<i>Lactobacillus rhamnosus </i>RHT-3201 conjugated to polysaccharide polymer binder, and use thereof for prevention or treatment of atopic diseases
11268064 · 2022-03-08
Assignee
Inventors
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A23V2002/00
HUMAN NECESSITIES
A23L33/00
HUMAN NECESSITIES
International classification
A23L33/00
HUMAN NECESSITIES
Abstract
The present invention relates to heat-killed Lactobacillus rhamnosus conjugated to a polysaccharide polymer binder, a preparation method therefor and a use thereof. The heat-killed Lactobacillus rhamnosus conjugated to a polysaccharide polymer binder of the present invention has an excellent therapeutic effect for atopic diseases, and particularly has high industrial applicability because membrane adhesion competitiveness, which is an advantage of existing lactic acid bacteria, is significantly improved, thereby exhibiting dermatitis preventing, alleviating and treating effects of the same level as steroid-based drugs.
Claims
1. A heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder prepared by a method comprising the steps of: separating a fermentation culture medium prepared by culturing Lactobacillus rhamnosus into bacteria and a fermentation filtrate, wherein the Lactobacillus rhamnosus is Lactobacillus rhamnosus IDCC 3201 (KCTC 10833BP); selecting at least one polymeric polysaccharide from the group consisting of hyaluronic acid, alginate, maltodextrin, and chitosan; mixing the fermentation filtrate and the at least one polymeric polysaccharide to prepare a polymeric polysaccharide binder, wherein the at least one polymeric polysaccharide is mixed with the fermentation filtrate at a ratio of 0.0001% (w/v)-10% (w/v); heat-killing the bacteria, wherein the heat-killed bacteria comprises an adhesion promoter of the heat-killed bacteria, wherein the adhesion promoter is selected from the group consisting of lipoteichoic acid and peptidoglycan; and conjugating the polymeric polysaccharide binder to a surface of the heat-killed bacteria.
2. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the at least one polymeric polysaccharide is hyaluronic acid.
3. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 2, wherein the hyaluronic acid is mixed with the fermentation filtrate at a ratio of 0.0001-1% (w/v).
4. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the heat-killing is performed at a temperature range of 60−100° C.
5. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the heat-killing is performed for 10-120 minutes.
6. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the step of mixing further comprises a concentration process to obtain a highly concentrated polymeric polysaccharide binder.
7. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the at least one polymeric polysaccharide is mixed with the fermentation filtrate at a ratio of 0.0001% (w/v)-1% (w/v) in the step of mixing.
8. The heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1, wherein the at least one polymeric polysaccharide is mixed with the fermentation filtrate at a ratio of 0.0001% (w/v) 0.01% (w/v) in the step of mixing.
9. A composition comprising the heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1 as an active ingredient.
10. The composition of claim 9, wherein the composition is a pharmaceutical composition or a food composition.
11. The composition of claim 9, wherein the composition contains the heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder at a concentration of 10.sup.8 to 10.sup.10 CFU/g.
12. The composition of claim 10, wherein the food is selected from the group consisting of fermented milk, yogurt, beverages, milk beverages, food additives, and health functional food.
13. A method for treating or alleviating atopy or atopic dermatitis in a subject in need thereof, the method comprising administering a composition comprising the heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder of claim 1 as an active ingredient to the subject in an amount effective for treating or alleviating atopy or atopic dermatitis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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MODE FOR CARRYING OUT THE INVENTION
(27) Hereinafter, the present invention will be described in detail.
(28) However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
(29) Evaluation on Effects of Binders by Type
(30) <1-1> Preparation of Various Types of Binder Samples
(31) Lactobacillus rhamnosus IDCC 3201 (KCTC 10833BP) were cultured in MRS medium containing 0.1-1% whey powder at 37° C. for 24 hours. After culturing, bacteria and a culture filtrate were separated via centrifugation. Then, hyaluronic acid, alginate, maltodextrin, chitosan, and polyethylene glycol were added at 0.1% (w/v) to the culture filtrate, respectively, followed by stirring for 1 hour and then concentration, thereby preparing binders which were then freeze-dried. Each sample was diluted with a phosphate buffer solution to prepare a final 1 μg/ml of sample. Skimmed milk powder was used as a control.
(32) <1-2> Preparation of Spleen Cells
(33) 5-week old female BALB/c mice were boosted with 2 mg of aluminum hydroxide and 1 mg of ovalbumin through intraperitoneal injection, and second-boosted in the same manner after six days. On day 13, the spleen was harvested from each mouse and spleen cells were extracted, thereby preparing spleen cell liquid. Preferably, the spleen cells were prepared by the following method.
(34) The mouse spleen was aseptically harvested, and 10 Ml of Hanks' balanced salt solution (HBSS) was dropwise added. It was burst using tweezers on a 60-mesh net to collect a cell suspension, and then allowed to overlap 2.5 ml of fetal bovine serum, followed by standing for 10 minutes, thereby precipitating large masses. The large masses were suspended in an NH.sub.4Cl solution (pH 7.0) for 3-4 minutes to achieve the hemolysis of red blood cells. Then, the suspension was mixed with 2.5 ml of fetal bovine serum, followed by centrifugation at 1,500 RPM for 5 minutes. The precipitate was washed twice with HBSS, and then dispensed in DMEM medium containing 10% fetal bovine serum and 1 mg/ml ovalbumin at 5×10.sup.6 cells/ml, thereby preparing spleen cells.
(35) <1-3> Culture of Spleen Cells and Measurement of Cytokines
(36) 200 μl of spleen cell suspension (5×10.sup.6 cells/ml) and 10 μl (1 μg/ml) of each sample liquid prepared in <1-1> were added to each well of a 96-well plate, followed by culturing in a 5% CO.sub.2 incubator for 7 days. After the completion of the culture, IL-4 and IL-12 levels in the culture liquid were measured using the Cytoset kit (Biosource). The levels of IL-4 and IL-12 production by each sample were expressed as a rate of increase compared with the control as shown in Table 1.
(37) TABLE-US-00001 TABLE 1 Determination of binders Increase (%) compared with control IL-4/IL-12 Type of Binder IL-4 IL-12 ratio Hyaluronic acid 5 52 0.09 Alginate 15 29 0.51 Maltodextrin 9 36 0.25 Chitosan 17 38 0.44 Polyethylene 13 22 0.59 glycol
(38) As test results, the hyaluronic acid binder showed a 5% increase in IL-4 production and a 52% increase in IL-12 production. The alginate binder showed a 15% increase in IL-4 production and a 29% increase in IL-12 production. The maltodextrin binder showed a 9% increase in IL-4 production and a 36% increase in IL-12 production. The chitosan binder showed a 17% increase in IL-4 production and a 38% increase in IL-12 production. The polyethylene glycol binder showed a 13% increase in IL-4 production and a 22% increase in IL-12 production. Therefore, when the binder was prepared using a polymeric polysaccharide, the increase rate of IL-12 was relatively high, while the increase rate of IL-4 was relatively low. Of these, the hyaluronic acid showed the highest increase rate of IL-12 and a relatively low increase rate of IL-4. Following hyaluronic acid, maltodextrin, chitosan, and alginate showed favorable results in that order.
Example 2
(39) Evaluation on Effects of Binders by Concentration
(40) <2-1> Preparation of Samples by Concentration
(41) The effects of binders by concentration were evaluated while, among the polymeric polysaccharide materials, the hyaluronic acid showing the best effect in Example 1 was used as a representative material. The hyaluronic acid was added at an amount of 0.0001-1% (w/v) to the culture filtrate, followed by stirring for 1 hour, and then the mixture was concentrated under reduced pressure, thereby preparing hyaluronic acid binders with different concentrations, respectively, and these binders were freeze-dried. Each sample was diluted with a phosphate buffer to prepare a final 1 μg/ml of sample. Skimmed milk powder was used as a control.
(42) <2-2> Test on Cytokine Production
(43) The cytokine production rate in the spleen cells was investigated for the binder samples prepared in Example <2-1> by the same method as in Example <1-3>. The results are shown in Table 2 below.
(44) TABLE-US-00002 TABLE 2 Determination of concentration of hyaluronic acid added Incease (%) compared Hyaluronic acid with control IL-4/IL-12 concentration (w/v) IL-4 IL-12 ratio 0.0001 9 46 0.19 0.001 7 52 0.13 0.01 10 37 0.27 0.1 12 33 0.36 1 15 25 0.60
(45) As test results, as shown in Table 2 above, the optimal concentration of hyaluronic acid for preparing a hyaluronic acid binder was 0.001% (w/v), and here, the production of IL-4 was reduced compared with that in the control, and the production of IL-12 was increased compared with that in the control.
Example 3
(46) Evaluation on Effects of Bacteria by Heat-Killing Condition
(47) In order to determine the optimal bacteria heat-killing conditions, the heat-killing conditions according to the temperature and time were optimized and the adhesive ability to Caco-2 cells was evaluated, thereby selecting optimal conditions. More details for the test were as follows.
(48) <3-1> Preparation of Heat-Killed Bacteria Samples
(49) Lactobacillus rhamnosus bacteria were heat-killed at 60-120° C. for 10, 20, 30, 40, 50, and 60 minutes (Heater, EYELA OSB-2100, China), and cooled at 30° C. before being used as test samples. Each sample was washed twice with a phosphate buffer solution, re-suspended in a 1 ml of the same buffer solution, and diluted to 1×10.sup.8 cells/ml in serum-free DMEM, before being used for the test.
(50) <3-2> Evaluation on Adhesive Ability to Intestinal Mucosa
(51) The Caco-2 cell monolayer was prepared by inoculating Caco-2 cells (Korean Cell Line Bank) at 1.2×10.sup.5 cells/ml in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone, USA) supplemented with 10% (v/v) fetal calf serum and 20 μl/ml gentamicin, dispensing 1 ml of the resultant medium in each well of the 6-well tissue culture plate, culturing the cells for 7 days, and washing twice with a phosphate buffer solution.
(52) 1 ml of the sample liquid prepared in Example <3-1> was put into each well in which the Caco-2 monolayer was formed, followed by reaction for 90 minutes. 1 ml of DMEM, instead of heat-killed lactic acid bacteria, was used as a control. After the reaction, the supernatant was removed, and 1 ml of 0.04% (w/v) Tween 80 was added to recover the heat-killed lactic acid bacteria adhering to the Caco-2 cells, and the bacterial count was measured using a hemocytometer. The adhesion efficiency was calculated as a ratio of the adhering bacterial count to the initial bacterial count (see Table 3).
(53) TABLE-US-00003 TABLE 3 Adhesion rate according to heat-killing condition of bacteria Adhesion rate (%) to Caco-2 cells 60° C. 70° C. 80° C. 90° C. Heat killing time 10 44 50 56 45 (minutes) 20 48 53 68 49 30 56 63 71 54 40 60 66 76 42 50 62 71 80 35 60 63 72 85 32
(54) As shown in Table 3, the adhesion efficiency was most favorable when the heat-killing conditions of 80° C. and 60 minutes were applied. The constituent elements, such as lipoteichoic acid, existing on the cellular wall of lactic acid bacteria, inhibit the adhesion of harmful bacteria, such as E. coli and Salmonella, to the intestinal mucosal surface, and bind with TLR-2, which is one of the receptors of dendritic cells among intestinal mucosal cells, to be involved in the activation of human intestinal tract immunity. Therefore, the optimal bacteria heat-killing conditions are established to facilitate the emission of related factors existing on the cellular wall, so that the heat-killed lactic acid bacteria are allowed to adhere to the intestinal tract while being in competition with resident flora of the intestinal tract, and thus, ultimately, the activation of the intestinal tract immunity can be expected.
Example 4
(55) Preparation of Heat-Killed Lactobacillus rhamnosus Conjugated to Polymeric Polysaccharide Binder
(56) The bacteria heat-killed at 80° C. for 60 minutes in Example 3 was mixed with 0.001% of the hyaluronic acid binder prepared in Example 2 in which the Lactobacillus rhamnosus culture filtrate was concentrated, followed by reaction. A carrier, such as starch, as a vehicle, was mixed with the resultant reaction material, followed by a drying process, thereby preparing heat-killed Lactobacillus rhamnosus conjugated to a polymeric polysaccharide binder (hereinafter, expressed by “RHT-3201”). The immunity promotion effect was relatively compared between RHT-3201 prepared as above and Lactobacillus rhamnosus IDCC 3201 as a probiotic, by the same method as in Example <1-3> (see Table 4).
(57) TABLE-US-00004 TABLE 4 Comparison of immunity promotion effect between conventional probiotic bacterium and RHT-3201 according to the present invention RHT-3201 Probiotic preparation IL-4 increase (%) compared 3 20 with control IL-12 increase (%) compared 67 29 with control IL-4/IL-12 ratio 0.04 0.68
(58) As shown in Table 4, it was verified that the regulating effect of cytokines involved in Th1/Th2 was 17-fold higher in RHT-3201 lactic acid bacteria of the present invention, compared with an existing probiotic. It is suggested that the RHT-3201 was prepared in such a form that it favorably binds to a corresponding receptor of the intestinal mucosa, such as TLR-2 during the preparing process.
Example 5
(59) Structural Analysis Through Electron Microscope (FE-SEM)
(60) As for the phase of RHT-3201 prepared in Example 4, structural analysis was conducted through photographing using an electron microscope (FE-SEM, Model: LEO SUPRA 55, GENESIS 2000 (Carl Zeiss, EDAX)) according to each preparation process. The structural analysis results using the electron microscope according to the RHT-3201 preparation steps are shown in
(61) As shown in
Example 6
(62) Mass Production of RHT-3201
(63) In order to investigate reproducibility when RHT-3201 was mass-produced by the preparation method in Example 4 above, the intestinal adhesion rate was compared between a probiotic and mass-produced RHT-3201 (see Table 5).
(64) Specifically, Lactobacillus rhamnosus IDCC 3201 was cultured at 37° C. overnight in a fermentation tank with a production scale of 1 ton. The culture filtrate and the bacteria were separated through centrifugation, and hyaluronic acid having 0.001% (w/v) of the volume of the culture liquid was added to the culture filtrate, followed by concentration under reduced pressure at 50° C., thereby preparing a binder, which is to be conjugated to the surface of the heat-killed bacteria. The bacteria were subjected to a heat treatment at 80° C. for 60 minutes, thereby preparing heat-killed bacteria. In addition, the binder and the heat-killed bacteria were mixed, homogenized, and dried, thereby preparing RHT-3201. The Lactobacillus rhamnosus IDCC 3201 probiotic used as a control was prepared according to an ordinary process for preparing probiotic raw materials. Specifically, the bacteria were obtained from the same culture liquid, suspended in 20 ml of a phosphate buffer solution, and freeze-dried, thereby preparing a bacterial powder.
(65) TABLE-US-00005 TABLE 5 Comparison of adhesion rates bewteen conventional probiotic preparation and RHT-3201 according to the present invention RHT-3201 Probiotic preparation Adhesion rate (%) 85 54
(66) As shown in Table 5, it was verified that the adhesion rate of RHT-3201 showed a 57% improvement compared with the Lactobacillus rhamnosus probiotic preparation.
Example 7
(67) Evaluation on Intake Stability
(68) <7-1> Acid Stability
(69) A lactic acid bacteria preparation is exposed to gastric acids when passing through the stomach of the digestive tract. While this environment was reproduced in vitro conditions, the comparison of acid stability between the probiotic bacterium and RHT-3201 of the present invention was conducted. More specifically, 10% HCl was dropped into MRS medium to adjust pH to 2.3 and 2.5, and then the MRS was sterilized for use. 1 g of the probiotic and RHT-3201 samples were respectively put into the pH-adjusted MRS medium, followed by reaction for 0, 1, and 2 hours. The adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells by the same method as in Example <3-2>. Here, the lactic acid bacterium used in the test was Lactobacillus rhamnosus IDCC 3201. RHT-3201 prepared by the preparing process in Example 6 and the probiotic were used for the test.
(70) TABLE-US-00006 TABLE 6 Acid stability measurement results of RHT- 3201 according to the present invention Ahe- Adhe- pH sion pH sion Test 2.3(×10.sup.8 CFU/ml) rate 2.5(×10.sup.8 CFU/ml) rate sample 0 1 h 2 h (%) 0 1 h 2 h (%) Probiotic 1 0.1 0.04 4 1 0.19 0.07 7 bacterium RHT-3201 1 0.7 0.34 34 1 0.83 0.48 48
(71) As shown in Table 6, it was verified that, compared with the probiotic bacterium, the adhesion efficiency of RHT-3201 of the present invention maintained stable even when exposed to an acid for 2 hours.
(72) <7-2> Bile Stability
(73) The enterohepatic circulation of bile acids is attained such that the bile acids are produced from the liver, leak through the biliary tract, flow into the small intestine, are then 95% absorbed into the ileum, which is the end section of the small intestine, and again enter the liver. The bile acids affect the adhesive ability of lactic acid bacteria settled in the small intestine. Therefore, the difference in the adhesion rate between the probiotic bacterium and RHT-3201 of the present invention when exposed to bile acids was compared. More specifically, the MRS media with and without 0.3% bile acids added thereto was used after being sterilized. Then, 1 g of the probiotic bacterium and RHT-3201 of the present invention were inoculated in the media, respectively, followed by reaction for 2 hours. Subsequently, the adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells by the same method as in Example <3-2>.
(74) Here, the lactic acid bacterium used in the test was Lactobacillus rhamnosus IDCC 3201. RHT-3201 prepared by the preparing process in Example 6 and the probiotic were used for the test.
(75) TABLE-US-00007 TABLE 7 Bile stability results of RHT-3201 according to the present invention MRS MRS + 0.3% Adhesion Test sample (×10.sup.8 CFU/ml) bile (×10.sup.8 CFU/ml) rate (%) Probiotic 1 0.23 23 bacterium RHT-3201 1 0.57 57
(76) As shown in Table 7, it was verified that, when exposed to bile acids for 2 hours, the adhesion efficiency of RHT-3201 maintained at 57%, which was about 2.5-fold higher than that of the probiotic bacterium.
Example 8
(77) Evaluation on Stability Over Time by Temperature
(78) <8-1> Comparative Test of Stability Over Time at 4° C. Between RHT-3201 and a Probiotic Bacterium
(79) RHT-3201 of the present invention prepared in Example 6 above and a probiotic bacterium were stored in a refrigerator at 4° C. for 365 days, and then the adhering bacterial count per 1 g of a raw material was measured over time. The results are shown in Table 8. The adhering bacterial count was measured by the same method as in Example <3-2>. The adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells.
(80) TABLE-US-00008 TABLE 8 Comparative test of stability over time at 4 C. between RHT- 3201 according to the present invention and a probiotic bacterium Stability over time at 4
0 60 180 365 Residual Test sample day days days days adhesion rate (%) Probiotic 65% 52% 34% 19% 29.2 bacterium RHT-3201 82% 71% 52% 45% 54.8
(81) <8-2> Comparative Test of Stability with Time at 15° C. Between RHT-3201 and Probiotic
(82) RHT-3201 of the present invention prepared in Example 6 above and a probiotic were stored in a refrigerator at 15° C. for 365 days, and then the adhering bacterial count per 1 g of a raw material was measured over time. The results are shown in Table 9. The adhering bacterial count was measured by the same method as in Example <3-2>. The adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells.
(83) TABLE-US-00009 TABLE 9 Comparative test of stability over time at 15° C. between RHT-3201 according to the present inventio and a probiotic bacterium Stability over time at 15 0 60 180 365 Residual Test sample day days days days ahesion rate (%) Probiotic 65% 46% 29% 15% 23.1 bacterium RHT-3201 82% 66% 59% 38% 46.3
(84) <8-3> Comparative Test of Stability Over Time at 25° C. Between RHT-3201 and a Probiotic Bacterium
(85) RHT-3201 of the present invention prepared in Example 6 above and a probiotic bacterium were stored in a refrigerator at 25° C. for 365 days, and then the adhering bacterial count per 1 g of a raw material was measured over time. The results are shown in Table 10. The adhering bacterial count was measured by the same method as in Example <3-2>. The adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells.
(86) TABLE-US-00010 TABLE 10 Comparative test of stability over time at 25° C. between RHT-3201 according to the present invention and a probiotic bacterium Stability with time at 25 0 60 180 365 Residual Test sample day days days days adhesion rate (%) Probiotic 65% 39% 25% 12% 18.4 bacterium RHT-3201 82% 56% 49% 35% 42.6
(87) <8-4> Comparative Test of Stability Over Time at 37° C. Between RHT-3201 and a Probiotic Bacterium
(88) RHT-3201 of the present invention prepared in Example 6 above and a probiotic were stored in a refrigerator at 37° C. for 365 days, and then the adhering bacterial count per 1 g of a raw material was measured over time. The results are shown in Table 11. The adhering bacterial count was measured by the same method as in Example <3-2>. The adhesion rate was analyzed by measuring the adhering bacterial count to Caco-2 cells.
(89) TABLE-US-00011 TABLE 11 Comparative test of stability over time at 37° C. between RHT-3201 according to the present invention and a probiotic bacterium Stability over time at 37 0 60 180 365 Residual Test sample day days days days adheson rate (%) Probiotic 65% 29% 15% 8% 12.3 bacterium RHT-3201 82% 46% 44% 32% 39.0
Example 9
(90) Regulation of Cytokine Production by RHT-3201 in Mild Atopic Dermatitis-Induced In Vitro Models
(91) In mild atopic dermatitis-induced mouse models, the effects of RHT-3201 according to the present invention, a probiotic, and a killed bacterial preparation, which were produced from the same lactic acid bacteria, were evaluated, while the specific test method was as follows.
(92) <9-1> Preparation of Samples
(93) RHT-3201 of the present invention and a probiotic preparation were prepared by the same method as in Example 6 above, using a culture liquid prepared by culturing Lactobacillus rhamnosus IDCC 3201 in MRS medium containing 0.1-1% whey powder at 37° C. for 24 hours.
(94) For killed bacteria, bacteria was obtained from the culture liquid, suspended in 500 μl of a phosphate buffer solution, and dried through freeze-drying. The dried bacteria was weighed, and suspended in a sterile phosphate buffer to reach 300 mg/ml. The suspension was heat-treated at 100° C. for 30 minutes, so the bacteria were killed. The three types of samples were diluted with a phosphate buffer solution to prepare 1 μg/ml of final samples, respectively. Skimmed milk powder was used as a control.
(95) <9-2> Culture of Spleen Cells and Measurement of Cytokines
(96) Spleen cells were prepared by the same method as in Example <1-2>. 200 μl of spleen cell suspension (5×10.sup.6 cells/ml) and 10 μl (1 μg/ml) of each sample liquid prepared in Example <9-1> were added to each well of a 96-well plate, followed by culturing in a 5% CO.sub.2 incubator for 7 days. After the completion of the culture, IL-4 and IL-12 levels in the culture liquid were measured using the Cytoset kit (Biosource). The levels of IL-4 and IL-12 production by each sample were expressed as a rate of increase thereof compared with the control as shown in Table 12.
(97) TABLE-US-00012 TABLE 12 Increase rate of cytokines from spleen cells upon the addition of lactic acid bacteria sample Increase (%) compared with control IL-4/IL-12 Sample IL-4 IL-12 ratio Lactobacillus rhamnosus IDCC 20 29 0.68 3201 (probiotic bacterium) Lactobacillus rhamnosus IDCC 12 34 0.35 3201 (killed bacterium) RHT-3201 3 67 0.04
(98) As test results, a 20% increase in IL-4 production and a 29% increase in IL-12 production were shown in the Lactobacillus rhamnosus IDCC 3201 probiotic bacterium. A 12% increase in IL-4 production and a 34% increase in IL-12 production were shown in the killed bacteria. A 3% increase in IL-4 production and a 67% increase in IL-12 production were shown in RHT-3201. Therefore, it was verified that RHT-3201 of the present invention, which showed a significant increase in IL-12 production and a relatively mild increase in IL-4 production, had the most excellent effect in the regulation of cytokine production.
Example 10
(99) Therapeutic Effect of RHT-3201 According to the Present Invention in Severe Atopic Dermatitis-Induced In Vivo Models
(100) <10-1> Preparation of Test Material and Inducible Antigen
(101) RHT-3201 according to the present invention was used as a test material, while steroid-based dexamethasone (Sigma Co. Ltd), which is used as a main therapeutic agent for severe atopic dermatitis, was used as a positive control. An extract of Dermatophagoides farina, which is a major species of house dust mite, was used as an atopy inducing material. The extract was purchased in an ointment form from Central Lab. Animal Inc.
(102) The dexamethasone was prepared by being dissolved in ethanol into a concentration of 0.1% (w/v), and RHT-3201 of the present invention was prepared by being dissolved in distilled water to doses of 1×10.sup.8 CFU/0.5 ml/mouse, 1×10.sup.9 CFU/0.5 ml/mouse, and 1×10.sup.10 CFU/0.5 ml/mouse, respectively. All the samples to be administered were prepared on the testing day. The dexamethasone was coated on the skin of a testing subject at 100 μl for each subject twice a week, while RHT-3201 according to the present invention was orally administered at 0.5 ml for each subject using a mouse zonde once a day. Distilled water was administered for a normal group and an atopy control group. The administration was conducted for 8 weeks in all the test groups.
(103) <10-2> Preparation and Breeding of Test Animals
(104) Female NC/Nga mice (6-week aged) were purchased from Central Lab. Animal Inc., and acclimated for 1 week before use. The breeding environment was as follows: constant temperature (22±2° C.) and constant humidity (50-60%) were maintained, while cycles of light (08:00˜20:00) and dark (20:00˜08:00) were controlled at an interval of 12 hours. Three animals were allocated and bred in each polysulfone cage, being fed with free access to a test diet and water for 24 hours.
(105) <10-3> Induction of Severe Atopic Dermatitis
(106) The dosal areas up to the upper portions of auricles of the NC/Nga 7-week aged mice were completely shaved, and 150 μl of an aqueous solution of 4% sodium dodecyl sulfate (SDS) was then sprayed on the shaved parts. After complete drying, 100 mg of an ointment for dust mites (mite extract) was uniformly coated on the shave parts. The ointment for dust mites (mite extract) was coated twice a week for three weeks, a total of six times, to induce moderate or more severe dermatitis. Thereafter, the ointment was coated once a week during an administration period to the test groups (8 weeks), thereby maintaining severe atopic dermatitis.
(107) TABLE-US-00013 TABLE 13 Sever atopic dermatitis inductin scores in each model before treatement with test materials Severe atopic dermatitis score Mean ± SE (n = 6) Normal group 0 Atopy control (Vehicle) 11.0 ± 0.4 Dexamethasone (Postive control) 10.8 ± 0.4 RHT-3201 1 × 10.sup.8 CFU/mouse 10.8 ± 0.4 1 × 10.sup.9 CFU/mouse 10.7 ± 0.3 .sup. 1 × 10.sup.10 CFU/mouse 10.7 ± 0.3
(108) As shown in Table 13, as a result of inducing severe atopic dermatitis by coating the ointment for dust mites (mite extract) on the NC/Nga mice for three weeks, the mean score for each group was 10 or higher, so the NC/Nga mice with severe atopic dermatitis were prepared.
(109) <10-4> Evaluation of Atopic Dermatitis
(110) In the present evaluation, the severity of atopic dermatitis was expressed as a total of evaluation scores of five items, using SCORAD (SCORing Atopic Dermatitis), which is a generally used method for clinical macroscopic evaluation. The items were erythema, dry skin, edema, excoriation, and lichenification. The scores for the five items (no symptom (score 0), mild symptom (score 1), moderate symptom (score 2), severe symptom (score 3) for each item) were added up, while the evaluation score was determined between the lowest score of 0 (a state in which there is no symptom) and the highest score of 15 (a state in which the symptoms for all items were severe). The severity of the mouse skin lesions was evaluated every week (see
(111) In
(112) As a result of investigating the effect of alleviating the severe atopic dermatitis after 8-week treatment, it was verified from the results in
(113) It has been generally known that lactic acid bacteria preparations need to be taken in large quantities without any report about optimal concentrations of such preparations for obtaining the effect of alleviating the severe atopic dermatitis. The reason is that, as for lactic acid probiotics, the lactic acid bacteria are killed in large quantities by gastric and bile acids while passing through the gastrointestinal tract, causing the reduction in adhesion efficiency to the intestinal mucosa capable of exhibiting immune actions, and thus are excreted from the body. Unlike this, RHT-3201 of the present invention was shown to definitely possess a therapeutic effect at its different concentrations. The ×10.sup.9 and ×10.sup.10 intake units showed similarity within an error range regarding the efficacy of alleviating the severe atopic dermatitis. The reason is that the combination number with the toll-like receptor distributed in dendritic cells of the intestinal mucosa is saturated to have a threshold.
(114)
(115) <10-5> Scratching Behavior and Body Weight Measurement
(116) The number of scratching behaviors for 15 minutes was counted once a week from week 1 of administration of a test drug. Only the number of scratching behaviors using a hind leg was counted in order to avoid counting scratching behaviors due to the other causes besides scratching behaviors due to pruritus. Meanwhile, the weight loss tends to occur due to side effects of drugs and stress at the time of inducing severe atopic dermatitis. In order to investigate whether the side effect of weight loss occurs by the test materials of the present invention, the test animals were weighed during the administration period of the test materials.
(117) As shown in
(118) In addition, as shown in
(119) <10-6> Measurement of Serum IgE Level
(120) After the administration of test drugs for eight weeks, the blood was taken through the retro-orbital venous plexus, and centrifuged at 12,000 RPM for 10 minutes to separate the serum. The serum IgE level was measured using an ELISA kit (SHIBAYAGI, Japan).
(121) As shown in
(122) <10-7> Cytokine Producing Ability of Lymph Node Cells
(123) After the end of administration of test materials, each of the severe atopic dermatitis-induced NC/Nga mice was sacrificed. The size and weight of the axillary lymph nodes were measured. In addition, the cell suspensions obtained from the axillary lymph nodes were dispensed in the RPMI-1640 medium (containing 10% FBS, 1% penicillin, and 1% streptomycin) to a concentration of 5×10.sup.6 cells/ml. The extract of dust mite (D. farina) was added to obtain a final concentration of 10 μg/ml, and cultured for 48 hours in a 5% CO.sub.2 incubator at 37° C. The supernatant was taken to measure IFN-γ, IL-4, IL-10, and IL-12 production yields using an ELISA kit (IFN-γ & IL-4 manufacturer: R&D SYSTEMS, USA; and IL-10 & IL-12 manufacturer: SIGMA ALDRICH, USA).
(124) As shown in
(125) For the confirmation of the cytokine producing ability according to the administration of RHT-3201, the axillary lymph node in the severe atopic dermatitis-induced NC/Nga mice was exposed to the dust mite antigen, and then the levels of cytokines involved in the immune response were measured. The results are shown in FIGS. 14 to 21. It was verified that the administration of RHT-3201 of the present invention significantly reduced Th1-type cytokine IFN-γ (see
(126) For the comparison of action mechanism of RHT-3201 on severe atopic dermatitis, the ratio of Th1-type cytokine/Th2-type cytokine was shown for the levels of cytokines expressed in each administration group. As a result, as shown in
(127) For the confirmation that the action factor involved in the inhibition of regulatory T cells (Treg) as another action mechanism of RHT-3201 of the present invention on severe atopic dermatitis is cytokine IL-10, the IFN-γ/IL-10 ratio (see
(128) <10-8> Histopathological Observation
(129) The dorsal skins of the NC/Nga mice were harvested, fixed in 10% formalin, and paraffin-embedded, and thinly cut to a thickness of 4 μm, thereby preparing a slide. Subsequently, the changes in the thickness of the epidermis and the dermis were observed by hematoxylin & eosin (H&E) staining, while the mast cells were confirmed by toluidine blue staining. The skin thickness and the number of mast cells were calculated through observation using an optical microscope of 100 and 400 magnifications.
(130) As a result of observing the dorsal skin tissues of the NC/Nga mice through H&E staining, as shown in
(131) As a result of observing the dorsal skin tissues of the NC/Nga mice through toluidine blue staining, it was verified that the mast cells further infiltrated into the skin tissues in the vehicle group with atopic dermatitis induced due to the exposure to dust mites, which is an atopic dermatitis inducing antigen material, compared with the normal group (see
INDUSTRIAL APPLICABILITY
(132) As set forth above, the present invention relates to heat-killed Lactobacillus rhamnosus conjugate to a polymeric polysaccharide binder, a method for preparing the same, and a use thereof. The heat-killed Lactobacillus rhamnosus conjugate to a polymeric polysaccharide binder of the present invention possesses an excellent therapeutic effect on atopic diseases, especially, significantly improves the adhesive competitiveness to the intestinal mucosa of conventional lactic acid bacteria preparations, and exhibits a preventing, alleviating, or treating effect on atopic dermatitis at an equivalent level to a steroid-based drug. Therefore, the present invention is highly industrial applicable.