Abstract:
A novel strain of  Lactobacillus rhamnosus  is disclosed, which is phylogenetically distinct from the published strains in the species and exhibits excellent probiotic properties. The medical and nutritional uses of the bacterial strain are also disclosed.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to a novel lactic acid-forming microorganism which exhibits excellent probiotic properties. The present invention also relates to the uses of the novel microorganism as a probiotic in food, beverage, animal feed and/or dietary supplement compositions, and as a medicament in controlling the colonization of undesirable intestinal microorganisms in the alimentary tract of a mammal.  
         [0003]     2. Description of the Related Art  
         [0004]     The oral administration of large numbers of  Lactobacillus rhamnosus , such as  L . ( casei  subsp.)  rhamnosus  GG (ATCC 53103), to a mammal has been found helpful to maintain or even enhance the healthy state of the mammal. It is believed that  L. rhamnosus , when ingested, would colonize transiently on the intestinal mucosa, which results in inhibition of the growth of pathogenic bacteria and viruses (such as rotavirus), stabilization of gut permeability, and suppression of allergic reactions in food hypersensitivity. The bacterium is particularly effective in alleviating the symptoms of gastroenteric disorders, such as diarrhea, by eliciting nonspecific humoral immune response in hosts.  
         [0005]      L. rhamnosus , reported in 1989 as a new species derived from  L. casei , shares similar phenotypes with two other members of the  Lactobacillus  genus, i.e.,  L. casei  and  L. paracasei . The three species can be further distinguished in terms of the differences in the genes encoding ribosomal RNAs. Approaches have been conducted based on this finding. For example, Rodtong et al. recognized the species-uniqueness of 16S rDNA and developed a ribotyping process to differentiate  Lactobacillus  strains (Rodtong, S. and Tannock, G. W. (1993)  Applied and Environmental Microbiology  59: 3480-3484). Taking advantage of the convenience and effectiveness of polymerase chain reaction (PCR), Ward et al. and Alander et al., on the other hand, used different sets of primers to identify  L. rhamnosus  based on the sequence polymorphism of 16S rDNA (Ward, L. J. H., and Timmins, M. J. (1999)  Letters in Applied Microbiology  29: 90-92; and Alander, M. et al.,  Applied and Environmental Microbiology  65: 351-354).  
         [0006]     According to the present invention, the inventor has identified a novel strain of  L. rhamnosus  (hereinafter referred to as strain Tcell-1) which is phylogenetically distinct from the published strains in the species and exhibits excellent probiotic properties.  
       SUMMARY OF THE INVENTION  
       [0007]     It is a primary object of the present invention to provide a novel strain of  L. rhamnosus . In the experiments performed in the invention, the inventor has characterized the phylogenetic distinction of the bacterial strain and demonstrated the desired probiotic properties thereof.  
         [0008]     Another object of the present invention is to provide a composition containing the bacterium strain according to the invention and a suitable excipient for the manufacture of foodstuffs, such as beverages, food, animal feed, and dietary supplements.  
         [0009]     Still another object of the present invention is to provide a pharmaceutical composition comprising the bacterium strain according to the invention, as well as to provide a method for the treatment or prophylaxis of gastroenteric disorders in a subject by administering such a composition to the subject. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]     These and other features and advantages of this invention will become apparent from the following detailed description of this invention, with reference to the accompanying drawings, in which:  
         [0011]     FIGS.  1 A-C demonstrate the enteroscopic sampling from the upper jejunum and rectum tissues of a volunteer;  
         [0012]      FIG. 2  is a fermentation profile of the bacterial strain according to the present invention;  
         [0013]      FIG. 3A  is a restriction map of the chromosomal DNA from the bacterial strain according to this invention;  
         [0014]      FIG. 3B  shows the result of Southern analysis of  FIG. 3A  using  E. coli  MRE600 16S+23S rDNA as the probe;  
         [0015]      FIG. 4  shows the result of PCR analysis using the primers designed by Ward &amp; Timmins, in which the DNA extracted from the bacterial strain according to the invention (lane 2) and water (lane 3; as a negative control) was subjected to PCR;  
         [0016]      FIG. 5  shows the result of PCR analysis using the primers designed by Alander et al., in which two sets of the species-specific primers, rham-rham2 (lanes 2-3) and rham-casei (lanes 4-5) were used in the PCR; and 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0017]     In accordance with the present invention, a strain of  L. rhamnosus  was isolated from the intestinal specimens donated by domestic volunteers. In a preliminary process, the microorganisms from the specimens were screened by a series of selective media, among which MRS agar medium and Rogosa SL agar medium exclusively allow the proliferation of  Lactobacillus . The bacteria selected according to the above procedure were subjected to a four-step screening strategy for identifying  L. rhamnosus:  
        Step 1: fermentation patterning using an API 50CHL kit (BioM&#39;erieux, Lyon, France);     Step 2: ribotyping according to the method described in Rodtong et al. (supra), in which the total DNAs extracted from the microorganisms were treated with restriction enzymes EcoRI, BclI, BglII or HindIII and detected by the rDNA probe of  Escherichia coli  subsequent to Southern blotting, so that the restriction fragment fingerprints of the suspected microorganisms can be obtained and compared with those derived from the  L. rhamnosus  DNA;     Step 3: PCR analysis according to the method described in Ward et al. (supra), in which a universal primer Y 2  (5′-CCCAC TGCTG CCTCC CGTAG GAGT-3′) and a species-specific primer rham (5′-TGCAT CTTGA TTTAA TTTTG-3′) were used in the reaction such that a major product of 290 bp will be produced when the chromosomal DNA of  L. rhamnosus  appears in the reaction mixture; and     Step 4: PCR analysis according to the method described in Alander et al. (supra), in which a pair of species-specific primers, rham (as indicated in Step 3) and rham2 (5′-CCGTC AATTC CTTTG AGTTT-3′), will amplify a specific product of 863 bp in the presence of  L. rhamnosus  DNA.        
 
         [0022]     Accordingly, the bacterial strain according to this invention was identified to belong to the species  L. rhamnosus . However, sequencing of the 863 bp product obtained in the Step 4 leads to a surprising finding that the bacterial strain according to this invention is phylogenetically distinct from all the published strains in the species  L. rhamnosus.    
         [0023]     The bacterial strain thus identified was designated as “ Lactobacillus rhamnosus  Tcell-1” and was deposited in the Culture Collection and Research Center (CCRC) of the Food Industry Research and Development Institute (FIRDI), Hsinchu, Taiwan, R.O.C. under the accession number CCRC 910145 (on Apr. 14, 2000). The bacterium was also deposited at the American Type Culture Center (ATCC) with accession number PTA-2406 on Aug. 22, 2000 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.  
         [0024]     Further studies concerning the probiotic properties of the strain Tcell-1 were also conducted. The results reveal that the bacterial strain according to the present invention can survive and grow well even in the stringent environment that an ingested bacterium would encounter in the gastrointestinal tracts, including extremely acidic pH and a high level of bile. The ability of  L. rhamnosus  Tcell-1 to resist certain antibiotics is apparently beneficial to administration of the bacterium to a subject who is required to take antibiotics. The superiority of the bacterial strain is further reinforced by its capability of inhibiting the growth of various enterobacteria.  
         [0025]     In view of the advantageous properties mentioned above, the bacterial strain according to the present invention is suitable for acting as a probiotic. According to the present invention, the strain Tcell-1 can be formulated into a broad variety of edible materials, including beverages, such as fluid milk, fermented milk, yogurts, fruit juices and sports drinks; food, such as ice cream, cheese and snacks; animal feed; dietary supplements; and infant formulas. Apparently, it is appreciable to those skilled in the art that the bacterial strain of this invention can be formulated in any suitable form by conventional methods for human or non-human animal&#39;s uptake. More preferably, the bacterial strain of this invention is formulated into the edible material in combination with other probiotic organisms, such as  L. acidophilus, L. brevis, L. casei, L. plantarum, L. salivarius, L. bifidus, L. bulgaricus, L. causasicus, Streptococcus lactis  and other strains of  L. rhamnosus , or a combination thereof. In addition,  L. rhamnosus  Tcell-1 is preferably formulated together with lactosucrose, chitin, chitosan, manitol, yogurt powder or a combination thereof.  
         [0026]      L. rhamnosus  Tcell-1 can also be used alone or with other active ingredients as a medicament in controlling the colonization of undesirable intestinal microorganisms in the alimentary tract of a mammal, to alleviate the conditions caused thereby. The composition can be formulated in solution, emulsion, powder, tablet, capsule or other adequate forms for oral administration.  
         [0027]     The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.  
       THE PREFERRED EMBODIMENTS OF THE INVENTION  
     Example 1  
     Isolation of  L. rhamnosus  Tcell-1  
       [0028]     Six healthy adults, aged from 25-45 and having no addiction to alcohol or smoking or chronic use of a drug, participated voluntarily in this experiment. None of them are vegetarians nor have abnormal dietary habit. The voluntary donors were subjected to fasting for 12 hours before enteroscopic sampling. Three biopsy specimens, each about 2 mm 2  in size, were picked up from different sites in the upper jejunum and rectum of each donor (FIGS.  1 A-C). The tissue specimens were then washed with physical saline (0.9% NaCl in distilled water) and stored in an ice-cold storage solution (0.9% NaCl, 0.1% Peptone, 0.1% Tween-80 and 0.02% Cysteine) for further analysis. The specimens were treated in an ultrasonic bath for 5 minutes and agitated vigorously for an additional 2 minutes. The obtained suspensions were undiluted or diluted in five- or ten-fold, and aliquots of the preparations were spread on the following solid media to obtain the profiles of enterobacteria contained therein (see also Johansson et al.,  Applied and Environmental Microbiology  59(1): 15-20).  
         [0029]     1. Brain heart infusion agar (purchased from Difco) which is an enriched medium for aerobically or anaerobically culturing the entire population of enterobacteria at 37° C. for 3 days;  
         [0030]     2. MRS agar (Difco) for anaerobically culturing  Lactobacillus  at 37° C. for 5 days;  
         [0031]     3. Phenol ethanol agar (Difco) for aerobically or anaerobically culturing the Gram(+) bacteria at 37° C. for 3 days;  
         [0032]     4. Azide blood agar (purchased from Oxoid) for aerobically culturing  Streptococcus  at 37° C. for 2 days;  
         [0033]     5. Slanetz-Bartley agar (Oxoid) for anaerobically culturing  Enterococcus  at 37° C. for 2 days;  
         [0034]     6. Violet red bile glucose agar (Oxoid) for culturing  Enterobacteriaceae  at 37° C. for 2 days;  
         [0035]     7. Rogosa SL agar (Difco) for anaerobically culturing  Lactobacillus  at 37° C. for 5 days; and  
         [0036]     8. Reinforced clostridial agar (Difco) for anaerobically culturing  L. bifidus  at 37° C. for 5 days.  
         [0037]     The results are shown in Table 1.  
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           TABLE 1                           In the upper jejunum specimens:            Name   Medium †              of the   BHIA   MRS   PEA   ABA   SBA            Donor   1x ‡     5x   10x   1x   5x   10x   1x   5x   10x   1x   5x   10x   1x   5x   10x                    JF   3   0   0   0   0   0   0   0   0   1   0   0   0   0   0       JS   M §     30   1   0   0   0   0   0   0   M   26   0   52   6   3       HK   0   0   0   0   0   0   0   0   0   0   0   6   0   0   0       V   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0       B   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0       RG   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0                    Name   Medium            of the   VRBGA   RA   RCA            Donor   1x   5x   10x   1x   5x   10x   1x   5x   10x                    JF   0   0   0   0   0   0   M   0   0       JS   0   0   0   0   0   0   M   0   21       HK   0   0   0   0   0   0   0   0   0       V   0   0   0   0   0   0   0   0   0       B   0   0   0   0   0   0   0   0   0       RG   0   0   0   0   0   0   0   0   0                    In the rectum specimens:            Name   Medium            of the   BHIA   MRS   PEA   ABA   SBA            Donor   1x   5x   10x   1x   5x   10x   1x   5x   10x   1x   5x   10x   1x   5x   10x                    JF   M   47   6   M   23   1   0   9   0   M   1   0   28   0   0       JS   M   15   1   M   8   0   0   12   0   M   0   1   0   1   0       HK   M   1   0   0   0   0   0   0   0   12   0   0   0   0   0       V   M   0   0   0   0   0   0   0   0   30   0   0   0   0   0       B   M   28   11   M   67   3   0   10   0   M   14   0   M   4   4       RG   M   0   0   0       0   0   0   0   1   0   0   0   0   0                    Name   Medium            of the   VRBGA   RA   RCA            Donor   1x   5x   10x   1x   5x   10x   1x   5x   10x               JF   M   M   M   0   0   0   M   M   M       JS   M   15   0   0   0   0   M   M   0       HK   0   0   0   0   0   0   M   0   0       V   0   0   0   0   0   0   M   0   0       B   M   M   M   M   0   0   M   M   M       RG   0   0   0   0   0   0   M   0   0                   † The abbreviation BHIA represents brain heart agar; MRS represents MRS agar; PEA represents phenol ethanol agar; ABA represents azide blood agar; SBA represents Slanetz-Bartley agar; VRBGA represents violet red bile glucose agar; RA represents Rogosa SL agar; and RCA represents reinforced clostridial agar.              ‡ 1X, 5X and 10X are the dilution folds of the bacterial suspensions.              § The letter M indicates that the number of bacterial colonies on the medium plate is higher than 100.             
 
         [0038]     As shown in Table 1, the profiles of enterobacteria in the upper jejunum and rectum specimens are quite different.  
       Example 2  
     Isolation of  Lactobacillus    
       [0039]     From the MRS and Rogosa SL agar media in Example 1, 200 colonies were picked up randomly and transferred separately to fresh MRS agar media containing 1% CaCO 3 . After incubation, the colonies surrounded by clear zones were picked up, and each of them was transferred to a basal MRS agar medium supplemented with 1% rhamnose and 0.05% chlorophenol red. Finally, the yellowish colonies, presumably constituted by  Lactobacillus , were picked up and further transferred to fresh MRS broth and incubated anaerobically at 37° C. for 2 days for further analysis.  
       Example 3  
     Identification of  L. rhamnosus  Tcell-1 as a New Strain  
       [heading-0040]     (a) Fermentation Pattern  
         [0041]     The  Lactobacillus  broth prepared in Example 2 was precipitated, washed with distilled water and resuspended in a defined amount of distilled water. The bacterial suspensions thus obtained were investigated using an API 50CHL kit according to the protocol provided by the manufacturer. Upon this procedure, a strain of  L. rhamnosus  was identified based on the fermentation pattern specific to the species ( FIG. 2 ) and designated as  L. rhamnosus  Tcell-1.  
         [0042]     Total DNA of the strain Tcell-1 was prepared from a 3 ml culture growing in the mid-log phase according to a conventional method described by Sambrook et al. (Sambrook, J. et al.,  Molecular Cloning, A Laboratory Manual,  2 nd  ed., Cold Spring Harbor Laboratory Press, 1989), and resuspended in 50 μl of TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0). The DNA solution thus obtained was used in the following analyses for further investigation of the strain Tcell-1.  
         [heading-0043]     (b) Ribotyping Analysis  
         [0044]     10 μl aliquots of the DNA were digested by restriction endonucleases, EcoRI, BclI, BglII and HindIII, respectively, for 3 hours. The digested products were loaded into the wells of a 0.8% agarose gel, and electrophoresis was carried out at 5 V/cm for 2 hours. The gel was then stained with ethidium bromide, and an image of the gel was obtained as shown in  FIG. 3A . The DNAs on the gel were denatured and transferred to a nylon-based membrane (Hybond-N + , Amersham) as described by Sambrook et al. (supra). To prepare the probe for Southern analysis, 1 μl of  E. coli  MRE600 16S+23S rRNA (purchased from Boehringer Mannheim) was used as the template which was amplified via incorporation of [α- 32 P] dCTP by AMV reverse transcriptase (Bethesda Research Laboratories) and random primers. Blots were hybridized at 68° C. for 16-24 hours in a hybridization solution containing 5×SSC, 1× Denhardt&#39;s solution, 1% SDS and 100 mg/ml of Harpin sperm DNA, washed properly to enhance the signal-to-noise ratio, and subjected to autoradiography. As shown in  FIG. 3B , the ribotype of the strain Tcell-1 was in perfect agreement with the typical pattern of  L. rhamnosus  as described by Rodtong et al. (supra).  
         [heading-0045]     (c) PCR Analysis Using the Ward &amp; Timmins&#39; Primers  
         [0046]     To an 1 ml eppendorf, 1 μl of Tcell-1 DNA harvested in Example 3(a), 1 μl of primer Y 2 , 1 μl of primer rham, 0.5 μl of DynaZymeII (Finnzymes Oy) and each dNTP (dATP, dTTP, dCTP and dGTP) at 100 μM were added. The reaction mixture was added with distilled water to a final volume of 50 μl and further overlaid with mineral oil. The reaction mixture was placed in a GeneAmp® PCR System 2400 thermocycler (Perkin Elmer) and thermocycled under the following conditions:  
                                                       Initial condition:   94° C. for 3 min.               45° C. for 45 sec.               72° C. for 1 min.           Thermocycling:   94° C. for 45 sec.               45° C. for 45 sec.               72° C. for 1 min.           Number of thermocycles:   30           Chain extension:   94° C. for 45 sec.               45° C. for 45 sec.               72° C. for 5 min.                      
 
         [0047]     Following thermocycling, the amplified products were separated on a 0.2% agarose gel. The gel was stained with ethidium bromide, and a major amplicon of 290 bp was observed on the gel under a UV light source ( FIG. 4 )  
         [heading-0048]     (d) PCR Analysis Using the Alander&#39;s Primers  
         [0049]     The PCR in Example 3 (c) was repeated except that the Ward &amp; Timmins&#39; primers were replaced with the rham and rham2 primers designed by Alander et al. (supra). The electrophoresis analysis on a 0.2% agarose gel revealed that the amplified products contain a major band of 863 bp ( FIG. 5 ), which, as described above, was reported to be a critical indicator to identify  L. rhamnosus.    
         [0050]     The experiments conducted in Examples 3(a)-(d) conclude that the biochemical and genetic traits of the strain Tcell-1 matched with those considered belonging to species  L. rhamnosus.    
         [heading-0051]     (e) Differentiation of Strain Tcell-1 from other  L. rhamnosus  Strains  
         [0052]     Using TOPO TA cloning™ kit (Invitrogen), the 863 bp product obtained in Example 3(d) was cloned into a pCR-TOPO™ vector according to the protocol provided by the manufacturer. The resultant plasmid was then introduced into TOP10 One Shot™ electrocompetent cells (Invitrogen) by electroporation. Following proliferation of the transformants in a selective medium, the plasmid was harvested and the 863 bp insert was sequenced.  
         [0053]     The sequence was used as a query sequence and searched against a nucleotide sequence database in the GenBank (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?CMD=search&amp;DB=nucleotide). The sequence alignment shown in Table 2 suggests that the strain Tcell-1 is phylogenetically distinct from all the six  L. rhamnosus  strains available in the GenBank based on the 16S rDNA sequences. The Tcell-1 DNA sequence shown in Table 2, which is 776 bp in size, was designated as SEQ ID No.1.  
                                 TABLE 2                           symbol comparison table: genetiq.dat; gap penalty: 4                            1       10        20        30        40        50                   TCELL1    TATACACTGGTACCTCCCTAAGTGGGATACATTGAAACAATCTATCCGCATAATCAAGA                              *** ***            *   *     * *    * *  *  *               AF21761      TTGTACACACCGCCC.GTCACACCATGAGAGTTTGTAACA...CCCGAAGCCGGTG                      *************** ************************   *************               E08782     CTTGTACACACCGCCC.GTCACACCATGAGAGTTTGTAACA...CCCGAAGCCGGTG                      *   *   * *   * *  *  *     **       ***   * **** *               AF18273   CCTTTCTAAGGAAACAGACTGAAAGTCTGACGGAAACCTGCACA...CACGAAACTTTGT                                          *  * *** ****     **               A61362                    CTAAGGAAACAGACTGAAAGTCTGACG................                                    ***************************               U32966                    CTAAGGAAACAGACTGAAAGTCTGACG................                                      *************************               AF12120                      AAGGAAACAGACTGAAAGTCTGACG................                                      1       10        20               consens               A C  CC A GA ACAGAC GAAA TCT AC    C C  A                   1       10        20        30        40        50        60                           70        80        90       100       110               TCELL1   CCGCATGTCTTGCTAAGATGCGTAACTATCGCTTTGGATGACCCCGCGTATAGCTAGTTG                    ** *       *     *  * * * *              **  *  *               AF21761   GCGTAA......CCCTTTTAGGGAGCGA.............GCCGTCTAAGGTGGGACAA                   ******      ** ***  **                    *     * *  **   **               E08782   GCGTAA......CCTTTTAGGGAGCGAG.............CCGTCTAAGGTGGGACAAA                                  **        *                        *               AF18273   TTAGTTTTGAGGGGATTACCCTCAAGCACCC.........TAGCGGGTGCGACTTTGTTC               A61362   .............................................................               U32966   .............................................................               AF12120   .............................................................               consens                           70        80        90       100       110       120                           130       140       150       160       170               TCELL1   TAAGTAACGCTCACCAAGCAATGATGCTAGCCAACTAAGTTGATCGCCACATTGGACTAA                       * * * * *    * **  * *    *  *  *       ** *    *   **               AF21761   ATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCC                       *  **   *      *****************************************               E08782   TGATTAGGGTGAAGTCGT.AACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCC                   *    *   ** *              *    *   *   **     *     *   *               AF18273   TTTGAAAACTGGATATCATTGTTGTAAATGTTTTAAATTGCCGAGAACACAGGCTATTTG               A61362   ............................................................               U32966   ............................................................               AF12120   ............................................................               consens               A                       A          C         T                           130       140       150       160       170       180                             190       200       210       220       230               TCELL1   ACACGGCC.CAAACTCTACGGAGGCAGCAGTAGGAATCTTCCACAATGGACGCAAGTCTG                      *      ****   ** **                   *  **        *  *               AF21761   TTTCTAAG.GAAACAG.ACTGAAAGTCTGA...........CGGAAACCTGCACACACGA                   ******** ******* *************           *******************               E08782   TTTCTAAG.GAAACAG.ACTGAAAGTCTGA...........CGGAAACCTGCACACACGA                   * *        ** *  *   *    *                ** * * * **  ** * *               AF18273   TATGAGTTTCTAATAATAGAAATTCGCAT............CGCATAACCGCTGACGCAA                                                               * * * **  ** * *               A61362   ...........................................GAAACCTGCACACACGA                                                              *****************               U32966   ...........................................GAAACCTGCACACACGA                                                              *****************               AF12120   ...........................................GAAACCTGCACACACGA                                                                 30        40               consens              AA    A   A                   C GAAACCTGCACACACGA                          190       200       210       220       230       240                            250       260       270       280       290               TCELL1   ATGGAGCAACGCCGCGTGACTGAAGAAGGCTTTCGGGGCGTAAAACTCTGTTGTTGGAGA                   *                    **  *            *       *         **               AF21761   AACTTTGTTTAGTTTTGAGGGGATTACCCTCAAGCACCCTAGCGGGTG.......CGACT                   ************************ **************** ******       *****               E08782   AACTTTGTTTAGTTTTGAGGGGATCACCCTCAAGCACCCTAACGGGTG.......CGACT                     *  *      *         *   * * *            **          ***               AF18273   GTCAGTACAGGTTAAGTTACAAAGGGCGCACGGTGGATGCCTTGGCACTAGGAGCCGATG                     *  *      *         *   * * *            **               A61362   AACTTTGTTTAGTTTTGAGGGGATTACCCTCAAGCACCCTAGCGGGTG............                   ************************************************               U32966   AACTTTGTTTAGTTTTGAGGGGATTACCCTCAAGCACCCTAGCGGGTG............                   ************************************************               AF12120   AACTTTGTTTAGTTTTGAGGGGATTACCCTCAAGCACCCTAGCGGGTG............                         50        60        70        80        90               consens   AACTTTGTTTAGTTTTGAGGGGATTACCCTCAAGCACCCTAGCGGGTG        GA                          250       260       270       280       290       300                            310       320       330       340       350               TCELL1   AGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCT                                    *****                **      *     * *   *               AF21761   TTGT.....TCTTTGAAAACTGGATATCATTGTTGTAAATGTTTTAAATTGCCGAGAACA                   ****     ************************  *** *********************               E08782   TTGT.....TCTTTGAAAACTGGATATCATTGTATTAATTGTTTTAAATTGCCGAGAACA                     *      **   *    *     **   *   **   **      *   *  **               AF18273   AAGGACGGAACTAATACCGATATGCTTCGGGGAGCTATAAGTAAGCTTTGATCCGGAGAT                                                     ** *             *    *               A61362   ...............................CGACTTTGTTCTTTGAAAACTGGATATCA                                                  *****************************               U32966   ...............................CGACTTTGTTCTTTGAAAACTGGATATCA                                                  *****************************               AF12120   ...............................CGACTTTGTTCTTTGAAAACTGGATATCA                                                         100       110               consens                       T           GACT T T CTTT AAAA TCGA A CA                          310       320       330       340       350       360                            370       380       390       400       410               TCELL1   AACTCAGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGG                    *     *               *  *               AF21761   CAGCGTATTTGTATGAGTTTCTAATAA..............................TA                   *****   **        **     **                                *               E08782   CAGCG.TATTTGTATGAGTTTCTGAAA..............................AA                      **      * *      *    *               AF18273   TTCCGAATGGGGGAACCCAGTACACATCAGTGTATT.....................GC                   **               A61362   TTGTTGTAA.................................................AT                   *********                                                 **               U32966   TTGTTGTAA.................................................AT                   *********                                                 **               AF12120   TTGTTGTAA.................................................AT                                                                            130               consens   TTGT  TA                                                  A                          370       380       390       400       410       420                            430       440       450       460       470               TCELL1   CGTAAAGCGAGCGCAGGCGGTTTTTTAACTCTGATGTGAAAGCCCTCGGCTTAACCGAGG                      *     * ****                        ** * **      *  *               AF21761   GAAATTCGCATCGCA.......................TAACCGCTGACGCAAGTC....                   ***************                       ******************               E08782   GAAATTCGCATCGCA.......................TAACCGCTGACGCAAGTC....                            *   **                       ** *   *               AF18273   CTGCAAGTGAATACA.......................TAGCTTGTTGGCGGCAGACGCG                    *   *        *               A61362   GTTTTAAATTGCCGA................................................                   ***************               U32966   GTTTTAAATTGCCGA................................................                   ***************               AF12120   GTTTTAAATTGCCGA................................................                          140               consens   GTT TA   AGC CA                       A     T                          430       440       450       460       470       480                            490       500       510       520       530               TCELL1   AAGTGCATCGGAAACTGGGAAACTTGAGTACAGAAGAGGACAGTGGAACTCCATGTGTAG                   *    **    *       *      *     ** *          *    *               AF21761   AGTACCAGGTAAGTTACAAAGGGCGCACGGTGGATGCCTTGGCACTAGGAGC.......C                   *****  * *******************************************       *               E08782   AGTACAGGTTAAGTTACAAAGGGCGCACGGTGGATGCCTTGGCACTAGGAGC.......C                    * *   *  *  *  **     **** *  *          * * *    *       *               AF18273   GGGAACTGAAACATCTCAGTACCCGCAGGAAGAGAAAGAAAACTCGATTCCCATAGTAGC                     ****  *      *             ** *  *  **  * *               A61362   ..GAACACAGCGTATTTGTATGAGTTTCTAATAATAGAAATTCGCATC............                     **********************************************               U32966   ..GAACACAGCGTATTTGTATGAGTTTCTAATAATAGAAATTCGCATC............                     **********************************************               AF12120   ..GAACACAGCGTATTTGTATGAGTTTCTAATAATAGAAATTCGCATC............                       150       160       170       180       190               consens   GAACA AG   ATT G A G  T ACTAA AATA  AA  C C A    C                          490       500       510       520       530       540                            550       560       570       580       590               TCELL1   CGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTA                        *     * *   ***  **    *  * * *    *                  *               AF21761   GATGAAGGACGGAACTAATACCGATATGCTTCGGGGAGCTATA................A                   *******************************************                *               E08782   GATGAAGGACGGAACTAATACCGATATGCTTCGGGGAGCTATA................A                   *   **  ** *     *   ** * *       *   ***               AF18273   GGCGAGCGAAGTGGGAAGAGCCCAAACCGAGAAGCTTGCTTCTCGGGGTTGTAGGACTGG                                * *  * * *  **  *      * *               A61362   .............GCATAACCGCTGACGCAAGTCAGTACAGG                                *****************************               U32966   .............GCATAACCGCTGACGCAAGTCAGTACAGG                                *****************************               AF12120   .............GCATAACCGCTGACGCAAGTCAGTACA                                      200       210       218               consens                GCATAA CGCA ACGCA   G GT CA                          550       560       570       580       590       600                            610       620       630       640       650               TCELL1   ACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCAT                        *  ***  *    *               *       *  **** *      **               AF21761   GTAAGCTTTGATCCGGAGATT...........TCCGAATGGGGGAACCCAGTA....CAC                   *********************           *********************    ***               E08782   GTAAGCTTTGATCCGGAGATT...........TCCGAATGGGGGAACCCAGTA....CAC                     *         **  ** *             *  * * *  *    * * *      *               AF18273   ACATTGGAGTTACCAAAGTTCG..........ACGTAGTCGAAGTCAGCTGGAAAGCTGC               A61362               U32966               AF12120               consens                C                                  C G                          610       620       630       640       650       660                            670       680       690       700       710               TCELL1   GCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCACTAACGC                     *          * ***  *    ** *    *  *                *  ****               AF21761   ATCAGTG.....TATTGCCTGCAAGTGAATACATAGCTTGT......TGGCGGCAGACGC                   *******       *  *    **   *   *    *              * *    *               E08782   ATCAGTGTGTTGCTTGTCAGTGAATACATAGCTGGCCGGCG......GCCAGACGCGGGG                     **  *          *  ** * **  * * *                  * *               AF18273   GCCATAGAAGGTGAAAGCCCTGTAAACGAAACGGCGGACTC....TCCGTCCAGGATCCT               A61362               U32966               AF12120               consens     C              C                          670       680       690       700       710       720                            730       740       750       760       770   776               TCELL1   ATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGG                       * *                        **   *  **               AF21761   GGGGAACT......................GAAACATCTAAG                        *                         *   *  **               E08782   AACTGAAA......................CATCTAAGTACCCGGA                    * *                           *     * *     *               AF18273   GAGTACGGCGGAACACGTGAAATTCCGTCGGAATCCGGGAGGACCATCT               A61362               U32966               AF12120               consens                                  A       A                          730       740       750       760       770   778                  
 
       Example 4  
     Characterization of  L. rhamnosus  Tcell-1  
       [heading-0054]     (a) Tolerance of Acid  
         [0055]     MRS liquid media were prepared at pH 2, 3, 4, 5 and 6, respectively, and supplemented with 0.3% bile salt. To 1.5 ml of each medium, 10 6  Tcell-1 cells were inoculated and incubated anaerobically at 37° C. Samples were collected at 0 and 4 hours after the inoculation, and the populations of the microorganisms in each culture were assessed with reference to the optical density at 620 nm. The results are shown in Table 3.  
                                                 TABLE 3                           Incubation   OD 620                  time   pH2   pH3   pH4   pH5   pH6                        0 hour   0.01   0.018   0.010   0.014   0.012           4 hour   0.01   0.018   0.016   0.025   0.030                      
 
         [0056]     As shown in Table 3, the growth rate of  L. rhamnosus  Tcell-1 remarkably reduced as the cultures were acidified to a pH at which the ingested substances would encounter in an animal stomach, i.e., a pH of below 3. Surprisingly, the cells incubated in such an acidic environment for 4 hours can still restore their normal growth if transferred to a fresh MRS medium at pH 6.0 (data not shown). The data indicate that  L. rhamnosus  Tcell-1 can tolerate the attack of gastric acid.  
         [heading-0057]     (b) Tolerance of Bile Salt  
         [0058]     Example 4(b) was repeated except that the MRS liquid media contained bile salts at concentrations of 0.1, 0.2, 0.3 and 0.4%, respectively, while the pH of the media was constantly set at 2.5. The results are shown in Table 4.  
                                             TABLE 4                           Incubation   OD 620                  time   0.1%   0.2%   0.3%   0.4%               0 hour   0.011   0.010   0.013   0.018       4 hour   0.028   0.025   0.023   0.032                  
 
         [0059]     From Table 4, it is demonstrated that the growth of  L. rhamnosus  Tcell-1 was sustained at a high level of bile.  
         [heading-0060]     (c) Resistance to Antibiotics  
         [0061]     2-3 ml aliquots of a bacterial suspension from an overnight culture of  L. rhamnosus  Tcell-1 were spread on MRS agar media added with 10 μg/ml of kanamycin, vancomycin, chloramphenicol or ampicillin. After incubation,  L. rhamnosus  Tcell-1, while its growth was attenuated in the media containing chloramphenicol or ampicillin, was found to be tolerant of kanamycin and vancomycin.  
         [heading-0062]     (d) Inhibition of the Colonization of Other Bacteria  
         [0063]     2-3 ml aliquots of a bacterial suspension from an overnight culture of  L. rhamnosus  Tcell-1 were spread on MRS agar media. Each of the plates was incubated at 30° C. for 22 hours, on which 7 ml of soft agar mixed with 100 μl suspension from one of the nine enterobacterial strains listed in Table 5 (purchased from the FIRDI) was poured.  
                           TABLE 5                                   Bacterium   Medium                             Enterobacter aerogenes     DIFCO 0001             Clostridium perfringens     brain heart infusion               (anaerobically cultured)             Klebsiella pneumoniae     DIFCO 0001             Yersinia enterocolitica     brain heart infusion             Listeria monocytogenes     brain heart infusion             Streptococcus mutans     brain heart infusion             Citrobacter freundii     DIFCO 0001             Shigella dysenteriae     DIFCO 0001             Yersinia ruckeri     DIFCO 0001                      
 
         [0064]     The obtained cultures were incubated for an additional 48 hours at 37° C. and observed with bare eyes. Based on the presence of inhibition rings around the colonies of  L. rhamnosus  Tcell-1, the inventor found that the strain Tcell-1 can significantly suppress the growth of  E. aerogenes, C. perfringens, L. monocytogenes, S. mutans  and  C. freundii . The results strongly suggest that  L. rhamnosus  Tcell-1 exhibits promising probiotic properties for controlling or inhibiting the colonization of the undesired bacteria in the bowel.  
       Example 5  
     Probiotic Formulations Containing  L. rhamnosus  Tcell-1  
       [0065]      L. rhamnosus  Tcell-1 can be utilized in various forms of foodstuffs, two examples of which are described as follows:  
         [heading-0066]     Formula 1:  
         [heading-0067]     Ten Strains of Lactic Acid-Forming Bacteria:  
         [none]    
       
         
           
               L. acidophilus, L. brevis, L. casei, L. plantarum, L. salivarius, L. bifidus, L. bulgaricus, L. causasicus, Streptococcus lactis  and  L. rhamnosus  Tcell-1; 
 
 Excipients: 
 
              lactosucrose oligo, manitol, chitin &amp; chitosan, yogurt powder; 
 
 Natural Condensates: 
 
              alfalfa, barley and wheat grass juice powder, pure soya lecithin, carrot juice powder, phosphatidyl choline, Hawaiian Spirulina pacifica, apple pectin powder, phosphatidyl inositol CGF chlorella, non-dairy probiotic culture: rhamnosus, acidophilus in a base of FOS, peace river bee pollen powder, stevia, freeze-dried mango, black currant, dandelion root extract 4:1, beetroot extract, Siberian ginseng extract 0.4%, pacific kelp 4:1 extract, artichoke 4:1 extract 2%, soya extract, bilberry extract 5:1, pineapple extract, cranberry juice extract 18:1, rosehip extract 4:1, lycopene, Milk Thistle Phytosome™, Ginkgo Biloba Phytosome™ and Grape Seed Phytosome™; 
 
 Other Ingredients: 
 
              Vitamin C, calcium, magnesium, zinc. 
 
 Formula 2: 
 
 Nine Strains of Lactic Acid-Forming Bacteria: 
 
               L. acidophilus, L. brevis, L. casei, L. plantarum, L. salivarius, L. bifidus, L. bulgaricus, L. causasicus  and  L. rhamnosus  Tcell-1; 
 
 Other Ingredients: 
 
              calcium lactate, lactosucrose oligo, lactose, dextrose, powered milk, vegetable oil and small amounts of an emulsifier and natural seasonings  
           
         
       
     
         [0080]     The formulation is coated on peanut and raisin granules to make up a probiotic healthy dessert.  
         [0081]     With this invention thus explained, it is apparent that numerous modifications and variations can be made without departing from the scope and spirit of this invention. It is therefore intended that this invention be limited only as indicated by the appended claims.