Description:TECHNICAL FIELD

The present application relates to postbiotic compositions, methods for preparing said compositions and applications of said compositions.

BACKGROUND

The research in past few decades has strongly suggested a close relationship between human microbiota and health. The composition and function of microbiota can vary based on various factors such as the age, sex, locations, race, and the diet of the host. An imbalance in the gut microbiota, can lead to diseases and may sometimes even have serious consequences. Therefore, prevention and therapeutic strategies that maintain a healthy gut microbiota have become popular. Prebiotics, probiotics and synbiotics are said to modulate the composition and function of the microbiota. As a result, recent years have seen a surge in products and diet regimens that include prebiotics, probiotics, or synbiotics. Examples of such products include supplements, nutraceuticals, foods, drugs, and medical devices. Postbiotics, are the newest addition to this family of “-biotics”. The postbiotics circumvent the technical challenge of colonization efficiency and maintaining the viability, stability, and functionality of the microorganisms in the product. They facilitate delivery of the active ingredients at the desired location in the intestine. They have better shelf-life and may simplify packaging and transport. The postbiotics can also be used in situations in which it is harder to control and maintain production and storage conditions such as in developing countries.

SUMMARY

The present disclosure relates to a method for preparing a postbiotic composition. The method comprises culturing Lactobacillus bacteria in a culture medium containing short chain fructooligosaccharides and an extract of Ocimum sanctum; and subjecting the culture to filtration to obtain the postbiotic composition.
The present disclosure also relates to the postbiotic compositions obtained from said method.

BREIF DESCRIPTION OF DRAWINGS

FIG. 1 is an HPLC chromatogram of Comparative Composition CC-3.

FIG. 2 is an HPLC chromatogram of Fermented Composition 1 (FC-1).

FIG. 3 is an HPLC chromatogram of FC-2 Fermented Composition 1 (FC-2).

FIG. 4 is an HPLC chromatogram of Inventive Composition PC-A in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments of the present disclosure. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner, simply because it is being utilized in conjunction with a detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several features, no single one of which is solely responsible for its desirable attributes, or which is essential to practicing the inventions herein described.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.

The terms “a,” “an,”, and “the” are used to refer to “one or more” (i.e., to at least one) of the grammatical object of the article.
Reference throughout this specification to “an aspect”, “another aspect” 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 invention.

The terms "comprise", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion and are not intended to be construed as “consists of only”, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

Likewise, the terms “having” and “including”, and their grammatical variants are intended to be non-limiting, such that recitations of said items in a list are not to the exclusion of other items that can be substituted or added to the listed items.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

The present disclosure relates to a method for preparing a postbiotic composition. The method comprises culturing Lactobacillus bacteria in a culture medium containing short chain fructooligosaccharides (scFOS) and an extract of Ocimum sanctum; and subjecting the culture to filtration to obtain the postbiotic composition.

In accordance with an embodiment, the Lactobacillus bacteria is selected from the group consisting of Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus reuteri and a combination thereof.

In accordance with an embodiment. the Lactobacillus bacteria is in the range of 108 to 109 Colony Forming Units (CFUs).

In an embodiment, the Lactobacillus bacteria is Lactobacillus fermentum. In a specific embodiment, Lactobacillus bacteria is a newly discovered strain of Lactobacillus fermentum, strain FS-10. The strain was discovered from Maharashtra. The strain FS-10 was deposited under the Budapest Treaty at the National Centre for Microbial Resource (NCMR) affiliated to the National Centre for Cell Science (NCCS), Pune, India, on April 04, 2022, with the Deposit Accession No. MCC 0567.

In an embodiment, the Lactobacillus bacteria is Lactobacillus plantarum. In a specific embodiment, Lactobacillus bacteria is a newly discovered strain of Lactobacillus plantarum, strain LPL-IC. The strain was discovered from Maharashtra. The strain LPL-IC was deposited under the Budapest Treaty at the NCMR-NCCS, Pune, India, on November 16, 2021, with the Deposit Accession No. MCC 0238.

The culturing is carried out for a period sufficient for the bacteria to effectively ferment the scFOS and the extract of Ocimum sanctum. In accordance with an embodiment, the culturing is carried out for 18 to 28 hours.

The extract of Ocimum sanctum can be prepared by any suitable conventional method. Examples of suitable methods include but are not limited to Soxhlet extraction, maceration, infusion, digestion, percolation, microwave-assisted extraction, ultrasound assisted extraction and decoction. In accordance with an embodiment, the extract is a methanolic, ethanolic or aqueous extract. The extract may be in solid or a liquid form. In accordance with an embodiment, the extract is in a powder form. In accordance with an embodiment, the extract is derived from fresh or dried leaves and/or stems of Ocimum sanctum. In accordance with an embodiment, the extract is derived from the fresh or dried leaves.

In accordance with an embodiment, the culture medium is selected from the group consisting of deMan, Rogosa and Sharpe (MRS) medium, reinforced clostridial medium, and tryptone based customized MRS medium. In a specific embodiment, the medium is MRS medium.

In accordance with an embodiment, the scFOS are in the range of 0.1 to 2% w/v.

In accordance with an embodiment, the extract of Ocimum sanctum is in the range of 0.01 to 0.1 % w/v.

In accordance with an embodiment, the culturing is carried out at a temperature in the range of 35 to 39°C.

The filtration yields the postbiotic composition as a filtrate. The filtration may be carried out using a filter having a suitable pore size. In accordance with an embodiment, the pore size is in the range of 0.22 to 0.45µm. In accordance with an embodiment, the filtration is ultrafiltration. Any suitable filter may be used. Examples of the suitable filters include but are not limited to a syringe filter, hydrophilic polyethersulfone, and polyvinylidene fluoride membrane. In accordance with an embodiment, the filtration removes all or substantially all bacteria and/or bacterial debris.

In accordance with an embodiment, prior to the filtration the culture is subjected to centrifugation. The supernatant obtained after the centrifugation is then subjected to the filtration.

In accordance with an embodiment, prior to the culturing, the Lactobacillus bacteria may be grown under microaerophilic conditions at a temperature in the range of 35 to 39°C for 18 to 24 hours. The Lactobacillus bacteria may be grown in a medium selected from the group consisting of MRS medium, reinforced clostridial medium, and tryptone based customized MRS medium.

The composition so obtained from the disclosed method may be subjected to further processing.

In accordance with an embodiment, the postbiotic composition so obtained is subjected to sterilization. Any suitable method may be used for the sterilization. Examples of such methods include applying pressure, heat, chemicals and/or radiation. Specific examples of sterilization are autoclaving, and X-ray or UV-light treatment.

In accordance with an embodiment, the postbiotic composition is dried to a moisture content of less than about 5% w/w. In accordance with any embodiment, the composition is dried by freeze drying. In an embodiment, the dried composition is subjected to grinding to obtain a fine powder.

The present disclosure also relates to a postbiotic composition obtained from the method as disclosed above. The postbiotic composition comprises fermented extract of Ocimum sanctum, fermented scFOS, lactic acid, acetic acid, and a fermented culture medium. In accordance with an embodiment, the fermented extract of Ocimum sanctum includes component(s) or ingredient(s) of the extract fermented by the bacteria.

In accordance with an embodiment, the composition comprises 0.025-0.5% w/w of the fermented components of the extract of Ocimum sanctum; 0.05-1% w/w of the fermented scFOS; 100-160 ppm lactic acid; 25-55 ppm acetic acid; and 1.0-3.0% the fermented culture medium.
In accordance with an embodiment, the composition is completely or substantially devoid of bacterial cells.

The composition may include one or more additional components. Examples of such components include but are not limited to water, vitamin(s), mineral(s), anti-oxidizing agent(s), preserving agent(s), fragrance(s), flavoring agent(s), sweetener(s), plant extract(s), essential oil(s), diluent(s), carrier(s), excipient(s), filler(s) and a combination thereof.

In accordance with an embodiment, the composition may be formulated in any suitable form. Examples of the suitable forms include but are not limited to solid, aerosol, pill, capsule, tablet, paste, powder, gel, lotion, granules, and liquid.

The present disclosure also relates to a dietary supplement, nutritional composition, food, or beverage comprising the disclosed composition.

The present disclosure also concerns a method for treating an inflammatory disorder in a subject in need thereof. In accordance with an embodiment, the method comprises orally administering, to said subject in need thereof, a therapeutically effective amount of the disclosed composition. In accordance with an embodiment, the method comprises topically applying the disclosed composition.

The phrase “inflammatory disorder” includes any disease or disorder characterized by an abnormal and/or excessive inflammatory response. Examples of the inflammatory disorders include, without limitation, diabetic retinopathy, atherosclerosis, metabolic syndrome, inflammatory bowel disease, Crohn's disease, non-alcoholic fatty liver disease, osteoarthritis, congestive heart failure, neurodegenerative diseases, coronary artery disease, rheumatoid arthritis, osteoporosis, nephropathy in diabetes mellitus, alopecia areata, Graves' disease, systemic lupus erythematosus, lichen sclerosus, ulcerative colitis, periodontal disease, juvenile chronic arthritis, chronic iridocyclitis, psoriasis, insulin dependent diabetes, diabetic complications, multiple sclerosis, rosacea, asthma and any other diseases with an inflammatory component.

The present disclosure also concerns a method of making a medicament intended to treat an inflammatory disorder, using the disclosed composition.

EXAMPLES

The examples below illustrate embodiments of the inventive compositions, the method of making and using them, and the advantages achieved. The present disclosure is not intended to be limited by the examples, and it is to be appreciated that one skilled in the art would understand that a variety of compositions can be prepared, by following the teachings herein, which would achieve the same results.

The extract of Ocimum sanctum used in the Examples below was commercially procured from Prakruti Products Pvt Ltd. in a powder form.

Human monocytic cell line THP-1 used in the Examples below was obtained from American Type Culture Collection (ATCC).

Example 1: Preparation of Postbiotic Composition A (PC-A)

For preparation of the postbiotic composition, FS-10 strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria were then cultured in a customized MRS medium containing 0.5% w/v scFOS and 0.05% w/v the extract of Ocimum sanctum. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe filter to obtain PC-A.

Example 2: Preparation of Fermented Composition 1 (FC-1)

FS-10 strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria was then cultured in a customized MRS medium containing 0.5% w/v scFOS. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe to obtain FC-1.

Example 3: Preparation of Fermented Composition 2 (FC-2)

FS-10 strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria was then cultured in a customized MRS medium containing 0.05% w/v the extract of Ocimum sanctum. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe filter to obtain FC-2.

Example 4: Evaluation of anti-inflammatory activity of PC-A

a) Study Design: The study design was as follows:
- Test System: Human monocytic cell line THP-1.
- Control A: THP-1 cells treated with (Phorbol Myristate Acetate) and lipopolysaccharide (LPS).
- Test Items: PC-A diluted in Dulbecco's Modified Eagle's medium at 12% concentation.
- Comparative Compositions: The details of the comparative compositions are provided in Table 1 below. The comparative compositions were diluted in Dulbecco's Modified Eagle's medium at 12% concentration.

Table 1: Details of the Comparative Compositions of Example 4
S. No. Comparative Composition Details of the Comparative Composition
1. CC-1 MRS medium containing 0.2% w/v scFOS.
2. CC-2 MRS medium containing 0.05% w/v the extract of Ocimum sanctum.
3. CC-3 FS-10 strain cultured in MRS medium for 18 hours.
4. CC-4 FC-1
5. CC-5 MRS medium containing 0.2% w/v scFOS and w/v 0.05% w/v Ocimum sanctum extract powder.
6. CC-6 FC-2

b) Procedure: Inflammation was induced in human monocytic cell line THP-1 using Phorbol-myristate acetate (PMA) and lipopolysaccharide (LPS). One batch of cells was treated with 6% PC-A. Another batch was treated with 12% PC-A. Similarly, separate batches of cells were treated with the 6% and 12% dilutions of the comparative compositions CC-1 to CC-6. After 18 hours of treatment the level of anti-inflammatory cytokine IL-10 in Control A and the treated cell batches was measured.

c) Results and Observation: Table 2 provides IL-10 levels in Control A and the cell batches treated with the test items and the comparative compositions.

Table 2: The IL-10 Levels
S. No. Test Item/Comparative Composition The IL-10 Level (pg/ml)
1. Control A 175.78
2. 12% PC-A 595.08
3. 12% CC-1 113.08
4. 12% CC-2 75.74
5. 12% CC-3 361.28
6. 12% CC-4 392.08
7. 12% CC-5 79.74
8. 12% CC-6 382.28

IL-10 levels were found to be higher in cell batches treated with 12% PC-A as compared to 12% comparative compositions.

Example 5: Preparation of Postbiotic Composition B (PC-B)

LPL-IC strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria was then cultured in a customized MRS media containing 0.5% w/v scFOS and 0.05% w/v the extract of Ocimum sanctum. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe filter to obtain PC-B.

Example 6: Preparation of Fermented Composition 3 (FC-3)

LPL-IC strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria was then cultured in a customized MRS medium containing 0.5% w/v scFOS. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe filter to obtain FC-3.

Example 7: Preparation of Fermented Composition 4 (FC-4)

LPL-IC strain was first grown in MRS medium at 37°C for 24 hours under microaerophilic conditions. The 108 to 109 CFUs of the bacteria was then cultured in a customized MRS medium containing 0.05% w/v the extract of Ocimum sanctum. After growing the culture for 18 hours, the culture was subjected to centrifugation. The supernatant obtained after centrifugation was subjected to filtration using a 0.22µm syringe filter to obtain FC-4.

Example 8: Evaluation of anti-inflammatory activity of PC-B

a) Study Design: The study design was as follows:
- Test System: Human monocytic cell line THP-1.
- Control B: THP-1 cells treated with PMS and LPS.
- Test Item: PC-B diluted in Dulbecco's Modified Eagle's Medium at 12% concentration.
- Comparative Compositions: The details of the comparative compositions are provided in Table 3 below. The comparative compositions were diluted in Dulbecco's Modified Eagle's medium at 12% concentration.

Table 3: The Details of the Comparative Compositions
S. No. Comparative Composition Details of the Comparative Composition
1. CC-1 MRS medium containing 0.2% w/v scFOS.
2. CC-2 MRS medium containing 0.05% w/v the extract of Ocimum sanctum.
3. CC-5 MRS medium containing 0.2% w/v scFOS and 0.05% w/v the extract of Ocimum sanctum.
4. CC-7 LPL-IC strain cultured in MRS medium for 18 hours.
5. CC-8 FC-3
6. CC-9 FC-4

d) Procedure: Inflammation was induced in human monocytic cell line THP-1 using PMA and LPS. A batch of cells was treated with 12% PC-B. Similarly, separate batches of cells were treated with the 12% dilution of the comparative compositions.

e) Results and Observation: Table 4 provides IL-10 levels in Control B as well as the cell batches treated with the test item and the comparative compositions.

Table 4: IL-10 Levels
S. No. Test Item/Comparative Composition IL-10 Level (pg/ml)
1. Control B 157.40
2. 12% CC-1 113.08
3. 12% CC-2 75.74
4. 12% CC-5 79.74
5. 12% CC-7 135.24
6. 12% CC-8 138.48
7. 12% CC-9 219.56
8. 12% PC-B 304.97

IL-10 levels were found to be significantly higher in cell batches treated with 12% PC-B.

Example 9: Identification of Metabolites Present in PC-A

a) Procedure: PC-A was subjected to HPLC analysis for identification of bacterial metabolites present in it.

a) Results and Observation: The two main metabolites identified were lactic acid and acetic acid. The quantity of lactic acid and acetic acid was significantly higher for PC-A compared to the comparative compositions CC-3, FC-1, and FC-2. HPLC chromatograms of CC-3, FC-1, FC-2, and PC-A are depicted in FIG. 1, 2, 3 and 4 respectively.

Table 5: Metabolites
Composition Lactic Acid (ppm) Acetic Acid (ppm)
CC-3 29.88257768 9.513354282
FC-1 77.21623077 22.36713757
FC-2 28.67005953 11.15606213
PC-A 123.1913404 39.55783451

Example 10: Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of PC-A

PC-A was subjected to LC-MS analysis (LCMS AgilentQ-ToF HRMS) for the identification of metabolites, phytochemicals or any other bioactive compounds present. The analysis was conducted at GeneomBio technologies, Pune.

a) Procedure: Sample preparation was done in 100% methanol. The chromatography was carried out using Agilent Zorbax Eclipse XBD-C18 column. Mobile phase used were:
- 0.1% formic acid in water; and
- 0.1% formic acid in acetonitrile with flow rate 0.3ml/min at column temperature of 40°C.
The sample was run using positive and negative mode. The M/Z scan range was 50 to 1700.

b) Results and observation: The analyses showed presence of different bioactive compounds in PC-A. Table 6 lists the compounds identified.

Table 6: Bioactive Compounds
S. No. Compound M/Z
1 Astragalin 689.1346
2 S-Curzeone 251.056
3 Quillaic acid 1637.6908
4 16-Hydroxy-10-oxohexadecanoic acid 304.2496
5 Saponins Tragoposaponin 1106.557
Basellasaponin 969.4328
Matesaponin 1076.5988
6 Palmitine 370.1885

INDUSTRIAL APPLICABILITY

The disclosure provides postbiotic compositions and method of obtaining such compositions. The compositions offer health benefits. The compositions are effective against inflammation. The compositions are suitable for incorporating into various types of formulations, nutritional supplements, food, feed, and beverage.
, Claims:1. A method for preparing a postbiotic composition, the method comprising:
culturing Lactobacillus bacteria in a culture medium comprising short chain fructooligosaccharides and an extract of Ocimum sanctum; and
subjecting the culture to filtration to obtain the postbiotic composition.

2. The method as claimed in claim 1, wherein the Lactobacillus bacteria is selected from the group consisting of Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus reuteri and a combination thereof.

3. The method as claimed in claim 1 or 2, wherein the Lactobacillus bacteria is Lactobacillus fermentum strain FS-10.

4. The method as claimed in claim 1 or 2, wherein the Lactobacillus bacteria is Lactobacillus plantarum strain LPL-IC.

5. The method as claimed in any of the preceding claims, wherein the culturing is carried out for 18 to 24 hours.

6. The method as claimed in any of the preceding claims, wherein the culture medium is selected from the group consisting of deMan, Rogosa and Sharpe medium, reinforced clostridial medium, and tryptone based customized deMan, Rogosa and Sharpe medium.

7. The method as claimed in any of the preceding claims, wherein the culture medium comprises the short chain fructooligosaccharides in the range of 0.1 to 2% w/v.

8. The method as claimed in any of the preceding claims, wherein the culture medium comprises the extract of Ocimum sanctum in the range of 0.01 to 0.1 % w/v.

9. The method as claimed in any of the preceding claims, wherein the Lactobacillus bacteria is in the range of 108 to 109 CFUs.

10. The method as claimed in any of the preceding claims, wherein the culturing is carried out at a temperature in the range of 35 to 39°C.

11. The method as claimed in any of the preceding claims, wherein prior to the filtration the culture is subjected to centrifugation.

12. The method as claimed in any of the preceding claims, wherein the filtration removes bacterial cells from the culture.

13. The method as claimed in any of the preceding claims, wherein prior to the culturing the Lactobacillus bacteria is grown under microaerophilic conditions at a temperature in the range of 35 to 39°C for 18 to 24 hours.

14. The method as claimed in claim 13, wherein the Lactobacillus bacteria is grown in a medium selected from the group consisting of deMan, Rogosa and Sharpe medium, reinforced clostridial medium, tryptone based customized deMan, Rogosa and Sharpe medium.

15. A postbiotic composition obtained from the method as claimed in any of the preceding claims.

16. A postbiotic composition as claimed in claim 15, comprising:
0.025-0.5% w/w of fermented extract of Ocimum sanctum;
0.05-1% w/w of fermented short chain fructooligosaccharides;
100-160 ppm lactic acid;
25-55 ppm acetic acid; and
1.0-3.0% a fermented culture medium.

17. The composition as claimed in claim 15 or 16, further comprising one or more of a component(s) selected from the group consisting of water, a vitamin, a mineral, an anti-oxidizing agent, a preserving agent, a fragrance, a flavoring agent, a sweetener, one or more plant extract, a diluent, a carrier, an excipient, a filler, an essential oils and a combination thereof.

18. The composition as claimed in any of claims 15-17, wherein the composition is in a form selected from the group consisting of a solid, an aerosol, a pill, a capsule, a tablet, a paste, a powder, a gel, a lotion, granules, and a liquid.

19. A dietary supplement, nutritional composition, food, feed, or beverage comprising the composition of any of claims 15-18.