LEE CHIA-CHIA (CN)
LEE TING-YU (CN)
HSU HAN-YIN (CN)
WANG JIU-YAO (CN)
CN107308190A | 2017-11-03 | |||
US20100278795A1 | 2010-11-04 | |||
EP2223697A1 | 2010-09-01 | |||
CN111195267A | 2020-05-26 | |||
CN103314099A | 2013-09-18 |
WHAT IS CLAIMED IS: 1. An isolated strain of Lactobacillus delbrueckii subsp. lactis LDL557, which is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under an accession number DSM 33617. 2. A composition, comprising an isolated strain of Lactobacillus delbrueckii subsp. lactis LDL557 as claimed in Claim 1. 3. The composition as claimed in Claim 2, which is formulated as a food product. 4. The composition as claimed in Claim 2, which is formulated as a pharmaceutical composition. 5. A method for alleviating an inflammation-related disorder, comprising administering to a subject in need thereof a composition as claimed in Claim 2. 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 administered by a route selected from the group consisting of oral administration, parenteral administration, respiratory tract administration, and topical administration. 8. The method as claimed in Claim 5, 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. 9. Use of an isolated strain of Lactobacillus delbrueckii subsp. lactis LDL557 as claimed in Claim 1 in the manufacture of a medicament or a food product for alleviating an inflammation- related disorder in a subject. 10.The use as claimed in Claim 9, wherein the medicament is administered by a route selected from the group consisting of oral administration, parenteral administration, respiratory tract administration, and topical administration. 11.The use as claimed in Claim 9, 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. 12.An isolated strain of Lactobacillus delbrueckii subsp. lactis LDL557 for use in alleviating an inflammation-related disorder in a subject, wherein the isolated strain of Lactobacillus delbrueckii subsp. lactis LDL557 is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under an accession number DSM 33617. 13.The isolated strain for use as claimed in Claim 12, 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. |
Table 1 Example 2. Screening of LAB isolates having in vitro anti-allergic activity Materials: A. Preparation of heat-killed bacterial suspension of Lactobacillus paracasei 33 (LP33) A heat-killed bacterial suspension of Lactobacillus paracasei 33 (having a bacterial concentration of 10 9 CFU/mL) was prepared according to the procedures described in the abovementioned section B of Example 1. Methods: The in vitro anti-allergic activity was analyzed using a method slightly modified from that described by Lee J. et al. (2013), J. Microbiol. Biotechnol., 23:724-730. Briefly, the female BALB/c mice were divided into nine groups, including a normal control group, a pathological control group, a comparative group, and six experimental groups (i.e., experimental groups 1 to 6)(n=12 per group). The mice of the pathological control group, comparative group, and six experimental groups were intraperitoneally injected with the OVA emulsion prepared in section 4 of “General Experimental Materials” at a dose of 200 μL/mouse. The mice of the normal control group were intraperitoneally injected with PBS at a dose of 200 μL/mouse. The mice in each group was subjected to the once-a-week injection for a total period of 2 weeks. After the 2-week experimental period, the mice in each group were anesthetized using CO 2 , and were subsequently sacrificed. Thereafter, the spleen tissue was obtained from each mouse carcass, followed by grinding. Each group of the resultant milled spleen sample was incubated in a respective well of a 24-well culture plate containing a suitable amount of an RPMI 1640 medium (supplemented with 10% FBS), followed by cultivation in an incubator (37°C, 5% CO 2 ) for 24 hours. Subsequently, each group of the spleen cells thus obtained was incubated in a respective well of a 24- well culture plate containing 1 mL of an RPMI 1640 medium (supplemented with 10% FBS and 100 μg/mL OVA) at 2×10 6 cells/well, followed by cultivation in an incubator (37°C, 5% CO 2 ) for 48 hours. Afterwards, each of the cell cultures of the experimental groups 1 to 6 was added with 100 μL of a respective one of the heat-killed bacterial suspensions of LAB isolates 008, 221, 554, 556, 557, and 558 prepared in the abovementioned section B of Example 1. In addition, the cell culture of the comparative group was added with 100 μL of the heat-killed bacterial suspension of Lactobacillus paracasei 33, and the cell cultures of the normal control group and pathological control group received no treatment. The treating agents for all the groups are summarized in Table 2 below. Table 2 Each group was cultivated in an incubator (37°C, 5% CO 2 ) for 48 hours. After centrifugation at 3,000 rpm for 15 minutes, the resultant supernatant was collected, and was subjected to determination of IL- 4, IL-6, IL-13, and IL-17A contents using an IL-4 ELISA kit (Invitrogen, Cat. No. BMS613TEN), an IL-6 ELISA kit (Invitrogen, Cat. No. 88-7064-88), an IL- 13 ELISA kit (Invitrogen, Cat. No. 88-7137-86), and an IL-17A ELISA kit (Invitrogen, Cat. No. BMS6001TEN) in accordance with the manufacturer’s instructions. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Results: Referring to FIGS. 1 to 4, the contents of IL-4, IL-6, IL-13, and IL-17A determined in the experimental group 5 were apparently or significantly lower than those determined in the experimental groups 1 to 4 and 6, the comparative group, and the pathological control group, indicating that LAB isolate 557 had the best in vitro anti-allergic activity. Therefore, LAB isolate 557 showed more potential for development, and was subjected to characteristic analysis described below. Example 3. Characteristic analysis of LAB isolate 557 In order to identify the bacterial species of LAB isolate 557, the following preliminary characteristic determination, 16S rDNA sequence analysis, and carbohydrate fermentation profiling were conducted. A. Preliminary tests Items of the preliminary tests conducted for LAB isolate 557 include: gram staining, morphological observation, mobility, catalase test, growth under aerobic and anaerobic conditions, and ability to produce an endospore. The results of the aforesaid preliminary tests indicate that LAB isolate 557 is gram-positive, non- motile, catalase-negative, grows under anaerobic conditions, and non-endospore forming. B. 16S rDNA sequence analysis Genomic DNA of LAB isolate 557 was extracted using Genomic DNA Mini Kit (Geneaid Biotech Ltd., Cat. No. GB300). 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 474 bp.
Table 3 The resultant PCR product was subjected to 2% agarose gel electrophoresis analysis for molecular weight verification. Thereafter, the PCR product was verified by sequencing analysis which was entrusted to Genomics BioSci & Tech Co., Ltd., Taiwan, so as to obtain the 16S rDNA sequence (SEQ ID No: 3) of LAB isolate 557. Through comparison with the data in the NCBI's gene database, it was found that the 16S rDNA sequence of LAB isolate 557 is most homologous to that of Lactobacillus delbrueckii subsp. lactis. In view of the aforesaid experimental results, LAB isolate 557 of the present disclosure is identified as Lactobacillus delbrueckii subsp. lactis. In order to confirm whether Lactobacillus delbrueckii subsp. lactis strain LDL557 (i.e. LAB isolate 557) is a novel Lactobacillus delbrueckii subsp. lactis strain, the following experiment was conducted. B. Carbohydrate fermentation profiling The carbohydrate fermentation profile of Lactobacillus delbrueckii subsp. lactis strain LDL557 was determined using API® 50 CHL identification system (bioMérieux). The result is shown in Table 4 below. Table 4
Note:“+” indicates that Lactobacillus delbrueckii subsp. lactis strain LDL557 is capable of fermenting the carbohydrate tested to produce an acid, whereas “-” indicates that the strain has no such capability. The aforesaid result was subjected to comparison with the data in the APIWEB ™ on-line bacteria and yeast database, and it was found that the carbohydrate fermentation profile of Lactobacillus delbrueckii subsp. lactis strain LDL557 of the present disclosure has 84.9% identity to that of Lactobacillus delbrueckii subsp. lactis, suggesting that the Lactobacillus delbrueckii subsp. lactis strain LDL557 characterized thus far by the applicant is different from conventionally known strains of Lactobacillus delbrueckii subsp. lactis. Based on the aforementioned characterization results, the applicant believes that the Lactobacillus delbrueckii subsp. lactis strain LDL557 is a novel strain of Lactobacillus delbrueckii subsp. lactis. As such, Lactobacillus delbrueckii subsp. lactis strain LDL557 has been deposited at the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI), Taiwan under an accession number BCRC 910780 since May 17, 2017, and has also been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under an accession number DSM 33617 since August 10, 2020 in accordance with the Budapest Treaty. Example 4. Evaluation of the ability of Lactobacillus delbrueckii subsp. lactis LDL557 to stimulate secretion of IL-6 and IL-10 by macrophages A. Preparation of heat-killed bacterial suspension of Lactobacillus delbrueckii strain A respective one of Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure and eleven comparative strains of Lactobacillus delbrueckii subspecies screened by the applicant (as shown in Table 5) was subjected to the procedures described in the abovementioned section B of Example 1, so as to obtain twelve heat-killed bacterial suspensions (each of the bacterial suspensions had a bacterial concentration of 10 8 CFU/mL). The resultant heat-killed bacterial suspensions were subjected to the following experiments. Table 5 B. Determination of contents of IL-6 and IL-10 Human acute monocytic leukemia cell line THP-1 (BCRC 60430) was purchased from the BCRC of the FIRDI (Taiwan). THP-1 cells were incubated in a respective well of a 24-well culture plate containing 1 mL of an RPMI 1640 medium (supplemented with 10% FBS and 10 ng/mL phorbol 12-myristate-13-acetate (PMA)) at 5×10 5 cells/well, followed by cultivation in an incubator (37°C, 5% CO 2 ) for 48 hours, so as to induce differentiation of THP-1 cells into macrophages. Afterwards, the resultant cell cultures were divided into 12 groups, including one experimental group and eleven comparative groups (i.e., comparative groups 1 to 11). The culture medium in each well was removed. Subsequently, the cell culture of the experimental group was added with 900 μL of an RPMI 1640 medium and 100 μL of the heat-killed bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557. In addition, each of the cell cultures of the eleven comparative groups was added with 900 μL of an RPMI 1640 medium and 100 μL of a respective one of the eleven heat-killed bacterial suspensions of the comparative strains of Lactobacillus delbrueckii. Each group was cultivated in an incubator (37°C, 5% CO 2 ) for 24 hours. After centrifugation at 3,500 rpm for 15 minutes, the resultant supernatant was collected, and was then subjected to determination of IL-6 and IL-10 contents using an IL-6 ELISA kit (Cat. No. 88-7066-88, Invitrogen) and an IL-10 ELISA kit (Cat. No. 88-7106-88, Invitrogen) in accordance with the manufacturer’s instructions. Referring to FIGS. 5 and 6, the contents of IL-6 and IL-10 determined in the experimental group were higher than those determined in the comparative groups 1 to 11, indicating that the ability of Lactobacillus delbrueckii subsp. lactis LDL557 to stimulate secretion of IL-6 and IL-10 from macrophages is better than that of other known strains of Lactobacillus delbrueckii. Example 5. In vivo anti-allergy test on Lactobacillus delbrueckii subsp. lactis LDL557 A. Preparation of bacterial suspensions of Lactobacillus delbrueckii subsp. lactis LDL557 and Lactobacillus rhamnosus GG (LGG) A respective one of Lactobacillus delbrueckii subsp. lactis LDL557 and LGG was inoculated in MRS broth, and was then cultured at 37°C for 16 hours to 18 hours. After centrifugation at 5,000 rpm and 4°C for 10 minutes, the respective resultant cell pellet was collected, and was washed with phosphate-buffered saline (PBS), followed by a freeze-drying treatment, so as to obtain a freeze-dried powder of Lactobacillus delbrueckii subsp. lactis LDL557 and a freeze-dried powder of LGG. A respective one of the two freeze-dried powders was mixed with a suitable amount of a 0.85% physiological salt solution, so as to obtain a bacterial suspension having a bacterial concentration of 10 9 CFU/mL. The respective one of the resultant bacterial suspensions was used for the following experiments. B. Induction of allergy and administration of bacterial suspensions The induction of allergy was conducted using a method slightly modified from that described by Lee J. et al. (2013), supra. Briefly, the female BALB/c mice (6 weeks old, with a body weight of approximately 19-20 g) were divided into four groups, including a normal control group, a pathological control group, a comparative group, and an experimental group (n=6 per group). The mice of the pathological control group, comparative group, and experimental group were intraperitoneally injected with the OVA emulsion prepared in section 4 of “General Experimental Materials” at a dose of 200 μL/mouse. The mice of the normal control group were intraperitoneally injected with Imject™ alum adjuvant (Cat. No. 77161, Thermo Fisher Scientific) at a dose of 200 μL/mouse. On the 14 th day after the injection of the OVA emulsion or the Imject™ alum adjuvant, the mice in each group were injected once more in the same manner as described above. Thereafter, the mice of the experimental group were fed, via oral gavage, with the bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of this example at a dose of 9-10 log CFU/kg, and the mice of the comparative group were fed, via oral gavage, with the bacterial suspension of LGG prepared in section A of this example at a dose of 9-10 log CFU/kg. In addition, each of the mice of the normal control group and pathological control group was fed, via oral gavage, with 0.2 mL of a 0.85% sodium chloride solution. Each mouse was fed once daily for a 21-day treatment period. C. Determination of IgA content in fecal sample After the 21-day treatment period, all the mice were sacrificed by virtue of 95% CO 2 asphyxiation, and the fecal sample was obtained from each mouse carcass. A suitable amount of PBS was added to the respective fecal sample to reach a final concentration of 100 mg/mL. The respective diluted fecal sample was subjected to determination of IgA content using an IgA ELISA kit (Cat. No. E90-103, Bethyl Laboratories Inc.) according to the manufacturer’s instructions. Referring to FIG. 7, the IgA content determined in the experimental group was higher than those determined in the comparative group and the pathological control grou. This result suggests that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure is effective in inducing IgA secretion, and hence can exhibit anti-allergic activity. Example 6. In vivo anti-asthma test on Lactobacillus delbrueckii subsp. lactis LDL557 A. Preparation of emulsion containing Dermatophagoides pteronyssinus (DerP) DerP (Allergon, Sweden) was subjected to an ultrasonic vibration treatment. 50 ^g of the resultant Derp powder was dissolved in 200 μL of Imject™ Alum adjuvant (Cat. No. 77161, Thermo Fisher Scientific), so as to prepare a Derp emulsion. B. Induction of asthma and administration of bacterial suspensions The female BALB/c mice (6 weeks old, with a body weight of approximately 18-22 g) were divided into six groups, including a normal control group, a pathological control group, a comparative group, and three experimental groups (i.e., experimental groups 1 to 3)(n=12 per group). The mice of the experimental group 1 were fed, via oral gavage, with the bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 5 at a dose of 5×10 9 CFU/kg, the mice of the experimental group 2 were fed, via oral gavage, with the bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 5 at a dose of 5×10 10 CFU/kg, and the mice of the experimental group 3 were fed, via oral gavage, with the heat-killed bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 4 at a dose of 5×10 10 CFU/kg. In addition, the mice of the comparative group were fed, via oral gavage, with dexamethasone at a dose of 1 mg/kg, and each of the mice of the normal control group and pathological control group was fed, via oral gavage, with 0.2 mL of a 0.85% physiological salt solution. Each mouse was fed once daily for a 30-day treatment period. On the 14 th and 21 st days, after the treatment of the bacterial suspension, the physiological salt solution, or dexamethasone, the mice of the pathological control group, comparative group, and three experimental groups were intraperitoneally immunized with the Derp emulsion prepared in section A of this example at a dose of 100 μL/mouse. On the 22 nd day, after the treatment of the bacterial suspension, the physiological salt solution, or dexamethasone, the mice of the pathological control group, comparative group, and three experimental groups were intranasally instilled with the Derp emulsion prepared in section A of this example at a dose of 20 μL/mouse. Each mouse was intranasally instilled once daily for a total period of 5 days. On the 28 th day, after the treatment of the bacterial suspension, the physiological salt solution, or dexamethasone, the mice of the pathological control group, comparative group, and three experimental groups were intratracheally instilled with the Derp emulsion prepared in section A of this example at a dose of 40 μL/mouse, so as to induce asthma. In addition, the mice of the normal control group received no Derp emulsion . C. Determination of respiratory parameters After the 30-day treatment period, each mouse was anesthetized by virtue of Zoletil ® , followed by conducting tracheotomy. Thereafter, the respective mouse was intubated and ventilated using a FlexiVent instrument (SCIREQ Inc.), followed by causing methacholine-induced airflow obstruction in the respective mouse using increasing concentrations (0, 1, 2, 4, and 8 mg/mL) of aerosolized methacholine (Sigma-Aldrich, Cat. No. PHR1943). The respiratory system resistance (Rrs), respiratory system elastance (Ers), and tissue damping (G) of each mouse were determined at the different concentrations of aerosolized methacholine. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIGS. 8 to 10, the Rrs, Ers, and G determined in each of the experimental groups 1 to 3 were respectively lower than those determined in the pathological control group, indicating that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate airway obstruction. D. Determination of IL-10 content in bronchoalveolar lavage fluid (BALF) After completion of the lung function test as described in section C of this example, the respective mouse was sacrificed by virtue of 95% CO 2 asphyxiation. 2 mL of sterile saline was injected into the lung of the respective mouse using a bronchoscope, followed by collecting the BALF thus formed. After centrifugation at 300 g and 4°C for 15 minutes, the resultant supernatant was collected, and was used as a BALF sample. 100 μL of the respective BALF sample was added to a corresponding well of a 96-well culture plate, and was then subjected to determination of IL-10 content using an IL-10 ELISA kit (Cat. No. DY417, R&D Systems Inc.). The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIG. 11, the IL-10 contents determined in the experimental groups 1 to 3 were each significantly lower than that determined in the pathological control group, indicating that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate airway inflammation. E. Histopathologic analysis After completion of the determination of IL-10 content as described in section D of this example, the lung tissue was obtained from each mouse carcass, followed by fixation with a 10% paraformaldehyde solution (in PBS) at room temperature for 48 hours. The fixed tissue sample was then embedded with paraffin, followed by slicing to obtain a tissue section having a thickness of 5 μm. The tissue section was stained with hematoxylin and eosin (H&E) using a staining protocol well-known to those skilled in the art, and was observed under an Olympus upright microscope equipped with a transmitted light source at 100x magnification. 144 areas of the respective tissue section were randomly selected and photographed, and the cellular infiltration index of each group was determined generally according to the method described in Sung J.E. et al. (2017), Int. J. Mol. Med., 40:1365-1376. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIG. 12, the cellular infiltration indices determined in the experimental groups 1 to 3 were each significantly lower than that determined in the pathological control group, indicating that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate lung infiltration. Summarizing the above test results, it is clear that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure is effective in alleviating asthma. Example 7. In vivo anti-colitis test on Lactobacillus delbrueckii subsp. lactis LDL557 A. Induction of colitis and administration of bacterial suspensions The induction of colitis was conducted using a method slightly modified from that described by Im E. et al. (2009), J. Nutr., 139:1848-1854. The female C57BL/6 mice (7 weeks old, with a body weight of approximately 19-21 g) were divided into four groups, including a normal control group, a pathological control group, and two experimental groups (i.e., experimental groups 1 to 2)(n=8 per group). The mice of the experimental group 1 were fed, via oral gavage, with the bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 5 at a dose of 9-10 log CFU/kg, and the mice of the experimental group 2 were fed, via oral gavage, with the heat-killed bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 4 at a dose of 9-10 log CFU/kg. In addition,each of the mice of the normal control group and pathological control group was fed, via oral gavage, with 0.2 mL of a 0.85% physiological salt solution. Each mouse was fed once daily for a 21-day treatment period. Prior to the start of the treatment of the bacterial suspension or the physiological salt solution on the 14 th , 16 th , 18 th , and 20 th days, the drinking water for the mice of the pathological control group and two experimental groups was replaced with water containing 2% dextran sodium sulfate (DSS) (MP biomedical, Cat. No. 9011-18-1), so as to induce the occurrence of colitis. The drinking water provided for the mice of the normal control group throughout the treatment period contained no DSS. B. Analysis of intestinal permeability On the 20 th day, after the treatment of the bacterial suspension or the physiological salt solution, the respective mouse was subjected to determination of intestinal permeability using a method slightly modified from that described by Woo J.K. et al. (2016), BMC Complement Altern. Med., doi: 10.1186/s12906-016-1479-0. Briefly, the mice of each group were fed, via oral gavage, with a 0.85% physiological salt solution containing 2 mg/mL fluorescein isothiocyanate-dextran 4000 (FITC-D4000) at a dose of 10 mL/kg. At the 4 th hour after the administration of FITC-D4000, a blood sample was collected from the facial vein of each mouse through puncture, and was then subjected to centrifugation at 10,000 rpm and 4°C for 5 minutes. The serum sample thus obtained was subjected to fluorescence spectroscopy. The fluorescence intensity of each group was detected using a fluorescence spectrometer (Hitachi F7000) at an excitation wavelength of 492 nm and an emission wavelength of 525 nm. The fluorescence intensity thus obtained was subsequently converted to concentration expressed in μg/mL based on a correlation curve previously prepared by plotting different known concentrations (40, 20, 10, 5, 2.5, 1.25, and 0.625 μg/mL) of FITC- D4000 standards versus their fluorescence intensities. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIG. 13, the FITC-D4000 concentrations determined in the experimental groups 1 and 2 were each lower than that determined in the pathological control group, indicating that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate colitis, and can maintain the intestinal barrier function and the intestinal permeability. C. Histopathologic analysis After the 21-day treatment period, the respective mouse was sacrificed by virtue of 95% CO 2 asphyxiation, and the colon tissue was obtained from the respective mouse carcass. The colon tissue was washed with an ice-cooled physiological salt solution, followed by determining the length and photographing. A colon tissue sample having a length of about 1-2 cm was obtained from the respective washed colon tissue, followed by fixation with a 10% paraformaldehyde solution (in PBS) at room temperature for 24 hours. The fixed tissue sample was then embedded with paraffin, followed by slicing to obtain a tissue section having a thickness of about 3-5 μm. The tissue section was stained with hematoxylin and eosin (H&E) using a staining protocol well-known to those skilled in the art, and was then observed under an optical microscope (Carl Zeiss, Oberkochen, Germany) at 200x magnification. One area of the respective tissue section was randomly selected and photographed, and the pathological change in the respective tissue section was assessed according to the method described in Liu Y.W. et al. (2011), Int. Immunopharmacol., 11:2159-2166. The degree of colitis was ranked with a score ranging from 0 to 12, and the higher scale indicated the higher severity of colitis. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIGS. 14 and 15, the lengths of the colon tissues determined in the experimental groups 1 and 2 were each longer than that determined in the pathological control group, and the scores of colitis determined in the experimental groups 1 and 2 were each apparently or significantly lower than that determined in the pathological control group. These results indicate that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate colitis. D. Determination of tumor necrosis factor-α (TNF-α) content in colon tissue A colon tissue sample having a length of 1 cm was obtained from the respective colon tissue obtained in section C of this example, followed by washing with sterile PBS containing protease inhibitor (Sigma, Cat. No. P1860). The respective washed colon tissue sample was incubated with 1 mL of an RPMI 1640 medium (supplemented with 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 0.25 μg/mL amphotericin B, protease inhibitor, and 1% L-glutamate), followed by cultivation in an incubator (37°C, 5% CO 2 ) for 24 hours. After centrifugation at 3,000 rpm for 15 minutes, the resultant supernatant was collected, and was then subjected to determination of TNF-α content using a TNF-α ELISA kit (Cat. No. BMS607-3TEN, Invitrogen). The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIG. 16, the TNF-α contents determined in the experimental groups 1 and 2 were each lower than that determined in the pathological control group. This result suggests that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively reduce inflammatory markers in colon tissues. Example 8. In vivo anti-arthritis test on Lactobacillus delbrueckii subsp. lactis LDL557 A. Preparation of bovine type II collagen (CII) emulsion CII (Chondrex Inc., Cat. No. 20022) was mixed with incomplete freund adjuvant (IFA)(Sigma-Aldrich, Cat. No. F5506) at a ratio of 1:1 (v/v), so as to obtain a CII emulsion. B. Induction of arthritis and administration of bacterial suspensions The female SD rats (6 weeks old, with a body weight of approximately 160-180 g) were divided into four groups, including a normal control group, a pathological control group, and two experimental groups (i.e., experimental groups 1 to 2)(n=12 per group). The rats of the experimental group 1 were fed, via oral gavage, with the bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 5 at a dose of 5×10 8 CFU/kg, and the rats of the experimental group 2 were fed, via oral gavage, with the heat-killed bacterial suspension of Lactobacillus delbrueckii subsp. lactis LDL557 prepared in section A of Example 4 at a dose of 5×10 8 CFU/kg. In addition, each of the rats of the normal control group and pathological control group was fed, via oral gavage, with 1 mL of a 0.85% physiological salt solution. Each mouse was fed once daily for a 42-day treatment period. On the 14 th day, after the treatment of the bacterial suspension or the physiological salt solution, 200 μL of the CII emulsion prepared in section A of this example was injected subcutaneously into the tail of the respective one of the rats of the pathological control group and two experimental groups. The rats were given a boost injection on the 7 th day after the initial immunization, so as to induce the occurrence of arthritis. In addition, the rats of the normal control group were injected with PBS. C. Morphological observation On the 28 th day, after the treatment of the bacterial suspension or the physiological salt solution, changes (i.e., swelling and enlargement) in the joints of the four paws of each rat were assessed visually. Based on the result of the morphological observation (data not shown), it was found that in the pathological control group, severe swelling and enlargement in the joints of the four paws were observed. In contrast, in the experimental groups 1 and 2, swelling and enlargement in the joints of the four paws were sufficiently ameliorated. D. Determination of CII-specific IgG level in serum sample After the 42-day treatment period, a blood sample was collected from the tail artery of each rat through arterial puncture, and was then subjected to centrifugation at 5,000 rpm and 4°C for 15 minutes. The serum sample thus obtained was diluted 10 6 -fold with phosphate-buffered saline (PBS). 100 μL of a 5 μg/mL CII solution (in PBS) was added to a respective well of a 96-well culture plate, followed by being left standing at room temperature overnight. Afterwards, 100 μL of the respective diluted serum sample was added to the corresponding well, followed by being left standing at room temperature for 2 hours. Thereafter, the contents of CII-specific IgG, IgG2a, and IgG2b were determined using an IgG (total) rat uncoated ELISA Kit (Invitrogen, Cat. No. 88-50490-88), an IgG2a rat uncoated ELISA Kit (Invitrogen, Cat. No. 88-50510- 88), and an IgG2b rat uncoated ELISA Kit (Invitrogen, Cat. No. 88-50520-88) in accordance with the manufacturer’s instructions. The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIGS. 17 to 19, the OD 450 (optical density at 450 nm) values of CII-specific IgG2a, IgG2b, and IgG determined in each of the experimental groups 1 and 2 were apparently or significantly lower than those determined in the pathological control group. The results indicate that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively reduce the CII-specific IgG2a, IgG2b, and IgG levels in the serum. E. Expression profile of sirtuin-1 (SIRT1) and cyclooxygenase-2 (COX-2) in ankle tissue After completion of the determination of CII- specific IgG level as described in section D of this example, the respective rat was sacrificed by virtue of 95% CO 2 asphyxiation. An ankle tissue was obtained from the respective rat carcass, and was then ground using liquid nitrogen, so as to obtain a ground ankle tissue sample. 0.2 g of the respective ground ankle tissue sample was mixed with 300 μL of a RIPA lysis buffer (VWR Life Science, Cat. No. N653) containing protease inhibitor cocktail (VWR Life Science, Cat. No. M221), phosphatase inhibitor cocktail (BioVision, Cat. No. K282), and EDTA. The resultant mixture was left to stand at 4°C for 30 minutes. After centrifugation at 14,000 rpm and 4°C for 15 minutes, the supernatant thus obtained served as a total protein sample. The protein concentration in the total protein sample was determined using Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, USA). The total protein sample of the respective rat was subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western Blotting analysis for detection of SIRT1 and COX-2 by virtue of the technique well known to and routinely used by one skilled in the art. In addition, β-actin was used as an internal control. The instruments and reagents used for SDS-PAGE analysis and Western Blotting analysis are as follows: (1) SDS-PAGE analysis was performed using a Mini- PROTEAN® electrophoresis system (Bio-Rad). (2) Protein transfer was performed using a Mini Trans- Blot® cell (Bio-Rad) and a polyvinylidene difluoride (PVDF) membrane. (3) In Western Blotting analysis, primary and secondary antibodies used for detecting each protein are shown in Table 6. Table 6 (4) Chemiluminescence staining was performed using SuperSignal™ West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Cat. No. TG268240A), and signal detection was performed using ChemiDoc™ imaging system (Bio-Rad). Subsequently, ImageJ Imaging Software was used for semi-quantitatively calculating the corresponding protein expression level. The expression level of the respective one of SIRT1 and COX-2 in each group was normalized by the expression level of corresponding β-actin thereof. The relative expression level was calculated using the following Equation (I): A=B/C (I) where A=relative expression level of SIRT1 or COX- 2 B=normalized expression level of SIRT1 or COX-2 of respective group C=normalized expression level of SIRT1 or COX-2 of normal control group The data thus obtained were analyzed according to the method described in section 1 of “General Procedures”. Referring to FIGS. 20 and 21, the relative expression levels of SIRT1 determined in the experimental groups 1 and 2 were each higher than that determined in the pathological control group, and the relative expression levels of COX-2 determined in the experimental groups 1 and 2 were each lower than that determined in the pathological control group. The results indicate that Lactobacillus delbrueckii subsp. lactis LDL557 of the present disclosure, whether in the form of live cells or dead cells, can effectively alleviate arthritis. 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.