Stable probiotic composition for the management of lactose intolerance

Abstract

The present invention discloses a stable probiotic composition comprising Bacillus coagulans individually and/or in combination with multi-enzyme complex for the utilization of lactose and therapeutic management of lactose intolerance. It also discloses a method for the management of lactose intolerance using composition comprising Bacillus coagulans individually and/or in combination with multi-enzyme complex.

Claims

1. A method of increasing utilization of lactose by probiotic bacteria Bacillus coagulans MTCC5856 from a food containing lactose, said method comprising a step of bringing into contact said food containing lactose with an effective dose of a composition comprising probiotic bacteria Bacillus coagulans MTCC5856 and enzymes comprising α-amylase, cellulase, lipase, lactase and neutral or acid protease, to bring about the effect of increasing utilization of lactose by Bacillus coagulans MTCC5856 by metabolizing the lactose in food in synergistic manner.

2. The method as in claim 1, wherein the effective dose of Bacillus coagulans is 1×10.sup.6 to 1×10.sup.14 colony forming units (cfu) per unit dose.

3. The method as in claim 1, wherein the effective dose of Bacillus coagulans is 2×10.sup.9 colony forming units (cfu) per unit dose.

4. The method according to claim 1, wherein the enzymes comprise a) α-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.

5. The method according to claim 1, wherein the food containing lactose is selected from the group consisting of milk, sour cream, buttermilk, butter, yogurt, cheese, ice cream, bread, baked goods, waffles, pancakes, biscuits, cookies, breakfast mixes, breakfast cereals, instant potatoes, soups, potato chips and corn chips.

6. The method as in claim 1, wherein the increasing utilization of lactose by Bacillus coagulans MTCC5856 and enzyme blend is effective in managing symptoms of lactose intolerance, selected from the group consisting of flatulence, diarrhea, bloated stomach, stomach cramps and pains, stomach rumbling, nausea, borborygmi, and vomiting.

7. The method as in claim 6, wherein symptoms of lactose intolerance occurs in conditions selected from the group consisting of lactase deficiency, congenital alactasia, lactose ingestion, and lactose malabsorption.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1a and 1b shows the graphical representation of utilization of lactose g/L (FIG. 1a) and percentage of reduction of lactose (FIG. 1b) in MRS media supplemented with 2.5, 5.0, 10 and 20 g/L by the probiotic strain B. coagulans MTCC 5856

(2) FIG. 2 shows the graphical representation of utilization of lactose in dairy food (skimmed milk) by B. coagulans MTCC 5856 after 36 h of fermentation

(3) FIG. 3 shows the graphical representation of strain comparison of different strains of B. coagulans for lactose utilization and lactase enzyme production

(4) FIG. 4 shows the graphical representation of effect of pH on the lactase (β-galactosidase) enzyme activity from Bacillus coagulans MTCC 5856

(5) FIG. 5 shows the graphical representation of effect of temperature on the Lactase (β-galactosidase) enzyme activity from Bacillus coagulans MTCC 5856 was investigated at a temperature range from 10 to 70° C.

(6) FIG. 6 shows the graphical representation of effect of gastric acid on the stability of lactase (β-galactosidase) activity was evaluated from the range of pH 1.5 to 7.0 for different time intervals up to 180 min

(7) FIG. 7 shows the graphical representation of thermo stability of Lactase enzyme was evaluated from the range of 37 to 70° C.

(8) FIG. 8 shows the graphical representation of evaluation of utilization of Lactose by B. coagulans MTCC 5856 in media with multi enzyme complex (50, 100 and 150 mg) and without multi enzyme complex (as control) was performed

(9) FIG. 9 shows the graphical representation of evaluation of utilization of lactose by B. coagulans MTCC 5856 in media containing skimmed milk with multi enzyme complex (50, 100 and 150 mg) and without multi enzyme complex (as control) was performed

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

(10) In the most preferred embodiment, present invention relates to a method of increasing lactose utilization said method comprising step of bringing into contact foods containing lactose with probiotic bacteria Bacillus coagulans to bring about the effect of increased lactose utilization. In a related embodiment, Bacillus coagulans strain is specifically MTCC 5856. In another related embodiment, the foods containing lactose is selected from the group comprising milk, sour cream and buttermilk, butter, yogurt, cheese, ice cream, bread and baked goods like waffles, pancakes, biscuits, cookies and breakfast mixes, breakfast cereals, instant potatoes, soups, potato chips and corn chips and/or foodstuff wherein lactose is present.

(11) In another most preferred embodiment, invention relates to a method of increasing lactose utilization, said method comprising step of bringing into contact foods containing lactose with probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to bring about the effect of increased lactose utilization in synergistic manner. In yet another preferred embodiment, Bacillus coagulans strain is specifically MTCC 5856. In related embodiment, the food containing lactose is selected from the group comprising milk, sour cream and buttermilk, butter, yogurt, cheese, ice cream, bread and baked goods like waffles, pancakes, biscuits, cookies and breakfast mixes, breakfast cereals, instant potatoes, soups, potato chips and corn chips and/or foodstuff wherein lactose is present. In yet another related embodiment, the effective dose of Bacillus coagulans is 1×10.sup.6 to 1×10.sup.14 colony forming units (cfu) per unit dose. In further related embodiment, Bacillus coagulans is preferably 2×10.sup.9 colony forming units (cfu) per unit dose. In yet another related embodiment, the multi-enzyme complex comprises of a) α-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/a. In yet another related embodiment, the symptoms of lactose intolerance are selected from the group consisting of flatulence (wind), diarrhea, bloated stomach, stomach cramps and pains, stomach rumbling, nausea, feeling sick, borborygmi, and vomiting. In another related embodiment, lactose intolerance are selected from the group consisting of lactase deficiency, or hypolactasia, congenital alactasia, lactose ingestion, lactose malabsorption.

(12) In yet another most preferred embodiment, invention relates to a method for the therapeutic management of lactose intolerance in mammals, said method comprising step of administering a composition comprising probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to mammals in need of such therapy. In another preferred embodiment, mammal is human. In related embodiment, Bacillus coagulans strain is specifically MTCC 5856. In yet another related embodiment, the effective dose of Bacillus coagulans is 1×10.sup.6 to 1×10.sup.14 colony forming units (cfu) per unit dose. In further related embodiment, Bacillus coagulans is preferably 2×10.sup.9 colony forming units (cfu) per unit dose. In yet another related embodiment, the symptoms of lactose intolerance are selected from the group consisting of flatulence (wind), diarrhea, bloated stomach, stomach cramps and pains, stomach rumbling, nausea, feeling sick, borborygmi, and vomiting. In another related embodiment, lactose intolerance are selected from the group consisting of lactase deficiency, or hypolactasia, congenital alactasia, lactose ingestion, lactose malabsorption. In yet another related embodiment, the multi-enzyme complex comprises of a) α-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, composition containing Bacillus coagulans and multi-enzyme complex is formulated with pharmaceutically/nutraceutically acceptable 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.

(13) The specific examples included herein below illustrate the aforesaid most preferred embodiments of the present invention.

EXAMPLES

Example 1: Utilization of Lactose by Bacillus coagulans MTCC 5856 in Media

(14) B. coagulans MTCC 5856 was grown in media (Compositions: 10 g/l Soya Peptone, 6.5 g/l Yeast extract, 2.5 to 20 g/l Lactose, 1.0 g/l Dipotassium phosphate, 1.0 g/l Monopotassium phosphate, 0.3 g/l Magnesium sulfate and 0.3 g/l sodium chloride, pH 6.5) for 24 h. After 24 h of incubation, seed was transferred to fresh sterile media (Compositions: 10 g/l Soya Peptone, 6.5 g/l Yeast extract, 2.5 to 20 Lactose, 1.0 g/l Dipotassium phosphate, 1.0 Monopotassium phosphate, 0.3 g/l Magnesium sulfate and 0.3 g/l sodium chloride, pH 6.5) and incubated at 37° C. for 96 h with 180 rpm. After every 24 h of incubation, the fermented broth was checked for enzyme activity. Further, the supernatant was collected and carried out further for lactase enzyme assay. Lactase activity was determined as per standard method of Food Chemicals Codex (FCC) by using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate (Institute of Medicine. 2003. Food Chemicals Codex: Fifth Edition. Washington, D.C.: The National Academies Press). Lactose content was determined as per the 3, 5-Dinitrosalicylic acid (DNSA) method (Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426-428).

Example 2: Utilization of Lactose by Bacillus coagulans MTCC 5856 in Dairy Foods (Skimmed Milk)

(15) The utilization of lactose in dairy foods by Bacillus coagulans MTCC 5856 was evaluated by growing Bacillus coagulans MTCC 5856 in skimmed milk media. Bacillus coagulans MTCC 5856 was grown in media containing skimmed milk (20 g/L), soya peptone (5 g/L), calcium carbonate (0.05 g/L), Manganese sulfate (0.1 g/L), ammonium sulphate (1.46 g/L) using 2.5 L fermenter (Bioengineering AG Sagenrainstrasse, Wald, Switzerland). The fermented broth was checked for enzyme activity at different intervals. Further, the supernatant was collected and carried out further for lactase enzyme assay. Lactase activity was determined as per standard method of Food Chemicals Codex (FCC) by using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate (Institute of Medicine. 2003. Food Chemicals Codex: Fifth Edition. Washington, D.C.: The National Academies Press). Lactose content was determined as per the 3, 5-Dinitrosalicylic acid (DNSA) method (Miller, G. L. (1959) by using lactose as standard. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426-428).

Example 3: Lactase Enzyme Assay (O-Nitrophenol Beta-Galactosidase Enzyme Assay)

(16) The supernatant collected from the fermented broth was tested for lactase activity. O-nitrophenyl-β-Dgalactopyranoside (ONPG) enzyme assay was used for testing the lactase activity. 4 ml of 3.7 mg/ml ONPG in acetate buffer-(Dilute 5.8 ml of glacial acetic acid in 1 L DM water, adjust the pH to 4.5) was pre-incubated for 10 min in 37° C. water bath, to that 1 ml of the sample (supernatant of broth and cell free extract) was added and further incubated at 37° C. for 15 min. To stop the reaction 1 ml of 1% Na.sub.2CO.sub.3 was added and then 8 ml of DM water was added. OD of samples was recorded at 420 nm. Lactase activity is expressed in ALU/ml or ALU/g. Lactase (ALU) “Acid Lactase unit”—One FCC Acid Lactase Unit (ALU) is defined as the quantity of enzyme that will liberate one micromole of o-nitrophenol per minute at 37° C. and a pH of 4.5. It is based on a 15-minute hydrolysis of an o-nitrophenol-beta D-galactopyranoside substrate.

Example 4: Effect of pH on the β-Galactosidase Activity from Bacillus coagulans MTCC 5856

(17) The effect of pH on the lactase activity was determined by testing the enzyme activity at different pH values ranging from 3.0 to 10 using 0.05 M of the following buffer systems: sodium acetate (3.0, 3.5, 4.0, 4.5, 5.0, 5.5), sodium phosphate (pH 6.0, 6.5, 7.0, 7.5) and tris-HCl (pH 8.0, 8.5, 9.0, 9.5, 10). Lactase activity was determined as per standard method (FCC) by using ONPG as substrate (Institute of Medicine. 2003. Food Chemicals Codex: Fifth Edition. Washington, D.C.: The National Academies Press).

Example 5: Effect of Temperature on the β-Galactosidase Activity from Bacillus coagulans MTCC 5856

(18) The effect of temperature on the activity of lactase was determined by performing the standard assay procedure at different temperatures ranging from 10 to 70° C. (10, 20, 30, 40, 50, 60, and 70° C.). Substrate was pre-incubated at the respective temperatures for 5 minutes followed by the enzyme activity determination as per the method of Food Chemicals Codex (FCC) using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate.

Example 6: Gastric Acid and Thermo-Stability of the β-Galactosidase Activity from Bacillus coagulans MTCC 5856

(19) The gastric stability of Lactase enzyme was evaluated by pre-incubating lactase enzyme produced by the Bacillus coagulans MTCC 5856 in buffer pH of 1.5 and 3.0. Samples were taken at 0, 30, 60, 90, 120 and 180 minutes of incubation. Lactase activity was determined as per standard method (FCC) by using ONPG as substrate (Institute of Medicine. 2003. Food Chemicals Codex: Fifth Edition. Washington, D.C.: The National Academies Press). Thermostabilty of lactase enzyme produced by the Bacillus coagulans MTCC 5856 was performed while incubating lactase enzyme in buffer at different temperature (50, 60 and 70° C.). Samples were taken at different time intervals (0, 5, 10, 20, 30, 40, 60 and 90 minutes). Lactase activity was determined as per standard method of Food Chemicals Codex (FCC) by using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate (Institute of Medicine. 2003. Food Chemicals Codex: Fifth Edition. Washington, D.C.: The National Academies Press).

Example 7: Combination Study of Bacillus coagulans MTCC 5856 and Multi-Enzyme Complex for the Utilization of Lactose in Media and in Dairy Foods (Skimmed Milk)

(20) B. coagulans MTCC 5856 was grown in media (Compositions: 10 g/l Soya Peptone, 6.5 g/l Yeast extract, 2.5 to 20 g/l Lactose, 1.0 g/l dipotassium phosphate, 1.0 g/l monopotassium phosphate, 0.3 g/l Magnesium sulfate and 0.3 g/l sodium chloride, pH 6.5) and various concentrations of multi-enzyme complex (50, 100 and 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 every 24 of incubation, lactose content was quantified by following DNSA method using lactose as standard. One group without multi-enzyme complex was also taken in this experiment. Another experiment was performed using media containing skimmed milk (20 g/L), soya peptone (5 g/L), calcium carbonate (0.05 g/L), Manganese sulfate (0.1 g/L), ammonium sulphate (1.46 g/L) and various concentrations of multienzyme complex (50, 100 and 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 every 24 of incubation, lactose content was quantified by following DNSA method using lactose as standard. One group without multi-enzyme complex was also taken in this experiment.

(21) TABLE-US-00001 TABLE 1 Composition of the multi-enzyme complex (MEC) Sr. No. Enzyme Activity (Unit/g) 1 α-Amylase 24000 DU 2 Neutral Protease 6000 PC 3 Cellulase 1100 CU 4 Lactase 4000 ALU 5 Lipase 200 FIP

(22) DU, Dextrinizing Unit; PU, Protease Unit; CU, Cellulase Unit; ALU, Acid Lactase Unit; FIP, Federation Internationale de Pharmaceutiques Unit

Example 8: Formulations Containing Bacillus coagulans and Multi-Enzyme Complex for Lactose Intolerance

(23) 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 of alcohol induced and drug hepatotoxicity. The following tables provide examples of different Bacillus coagulans and multi-enzyme complex compositions.

(24) Tables 2-5 Provide illustrative examples of formulations containing Bacillus coagulans MTCC 5856 (LACTOSORE®) for the treatment/management of lactose intolerance.

(25) TABLE-US-00002 TABLE 2 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

(26) TABLE-US-00003 TABLE 3 Bacillus coagulans capsule Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE ®) Excipients Maltodextrin * ®-Registered trade mark of Sabinsa Corporation, USA

(27) TABLE-US-00004 TABLE 4 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

(28) TABLE-US-00005 TABLE 5 Bacillus coagulans + multi-enzyme complex (DigeZyme ®) tablet Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE ®) Multi-enzyme complex (DigeZyme ®) Excipients Microcyscystalline cellulose, Colloidal Silica, Magnesium Stearate * ®-Registered trade mark or Sabinsa Corporation, USA

(29) TABLE-US-00006 TABLE 6 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

(30) TABLE-US-00007 TABEL 7 Bacillus coagulans + multi-enzyme complex (DigeZyme ®) capsule Active Ingredients Bacillus coagulans MTCC 5856; 2 billion cfu (LACTOSORE ®) Multi-enzyme complex (DigeZyme ®) Excipients Maltodextrin * ®-Registerd trade mark of Sabinsa Corporation, USA

(31) The above formulations are merely illustrative examples; any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.

(32) 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.