TWI450722B - Lactic acid bacteria isolated and their use - Google Patents

Description

Isolated lactobacillus and use thereof

The present invention relates to an isolated Lactobacillus strain, and more particularly to a Lactobacillus strain having an immunomodulatory function.

In recent years, studies on lactic acid bacteria and immune regulation have been continuously explored. These special lactic acid bacteria strains play an important role in the activation and regulation of various immune cells or in improving immune imbalance. Since many immune cells in an organism have the ability to produce cytokines or are regulated by cytokines, many studies have confirmed that lactic acid bacteria have the function of promoting and regulating the secretion of cytokines. However, the immune responses caused by different lactic acid bacteria strains are also different. Therefore, most studies have examined the types and secretion of cytokines against different cells and tissues, and explored their immunomodulatory responses.

Kefir grain is a microbial phase composed of compound lactic acid bacteria and yeast flora. It is a natural fungus for making Kefir fermented milk. It contains lactic acid bacteria not only in Kraft's sticky texture, sourness and function. It plays an important role in the characteristics, and is also closely related to the formation and proliferation of Kefir particles, which are the key microorganisms for the aroma and flavor generation of the products. In recent years, studies on the functional properties of Kefir fermented milk have been proposed, and studies have confirmed that Kefir fermented milk has a significant effect on immune function regulation. For example, the effect of lactic acid bacteria fermented milk on reducing allergic reactions, Master of Peng Yudi Thesis; discussion on the antioxidant capacity and immune function of probiotics and their fermented milk, Liu Yuru's master thesis; and de LeBlanc et al., 2006, Study of cytokines involved in the prevention of a murine experimental breast cancer by kefir. Cytokine 34:1 -8..

Although the results of the current study confirm that the Kefir fermented milk has immunomodulatory function, the microbial flora in the Kefir granule is complicated, and there is no relevant research showing that the Kefir immunomodulatory ability and the strain in the Kefir granule Relationship.

For the purpose of achieving the above and other objects, the present invention provides a Lactobacillus M1 ( Lb. kefiranofaciens M1) and Lactobacillus faecalis M2 ( Lb. kefiri M2) isolated from Kefir particles, which has a cytokine that slows down the pre-inflammatory response. Th2 cytokines and Th17 cytokine secretion, and can enhance spleen-regulated T cells, reduce OVA-specific IgE, which has anti-allergic and therapeutic effects on intestinal inflammation, can be used to make anti-allergic drugs, improve allergy symptoms Healthy food or health food, as a food additive; or as a feed additive for animal feed or pet feed.

The embodiments of the present invention are described by way of specific examples, and those skilled in the art can readily understand the advantages and functions of the present invention from the disclosure. The present invention may be embodied or applied by other different embodiments, and the various details of the present invention may be variously modified and changed without departing from the spirit and scope of the invention.

Kefir granules are composed of complex lactic acid bacteria and yeast strains. They are natural fungi for making Kefir fermented milk. The lactic acid bacteria not only play an important role in the viscous texture, sourness and functional properties of Kefir, but also The formation of Fol particles is closely related to hyperplasia and is the main key to the functionality of Kefir fermented milk. According to the experimental results, the strain in the Kefuer fermentation bacterium Kefir granule is the key to the function of Kefir fermented milk.

The invention tests the Kefuer particles of three different sources in the Hsinchu group, the Mongolian group and the Yilan group, and uses Harrison's disc method to select representative strains from each group of Kefir particles, and observes the morphology and biochemistry of the strain by traditional identification methods. Characteristics, and the use of denaturing gradient colloidal electrophoresis testing techniques and DNA sequencing and other molecular biotechnology to identify strain identity. The results showed that Lb. kefiri accounted for the most 53% of the K. sinensis granules, followed by Lb. kefiranofaciens accounted for 43%, Leuconostoc mesenteroides accounted for 3%, and Lactococcus lactis accounted for 1%. Among the Mongolian Kefuer particles, Lb. kefiri accounted for 58%, followed by Lb. kefiranofaciens accounted for 24%, Leu. mesenteroides accounted for 16%, and Lc. lactis (2%). Among the Yilan Kefuer particles, only three lactic acid bacteria were found to be Lb. kefiri accounted for 58%, Lb. kefiranofaciens accounted for 40%, and Leu. mesenteroides accounted for 2%. The four groups of Kefuer supernatant and RAW264.7 cells were co-cultured in the Taipei group, the Hsinchu group, the Mongolian group and the Yilan group. The results are shown in Fig. 1. The Mongolian group Kefir particles secrete tumor necrosis for RAW264.7. The effect of factor-α (tumor necrosis factor (TNF)-α) is most significant.

Compared with other Kefuer grains, Mongolian Kefuer particles have larger granules and a more viscous appearance. The Kefir fermented milk made by the Mongolian Kefuer grain fermentation has significant flavor and other Kefu. Different fermented milk has higher alcohol content. Although the group Hsinchu, Ilan and Mongolia groups are three kinds of Kefu Er grains containing Lb. kefiranofaciens, but the Mongolian group Lb. kefiranofaciens the lowest proportion of grain Kefu Er. Further analysis of the 16S rDNA sequence revealed that there were differences in the 16S rDNA of Lb. kefiranofaciens from 3 different sources.

The present invention analyzes the full-length sequence of 16S rDNA and identifies the lactic acid bacteria strains M1 and M2 isolated from the Mongolian group Kefir. Using the primer pair (8F and 1512R), the 16S rDNA gene fragment was amplified by polymerase chain reaction (PCR), and the PCR product was purified by a purification kit. The sequencing results of the lactic acid bacteria strains M1 and M2 isolated from the Mongolian group Kefir particles are SEQ ID NO. 1 and SEQ ID NO. 2, respectively, as shown in the sequence listing attached to the specification, using the Basic Local Alignment Search Tool (BLAST). ) ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) functions in the Genbank alignment sequence identity. It was confirmed that the Lactobacillus strains M1 and M2 isolated from the Mongolian group Kefir particles were Lb. kefiranofaciens M1 (registered number BCRC 910502) and Lb. kefiri M2 (registered number BCRC 910496), respectively, and the similarity was 99%.

The Lactobacillus strains M1 and M2 isolated from the Mongolian group Kefir particles have a pro-inflammatory cytokine [including interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α], T helper cells Type 2 (T helper cell type II, Th2) cytokines (IL-4, IL-5 and IL-13), and T helper cell type 17, Th17 cytokine secretion, promotion The spleen-regulated T cell (Treg) content, as well as the reduction of the effect on ovalbumin (OVA)-specific immunoglobulin (Ig) E, can be used to make anti-allergic or asthma drugs. The Lactobacillus isolated from the Mongolian group Kefir particles, the heat-inactivated strain still has the ability to regulate the secretion of cytokines by the spleen and ascites cells, and can be used for making anti-allergic drugs related to the first type of allergy.

The features and effects of the present invention are further illustrated by the following specific examples, but are not intended to limit the scope of the invention.

Example Example 1 Effect of Kefuer Fermentation Upper Layer on Secretion of Cytokines in RAW 264.7 Cell Line

(A) Preparation of Kefuer fermentation supernatant

The Mongolian Kefuer grain was used as the mushroom element, and the fermentation was carried out with the milk of the Agricultural University of Taiwan University. The addition amount was 5% (W/V), and the fermentation was carried out at 20 ° C. The fermentation time was 8, 16 respectively. 24, 48, and 72 hours. After the completion of the fermentation, centrifugation was carried out at 6000 x g for 30 minutes, the supernatant was taken, and then centrifuged at 13,000 x g for 30 minutes, and filtered using a 0.22 μm filter to obtain a Kefir supernatant.

(B) Preparation of mouse macrophage cell line

Mouse macrophage cell line (RAW 264.7, ATCC TIB71, culture medium is DMEM) was added, 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria) and 1% antibiotic [100] were added. U/ml penicillin (penicillin) and 100 μg/ml streptomycin (Sigma) were placed in a 5% CO ₂ cell culture incubator and cultured at 37 ° C to accumulate cells. Next, rinse with 10 ml of PBS, remove PBS, add 10 ml of the culture solution, scrape the cells with cell scraping and fully suspend and disperse, and then use the staining method (0.5% Trypan-blue, Invitrogen, Grand Island, NY) ) Perform cell number calculations. Finally, the culture medium (10% heat-inactivated fetal bovine serum was added to DMEM, and the serum was heated at 56 ° C for 30 minutes) to adjust the cell concentration to 5 × 10 ⁵ cells / ml, and add 1 ml to 24-well plate. spare.

(C) Inducing cytokine secretion test

After the cells were attached to the incubator at 37 ° C for 2 hours, 5 μl of the Kefuer supernatant (fermentation for 48 hours) was added to activate the RAW 264.7 cell line, and the cells were cultured at 37 ° C for 1, 3, 6, and 12, respectively. After 24 hours, the concentration of cytokines was measured by an enzyme-linked immunosorbent assay (ELISA) using a mouse cytokine kit (R&D system, Mckinley, MN). (A) to 2 (D) are shown.

According to the results of the 2(A) and 2(B) graphs, the Kefuer supernatant and the RAW 264.7 cell line were cultured for 24 hours, and the cytokines IL-6 and TNF-α were secreted. The effect is also significantly improved, and the fermentation has a better effect for 48 hours. On the other hand, the results of the second (C) and 2 (D) graphs showed that the Kefuer supernatant obtained by fermentation for 48 hours was cultured for 6 hours with the RAW 264.7 cell line, and the cytokines IL-1β and IL- were significantly increased. 12 secretion effect.

Example 2 - Effect of isolated Lactobacillus strain on secretion of cytokines in RAW 264.7 cell line

The lactic acid strains were isolated using Lb. kefiranofaciens M1 (Accession No. BCRC 910502), Lb. kefiri M2 (Accession No. BCRC 910496), Leu. mesenteroids M3 and Lc. lactis M4 isolated from Mongolian Kefuer. On the other hand, Lb. kefiri BCRC 14011 and Lb. kefiranofaciens BCRC 16059 were used as standard strains (purchased from the Center for Biological Resource Conservation and Research of the Food Industry Development Research Institute). The strain was activated with Lactobacilli MRS medium (Difco Laboratories, Detroit, MI), except that Lb. kefiri was cultured in an incubator at 37 ° C, and the remaining strains were cultured at 30 ° C. After the activation was completed, the cells were washed with a PBS solution, and the concentration was adjusted to 10 ⁷ CFU/ml to obtain a strain test sample.

Repeat step (C) of Example 1 to separate 5 liters of lactic acid bacteria strains M1, M2, M3, M4 test samples, standard strains BCRC 14011 (standard strain L1) and BCRC 16059 (standard strain L2) test samples, and control The Kefuer supernatant was co-cultured with RAW 264.7 cell line, and after culturing for 24 hours in a 37 ° C cell incubator, the concentration of cytokines was measured, and the results are shown in Figures 3(A) to 3(E).

Figures 3(A) to 3(E) show the four strains of lactic acid bacteria (M1, M2, M3, M4) isolated from the Mongolian group, the standard strains (L1, L2), and the upper layer of Kefir. The liquid control group induced the secretion of cytokines IL-6, IL-1β, IL-10, TNF-α, and IL-12.

Example 3 Effect of Lactobacillus fermented milk on secretion of cytokines in RAW 264.7 cell line

Lb. kefiranofaciens M1, Lb. kefiri M2, Leu. mesenteroids M3 and Lc. lactis M4 isolated from lactic acid strains isolated from Mongolian Kefuer particles; and Lb. kefiri BCRC 14011 and Lb. kefiranofaciens BCRC 16059 The standard strain was added to 10 ml of milk (10 ⁷ CFU) for fermentation for 48 hours. After the completion of the fermentation, centrifugation and filtration were carried out according to the procedure (A) of Example 1, to obtain a fermentation supernatant liquid of the strain.

Repeat step (C) of Example 1 to separate the fermentation supernatant of the lactic acid bacteria strains M1, M2, M3, and M4 with 5 μl of the fermentation supernatant of the standard strains BCRC 14011 (standard strain L1) and BCRC 16059 (standard strain L2), respectively. The liquid and the control Kefuer supernatant were tested with the RAW 264.7 cell line, and after culturing for 24 hours in a 37 ° C cell culture chamber, the concentration of the cytokine was measured, and the results were as shown in Figures 4(A) to 4(D). Show.

Figures 4(A) to 4(D) show the fermentation supernatants of the four strains of lactic acid bacteria (M1, M2, M3, M4) and the standard strains (L1, L2) isolated from the Mongolian group of Kefir, respectively. The Kefuer supernatant control group induced the secretion of cytokines IL-6, IL-1β, TNF-α, and IL-12.

Example 4 Effect of Lactobacillus M1 and Fermented Milk on Secretion of Cytokines by Mouse Macrophages

The test was carried out in BALB/c female mice of six weeks old. Immune cells were aggregated by intraperitoneal injection of 2 ml of 3% thioglycollate. After 72 hours, the mice were sacrificed. In the aseptic table, 5 ml of PBS (phosphate buffered saline) was injected into the peritoneum, and the immune cells between the peritoneal organs were uniformly suspended in PBS, and the peritoneal cells were extracted with a 5 ml empty syringe. Two times, 8 ml of ascites fluid was obtained. The ascites was centrifuged at 1500 rpm for 10 minutes at low speed, the supernatant was discarded, the cell pellet was patted to disperse, and RPMI 1640 containing 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria) was added. The culture medium (HyClone, Logan, UT), the cells were suspended and cultured in a 24-well culture dish, and after standing at 37 ° C for 3 hours in a cell culture incubator, the cells which were not attached to the suspension were washed away, and then 10% heat inactivation was added. FBS RPMI 1640 medium, the attached cells are scraped with cell scraping. The number of cells was calculated by Trypan-blue staining to be 5 x 10 ⁶ cells/ml. 1 ml of the cell suspension was cultured in a 24-well culture dish, and cultured in a 37 ° C cell incubator for 3 hours, and the cells were attached and washed for use.

According to the step (C) of Example 1, 5 μl of the lactic acid bacteria strain M1 test sample (M1c), the lactic acid bacteria strain M1 fermentation supernatant (M1s), and the control group PBS were respectively tested with the mouse ascites macrophage. After culturing for 24 hours in a 37 ° C cell culture incubator, the concentration of cytokines was measured, and the results are shown in Fig. 5.

Example 5 - Effect of isolated Lactobacillus strain on cytokine secretion by macrophages

(A) Source, culture and preparation of lactic acid bacteria strain

Mongolian use of lactic acid bacteria isolated particles Ke Fuer Lb. kefiranofaciens M1 (Accession No. BCRC 910502) and Lb. kefiri M2 (Accession No. BCRC 910496), using Lactobacilli MRS broth (Difco Laboratories, Detroit, MI) activated strain, respectively Incubate in an incubator at 30 ° C and 37 ° C. The activated strain was cleared of the bacterial body with a PBS solution, and the concentration was adjusted to 1.0 × 10 ⁶ CFU/ml, which is the strain samples M1 and M2.

The heat-inactivated strain was suspended in PBS solution and heated to 85 ° C in a constant temperature water bath for 40 minutes to thermally inactivate the strain, respectively, Lb. kefiranofaciens M1 (Accession No. BCRC 910502), heat-inactivated strain sample HI- Heat inactivated strain sample HI-M2 of M1, and Lb. kefiri M2 (registration number BCRC 910496).

(B) Separation of macrophages

The test was carried out in BALB/c female mice of six weeks old. Immune cells were aggregated by intraperitoneal injection of 2 ml of 3% thioglycollate. After 72 hours, the mice were sacrificed. In the aseptic table, 5 ml of PBS was injected into the peritoneum, and the immune cells between the peritoneal organs were uniformly suspended in PBS, and the peritoneal cells were extracted with a 5 ml empty syringe, and repeated twice to obtain 8 ML of ascites fluid. The ascites was centrifuged at 1500 rpm for 10 minutes at low speed, the supernatant was discarded, the cell pellet was patted to disperse, and RPMI 1640 containing 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria) was added. The culture medium (HyClone, Logan, UT), the cells were suspended and cultured in a 24-well culture dish, and after standing at 37 ° C for 3 hours in a cell culture incubator, the cells which were not attached to the suspension were washed away, and then 10% heat inactivation was added. FBS RPMI 1640 medium, the attached cells are scraped with cell scraping. The number of cells was calculated by Trypan-blue staining to be 5 x 10 ⁶ cells/ml. 1 ml of the cell suspension was cultured in a 24-well culture dish, and cultured in a 37 ° C cell incubator for 3 hours, and the cells were attached and washed for use.

(C) Cytokine assay

The strain samples M1, M2, and the heat-inactivated strain samples HI-M1, HI-M2 were co-cultured with mouse macrophages, and the control group was PBS. The concentration of the strain sample was 1.0×10 ⁶ CFU/ml, and the culture solution was collected by culturing at 37 ° C for 24 hours. The concentration of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) using a mouse cytokine kit (R&D system, Mckinley, MN). The results are shown in Figures 6(A) to 6(E). Show.

The results showed that the strains M1 and M2 in the viable state or the heat-inactivated strain samples HI-M1 and HI-M2 were co-cultured with mouse macrophages, and the inactivated strain samples HI-M1 and HI-M2 were heated. Th1 cytokines such as IL-12 and interferon (IFN)-γ were significantly increased, and there was no significant difference between the cells and the live bacteria, and there was no significant difference between Lb. kefiranofaciens M1 and Lb. kefiri M2. In the pre-inflammatory cytokine secretion of mouse macrophages, the heat-inactivated strain induced IL-1β secretion significantly lower than that of live strains, and stimulated the ability of mouse macrophages to secrete TNF-a and heat-inactivated strains. It was significantly higher than the live strain, while IL-6 induced secretion, and there was no significant difference between the two strains.

Example 6 - Effect of isolated Lactobacillus strain on cytokine secretion in spleen cells

(A) Source, culture and preparation of lactic acid bacteria strain

Mongolian use of lactic acid bacteria isolated particles Ke Fuer Lb. kefiranofaciens M1 (Accession No. BCRC 910502) and Lb. kefiri M2 (Accession No. BCRC 910496), using Lactobacilli MRS broth (Difco Laboratories, Detroit, MI) activated strain, respectively Incubate in an incubator at 30 ° C and 37 ° C. The activated strain was cleared of the bacterial body with a PBS solution, and the concentration was adjusted to 1.0 × 10 ⁶ CFU/ml, which is the strain samples M1 and M2.

The heat-inactivated strain was suspended in PBS solution and heated to 85 ° C in a constant temperature water bath for 40 minutes to thermally inactivate the strain, respectively, Lb. kefiranofaciens M1 (Accession No. BCRC 910502), heat-inactivated strain sample HI- Heat inactivated strain sample HI-M2 of M1, and Lb. kefiri M2 (registration number BCRC 910496).

(B) Separation of spleen cells

The spleen of the mouse was removed in a sterile workstation, and the spleen was placed on a 70 micron Nylon cell strainer (BD Falcon, Franklin Lakes, NJ), which was ground at the end of the sterile syringe and filtered to remove connective tissue. Rinse with 10 ml of sterile PBS, centrifuge at 1200 rpm for 5 minutes, then aspirate the supernatant and add 5 ml of red blood cell (RBC) lysis solution, Qiagen, Germantown, Maryland, at room temperature. Mix well for 2 minutes to completely dissolve the red blood cells. After centrifugation to remove the supernatant under the conditions described above, 10 ml of PBS was added to wash away the residual RBC lysis solution, and finally 5 ml of 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria) RPMI 1640. The culture medium (HyClone, Logan, UT) was fully suspended, and the spleen cells were adjusted to 1 × 10 ⁷ cells/ml by Trypan-blue staining.

(C) Cytokine assay

The strain samples M1, M2, and the heat-inactivated strain samples HI-M1, HI-M2 were co-cultured with mouse spleen cells, and the control group was PBS. The concentration of the strain sample was 1.0×10 ⁶ CFU/ml, and the culture solution was collected by culturing at 37 ° C for 24 hours. The concentration of cytokines was measured by enzyme-linked immunosorbent assay using a mouse cytokine kit (R&D system, Mckinley, MN) and the results are shown in Figures 7(A) and 7(B).

The results showed that in the culture of spleen cells, the live strain and the heat-inactivated strain had the same tendency for Th1 cell hormone secretion, showing that the ability of Lb. kefiranofaciens M1 and Lb. kefiri M2 to activate immunity was partly derived from the bacterial cells.

Example 7 - Sensitized mouse test: Effect of heat-inactivated Lactobacillus strain on serum IgE and spleen cell cytokine secretion in mice

(A) Heat-inactivated lactic acid bacteria strain and lactic acid bacteria strain fermented milk supernatant

The fermented milk supernatant (whey) of the strain sample M1 was obtained by performing fermentation according to Example 3 using the heat-inactivated strain sample HI-M1 of the lactic acid bacteria Lb. kefiranofaciens M1 (registered number BCRC 910502) isolated from the Mongolian group Kefir.

(B) Sensitization process

The test was performed on 6-week-old BALB/c female mice, and sensitized on days 0, 13, 20, and 27, and the sensitization mode was allergic to 20 μg of ovalbumin (OVA, Sigma, St. Louis, MO). Originally, and with adjuvant 2 mg of aluminum hydroxide (Al(OH) ₃ , Sigma), 200 μl was injected via intraperitoneal injection. During the 28th experiment, different concentrations of Lb. kefiranofaciens M1 heat-inactivated strain samples HI-M1 300 μl were fed by tube feeding, and the final number of lactic acid bacteria was 1.5 x 10 ⁷ , 3 x 10 ⁷ and 6 x 10 ⁷ CFU/ml.

(C) serum acquisition

Cheek-pouch blood collection was performed on days 0, 7, 14, 21, and 28, and blood was placed in blood collection tubes (BD microtainer chemisty tubes, BD, Franklin Lakes, NJ) after centrifugation at 6000 xg for 90 seconds. Serum was obtained, stored at -80 ° C and the concentrations of total IgE (mouse IgE ELISA set, BD science) and OVA-specific IgE (DS mouse IgE ELISA OVA, DS Pharma Biomedical, Osaka, Japan) were measured. The control group was PBS, and the results are shown in Fig. 8 and Fig. 9(A) and Fig. 9(B).

According to Fig. 8, after the OVA sensitized mice were fed with the heat-inactivated strain sample HI-M1 for 28 days, as the concentration of the cells increased, the IgE effect of the serum of the sensitized mice was reduced, and the daily feeding was 6 x 10 ^{7 .} CFU significantly inhibited the total IgE content in the serum of sensitized mice, whereas the fermented milk supernatant (whey) fed the strain sample M1 did not. It is shown that the concentration of lactic acid bacteria needs to reach a certain concentration in order to trigger an immune response and achieve the effect of reducing the IgE content in serum.

Figure 9 shows that the HI-M1 group of 6 x 10 ⁷ CFU of heat-inactivated strains was fed daily, and the third dose (Day 20) of OVA allergens significantly inhibited OVA specificity in serum after intraperitoneal injection. IgE secretion; while the total IgE content, the heat-inactivated strain sample HI-M1 was significantly lower in the second dose (day 13) after OVA injection than the OVA control in Figure 9 (A) group. This partial in vivo test confirmed that heat-inactivated Lb. kefiranofaciens M1 significantly improved the secretion of IgE and OVA-specific IgE on the 14th day of serum in OVA-sensitized mice.

(D) spleen cells

On the 28th day of the experiment, the mice were sacrificed, and the spleen cells were isolated in the aseptic table (the same method as in Example 6). The number of spleen cells was adjusted to 1 x 10 ⁷ cells/ml by Trypan-blue staining, and 1 ml of the cell suspension was taken [RPMI]. Add 10% FBS and 1% antibiotic (100 U/ml penicillin and 100 μg/ml streptomycin, Sigma) to 1640. In 24-well plate, add 0.22 μm sterile The OVA solution of the filter was stimulated, and the final OVA concentration of the cell culture solution was 100 μg/ml. After incubating in a 37 ° C incubator for 48 hours, the cells were centrifuged at 8000 rpm for 3 minutes to remove the spleen and the supernatant was collected and stored at -80. The cytokine was measured at ° C. The results are shown in Figures 10(A) to 10(C).

The results showed that the IL-12 of the HI-M1 group in which the heat-inactivated strain sample was fed 6 x 10 ⁷ CFU per day was significantly higher than the control group of OVA, while there was no significant difference in IFN-γ. On the other hand, the Th2 cytokines (IL-5) secreted by the mouse spleen cells were also significantly lower than the control group when the heat-inactivated strain sample HI-M1 was fed. The results confirmed that the sensitized mice were fed with the heat-inactivated strain sample HI-M1 to regulate the secretion of cytokines from spleen cells. When the spleen cells were stimulated again by OVA allergens in vitro, they could significantly reduce Th2 cytokines and enhance Th1 cells. hormone.

Example 8 - Sensitized mouse test: Effect of heat-inactivated Lactobacillus strain on regulatory T cells and B cells in spleen of sensitized mice

According to the procedure of Example 7, step (D), spleen cells not co-cultured with OVA were stained with a cell number of 5× ^{10 6} cells/ml, using anti-mouse PE-CD4 antibody (clone GK1.5), anti-murine FITC-CD25 antibody ( Clone 7D4) was stained with anti-mouse FITC-CD19 antibody (clone 6D5) (Beckman Coulter, Fullerton, 60 CA) at 4 ° C in the dark, washed twice with 4 ml of PBS to wash away excess dye on the cells, and finally 1 ml of PBS suspension cells were filtered through a 70 μm Nylon cell strainer (BD Falcon) and analyzed for CD4 ⁺ CD25 ⁺ regulated by flow cytometry (EPICS XL flow cytometer, Beckman Coulter, Fullerton, CT). The number of T cells and CD19 ⁺ B cells.

According to the figures 11(A) to 11(C), the heat-inactivated Lactobacillus strain HI-M1 was fed to the sensitized mice for 28 consecutive days, and the number of regulatory T cells in the spleen was significantly increased. According to Fig. 11(D), after feeding the heat-inactivated Lactobacillus strain HI-M1, the number of CD19 ⁺ B cells was significantly reduced as compared with the OVA control group. It was confirmed that continuous feeding of heat-inactivated Lb. kefiranofaciens HI-M1 significantly increased the number of regulatory T cells in the spleen of OVA-sensitized mice and decreased the number of CD19 ⁺ B cells.

Example 9 - Sensitized mouse test: Effect of heat-inactivated Lactobacillus strain on the regulation of Peyer's patch gene expression in sensitized mice

After the sacrifice of the mouse, the method of Torii et al. (2007) was used. After removing the duodenum from the mouse to the end of the ileum, the white protrusion was observed as the target Peyer's patch, and each mouse took 6-8, after washing with PBS. Stored in liquid nitrogen. RNA extraction was subsequently performed with TRIzol reagent (Invitrogen, Carlsbad, CA).

Peyer's patches RNA samples were quantified by ultra-micro spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE) and quality judged by Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA) (OD 260/280 ratio required) Above 2.0, the ratio of Bioanalyzer 28s/18s needs to be greater than 1.2, and the RIN value needs to be higher than 8). The quality-determined RNA was subjected to an Amp rapid calibration kit (Agilent Technologies). After the RNA sample was reverse transcribed into cDNA, the experimental group (feeding the heat-inactivated Lactobacillus strain HI-M1) was calibrated with cyanine 3, and The OVA control group was calibrated with cyanine 5. After calibration, the two sets of samples were subjected to a heterozygous reaction on a mouse whole gene 4 x 44k oligo nucleic acid wafer (G4122F-014868, Agilent Technologies). After the sample was heated at 60 ° C for 30 minutes, it was placed in a rotary oven at 4 rpm for reaction (protected from light and reacted at 65 ° C for 17 hours). After the reaction was completed, it was washed and dried, and the wafer was finally scanned by an Agilent microarray scanner (G2565CA, Agilent Technologies) and analyzed by software 10.5.1.1 (Agilent Technologies).

The results showed that the performance of a total of 1034 genes was significantly decreased, and the performance of 534 genes was significantly improved. The genes related to immunity were listed in Table 1. The results showed that feeding the heat-inactivated Lactobacillus strain HI-M1 significantly inhibited the overexpression of the complement system in sensitized mice, such as C1r, C1s, C2 and C3 , while enhancing Th1 ( Ifnr and Stat4 ) and pre-inflammatory responses ( Ccr7 ) Genes associated with cell surface signaling molecules ( Cd2, Cd3 and Cd28 ), which were also confirmed by real-time PCR.

Example 10 - Asthmatic mouse mode: Lactobacillus strains improve allergic asthma

(A) Preparation and treatment of lactic acid bacteria

Lb. kefiranofaciens M1 (Accession No. BCRC 910502) isolated from Mongolian Keefer granules was subcultured with Lactobacilli MRS medium (Difco Laboratories, Detroit, MI) and washed with PBS and adjusted to a concentration of 10 ⁷ before the experiment. , 10 ⁸ and 10 ⁹ CFU/ml, which is the strain sample M1.

The strain was suspended in a PBS solution and heated to 85 ° C for 40 minutes in a constant temperature water bath to thermally inactivate the strain, which is a heat-inactivated strain sample HI-M1 of Lb. kefiranofaciens M1 (Accession No. BCRC 910502).

(B) sensitization and antigen inhalation in asthmatic mice

Six-week old BALB/c female mice (purchased at National Taiwan University Experimental Animal Center, National Laboratory Animal Center, Taipei, Taiwan) were used. Sensitization of the mouse asthmatic model was performed by Lee et al. (2007) for a total of 32 days. On the 1st and 14th day, 20 micrograms of ovalbumin (OValbumin, OVA, Sigma, St. Louis, MO) with 2 mg of Al(OH) ₃ (aluminum hydroxide, Sigma) with a total injection volume of 200 μl and placed the mice on an ultrasound sprayer on August 28, 29 and 30 (Aerogen Ireland Ltd, Galway) In 20 minutes, the mice were given inhalation of OVA dust (1% OVA in PBS solution) to cause respiratory allergy. On the 31st day, the airway resistance test was performed. On the 32nd day, the mice were sacrificed and the spleen cells and bronchial washing fluid were separated. serum.

(C) Lactic acid bacteria feeding treatment

The feeding treatment was divided into three groups, and the concentration of the feeding bacteria was 10 ⁸ CFU/ml per mouse per day, and was fed by a tube feeding method with a total volume of 200 μl.

Group I - 32 days of trial, daily feeding (course A)

Group II - from day 1 to day 14, continuous feeding for 14 days (course B)

Group III - Days 28, 29, and 30, feeding bacteria one hour before inhalation of mouse dust (course C)

Positive control group - OVA asthma mice fed with 200 μl PBS (positive control, PC)

Negative control group - PBS-sensitized and inhaled mice fed with 200 μl PBS (negative control, NC)

(D) Respiratory resistance test

24 hours after the last inhalation of OVA dust, the naturally breathed mice were placed in a chamber of a whole body plethysmography (Buxco WBP, Buxco. Electronics, Wilmington, NC) and allowed to stand for 5 minutes. After the mice were calmed, the mice were subjected to airway hypperresponsiveness with increasing concentrations (12.5, 25 and 50 mg/ml) of methacholine (Sigma). The instrument will measure enhanced pause (Penh). ) as an indicator of airway obstruction (Peak et al., 1987).

(E) Obtaining bronchial washing fluid and lung section

For the method of obtaining bronchoalveolar lavage fluid (BAL), refer to Forsythe et al. (2007), and fill 1 ml of PBS into the respiratory tract via tracheal cannulation to obtain BAL. The same procedure is repeated three times to obtain the bronchi. The rinse was centrifuged at 200 xg for 15 minutes, and the supernatant was stored in a -80 ° C refrigerator for subsequent determination of cytokines.

The lungs were fixed in 10% formalin (Wako, Chuo-Ku, Osaka, Japan) for 24 hours, then embedded in paraffin, and then sliced and then H&E stained (hematoxylin and eosin, eosin). Cell staining was performed by IMEB Inc., San Marcos, CA). Sections were additionally stained with goblet cells and mucus by PAS staining (periodic acid-Schiff, IMEB Inc., San Marcos, CA).

(F) Isolation of spleen cells and analysis of CD4 ⁺ CD25 ⁺ regulatory T cells

Isolation of spleen cells in a sterile console (method as described above), using Trypan-blue staining to adjust the number of spleen cells to 1 x 10 ⁷ cells/ml, and take 1 ml of cell suspension [RPMI 1640 plus 10% FBS and 1 % antibiotic (100 U/ml penicillin and 100 μg/ml streptomycin, Sigma). Stimulated in a 24 well plate and then added to a 0.22 micron sterile filter in OVA. The cell culture medium was finally subjected to an OVA concentration of 100 mg/ml. After incubating for 48 hours in a 37 ° C cell culture incubator (Revco, Santa Fe Springs, CA), centrifugation was performed at 8000 rpm for 3 minutes to remove the spleen and collect the supernatant, and stored in -80 ° C refrigerator to be determined by cytokines.

Spleen cells not co-cultured with OVA were stained with 5× ^{10 6} cells/ml of cells, using anti-mouse PE-CD4 antibody (clone GK1.5), anti-murine FITC-CD25 antibody (clone 7D4) (Beckman Coulter, Fullerton) , CA), stained at 4 ° C in the dark, washed twice with 4 ml of PBS to wash away excess stain on the cells, and finally suspend the cells in 1 ml of PBS through a 70 μm Nylon cell filter (BD Falcon, Franklin Lakes , NJ) After filtering the cells, the CD4 ⁺ CD25 ⁺ regulatory T cells were analyzed by flow cytometry (EPICS XL79 flow cytometer, Beckman Coulter, Fullerton, CT).

(G) serum

On the 32nd day, the mice were sacrificed, the blood was collected from the cheeks and the serum was separated. The blood was placed in a blood collection tube (BD Microtainer Chemisty Tubes, BD, Franklin Lakes, NJ) and centrifuged at 6000 xg for 90 seconds to obtain serum, which was stored at -80 ° C and measured. Total IgE (mouse IgE ELISA set, BD) and concentration of OVA-specific IgE (DS mouse IgE ELISA OVA, DS Pharma Biomedical, Osaka, Japan).

(H) cytokine determination

The enzyme-linked immunosorbent assay (ELISA) was used. TNF-α, IFN-γ, IL-6, IL-12, IL-1β, IL-10, IL-4, IL were measured using a mouse cytokine kit (R&D system, Mckinley, MN). -5, IL-17, IL-17F, concentration of CCL-20 and IL-13.

Figures 12 through 14 show the effect of different feeding regimes on asthmatic mice. Sections 12(A), 12(B), and 12(C) show the results of the HI-M1 feeding of the heat-inactivated strain samples of Groups I, II, and III, respectively, and the results of the secretion of cytokines by the spleen cells in the positive control group. The results showed that the whole feeding strain (Group I feeding mode) significantly inhibited Th2 cytokines (IL-4 and IL-13) and pre-inflammatory cytokines (IL-6 and TNF-α); IL-6 and TNF-α were also significantly reduced, but there was no significant inhibitory effect on the secretion of IL-4 and IL-13.

Sections 13(A), 13(B), and 13(C) show the results of the HI-M1 feeding of the heat-inactivated strain samples of Groups I, II, and III, respectively, and the results of the bronchial washing solution in the positive control group. The results showed that the whole feeding strain (Group I feeding mode) significantly reduced the proinflammatory cytokines (TNF-α, IL-1β and CCL20) and Th2 cytokines (IL-5 and IL-13) in the respiratory tract of asthmatic mice. In contrast, the other two feeding treatment groups were unable to reduce the inflammatory response and Th2 cytokines in the respiratory tract of asthmatic mice.

14(A), 14(B), and 14(C) show the manner in which the heat-inactivated strain samples HI-M1 were administered to groups I, II, and III, respectively, and the positive control group for regulatory T cells in the spleen of asthmatic mice. The impact of quantity. The results showed that the number of regulatory T cells in the spleen was significantly increased in the whole feeding group (Group I feeding mode), but there was no significant difference between the other two treatment groups and the positive control group.

In the three feeding treatment groups, the whole process of feeding the heat-inactivated strain sample HI-M1 not only significantly inhibited the anterior inflammatory reaction and Th2 cytokine in the spleen and bronchial washing fluid of asthmatic mice, but also increased the number of regulatory T cells in the spleen.

Figures 15 to 19 show the effect of different doses of heat-inactivated strain samples HI-M1 on asthmatic mice. Figures 15(A) to 15(F) show the whole process of feeding heat-inactivated strain sample HI-M1, feeding different doses of heat-inactivated strain sample HI-M1, and positive control group and negative control group for bronchial washing of asthmatic mice. The effect of cytokine secretion in fluid. The results showed that high levels of Th2 (IL-4, IL-5 and IL-13) secreted by the spleen cells of asthmatic mice were associated with proinflammatory responses (IL-1β, IL-6 and TNF-α) cytokines daily. Feeding of 10 ⁸ and 10 ⁹ CFU heat-inactivated strain samples was significantly inhibited at the dose of HI-M1, whereas the asthmatic mice fed the strain sample M1 fermented milk showed no significant difference compared with the positive control group.

Figures 16(A) and 16(B) show that the heat-inactivated strain sample HI-M1 was fed throughout, fed different doses of heat-inactivated strain sample HI-M1, and the positive control group and the negative control group for asthmatic bronchi The effect of Th17 cell hormone secretion in the rinse solution. The results showed that daily feeding of 10 ⁷ , 10 ⁸ and ¹⁰⁹ CFU significantly inhibited the secretion of IL-17. For IL-17F, the group fed with 10 ⁹ CFU was significantly lower than the positive control group.

Figure 17 shows the effect of feeding the heat-inactivated strain sample HI-M1, feeding different doses of heat-inactivated strain sample HI-M1, and positive control group and negative control group on OVA-specific IgE in asthmatic mice. The results showed that HI-M1, a heat-inactivated strain sample fed more than 10 ⁷ CFU per day, significantly inhibited the content of OVA-specific IgE, feeding the strain sample M1 fermented milk asthmatic mice, and OVA-specific IgE in the serum of asthmatic mice. There is no significant difference from the positive control group.

Figures 18(A) to 18(F) show that the heat-inactivated strain sample HI-M1 was fed throughout, fed different doses of heat-inactivated strain sample HI-M1, and the positive control group and the negative control group for the lungs of asthmatic mice. The effect of immune cell infiltration. Figure 18(A) shows the negative control group. The results show that compared with the positive control group (Fig. 18(B)), the fed strain sample M1 fermented milk (Fig. 18(C)) and 10 ⁷ CFU The group (Fig. 18(D)) did not significantly improve the infiltration of immune cells into the respiratory tract; the daily feeding concentration was increased to 10 ⁸ CFU (Fig. 18(E)) and 10 ⁹ CFU (Fig. 18(F)). It can significantly improve the infiltration of immune cells into the respiratory tract (mainly mononuclear spheres).

Fig. 19(A) to Fig. 19(F) show the case where the goblet cells secrete mucus by PAS staining, and the negative control group in Fig. 19(A), and the results show that the control group of asthmatic mice (19 (B) ))), the respiratory tract is filled with mucus, and HI-M1, a heat-inactivated strain sample fed with different doses of 10 ⁷ , 10 ⁸ and 10 ⁹ CFU, respectively, can reduce the secretion of mucus in the respiratory tract of asthmatic mice (19(D), ( E), (F) map), which is the most significant improvement in the group fed with 10 ⁹ CFU per day (Fig. 19(F)).

Figure 20 shows the effect of feeding heat-inactivated strain sample HI-M1 on lung function in asthmatic mice. The asthmatic mice were stimulated with different concentrations of methylcholine. Compared with the positive control group (PC), mice fed the 10 ⁹ CFU heat-inactivated strain sample HI-M1 at a concentration of 25 and 50 mg of methylcholine. Under the stimulation of milliliters, the increase in Penh value can be significantly improved, and the resistance of the respiratory tract can be alleviated.

The above-described embodiments are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and scope of the invention will be covered by the appended claims.

<110> National Taiwan University

<120> Isolated Lactobacillus and its use

<160> 2

<170> PatentIn Version 3.5

<210> 1

<211> 1460

<212> 16S rDNA

<213>Lactobacillus kefiranofaciens

<400> 1

<210> 2

<211> 1460

<212> 16S rDNA

<213>Lactobacillus kefiranofaciens

<400> 2

Figure 1 shows the results of co-cultivation of four groups of Kefuer supernatant and RAW264.7 cells;

Figures 2(A) to 2(D) show the effect of Kefuer fermentation supernatant on the secretion of cytokines by RAW 264.7 cell line;

Figures 3(A) to 3(E) show the effect of the isolated Lactobacillus strain on the secretion of cytokines by RAW 264.7 cell line;

Figures 4(A) to 4(D) show the effect of Lactobacillus fermented milk supernatant on the secretion of cytokines by RAW 264.7 cell line;

Figure 5 shows the effect of Lactobacillus M1 and its fermented milk supernatant on the secretion of cytokines by mouse macrophages;

Figures 6(A) to 6(E) show the effect of isolated Lactobacillus strains on cytokine secretion by macrophages;

Figures 7(A) and 7(B) show the effect of isolated Lactobacillus strains on cytokine secretion by spleen cells;

Figure 8 is a graph showing the effect of feeding heat-inactivated strains on serum IgE in sensitized mice;

Figures 9(A) and 9(B) show the effect of feeding heat-inactivated strains on total IgE and OVA-specific IgE in serum of sensitized mice;

Figures 10(A) to 10(C) show the effect of feeding heat-inactivated strains on the secretion of cytokines by spleen cells of sensitized mice;

Figures 11(A) to 11(D) show the effects of feeding heat-inactivated strains on CD4 ⁺ CD25 ⁺ regulatory T cells and CD19 ⁺ B cells in spleen cells of sensitized mice;

Figures 12(A) to 12(C) show the results of feeding the heat-inactivated strains in different ways, and secreting cytokines from the spleen cells of asthmatic mice;

Figures 13(A) through 13(C) show the cytokine changes in the bronchial washings of asthmatic mice by feeding the heat-inactivated strains in different ways;

Figures 14(A) through 14(C) show the number of regulatory T cells in the spleen of asthmatic mice fed differently by heat inactivated strains;

Figures 15(A) through 15(F) show the effect of feeding different doses of heat-inactivated strains on cytokine secretion by bronchial washings in asthmatic mice;

Figures 16(A) and 16(B) show the effect of feeding different doses of heat-inactivated strains on Th17 cytokine secretion in asthmatic bronchial washings;

Figure 17 shows the effect of feeding different doses of heat-inactivated strains on OVA-specific IgE in the serum of asthmatic mice;

Figures 18(A) through 18(F) show the effect of feeding different doses of heat-inactivated strains on immune cell infiltration in the lungs of asthmatic mice;

19(A) to 19(F) shows the observation of the secretion of mucus by goblet cells by PAS staining;

Figure 20 shows the effect of feeding heat-inactivated strains on lung function in asthmatic mice.

Claims (15)

An isolated lactobacillus is a heat-inactivated strain, and the lactobacillus is deposited in the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Institute, and is registered with Lactobacillus kefiri M2, number BCRC 910496. The lactobacillus according to claim 1, wherein the lactobacillus is obtained by separating Kefir particles. The lactobacillus according to claim 2, wherein the Kefir granule is a Mongolian group of Kefir. A fermentation composition comprising Lactobacillus according to Item 1 of the patent application is formed by fermenting Mongolian Kefumycin in cow's milk for 24 to 72 hours. A composition for promoting secretion of cytokines in a pre-inflammatory reaction, comprising Lactobacillus as claimed in claim 1. The composition according to claim 5, wherein the pro-inflammatory cytokine comprises IL-6, IL- 1β , TNF-α and IL-12. A composition for slowing secretion of Th2 cell hormones, comprising Lactobacillus as claimed in claim 1. The composition of claim 7, wherein the Th2 cytokine comprises IL-4, IL-5 and IL-13. A composition for slowing secretion of Th17 cell hormones, comprising Lactobacillus as claimed in claim 1. A composition for increasing the content of spleen-regulated T cells, including Lactobacillus as claimed in claim 1. A composition for reducing OVA-specific IgE, including Lactobacillus as claimed in claim 1. A use of Lactobacillus as claimed in claim 1 for the manufacture of anti-allergic or anti-asthmatic drugs. The use according to claim 12, wherein the allergic type is allergic to the first type. A use of Lactobacillus as claimed in claim 1 for the production of food additive ingredients. A use of Lactobacillus as claimed in claim 1 for the production of animal or pet feed.