium from fermented milks: Survival during gastric transit.J. Dairy Sci. 74: 409-413).
    [24] In this example, the resistance of the strain of the present invention in artificial gastric juice was adjusted to pH 2.5 in view of the above.
    [25] Artificial gastric fluid was used by adding 1% pepsin to each broth adjusted to pH 2.5 using 1N HCl according to the method of Kobayashi et al. (Kobayashi et al. 1974. Tolerance of the multiple antibiotic resistant strain, L. casei PSR 3002, to artificial digestive fluids.Jpn. J. Microbiol. 29: 691-197). In order to test the resistance to artificial gastric juice, the culture solution of each strain was placed in 1 ml of an eppendorf tube, centrifuged at 6000 rpm for 10 minutes, and the culture supernatant was discarded and only the cells were recovered. The recovered cells were added in the same amount as the supernatant and added to the 9 ml test tube artificial gastric juice and incubated at 37 ° C. for 2 hours to measure the total bacterial count. The control was subjected to the same method as above using each broth without adjusting pH and adding pepsin.
    [26] As a result, as shown in Figure 1, the HY701 bacteria showed a very high survival rate of 77.6% of the bacteria after 2 hours, it can be seen that it is resistant to artificial gastric juice.
    [27] Comparison of Gastric Tolerance between Strains of the Invention and Commercial Strains Strain name time (hour)Inventive strain (HY701)Commercial Strain 1 (DH65)Commercial Strain 2 (KM332)Commercial Strain 3 (PA212) 02.3 × 10 8 2.0 × 10 8 1.8 × 10 8 1.9 × 10 8302.1 × 10 8 8.9 × 10 7 9.2 × 10 7 7.8 × 10 7601.9 × 10 8 8.7 × 10 7 6.4 × 10 7 6.8 × 10 6901.8 × 10 8 4.0 × 10 5 8.5 × 10 4 3.1 × 10 51201.8 × 10 8 1.6 × 10 3 2.3 × 10 3 8.5 × 10 2[Note] (Unit: colony forming unit / ml)
    [28] Example 3: Resistance to Artificial Bile Acids of Probiotic Probiotics of the Invention
    [29] Ingested probiotic bacteria pass through the stomach and pancreas and duodenum to reach the intestines. Resistance to the bile secreted from this site is also an important property of probiotic probiotics. Gilliland et al. Reported that the resistance to bile for probiotic probiotics should be able to grow in medium containing 0.3% oxgall (Gilliland et al. 1984. Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J. Dairy Sci. 67: 3045-3051).
    [30] In view of the above, the artificial bile solution was used by adding 1% sterilized 10% oxgall (Difco) solution to each sterilized broth. In order to test the resistance of artificial bile acid, the culture medium subjected to artificial gastric juice was added to a test tube containing artificial bile and incubated for 24 hours at 37 ° C. . Controls were tested using the same method as above for each broth without oxgall added.
    [31] After a test for resistance to artificial bile acid, resistance to artificial bile acid that has passed through the artificial gastric juice as shown in the resistance of a rough artificial bile acid the lactic acid bacteria of the artificial gastric juice Figure 2 showed a somewhat lower survival rate compared to control most 10 8 It is above the CFU / ml level and is higher than the initial cell count.
    [32] Therefore, the probiotic probiotic HY701 of the present invention is resistant to artificial bile acids as well as artificial gastric juice and reaches the target site of the probiotic, which can serve as a probiotic probiotic.
    [33] Example 4 Microbiological Identification of Probiotic Probiotics of the Invention
    [34] Currently widely used bacterial classification methods include Bergy's Manual of Determinative Bacteriology in the United States, Krassilinikov's (1949) classification used in the Soviet Union and Eastern Europe, as well as Prevot (used in France, parts of Europe and Africa). 1961).
    [35] In our country, Begey's taxonomy is used. Bergey's manual is edited by the American Microbiological Editor's Committee, which has been shared by many scholars.
    [36] In this example, Lactobacillus HY701 strains were identified by examining the availability of 49 carbon sources by Gram staining and API CHL (bioMereux Co., France) (Kim, WJ 1996. Screening of bacteriocinogenic lactic acid bacteria and their antagonistic effects in sausage fermentation.J. Microbiol.Biotechnol. 6: 461-467).
    [37] As a result of examining the carbon source availability by the API CHL kit, as shown in Table 2, the HY701 strain was identified as Lactobacillus fermentum with 96.6% similarity.
    [38] Microbiological Identification of Probiotic Strain HY701 using API 50 CHL kit Test itemsStrain HY701 ContrastGlycerol- Erythritol- D-Arabinose- L-Arabinose- Ribose+ D-Xylose+ L-Xylose- Adonitol- β-methylxyloxide (β-Methylxyloside)- Galactose- Glucose+ Fructose+ Mannose- Sorbose- Rhamnose- Dulcitol- Inositol- Mannitol- Sorbitol- α-Methyl-D-mannoside- α-Methyl-d-glucoside- N-acetylglucosamine- Amygdaline- Arbutin- Esculin- Salincin- Cellobiose- Maltose- Lactose+ Melibiose+ Saccharose+ Trehalose+ Inulin- Melezitose- Raffinose- Starch+ Glycogen- Xylitol- Gentiobiose- D-Turanose- D-Lyxose- D-Tagatose- D-Fucose- L-Fucose- D-Arabitol- L-Arabitol- Gluconate+ 2-ketogluconate- 5-ketogluconate- [Note] +: utilized,-: not utilized
    [39] Example 5 Determination of Enzymatic Activity of Probiotic Probiotics of the Invention
    [40] Among the properties that probiotic probiotics have, the enzymes they produce are also an important part. For example, β-glucuronidase, also known as a carcinogen, causes carcinogens such as benzopyrene (Benzo (a) pyrene) to enter the body with glucuronic acid in the liver for detoxification. It is excreted in the intestine together with bile and detoxified by the intestinal bacteria β-glucuronidase (Cole et al. 1989. Effect of probiotic supplements of Lactobacillus acidophilus and Bifidobacterium adolescentis 2204 on β- glucosidase and β-glucuronidase activity in the lower gut of rats associated with a human fecal flora.Microb.Ecol.Health Dis. 2: 223-225). Therefore, it is not preferable that the bacteria used as probiotic live bacteria have the activity of β-glucuronidase, a carcinogenic enzyme. In addition, the beneficial enzymes of probiotic probiotics prevent humans and animals from ingesting or disintegrating the stomach and intestines, causing abnormal fermentation in the intestines.
    [41] In order to measure the enzyme activity, the selected strains were passaged three times in each broth, and the culture medium was centrifuged to recover only the cells and washed twice with sterile water. After washing, it was suspended in sterile water to a concentration of about 10 6 CFU / ml. The suspension was inoculated with API ZYM (bioMerieux Co.) and incubated in the dark at 37 ° C. for 4 hours. ZYM A and B reagents were added dropwise to each chapel to help increase expression activity and to dissolve, and then reacted in a bright place for about 5 minutes.
    [42] Lactobacillus HY701 bacteria selected in this study were confirmed by the API ZYM kit, and as shown in Table 3, there was no activity against the oncogenic enzyme β-glucuronidase. These results indicate that there is no risk of carcinogenesis that may occur due to probiotic probiotics. Meanwhile, β-galactosidase, which can alleviate lactose intolerance, produces a strong probiotic probiotics.
    [43] Determination of Enzyme Activity of Probiotic HY701 Strains by API ZYM Kit enzymeStrain HY701 Control0 Alkaline phosphatase0 Esterase (C 4 )3 Esterase Lipase (C 8 )2 Lipase (C 14 )0 Leucine arylamidase3 Valine arylamidase0 Christine arylamidase0 Trypsin0 α-chymotrypsin0 Acid phosphatase4 Naphthol-AS-BI-phosphohydrolase3 α-galactosidase4 β-galactosidase5 β-glucuronidase0 α-glucuronidase (α-Glucosidase)0 β-glucuronidase (β-Glucosidase)0 N-Acetyl-β-glucosaminidase0 α-Mannosidase0 α-fucosidase0 0: 0 nmole, 1: 5 nmole, 2: 10 nmole, 3: 20 nmole, 4: 30 nmole, 5: ≥ 40 nmole
    [44] Example 6 Investigation of Resistance of Antibiotic to Probiotic Probiotics of the Invention
    [45] The paper disc method was used to measure antibiotic resistance. That is, each strain is incubated at the optimal temperature for 12 hours, and 100 μl of the culture solution is inoculated on each soft agar (0.7%) and overlayed on the agar plate. Antibiotic resistance was measured by placing a paper disc wetted with antibiotics (nisin, tetracycline, streptomycin, etc.) on each overlay plate for 24 hours.
    [46] As shown in Table 4 below, the HY701 strain was inhibited at concentrations of erythromycin 15, 30 μg / ml, 20, 30 μg / ml of roxyromycin, and 20 μg / ml of tetracycline, and was resistant to the other nine antibiotics. Have These results were higher than those reported by Lee et al. (Lee, SH and No, MJ 1997. Viability in artificial gastric and bile juice and antimicrobial activity of some lactic acid bacteria isolated from Kimchi. Appl. Microbiol.Biotechnol. 6: 617-622).
    [47] Antibiotic Resistance of Probiotic HY701 Strains Antibiotic (µg / ml)Strain HY701 Nisin0+ 25+ 50+ 100+ Streptomycin0+ 5+ 10+ 20+ Tetracycline0+ 5+ 10+ 20- Rifamycin0+ 5+ 10+ 20+ Ampicillin0+ 10+ 20+ 30+ Cyprofloxacin0+ 5+ 10+ 20+ Doxycycline0+ 10+ 20+ 30+ Roxithromycin0+ 10+ 20- 30- Nystatin0+ 5+ 10+ 20+ Gentamycin-Sulfate0+ 5+ 10+ 20+ Chloramphenicol0+ 10+ 20+ 30+ Erythromycin0+ 10+ 15- 30-
    [48] Example 7 Determination of Antimicrobial Activity of Probiotic Probiotics of the Invention
    [49] Listeria monocytogenes ATCC 19111 and E. coli JM109 were used to determine the antimicrobial activity of pathogenic bacteria of the strains of the present invention.
    [50] Listeria, a pathogenic bacterium, is a Gram-negative despoiler bacillus that causes livestock (cows, horses, pigs, goats, etc.) and poultry (chicken, ducks, etc.) and sometimes humans. Humans are often in direct contact with infected animals and orally infected with contaminated meat or dairy products, sometimes through contaminated dust. The incubation period is three to several weeks and the symptoms cause meningitis, sepsis and endometritis (pregnant women) (Oh Seung Hee, Kim Dong Won, 1997. The latest food hygiene, pp. 241).
    [51] Listeria used to measure antimicrobial activity was used in three passages for 24 hours at 37 ℃ in TSB medium, Listeria Selective Agar base manufactured by Unipath UK was used as the selection medium.
    [52] In order to measure the antimicrobial activity, HY701 strain, L. monocytogenes ATCC 19111 and E. coli JM109 were incubated for 12 hours, respectively, and the number of bacteria was diluted to 10 3 to 10 4 CFU / ml and the test tubes were added at 1: 1 volume ratio. 5 ml of the mixture was cultured in a shaking water bath at 37 ℃. During incubation, samples were taken at intervals of 1, 2, 4, 8, and 24 hours, and the number of bacteria was measured using a Listeria selective medium.
    [53] As shown in FIG. 3, the lactic acid bacteria HY701 strain increased the number of bacteria of Listeria (A) up to 4 hours and began to decrease after 8 hours and significantly decreased compared to the control after 24 hours (reduced to less than log 2). . In the case of E. coli (B), it increased up to about 2 hours, but decreased after 4 hours. The main cause of this inhibition appears to be due to organic acids produced by lactic acid bacteria and lowering of pH by lactic acid and substances such as bacteriocin (Hong et al. 1996. Growth of Lactobacillus acidophilus in whey-based medium and preparation of cell concentrate for production of probiotics.J. Appl.Microbiol.Biotechnol. 2: 128-131).
    [54] According to the above results, if infected with Listeria and Escherichia coli, it can be strongly inhibited by probiotic strains resident in the intestine.
    [55] As described above, the probiotic probiotic Lactobacillus fermentum HY701 of the present invention has a high resistance to gastric juice and bile acids, and thus has excellent intestinal viability, and produces β-galactosidase, thereby reducing lactose intolerance. There is an effective effect to suppress the Listeria and E. coli infections, and is superior to the existing strains, so it is a very useful invention in the food industry and the medical industry because of its potential as a functional food.
    权利要求:
    Claims (4)
    [1" claim-type="Currently amended] Lactobacillus fermentum HY701 strain (Accession No. KCCM 10351) which has excellent resistance to gastric juice and bile acids, produces β-galactosidase and has antimicrobial activity against Listeria and E. coli.
    [2" claim-type="Currently amended] The composition for treating lactose intolerance, comprising the probiotic probiotic of claim 1 as an active ingredient.
    [3" claim-type="Currently amended] Functional health food composition, characterized in that the probiotic probiotic of claim 1 as an active ingredient.
    [4" claim-type="Currently amended] A composition for inhibiting Listeria and Escherichia coli infection, comprising the probiotic probiotic of claim 1 as an active ingredient.
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    同族专利:
    公开号 | 公开日
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2002-01-25|Application filed by 주식회사 푸코
    2002-01-25|Priority to KR1020020004436A
    2003-07-31|Publication of KR20030064030A
    优先权:
    申请号 | 申请日 | 专利标题
    KR1020020004436A|KR20030064030A|2002-01-25|2002-01-25|Novel probiotic strain isolated from Korean feces having gastric juice-resistance, bile acid-resistance and its use|
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