KR100629397B1 - Probiotic weissella which prevent and cure urinary tract infections - Google Patents

Abstract

The present invention provides probiotic strain Weissella kimchi PL 9023 (KACC 91139) for urinary health, especially female health. The lactic acid bacterium of the present invention can be used as a prophylactic and therapeutic agent for various urinary tract diseases, and can be used directly for the preparation of a prophylactic and therapeutic agent for urinary diseases such as oral administration, vaginal preparations, urinary hepatic inserts, artificial urinary catheter coatings, or It can be used as an additive in the functional food and beverages.

Treatment of vaginal infections, urethra infections, urinary infections, lactobacillus, wasella, probiotics, urinary infections

Description

Probiotic Weissella which prevent and cure urinary tract infections}

Figure 1.Sequence of 16S rRNA of Weissella kimchii Strain PL9023

Figure 2. Growth inhibition ability of various urinary pathogenic bacteria and resistant bacteria of Weissella kimchii Strain PL9023 culture extract

Figure 3. Colony shape in TMB-PLUS medium of Weissella kimchii Strain PL9023

Figure 4. Self-aggregation reaction of Weissella kimchii Strain PL9023

Fig. 5.Agglutination of Weissella kimchii Strain PL9023 with C. albicans

6. Vaginal cell adhesion ability of Weissella kimchii Strain PL9023

7. Vaginal cell adhesion of urinary pathogenic bacteria

The present invention provides lactic acid bacteria that can prevent or treat urinary infections.

The present invention also provides a food additive having a prophylactic and therapeutic effect including vaginal infections.

In another aspect, the present invention provides a food additive having a prophylactic and therapeutic effect, including live bacteria and culture.

In another aspect, the present invention provides a food and beverage having a female health function enhancement effect using the lactic acid bacteria having the genital health effect of the female as a seed.

The present invention also provides a dosage form containing lactic acid bacteria having a female genital health effect.

In pre-pubertal women, the vagina retains a neutral pH of 6-8, similar to the skin, but during puberty, vaginal epithelial cells accumulate glycogen due to the release of sex hormones. Decomposed vaginal acidity is maintained at 4.5. The six most common bacteria in the vagina are lactobacillus, the survival of which is affected by the vaginal acidity and the availability of glucose. Vaginal epithelial cells contain large amounts of glycogen by female hormones, and Lactobacillus bacteria break down them to produce lactic acid. If vaginal acidity is not maintained or metabolic disorders occur, the disease is caused by a decrease in normal flora.

Lactobacillus resident in the vagina of healthy premenopausal women is dominated by L. acidophilus group and L. fermentum at 10 ⁷ to 10 ⁸ CFU / g. L. plantarum, L. brevis, L. jensenii, L. casei , L. delbrueckii and L. salivarius are also reported. Genitourinary pathogens that cause vaginitis include Trichomonas vaginalis, Gardnerella vaginalis, Candida albicans, GroupB Streptococcus agalactiae, Staphylococcus aureus, Neisseria gonorrore, and E coli . Most of these infections begin with urinary tract infections (Reid, G., Jass, J., Sebulsky, T., and MaCormick, JK 2003. Potential uses of probiotics in clinical practice.Clinical Microbiology Reviews 16: 658-672).

Looking at the mechanism of inhibition of lactobacilli against pathogens: 1. Competitively inhibiting the binding of pathogens to the genitourinary surface; Exposure to antibacterial substances. Among them, lactobacillus adhesion mechanism is related to non-specific factors (especially hydrophobicity and surface ionization degree) and specific components such as protein and carbohydrates on the cell surface (Lorca, G., Tonino, MI, de Valdez, GF, Ljungh) , A. 2002. Lactobacilli express cell surface proteins which mediate binding of immobilized collagen and fibrogen.FEMS Microbiol.Letters 206: 31-37).

Lactobacillus, which can be used as a probiotic for excellent urinary health of women, should be excellent in inhibiting the growth of pathogens because of its ability to produce lactic acid and hydrogen peroxide, which have bactericidal activity, and to inhibit pathogen growth. And ability to inhibit the attachment of bladder cells should be excellent. In addition, it should pass the human safety test by confirming that there is no production of various harmful products and no harmful enzyme production.

Accordingly, the present invention is to provide a lactic acid bacteria that can protect urinary health.

This lactic acid bacterium was deposited as Weccella kimchii Strain PL9023 as KACC 91139.

In addition, the lactic acid bacterium of the present invention is to inhibit the growth of urinary infection bacteria as a lactic acid bacteria having a function of excessively generating hydrogen peroxide having a bactericidal effect.

In addition, the lactic acid bacteria of the present invention is to prevent and treat urinary infections as lactic acid bacteria that generate bacteriocin, lactic acid, and other inhibitors that have a bacteriostatic effect.

In addition, the lactic acid bacteria of the present invention is to suppress the infection by inhibiting the adhesion of vaginal cells and bladder cells of urinary infection bacteria.

In another aspect, the present invention is to provide a lactic acid bacterium safe for human use in the form of oral administration or vaginal suppository.

It is another object of the present invention to provide a lactic acid bacterium capable of promoting various female health.

The lactic acid bacteria can be formulated into various compositions, and preferably the composition is preferably in the form of pharmaceutical compositions such as capsules, tablets and powders, suppositories, and forms that are easy to add various foods. Such formulations may be prepared using known pharmaceutically acceptable carriers, excipients, solvents or auxiliaries by known methods. Such methods and ingredients are well known and are described in detail in standard texts and manuals, such as, for example, Remington. 1995. The Science and Practice of Pharmacy. Mack Publising Co, Easton, PA 18042, USA. It is.

In addition, formal bacteria can be prepared in food by methods generally known in the art.

It can also be used as a spawn in fermented foods, including fermented dairy products, by methods well known in the art to promote women's health.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention provides lactic acid bacteria of the urinary infection prevention and treatment function.

The present invention also provides Weissella kimchii Strain PL9023 for urinary infection and treatment.

In addition, the present invention provides a prophylactic and therapeutic agent that uses 1 × 10 ⁷ CFU to 1 × 10 ¹¹ CFU or suppository of Weissella kimchii Strain PL9023 for urinary infection and treatment once or twice a day. to provide.

The present invention also provides food and beverage additives including Weissella kimchii Strain PL9023 for urinary infection and treatment.

The present invention also provides a fermentation product for the purpose of preventing and treating urinary infection, including Weissella kimchii Strain PL9023 for urinary infection and treatment.

Hereinafter, preferred embodiments of the present invention are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

Experimental Example 1 Weissella kimchii  Isolation and Identification of Strain PL9023

<Materials and Test Methods>

Samples were taken from the vagina of healthy premenopausal women and inoculated in MRS (Man-Rogosa-Sharpe liquid reference medium) medium containing 0.001% bromophenol blue, where well formed colonies were used for identification. Biochemical properties were identified using the API CH50.CHL kit and confirmed using 16S rRNA sequencing. In order to analyze 16S rRNA sequences, genomic DNA was isolated to amplify the 16S rDNA portion, and the amplified DNA fragments were confirmed by electrophoresis. DNA fragments from gels were purified by Qiagen PCR purification kit (Giagen, Hilden, Germany), mixed with a reaction solution containing d-Rhodamine dye-labeling dd-NTP, and subjected to sequencing PCR. Purification using The purified DNA was dissolved in a Template Suppression Reagent (TSR) and analyzed by ABI prism 310 Genetic Analyzer (PE Applied Biosystems, USA), and analyzed by Genebank ( http://www.ncbi.nlm.nih.gov/ ). It was identified using. ARDRA was used to distinguish Weissella confusa from W. kimchii .

<Results and discussion>

L. coprophilus was identified using the API 50CH kit. As a result of API identification, Weissella confusa and Weissella kimchii are both identified as L. coprophilus . However, Weissella confusa and Weissella kimchii showed the difference in fermentation ability of Galactose, β-Gentiobiose and Gluconate. The 16S rRNA sequence (FIG. 1) was named Weissella kimchii Strain PL9023 in 100% concordance with Weissella kimchii (Accession No. AY190629) of Genbank DB (FIG. 2).

Experimental Example 2 Safety Inspection

The use of Lactic Acid Bacteria (LAB) has a long history and most lactic acid bacteria have been known to have no pathogenic potential. Thus, most of the genus lactobacillus is recognized as "Generally Recognized as Safe (GRAS) status". However, as safety of lactic acid bacteria has recently been raised, safety tests have become mandatory for the use of lactic acid bacteria. Therefore, hemolysis test, gelatin liquefaction test, ammonia production test, indole production test, phenylalanine deamination test and harmful enzyme production test (β-glucosidase, β-glucuronidase, nitroreductase activity) were performed on PL9023.

<Materials and Experimental Methods>

Hemolysis Test (Hemolysis)

Weissella kimchii Strain PL9023 was inoculated in sheep blood Agar medium and incubated at 37 ° C for 24 hours to observe hemolysis (Isenberg, HD 1992. Hemolysis on sheep blood agar. Clinical Microbiology Procedures Handbook Vol. 1, pp. 1.20.16 -1.20.17.American Society of Microbiology, Washingtong DC, USA).

Gelatin Liquefaction Test

Sloped media were made with MRS gelatin medium (0.3 g beef extract, 0.5 g peptone, 12 g gelatin, 100 ml MRS broth), inoculated with Weissella kimchii Strain PL9023, and incubated at 35 ° C for 6 weeks. The gel was liquefied by cooling for 4 hours at 4 ° C with the control group not inoculated (Isenberg, HD 1992. Gelatin liquefaction test. Clinical Microbiology Procedures Handbook Vol. 1, pp. 1.19.42-1.19.43. of Microbiology, Washingtong DC, USA).

Confirmation of Ammonia Generation

Filter and sterilize the urea agar medium (20 g urea, 5 g NaCl, 2 g KH2PO4, 1 g peptone, 1 g glucose, 12 mg phenol red, 100 ml distilled water), and then dissolve 15 g of agar in 900 ml of distilled water. After mixing, adjust the total volume to 1 l (pH 6.9). Weissella kimchii Strain PL9023 was inoculated and cultured at 37 ° C. for 12 hours to confirm the change of medium color (Isenberg, HD 1992. Urease. Clinical microbiology procedures handbook.Vol. 1, pp. 2.6.8.American Soceity of Microbiology, Washington D, C., USA).

Indol generation confirmation

Inoculate Weissella kimchii Strain PL9023 in MRS agar containing 0.1% Tryptone and incubate for 18 hours. Five drops of Kovac's reagent (10 g p-dimethylaminobenzaldehyde, 150 ml buthanol, 50 ml hydrocholic acid) was added to observe the color change (Isenberg, HD 1992. Indole test. Clinical Microbiology Procedures) Handbook Vol. 1, pp.1.19.13-1.19.15.American Society of Microbiology, Washingtong DC, USA).

Phenylalanine Deamine Probe

0.2% D, L-phenylalanine was added to MRS medium to inoculate Weissella kimchii Strain PL9023 and incubated for 24 hours. Add 5-10 drops of 10% ferric chloride and let it flow down the medium, and observe the color change within 1-5 minutes. If positive, the resulting phenylpyruvic acid reacts with 10% ferric chloride to turn green. v was negative (Ishibashi N. and S. Yamazaki. 2001. Probiotics and safety. Am. J. Clin. Nutr. 73: 465S-470S).

β-glucuronidase Production Confirmation Test

p-nitrophenyl-β-D-glucuronide was dissolved in 0.1 M sodium phosphate buffer, pH 6.0 to 0.2%. Weissella kimchii Strain PL9023 was suspended in phosphate buffer with Ab ₆₀₀ = 4 to make a suspension, and 200 µl suspension was added with 200 µl of substrate-based buffer for 16 hours at 37 ° C. If the color of the culture turns yellow, read it positively (Mastromarino, A., BS Reddy, and EL Wynder. 1976. Metabolic epidermiology of colon cancer: enzymic activity of fecal flora. Am. J. Clin. Nutr. 29: 1455- 1460).

Nitroreductase activity confirmation test

Weissella kimchii Strain PL9023 cultured overnight in MRS liquid medium was centrifuged at 3,000X g for 10 minutes to collect the cells and sonicated for 5 minutes. 4-nitrobenzoic acid (final conc. 30 ㎍ / ml) and trichloroacetic acid (final conc. 0.21%) were added to the supernatant and treated at 37 ° C for one hour, sodium nitrite (final conc. 0.007%) was added, and at room temperature. 20 minutes treatment. Ammonium sulfamate (final conc. 0.04%) is added and treated at room temperature for 3 minutes. NEDD (N- (1-naphtyl) ethylenediamine dihydrochiolide) (final conc. 0.35%) was added, followed by color development at 4 ° C, and measured with a 540 nm spectrophotometer. Positive reactions were compared by adding 1 μg / ml 4-aminobenzoic acid (Rafii, F., W. Franklin, RH Heflish, and CE Cerniglia. 1991. Reduction of nitroaromatic compounds by anaerobic bacterial isolated from the human gastrointestinal tract. Appl. Environ.Microbiol. 57: 962-968).

Antibiotic resistance

Probiotics are more resistant to antibiotics, which means better intestinal viability. However, the transmission of these resistances can cause resistance problems (Gevers, D., G. Huys, and J. Swings. 2003. In vitro conjugal transfer of tetracycline resistance from Lactoacillus isolates to other Gram-positive bacteria.FEMS Microb Letters 225: 125-130) The transfer of resistance to other organisms was confirmed.

Table 2. Antibiotic Resistance of Weissella kimchii Strain PL9023 Using Disc Diffusion

Confirmation of metastasis of antibiotic resistance

A filter binding assay was performed to identify antibiotic resistance transfer (givers, D., G. Huys, and J. Swings. 2003. In vitro conjugal transfer of tetracycline resistance from lactobacillus islates to other Gram-positive bacteria.FEMS Microb Letters 225: 125-130. Incubate the Weissella kimchii Strain PL9023 to the mid-exponential phase (approximately 4-5 hours), mix 1 ml of it with 1 ml of E. faecalis CCARM 5110, filter it through a sterile cellulose acetate filter and filter it with peptone physiological saline solution (PPS). Washed. The filter paper is placed on a non-selective agar medium and incubated at 37 ° C for 16 hours. Cells grown on filter paper were washed and washed with 2 ml of PPS, diluted again, and then inoculated in Enterococcosal selective medium containing each antibiotic to incubate at 37 ° C. for 24-48 hours to identify resistant E. faecalis .

<Results and discussion>

Weissella kimchii Strain PL9023 did not produce any harmful products or enzymes, and antibiotic resistance transfer experiments showed no resistance to E. faecalis .

Experimental Example 3 Hazardous Bacteria Growth Inhibition Test

<Materials and Experimental Methods>

W. kimchii PL9023 was incubated for 24 hours at 37 degrees to reach the stationary phase in MRS liquid medium. Pathogens isolated from the vagina were used to confirm the growth inhibitory effects of pathogenic bacteria. Candida albicans, Staphylococcus aureus and Escherichia coli were incubated in potato dextrose agar (PDA), mannitol salt agar and MacCONKEY agar, respectively. Streptococcus agalactiae was inoculated into Blood agar and cultured in a 10% CO ₂ incubator. Pathogenic bacteria were each inoculated in Muller Hinton solid medium medium, and 10 μl of the solution of W. kimchii PL9023 extracted with ethyl acetate was placed to determine whether growth inhibitory rings were formed. Ethyl acetate extract of the culture solution is prepared by dissolving the culture solution with the same amount of ethyl acetate dissolved in 100% distilled water.

<Results and discussion>

As shown in Figure 2, the ethyl acetate extract of W. kimchii PL9023 was isolated from vaginal C. albicans, E. coli, S. aureus, Strep. Agalactiae also inhibited the growth of antibiotic resistant MRSA.

Experimental Example 4 Production of Hydrogen Peroxide: Quantitative and Qualitative Methods

<Materials and Experimental Methods>

TMB-Plus medium (Brucella agar base plate supplemented with horseradish peroxidase aud 3,3 ', 5,5'-tetramethylbendine dihydrochloride (TMB)) was used to confirm the hydrogen peroxide production capacity. This method uses horseradish peroxidase-H ₂ O ₂ complex to change the color of chromogen that acts as hydrogen donor. In other words, when lactic acid bacteria are incubated under anaerobic conditions and exposed to air for a short time for about 30 minutes, hydrogen peroxide is produced, which causes TMBs to be oxidized to form a blue color. Qualitative experiments were measured using Amplex Red Hydrogen Peroxide / Peroxidase Assay Kit (Molicular Probes, Eugene, Oregon, USA. A-22188). The lactic acid bacteria were inoculated in 1% MRS liquid medium and incubated overnight. Next, the bacterial culture was washed twice with Brucella liquid medium, and the turbidity of the cells was adjusted to OD = 4 at Ab.600, incubated at 37 ° C for 4 hours, centrifuged, and 50 µl of the supernatant was transferred to a 96 well plate. 50 μl of color reagent (100 uM amplex red reagent, 0.2 U / ml horseradish peroxidase) was added, followed by color development at room temperature for 30 minutes and measurement at 560 nm.

<Results and discussion>

Weissella kimchii PL9023 showed the formation of hydrogen peroxide by forming blue colonies in TMB-Plus medium (FIG. 3). When the amount of production was measured, as shown in Table 4 below, it produced much more hydrogen peroxide than other strains.

Table 4. Hydrogen Peroxide Production

Experimental Example 5 Coagulation Test

In order for the lactic acid bacteria to survive in the body, it must have its own cohesion. In addition, when there is cohesion with pathogens, various bacterial inhibitors such as hydrogen peroxide, lactic acid and bacteriocin produced by lactic acid bacteria act directly to increase the ability to inhibit pathogens.

<Materials and Experimental Methods>

To determine the cohesive capacity of W. kimchii PL9023 itself, first, W. kimchii PL9023 is incubated overnight at 37 in MRS medium. Collect this by centrifugation for 10 minutes and wash twice with brine to make a suspension with 0.6 absorbance in MEM medium. This was incubated at 37 degrees, 100 rpm for 4 hours, and then stained with Gram staining and observed under a microscope. In the case of agglutination reaction with pathogens, experiment by adding pathogens and lactic acid bacteria simultaneously.

<Results and Reasoning>

As shown in Figure 4 W. kimchii PL9023 self-aggregation occurred only at pH 4.4. This shows that Weissella kimchii PL9023 can exist as a normal flora in the human body. When cultured with urinary infection pathogenic bacteria, W. kimchii PL9023 showed coaggregation with C. albicans at all pHs and did not show a clear aggregation reaction with other pathogenic bacteria (FIG. 5). This shows that other bacteria are not easily attached to vaginal cells by W. kimchii PL9023.

Experimental Example 6 Vaginal Cell Adhesion Test of Lactic Acid Bacteria

There are two mechanisms in which lactic acid bacteria inhibit cell adhesion of pathogens, and lactic acid bacteria adhere to cells competitively with pathogens and lactic acid bacteria aggregate with pathogens to inhibit the attachment of pathogens. First, the cell adhesion of the lactic acid bacteria itself was measured.

<Materials and Test Methods>

Isolation of Vaginal Cells

Pre-menopausal women's vaginal lining is scraped three times in a row with a pop brush to obtain cells and make a suspension in 10 ml minimal essential medium. Bacteria present in the vaginal cells are shaken for 30 seconds in 10 ml of MEM and then washed three times by centrifugation (750 X g) for 5 minutes. Next, the resultant was filtered with filter paper (8 μm), and the cells of the filter paper were adjusted to the concentration of 2 × 10 ⁴ cells / ml in the medium again.

Measurement of Vaginal Cell Attachment of W. kimchii PL9023

Mix W. kimchii PL9023 (10 ⁸ cells / ml) and vaginal cells (2 x 10 ⁴ cells / ml) prepared in the same amount of MEM medium and shake for 37 to 30 minutes in an orbital shaker (100 rpm / min). It is filtered through filter paper (8 μm) to remove bacteria and washed with the same amount of MEM. Cells attached to the filter paper are placed on a glass slide coated with aluminum, fixed with methanol, stained with Gram, and observed under a microscope.

<Results and discussion>

As shown in FIG. 6, W. kimchii PL9023 had excellent adhesion to vaginal cells and had an average of 100 or more attachments per vaginal cell even at pH 7.2 and the highest number at pH 4.4.

Experimental Example 7 W. kimchii  Human vaginal cell adhesion inhibition test of harmful bacteria by strain L9023

When W. kimchii PL9023 was bound first, the following experiment was performed to determine how the adhesion of pathogens was reduced (exclusion test). W. kimchii PL9023 (10 ⁸ cells / ml) and vaginal cells (2 x 10 ⁴ cells / ml) in the same amount of MEM medium are mixed and shaken for 37 to 30 minutes in an orbital shaker (100 rpm / min). Add each pathogen (1-2 x 10 ⁶ cells / ml) and shake again for 30 minutes. Then filter by filter paper (8 ㎛) to remove bacteria and washed with the same amount of MEM. Cells attached to the filter paper were placed on a glass slide coated with aluminum, fixed with methanol, stained with gram, and observed under a microscope.

To measure the effect of dropping already attached bacteria (Displacement test), first incubate pathogens and vaginal cells, and after 30 minutes, incubate with W. kimchii PL9023, remove bacteria in the same way as above, and then add the number of bacteria. Count. At this time, 20 fields in each culture dish were randomly selected for statistical processing, and the number of bacteria present in each field was counted. Standard deviation was calculated using Sigma Plot (SPSS Inc., Chicago, USA).

<Results and discussion>

Table 3. Degree of W. kimchii PL9023 Inhibition of Vaginal Cell Adhesion in Various Urinary Infectious Bacteria

1. Number of bacteria attached to one C. albicans vaginal cell

2. E. coli

3. S. aureus

4. Strep. agalactiae

Pathogenic bacteria average 7 E. coli and 8 Strep. Per vaginal cell . agalactiae , 13 C. albicans and 16 S. aureus attached and less attached at low pH (FIG. 7). In the case of C. albicans , the adhesion was inhibited by W. kimchii Strain PL9023 at 50% compared to the control group. In particular, at pH 4.4, W. kimchii Strain PL9023 exhibited significant inhibitory activity under each test condition.

As mentioned above, Weissella kimchii Strain PL9023 (KACC 91139) of the probiotic strain of the present invention has been confirmed human stability, it is excellent in the sexual inhibition and adhesion inhibitory to female texturitis bacteria Confirmed. Weissella kimchii strain PL9023, KACC 91139 of the probiotic strain of the present invention can be used as a prophylactic and therapeutic agent for various urinary tract diseases, oral administration, vaginal suppuration, urinary liver insertion, artificial urine It can be used directly in the manufacture of preventive and therapeutic agents for urinary diseases, such as catheter coatings, or as an additive or fermentation agent for functional foods and drinks for this purpose.

Claims (7)

delete Weissella kimchii Strain PL9023, KACC 91139 of Accession No. KACC 91139, which inhibits the growth and vaginal cell adhesion of female urinary tract bacteria. Weissella kimchii Strain PL9023 (KACC 91139) of the accession number KACC 91139 composition for preventing or treating urinary tract infections. The composition of claim 3, wherein the composition for preventing or treating urinary infection is an oral dosage form, a suppository, or an ointment. A food additive for preventing or treating urinary infections in women, including Weissella kimchii Strain PL9023, KACC 91139, deposited under accession number KACC 91139. 6. The food additive of claim 5, wherein the food additive is added to yogurt, baby food, dairy, cheese, kimchi or beverage. Dairy or fermented foods for the prevention or treatment of urinary infections, prepared using Weissella kimchii Strain PL9023 (KACC 91139) of accession number KACC 91139

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