DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to sustained release formulations comprising biological or bioactive agents or derivatives thereof of natural, synthetic or semi synthetic origin material along with suitable binders and/or one or more carriers or nutrient sources to achieve sustained release of the said biological or bioactive agents or their derivatives.

BACKGROUND AND PRIOR ART OF THE INVENTION:
Microorganisms along with other bioactive compounds are the most abundant of all biota in soil and are responsible for driving nutrient, organic matter cycling, soil fertility, soil restoration, plant health and ecosystem primary production.

Soil microorganisms play a crucial role in agriculture, be it bacteria, actinomycetes, fungi, algae or protozoa; each one has a definite role to play. Certain micro-organisms produce compounds such as auxins, gibberellins and antibiotics that ultimately promote plant growth, others have the ability to fix nitrogen; some can solubilize phosphate and mobilize potash in the soil. Additionally, there are few soil microbes which have potent insecticidal, anti-fungal, anti-protozoal, drought tolerant, heat and salt tolerant properties.

Micro-organisms are also reported to impart plants with enhanced immunity. Beneficial soil microorganisms include those that create symbiotic associations with plant roots (rhizobia, mycorrhizal fungi, actinomycetes, diazotrophic bacteria), promote nutrient mineralization and availability, produce plant growth hormones, and are antagonists of plant pests, parasites or diseases therefore serving as biocontrol agents. Many of these organisms are naturally present in the soil, although in some situations it may be beneficial to increase their populations by either inoculation or by applying various agricultural management techniques that enhance their abundance and activity.

Recent practices for using these microbes or any other biological or bioactive agent or their derivatives for agricultural benefits involve preparation of liquid or solid powdered formulations. These practices encounter the disadvantage of being used repeatedly on field to maintain the required concentration of these substances, as they tend to run off due to irrigation practices and variable soil textures. In case of microbes, many times, the soil is not nutritionally competent enough to support the growth of microbes that are being dosed in soil. Hence, an additional source of nutrition also needs to be provided. Further, accurate application of dosages cannot be achieved with either liquid or powdered formulations comprising any biological or active agent.

PCT Publication No.WO2010/119032 reports a controlled release system in the form of a tablet comprising at least one agrochemical compound and a polymer, lipid or wax, either alone or in mixtures, wherein the agrochemical compound is a nematicidally active biological organism which is a bacterium or a fungus.

However, the controlled release system reported therein demonstrated seeds to be encapsulated with hydroxypropyl methyl cellulose and pullulan. The formulation disclosed in the said PCT publication fails to demonstrate disintegration studies indicating its extended release time duration, therefore raising ambiguity as to the application of the said formulation.

Prior art literature reports the use of binders in sustained release formulations, however they do not provide formulations comprising micro-organisms having sustained release attributes.

PCT Publication No.WO2014/047793 reports a method for preparing slow release fertilizer with a coated surface. The resultant coating layer of the formulation comprises lignin, alkenyl succinic anhydride, stearic acid, polyethylene glycol (PEG)-2000 and kaolin.

In view of the urgent need in the art to provide an effective solution to encounter hazardous issues relating to persistent use of chemical fertilizers in agriculture, the present invention provides an environmentally safe agricultural formulation comprising microorganisms or bioactive agents that possesses sustained release attributes over a period of time as a result of the presence of biodegradable binders in the said formulation.

OBJECT OF THE INVENTION:
It is an object of the present invention to provide an environmentally safe agricultural formulation comprising biological or bioactive agents or their derivatives thereof of natural, synthetic or semi-synthetic origin material, such as beneficial soil micro-organisms having a sustained release profile for an extended duration in soil as well as in water.

It is another object of the present invention to provide a process for the preparing a sustained release formulation.

SUMMARY OF THE INVENTION:
In an aspect, the present invention provides sustained release formulations comprising biological or bioactive agents or derivatives thereof of natural, synthetic or semi synthetic origin and suitable binders in varying concentrations to achieve sustained release of the said biological or bioactive agents or their derivatives. The said sustained release formulation comprises the said biological or bioactive agents or derivatives thereof of natural, synthetic or semi synthetic origin and binder in a ratio ranging from about 20:1 to about 1:10, respectively.

Further, the present single unit sustained release formulation comprising micro-organisms as the biological agent, optionally comprises a nutrient source. The nutrient source accelerates the growth of microbes being delivered to soil/water for agricultural benefits. Accordingly, in an optional aspect, the single unit sustained release formulation comprises a combination consisting of at least one beneficial micro-organism or a consortium of micro-organisms; and a nutrient source in a ratio ranging from about 1:50 to about 50:1. The combination of micro-organisms and nutrient source is later subjected to compression with a binder in a ratio ranging from 20:1 to 1:10, respectively.

In another aspect, the present invention provides a single unit sustained release formulation comprising; compacting a combination of bacterium or a bacterial consortia, and a binder in a ratio ranging from about 20:1 to about 1:10, respectively, by subjecting the said combination to high pressure compression followed by subjecting to temperatures ranging from about 20°C to about 100°C to allow interaction of the binder molecules with each other, thereby resulting in a stable sustained release formulation.

The said sustained release formulation is having a disintegration rate ranging from 1 hour to 96 hours in water and 5 days to about two months in soil.

In yet another aspect, the present invention provides a sustained release formulation for use in agriculture for nourishing plants as well as soil and providing nutrients.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In the most preferred embodiment, the present invention provides sustained release formulations comprising biological or bioactive agent(s) or derivative(s) thereof of natural, synthetic or semi synthetic origin material and a suitable binder in varying concentrations to achieve sustained release of the bioactive agents.

Accordingly, the said sustained release formulation comprises the said biological or bioactive agents or derivatives thereof of natural, synthetic or semi synthetic origin and a binder in a ratio ranging from about 20:1 to about 1:10, respectively.

The bioactive agents that can be used in the formulation can be selected from chemically synthesized plant growth promoters or regulators (such as salicyclic acid, indole acetic acids, gibberllins), fertilizers, micro or macro nutrients in desired concentration.

The biological agent(s) or bioactive agent(s) of natural origin is selected from the group comprising micro-organisms, herbal extracts, enzymes, compounds extracted from microbial/herbal sources.

The bioactive agents of synthetic/semi-synthetic origin are selected from the group comprising of fertilizer, plant growth promoting or protecting chemicals, pesticides and weedicides.

The derivatives of bioactive agents is selected from the group comprising polymer, enzymes, primary or secondary metabolites, chelating agents or any other product produced by the biological or bioactives agents mentioned above.

The present invention provides a single unit sustained release formulation comprising at least one micro-organism or a consortium of micro-organisms and a binder, wherein the said formulation has a protracted release profile resulting in the release of beneficial micro-organisms and nutritional sources in agricultural fields over a period ranging from 1 to 96 hours in water and 5 days to about two months in soil. However, the release pattern can be changed by modifying the concentrations of each component mentioned in the formulation.

In a preferred embodiment, the present invention provides a sustained release formulation comprising at least one bacterium or a consortium of bacteria and a binder for agricultural purposes.

In accordance with the preferred embodiment, the present invention provides a single unit sustained release formulation comprising at least one bacterium, and a binder, wherein the said formulation is prepared by a process comprising compacting a combination of at least one beneficial micro-organism or a consortium of micro-organism and a binder in a ratio ranging from about 20:1 to about 1:10 respectively.

The bacteria and binder combination is then subjected to high pressure compression at 1 ton to 5 ton pressure followed by subjecting the said combination to temperatures ranging from about 20°C to about 100°C to allow interaction of the binder molecules resulting in a stable sustained release formulation having a disintegration rate ranging from 1 to 96 hours in water and 5 days to about two months in soil.

The micro-organism used in the sustained release formulation is selected from the group consisting of bacteria, actinomycetes, yeast, mould, parasitic cultures having plant growth promoting, anti-nematode, anti-bacterial, anti-fungal, antiviral or nutrient mobilization properties. More preferably, a bacterium or a consortium of bacteria is used in the sustained release formulation of the present invention.

In an embodiment, the present invention provides the single unit sustained release formulation to preferably comprise atleast one bacterium selected from the group comprising Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium. The said bacteria are formulated either alone or in a consortium.

The bacterial species used in the present invention were procured from the American Type Culture Collection (ATCC), viz. Bacillus pumilus ATCC 14884, Bacillus megaterium ATCC 9885, Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580.

The formulations of the present invention comprising a consortium of the bacteria, the bacteria were tested for compatibility. Accordingly, Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium were all determined to be compatible with each other. None of the micro-organisms inhibited the growth of the other.

Bacteria having plant growth promoting abilities selected from the group consisting of, but not limited to Bradyrhizobium sp , Bacillus sp, Pseudomonas sp, Klebsiella sp, Trichoderma sp, Herbaspirillum sp , Azospirillum sp, Azotobacter sp, Pantoea sp, Enterobacter sp, Escherichia sp, Grimontella sp, Klebsiella sp, Pantoea sp, Rahnella sp, Stenotrophomonas sp, Rhizobium sp, Herbaspirillum sp, Lactobacillussp, Rhodobacter sp and Citrobacter sp, is employed in the sustained release formulation of the present invention to satisfy specific requirements for a particular crop. The aforesaid strains of bacteria are procured from ATCC.

In an embodiment, the concentration of bacteria used in the present formulation is in the range of about 102cfu/gm (Colony Forming Unit per Gram) to about 1011cfu/gm. Alternatively, spores of the said bacteria are used in the present formulation.

The bacteria or the consortium employed in the present sustained release formulation is used as a dried powdered mixture. Drying of the bacteria is achieved by conventional techniques including spray drying, tray drying, vacuum drying, microwave-vacuum drying, freeze drying, super critical drying, or by natural air drying techniques.

In another preferred embodiment, the binder used in the formulation of the present invention is selected from the group comprising long chain fatty acids or their salts or their derivatives, clay, bentonite, gelatin, cellulose and their derivatives, simple or complex sugar of their derivatives and synthetic polymers such as polyvinylpyrrolidone or polyethylene glycol.

Effect of compression on bacteria when determined showed no reduction in counts before and after applying pressure for preparation of the present formulation, therefore, suggesting that a single bacterium as well as the consortia of bacteria can withstand pressure as high as 5 tons employed for preparing the formulation (Table 2). The viability testing of the bacteria after subjecting it to high temperatures showed negligible reduction in the cell count (Table 3).

In an optional embodiment, the present invention provides a bacteria or bacterial consortia along with a nutrient source, which is later compacted with a binder. Accordingly, the said sustained release formulation comprises a combination of at least one beneficial micro-organism or a consortium of micro-organisms and a source of nutrition in a ratio of about 1:50 to about 50:1, respectively.

The single unit sustained release formulation comprises at least one beneficial micro-organism or a consortium of micro-organism and a source of nutrition in a ratio ranging from about 1:50 to 50:1.

In another embodiment, the nutrients used in the present formulation are selected from simple and complex carbon sources such as starch, cellulose, hemicellulose, maltose, sucrose, glucose and natural and/or synthetic nitrogen sources such as salts of nitrate, yeast extract and peptones. Alternatively, complex sources of nutrients such as extracts plants of their derivatives can also be supplied. The nutrients used in the formulation support growth of microorganisms used in the formulation.

Additionally, the present formulation comprises a chemical fertilizer selected from the group consisting of but not limited to urea, DAP, ammonium sulphate, calcium ammonium nitrate, nitro-phosphates; micronutrients selected from the group consisting of but not limited to boron, copper, iron, manganese, molybdenum, zinc, chlorine, nickel; macronutrients selected from nitrogen, phosphorous, potassium and sulphur, natural or synthetic plant growth promoters selected from amino acids, growth promoting compounds, vitamins or even bio-attractants to meet differential requirements in agriculture can be introduced in the present formulation.

In order to impart rigidity to the solid dosage formulation prepared, differential pressure in the range of 1 ton to 5 ton was applied to the said formulations.

In order to determine robustness of the formulation, sustained release pattern was studied under different atmospheric conditions of pH, temperature and salinity. The formulation was observed to be giving similar sustained release even under extremes of pH, temperature and salinity in both soil and water as well.

In an another embodiment, sustained release formulation could exert positive effect on growth of variety of plant on different plant growth attributes such as root length, shoot length, height of plant, tiller formation, etc.

In yet another embodiment, the present invention provides a sustained release formulation prepared in varying sizes taking different quantities during preparation process.

In a further embodiment, the present invention provides a carrier selected from the group comprising soluble salts, soluble or insoluble nutrients and insoluble inert material like clay, bentonites, silicates, talc, sawdust etc.

The form in which the sustained release solid dosage formulation can be prepared as pellets, balls, granules, blocks, tablets, bricks, slabs, bullets and the like. The different formulations of the present sustained release formulation should not be construed to be limited to the aforesaid forms.
In another optional embodiment, embodiment, the present invention provides a process for the preparation of the sustained release formulation comprising;
(a) preparing a mixture of micro-organisms or bioactive agents with optional nutrients source in a ratio ranging from about 1: 50 to about 50:1;
(b) adding to the mixture obtained in step (a), a binder in a ratio ranging from about 20:1 to about 1:10 respectively, and compacting the mixture to form a formulation; and
(c) subjecting the combination to high temperatures to achieve the interaction of the binder molecules with each other.

Additionally, soil conditioners, chemical fertilizers, micro and macro nutrients, growth promoters and bio-attractants may be added to the present formulation as additives, prior to compression of the powdered mixture comprising the combination of micro-organisms and source of nutrition and the binder or the mixture of binders.

The formulation can be prepared using hydraulic press, tablet press, by casting or molding techniques with or without the application of pressure.

In yet another embodiment, the present invention provides a process wherein the formulation is exposed to temperature ranging from about 20°C to about 100°C resulting in extended sustained release in soil as well as water over protracted quantum of time.

The present sustained release formulation is applied direct to soil in the rhizosphere, additionally it may be used in hydroponics, in coco peat, vermiculite or any natural, synthetic or semi-natural growth medium.
In yet another preferred embodiment, the present invention provides a method of delivering the measured dose to the plants for foliar application by reducing the release time of the formulation. Accordingly, the present sustained release formulation with shortest release time can be sprayed across agricultural fields in a concentration ranging from 0.5 kg/ha to 5 kg/ha. The formulation can be applied or sowed into the rhizosphere vicinity of crops in the same proportion as mentioned above. Whereas while being used in hydroponics or aquaponics, it can be suspended in a suitable liquid medium for appropriate period of time.

Advantages of the invention:
• The application of the present formulation is less time consuming as well as less laborious.
• As the formulation optionally contains a source of nutrient, it accelerates the growth of microorganisms being supplemented in the formulation.
• The concept used in the formulations can also be extended to sustained release of any microbial or non-microbial components (such as auxins, gibberellic acid, growth promoters, chemical or biological fertilizers) which can positively impact soil health and crop yield.
• Controlled release of micro-organisms offer consistent supply of good bacteria for efficient performance irrespective of soil type and irrigation practices.
• Using the formulation of the present invention, measured dosages can either be applied to the soil or sprayed onto foliage.

Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1: Sustained release formulations prepared using different binders
Different binders belonging to long chain fatty acids or their salts/derivatives were tested for their stability in the preparation of the present sustained release formulation. Each of the binders mentioned in Table 1 were mixed with the powdered bacterial consortium in a ratio of 1:7 and the mixtures were compressed in a hydraulic press. The release pattern and duration of the said sustained release formulation was thus determined.

Table 1: Evaluation of different binders for preparation of sustained release formulations
Binders used Binding efficiency Release Pattern Duration of Disintegration of formulation in 1L water (hrs)
Stearic acid Very good binding property Sustain release achieved 6
Palmitic acid Good binding property Early disintegration of formulation. 1
Glycerol Mono stearate Good binding property Sustain release achieved 4
Lauric acid No rigid binding. Early disintegration of formulation. 1
Myristic acid No rigid binding. Early disintegration of formulation 1
The presence of stearic acid in the present formulation resulted in maximum duration of sustained release compared to other binders. Additionally, the formulation so prepared has a smoother surface and was found to be scalable in a larger facility such as a tablet press or rotary press. Glycerol monostearate also resulted in moderate duration of sustained release. In view of the observations noted in table 1, stearic acid was considered to be the most preferable binder to be used in the preparation of the present sustained release formulation.

Example 2: Effect of pressure on rigidity of the formulations
Differential pressure was applied for determining the rigidity imparted to the solid dosage formulation prepared comprising dry powder. The values of pressure ranged from 0.5 to 5 tons. The extent of solubility of the formulations was determined by suspending the solid dosage in water as well as soil and observing the time for its complete disintegration. The Table 2 shows rate of disintegration of formulations compressed using different pressure.
Table 2: Rate of disintegration of formulation compressed using differential pressure
Pressure value (Tons) Time taken for complete disintegration in water Duration of complete disintegration in soil (Days)
0.5 1 hours 6
1 2 hours 7
2 2 hours 8
3 2 hours 10
4 2 hours 10
5 2 hours 10

It was observed that a pressure of 1-5 tons can be used for imparting the required rigidity to the prepared formulations, 1 ton being the minimum value to impart rigidity to the present solid dosage formulation.

Example 3: Compatibility testing of bacteria to be used in consortium
Gram positive spore formers such as Bacillus pumilus ATCC 14884, Bacillus megaterium ATCC 9885, Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 were used in the formulations of the present invention. The compatibility of bacteria with each other was determined by preparing suspension of each bacterium in the exponential growth phase. In a nutrient agar plate, the four bacterial suspensions were streaked with a cotton swab in such a way that each streak line crosses the other three either vertically or horizontally. Nutrient agar plate was incubated for 48-72 hours and was observed for growth inhibition of one culture with the other. It was observed that none of the cultures inhibited growth of other cultures or were inhibited by the presence of other bacteria. Hence, the four bacterial cultures tested were identified to be compatible with each other and were suitable to be incorporated in a consortium.

Example 4: Effect of compression on bacteria
Each bacterium and the consortium of said bacteria used in the formulation were evaluated for their robustness under high pressure used for compression. The stability of bacteria at 5 ton pressure was determined by counting the number of bacteria in terms of CFU/gm before application of pressure (i.e. when it is in the powdered form) and after application of pressure (i.e. compressed in formulations). The study was conducted individually for each culture and for the consortium as well.

Table 3: Population of bacteria before and after applying pressure
Culture/consortium name CFU/gm of bacteria in powder (without pressure application) CFU/gm of bacteria in solid formulation (after application of pressure)
Bacillus pumilus 2 x 105 1.8 x 105
Bacillus subtilis 3.1 x 105 3.0 x 105
Bacillus licheniformis 1.7 x105 2.2 x 105
Bacillus megaterium 2.6 x 105 1 x105
Consortium of all above mentioned bacteria 8.7 x 105 5.4 x105

It was observed that there is no log reduction in counts before and after applying pressure of 1 ton for preparation of the formulation; thereby suggesting that individual bacteria and consortia can withstand pressure as high as 5 tonnes used for making the formulation.

Example 5: Determination of optimum conditions for interaction of binder molecules in sustained release formulation
The present inventors aimed to increase the duration of complete disintegration of the formulation by inducing interaction of stearic acid. Stearic acid molecules have a property to interact with each other at a temperature of 70°C. The pattern and degree of interaction at different durations was studied by subjecting the present formulation comprising the binder and microbial consortium in a ratio of 1:9 by exposing to the temperature of 70°C for varying durations. The present formulation was cooled to room temperature after exposure.
Table 4: Evaluation of formulation after exposure 70°C for different durations
Duration of exposure to 70°C (minutes) Binding property Observations after heat exposure to the formulation
1 unsatisfactory No complete matrix formation observed when formulation was diagonally broken in two pieces.
2 satisfactory
4 satisfactory
6 satisfactory
8 satisfactory
10 satisfactory Complete Matrix formation observed without de-structuring of formulation
12 unsatisfactory Melting of the binder, de-structuring of formulation & sticking of the components to container pan
14 unsatisfactory
16 unsatisfactory
It was observed from the aforesaid Table that exposure of formulation for 10 minutes resulted in efficient polymerization; this duration was considered to be optimum for complete and even interaction resulting in interaction of the binder molecules within the formulation.

Example 6: Preparation of the sustained release formulation
Formulation 1: Spray dried or tray dried or air dried or freeze dried powder of bacteria with plant growth promoting activity was enriched with nutrient sources such as glucose, bacteriological peptone, soya peptone, tryptone, yeast extract etc. in 1:1 proportions. This mixture comprised an inert filler such as clay or benotinte in a ratio of 9:1 and was compressed in a hydraulic press to form a pellet. The formulation so prepared was evaluated for its sustained release potential for direct application in soil as well as application in water.

Formulation 2: Spray dried powder of bacteria having plant growth promoting activity was enriched with nutrient sources such as glucose, bacteriological peptone, soya peptone, tryptone, yeast extract etc. in in 1:1 proportions. This mixture was mixed with a binder (stearic acid) in a ratio of 9:1. This mixture was compressed using hydraulic press to form a pellet. The formulation so prepared was evaluated for its sustained release potential for direct application in soil as well as application in water.

Formulation 3: Spray dried powder of bacteria having plant growth promoting activity was enriched with nutrient sources such as glucose, bacteriological peptone, soya peptone, tryptone, yeast extract etc. in 1:1 proportions, respectively. To this mixture, binder (stearic acid) was added in a ratio of 9:1. This blended formulation mixture was compressed using hydraulic press at 1 ton pressure to form a pellet. This compacted pellet was kept in an oven at 70°C for 10 minutes to allow physical changes in the binder molecules. The formulation so prepared was evaluated for its sustained release potential for direct application in soil as well as application in water.

Formulation 4: Spray dried powder of bacteria having plant growth promoting activity was mixed with inert carrier; bentonate in 1:1 proportion. To this mixture, binder (stearic acid) was added in a ratio of 9:1. This mixture was compressed using hydraulic press at 1 ton pressure to form a pellet. This compacted pellet was kept in an oven at 70°C for 10 minutes to allow physical changes in the binder molecules. The formulation so prepared was evaluated for its sustained release potential for direct application in soil as well as application in water.

Formulation 5: Salicyclic acid and its salts are most commonly referred as plant growth enhancer used in agricultures. Chemically synthesized salt of salicyclic acid was used in the formulation. Sodium salt of salicyclic acid was mixed with dextrose and this mixture containing final concentration of 500 mg/kg of sodium salt of salicyclic acid was mixed with binder stearic acid in 9:1 proportion. This mixture was compressed using hydraulic press at 1 ton pressure to form a pellet. This compacted pellet was kept in an oven at 70°C for 10 minutes to allow physical changes in the binder molecules. The formulation so prepared was evaluated for its sustained release potential for direct application in water as well as in soil.
Though sodium salt of salicyclic acid is used as bioactive compound in the above formulation it can be replaced by any other bioactive compound or their derivatives thereof of natural, synthetic or semi synthetic origin material and their concentration in the formulation can be changed as per requirement.

Example 7: Evaluation of sustained release potential of the formulations in Example 6
Example 7(a): Sustained release of the present formulation in water
Individual formulation was wrapped in a metal mesh and tied with a string and weighed (W1). This assembly was dipped in a stationary flask containing water keeping one end of the string outside the beaker. The assembly was taken out of the water at definite time intervals, dried in an vacuum oven at 600C till it attains constant weight and weighed (W2) to obtain the quantity of formulation remaining after its partial disintegration. The above step was repeated at regular intervals until complete disintegration of the formulation in water was observed. The percentage (%) disintegration of the formulation in respective time was calculated using the formula given below:
% disintegration of the formulation = 100 – [(W1-W2)/W1]
Results: Formulations 1, 2 and 3 were prepared and evaluated for its sustained release mechanism in water, it was observed that the duration of complete disintegration of formulations 2 was completely dissolved within 2 hours whereas formulation 3, 4 and 5(in Example 6) took 96 hours for complete disintegration. However, the control formulation 1 was completely dissolved within 40-50 minutes.

Example 7(b): Sustained release of the present formulation in soil
Each compacted formulation was wrapped in a metal mesh and tied with a string and weighed (W1). This assembly was buried 4-5 cm below in soil in a pot, keeping one end of the string outside the soil pot. The pot was watered daily with definite amount of water. After regular intervals, the formulation was taken out of the soil pot and cleaned to get rid of adhering soil. The assembly was dried in vacuum oven at 600C till it attains constant weight and weighed (W2) to determine quantity of formulation after its partial disintegration. The above step was repeated at regular intervals until complete disintegration of the formulation was obtained. Percentage disintegration was calculated using the formula mentioned in example 7(a).
Results: Formulations 1 and 2 and 3 were prepared and studied for their sustained release in soil, it was observed that the duration of complete disintegration of Formulation 2 was 8 days whereas formulation 3, 4 and 5 took 45 days for complete disintegration. However, the control formulation prepared in formulation 1 was completely disintegrated within 10-20 hours.

Example 8: Preparation of formulation comprising varying ratios of binder and bacterial consortium
The binder selected in Example 1 i.e. stearic acid was added with different proportions of powdered bacterial consortia. The release pattern of each formulation was studied to determine the duration of sustained release. The said formulation was prepared by employing varying ratios of binder to microbial consortia, i.e. 1:1, 1:2, 1:7, 1:8 and 1:9 with binder. Each mixture was compressed in a hydraulic press at 1 ton pressure to make a pellet and the compacted pellet was subjected to heat treatment at 70°C for 10 minutes. The formulations were dispensed in 1L water for 20 days to study the disintegration.

Table 5: Evaluation of sustained release potential of the present sustained release formulation comprising binder together with varying concentrations of microbial consorita
Duration of release percentage disintegration of the formulation in water
1:1 1:2 1:6 1:7 1:8 1:9
0 day 0 0 0 0 0 0
1 day 0 0 0 10 15 25
2 day 0 0 10 20 30 50
3 day 0 0 20 30 45 75
4 day 0 0 30 35 60 100
5 day 0 0 35 40 75 -
6 day 0 0 40 45 80 -
7 day 0 0 45 50 85 -
8 day 0 0 50 55 90 -
9 day 0 0 55 60 95 -
10 day 0 0 60 65 100 -
11 day 0 0 65 70 - -
12 day 0 0 70 75 - -
13 day 0 0 75 80 - -
14 day 0 0 80 85 - -
15 day 0 0 85 90 - -
16 day 0 0 90 95 - -
17 day 0 0 95 100 - -
18 day 0 0 100 - - -
19 day 0 0 - - - -
20 day 0 0 - - - -

Formulations comprising the binder and microbial consortia in a ratio of 1:1 and 1:2 show no disintegration of the formulation due to the binder being at a higher or near equal concentration of the bacterial consortia, thereby resulting in an extremely rigid formulation. The formulation comprising the binder and the bacterial consortia in 1:7 and 1:6 ratios, showed moderate early disintegration for a duration of 17 and 18 days, respectively. The ratio of 1:9 resulted in complete disintegration and subsequent discharge of active ingredients in the formulation by a duration of 5 days. Even though a sustained release formulation comprising the binder and microbial consortia in a ratio of 1:9 was employed in the present invention, the ratio can be changed accordingly to achieve complete disintegration.

Example 9: Effect of temperature on viability of bacterial cultures
Referring to Example 5, the temperature of 70°C is used for one of the formulations. The effect of this temperature on the viability of bacterial cultures used in the consortium was evaluated by exposing the dry powder of bacterial consortium to 70°C for a maximum of 20 minutes. Count of the consortium before and after heat treatment was carried out and documented in table below.

Table 6: Count of consortium before and after heat treatment
Count of consortium (CFU/gm)
Before heat treatment 3.4 x 107
After heat treatment 2.8 x 107
There was no reduction in the viable count of dry powdered bacterial consortium after heat treatment suggesting that the consortium is stable at temperatures as high as 70°C. Hence, method of preparation of Formulation 3 and 4 in Example 6 neither affects the microbial viability nor the performance of the bacterial cultures.

Example 10: Study of sustained release of active ingredients in formulations
a) Sustained release of microbial consortium:
Formulation 2 cited in Example 6 was selected to study the release pattern of the bacterial consortium comprised therein. The formulation was tied in a wired mesh and suspended in water. The tied formulation was transferred to another container having same volumes of water after every 24 hours until its complete disintegration was achieved. Microbial count of the leachate (water where the Formulation was suspended) was conducted after every transfer.
Table 7: Microbial count of the leachate
Duration of leaching Count
0 hours 0
24 hours 2.5 x106 CFU/ml of leachate
48 hours 8 x105 CFU/ml of leachate
72 hours 6.7 x 105 CFU/ml of leachate
96 hours 7.5 x 105 CFU/ml of leachate

It was observed that there is a consistent count of 105 CFU/ml in the leachate sample from 24 hours till its complete disintegration.

b) Sustained release of nutrient (eg. Glucose):
Formulation 2 cited in Example 6 was selected to study the release pattern of one of the source of nutrients in the formulation i.e. dextrose. The formulation was tied in a wired mesh and suspended in water. The tied formulation was transferred to another container having same volumes of water after every 24 hours until there is complete disintegration of the formulation. The estimation of dextrose was done as per DNSA method of sugar estimation. The values of sugar concentration in the leachate have been noted in the table below.
Table 8:
Duration of leaching Concentration of sugar in leachate
0 hours 0
24 hours 60 mg/ml of leachate
48 hours 40 mg /ml of leachate
72 hours 35 mg/ml of leachate
96 hours 42 mg /ml of leachate

It was observed that approximately 40 mg sugar per ml of leachate is being released consistently from 24 hours till its complete disintegration

b) Sustained release of active ingredient sodium salicylate
Formulation 5 was subjected to study the release pattern of the bioactive agent i.e. sodium salt of salicyclic acid therein. The formulation was tied in a wired mesh and suspended in water. The tied formulation was periodically transferred to another container having same volumes of water after every 24 hours until its complete disintegration was achieved. Spectrophotometric estimation of sodium salt of salicylic acid using Fe (III) method was conducted after every transfer as per the method described below.
Table 9: Sodium Salicylate content of the leachate
Duration of leaching Salicyclic acid per ml of leachate
0 hours 0
24 hours 179 mg/L of leachate
48 hours 162 mg/L of leachate
72 hours 150 mg/L of leachate
96 hours 152 mg/L of leachate

It was observed that around 150mg salicyclic acid per Litre of leachate was being released consistently from the formulation from 24 hours till its complete disintegration.

Example 11: Effect of nutrients (eg: glucose) on bacterial consortia in the present formulation
Formulation to be used in the experiment was prepared by enriching spray dried powder of bacteria having plant growth promoting activity with nutrient sources such as bacteriological peptone, soya peptone, tryptone, yeast extract etc. in 1:1 proportions. This nutrient and bacterial mixture was mixed with binder stearic acid in a ratio of 9:1 respectively. The said mixture was compressed using a hydraulic press at 1 ton pressure. The resultant solid formulation was subjected to high temperature at 70°C for 10 minutes to allow physical changes in the binder molecules. A control pellet (formulation 4) was also prepared by replacing the nutrient source with an inert material while keeping rest of the method of preparation unaltered.

To demonstrate the effect of nutrients on the growth of bacterial consortia present in the formulation, a pellet comprising the present formulation (with nutrients), and another pellet (without a nutrient source); each weighing 5 gms were buried 4-5 cm below soil in two different pots of same size. The sterilized soil was used for this purpose which was devoid of any microbes. . Both the sets were watered on a daily basis to facilitate the release of the formulation. A portion of 1 gm soil was withdrawn after an interval of 7 days from each set and a total viable count was performed.
Table 10: Viable count of bacteria indicating effect of presence of nutrients
Duration of sampling
Total viable count (CFU/gm of soil)
Experimental set (Formulation with presence of nutrients
Control set
(Formulation with absence of nutrients)
0 day 0 0
7 days 7.5 X 104 3.94 X 104
14 days 3 X 105 2.5 X 104
21 days 6 X 106 9 X 104
28 days 3.1X 107 1.1 X 104
45 days 5.2X 106 6.73 X 103

During the first 7 days after application of the formulation, there is an increase in the total viable bacterial count using the pellet comprising nutrients. However, after an interval of 15 days, there is a slight increase in the bacterial population and it remains maintained until the complete disintegration which took approximately 45 days.

In case of the control experiment, where no nutrient source was supplied in the formulation, the total viable count was found to decline with time. The complete disintergration of the pellet was achieved at around 28-45 days and the total viable count at that time was reduced till 103 CFU/gm. The observation in the experiment justifies the use of nutrients added in the formulation along with the bacterial consortium which accelerates the growth; however, the growth of microbes can take place without adding nutrients in the formulation, as it relies on the nutrients present in soil. Similar advantages were observed when the present formulation is used in hydroponics.

Example 12: Sustained release of formulations at varying soil pH
The pH of agricultural soil is usually neutral but is often found to be in the range of 5 to 9. The application of the present formulation in diverse soil types was analysed to determine disintegration patterns of formulation at different values of pH in soil. The formulation was prepared as per the method in Example 11. Quantity of the dissolved formulation was determined by retrieving the formulation after every 7 days and its dry weight was calculated.
percentage reduction was calculated by the formula:
% of formulation remained = [(A-B)/A] x100
where A: Initial dry weight of the formulation
B: Dry weight of the formulation retrieved after definite interval

Table 11: Release/disintegration pattern of formulation in soil at different values of pH
Duration
(in days)
Percentage of formulation remained in soil
pH 5 pH 6 pH 7 pH 8 pH 9
0 100 100 100 100 100
7 85 70 73 75 75
14 60 62 60 50 53
21 40 45 40 40 40
28 10 12 10 5 5
45 0 0 0 0 0

There is a slightly early disintegration of the formulation at pH values higher than neutral. However, there is no significant difference in overall time required for complete disappearance of the pellet in the soil due to disintegration. Hence, the present formulation can be applied to diverse agricultural soils.

Example 13: Sustained release of formulations at different salt concentration
Average salinity of agricultural soil ranges from 0 to 3 gm/lit. However, in temperate regions, the salinity of the soil increases as an effect of evaporation of soil moisture. The soil salinity is also found to increase due to irrational use of chemical fertilizers as well. This experiment was performed to determine the efficiency of sustained release formulation at different values of salt concentration in soil. Generally, salinity is contributed by the presence of Na+, K+, Ca2+, Mg2+ and Cl- Ions. To simulate such conditions, 3.5 % solution of commercially available sea salt containing NaCl, CaCl2, CaSO4 and Na2SO4 was used in these experiments. Formulation was prepared by the method as mentioned in Example 11 and percentage formulation remaining in salt water was calculated using the formula mentioned in Example 12.

Table 12: Disintegration pattern of the formulation in normal and saline water
duration (in days) percentage of formulation remaining in water.
Normal tap water Saline water
1 100 100
2 75 85
3 50 60
4 20 35
5 0 5
It can be concluded from the data that at higher values of salinity, the release pattern of the formulation is slowed down by approximately 20%. However, complete disintegration can be achieved irrespective of the salinity of the soil.

Example 14: Sustained release of the formulations at varying temperatures
The soil temperatures vary throughout the year owing to different weather conditions in different seasons. In this experiment, the release pattern of the best formulation as an effect of different temperatures was studied. To do so, the formulation was prepared by the method described in Example 11 and it was applied to soil as well as added to water. One set of each was kept in incubators at different temperatures such as 25°C, 30°C and 35°C. Disintegration pattern of the formulation in each set was studied and observations are noted in the table below:

Table 13: Sustained release of formulation in water at different temperatures
Temperature of water percentage of formulation remaining in water
25°C 30 °C 35°C
Days
0 day 100 100 100
1 day 75 70 62
2 day 50 50 48
3 day 20 25 30
4 day 0 0 15
5 day 0 0 3

Table 14: Sustained release of formulation in soil at different temperatures
percentage of formulation remaining in soil
Temperature of pot soil 25°C 30°C 35 °C
0 day 100 100 100
7 days 85 75 75
14 days 70 65 62
21 days 45 45 35
28 days 10 12 5
45 days 0 0 0

The aforesaid data indicates that the temperature does not impose any adverse effect on disintegration of the formulation in water. While in soil, there is a slight delay in release of the formulation as an effect of low temperature values. However, it does not seem to affect the total disintegration time required for complete disintegration of the formulation.

Example 15: Demonstration of benefits of bacteria used in the consortium
The bacteria used in the consortium were intended to be beneficial to plants in terms of increasing yield as well as in improvement of the quality of agricultural produce. In vitro plant growth promotion potential of the cultures used in the formulation was assessed in the laboratory studies.

Table 15: Study of plant growth promoting potential of the cultures used in the formulation
Bacterial
culture Nitrogen Fixation Phosphate solubilization Potash solubilization Zinc solubilization Siderophore production Thiosulphate reduction Indole acetic acid production
B. licheniformis + - - - - + +
B. megaterium - - - - - + +
B. subtilis - + + - - + -
B. pumilis - - - - - + -
(‘+’ denotes to presence and ‘-‘ denotes absence of plant growth promoting potential)

The table depicts that each bacterium qualifies at least one of the plant growth promoting attributes. Synergism is expected to occur after application of all these bacteria onto soil. The observations in the table also justify the need to using consortium over single bacterium.

Example 16: Pot experiment studies to demonstrate beneficial effect of sustained release formulation
The efficiency of the present sustained release formulation was demonstrated through pot experiments by employing three different plant families; monocotyledons such as wheat, dicotyledons such as Bengal gram and vegetables such as okra (Abelmoschus esculentus). The experiments were performed in a set-up, wherein 10 seeds of each of the plants were sown in soil measuring 1 cubic feet of volume. One set of potted plants, i.e. Set C was treated with 5 gm of the present sustained release pellet which was sown at around 5 cm deep from the surface.

In another set i.e. Set B, 2.25 gm of powdered bacterial consortium was sprinkled onto the soil at the start of experiments; while another set of untreated potted plants, i.e. Set A was referred to as the control. The pots were sown with seeds and watered on alternate days with equal quantities of water.

Different growth attributes such as height of the plants, leaves per branches, internodal distance and number of root hair were amongst the varying growth attributes evaluated during the study. All experiments were conducted at least six times and values mentioned in each table represent the mean of all six values.

In the results enlisted in the tables below, “A” represents the control set, “B” represents a set where powdered bacterial consortium was sprinkled while “C” represents a set where the present sustained release formulation was used.

Table 16: Plant growth attributes for Bengal gram (dicotyledons)
Duration of growth Set Height (mm) Number of branches Leaf per branch Internode Dist (mm) Root hairs branches
7 Days A 74 3 6 19 NA
B 98 4 8 21 NA
C 113 4 8 23 NA

15 Days A 139 8 12 23 NA
B 147 9 12 25 NA
C 169 9 12 26 NA

21 days A 178 11 12 23 NA
B 206 11 13 26 NA
C 207 12 13 30 NA

28 Days A 203 11 12 23 6
B 215 11 14 26 12
C 240 12 16 33 25

Table 17: Plant growth attributes for wheat (monocotyledon) after 28 days
Set Height (mm) Leaf per Plant Root hairs branches per plant Leaf Weight (Gms) Root weight (Gms)
A 250 3 6 8.25 7.2
B 280 3 12 8.5 7.9
C 320 5 25 11.8 9.45

Table 18: Plant growth attributes for Okra after 30 days
Set Height (mm) Leaf per Plants Root length (mm) Root hairs branches per plant
A 150 3 32 8
B 182 3 45 17
C 213 4 83 39

It is imperative from the data presented in the tables that use of sustained release formulation exerts beneficial effect on different attributes captured during the growth periods than only using bacterial consortium. Hence, use of sustained release formulation may exert its beneficial effect in fields as well; thus increasing the total yield of the crop.
,CLAIMS:1. A single unit sustained release formulation comprising biological or bioactive agent(s) and binder.

2. The single unit sustained release formulation as claimed in claim 1, wherein, the biological agent(s) or bioactive agent(s) is selected from the group comprising micro-organisms, chemically synthesized fertilizer, plant growth promoting/protecting substances, pesticides, weedicides and micronutrients.

3. The single unit sustained release formulation as claimed in claims 1 and 2, wherein the biological agent(s) or bioactive agent(s) and a binder is in a ratio ranging from about 20:1 to about 1:10.

4. The single unit sustained release formulation as claimed in claim 3, wherein, the said biological agent is a bacterium or a bacterial consortium.

5. The single unit sustained release formulation as claimed in claim 3, wherein, the said bioactive agent is salicyclic acid.

6. The single unit sustained release formulation as claimed in claim 4, wherein the bacteria are selected from the group of Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.

7. The single unit sustained release formulation as claimed in claim 6, wherein the concentration of bacteria is ranging from about 102cfu/gm to about 1011cfu/gm.

8. The single unit sustained release formulation as claimed in claim 1, wherein the binder is selected from the group comprising long chain fatty acids or their salts or their derivatives, gelatin, cellulose and their derivatives, simple or complex carbohydrates or their derivatives and synthetic polymers such as polyvinylpyrrolidone or polyethylene glycol.

9. The single unit sustained release formulation as claimed in claim 8, wherein the long chain fatty acids is preferably selected from the group comprising stearic acid, palmitic acid, glycerol mono stearate, lauric acid and myristic acid.

10. The single unit sustained release formulation as claimed in claim 1, wherein the formulation optionally comprises a nutrient source selected from the group comprising starch, cellulose, hemicellulose, maltose, sucrose, glucose and their derivatives natural and/or synthetic nitrogen sources such as salts of nitrate, yeast extract and soya or plant derived peptones or proteins.

11. The single unit sustained release formulation as claimed in claim 10, wherein the ratio of bacteria to the source of nutrition is in the range of 1:50 to 50:1, respectively.

12. The single unit sustained release formulation as claimed in claim 1, wherein carrier is further selected from the group comprising soluble salts, soluble or insoluble nutrients and insoluble inert material like clay, bentonites, silicates, talc and sawdust.

13. The single unit sustained release formulation as claimed in claim 1, wherein the said formulation is in various geometric forms selected from the group comprising pellets, balls, granules, blocks, tablets, bricks, slabs, and bullets.

14. A process for the preparing a single unit sustained release formulation as claimed in claim 1, wherein the said process comprising compacting a combination of at least one biological or bioactive agent and a binder at 1 ton pressure followed by subjecting the compacted form to temperature ranging from 20°C to 100°C to allow interaction of the binder molecules resulting in the said sustained release formulation.

15. The process as claimed in claim 14, wherein at least one bacterium or a bacterial consortium and a binder is in a ratio ranging from about 20:1 to 1:10.