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

Background of the invention:
Biopesticides can be obtained from microbial, animal and plant sources. Most common sources of biopesticides are microorganism. The active ingredients of microbial based pesticides are spores, endotoxins and enzymes produced by the microorganisms i.e. bacteria and fungi. Amongst the microbial source, Bacillus thuringiensis is commonly used as biopesticides. It is used against different types of insect pests in agriculture.

Bacillus thuringiensis is a Gram-positive, soil-dwelling bacterium. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic environments, animal faeces, insect-rich environments, and flour mills and grain-storage facilities. It has also been observed to parasitize other moths such as Cadra calidella. B. thuringiensis based products are used on crops and ornamental plants. It is also used in and around buildings, in aquatic settings, and in aerial applications. These products are commonly used as sprays, dusts, granules, and pellets. Some of these products are approved for use in organic agriculture. It is mainly used against Lepidoptera pests.

Moreover, the production approaches for microbial based pesticides are diverse and inoculation techniques are based on application in liquid forms such as sprays and drenches or solid forms such as lyophilizates delivered to soil as foliar surfaces substrate.

Generally, biopesticides are manufactured by fermentation followed by harvesting of broth. In liquid formulation, fermented broth is harvested and blended with stabilizers and bottled. For dry preparation, fermented broth is blended with charcoal, talc and clay or peat to make dry, afterwards it is milled and packed. The major concern in the preparation and storage of such biopesticides is to achieve and maintain high viability of microorganisms and shelf life.

European patent Publication no. EP1306008 discloses of a formulation of an insecticide of Bacillus thuringiensis for the treatment of mosquito breeding site against mosquito larvae, the formulation comprising an insecticidal component derived from Bacillus thuringiensis M-H-14, a carrier component selected form the group consisting of peat, corn cobs, corn grain, wheat strain, cork and palm seed grain, and a component for the protection against sun-light and optionally feeding filler and excipients. The pourable concentrate of the Bacillus thuringiensis M-H-14 formulation with the smallest mean particle size, settled more slowly than the wettable powders and consequently provided a more prolonged control.

Lithuanian patent document no. LT 4541 discloses bacteria production and the invention comprises a method of production of lactobacilli and lyophilization. The formulation consisting of fat free milk powder, sodium glutamate, glucose, sodium bicarbonate which was prepared in water base.

CA2812049 A1 discloses rapid lyophilized water soluble biopesticide granules of Bacillus thuringiensis. The invention provides an effervescent granule containing the active ingredient in water with release of carbon dioxide. The invention relates to a granulated lyophilized effervescent biopesticide granule. In the invention, biopesticide and milk are mixed respectively. After lyophilization process, effervescent granules are obtained upon mixing acid and carbonate salt. The obtained granules are in the form of a single use dosage as powder or granules which do not have harmful effect on environment and human health, and are economical.

Lyophilization is well known and widely used technique for extending microbial cell viability. Lyophilization is a drying and preservation process carried out at low/ freezing temperature under pressure that enhance the shelf life of product. This process removes water by sublimation for drying the substance. Additionally, it is useful approach for preparation of ready to use water soluble formulations with easy and economic transportation and low storage space requirement.

However, the initial concern with cell viability is their ability to withstand the freezing and drying procedures. The irreversible damage to the bacterial cells caused by the rigors undergone during the lyophilization procedure results in a large proportion of the cells being killed during freeze-drying.

The present invention obviates the drawbacks in the prior art and provides a lyophilized, water-soluble formulation of B. thuringiensis and a method of production thereof 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 thuringiensis.

Another object of the present invention is to provide a water-soluble formulation of Bacillus thuringiensis 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 thuringiensis.

Yet another object of the present invention is to provide a method of production of a water-soluble lyophilized formulation of Bacillus thuringiensis 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 thuringiensis, 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 thuringiensis. More particularly, the present invention provides to a water-soluble lyophilized formulation of Bacillus thuringiensis 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 thuringiensis to achieve and maintain high viability of microorganisms and shelf life.

The term Bacillus thuringiensis in the specification refers to and includes the microorganism, its spores, endospores and exotoxins and endotoxins.

In a preferred embodiment, the active ingredient of the formulation comprises Bacillus thuringiensis of viable cell count of at least 5×1012 cfu/ml with at least one cryoprotectant. Said at least one cryoprotectant is selected from maltodextrin or trehalose or skim milk powder, or any combination thereof.

In another embodiment, the formulation comprises the active ingredient of the Bacillus thuringiensis with maltodextrin or trehalose and skim milk powder or a combination thereof.

In one of the embodiments, the formulation comprises the active ingredient of the Bacillus thuringiensis with maltodextrin and trehalose and skim milk powder.

In yet another embodiment, the formulation comprises the active ingredient of the Bacillus thuringiensis, and lyophilization mixture comprising at least one cryoprotectant and at least one buffering agent or wetting agent or polysaccharides or any combination thereof.

In one of the embodiments, the lyophilization mixture comprises maltodextrin or trehalose or a combination thereof, and carboxymethycellulose or glycerol or lactose or non-fat dry milk or a milk powder or any combination thereof.

In a further embodiment, the lyophilization mixture comprises trehalose and lactose. This cryoprotectant mixture of trehalose and lactose is preferred due to solubility characteristics and low cost.

The formulation is a water-soluble powder product.

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

The method of production of the formulation of water soluble lyophilized formulation of B. thuringiensis comprises batch fermentation followed by concentration of biomass of microorganism by centrifugation/filtration, addition of cryoprotectants to make a mixture of microorganism and cryoprotectants. The mixture is then freeze dried.

The bacteria is grown in a culture medium consisting of sugar as the carbon and energy source along with nitrogen source, vitamins and minerals. The cultured B. thuringiensis 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 fermented biomass without any microbial contamination from the environment. The fermentation is complete when the optimum cfu count per ml is achieved. The cfu/ml of B. thuringiensis after fermentation is in the range from 5 × 108 to 1 × 1010 cfu/ml and more preferably 5 × 109 cfu/ml.

When the fermentation is complete, the fermented biomass is concentrated using centrifugation or any other known technique and sent to a tank of previously prepared cryoprotectants. The fermented biomass is mixed with the cryoprotectants and frozen in liquid nitrogen to obtain frozen biomass. The frozen biomass is recovered and packed in sterile containers and stored at -60?C to -80?C until freeze drying.

For freeze drying, the stored frozen biomass is placed in freeze dryer for 2-3 days to obtain freeze dried biomass. The freeze dried biomass is harvested and milled to obtain final product. The final product is packed in bags and stored at -20?C. As and when required, the final product is packed in sachet at pre-defined potency. The sachets containing the final product 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. 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 the drawings:
Figure 1 depicts CFU count result of storage stability at accelerated conditions (37°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A) and dextrose (Formulation B).
Figure 2 depicts viability (%) and log10 cfu/g results of storage stability at accelerated conditions (37°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A).
Figure 3 depicts viability (%) and log10 cfu/g results of storage stability at accelerated conditions (37°C) of selected strain of Bacillus thuringiensis formulated in dextrose (Formulation B).
Figure 4 depicts CFU count result of storage stability at ambient conditions (25°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A) and dextrose (Formulation B).
Figure 5 depicts viability (%) and log10 cfu/g results of storage stability at ambient conditions (25°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A).
Figure 6 depicts viability (%) and log10 cfu/g results of storage stability at ambient conditions (25°C) of selected strain of Bacillus thuringiensis formulated in dextrose (Formulation B).
Figure 7 depicts CFU count result of storage stability at refrigerated conditions (4°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A) and dextrose (Formulation B).
Figure 8 depicts viability (%) and log10 cfu/g results of storage stability at refrigerated conditions (4°C) of selected strain of Bacillus thuringiensis formulated in maltodextrin (Formulation A).
Figure 9 depicts viability (%) and log10 cfu/g results of storage stability at ambient conditions (4°C) of selected strain of Bacillus thuringiensis formulated in dextrose (Formulation B).

Detailed Description of the invention with non-limiting embodiments, illustrations and examples:
Accordingly, the present invention provides a water-soluble lyophilized formulation of Bacillus thuringiensis, comprising selected strain of B. thuringiensis viable cells in suitable excipients/carrier. The viable cell count of Bacillus thuringiensis in the formulation is in the range from 1 × 108 to 5 × 1010 cfu/g. More preferably, the viable cell count of Bacillus thuringiensis in the formulation is 1 × 1010 cfu/g of viable cells of selected strain of B. thuringiensis.

The selected strain of B. thuringiensis has been obtained from soil samples of Heliothis infected field in Bijnor, Uttar Pradesh, India. The microbe has been deposited at MTCC (Microbial Type Culture Collection) with Deposit No. MTCC 25575 and accession date 3 December 2021.

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 thuringiensis. The isolated pure culture of the selected strain of Bacillus thuringiensis cells has been cultivated and grown in optimized fermentation conditions using optimized nutrient media (Table 1) to obtain fermented broth. The fermented broth contains the B. thuringiensis viable cell count in the range of 5 × 108 to 1 × 1010 cfu/ml and more preferably 5 × 109 colony forming units (cfu/ml). The B. thuringiensis viable cells have been harvested from the fermented broth to get a concentrate comprising the B. thuringiensis selected strain and water. The concentrate comprises in the range from 1 × 1010 to 5 × 1012 cfu/g and more preferably at least 5× 1011 cfu/g dry matter of the concentrate of B. thuringiensis viable cells.

Table 1 Optimized nutrient media composition for the fermentation of selected strain of B. thuringiensis.
S.No. Description Concentration (%, w/v)
1 Carbon source (selected from dextrose, jaggery, sucrose, glycerol, soluble starches or any combination thereof) 0.1 to 5%
2 Nitrogen source I (selected from fish meal, corn meal, soyabean meal, oatmeal or any combination thereof) 0.1 to 3%
3 Nitrogen source II (selected from soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone or any combination thereof) 0.1 to 3%
4 Nitrogen source III (selected from yeast extract, rice bran extract, barley extract, meat extract or any combination thereof) 0.1 to 3%
5 Salts (selected from magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate or any combination thereof) 0.001 to 5 %
6 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. thuringiensis viable cell count ranging from 5 × 108 to 1 × 1010 cfu/ml and more preferably at least 5 × 109 colony forming units (cfu/ml). The viable cells of bacterial culture have been harvested using centrifugation at 8000 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 for the freeze drying of selected strain of B. thuringiensis to form a slurry. The composition of lyo-protectant solution is mentioned in the Table 2.

Table 2. The composition of lyoprotectant solution used for the freeze drying of selected strain of B. thuringiensis
Groups CMC Trehalose Lactose MSG Skim Milk Maltodextrin Ascorbic acid Glycerol PEG
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
CMC - carboxymethyl cellulose; MSG - monosodium glutamate; PEG - polyethylene glycol

The lyo-protectant solution is prepared by hydrating carboxymethyl cellulose into water for 30 minutes to 60 minutes and then other ingredients such as trehalose, lactose, monosodium glutamate, maltodextrin, ascorbic acid, glycerol and polyethylene glycol are added. This fraction is sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. Then, milk fraction was prepared by adding milk powder is into water and is allowed to form uniform suspension. This skimmed milk fraction is also separately sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. After autoclaving, both the fractions are allowed to cool down to room temperature (25°C to 30°C) and then both the fractions are mixed and used as lyo-protectant solution in the invention.

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. thuringiensis is loaded into the freeze drier for primary drying and secondary drying to obtain dry solid frozen mass.

The primary drying of B. thuringiensis 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 to obtain solid frozen mass. Then, secondary drying of solid frozen mass 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 dry solid frozen mass of the formulation is then milled and sieved to obtained fine free flowing powder formulation and then standardized/diluted in suitable excipients/carrier to contain minimum 1 × 1010 cfu/g viable cell counts of selected strain of B. thuringiensis.

The suitable excipients/carrier to standardize the cfu count of B. thuringiensis is selected from maltodextrin and dextrose. The formulation prepared using maltodextrin as excipient is referred to as “Formulation A” and the formulation prepared using dextrose as excipient 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. thuringiensis 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. thuringiensis 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. thuringiensis, 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 (CFU). The result has been reported as average CFU/g of B. thuringiensis sample, calculated from the duplicates. The results are shown in Tables 3-5 and figures 1-9.

Table 3 shows storage stability results (CFU/g) of the Formulation A and Formulation B at the accelerated conditions. Table 4 shows storage stability results (CFU/g) of the Formulation A and Formulation B at real time conditions. Table 5 shows storage stability results (CFU/g) of the Formulation A and Formulation B at the refrigerated conditions.

Table 3. Storage stability results (CFU/g) of selected strain of Bacillus thuringiensis powder diluted in maltodextrin and dextrose at 37 °C.
Time (day) Formulation A Formulation B
0 day 3.5 × 1010 2.6 × 1010
7 days 2.0 × 1010 1.6 × 1010
14 days 2.0 × 1010 1.7 × 1010
21 days 2.1 × 1010 1.7 × 1010
28 days 3.0 × 1010 1.0 × 1010
35 days 1.8 × 1010 2.0 × 1010
42 days 2.0 × 1010 1.3 × 1010
49 days 1.8 × 1010 1.0 × 1010
56 days 2.0 × 1010 2.0 × 1010
63 days 1.7 × 1010 1.6 × 1010
70 days 1.8 × 1010 1.6 × 1010
77 days 1.4 × 1010 1.5 × 1010
84 days 1.9 × 1010 1.0 × 1010

Table 4. Storage stability results (CFU/g) of selected strain of Bacillus thuringiensis powder diluted in maltodextrin and dextrose at 25°C.
Time (months) Formulation A Formulation B
0 day 3.5 × 1010 2.6 × 1010
1st month 2.0 × 1010 1.3 × 1010
2nd month 1.9 × 1010 1.8 × 1010
3rd month 2.0 × 1010 1.7 × 1010
4th month 1.9 × 1010 2.1 × 1010
5th month 1.2 × 1010 2.0 × 1010
6th month 3.0 × 1010 1.3 × 1010
7th month 3.0 × 1010 2.0 × 1010
8th month 1.8 × 1010 2.8 × 1010
9th month 2.0 × 1010 2.0 × 1010
10th month 1.2 × 1010 2.1 × 1010
11th month 2.0 × 1010 1.3 × 1010
12th month 2.8 × 1010 2.0 × 1010
Table 5. Storage stability results (CFU/g) of selected strain of Bacillus thuringiensis powder diluted in maltodextrin and dextrose at 4 °C.
Time (months) Formulation A Formulation B
0 day 3.5 × 1010 2.6 × 1010
1st month 2.0 × 1010 1.1 × 1010
2nd month 1.1 × 1010 1.3 × 1010
3rd month 1.9 × 1010 2.1 × 1010
4th month 3.0 × 1010 2.0 × 1010
5th month 2.5 × 1010 2.9 × 1010
6th month 1.0 × 1010 1.0 × 1010
7th month 3.0 × 1010 2.0 × 1010
8th month 1.5 × 1010 1.0 × 1010
9th month 3.0 × 1010 1.4 × 1010
10th month 1.0 × 1010 2.9 × 1010
11th month 1.1 × 1010 1.0 × 1010
12th month 2.0 × 1010 2.2 × 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. thuringiensis bearing MTCC deposit number MTCC 25575 dated 3 December 2021 in suitable excipients/carrier.
2. The formulation as claimed in claim 1, wherein active ingredient of said formulation is viable cells of B. thuringiensis in the range of 1 × 108 to 5 × 1010 cfu/g .
3. The formulation as claimed in claim 1, wherein active ingredient of said formulation is viable cells of B. thuringiensis having 1 x 1010 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 the formulation is in the form of dry powder requiring low storage space, thereby making the formulation economic, cost-effective and user friendly.
6. A method to obtain the formulation of claim 1, wherein the method comprises:
- isolating pure culture of the selected strain of Bacillus thuringiensis cells,
- cultivating the isolated cells in optimized fermentation conditions using optimized nutrient media to obtain fermented broth containing the B. thuringiensis viable cell count ranging from 5 × 108 to 1 × 1010 cfu/ml and more preferably at least 5 × 109 colony forming units (cfu/ml),
- harvesting the B. thuringiensis viable cells from the fermented broth under specific conditions to get a concentrate comprising the B. thuringiensis viable cells and water, wherein the concentrate comprises in the range from 1 × 1010 to 5 × 1012 cfu/g and more preferably 5 × 1011 cfu/g dry matter of the concentrate of B. thuringiensis viable cells,
- mixing the concentrate with a lyo-protectant solution 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,
- standardizing the dry powder formulation in suitable excipients/carrier to contain minimum 1×108 cfu/g to 5×1010 cfu/g, preferably 1×1010 cfu/g viable cell counts of the selected strain of B. thuringiensis,
- storing the standardized dry powder formulation at a temperature of -20?C.
7. The method as claimed in claim 6, 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).
8. The method as claimed in claim 7, wherein said carbon source is selected from dextrose, jaggery, sucrose, glycerol, soluble starches or any combination thereof.
9. The method as claimed in claim 7, wherein nitrogen source I is selected from fish meal, corn meal, soyabean meal, oatmeal or any combination thereof.
10. The method as claimed in claim 7, 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.
11. The method as claimed in claim 7, wherein nitrogen source III is selected from yeast extract, rice bran extract, barley extract, meat extract or any combination thereof.
12. The method as claimed in claim 7, wherein salt is selected from magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate or any combination thereof.
13. The method as claimed in claim 6, 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.
14. The method as claimed in claim 6, wherein the specific conditions for harvesting the B. thuringiensis viable cells are centrifugation at 8000rpm for 10 min at 4°C aseptically.
15. The method as claimed in claim 6, wherein lyoprotectant solution comprises carboxymethyl cellulose in the range of 0%-1.5%, trehalose in the range of 0.1%-5.5%, lactose in the range of 0.1%-12%, monosodium glutamate in the range of 0.1%-4.5%, skim milk in the range of 0.1%-9.4%, maltodextrin in the range of 0.1%-8.9%, ascorbic acid in the range of 0.1%-2.0%, glycerol in the range of 0.1%-3.1%, and polyethylene glycol in the range of 0.1%-1.9%.
16. The method as claimed in claim 15, wherein the lyoprotectant solution is prepared by
- hydrating carboxymethyl cellulose into water for 30 minutes to 60 minutes,
- adding trehalose, lactose, monosodium glutamate, maltodextrin, ascorbic acid, glycerol and polyethylene glycol to obtain a mixture,
- sterilizing the mixture by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain sterilized mixture and then cooling down the sterilized mixture to room temperature,
- preparing milk fraction by adding milk powder into water to form an uniform suspension and sterilizing by autoclaving at 121°C and 15 lbs pressure for 15 minutes to obtain sterilized milk fraction and then cooling down the sterilized milk fraction to room temperature,
- mixing the cooled sterilized mixture and the cooled sterilized milk fraction.
17. The method as claimed in claim 6, 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.
18. The method as claimed in claim 6, 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.
19. The method as claimed in claim 6, wherein the suitable excipient/carrier is selected from maltodextrin and dextrose.