DESC:NANOCOMPOSITE FEED ADDITIVE FOR LIVESTOCK AND
POULTRY AND PREPARATION METHOD THEREOF

CROSS REFERENCE TO RELATED APPLICATION
[001] The present application claims priority from the Provisional Application No. IN 202441058823 filed on Aug 03, 2024, the full disclosure of which is hereby incorporated by reference herein.
FIELD OF THE INVENTION
[002] The present invention relates to the field of nano technology more particularly to a calcium - phosphorous based nanocomposite feed additive along with other essential minerals for use in livestock and poultry to achieve bone strength, good health and improved immunity. Further, the invention is directed towards the process or method of preparation of the nanocomposite feed additive that provides a comprehensive nutritional solution to support the physiological and immunological needs of livestock and poultry.
BACKGROUND OF THE INVENTION
[003] Calcium (Ca) and Phosphorous (P) are important nutrients required for poultry and livestock. In poultry, Calcium is essential for bone development, and eggshell formation while phosphorous is a significant component in eggshell and plays an important role in energy metabolism. Besides Calcium and Phosphorous, other minerals such as magnesium, iron, zinc, copper, selenium, and manganese are vital for bone health, enzyme function, oxygen transport, and immune support. Essential minerals, micronutrients and amino acids as a balanced diet have a symbiotic relationship with each other that promises healthy bones, better growth, improved immunity and general functioning in turn promoting livestock and poultry markets.
[004] Calcium and Phosphorous requirement for livestock are huge as they directly influence the milk yield and the deficiency symptoms are fatal in many cases. Low calcium absorption in livestock can result in rickets, milk fever, osteomalacia, osteoporosis etc., where the major source of calcium and phosphorous used are mineral-based limestone, dicalcium phosphate or diammonium phosphate, and these Ca-P sources have bioavailability of less than 50%. Calcium metabolism is also greatly influenced by Vitamin D content. The site of absorption of calcium is the small intestine in most livestock and the solubility of calcium greatly influences its bioavailability.
[005] The most used sources of Calcium and Phosphorous includes dicalcium phosphate (DCP), monocalcium phosphate (MCP), tricalcium phosphate (TCP), monosodium phosphate (MSP), Calcium Carbonate (limestone), and plant-based phosphorous. The last time the National Research Council (NRC) established the mineral requirements for poultry was over 25 years ago. In the interim, diets have evolved, housing and management have improved, and genetics pertaining to broiler and layer chickens have seen significant changes. Many recent studies support the theory of high percentage Calcium and Phosphorous in poultry diet than the required concentrations.
[006] The major ignored concept is the difference between "absorption" and "digestion". Since there is no enzymatic hydrolysis for minerals, including Ca, absorption may be the most acceptable scientific word. Solubilization is the most crucial stage across the path of the intestinal epithelium, while Ca's solubility in the gastrointestinal tract of chickens is pH and size dependent. Ca is more soluble at lower pH values, where there is a negative correlation between solubility and pH. The gizzard and proventriculus are crucial components in calcium absorption while the fact that large particles are held longer in the gizzard slightly offsets the apparent disadvantage in the dissolving rate of large particulate Ca sources. The soluble calcium should be dissolved in the proventriculus and gizzard before reaching the small intestine, where the rise in pH poses a risk of precipitation with anions such as Ca, P and phytic acid before absorption.
[007] Another major bottleneck in the case of phosphorous is that most of the meals for non-ruminants include cereal grains containing high P concentrations but extremely low Ca concentrations. Still, much of the P is bound as phytate and is not biologically accessible. Thus, a need for non-phytate phosphorous is the point of focus which is mainly of mineral source. In addition, there is the issue of worldwide P scarcity and concerns over P-related environmental pollution, particularly from animal production facilities. An ideal feed formulation that minimizes P excretion could allay such concerns.
[008] Thus, considering all the research gaps, the nanocomposite feed additive based on Calcium-Phosphorous and other essential minerals is formulated with an appropriate proportion of calcium and phosphorous to optimize nutrient balance. Phosphorous is derived from a non-phytate form, enhancing its bioavailability as it avoids the inhibitory effects of phytates on mineral absorption. The calcium component is surface modified to ensure high solubility, addressing the common challenge of poor solubility in traditional calcium supplements. The nano-sized formulation further supports superior absorption, not just digestion, by increasing the surface area and pore volume and facilitating easier passage through cell membranes, ultimately leading to better overall nutrient uptake and efficacy.
OBJECT OF THE INVENTION
[009] The primary objective of this invention is to develop a calcium-phosphorus based nanocomposite that enhances nutrient bioavailability and effectiveness in livestock and poultry, thereby improving their overall health, well-being, growth, and productivity.
[0010] It is another object of the present invention to surface modify the calcium component in calcium-phosphorous based nanocomposite to ensure high solubility, overcoming the common issue of poor calcium absorption in traditional supplements.
[0011] It is yet another object of the present invention to downsize the calcium and phosphorous components to nano-range to support better absorption in small intestine, gizzard and proventriculus.
[0012] It is a further object of the present invention to formulate the calcium-phosphorous based nanocomposite with an appropriate proportion of calcium and phosphorous to optimize the nutrient balance for better health outcomes.
[0013] It is another object of the present invention to enrich the composite with vitamins and other essential nutrients or minerals in appropriate proportions to provide a balanced nutrition to livestock and poultry. The enriched calcium-phosphorous based nanocomposite is developed not only to enhance absorption but also to support efficient digestion, ensuring that the nutrients are effectively utilized.
[0014] Other objects of the invention will be apparent from the description of the invention herein below.
SUMMARY OF THE INVENTION
[0015] The aspects of the present invention can overcome the problems mentioned above and other problems in the prior art by providing a stable calcium - phosphorous based nanocomposite to be used as a feed additive in livestock and poultry to achieve bone strength, good health and improved immunity.
[0016] Accordingly, in one aspect, the present invention provides a calcium-phosphorous based nanocomposite feed additive comprising 80-90% w/w of one or more sources of calcium and phosphorus, 5-10% w/w of non-ionic surfactants, 1-5% w/w of synthetic polyelectrolyte anionic polymers, 1-5% w/w of natural or synthetic polyelectrolyte cationic polymers and 1-5% w/w of vitamins and micronutrients.
[0017] In another aspect, the invention provides a method of preparation of calcium-phosphorous based nanocomposite feed additive comprising steps of (a) preparing a core nanocomposite dispersion comprising calcium and phosphorous sources, vitamins, micronutrients and non-ionic surfactants; (b) preparing polyelectrolyte anionic and cationic polymer solutions; (c) layer-by layer encapsulating the calcium - phosphorous nanocomposite dispersion by anionic and cationic polymer solution; and (d) spray drying and micro milling the encapsulated calcium- phosphorous nanocomposite dispersion to obtain a dry powder of the calcium- phosphorous based nanocomposite.
[0018] These and other features, aspects and advantages of the present invention will become better understood with reference to the following drawings, description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1A illustrates the Fourier Transform Infrared (FTIR) spectroscopic analysis after first layer encapsulation with polyanionic polymer in accordance with an embodiment of the present invention;
[0020] Figure 1B illustrates the Fourier Transform Infrared (FTIR) spectroscopic analysis after second layer encapsulation with polycationic polymer in accordance with an embodiment of the present invention;
[0021] Figure 2A illustrates the zeta potential analysis after first layer encapsulation with polyanionic polymer in accordance with an embodiment of the present invention;
[0022] Figure 2B illustrates the zeta potential analysis after second layer encapsulation with polycationic polymer in accordance with an embodiment of the present invention;
[0023] Figure 3 illustrates the particle size distribution of the calcium-phosphorus based nanocomposite under Field Emission Scanning Electron Microscopy (FESEM) in accordance with an embodiment of the present invention;
[0024] Figure 4 illustrates the particle size distribution of the calcium- phosphorus based nanocomposite using Dynamic Light Scattering (DLS) in accordance with an embodiment of the present invention;
[0025] Figure 5 illustrates the surface area analysis of the spray-dried calcium- phosphorus based nanocomposite using Brunauer–Emmett–Teller (BET) surface analyser in accordance with an embodiment of the present invention;
[0026] Figure 6 illustrates the X-Ray diffraction pattern of the spray-dried calcium-phosphorus based nanocomposite in accordance with an embodiment of the present invention;
[0027] Figure 7A illustrates the bioavailability of calcium-phosphorous based nanocomposite in the gastric phase in accordance with an embodiment of the present invention;
[0028] Figure 7B illustrates the bioavailability of calcium-phosphorous based nanocomposite in the intestinal phase in accordance with an embodiment of the present invention;
[0029] Figure 8A illustrates the stable dispersion of calcium phosphorus (Ca-P) clusters within the non-ionic surfactant in accordance with an embodiment of the present invention;
[0030] Figure 8B illustrates the encapsulation of stabilized of Ca-P dispersion through layer-by-layer coating of polyelectrolyte polymers in accordance with an embodiment of the present invention; and
[0031] Figure 9 illustrates the average weight gain in poultry birds as a positive result of Ca-P nanocomposite when given as feed additive in accordance with an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] For a better understanding of the objects, technology and advantages of the present invention, the instant invention will be further explained in detail with respect to embodiments and accompanying figures as given above. It should be understood that the specific embodiments described herein are only to be used for explaining the present invention but not used to limit the present invention.
[0033] Broadly, the embodiments of the present invention provide a calcium-phosphorus nanocomposite to be used as a feed additive in poultry and livestock to achieve bone strength, good health and improved immunity. A process/method of synthesis/preparation of the calcium phosphorous nano composite includes using calcium and phosphorous sources at appropriate proportions and reducing the size by physical techniques of nanomaterial synthesis further modifying the surface with monolayers and bilayers of anionic and cationic polymers for better solubility in digestive tract.
[0034] The calcium - phosphorous based nanocomposite feed additive of the present invention with effective surface chemistry results in better bioavailability of calcium (Ca) and phosphorous (P).
[0035] The present invention, in an aspect, provides a calcium-phosphorous (Ca-P) based nanocomposite feed additive comprising calcium and phosphorus sources, non-ionic surfactants, synthetic polyelectrolyte anionic polymers, natural or synthetic polyelectrolyte cationic polymers and vitamins and micronutrients.
[0036] In an embodiment, one or more sources of calcium and phosphorus are selected from the group consisting of calcium carbonate, monocalcium phosphate, and dicalcium phosphate.
[0037] In an embodiment, the calcium sources are selected from calcium carbonate and dicalcium phosphate.
[0038] In an embodiment, the phosphorous sources are selected from monocalcium phosphate and dicalcium phosphate.
[0039] In an embodiment, the calcium and phosphorus sources are present in an amount of 80-90% w/w of the total weight of the nanocomposite.
[0040] In an embodiment, the calcium and phosphorus sources are in a weight ratio of 2.5:1, 2:1, or 1:1
[0041] In an embodiment, the non-ionic surfactants are selected from the group consisting of polyethylene glycol, propylene glycol, polysorbate 20, and polysorbate 80.
[0042] In an embodiment, the non-ionic surfactants are present in an amount of 5-10% w/w of the total weight of the nanocomposite.
[0043] In an embodiment, the polyelectrolyte anionic polymer is synthetic.
[0044] In an embodiment, the synthetic polyelectrolyte anionic polymers are selected from the group consisting of polyacrylic acid, sodium alginate, and poly-L-glutamic acid.
[0045] In an embodiment, the synthetic polyelectrolyte anionic polymers are present in an amount of 1-5% w/w of the total weight of the nanocomposite.
[0046] In an embodiment, the polyelectrolyte cationic polymer is natural or synthetic.
[0047] In an embodiment, the natural or synthetic polyelectrolyte cationic polymers are selected from the group consisting of chitosan, polyethyleneimine, and poly allylamine hydrochloride.
[0048] In an embodiment, the source and geographical origin of chitosan is Marine Hydrocollides, Santo Gopalan Road, Chullickal, Cochin - 682 005, Kerala, India.
[0049] In an embodiment, the natural or synthetic polyelectrolyte cationic polymers are present in an amount of 1-5% w/w of the total weight of the nanocomposite.
[0050] In an embodiment, the vitamins are selected from the group consisting of vitamin A, D, E, K, and B.
[0051] In an embodiment, the vitamins include equal parts of vitamin A, D, E, K, and B.
[0052] In an embodiment, the micronutrients are selected from the group consisting of zinc, iron, copper, and manganese.
[0053] In an embodiment, the vitamins and micronutrients are present in an amount of 1-5% w/w of the total weight of the nanocomposite.
[0054] In an embodiment, the vitamins and micronutrients are in a weight ratio of 4:1.
[0055] In a non-limiting embodiment, the particle size of the nano composite is 200 to 600 nm as characterized by Field Emission Scanning Electron Microscopy (FESEM) and Dynamic Light Scattering (DLS).
[0056] In an embodiment, the zeta potential of the nanocomposite is >+25 mV.
[0057] In a non-limiting embodiment, the zeta potential of the nanocomposite is +25.3 mV.
[0058] In an embodiment, the calcium-phosphorus based nanocomposite is characterized by Fourier Transform Infrared (FTIR) spectroscopy.
[0059] In a non-limiting embodiment, the Fourier Transform Infrared (FTIR) spectroscopy having spectra peaks at 3330cm?¹, confirms the hydrogen bonding from polyacrylic acid (PAA), while prominent peaks at 1635cm?¹, 1413cm?¹ confirms the -COO- groups in PAA reacting with Ca2+. The significant peak at 1022 cm?¹ is linked to phosphate (PO4³?) stretching vibrations while the peak at 628 cm?¹ also indicates the existence of phosphate-metal interactions or P–O–P. A band at 1635 cm?¹ attributes to the asymmetric stretching of carboxylate groups (–COO?) from PAA, suggesting ionic interactions between protonated –NH3? groups of chitosan and –COO? groups of the underlying PAA layer.
[0060] In a non-limiting embodiment, the nanocomposite is characterized by X-ray diffraction (XRD) showing a crystalline nature with characteristic peak at 29.3°.
[0061] In an embodiment, the surface area of the nanocomposite of the present invention is analysed using Brunauer–Emmett–Teller (BET) surface analyser.
[0062] In a non-limiting embodiment, the surface area of the nanocomposite is 23.434 m2/g.
[0063] In an embodiment, the present nanocomposite feed additive can be formulated in the form of powder, pellet, granule or liquid dispersions.
[0064] The present invention, in another aspect, provides the method of preparation of calcium-phosphorous based nanocomposite feed additive comprising the steps of:
a) preparing a core nanocomposite dispersion by dispersing calcium and phosphorous sources, vitamins and micronutrients in distilled water and mixing using a high shear mixer at speed 400-500 rpm for 60-80 minutes to obtain aqueous dispersed solution; subjecting the aqueous dispersed solution to ultrasonication at 15-30 kHz for 80-120 minutes; and adding non-ionic surfactants and mixing using a high shear mixer at speed 200-300 rpm for 60-80 minutes to obtain the calcium – phosphorous nanocomposite dispersion ( Solution A);
b) preparing polyelectrolyte polymer solutions by dissolving anionic polymers in water maintaining at alkaline pH; and dissolving cationic polymers in water maintained at acidic pH to obtain Solution B and Solution C respectively;
c) layer-by layer encapsulating the calcium -phosphorous nanocomposite dispersion by i) adding Solution A to Solution B and stirring using high sheer agitator at 250 to 400 rpm for 30-40 min followed by filtration to separate excess polymer and redispersed particles; and ii) adding redispersed particles to Solution C and stirring using high sheer agitator at 250 to 400 rpm for 30-40 min to obtain an encapsulated calcium- phosphorous nanocomposite dispersion; and
d) forming a powder nanocomposite by spray drying the encapsulated calcium- phosphorous nanocomposite dispersion with the inlet temperature of 250-300?C and an outlet temperature of 140-170?C followed by micro milling for 60-90 minutes using 1mm-3mm zirconium and ceramic balls at 400-500 rpm to obtain a dry powder of the calcium- phosphorous based nanocomposite.
[0065] In an embodiment, in step (a) one or more sources of calcium and phosphorus are selected from the group consisting of calcium carbonate, monocalcium phosphate, and dicalcium phosphate and the weight percentage of calcium and phosphorous sources is 80-90%.
[0066] In an embodiment, in step (a) the vitamins are selected from the group consisting of vitamin A, D, E, K, and B, and the micronutrients are selected from the group consisting of zinc, iron, copper, and manganese and the weight percentage of vitamins and micronutrients is 1-5%.
[0067] In an embodiment, in step (a) the non-ionic surfactants are selected from the group consisting of polyethylene glycol, propylene glycol, polysorbate 20, and polysorbate 80 and the weight percentage of non-ionic surfactants is 5-10%.
[0068] In an embodiment, in step (b) the synthetic anionic polymers are selected from the group consisting of polyacrylic acid, sodium alginate, and poly-L-glutamic acid and the weight percentage of anionic polymers is 1-5%.
[0069] In an embodiment, in step (b) the natural or synthetic cationic polymers are selected from the group consisting of chitosan, polyethyleneimine, and poly allylamine hydrochloride and the weight percentage of cationic polymers is 1-5%.
[0070] In an embodiment, the step (c) comprises filtration selected from automated crossflow filtration and tangential flow filtration.
[0071] The present invention, in yet another aspect, provides the method for improving health and immunity in livestock and poultry, comprising incorporating an effective amount of the nanocomposite additive of the present invention into the feed of said animals.
[0072] In an embodiment, the calcium-phosphorus-based nanocomposite feed additive is incorporated into the livestock or poultry feed at a concentration of 5-10 kg per ton of feed.
[0073] In an embodiment, the livestock is selected from the group consisting of cattle, pigs, sheep and goats.
[0074] In an embodiment, poultry is selected from the group consisting of chickens, turkey, duck, geese and quails.
[0075] The above description of the invention, together with the below accompanying examples should not be construed as limiting the invention because those skilled in the art to which this invention pertains will be able to devise other forms thereof within the ambit of the appended claims.
EXAMPLES
Example 1: Method of Preparation of the calcium-phosphorous (Ca-P) based nanocomposite of the present invention
[0076] A stable calcium – phosphorous based nanocomposite prepared by the method as below is used as a feed additive that provides a comprehensive nutritional solution to support the physiological and immunological needs of poultry and livestock.
Step(a): Preparation of Core Nanocomposite Dispersion
[0077] A dispersed aqueous mixture was prepared by combining 24.60 wt% calcium carbonate, 10 wt% dicalcium phosphate, and 51.4 wt% monocalcium phosphate in distilled water. The mixture was subjected to high-shear mixing for uniform dispersion at 400-500 rpm for 60-80 minutes. To it, 4% of micronutrient salts of zinc sulphate, manganese sulphate, ferrous sulphate and copper sulphate in the ratio of 1:1:1:1 was added. The vitamin blend of A, B complex, D and E was added at a concentration of 1% to the dispersion and mixed thoroughly.
[0078] The dispersion was then subjected to ultrasonication at 20 kHz for 90 minutes to achieve uniform nanoscale dispersion. After ultrasonication, a 5% non-ionic surfactant blend of polyethylene glycol (PEG) and propylene glycol in a 1:1 weight ratio was added to the dispersion to obtain the calcium – phosphorous nanocomposite dispersion (Solution A). This blend via hydrogen bonding, van der waals based surface adsorption, and viscosity modulation, effectively prevents aggregation and enhances dispersion stability. The pH of the dispersion solution was adjusted to 8.5 using a buffer solution to facilitate the formation of Ca-P clusters.
Step (b): Preparation of Polyelectrolyte Polymer Solutions
[0079] Solution B; A 1.5 wt% solution of polyacrylic acid (PAA) in water, adjusted to pH 8.5 using buffer solution to ensure complete ionization of carboxyl groups (–COO?) present in the polymer.
[0080] Solution C: A 1.5 wt% solution of chitosan in 0.5% glacial acetic acid solution (pH 3) to ensure protonation and facilitate solubilization of the polymer.
Step (c): Layer-by Layer encapsulation of the calcium -phosphorous nanocomposite dispersion
[0081] Solution A was added slowly at a flow rate of 5-10 liter per minute to Solution B (PAA) under controlled agitation at 300 to 350 rpm in a high-shear agitator. The pH of the solution was maintained at 8.5–9 to facilitate electrostatic adsorption of the anionic polymer via interaction between –COO? groups of PAA and Ca²? ions on the nanoparticle surface. After 30-40 minutes of mixing, the particles were separated from the excess polymer solution using automated crossflow filtration and then redispersed in deionized water.
[0082] The redispersed particles were then added to Solution C (chitosan) with the pH adjusted to 4.5 to 5.5 to ensure protonation of –NH2 groups to –NH3?. Stirring was continued for 30-40 minutes at 300 to 350 rpm to promote electrostatic interaction between the –NH3? groups of chitosan and the –COO? groups from the previously adsorbed PAA layer, resulting in the formation of a uniform second (cationic) layer.
Step (d) Formation of powder nanocomposite
[0083] The encapsulated Ca-P nanocomposite dispersion was subjected to spray drying. The inlet temperature was maintained at 270°C and the outlet temperature at 155°C followed by micro milling for 60-90 minutes using 1mm-3mm zirconium and ceramic balls at 400-500 rpm to obtain a dry powder of the calcium- phosphorous based nanocomposite.
Example 2: Fourier Transform Infrared (FTIR) spectroscopic analysis of layer-by-layer encapsulation in the process of present invention
[0084] The FTIR analysis is done to confirm the anionic polymer encapsulation layer in the Ca-P stable dispersion solution of the present invention (Fig 1A). The FTIR spectrum of the calcium phosphorous (Ca-P) dispersion after reaction with poly (acrylic acid) (PAA) confirms the effective interaction between the anionic polymer and the surface of the Ca-P stable nano dispersion. A wide absorption band near 3330cm?¹ relates to O–H stretching vibrations, signifying the existence of hydrogen-bonded hydroxyl groups from either water or PAA. Significantly, prominent peaks at 1635cm?¹ and 1413cm?¹ correspond to the asymmetric and symmetric stretching vibrations of carboxylate (–COO?) groups, respectively. These bands are indicative of PAA's interaction with calcium ions, implying the development of ionic complexes via –COO?–Ca²? coordination. The gap between these two peaks signifies bidentate or bridging coordination modes. Moreover, the significant peak at 1022 cm?¹ is linked to phosphate (PO4³?) stretching vibrations, supporting the existence of the calcium phosphate structure. The peak at 628 cm?¹ also indicates the existence of phosphate-metal interactions or P–O–P bending modes. In summary, the spectrum clearly indicates PAA attachment to the Ca-P nanoparticles via electrostatic and coordination interactions, resulting in a stable, polymer-encased nanocomposite.
[0085] The FTIR analysis is done to confirm the cationic polymer encapsulation as second layer is formed above the Ca-P stable dispersion solution of the present invention (Fig 1B). The FTIR spectrum of the calcium phosphorous (Ca-P) dispersion encapsulated first with poly(acrylic acid) (PAA) and subsequently with chitosan confirms the successful formation of a bilayer nanocomposite. A broad absorption band around 3318 cm?¹ corresponds to overlapping O–H and N–H stretching vibrations, indicating the presence of hydroxyl groups from the phosphate surface and the polysaccharide backbone, along with primary amine groups from chitosan. A prominent band at 1635 cm?¹ can be attributed to either the amide I region of chitosan or the asymmetric stretching of carboxylate groups (–COO?) from PAA, suggesting ionic interactions between protonated –NH3? groups of chitosan and –COO? groups of the underlying PAA layer. Additional peaks at 1116 cm?¹ and 1029 cm?¹ correspond to C–O stretching vibrations of the chitosan backbone and P–O stretching of phosphate groups in the Ca-P core, respectively. The band at 625 cm?¹ reflects P–O–P bending or Ca–O lattice vibrations, confirming that the structural integrity of the Ca-P core is retained after polymer layering. Overall, the spectral features validate the effective layer-by-layer encapsulation of Ca-P with both PAA and chitosan, demonstrating successful surface functionalization via electrostatic and hydrogen bonding interactions.
Example 3: Zeta potential analysis of the layer-by-layer encapsulation in the process of present invention
[0086] The zeta potential analysis is done to confirm the anionic polymer encapsulation layer which is formed above the Ca-P stable dispersion solution (Fig 2A). A zeta potential of –32.7 mV for the calcium phosphorous (Ca-P) dispersion after reacting with poly (acrylic acid) (PAA) indicates successful surface functionalization with the anionic polymer. The negative charge depicts the carboxylate (–COO?) groups of PAA that bind to the Ca²? ions in the Ca-P dispersion core, leaving an excess of negatively charged groups exposed. This value reflects the electrostatic stabilization, sufficient to prevent particle aggregation and prevents precipitation. Furthermore, the negative surface charge confirms the readiness of the PAA-coated nanoparticles for subsequent layer-by-layer encapsulation with a cationic polymer, ensuring effective electrostatic interaction in the next coating step.
[0087] The zeta potential is done to confirm the cationic polymer encapsulation as second layer which is formed above the Ca-P stable dispersion solution (Fig 2B). A zeta potential of +25.3 mV for the calcium phosphorous (Ca-P) dispersion after coating with chitosan over the PAA-encapsulated Ca-P core indicates a successful reversal of surface charge, confirming the effective adsorption of the cationic polymer. Chitosan, containing protonated amine groups (–NH3?) under mildly acidic conditions, binds electrostatically to the negatively charged carboxylate groups (–COO?) of the PAA layer. The +25.3-mV reading suggests electrostatic repulsion between particles and further validates the formation of a stable bilayer nanocomposite structure through layer-by-layer encapsulation.
Example 4: Particle size distribution of the Ca-P nanocomposite of the present invention under Field Emission Scanning Electron Microscopy (FESEM)
[0088] The spray dried sample of Ca-P nanocomposite for analysis under FESEM is sprinkled on a carbon tape to avoid charging effects during electron beam scanning. To enhance conductivity and image resolution, a thin layer of conductive coating (gold) was sputtered on the surface. The parameters of the FESEM, such as accelerating voltage and magnification was adjusted to optimize imaging for particle dispersion and individual particle morphology. The FESEM image obtained (Fig 3) shows well-dispersed particles with an average size distribution between 200 to 600 nm, as indicated by measured dimensions. The particles appear to have a relatively uniform size distribution, suggesting successful encapsulation of well-dispersed nutrient ions. The magnification of 50,000× highlights individual particle boundaries, while the absence of significant aggregation indicates good dispersion in the preparation. The scale bar of 200 nm and clear resolution further confirm the morphology characteristics of the nanocomposite.
Example 5: Particle size distribution of the Ca-P nanocomposite of the present invention dispersed in water using Dynamic Light Scattering (DLS)
[0089] The objective of this study is to determine the particle size distribution of the Ca-P nanocomposite using ASTM E3247 guidelines, which standardize the determination of nanoparticle size through batch-mode dynamic light scattering in aqueous suspensions. Sample preparation begins with the cleaning of glassware and sample cavities to ensure minimal contamination, involving rinsing with a cleaning agent and deionized water, followed by thorough drying with lint-free tissue. The test sample is prepared using deionized water as the diluent, with dilution of 1% (1 g dissolved in 100 ml). The diluted sample is filtered through a 0.45-micron filter, ensuring uniformity. Prior to analysis, the disposable or quartz cells are thoroughly rinsed with deionized water and test solution to avoid contamination. The filtered sample is then loaded into the cell using a clean syringe and analysed to determine the hydrodynamic diameter of nanoparticles in water suspensions.
[0090] The particle size distribution was found to be between 200-600 nm (Fig 4) with a poly dispersity index of 0.2 to 0.3 indicating a uniform distribution of nano particles even after dispersed in water.
Example 6: Surface area analysis of the Ca-P nanocomposite of the present invention using Brunauer–Emmett–Teller (BET) surface analyser
[0091] The BET analysis results indicate that the Ca-P nanocomposite resulted in higher surface area (Fig 5). The increased surface area of 23.434 m2/g could enhance the absorption of those minerals (Ca and P) in the digestive system of poultry/livestock animals when compared to the surface area of 6.979 m2/g of conventional feed grade dicalcium phosphate (DCP). The larger pore diameter of 20.6069 nm, which could improve the digestibility and bioavailability of Ca and P when included in livestock and poultry diets and the higher pore volume of 0.0589 cm3g results in enhanced release rate of nutrients over time which benefits better absorption.
[0092] The elevated surface area observed in the nano composite is a defining characteristic of nano-scale materials and directly correlates with enhanced bioavailability.
Example 7: X-Ray Diffraction analysis of the Ca-P nanocomposite of the present invention
[0093] The X-ray diffraction (XRD) pattern of the spray-dried calcium phosphorous (Ca-P) nanocomposite shows distinct crystalline peaks, with the most prominent diffraction angles observed at 2? ˜ 11.6°, 29.3°, 32.4°, 42.8°, and 48°. The intense peak at 29.3° suggests a dominant crystalline phase, typically associated with hydroxyapatite (Ca10(PO4)6(OH)2) or dicalcium phosphate dihydrate (DCPD), depending on peak matching with standard reference patterns (Fig 6). The peak at 11.6° is characteristic of DCPD (brushite), confirming the presence of a hydrated calcium phosphate phase in the nanocomposite. Peaks near 32.4° and 48° are also commonly attributed to hydroxyapatite reflections, supporting partial crystallization or transformation of phosphate phases during spray drying. The overall pattern—with sharp peaks superimposed on a broader background—indicates a mixed-phase material with both crystalline and amorphous regions, likely due to the rapid solvent evaporation during spray drying and the presence of stabilizing polymers. This confirms the successful synthesis of a structurally organized Ca-P nanocomposite.
Example 8: Analysis of Ca and P content in the nano composite feed additive of the present invention
[0094] Calcium analysis: The determination of calcium content in samples was carried out using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) in accordance with IS 3025, Part-65:2022. The procedure began with the thorough cleaning of all glassware and Teflon digestion vessels using a sequential wash of detergent, tap water, nitric acid, and deionized water to prevent contamination. For sample preparation, a 45 mL aliquot of the aqueous sample was digested with 5 mL of concentrated nitric acid in a Teflon microwave digestion vessel. The digestion was performed using a microwave digestion system under controlled temperature conditions as per the equipment manufacturer’s guidelines. After cooling, the digested sample was either filtered or settled before analysis. The ICP-OES instrument was initialized by igniting the plasma and allowing it to stabilize for 30–60 minutes. Calibration was performed using a blank and a working standard, with the instrument software set according to the manufacturer's protocol. Calcium was then quantified based on the emission intensities, with results directly reported in mg/L. If sample concentrations exceeded the linear range of detection, appropriate dilutions with nitric acid were made and reanalysis was conducted to ensure accuracy.
[0095] Phosphorous analysis: The objective of this analysis is to determine the percentage of phosphorus in spray dried Ca-P nano composite using the Gravimetric Quinoline Molybdate Method as per the FCO, Schedule-2, Part-B:1985 guidelines. The process involves sample digestion with nitric and perchloric acids to oxidize organic matter, followed by precipitation using the Quimociac reagent. After careful cooling and filtration, the precipitate, consisting of (C6H7N)3H3PO4·12MoO3, is dried to constant weight in a crucible at 250°C. The weight difference between the crucible with precipitate (G1) and the empty crucible (G2) is used to calculate P2O5 using the formula:
% P2O5 (w/w) = (G1-G2) x 3.2074 x V2
G3 x V1
where V1 is the aliquot volume, V2 is the sample dilution volume, and G3 is the sample weight. This method ensures accurate quantification of phosphorus content, essential for quality assessment.
[0096] Based on the above-mentioned methods the results obtained are as follows:
Total Calcium % w/w = 25.11
Total Phosphorous % w/w = 18.46
Example 9: In-vitro Bioavailability of Ca-P based nanocomposite feed additive of the present invention in gastric phase and intestinal phase in the digestive tract of the chicken
[0097] The in-vitro bioavailability of the calcium phosphorous nanocomposite was evaluated under simulated gastric and intestinal conditions mimicking the digestive environment of poultry birds. In the gastric phase, the nanocomposite was incubated with pepsin in an acidic medium (0.1 M HCl) at 37°C for 2 hours, followed by neutralization with sodium bicarbonate. This setup replicates stomach digestion, where pepsin aids in breaking down proteins, enhancing the release and solubility of minerals like calcium and phosphorus. The results indicated that the nanocomposite significantly improved gastric phase bioavailability (Fig 7A), with calcium and phosphorus concentrations being markedly higher than conventional DCP used as feed, suggesting enhanced solubilization due to the smaller particle size and higher surface area of the nanocomposite. In the intestinal phase simulation, the nanocomposite was incubated with pancreatin enzymes and bile salts at pH 7.2, simulating the small intestine's enzymatic activity. Again, the nanocomposite outperformed conventional feed grade DCP, with calcium bioavailability being 2.3 times higher and phosphorus 1.75 times higher (Fig 7B). This improvement is attributed to the increased solubility and interaction potential of the nano-sized calcium and phosphorous in biological fluids, leading to superior absorption. These results support the nanocomposite's enhanced efficiency and suitability for poultry feed supplementation at reduced dosages.
Example 10: Synergistic activity or the intermolecular interactions in Ca-P nanocomposite of the present invention
[0098] The preparation of Ca-P nanocomposite involves preparing a stable dispersion of Ca and P sources using ultrasonication and addition of non-ionic surfactant following a layer-by-layer encapsulation of anionic and cationic polymer in sequence. The molecular docking image (Fig 8A) illustrates the encapsulation of Ca-P clusters within a polyethylene glycol (PEG) shell, demonstrating successful surface interaction and stabilization. In step (a) of the method, the dispersion of Ca-P is stabilized through ultrasonication followed by the addition of non-ionic surfactants like PEG. The dissolution of precursors such as CaCO3, CaHPO4, and Ca(H2PO4)2 generates Ca²? and phosphate ions. Upon adjustment to an alkaline pH (8–9), phosphate equilibria shift to produce more PO4³?, favoring the precipitation of Ca-P. PEG interacts with the Ca-P through hydrogen bonding (PEG–O?H–OPO3²?) and van der waals forces, creating steric stabilization that prevents particle aggregation. Propylene glycol, used as a viscosity modifier, further enhances this stabilization by inhibiting precipitation. The molecular docking image highlights these interactions with PEG forming a surrounding matrix around the Ca-P core, consistent with the encapsulation and surface interactions described.
[0099] In step (c) (Fig 8B), the stabilized Ca-P dispersion undergoes further functionalization through layer-by-layer polyelectrolyte encapsulation. Initially, an anionic polyelectrolyte containing –COOH/–COO? groups bind to the Ca²? ions on the Ca-P surface via ionic interactions. This process is pH-dependent and is optimized at alkaline pH (8–9) to ensure maximum binding. Subsequently, a cationic polyelectrolyte layer is added, where –NH2/–NH3? groups form electrostatic bonds with the negatively charged –COO? groups of the first layer. For effective cationic polymer binding, the pH is adjusted to slightly acidic conditions (pH 4–6) to protonate amine groups. This layer-by-layer assembly strategy results in enhanced stability, biocompatibility, and surface functionalization of Ca-P nanoparticles. The molecular docking image reinforces this mechanism by showing close spatial orientation and surface interaction of PEG with the Ca-P core, indicating successful encapsulation and stabilization prior to polyelectrolyte coating.
Example 11: Efficacy of the Ca-P based nanocomposite feed additive of the present invention in farm trials
[00100] The trial conducted at Karnataka Veterinary, Animal and Fisheries University demonstrated that Ca-P based nanocomposite can effectively replace 40–50% of conventional dicalcium phosphate (DCP) in broiler feed without compromising performance. Birds fed with reduced levels of Ca-P nanocomposite feed additive (up to 60%) showed comparable or improved metrics in body weight, average daily gain, and feed conversion ratio compared to those on 100% conventional feed grade DCP. Figure 9 illustrates the gain in average body weight (in grams) as an effect of Ca-P nanocomposite with different inclusion rates compared with conventional DCP. The different concentrations of Ca-P nanocomposite as feed additive tested are as follows:
T1: Control- 8 Kg of Conventional DCP per ton of feed
T2- 7.2 Kg of Ca-P nanocomposite as an additive per ton of feed
T3- 6.4 Kg of Ca-P nanocomposite as an additive per ton of feed
T4- 5.6 Kg of Ca-P nanocomposite as an additive per ton of feed
T5- 4.8 Kg of Ca-P nanocomposite as an additive per ton of feed
T6- 4 Kg of Ca-P nanocomposite as an additive per ton of feed

[00101] Notably, birds fed with feeds containing 70% and 60% of Ca-P nanocomposite feed additive inclusion performed as well as or better than the control group (fed with 100% dose of conventional DCP), indicating that the nanoform ensures efficient mineral utilization at lower inclusion rates.
[00102] Further analysis of carcass characteristics, organ weights, serum mineral levels, and tibia bone parameters confirmed that there were no significant adverse effects from reducing the feed as nanoform Ca-P. Bone strength and length, serum calcium and phosphorus, and immune responses (ND and IBD titres) remained within acceptable ranges across all treatment groups. This suggests that the nanoform's higher bioavailability enables better absorption and utilization, allowing for cost-effective feed formulation. Overall, the trial supports the viability of Ca-P nanocomposite of the present invention in reducing feed mineral input while maintaining poultry health and performance.
[00103] While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method to implement the inventive concept as taught herein.
[00104] Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open-ended as opposed to limiting. As examples of the fore going: the term “including” should be read as mean “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive limiting list thereof; and adjectives such as “conventional,” “traditional,” “standard,” “known” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or standard technologies that may be available or known now or at any time in the future. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group but rather should also be read as “and/or” unless expressly stated otherwise. Furthermore, although item, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.
[00105] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
,CLAIMS:We claim:
1. A calcium-phosphorous based nanocomposite feed additive comprising:
a) 80-90% w/w of one or more sources of calcium and phosphorus, wherein the sources are selected from the group consisting of calcium carbonate, monocalcium phosphate, and dicalcium phosphate and the calcium and phosphorus sources are in a weight ratio of 2.5:1, 2:1, or 1:1;
b) 5-10% w/w of non-ionic surfactants selected from the group consisting of polyethylene glycol, propylene glycol, polysorbate 20, and polysorbate 80;
c) 1-5% w/w of synthetic polyelectrolyte anionic polymers selected from the group consisting of polyacrylic acid, sodium alginate, and poly-L-glutamic acid;
d) 1-5% w/w of natural or synthetic polyelectrolyte cationic polymers selected from the group consisting of chitosan, polyethyleneimine, and poly (allylamine hydrochloride); and
e) 1-5% w/w of vitamins and micronutrients, wherein the vitamins are selected from the group consisting of vitamin A, D, E, K, and B, and the micronutrients are selected from the group consisting of zinc, iron, copper, and manganese, and the micronutrients and vitamins are in a weight ratio of 4:1.
2. The calcium-phosphorous based nanocomposite feed additive as claimed in claim 1 wherein the particle size of the nano composite is 200 to 600 nm.
3. The calcium-phosphorous based nanocomposite feed additive as claimed in claim 1, wherein the zeta potential of the nanocomposite is >+25 mV.
4. The calcium-phosphorous based nanocomposite feed additive as claimed in claim 1, wherein the nanocomposite is characterized by:
Fourier Transform Infrared (FTIR) spectroscopy having spectra peaks at 3330cm?¹, 1635cm?¹, 1413cm?¹, 1022 cm?¹, 628 cm?¹ and 1635 cm?¹;
X-ray diffraction (XRD) pattern showing a crystalline nature with characteristic peak at 29.3°;
Brunauer–Emmett–Teller (BET) isotherm showing a surface area of 23.434 m2/g; or
Field Emission Scanning Electron Microscopy image (FESEM) showing particle size distribution of the nanocomposite as depicted in figure 3.
5. A method of preparation of calcium-phosphorous based nanocomposite feed additive comprising the steps of:
a) preparing a core nanocomposite dispersion by dispersing calcium and phosphorous sources, vitamins and micronutrients in distilled water and mixing using a high shear mixer at 400-500 rpm for 60-80 minutes to obtain aqueous dispersed solution; subjecting the aqueous dispersed solution to ultrasonication at 15-30 kHz for 80-120 minutes; and adding non-ionic surfactants and mixing using a high shear mixer at speed 200-300 rpm for 60-80 minutes to obtain the calcium – phosphorous nanocomposite dispersion( Solution A);
b) preparing polyelectrolyte polymer solutions by dissolving anionic polymers in water maintaining at alkaline pH; and dissolving cationic polymers in water maintained at acidic pH to obtain Solution B and Solution C respectively;
c) layer-by layer encapsulating the calcium -phosphorous nanocomposite dispersion by i) adding Solution A to Solution B and stirring using a high sheer agitator at 250 to 400 rpm for 30-40 min followed by filtration to separate excess polymer and redispersed particles; and ii) adding redispersed particles to Solution C and stirring using a high sheer agitator at 250 to 400 rpm for 30-40 min to obtain an encapsulated calcium- phosphorous nanocomposite dispersion; and
d)forming a powder nanocomposite by spray drying the encapsulated calcium- phosphorous nanocomposite dispersion at an inlet temperature of 250-300?C and an outlet temperature of 140-170?C followed by micro milling for 60-90 minutes using 1mm-3mm zirconium and ceramic balls at 400-500 rpm to obtain a dry powder of the calcium- phosphorous based nanocomposite.
6. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein in step (a) one or more sources of calcium and phosphorus are selected from the group consisting of calcium carbonate, monocalcium phosphate, and dicalcium phosphate and the weight percentage of calcium and phosphorous sources is 80-90%.
7. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein in step (a) the vitamins are selected from the group consisting of vitamin A, D, E, K, and B, and the micronutrients are selected from the group consisting of zinc, iron, copper, and manganese and the weight percentage of vitamins and micronutrients is 1-5%.
8. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein in step (a) the non-ionic surfactants are selected from the group consisting of polyethylene glycol, propylene glycol, polysorbate 20, and polysorbate 80 and the weight percentage of non-ionic surfactants is 5-10%.
9. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein in step (b) the synthetic anionic polymers are selected from the group consisting of polyacrylic acid, sodium alginate, and poly-L-glutamic acid and the weight percentage of anionic polymers is 1-5%.
10. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein in step (b) the natural or synthetic cationic polymers are selected from the group consisting of chitosan, polyethyleneimine, and poly allylamine hydrochloride and the weight percentage of cationic polymers is 1-5%.
11. The method of preparation of calcium-phosphorous based nanocomposite feed additive as claimed in claim 5, wherein step (c) comprises filtration selected from automated crossflow filtration and tangential flow filtration.