The invention disclosed in this application relates to an improved composition (medium) which is useful for assay of a probiotic and a process for its preparation.
Probiotics are single bacteria or a mixture of microorganisms which have been mutated or rendered non-pathogenic. They are administered in oral form to patients who may have disturbances in the normal microbiota of the gastro-intestinal tract due to infections or antibiotic therapy. Probiotics are also used to treat infections in place of antibiotics and hence have been postulated to have inhibitory effects on the growth of pathogenic microorganisms.
The action of probiotics is studied in vitro by using bacteriological media similar to those used to study antibiotic susceptibility such as Mueller Hinton agar/ broth, which has a low content of thymidine and cations both of which are known to decrease the activity of some antibiotics. Such a medium however is not ideal to test the activity of a probiotic which consists of a mixture of organisms all of which may have different growth requirements. Meat particles are known to produce a low 0/R (oxidation reduction potential) that facilitates the growth of anaerobes.
Prior art
Bacteriological media containing meat particles are conmiercially available and are used to cultivate facultative and strict anaerobes. Such a medium however is not used for antibiotic assays since it is not free of all substances that may be inhibitory to some antibiotics. Mueller Hinton medium/ broth has been used to assay probiotics and antibiotics; however it supports the growth of only aerobes and facultative anaerobes and may not be ideal if the probiotic consists of a mixture of organisms of different oxygen requirements.

At present, no composition (medium) is available globally in which varied types of probiotic organisms can be grown in order to study their antimicrobial activity. If such a composition is made available it can be used to test the activity of a variety of probiotics.
Accordingly we directed our R & D towards development of such a composition (media) which can be useful for assay of probiotics which will overcome the above mentioned difficulties experienced in the prior art and consequently benefit in the development of probiotic assays.
Therefore the main objective of the present invention is to provide an improved composition (media) which is useful for assay of a probiotic, for the support of the growth of different types of probiotic organisms.
Another objective of the present invention is to provide an improved composition (medium) which allows the individual probiotic organisms to metabolize and produce antibiotic like substances.

Still another objective of the present invention is to provide an improved composition (medium) which will not be inhibitory to those antibiotic-like substances and facilitate assay of such substances.
Yet another objective of the present invention is to provide an improved composition (medium), which can be used with ease in a clinical laboratory without use of additional equipment such as anaerobic jars etc.
Still another objective of the present invention is to provide a process for the preparation of an improved composition (medium) which will support the growth of different types of probiotic organisms.
The composition (medium) of the present invention can support the growth of different kinds of organisms of varied oxygen and nutritional requirements and is also suitable for probiotic and antibiotic assays. As mentioned above there is no composition (medium) known anywhere in the world which can serve both functions.
We observed during our R & D work carried out that, by mixing appropriately selected ingredients such as Meat pieces. Peptone, sugars, NaCl, Casein hydrolysate, beef infusion, starch etc in appropriate proportions, the resulting composition (media) due to synergistic activity of the ingredients employed develops a property which can be utilized for probiotic assay.

Accordingly, the present invention provides an improved composition (media) useful for probiotic assay. The probiotic assay medium comprises a meat component and a broth component. The composition (media) comprises (i) Meat pieces in an amount in the range of 5 to 50 % w/v (weight/volume) of the broth (ii) The Broth in turn is made up of Meat inftision in the range of 10-50% v/v (volume/volume), Protein hydrolysate in an amount in the range of 0.1 to 10% w/v, Sugar in an amount in the range of 0.1 to 5% w/v and Starch in an amount in the range of 0.05 to 5% w/v (iii) The Meat infusion is an aqueous extract of meat supplemented with Peptone in an amount in the range of 0.5 to 10% w/v and Salt (NaCl) in an amount in the range of 0.1 to 5.0 % w/v (iv) the pH of the resulting composition (media) being in the range of 7.4 + / - 0.2.
According to a preferred embodiment of the invention the amount of Meat pieces used ranges from 10-40% w/v in the broth (ii) The Broth in turn is made up of Beef inftision in the range of 25-45% v/v, Casein hydrolysate in an amount in the range of 1-2% w/v. Dextrose in an amount in the range of 1-2% w/v and Soluble Starch in an amount in the range of 0.1-0.2% w/v (iii) The Beef inftision is an aqueous extract of meat supplemented with Proteose Peptone in an amount in the range of 1-8% w/v and Sodium chloride in an amount in the range of 0.2 - 0.7% w/v (iv) the pH of the resulting composition (media) being in the range of 7.4 + / - 0.2.
Sterile probiotic assay medium when inoculated with mixed probiotic organisms - allow the growth of aerobes in the broth and the growth of anaerobes on the meat particles. After an incubation period of 24-72 hours at 37° C in an atmosphere of about 7 - 10 % CO2 the antimicrobial activity can be studied using the supernatant that contains the metabolites against the pathogens of interest.
According to the invention there is provided a process for the preparation of an improved composition (media) useful for probiotic assay which comprises addition of Meat pieces (bullock heart/ meat) to constitute 15-25% w/v of the broth (ii) The Broth in turn is made up by addition of Beef infusion in the range of 30-40% v/v, Casein hydrolysate in an amoimt in the range of 0.5-2% w/v, Dextrose in an amount in the range of 0.5-2% w/v, and Soluble Starch in an amount in the range of 0.1-0.2% w/v in distilled water (iii) The Beef inftision is an aqueous extract of meat (prepared by standard methods) and is

supplemented with Proteose Peptone in an amount in the range of 1-5% w/v and Sodium chloride in an amount in the range of 0.3-0.6% w/v (iv) the pH of the resulting composition (media) is adjusted to fall in the range of 7.4 + / - 0.2. The medium is autoclaved at a temperature in the range of 115° C - 120° C, at a pressure in the range of 10 lbs to 15 lbs of pressure for a period in the range of 15 to 20 minutes, following which the resulting composition (medium) is cooled to room temperature.
According to an embodiment of the present invention the autoclaving may be effected at a temperature in the range of 120° C to 135 ° C at a pressure in the range of 10 to 30 lbs of pressure for a period in the range of 15 to 30 minutes.
The composition can be stored at 4° C until required. The probiotic(s) is inoculated into the medium and incubated at 37°C for 48-72 hours.
It is observed that Probiotic growth in the medium happens best at 37°C, for 72 hours of incubation in a 5-10 % CO2 atmosphere.
The ingredients (Proteose peptone, NaCl, Dextrose, Casein hydrolysate. Starch) used in the composition of the present invention are available in a dehydrated form from microbiological media suppliers. They should be reconstituted in distilled water.
The Composition (medium) of the present invention can be inoculated with the contents of a commercial probiotic capsule under sterile conditions. The inoculated medium should be incubated at 37°C for 72 hours. The cell free supernatant can be obtained by centrifUgation at 5000 rpm and its antimicrobial efficacy can be tested against broth cultures of the pathogens of interest.
The details of the invention are given in the Examples given below which are provided for illustration only and therefore cannot be construed to limit the scope of the invention.

Example 1 Medium preparation
The medium comprises beef infusion combined with supplements and meat particles.
Preparation of beef infusion CI litre stock)
To prepare the beef infusion, 500g of lean meat is minced finely and allowed to extract in
1 litre of water for 24 hours in the cold. The extract is strained from the meat through a
muslin cloth. It is boiled for 15 minutes after which it turns turbid and brown. It is filtered
through Whatman 1 filter paper. The filtrate obtained is yellow and clear.
Proteose Peptone (20g), Sodium chloride (5g) are added and the total volume which
lessens after the boiling step is made up to 1 litre. The pH is adjusted to 7.5 and the
medium autoclaved at 121° C for 15 minutes.
Preparation of meat particles
Minced bullock heart (500g) is placed in alkaline boiling water (1.5ml of NaOH Imol/L in 500ml distilled water) and the contents simmered for 20 min. While still hot, the liquid is drained off through a muslin filter. The meat is then dried further on filter paper.
Probiotic Testing Medium The Broth component
Beefinfiision 300ml (30% v/v)
Casein hydrolysate 17.5g (1.75% w/v)
Soluble Starch 1.5g (0.15% w/v)
Dextrose 20.0g (2% w/v)
The contents are mixed and made up to a volume of 1 litre with distilled water. The broth
is boiled gently at 100° C with gentle agitation, until all the contents are well dissolved.
The pH is adjusted to 7.4
The Meat component
3g of meat particles (20% w/v) are added to volumes of 15ml of the broth dispensed in culture tubes. The tubes are cotton plugged and are autoclaved at 121° C for 15 minutes.

Example 2 Medium preparation
The medium comprises beef infusion combined with supplements and meat particles.
Preparation of beef infusion (1 litre stock)
To prepare the beef infusion, 500g of lean meat is minced finely and allowed to extract in
1 litre of water for 24 hours in the cold. The extract is strained from the meat through a
muslin cloth. It is boiled for 15 minutes after which it turns turbid and brown. It is filtered
through Whatman 1 filter paper. The filtrate obtained is yellow and clear.
Proteose Peptone (20g), Sodium chloride (5g) are added and the total volume which
lessens after the boiling step is made up to 1 litre. The pH is adjusted to 7.5 and the
medium autoclaved at 121° C for 15 minutes.
Preparation of meat particles
Minced beef meat (500g) is placed in alkaline boiling water (1.5ml of NaOH Imol/L in 500ml distilled water) and the contents simmered for 20 min. While still hot, the liquid is drained off through a muslin filter. The meat is then dried further on filter paper.
Probiotic Testing Medium The Broth component
Beef infiision 300ml (30% v/v)
Casein hydrolysate 17.5g (1.75% w/v)
Soluble Starch 1.5g (0.15% w/v)
Dextrose 20.0g (2% w/v)
The contents are mixed and made up to a volume of 1 litre with distilled water. The broth
is boiled gently at 100° C with gentle agitation, until all the contents are well dissolved.
The pH is adjusted to 7.4.
The Meat component
1.5g of meat particles (10% w/v) are added to volumes of 15ml of the broth dispensed in culture tubes. The tubes are cotton plugged and are autoclaved at 121° C for 15 minutes.

ADVANTAGES OF THE INVENTION:
♦ The composition (medium) of the invention can allow the simultaneous growth of multiple probiotic organisms of varied oxygen requirements.
♦ Cell free supematants of the culture can be tested for antimicrobial activity against pathogens of interest using the composition.
♦ Also, pathogens can be grown along with the probiotic organisms and viable counts from the medium can be made over a time period to check if the pathogens growth is inhibited or enhanced.
♦ Since each organism is able to grow optimally and metabolise, the metabolic by¬products accumulate in the supernatant, which can easily be assayed. Toxic by¬products that may inhibit the metabolites are adsorbed by certain components (starch) of the medium. Hence probiotic organisms grow well.

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We Claim
1. An improved composition (media) useful for probiotic assay which comprises (i) Meat pieces in an amount in the range of 5-50% w/v in a broth (ii) The Broth in turn is made up of Meat infusion in the range of 10-50% v/v, Protein hydrolysate in an amoimt in the range of 0.1 to 10% w/v, Sugar in an amount in the range of 0.1 to 5% by w/v and Starch in an amount in the range of 0.05 to 5% w/v in distilled water (iii) The Meat infusion is an aqueous extract of meat supplemented with Peptone in an amount in the range of 0.5 to 10% w/v and Sah (NaCl) in an amount in the range of 0.1 to 5.0 % w/v (iv) the pH of the resulting composition (media) being in the range of 7.4 + / - 0.2.
2. An improved composition (media) as claimed in claim 1 wherein the amount of Meat pieces used ranges from 10-40% w/v of the Broth.
3. An improved composition (media) as claimed in claim 1 to 2 wherein the volume of Meat infusion in the Broth ranges from 20-40% v/v.
4. An improved composition (media) as claimed in claims 1 to 3 wherein the amount of Casein hydrolysate in the Broth ranges from 1 to 2 % w/v.
5. An improved composition (media) as claimed in claims 1 to 4 wherein the amount of Dextrose used in preparation of the Broth ranging from 1 to 2 % w/v.
6. An improved composition (media) as claimed in claims 1 to 5 wherein the amount of soluble starch used ranging from 0.1 to 0.2 % by w/v.
7. An improved composition (media) as claimed in claims 1 to 6 wherein the amount of Proteose peptone used in the preparation of Meat infusion (a component of the broth) ranges from 0.5-10% w/v.
8. An improved composition (media) as claimed in claims 1 to 7 wherein the amount of NaCl also used in the preparation of Meat infusion ranges from 0.2 to 0.7 % w/v.
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9. An improved composition (media) as claimed in claims 1 to 8 wherein the pH of the
composition (media) is set to 7.5.
10. A process for the preparation of an improved composition (media) as claimed in claims 1 to 8 which comprises addition of Meat pieces (Bullock heart/ Beef meat) to comprise 15-25% w/v of a broth (ii) The Broth is prepared by adding Beef infusion in the range of 30-40% v/v, Casein hydrolysate in an amount in the range of 0.5-2% w/v. Dextrose in an amount in the range of 0.5-2% w/v and Soluble Starch in an amount in the range of 0.1-0.2% w/v to distilled water (iii) The Beef infusion (a component of the broth) is an aqueous extract of meat (prepared by standard methods) supplemented with Proteose Peptone in an amount in the range of 1-5% w/v and Sodium chloride in an amount in the range of 0.3-0.6% w/v (iv) the pH of the resulting composition (media) is adjusted to fall in the range 7.4 + / - 0.2. The medium is autoclaved at a temperature in the range of 115° C - 120° C/ at a pressure in the range of lOlbs to 15 lbs of pressure for a period in the range of 15 to 20 minutes, following which the resulting composition (medium) is cooled to room temperature.
11. A process as claimed in claim 10 wherein the autoclaving is effected at a temperature in the range of 120° C to 135° C at a pressure in the range of 10 to 15 lbs of pressure for a period in the range of 15 to 20 minutes.
12. An improved composition (media) as claimed in claim 10 and 11 wherein the amount of Meat pieces (Bullock heart/ Beef meat) used ranges from 15-20% w/v of the Broth.
13. An improved composition (media) as claimed in claims 10 to 12 wherein the amoimt of Casein hydrolysate in the Broth ranges from 1 to 2 % by w/v.
14. An improved composition (media) as claimed in claims 10 to 13 wherein the amount of Dextrose used in preparation of the Broth ranging from 1 to 2 % w/v.
15. An improved composition (media) as claimed in claims 10 to 14 wherein the amount of soluble starch used ranging from 0.1 to 0.2 % by w/v.
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16. An improved composition (media) as claimed in claim 10 to 15 wherein the volume of
Beef infUsion in the Broth ranges from 30-40% v/v.
17. An improved composition (media) as claimed in claims 10 to 16 wherein the amount of Proteose peptone used in the preparation of Beef infusion (a component of the Broth) ranges from 1 -4% w/v.
18. An improved composition (media) as claimed in claims 10 to 17 wherein the amount of NaCl also used in the preparation of Beef infusion (a component of the Broth) ranges from 0.3 to 0.5 % w/v.
19. An improved composition (media) as claimed in claims 10 to 18 wherein the pH of the composition (media) is set to 7.5.
20. An improved composition (media) useful for probiotic assay substantially as herein
described with reference to the Examples 1 & 2.
21. A process for the preparation of an improved composition (media) useful for probiotic
assay substantially as herein described with reference to the Examples 1 8c 2.

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