Claims:We Claim:
1. A liquid emulsion comprising an oil phase, an aqueous phase, surface active agent, lipid moiety and at least two microorganisms as active ingredient and optionally any other pharmaceutically acceptable excipients.
2. A liquid emulsion according to claim 1 comprise at least two microorganisms including, but not limited to,Lactobacillus rhamnosus, Bacillus coagulans and Bifidobacterium bifidum.
3. A liquid emulsion according to preceding claims, wherein the Cell count of each microorganism is not less than 5 billion per 5 mL.
4. A liquid emulsion which is stable for a period of 6 months and during its shelf life.
5. A liquid emulsion according to preceding claims,wherein the emulsion is stable for its shelf life at 25°C.
6. A liquid emulsion according to preceding claims,wherein the surface active agent is Xanthan gum.
7. A liquid emulsion according to preceding claims,wherein the lipid moiety is essentially Soybean lecithin.
8. A liquid emulsion according to preceding claims,having the pH value ranges from 4.0 to 7.0.
9. A liquid emulsion according to preceding claims,wherein the viscosity of the emulsion is less than 2000 cp.
10. A liquid emulsion comprising an oil phase, an aqueous phase, surface active agent, lipid moiety and at least two microorganisms as active ingredient and optionally any other pharmaceutically acceptable excipients supplied in a blow fill container.
, Description:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE

Specification
(Section 10, rule 13)

TITLE: A stable mixed probiotic composition and preparation thereof

VIRCHOW BIOTECH PRIVATE LIMITED
Gagillapur, Quthbullapur, Mandal, R.R. Dist.
Hyderabad, India. Phone: +91-40-23119481 &
Cell: +91- 9000104180, +91 8297419169. Fax: +91-40-23119486

The following specification particularly describes the nature of this invention and the manner in which it is to be performed.

A stable mixed probiotic composition and preparation thereof
Field of Invention
The present invention relates to probiotic compositions, and more particularly to a method of preparing a probiotic composition comprising mixed non-pathogenic bacteria strains which may be stored in biologically active form for long periods of time, as well as compositions thereof. In particular, the present invention relates to probiotic compositions, and more particularly to a method of preparing a probiotic composition comprising mixed non-pathogenic bacteria strains which may be stored in biologically active form for long periods of time, as well as compositions thereof wherein the Probiotic composition is in the form of emulsion and comprising of more than one bacterial strains to modify the gut flora and to replace harmful microbes with useful microbes in a Blow-Fill-Seal container making the invention cost effective and sterile.
Background of the Invention
Probiotic bacteria are those which are beneficial to humans and/or animals. The use of probiotic bacteria is known in the art for improving the microbial balance in the intestinal tract of mammals, in order to prevent or treat gastro-enteric infections and other diseases or disorders involving and/or causing changes in or to the intestinal micro flora composition, and/or resulting in any change to the microflora composition, and/or maintaining such changes, as well as changes to the microflora composition which actively cause or potentiate such diseases or disorders. However, the results of studies carried out to date have been inconsistent and/or ambiguous. For example, in some studies, the use of probiotic bacteria alone to treat "traveler's diarrhea" was not sufficient to provide a significant effect in patients as opposed to placebo, yet the combination of the probiotic treatment with antibiotics proved to be highly effective. Other studies have shown that probiotic treatment alone had a beneficial effect, yet such an effect often required 3-6 months to be felt (see also J. JAMA, 1996, vol. 275, No 11, US patent 5 433 826, 1995, US patent 5 589 168, 1996 and others). Recent studies have been directed towards investigation of the effects of various types of probiotic bacteria, either alone or in combination; improvement of the survival rate of probiotic bacteria and methods of enabling long-term preservation; biomass accumulation, and the use of probiotic bacteria in prophylaxis and treatment of humans and animals. Approximately 400 different kinds of bacteria and bacteroids are known to exist in the digestive tract of humans and other mammals, which may provide about 30-40% of excrement volume. The characteristics and functions of only about 15 of these known types have been studied in any detail. Each of these types of bacteria occupies its own ecological niche in the digestive tract, each having particular conditions for optimal survival and multiplication rate. Pathogenic bacteria, which may cause various diseases or disorders, also occupy their own particular environmental niches or habitats. Competition between pathogenic and probiotic bacteria may occur under various conditions, but maximal competitive effect occurs when the conditions for optimal survival and multiplication rate of both pathogenic and probiotic bacteria are similar. Under such conditions, survival depends upon more stringent competition for nutrients or growth factors, a well as upon synergistic nutrient utilization and competition for receptor sites. Factors such as production of antimicrobial substances, intensity of multiplication, and creation of restrictive environment, including induction of immunological processes and stimulation of epithelial cell turnover also have great significance under such conditions. Probiotic compositions have been developed using cultures of non-pathogenic E. coli with other non-pathogenic bacteria (US Patent No. 5,340,577; US Patent No. 5,443,826; US Patent No. 5,478,557; US Patent No. 5,604,127). Lactose-positive non-pathogenic E. coli strain having high antagonistic activity have been produced as freeze-dried preparations in Germany and Russia (e.g. use of freeze-dried preparation Colibacterinsiccum of E. coli Ml 7, described in Vidal Handbook: Pharmaceutical preparations in Russia, Astra Pharm Service, 1997, Moscow). In recent years, there has been a move towards developing probiotic preparations comprising a large number (up to 30) different kinds of bacteria and bacteroids (for example, US Patent No. 5,443,826; US Patent No. 5,478,557).
The protection and delivery of probiotic cultures remain a challenge for the food and pharmaceutical industries. In order, to produce health benefits probiotic strains should be present in viable form at a suitable level during the product is shelf life until consumption and maintain high viability throughout the gastrointestinal tract.
Cocktails of various micro-organisms, particularly species of Lactobacillus and Bifidobacterium, have traditionally been used in fermented dairy products and medicines to promote health. However to be effective, said probiotics must not only survive manufacturing processing, packaging and storage conditions, but also then must survive transit through the gastrointestinal tract so the probiotic material remains viable to have a positive health effect that all the existing delivery systems were unable to provide.
Again some of the microorganisms are of hydrophobic nature while others are of hydrophilic nature. While using a mixed probiotic composition one must take this stability and solubility properties of those microorganisms in to consideration. A suitable dosage form that can accommodate both types of microorganisms at their most efficient way will be highly appreciated.
So there was a constant need for a stable probiotic composition comprising of more than one microorganism with improved viability and more efficacy.Inventors of present invention surprisingly formulated a new delivery system based on gums added to the aqueous phase of water – in- oil emulsion. Emulsion is defined as dispersion of two immiscible liquids stabilized by adding one or more emulsifier(s). When the core phase is aqueous, it is termed as a water- in-oil (w/w) emulsion however, for a hydrophobic core, oil-in-water emulsion (o/w). The emulsification technique has widely used for encapsulation of various microbes. Aqueous suspension of cells and/or polymer (dispersed phase) dispersed in an oil in organic phase (continuous phase), are homogenized to form a w/o emulsion with the aid of suitable emulsifier. The present invention is supplied in a Blow-Fill-Seal container making the invention a cost effective and sterile.
Summary of the Invention
The present invention disclosure provides a liquid emulsion comprising at least two probiotic, preferably three microorganisms including, but not limited to, Lactobacillus rhamnosus, Bacillus coagulans and Bifidobacterium bifidum.
Another object of the invention is to provide a liquid formulation containing aqueous unstable microorganisms that can now be dispensed in a known dose for a therapeutic use.
Another object of the invention is to provide a liquid formulation containing aqueous unstable microorganisms that can now be dispensed in the form of an emulsion preferably lipid emulsion.
Another object of the invention is to provide a liquid formulation containing at least one lipid moiety wherein the lipid moiety is selected from Lecithin, Cholesterol, triacylglycerols, mono- and diacylglycerols and like and combinations thereof.
Another object of the invention is to provide a liquid formulation containing one emulsifying agent wherein this emulsifying agent is selected from Xanthan Gum,Tragacanth Gum, Oat Gum, Locust Bean Gum, Karaya Gum, Guar Gum, Gellan Gum, Gum Arabic, Acacia Gum and like and combinations thereof.
The present disclosure provides a method of manufacturing an emulsion comprising at least two probiotic. For example, the present invention may maintain about 50% or greater of the CFU’s.
The present disclosure further provides compositions, uses, and process relating to mixed probiotic emulsion.
The present invention also discloses the probiotic emulsion formulation that might be stored in Blow-Fill-Seal Container.
The present invention also discloses the probiotic emulsion formulation that might be stored in Blow-Fill-Seal Container wherein the container is preferably made of thermoplastic, more preferably polyethylene, most preferably Low Density Polyethylene.
An embodiment of present invention provides a liquid formulation comprising an Oil phase, an aqueous phase, surface active agent, lipid moiety and optionally any other pharmaceutical acceptable excipients for a liquid formulation as per the known art and combinations thereof.
An embodiment of present invention provides a liquid formulation comprising an Oil phase, an aqueous phase, surface active agent, lipid moiety and optionally any other pharmaceutical acceptable excipients rendering pH 4.0 to 7.0 and viscosity less than 2000 cp by using Brookfield Viscometer.
This summary does not necessarily describe all features of the invention. Other aspects, features and advantages of the invention will be apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.
Detailed Description of the Invention
The present invention now is described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, this embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventionto those skilled in the art.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.
As used herein, the term “Probiotic” means live microorganisms which when administered in adequate amounts may confer a health benefit to the subject.
As used herein, the term “subject” is not limited to a specific species or sample type. For example, the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian species.
As used herein, “a” or “an” means “at least one” or “one or more”.
The term “emulsion” as used in this specification denotes a two-phase system in which one phase is finely dispersed in the other phase. The term emulsifier, as used by this invention, denotes an agent that can reduce and/or eliminate the surface and the interfacial tension in a two-phase system. The emulsifier agent, as used herein, denotes an agent possessing both hydrophilic and lipophilic groups in the emulsifier agent. The term solution, as used herein, indicates a chemically and physically homogenous mixture of two or more substances. The term solubility, as used herein, denotes a solid brought into contact with a liquid, whereby molecules of the solid establish equilibrium with the liquid leaving the solid and returning to it. The term slightly soluble, as used herein, denotes 100 to 1,000 parts of solvent for 1 part of solute, very slightly soluble from 1,000 to 10,000 parts of solvent for 1 part of solute, and practically insoluble, or insoluble denotes more than 10,000 parts of solvent to 1 part of solvent.
Probiotics are live microbes, which are nonpathogenic administered to improve intestinal microbial balance. They consists of Saccharomyces boulardii yeast or lactic acid bacteria, such as Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium species etc., and are adapted as comestible supplements, foods and drugs. Probiotics have received renewed attention in the 21st century from product manufacturers, research studies and consumers. The history of probiotics can be traced to the first use of cheese and fermented products that were well known to the Greeks and Romans. The original modern hypothesis of the positive role played by certain bacteria was first introduced by Russian scientist and Nobel laureate Elie Metchnikoff, who in 1907 suggested that it would be possible to modify the gut flora and to replace harmful microbes with useful microbes.
Sales of probiotic products have a rising trend from 2010 to 2014, increasing globally by 35% from US$23.1billion to $31.3 billion. Some regions have increased their use by even more than the average, including Eastern Europe (67%), Asia Pacific (67%), and Latin region, and the leading consumers nearly half of probiotics sold globally in 2014.
The present invention is directed to an emulsified or dispersion drug delivery system containing a mixture of oil, surfactant, co-surfactant and at least two probiotic.
Examples of non-toxic oils comprise a member selected form the group consisting of Soya oil, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, almond oil, mineral oil, castor oil, coconut oil, palm oil, cocoa butter, safflower, a mixture of mono- and diglycerides of 16 to 18 carbon atoms, unsaturated fatty acids, fractionated triglycerides derived from coconut oil, fractionated liquid triglycerides derived from short chain 10 to 15 carbon atoms fatty acids, acetylated monoglycerides, acetylated diglycerides, acetylated triglycerides, olein known also as glyceraltrioleate, palmitin known as glyceryl tripalmitate, stearin known also as glyceryl tristearate, lauric acid hexylester, oleic acid oleylester, glycolyzedethoxylated glycerides of natural oils, branched fatty acids with 13 molecules of ethyleneoxide, and oleic acid decylester. The concentration of oil, or oil derivative in the emulsion formulation is 1 wt % to 40 wt %, with the wt % of all constituents in the emulsion preparation equal to 100 wt %. The oils are disclosed in Pharmaceutical Sciences by Remington, 17th Ed., pp. 403-405, (1985) published by Mark Publishing Co., in Encyclopedia of Chemistry, by Van Nostrand Reinhold, 4th Ed., pp. 644-645, (1984) published by Van Nostrand Reinhold Co.; and in U.S. Pat. No. 4,259,323 issued to Ranucci.
The oils in the emulsion are present in a preferred amount of 5 to 20 wt. %. The pharmacologically active present in the emulsion is preferably a poor water soluble compound, preferably selected from the group comprising drugs or dietary supplements, or nutraceuticals, preferably probiotics.
In the present invention, the term ‘surfactant’ refers to non-ionic, cationic, anionic, and zwitterionic surface-active agents. Useful non-ionic surface-active agents include ethylene glycol stearates, propylene glycol stearates, diethylene glycol stearates, glycerol stearates, sorbitan esters (e.g., Span™ products) and polyhydroxyethylenically treated sorbitan esters (e.g., Tween™ products), aliphatic alcohols and poly(ethylene glycol) ethers, and phenol and PEG ethers. Useful cationic surface-active agents include quaternary ammonium salts (e.g. cetyltrimethylammonium bromide) and amine salts (e.g. octadecylamine hydrochloride). Useful anionic surface-active agents include sodium stearate, potassium stearate, ammonium stearate, and calcium stearate, triethanolamine stearate, sodium lauryl sulphate, sodium dioctylsulphosuccinate, and sodium dodecylbenzenesulphonate. Natural surface-active agents may also be used, such as for example phospholipids, e.g. diacylphosphatidylglycerols, diaceylphosphatidylcholines, and diaceylphosphatidic acids, the precursors and derivatives thereof, such as, for example, soya lecithin and egg yolk.
The term “Co-surfactant”, used herein, refers to emulsifying agent that is a compound that concentrates at the interface of two immiscible phases, usually an oil and water. It lowers the interfacial free energy, reduces the interfacial tension between the phases, and forms a film or barrier around the droplets of the immiscible, discontinuous phase as they are formed, preventing the coalescence of the droplets.Various emulsifying agents can be used in processes of preparation ofpharmaceutical compositions of the present invention including, but not limited to, Acacia Gum, Acacia Gum modified with octenyl succinic, anhydride (OSA), Acetylated Mono- glycerides, Acetylated Tartaric, Acid Esters of Mono- and Di-glycerides, Agar, Algin, Alginic Acid, Ammonium Alginate, Ammonium Carrageenan, Ammonium Furcelleran, Ammonium Salt of Phosphorylated Glyceride, Arabino-galactan, Baker's yeast, Glycan, Calcium Alginate, Calcium Carbonate, Calcium Carrageenan, Calcium Citrate, Calcium Furcelleran, Calcium Gluconate, Calcium Glycerophosphate, Calcium Hypophosphite, Calcium Phosphate dibasic, Calcium Phosphate tribasic, Calcium Sulphate, Calcium Tartrate, Carboxymethyl Cellulose, Carob Bean Gum, Carrageenan, Cellulose Gum, Citric Acid Esters of Mono- and Di-glycerides, Furcelleran, Gelatin, Gellan Gum, Guar Gum, Gum Arabic, Hydroxylated Lecithin, Hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose, Irish Moss Gelose, Karaya Gum, Lactylated Mono- and Di-glycerides, Lactylic Esters of Fatty Acids, Locust Bean Gum, Methylcellulose, Methyl Ethyl Cellulose, Monoglycerides, Mono- and Di-glycerides, Monosodium Salts of Phosphorylated Mono- and Di-glycerides, Oat Gum, Pectin, Polyglycerol Esters of Fatty Acids, Polyglycerol Esters of InteresterifiedCastor Oil Fatty Acids, Potassium Alginate, Potassium Carrageenan, Potassium Furcelleran, Quillaia extract (Type 2), Tragacanth Gum and Xanthan Gum.
Various solvents can be used in processes of preparation ofpharmaceutical compositions of the present invention including, but not limited to, water, methanol, ethanol, acidified ethanol, acetone, diacetone, polyols, polyethers, oils, esters, alkyl ketones, methylene chloride, isopropyl alcohol, butyl alcohol, methyl acetate, ethyl acetate, isopropyl acetate, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol,monoethyl ether, dimethylsulphoxide, ?,?-dimethylformamide, tetrahydrofuran, and any mixtures thereof. In general, liquids that are used as solvents and vehicles for the various processing operations will not be present in a finished formulation, as they will be evaporated when they are no longer needed.
The present invention comprises a probiotic composition, preferably mixture of at least two microorganisms. Any suitable mixture of probiotic may be used herein. See for example US 2010/0221226, US 2010/0266727, US 2010/0266560 and US Pat. No.6,060,050. Various probiotics are known such as, for example, Bifidobacterium adolescentis, Fidobacteriumanimalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium infantis, Lactobacillus amylovorus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Streptococcus thermophilus, Streptococcus salivarius, Streptococcus oralis, Streptococcus uberis, Streptococcus rattus, Escherichia coli, Bacillus coagulans, Bacillus lansii, Saccharomyces cerevisiae, Saccharomyces boulardii and combinations thereof. Preferred probiotics for use herein include Lactobacillus rhamnosus, Bacillus coagulans and Bifidobacterium bifidum.
Some exemplary embodiments of the present invention relates to an emulsion having
a surfactant, preferably a phospholipid, more specifically a Soybean lecithin, preferably between about 0.01% to about 10%, more preferably between about 0.5% to about 5% and most preferably between about 1% to about 2% by weight.
at least one emulsifying or gelling agent, preferably a Gum, preferably between about 0.1% to about 0.5% by weight.
an oil , preferably volatile oil, preferably about 5-20% by weight and
Water
According to present invention, the process for preparation of the emulsion comprises following steps:
Preparation of Aqueous Phase:
Step 1: Xanthan gum was added to water and mixed in cyclo mixer for 30minutes.
Step 2:Xanthan gum solutionwas transferred in to a Scott Durand Bottle and stirred for 60 minutes at 60 degree Celsius with a magnetic Stirrer.
Step 3: Meanwhile Soya lecithin was added to Xanthan solution and stirring was continued.
Preparation of Oil Phase:
Step 1: 20ml of soya oil (autoclaved) is taken in a scottduran bottle. Probiotics was added to Soya oil and mixed it properly with cyclo mixer for 5minutes.
Step 2: This probiotic mixed oil was kept in water bath for 10 minutes at a temperature of 60 degree Celsius.
Preparation of Emulsion:
Step 1: Aqueous phase was kept under homogenization at 8000 rpm.
Step 2: Oil phase was added to Phase A. Homogenization was continued for 5 minutes at 8000 rpm.
Step 3: Sweetener was added to the mixture.
Step 4: Step 3 mixture was incubated at room temperature for stability studies.
Step 5: pH was checked to be within 4.0 to 7.0.And Viscosity was checked to be less than 2000 cp.

VIABILITY TESTS:
The viability of probiotic microorganisms in the final product until the time of consumption has been proposed with the descriptor minimum of biovalue (MBV). MBV is the minimum of viable probiotic cells per gram or milliliter of probiotic product, and is the most important qualitative parameter of probiotic products as it determines their pharmaceutical effectiveness. The viability of probiotic microorganisms is affected by factors such as the strain of probiotic bacteria, interactions among present species, pH, production of organic acids and volatile compounds (e.g., lactic acid, acetic acid, orotic acid, succinic acid, uric acid, citric acid, ethanol, pyruvate, acetaldehyde, diacetyl and acetoin). Other important factors are the metabolic products and acids produced during refrigerated storage, concentration of hydrogen peroxide and dissolved oxygen in fermented milks, concentration of sodium chloride in the media, inoculation level, incubation temperature and time, growth promoters (nutrients availability) and inhibitors, buffering capacity of the media, storage temperature, heat treatments, homogenization and packaging materials and conditions.
Procedure
Step 1: The samples were taken from prepared serial dilution at 10-8 dilution.
Step 2: The strains grow on selective media.
Step 3: The strains are incubated at 35 ± 2 degree Celsius.
Step 4: Step 1-3 were performed in replicate plates, by denoting R1, R2, R3 and R4 for accuracy.
Calculation:
x = in 10-8 dilution CFU/ml =R1+R2×10÷2 million/ml
y = in 10-7dilution CFU/ml =R3+R4×100÷2 million/ml
z = average CFU/ml = (X+Y)/2million/ml
Average CFU/5ml = 5×z×1000billion

Acceptance Criteria: 5 billion/5ml (Each Strain individually)

STABILITY STUDIES
From a practical point of view, measurement of stability is one of the most important tests, the mixed probiotic suspension was formulated, and pure vegetable oils was used as stabilizing agents at room temperature and stored at Room temperature (25°C ± 2°C), 5 ± 3°C and at 40 ± 2°C and Stability Studies were performed.

Storage Conditions for Stability Study
Storage Condition Time
5 ± 3°C 0th months, 3 months, 6 months
25°C ± 2°C 0th months, 3 months, 6 months
40°C ± 2°C 0th months, 1 months, 2 months and 3 months

Different methods and procedures were followed by various laboratories, whereas no standard method is available in the literature for determining the stability of the probiotics in oils.

Stability Studies at 5 ± 3°C
The Stability studies were conducted at5 ± 3°C. Results as follows for the mixed emulsion suspension:

Time Colonies Observed
0 months Lactobacillus rhamnosus- 5.7 × 109
Bacillus coagulans - 5.4 × 109
Bifidobacterium bifidum - 5.2 × 109
3 months Lactobacillus rhamnosus - 5.5 × 109
Bacillus coagulans- 5.3 × 109
Bifidobacterium bifidum - 5.2 × 109
6 months Lactobacillus rhamnosus – 5.2 × 109
Bacillus coagulans - 5.2 × 109
Bifidobacterium bifidum- 5.1 × 109

Stability Studies at 25°C ± 2°C
The Stability studies were conducted at25°C ± 2°C. Results as follows for the mixed emulsion suspension:
Time Colonies Observed
0 months Lactobacillus rhamnosus- 5.8 × 109
Bacillus coagulans - 5.5 × 109
Bifidobacterium bifidum - 5.4 × 109
3 months Lactobacillus rhamnosus - 5.8 × 109
Bacillus coagulans - 5.5 × 109
Bifidobacterium bifidum - 5.3 × 109
6 months Lactobacillus rhamnosus – 5.7 × 109
Bacillus coagulans - 5.5 × 109
Bifidobacterium bifidum - 5.3 × 109

Stability Studies at 40°C ± 2°C
Time Colonies Observed
0 months Lactobacillus rhamnosus- 5.8 × 109
Bacillus coagulans - 5.5 × 109
Bifidobacterium bifidum - 5.4 × 109
1 months Lactobacillus rhamnosus - 5.5 × 109
Bacillus coagulans - 5.4 × 109
Bifidobacterium bifidum - 5.3 × 109
2 months Lactobacillus rhamnosus – 5.3 × 109
Bacillus coagulans– 5.1 × 109
Bifidobacterium bifidum - 5.2 × 109
3 months Lactobacillus rhamnosus – 5.2 × 109
Bacillus coagulans– 5.0 × 109
Bifidobacterium bifidum - 5.1 × 109

The product has been found stable for period of 24 months duration of its shelf life and the count remains within the acceptance criteria i.e., not less than 5 billion per 5 mL for each individual strain.

Filling of Final Product
After the preparation of the final stable liquid emulsion formulation, that is sent to BFS filling-sealing machine in an aseptic condition. The filling process is disclosed herein. Thermoplastic, specifically Polyethylene, most specifically Low Density Polyethylene, resin is extruded as hollow tube known as parison. When the parison reached to a proper length the holding jaw is closed and the parison is cut. The bottom of the parison is pinched closed, while the top is held in place. The blow fill nozzle is lowered into the parison forming a seal with the neck of the mold. Sterile, filtered compressed air is blown into the parison expanding it then vented out. The sterile product can be metered in through the fill Nozzles. Separate sealing molds close to form the top and hermetically seal the container. The mould then opens and the formed, filled and sealed containers are conveyed out of the machine.
The invention having been disclosed in connection with the foregoing embodiments, additional variations will now be apparent to persons skilled in the art. Various modifications and variations to the above described development of anti-snake venom of high potent can be made without departing from the scope of the invention.
From the foregoing it will be understood that the embodiments of the present invention described above are well suited to provide the advantages set forth, and since many possible embodiments may be made of the various features of this invention and as the process herein described may be varied in various parts, all without departing from the scope of the invention, it is to be understood that all matter hereinbefore set forth or shown in the accompanying process is to be interpreted as illustrative and that in certain instances some of the features may be used without a corresponding use of other features, all without departing from the scope of the invention.

Date: Day 30th December, 2017