Claims:1. A composition for controlling Prevotella copri in gut, the composition comprising green tea extract, curcumin and a fructo-oligosaccharide in a weight ratio of 1:1:2 to 3:1:12.

2. The composition as claimed in claim 1, wherein the green tea extract, the curcumin and the fructo-oligosaccharide are in the weight ratio of 1:1:2 to 2:1:12.

3. The composition as claimed in any of claims 1 or 2, wherein the green tea extract and the curcumin are homogenously dispersed in the fructo-oligosaccharide.

4. The composition as claimed in any of claims 1 to 3, wherein green tea extract comprises epigallocatechin gallate.

5. The composition as claimed in any of claims 1 to 3, further comprising an ingredient selected from the group consisting of galacto-oligosaccharides (GOS), maltodextrin, starch, polyols, saccharides, artificial sweeteners and natural sweeteners.

6. The composition as claimed in any of claims 1 to 5, wherein the composition is in granular form.

7. The composition as claimed in any of claims 1 to 6, wherein the composition is in an oral dosage form selected from the group consisting of a tablet, a capsule, and a powder for oral administration.

8. The composition as claimed in claim 6, wherein the composition comprises granules having a particle size in the range of 1 to 1000 micron.

9. A food matrix comprising about 0.01 to 5 %w/w of the composition as claimed in any of claims 1 to 8.

10. A process for preparing a composition, the process comprising:
a) blending the green tea extract, the curcumin and the fructo-oligosaccharide in a ratio of 1:1:2 3:1:12 to form a homogenized blend;
b) adding a solvent to the homogenized blend to form a slurry;
c) subjecting the slurry to evaporation at a temperature in the range of 40 to 70? C and pressure in the range of 5 to 400 millibar to form a solid mass;
d) grinding the solid mass to form the composition.

11. The process as claimed in claim 11, wherein the solvent is a polar protic solvent selected from the group consisting of water, ethanol, methanol and a combination thereof.
, Description:FIELD OF INVENTION
The present disclosure relates to compositions for controlling Prevotella copri in the gut. Specifically, the present disclosure relates to compositions comprising green tea extract, curcumin, and a fructo-oligosaccharide and use of said compositions in controlling Prevotella copri.

BACKGROUND
Prevotella is a large genus that includes different species with a vast genomic diversity. Most of them is found in the oral cavity and the gut, with Prevotella copri being the most abundant species in the gut. Prevotella copri has been reported to be associated with opportunistic infections, e.g. periodontitis, bacterial vaginosis and other infections. Recent studies have linked higher intestinal abundance of Prevotella copri to rheumatoid arthritis, metabolic syndrome. Studies suggest that Prevotella copri may trigger and/or worsen inflammatory diseases.
Use of the antibiotics is an established therapy for the treatment of infections triggered by Prevotella copri. However, due to the adverse effects associated with the use of antibiotics, attention is increasingly focussed on natural compounds. Plant based derivatives can provide an alternate to the antibiotics. Plants provide an excellent source of drugs and a large proportion of currently available rugs have been either directly or indirectly from the plant sources. The use of plant-derived natural products in medical treatments is attracting more attention due to its potential efficacy and no side effects.
Therefore, there is a need of a plant-based composition for controlling Prevotella copri that are effective and are not accompanied by any adverse side effects.

SUMMARY
The present disclosure relates to a composition for controlling Prevotella copri in gut. The composition comprising green tea extract, curcumin, and a fructo-oligosaccharide in a w/w ratio of 1:1:2 to 3:1:12.

The present disclosure also relates to a process for preparing the composition. The process comprising blending the green tea extract, the curcumin and the fructo-oligosaccharide in a ratio of 1:1:2 3:1:12 to form a homogenized blend; adding a solvent to the homogenized blend to form a slurry; subjecting the slurry to evaporation at a temperature in the range of 40 to 70? C and pressure in the range of 5 to 400 millibar to form a solid mass; grinding the solid mass to form the composition.

BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates the results of the comparative sensory analysis, in accordance with Example 2 of the present disclosure.
Fig.2 illustrates the inhibitory activity of the curcumin-green tea extract (D2), green tea extract-fructooligosaccharide (D3) as compared with the 2.5 µg of Ciprofloxacin standard drug (S) and 1% dimethyl sulfoxide solution as control (C) against Prevotella Copri.
Fig. 3 illustrates the inhibitory activity of the green tea extract-fructooligosaccharide (M1), green tea extract (M2) and fructooligosaccharide (W1) as compared with the 2.5 µg of Ciprofloxacin, the standard drug (S), and 1% dimethyl sulfoxide solution as control (C) and water (C1) against Prevotella Copri.

DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alterations and further modifications in the disclosed composition and method, and such further applications of the principles of the disclosure therein being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof.

Reference throughout this specification to “one embodiment” “an embodiment” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The present disclosure relates to compositions for controlling Prevotella copri in the gut. The present disclosure also relates to methods for controlling Prevotella copri using such compositions.
In the context of the present disclosure, the term “controlling” refers to the ability to kill or inhibit the growth of Prevotella Copri.
The present disclosure particularly relates to a composition comprising green tea extract, curcumin, and a fructooligosaccharide. In an embodiment, the disclosed composition comprises the green tea extract, the curcumin, and the fructooligosaccharide in a weight ratio in a range from 1:1:2 to 3:1:12.
In a specific embodiment, the disclosed composition comprises the green tea extract, the curcumin and the fructooligosaccharide in the weight ratio of 1:1:2 to 2:1:12.
In an embodiment, the disclosed composition comprises the green tea extract, and the curcumin in a weight ratio of 1:1 to 3: 1. In an embodiment, the fructooligosaccharide is present in an amount of two to twelve times the total amount of the green tea extract and the curcumin in the composition.
The green tea extract can be derived from the leaves and buds of Camella Sinensis. The applicant has obtained the green tea extract from commercial sources, namely, Vidya Herbs Private Ltd., Bangalore.
The green tea extract comprises chemical constituents such as flavonoids, vitamins, epigallocatechin gallate (EGCG) epicatechin 3-gallate (EGC), epicatechin 3-gallate (EGC), epigallocatechin (ECG), and epicatechin (EC). In a specific embodiment, the green tea extract contains epigallocatechin gallate (EGCG).

In an embodiment, the green tea extract comprises epigallocatechin gallate (EGCG) in the range of 30 to 95% w/w based on the total weight of green tea extract.
Curcumin is derived from the rhizomes of Curcuma longa. The applicant has obtained the curcumin from commercial sources, namely, Vidya Herbs Private Ltd, Bangalore.
In accordance with an aspect, the fructo-oligosaccharide in the composition is a mixture of oligosaccharides consisting of a sucrose molecule (fructose-fructose disaccharide, GF1) linked to one (GF2), or two (GF3) or three (GF4) additional fructose units added by ß-2-1 glycosidic linkages to the fructose unit of sucrose. The GF2, GF3 and GF4 oligosaccharides, are also called 1-kestose, nystose and fructofuranosylnystose, respectively.
In accordance with an embodiment, the fructo-oligosaccharide in the FOS composition comprises about 35 to 55% of 1-kestose (GF2), 35 to 55% nystose (GF3) and 5 to 20% fructofuranosylnystose (GF4).
Examples of the fructo-oligosaccharide includes but are not limited to short-chain fructooligosaccharides, long-chain oligofructose and inulin. The applicant has obtained the fructooligosaccharide from commercially available brands, namely, Fossence (by Tata Chemicals Ltd.).
The disclosed composition comprising the green tea extract and the curcumin are homogeneously dispersed in the fructo-oligosaccharide. In the context of the present disclosure the term “homogenously dispersed” means that the curcumin and the green tea extract is distributed uniformly in the fructo-oligosaccharide in the disclosed composition.
In an embodiment, the disclosed composition is in granular form.
In accordance with an embodiment, the composition comprises granules having a particle size in the range of 1 to 1000 microns. In a specific embodiment, the granules have the particle size in the range of 10 to 800 microns.
In accordance with an embodiment, the disclosed composition further comprises one or more ingredient selected from the group comprising of galacto-oligosaccharides (GOS), maltodextrin, starch, polyols, saccharides, artificial sweeteners and natural sweeteners.
The granules can be formulated in any suitable dosage form. In accordance with an embodiment, the composition is in the form of a solid dosage form or a liquid dosage form. In accordance with an embodiment, the solid dosage form is selected from the group consisting of a tablet, a capsule, and a powder for oral administration.
The disclosed composition may further comprise one or more pharmaceutically acceptable excipient(s) selected from soluble and insoluble lubricants, disintegrants, binders, glidants, or combination thereof. Examples of suitable excipients include, but are not limited to starch, polysaccharides, natural and natural like flavors, gums, silica, polyols, artificial sweeteners.
Examples of binders include, but are not limited to, corn starch, pregelatinized starch, microcrystalline cellulose, silicified MCC (e.g., Prosolv® HD 90), methyl cellulose, hydroxypropyl cellulose (HPC-L), methylcellulose, carboxymethyl cellulose sodium, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and mixtures thereof.
Examples of disintegrants include, but are not limited to, cross-linked polyvinyl pyrrolidone, corn starch, modified starches, agar-agar, calcium carbonate, sodium carbonate, alginic acids, cross-carmellose sodium, sodium starch glycolate (SSG), microcrystalline cellulose, hydroxypropyl cellulose (L-HPC), and mixtures thereof.
Examples of anti-adherents include, but are not limited to, magnesium stearate, talc, calcium stearate, glyceryl behenate, stearic acid, and mixtures thereof.
Examples of lubricants and glidants include, but are not limited to, colloidal anhydrous silica, stearic acid, magnesium stearate, calcium stearate, talc, microcrystalline wax, yellow beeswax, white beeswax, sodium stearyl fumarate, and mixtures thereof.
Examples of suitable excipients include, but are not limited to, Microcrystalline Cellulose (MCC) such as MCC101, Sodium Starch Glycolate (SSG), polyvinylpyrrolidone such as PVP K30, magnesium stearate, and a combination thereof.
The present disclosure also relates to a process for preparing a composition comprising green tea extract, curcumin, and a fructooligosaccharide.

The process comprises blending the green tea extract, the curcumin and the fructooligosaccharide in a ratio from 1:1:2 to 3:1:12 to form a homogenized blend; adding a solvent to the homogenized blend to form a slurry; subjecting the slurry to reduced temperature and pressure to cause evaporation to form a solid mass; followed by grinding the solid mass to form the composition.
The green tea extract, the curcumin and the fructo-oligosaccharide can be blended together sequentially in any order or simultaneously. In an example, the green tea extract, and the curcumin are blended prior to the addition of the fructo-oligosaccharide.
In accordance with an embodiment, the evaporation of the slurry is carried out at a temperature in the range of 40 to 70 ?C and pressure in the range of 5 to 400 millibar for a time period in the range of 0.5 to 12 hours to form the solid mass.
Any suitable equipment can be used for evaporating the slurry to form the solid mass. Such equipment includes but is not limited to a rotary evaporator.
In accordance with an embodiment, in the homogenized blend, the green tea extract and, the curcumin are uniformly dispersed in the fructo-oligosaccharide.
In accordance with an embodiment, the solvent added in the homogenized blend is a polar protic solvent. The solvent is selected from the group consisting of water, ethanol, methanol or a combination thereof.
The solid mass obtained after the evaporation process is then ground to obtain the desired granule size. The grinding may be carried out in any suitable apparatus for grinding solid mass. Such apparatus includes but is not limited to mortar mills, vibrator mills or ball mills. The grinding of the solid mass is carried out for 1 to 120 minutes.
In accordance with an embodiment, the grinding is done to obtain the composition comprising granules having a particle size in the range of 1 to 1000 microns. In a specific embodiment, the granules obtained from the above process have the particle size in the range of 10 to 800 microns.
In accordance with an embodiment, the homogenized blend of the green tea extract, the curcumin and the fructo-oligosaccharide are co-processed with an ingredient selected from the group comprising of galacto-oligosaccharides (GOS), maltodextrin, starch, polyols, saccharides, artificial sweeteners and natural sweeteners.
The granules obtained from the above process can be formulated in any suitable dosage form. In accordance with an embodiment, the composition is an oral dosage form selected from the group consisting of a tablet, a capsule, and a powder for oral administration.
The disclosed composition obtained from above process may further comprise one or more pharmaceutically acceptable excipient(s) selected from soluble and insoluble lubricants, disintegrants, binders, glidants, or combination thereof. Examples of suitable excipients include, but are not limited to starch, polysaccharides, flavor, stabilizers, bulking agent, gums, silica, polyols, and artificial sweeteners.
Examples of suitable binders that can be used in the process include, but are not limited to, corn starch, pregelatinized starch, microcrystalline cellulose, silicified MCC (e.g., Prosolv® HD 90), methyl cellulose, hydroxypropyl cellulose (HPC-L), methylcellulose, carboxymethyl cellulose sodium, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and mixtures thereof.
Examples of suitable disintegrants that can be used in the process include, but are not limited to, cross-linked polyvinyl pyrrolidone, corn starch, modified starches, agar-agar, calcium carbonate, sodium carbonate, alginic acids, cross-carmellose sodium, sodium starch glycolate (SSG), microcrystalline cellulose, hydroxypropyl cellulose (L-HPC), and mixtures thereof.
Examples of suitable anti-adherents that can be used in the process include, but are not limited to, magnesium stearate, talc, calcium stearate, glyceryl behenate, stearic acid, and mixtures thereof.
Examples of lubricants and glidants that can be used in the process include, but are not limited to, colloidal anhydrous silica, stearic acid, magnesium stearate, calcium stearate, talc, microcrystalline wax, yellow beeswax, white beeswax, sodium stearyl fumarate, and mixtures thereof.
Examples of suitable excipients that can be used in the process include, but are not limited to, Microcrystalline Cellulose (MCC) such as MCC101, Sodium Starch Glycolate (SSG), polyvinylpyrrolidone such as PVP K30, magnesium stearate, and a combination thereof.
The present disclosure also relates to a method for controlling Prevotella copri in the gut. The method comprising administering to the subject in need thereof a therapeutically effective amount of the disclosed composition. It has been found that by administering the composition as disclosed, Prevotella copri is controlled in the gut. The fructo-oligosaccharide in the disclosed composition helps in the modulation of gut by stimulating the growth of nonpathogenic intestinal microflora.
The present disclosure also relates to a food matrix comprising from about 0.1 to 5 % w/w of the composition. Such food matrix includes but are not limited to cereals, cookies, doughnuts, biscuits, pizza dough, pasta, bread, other baked goods, juices, salads, sauces, and candies.

Specific embodiments are disclosed below.
A composition for controlling Prevotella copri in gut, the composition comprising green tea extract, curcumin and a fructo-oligosaccharide in a weight ratio of 1:1:2 to 3:1:12.
Such composition (s), wherein the green tea extract, the curcumin and the fructo-oligosaccharide are in the weight ratio of 1:1:2 to 2:1:12.
Such composition (s), wherein the green tea extract and the curcumin are homogenously dispersed in the fructo-oligosaccharide.
Such composition (s), wherein green tea extract comprises epigallocatechin gallate.
Such composition (s), further comprising an ingredient selected from the group consisting of galacto-oligosaccharides (GOS), maltodextrin, starch, polyols, saccharides, artificial sweeteners and natural sweeteners.
Such composition (s), wherein the composition is in granular form.
Such composition (s), wherein the composition is in an oral dosage form selected from the group consisting of a tablet, a capsule, and a powder for oral administration.
Such composition (s), wherein the composition comprises granules having a particle size in the range of 1 to 1000 micron.
A food matrix comprising about 0.01 to 5 %w/w of the composition.
A process for preparing a composition, the process comprising:
blending the green tea extract, the curcumin and the fructo-oligosaccharide in a ratio of 1:1:2 3:1:12 to form a homogenized blend;
adding a solvent to the homogenized blend to form a slurry;
subjecting the slurry to evaporation at a temperature in the range of 40 to 70? C and pressure in the range of 5 to 400 millibar to form a solid mass;
grinding the solid mass to form the composition.
Such process, wherein the solvent is a polar protic solvent selected from the group consisting of water, ethanol, methanol and a combination thereof.

EXAMPLES
The following examples are provided to explain and illustrate the preferred embodiments of the present disclosure and do not in any way limit the scope of the disclosure as described.

Example 1: Process of preparation of the composition in accordance with the embodiment of the present disclosure:
Table 1: Composition formulation:
Sr. No. Components Quantity for 7.187grams
1 Green Tea extract 1 gram
2 Curcumin 0.187 gram
3 FOS 6 grams
Total 7.187 grams

1 gram of green tea extract,0.187 grams of curcumin and 6 grams of fructo-oligosaccharide were blended to form a mixture. The mixture was taken in a round bottom flask and 10 ml (~10 grams) of water was added. This mixture was first mixed in a rotatory evaporator for few minutes at 50 °C. After it became slurry and was completely mixed, water of this syrup like slurry material was evaporated at 55-60 °C at reduced pressure of ~50 millibar. After evaporation of water, this co-processed solid mass was taken out from flask with help of spatula and was ground for further process.

Example 2: Process of preparation of the composition in accordance with the embodiment of the present disclosure:
Table 2: Composition formulation:
Sr. No. Components Quantity for 28.75 grams batch
1 Green Tea extract 4 grams
2 Curcumin 0.75 grams
3 FOS 24 grams
Total 28.75 grams

4 grams of green tea extract, 0.75 grams of curcumin and 24 grams of fructo-oligosaccharide were blended to form a mixture. The mixture was taken in a round bottom flask and 20 ml (~20 gm) of water was added. This mixture was first mixed in a rotatory evaporator for few minutes at 50 °C. After it became slurry and was mixed completely, water of this syrup like slurry material was evaporated at 55-60 °C at reduced pressure of ~50 millibar. After evaporation of water, this co-processed solid mass was taken out from flask with help of spatula and was ground for further process.
Example 3: Comparative sensory analysis of cookies comprising (i) admixture of green tea extract, curcumin and fructo-oligosaccharide, and (ii) composition prepared in Example 2, as provided in Table 2.
Table 3: Cookie Formulation
Composition Admixture
% (w/w) Composition % (w/w)
S. No
1 Whole Wheat Flours 48.31 48.31
2 Fat (Marvo) 22.00 22.00
3 Sugar 21.00 21.00
4 Skim Milk Powder 0.87 0.87
5 Baking powder 0.10 0.10
6 Ammonium Bicarbonate 0.35 0.35
7 Salt 0.06 0.06
8 Vanilla 0.12 0.12
9 Green Tea Extract-curcumin 1.19 0.00
10 FOS 6.00 0.00
11 Composition of Example 2 0.00 7.19
Total 100 100
Cookies were prepared according to process given below:
1. All the ingredients were weighed accurately.
2. Salt was dissolved in little amount of water.
3. Fat, fructo-oligosaccharide, milk powder and sugar were added.
4. The ingredients were blended for 1 minute at low speed.
5. The ingredients were blended for 3 minutes at high speed.
6. After 3 minutes, water was added slowly.
7. The ingredients were blended for another 4 minutes at high speed.
8. All dry ingredients were added and further mixed for 1 to 2 minutes.
9. Cookies were cut as per required size.
10. Wet cookie weight was found to be 16 grams.
11. Cookies were baked at 160 0C for 15 to 20 minutes.
12. Cookies were packed in laminated aluminum packaging and sealed.
Sensory Analysis: Sensory analysis was done by 12-member panelists with 5 point hedonic scale in terms of bitterness taste “not detectable” and “very unpleasant”.
The results were evaluated in accordance with the criteria for scoring as given below:
1: not detectable
5: very unpleasant

Table 4: Results of the responses of the panelists to the admixture sample and the composition of the present disclosure.

Trained Panel Member Admixture sample Composition
Member 1 4 1
Member 2 4 1
Member 3 3 1
Member 4 4 1
Member 5 4 1
Member 6 3 1
Member 7 5 1
Member 8 5 1
Member 9 5 1
Member 10 4 1
Member 11 2 2
Member 12 2 1
Total 45 13
Average 3.75 1.08
Standard Error 0.29 0.08

Observations: Results of sensory analysis shows that disclosed composition effectively masked the bitter taste of green tea extract as compared to the simple admixture sample. Simply adding the mix of green tea extract, curcumin and fructo-oligosaccharide does not provide any bitterness masking. On the other hand, the composition of example 2 effectively masked the bitter taste of the green tea extract. Table 4 and Figure 1 shows the results of sensory analysis.
Example 4: Evaluation of anti-microbial activity of the disclosed composition against Prevotella copri by well diffusion assay.

Test organisms: Prevotella copri

Test compounds:
1. Fructo-oligosaccharide (FOS)
2. Green tea extract (GTE)- fructooligosaccharide (FOS) (1:1)
3. Green tea extract (GTE)
4. Curcumin
5. Curcumin-green tea extract (GTE) (D2) (1:1)
6. Curcumin-green tea extract (GTE)-fructooligosaccharide (FOS) (D3) (1:1:2)

Standard compound (S): Ciprofloxacin (2.5 µg)
Control (C): 1% DMSO

Preparation of stock solution: Fructo-oligosaccharide stock solution (100mg/ml) was prepared in water. Green tea extract-fructooligosaccharide stock solution (100mg/ml) and green tea extract (100 mg/ml) were prepared in methanol respectively. Curcumin, curcumin- green tea extract (GTE), and curcumin- green tea extract (GTE) – fructooligosaccharide (FOS) were prepared in 1% dimethyl sulfoxide (DMSO) respectively.

Table 5: Stock solution of the compounds

S.No Compound Concentration (mg/ml) Solvent
1 FOS (L95) 100 Water
2 GTE FOS 100 Methanol
3 Green Tea Extract 100 Methanol
4 Curcumin 100 1% DMSO
5 Curcumin-GTE 100 1% DMSO
6 Curcumin-GTE -FOS 100 1% DMSO

Culture media: Columbia agar media for bacteria was weighed accurately, dissolved in 44 gm per liter of distilled water and autoclaved. The culture media was supplemented with heat inactivated sheep blood (5%).

Procedure for the determination of the antimicrobial activity:
100 µl inoculum of test cultures was inoculated on Columbia agar plates (90 mm). Test compounds (10µl) each were added to 5mm wells on Columbia agar plates. The plates were incubated at 35?C for 24 to 48 hours and observed for the zone of inhibition around the well. The plates were observed for zone of inhibition and diameter was measured by a ruler and compared with standard compound used.

Results:
Table 6: Inhibitory activity of the compounds against Prevotella copri

S.No Test Sample Concentration (µg/well) Zone of inhibition
(mm)

1 Methanol (Control) 10 µL -
2 Water (Control) 10 µL -
3 1% DMSO (Control) 10 µL -
4 Ciprofloxacin (Standard) 10µg 30.00 ± 0.00
5 Curcumin 1000 µg -
6 Curcumin-GTE 1000 µg 15.00 ± 0.00
7 Curcumin-GTE-FOS 1000 µg 14.00 ± 0.50
8 FOS 1000 µg -
9 GTE-FOS 1000 µg 15.00 ± 0.00
10 Green Tea Extract 1000 µg 17.00 ± 0.00

Observations: As it can be observed from Table 6, curcumin and fructo-oligosaccharide does not have any inhibitory activity on Prevotella copri. The green tea extract, curcumin-green tea-fructooligosaccharide, green tea extract-fructooligosaccharide, and curcumin-green tea extract showed inhibitory activity with a zone of inhibition of 17 mm, 14 mm, 15 mm and 15 mm respectively. Fig.2 shows the inhibitory activity of the curcumin-green tea extract (D2), green tea extract-fructooligosaccharide (D3) as compared with the ciprofloxacin standard drug (S) and 1% dimethyl sulfoxide solution as control (C) against Prevotella Copri. Fig. 3 shows the inhibitory activity of the green tea extract-fructooligosaccharide (M1), green tea extract (M2) and fructooligosaccharide (W1) as compared with ciprofloxacin, the standard drug (S), and 1% dimethyl sulfoxide solution control (C) and water (C1) against Prevotella Copri.

Example 5: Minimum inhibitory concentration (MIC) of the test compounds by micro broth dilution technique as per National Committee for Clinical Laboratory Standards (NCCLS) method.

Test organisms: Prevotella copri

Test compounds:

1. Curcumin-green tea extract (1:1)
2. Curcumin-green tea extract – fructooligosaccharide (1:1:2)
3. Green tea extract- fructooligosaccharide (1:1)
4. Green tea extract
Standard compound (S): Ciprofloxacin
Inoculum: Cell suspension prepared from bacterial cultures grown on Columbia broth adjusted to 1-2 x 105 cells/ml.

Drug concentrations:
Ciprofloxacin: 0.25-16 µg/ml (two-fold dilutions) in Columbia broth.
Test compounds: 15.625– 1000 µg/ml of samples at 2-fold dilutions in Columbia broth
Control: Columbia broth inoculated with culture and without test compound.

Procedure for the determination of the minimum inhibitory concentration (MIC) of the test compounds.

1. 90µl of solution with standard drug (0.25-16 µg/ml) or test compounds having different test concentration (15.625– 1000 µg/ml) was added in triplicate to 96 well culture plate containing 10µL inoculum.
2. 100µl of Columbia broth without standard drug and test compounds was added in triplicates to wells containing 10µl of inoculum.
3. Treated bacterial cultures was incubated at 37?C in an anaerobic jar and were observed after 24 to 48 hours and optical density (O.D) at the wavelength of 590 nm was measured in Tecan plate reader.
4. Minimum Inhibitory Concentration (MIC) of the standard drug and the test compounds were determined and compared with control.

Table 7: Minimum Inhibitory concentration (MIC) of the test compounds

S. No. Test compounds Minimum Inhibitory Concentration (MIC) (µg/mL)
1. Ciprofloxacin 1
2. Green tea extract (125 µg/mL) 125
3. Curcumin-green tea extract (62.5+62.5 µg/mL) 125
4. Curcumin-green tea extract –fructooligosaccharide (62.5+62.5+125 µg/mL) 250
5. Green tea extract –fructooligosaccharide
(125+125 µg/mL) 250

Observations: Based on the results of Table 7, it can be concluded that when green tea extract is used in combination with curcumin, same inhibitory activity of ‘green tea extract alone’ can be achieved by using half amount of green tea extract. Curcumin in combination with half the amount of the green tea extract shows synergistic effect.

INDUSTRIAL APPLICABILITY
The disclosed composition is easy to make and is inexpensive. The disclosed composition helps in controlling Prevotella Copri in the gut. In the disclosed composition, the curcumin and the green tea extract shows the same inhibitory effect of the green tea extract alone by using half the amount of the green tea extract. Curcumin in combination with half the amount of the green tea extract shows synergistic effect in inhibition of Prevotella copri. The disclosed composition facilitates oral administration of green tea extract by effectively masking the bitter taste of green tea extract. The disclosed composition exhibits substantial bitterness masking as compared to a mere admixture of green tea extract, curcumin and fructo-oligosaccharide. Fructooligosaccharide in the disclosed composition acts as a prebiotic that beneficially affect the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. Fructooligosaccharide in the composition also does not interfere the inhibition activity of the green tea extract or the curcumin. The disclosed composition provides a safer solution for effectively controlling the growth of Prevotella Copri in the gut.
The disclosed granular composition can be used in food matrix including but not limited to cereals, cookies, crackers, doughnuts, biscuits, pizza dough, pasta, bread, other baked goods, juices, salads, sauces, and candies. The disclosed composition can also be used as dietary supplements and nutraceuticals.