DESC:This application is a cognate of application no. 202341064584 filed on 26-09-2023 and application no. 202341064582 filed on 26-09-2023.
FIELD
The present disclosure relates to a method for obtaining multiple metabolite concentrates. Particularly, the present disclosure relates to a method for obtaining a multiple metabolite concentrates from bacterial species.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicate otherwise.
Multiple metabolite concentrate (MMC): The term “multiple metabolite concentrate” herein refers to the various metabolites obtained from the microbes.
Microbial consortium: The term “microbial consortium” refers to the mixture of micro-organisms.
BACKGROUND
The background information herein below relates to the present disclosure but is not necessarily prior art.
Synthetic plant protection chemicals (PPPs) used for treating the plant pests pose a threat to the environment and due to the repeated use of such synthetic plant protection chemicals (PPPs), the pests become resistant. Due to the drawback of the synthetic plant protection chemicals (PPPs), the biopesticides are considered as important components of the integrated pest management (IPM) programs and have received much attention as substitutes for synthetic plant protection chemicals (PPPs) in the recent years.
Biopesticides have been defined as a type of pesticide based on microorganisms or natural products. Typically, biopesticides are prepared by growing and concentrating naturally occurring organisms and/or their metabolites including bacteria and other microbes, fungi, viruses, nematodes, proteins and the like.
Further, microbial metabolites are one of the promising sources having therapeutic potential, wherein some of the microbial metabolites are antagonistic and inhibitory in nature towards insects, pests and plant pathogens. Several bacterial metabolites have been studied for the pesticidal activity. However, very few have been identified to have commercial potential. Furthermore, the yield of MMC by using traditional methods is very low and the final product has impurities. Conventional methods are also time-consuming.
There is, therefore, felt a need to develop a method for obtaining multiple metabolite concentrates (MMC) from the bacterial species that mitigates the drawback mentioned herein above or at least provides a useful alternative.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
It is an object of the present disclosure to ameliorate one or more problems of the background or to at least provide a useful alternative.
An object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC).
Another object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC) from bacterial species.
Still another object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC) from microbial consortium.
Yet another object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC) from Bacillus velezensis and Priestia megaterium.
Still another object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC) which is simple and economic.
Yet another object of the present disclosure is to provide a method for obtaining multiple metabolite concentrates (MMC) that provides MMC with no impurities.
Still another object of the present disclosure is to provide a time-saving method for obtaining a MMC.
Yet another object of the present disclosure is to provide multiple metabolite concentrates of a microbial consortium that is effective against pests.
Still another object of the present disclosure is to provide a culture medium composition that provides increased growth of bacterium species or microbial consortium.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates a method for obtaining multiple metabolite concentrates. The method comprises the step of isolating at least two bacterial species. Separately, the at least two bacterial species are cultured in a separate culture medium at a first predetermined temperature for a first predetermined time period to obtain at least two mother cultures. The at least two mother cultures are transferred into a separate fermenter and fermenting the cultures separately at a second predetermined temperature for a second predetermined time period to obtain at least two bacterial broths of the at least two cultures. The at least two bacterial broths are mixed with a fluid medium separately in a predetermined mass ratio and allowed to undergo lysis of the at least two bacterial broths by stirring at a predetermined speed at a third predetermined temperature for a third predetermined time period to obtain two mixtures comprising lysed bacterial cells and metabolites and the mixtures are concentrated separately to obtain the multiple metabolite concentrates.
The bacterial species are selected from the species of Bacillus and Priestia.
The Bacillus species is selected from the group consisting of Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus aryabhattai and the Priestia species is Priestia megaterium, wherein the Bacillus velezensis is deposited as MTCC Accession No. 25698 having its 16S rDNA sequence designated as SEQ ID No.1 and the Priestia megaterium is deposited as MTCC Accession No. 25699 having its 16S rDNA sequence designated as SEQ ID No. 2.
The first predetermined temperature is in the range of 25ºC to 40 ºC, the first predetermined time period is in the range of 20 hours to 40 hours, the second predetermined temperature is in the range of 25 ºC to 40 ºC; the second predetermined time period is in the range of 100 hours to 130 hours; the third predetermined temperature is in the range of 25 ºC to 40 ºC, the third predetermined time period is in the range of 20 hours to 40 hours and the predetermined speed is in the range of 50 rpm to 300 rpm.
The predetermined mass ratio of the bacterial broth to the fluid medium is in the range of 1:1 to 1:5.
The fluid medium is selected from the group consisting of methanol, ethanol, acetone, chloroform, butanol and ethyl acetate.
The culture medium comprises 2 mass% to 4 mass% of at least one legume or cereal flour, 0.1 mass% to 0.5 mass% at least one inorganic phosphate, 0.1 mass% to 0.5 mass% at least one saccharide, 0.01 mass% to 2 mass% at least one inorganic sulfate and q.s water, wherein the mass% of each ingredient is with respect to the total mass of the culture medium.
The legume or cereal flour is selected from the group consisting of wheat flour, ragi flour and soya flour, defatted soya flour, full-fat soya flour, high-protein soya flour, toasted soya flour, enzyme-active soya flour and specialty soya flour.
The inorganic phosphate is dipotassium phosphate.
The saccharide is selected from the group consisting of dextrose, lactose, glucose and galactose.
The inorganic sulfate is selected from the group consisting of magnesium sulfate, ferrous sulfate and manganese sulfate.
The at least two multiple metabolite concentrates are present in a weight ratio in the 1:4 to 4:1.
The at least two multiple metabolite concentrates are present in a weight ratio of 1:1.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1 illustrates (A) image of soil samples sourced from agricultural fields; and (B) image of bacterial isolates from the soil samples in accordance with the present disclosure;
Figure 2 illustrates graphical representation of (A) growth kinetics of Bacillus velezensis in various media; and (B) growth kinetics of Priestia megterium in various media in accordance with the present disclosure;
Figure 3 illustrates (A) image of ingredients of a culture medium; (B) image of a nutrient broth; and (C) image of a culture medium based on soy flour in accordance with the present disclosure;
Figure 4 illustrates (A) image of evaporation of solvent extract by oven method and (B) image of evaporation of solvent extract by rotary evaporator in accordance with the present disclosure; and
Figure 5 illustrates (A) the sequence of 16S rDNA of Bacillus velezensis having SEQ ID No. 1 and (B) the sequence of 16S rDNA of Priestia megaterium having SEQ ID No. 2 in accordance with the present disclosure.
DETAILED DESCRIPTION
The present disclosure relates to a method for obtaining multiple metabolite concentrates. Particularly, the present disclosure relates to a method for obtaining multiple metabolite concentrates from bacterial species.
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed elements.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
Synthetic plant protection chemicals (PPPs) used for treating the plant pests pose a threat to the environment and due to the repeated use of such synthetic plant protection chemicals (PPPs), the pests become resistant. Due to the drawback of the synthetic plant protection chemicals (PPPs), the biopesticides are considered as important components of the integrated pest management (IPM) programs and have received much attention as substitutes for synthetic plant protection chemicals (PPPs) in the recent years.
Biopesticides have been defined as a type of pesticide based on microorganisms or natural products. Typically, biopesticides are prepared by growing and concentrating naturally occurring organisms and/or their metabolites including bacteria and other microbes, fungi, viruses, nematodes, proteins and the like.
Further, microbial metabolites are one of the promising sources having therapeutic potential, wherein some of the microbial metabolites are antagonistic and inhibitory in nature towards insects, pests and plant pathogens. Several bacterial metabolites have been studied for the pesticidal activity. However, very few have been identified to have commercial potential. Furthermore, the yield of MMC by using traditional methods is very low and the final product has impurities. Conventional methods are also time-consuming.
The present disclosure provides a method for obtaining multiple metabolite concentrates. Particularly, the present disclosure provides a method for obtaining multiple metabolite concentrates from bacterial species.
In accordance with the present disclosure, the present disclosure provides a method for obtaining multiple metabolite concentrates. The method comprises the step of isolating at least two bacterial species. Separately, the at least two bacterial species are cultured in a separate culture medium at a first predetermined temperature for a first predetermined time period to obtain at least two mother cultures. The at least two mother cultures are transferred into a separate fermenter and fermenting the cultures separately at a second predetermined temperature for a second predetermined time period to obtain at least two bacterial broths of the at least two cultures. The at least two bacterial broths are mixed with a fluid medium separately in a predetermined mass ratio and allowing to undergo lysis of the at least two bacterial broth by stirring at a predetermined speed at a third predetermined temperature for a third predetermined time period to obtain two mixtures comprising lysed bacterial cells and metabolites and the mixtures are concentrated separately to obtain multiple metabolite concentrates.
The method is explained in detail.
In a first step, at least two bacterial species are isolated.
In an embodiment of the present disclosure, at least two bacterial species are isolated from soil samples collected from agricultural fields nearby Bangalore.
Multiple bacteria are isolated from the soil samples by standard serial dilution technique, followed by selection of colonies and identification through molecular tools. Further, the isolated bacteria are screened against plant pests such as white flies, mealy bugs and the like. Such bacteria having pesticidal activity against pests are identified with molecular tools and taxonomically named.
In a second step, separately, at least two bacterial species are cultured in a separate culture medium at a first predetermined temperature for a first predetermined time period to obtain at least two mother cultures.
In an embodiment of the present disclosure, the bacterial species are selected from the group of Bacillus species and Priestia species.
The Bacillus species is selected from the group consisting of Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus aryabhattai. The Priestia species is Priestia megaterium. In an exemplary embodiment of the present disclosure, the Bacillus species is Bacillus velezensis and the Priestia species is Priestia megaterium.
Bacillus velezensis and Priestia megaterium are Gram-positive, spore-forming bacteria. The combination of these bacteria enhances the efficiency of the product due to complimentary effect from the multiple metabolite concentrate (MMC).
The Bacillus velezensis is deposited as MTCC Accession No. 25698 having its 16S rDNA sequence designated as SEQ ID No. 1 and the Priestia megaterium is deposited as MTCC Accession No. 25699 having its 16S rDNA sequence designated as SEQ ID No. 2.
Figure 5 illustrates (A) the sequence of 16S rDNA of Bacillus velezensis having SEQ ID No. 1 and (B) the sequence of 16S rDNA of Priestia megaterium having SEQ ID No. 2.
In an embodiment of the present disclosure, the culture medium comprises 2 mass% to 4 mass% of at least one legume or cereal flour, 0.1 mass% to 0.5 mass% of at least one inorganic phosphate, 0.1 mass% to 0.5 mass% of at least one saccharide, 0.01 mass% to 2 mass% of at least one inorganic sulfate and q.s.water, wherein the mass% of each ingredient is with respect to the total mass of the culture medium.
In an exemplary embodiment of the present disclosure, the legume or cereal flour is present in an amount of 3 mass%, the inorganic phosphate is present in an amount of 0.2 mass%, the saccharide is present in an amount of 0.2 mass%, the inorganic sulfate is present in an amount of 0.05 mass% and 96.5 mass% of water, wherein the amounts are with respect to the culture medium.
In an embodiment of the present disclosure, the legume or cereal flour is selected from the group consisting of wheat (Triticum aestivum) flour, ragi (Finger millet) flour and soya (Glycine max) flour, defatted soya flour, full-fat soya flour, high-protein soya flour, toasted soya flour, enzyme-active soya flour and specialty soya flour. In an exemplary embodiment of the present disclosure, the legume cereal flour is high-protein soya flour.
In an embodiment of the present disclosure, the inorganic phosphate is dipotassium phosphate (K2HPO4).
In an embodiment of the present disclosure, the saccharide is selected from the group consisting of dextrose, lactose, glucose and galactose. In an exemplary embodiment of the present disclosure, the saccharide is dextrose.
In an embodiment of the present disclosure, the inorganic sulfate is selected from the group consisting of magnesium sulfate (MgSO4), ferrous sulfate (FeSO4) and manganese sulfate (MnSO4). In an exemplary embodiment of the present disclosure, the inorganic sulfate is magnesium sulfate.
The culture medium is critically designed which is based on natural materials and supplemented by selected chemicals in minimal quantities. This helps in augment the potency of the active ingredients fostering optimal and opulent growth conditions for the growth of the bacterial culture. The culture medium of the present disclosure is conducive to luxurious growth of the bacteria.
In an embodiment of the present disclosure, the first predetermined temperature is in the range of 25 ºC to 40 ºC. In an exemplary embodiment of the present disclosure, the predetermined temperature is 37 ºC.
In an embodiment of the present disclosure, the first predetermined time period is in the range of 20 hours to 30 hours. In an exemplary embodiment of the present disclosure, the predetermined time period is 24 hours.
In a third step, the at least two mother cultures are transferred to a separate fermenter and the cultures are fermented separately at a second predetermined temperature for a second predetermined time period to obtain at least two bacterial broths of the at least two cultures.
The fermenter is in controlled conditions. The sample is checked intermittently for contamination and other quality control parameters.
In an embodiment of the present disclosure, the second predetermined temperature in the range of 20 ºC to 40 ºC. In an exemplary embodiment of the present disclosure, the second predetermined temperature is 37 ºC.
In an embodiment of the present disclosure, the second predetermined time period is in the range of 100 hours to 130 hours. In an exemplary embodiment of the present disclosure, the second predetermined time period is 120 hours.
In a fourth step, the at least two bacterial broths are mixed with a fluid medium separately in a predetermined mass ratio and allowed to undergo lysis of the at least two bacterial broths by stirring at a predetermined speed at a third predetermined temperature for a third predetermined time period to obtain two mixtures comprising lysed bacterial cells and metabolites.
In an embodiment of the present disclosure, the fluid medium is selected from the group consisting of methanol, ethanol, acetone, chloroform, butanol and ethyl acetate. In an exemplary embodiment of the present disclosure, the fluid medium is ethyl acetate.
The grown bacterial mass having desired optical density (OD) value (colony forming units-CFU), is subjected to lysis by solvent extraction by using ethyl acetate (fluid medium).
In an embodiment of the present disclosure, the predetermined mass ratio of the bacterial broth to the fluid medium is in the range of 1:1 to 1:5. In an exemplary embodiment of the present disclosure, the predetermined mass ratio of the bacterial broth to the fluid medium is 1:4.
The so obtained mixtures comprising lysed bacterial cells and metabolites are processed further.
In an embodiment of the present disclosure, the predetermined stirring speed is in the range of 50 rpm to 300 rpm. In an exemplary embodiment of the present disclosure, the predetermined stirring speed is 150 rpm.
In an embodiment of the present disclosure, the third predetermined temperature is in the range of 25 ºC to 40ºC. In an exemplary embodiment of the present disclosure, the third predetermined temperature is 37ºC.
In an embodiment of the present disclosure, the third predetermined time period is in the range of 20 hours to 30 hours. In an exemplary embodiment of the present disclosure, the third predetermined time period is 24 hours.
The so obtained mixtures are processed in the fifth step.
In a fifth step, the mixtures are concentrated to obtain the at least two multiple metabolite concentrates.
In an embodiment of the present disclosure, the concentration of the mixtures is carried out by evaporating in a rotary evaporator.
In an embodiment of the present disclosure, 1000 litres of mixture (mixture comprising lysed bacterial mass, metabolites along with the broth and fluid medium) is concentrated to 225 litres of the mixture.
The Rota evaporation technique is used with controlled temperature, boiling point, speed and vacuum for the better evaporation. The evaporation process is carried out at a temperature/boiling point of 80? at a speed of 120 rpm for desired results.
The Rota evaporation technique is advantageous over conventional oven drying for concentrating the mixture and it overcomes the disadvantages of oven drying such as chances of contamination, consumption of more time and the like.
In an embodiment of the present disclosure, the at least two multiple metabolite concentrates are present in a weight ratio in the range of 1:4 to 4:1.
In an embodiment of the present disclosure, the at least two multiple metabolite concentrates are present in a weight ratio of 1:1.
The multiple metabolite concentrate prepared by using the method of the present disclosure remains stable up to 3 years at room temperature in dry place in closed condition.
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment but are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further described in light of the following experiments which are set forth for illustration purposes only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial scale.
EXPERIMENTAL DETAILS:
Experiment 1: Method for obtaining multiple metabolite concentrates in accordance with the present disclosure
Step i): Isolation of bacteria:
10 g of soil (collected from agricultural fields nearby Bangalore) was suspended in 100 ml of sterilized saline and serially diluted. Specific serially diluted sample was plated on nutrient agar, followed by incubation at 37 ºC for overnight. The isolated colonies were taken for further testing, wherein the bacteria were selected which showed the pesticidal activity.
With the use of molecular techniques and colony morphological analysis, the selected bacteria were identified as Bacillus velezensis and Priestia megaterium. The isolated Bacillus velezensis was deposited as MTCC Accession No. 25698 and the Priestia megaterium was deposited as MTCC Accession No. 25699.
Figure 1 illustrates (A) image of soil samples sourced from agricultural fields; and (B) image of bacterial isolates from the soil samples in accordance with the present disclosure.
Step ii): Culturing of isolated bacteria:
The isolated colonies of Bacillus velezensis and Priestia megaterium were separately inoculated in a culture medium at 37 ºC for 24 hours to obtain mother cultures of Bacillus velezensis and Priestia megaterium having desired OD (optical density).
Separately the culture medium was prepared by using the ingredients provided in Table 1.
Experiment 2(B): Optimization of the culture media in accordance with the present disclosure
Different culture media were prepared by varying the ingredients as enlisted in below Table 1 for optimizing the culture media for the growth of the bacteria in accordance with the present disclosure.
Table 1: Ingredients optimization of the culture medium
Sr. No. Ingredients Culture Medium 1 Culture Medium 2 Culture Medium 3
1. Soya Flour 30 --- ---
2. Wheat flour --- 30 ---
3. Ragi flour --- --- 30
4. K2HPO4 2 2 2
5. Dextrose 2 2 2
6. MgSO4 0.5 0.5 0.5
7. Water 965.5 965.5 965.5
pH of all the culture media was kept constant to 7.
The culture media summarized in Table 1 were further tested for the growth of two bacterial cultures, Bacillus velezensis and Priestia megaterium.
Figure 2 illustrates graphical representation of (A) growth kinetics of Bacillus velezensis in various culture media; and (B) growth kinetics of Priestia megterium in various culture media in accordance with the present disclosure.
Soy flour gave the best results and showed optimum growth of the two bacteria. Hence, the culture medium based on soy flour was used for further experimentation.
Table 2: Composition of the culture medium containing soya flour
Sr. No. Ingredients Function Amount (g/L)
1 Soya Flour Source of Protein, Nitrogen which is required for the luxurious growth and enhancement of metabolite. 30
2 K2HPO4 To enhance the metabolite 2
3 Dextrose Source of Carbohydrate 2
4 MgSO4 micronutrient 0.5
5 Water ---- 965.5
pH of the culture medium was 7.
Figure 3 illustrates (A) image of ingredients of a culture medium; (B) image of a nutrient broth; and (C) image of a culture medium based on soy flour in accordance with the present disclosure.
Step iii): Preparation of a bacterial mass:
After the desired OD of the cultures (mother cultures) of Bacillus velezensis and Priestia megaterium were obtained, the cultures were transferred to a separate fermenter containing the culture medium respectively to obtain a bacterial broth of respective cultures. The fermenter was operated at 37 ºC for 120 hours.
Sample from the fermenter was checked intermittently for contamination and quality control parameters.
Step iv): Lysis of the bacterial mass:
After the desired growth of the bacterial broth (microbial consortium) containing Bacillus velezensis and Priestia megaterium, 250 litres of ethyl acetate (fluid medium) was added to the fermenters separately and mixed for overnight (8 hours) at 150 rpm at 27oC to obtain a mixture of lyzed bacteria of Bacillus velezensis and metabolites and a mixture of lyzed bacteria of Priestia megaterium and metabolites separately. Ethyl acetate lysed the bacterial cells. The mass ratio of the bacterial broth to ethyl acetate was 1:4.
Step v): Concentration of the mixture containing lysed bacteria:
The mixture was concentrated under vacuum in a Rota evaporator.
1000 litres of the bacterial broth and 250 litres of ethyl acetate was concentrated to 225 litres to obtain the multiple metabolite concentrate (MMC).
The so obtained MMC was further formulated with suitable surfactants and preservatives to obtain pesticidal formulation to be used against plant pests such as white flies and mealy bugs.
Experiment 2: Optimization of the fluid medium for lysis in accordance with the present disclosure
Various fluid media were used for bacterial cell lysis and extraction of metabolites to identify the fluid medium that gives optimum results.
The list of fluid media tested for the study has been enlisted in Table 2 below.
Table 2: List of the fluid media and results
Solvent Type Solvent Name Boiling Point Pressure Results Remark

Polar Solvents

Methanol 64.7 ? 111.7 kPa No Significant results with respect to separation. Separation issues occurred with Polar Solvents.

Ethanol 78.4 ? - No Significant results with respect to separation. Separation issues occurred with Polar Solvents.
Acetone 56 ? - No Significant results with respect to separation. Separation issues occurred with Polar Solvents.

Non-Polar
Solvents

Chloroform 61.2 ? - - Lysis issue

Butanol 117.7 ? 101.3 kPa - Lysis issue

Ethyl Acetate
77.1 ºC 96.2 kPa Lysis was achieved up-to expected level with in short time Ethyl Acetate was the efficient solvent for lysis and extraction than the other non-polar solvents
From Table 2, it was observed that ethyl acetate was the efficient fluid medium for lysis and extraction of metabolites than the other non-polar fluid media.
Experiment 2(A): Optimization of the temperature during the evaporation in accordance with the present disclosure
Concentration of the mixture obtained in Experiment 1 (last step) was carried out by using Rota evaporation technique at various temperatures in order to determine the optimum temperature. The results of the study are summarized in Table 3 below.
Table 3: Results of different temperatures during concentration of metabolites
Sr.No. Temperature (?) under vacuum Evaporation Process Yield Remark

1 40 oC Started Evaporation Nil As the temperature increased, there was increases in the evaporation rate

2 50 oC Solvent evaporation Nil As the temperature increased, there was increases in the evaporation rate

3 60 oC Broth Evaporation Nil As the temperature increased, there was increases in the evaporation rate
4 75 oC Complete evaporation 17g/L As the temperature increased, there was increases in the evaporation rate
5 80 oC Complete evaporation ---- Degradation of the broth ingredients
From Table 3, it was observed that after 75 oC, the ingredients of the bacterial broth started degradation.
TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of;
? a multiple metabolite concentrate that:
• has pesticidal activity; and
• is stable up to 3 years; and
? a method for obtaining a multiple metabolite concentrate that is
• simple, economic, easy to scale up and environment friendly.
The embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
The foregoing description of the specific embodiments so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired object or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation. ,CLAIMS:WE CLAIM:
1. A method for obtaining multiple metabolite concentrates, said method comprises the following steps:
(i) isolating at least two bacterial species;
(ii) separately, culturing said at least two bacterial species in a separate culture medium at a first predetermined temperature for a first predetermined time period to obtain at least two mother cultures;
(iii) transferring said at least two mother cultures into a separate fermenter and fermenting said cultures separately at a second predetermined temperature for a second predetermined time period to obtain at least two bacterial broths of said at least two cultures;
(iv) mixing said at least two bacterial broths with a fluid medium separately in a predetermined mass ratio and allowing to undergo lysis of said at least two bacterial broths by stirring at a predetermined speed at a third predetermined temperature for a third predetermined time period to obtain two mixtures comprising lysed bacterial cells and metabolites; and
(v) concentrating said mixtures separately to obtain said multiple metabolite concentrates.
2. The method as claimed in claim 1, wherein said bacterial species are selected from the species of Bacillus and Priestia.
3. The method as claimed in claim 2, wherein
• said Bacillus species is selected from the group consisting of Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus aryabhattai; and
• said Priestia species is Priestia megaterium,
wherein
• said Bacillus velezensis is deposited as MTCC Accession No. 25698 having its 16S rDNA sequence designated as SEQ ID No. 1; and
• said Priestia megaterium is deposited as MTCC Accession No. 25699 having its 16S rDNA sequence designated as SEQ ID No. 2.
4. The method as claimed in claim 1, wherein
• said first predetermined temperature is in the range of 25 ºC to 40 ºC;
• said first predetermined time period is in the range of 20 hours to 30 hours;
• said second predetermined temperature is in the range of 25 ºC to 40 ºC;
• said second predetermined time period is in the range of 100 hours to 130 hours;
• said third predetermined temperature is in the range of 25 ºC to 40 ºC;
• said third predetermined time period is in the range of 20 hours to 40 hours; and
• said predetermined speed is in the range of 50 rpm to 300 rpm.
5. The method as claimed in claim 1, wherein said predetermined mass ratio of said bacterial broth to said fluid medium is in the range of 1:1 to 1:5.
6. The method as claimed in claim 1, wherein said fluid medium is selected from the group consisting of methanol, ethanol, acetone, chloroform, butanol and ethyl acetate.
7. The method as claimed in claim 1, wherein said culture medium comprises:
a. 2 mass% to 4 mass% of at least one legume or cereal flour;
b. 0.1 mass% to 0.5 mass% at least one inorganic phosphate;
c. 0.1 mass% to 0.5 mass% at least one saccharide;
d. 0.01 mass% to 2 mass% at least one inorganic sulfate; and
e. q.s. water,
wherein said mass% of each ingredient is with respect to the total mass of said culture medium.
8. The method as claimed in claim 7, wherein
• said legume or cereal flour is selected from the group consisting of wheat flour, ragi flour and soya flour, defatted soya flour, full-fat soya flour, high-protein soya flour, toasted soya flour, enzyme-active soya flour and specialty soya flour;
• said inorganic phosphate is dipotassium phosphate;
• said saccharide is selected from the group consisting of dextrose, lactose, glucose and galactose; and
• said inorganic sulfate is selected from the group consisting of magnesium sulfate, ferrous sulfate and manganese sulfate.
9. The method as claimed in claim 1, wherein said at least two multiple metabolite concentrates are present in a weight ratio in the range of 1:4 to 4:1.
10. The method as claimed in claim 9, wherein said at least two multiple metabolite concentrates are present in a weight ratio of 1:1.

Dated this 8th day of May, 2024

_______________________________
MOHAN RAJKUMAR DEWAN, IN/PA – 25
OF R. K. DEWAN & CO.
AUTHORIZED AGENT OF APPLICANT

TO,
THE CONTROLLER OF PATENTS
THE PATENT OFFICE, AT CHENNAI