METHOD AND COMPOSITION FOR THERAPEUTIC MANAGEMENT OF GLUTEN INTOLERANCE

Abstract

The present invention discloses the potential of probiotic bacteria Bacillus coagulans individually or in combination with multi-enzyme complex for reducing gluten content in foods rich in gluten and for the increased utilization of gluten. The invention further discloses a method for the management of gluten intolerance using composition comprising Bacillus coagulans and multi-enzyme complex in mammals.

Claims

1. A method of reducing gluten content in foods, said method comprising step of bringing into contact foods containing gluten with probiotic bacteria Bacillus coagulans individually or in combination with multi enzyme complex to bring about the effect of reducing gluten content.

2. The method as in claim 1, where the type of gluten is selected from the group consisting of Gliadin, Hordeins, Avenins, and Secalins.

3. The method as in claim 1, wherein foods containing gluten is selected from the group consisting of barley, wheat, rye, and oats.

4. The method as in claim 1, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 5856.

5. The method as in claim 1, wherein the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu).

6. The method as in claim 1, wherein the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FIP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC/g.

7. A method of increasing gluten utilization in mammals, said method comprising step of administering probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to said mammals to bring about an effect of increased utilization of gluten.

8. The method as in claim 7, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 5856.

9. The method as in claim 7, wherein the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu).

10. The method as in claim 7, wherein the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FIP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC/g.

11. The method as in claim 7, wherein the mammal is human.

12. A method for the therapeutic management of gluten intolerance and related conditions in mammals by enhancing gluten utilization, said method comprising step of administering a composition comprising probiotic bacteria Bacillus coagulans individually or in combination with multi-enzyme complex to mammals in need of such therapy to bring about a reduction is symptoms of gluten intolerance.

13. The method as in claim 12, wherein the Bacillus coagulans strain is Bacillus coagulans MTCC 5856.

14. The method as in claim 12, wherein, the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu).

15. The method as in claim 12, wherein the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 Cu/g, c) lipase: not less than 200 FP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC/g.

16. The method as in claim 12, wherein the related conditions of gluten intolerance are selected from the group consisting of autoimmune disorders like celiac disease, dermatitis herpetiformis, gluten ataxia, non-celiac gluten sensitivity (NCGS), gluten ingestion, Gluten mal-absorption.

17. The method as in claim 12, wherein the mammal is human.

18. The method as in claim 12, wherein the composition comprising Bacillus coagulans individually or in combination with multi-enzyme complex is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 shows the graphical representation of growth of Bacillus coagulans MTCC 5856 spores and vegetative cells in media containing gluten.

[0021] FIG. 2 shows the graphical representation of Bacillus coagulans MTCC 5856 showing the ability to reduce gluten content in the media.

[0022] FIG. 3 shows the graphical representation of reduction in gliadin content from wheat flour (WF) by Bacillus coagulans MTCC 5856

[0023] FIG. 4 shows the graphical representation of reduction in gliadin content from wheat gluten by Bacillus coagulans MTCC 5856

[0024] FIG. 5 shows the graphical representation of reduction in gliadin content from wheat gluten by Bacillus coagulans MTCC 5856 and MEC (Multi enzyme complex)

[0025] FIG. 6 shows the graphical representation of reduction in gliadin content from wheat flour by Bacillus coagulans MTCC 5856 and MEC (Multi enzyme complex)

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In the most preferred embodiment, present invention relates to a method of reducing gluten content in foods, said method comprising step of bringing into contact foods containing gluten with probiotic bacteria Bacillus coagulans individually or in combination with multi enzyme complex to bring about the effect of reducing gluten content. In a related embodiment, the type of gluten is selected from the group comprising, Gliadin, Hordeins, Avenins, and Secalins. In another related embodiment, the foods containing true gluten is barley, wheat, rye, and oats. In another related embodiment, the foods containing gluten is selected from but not limited to glutens in flours of Barley. Wheat, Rye, and Oats. In another related embodiment, the foods containing gluten is selected from but not limited to wheat, all purpose flour, almond flour, rice flour, corn flour, graham flour, semolina, Farro flour. In another related embodiment, the gluten from wheat is selected from different types of wheat species mainly Farro medio or Emmer wheat, Farro piccolo or Einkom wheat, Farro grande or Spelt wheat, triticale, durum, khorsan, kamut. In a related embodiment, Bacillus coagulans strain is specifically MTCC 5856. In yet another related embodiment, the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu). In yet another related embodiment, the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FIP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 600 PC/g.

[0027] In another most preferred embodiment, invention relates to a method of increasing gluten utilization in mammals, said method comprising step of administering probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to said mammals to bring about an effect of increased utilization of gluten. In a related embodiment, Bacillus coagulans strain is specifically MTCC 5856. In yet another related embodiment, the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu). In yet another related embodiment, the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FIP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC/g. In another related embodiment, the mammal is human.

[0028] In yet another most preferred embodiment, invention relates to a method for the therapeutic management of gluten intolerance and related conditions in mammals by enhancing gluten utilization, said method comprising step of administering a composition comprising probiotic bacteria Bacillus coagulans individually or in combination with multi-enzyme complex to mammals in need of such therapy, to bring about a reduction is symptoms of gluten intolerance. In related embodiment, Bacillus coagulans strain is specifically MTCC 5856. In yet another related embodiment, the effective dose of Bacillus coagulans is 210.sup.9 colony forming units (cfu). In yet another related embodiment, the multi-enzyme complex comprises of a) -amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FIP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC % g. In yet another related embodiment, the symptoms of gluten intolerance are selected from the group comprising, but not limited to, chronic diarrhea, bloated stomach, stomach cramps and pains, joint pain, canker sores inside mouth, missed menstrual periods, headache, anemia, fatigue, depression and anxiety, nausea and vomiting, nasal congestion, difficulty breathing or anaphylaxis, eye irritation, hives and rash, abdominal bloating and gas, constipation, pale and foul-smelling stool. In another related embodiment, related conditions of gluten intolerance are selected from the group consisting of autoimmune disorders like celiac disease, dermatitis herpetiformis, gluten ataxia, non-celiac gluten sensitivity (NCGS), gluten ingestion, Gluten mal-absorption. In another related embodiment, composition comprising Bacillus coagulans alone or in combination with multi-enzyme complex is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

[0029] The specific examples included herein below illustrate the aforesaid most preferred embodiments of the present invention.

Example 1: Utilization of Gluten (GL) by Bacillus Coagulans MTCC 5856 in Media

Methodology

[0030] B. coagulans MTCC 5856 was grown in media (Compositions: 5 g/l Peptic digest of animal tissue, 5 g/l Yeast extract, 2 g/l Dextrose, 0.5 g/l Dipotassium phosphate, 0.5 g/l Monopotassium phosphate, 0.3 g Magnesium sulfate and 0.3 g/l sodium chloride, 0.1 g/l Manganese sulphate pH 6.5). After 48 h of incubation, a seed of 250 ml was washed with saline twice and final pallet was added with 10 ml of saline and transferred to fresh sterile media.

[0031] After 24 h of incubation, the seed was transferred to fresh sterile media (Composition: 2.5 to 10 g/l gluten from wheat (sigma-Aldrich), 2.5 g/l Dextrose, 1.0 g/l Dipotassium phosphate, 1.0 g/l Monopotassium phosphate, 0.5 g/l Magnesium sulfate and 2 g/l sodium chloride, pH 6.5) and incubated at 37 C. for 72 h with 180 rpm. After every 24, 48 and 72 h of incubation, the fermented broth was collected and freeze dried (VirTis 2 K Freeze Dryer, SP Industries, Inc., Warminster, Pa. USA). Further, the powder was collected and gliadin content analysis carried out using Veratox for Gliadin R5 kit. The assays were performed according to the manufacturers' instructions as described.

[0032] Veratox for Gliadin R5 is a sandwich enzyme-linked immunosorbent assay (S-ELISA). Gliadin is extracted from samples with a 60% ethanol solution by shaking in a shaker. Added 150 L controls and extracted samples to transfer wells. Then, transferred 100 L to the antibody wells and incubated for 10 minutes and mixed for 20 seconds by sliding back and forth on a flat surface and dump liquid from antibody wells. Transferred 100 L conjugate from reagent boat to antibody wells using 12-channel pipettor, mixed for 20 seconds by sliding back and forth on a flat surface and incubated for 10 minutes. After incubation, the solution was added with 100 L substrate from reagent boat to antibody wells using 12-channel pipettor and incubated for 10 minutes. Mixed well for 20 seconds by sliding back and forth on a flat surface. Further, transferred (10 L red Stop from reagent boat to antibody wells and mixed well and read results in a microwell reader. The development of blue color indicates that the samples contain high levels of gliadin while purple or red samples contain little or no gliadin. The optical densities of the controls form a standard curve, and the sample optical densities are plotted against the curve to calculate the exact concentration of gliadin in parts per million (ppm).

Results

[0033] Data of the study suggested that Bacillus coagulans MTCC 5856 has ability to grow while fermenting Wheat gluten as nutritional source (FIG. 1)

[0034] With regard to the potential of Bacillus coagulans to remove gliadin content, both the spores and vegetative cells of Bacillus coagulans MTCC 5856 significantly removed the gliadin content from the media (FIG. 2)

Example 2: Utilization of Gluten by Bacillus coagulans MTCC 5856 in Wheat Flour (WF)

Methodology

[0035] B. coagulans MTCC 5856 was grown in media (Composition: 5 g/l Peptic digest of animal tissue, 5 g/l Yeast extract, 2 g/l Dextrose, 0.5 g/l Dipotassium phosphate, 0.5 g/l Monopotassium phosphate, 0.3 g/l Magnesium sulfate and 0.3 g/l sodium chloride, 0.1 g/l Manganese sulphate pH 6.5). After 48 h of incubation, seed of 250 ml was washed with saline twice and final pallet was mixed with 10 ml of saline and transferred to fresh sterile media.

[0036] After 24 h of incubation, seed was transferred to fresh sterile media (Composition: 2.5 to 10 g/l wheat flour in 1000 ml of Potassium phosphate buffer (0.1 M, pH 6.5) and incubated at 37 C. for 72 b with 180 rpm. After every 24, 48 and 72 h of incubation, the fermented broth was collected and freeze dried. Further, the powder was collected and carried out gliadin content analysis using Veratox for Gliadin R5 kit as per the procedure described in example 1.

Results

[0037] Spores and vegetative cells of Bacillus coagulans MTCC 5856 were incubated with whole wheat flour. Both spores and vegetative cells of Bacillus coagulans MTCC 5856 significantly removed the gliadin content from the wheat flour. (FIG. 3)

[0038] Further, the ability of Bacillus coagulans MTCC 5856 to remove the gliadin content from wheat was compared with other strains was Bacillus coagulans (Bacillus coagulans ATCC 31284). Bacillus coagulans MTCC 5856 was much more effective in removing the gliadin content from wheat compared to Bacillus coagulans ATCC 31284 (Table 1).

[0039] Table 1: Comparison of reduction of gliadin content (ppm) from wheat flour (WF) by B.coagulans MTCC 5856 and B.coagulans ATCC 31284 Gliadin content in Gliadin content in Wheat flour Gliadin content in Wheat flour Wheat flour with B.coagulans MTCC 5856 with B.coagulans ATCC 31284 Untreated (ppm) (ppm) (ppm)

TABLE-US-00001 TABLE 1 Comparison of reduction of gliadin content (ppm) from wheat flour (WF) by B.coagulans MTCC 5856 and B.coagulans ATCC 31284 Gliadin content in Gliadin content in Wheat Gliadin content in Wheat Wheat flour flour with B.coagulans flour with B.coagulans Untreated MTCC 5856 ATCC 31284 (ppm) (ppm) (ppm) 929 29.84 ND 2457 19.58 1102 4685 11.57 2315 NDNot done

[0040] Similarly, B.coagulans MTCC 5856 was much effective in removing the gliadin content from wheat gluten (FIG. 4 and table 2) compared to B.coagulans ATCC 31284.

TABLE-US-00002 Comparison of reduction of gliadin content (ppm) from wheat gluten (WG) by B.coagulans MTCC 5856 and B.coagulans ATCC 31284 Gluten Untreated Gluten with B.coagulans Gluten with B.coagulans (ppm) MTCC 5856 (ppm) ATCC 31284 (ppm) 586 12.45 ND 1914 35.24 906 3681 78.6 1912 NDNot done

Example 3: Combination Study of Bacillus coagulans MTCC 5856 and Multi-Enzyme Complex for the Utilization of Gluten (GL) in Media and in Wheat Flour (WT)

[0041] B.coagulans MTCC 5856 was grown in media (Compositions: 10 g/l gluten from wheat (sigma-Aldrich), 2.5 g/l Dextrose, 1.0 g/l Dipotassium phosphate, 1.0 g/l Monopotassium phosphate, 0.5 g/l Magnesium sulfate and 2 g/l sodium chloride. pH 6.5) and various concentrations of multi-enzyme complex (150 mg/L) were added after the media sterilization and along with B. coagulans MTCC 5856 and incubated at 37 C. for 72 h. After 72 h of incubation, the fermented broth was collected and freeze dried (VirTis 2 K. Freeze Dryer, SP Industries, Inc., Warminster, Pa. USA). Further, the powder was collected and carried out gliadin content analysis. One group without multi-enzyme complex was also taken in this experiment. The contents of the multi-enzyme complex is disclosed in table 3.

TABLE-US-00003 TABLE 3 Composition of the multi-enzyme complex (MEC) Sr. No. Enzyme Activity (Unit/g) 1 -Amylase 24000 DU 2 Neutral Protease 6000 PC 3 Cellulase 1100 CU 4 Lactase 4000 ALU 5 Lipase 200 FIP DU, Dextrinizing Unit; PU, Protease Unit; CU, Cellulase Unit; ALU, Acid Lactase Unit; FIP, Federation Internationale de Pharmaceutiques Unit

[0042] Another experiment was performed using media containing Composition: 10 g/l wheat flour in 1000 ml of Potassium phosphate buffer (0.1 M, pH 6.5) and incubated at 37 C. for 72 h with 180 rpm. After every 24, 48 and 72 h of incubation, the fermented broth was collected and freeze dried. Further, the powder was collected and carried out gliadin content analysis and multienzyme complex (150 mg/L) were added after the media sterilization and along with B. coagulans MTCC 5856 and incubated at 37 C. for 72 h. After 72 h of incubation, the fermented broth was collected and freeze dried. Further, the powder was collected and carried out gliadin content analysis. One group without multi-enzyme complex was also taken in this experiment.

Results

[0043] The results are given in Table 4.

TABLE-US-00004 TABLE 4 Reduction of gliadin content from wheat flour and wheat gluten by B. coagulans MTCC 5856 and MEC (Multi enzyme complex) Gliadin Composition content (ppm) Wheat Gluten 10 g/L (Untreated control) 3678 Wheat Gluten 10 g/L + MEC 150 mg 860 Wheat Gluten 10 g/L + MEC 150 mg + B. coagulans 3.78 5856 Wheat flour 10 g/L (Untreated control) 4671 Wheat flour 10 g/L + MEC 150 mg 855 Wheat flour 10 g/L + MEC 150 mg + B. coagulans 5856 4.04

[0044] The combination of B.coagulans and multi-enzyme complex shows synergistic effect on lowering the gliadin content in both whole wheat grains and wheat flour as compared to multi enzyme complex individually as shown in Table 4 and FIG. 5 & FIG. 6.

Example 4: Formulations Containing Bacillus coagulans and Multi-Enzyme Complex for Lactose Intolerance

[0045] Bacillus coagulans and multi-enzyme complex is formulated with pharmaceutically/nutraceutically acceptable compositions with excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or combined with other hepatoprotective compositions and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables and administered for treatment gluten intolerance. The following tables provide examples of different Bacillus coagulans and multi-enzyme complex compositions.

[0046] Tables 5-10 Provide illustrative examples of formulations containing Bacillus coagulans MTCC 5856 (LACTOSORE) for the treatment/management of gluten intolerance.

TABLE-US-00005 TABLE 5 Bacillus coagulans tablet Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Excipients Microscystalline cellulose, Colloidal Silica, Magnesium Stearate *-Registered trade mark of Sabinsa Corporation, USA

TABLE-US-00006 TABLE 6 Bacillus coagulans capsule Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Excipients Maltodextrin *-Registered trade mark of Sabinsa Corporation, USA

TABLE-US-00007 TABLE 7 Bacillus coagulans drink mix Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Excipients Maltodextrin, Taurin, Citric acid, Sucralose, Flavouring agent, Vitamin B6 and B12 *-Registered trade mark of Sabinsa Corporation, USA

TABLE-US-00008 TABLE 8 Bacillus coagulans + multi-enzyme complex (DigeZyme) tablet Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Multi-enzyme complex (DigeZyme) Excipients Microscystalline cellulose, Colloidal Silica, Magnesium Stearate *-Registered trade mark of Sabinsa Corporation, USA

TABLE-US-00009 TABLE 9 Bacillus coagulans + multi-enzyme complex (DigeZyme) drink mix Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Multi-enzyme complex (DigeZyme) Excipients Maltodextrin, Taurin, Citric acid, Sucralose, Flavouring agent, Vitamin B6 and B12 *-Registered trade mark of Sabinsa Corporation, USA

TABLE-US-00010 TABLE 10 Bacillus coagulans + multi-enzyme complex (DigeZyme) capsule Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE) Multi-enzyme complex (DigeZyme) Excipients Maltodextrin *-Registered trade mark of Sabinsa Corporation, USA

[0047] The above formulations are merely illustrative examples; any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.

[0048] Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention. The scope of the invention is to be interpreted only in conjunction with the appended claims.