DESC:Field of the invention:
The present invention relates to a water-soluble lyophilized formulation of microorganism Bacillus subtilis. More particularly, the present invention relates to a water-soluble lyophilized formulation of Bacillus subtilis with high viability of microorganism and extended shelf life. The present invention also relates to a method to obtain a water-soluble lyophilized formulation of microorganism Bacillus subtilis to achieve and maintain high viability of microorganisms and shelf life.

Background of the invention:
Bacillus subtilis is a Gram positive bacterium widespread in soil and on plant surfaces. It is known to be strictly aerobic or facultative anaerobic. It produces stress-tolerant endospores. The members of Bacillus subtilis group are generally regarded as safe (GRAS) for animals and humans. Many strains of Bacillus subtilis strains have been reported to produce antibiotics greatly inhibitory to plant pathogenic bacteria or fungi. Bacillus subtilis has been used in feed additives, seed protecting formulations, source of enzymes as well as plant disease control agent for many years. Bacillus subtilis is used on agricultural seeds of vegetables, soybeans, cotton, and peanuts and flower and ornamental seeds.

Commercially, the product of B. subtilis are present in liquid and dry form i.e. spray drying. The major concern in the preparation and storage of such products is to achieve and maintaining high viability and recovery of microorganisms. Several failures remain with liquid and spray drying of microbial based formulation i.e. yield and shelf life.

WO2006029467A1 discloses a process for freeze drying, particularly freeze drying samples containing biological material. The rapid freeze drying a sample includes freezing the sample followed by freeze drying the sample under temperature and vacuum conditions such that the sample is dried close to the collapse temperature of the sample. T collapse temperature of the sample has been defined as the temperature during freezing that results in the collapse of the structural integrity of the product. Further, 'close to the collapse temperature' means a temperature about 0.1 to 10°C below the collapse temperature. Biological materials include, but not limited to, microorganisms such as bacteria, yeasts, fungi, viruses; proteins such as antibodies, growth factors, hormones and the like; nucleic acids such as DNA and RNA. The cryoprotectant is selected from the group consisting of glycerol, 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, dimethyl sulfoxide (DMSO), methanol, 1 ,2-propanediol, proline, and ethylene glycol. The formulation is helpful in rapid drying and inhibition of sudden collapse during lyophilization.

CN107446857A discloses a kind of preparation method of Bacillus subtilis freeze-dried powder. The concentration of Bacillus subtilis suspension comprises of 4-8 × 108 cfu/mL and formulation components are trehalose, mannitol and sodium thiosulfate. The concentration of trehalose, mannitol and sodium thiosulfate was in the range of 0.15~ 0.5mol/L, 0.05~0.4mol/L, and 0.05~0.5mol/L, respectively.

Lyophilization technique is used in order to improve shelf life of product and maintain viability of biological materials. However, during lyophilization, microbial spores/ vegetative cells are processed under low temperature and pressure, which may lead to loss of viability and recovery of product.

The present invention obviates the drawbacks in the prior art by providing a formulation of Bacillus subtilis biomass blended with specific cryoprotectants to overcome the losses due to freezing temperature, thereby extending the viability and maintaining the functionality of the microbes in the formulation. The present invention also provides a method of production of lyophilized, water-soluble formulation of B. subtilis for use in organic agriculture.

Object of the invention:
The main object of the present invention is to provide a water-soluble lyophilized formulation of microorganism Bacillus subtilis.
Another object of the present invention is to provide a water-soluble formulation of Bacillus subtilis with high viability of microorganism and extended shelf life.

Yet another object of the present invention is to provide a method of production of a water-soluble lyophilized formulation of microorganism Bacillus subtilis.

Yet another object of the present invention is to provide a method of production of a water-soluble lyophilized formulation of Bacillus subtilis with high viability of microorganism and extended shelf life.

Yet another object of present invention is to provide a method of production of a water-soluble lyophilized formulation of Bacillus subtilis, thereby facilitating easy transportation and low storage space requirement, making the formulation cost effective and user friendly.

Summary of the present invention:
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, 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 a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Accordingly, the present invention provides a water-soluble lyophilized formulation of microorganism Bacillus subtilis. More particularly, the present invention provides to a water-soluble lyophilized formulation of Bacillus subtilis with high viability of microorganism and extended shelf life. The present invention also provides a method to obtain a water-soluble lyophilized formulation of microorganism Bacillus subtilis to achieve and maintain high viability of microorganisms and shelf life.

The term Bacillus subtilis in the specification refers to and includes the microorganism, it’s spores, endospores and exotoxins and endotoxins.

The formulation of the present invention comprises Bacillus subtilis having viable cell count in the range of 1×108 to 5×1010 cfu/g, with at least one excipient/carrier. More preferably, the formulation comprises Bacillus subtilis having viable cell count of 1×109 cfu/g with at least one excipient/carrier.

The excipient/carrier is selected from maltodextrin, or dextrose or trehalose, or a combination thereof.

In a preferred embodiment, the excipient/carrier is maltodextrin.

In an embodiment, the formulation comprises active ingredient of the selected strain of Bacillus subtilis and maltodextrin.
In yet another embodiment, the formulation comprises active ingredient of the selected strain of Bacillus subtilis, a lyoprotectant and excipient/carrier.

In a preferred embodiment, the formulation of the present invention comprises pure culture of selected strain of B. subtilis having viable cell count in the range of 1×108 to 5×1010 cfu/g, a lyoprotectant and suitable excipient/carrier.

In another preferred embodiment, the formulation of the present invention comprises pure culture of selected strain of B. subtilis having viable cell count of 1×109, a lyoprotectant and suitable excipient/carrier.

The another embodiment, the formulation of the present invention comprises pure culture of selected strain of B. subtilis having viable cell count of 1x1010 cfu/g , a lyoprotectant and suitable excipient/carrier.

The formulation is a water-soluble powder product.

The formulation is stable at low and high temperatures and also at room temperature for at least 24 months.

The method of production of the formulation of water soluble lyophilized formulation of B. subtilis comprises batch fermentation followed by concentration of biomass of microorganism by centrifugation/filtration, treatment with a lyoprotectant and standardization with excipient/carrier to contain viable cell count in the range of 1×108 to 5×1010 cfu/g, more preferably 1×109.

The bacteria is grown in a culture medium consisting of sugar and/or starch as the carbon and energy source along with nitrogen source, vitamins and minerals. The cultured B. subtilis is maintained as a Cell Bank System. Working cell is prepared when needed, starting from a vial of a Master Cell stock. The working cell of microorganism is then grown under standard fermentation techniques under suitable conditions to obtain fermented biomass.

All fermentations are carried out under controlled hygienic aseptic conditions in order to get pure culture of fermented biomass without any microbial contamination from the environment. The fermentation is complete when the optimum cfu count per ml is achieved. The optimum cfu/ml of B. subtilis after fermentation is in the range of 1×109 to 1×1010 cfu/ml, more preferably 5×109 cfu/ml.

When the fermentation is complete, the fermented biomass is concentrated using centrifugation or any other known technique. Suitable amount of the lyoprotectant is mixed with the concentrate to form a slurry. The slurry is freeze dried to form solid frozen mass in a tray. The tray loaded with solid frozen mass is kept in a freeze drier for primary drying followed by secondary drying under specific conditions. Concentrated freeze dried mass obtained after secondary drying milled to obtain final product in the form of fine free flowing powder. The final product is standardized/diluted in suitable excipient/carrier to contain in the range of 1×108 to 5×1010 cfu/g, preferably 1×109 cfu/g.

The formulation of the present invention has high viability of microorganisms and high shelf life of at least 24 months. The formulation is easy to transport and requires low storage space. Thereby, the formulation of the present invention is economic, cost-effective and user friendly.

BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 CFU result of storage stability at accelerated conditions (37°C) of Formulation A and Formulation B.
Fig. 2 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (37°C) of Formulation A.
Fig. 3 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (37°C) of Formulation B.
Fig. 4 CFU result of storage stability at accelerated conditions (25°C) of Formulation A and Formulation B.
Fig. 5 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (25°C) of Formulation A.
Fig. 6 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (25°C) of Formulation B.
Fig. 7 CFU result of storage stability at accelerated conditions (4°C) of Bacillus subtilis of Formulation A and Formulation B.
Fig. 8 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (4°C) of Formulation A.
Fig. 9 Viability (%) and log10 cfu/g results of storage stability at accelerated conditions (4°C) of Formulation B.

DETAILED DESCRIPTION OF INVENTION WITH NON-LIMITED EMBODIMENTS AND EXAMPLES
Accordingly, the present invention provides a water-soluble lyophilized formulation of Bacillus subtilis, comprising selected strain of B. subtilis viable cells in suitable excipients/carrier. The formulation contains viable cells of selected strain of B. subtilis in the range of 1×108 to 5×1010 cfu/g. More preferably, the formulation of the present invention comprises selected strain of B. subtilis having viable cell count of 1×109 cfu/g.

The selected strain of B. subtilis has been isolated from rhizosphere of vegetable garden of Bhagpat, Uttar Pradesh, India. The microbe has been deposited at MTCC (Microbial Type Culture Collection) with deposit no. MTCC5728; accession date 10 January 2013.

The formulation shows extended shelf life of at least 24 months with maintaining the high viability of at least 1×1010 cfu/g of viable cell at ambient temperatures and lower temperatures, thereby maintaining high efficacy of the formulation in the fields.
The present invention also provides a method to obtain the water-soluble lyophilized formulation of selected strain of Bacillus subtilis. The pure culture of the selected strain of Bacillus subtilis spores has been isolated and cultivated in optimized fermentation conditions using optimized nutrient media (Table 1) to obtain fermented cells. The fermented cells have been harvested to get a concentrate comprising the B. subtilis viable cells and water. The concentrate comprises from 1×1010 to 5×1012 cfu/g dry matter of the concentrate of B. subtilis viable cells. isolating pure culture of the selected strain of Bacillus subtilis spores.

Table 1. Optimized nutrient media composition for the fermentation of selected strain of B. subtilis
S. No. Description Concentration (%, w/v)
1 Carbon source (dextrose, jaggery, sucrose, glycerol, soluble starches) 0.1 to 5%
2 Nitrogen source I (fish meal, corn meal, soyabean meal, oatmeal) 0.1 to 3%
3 Nitrogen source II (soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate , proteose peptone, bacteriological peptone) 0.1 to 3%
4 Nitrogen source III (yeast extract, rice bran extract, barley extract, meat extract) 0.1 to 3%
5 Salts (magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate) 0.001 to 5 %
12 Water q.s.

The optimized fermentation condition comprises temperature ranging from 25°C to 40°C and pH ranging from 5.5 to 8.0 for a period of time to achieve above 90% sporulation.

The fermented broth contains B. subtilis viable cell count ranging from 1×109 to 1×1010 cfu/ml, more preferably 5×109cfu/ml (colony forming units per ml). The viable cells of bacterial culture have been harvested using centrifugation at 10000 rpm for 10 min at 4°C aseptically to obtain concentrate of cell biomass.

The concentrate of cell biomass is thoroughly mixed with a lyo-protectant solution to form a slurry. The lyo-protectant is selected from lyo-protectant 1 or lyo-protectant 2. The composition of lyo-protectant 1 and lyo-protectant 2 is mentioned in the Table 2 and 3.

Table 2 The composition of lyoprotectant 1 used for the freeze drying of selected strain of B. subtilis
Groups Maltodextrin Skim Milk MSG Lactose Trehalose Ascorbic acid Glycerol PEG Gelatin
Concentration (%)
1 0.1 1.6 7.2 1.5 1.1 0.1 2.0 0.1 1.7
2 0.0 5.5 8.1 2.0 1.7 8.9 0.5 0.9 1.9
3 0.3 0.9 7.5 3.0 6.8 6.4 1.9 0.7 1.1
4 0.4 0.8 5.5 4.5 1.6 0.9 1.2 0.3 1.2
5 0.6 0.1 1.9 0.1 3.7 8.7 1.0 1.9 1.5
6 0.5 5.0 5.0 1.0 5.0 5.0 0.5 1.0 0.5
7 0.1 2.5 1.8 0.9 0.1 4.5 0.7 1.5 0.9
8 0.9 3.5 12.0 1.9 9.4 6.1 0.9 2.0 0.1
9 1.0 1.5 8.0 2.6 4.6 7.5 0.6 3.1 1.2
10 1.5 10. 0.1 3.5 6.4 0.2 0.1 1.5 1.0
MSG, monosodium glutamate; PEG, polyethylene glycol;
Table 3. The composition of lyoprotectant 2 used for the freeze drying of selected strain of B. subtilis
Groups Maltodextrin Skim Milk MSG Lactose Trehalose Ascorbic acid Glycerol PEG Gelatin CMC
Concentration (%)
1 0.1 1.6 7.2 1.5 11 0.1 2.0 0.1 1.7 0.2
2 0.0 5.5 8.1 2.0 1.7 8.9 0.5 0.9 1.9 0.5
3 0.3 0.9 7.5 3.0 6.8 6.4 1.9 0.7 1.1 0.9
4 0.4 0.8 5.5 4.5 1.6 0.9 1.2 0.3 1.2 1.0
5 0.6 0.1 1.9 0.1 3.7 8.7 1.0 1.9 1.5 0.4
6 0.5 5.0 5.0 1.0 5.0 5.0 0.5 1.0 0.5 0.8
7 0.1 2.5 1.8 0.9 0.1 4.5 0.7 1.5 0.9 0.5
8 0.9 3.5 12.0 1.9 9.4 6.1 0.9 2.0 0.1 0.7
9 1.0 1.5 8.0 2.6 4.6 7.5 0.6 3.1 1.2 0.9
10 1.5 10. 0.1 3.5 6.4 0.2 0.1 1.5 1.0 1.5
MSG, monosodium glutamate; PEG, polyethylene glycol; CMC, carboxymethyl cellulose.

The lyo-protectant 1 solution has been prepared by dispersing accurately weighed amount of gelatin into water and allowing it to hydrate for 30 to 60 minutes and then accurately weighed amounts of maltodextrin, monosodium glutamate, lactose, trehalose, ascorbic acid, glycerol and polyethylene glycol have been added. This fraction has been sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain first fraction of lyo-protectant 1. Further, accurately weighed amount of skimmed milk powder has been dispersed into water and allowed to form suspension while stirring it occasionally. Skimmed milk fraction has also been separately sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain second fraction of lyo-protectant 1. After autoclaving, both the fractions have been allowed to cool down to room temperature (25 to 30°C) and then both the fractions have been mixed and used as lyo-protectant 1.

In a similar procedure, lyo-protectant 2 has been prepared by dispersing carboxymethyl cellulose and gelatin in water and allowing to hydrate, followed by addition of maltodextrin, monosodium glutamate, lactose, trehalose, ascorbic acid, glycerol and polyethylene glycol. This fraction has been sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain first fraction of lyo-protectant 2. Further, accurately weighed amount of skimmed milk powder has been dispersed into water and allowed to form suspension while stirring it occasionally. Skimmed milk fraction has also been separately sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain second fraction of lyo-protectant 2. After autoclaving, both the fractions have been allowed to cool down to room temperature (25 to 30°C) and then both the fractions have been mixed and used as lyo-protectant 2.
The slurry is loaded onto a tray and frozen between a temperature of -45°C to -80°C to obtain frozen slurry.

The tray containing frozen slurry of B. subtilis is loaded into the freeze drier for primary drying and secondary drying to obtain dry solid frozen mass.

The primary drying of B. subtilis frozen slurry has been carried out under a vacuum pressure ranging from 0.5 to 4 millibar (mbar), at a temperature ranging from -35 to + 40°C for the duration until 90% of water is evaporated/removed. Then, secondary drying has been performed under a vacuum pressure ranging from 0.01 to 0.5 millibar (mbar), at a temperature ranging from +4°C to +40°C for a period of time sufficient to reduce the moisture content <2% or water activity below 0.3, thereby obtaining a dry solid frozen mass of the formulation. The solid frozen mass of the formulation is then milled and sieved to obtained fine free flowing powder and then standardized/diluted in suitable excipients/carrier to contain viable cell counts of selected strain of B. subtilis in the range of 1×108 to 5×1010, preferably of 1×109 cfu/g.

The suitable excipients/carrier to standardize the cfu count of B. subtilis is selected from maltodextrin and dextrose.

In a preferred embodiment, the suitable excipients/carrier to standardize the cfu count of B. subtilis is maltodextrin.

As a non-limiting example of the present invention, the stability studies of the formulation have been conducted on the formulation comprising B. subtilis and maltodextrin. The formulation of present invention comprising B. subtilis and maltodextrin, prepared using lyo-protectant 1 is referred to as “Formulation A” and the formulation prepared using lyo-protectant 2 is referred to as “Formulation B”

Stability studies

The stability studies of the formulation of the present invention have been conducted at three different conditions i.e accelerated (37°C), real time (25°C) and refrigerated conditions (4°C). The samples of the B. subtilis freeze dried powder have been optimized with the excipient selected from maltodextrin and dextrose in order to obtain = 1 × 1010 cfu/g. The standardized samples of lyophilized formulation of B. subtilis soluble powder have been packed in sealed aluminum bags. These samples have been tested for the colony-forming units (CFU) per gram, using the assay.

Viable cell counts (cfu) have been determined in freeze-dried powder immediately after freeze-drying and also at selected time points during the stability studies. A standard spread plating method has been used. The sample has been suspended in sterile saline and mixed well by vortexing. After 30 minutes of revitalization, the cell suspension has been serially diluted in sterile saline. For the cfu of the selected strain of B. subtilis, the dilutions are plated in duplicates. The agar plates have been incubated aerobically for three days at 37°C. Plates with 30 - 300 colonies have been chosen for counting of colony forming units. The result has been reported as average cfu/g of B. subtilis, calculated from the duplicates. The results are shown in Tables 4-6 and figures 1-9.

Table 4 shows storage stability results (CFU/g) at the accelerated conditions (37 °C). Table 5 shows storage stability results (CFU/g) at real time conditions (25 °C). Table 6 shows storage stability results (CFU/g) at the refrigerated conditions
(4 °C).

Table 4. Storage stability of Formulation A and Formulation B at 37 °C.

Time (days)
Formulation A Formulation B
0 day 2.3 × 1010 2.8 × 1010
7 days 1.4 × 1010 1.0 × 1010
14 days 1.1 × 1010 1.0 × 1010
21 days 1.1 × 1010 1.0 × 1010
28 days 1.0 × 1010 1.0 × 1010
35 days 1.0 × 1010 1.0 × 1010
42 days 1.4 × 1010 1.0 × 1010
49 days 2.1 × 1010 1.1 × 1010
56 days 1.7 × 1010 1.0 × 1010
63 days 2.3 × 1010 2.0 × 1010
70 days 1.6 × 1010 2.0 × 1010
77 days 1.5 × 1010 1.0 × 1010
84 days 2.0 × 1010 1.0 × 1010

Table 5. Storage stability of Formulation A and Formulation B at 25°C.
Time (Months)
Formulation A Formulation B
0 day 2.3 × 1010 2.8 × 1010
1st month 2.0 × 1010 1.7 × 1010
2nd month 1.3 × 1010 1.2 × 1010
3rd month 2.0 × 1010 1.1 × 1010
4th month 1.0 × 1010 2.0 × 1010
5th month 1.0 × 1010 1.0 × 1010
6th month 2.0 × 1010 1.3 × 1010
7th month 2.0 × 1010 1.0 × 1010
8th month 1.1 × 1010 2.3 × 1010
9th month 2.0 × 1010 2.0 × 1010
10th month 3.0 × 1010 2.7 × 1010
11th month 1.6 × 1010 1.5 × 1010
12th month 2.0 × 1010 2.0 × 1010

Table 6. Storage stability of Formulation A and Formulation B at 4 °C.
Time (Months)
Formulation A Formulation B
0 day 2.3 × 1010 2.8 × 1010
1st month 1.6 × 1010 1.8 × 1010
2nd month 1.7 ×1010 2.0 × 1010
3rd month 1.8 × 1010 1.0 × 1010
4th month 2.0 × 1010 1.0 × 1010
5th month 3.0 × 1010 1.0 × 1010
6th month 2.0 × 1010 1.0 × 1010
7th month 1.2 × 1010 2.0 × 1010
8th month 1.5 × 1010 1.0 × 1010
9th month 3.0 × 1010 1.4 × 1010
10th month 2.0 × 1010 1.5 × 1010
11th month 2.1 × 1010 3.0 × 1010
12th month 2.9 × 1010 2.0 × 1010

The dry powdered formulation is packed in bags and stored at -20?C. As and when required, the formulation is packed in sachet at pre-defined potency. The sachets containing the formulation are stored at -80?C until delivery.

The formulation of the present invention has high viability of microorganisms and high shelf life of at least 24 months at lab scale. The studies on high viability of microorganisms and high shelf life at pilot scale have been conducted for 12 months and data have been provided herein. The experiments on high viability of microorganisms and high shelf life are still on and further data will be generated in due course. The formulation is easy to transport and requires low storage space.

Therefore, the formulation of the present invention is economic, cost-effective and user friendly.

CLAIMS:

We claim:
1. A water-soluble lyophilized formulation, said formulation comprises viable cells of B. subtilis bearing MTCC deposit number 5728 dated 10 January 2013 in suitable excipients/carrier.
2. The formulation as claimed in claim 1, wherein said formulation comprises viable cells of B. subtilis in the range of 1×108 to 5×1010 cfu/g in suitable excipients/carrier.
3. The formulation as claimed in claim 1, wherein said formulation comprises viable cells of B. subtilis having 1×109 cfu/g.
4. The formulation as claimed in claim 1, wherein said formulation shows extended shelf life of at least 24 months with maintaining the high viability of at least 1 × 1010 cfu/g of viable cell at ambient temperatures and lower temperatures, thereby maintaining high efficacy of the formulation in the fields.
5. The formulation as claimed in claim 1, wherein said excipient/carrier is selected from maltodextrin, or dextrose, or trehalose, or a combination thereof.
6. The formulation as claimed in claim 1, wherein said excipient/carrier is maltodextrin.
7. The formulation as claimed in claim 1, wherein the formulation is in the form of dry powder requiring low storage space, thereby making the formulation economic, cost-effective and user friendly.
8. A method to obtain the formulation of claim 1, wherein the method comprises:
- isolating pure culture of the selected strain of Bacillus subtilis spores,
- cultivating the isolated cells of the pure culture in optimized fermentation conditions using optimized nutrient media to obtain fermented broth containing the B. subtilis viable cell count ranging from 1x108 to 1 × 1010 cfu/ml, more preferably 5x109 cfu/ml,
- harvesting the B. subtilis viable cells from the fermented broth under specific conditions to get a concentrate comprising the B. subtilis viable cells and water, wherein the concentrate comprises viable cells of B. subtilis in the range from 1 × 1010 to 5 × 1012 cfu/g, preferably of 5×1011 cfu/g.
- preparing lyoprotectant 1 and lyo-protectant 2,
- mixing the concentrate with the lyo-protectant 1 or lyo-protectant 2 to form a slurry,
- loading the slurry onto a tray and frozen between a temperature of -45°C to -80°C to obtain frozen slurry,
- loading the tray containing frozen slurry into the freeze drier for primary drying for a period until 90% of water is evaporated to obtain solid frozen mass,
- carrying out secondary drying of solid frozen mass to obtain dry solid frozen mass for a period sufficient to reduce moisture content <2% or water activity below 0.3,
- milling and sieving the dry solid frozen mass to obtain fine free flowing dry powder formulation comprising viable cells in the range of 1×1010 to 5×1012 cfu/g, more preferably 5×109 cfu/g,
- standardizing the dry powder formulation in suitable excipients/carrier to contain viable cell counts of the selected strain of B. subtilis in the rage of 1x108 to 5x1010 cfu/g, preferably of 1X109 cfu/g,
- storing the standardized dry powder formulation at a temperature of -20?C.
9. The method as claimed in claim 8, wherein the optimized nutrient media composition for the fermentation of selected strain of B. thuringiensis comprises carbon source in the range of 0.1% to 5% (w/w), nitrogen source I in the range of 0.1% to 3% (w/w), nitrogen source II in the range of 0.1% to 3% (w/w), nitrogen source III in the range of 0.1% to 3% (w/w), and salt in the range of 0.001% to 5% (w/w).
10. The method as claimed in claim 9, wherein said carbon source is selected from dextrose, jaggery, sucrose, glycerol, soluble starches or any combination thereof.
11. The method as claimed in claim 9, wherein nitrogen source I is selected from fish meal, corn meal, soyabean meal, oatmeal or any combination thereof.
12. The method as claimed in claim 9, wherein nitrogen source II is selected from soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone or any combination thereof.
13. The method as claimed in claim 9, wherein nitrogen source III is selected from yeast extract, rice bran extract, barley extract, meat extract or any combination thereof.
14. The method as claimed in claim 9, wherein salt is selected from magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate or any combination thereof.
15. The method as claimed in claim 8, wherein the optimized fermentation condition comprises temperature ranging from 25°C to 40°C and pH ranging from 5.5 to 8.0 for a period of time to achieve above 90% sporulation.
16. The method as claimed in claim 8, wherein the specific conditions for harvesting the B. subtilis viable cells are centrifugation at 8000 rpm for 10 min at 4°C aseptically.
17. The method as claimed in claim 8, wherein lyoprotectant 1 comprises maltodextrin in the range of 0%-1.5%, skim milk in the range of 0.1%-10%, monosodium glutamate in the range of 0.1%-12%, lactose in the range of 0.1%-4.5%, trehalose in the range of 0.1%-9.4%, ascorbic acid in the range of 0.1%-8.9%, glycerol in the range of 0.1%-2.0%, polyethylene glycol in the range of 0.1%-3.1%, and gelatin in the range of 0.1% to 1.9%.
18. The method as claimed in claim 8, wherein lyoprotectant 2 comprises maltodextrin in the range of 0%-1.5%, skim milk in the range of 0.1%-5.5%, monosodium glutamate in the range of 0.1%-12%, lactose in the range of 0.1%-4.5%, trehalose in the range of 0.1%-11%, ascorbic acid in the range of 0.1%-8.9%, glycerol in the range of 0.1%-2.0%, polyethylene glycol in the range of 0.1%-3.1%, gelatin in the range of 0.1% to 1.9%, and carboxymethyl cellulose in the range of 0.2%-1.5%.
19. The method as claimed in claim 17, wherein the lyoprotectant 1 is prepared by
- dispersing accurately weighed amount of gelatin into water and allowing it to hydrate for 30 to 60 minutes,
- adding accurately weighed amounts of maltodextrin, monosodium glutamate, lactose, trehalose, ascorbic acid, glycerol and polyethylene glycol and sterilizing by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain 1st fraction of lyo-protectant 1.
- Dispersing accurately weighed amount of skimmed milk powder in water and allowing to form suspension while stirring it occasionally and sterilizing by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain second fraction of lyo-protectant 1,
- Cooling down the first fraction and second fraction of lyo-protectant 1 to room temperature,
- Mixing the cooled first fraction and second fraction of lyo-protectant 1 to obtain lyo-protectant 1.
20. The method as claimed in claim 18, wherein the lyoprotectant 2 is prepared by
- dispersing accurately weighed amount of carboxymethyl cellulose and gelatin in water and allowing to hydrate,
- adding accurately weighed amount of maltodextrin, monosodium glutamate, lactose, trehalose, ascorbic acid, glycerol and polyethylene glycol, then sterilizing by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain first fraction of lyo-protectant 2,
- dispersing accurately weighed amount of skimmed milk powder into water and allowing to form suspension while stirring it occasionally and sterilizing by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain second fraction of lyo-protectant 2,
- Cooling down the first fraction and second fraction of lyo-protectant 2 to room temperature,
- Mixing the cooled first fraction and second fraction of lyo-protectant 1 to obtain lyo-protectant 1.
21. The method as claimed in claim 8, wherein the primary drying of frozen slurry is carried out under a vacuum pressure ranging from 0.5 to 4 millibar (mbar), at a temperature ranging from -35°C to + 40°C.
22. The method as claimed in claim 8, wherein the secondary drying of solid frozen mass is carried out under a vacuum pressure ranging from 0.01 to 0.5 millibar (mbar), at a temperature ranging from +4°C to +40°C.
23. The method as claimed in claim 8, wherein the suitable excipient/carrier is selected from maltodextrin, or dextrose or trehalose, or a combination thereof.
24. The method as claimed in claim 8, wherein the suitable excipient/carrier is maltodextrin.