Description:TECHNICAL FIELD
The present invention relates to a method of extraction of phytochemicals from natural sources and adoption of the same in the preparation of nutrition enriched supplements for livestock. The method comprises adoption of a unique medium- a synergistic formulation for easy extraction of phytochemicals from natural sources comprising- biomass and herbs. The extraction method of present invention is combined with biotransformation, bioenhancement and stabilization steps, for developing nutrition enriched supplement for livestock.
BACKGROUND AND PRIOR ART
Meeting farmed livestock nutritional requirements is extremely important while rearing livestock. Nutrition affect their growth rate, milk production, reproduction rate, immunity, and over all well-being. Farmed livestock solely depend on feed and feed supplements therewith given by humans and hence it becomes important to design and develop feed and feed supplements that provides balanced nutrition, depending on the livestock.
Most effective way to supply nutrition to livestock is to have a system which can help in selective extraction of intended phytoconstituent/phytochemicals from targeted natural source used to feed livestock and bio-transform the phytoconstituent/phytochemicals to easily digestible constituents so that it can enhance the bioavailability in livestock, thus increasing the nutritional value.
Conventional methods used for extraction of intended phytoconstituents/ phytochemicals is organic solvent medium. Main factor for extraction depends on the nature of solvent selected e.g. hydrophilic or lipophilic. These types of extraction do not ensure breaking down of insoluble fibrous material like lignocellulosic components, which may be either holding required phytochemicals or molecules of interest. Further these methods of extraction may lead to solvent residue in the extract contributing to toxicity issues, fire and environmental hazards, loss of solvent during extraction and recovery phase which will lead to cost escalation. To increase the efficiency of extraction, high temperature and pressure are required which will result in degradation or deactivation of thermolabile phytochemicals.
To overcome the problem faced in the adoption of conventional method, novel class of solvent known as deep eutectic solvents (DES) and natural deep eutectic solvent (NADES) are used. These systems are eco-friendly, non-toxic and biodegradable organic compounds, low cost, being easy to produce. However, both DES and NADES suffers limitation of using high volume of the solvent system (up to 20 times with respect to substrate), energy intensive recovery process, which makes them less attractive for the large-scale production.
Present invention aims to address the above mentioned problem by adopting a unique system comprising a medium for extraction of phytochemicals from natural sources comprising biomass and herbs. It is also proposed to use the system for enhancing the nutrition value of the feed supplements for livestock by following steps of biotransformation, bioenhancement and stabilization.

SUMMARY OF INVENTION
Accordingly, the present invention provides a unique formulation of compounds that presents itself as a unique medium for easy extraction of phytochemicals from natural sources.
The present invention provides synergistic formulation as a medium for extraction of phytochemicals from natural source , comprising nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%.
The invention also provides a method of preparation of synergistic formulation as a medium for extraction of phytochemicals from biomass, comprising nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%; said method comprising acts of
a) mixing the nitrogen containing compound, natural organic compound, edible acid, alkali or alkaline earth metal compound and water in a blender to obtain a mixture; and
b) heating the mixture at a temperature of 50-140°C for about 15 minutes to 25 hours to obtain the synergistic formulation.
The present invention also adopts the medium for solubilisation of phytochemical from natural sources- biomass, herbs and said step in combination with biotransformation, bioenhancement, stabilization to provide feed supplement which is easily digestible and enhanced in nutritional value for livestock.
The present invention provides a method of preparation of livestock feed supplements enhanced in nutritional value.
The invention also relates to a feed supplement for livestock, comprising synergistic formulation as a medium for extraction of phytochemicals from biomass, wherein the synergitic formulation comprises nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60% , said feed prepared by a method comprising acts of –
(a) preparation the synergistic formulation as the medium for extraction;
(b) charging the natural source into the medium and heating at a temperature ranging from 80-90°C for 2-15 hours and cooling to solubilize the phytochemicals from biomass;
(c) taking solubilized natural source through steps of biotransformation and bioenhancement; and
(d) stabilization of the bioenhanced natural source by mixing with a stabilizer to obtain the feed supplement for livestock.
The invention relates to a method of preparation of feed supplement for livestock comprising synergistic formulation as a medium for extraction of phytochemicals from biomass, wherein the synergistic formulation comprises nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%, said method comprising acts of;
(a) preparation the synergistic formulation as the medium for extraction, comprising steps of –
(i) mixing the nitrogen containing compound, natural organic compound, edible acid, alkali or alkaline earth metal compound and water in a blender to obtain a mixture; and
(ii) heating the mixture at a temperature of 50-140°C for about 15 minutes to 25 hours to obtain the synergistic formulation;
(b) charging the natural source into the medium and heating at a temperature ranging from 80-90°C for 2-15 hours and cooling to solubilize the phytochemicals from natural source;
(c) Taking solubilized natural source through steps of biotransformation and bioenhancement; and
(d) stabilization of the bioenhanced natural source to obtain the feed supplement for livestock.
BRIEF DESCRIPTION OF FIGURE
The figure provided herein is for exemplifying only and should not be considered to be restricting in terms of scope of invention.
Figure 1: shows the graph on milk yield enhancement in cows by the usage of feed supplement of present invention.
DESCRIPTION OF DISCLOSURE
The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the figures, chemicals used in the invention, description and claims. It may further be noted that as used herein and in the appended claims, the singular “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.

Definition:
a) Nitrogen containing compound: A compound selected from a group comprising polyalkyl ammonium salts and amides.
b) Natural organic compound: A compound selected from a group comprising polyols, sugars, hydroxy acids, amino acids, and peptides.
c) Alkali or alkaline earth metal compound: A compound- salt or base of an alkali or alkaline earth metal.
d) Livestock: an animal selected from a group of cattle, horses, sheep, goats, pigs, and the like.
e) Phytochemicals: Mixture of compounds comprising carbohydrates, proteins, lignins, flavonoids, terpenes, tannins, alkaloids, organic acids, vitamins, polyols of plant origin.

Source of Biological materials:
Paddy straw: obtained from a farmer at KR Nagar Bangalore.
Wheat bran: is purchased from Krishna Floor mills, Bangalore.
Lawsonia innermis: is purchased from Malhar Enterprises, Navi Mumbai
Herbs: All herbs/herbal materials are purchased from Natural Holistics, Bangalore
Acetobactor aciti is purchased from Hi-media, Bangalore
Propionobacter is purchased from Bio Fermen pvt ltd, Bangalore
Lactobacillus species is purchased from Mystical biotech, Bangalore
Enzymes and bacterial species purchased from Mystical biotech, Bangalore
Wax: SD fine chem, Mumbai
copra cake: Feed ingredients stores, Doddaballapur, Karnataka

The present invention provides a unique synergistic formulation that can be adopted as a medium for extraction of phytochemicals, nutrients; in one pot process for developing nutrition enriched feed supplement for farmed livestock. The components of medium also offers the advantage of being adopted as part of feed supplement.
In an embodiment, the synergistic formulation comprises nitrogen containing compound (polyalkyl ammonium salt or amide), natural organic compound (polyol, sugar, amino acid, or peptide), edible acid, alkali or alkaline earth metal compound and water for the extraction of phytochemicals from natural sources-biomass and herbs.
In another embodiment of present invention comprises a method of preparation of the synergistic formulation by mixing specific proportion of nitrogen containing compound, natural organic compound, edible acid, alkali or alkaline earth metal compound and water with one another in a blender and heating at a temperature ranging from 50-140°C for about 15 minutes to 25 hours.
In another embodiment the feed supplement constitutes phytochemicals extracted from natural source selected from biomass and/or herbs, solubilised in the medium, the synergistic formulation comprising polyalkyl ammonium salt or amide, natural organic compound (polyol, sugar, amino acid, or peptide), edible acid, alkali or alkaline earth metal compound and water; Biotransformer, Bioenhancer, and Stabilizer.
In another embodiment, the process of developing the feed supplement comprises steps of- solubilisation involving unique medium- a synergistic formulation comprising nitrogen containing compound (polyalkyl ammonium salt or amide), natural organic compound (polyol, hydroxy acids, organic acids sugar, amino acid, or peptide edible acid), alkali or alkaline earth metal compound and water for the extraction of phytochemicals from natural source; saccharification of the extracted phytochemical by Biotransformation, Bioenhancement; and Stabilization by crosslinking and or coating.
In an embodiment the medium, the synergistic formulation comprises nitrogen containing compound (polyalkyl ammonium salt or amide) in the range of 1-25%wt/wt, natural organic compound (polyol, hydroxy acids, organic acids sugar, amino acid, or peptide) in the range of 1-20% wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%.
In an embodiment biomass is selected from a group comprising paddy straw, wheat bran, rice bran, soy hulls, wheat straw, corn cob, maize straw, rice bran, sugarcane-bagasse and the like.
In an embodiment herb is selected from a group comprising Garlic, Ginger, Guggul, Amla, Lawsonia, Black pepper, Cinnamon, Osmium sanctum,Andrographis and the like.
In an embodiment polyalkyl ammonium salt or amides are selected from a group comprising of choline acetate, 2-(trimethylazaniumyl)acetate3-carboxy-2-(carboxymethyl)-2-hydroxypropanoate, Trimethylammoniumchloride hydrochloride, Guar hydroxypropyltrimonium chloride, tetrabutylammonium bromide, N,N,N-Tri(octyl)octan-1-aminium bromide, Ethyltrimethylammonium hydroxide, Ethanaminium-2-hydroxy-N,N,N-trimethyl-,(2R,3R)-2,3-dihydroxybutanedioate, Ethanaminium-2-hydroxy-N,N,N-trimethyl-2-hydroxy-1,2,3 propanetricarboxylate, (2-hydroxyethyl) trimethylazaniumchloride, 2-hydroxyethyl(trimethyl) azaniumacetate, N-Methyl-N,N,N-trioctylammonium chloride, N-Benzyl-N,N-dimethylhexadecan-1-ammonium chloride, choline chloride, betaine, choline citrate, urea, 2-(trimethylazaniumyl)acetate hydrochloride, Triethyl(2-hydroxyethyl)ammonium chloride, thiourea, or mixture thereof.
In another embodiment of present invention natural organic compounds comprising polyols, sugars, amino acids, peptides are selected from a group comprising sucrose, maltose, galactose, glycerol, propylene glycol, soy protein hydrolysates, yeast protein hydrolysates, cane sugar, honey, maple syrup, yeast peptides, silk peptides, milk peptides, maize peptides, and soy peptides.
In an embodiment edible acid is selected from a group comprising citric acid, phosphoric acid, methanoic acid, acetic acid, propanoic acid, butanoic acid, palmitic acid, stearic acid, tartaric acid, malic acid, maleic acid, pentanoic acid, adipic acid, lactic acid, alfa linolenic acid, sorbic acid, fumaric acid, pyruvic acid, 2-hydroxy-4-(methylthio)butanoic acid, oxalic acid, eicosapentaenoic acid, docosahexaenoic acid, valeric acid, palm fatty acids, hydrogenated palm fatty acids, fatty acids with carbon chain length of C4 to C12 from coconut oil and the like.
In an embodiment alkaline or alkaline earth metal compound are selected from a group comprising sodium acetate, sodium propionate, sodium butanoate, calcium acetate, calcium propionate, calcium butanoate, sodium citrate, calcium citrate, sodium valerate, calcium valerate, sodium chloride, sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, sodium hydrogen carbonate.
An embodiment of the present invention also relates to process of preparation feed supplement involving-
(a) Preparation of medium, the synergistic formulation comprising mixing the ingredients-polyalkyl ammonium salt or amides in the range of 1-25%, natural organic compounds (polyols, sugars, amino acids, peptides) in the range of 1-20%, edible acids in the range of 1-50%, salt or corresponding base in the range of 0.1-15% and water in the range of 2-60% in to a blender and heating at a temperature ranging from 50-80°C;
(b) Solubilization of the phytonutrients from natural source selected from biomass and herbs in the medium by charging the biomass into the medium and heating at a temperature ranging from 80-90°C for 2-15 hours and cooling; typically the ratio of the medium and biomass is in the range of 10:1 to 1:1;
(c) Biotransformation and bioenhancement: Typically the biotransformation involves addition of compound selected from a group comprising including cellulase, hemicellulase, amylase, alpha galactosidase, alpha amylase, beta glucanase, hemicellulase, glucoamylase, bacterial alpha amylase, pectinase, mannose to the solubilised mass, maintaining the temperature at 35-70°C for 1-15 hours, then adding bioenhancers and mixing for about 20 minutes.
In an embodiment the Biotransformation may also constitute bacteria selected from a group comprising Acetobacter, lactobacillus, Bifidobacterium. Propionibacterium, bacillus, Saccharomyces, enterococcus and enzyme selected from a group comprising Cellulases, hemicellulases, glucoamylase, amylopectin, beta glucanase, protease, lipase, mannanase, pectinase, papain, alfa galactosidase, phytase.
In another embodiment Bioenhancer may constitute shatavari, copra cake, milk thistle, fenugreek and like.
(d) Stabilization step involve mixing the product prepared in biotransformation step with a stabilizer compound selected from a group comprising formaldehyde, modified clay, beta cyclodextrin, trans glutaminase, paraformaldehyde, calcium hydroxide, calcium carbonate, calcium oxide, calcium bentonite, sodium bentonite, hydrogenated fat from soya, palm, sunflower, ground nut, til seeds, Bees wax, Carnauba wax, soya oil, sunflower oil, coconut oil, palm oil, til oil, mustard oil, rice bran oil, ground nut oil, dextrins, sepiolite, zeolite and mixture thereof at a temperature ranging from about 30-80°C.
EXPERIMENTATION
Example 1:
Step 1: Preparation of medium- synergistic formulation
7.5% (wt/wt) steric acid, 0.5% (wt/wt) citric acid, 15% (wt/wt) urea, 14% (wt/wt) cane sugar 4.7% (wt/wt) calcium hydroxide, 58.3% (wt/wt) water are mixed in a blender and heated to 50-80 °C for a period of 2 hours to get the synergistic formulation that is further adopted in solubilization.
Step 2: Solubilization step
50% (wt/wt) paddy straw and 50% (wt/wt) medium are mixed at 80-90 °C for 2-15 hours and cooled to 25-50 °C.
Step 3: Biotransformation (saccharification) and bioenhancement
82.3%(wt/wt) product obtained by solubilization and 1% (wt/wt) cellulase enzyme and maintained at 35 to 70 °C for 1-15 hours and then 0.2% (wt/wt) shatavari, 16.5% (wt/wt) copra cake are added and mixed for 20 minutes.
Step 4: Stabilization
92% (wt/wt) product obtained in biotransformation & bioenhancement step and 2% (wt/wt) formal dhehyde are mixed and maintained for 5 hours at 20-50 °C.
The biomass obtained in the stabilization step is rolled in to uniform sized small balls or cakes and dried at 40-80 °C and cool to 25-50 °C. The surface is coated with coconut oil.
The feed supplement prepared as in example 1 is compared with the control 1- water and control 2- mixture of water and ethyl alcohol in the ratio 1:1, for its properties and tabulated in table 1 .
Table 1: Comparison of the treatment with Control
Step Criteria Treatment medium Control 1 Water Control 2 Mixture of water and alcohol (1:1)
Nature of the components before medium preparation Physical state Mix of liquids and solids Liquid Liquid
Extraction medium (After medium preparation) Physical state Viscous liquid Liquid Liquid
Solubilisation
% solubility 40-60% Not soluble 5%
Biotransformation Reducing sugars 30% 5% 7%
Stabilisation Percentage of urea/amide released in 24 hours 0.6% Nil Nil

Example 2:
Step 1: Preparation of medium- synergistic formulation
8.75% (wt / wt) choline chloride, 26.25% (wt / wt) citric acid, 3.25% (wt / wt) acetic acid, 3.25% (wt / wt) sodium acetate, 3.25% (wt / wt) calcium hydroxide, 3.25%(wt / wt) glycerol, 52% (wt / wt) water are mixed in a blender and heated to 50-80 °C for a period of 3 hours to get medium.
Step 2: Solubilization
35% (wt/wt) wheat bran and 65% (wt / wt) medium- the synergistic formulation are mixed at 60-90 °C for 2-15 hours and cooled to 25-60 °C.

Step 3: Biotransformation (saccharification and fermentation)
96% (wt / wt) biomass obtained in step 2 and 1% (wt / wt) cellulase and hemicellulase are mixed and maintained at 35 to 60 °C for 4-30 hours and then 1% (wt / wt) Lactobacillus species is added to the above mass at temperature of about 37°C and stirred for 10-20 hours.
Step 4: Stabilization
50% (wt/wt) biomass obtained in step 3 and 50% (wt / wt) modified clay are mixed and dried at temperature 30 °C for 15 hours to obtain the product, which can be used as feed supplement.
The feed supplement prepared as in example 2 is compared with the control 1- water and control 2-mixture of water and ethyl alcohol in the ratio 1:1 for its properties and tabulated in table 2.
Table 2: Comparison of the treatment with Control
Step Criteria Treatment medium Control 1 Water Control 2 mixture of Water-alcohol (1:1)
Nature of the components before medium preparation Physical state Mix of liquids and solids Liquid Liquid
Extraction medium (After medium preparation) Physical state Viscous liquid Liquid Liquid
Solubilisation
% Solubility 60-80% 30% 8%
Biotransformation % of acids 50% 15% 7%
Stabilisation Flowability Free flowing Free flowing Free flowing

Example 3:
The effect of solubility of the biomass is analysed with the medium of present invention and compared with the individual components of the invention to show the synergy amongst the components which results in enhanced solubility. The results are tabulated in table 3, 4 & 5.
Table 3
Components of the medium Temperature in °C Biomass % solubility
Citric acid + water 90-100 Wheat bran 10.9
Acetic acid + water 90-100 Wheat bran 7.4
choline chloride- water 90-100 Wheat bran 10.1
Calcium hydroxide +water 90-100 Wheat bran 3.2%
Glycerol 90-100 Wheat bran Nil
Sodium acetate + water 90-100 Wheat bran Nil

8.75%wt/wt Citric acid+ 26.75% wt/wt choline chloride+3.25% wt/wt Acetic acid+3.25% wt/wt Glycerol +3.25% wt/wt sodium acetate+3.25% wt/wt calcium hydroxide +52% wt/wt water 90-100 Wheat bran 53

Table 4
Components of the medium Temperature in °C Biomass % solubility
Steric acid +water 90-100 Paddy straw 3.4
Citric acid +water 90-100 Paddy straw 7.9
Urea + water 90-100 Paddy straw 2.1
Calcium hydroxide 90-100 Paddy straw 2.1
Cane sugar +water 90-100 Paddy straw 3.2
7.5% wt/wt steric acid, 0.5% wt/wt citric acid, 15% wt/wt urea, 14% wt/wt cane sugar,4.7% wt/wt Calcium hydroxide 58.3% wt/wt
water
90-100 Paddy straw 37

Table 5
Components of the medium Temperature in °C Biomass % solubility
Adipic acid + water 90-100 Paddy straw Nil
Urea + water 90-100 Paddy straw Nil
Cysteine +water 90-100 Paddy straw 4%
18% wt/wt Adipic acid+23% wt/wt Cysteine+19% wt/wt urea+60% wt/wt water 90-100 Paddy straw 33%

Table 6: Comparison of paddy straw solubility after treatment with medium
Components of medium Extraction condition Solubility
Water 90-100°C, 3 hours 0%
Water- alcohol 80-90°C, 3 hours 5%
7.5% wt/wt steric acid, 0.5% wt/wt citric acid, 15% wt/wt urea, 14% wt/wt cane sugar,4.7% wt/wt Calcium hydroxide 58.3% wt/wt water 90-100 C°, 3 hours 43.1%
Example 4: The effect of temperature on solubility of the biomass is analysed with the medium of present invention and compared with the water & water-alcohol medium which is presently used for extraction of phytochemicals as shown in Table 6 & Table 7

Table 7: Comparison of wheat bran solubility after treatment with medium
Components of medium Extraction condition Solubility
Water 90-100 C°, 3 hours 30%
Water –alcohol 90-100 C°, 3 hours 8%

8.75% wt/wt Citric acid + 26.75% wt/wt choline chloride + 3.25% wt/wt Acetic acid + 3.25%wt/wt Glycerol + 3.25% wt/wt sodium acetate + 3.25% wt/wt calcium hydroxide + 52% wt/wt water 90-100 C°, 3 hours 62%

Example 5:
The experiment is also carried out using various proportion of Adipic acid, cysteine, urea, water for the preparation of the medium of present invention and Paddy straw is adopted as the solute material, the results are tabulated in the Table 8.
Table 8: Results of solubility comprising Paddy straw as the solvent.
Components of the medium in % w/w
Time in hours Temperature in °C Paddy straw solubility in %
Adipic acid Cysteine Urea water 90-100
40 30 30 0 3 90-100 15.5
40 10 0 50 3 90-100 3.1
40 0 10 50 3 90-100 10.6
40 25 25 50 3 90-100 5.2
20 15 15 50 3 90-100 43.1
20 15 15 50 4 90-100 42.8
20 15 15 50 2 90-100 35.8
20 15 15 50 3 80-90 31.3

Example 6:
Step 1: Preparation of medium- synergistic formulation
15% (wt / wt) choline citrate, 20 (wt / wt) tartaric acid, 5% w/w glycerol and water are mixed in a blender and heated to 50-90 °C for a period of 1 hour to get medium.
Step 2: Solubilization
48% (wt/wt) of garlic with 2% Lawsonia inermis and 50% (wt / wt) medium- the synergistic formulation are mixed at 30-90 °C for 2-15 hours and cooled to 25-60 °C.

Step 3: Stabilization
29% (wt/wt) biomass obtained in step 2 and 71% (wt / wt) modified clay are mixed and dried at temperature 30 – 90 °C for 15 hours to obtain the product, which can be used as feed supplement.
Example 7:
Step 1: Preparation of medium- synergistic formulation
25% (wt / wt) betaine, 15% (wt / wt) glycerol, 2% w/w methionine and 48% water are mixed in a blender and heated to 40-90 °C for a period of 1-4 hour to get medium.
Step 2: Solubilization
43% (wt/wt) of ginger with 2% cinnamon and 55% (wt / wt) medium- the synergistic formulation are mixed at 30-90 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Bioenhancement
80% wt/wt of product obtained in step 2 is mixed with a bioenhancer Lawsonia inermis 20% wt/wt.
Step 4: Stabilization
35% (wt/wt) biomass obtained in step 3 and 65% (wt / wt) modified clay are mixed and dried at temperature 30 – 90 °C for 12 hours to obtain the product, which can be used as feed supplement.

Example 8:
Step 1: Preparation of medium- synergistic formulation
35% (wt / wt) choline chloride, 28% (wt / wt) Lactic acid, 2% w/w propylene glycol and 35% water are mixed in a blender and heated to 40-90 °C for a period of 1-4 hour to get medium.
Step 2: Solubilization
56% (wt/wt) of Ginger with 4% Lawsonia inermis and 40% (wt / wt) medium- the synergistic formulation are mixed at 30-90 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Stabilization
45% (wt/wt) biomass obtained in step 2 and 55% (wt / wt) modified clay are mixed and dried at temperature 30 – 90 °C for 10 hours to obtain the product, which can be used as feed supplement.
Example 9:
Step 1: Preparation of medium- synergistic formulation
40% (wt / wt) choline bi tartrate, 34% (wt / wt) butyric acid, 6% w/w sorbitol and 20% water are mixed in a blender and heated to 40-90 °C for a period of 1-4 hour to get medium.
Step 2: Solubilization
56% (wt/wt) of Amla powder with 2% Psoralia powder and 40% (wt / wt) medium- the synergistic formulation are mixed at 30-95 °C for 1-18 hours and cooled to 25-70 °C.

Step 3: Stabilization
60% (wt/wt) biomass obtained in step 2 and 40% (wt /wt) modified clay are mixed and dried at temperature 30 – 90 °C for 10 hours to obtain the product, which can be used as feed supplement.
Example 10:
Step 1: Preparation of medium- synergistic formulation
10% (wt/wt) lauric acid acid, 7.5% (wt/wt) citric acid,12.5 % (wt/wt) lysine HCl, 20% (wt/wt) sucrose, 5% calcium carbonate, 45% (wt/wt) water are mixed in a blender and heated to 40-90 °C for a period of 1-5 hours to get the synergistic formulation that is further adopted in solubilization.
Step 2: Solubilization step
30% (wt/wt) millet straw and 70% (wt/wt) medium are mixed at 80-90 °C for 1-13 hours and cooled to 25-50 °C.
Step 3: Biotransformation (saccharification)
82.3%(wt/wt) product obtained by solubilization and 1% (wt/wt) cellulase and hemicellulase enzyme and maintained at 35 to 70 °C for 1-15 hours and then 0.2% (wt/wt) jeevanti, 16.5% (wt/wt) groundnut cake are added and mixed for 20 minutes.
Step 4: Stabilization
90% (wt/wt) product obtained in biotransformation step and 3% (wt/wt) formaldhehyde are mixed and maintained for 5 hours at 20-50 °C.
The biomass obtained in the stabilization step is rolled in to uniform sized small balls or cakes and dried at 40-80 °C and cool to 25-50 °C. The surface is coated with groundnut oil.
Example 11:
Step 1: Preparation of medium- synergistic formulation
10% (wt/wt) urea, 7.5% (wt/wt) methionine, 12.5 % (wt/wt) butyric acid , 20% (wt/wt) sucrose, 5% calcium carbonate, 45% (wt/wt) water are mixed in a blender and heated to 40-90 °C for a period of 1-5 hours to get the synergistic formulation that is further adopted in solubilization.
Step 2: Solubilization step
30% (wt/wt) sorghum husk and 70% (wt/wt) medium are mixed at 80-90 °C for 1-13 hours and cooled to 25-50 °C.
Step 3: Biotransformation (saccharification)
82.3% (wt/wt) product obtained by solubilization and 2% (wt/wt) cellulase and hemicellulase enzyme and maintained at 35 to 70 °C for 1-15 hours and then 0.2% (wt/wt) jeevanti, 15.5% (wt/wt) groundnut cake are added and mixed for 20 minutes.
Step 4: Stabilization
90% (wt/wt) product obtained in biotransformation step and 3% (wt/wt) formaldhehyde are mixed and maintained for 5 hours at 20-50 °C.
The biomass obtained in the stabilization step is rolled in to uniform sized small balls or cakes and dried at 40-80 °C and cool to 25-50 °C. The surface is coated with palm fatty acid.
Example 12:
Step 1: Preparation of medium- synergistic formulation
40% (wt / wt) choline citrate, 25% (wt / wt) ethanoic acid , 4% (wt / wt) soy protein hydrolysate, 3.0 (wt / wt) sodium propionate, 3% (wt / wt) calcium hydroxide, 5% w/w propylene glycol, 20% (wt / wt) water are mixed in a blender and heated to 50-80 °C for a period of 3 hours to get medium.
Step 2: Solubilization
25% (wt/wt) rice bran and 75% (wt / wt) medium- the synergistic formulation are mixed at 40-96 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Biotransformation (saccharification and fermentation)
96% (wt / wt) biomass obtained in step 2 and 1% (wt / wt) cellulase and hemicellulase are mixed and maintained at 35 to 60 °C for 1-30 hours and then 3% (w / w) propionibacterium species is added to the above mass at temperature of about 37°C and stirred for 10-20 hours.
Step 4: Stabilization
35% (wt/wt) biomass obtained in step 3 and 65% (wt / wt) bentonite are mixed and dried at temperature 30 °C for 15 hours to obtain the product, which can be used as feed supplement.
Example 13:
Step 1: Preparation of medium- synergistic formulation
35% (wt / wt) choline citrate, 26% (wt / wt) methionine hydroxy analogue, 4% (wt / wt) glycine 2.0 (wt / wt) sodium propionate, 3% (wt / wt) calcium hydroxide, 10% w/w sorbitol, 20% (wt / wt) water are mixed in a blender and heated to 50-80 °C for a period of 3 hours to get medium.
Step 2: Solubilization
10% (wt/wt) wheat bran and 90% (wt / wt) medium- the synergistic formulation are mixed at 40-96 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Biotransformation (saccharification and fermentation)
96% (wt / wt) biomass obtained in step 2 and 2% (wt / wt) cellulase and hemicellulase are mixed and maintained at 30 to 60 °C for 1-30 hours and then 3% (wt / wt) acetobacter species is added to the above mass at temperature of about 37°C and stirred for 10-20 hours.
Step 4: Stabilization
25% (wt/wt) biomass obtained in step 3 and 75% (wt / wt) zeolite are mixed and dried at temperature 30 °C for 15 hours to obtain the product, which can be used as feed supplement.
Example 14:
Step 1: Preparation of medium- synergistic formulation
10% (wt/wt) lysine, 5% (wt/wt) Methionine hydroxy analogue, 15% w/w choline chloride, 20% (wt/wt) sucrose, 5% calcium carbonate, 45% (wt/wt) water are mixed in a blender and heated to 40-90 °C for a period of 1-5 hours to get the synergistic formulation that is further adopted in solubilization.
Step 2: Solubilization step
29% (wt/wt) amla powder, 20% garlic bulbs and 1% garcinia powder and 50% (wt/wt) medium are mixed at 80-90 °C for 1-13 hours and cooled to 25-50 °C.
Step 3: Stabilization
50% of the above mass is mixed 50% sepiolite and dried at 30-90 deg C to get dry powder which is a feed supplement powder.
Example 15:
Step 1: Preparation of medium- synergistic formulation
10% (wt / wt) Betaine tartarate, 25% (wt / wt) propylene glycol, 10% w/w cysteine , 55% water in blender and heated to 30-80 °C for a period of 0.5-3 hours to get medium.
Step 2: Solubilization
2.5% (wt/wt) of black pepper 2.5% w/w of Cinamon and 95% (wt /wt) medium-the synergistic formulation are mixed at 40-96 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Stabilization
25% (wt/wt) biomass obtained in step 2 along with 10% lemongrass oil was added to medium containing 20% polysorbate and 45% water. Mixed vigorously to get homogeneous solution.
Example 16:
Step 1: Preparation of medium- synergistic formulation
10% (wt / wt) choline tartarate, 25% (wt / wt) sorbitol, 65% water in blender and heated to 30-80 °C for a period of 0.5-3 hours to get medium.
Step 2: Solubilization
2.5% (wt/wt) of Osmium sanctum leaves 2.5% w/w of Andrographis and 95% (wt / wt) medium- the synergistic formulation are mixed at 40-96 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Stabilization
25% (wt/wt) biomass obtained in step 2 along with 10% Thymol was added to medium containing 30% polysorbate and 35% water. Mixed vigorously to get homogeneous solution/suspension.
Example 17:
Step 1: Preparation of medium- synergistic formulation
10% (wt / wt) Lysine, 20% (wt / wt) Glycine, 35% Betaine tartarate and 35% water in blender and heated to 30-80°C for a period of 0.5-3 hours to get medium.
Step 2: Solubilization
35 % (wt/wt) of Guggul powder 5% w/w of Amla and 70% (wt / wt) medium- the synergistic formulation are mixed at 40-96 °C for 2-15 hours and cooled to 25-60 °C.
Step 3: Stabilization
40% (wt/wt) biomass obtained in step along with precipitated silica is dried at 30-90 deg C for a period of 1-16 hours resulting in free flowing powder.
Example 18:
The observe the effect of the feed as developed according to example 1, it is fed to dairy Jersy cows to check on the yield of milk. Cows in the mid lactation period are selected and fed with 80g in the morning and 80g in the evening. Trial is run for 30 days in 5 Jersey cows and increase in milk yield is recorded daily basis.
Treatment description Average milk yield /day/animal
Baseline ( before feeding) 8 liters
Treatment 8.9- 9 liters

It is observed that the average milk yield went up by about 10-12% /day. The figure 1 provides the graph plotted on the yield of milk.
Example 19:
Product of example 2 is tested for its efficacy against pathogen. Feed samples are moistened with 15 % water. Said samples are treated with product of example 2. Sample is stored for 3 days, later extracted into phosphate buffer to determine the total yeast and mold count.

Treatment description Yeast and mold count
Control Too numerous to count
Treatment <10 CFU/100g

The present invention thus provides a synergistic formulation that can be used as a medium for extraction and adopts the same for providing feed supplement with enriched nutrition value for livestock. The method is comprise simple process and economical for adoption.
, Claims:We Claim

1. A feed supplement for livestock, comprising synergistic formulation as a medium for extraction of phytochemicals from natural source, wherein the synergistic formulation comprises nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%, said feed prepared by a method comprising acts of –
(a) preparing the synergistic formulation as the medium for extraction;
(b) charging the natural source into the medium and heating at a temperature ranging
from 80-90°C for 2-15 hours and cooling to solubilize the phytochemicals from biomass;
(c) taking solubilized natural source through steps of biotransformation and bioenhancement; and
(d) stabilization of the bioenhanced natural source by mixing with a stabilizer to obtain
the feed supplement for livestock.
2. The synergistic formulation as claimed in claim 1, wherein the natural source is selected from a group of biomass and herbs.
3. The synergistic formulation as claimed in claim 1, wherein the biomass is selected from a group comprising paddy straw, wheat bran, rice bran, soy hulls, wheat straw, corn cob, maize straw, wheat straw, sorghum husk, bagasse and the like.
4. The synergistic formulation as claimed in claim 1, wherein the herb is selected from Garlic, Ginger, Guggul, Amla, Lawsonia, Black pepper, Cinnamon, Ocimumsanctum, Andrographis.
5. The feed supplement for livestock as claimed in claim 1, wherein the medium and
natural source are in the ratio in the range of 10:1 to 1:1.
6. A method of preparation of feed supplement for livestock comprising synergistic formulation of claim 1 as a medium for extraction of phytochemicals from biomass, wherein the synergistic formulation comprises nitrogen containing compound in the range of 1-25%wt/wt, natural organic compound in the range of 1-20%wt/wt, edible acid in the range of 1-50%wt/wt, alkali or alkaline earth metal compound in the range of 0.1-15%wt/wt and water in the range of 2-60%, said method comprising acts of-
(a) preparing the synergistic formulation as the medium for extraction, comprising steps of –
(i) mixing the nitrogen containing compound, natural organic compound, edible acid, alkali or alkaline earth metal compound and water in a blender to obtain a mixture; and
(ii) heating the mixture at a temperature of 50-140°C for 15 minutes to 25 hours to obtain the synergistic formulation;
(b) charging the natural source into the medium and heating at a temperature ranging from 80-90°C for 2-15 hours and cooling to solubilize the phytochemicals from biomass;
(c) taking solubilized natural source through steps of biotransformation and bio enhancement; and
(d) stabilization of the bioenhanced natural source to obtain the feed supplement for livestock.