<i>Lactobacillus acidophilus </i>TW01 isolate and use thereof

Abstract

Disclosed herein is an isolated strain of Lactobacillus acidophilus TW01, which is deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH under an accession number DSM 33990. Also disclosed herein are uses of the isolated strain of Lactobacillus acidophilus TW01 for alleviating an inflammation-related disorder and for improving gut health.

Claims

1. A method for alleviating an inflammation-related disorder, comprising administering to a subject in need thereof a composition including an isolated strain of Lactobacillus acidophilus TW01, wherein the isolated strain of Lactobacillus acidophilus TW01 is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under an accession number DSM 33990.

2. The method as claimed in claim 1, wherein the composition is a food product or a pharmaceutical composition.

3. The method as claimed in claim 2, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

4. The method as claimed in claim 1, wherein the inflammation-related disorder is selected from the group consisting of allergy, asthma, arthritis, psoriasis, atopic dermatitis, systemic lupus erythematosus, inflammatory bowel disease, and combinations thereof.

5. A method for improving gut health, comprising administering to a subject in need thereof a composition including an isolated strain of Lactobacillus acidophilus TW01, wherein the isolated strain of Lactobacillus acidophilus TW01 is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under an accession number DSM 33990.

6. The method as claimed in claim 5, wherein the composition is a food product or a pharmaceutical composition.

7. The method as claimed in claim 6, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form and a parenteral dosage form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other features and advantages of the present disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which:

(2) FIG. 1 shows the interleukin-10 (IL-10) content in each group of Example 3, infra; and

(3) FIG. 2 shows the IL-12 content in each group of Example 3, infra.

DETAILED DESCRIPTION

(4) It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Taiwan or any other country.

(5) For the purpose of this specification, it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning.

(6) Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of this disclosure. Indeed, this disclosure is in no way limited to the methods and materials described.

(7) The present disclosure provides an isolated strain of Lactobacillus acidophilus TW01, which has been deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (No. 331, Shih-Pin Rd., Hsinchu City 300, Taiwan) under an accession number BCRC 911039 since Mar. 5, 2021, and which has also been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under an accession number DSM 33990 since Aug. 2, 2021 in accordance with the Budapest Treaty.

(8) According to the present disclosure, the isolated strain of Lactobacillus acidophilus TW01 may be live cells or dead cells, concentrated or non-concentrated, a liquid, a paste, a semi-solid, a solid (e.g., a pellet, a granule, or a powder), and may be heat-inactivated, frozen, dried, or freeze-dried (e.g., may be in freeze-dried form or spray/fluid bed dried form). In an exemplary embodiment, the isolated strain of Lactobacillus acidophilus TW01 is in a liquid form. In another exemplary embodiment, the isolated strain of Lactobacillus acidophilus TW01 is present in the form of live cells.

(9) The present disclosure also provides a method for alleviating an inflammation-related disorder, which includes administering to a subject in need thereof a composition including the aforesaid isolated strain of Lactobacillus acidophilus TW01.

(10) As used herein, the term “alleviating” or “alleviation” refers to at least partially reducing, ameliorating, relieving, controlling, treating or eliminating one or more clinical signs of a disease or disorder; and lowering, delaying, stopping or reversing the progression of severity regarding the condition or symptom being treated and preventing or decreasing the likelihood or probability thereof.

(11) As used herein, the term “administering” or “administration” means introducing, providing or delivering the abovementioned composition to a subject showing condition(s) or symptom(s) of an inflammation-related disorder by any suitable routes to perform its intended function.

(12) As used herein, the term “subject” refers to any animal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats. In certain embodiments, the subject is a human.

(13) As used herein, the terms “inflammation-related disorder” and “immune-related disorder” can be used interchangeably.

(14) According to the present disclosure, the inflammation-related disorder may be selected from the group consisting of allergy (e.g., allergic rhinitis), asthma, arthritis, psoriasis, atopic dermatitis, systemic lupus erythematosus, inflammatory bowel disease (IBD) (e.g., colitis and Crohn's disease), and combinations thereof.

(15) According to the present disclosure, the composition may be formulated as a food product using a standard technique well known to one of ordinary skill in the art. For example, the composition may be directly added to an edible material or may be used to prepare an intermediate composition (e.g., a food additive or a premix) suitable to be subsequently added to the edible material.

(16) As used herein, the term “food product” refers to any article or substance that can be ingested by a subject into the body thereof. Examples of the food product may include, but are not limited to, fluid milk products (e.g., milk and concentrated milk), fermented milk (e.g., yogurt, sour milk, and frozen yogurt), milk powder, butter, beverages (e.g., tea and coffee), functional beverages, flour products, baked foods, confectionery, candies, health foods, animal feeds, and dietary supplements.

(17) According to the present disclosure, the composition may be prepared in the form of a pharmaceutical composition. The pharmaceutical composition may be formulated into a suitable dosage form for oral, parenteral or topical administration using technology well known to those skilled in the art.

(18) According to the present disclosure, the suitable dosage form for oral administration includes, but is not limited to, sterile powders, tablets, troches, lozenges, sustained film-coated tablets, oral ointments, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrup, elixir, slurry, drops, and the like.

(19) For parenteral administration, the pharmaceutical composition according to the present disclosure may be formulated into an injection, e.g., a sterile aqueous solution or a dispersion.

(20) The pharmaceutical composition according to the present disclosure may be administered via one of the following parenteral routes: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration. In certain embodiments, the pharmaceutical composition may be administered via intralesional injection.

(21) According to the present disclosure, the pharmaceutical composition may be formulated into an external preparation suitable for topical application to the skin using technology well known to those skilled in the art. The external preparation includes, but is not limited to, emulsions, gels, ointments, creams, patches, liniments, powder, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

(22) The pharmaceutical composition according to the present disclosure may further include a pharmaceutically acceptable carrier widely employed in the art of drug-manufacturing. For instance, the pharmaceutically acceptable carrier may include one or more of the following agents: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, fillers, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The choice and amount of the aforesaid agents are within the expertise and routine skills of those skilled in the art.

(23) The present disclosure further provides a method for improving gut health, which includes administering to a subject in need thereof the aforesaid composition.

(24) In certain embodiments, the composition for improving gut health is a pharmaceutical composition. The pharmaceutical composition may be formulated into a suitable dosage form for oral or parenteral administration. The oral dosage form, parenteral dosage form, and pharmaceutically acceptable carrier of this pharmaceutical composition are similar to those described above for the pharmaceutical composition for alleviating an inflammation-related disorder.

(25) In other embodiments, the composition for improving gut health is a food product as described above.

(26) The dose and frequency of administration of the composition according to the present disclosure may vary depending on the following factors: the severity of the illness or disorder to be treated, routes of administration, and age, physical condition and response of the subject to be treated. In general, the composition may be administered in a single dose or in several doses.

(27) The present disclosure will be further described by way of the following examples. However, it should be understood that the following examples are intended solely for the purpose of illustration and should not be construed as limiting the present disclosure in practice.

EXAMPLES

(28) General Experimental Materials:

(29) 1. Ox-bile (dehydrated, purified fresh bile), sodium chloride, and glycerol used in the following experiments were purchased from Sigma-Aldrich. 2. Agar, BD Difco™ Lactobacilli MRS (De Man, Rogosa and Sharpe) broth, and tryptic soy broth used in the following experiments were purchased from BD (Becton, Dickinson and Company) Biosciences. 3. API® 50 CHL microbial identification kit was purchased from Creative Life Science Co., Ltd., Taiwan. 4. The lactobacilli MRS agar medium used in the following experiments was prepared by adding 1.5% agar to BD Difco™ Lactobacilli MRS broth. 5. Murine macrophage cell line RAW 264.7 was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (No. 331, Shih-Pin Rd., Hsinchu City 300, Taiwan). The RAW 264.7 cells (BCRC 60001) were grown in a 10-cm Petri dish containing Dulbecco's Modified Eagle's Medium (DMEM) (Cat. No. D0819, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS). The RAW 264.7 cells were cultivated in an incubator with culture conditions set at 37° C. and 5% CO.sub.2. Medium change was performed every two to three days. Cell passage was performed when the cultured cells reached 80%-90% of confluence.
General Procedures:
1. Statistical Analysis

(30) The experimental data are expressed as mean±standard deviation (SD). All the data were analyzed using SAS software, so as to evaluate the differences between the groups.

Example 1. Preliminary Screening of Lactic Acid Bacteria (LAB) Isolates

(31) A. Source and Isolation of Tested Strains

(32) Coffee grounds purchased from Gukeng Township (Yunlin, Taiwan) were subjected to a fermentation reaction, and the resultant coffee fermented broth was then mixed with a suitable amount of a 0.85% saline solution, so as to obtain a dilution (prepared using a dilution factor of 10.sup.5). 0.1 mL of the dilution was evenly spread onto Lactobacilli MRS agar medium, followed by cultivation under an anaerobic condition in an incubator at 37° C. for 72 hours.

(33) Six LAB isolates were randomly selected from the MRS agar medium, and were designated as F2-2, F3-4, S2-1, K1-2, B-1, and D-1, respectively. These isolates were subjected to the analyses below.

(34) B. Preparation of Bacterial Suspension of LAB Isolate

(35) A respective one of the six LAB isolates obtained in section A of this example was inoculated in BD Difco™ Lactobacilli MRS broth, and was then cultivated under an anaerobic condition in an incubator at 37° C. for 24 hours to obtain a culture. After centrifugation at 1,200 g for 10 minutes, the resultant cell pellet was collected, and was washed with a 0.85% sterile saline solution, followed by centrifugation at 1,200 g for 10 minutes. The aforesaid washing and centrifugation steps were repeated twice.

(36) After removal of the supernatant, the bacterial cells were re-suspended in a suitable amount of a 0.85% sterile saline solution, so as to obtain a bacterial suspension having a bacterial concentration of 1×10.sup.8 CFU/mL. The six bacterial suspensions thus obtained were used for the following experiment.

Example 2. Acid Tolerance Test and Bile Salt Tolerance Test on LAB Isolates

(37) Experimental Procedures:

(38) A. Acid Tolerance Test

(39) A 0.85% sterile saline solution was adjusted to pH 2.0 through addition of a 5 N hydrochloric acid solution and a 0.1 N sterile sodium hydroxide solution, so as to obtain an acid resistance test solution. 1 mL of a respective one of the six bacterial suspensions prepared in section B of Example 1 was mixed with 1.5 mL of a 0.85% sterile saline solution and 5 mL of the acid resistance test solution, followed by cultivation in an incubator (37° C.) for a total period of 60 minutes. On the 30th and 60th minutes after cultivation, 100 μL of the resultant cell culture was collected, and the number of surviving bacterial cells was counted using a spread plate protocol well-known to those skilled in the art. The log value of the colony forming unit (CFU) was further calculated and the viable cell count was indicated by log CFU/mL.

(40) B. Bile Salt Tolerance Test

(41) A sterile saline solution containing 0.3% (w/v) Ox-bile (dehydrated, purified fresh bile) was adjusted to pH 8.0 through addition of a 0.1 N sterile sodium hydroxide solution, so as to obtain a simulated bile salt solution. 1 mL of a respective one of the six bacterial suspensions prepared in section B of Example 1 was mixed with 1.5 mL of a 0.85% sterile saline solution and 5 mL of the simulated bile salt solution, followed by cultivation in an incubator (37° C.) for 240 minutes. Thereafter, 100 μL of the resultant cell culture was collected, and the number of surviving bacterial cells was counted using a spread plate protocol well-known to those skilled in the art. The log value of the colony forming unit (CFU) was further calculated and the viable cell count was indicated by log CFU/mL.

(42) Results:

(43) As shown in Table 1 below, on the 30th minute after cultivation in an acidic environment (pH 2), the viable cell count of LAB isolate B-1 was higher than those of LAB isolates F3-4, S2-1, K1-2, and D-1. On the 60.sup.th minute after cultivation in an acidic environment (pH 2), LAB isolates F3-4, K1-2, and B-1 still exhibited good survivability.

(44) In addition, after cultivation in an environment containing 0.3% (w/v) Ox-bile for 240 minutes, LAB isolates F2-2, F3-4, S2-1, B-1, and D-1 exhibited excellent survivability. These results indicate that LAB isolates F3-4, S2-1, B-1, and D-1 are able to overcome the environmental pressure posed by the human gastrointestinal tract, and hence can reach and colonize the intestine(s) after ingestion.

(45) According to these results, the Applicant selected LAB isolates F3-4, S2-1, B-1, and D-1 for further experimentation to evaluate the anti-inflammatory activities of these strains.

(46) TABLE-US-00001 TABLE 1 Bile salt Acid tolerance test tolerance test Viable cell count LAB (log CFU/mL) isolate 30.sup.th minute 60.sup.th minute 240.sup.th minute F2-2 Not determined Not determined 6.66 ± 0.11 F3-4 4.11 ± 0.12 3.94 ± 0.03 7.17 ± 0.08 S2-1 3.20 ± 0.10 Not determined 7.78 ± 0.10 K1-2 3.52 ± 0.15 1.49 ± 1.07 Not determined B-1 7.68 ± 0.03 1.33 ± 0.94 6.06 ± 0.13 D-1 2.78 ± 0.13 Not determined 6.56 ± 0.10

Example 3. Evaluation of the Ability of LAB Isolates to Stimulate Secretion of Interleukin-10 (IL-10) and Interleukin-12 (IL-12) by Macrophages

(47) Experimental Procedures:

(48) A. Preparation of Test Sample of LAB Isolate

(49) Each of the bacterial suspensions of LAB isolates F3-4, S2-1, B-1, and D-1 prepared in section B of Example 1 was subjected to a freeze-drying treatment, so as to obtain a freeze-dried powder of LAB isolate F3-4, a freeze-dried powder of LAB isolate S2-1, a freeze-dried powder of LAB isolate B-1, and a freeze-dried powder of LAB isolate D-1. A respective one of the four freeze-dried powders was mixed with a suitable amount of BD Difco™ Lactobacilli MRS broth, and the resultant test sample was subjected to the following experiments.

(50) B. Determination of Contents of IL-10 and IL-12

(51) The RAW 264.7 cells prepared in section 5 of “General Experimental Materials” were divided into 10 groups, including one blank control group, one positive control group, and eight experimental groups (i.e., experimental groups 1 to 8). Each group of the RAW 264.7 cells was incubated in a respective well of a 24-well culture plate containing 5 mL of DMEM supplemented with 10% FBS at 6.25×10.sup.5 cells/well, followed by cultivation in an incubator (37° C., 5% CO.sub.2) for 48 hours. Afterwards, each of the cell cultures of the eight experimental groups and positive control group was treated with the corresponding treating agent so that the cell culture of each group had a final concentration of the corresponding treating agent shown in Table 2. The cell culture of the blank control group received no treatment.

(52) Each group was cultivated in an incubator (37° C., 5% CO.sub.2) for a total period of 48 hours. On the 24.sup.th and 48.sup.th hours after cultivation, 5 mL of the respective resultant cell culture was collected, and was then subjected to centrifugation at 3,000 rpm for 15 minutes. The resultant supernatant was collected, and was then subjected to determination of IL-10 and IL-12 contents using an IL-10 enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. EHIL10, Invitrogen) and an IL-12 ELISA kit (Cat. No. KAC1568, Invitrogen) in accordance with the manufacturer's instructions.

(53) TABLE-US-00002 TABLE 2 Final concentration Group Treating agent (pg/mL) Blank control — 0 group Positive Lipopolysaccharide 100 pg/mL control group (LPS) Experimental Test sample of LAB 100 pg/mL group 1 isolate S2-1 Experimental Test sample of LAB 200 pg/mL group 2 isolate S2-1 Experimental Test sample of LAB 100 pg/mL group 3 isolate F3-4 Experimental Test sample of LAB 200 pg/mL group 4 isolate F3-4 Experimental Test sample of LAB 100 pg/mL group 5 isolate B-1 Experimental Test sample of LAB 200 pg/mL group 6 isolate B-1 Experimental Test sample of LAB 100 pg/mL group 7 isolate D-1 Experimental Test sample of LAB 200 pg/mL group 8 isolate D-1
Results:

(54) Referring to FIGS. 1 and 2, the contents of IL-10 and IL-12 determined in the experimental groups 5 and 6 were higher than those determined in the experimental groups 1 to 4 and 7 to 8 and the positive control group, indicating that the ability of LAB isolate B-1 to stimulate secretion of IL-10 and IL-12 from macrophages is better than that of other LAB isolates. Therefore, LAB isolate B-1, having a greater potential for development, was subjected to characteristic analysis described below.

Example 4. Characteristic Analysis of LAB Isolate B-1

(55) In order to identify the bacterial species of LAB isolate B-1, the following preliminary characteristic determination, 16S rDNA sequence analysis, and carbohydrate fermentation profiling were conducted.

(56) A. Preliminary Tests

(57) Items of the preliminary tests conducted for LAB isolate B-1 included: gram staining, morphological observation, mobility, catalase test, growth under aerobic and anaerobic conditions, and ability to produce an endospore.

(58) The results of the aforesaid preliminary tests indicate that LAB isolate B-1 is gram-positive, non-motile, and catalase-negative, grows under anaerobic conditions, and is non-endospore forming. The cells of LAB isolate B-1 are coccus-shaped or rod-shaped.

(59) B. 16S rDNA Sequence Analysis

(60) Genomic DNA of LAB isolate B-1 was extracted using Genomic DNA Purification Kit (BioVision, Cat. No. K1457). The thus obtained genomic DNA was used as a template and was subjected to polymerase chain reaction (PCR) that was performed using a designed primer pair specific for 16S ribosomal DNA (rDNA) and the reaction conditions shown in Table 3, thereby obtaining a PCR product having a size of approximately 1460 bp.

(61) TABLE-US-00003 TABLE 3 Volume Contents (μL) Genomic DNA of LAB isolate B-1 (10 ng) 1 16S rDNA- Forward primer 27F (10 μM): 0.5 specific 5′-agagtttgatcctggctcag-3′ primer pair (SEQ ID NO: 1) Reverse primer 1492R (10 μM): 0.5 5′-ggttaccttgttacgact-3′ (SEQ ID NO: 2) dNTPs (10 mM) 0.5 10X buffer 2.5 Tag DNA polymerase (5 U/μL) 0.5 ddH.sub.2O 18.5 Operation conditions: denaturation at 94° C. for 5 min, followed by 30 cycles of the following reactions: denaturation at 95° C. for 60 sec, primer annealing at 50° C. for 60 sec, and extension at 72° C. for 60 sec; and lastly, elongation at 72° C. for 8 min.

(62) The resultant PCR product was subjected to 2% agarose gel electrophoresis analysis for molecular weight verification.

(63) Thereafter, the PCR product was verified by sequencing analysis which was entrusted to Seeing Bioscience Co., Ltd., Taiwan, so as to obtain the 16S rDNA sequence (SEQ ID No: 3) of LAB isolate B-1. Through comparison with the data in the NCBI's gene database, it was found that the 16S rDNA sequence of LAB isolate B-1 is most homologous to that of Lactobacillus acidophilus.

(64) In view of the aforesaid experimental results, LAB isolate B-1 of the present disclosure is identified as Lactobacillus acidophilus.

(65) C. Carbohydrate Fermentation Profiling

(66) The carbohydrate fermentation profile of Lactobacillus acidophilus strain TW01 (i.e. LAB isolate B-1) was determined using API® 50 CHL identification system (bioMérieux). The result is shown in Table 4 below.

(67) TABLE-US-00004 TABLE 4 Capability of fermenting carbohydrate tested to Carbohydrate produce acid Glycerol − Erythritol − D-Arabinose − L-Arabinose − D-Ribose − D-Xylose + L-Xylose + D-Adonitol − Methyl-β-D-xylopyranoside − D-Galactose − D-Glucose + D-Fructose + D-Mannose + L-Sorbose + L-Rhamnose + Dulcitol + Inositol − D-Mannitol − D-Sorbitol + Methyl-α-D-mannopyranoside + Methyl-α-D-glucopyranoside − N-Acetylglucosamine + Amygdalin + Arbutin + Esculin + Salicin + D-Cellobiose + D-Maltose + D-Lactose + Melibiose + Sucrose − Trehalose + Inulin + Melezitose − Raffinose + Amidon + Glycogen − Xylitol − Gentiobiose − D-Turanose + D-Lyxose + D-Tagatose − D-Fucose + L-Fucose − D-Arabitol − L-Arabitol − Gluconate − Note: “+” indicates that Lactobacillus acidophilus strain TW01 is capable of fermenting the carbohydrate tested to produce an acid, whereas “−” indicates that the strain has no such capability.

(68) Based on the aforementioned characterization results, the applicant believes that the Lactobacillus acidophilus strain TW01 is a strain of Lactobacillus acidophilus. As such, Lactobacillus acidophilus strain TW01 has been deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (No. 331, Shih-Pin Rd., Hsinchu City 300, Taiwan) under an accession number BCRC 911039 since Mar. 5, 2021, and has also been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (Inhoffenstraße 7B, 38124 Braunschweig, Germany) under an accession number DSM 33990 since Aug. 2, 2021 in accordance with the Budapest Treaty.

(69) Summarizing the above test results, it is clear that Lactobacillus acidophilus TW01 of the present disclosure can increase the anti-inflammation-associated IL-10 and IL-12, and hence is capable of alleviating an inflammation-related disorder and modulating gut immunity.

(70) In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiments. It will be apparent, however, to one skilled in the art, that one or more other embodiments may be practiced without some of these specific details. It should also be appreciated that reference throughout this specification to “one embodiment,” “an embodiment,” an embodiment with an indication of an ordinal number and so forth means that a particular feature, structure, or characteristic may be included in the practice of the disclosure. It should be further appreciated that in the description, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects, and that one or more features or specific details from one embodiment may be practiced together with one or more features or specific details from another embodiment, where appropriate, in the practice of the disclosure.

(71) While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

(72) TABLE-US-00005 SEQUENCE LISTING <110> I EATING LIGHT LTD. <120>  LACTOBACILLUS ACIDOPHILUS TW01 ISOLATE AND USE THEREOF amplification of <130> PE-65633-AM <160>    3 <170> PatentIn version 3.5 <210>    1 <211>   20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer 27F for PCR amplification of bacterial 16S rDNA fragment <400>    1 agagtttgat cctggctcag 20 <210>    2 <211>   18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1492R for PCR amplification of bacterial 16S rDNA fragment <400>    2 ggttaccttg ttacgact 18 <210>    3 <211> 1468 <212> DNA <213> Lactobacillus acidophilus <400>    3 atgaagaaaa atagaaaatt tttaggttta gctgctgctg cattgttagc agttgcacct   60 gttgtaacta gtgccgtacc tgtaagtgct gacacaccaa cggtggaccc ggggttgtcg  120 aaacctgtaa attctccagc acaatcacaa gttactggtg ctactccatt cttctcatat  180 cagaatggta acccaattta ttctgccggt gaaocaccad acattaatgc tggttcattt  240 actactatcg gccaaattgt agatgcaatt aataagaaca ttgtctttgg tgaagctggc  300 tcaactggaa caactcgtca agaagatatt tcagctgcag aagtaattag acaattaaaa  360 gccgacagca agagtgttga aattaaaggc aacgatgcaa aggccacagt ttcaaaatta  420 ccagcaaact ttgtaattac tttgaagcac actgtaaatg gtcaagctaa tactttgaat  480 gttcgtttct acactacttc tcaaccaaca gaatetgtag ataagtctgc tccagtattc  540 tacgtaactg aaggttcatc agctgctaag caagctactt caggtgcata ctaccaagta  600 gctgcaggct caaacttcaa cccattaagc ttcgtaaata gtaatggtga aactgtatca  660 ttctcagctc gtccagctga tggtaataac gctggtgcaa ctgtaagtgt tgcttctaac  720 ccagtagata ctaccaacgc aggtcgtttc tacactgtta ctttgactgc tactaacact  780 tcaaacaaga ctagccgcta ctcatacact gtattgattg tttcaaacgg tttacaaaaa  840 gtttatgata aatggtgcta gttcagcagc aacttacagc atttacggta accaagtttc  900 atcaaactca actaccttta aggatggtca agaagttcac gtaggtaaca ctacaagaac  960 tattaacaat gtatcatact caaaggtatc aactaagtct aaggcagatg ctgaccaagg 1020 taacctttgg attcaaactt cagctttgac tcaaactacc ccaaccactc cttcagacag 1080 caatgctgaa actcataatg taatggttga ctcacgtgct tacgacaagg acggtaacta 1140 cttaggccac atgtactacg catatgacaa cattgatatc gttccaactg ttgtaaccat 1200 caacggcaag acttactaca aggttgctaa caaggatgaa tatgtcagcg tcaccaacat 1260 caccggccac caacgtactt tacgtcacaa cgcttacatt tactggtcat cataccgtcg 1320 taccccaggt actggcaaga tgtacagagg ccaaactgta actacttacg gtcctgcaat 1380 gagattcaag aacggtaaga agtactacag aatccaaggc tgcagaaaca acaacaagcg 1440 ttacatcaag gctgcaaact tctattaa 1468