A biological agrochemical composition and method of synthesis thereof

Abstract

A biological agrochemical composition and method of synthesis thereof, are disclosed. Said biological agrochemical composition broadly comprises: a filtrate of Lactobacillus acidophilus; a filtrate of Aspergillus niger; neem oil; and polyoxyethylene (20) sorbitan monolaurate. The disclosed biological agrochemical composition offers at least the following synergistic advantages and effects: is efficient; is non-toxic; is environment- friendly; improves soil fertility; and / or is effective against a broad range of weeds.

Description

[0001] TITLE OF THE INVENTION: A BIOLOGICAL AGROCHEMICAL

[0002] COMPOSITION AND METHOD OF SYNTHESIS THEREOF

[0003] FIELD OF THE INVENTION

[0004] The present disclosure is generally related to agrochemical compositions. The present disclosure is particularly related to biological agrochemical compositions. The present disclosure is more particularly related to a biological agrochemical composition; and method of synthesis thereof.

[0005] BACKGROUND OF THE INVENTION

[0006] Agrochemical compositions (for example, chemical pesticides) are known in the art. However, they suffer from various drawbacks, including, but not limited to: result in residue accumulation that leads to pollution; exhibit non-selectivity; cause detrimental effects, on non-target plants and organisms; pose toxicity risks to human beings; and / or adversely impact soil surface and / or soil fertility. Further, in the domain of agrochemical compositions, the quest for improvements is never ending.

[0007] There is, therefore, a need in the art, for: a biological agrochemical composition; and method of synthesis thereof, which overcomes the aforementioned drawbacks and shortcomings.

[0008] SUMMARY OF THE INVENTION

[0009] A biological agrochemical composition, and method of synthesis thereof, are disclosed. Said biological agrochemical composition broadly comprises: a filtrate of Lactobacillus acidophilus', a filtrate of Aspergillus niger, neem oil; and polyoxyethylene (20) sorbitan monolaurate.

[0010] Said filtrate of Lactobacillus acidophilus and said filtrate of Aspergillus niger are in a ratio of about 1: 1. Said filtrate of Lactobacillus acidophilus and said neem oil are in a ratio of about 1:1. Said filtrate of Lactobacillus acidophilus and said polyoxyethylene (20) sorbitan monolaurate are in a ratio of about 3: 1. The disclosed biological agrochemical composition offers at least the following synergistic advantages and effects: is efficient; is non-toxic; is environment-friendly; improves soil fertility; and / or is effective against a broad range of weeds.

[0011] BRIEF DECRIPTION OF THE DRAWINGS

[0012] Figure 1, Figure 2, and Figure 3 illustrate results of trials conducted on different types of weeds with about 50 mL / L of a synthesised biological agrochemical composition, at different time intervals, in accordance with an embodiment of the present disclosure;

[0013] Figure 4 and Figure 5 illustrate results of trials conducted at a test estate, with a dosage of about 25 mL / L and about 50 mL / L, respectively, of a synthesised biological agrochemical composition, in accordance with an embodiment of the present disclosure;

[0014] Figure 6 illustrates results of trials conducted at another test estate, with a dosage of about 50 mL / L, of a synthesised biological agrochemical composition, in accordance with an embodiment of the present disclosure;

[0015] Figure 7 illustrates results of trials conducted at another test estate, with a dosage of about 10 mL / L of a control herbicide composition, in accordance with an embodiment of the present disclosure; and

[0016] Figure 8 illustrates results of trials conducted at yet another test estate, with a dosage of about 50 mL / L of a synthesised biological agrochemical composition, in accordance with an embodiment of the present disclosure.

[0017] DETAILED DESCRIPTION OF THE INVENTION

[0018] Throughout this specification, the use of the words “comprise” and “include”, and variations, such as “comprises”, “comprising”, “includes”, and “including”, may imply the inclusion of an element (or elements) not specifically recited. Further, the disclosed embodiments may be embodied, in various other forms, as well.

[0019] Throughout this specification, the use of the phrase “biological agrochemical”, and its variations, is to be construed as “an agricultural chemical product, where the active constituent comprises, or is derived from a living organism”. Throughout this specification, the use of the phrase “biological agrochemical”, and its variations, is to be construed as being inclusive of: herbicides and / or weedicides.

[0020] Throughout this specification, the use of the word “samples” is to be construed as: “soil samples; weed samples; and / or the like”, as the case may be.

[0021] Throughout this specification, the use of the acronym “BLQ”, is to be construed as: "below the limit of quantification."

[0022] Throughout this specification, the use of the acronym “RPM” is to be construed as: “Revolutions Per Minute”.

[0023] Throughout this specification, the use of the phrase “Lactobacillus sp.” is to be construed as: “Lactobacillus acidophilus” .

[0024] Throughout this specification, the use of the phrase “Aspergillus sp.” is to be construed as: “Aspergillus niger”.

[0025] Throughout this specification, the disclosure of a range is to be construed as being inclusive of: the lower limit of the range; and the upper limit of the range.

[0026] Throughout this specification, the words “the” and “said” are used interchangeably.

[0027] Also, it is to be noted that embodiments may be described as a method. Although the operations, in a method, are described as a sequential process, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A method may be terminated, when its operations are completed, but may also have additional steps.

[0028] A biological agrochemical composition (hereafter, also referred as “composition”), and a method of synthesis thereof, are disclosed. In an embodiment of the present disclosure, the biological agrochemical composition broadly comprises:

[0029] A biological agrochemical composition and method of synthesis, are disclosed. Said biological agrochemical composition broadly comprises: about 30 mL / L of a filtrate of Lactobacillus acidophilus', about 30 mL / L of a filtrate of Aspergillus niger, about 30 mL / L of neem oil; and about 10 mL / L polyoxyethylene (20) sorbitan monolaurate. Said filtrate of Lactobacillus acidophilus and said filtrate of Aspergillus niger are in a ratio of about 1: 1. Said filtrate of Lactobacillus acidophilus and said neem oil are in a ratio of about 1:1. Said filtrate of Lactobacillus acidophilus and said polyoxyethylene (20) sorbitan monolaurate are in a ratio of about 3: 1.

[0030] The disclosed method for synthesis of the biological agrochemical composition broadly comprises following steps.

[0031] Initially, the Lactobacillus sp. was grown on Luria Bertani (LB) broth at about 37°C for about 48 hours, whereas the Aspergillus sp. was grown on Potato Dextrose Broth at about 37°C, for about 4 days to about 5 days, to obtain respective starter cultures for fermentation.

[0032] Fermented media were prepared with ingredients that are provided in the table given below. A person skilled in the art will appreciate the fact that the growth media could be of any suitable type.

[0033] A person skilled in the art will appreciate the fact that the mineral salt may be of any suitable type known in the art. For example, the mineral salt may be: dipotassium phosphate; magnesium sulphate (or magnesium sulfate); and / or manganese (II) sulphate (or manganese (II) sulfate).

[0034] The sterilised fermentation media were supplemented with about 0.5% v / v of the respective starter culture in a fermenter and incubated at about 37°C for about 72 hours. After incubation, the fermented media were heated at about 70°C to kill live organisms and allowed to cool for about 24 hours, to obtain heat-killed fermented media. Then, the heat-killed fermented media were filtered using a membrane filter (for example, a size of about 0.2 microns) to remove physical contaminants and debris, to obtain a filtrate (liquid portion) of Lactobacillus sp. and a filtrate of Aspergillus sp. The filtrate of Lactobacillus sp. and the filtrate of Aspergillus sp. were finally blended together, in a ratio of about 1: 1, along with neem oil (about 30% v / v) and an adjuvant (about 10% v / v). The neem oil was procured from Ecobiocides and Botanicals Private Limited, Theni, Tamil Nadu, India.

[0035] In another embodiment of the present disclosure, the adjuvant is polyoxyethylene (20) sorbitan monolaurate.

[0036] The obtained biological agrochemical composition was stored at cool and dry condition for further use.

[0037] The disclosed biological agrochemical composition has been tested and results were explained as follows.

[0038] Soil samples from various fields in which the weeds were treated with the disclosed composition as well as the commercial herbicide were collected and analysed for soil nutrients (Nitrogen (N), Phosphorus (P), Potassium (K)), and Total Organic Carbon (TOC) using Kjeldahl method, Olsen method, Ammonium acetate method, and Walkley-Black method, respectively.

[0039] Comparative studies for herbicide residues (2,4-Dichlorophenoxyacetic acid or 2,4-D), glyphosate, oxyfluorfen, and paraquat) in soil samples and weed samples are detailed as follows.

[0040] Herbicide Residue Analyses in Soil Samples

[0041] For sample preparation, the soil samples were freeze-dried to remove moisture. After freeze-drying, said samples were ground to a fine powder to ensure uniformity. The prepared samples were then stored at about -20°C to preserve their integrity until further analyses. Sample Preparation

[0042] To prepare the soil sample for analyses, first the soil samples were air-dried at a temperature of about 25 °C. Once dried, the soil samples were sieved through a mesh (about 2 mm) to ensure uniform particle size. After sieving, the soil samples were homogenised thoroughly and stored at about 4°C for further analyses.

[0043] For determining moisture content, about 10 g soil samples were weighed before drying and after drying. Such measurements were utilised to calculate the moisture content and dry weight correction in subsequent analyses.

[0044] 2,4-D Analyses

[0045] Initially, about 20 mL of acidified acetone (about 0.1 % acetic acid) was added to about 10 g of soil samples to obtain a mixture. The mixture was then sonicated for about 15 minutes, followed by centrifugation at about 3,000 rpm for about 10 minutes, to obtain liquid extracts.

[0046] Subsequently, a C18 solid-phase extraction (SPE) cartridge was conditioned by passing about 5 mL of methanol, followed by about 5 mL of water and loading the samples onto the cartridge. Said cartridge was then washed with additional water (about 5 mL) to remove impurities, followed by eluting a target analyte (herbicide residue) with about 10 mL of methanol, to obtain eluted solutions. The eluted solutions were then concentrated under a nitrogen stream for further analyses.

[0047] For HPLC analyses, a C18 column (for example, about 250 x 4.6 mm, about 5 pm particle size) was used. The mobile phase consisted of a mixture of acetonitrile and water, in a ratio of about 60:40, with a flow rate of about 1.0 mL / min. About 20 pL of concentrated samples were injected into the HPLC system, followed by detection using UV light at a wavelength of about 280 nm, and total run time for the analyses was about 15 minutes.

[0048] Glyphosate Analyses

[0049] About 20 mL of about 0.6 M potassium hydroxide (KOH) was added to about 10 g of soil samples, to obtain a mixture. The mixture was shaken for about 2 hours to facilitate extraction of glyphosate, and centrifuged at about 4,000 rpm to separate s liquid extracts from solid residues.

[0050] Next, a derivatization step was carried out. The pH of the liquid extracts, from the previous step, was adjusted to about 9.0, and about 0.5 mL of FMOC-C1 (fluorenylmethyloxycarbonyl chloride) solution was added. The reaction was allowed to proceed for about 2 hours at room temperature, after which it was stopped by adding phosphoric acid.

[0051] For a clean-up phase, a strong anion exchange Solid-Phase Extraction (SPE) cartridge was utilised. The target analyte was eluted with about 0.1 M hydrochloric acid (HC1). Finally, eluted samples were analysed using High-Performance Liquid Chromatography with fluorescence detection (HPLC-FLD), by setting an excitation wavelength to about 266 nm and an emission wavelength of about 305 nm.

[0052] Oxyfluorfen Analyses

[0053] To analyse oxyfluorfen in soil samples, about 30 mL of acetonitrile was added to about 10 grams of soil samples, to obtain a mixture. The mixture was shaken for about 1 hour and centrifuged at about 3,500 rpm. This extraction step was repeated twice to ensure thorough extraction.

[0054] Following extraction, a clean-up phase was performed. The extracts obtained in the previous step were passed through a magnesium silicate cartridge, and the target analyte was eluted using a mixture of hexane and acetone, in a ratio of about 90: 10. The eluted solution was then concentrated to a final volume of about 1 mL.

[0055] Further, Gas Chromatography-Mass Spectrometry (GC-MS) analyses was performed using a GC-MS column. Chromatography was started at an initial temperature of about 60°C for about 2 minutes, followed by a ramping phase, where the temperature was increased at a rate of about 20°C per minute until about 280°C, which was held for about 10 minutes. Sample injection volumes of about 1 pL were used to perform the analyses. The Mass Spectrometry (MS) conditions were set with an ion source having temperature of about 230°C, a transfer line temperature of about 280°C, and a scan range from about 50 m / z to about 500 m / z. Paraquat Analyses

[0056] To analyse paraquat in soil samples, about 20 mL of about 0.5M sulphuric acid (H2SO4) was added to about 10 grams of soil samples, to obtain mixtures. The mixtures were shaken for about 1 hour and centrifuged at about 4,000 rpm. Resulting liquid extracts were filtered through a filter (about 0.45 pm) to remove any solid particles.

[0057] Following extraction, a clean-up phase was performed using strong cation exchange Solid-Phase Extraction (SPE). The SPE cartridge was conditioned with methanol followed by water and the target analyte (paraquat) was then eluted with an ammonium chloride solution.

[0058] For the final analysis, a High-Performance Liquid Chromatography (HPLC) was conducted. The mobile phase comprised of: about 0.1% formic acid in water; and acetonitrile. A gradient elution method was employed during the analysis, and detection of paraquat was carried out at a wavelength of about 257 nm.

[0059] Total Plate Count (TPC) Analyses

[0060] Sample Collection and Preparation

[0061] Field Sampling

[0062] Soil samples were taken from a depth of about 5 cm to about 15 cm, with multiple samples collected in a W-pattem across fields. The collected samples were transported in a cooler at about 4°C and processed within about 24 hours of collection.

[0063] Initial Processing

[0064] The samples were homogenised aseptically and visible debris and stones were removed. Then, the samples were passed through a sterile sieve (about 2 mm). If immediate analysis was not possible, the processed samples were stored at about 4°C. Serial Dilution Preparation

[0065] Initial Suspension

[0066] About 90 mL of sterile physiological saline (about 0.85% NaCl) was added to about 10 g of soil samples in a sterile flask, to obtain mixtures. The mixtures were shaken for about 10 minutes at about 150 rpm, then allowed to settle for about 2 minutes, resulting in about 101dilution.

[0067] Dilution Series

[0068] Sterile tubes were labelled for dilutions ranging from about 102to about 107. About 9 mm of sterile saline was added to each tube, and about 1 mL from the about 101dilution was transferred to the about 102tube (likewise, subsequent dilutions were carried out). Each tube was vortexed for about 10 seconds, and this process continued through to the 107dilution, ensuring that pipette tips were changed between dilutions, to avoid crosscontamination.

[0069] Plating Procedure

[0070] Media Preparation’.

[0071] Plate Count Agar (PCA) was prepared by suspending about 23.5 grams in about 1 L of distilled water, to obtain a mixture. The mixture was heated to dissolve completely and then autoclaved at about 121°C for about 15 minutes. After cooling to about 45-50°C to obtained cooled OCA, about 15-20 mL of the cooled PCA was poured onto each plate and allowed to solidify. The plates were stored inverted at about 4°C, and a quality control check of the media was included.

[0072] Sample Plating:

[0073] Plates were labelled with the dilution factor and sample ID. From each dilution, about 0.1 mL was pipetted onto the plates, and a sterile spreader was used to distribute the samples evenly. Duplicate plates were prepared for each dilution, and a blank plate was included as a negative control. Incubation

[0074] The plates were inverted and incubated at about 28°C for soil bacteria. They were checked after about 24 hours, with final counts taken at about 48 hours to about72 hours.

[0075] Colony Counting and Calculation

[0076] Plates with between about 30 colonies to about 300 colonies were selected for counting. All visible colonies were counted, using a colony counter, with each counted colony marked for reference.

[0077] Colony Forming Unit (CFU) was calculated using the following equation given below.

[0078] CFU / g = N x D x V where,

[0079] N = Number of colonies per plate

[0080] D = Dilution factor

[0081] V = Volume factor (about 10 for about O.lmL plating)

[0082] Results of TPC analyses were reported as CFU / g of soil, with the mean calculated from duplicate plates expressed in scientific notation along with the standard deviation.

[0083] Various weed samples that were treated with the disclosed composition as well as commercial herbicide were analysed for soil nutrients (Nitrogen (N), Phosphorus (P), Potassium (K)), and Total Organic Carbon (TOC), using Kjeldahl method, Olsen method, ammonium acetate method, and Walkley-Black method, respectively.

[0084] Herbicide Residue Analyses in Weed Samples

[0085] For sample preparations, weed samples were freeze-dried to remove moisture. After freeze-drying, said weed samples were ground to fine powder. The powdered samples were then stored at about -20°C to preserve their integrity until further analyses.

[0086] 2,4-D Analyses

[0087] To analyse 2,4-D in weed samples, about 5 grams of weed samples were weighed. A mixture of about 20 mL of methanol and water in a ratio of about 80:20 was added to the weighed weed samples. The weed samples were homogenised for about 2 minutes to ensure thorough mixing and centrifuged at about 4,000 rpm. Resulting liquid was filtered through a filter (about 0.45 pm fdter) to remove remaining particulates.

[0088] For Liquid Chromatography-Mass Spectrometry (LC-MS / MS) analysis, a suitable column was used. The mobile phase comprised: about 0.1% formic acid in water; and about 0.1% formic acid in methanol. Flow rate was set to about 0.3 mL / min. The mass spectrometry parameters were configured to operate in negative ion mode, utilising Multiple Reaction Monitoring (MRM) transitions. Quantifier transition was set to about 219> 161 , while qualifier transition was set to about 219> 125.

[0089] Glyphosate Analyses

[0090] To analyse glyphosate in weed samples, about 20 mL of a water and chloroform mixture in a ratio of about 50:50 was added to the weed samples. The weed samples were shaken for about 1 hour. After shaking, an aqueous layer was separated from an organic layer. The organic layer was then re-extracted (using the same procedure as mentioned above) and the aqueous extracts were combined for further processing.

[0091] Oxyfluorfen Analyses

[0092] To analyse oxyfluorfen in weed samples, about 30 mL of a mixture of acetone and hexane in the ratio of about 1: Iwas added to the weed samples. The weed samples were sonicated for about 15 minutes After sonication, the samples were centrifuged, and a supernatant was collected for analyses. This extraction step was repeated twice.

[0093] For GC-MS analyses, ions corresponding to m / z values of about 361, about 300, and about 252, were monitored.

[0094] Paraquat Analyses

[0095] To analyse paraquat in weed samples, about 20 mL of a mixture of methanol and water in a ratio of about 50:50 was added to the weed samples. The weed samples were then acidified with formic acid and shaken for about 30 minutes to ensure thorough mixing and extraction of paraquat. Following shaking, the samples were centrifuged, and filtered, to obtain pure extracts. The results of soil samples and weed samples treated with the disclosed composition and a commercial herbicide are provided in the table given below.

[0096] The soil samples A, B, and C are the samples that were treated with the disclosed composition or the soil samples taken from a field where the disclosed composition was sprayed and / or applied.

[0097] The analyses of soil samples treated with the disclosed composition and commercial herbicide revealed the following findings.

[0098] Total Nitrogen (N): The commercial herbicide-treated soil had the highest nitrogen content (about 531 mg / kg), while soil sample B was close at about 516 mg / kg.

[0099] Phosphorous (P): The soil sample B exhibited the highest phosphorous level at about 248 mg / kg, followed closely by the commercial herbicide-treated soil sample (about 238 mg / kg). Potassium (K): The highest potassium level was seen in soil sample C (about

[0100] 482 mg / kg), while the commercial-herbicide treated soil sample had about 386 mg / Kg. Total Organic Carbon (TOC): The soil sample C showed the highest TOC at about 0.58%, followed by the soil sample B at about 0.53%. The commercial herbicide-treated soil sample had a TOC of about 0.40%.

[0101] Presence of herbicide residue (2,4-D, Glyphosate, Oxyfluorfen, Paraquat): All the soil samples except the control showed below the limit of quantification for 2,4-D and glyphosate.

[0102] Total Plate Count: The soil sample B and soil sample C showed the highest microbial counts at about 9,00,000 CFU / g, suggesting higher levels of soil activity, compared to the commercial herbicide-treated soil sample at about 6,00,000 CFU / g.

[0103] The disclosed biological agrochemical composition doesn’t have active ingredients of glyphosate and 2,4, D-amine salts, as per tests done by UPASI (United Planters' Association of Southern India) Tea Research Foundation, Tamil Nadu, India.

[0104] Results of various field trials carried out for testing efficiency of said composition against different types of weeds are detailed as follows.

[0105] As illustrated, in Figure 1, it was observed that the composition effectively controlled multiple weeds at dosages of about 50ml / L. The weeds evaluated in the trial include: nut grass (Cyperus rotundas)', Chromolaena odorata', parthenium; Digitaria sp.; Spermacoce ocymoides', touch-me-not (Mimosa pudica)', and Cynodon dactylon.

[0106] Soil pH and nitrogen (N%) evaluated before and after the application of the synthesised biological agrochemical composition are given in the below table.

[0107] As illustrated, in Figure 2, it was observed that the synthesised biological agrochemical composition effectively controlled multiple weeds at dosages of about 50ml / L. The weeds evaluated in the trial include: Alternanthera sessilis', Euphorbia heterophylla', Ipomoea purpurea', goosegrass; Portulaca oleracew, parthenium; Cynodon dactylow, Amaranthus viridis', and Solarium scabrum.

[0108] The soil pH and nitrogen (N%) evaluated before and after the application of the synthesised biological agrochemical composition are given in the below table.

[0109] As illustrated, in Figure 3, it was observed that the synthesised biological agrochemical composition effectively controlled multiple weeds, at dosages of about 50ml / L. The weeds evaluated in the trial include: Euphorbia hirtw, Euphorbia heterophyllw, Chromolaena odorala', Sida acuta', Koeleria glaucw, Passiflora foetida', Senna occidentalism nut grass (Cy erus rotundas)', touch-me-not (Mimosa pudicay, and Croton bonplandianus .

[0110] The soil pH and nitrogen (N%) evaluated before and after the application of the synthesised biological agrochemical composition are given in the below table.

[0111] As illustrated, in Figure 4 and Figure 5, it was observed that the synthesised biological agrochemical composition effectively controlled weeds, at temperatures of about 15- 20°C and a humidity of about 45%-50%.

[0112] As illustrated, in Figure 6, it was observed that the synthesised biological agrochemical composition effectively controlled multiple weeds, at dosages of about 50 mL / L of the synthesised biological agrochemical composition.

[0113] Figure 7 illustrates trials conducted at another test estate, with a dosage of about 10 mL / L of a control herbicide composition. As illustrated, in Figure 8, it was observed that the synthesised biological agrochemical composition effectively controlled weeds which are more than about 3 feet tall and very thick, for about 40 days, with no pre-emergence occurring over about 35days.

[0114] The disclosed biological agrochemical composition offers at least the following synergistic advantages and effects: is efficient; is non-toxic; is environment-friendly; improves soil fertility; and / or is effective against a broad range of weeds.

[0115] It will be apparent to a person skilled in the art that the above description is for illustrative purposes only and should not be considered as limiting. Various modifications, additions, alterations, and improvements, without deviating from the spirit and the scope of the disclosure, may be made, by a person skilled in the art. Such modifications, additions, alterations, and improvements should be construed as being within the scope of this disclosure.

Claims

CLAIMSWe Claim:

1. A synergistic biological agrochemical composition comprising: a filtrate of Lactobacillus acidophilus', a filtrate of Aspergillus niger, neem oil; and polyoxyethylene (20) sorbitan monolaurate, with: said filtrate of Lactobacillus acidophilus and said filtrate of Aspergillus niger being in a ratio of 1 : 1 ; said filtrate of Lactobacillus acidophilus and said neem oil being in a ratio of 1 : 1 ; and said filtrate of Lactobacillus acidophilus and said polyoxyethylene (20) sorbitan monolaurate being in a ratio of 3: 1.

2. The synergistic biological composition, as claimed in claim 1, wherein: concentration of said filtrate of Lactobacillus acidophilus is 30 mL / L; concentration of said filtrate of Aspergillus niger is 30 mL / L; concentration of said neem oil is 30 mL / L; and concentration of said polyoxyethylene (20) sorbitan monolaurate is 10 mL / L.

3. A method of synthesising a biological agrochemical composition, said method comprising steps of: cultivating Lactobacillus acidophilus in Luria Bertani broth at 37°C for 48 hours to obtain a Lactobacillus starter culture; cultivating Aspergillus niger in Potato Dextrose Broth at 37°C for 4 to 5 days to obtain an Aspergillus starter culture; preparing and sterilising a Lactobacillus medium comprising: 1% of glucose; 0.25% of yeast extract; and a mineral salt; preparing and sterilising an Aspergillus medium comprising: 2% of glucose; 0.2% of ammonium sulphate; and 0.002% of dipotassium phosphate;supplementing the Lactobacillus medium with 0.5% v / v of the Lactobacillus starter culture; supplementing the Aspergillus medium with 0.5% v / v of the Aspergillus starter culture; incubating the Lactobacillus medium and the Aspergillus medium at 37°C for 72 hours, followed by heating at 70°C to kill live organisms and cooling for 24 hours, to obtain heat-killed media of Lactobacillus and Aspergillus, respectively; filtering the heat-killed media of Lactobacillus and Aspergillus to remove physical contaminants and debris, to obtain a filtrate of Lactobacillus acidophilus and a filtrate of Aspergillus niger, respectively; and blending said filtrate of Lactobacillus acidophilus with said filtrate of Aspergillus niger, neem oil, and polyoxyethylene (20) sorbitan monolaurate, with: said filtrate of Lactobacillus acidophilus and said filtrate of Aspergillus niger being in a ratio of 1 : 1 ; said filtrate of Lactobacillus acidophilus and said neem oil being in a ratio of 1 : 1 ; and said filtrate of Lactobacillus acidophilus and said polyoxyethylene (20) sorbitan monolaurate being in a ratio of 3: 1.

4. The method of synthesising a biological composition, as claimed in claim 3, wherein: concentration of said filtrate of Lactobacillus acidophilus is 30 mL / L; concentration of said filtrate of Aspergillus niger is 30 mL / L; concentration of said neem oil is 30 mL / L; and concentration of said polyoxyethylene (20) sorbitan monolaurate is 10 mL / L.

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