A LIQUID PROBIOTIC COMPOSITION STABLE AT AMBIENT TEMPERATURE

Abstract

The present invention provides a stable liquid syrup composition compositions comprising probiotic bacteria, retaining their cellular viability at ambient storage conditions for at least two years.

Claims

1. A liquid composition comprising probiotic bacteria and at least 60% sweetener weight by volume, wherein the said probiotic bacteria retains at least 75% of its cellular viability of the original value as measured by the spore count.

2. A liquid composition comprising probiotic bacteria, at least 60% sweetener weight by volume, and one more or more excipients selected from the group consisting of hydrophilic vehicles, solubilizer, pH modifier, buffer, viscosity modifier, preservatives, and stabilizers, wherein the said probiotic bacteria retains at least 75% of its cellular viability of the original value as measured by the spore count.

3. The liquid composition comprising a probiotic bacteria as claimed in claim 1, wherein the cellular viability as measured by the spore count is retained to at least 75% of the original value, when the said liquid composition is stored at ambient storage conditions for a period of at least 6 to 24 months.

4. The liquid composition comprising a probiotic bacteria as claimed in claim 1, wherein the said probiotic bacteria belongs to the genus Bacillus, genus Lactobacillus or a mixture of both.

5. The liquid composition comprising a probiotic bacteria as claimed in claim 4, wherein the said probiotic bacteria is selected from the species Bacillus subtilus, Bacillus clausi and Bacillus coagulans.

6. The liquid composition comprising a probiotic bacteria as claimed in claim 4, wherein the said probiotic bacteria is selected from the species lactobacillus acidophilus and lactobacillus casei.

7. The liquid composition comprising a probiotic bacteria as claimed in claim 1, wherein the spore count of the composition prepared is in the range 0.210.sup.9 cfu/5 ml to 1010.sup.9 cfu/5 ml.

8. The liquid composition comprising a probiotic bacteria as claimed in claim 1, wherein the sweetener is a sugar.

9. The liquid composition comprising a probiotic bacteria as claimed in claim 8, wherein the sugar is selected from the group consisting of sucrose, glucose, and fructose.

10. The liquid composition comprising a probiotic bacteria as claimed in claim 1, wherein the sweetener is a mixture of sugar and sugar alcohol at ratio of sugar to sugar alcohol in the range of 0.85 to 2.25.

11. The liquid composition comprising a probiotic bacteria as claimed in claim 10, wherein the sugar alcohol is mannitol, sorbitol, xylitol or a mixture thereof.

12. A liquid composition comprising yeast, preferably, saccharomyces boulardii and at least 60% sweetener by weight, and one more or more excipients selected from the group consisting of hydrophilic vehicles, solubilizer, pH modifier, buffer, viscosity modifier, preservatives, and stabilizers, wherein the said probiotic bacteria retains at least 75% of its cellular viability of the original value as measured by the spore count.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0023] The objectives of the present invention are accomplished and the shortcomings associated with the probiotic compositions of the prior art are overcome by the present invention as described below in the preferred embodiments.

[0024] The present invention encompasses liquid compositions of probiotic bacteria that are stable at ambient conditions. The bacteria of the invention is selected from the group consisting of genera bacillus.

[0025] In an embodiment the percentage of sweetener in the formulation is preferably 60%, 70% or 80%.

[0026] In an embodiment, the formulation of the invention comprises at least 60% sweetener, which does not promote the growth of bacterial spores.

[0027] In another embodiment of the invention, the probiotic compositions of the present invention have pH in the range 3.5 to 6.0.

[0028] In yet another embodiment the present invention provides probiotic compositions comprising 1) one or more Bacillus species in spore form, 2) a hydrophilic vehicle 3) a solubilizer which is a surfactant 4) pH modifiers and buffering agents 5) viscosity modifying agents 6) preservatives 7) stabilizers and 8) sweeteners

[0029] In yet another embodiment, the present invention also provides probiotic compositions comprising 1) one or more Lactobacillus species, 2) a hydrophilic vehicle 3) a solubilizer which is a surfactant 4) pH modifiers and buffering agents. 5) Viscosity modifying agents 6) preservatives 7) stabilizers and 8) sweeteners.

[0030] In another embodiment of the invention are provided probiotic compositions which when stored at ambient storage conditions for 24 months do not promote E coli growth.

[0031] In another embodiment of the invention, the present invention provides probiotic compositions wherein the pH of the composition when stored at ambient storage conditions for 24 months does not decrease by more than 0.1.

[0032] In another embodiment, the invention provides a liquid composition comprising probiotic bacteria which belongs to the genus Bacillus.

[0033] In another embodiment the invention provides a liquid composition comprising probiotic bacteria selected from Bacillus subtilus, Bacillus clausi and Bacillus coagulans.

[0034] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria which belongs to the genus Lactobacillus.

[0035] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria selected from lactobacillus acidophilus and lactobacillus casei

[0036] In another embodiment, the invention provides a liquid composition comprising probiotic bacteria which is a mixture of probiotic bacteria selected from the genus bacillus and lactobacillus.

[0037] In another embodiment, the invention provides a liquid composition comprising a probiotic bacteria and a sweetener.

[0038] In another embodiment, the invention provides a liquid composition comprising a probiotic bacteria and a sweetener wherein the sweetener is a sugar.

[0039] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria and a sugar selected from sucrose, glucose, and fructose.

[0040] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria and a sugar wherein the sugar is selected from a mixture of sucrose, glucose, and fructose.

[0041] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria and a sweetener wherein the sweetener is a mixture of sugar and sugar alcohol.

[0042] In another aspect of the invention, the liquid compositions comprising probiotic bacteria, which are not stable in the presence of sugar alcohol alone, are stable in the presence of sugar alcohols and sugars.

[0043] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria and a mixture of sugar and sugar alcohol, wherein the sugar alcohol is selected from mannitol, sorbitol and xylitol.

[0044] In another embodiment the invention provides a liquid composition comprising a probiotic bacteria and a sugar and sugar alcohol wherein the ratio of sugar to sugar alcohol ranges from 0.85 to 2.25.

[0045] In a preferred embodiment the compositions of the invention retain cellular viability under ambient storage conditions for at least two years for use as probiotic syrup in food, food supplement and pharmaceutical composition, particularly for health care.

[0046] The bacteriostatic preservative in the formulation is selected from the group consisting of bronidiol, a combination of methyl paraben and propyl paraben, sodium salt of parabens. Preferably, the preservative is bronidiol.

[0047] The present invention describes probiotic compositions having pH in the range 3.5 to 6.0. Citric acid serves as the pH modifier in the instant formulation.

[0048] The present invention also envisages a liquid composition comprising yeast and at least 60% sweetener by weight, and one more or more excipients selected from the group consisting of hydrophilic vehicles, solubilizer, pH modifier, buffer, viscosity modifier, preservatives, and stabilizers, wherein the said probiotic bacteria retains at least 75% of its cellular viability of the original value as measured by the spore count. The yeast of the invention is preferably saccharomyces boulardii.

[0049] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description only. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.

Example 1

[0050] Method of Preparation of Probiotic Composition

[0051] The compositions of the present invention are prepared as per the procedure described below: Water is heated in a vessel to 70 C. Vehicle and sweetener are added with stirring. After a homogenous solution is formed, the same is filtered to clarify and cooled to 45 C. The probiotic powder is added with stirring to form a homogenous solution. All other components of the formulation except the bacteriostatic preservative are added at this stage. Finally the bacteriostatic preservative, for example, Bronidiol is added and the volume of the composition is made up to 1000 ml using requisite quantity of water.

Example 2

[0052] Method for the Determination of Spores Count [0053] 1. Take 1 gram of sample and suspend in 99 ml of saline+0.5% Tween 80 in an Erlenmeyer flask (This is 100 times dilution) [0054] 2. Keep it on a rotary shaker (200 rpm) at 37 C. for 30 mins. [0055] 3. Transfer 0.5 ml from the above flask to 4.5 ml of saline and carry out serial dilutions up to 10.sup.9 [0056] 4. Surface spread 0.1 ml of the last 3 (10.sup.7, 10.sup.8, 10.sup.9) dilutions in duplicate on sterile Luria Bertani agar or Soybean casein Digest media (SCDM) agar plate using sterile spreader. [0057] 5. Incubate the plates at 37 C. and count the number of colony forming units.

Spore count CFU/gm=Number of coloniesDilution factorvolume of sample used [0058] For example If 10.sup.7 dilution plate gave 10 colonies, then [0059] Spore count CFU/gm 1010.sup.710=1.010.sup.9 cfu/ml or 5.010.sup.9 cfu/5 ml [0060] Media composition

TABLE-US-00001 1) Diluent Sodium chloride 0.85 gm Tween 80 0.5 gm Distilled water 100 ml 2) Phenol red dextrose broth Phenol red dextrose broth (Hi media MO56) 2.1 gm Distilled water 100 ml 3) Soya bean casein digest medium (SCDM) Soya bean casein digest medium broth (Himedia MO11) 3 gm Agar powder (Himedia -RM026) 2 gm Distilled water 100 ml 4) Luria Bertani agar Tryptone (Casein hydrolysate) 1 gm Yeast extract 0.5 gm Sodium chloride 0.5 gm Distilled water 100 ml pH 7.5 Agar powder (Himedia -RM026) 2 gm

[0061] For all spore count measurements to evaluate stability following conditions were used.

[0062] Ambient Storage Conditions

[0063] Temperature 30 C.2 C., Humidity 75% RH5% RH

[0064] RH=Relative Humidity

[0065] Storage Conditions for Accelerated Aging Test

[0066] Temperature 40 C.2 C., Humidity 65% RH5% RH

Example 3

[0067] Formulation of the Probiotic Composition

TABLE-US-00002 Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml

[0068] Results of Accelerated Stability Test

TABLE-US-00003 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 3.8 3.1 2.85 4.1 3.7

[0069] Results of Stability Test Under Ambient Storage Conditions

TABLE-US-00004 Months 0 1 2 3 6 12 18 24 Spore count 10.sup.9/5 ml 2.7 2.4 3.6 2.6 3.1 2.5 2.2 2.6 pH 3.5 3.5 3.4 3.6 3.5 3.4 3.5 3.6

[0070] The comparative results show that results of stability test under ambient storage conditions are adequately simulated by the results of the accelerated stability test.

Example 4

[0071] Formulation of the Probiotic Composition with Change in Bacterial Concentration

TABLE-US-00005 Sucrose 600 gm B. subtilis 0.2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Sucrose 600 gm B. subtilis 10 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml

Example 5

[0072] Formulation of the Probiotic Composition with Change in Sweetener

TABLE-US-00006 High fructose corn syrup 780 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Sucrose + High fructose corn syrup 300 gm + 390 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml

Example 6

[0073]

TABLE-US-00007 Sucrose 600 gm Sorbitol 150 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Sucrose 600 gm Glycerol 50 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml

Example 7

[0074] Formulation of the Probiotic Composition with pH Modifier

TABLE-US-00008 Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Citric acid 0.1%

Example 8

[0075] Formulation of the Probiotic Composition with Change in Preservative

TABLE-US-00009 Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Methyl Paraben + Propyl Paraben sodium salt 0.075% + 0.075% Water q.s. 1000 ml Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Sodium Benzoate 0.1% Water q.s. 1000 ml

Example 9

[0076] Formulation of the Probiotic Composition with a Thickener

TABLE-US-00010 Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Methyl cellulose 0.5% Sucrose 600 gm B. subtilis 2 10.sup.9/5 ml Flavour strawberry 5 gm Colour FDC Red 0.2 gm Bronidiol 1 gm Water q.s. 1000 ml Carboxy methyl cellulose 1%

[0077] In the following examples only the contents of the probiotic bacteria and the sugar/sugar alcohol are mentioned. Other ingredients are as per example 3

Example 10

[0078]

TABLE-US-00011 Sucrose 300 gm Sorbitol 350 gm

[0079] Results of Accelerated Stability Test

TABLE-US-00012 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 2.7 2.4 3.6 2.6 3.1

Example 11

[0080]

TABLE-US-00013 Sorbitol 650 gm

[0081] Composition not Stable

Example 12

[0082]

TABLE-US-00014 Sucrose 250 gm Mannitol 400 gm

[0083] Results of Accelerated Stability Test

TABLE-US-00015 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 3.1 2.4 2.9 3.6 3.4

Example 13

[0084]

TABLE-US-00016 Mannitol 650 gm

[0085] Composition not Stable

Example 14

[0086]

TABLE-US-00017 Sucrose 550 gm Glycerol 100 gm B. subtilis 6.47 10.sup.9/5 ml

[0087] Results of Accelerated Stability Test

TABLE-US-00018 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 3.8 3.1 2.85 4.1 3.7

Example 15

[0088]

TABLE-US-00019 Glycerol 650 gm B. subtilis 6.2 10.sup.9/5 ml

[0089] Composition not Stable

Example 16

[0090]

TABLE-US-00020 Sucrose 350 gm Glucose 100 gm Sorbitol 200 gm B. subtilis 6.4 10.sup.9/5 ml

[0091] Results of Accelerated Stability Test

TABLE-US-00021 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 2.7 2.4 3.6 2.6 3.1

Example 17

[0092]

TABLE-US-00022 Sucrose 250 gm Glucose 250 gm Glycerol 150 gm B. subtilis 5.8 10.sup.9/5 ml

[0093] Results of Accelerated Stability Test

TABLE-US-00023 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 3.1 2.4 2.9 3.6 3.4

Example 18

[0094] In this and all subsequent examples the liquid probiotic composition as in example 3 is used. The bacteria species and content is varied as indicated

[0095] Species Bacillus clausi: Content 3.810.sup.9/5 ml

[0096] Results of Accelerated Stability Test

TABLE-US-00024 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 3.8 3.5 2.9 3.1 3.2 pH 3.6 3.5 3.6 3.6 3.6

[0097] Results of Stability Test Under Ambient Conditions

TABLE-US-00025 Months 0 1 2 3 6 12 18 24 Spore count 10.sup.9/5 ml 3.8 3.1 3.3 3.1 3.2 3.6 3.7 3.5 pH 3.6 3.6 3.5 3.6 3.5 3.5 3.6 3.5

Example 19

[0098] Species Bacillus coagulans: Content 2.410.sup.9/5 ml

[0099] Results of Accelerated Stability Test

TABLE-US-00026 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 2.4 2.2 2.1 2.4 2.2 pH 3.6 3.6 3.6 3.5 3.6

[0100] Results of Stability Test Under Ambient Conditions

TABLE-US-00027 Months 0 1 2 3 6 12 18 24 Spore count 10.sup.9/5 ml 2.4 2.2 2.1 2.4 2.2 2.6 2.1 2.5 pH 3.6 3.6 3.6 3.6 3.5 3.5 3.6 3.6

Example 20

[0101] Species: Lactobacillus acidophilus Content 2.810.sup.9/5 ml

[0102] Results of Accelerated Stability Test

TABLE-US-00028 Months 0 1 2 3 6 Spore count 10.sup.9/5 ml 2.4 2.2 2.1 2.4 2.2

Example 21

[0103] Species: Bacillus subtilis Content 3.810.sup.9/5 ml+Lactobacillus casei Content 3.410.sup.9/5 ml

[0104] Results of Accelerated Stability Test

TABLE-US-00029 Months 0 1 2 3 6 Bacillus subtilis Spore 3.8 3.1 2.85 4.1 3.7 count 10.sup.9/5 ml Lactobacillus casei 3.4 3.2 3.1 3.6 3.1 Spore count 10.sup.9/5 ml pH 3.5 3.5 3.5 3.5 3.6