Patent Grant
8021868
1. Field of the Invention
The invention relates to novel strains of lactic acid bacteria. The composition comprising of Lactobacillus acidophilus PM-A0002, Lactobacillus gasseri PM-A0005, Lactobacillus salivarius PM-A0006, Lactobacillus johnsonii PM-A0009 and Lactobacillus acidophilus PM-A0013 and their use for treating allergy related diseases.
2. Background
The recent increase in allergic diseases such as atopic dermatitis, atopic eczema, and allergic rhinitis has been, and continues to be, a serious social problem in many countries. There is a theory which implies that allergy and asthma have increased during the last 20 to 50 years because of a reduced exposure in childhood to bacterial and viral infections brought about by improvements in public health measures such as vaccination and sanitation. Allergic diseases are reported to be caused by a skew in the balance between T helper type 1 (Th1) and 2 (Th2) cells. Classical allergy is a type 2 hypersensitivity reaction mediated by the interaction of mast cells and eosinophils coated with allergen-specific IgE and a cross-lining allergen. Few lactic acid bacteria have been shown to stimulate Th1 related cytokines secretion, they have the potential to either prevent or ameliorate disease conditions or both.
The term probiotic is derived from the Greek and literally translates as ‘for-life’. It was first used by Lilly et al in 1965 (Lilly D M and Stillwell R H, 1965, Science, 14: 747-748). Probiotics are live microbial food supplements that can change either the composition and/or the metabolic activities of the microbiota or modulate immune system reactivity in a way that benefits health. Rarker described probiotics as “organisms and their secreted substances which contribute to an intestinal microbial balance.” A recent detailed definition of probiotics is “a preparation of or a product containing viable, defined microorganisms in sufficient numbers to alter the existing microflora (by displacement or colonization) in the intestine of the host and thereby exert beneficial health effects (Schrezenmeir J, de Vrese M, 2001, Am J Clin Nutr, 73: 361S-364S). Probiotics are now commonly available over the counter and in the chiller cabinet of every supermarket as bio-yoghurts, probiotic drinks or food supplements. Few different microorganisms have been used as probiotics, the most common being the lactic acid bacteria. Lactic acid bacteria are members of the commensal microflora of a healthy human colon. They can be found on food particles in the lumen of the gut and in the mucus overlying the epithelial cell barrier, putting them in very close proximity to the human host (Macfarlanc S, Furrie E, Cummings J H and Macfarlanc G T, 2004, Clinical Infectious Diseases, 38: 1690-1699). The possible function of probiotics are varied and include the production and secretion of antimicrobial substances, a stimulus to the host's immune responses, and displacement of pathogen colonization. They provide health benefits to the host by stimulating metabolic activities or by protecting against conditions such as intestinal infection, food allergies, and colon cancer.
Otherwise, the current state of evidence suggests that probiotic effects are strain specific. Strain identity is important to link a strain to a specific health effect as well as to enable accurate surveillance and epidemiological studies. So we must use in vitro tests that to screen potential probiotic strains. In vitro tests are useful to gain knowledge of strains and the mechanism of the probiotic effect. However, it was noted that the currently available tests are not fully adequate to predict the functionality of probiotic microorganisms in the human body. It was also noted that in vitro data available for particular strains are not sufficient for describing them as probiotic. Probiotics for human use will require substantiation of efficacy with human trials. Appropriate target-specific in vitro tests that correlate with in vivo results are recommended. For example, in vitro bile salts resistance was shown to correlate with gastric survival in vivo. The seven points of the main currently used in vitro tests for the study of probiotic strains are first is resistance to gastric acidity, second is bile acid resistance, third is adherence to mucus and/or human epithelial cells and cell lines, forth is antimicrobial activity against potentially pathogenic bacteria, fifth is ability to reduce pathogen adhesion to surfaces, sixth is bile salt hydrolase activity, seventh is resistance to spermicides (Guidelines for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002).
Allergic children in Estonia and Sweden were found to be less often colonized with lactobacilli compared with nonallergic children (Björksten B, Naaber P, Sepp E and Mikelsaar M, 1999, Clinical and Experimental Allergy, 29: 342-346).
Lactobacilli are thought to induce Th1 reaction and improve allergic diseases (Cross M L, Stevenson L M and Gill H S, 2001, International Immunopharmacology, 1: 891-901).
Furthermore, orally administered heat-treated Lactobacillus casei (strain Shirota) was found to inhibit IgE production induced by ovalbumin in mice serum (Matsuzaki T, Yamazaki R, Hashimoto S and Yokokura T, 1998, Journal Dairy Science, 81: 48-53).
Moreover, intraperitoneally injected heat-treated Lactobacillus plantarum L-137 was demonstrated to suppress IgE production in response to a casein allergy in mice (Murosaki S, Yamamoto Y, Ito K, Inokuchi T, Kusaka H, Ikeda H and Yoshikai Y, 1998, J. Allergy Clin. Immunol, 102: 57-64).
Oral administration of lysed Enterococcus faecalis FK-23 resulted in a decrease of peritoneal accumulation of eosinophils induced by ragweed pollen (Shimada T, Cheng L, Ide M, Fukuda S, Enomoto T, Shirakawa T, 2003, Clin Exp Allergy, 33: 684-687).
In humans, Lactobacillus rhamnosus strain GG administered in the perinatal period reduced the incidence of atopic eczema in children at risk during the first 2 years of life (Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P and Isolauri E, 2001, Lancet 357: 1076-1079) and beyond infancy (Kalliomäki M, Salminen S, Poussa T, Aivilommi H and Isolauri E, 2003, Lancet 361: 1869-1871).
Lactobacillus rhamnosus 19070-2 and Lactobacillus reuteri DSM 122460 improved moderately the clinical severity of eczema in children with atopic dermatitis (Rosenfeldt V; Benfeldt E, Nielsen S D, Michaelsen K F, Jeppesen D L, Valerius N H and. Paerregaard A, 2003, J. Allergy Clin. Immunol. 111:389-395).
The invention related to novel lactic acid bacterial strains comprising of Lactobacillus acidophilus PM-A0002, Lactobacillus gasseri PM-A0005, Lactobacillus salivarius PM-A0006, Lactobacillus johnsonii PM-A0009, Lactobacillus acidophilus PM-A0013.
In another aspect, the invention may be said broadly to consist of a composition of a biologically pure culture of any one of Lactobacillus acidophilus PM-A0002, China Center for Type Culture Collection (CCTCC) deposit number M 207038 dated Apr. 6, 2007, Lactobacillus gasseri PM-A0005, CCTCC deposit number M 207039 dated Apr. 6, 2007, Lactobacillus salivarius PM-A0006, CCTCC deposit number M 207040 dated Apr. 6, 2007, Lactobacillus johnsonii PM-A0009, CCTCC deposit number M 207041 dated Apr. 6, 2007, Lactobacillus acidophilus PM-A0013, CCTCC deposit number M 207042 dated Apr. 6, 2007 in an anti-allergy stimulating concentration, with a physiologically acceptable excipient or diluent.
In one embodiment said composition contains any one or more of said strains.
Preferably said physiologically acceptable excipient or diluent is a food.
Preferably said food is any one of cultured milk, yoghurt, cheese, milk drink, milk powder, coffee or tea.
Alternatively said composition is a pharmaceutical composition and said excipient or diluent is pharmacologically acceptable excipient or diluent.
In another aspect, the invention may be said broadly to consist of a method of enhancing IL12 or IFN-gamma which are Th1 cytokines and modifying conditions of allergy which comprise administering to a mammal any one of the above biologically pure cultures at an anti-allergy stimulating dosage rate.
In one embodiment said composition contains any one or more of said strains.
Preferably said physiologically acceptable excipient or diluent is a food.
Preferably said food is any one of cultured milk, yoghurt, cheese, milk drink, milk powder, coffee or tea.
Alternatively said composition is a pharmaceutical composition and said excipient or diluent is pharmacolohically acceptable excipient or diluent.
Anti-allergy, physiologically acceptable, biologically pure strains of homologues or mutants of any one of the strains:
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
In another embodiment the invention may be said broadly to consist of a method of anti-allergy which comprises administering to a mammal any one of the above biologically pure cultures at an immunostimulating dosage rate.
In another embodiment substantially biologically pure cultures of one or more of the above-defined strains are present.
Preferably said culture is administered in the form of a composition with a physiologically acceptable excipient or diluent.
Preferably said physiologically acceptable excipient or diluent is a food.
Preferably said food is cultured milk, yoghurt, cheese, milk drink or milk powder.
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any one or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
The invention may be said broadly to consist of a composition of a biologically pure culture of any one of Lactobacillus acidophilus PM-A0002, CCTCC deposit number M 207038 dated Apr. 6, 2007, Lactobacillus gasseri PM-A0005, CCTCC deposit number M 207039 dated Apr. 6, 2007, Lactobacillus salivarius PM-A0006, CCTCC deposit number M 207040 dated Apr. 6, 2007, Lactobacillus johnsonii PM-A0009, CCTCC deposit number M 207041 dated Apr. 6, 2007, Lactobacillus acidophilus PM-A0013, CCTCC deposit number M 207042 dated Apr. 6, 2007.
The invention comprising of five latic acid bacterial strains are deposited at China Center for Type Culture Collection. This deposited center address is Wuhan University, Wuhan, China. Zip Code is 430072. Table 1 shows the detailed deposited data.
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
These five Lactobacilli were discovered special function which is decrease and modify condition of allergy. The allergy conditions which contain airway hyperreactivity and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, hypersensitive pneumonia, extrinsic allergic alveolitis, urticaria, eczema, anaphylaxis, angioedema, allergic and migraine headache, certain gastrointestinal disorders, and asthma.
In Vitro Test
Preparation of Human Peripheral Blood Mononuclear Cells (PBMC) and Determination of Cytokines.
PBMC were obtained from healthy donors by centrifugation with Ficoll-Hypaque, and the light-density fraction from the 42.5-50% interface was recovered. Cytokines including and IFN-gamma in the culture supernatants from PBMC and lactobacillus cells were cultured together. PBMC (1×106 cells/well) and lactobacillus cells were incubated at a ratio of 1:10 at 37° C. for 48 h. The culture supernatant was obtained from 48 h cultures. The content of IFN-gamma in the culture supernatants was assayed by the sandwich ELISA method.
Preparation of Human Dendritic Cells and Determination of Cytokines
Human dendritic cells were generated from PBMC. CD14+ cells were purified by positive selection using anti-CD14+ microbeads in conjunction with the MiniMACS system by following the manufacturer's instructions (Miltenyi Biotec., Auburn, CA). The CD14+ cells were cultured at 1×106 cells per 1 mL of RPMI-1640 containing 10% fetal bovine serum in 24-well plates with human granulocyte macrophage-colony stimulating factor (hGM-CSF; 800 U/mL) and human IL-4 (500 U/mL). Fresh medium containing hGM-CSF and IL-4 was added every 2-3 days. Human monocyte-derived dendritic cells were used routinely at day 6 of culture. Cytokines including IL-12 in the culture supernatants from dendritic cells and lactobacillus cells were cultured together. Dendritic cells (1×106 cells/well) and lactobacillus cells were incubated at a ratio of 1:10 at 37° C. for 48 h. The culture supernatants were obtained from 48 h cultures. The content of IL-12 in the culture supernatants was assayed by the sandwich ELISA method.
Acid and Bile Tolerance
Acid and bile tolerance of the lactic acid bacteria were studied by incubating in MRS broth supplemented with pH 2.5 and 1.5% oxgall. The pH was adjusted to 2.5 with hydrochloric acid and lactic acid bacteria were incubated at 37° C. for 3 h. The 1.5% oxgall cultures were incubated at 37° C. for 4 h. Each of these five lactobacilli were subcultured at least three times before experimental use, followed by centrifugation after the final subculture, inoculation into the broth, and growth monitoring using the plate count method. Acid and bile tolerance were determined by comparing the final plate count after 24 h.
Adhesion to Intestinal Cells
Caco-2 cells in a monolayer were washed twice with PBS, 1.5 mL of MEM was added to each dish, and the dishes were incubated for 1 h before inoculation of lactic acid bacteria. Overnight cultures of lactic acid bacteria were appropriately diluted with MEM to give a lactobacillus cells concentration of approximately 108 cfu/mL, and 1.5 mL was used to inoculate the Caco-2 cells. After incubation for 1 h at 37° C., all of the dishes were washed four times with PBS to release unbound lactic acid bacteria. The lactic acid bacteria were then fixed with 3 mL of methanol and incubated for 5 to 10 min at room temperature. After removal of the methanol, the cells were stained with Gram's stain. Each adhesion assay was performed in triplicate with cells from three successive passages (8 to 13 cell passages).
In Vivo Test
Animals Model for Asthma and Study Protocol
Female BALB/c mice, aged between six and eight weeks, were obtained from the College of Medicine Laboratory Animal Center, National Taiwan University (they originating from the Jackson Laboratory, Bar Harbor, Me., USA), and were divided into different groups for each experiment. There were 14 test mice in each group. Animals were actively sensitized by intra-peritoneal injection of 50 μg ovalbumin emulsified in 4 mg aluminum hydroxide in the total volume of 100 μL on Days 2, 16, 30 and 44. After actively sensitized, animals were induced airway responsiveness by intra-nasal dripping of 100 μg/10 μL ovalbumin on Days 54 and 55. The process was shown in
Determination of Ovalbumin-Specific IgE
The level of ovalbumin-specific IgE was determined by ELISA. Protein high-binding plates were coated with 100 μL of 0.5 mg ovalbumin diluted in coating buffer (0.1 M NaHCO3, pH=8.2). Following overnight incubation at 4° C., plates were washed three times and blocked with 1% (wt/vol) BSA-PBS buffer for 2 h at 25° C., plates were washed three times. Sera were used for ovalbumin-specific IgE measurement. Following overnight incubation at 4° C., plates were washed four times. Biotin-conjugated monoclonal rat anti-mouse IgE was used at a 1:100 dilution and was added for incubation for 1-2 h at 25° C., plates were washed five times. Avidin-horseradish peroxidase conjugated (1:1000) was then added and incubation continued for 1 h at 25° C., plates were washed six times. The color reaction was developed with the addition of ABTS (2,2′-Azino-bis-3-Ethylbenzthiazoline-6-Sulfonic Acid) for 30 minutes at 25° C. To add 5% SDS for stopped reaction. Plates were read in a microplate autoreader at a wavelength of 450 nm. The results were expressed by ELISA unit. ELISA unit were calculated by the following equations:
ELISA unit=(Abs.sample−Abs.blank)/(Abs.positive control−Abs.blank)
Determination of Airway Responsiveness
Using barometric whole-body plethysmography (WBP; Biosystem X A, Buxco Electronics Inc. Sharon, Conn., USA), the response to inhaled methacholine for conscious. Mice were obtained and averaged for 3 min. Aerosized saline, followed by increasing concentrations of methacholine (ranging from 0, 6.25, 12.5, 25 and 50 mg/mL), was nebulized for 3 min, following which reading were taken and averaged for 3 min, this occurring subsequent to each nebulization event. Airway responsiveness was expressed as the Penh value per dose of methacholine.
Assessment of Cells and Supernatant in Bronchoalveolar Lavage Fluids (BALF)
Following the measurement of lung-function parameters, mice were cannulated and lavaged with 1 mL aliquots of 2% BSA in HBSS (Hank's balanced salt solution) buffer through a polyethylene tube introduced through the tracheostomy. Lavage fluid was collected and then centrifuged (500×g for 10 min at 4° C.), and the cell pellet so obtained was resuspended in 1 mL of 2% BSA in HBSS buffer. Cells supernatant was collected and determined eotaxin and PGE2 by ELISA. Total cell counts were conducted by adding 10 μL of the cell suspension to 90 μL of 0.4% trypan blue following which the cells were counted under a light microscope in a chamber. Differentiated cell counts were made from cytospin preparations stained by Liu's stain. Cells were identified and differentiated into the following groups: eosinophils, lymphocytes, neutrophils, and macrophages by standard morphological techniques, for which 500 cells needed to be counted under 1000-fold magnification and the percentage and absolute number of each cell type was estimated.
Preparation of Spleen Cells Suspension and Determination of Cytokines
Mice were sacrificed by cervical dislocation following deep anesthesia. Their spleens were aseptically removed. Single-cell suspensions were then prepared by gently tearing each spleen against sterile glass slide and removing the red blood cells using Tris-buffered NH4Cl solution. The cells were washed three times in cold HBSS (Hank's balanced salt solution) buffer and then resuspended in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin mixture, and 5 μg/mL ConA. Splenocytes (5×106 cells/well) were incubated at 37° C. for 48 h. The culture supernatant was obtained from 48 h culture. The content of IFN-gamma in the culture supernatants was assayed by the sandwich ELISA method. Briefly, microtiter plates were coated with 1 μg/mL of anti-IFN-gamma in 50 mM carbonate buffer (pH 9.6) overnight at 4° C., and then the wells were washed three times. After blocking with 1% (wt/vol) BSA-PBS buffer for 1 h at 25° C., samples were added to each well and the plates were incubated for 2 h at 37° C. The wells were then washed four times. Bound IFN-gamma was detected by biotin-conjugated anti-IFN-gamma antibody and streptoavidine-conjugated peroxidase. After the wells were washed five times, TMB (Tetramethylbenzidine) substrate was added to each well. The optical density (OD) was measured at 450 nm.
Statistical Analysis
The data were present by Means ±SDs (in vitro data) or Means ±SEMs (in vivo data). The significance of differences in the data was estimated using Student's t-test, with the significance level set at P<0.05, with difference level set at P<0.1.
These five lactobacilli species were confirmed its character by 16S rDNA sequence and API identification system result in taxonomy. The PM-A0002 which is ProMD Biotech Co., Ltd. number was identified as Lactobacillus acidophilus. The PM-A0005 which is ProMD Biotech Co., Ltd. number was identified as Lactobacillus gasseri. The PM-A0006 which is ProMD Biotech Co., Ltd. number was identified as Lactobacillus salivarius. The PM-A0009 which is ProMD Biotech Co., Ltd. number was identified as Lactobacillus johnsonii. The PM-A0013 which is ProMD Biotech Co., Ltd. number was identified as Lactobacillus johnsonii. Table 2 shows these five lactobacilli detailed structure and general property data.
Lactobacillus acidophilus PM-A0002:
Lactobacillus gasseri PM-A0005:
Lactobacillus salivarius PM-A0006:
Lactobacillus johnsonii PM-A0009:
Lactobacillus acidophilus PM-A0013:
Detection these five lactobacilli: Lactobacillus acidophilus PM-A0002, Lactobacillus gasseri PM-A0005, Lactobacillus salivarius PM-A0006, Lactobacillus johnsonii PM-A0009, Lactobacillus acidophilus PM-A0013 which cocultured with PBMC can increase Th1 pathway cytokine, such as IFN-gamma. This method can screen anti-allergy lactobacilli.
The result is shown in table 3 and
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
Detection these five lactobacilli: Lactobacillus acidophilus PM-A0002, Lactobacillus gasseri PM-A0005, Lactobacillus salivarius PM-A0006, Lactobacillus johnsonii PM-A0009, Lactobacillus acidophilus PM-A0013 which cocultured with dendritic cell can increase Th1 pathway cytokine, such as IL-12. This method can screen anti-allergy lactobacilli.
The result is shown in table 4 and
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
The effect of acid on the viability of lactobacilli is shown in table 5 and
The effect bile acid on the viability of lactobacilli is shown in table 6 and
Lactobacillus strain
Lactobacillus
acidophilus PM-A0002
Lactobacillus gasseri
Lactobacillus salivarius
Lactobacillus johnsonii
Lactobacillus
acidophilus PM-A0013
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
The adhesive results were shown in table 7 and
Lactobacillus acidophilus PM-A0002
Lactobacillus gasseri PM-A0005
Lactobacillus salivarius PM-A0006
Lactobacillus johnsonii PM-A0009
Lactobacillus acidophilus PM-A0013
Animals were intrapetitoneally sensitized with ovalbumin allergen and orally treated with Lacrobacillus salivarius PM-A0006 which had anti-allergy effect for 56 days. The moderated allergy evaluation was proceeded by the asthma animal model.
Region 1
Determination of ovalbumin-specific IgE.
The ovalbumin-specific IgE results were shown in table 8 and
Region 2
Suppression of hyperreactivity (AHR) in vivo.
The AHR results were shown in table 9 and
Region 3
Assessment of cells in bronchoalveolar lavage fluids (BALF).
The cell number of eosinophils in BALF results was shown in table 10 and
Region 4
Assessment of supernatant in bronchoalveolar lavage fluids (BALF).
The cytokine and chemokine of supernatant in the BALF results were shown in table 11,
Region 5
Augmentation of IFN-gamma production from splenocytes which were PM-A0006-treated mice by the addition of ConA in vitro.
The IFN-gamma production results were shown in table 12 and
| Number | Date | Country | Kind |
|---|---|---|---|
| 96100486 A | Jan 2007 | TW | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6379663 | Gill et al. | Apr 2002 | B1 |
| 6994848 | Hsu et al. | Feb 2006 | B2 |
| 7179460 | Dennin et al. | Feb 2007 | B2 |
| 7183108 | Cayuela et al. | Feb 2007 | B1 |
| Number | Date | Country |
|---|---|---|
| WO-0134186 | May 2001 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20080166331 A1 | Jul 2008 | US |
