Probiotics for inhibiting and preventing progression of renal diseases, and compositions for inhibiting and preventing progression of renal diseases comprising same
11369647 · 2022-06-28
Assignee
Inventors
- Su Ae Kim (Seongnam-si, KR)
- Chang Taek Oh (Seongnam-si, KR)
- Jong Hun Lee (Seongnam-si, KR)
- Su Hwan Lim (Seongnam-si, KR)
- Jeom Yong Kim (Seongnam-si, KR)
- Sun Kyu Park (Seongnam-si, KR)
- Min Jung Jang (Seongnam-si, KR)
- Min Ju Lim (Seongnam-si, KR)
Cpc classification
A23L33/135
HUMAN NECESSITIES
International classification
Abstract
A probiotic for inhibiting and preventing the progression of a renal disease and a composition containing the same are disclosed. The probiotic contains a novel Lactobacillus acidophilus BP105 strain, a novel Lactobacillus salivarius BP121 strain. The novel probiotics, the Lactobacillus acidophilus BP105 strain and the Lactobacillus salivarius BP121 strain exhibit excellent indoxyl sulfate removal ability, p-cresol removal ability, and phosphorus absorption ability.
Claims
1. A method for inhibiting and preventing the progression of a renal disease in an animal, comprising administering a composition comprising one or more selected from the groups consisting of isolated Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P), a culture thereof, a concentrate thereof, a paste thereof, a spray-dried material thereof, a lyophilisate thereof, a vacuum-dried material thereof, a drum-dried material thereof, a liquid thereof, and a dilution thereof, and a homogenate thereof in an effective amount to the animal, wherein the composition does not comprise a prebiotic inulin.
2. A method of claim 1, wherein the method comprises further administering one or more selected from the groups consisting of isolated Lactobacillus acidophilus BP105 strain (Accession No. KCCM12169P), a culture thereof, a concentrate thereof, a paste thereof, a spray-dried material thereof, a lyophilisate thereof, a vacuum-dried material thereof, a drum-dried material thereof, a liquid thereof, a dilution thereof, and a homogenate thereof.
3. A method of claim 1, wherein the Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P) is isolated from feces of an infant.
4. A method of claim 1, wherein the Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P) has indoxyl sulfate removal ability, p-cresol removal ability, and phosphorus absorption ability.
5. A method of claim 1, wherein the composition is a pharmaceutical composition, a dietary supplement, or a foodstuff.
6. A method of claim 1, wherein the composition is a dietary supplement or a foodstuff.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
BEST MODE
(9) Hereinafter, the present invention will be described in more detail.
(10) Unless defined otherwise, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, preferred methods or samples are described in the present specification, but similar or equivalent ones are included in the scope of the present invention. The contents of all publications described by reference in the present specification are incorporated in the present specification by reference in their entirety.
(11) The present invention relates to probiotics for inhibiting and preventing the progression of a renal disease, comprising a strain isolated from human feces, preferably feces of infants by a method comprising the steps of:
(12) (a) diluting feces of infants 12 months or less of age with sterile water and inoculating MRS medium with the feces to incubate them;
(13) (b) subculturing a milky white single colony produced in the MRS medium; and
(14) (C) selecting and obtaining lactic acid bacteria from the colonies subcultured in step (b).
(15) The probiotics include 15 strains as shown in Table 1 below:
(16) TABLE-US-00001 TABLE 1 Identified Strains BP101 BP103 BP104 BP105 BP107 BP109 BP110 BP111 BP115 BP121 BP122 BP123 BP131 BP132 BP133
(17) The strains were identified by 16s rRNA analysis of the probiotics of the present invention.
(18) In particular, the probiotics are characterized by including a Lactobacillus acidophilus BP105 strain deposited under Accession No. KCCM12169P and Lactobacillus salivarius BP121 deposited under Accession No. KCCM12170P.
(19) The novel strains, the Lactobacillus acidophilus BP105 strain and the Lactobacillus salivarius BP121 strain were deposited, according to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the Korean Culture Center of Microorganisms on Nov. 16, 2017 under the names as described above. The accession numbers are KCCM12169P for Lactobacillus acidophilus BP105 and KCCM12170P for Lactobacillus salivarius BP121. Korean Culture Center of Microorganisms is located at the following address: Yurim B/D, 45 Hongjenae 2ga-gil, Hongje-dong, Seodaemun-gu, Seoul 120-861, South Korea.
(20) Each of the 15 strains exhibits excellent effects on indoxyl sulfate removal and p-cresol removal. In particular, the Lactobacillus acidophilus BP105 strain and the Lactobacillus salivarius BP121 strain exhibit very excellent effects on indoxyl sulfate removal and p-cresol removal, and also exhibit very excellent effects on phosphorus absorption rate.
(21) The probiotics of the present invention which may be used have, but are not limited to, a live bacteria content of 1×10.sup.1 to 1×10.sup.15 CFU/g.
(22) In addition, the present invention relates to a composition for inhibiting and preventing the progression of a renal disease, comprising one or more selected from the groups consisting of the probiotics, cultures thereof, concentrates thereof, pastes thereof, spray-dried materials thereof, lyophilisates thereof, vacuum-dried materials thereof, drum-dried materials thereof, liquids thereof, dilutions thereof, and crushes thereof.
(23) The composition for inhibiting and preventing the progression of a renal disease may be a pharmaceutical composition or a food composition.
(24) The pharmaceutical composition may be granules, limonades, powders, syrups, liquids and solutions, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills, or soft or hard gelatin capsules.
(25) The type of the food composition is not particularly limited, and includes a general food composition as well as a health functional food composition.
(26) In addition, the present invention relates to a Lactobacillus acidophilus BP105 strain deposited under Accession No. KCCM12169P.
(27) The strain has excellent indoxyl sulfate removal ability, p-cresol removal ability, and phosphorus absorption ability.
(28) In addition, the present invention relates to a composition for inhibiting and preventing the progression of a renal disease, comprising one or more selected from the groups consisting of the Lactobacillus acidophilus BP105 strain, cultures thereof, concentrates thereof, pastes thereof, spray-dried materials thereof, lyophilisates thereof, vacuum-dried materials thereof, drum-dried materials thereof, liquids thereof, dilutions thereof, and crushes thereof.
(29) The composition for inhibiting and preventing the progression of a renal disease may be a pharmaceutical composition or a food composition.
(30) The pharmaceutical composition may be granules, limonades, powders, syrups, liquids and solutions, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills, or soft or hard gelatin capsules.
(31) The type of the food composition is not particularly limited, and includes a general food composition as well as a health functional food composition.
(32) In addition, the present invention relates to a Lactobacillus salivarius BP121 strain deposited under Accession No. KCCM12170P.
(33) The strain has excellent indoxyl sulfate removal ability, p-cresol removal ability, and phosphorus absorption ability.
(34) In addition, the present invention relates to a composition for inhibiting and preventing the progression of a renal disease, comprising one or more selected from the groups consisting of the Lactobacillus salivarius BP121 strain, cultures thereof, concentrates thereof, pastes thereof, spray-dried materials thereof, lyophilisates thereof, vacuum-dried materials thereof, drum-dried materials thereof, liquids thereof, dilutions thereof, and crushes thereof.
(35) The composition for inhibiting and preventing the progression of a renal disease may be a pharmaceutical composition or a food composition.
(36) The form of the pharmaceutical composition may be granules, limonades, powders, syrups, liquids and solutions, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills, or soft or hard gelatin capsules, but is not limited thereto.
(37) The type of the food composition is not particularly limited, and includes a general food composition as well as a health functional food composition.
(38) In the present invention, the pharmaceutical composition as described above may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers comprised in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
(39) The pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the ingredients as described above.
(40) The pharmaceutical composition of the present invention may be administered orally or parenterally.
(41) The pharmaceutical composition of the present invention may be formulated in a single-dose form or in multi-dose vessels using a pharmaceutically acceptable carrier and/or excipient, according to a method that may be easily carried out by those skilled in the art.
(42) In the present invention, the food composition as described above may comprise, in addition to the active ingredient, ingredients that are commonly added during food preparation, and, for example, may comprise proteins, carbohydrates, fats, nutrients, seasonings, sweeteners and flavoring agents, but is not limited thereto. Examples of the carbohydrates include conventional sugars, such as monosaccharides, for example, glucose, fructose, and the like; disaccharides, for example, maltose, sucrose, oligosaccharide, and the like; and polysaccharides, for example, dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweeteners, natural sweeteners (taumatin, stevia extract, rebaudioside A, glycyrrhizin, and the like) and synthetic sweeteners (saccharin, aspartame, and the like) may be used.
(43) Examples of the food composition may include patient nutrition, meat, cereals, caffeine drinks, general drinks, dairy products, chocolates, bread, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohols, vitamin complexes, other health supplement foods, and the like, but are not limited thereto. When prepared in the form of the food composition as described above, it is preferable because patients suffering from diseases caused by an increase in uremic toxins in the blood and an increase in the concentration of phosphorus in the blood may take it conveniently and easily.
(44) In the present invention, the dosage of the probiotics, Lactobacillus acidophilus BP105 strain and Lactobacillus salivarius BP121 strain is preferably determined in consideration of the administration method, the age, sex, body weight and severity of the disease of the patient, and the like.
(45) By way of an embodiment, the probiotics, Lactobacillus acidophilus BP105 strain and Lactobacillus salivarius BP121 strain may be administered with a living bacteria content of 1×10.sup.1 to 1×10.sup.15 CFU/g per day and it can be administered in more than once a day.
(46) In addition, the pharmaceutical composition or the food composition comprising the above ingredients may be administered one or more times per day with a living bacteria content of 1×10.sup.1 to 1×10.sup.15 CFU/g, based on the active ingredients.
(47) However, the above dosage is only an example, and may be changed by a doctor's prescription according to the patient's condition.
MODE FOR CARRYING OUT THE INVENTION
(48) Hereinafter, the present invention will be described in detail by way of the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the following examples.
Example 1: Isolation of Probiotics
(49) The feces of infants of 1 month of age were diluted 10 steps in sterile water to isolate the strains by the dilution plating method. The diluted fecal sample was smeared into MRS medium (MRS broth agar; BD Difco), and then incubated anaerobically at 37° C. for 72 hours. A milky white single colony appearing on the MRS agar plate was subcultured to purely isolate the probiotics of the present invention.
Example 2: Identification of Strains Included in Probiotics
(50) Chromosomal DNA extraction and purification were performed on the strain purely isolated in Example 1 above. Two universal primers, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) were used to perform amplification of the 16s rRNA gene, and then sequencing analysis of the amplified 16s rRNA gene was performed. Using the analyzed 16s rRNA sequence data and EzTaxon server (http://www.ezbiocloud.net), only 15 strains corresponding to GRAS (Generally Recognized as Safe) were selected and shown in Table 2 below.
(51) TABLE-US-00002 TABLE 2 Identified Strains BP101 BP103 BP104 BP105 BP107 BP109 BP110 BP111 BP115 BP121 BP122 BP123 BP131 BP132 BP133
(52) The results of 16S rRNA sequencing analysis for BP105—L. acidophilus were as follows:
(53) TABLE-US-00003 <16S rRNA of bacterial strain, L. acidophilus BP105> (SEQ ID NO: 1) CTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCT GAACCAACAGATTCACTTCGGTGATGACGTTGGGAACGCGAGCGGCGGAT GGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGG AAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATA AAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTA GTTGGTAGGGTAACGGCCTACCAAGGCAATGATGCATAGCCGAGTTGAGA GACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAG GCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGC CGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAG AAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAG TCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCG TTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCT GATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTT CTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCG TAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAAC TGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGG TAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTC TCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGC AAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCT AGTGCAATCCGTAGAGATACGGAGTTCCCTTCGGGGACACTAAGACAGGT GGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG CAACGAGCGCAACCCTTGTCATTAGTTGCCAGCATTAAGTTGGGCACTCT AATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTC ATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGTACA ACGAGGAGCAAGCCTGCGAAGGCAAGCGAATCTCTTAAAGCTGTTCTCAG TTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAA TCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACC GGCCCGTCACACCATGGGAAGTCTGCAATGCCCCAAACCCGG
(54) The results of 16S rRNA sequencing analysis for BP121—L. salivarius were as follows:
(55) TABLE-US-00004 <16S rRNA of bacterial strain, L. salivarius BP121> (SEQ ID NO: 2) CCTAGATATAGTTTTTTTAATGCTCAGGACGAACGCTGGCGGCGTGCCTA ATACATGCAAGTCGAACGAAACTTTCTTACACCGAATGCTTGCATTCATC GTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTA AAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAA GGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATG GACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGAT GATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACAC GGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACG CAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTA AAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGAT GACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGG TAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAAC GCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTA GTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCC ATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAA GCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCA AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGG TGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTC CGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGG CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAC CTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCT TCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTG AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCC AGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAA GGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACAC GTGCTACAATGGACGGTACAACGAGTCGCGAGACCGCGAGGTTTAGCTAA TCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACAT GAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGT T
Test Example 1: Evaluation of Indoxyl Sulfate Removal Ability
(56) To isolate microorganisms that remove and degrade indoxyl sulfate, which is known to cause oxidative stress in the kidney, the indoxyl sulfate removal ability was evaluated on 15 strains isolated in Example 2 above. After pre-incubation for 24 hours each in MRS broth, each 1% was inoculated in MRS broth to which indoxyl sulfate at a concentration of 60 μg/ml was added, and incubated for 24, 48, and 72 hours.
(57) After obtaining the bacteria incubated for 24, 48, and 72 hours, respectively, centrifugation was performed at 12,000 rpm for 10 minutes to take the supernatant from which the bacteria were removed. Indican assay (Sigma Co., Ltd.) was used to measure residual indoxyl sulfate in the culture supernatant from which the bacteria were removed.
(58) The measured results of residual indoxyl sulfate in the culture supernatant from which the bacteria were removed were shown in Table 3 below:
(59) TABLE-US-00005 TABLE 3 Removing Rate (%) of Indoxyl Sulfate Incubation (hrs) Strains 24 48 72 BP101 2.3 ± 2.4 10.3 ± 0.9 16.9 ± 1.6 BP103 7.3 ± 1.8 8.3 ± 2.0 15.1 ± 7.7 BP104 0.4 ± 0.2 3.3 ± 0.5 7.9 ± 2.4 BP105 8.1 ± 1.9 21.2 ± 6.2 27.9 ± 4.4 BP107 2.8 ± 0.7 8.3 ± 1.4 15.9 ± 1.3 BP109 −4.3 ± 0.9 1.4 ± 1.9 9.4 ± 2.0 BP110 −6.4 ± 1.4 0.5 ± 3.5 6.6 ± 1.7 BP111 7.9 ± 2.3 11.0 ± 0.7 21.1 ± 3.2 BP115 2.1 ± 3.0 11.1 ± 1.0 15.7 ± 1.7 BP121 14.7 ± 0.9 24.7 ± 0.6 30.1 ± 0.5 BP122 6.3 ± 0.8 11.9 ± 0.8 19.3 ± 1.3 BP123 6.4 ± 0.2 12.7 ± 1.3 18.5 ± 1.6 BP131 2.7 ± 1.0 10.9 ± 1.3 17.1 ± 1.1 BP132 5.4 ± 2.3 10.9 ± 0.9 19.0 ± 1.7 BP133 7.6 ± 1.1 9.9 ± 1.1 18.6 ± 5.3
(60) As confirmed in Table 3 above, as a result of analyzing indoxyl sulfate in the culture supernatant taken by time, significant reduction in indoxyl sulfate when incubated for 24 hours was confirmed in 7 strains out of 15 strains compared to the medium that was not inoculated with bacteria. In particular, when incubated for 72 hours, it was confirmed that the Lactobacillus acidophilus BP105 strain removed 27.9±4.4% of indoxyl sulfate, and the Lactobacillus salivarius BP121 strain removed 30.1±0.5% of indoxyl sulfate, which was relatively superior.
Test Example 2: Evaluation of p-Cresol Removal Ability
(61) To select for microorganisms that remove and decompose p-cresol known to cause cardiovascular disease, 15 microorganisms isolated in Example 2 above were pre-incubated in each MRS broth. 1% of the pre-incubated microorganisms were incubated for 24, 48, and 72 hours in MRS broth to which p-cresol at a concentration of 250 μg/ml was added.
(62) After incubation for 24, 48, and 72 hours, respectively, a culture solution for each time was obtained and centrifugation was performed at 12,000 rpm for 10 minutes. To take the supernatant of the centrifuged bacteria and measure the amount of residual p-cresol, high-performance liquid chromatography (HPLC) analysis was performed. Prior to high performance liquid chromatography (HPLC) analysis, all samples were pretreated through a 0.22 μm filter (Millipore). The high performance liquid chromatography (HPLC) used in this experiment was a Waters Alliance e2695 system, and analysis was performed using a Phenomenex kintex C18 (5 μm, 4.6×250 mm) column. At this time, a UV detector (220 nm) was used as a detector, and analysis was performed under isocratical elution of a mobile phase using acetonitrile (ACN) 30%, a flow rate of 1 ml/min, and a column temperature of 35° C., and the results were shown in Table 4 below.
(63) TABLE-US-00006 TABLE 4 Removing Rate (%) of p-Cresol Incubation (hrs) Strains 24 48 72 BP101 1.90 ± 1.05 2.45 ± 0.27 4.37 ± 1.64 BP103 2.93 ± 1.23 3.38 ± 1.51 2.40 ± 1.87 BP104 0.92 ± 1.97 1.66 ± 1.44 3.26 ± 2.10 BP105 3.62 ± 1.80 2.56 ± 2.57 3.03 ± 1.40 BP107 1.46 ± 1.69 2.12 ± 0.87 1.90 ± 1.47 BP109 2.74 ± 2.03 2.22 ± 0.97 3.08 ± 1.33 BP110 2.95 ± 4.24 3.18 ± 0.54 2.83 ± 0.88 BP111 0.42 ± 0.81 1.86 ± 0.68 1.57 ± 0.27 BP115 0.60 ± 1.77 3.73 ± 1.40 3.12 ± 1.55 BP121 4.70 ± 0.43 5.40 ± 1.96 5.89 ± 1.24 BP122 1.37 ± 0.71 3.92 ± 1.40 2.96 ± 2.27 BP123 4.54 ± 0.99 3.61 ± 1.19 3.42 ± 2.41 BP131 8.80 ± 2.57 9.01 ± 0.18 9.06 ± 0.23 BP132 0.66 ± 4.93 4.57 ± 2.49 2.43 ± 1.22 BP133 0.74 ± 0.16 1.06 ± 0.91 2.38 ± 0.45
(64) As confirmed in Table 3 above, as a result of analyzing the residual p-cresol in the culture supernatant for each time, significant reduction when incubated for 24 hours was shown in total 6 strains of BP101, BP103, BP105, BP121, BP123, and BP131 compared to the medium that was not inoculated with bacteria. In particular, when cultured up to 72 hours, it was confirmed that the Lactobacillus BP121 strain reduced 5.89±1.24% of p-cresol, and the Lactobacillus BP131 strain decreased 9.06±0.23% of p-cresol, which was relatively superior.
Test Example 3: Measurement of Phosphorus Absorption Rate
(65) The Lactobacillus acidophilus BP105 strain (Accession No. KCCM12169P) and the Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P), which are strains that have been confirmed to have indoxyl sulfate inhibitory ability and p-cresol inhibitory ability, were evaluated for their phosphorus absorption ability, which causes hyperphosphatemia when renal function decreases. The phosphorus absorption ability of microorganisms was carried out according to the colorimetric method using 5-bromo-4-chloro-3-indolyl phosphate disodium salt (Sigma Co., Ltd.). According to the colorimetric method, in the case of microorganisms using phosphate for growth, it shows a blue-green color. The experimental results are shown in
(66) As confirmed in
Test Example 4: Evaluation of Phosphorus Absorption Rate
(67) (1) Measurement of Phosphorus Absorption Rate Per Strain
(68) To quantitatively evaluate the phosphorus absorption ability confirmed in Test Example 3, the phosphorus absorption rate for each strain was evaluated. Each of Lactobacillus acidophilus BP105 (Accession No. KCCM12169P) and Lactobacillus salivarius BP121 (Accession No. KCCM12170P) strains was pre-incubated in MRS broth, and then was inoculated with MRS medium containing 20 mM phosphate to have an OD value of 1.0.
(69) First, the incubation is performed for 3 hours to measure the phosphorus absorption amount of the strain, and after 3 hours of incubation, the OD value was measured to derive the phosphorus absorption rate per strain by dividing the phosphorus absorption amount by the difference value (corresponding to the number of strains) from the original OD value. The phosphorus absorption amount was measured using Cedex Bio (Roche).
(70) As a result of the experiment, the Lactobacillus acidophilus BP105 strain (Accession No. KCCM12169P) showed a phosphorus absorption rate of 0.0955 mM per hour, and the Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P) showed a phosphorus absorption rate of 0.2384 mM per hour. As a result of the experiment, it was confirmed that the BP121 strain has a 2.5 times higher phosphorus absorption ability than the BP105 strain (see
(71) (2) Evaluation of Phosphorus Absorption Rate Over Time
(72) To measure the phosphorus absorption rate over time of Lactobacillus acidophilus BP105 (Accession No. KCCM12169P) and Lactobacillus salivarius BP121 (Accession No. KCCM12170P), the strains were incubated for 9 hours in the same manner as above. As a result, it was confirmed that the BP105 strain absorbed 1.63 mM phosphorus until 9 hours (see
Test Example 5: Evaluation of Indoxyl Sulfate Inhibitory Efficacy in Acute Renal Disease Model
(73) Acute renal disease model was established by intraperitoneal administration of 7 mg/kg cisplatin to male rats (SD rats). 1×10.sup.9 CFU of each of Lactobacillus acidophilus BP105 strain (Accession No. KCCM12169P and Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P), and a mixture prepared at a concentration of 1×10.sup.9 CFU by mixing both strains at a ratio of 1:1 were orally administered 10 days before induction and 4 days after induction of cisplatin, for a total of 14 days. The experiment was conducted for 2 weeks, and experimental animals were sacrificed on the 14.sup.th day. To evaluate in vivo indoxyl sulfate inhibitory efficacy of BP105 and BP121 by inducing acute nephrotoxicity with cisplatin, the final blood indoxyl sulfate concentration was measured on the 14.sup.th day. As a result, the groups administered with BP105, BP121, and mixtures thereof all showed a significant decrease in indoxyl sulfate compared to the acute nephrotoxicity-inducing group (see
Test Example 6: Evaluation of Renal Protective Efficacy in Acute Renal Disease Model
(74) To evaluate the renal protective effect of the Lactobacillus salivarius BP121 strain (Accession No. KCCM12170P), male rats (SD rats) were intraperitoneally administered with 7 mg/kg cisplatin to induce acute renal disease, and then were evaluated for renal function. BP121 was administered in 3 groups of 1×10.sup.8, 1×10.sup.9, and 1×10.sup.10 CFU, respectively, and Kremezin, a uremic agent, was administered orally at 0.5 g/rat as a positive control. Each group was administered orally 10 days before induction and 4 days after induction of cisplatin, for a total of 14 days, and the same amount of PBS was used in the control group.
(75) To evaluate the renal protective effect, the concentrations of blood urea nitrogen (BUN) and creatinine, which are the renal function indicators, were measured. As a result, it was confirmed that in the high concentration of BP121-administered group, BUN decreased by 26% and creatinine decreased by 32%, all of which had significant efficacy. As a result of evaluating the concentration of indoxyl sulfate in the blood, it was confirmed to have a significant decrease of about 26%, but did not reach the uremic agent cremezin, which was confirmed by a 38% reduction (see
(76) As a result of confirming the inflammation indicator according to the administration of BP121 in blood and renal tissue, respectively, serum TNF-α decreased by 37% in the blood, showing a tendency to decrease, and decreased by 32% in the renal tissue, showing a significance. As a result of confirming the expression level of the inflammatory cytokine IL-6, a 26% reduction in serum and a 41% of marked reduction in renal tissue were confirmed compared to cisplatin-induced acute nephrotoxicity model. Malondialdehyde is a final product of lipid peroxidation, and its amount is significantly increased due to cisplatin administration, whereas the high concentration of BP121-administered group shows a significant tendency to decrease by 41%, confirming that the administration of BP121 plays an antioxidant role (see
(77) To confirm changes in the body of short-chain fatty acids known as anti-inflammatory agents, which provide a renal protective effect and an improvement effect of the intestinal environment, feces were collected on the last day of administration of BP121 to analyze the amount of short-chain fatty acids. As a result, acetic acid, propionic acid, and butyric acid were all confirmed to be significantly increased, and the total short-chain fatty acids were confirmed to be 4.1 times higher than the cisplatin-induced group (see
Test Example 7: Evaluation of Short-Chain Fatty Acid Production Ability Over Time
(78) To confirm whether the change in the amount of short-chain fatty acids discharged to feces upon administration of Lactobacillus salivarius BP121 strain in Test Example 6 is an effect of BP121, the amount of short-chain fatty acids over time was measured through a single culture of the Lactobacillus acidophilus BP105 strain and the Lactobacillus salivarius BP121 strain. After pre-incubation for 24 hours in MRS broth, respectively, each of BP105 and BP121 was inoculated at 1% in MRS broth and incubated for 48 hours to measure culture supernatant at 0, 6, 12, 24, and 48 hours.
(79) As a result, it was confirmed that the amount of each short-chain fatty acid was increased over time, and it was confirmed that the short-chain fatty acids were not measured in the control group that was not inoculated with bacteria (see