A biostimulant composition and method of preparation thereof

Abstract

The present invention provides a bio-stimulant composition for promoting plant growth and development. The biostimulant composition comprises seaweed extract, milk solids, one or more amino acid and optionally one or more of the ingredients selected from protein hydrolysate, humic substance, macronutrient and micronutrient. The present invention also provides a method for preparing the bio-stimulant composition.

Description

[0001] A BIOSTIMULANT COMPOSITION AND METHOD OF PREPARATION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a bio-stimulant composition for promoting plant growth and development. The present invention also relates to a method for preparing the bio-stimulant composition.

[0004] BACKGROUND OF THE INVENTION

[0005] Bio-stimulants, also termed as plant conditioners or bio effectors are used to promote the growth of crop plants that encompass a wide array of natural substances, which, when applied to crop plants, promote growth, development, and stress tolerance. Biostimulants are important tools for sustainable agriculture due to their ability to enhance tolerance to abiotic stress, nutrient use efficiency, crop performance, and quality. They are complementary to mineral fertilizers by improving availability, assimilation, translocation, and use of certain plant nutrients. However, unlike traditional fertilizers that provide essential nutrients directly to plants, bio-stimulants exert their effects through indirect mechanisms, often involving interactions with the plant's physiology, soil microorganisms, and nutrient uptake processes. A growing number of regulatory frameworks around the globe recognize plant bio-stimulants as a distinct product category related to the broad concept of crop physiology, plant nutrition, and soil fertility.

[0006] Use of bio-stimulants in agriculture is increasingly more frequent because of the nutritional demand of high yield crops, where the objective is generally to make up for the nutritional requirements in critical times, to shorten or delay plant cycles and to induce specific phonological stages, in addition, to counteract plant stress conditions, energy intake in productive stages or foliar nutrition for plant health purposes. Further it has gained more significance in recent times as the excessive use of synthetic chemicals and fertilizers imposes a global health hazard and pollutes agricultural lands worldwide. Thus, promoting the use of natural plant growth regulators is highly desirable.

[0007] Currently seaweed extracts are widely used as plant bio-stimulants, which constitutes more than 33% of the total bio-stimulant market worldwide. Seaweed extracts contain plant growth promoters, trace minerals, vitamins, and polysaccharides that positively impact plant physiology. It is estimated that seaweeds or macroalgae comprise nearly 10,000 species, which are subdivided mainly into three categories based on their pigmentation, Phaeophyta (Brown), Rhodophyta (Red), and Chlorophyta (Green).

[0008] Though seaweed extracts are currently the dominant category of the plant biostimulants segment, there are several challenges associated with the use of seaweed as bio-stimulant. A review of the literature available in this field shows that the results obtained from experiments where seaweed extracts were applied are not always positive. In a few published cases, crops treated with seaweed extracts and grown under abiotic stress conditions showed no significant differences in crop yield (Di Stasio et al., 2018) or even inhibitory germination responses (Masondo et al., 2018). In most cases, crop productivity was still reduced under severe stress conditions even with seaweed extract treatment, albeit to a lesser extent than in nontreated plants (Hashem et al., 2019; Murtic et al., 2018; Trivedi et al., 2018). Thus, seaweed extracts are not always able to completely reverse or eliminate stress-induced damage and may not always provide the evident growth results especially under severe abiotic stress, though they result in an improved resilience to abiotic stress in these crops, and biostimulants should therefore not be sold as ‘miracle’ inputs (Ayodeji O. Deolu-Ajayi et.al., Plant Cell Environ. 2022; 45: 2537- 2553.). Attempts have also been made to fortify seaweed extracts along with other potential nourishing substances such as organic acids, inorganic salts, chitin, chitosan derivatives, and biopolymers among others, to improve plant growth and yield. Despite such attempts, product performance is still a barrier, and satisfaction is low among the users. It is also reported that often, the seaweed products, particularly liquid formulations, become less stable over time (Plants 2021, 10, 531).

[0009] Another major challenge is that the price of many of the bio-stimulant products that are currently available in the market remains high and clearly not affordable for all categories of farmers. Further, as the currently used methodology in macroalgae harvesting could ultimately lead to product inconsistency due to seasonal and environmental variations, there exists a need for improved solutions implementing a solid industry allowing the development of consistent, efficient and economically viable products as bio stimulants.

[0010] Accordingly, there is a need to develop a stable bio-stimulant composition that exerts a substantial effect in stimulating plant growth, and which is more economical and affordable by all categories of farmers.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to a bio stimulant composition comprising seaweed extract, milk solids, one or more amino acid and optionally one or more of the ingredients selected from protein hydrolysate, humic substance, macronutrient and micronutrient.

[0013] In an embodiment, the composition may comprise seaweed extract, milk solids, amino acids, protein hydrolysate and humic substance.

[0014] In an embodiment, the composition may comprise seaweed extract, milk solids, amino acids, micronutrients and macronutrients.

[0015] The composition may further comprise one or more of the ingredients selected from stabilizer, emulsifier, spreading agent and preservative,

[0016] In an embodiment, the seaweed extract and the milk solids in the composition may be fermented seaweed extract and fermented milk solids respectively.

[0017] The seaweed extract and the milk solids may be fermented by an inoculum / culture of lactic acid bacteria (LAB).

[0018] The Lactic acid bacteria (LAB) may be selected from Lactobacillus spp. , Streptococcus spp., Pediococcus spp., Leuconostoc spp and combination thereof.

[0019] The bacterial inoculum used may be in an amount of 0.005 v / v % to 0.9 v / v %.

[0020] The present invention, the seaweed extract may be in an amount of 0.6 w / w% to 75w / w%, preferably 0.6 w / w% to 50 w / w%, milk solids in an amount of 0.5 w / w% to 55w / w%, amino acid in an amount of 0.02 w / w%to 32 w / w%, stabilizers in an amount of 0. 1 w / w% to 20w / w%, protein hydrolysate in an amount of 0w / w% to 18w / w%, humic substances in an amount of 0.2 w / w% to 5w / w%, macronutrients in an amount of 0.001 to 15 w / w%, micronutrients in an amount of 0.001 to 10 w / wt%, emulsifiers in an amount of 0.2 w / w% to 6 w / w%, preservatives in an amount of 0. 1 w / w% to 4 w / w%.

[0021] The biostimulant composition may comprise one or more substances selected from alginic acid, betaines, amino acids, fucoidan and mannitol, preferably alginic acid.

[0022] The alginic acid content may be in an amount of 0.001 w / w% to 15w / w% per total weight of the composition.

[0023] The seaweed may be selected from Ascophyllum nodosum, Ecklonia maxima, Fucus vesiculosus, Sargassum sp., Hydroclathrus spp., Laminaria digitata, Macrocystispyrifera, Nereocystis Sp. and Cystoseira Sp and combination thereof. Preferably the seaweed may be Ascophyllum nodosum.

[0024] The milk solids may be selected from fermented milk powder, dried milk powder, whey powder, whey proteins, caseinates, milk proteins, skim milk and combination thereof.

[0025] The amino acid may be selected from L-lysine, L-arginine, L-leucine, L-valine, L- glutamic acid, L-glycine, L-methionine, L-proline, L-tryptophan, L-threonine, L- serine, L-isoleucine, L-phenyl-alanine, L-alanine, L- cysteine, L-tyrosine, L-histidine, L-aspartic acid and combination thereof.

[0026] The protein hydrolysate may be selected from soybean meal, microalgal extracts, com gluten, fish meal, chicken feather meal, slaughterhouse waste and untreated hairs / wool.

[0027] The humic substance may be selected from potassium humate, fillvic acid, humin, hymetomalonic acid and combination thereof.

[0028] The macronutrient may be one or more selected from nitrogen (N) in an amount of 0. 1 to 10w / w% , phosphoms(P) in an amount of 0.01 to lOw / w, iron (Fe) in an amount of 0.001 to 15 w / w%, potassium(K) in an amount of 0.01 w / w%to 10 w / w%, copper (Cu) in an amount of 0.001 to 10w / w%, and combination thereof. The micronutrient may be selected from boron (B) in an amount of 0.001 to 10w / w%, manganese (Mn) in an amount of 0.001 to 10w / w%, zinc (Zn) in an amount of 0.001 to 10w / w%, magnesium (Mg) in an amount of 0.001 to 5w / w%, molybdenum (Mb) in an amount of 0.001 to 10 w / w% and combination thereof.

[0029] The composition may be in liquid form, free flowing powder from, granular form or in the form of tablets, noodles, sticks, pellets, or capsules.

[0030] In another embodiment, the present invention provides a method of preparing a bio stimulant composition. The method comprises the steps of dissolving milk solids in water and heating the mixture, stirring said mixture for sufficient time and cooling the mixture, adding stabilizers to said mixture and maintaining the solution at suitable pH, adding an inoculum containing lactic acid bacteria (LAB) and fermenting the solution for sufficient time, at suitable pH, to form fermented milk solids adding seaweed extract, adding amino acids to the mixture further fermenting the solution obtained, optionally adding a fermentation arrester to said mixture, sieving the solution to obtain free-flowing viscous liquid biomass, optionally adding one or more of humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients.

[0031] Said method may comprises the steps of dissolving milk solids in water and heating the mixture at 80°C to 90°C for 1 to 3 hours, stirring said mixture at a 100- 150 RPM for 2 to 3 hours and cooling at temperature 30 to 36°C, adding 0.1 to 20 w / w% of stabilizers to said mixture and maintaining the pH of solution at 4 to 5, adding 0.005 w / w% to 0.9 % w / w% of inoculum containing lactic acid bacteria (LAB) and fermenting the solution for 12 to 15 hours, at pH 2 to 5, adding 0.6 w / w% to 75w / w% seaweed extract at pH 2 to 5, adding 0.02 % to 32%w / w amino acids to the mixture, further fermenting the solution at temperature 30 to 32°C for 2 to 72 hours, adding 0.1% w / w%, to 20% w / w% of fermentation arrester to said mixture, sieving the solution obtained, through 200 to 250-micron size sieve and obtaining a free-flowing viscous liquid biomass, optionally adding one or more of, humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients. The spreading agent may be selected from Tween 20 (Polysorbate 20). Tween 80 (Polysorbate 80), Span 20 / 60 / 80 (Sorbitan esters, less hydrophilic), Ethoxylated castor oil (PEG castor oil derivatives), Alkyl polyglucosides, Fatty acid ethoxylates (PEG-6 caprylic / capric glycerides) and Polyalkyleneoxide modified Heptamethyl Trisiloxane

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a stable bio-stimulant composition that can be applied to various crop plants, which demonstrates good performance with respect to various parameters related to the growth and development of the plants to which it is applied, at an affordable cost. It is a further object of the invention to provide a bio-stimulant composition that enhances root and shoot development, nutrient absorption, crop yield and quality, resistance to biotic and abiotic stress, disease resistance and resistance to pest attacks. It is also the objective of the invention to provide a method to produce such bio-stimulant composition.

[0034] Accordingly, in an embodiment, the present invention provides a stable and sustainable bio-stimulant composition comprising seaweed extract and milk solids that substantially stimulates plant growth and development. In an embodiment, the present invention also provides a method to produce said bio-stimulant composition.

[0035] An embodiment of the present invention provides a stable and sustainable bio-stimulant composition comprising seaweed extract and milk solids along with micronutrients and macronutrients that substantially stimulate plant growth and development.

[0036] In one embodiment, the bio stimulant composition comprises a seaweed extract, milk solids, at least one amino acid, and optionally comprises protein hydrolysate, humic substances, macronutrients, and micronutrients. The composition may be formulated for agricultural use to enhance plant growth and health.

[0037] The bio-stimulant composition of the present invention surprisingly results in significant improvement in various aspects of plant growth and development and has a very good shelf life. The said bio-stimulant composition releases its nutrients in small and simple molecular forms which promotes faster absorption of nutrients when applied on plants (stomata / leaves / roots / branches / axial buds / fruit / flower). During fermentation, microorganisms break down complex organic molecules into simpler forms, releasing bioactive compounds such as amino acids, peptides, and organic acids. Due to the fast absorption of nutrients (in free form), faster growth results are observed in fields. Thus, the bio-stimulant composition of the present invention results in a synergistic effect.

[0038] This effect is surprising because, though milk has long been valued for its nutritional content, the effect of milk as plant bio stimulant has been less explored. Though milk may have the potential to act as natural growth promoters and stress alleviators, the application of milk as an effective plant bio stimulant has not yet been demonstrated and has not been found promising.

[0039] In an embodiment, the composition comprises seaweed extract, milk solids, amino acids, protein hydrolysate, and humic substances.

[0040] In another embodiment, the composition comprises seaweed extract, milk solids, amino acids, micronutrients, and macronutrients. This formulation is tailored to supply essential nutrients for optimal plant development.

[0041] In another embodiment, the composition further comprises one or more of the ingredients selected from stabilizer, emulsifier, spreading agent and preservative to enhance shelf life, stability, and ease of application and effectiveness of the composition.

[0042] In one embodiment, the milk solids and / or the seaweed extract are fermented by employing an inoculum or culture of lactic acid bacteria (LAB) to improve the bioavailability of nutrients and promote beneficial microbial activity. The lactic acid bacterium may be selected from Lactobacillus spp., Streptococcus spp., Pediococcus spp., Leuconostoc spp and combination thereof. The Lactic acid bacteria (LAB) used for fermentation may be selected from one or more of Lactobacillus delbrueckii subsp. Bulgaricus, Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Streptococcus lactis, Streptococcus cremoris, Leuconostoc mesenteroides, Leuconostoc lactis, Pediococcus acidilactici, Pediococcus pentosaceus or mixed cultures comprising of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc spp. or other suitable species known for their fermentative capabilities. In an embodiment, the lactic acid bacteria used for fermentation may be of the genus Lactobacillus.

[0043] In an embodiment, the bacterial inoculum used is in an amount of 0.005 v / v % to 0.9 v / v%, ensuring effective fermentation and nutrient conversion.

[0044] In an embodiment, the seaweed extract may be present in an amount of 0.6 w / w% to 75 w / w%, preferably 0.6 w / w% to 50 w / w%, milk solids in an amount of 0.5 w / w% to 55w / w%, amino acid in an amount of 0.02 w / w% to 32 w / w%, protein hydrolysate in an amount of 0w / w% to 18w / w%, humic substances in an amount of 0.2 w / w% to 5w / w%, macronutrients in an amount of 0.001 to 15 w / w%, micronutrients in an amount of 0.001 to 10 w / wt%, stabilizers in an amount of 0. 1 w / w% to 20w / w%, emulsifiers in an amount of 0.2 w / w% to 6 w / w%, preservatives in an amount of 0.1 w / w% to 4 w / w%.

[0045] In an embodiment of the present invention, the seaweed extract used in the biostimulant composition may be obtained from brown seaweed. In an embodiment, the seaweeds may be selected from one or more of Ascophyllum nodosum, Ecklonia maxima, Fucus vesiculosus, Sargassum sp., Hydroclathrus spp., Laminaria digitata, Macrocystispyrifera, Nereocystis Sp. and Cystoseira Sp. In an embodiment of the invention, the brown seaweed is Ascophyllum nodosum.

[0046] In an embodiment, the composition may comprise one or more substances selected from alginic acid, betaines amino acids, fucoidan, mannitol and uronic acid, which may be contained in the sea weed extract.

[0047] In an embodiment, the alginic acid may be present in an amount of 0.001 w / w% to 15w / w% of the total weight of the composition. Alginates or alginic acid is a hydrophilic anionic polysaccharide. Alginic acid has good bioactivity and biocompatibility, and understood to have various health benefits. Brown seaweed serves as the primary source of alginates.

[0048] In an embodiment of the invention, the milk solids may comprise milk powder, dried milk products such as whey, whey powder, whey proteins, caseinates, milk proteins, skim milk etc.

[0049] In an embodiment, the L- Amino acids may be at least one from the group consisting of L-Lysine, L-Arginine, L-Leucine, L-Valine, L- Glutamic acid, L-Glycine, L- Methionine, L-Proline, L-Tryptophan, L-Threonine, L-Serine, L-Isoleucine, L- Phenyl-alanine, L-Alanine, L- Cysteine, L-Tyrosine, L-Histidine and L-Aspartic acid and combination thereof. In a preferred embodiment, L-Glycine may be used.

[0050] In an embodiment, the protein hydrolysates may be sourced from at least one from the group consisting of soybean meal, microalgal extracts, com gluten, fish meal, chicken feather meal, slaughterhouse waste and untreated hairs / wool.

[0051] In an embodiment, the humic substances may be selected from at least one of potassium humate, fulvic acid, humin, hymetomalonic acid. The humic substances may be preferably potassium humate.

[0052] In an embodiment, the macronutrient may be selected from nitrogen (N), phosphorus (P), iron (Fe), potassium (K) and copper (Cu). Nitrogen (N) may be present in an amount of 0. 1 to I0w / w%, phosphorus in an amount of 0.01 to 10w / w%, iron (Fe) in an amount of 0.001 to 15 w / w%, potassium in an amount of 0.5 w / w% to 10 w / w and copper (Cu) in an amount of 0.001 to 10w / w%.

[0053] The composition of the invention may contain micronutrients such as boron (B), manganese (Mn), zinc (Zn), magnesium (Mg), molybdenum (Mb). Boron (B) may be present in an amount of 0.001 to 10w / w%, manganese (Mn) in an amount of 0.001 to 10w / w%, zinc (Zn) in an amount of 0.001 to 10w / w%, magnesium (Mg) in an amount of 0.01 to 10w / w% and molybdenum (Mb) in an amount of 0.001 to 10 w / w%. These may be in the elemental form or in the form of complexes or compounds such as Cu-EDTA, Fe-EDTA, Zn-EDTA, Mn-EDTA, Mg-EDTA, zinc sulphate heptahydrate, ferrous sulphate heptahydrate, manganese sulphate monohydrate, copper sulphate pentahydrate, potassium nitrate, solubor® (20% Disodium Octaborate Tetrahydrate), sodium molybdate, calcium propionate, glycerine / glycerol, lactic acid, urea etc.

[0054] In an embodiment, the bio-stimulant composition may be prepared in liquid form which may be used as foliar spray, seed treatment or soil drench.

[0055] In a further embodiment, the bio-stimulant composition may be prepared in granular form.

[0056] In still another embodiment, the bio-stimulant composition may be prepared in powder form.

[0057] The foliar application of the present bio-stimulant composition on ornamental crops with larger leaf area, directly delivers the composition to the target crop region through stomata and cell walls on the surface of the leaf thereby leading to rapid absorption and transportation of nutrients, increasing plant height, stem diameter, dry weight, enhancing photosynthesis rate in plants by increasing the leaf area, enhances stomata conductance by increased carbon assimilation rate per unit leaf area, and thus have an improved effect on plant height and root length. It further increases their resistance against water stress, salt alkali stress, and low temperature stress. It improves the quality of crops, their taste, nutrition, color characteristics, enhances the nutritional value of crops by increasing the protein content, amino acid content, vitamin content, and increases their antioxidant enzyme activity.

[0058] Moreover, the present bio-stimulant composition stimulates seed germination, enhances root growth, their nutrient uptake capacity and their ability to use stored energy, enhances soil microbial and enzymatic activities leading to an improvement in biological fertility of plant, enhances cell membrane stability, provides better root to shoot ratio, accumulates osmolytes, stimulates antioxidant defense system, improves fruiting and shoot growth in plants. It also improves color, texture, and nutritional value (e.g., phytochemicals) of fruits and vegetables, reduces crop loss caused by adverse soil conditions and environmental stress. Also promotes stress tolerance under drought conditions.

[0059] It also reduces the requirement of nitrogen administration to the soil and stimulates plant growth and yield by increasing nutrient uptake, increasing cellular respiration, photosynthesis, protein synthesis, and enzyme activity. It further promotes protection of plants against stress events, increases the activity of peroxidase (POX), catalase (CAT), and phenyl alanine ammonium lyase (PAL) enzymes, improves fruit yield and quality by increasing total sugar content and the amount of oil, protein, and fibre, among others, improves soil structure and fertility, improves root development.

[0060] An embodiment of the present invention provides method to entrap the developed biostimulant within the matrix of bio-available mineral carriers such as dolomite and zeolite materials to compose granular formulations.

[0061] The present invention discloses the combination of milk solids and seaweeds along with other ingredients, in a defined range which targets the bio-synthetic pathways within the plants to generate specific plant growth promoting molecules.

[0062] The invention provides a stable bio-stimulant composition that may be applied to a plant or a part thereof including seed, leaves, roots, shoots, flowers, fruits and so on. The composition of the present invention promotes germination of treated seeds, enhances white root developments, increases root length, shoot length, height, stem diameter, dry weight leaf area, relative water and chlorophyll content, promotes early maturation, increases no. of flowers, improves quality of fruits, increases yield, and combinations thereof. It also increases protein, amino acid and vitamin content thereby enhancing the nutritional value, increasing sugar and vitamin content of the fruit.

[0063] Further the bio-stimulant composition of the present invention exerts its effects on plant health by influencing internal factors such as increase in chlorophyll content which in turn leads to enhanced rate of photosynthesis thereby promoting overall health of the plant, increased proline concentrations leading to the plant’s ability to fight draught stress. Moreover, the composition of the present invention increases resistance to abiotic stress, like water stress, salt alkali stress, and low temperature stress as shown by the study of biochemical and physiological parameters such as glycine betaine, total soluble sugar, lipid peroxidation, salt tolerance index, and increased hormone synthesis of the plants treated with the present bio-stimulant composition. The application of the bio-stimulant composition further reduces the incidence of diseases and thereby improves the yield and quality of crop plants.

[0064] In an embodiment, the bio-stimulant composition comprises seaweed extract in an amount of 0.6 w / w% to 75w / w%, preferably 3 w / w% to 50 / w% and further preferably 6 w / w% to 45 w / w%.

[0065] The bio-stimulant composition of the present invention may contain substances such as alginate / alginic acid, betaines, amino acids, fucoidan and mannitol from the seaweed extract. The alginic acid content may be 0.001 w / w% to 15w / w%.

[0066] In an embodiment, the bio-stimulant composition comprises milk solids in an amount of 0.5 w / w% to 55 w / w% of the total weight of the composition. The milk solids of the present composition are fermented using inoculation of 0.005 % to 0.9 % of Lactic Acid Bacteria (LAB).

[0067] In another aspect the bio-stimulant composition of the present invention may further comprise 0.2 % to 5 % of humic substances, 0 % to 18 % of hydrolysed proteins; 1 % to 20 % of stabilizers; 0.2 % to 6 % of emulsifiers; 0.1 % to 4 % of preservatives; 1% to 18 % of buffering agent; 2% to 20% of fermenter arrester.

[0068] Humic substances are complex organic compounds formed from the decomposition of plant and animal residues. The humic substance in the present invention may be potassium humate, fillvic acid or humin, hymetomalonic acid, preferably potassium humate.

[0069] The bio stimulant composition of the invention may contain stabilizer which may protect the active ingredients from degradation and oxidation up on storage. The stabilizers may be selected from group consisting of pectin, chitosan, carboxymethyl cellulose, sago starch, agar-agar, sodium alginate, carrageenan, propylene glycol alginate, gelatine, silica, polyvinyl alcohol, citric acid and ethyl cellulose.

[0070] The bio stimulant composition of the invention may contain an emulsifier, may help the components to mix with water. The emulsifiers may be selected from polysorbate 20, 60, 80, lecithin, xanthum gum, sorbitan esters, glycerol monostearate, mono / di glycerides, and PEG-40.

[0071] The preservatives may be selected from calcium propionate, nipacide BIT 20, lactic acid, sodium benzoate, potassium sorbate, sorbic acid, methyl paraben, propyl paraben, phenoxy ethanol, benzyl alcohol, gluconolactone, isothiazolinone, sodium metabisulfite, and caprylyl glycol.

[0072] The buffering agents may be at least one or combinations of dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium citrate, sodium fumarate, sodium carbonate and 2-hydroxypropanoic acid.

[0073] The fermentation arrester may be at least one from the group of potassium sorbate, methyl hydroxyl benzoate, ethyl hydroxyl benzoate, propyl hydroxyl benzoate, and sodium benzoate.

[0074] In an embodiment, the composition contains seaweed extract in an amount of 0.6 w / w% to 75 w / w%, preferably 0.6 w / w% to 50 w / w%, milk solids in an amount of 0.5 w / w% to 55 w / w%, amino acids in an amount of 0.02 w / w% to 32 w / w%, stabilizers in an amount of 0.1 w / w% to 20 w / w%, protein hydrolysate in an amount of 0 w / w% to 18 w / w%, humic substances in an amount of 0.2 w / w% to 5 w / w%, emulsifiers in an amount of 0.2 w / w% to 6 w / w%, preservatives in an amount of 0. 1 w / w% to 4 w / w%, and the balance amount of demineralized water.

[0075] In a further embodiment, the composition contains seaweed extract in an amount of 0.6 w / w% to 75 w / w%, preferably 0.6 w / w% to 50 w / w%, milk solids in an amount of 0.5 w / w% to 55 w / w%, amino acids in an amount of 0.5 w / w% to 32 w / w%, stabilizers in an amount of 0.1 w / w% to 20 w / w%, protein hydrolysate in an amount of 0 w / w% to 18 w / w%, macronutrients in an amount of 0.001 - 15 w / w%, micronutrients in an amount of 0.001-10w / w% emulsifiers in an amount of 0.2 w / w% to 6 w / w%, preservatives in an amount of 0. 1 w / w% to 4 w / w%, and the balance amount of demineralized water.

[0076] In an embodiment, the macronutrient may be selected from nitrogen (N), phosphorus (P), iron (Fe), potassium (K) and copper (Cu). Nitrogen (N) may be present in an amount of 0. 1 to 10w / w%, phosphorus in an amount of 0.01 to 10w / w%, iron (Fe) in an amount of 0.001 to 15 w / w%, potassium in an amount of 0.01 w / w% to 10 w / w and copper (Cu) in an amount of 0.001 to 10w / w% to provide essential nutrients for plant growth.

[0077] The composition of the invention may contain micronutrients such as boron (B), manganese (Mn), zinc (Zn), magnesium (Mg), molybdenum (Mb). Boron (B) may be present in an amount of 0.001 to 10w / w%, manganese (Mn) in an amount of 0.001 to 10w / w%, zinc (Zn) in an amount of 0.001 to 10w / w%, magnesium (Mg) in an amount of 0.001 to 10w / w% and molybdenum (Mb) in an amount of 0.001 to 10 w / w% to support plant physiological functions. The macronutrient and micronutrients may be in the elemental form or in the form of complexes or compounds such as Cu-EDTA, Fe- EDTA, Zn-EDTA, Mn-EDTA, Mg-EDTA, zinc sulphate heptahydrate, ferrous sulphate heptahydrate, manganese sulphate monohydrate, copper sulphate pentahydrate, potassium nitrate, solubor® (20% Disodium Octaborate Tetrahydrate), sodium molybdate, calcium propionate, glycerine / glycerol, lactic acid, urea etc.

[0078] In another embodiment, nitrogen (N) may be derived from amino acids, protein hydrolysates, technical grade urea , fermented seaweed extract, phosphorous (P) in the form of phosphorus pentoxide is (P2O5) from Dipotassium hydrogen phosphate, (Fe) from Fe-EDTA, Ferrous sulphate heptahydrate, potassium (K) from potassium humate, potassium sorbate, Dipotassium hydrogen phosphate, potassium nitrate, (Cu) from Copper sulphate pentahydrate, Cu-EDTA.

[0079] In another embodiment, the present invention provides a method of preparing a bio stimulant composition. The method comprises the steps of dissolving milk solids in water and heating the mixture, stirring said mixture for sufficient time and cooling the mixture, adding stabilizers to said mixture and maintaining the solution at suitable pH, adding an inoculum containing lactic acid bacteria (LAB) and fermenting the solution for sufficient time, at suitable pH, to form fermented milk solids adding seaweed extract, adding amino acids to the mixture further fermenting the solution obtained, optionally adding a fermentation arrester to said mixture, sieving the solution to obtain free-flowing viscous liquid biomass, optionally adding one or more of humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients.

[0080] In further embodiment the present invention provides the method for preparation of liquid bio-stimulant composition. The method comprises the steps of:

[0081] 1. Dissolving the milk solids in required quantity of water with constant stirring in a clean fermenter and heating the mixture at 80°C to 90°C for about 1-3 hours

[0082] 2. Stirring the mixture at 100-150 RPM obtained in step 1 for about 2 to 3 hours and cooling the mixture bringing the temperature down to 30 to 36°C and adding 1% to 20% of stabilizers to said mixture.

[0083] 3. Adding 0.1 to 20 w / w% of stabilizers to the solution of step 2 to maintain the pH of the solution around 4 to 5.

[0084] 4. Adding 0.005 % to 0.9 % w / w of inoculum containing lactic acid bacteria (LAB) to the solution obtained in step 3 and stirring the solution obtained thoroughly for 1 to 2 hours ensuring proper mixing

[0085] 5. Fermenting the solution in step 4 and concentrating the solution for about 12 to 15 hours and maintaining the pH of the solution at 2-5, ensuring proper fermentation of milk solids.

[0086] 6. Adding a required quantity of seaweed extract and amino acids to the fermented milk solid solution obtained in step 5 at pH 2-5 and obtaining a mixture of seaweed biomass and fermented milk solids and fermenting the mixture in the fermenter at temperature of about 30 to 32°C for 2 to 72 hours;

[0087] 7. adding 0.1% to 20% of fermentation arrester in said mixture obtained after 0 to 72 hours based on the requirement of the formulation.

[0088] 8. Passing the mixture obtained in step 6 or 7 through 250-micron size sieve to get a free-flowing viscous biomass

[0089] 9. optionally adding humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients as per the required composition resulting in the bio- stimulant composition. In an embodiment, the method further comprises drying, preferably spray drying the liquid bio stimulant composition at temperature 90 to 300°C and obtaining powdered bio stimulant with particle size of 10 to 100 microns.

[0090] In an embodiment, the method further comprises adding the liquid bio stimulant composition to a mineral carrier of size 1000-6000 micron in an amount of 2 w / w% to 8 w / w% and rotating with 75-150 RPM 2 to 4 hours followed by drying, preferably in a fluid bed dryer, to obtain the bio stimulant composition in granular form.

[0091] The mineral carrier may be selected from bentonite, zeolite, dolomite or mixture of zeolite and dolomite.

[0092] The pH may be maintained by the addition of buffering agent in an amount of lw / w% to 18w / w%, wherein the buffering agent is selected from dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium citrate, sodium fumarate, sodium carbonate, dipotassium hydrogen phosphate, 2-hydroxypropanoic acid, and combination thereof.

[0093] The fermentation arrester may be added in an amount of 2w / w% to 20w / w% wherein the fermenter arrester is selected from potassium sorbate, calcium propionate, sodium bicarbonate, methyl hydroxyl benzoate, ethyl hydroxyl benzoate, propyl hydroxyl benzoate, sodium benzoate and combination thereof.

[0094] The spreading agent can be selected from Tween 20 (Polysorbate 20). Tween 80 (Polysorbate 80). Span 20 / 60 / 80 (Sorbitan esters, less hydrophilic), Ethoxylated castor oil (PEG castor oil derivatives), Alkyl polyglucosides, Fatty acid ethoxylates (PEG-6 caprylic / capric glycerides) and Polyalkyleneoxide modified Heptamethyl Trisiloxane.

[0095] A spreading agent can enhance the water absorption efficiency of hard to wet soils by increasing their water infiltration, drainage, water retention and hydrophilicity. It can help in overcoming challenges including low uptake, poor absorption, leaching, and runoff of the broadcasted fertilizers, while improving performance and efficiency. It also improves soil-air exchange capacity - favourable aerobic conditions for soil microbes- metabolic processes. It provides better uniformity, soil wetting, and better nutrient uptake. It also reduces hydrophobicity of soils thereby boosting water infdtration and thus less water use. Low dose rates may be required thereby more effective in less applications. It can promote feeder root. Silicon-based super spreaders are highly effective in foliar applications due to their ability to enhance the performance of agrochemicals. Some of its advantages may be:

[0096] Reduced Surface Tension for Better Coverage: Super spreaders, typically formulated with silicone-based surfactants, significantly lower the surface tension of spray solutions. This allows the liquid to spread more evenly across plant surfaces, including waxy or hydrophobic leaves, ensuring uniform coverage. Thus the spreading agent enhances agrochemical performance by improving penetration, adhesion, and rainfastness. It reduces application volume, increases efficacy, and offers environmental and equipment benefits. Its versatility across crops and applications also supports better plant health.

[0097] The application of the biostimulant composition may be through foliar spray.

[0098] In an embodiment the composition of the present invention can promote germination of treated seeds, enhances white root developments, increases root length, shoot length, height, stem diameter, dry weight leaf area, relative water and chlorophyll content, promotes early maturation, increases no. of flowers, improves quality of fruits, increases yield, enhances the nutritional value, sugar and vitamin content of the fruit, increases protein, amino acid and vitamin content, increase in chlorophyll content, enhance rate of photosynthesis, promote overall health of the plant, increase proline concentrations, enhance plant’s ability to fight draught stress, increases resistance to abiotic stresses water stress, salt alkali stress, and low temperature stress, increase glycine betaine, total soluble sugar, lipid peroxidation, salt tolerance index, and increased hormone synthesis. Increases disease resistance, improves the yield and quality of crop plants.

[0099] The bio-stimulant composition as per the present invention stimulates vegetative growth, uniform panicle initiation and enhanced quality grains which includes bright and vivid colored grains, better nutrient composition, increased bulk density, pleasant odor, increased size and uniform shape. It further improves fruit appearance quality such as fruit shape, fruit size, fruit color, fruit gloss, flavour, aroma, sugar-acid ratio, nutrient composition, storage and transportation performance, etc. Ensures complete germination of seeds and fastens the root and shoot development from the seeds. The present composition further stimulates fine root growth and thereby increases the capacity of nutrient uptake by roots, enhances shoot length, increases leaf area, promotes early maturation, increases no. of flowers, increases crop yield, and combinations thereof.

[0100] The bio-stimulant composition prepared using the method, may be a free-flowing liquid, which may be a blackish brown liquid with pH ranging from about 3.5-10 with specific gravity ranging from about 1.0- 1.3 g / ml.

[0101] Physical and chemical parameters of the developed liquid bio stimulant composition may be as shown in Table A. Table A

[0102] In an aspect, the present invention provides the method to prepare the granular biostimulant composition. The method for granular bio stimulant includes preparing the liquid bio- stimulant as described in process steps above and feeding said liquid bio- stimulant in a vessel followed by below steps.

[0103] The method involves preparing granules of specific size of about 1000-6000 micron from Bentonite or Zeolite or Dolomite or mixture of Zeolite and Dolomite and feeding the same in the coating pan. Rotating the coating pan for about 2 to 4 hours along with the definite spray of said liquid composition. The liquid spray can be between 2 to 8 % w / w of granular formulation. The liquid composition is evenly coated on the granules and further dried in Fluid Bed Dryer to get bio-stimulant in granular form.

[0104] Physical and chemical parameters of the developed Bentonite based bio stimulant composition are as shown in Table B below. Table B

[0105] Physical and chemical parameters of the developed Zeolite and Dolomite based bio Stimulant composition are as shown in Table C below.

[0106] Table C

[0107]

[0108] In an aspect, the present invention provides the method to prepare a free-flowing fine powdered bio-stimulant composition. The method to produce the bio stimulant powder formulation includes preparing the liquid bio-stimulant as described above and spray drying the prepared liquid composition at a temperature ranging from 90-300°C. The particle size of the p o w de re d bio stimulant ranges from 10-100 microns. This powder may be further developed into various forms such as tablets, noodles, sticks, pellets, capsules, and any solid form in which biostimulants can be used. Physical and chemical parameters o f the developed powdered bio stimulant composition are as shown in Table D below. Table D

[0109] It should be understood from the above table that all the parameters included above are derived from seaweed fermented biomass in which the fermentation has taken place in the presence of milk solids. EXAMPLES

[0110] The examples below are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention.

[0111] Biological Material: The seaweed used in the present invention were sourced from Algea the Artic Company, Algea AS of Omagata 78, 6517, Kristiansund, Norway

[0112] Source for LAB: Bulteh 2000 Ltd, 19, Industrial area, Zagora, Bulgaria

[0113] Example 1 - Preparation of liquid bio stimulant composition

[0114] A composition was prepared in the following manner. 480 kg of Skim Milk powder were dissolved in 4KL demineralized water followed by heating the mixture to 85 °C for a duration of two hours. The heated mixture was subjected to continuous stirring at 1000 revolutions per minute (RPM) for two hours and subsequently cooled to a temperature of 32°C. Calcium propionate was added as a preservative, and the pH of the mixture was adjusted to 4.5. Thereafter, the mixture was inoculated with Lactobacillus acidophilus at a concentration of 0.1% w / w and fermented for 14 hours under controlled conditions, maintaining the pH at 3.5. The mixture in the fermenter is kept in closed conditions without contact of air and also kept undisturbed without any agitation. The above mixture obtained contained fermented milk solid. Upon completion of the initial fermentation, 1.2% w / w of the seaweed extract derived from Ascophyllum nodosum with an alginic acid content 0.05%w / w was added, with the pH maintained at 3.5, Glycine 0.7% was then added into the mixture, which was subjected to further fermentation for 24 hours at 32°C. This was followed by the addition of a 0.28%w / w Benzisothiazolinone (fermentation arrester) *

[0115] [* In another variation, the above method was performed only with the exception that the fermentation arrestor was not added] .

[0116] The resulting solution was filtered through a mesh of 220 microns to obtain a free- flowing, viscous liquid biomass. Ingredients selected from emulsifiers, stabilizers, preservatives, spreading agent, macronutrients, and micronutrients were added, as per the requirements of different formulations and thoroughly mixed to yield the final composition. The final compositions prepared as per the above protocol are designated as Formulations 1, 2, 3, 4 and 5. All formulations were prepared under identical controlled conditions to ensure consistency, with the primary variation being the nutrient composition.

[0117] Example 2 - Preparation of granular bio stimulant composition

[0118] Preparation of a granular bio-stimulant composition involved the following steps. First, a liquid bio-stimulant composition as described in Example 1 was prepared. It was subsequently fed into a coating vessel. Granules having a particle size ranging from approximately 1000 to 6000 microns were prepared from bentonite, zeolite, dolomite, or a mixture of zeolite and dolomite, and were introduced into a rotating coating pan. The coating pan was operated for a duration of 2 to 4 hours while the liquid biostimulant composition was sprayed uniformly onto the granules at a concentration ranging from 2% to 8% w / w relative to the granular formulation. The sprayed liquid composition was evenly distributed over the surface of the granules, which were then subjected to drying in a fluid bed dryer to yield a granular bio-stimulant product.

[0119] Example 3 - Preparation of powder bio stimulant composition

[0120] Preparation of free-flowing fine powdered bio-stimulant composition involved the following steps. First, a liquid bio-stimulant composition as described in Example 1 was prepared. The liquid bio stimulant was subsequently subjected to spray drying at a temperature ranging from 150°C -200°C. The resulting powdered bio-stimulant exhibited a particle size of 10 to 100 microns.

[0121] Example 4 - Formulations as per the present invention

[0122] Different formulations as per the present invention were prepared as described in Example 1. These formulations according to the invention are designated as Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, and Formulation 6. For comparison purposes, formulations were prepared and designated as Formulation A, Formulation B, Formulation C, Formulation D, Formulation E, and Formulation F respectively without fermented milk solids.

[0123] The details of the formulations are provided below. Table 1

[0124] Table 2

[0125] Table 4

[0126] Table 5 Table 6 The physical and chemical parameters of all the formulations of table 1-5 are given below in Table 7.

[0127] Table 7

[0128] 5 Example 5.1 - Field evaluation of the bio efficacy of the present Bio stimulant composition in corn crop

[0129] Formulation (FORMULATION 1) was used to conduct the experiment.

[0130] An experiment was conducted at Nashik - 422 303, Maharashtra in a randomized

[0131] 10 block design to assess the growth promoting activity of bio stimulant composition in sweet com crop of Namdhari sweet variety.

[0132] The experiment was conducted with present bio stimulant with five treatments comprising 3 doses of FORMULATION 1, along with FORMULATION A and 15 untreated control in four replications for comparison. For purposes of convenience, the present invention composition has been represented by the code FORMULATION 1 containing milk solids and FORMULATION A without milk solids. As per requirement of the treatments, product was applied as foliar spray as mentioned below.

[0133] 20 TREATMENT DETAILS:

[0134] Table 8

[0135] The following parameters were measured: 1) Plant height at 15 days after 2ndspray : ) Plant height at 15 days after 2ndspray :

[0136] 3) Number of leaves per plant at 15 days after 1stspray ) Numbers of grains per 30 cobs at harvest

[0137] 5) Yield (Q / ha) at harvest Data generated from above observations mentioned were compiled and used for statistical analysis

[0138] Table 9 - Effect of Bio stimulant composition (FORMULATION 1) on growth and yield parameters of corn

[0139] *Mean of four replications. Values in parentheses denote percent increase over untreated control.

[0140] Example 5.2 - Field evaluation of the bio efficacy of the present Bio stimulant composition in tomato crop

[0141] Formulation (FORMULATION 2) was used to conduct the experiment.

[0142] An experiment was conducted atNashik - 422 303, Maharashtra in a randomized block design to access the growth promoting activity of bio stimulant composition in tomato crop of Abhinav variety.

[0143] The experiment was conducted with present biostimulant with five treatments comprising 3 doses of FORMULATION 2, along with FORMULATION B and untreated control in four replications for comparison. For purposes of convenience, the present invention composition has been represented by the code FORMULATION 2 containing milk solids and FORMULATION B without milk solids. As per requirement of the treatments product was applied in soil after planting as mentioned below.

[0144] TREATMENT DETAILS:

[0145] Table 10

[0146] The following parameters were measured: ) Plant height at 80 days of planting ) Number of Branches at 80 days of planting ) Plant shoot biomass at 80 days of planting ) Plant root biomass at 80 days of planting ) Number of fruits per plant at harvest ) Number of fruits per plant at harvest

[0147] Data generated from above observations mentioned were compiled and used for statistical analysis.

[0148] Table 11 - Effect of Biostimulant composition (FORMULATION 2) on growth and yield parameters of Tomato

[0149] *Mean of four replications, values in parentheses denote percent increase over untreated control.

[0150] EXAMPLE 5.3 - Field evaluation of the bio efficacy of the present Biostimulant composition in potato crop

[0151] Formulation (FORMULATION 3) was used to conduct the experiments. An experiment was conducted at Meerut - 250 110, Uttar Pradesh in a randomized block design to access the growth promoting activity of biostimulant composition in potato crop of K Chipsona -3 variety.

[0152] The experiment was conducted with present biostimulant with five treatments comprising 3 doses of FORMULATION 3, along with FORMULATION C and untreated control in four replications for comparison. For purposes of convenience, the present invention composition has been represented by the code FORMULATION 3 containing milk solids and FORMULATION C without milk solids. As per requirement of the treatment product was applied to seed tubers uniformly just before planting.

[0153] TREATMENT DETAILS:

[0154] Table 12

[0155] The following parameters were measured: ) Emergence percentage at 30 days of planting ) Plant stand at Maturity ) Plant height at 60 days of planting ) Number of shoots at 60 days of planting ) Compound leaves per plant at 60 days of planting ) Chlorophyll content (SPAD value) at 60 days of planting ) Graded and total no. of potato (<25g , 25-75 g , >75 g) , 000 / ha at harvest ) Graded and total yield (<25g , 25-75 g , >75 g) , tons / ha at harvest ) Tuber dry matter content & yield at harvest 0) Dry biomass yield (t / ha)

[0156] Data generated from above observations mentioned were compiled and used for statistical analysis. Table 13- Effect of Biostimulant composition (FORMULATION 3) on growth parameters of Potato Var. K Chipsona-03

[0157] *Mean of four replications. Values in parentheses denote percent increase over untreated control.

[0158] Table 14 - Effect of Biostimulant composition (FORMULATION 3) on yield parameters of Potato Var. K Chipsona-03

[0159] (L,M,S - Large, Medium and Small tubers)

[0160] *Mean of four replications. Values in parentheses denote percent increase over untreated control. EXAMPLE 5.4 - Field evaluation of the bio efficacy of the present Biostimulant composition in Pomegranate crop

[0161] The obtained composition (FORMULATION 4) was used to conduct the various experiments. An experiment was conducted at Pandharpur - 413 304, Maharashtra in a randomized block design to access the growth promoting activity of biostimulant composition in 7- year-old uniform crop of Pomegranate crop of Bhagwa variety.

[0162] The experiment was conducted with present biostimulant with five treatments comprising 3 doses of FORMULATION 4, along with FORMULATION D and untreated control in four replications for comparison. For purposes of convenience, the present invention composition has been represented by the code FORMULATION 4 containing milk solids and FORMULATION D without milk solids. TREATMENT DETAILS:

[0163] Table 15

[0164] 5 The following parameters were tested:

[0165] 1) Flower numbers per tree: Total numbers of flowerers separately as male and bisexual were counted separately from each tree and from all replications. Mean value of each and total is used for statistical analysis. 0 2) Fruit set percentage: Fruit set was calculated using initial bisexual flowers and total fruits harvested per treatment and percentage value is used for statistical analysis.

[0166] 3) Fruit Size : Total fruits harvested were categorized into > 250 g weight and < 250 weight and percentage of > 250 g weight fruits were used for statistical analysis.

[0167] 4) Yield : The fruit yield was measured individual treatment wise and mean value from 5 all replications converted into kg per tree for statistical analysis.

[0168] 5) Juice percentage: The juice percentage from a concentrate was calculated by dividing the volume (wt) of the concentrate by the total volume (wt) of the diluted juice (concentrate + water) once mixed, then multiplied by 100 to get the percentage value. Mean value from 10 fruits per treatment and replication wise used for statistical analysis. 0

[0169] Table 16 - Effect of Biostimulant composition (FORMULATION 4) on quality and yield parameters of pomegranate crop

[0170]

[0171] *Mean of four replications. Values in parentheses denote percent increase value over untreated

[0172] EXAMPLE 5.5 - Field evaluation of the bio efficacy of the present Biostimulant composition in Rice crop An experiment was conducted at Ludhiana, Punjab state in a randomized block design to access the growth promoting activity of biostimulant composition in Rice crop on variety Pusa basmati 1121. The experiment was conducted with present biostimulant composition - 5 (FORMULATION 5) with five treatments comprising 3 doses of FORMULATION 5, along with FORMULATION E and untreated control in four replications for comparison. For purposes of convenience, the present invention composition has been represented by the code FORMULATION 5 containing milk solids and FORMULATION E without milk solids.

[0173] TREATMENT DETAILS:

[0174] Table 17

[0175] The following parameters were measured: ) Chlorophyll at flowering: A SPAD (Soil & Plant Analysis Development) value is a relative measurement of the amount of chlorophyll in a leaf. SPAD value (30 leaves / treatment) by recorded at flowering by SPAD 502 chlorophyll meter. Mean value from all replications used for statistical analysis ) Tillers / m2 at flowering: The total tillers of rice crop were counted in 1 m2 area per treatment and mean value from all replications used for statistical analysis. ) 1000 grain weight: The 1000 grain weight was counted from randomly selected grains at harvest, and the mean was expressed in g for statistical analysis. ) Grain yield: The grain yield was measured individual treatment wise and mean value from all replications converted into Q per hectare for statistical analysis.

[0176] Table 18: Effect of Biostimulant composition (FORMULATION 5) on growth and yield parameters of Rice

[0177] *Mean of four replications. Values in parentheses denote percent increase value over untreated. Example 6 - Synergistic activity of biostimulant composition

[0178] The following treatments were conducted to assess the synergistic potential of the developed biostimulant compositions.

[0179] Example 6.1 - Synergistic activity of biostimulant composition

[0180] FORMULATION 1 on maize

[0181] The synergistic activity of the biostimulant composition FORMULATION 1 was evaluated on maize (Zea mays) through a comparative study involving its individual constituent ingredients applied separately, as well as an untreated control.

[0182] A total of 10 distinct treatments were applied, with three replications, on maize variety PAC 751. The field experiment was conducted on plots measuring 5 x 5 m2, with the crop at 25 days after sowing (DAS) Total treatments- 10, Tri to TrlO. With 3 replications and values taken are mean of those 3 replicates.

[0183] The biostimulant was applied via foliar spray using a hollow cone nozzle . Agronomic practices including fertilization, irrigation, inter-culturing, and weeding were carried out as per the crop’s requirements.

[0184] Table 19 Synergistic Effects of Biostimulant FORMULATION 1 on Maize: SPAD

[0185] Value and Plant Height

[0186] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate: SPP: Soy protein

[0187] (hydrolysed)

[0188] Table 20 Synergistic Effects of Biostimulant FORMULATION 1 on Maize: Stem Diameter, Dry Weight, and Yield Parameters

[0189] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate : SPP: Soy protein

[0190] (hydrolysed)

[0191] The synergistic activity was calculated Colby formula.

[0192] (Colby’s Equation

[0193] Expected efficacy (EE) = X+Y+Z -(XY+YZ+XZ) / 100 + (X*Y*Z) / 10000

[0194] Where,

[0195] E= Expected % control by mixture of three products A, B & C in a defined dose.

[0196] X= Observed % control by product A

[0197] Y= Observed % control by product B

[0198] Z= Observed % control by product C)

[0199] Protein Hydrolysate and Fermented Milk Solids when used alone, showed no efficacy. Therefore, Colby formula for 3-way mixture has been used to calculate the expected efficacy of compound)

[0200] Example 6.2 - Synergistic activity of biostimulant composition

[0201] FORMULATION 2 on Tomato

[0202] The synergistic activity of the biostimulant composition FORMULATION 2 was evaluated on Tomato (Solanum lycopersicum) through a comparative study involving its individual constituent ingredients applied separately, as well as an untreated control..

[0203] A total of 10 distinct treatments were applied, with three replications, on tomato variety Abhinav. The field experiment was conducted on plots measuring 5 x 5 m2, with the crop at 25 days after sowing (DAS) . Total treatments- 10, Tri to TrlO. With 3 replications and values taken are mean of those 3 replicates.

[0204] The biostimulant was applied via foliar spray using a hollow cone nozzle . Agronomic practices including fertilization, irrigation, inter-culturing, and weeding were carried out as per the crop’s requirements.

[0205] Table 21 Synergistic Effects of Biostimulant FORMULATION 2 on Tomato: Root,

[0206] Shoot, and plant length

[0207] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate: SPP: Soy protein Table 22 Synergistic Effects of Biostimulant FORMULATION 2 on Tomato: Stem diameter, fresh weight, and yield parameters

[0208] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate : SPP: Soy protein Example 6.3 - Synergistic activity of bio stimulant composition FORMULATION 3 on Soybean

[0209] The synergistic activity of the biostimulant composition FORMULATION 3 was evaluated on Soybean (Glycine max) ) through a comparative study involving its individual constituent ingredients applied separately, as well as an untreated control.

[0210] A total of 10 distinct treatments were applied, with three replications, on variety Kimaya. The field experiment was conducted on plots measuring 5 x 5 m2, with the crop at 15 days after sowing (DAS) Total treatments- 10, Tri to TrlO. With 3 replications and values taken are mean of those 3 replicates.

[0211] The biostimulant was applied via foliar spray using a hollow cone nozzle. Agronomic practices including fertilization, irrigation, inter-culturing, and weeding were carried out as per the crop’s requirements.

[0212] Table 23 Synergistic Effects of Biostimulant FORMULATION 3 on Soybean :

[0213] Germination, Root and Shoot length

[0214] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate: SPP: Soy protein

[0215] Table 24 Synergistic Effects of Biostimulant FORMULATION 3 on Soybean: Stem diameter, fresh weight of Root, shoot and yield parameters

[0216] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH: Potassium Humate;SPP: Soy protein

[0217] Example 6.4 - Synergistic activity of bio stimulant composition FORMULATION 4 on Pomegranate

[0218] The synergistic activity of the biostimulant composition FORMULATION 4 was evaluated on Pomegranate (Punica granatum) through a comparative study involving its individual constituent ingredients applied separately, as well as an untreated control.

[0219] A total of 10 distinct treatments were applied, with three replications, on variety Kimaya. The field experiment was conducted on plots measuring 5 x 5 m2, with the crop age having lemon size fruit.

[0220] Total treatments-10, Tri to TrlO. With 3 replications and values taken are mean of those 3 replicates

[0221] The biostimulant was applied via foliar spray using a hollow cone nozzle . Agronomic practices including fertilization, irrigation, inter-culturing, and weeding were carried out as per the crop’s requirements.

[0222] Table 25 Synergistic Effects of Biostimulant FORMULATION 4 on Pomegranate:

[0223] Average fruit weight, SPAD and yield Parameters

[0224] AA- Amino acid; SWE: Sea weed extract; FMS: Fermented milk solids; PH :PotassiumHumate : SPP: Soy protein

[0225] Example 7 - Greenhouse experiment to evaluate the biostimulant composition on crops under abiotic stresses

[0226] This experiment aimed to assess the efficacy of different biostimulant formulations, namely FORMULATION A, FORMULATION 1, FORMULATION B, FORMULATION 2, FORMULATION C, FORMULATION 3, FORMULATION D, FORMULATION 4, FORMULATION E, and FORMULATION 5 in enhancing the tolerance of different crops such as com (Zea mays), tomato (Solarium lycopersicum), potato (Solarium tuberosum), pomegranate (Punica granatum), and rice (Oryza sativa) to abiotic stresses (drought, salinity, and heat). The study focused on key physiological and biochemical changes in plants while under stress and when treated with biostimulants with parameters such as total soluble sugar (TSS), reducing sugar (RS), proline synthesis (PS), relative water content (RWC), total phenol (TP), glycine betaine (GB), and lipid peroxidation rate (LPR).

[0227] Experimental Setup

[0228] 1. Location: Controlled greenhouse environment with adjustable temperature, humidity, and irrigation systems were maintained and green house experiments were conducted at Biostadt India Ltd, R&D centre at Bhavnagar, Gujarat.

[0229] 2. Plant Material: Uniform seedlings / plants of com, tomato, potato, pomegranate, and rice, were grown.

[0230] 3. Pots: 10 L pots filled with a standardized soil mix (70% loam, 20% sand, 10% compost) to ensure consistent nutrient availability.

[0231] 4. Replication: 4 replicates per treatment per crop

[0232] 5. Treatments: o Control: No biostimulant application. FORMULATION A (without fermented milk solids):. Application of biostimulant composition 1. Dosage: 0.25% (v / v) of FORMULATION A in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 1stspray: 3-4 leaf crop stage; 2ndspray: 9-10 leaf crop stage. FORMULATION 1 (with fermented milk solids)^ Application of enhanced biostimulant composition 2. Dosage: 0.25% (v / v) of FORMULATION 1 in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 1stspray: 3-4 leaf crop stage; 2ndspray: 9-10 leaf crop stage. FORMULATION B (without fermented milk solids):. Application of biostimulant composition 2. Dosage: 0.25% (v / v) of FORMULATION B in water. Water volume adjusted as plant’s size and root area at the time of application. Application time: Soil drenching with intervals of 20, 40 and 60 days after transplanting. FORMULATION 2 (with fermented milk solids)^ Application of enhanced biostimulant composition 2. Dosage: 0.25% (v / v) of FORMULATION 2 in water. Water volume adjusted as plant’s size and root area at the time of application. Application time: Soil drenching with intervals of 20, 40 and 60 days after transplanting. FORMULATION C (without fermented milk solids):. Application of biostimulant composition 3. Dosage: 1.4% (v / v) of FORMULATION C in water. Water volume adjusted as per tuber size. Application time: Seed potato tuber treatment and even coating before planting. FORMULATION 3 (with fermented milk solids)^ Application of enhanced biostimulant composition 3. Dosage: 1.4% (v / v) of FORMULATION 3 in water. Water volume adjusted as per tuber size. Application time: Seed potato tuber treatment and even coating before planting. FORMULATION D (without fermented milk solids);. Application of biostimulant composition 4. Dosage: 0.1% (v / v) of FORMULATION D in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 1stspray at 65 days of flowering followed by 2ndspray at 125 days of flowering. FORMULATION 4 (with fermented milk solids)^ Application of enhanced biostimulant composition 4. Dosage: 0.1% (v / v) of FORMULATION 4 in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 1stspray at 65 days of flowering followed by 2ndspray at 125 days of flowering. o FORMULATION E (without fermented milk solids):. Application of biostimulant composition 5. Dosage: 0.2% (v / v) of FORMULATION E in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 15-20 days interval after planting aligned with key growth stage as Seedling, Tillering, Bootleaf, and Early grain filling stage. o FORMULATION 5 (with fermented milk solids)^ Application of enhanced biostimulant composition 5. Dosage: 0.2% (v / v) of FORMULATION EFORMULATION 5 in water. Water volume adjusted as plant’s size and leaf area at the time of application. Application time: 15-20 days interval after planting aligned with key growth stage as Seedling, Tillering, Bootleaf, and Early grain filling stage.

[0233] 6. Biostimulant Application: Biostimulant applied at the onset of stress (Day 0), with repeated application depending on crop type, growth stage, plant size, leaf area to reinforce biostimulant effects.

[0234] Greenhouse Conditions

[0235] • Light: 14-hour photoperiod, 600 pmol / m2 / s PAR.

[0236] • Humidity: 50-60% (adjusted for heat stress).

[0237] • Irrigation: Automated drip system calibrated for stress treatments.

[0238] Stress Conditions

[0239] 1. Drought: Irrigation withheld to maintain soil moisture at 30% field capacity (FC) for stressed plants (control at 80% FC).

[0240] 2. Salinity: Irrigation with 150 mM NaCl solution (control with distilled water).

[0241] 3. Heat: Temperature maintained at 38°C daytime / 28°C nighttime (control at 25°C / 20°C).

[0242] Stress conditions were initiated on Day 0 after baseline measurements and maintained throughout the experimental period.

[0243] Experimental Design

[0244] • Type: Randomized Complete Block Design (RCBD) with crops, stress types, and treatments as factors.

[0245] • Time Points: Measurements taken at 0 days (baseline, pre-stress), 30 days, 60 days, and 90 days post-stress initiation in all crops except for pomegranate where measurements were taken at 0 days (baseline, pre-stress), 90 days, 180 days and 270 days post-stress initiation as per the specific crop cycle.

[0246] Sample Preparation for Biochemical and Physiological parameters assay: Leaves were collected at different time points as mentioned above and were packed in plastic bag and brought to the laboratory under ice cold conditions. Leaf tissues were isolated, cleaned, weighed and then transferred immediately to the respective medium for various biochemical and physiological analysis as mentioned below.

[0247] Parameters Measured Total Soluble Sugar (TSS): Quantified using the Dubois method. Seedlings (100 mg) were extracted with 5 ml of 80% ethanol and centrifuged at 3000 rpm for 10 minutes. Extraction was repeated 4 times with 80% ethanol and supernatants were collected into 25 ml volumetric flasks. Final volume of the extract was made to 25 ml with 80 % methanol. The extract (0.3 ml) was pipetted into separate test tubes and the tubes were placed in a boiling water bath to evaporate the methanol. One ml of millipore water and 1ml of 5% phenol was added in each test tube. Then 5 ml of sulphuric acid was added. The tubes were allowed to cool in ice-bath for 10-15 minutes. The intensity of colour was read at 490 nm on spectrophotometer. A standard curve was prepared using 10 mg glucose per 100 ml distilled water. Dubois et al. (1956). The amount of total soluble sugar present in the sample was calculated using the obtained standard curve. Reducing Sugar (RS): Measured via the dinitrosalicylic acid (DNSA) method which estimates glucose and galacturonic acid released by cellulose, polygalacturonase and P-1, 3 glucanase enzymes (Somogyi,1952). A known volume of aliquot was taken in test tube and final volume of 1.0 ml adjusted with distilled water. To this 0.5 ml DNSA reagent (1g DNSA + 200mg crystalline phenol + 50mg sodium sulphite in 100ml of 1% sodium hydroxide) was added and mixed properly. The content was heated in a boiling water bath for 5 min. When the contents of the tubes were still warm, 1.0 ml of 40% sodium potassium tartrate (Rochelle salt) solution was added. After cooling, the final volume was made 5.0 ml with distilled water. After that the tubes were read at 540 nm using spectrophotometer. Reagent blank was also performed by addition of 1.0 ml of distilled water in place of enzyme aliquot. A known concentration of standard (0.5 -2.5 pM) of glucose or galacturonic acid was carried out and was calibrated to calculate the amount of reducing sugar. Proline Synthesis: Determined spectrophotometrically using ninhydrin (pmol / g fresh weight). The sample (0.3 ml) and standard proline (0.1-0.6 ml from 0.05 mg / ml proline stock) were taken in a series of test tubes, and the volume was made up to 1.0 ml with distilled water. Then 2 ml glacial acetic acid and 2 ml acid ninhydrin reagent were added. Then tubes were kept in boiling water bath for 1 hr. The tubes were cooled in running water at room temperature. After that 4 ml toluene was added. The absorbance was recorded from toluene phase at 520 nm in spectrophotometer. The free proline was calculated as stated below and expressed as mg.g-1.

[0248] 4. Relative Water Content (RWC): Known weight (gm) of leaf sample was transferred in a petri dish, and to this 25 ml distilled water was added and kept for four hours. Then the leaves were taken out, dried by blotting paper and weighed (Turgid weight). The leaf was kept in oven at 84°C for 5 hr and weighted until constant weight was obtained. After this RWC were estimated as per formula and expressed as per cent relative water content (Weatherley, 1962).

[0249] Relative Water Content (%) = (Fresh weight (g) - Dry weight (g)) / Turgid weight (g) - Dry weight (g)) x 100

[0250] 5. Total Phenol (TP): 0.5 grams of leaf was extracted with 10 mb of 80% methanol. The mixture was sonicated for 30 minutes at room temperature and then filtered to obtain a clear supernatant. For the reaction, 0.5 mb of the extract was mixed with 2.5 mb of 10% Folin-Ciocalteu reagent and allowed to stand for 5 minutes. Subsequently, 2 mb of 7.5% sodium carbonate solution was added, and the mixture was incubated at room temperature for 30 minutes. The absorbance was measured at 760 nm using a UV-Vis spectrophotometer. A calibration curve was prepared using gallic acid standards ranging from 10 to 100 pg / mL. The TPC was calculated and expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE / g DW).

[0251] 6. Glycine Betaine (GB): Glycine betaine was done from fresh leaves as per the method of Hendawey (2015). Finely ground leaf material (0.5 g) was mechanically shaken with 20 ml of distilled water for 16 hrs. at 25 °C. The samples were then filtered, and the filtrate was stored in freezer until analysis. Thawed extracts were diluted 1 : 1 with 2 N sulphuric acid. Aliquot (0.5 ml) was measured into test tube and cooled in ice for 1 hour 0.2 ml of cold potassium iodide-iodine reagent [Iodine (15.7 g) and potassium iodide (20 g) were dissolved in 100 ml of water and kept in fridge at 4 °C] was added and the mixture was gently mixed with vortex mixture. The samples were stored at 0 to 4 °C for 16 hr. After the expiration of the period samples were transferred to centrifuge tubes and then centrifuged at 10,000 g for 15 min at 0 °C. The supernatant was carefully aspirated with 1 ml micropipette. The peridotite crystals were dissolved in 9 ml of 1,2-dichloroethane. Vigorous vortex mixing was done to effect complete solubility in developing solvent. After 2.0-2.5 hrs. an absorbance was measured at 365 nm. Reference standards of glycine-betaine (50- 200 pgml-1) were prepared in 2 N sulphuric acid and the amount of glycine betaine present in the sample was calculated by appropriate formula.

[0252] 7. Lipid Peroxidation Rate (LPR): Lipid peroxidation was measured using thiobarbituric acid (TBA) method (Heath and Packer, 1968). The level of lipid peroxidation was measured in the terms of malondialdehyde (MDA) content. One gram seedlings were ground in liquid nitrogen and homogenized in 0.1% (w / v) trichloroacetic acid (TCA) and 2% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 10,000 rpm for 15 min followed by addition of 2% PVP to supernatant of homogenate to remove excess polysaccharide, and again centrifuged at 10,000 rpm for 15 min. One ml of supernatant was taken and added into 4 ml of 20% TCA containing 1% thiobarbituric acid (TBA). The mixture was heated at 95°C for 30 min and then quickly cooled in an ice bath. The resulting mixture was centrifuged at 10,000 rpm for 15 min. The absorbance of MDA content was measured at 532 nm and 600 nm for the correction of non-specific turbidity. The level of lipid peroxidation was expressed in nano mol of MDA formed g-1 fr. wt using an extinction coefficient of 155 mM-1 cm-1.

[0253] Sampling and Analysis

[0254] • Sample Collection: Leaves were collected at above mentioned time points for analysis of different parameters.

[0255] • Tissue: Leaves (fully expanded) and roots collected for biochemical analysis.

[0256] • Statistical Analysis: Two-way ANOVA followed by Tukey’s HSD test (p < 0.05) to compare treatments, stress types, and time points.

[0257] Corn Lifecycle and Measurement points

[0258] Com has a distinct lifecycle with vegetative, reproductive, and grain-filling phases.

[0259] • 0 Days (Baseline, Seedling Stage): Seedlings were transplanted into 10 L pots, with initial root and shoot development. This was pre-stress exposure for abiotic stress studies.

[0260] • 30 Days (Vegetative Growth): Rapid stem elongation, leaf expansion, and root system development. Early abiotic stress (e.g., drought, salinity) effects may be applied or observed during this phase.

[0261] • 60 Days (Tasseling to Silking, Early Reproductive Phase): Critical phase with tassel emergence, pollen shed, and silk development. This is the peak period for abiotic stress impact, as reproductive development is sensitive to stressors like drought or heat.

[0262] • 90 Days (Grain Filling to Physiological Maturity): Kernel development, grain filling, and maturation. Sustained abiotic stress effects may reduce kernel number or weight, impacting yield.

[0263] Below is the bioefficacy trial data for a greenhouse experiment conducted on com crop under combined abiotic stresses (heat, salinity, and drought). The dataset compares the effects of two biostimulants, FORMULATION A and FORMULATION 1, against a control group, focusing on physiological parameters such as total soluble sugar (TSS), reducing sugar (RS), proline synthesis (PS), relative water content (RWC), total phenol (TP), glycine betaine (GB), and lipid peroxidation rate (LPR). Measurements were taken at intervals corresponding to key stages in the com lifecycle: 0 days (baseline), 30 days (early vegetative), 60 days (mid-vegetative to reproductive), and 90 days (near maturity). The data demonstrates that FORMULATION 1 provides better stress tolerance than FORMULATION A, as evidenced by higher values of beneficial parameters (TSS, RS, PS, RWC, TP, GB) and lower values of the stress damage indicator (LPR).

[0264] Table 26 Effect of Biostimulant FORMULATION 1 on stress tolerance in maize (corn)

[0265] • Baseline (0 days): All treatments start with identical values, representing unstressed conditions before the application of abiotic stresses and biostimulants.

[0266] • 30 days (Early Vegetative): Stress effects begin to appear. The control showed increased TSS, RS, PS, GB, TP, and LPR, with a drop in RWC. FORMULATION A improves these parameters compared to the control, and FORMULATION 1 further enhanced them, showing higher RWC and lower LPR.

[0267] • 60 days (Mid- Vegetative to Reproductive): Stress intensified. FORMULATION 1 consistently outperformed FORMULATION A, with greater accumulation of protective compounds (TSS, RS, PS, GB, TP), higher RWC, and reduced LPR, indicating less cellular damage.

[0268] • 90 days (Near Maturity): The differences were most pronounced. FORMULATION 1 maintained higher levels of beneficial osmolytes and antioxidants, preserves more water (RWC), and exhibits the lowest LPR, confirming superior stress tolerance compared to FORMULATION A and the control.

[0269] Brief Summary of FORMULATION 1 Effectiveness as a Biostimulant in Combating Stress in Corn: The bioefficacy trial data demonstrated that FORMULATION 1 was a more effective biostimulant than FORMULATION A in mitigating combined abiotic stresses (heat, salinity, and drought) in com, as evidenced by its superior performance across key physiological parameters measured at 0, 30, 60, and 90 days. FORMULATION 1 consistently outperformed both FORMULATION A and the control group, showing significantly higher values for beneficial parameters — Total Soluble Sugar (TSS), Reducing Sugar (RS), Proline Synthesis (PS), Relative Water Content (RWC), Total Phenol (TP), and Glycine Betaine (GB) — and lower values for the stress damage indicator, Lipid Peroxidation Rate (LPR).

[0270] Enhanced Osmoprotection and Energy Reserves: FORMULATION 1 exhibited greater accumulation of TSS (up to 93.9% increase at 90 days vs. 40.9% for FORMULATION A) and RS (up to 83.3% vs. 33.3% for FORMULATION A), indicating improved carbohydrate metabolism and energy availability under stress. Similarly, PS (up to 150.0% vs. 66.7% for FORMULATION A) and GB (up to 130.8% vs. 69.2% for FORMULATION A) were significantly higher, reflecting enhanced osmotic adjustment and cellular protection against dehydration and salinity.

[0271] Improved Water Retention: FORMULATION 1 maintained higher RWC (up to 36.4% increase at 90 days vs. 18.2% for FORMULATION A), suggesting better hydration status and turgor maintenance under drought and heat stress.

[0272] Stronger Antioxidant Defence: Higher TP levels (up to 146.2% vs. 92.3% for FORMULATION A) in FORMULATION 1 indicate a robust antioxidant response, reducing oxidative damage. This is corroborated by a greater reduction in LPR (up to 48.6% decrease vs. 28.6% for FORMULATION A), signifying less membrane damage and oxidative stress.

[0273] Consistent Superiority Over Time: The advantages of FORMULATION 1 were most pronounced at 60 and 90 days, corresponding to critical vegetative and reproductive stages, where stress impacts were severe. For instance, at 60 days, FORMULATION 1 achieved a 79.9% increase in TSS and 50.0% decrease in LPR compared to 34.8% and 33.3% for FORMULATION A, respectively.

[0274] These results confirm that FORMULATION 1 enhances stress tolerance more effectively than FORMULATION A by bolstering osmoprotection, water retention, and antioxidant defences, thereby reducing cellular damage and supporting com growth under abiotic stress conditions.

[0275] Tomato Lifecycle and Measurement Points

[0276] Tomato (Solanum lycopersicum) has a distinct lifecycle, and the measurement points are aligned with its key growth stages under greenhouse conditions:

[0277] • 0 days (Baseline, Seedling Stage): Post-transplanting, before significant stress exposure.

[0278] • 30 days (Vegetative Growth): Active leaf and stem development, early stress effects. • 60 days (Flowering to Early Fruit Set): Critical reproductive phase, peak stress impact.

[0279] • 90 days (Fruit Development to Maturity): Fruit ripening, sustained stress effects. These time points reflect typical greenhouse tomato growth, with stress effects becoming more pronounced overtime. • TSS, RS, PS, TP, GB: Increased under stress, with FORMULATION 2 showing the highest accumulation (e.g., TSS up to 80.3% increase at 90 days vs. 39.9% for FORMULATION B; TP up to 140.0% vs. 75.0% at 60 days), reflecting enhanced osmoprotection, energy reserves, and antioxidant activity in tomatoes.

[0280] • RWC: Decreased under stress, but FORMULATION 2 maintains higher values (up to 30.0% increase at 90 days vs. 16.7% for FORMULATION B), indicating better water retention critical for fruit development.

[0281] • LPR: Increased under stress, with Control showing the highest damage (30.00 nmol / g at 90 days), while FORMULATION 2 reduces it most effectively (46.7% decrease vs. 26.7% for FORMULATION B), indicating reduced oxidative damage.

[0282] • Tomato-Specific Adjustments with comparison to corn: Higher baseline TSS (20 mg / g vs. 15 mg / g in com) and TP (3 mg / g vs. 2 mg / g in com) reflect tomato’s fruitrich physiology. Lower LPR values (e.g., 30 nmol / g vs. 35 nmol / g in com at 90 days) suggest tomatoes may have less severe membrane damage under similar stress conditions.

[0283] • Statistical Reliability: Low SD and SE values indicated consistent replication data. CD values confirmed significant differences between treatments, with FORMULATION 2 consistently outperforming FORMULATION B and Control. FORMULATION 2 seems to be a superior biostimulant for enhancing stress tolerance in tomatoes, particularly during flowering and fruit development stages.

[0284] Brief Summary of FORMULATION 2 Effectiveness as a Biostimulant in Combating Stress in Tomato

[0285] The bioefficacy trial data demonstrated that FORMULATION 2 was a highly effective biostimulant compared to FORMULATION B in mitigating combined abiotic stresses (heat, salinity, and drought) in tomato crops, as evidenced by superior performance across key physiological parameters measured at 0, 30, 60, and 90 days, corresponding to seedling, vegetative, flowering / fruit set, and fruit maturity stages. FORMULATION 2 consistently outperformed FORMULATION B and the control, exhibiting higher values for beneficial parameters — Total Soluble Sugar (TSS), Reducing Sugar (RS), Proline Synthesis (PS), Relative Water Content (RWC), Total Phenol (TP), and Glycine Betaine (GB) — and lower values for Lipid Peroxidation Rate (LPR), indicating reduced stress damage. • Enhanced Osmoprotection and Energy Reserves: FORMULATION 2 significantly increased TSS (up to 80.3% at 90 days vs. 39.9% for FORMULATION B), RS (up to 100.0% vs. 50.0%), PS (up to 133.3% vs. 66.7%), and GB (up to 111.1% vs. 55.6%), supporting improved carbohydrate metabolism, osmotic adjustment, and cellular protection critical for tomato growth and fruit development under stress.

[0286] • Improved Water Retention: FORMULATION 2 maintained higher RWC (up to 30.0% increase at 90 days vs. 16.7% for FORMULATION B), ensuring better hydration and turgor maintenance during drought and heat stress, particularly vital during fruit set and ripening.

[0287] • Stronger Antioxidant Defence: FORMULATION 2 showed elevated TP levels (up to 140.0% increase at 60 days vs. 75.0% for FORMULATION B), enhancing antioxidant capacity, and reduced LPR (up to 46.7% decrease at 90 days vs. 26.7% for FORMULATION B), indicating less oxidative damage to cell membranes.

[0288] • Peak Performance in Critical Stages: The benefits of FORMULATION 2 were most pronounced at 60 days (flowering / fruit set) and 90 days (fruit maturity), with TSS (67.0% vs. 33.3%), RS (85.7% vs. 42.9%), and TP (140.0% vs. 75.0%) showing substantial improvements over FORMULATION B, supporting tomato yield under stress.

[0289] These results confirm that FORMULATION 2 enhanced stress tolerance in tomatoes more effectively than FORMULATION B by improving osmoprotection, water retention, and antioxidant defences, thereby reducing cellular damage and supporting robust growth and fruit production under abiotic stress conditions.

[0290] Potato Lifecycle and Measurement Points

[0291] Potato (Solanum tuberosum) has a distinct lifecycle under greenhouse conditions, and measurement points will align with its key growth stages:

[0292] • 0 days (Baseline, Sprouting): Post-planting of seed tubers, before significant stress exposure.

[0293] • 30 days (Vegetative Growth): Leaf and stem development, early stress effects.

[0294] • 60 days (Tuber Initiation): Onset of tuber formation, critical for yield, with pronounced stress impacts.

[0295] • 90 days (Tuber Bulking to Maturity): Tuber growth and maturation, sustained stress effects.

[0296] These time points reflect typical potato growth in a greenhouse, with stress effects intensifying overtime, particularly during tuber initiation and bulking.

[0297] Table 28 Effect of Biostimulant FORMULATION 3 on stress tolerance in Potato

[0298] • TSS, RS, PS, TP, GB: Increased under stress, with FORMULATION 3 showing the highest accumulation (e.g., TSS up to 67.0% increase at 90 days vs. 33.3% for FORMULATION C; TP up to 130.8% vs. 76.9%), reflecting enhanced osmoprotection, energy reserves, and antioxidant activity critical for tuber development.

[0299] • RWC: Decreased under stress, but FORMULATION 3 maintains higher values (up to 28.3% increase at 90 days vs. 16.7% for FORMULATION C), indicating better water retention essential for tuber bulking.

[0300] • LPR: Increased under stress, with Control showing the highest damage (32.00 nmol / g at 90 days), while FORMULATION 3 reduced it most effectively (43.8% decrease vs. 25.0% for FORMULATION C), indicating reduced oxidative damage.

[0301] • Statistical Reliability: Low SD and SE values indicate consistent replication data. CD values confirm significant differences between treatments, with FORMULATION 3 consistently outperforming FORMULATION C and Control. This demonstrates FORMULATION 3 as a superior biostimulant for enhancing stress tolerance in potatoes, particularly during tuber initiation and bulking stages.

[0302] Brief Summary of FORMULATION 3 Effectiveness as a Biostimulant in Combating Stress in Potato

[0303] The bioefficacy trial data demonstrates that FORMULATION 3 is a highly effective biostimulant compared to FORMULATION C in mitigating combined abiotic stresses (heat, salinity, and drought) in potato crops, as evidenced by superior performance across key physiological parameters measured at 0, 30, 60, and 90 days, corresponding to sprouting, vegetative growth, tuber initiation, and tuber bulking / maturity stages. FORMULATION 3 consistently outperformed FORMULATION C and the control, showing higher values for beneficial parameters — Total Soluble Sugar (TSS), Reducing Sugar (RS), Proline Synthesis (PS), Relative Water Content (RWC), Total Phenol (TP), and Glycine Betaine (GB) — and lower values for Lipid Peroxidation Rate (LPR), indicating reduced stress damage.

[0304] • Enhanced Osmoprotection and Energy Reserves: FORMULATION 3 significantly increased TSS (up to 67.0% at 90 days vs. 33.3% for FORMULATION C), RS (up to 80.0% vs. 40.0%), PS (up to 150.0% vs. 66.7%), and GB (up to 130.8% vs. 69.2%), supporting improved carbohydrate metabolism, osmotic adjustment, and cellular protection critical for tuber development under stress.

[0305] • Improved Water Retention: FORMULATION 3 maintained higher RWC (up to 28.3% increase at 90 days vs. 16.7% for FORMULATION C), ensuring better hydration and turgor maintenance during drought and heat stress, particularly vital during tuber bulking.

[0306] • Stronger Antioxidant Defence: FORMULATION 3 exhibited elevated TP levels (up to 130.8% increase at 90 days vs. 76.9% for FORMULATION C), enhancing antioxidant capacity, and reduced LPR (up to 43.8% decrease at 90 days vs. 25.0% for FORMULATION C), indicating less oxidative damage to cell membranes.

[0307] • Peak Performance in Critical Stages: The benefits of FORMULATION 3 were most pronounced at 60 days (tuber initiation) and 90 days (tuber bulking), with TSS (53.6% vs. 26.6%), RS (66.7% vs. 33.3%), and TP (133.3% vs. 66.7%) showing substantial improvements over FORMULATION C, supporting tuber yield under stress.

[0308] These results confirmed that FORMULATION 3 enhances stress tolerance in potatoes more effectively than FORMULATION C by improving osmoprotection, water retention, and antioxidant defenses, thereby reducing cellular damage and supporting robust tuber development under abiotic stress conditions.

[0309] Pomegranate Lifecycle and Measurement Points

[0310] Pomegranate (Punica granatum) is a perennial fruit crop with a distinct lifecycle under greenhouse conditions. • 0 days (Baseline, Vegetative Growth): Early season, post-pruning or new leaf flush, before significant stress exposure.

[0311] • 90 days (Flowering): Onset of flowering, critical for fruit set, with early stress effects. • 180 days (Fruit Development): Fruit growth and aril development, peak stress impact.

[0312] • 270 days (Fruit Maturity): Fruit ripening and harvest readiness, sustained stress effects.

[0313] These time points reflect typical pomegranate growth in a greenhouse, with longer intervals (90 days) compared to annual crops like com, tomato, and potato, as pomegranate is a woody perennial with a slower growth cycle. The intervals account for the extended duration of flowering, fruit development, and maturation.

[0314] Table 29 Effect of Biostimulant FORMULATION 4 on stress tolerance in Pomegranate

[0315] • TSS, RS, PS, TP, GB: Increased under stress, with FORMULATION 4 showing the highest accumulation (e.g., TSS up to 73.0% increase at 270 days vs. 36.3% for FORMULATION D; TP up to 108.0% vs. 60.0%), reflecting enhanced osmoprotection, energy reserves, and antioxidant activity critical for fruit development and aril quality in pomegranates.

[0316] • RWC: Decreased under stress, but FORMULATION 4 maintained higher values (up to 32.7% increase at 270 days vs. 18.2% for FORMULATION D), indicating better water retention essential for fruit development and ripening. • LPR: Increased under stress, with Control showing the highest damage (28.00 nmol / g at 270 days), while FORMULATION 4 reduces it most effectively (46.4% decrease vs. 28.6% for FORMULATION D), indicating reduced oxidative damage.

[0317] • Pomegranate-Specific Adjustments: Higher baseline TP (4 mg / g vs. 3 mg / g in tomato, 2.5 mg / g in potato) reflects pomegranate’s phenolic-rich arils. Moderate TSS (22 mg / g at baseline vs. 25 mg / g in potato) and lower LPR (28 nmol / g at 270 days vs. 32 nmol / g in potato) align with pomegranate’s woody perennial physiology and stress response.

[0318] • Statistical Reliability: Low SD and SE values indicated consistent replication data. CD values confirm significant differences between treatments, with FORMULATION 4 consistently outperforming FORMULATION D and Control. This dataset aligned with the pomegranate lifecycle and mirrors the structure of the com, tomato, and potato datasets, demonstrating FORMULATION 4 as a superior biostimulant for enhancing stress tolerance in pomegranates, particularly during fruit development and maturity stages.

[0319] Brief Summary of FORMULATION 4 Effectiveness as a Biostimulant in Combating Stress in Pomegranate

[0320] The bioefficacy trial data demonstrates that FORMULATION 4 is a highly effective biostimulant compared to FORMULATION D in mitigating combined abiotic stresses (heat, salinity, and drought) in pomegranate crops, as evidenced by superior performance across key physiological parameters measured at 0, 90, 180, and 270 days, corresponding to vegetative growth, flowering, fruit development, and fruit maturity stages. FORMULATION 4 consistently outperformed FORMULATION D and the control, showing higher values for beneficial parameters — Total Soluble Sugar (TSS), Reducing Sugar (RS), Proline Synthesis (PS), Relative Water Content (RWC), Total Phenol (TP), and Glycine Betaine (GB) and lower values for Lipid Peroxidation Rate (LPR), indicating reduced stress damage.

[0321] • Enhanced Osmoprotection and Energy Reserves: FORMULATION 4 significantly increased TSS (up to 73.0% at 270 days vs. 36.3% for FORMULATION D), RS (up to 93.3% vs. 50.0%), PS (up to 126.7% vs. 60.0%), and GB (up to 111.1% vs. 55.6%), supporting improved carbohydrate metabolism, osmotic adjustment, and cellular protection critical for fruit development and aril quality under stress. • Improved Water Retention: FORMULATION 4 maintained higher RWC (up to 32.7% increase at 270 days vs. 18.2% for FORMULATION D), ensuring better hydration and turgor maintenance during drought and heat stress, particularly vital during fruit development and ripening.

[0322] • Stronger Antioxidant Defense: FORMULATION 4 exhibited elevated TP levels (up to 108.0% increase at 270 days vs. 60.0% for FORMULATION D), enhancing antioxidant capacity, and reduced LPR (up to 46.4% decrease at 270 days vs. 28.6% for FORMULATION D), indicating less oxidative damage to cell membranes.

[0323] • Peak Performance in Critical Stages: The benefits of FORMULATION 4 were most pronounced at 180 days (fruit development) and 270 days (fruit maturity), with TSS (50.2% vs. 28.5%), RS (71.4% vs. 37.1%), and TP (93.3% vs. 50.0%) showing substantial improvements over FORMULATION D, supporting fruit yield and quality under stress.

[0324] These results confirm that FORMULATION 4 enhances stress tolerance in pomegranates more effectively than FORMULATION D by improving osmoprotection, water retention, and antioxidant defenses, thereby reducing cellular damage and supporting robust fruit development and quality under abiotic stress conditions.

[0325] Rice Lifecycle and Measurement Points

[0326] Rice (Oryza sativa) is an annual crop with a distinct lifecycle under greenhouse conditions.

[0327] • 0 days (Baseline, Seedling Stage): Post-germination, early vegetative growth, before significant stress exposure.

[0328] • 30 days (Tillering): Active tiller formation, early stress effects.

[0329] • 60 days (Panicle Initiation): Onset of reproductive phase, critical for grain set, with pronounced stress impacts.

[0330] • 90 days (Grain Filling to Maturity): Grain development and ripening, sustained stress effects.

[0331] These time points reflect typical rice growth in a greenhouse, with stress effects intensifying overtime, particularly during panicle initiation and grain filling. Table 30 Effect of Biostimulant FORMULATION 5 on stress tolerance in rice

[0332] 5

[0333] • TSS, RS, PS, TP, GB: Increased under stress, with FORMULATION EFORMULATION 5 showing the highest accumulation (e.g., TSS up to 75.5% increase at 90 days vs. 37.4% for FORMULATION E; TP up to 175.0% vs. 87.5%), reflecting enhanced osmoprotection, energy reserves, and antioxidant activity critical

[0334] 10 for grain development in rice.

[0335] . RWC: Decreased under stress, but FORMULATION EFORMULATION 5 maintains higher values (up to 26.2% increase at 90 days vs. 13.8% for FORMULATION E), indicating better water retention essential for rice’s semi- aquatic nature and grain fdling.

[0336] 15 • LPR: Increased under stress, with Control showing the highest damage (25.00 nmol / g at 90 days), while FORMULATION 5 reduces it most effectively (48.0% decrease vs. 28.0% for FORMULATION E), indicating reduced oxidative damage.

[0337] • Statistical Reliability: Low SD and SE values indicated consistent replication data. CD values confirm significant differences between treatments, with

[0338] 20 FORMULATION 5 consistently outperforming FORMULATION E and Control.

[0339] This demonstrates FORMULATION 5 as a superior biostimulant for enhancing stress tolerance in rice, particularly during panicle initiation and grain filling stages.

[0340] Brief Summary of FORMULATION 5 Effectiveness as a Biostimulant in Combating Stress in Rice

[0341] 25 The bioefficacy trial data demonstrated that FORMULATION 5 is a highly effective biostimulant compared to FORMULATION E in mitigating combined abiotic stresses (heat, salinity, and drought) in rice crops, as evidenced by superior performance across key physiological parameters measured at 0, 30, 60, and 90 days, corresponding to seedling, tillering, panicle initiation, and grain fdling / maturity stages. FORMULATION 5 consistently outperformed FORMULATION E and the control, showing higher values for beneficial parameters — Total Soluble Sugar (TSS), Reducing Sugar (RS), Proline Synthesis (PS), Relative Water Content (RWC), Total Phenol (TP), and Glycine Betaine (GB) — and lower values for Lipid Peroxidation Rate (LPR), indicating reduced stress damage.

[0342] • Enhanced Osmoprotection and Energy Reserves: FORMULATION 5 significantly increased TSS (up to 75.5% at 90 days vs. 37.4% for FORMULATION E), RS (up to 133.3% vs. 66.7%), PS (up to 180.0% vs. 80.0%), and GB (up to 133.3% vs. 66.7%), supporting improved carbohydrate metabolism, osmotic adjustment, and cellular protection critical for grain development under stress.

[0343] • Improved Water Retention: FORMULATION 5 maintained higher RWC (up to 26.2% increase at 90 days vs. 13.8% for FORMULATION E), ensuring better hydration and turgor maintenance, particularly vital for rice’s semi -aquatic nature during panicle initiation and grain filling.

[0344] • Stronger Antioxidant Defense: FORMULATION 5 exhibited elevated TP levels (up to 175.0% increase at 90 days vs. 87.5% for FORMULATION E), enhancing antioxidant capacity, and reduced LPR (up to 48.0% decrease at 90 days vs. 28.0% for FORMULATION E), indicating less oxidative damage to cell membranes.

[0345] • Peak Performance in Critical Stages: The benefits of FORMULATION 5 were most pronounced at 60 days (panicle initiation) and 90 days (grain filling), with TSS (60.3% vs. 29.9%), RS (100.0% vs. 50.0%), and TP (150.0% vs. 80.0%) showing substantial improvements over FORMULATION E, supporting grain yield under stress.

[0346] These results confirm that FORMULATION 5 enhances stress tolerance in rice more effectively than FORMULATION E by improving osmoprotection, water retention, and antioxidant defenses, thereby reducing cellular damage and supporting robust grain development under abiotic stress conditions.

[0347] The embodiments were chosen and described to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.

[0348] It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the present invention.

Claims

AMENDED CLAIMS received by the International Bureau on 10 March 2026 (10.03.2026)I Claim :

1. A bio stimulant composition comprising a. fermented seaweed extract b. fermented milk solids, c. one or more amino acid and, d. optionally one or more of the ingredients selected from, protein hydrolysate, humic substance, macronutrient and micronutrient. wherein the seaweed extract is present in an amount of 0.6 w / w% to 75 w / w%, the milk solids are present in an amount of 0.5 w / w% to 55w / w% and the amino acid is present in an amount of 0.02 w / w% to 32 w / w%.

2. The bio stimulant composition as claimed in claim 1, wherein the composition comprises seaweed extract, milk solids, amino acids, protein hydrolysate and humic substance.

3. The bio stimulant composition as claimed in claim 1, wherein the composition comprises seaweed extract, milk solids, amino acids, macronutrients and micronutrients.

4. The bio stimulant composition as claimed in claims 1 to 3, wherein it further comprises one or more of the ingredients selected from stabilizer, emulsifier, spreading agent and preservative.

5. The bio stimulant composition as claimed in claims 1 to 4, wherein the milk solids and seaweed extract are fermented by lactic acid bacteria (LAB).

6. The bio stimulant composition as claimed in claim 5, wherein the lactic acid bacterium is selected from Lactobacillus spp., Streptococcus spp., Pediococcus spp., Leuconostoc spp and combination thereof.

7. The bio stimulant composition as claimed in claims 1 to 6, wherein the bacterial inoculum is in an amount of 0.005 v / v % to 0.9v / v%.

8. The bio stimulant composition as claimed in claim 1, wherein the protein hydrolysate in an amount of 0 w / w% to 18w / w%, humic substances in an amount of 0.2 w / w% to 5w / w%,macronutrients in an amount of 0.001 to 15 w / w%, micronutrients in an amount of 0.001 to 10 w / w%, stabilizers in an amount of 0.1 w / w% to 20w / w%, emulsifiers in an amount of 0.2 w / w% to 6 w / w%, preservatives in an amount of 0.1 w / w% to 4 w / w%.

9. The bio stimulant composition as claimed in claim 1, wherein the seaweed extract is present in an amount of 0.6 w / w% to 50 w / w%.

10. The bio stimulant composition as claimed in claims 1 to 9, wherein the composition comprises one or more substances selected from alginic acid, betaines, amino acids, fucoidan, mannitol and uronic acid.

11. The bio stimulant composition as claimed in claim 10, wherein the alginic acid content is in an amount of 0.001 w / w% to 15w / w%.

12. The bio stimulant composition as claimed in claims 1 to 11 , wherein the seaweed is selected from Ascophyllum nodosum, Ecklonia maxima, Fucus vesiculosus, Sargassum spp., Hydroclathrus spp., Laminaria digitata, Macrocystispyrifera spp., Nereocystis spp. Cystoseira spp and combination thereof, preferably Ascophyllum nodosum.

13. The bio stimulant composition as claimed in claims 1 to 12, wherein the milk solids is selected from fermented milk powder, dried milk powder, whey powder, whey proteins, caseinates, milk proteins, skim milk and combination thereof.

14. The bio stimulant composition as claimed in claims 1 to 13, wherein the amino acid is selected from L-lysine, L-arginine, L-leucine, L-valine, L- glutamic acid, L-glycine, L- methionine, L-proline, L-tryptophan, L-threonine, L-serine, L-isoleucine, L-phenyl alanine, L-alanine, L- cysteine, L-tyrosine, L-histidine, L-aspartic acid and combination thereof.

15. The bio stimulant composition as claimed in claims 1 to 14, wherein the protein hydrolysate is selected from soybean meal, microalgal extracts, com gluten, fish meal, chicken feather meal, slaughterhouse waste and untreated hairs / wool.

16. The bio stimulant composition as claimed in claims 1 to 15, wherein the humic substance is selected from potassium humate, fillvic acid, humin, hymetomalonic acid and combination thereof.

17. The bio stimulant composition as claimed in claims 1 to 16, wherein the macronutrient is selected from nitrogen (N) in an amount of 0.1 to 10w / w%, phosphorus (P) in an amount of 0.01 to 10w / w%, potassium (K) in an amount of 0.01w / w% to 10 w / w%, iron (Fe) in an amount of 0.001 to 15 w / w%, copper (Cu) in an amount of 0.001 to 10w / w%, and combination thereof.

18. The bio stimulant composition as claimed in claims 1 to 17, wherein the micronutrient is selected from boron (B) in an amount of 0.001 to 10w / w%, manganese (Mn) in an amount of 0.001 to 10w / w%, zinc (Zn) in an amount of 0.001 to 10w / w%, magnesium (Mg) in an amount of 0.001 to 10w / w%, molybdenum (Mb) in an amount of 0.001 to 10 w / w% and combination thereof.

19. The bio stimulant composition as claimed in claims 1 to 18, wherein the composition is in liquid form, free flowing powder from, granular form or in the form of tablets, noodles, sticks, pellets, or capsules.

20. A method of preparing a bio stimulant composition, wherein the method comprises the steps of:(i) dissolving milk solids in water and heating the mixture;(ii) stirring and cooling the mixture;(iii) adding stabilizer to said mixture and maintaining at suitable pH;(iv) adding an inoculum containing lactic acid bacteria (LAB) and fermenting the solution to form fermented milk solids;(v) adding seaweed extract;(vi) adding amino acids to the mixture;(vii) further fermenting the solution obtained in step (vi) to form fermented seaweed extract;(viii) optionally adding a fermentation arrester to said mixture;(ix) sieving the solution obtained, to obtain free-flowing viscous liquid biomass;(x) optionally adding one or more of humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients.

21. The method as claimed in claim 20, comprising the steps of:(i) dissolving milk solids in water and heating the mixture at 80°C to 90°C for 1 to 3 hours;(ii) stirring said mixture at a 100-150 RPM for 2 to 3 hours and cooling at temperature 30°C to 36°C;(iii) adding 0.1 to 20 w / w% of stabilizers to said mixture and maintaining the pH of solution at 4 to 5;(iv) adding 0.005 w / w% to 0.9 % w / w% of inoculum containing lactic acid bacteria (LAB) and fermenting the solution for 12 to 15 hours, at pH 2 to 5 to form fermented milk solids;(v) adding 0.6 w / w% to 75w / w% seaweed extract at pH 2 to 5;(vi) adding amino acids to the mixture in an amount of 0.02 % to 32%w / w;(vii) further fermenting the solution obtained in step (vi) at temperature 30°C to 32°C for 2 to 72 hours to form fermented seaweed extract;(viii) adding 0.1% w / w%, to 20% w / w% of fermentation arrester to said mixture;(ix) sieving the solution obtained, through 200 to 250-micron size sieve and obtaining a free- flowing viscous liquid biomass;(x) optionally adding one or more of, humic substances, protein hydrolysates, preservatives, emulsifiers, stabilizers, spreading agent, micronutrients and macronutrients.

22. The method as claimed in claim 20 or 21, wherein the spreading agent is selected from Tween 20 (Polysorbate 20), Tween 80 (Polysorbate 80), Span 20 / 60 / 80 (sorbitan esters, less hydrophilic), ethoxylated castor oil (PEG castor oil derivatives), alkyl polyglucosides, Fatty acid ethoxylates (PEG-6 caprylic / capric glycerides) and Polyalkyleneoxide modified Heptamethyl Trisiloxane.

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