Claims:1. A method of increasing abundance of Faecalibacterium prausnitzii in gastro intestinal tract of a subject, the method comprising administering to said subject a composition comprising fructo-oligosaccharide, such that fructo-oligosaccharide is administered to the subject at a dose of at least 5grams per day, wherein said composition promotes an increase in abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the subject to a statistically significant level when measured by F-test.

2. The method as claimed in claim 1, wherein a dose of between 5 grams per day to 10grams per day of fructo-oligosaccharide is administered to the subject.

3. The method as claimed in claim 1, wherein the subject is a mammal and preferably the subject is a human being.

4. The method as claimed in claim 1, wherein the fructo-oligosaccharide composition is administered for 1 to 90 days.

5. The method as claimed in claim 1, wherein the composition of fructo-oligosaccharide comprises about 40-50% 1-kestose(GF2), 40-50% nystose(GF3) and 5-10% fructofuranosyl nystose (GF4).

6. The method as claimed in claim 1, wherein the composition of fructo-oligosaccharide is in the form of a liquid or a solid dosage form for oral administration.
7. A method of treating inflammatory bowel disease in a human subject, the method comprising increasing abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the human subject to a statistically significant level when measured by F-test by administering to said human subject a composition comprising fructo-oligosaccharide such that fructo-oligosaccharide is administered to the subject at a dose of least 5grams per day.

8. The method as claimed in claim 7, wherein a dose of between 5grams per day to 10grams per day of fructo-oligosaccharide is administered to the subject.

9. The method as claimed in claim 7, wherein the composition of fructo-oligosaccharide comprises about 40-50% 1-kestose(GF2), 40-50% nystose(GF3) and 5-10% fructofuranosylnystose (GF4).

10. The method as claimed in claim 7, wherein the fructo-oligosaccharide composition is administered for 1 to 90 days.

11. A composition for increasing abundance of Faecalibacterium prausnitzii in gastro intestinal tract of a subject, the composition comprising at least 5g of fructo-oligosaccharide, wherein said composition promotes increase in abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the subject to a statistically significant level when measured by F-test.

12. The composition as claimed in claim 11, wherein the composition comprises between 5g to 10g of fructo-oligosaccharide.
13. The composition as claimed in claim 11, wherein the fructo-oligosaccharide composition comprises about 40-50% 1-kestose(GF2), 40-50% nystose (GF3) and 5-10% fructofuranosylnystose (GF4).

14. The composition as claimed in claim 11, wherein the fructo-oligosaccharide composition is in the form of a liquid or a solid dosage form for oral administration.

15. The composition as claimed in claim 11, wherein the fructo-oligosaccharide composition further comprising other components selected from a group comprising Galacto-oligosaccharides, vitamins, minerals, plant extracts, pharmaceutically acceptable excipients or a mixture therefore.
, Description:FEILD OF THE INVENTION
The present disclosure relates to a method and composition for promoting growth of a bacteria in the gastro-intestinal tract of a subject. Specifically, the present disclosure relates to a method and a composition for increasing abundance of Faecalibacterium prausnitzii in gastro- intestinal tract of a mammal.

BACKGROUND
The gastro-intestinal tract or gut of a mammal harbours within itself a complex ecosystem that includes several bacterial species. For example, there are approximately 300 to 500 bacterial species in the intestinal tract of a human being (Quigley EMM. Gut Bacteria in Health and Disease. Gastroenterology & Hepatology. 2013;9(9):560-569). These bacteria provide beneficial effect to the hosts by maintaining a healthy gut and by increasing resistance against pathogenic bacteria.
Among the bacteria present in the gastro intestinal tract of a healthy human, it is reported that Faecalibacterium prausnitzii, a member of the Firmicutes phylum is the most abundant. This bacterium represents more than 5% of the total bacterial population in a healthy human. (Miquel et. al., 2013). Studies have indicated that this species is one of the highly functionally active members of the microbiome (Miquel et. al., 2013) and is known to improve gut health (Sokol et al.2009).
Faecalibacterium prausnitzii, is a butyrate-producing bacterium. Butyrate is known to play a major role in gut physiology and has pleiotropic effect in intestinal cell life cycle and has numerous beneficial effects for health. Butyrate provides protection against pathogen invasion, modulates the immune system and reduces cancer progression. Thus, by producing butyrate in the gut, Faecalibacterium prausnitzii is thought to have positive impact on physiological functions and overall health of the host. (Miquel et. al., 2013)
In addition to forming butyrate, Faecalibacterium prausnitzii, is found to exhibit anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-?? activation and IL-8 production. (Sokol et al.2009)
Recent studies suggest that relative abundance of Faecalibacterium prausnitzii is compromised in patients reporting Crohn’s disease and ulcerative colitis, two groups of inflammatory bowel disease. Relative abundance of Faecalibacterium prausnitzii is reported as 2.36 ± 1.75 and 4.24 ± 3.81 in subjects with Crohn’s disease and ulcerative colitis respectively as compared to 12.28 ± 7.43 in healthy subjects (Vrakas et al., 2017). It is also reported that reduction of Faecalibacterium prausnitzii is associated with a higher risk of postoperative recurrence of ileal Crohn's Disease (Sokol et al. PNAS, 2008, Vol.105, No.43, 16731-16736).
Thus, there is a need for method for enhancing the population of Faecalibacterium prausnitzii in the gastro-intestinal tract of a subject.

SUMMARY
A method of increasing abundance of Faecalibacterium prausnitzii in gastro-intestinal tract of a subject is provided. The method comprises administering to said subject a composition comprising fructo-oligosaccharide, such that fructo-oligosaccharide is administered to the subject at a dose of at least 5 grams per day, wherein said composition promotes an increase in abundance of Faecalibacterium prausnitzii in the gastro-intestinal tract of the subject to a statistically significant level when measured by F-test.
A method of treating inflammatory bowel disease in a human subject is also provided. The method comprises increasing abundance of Faecalibacterium prausnitzii in the gastro- intestinal tract of the human subject to a statistically significant level when measured by F-test by administering to said human subject a composition comprising fructo-oligosaccharide such that fructo-oligosaccharide is administered to the subject at a dose of least 5grams per day.
A composition for increasing abundance of Faecalibacterium prausnitzii in gastro- intestinal tract of a subject is further provided. The composition comprises at least 5 grams of fructo-oligosaccharide, wherein said composition promotes increase in abundance of Faecalibacterium prausnitzii in the gastro-intestinal tract of the subject to a statistically significant level when measured by F-test.

BRIEF DESCRPTION OF DRAWINGS
Figure 1 depicts a sample processing pipeline for microbiome annotation in accordance with an embodiment.
Figure 2 depicts the distribution of Faecalibacterium prausnitzii in fecal sample before and during fructo-oligosaccharide intervention in accordance with an embodiment.
Figure 3 depicts the relative abundance of Roseburia hominis in fecal samples before and during fructo-oligosaccharide intervention at a dose of 10 grams per day.
Figure 4 depicts the relative abundance of Roseburia hominis in fecal samples before and during fructo-oligosaccharide intervention at a dose of 5 grams per day.
Figure 5 depicts relative abundance of Bacteroides plebeius in fecal samples before and during fructo-oligosaccharide intervention at a dose of 10 grams per day.
Figure 6 depicts relative abundance of Bacteroides plebeius in fecal samples before and during fructo-oligosaccharide intervention at a dose of 5grams per day.
Figure 7 depicts the relative abundance of Prevotella copri in fecal samples before and during fructo-oligosaccharide intervention at a dose of 10 grams per day.
Figure 8 depicts the relative abundance of Prevotella copri in fecal samples before and during fructo-oligosaccharide intervention at a dose of 5 grams per day.
DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, 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 invention is thereby intended, such alterations and further modifications in the disclosed product and process, and such further applications of the principles of the invention therein being contemplated as would normally occur to one skilled in the art to which the invention 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 and are not intended to be restrictive.
Reference throughout this specification to “one embodiment” “an embodiment” or similar language means that a particular feature, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. 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 provides a method of increasing the abundance of Faecalibacterium prausnitzii in gastro-intestinal tract of a subject. The disclosure also provides a composition for increasing the abundance of Faecalibacterium prausnitzii in the gastro- intestinal tract of a subject.
The method of increasing the abundance of Faecalibacterium prausnitzii comprises administering to the subject a composition comprising fructo-oligosaccharide in an amount sufficient to increase the abundance of Faecalibacterium prausnitzii in the gastro-intestinal tract of the subject to a statistically significant level when measured by F-Test.
In accordance with an embodiment, the method results in an increase in absolute abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the subject to a statistically significant level when measured by F-Test.
As used herein ‘Absolute Abundance’ refers to the total count of the bacterium of interest. The total count or abundance of a bacterium may be determined most easily by quantification of the bacterium from a sample. Methods for quantification of bacteria include but are not limited to genomic sequencing, metagenomic sequencing or standard cell culturing methods based on optics.
In accordance with an alternative embodiment, the method results in an increase in relative abundance of Faecalibacterium prausnitzii in the gastro intestinal-tract of the subject to a statistically significant level when measured by F-Test.
As used herein ‘Relative Abundance’ refers to the count of bacterium of interest divided by the total count of all the measured bacteria, unless otherwise indicated. The relative abundance of a bacterium may be determined most easily by quantification of all bacteria from a sample. Methods for quantification of bacteria include but are not limited to genomic sequencing, metagenomic sequencing or standard cell culturing methods on optics.
In accordance with an embodiment, statistically significant level refers to a F-score reflective of a P-value less than or equal to 0.05.
In accordance with an aspect, the subject is a mammal. In accordance with an embodiment the mammal is a human being.
The amount of composition administered to the subject, is such that a dose of at least 5 grams per day of fructo-oligosaccharide is administered to the subject. In accordance with an embodiment, the amount of composition administered is between about 5 grams per day to about 10 grams per day of fructo-oligosaccharide to the subject. Preferably the amount of composition administered to the subject, is such that a dose of about 10 grams of fructo-oligosaccharide is administered on a daily basis. The daily amount may be consumed once per day or maybe divided into two or three portions that may or may not be equal.
In accordance with an embodiment, the dose of fructo-oligosaccharide required to increase the absolute abundance or relative abundance of Faecalibacterium prausnitzii may be determined based on the initial absolute abundance or relative abundance of Faecalibacterium prausnitzii in the gastro- intestinal tract of the subject.
In accordance with an aspect, the composition may be administered for 1 to 90 days till the required absolute abundance or relative abundance of Faecalibacterium prausnitzii is achieved. However, it is also possible that the composition may be administered for period longer than 90 days.
A composition for promoting the growth of Faecalibacterium prausnitzii in human gut is also disclosed. The composition comprising at least 5 grams of fructo-oligosaccharide, wherein said composition promotes an increase in abundance of Faecalibacterium prausnitzii in the gastro-intestinal tract of the subject to a statistically significant level when measured by F-test.
The composition comprises between 5 grams to 10 grams of fructo-oligosaccharide and preferably 10 grams of fructo-oligosaccharide.
In accordance with an aspect, the composition is a mixture of oligosaccharides consisting of a sucrose molecule (glucose -fructose disaccharide, GF1) linked to one (GF2), or two (GF3) or three (GF4) additional fructose units added by ß-2-l glycosidic linkages to the fructose unit of the 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 composition comprises about 40-50% 1-kestose (GF2), 40-50% nystose (GF3) and 5-10% fructofuranosylnystose (GF4).
The composition may contain other components including but not limited to galacto-oligosaccharides, vitamins, minerals, plant extracts. The composition may also contain pharmaceutically acceptable excipients.
The composition may be in any desirable formulation suitable for oral intake. In accordance with an embodiment, the composition is in the form of a solid dosage form or a liquid dosage form. In an example, the composition is in the form of oral syrup.
In accordance with an embodiment, the present disclosure also relates to a method of preventing or treating inflammatory bowel disease in a human subject. The method comprises of increasing the abundance of Faecalibacterium prausnitzii in the gastro-intestinal tract of the human subject to a statistically significant level when measured by F-test by administering to said human subject a composition comprising fructo-oligosaccharide such that fructo-oligosaccharide is administered to the subject at a dose of at least 5 grams per day.

EXAMPLES
Experimental design and sample collection
The study was conducted as per the pertinent requirements of the ICMR guidelines for Biomedical Research on Human Subjects, Good Clinical Practices for Clinical Research in India. The protocol was carried out in accordance with the approved guidelines, and was in agreement with Declaration of Helsinki principles.
The study was a random, double blinded design and was performed on a cohort of sixty (60) subjects. The group of sixty healthy adults were randomly divided into 3 groups: T1 group, T2 group, and T3 group depending upon the amount of fructo-oligosaccharide received/consumed. Table 1, provides the amount of fructo-oligosaccharide that is received/consumed by each of the groups.
Group Amount of fructo-oligosaccharide consumed per day
T1 2.5 grams
T2 5 grams
T3 10 grams

All participants consumed the assigned dosage of fructo-oligosaccharide on daily basis for three (3) months. DNA from the gut bacteria present in the fecal samples of the sixty subjects defining the cohort was extracted. For each of the sixty subjects, samples were collected at seven (7) time points–before the subject received the prebiotic on the -60th, -30th and 0th day and, during the three (3) month prebiotic intervention on the 1st, 30th, 60th, 90th day.

Sample processing
Each fecal sample was processed using standard nucleic acid extraction protocols. After the nucleic acid extraction, 16SrRNA gene based sequencing of the extracted DNA was performed. V4 region of the extracted 16S RNA gene was amplified and sequenced. Obtained sequenced data was processed using a standard pipeline (using Second Genome pipeline). Post processing through the mentioned pipeline, abundance of Faecalibacterium prausnitzii was computed and tabulated. An exemplary sample processing pipeline adopted to collect and process fecal samples for obtaining the microbiome data for microbiome annotation is shown in Figure 1.

Data analytics
Statistical assessment of abundance change of microorganism was performed using F-test. The test was performed on the R platform. Change in relative abundance of Faecalibacterium prausnitzii prior to intervention with fructo-oligosaccharide and during intervention across the arms of the study was assessed and reported.

Results:
It is observed that fructo-oligosaccharide could increase relative abundance of Faecalibacterium prausnitzii. Significant increase was observed at dosage of 10 grams per day and 5 grams per day. Fold change (increase) was 1.6, 1.38 and 1.04 at dosage 10 grams fructo-oligosaccharide per day, 5grams fructo-oligosaccharide per day and 2.5 grams fructo-oligosaccharide per day respectively. (Table 1).

Table 1: Summary of Faecalibacterium prausnitzii response at multiple dosages reported below.
Measure Dosage (grams per day)
2.5 5 10
F-Statistic 1.7 2.6 0.17
P-value of F-test 0.06 5.2*e-4 2.1*e-10
Fold change 1.04 1.38 1.6

Figure 2 reports response of Faecalibacterium prausnitzii on intervention with fructo- oligosaccharide. It is observed that with daily intervention of fructo-oligosaccharide at a dosage of 10 g per day relative abundance of Faecalibacterium prausnitzii is increased.
However, no change in relative abundance of other bacterium such as Roseburia hominis, Prevotella copri and Bacteroides plebeius was observed in subjects after intervention of fructo-oligosaccharide. Figures 3-8 report the relative abundance of some of these bacteria before and during intervention with fructo-oligosaccharide.
Figure 3 depicts the relative abundance of Roseburia hominis in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide with dosage 10 grams per day, no significant relative abundance change was observed (F-statistics = 0.64, P-value =0.09).
Figure 4 depicts the relative abundance of Roseburia hominis in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide at a dose of 5 grams per day, no significant relative abundance change was observed (F-statistics = 1.04, P-value =0.87).
Figure 5 depicts relative abundance of Bacteroides plebeius in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide at a dose of 10 grams per day, no significant relative abundance change was observed (F-statistics = 0.86, P-value =0.76).
Figure 6 depicts relative abundance of Bacteroides plebeius in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide at a dose of 5 grams per day, no significant relative abundance change was observed (F-statistics = 1.69, P-value =0.52).
Figure 7 depicts the relative abundance of Prevotella copri in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide at a dose of 10 grams per day, no significant relative abundance change was observed (F-statistics = 0.95, P-value =0.84).
Figure 8 depicts the relative abundance of Prevotella copri in fecal samples before and during fructo-oligosaccharide intervention. On intervention of fructo-oligosaccharide at a dose of 5 grams per day, no significant relative abundance change was observed (F-statistics = 0.82, P-value =0.44).

Specific Embodiments
A method of increasing abundance of Faecalibacterium prausnitzii in gastro intestinal tract of a subject is disclosed. The method comprising administering to said subject a composition comprising fructo-oligosaccharide, such that fructo-oligosaccharide is administered to the subject at a dose of at least 5 grams per day, wherein said composition promotes an increase in abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the subject to a statistically significant level when measured by F-test.
Such a method, wherein a dose of between 5 grams per day to 10 grams per day of fructo-oligosaccharide is administered to the subject.
Such a method, wherein the subject is a mammal and preferably the subject is a human being.
Such a method, wherein the fructo-oligosaccharide composition is administered for 1 to 90 days.
Such a method, wherein the composition of fructo-oligosaccharide comprises about 40-50% 1-kestose(GF2), 40-50% nystose(GF3) and 5-10% fructofuranosyl nystose (GF4).
Such a method, wherein the composition of fructo-oligosaccharide is in the form of a liquid or a solid dosage form for oral administration.
A method of treating inflammatory bowel disease in a human subject is disclosed. The method comprising increasing abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the human subject to a statistically significant level when measured by F-test by administering to said human subject a composition comprising fructo-oligosaccharide such that fructo-oligosaccharide is administered to the subject at a dose of least 5grams per day.
Such a method, wherein a dose of between 5grams per day to 10grams per day of fructo-oligosaccharide is administered to the subject.
Such a method, wherein the composition of fructo-oligosaccharide comprises about 40-50% 1-kestose(GF2), 40-50% nystose(GF3) and 5-10% fructofuranosylnystose (GF4).
Such a method, wherein the fructo-oligosaccharide composition is administered for 1 to 90 days.
A composition for increasing abundance of Faecalibacterium prausnitzii in gastro intestinal tract of a subject is disclosed. The composition comprising at least 5 grams of fructo-oligosaccharide, wherein said composition promotes increase in abundance of Faecalibacterium prausnitzii in the gastro intestinal tract of the subject to a statistically significant level when measured by F-test.
Such a composition, wherein the composition comprises between 5 grams to 10 grams of fructo-oligosaccharide.
Such a composition, wherein the fructo-oligosaccharide composition comprises about 40-50% 1-kestose(GF2), 40-50% nystose (GF3) and 5-10% fructofuranosylnystose (GF4).
Such a composition, wherein the fructo-oligosaccharide composition is in the form of a liquid or a solid dosage form for oral administration.
Such a composition, wherein the fructo-oligosaccharide composition further comprising other components selected from a group comprising Galacto-oligosaccharides, vitamins, minerals, plant extracts, pharmaceutically acceptable excipients or a mixture thereof.