RU2801965C2 - Pongamia compositions, methods of their production and analysis, as well as their application - Google Patents

Abstract

FIELD: oil and fat industry; feed industry.

SUBSTANCE: Pongamia composition, which is a meal, containing: karanjin at a concentration of less than or equal to 100 ppm (ppm); and at least one or more components selected from the group consisting of carbohydrates, proteins, fibres, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. A feed composition containing: at least 30% by weight of the Pongamia composition and the main feed. A method for producing a Pongamia composition, comprising: mechanical extraction of Pongamia oilseeds to obtain a defatted Pongamia seed cake, wherein the defatted Pongamia seed cake contains 8-30% oil by weight; mixing the defatted Pongamia seed cake with an alkyl alkanoate solvent to obtain an extraction mixture in which the alkyl alkanoate solvent optionally includes ethyl acetate; measuring the concentration of carangin in the alkylalkanoate extract; and separating the extraction mixture into a miscella and a Pongamia composition. Wherein, the Pongamia composition is a meal having (i) a concentration of carangin that is less than 20% of the concentration of carangin in the defatted cake of seeds of Pongamia, or (ii) a concentration of carangin that is less than or equal to 100 ppm. A method of feeding a ruminant, comprising providing the ruminant with a Pongamia composition or a Pongamia feed composition.

EFFECT: invention allows more complete and thorough extraction of karanjin and pongamol from untreated Pongamia seed cake, without reducing the amount of other components, including macronutrients (such as protein and carbohydrates), which are very important for achieving acceptable feed efficiency.

11 cl, 16 dwg, 25 tbl, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority and benefit of U.S. Provisional Application No. 62/741351, filed Oct. 4, 2018, the entire disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present disclosure relates generally to pongamia oilseed products, and more specifically to pongamia compositions having low concentrations of pongamia residual oil, caranjin, and pongamol, as well as methods for making and using such pongamia compositions. The present disclosure also relates to methods for analyzing compositions of pongamia obtained by the methods described here, as well as other processing methods known in this field. The present disclosure also relates to uses of pongamia compositions as feed for cattle and other ruminants.

BACKGROUND OF THE INVENTION

Pongamia seed cake, a by-product of oil extraction from pongamia oilseeds, is a potential renewable source of protein for feed applications. However, untreated pongamia seed cake contains residual oil and its own chemical constituents such as carangin and pongamol. It is desirable to reduce the amount of carangin and pongamol in the seed cake for use as a suitable feed source. In particular, carangin and pongamol have been found to be economically valuable for their own characteristics, and as a result, various processing methods have been studied to extract high purity caranjin and pongamol from the raw pongamia seed cake. However, existing methods often result in incomplete removal of residual oil, caranjin and pongamol from pongamia seed cake and thus preclude subsequent use of the seed cake itself.

There is currently a need for pongamia formulations with low levels of residual oil, carangin and pongamol, as well as improved methods for more complete and thorough extraction of caranjin and pongamol from untreated pongamia seed cake.

In addition, a major obstacle to the development of improved extraction methods has been the lack of a standardized method for estimating the concentrations of carangin and pongamol remaining in seed cake after such treatments. Although there are currently many methods for quantifying carangin and pongamol residues in pongamia seed cake after extraction treatments, these methods are often unreliable and/or inaccurate.

Most analytical methods measure the concentrations of carangin and pongamol in pongamia compositions by determining the concentrations of these chemical components present in the respective methanol or hexane solvent extracts, thus providing an indirect estimate of the concentrations in the pongamia composition. However, since such methods depend on the efficiency of extraction with methanol or hexane, and such efficiency will vary for each method depending on the nature of the analyzed material and its previous processing, these methods give unreliable quantitative values for carangin and other chemical compounds in pongamia seed cake. Moreover, different analytical methods give different concentrations of oil, carangin and pongamol in the same pongamia seed cake sample and often do not provide an internally consistent reference scale for different treatments. Therefore, the correct comparison of different processing methods based on existing analytical methods has been difficult in this area.

Thus, there is a need not only for pongamia compositions having low levels of residual oil, caranjin and pongamol, and alternative methods for preparing such pongamia compositions, but also for more accurate methods for analyzing pongamia compositions obtained by various processing methods in general.

SUMMARY OF THE INVENTION

In one aspect, a method is provided herein comprising: mixing a pongamia composition with an alkyl alkanoate solvent to form an extraction mixture; irradiating the extraction mixture with microwave radiation to obtain an irradiated mixture; separating the irradiated mixture into the extracted pongamia composition and the alkyl alkanoate extract; and measuring the concentration of carangin in the alkyl alkanoate extract.

In another aspect, a method is provided herein comprising: providing a first pongamia composition, wherein the first pongamia composition is a defatted pongamia seed cake obtained by mechanical extraction and contains 8-30% oil by weight; mixing the first pongamia composition with an alkyl alkanoate solvent to obtain an extraction mixture; and separating the extraction mixture into a miscella and a second pongamia composition, wherein the second pongamia composition has (i) a concentration of carangin that is less than 20% of the concentration of carangin in the first pongamia composition, or (ii) a concentration of carangin that is less than or equal to 100 ppm.

In yet another aspect, a pongamia composition is provided herein comprising: karangin; and at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. In some embodiments, the pongamia composition has a concentration of carangin less than or equal to 100 ppm. In some embodiments, the concentration of carangin is determined by treating the pongamia composition with an alkyl alkanoate solvent under microwave radiation.

In some aspects, pongamia compositions prepared in accordance with the methods described herein are provided. In another aspect, a food composition is provided herein comprising any of the pongamia compositions described herein.

In yet another aspect, a method of feeding a ruminant is provided, comprising providing the ruminant with any pongamia composition described herein or a food composition described herein.

BRIEF DESCRIPTION OF THE FIGURES

The present application may be understood with reference to the following description in conjunction with the accompanying figures.

Figure 1 shows an exemplary process for analyzing the composition of pongamia.

Figure 2 shows an exemplary process for preparing a composition of pongamia having a concentration of carangin less than or equal to 100 ppm.

Figures 3A and 3B are bar graphs comparing total concentrations of carangin and pongamol (in ppm adjusted for starting material) extracted from defatted pongamia seed cake using various methanol-based extraction methods.

4A and 4B are histograms comparing total concentrations of carangin and pongamol (in ppm corrected for starting material) extracted from defatted pongamia seed cake using various solvents (methyl tert-butyl ether, ethanol, hexane, toluene and ethyl acetate) in combination with various extraction methods.

5A and 5B are bar graphs comparing total concentrations of carangin and pongamol (in ppm corrected for starting material) extracted from defatted pongamia seed cake using microwave extraction or ethyl acetate or ionic liquid as solvent.

6A and 6B show a comparison of the total concentrations of carangin and pongamol (in ppm corrected for starting material) extracted from defatted pongamia seed cake for the various methods and solvents shown in FIGS. 3A-5B.

7A-7B is a histogram of observed total concentrations of carangin and pongamol (in ppm corrected for starting material) extracted from defatted pongamia seed cake using various alkyl alkanoate solvents in combination with microwave solvent extraction.

8A and 8B are histograms comparing carangin and pongamol residual concentrations (in ppm adjusted for starting material) in pongamia seed cake subjected to various mechanical treatments, as determined by ethyl acetate microwave extraction.

9A and 9B are histograms comparing the residual concentrations of carangin and pongamol (in ppm corrected for starting material) in pongamia seed cake subjected to various mechanical treatments in combination with solvent extraction treatments, as determined by ethyl acetate microwave extraction.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, exemplary methods, parameters, and the like are given. However, it should be understood that such a description is not intended to limit the scope of the present disclosure, but is instead presented as a description of exemplary embodiments.

The following description relates to pongamia compositions having low concentrations of carangin and pongamol, and methods for preparing and using pongamia compositions having low concentrations of carangin, as well as methods for analyzing pongamia compositions.

Methods for analyzing compositions from pongamia

In some aspects, methods for analyzing pongamia compositions are presented here. In some embodiments provided herein, methods are provided for determining the concentrations of caranjin and pongamol in pongamia.

Oilseeds harvested from pongamia (also known as 'Cytisus pinnatus', 'Dalbergia arborea', 'Derris indica', 'Galedupa pungum', 'karanj', 'Millettia pinnata', 'pongam', 'pongamia', 'Pongamia glabra' ”, “Pterocarpus flavus”, “Pongamia pinnata” and “Robinia mitis”, “Indian beech” and “mempari”) are highly valued as a renewable source of oil. For example, renewed interest in non-petroleum fuel sources has led to the use of pongamia oil as a feedstock for biodiesel production in many parts of the world.

The defatted pongamia seed cake left over from oil extraction from pongamia oilseeds has long been recognized as a potential renewable source of protein that can be used as a feed additive. However, defatted pongamia seed cake contains high concentrations of carangin and pongamol, which generally preclude the use of seed cake in feed products without any detrimental health effects. These compounds can make the seed cake inedible and potentially harmful to humans and animals. Previous attempts to develop edible compositions of pongamia have been unsuccessful, in part because the appropriate allowable maximum thresholds for concentrations of carangin and other anti-nutrients for consumption have not yet been established. Moreover, existing methods for the analysis of pongamia compositions were inaccurate and unreliable, so that the assessment of carangin concentrations in pongamia compositions, not to mention the further determination of the maximum allowable concentrations of carangin, is a difficult task. Thus, there remains a need for more accurate methods for determining the concentrations of carangin and other antinutrients present in pongamia formulations.

The present disclosure addresses this need by providing methods for analyzing pongamia compositions, namely methods for determining concentrations of carangin and other chemicals inherent in pongamia oilseeds, with greater accuracy and reliability than existing methods. In particular, in some aspects, the present disclosure provides an analytical microwave solvent extraction method for determining the concentrations of carangin and pongamol in pongamia compositions. Suitable solvents for use in such methods are described herein and may include solvents containing alkyl alkanoate(s).

It has surprisingly been found not only that the analytical methods of the present disclosure provide a more accurate measurement of carangin and pongamol in pongamia formulations than previously available methods, but also that similar prior methods using hexane and methanol based assays significantly underestimate the concentration residual carangin in the processed pongamia compositions. The use of an alkylalkanoate solvent containing at least one alkylalkanoate, including in combination with microwave irradiation, leads to an increase in the efficiency of extraction of carangine and pongamol from pongamia compositions and, consequently, to an improved quantitative determination of residual caranjin and pongamol remaining in pongamia compositions. after processing. Thus, the analytical methods described here provide a widely applicable yet reliable means of detecting and quantifying the presence of carangin and pongamol in various compositions derived from pongamia, and at concentrations lower than those that can be detected by traditional methods based on hexane and methanol.

In one aspect, a method is provided herein for analyzing pongamia compositions, wherein the method includes mixing the pongamia composition with an alkyl alkanoate solvent to form an extraction mixture, irradiating the extraction mixture with microwave radiation to form an irradiated mixture, separating the irradiated mixture into the extracted pongamia composition and the alkyl alkanoate extract, and measuring the concentrations of carangin and pongamol in the alkyl alkanoate extract and thus the corresponding concentrations in the pongamia composition indirectly. In another aspect, a method is provided herein for determining the concentrations of carangin and pongamol in a pongamia composition, comprising treating the pongamia composition with an alkyl alkanoate solvent under microwave radiation.

With reference to FIG. 1, process 100 is an exemplary process for analyzing a pongamia composition. At step 102, a pongamia composition is obtained. The pongamia composition is mixed with the alkyl alkanoate solvent in step 104, whereby an extraction mixture is obtained. The extraction mixture contains a pongamia composition and an alkyl alkanoate solvent. The extraction mixture is irradiated with microwave radiation in step 106 to obtain an irradiated mixture. The irradiated mixture is separated in step 108 to obtain an extracted pongamia composition and an alkyl alkanoate extract. In step 110, the alkyl alkanoate extract is analyzed.

It should be understood that in other embodiments, process 100 may include additional processing steps. In other embodiments, certain steps of process 100 may be omitted.

In one embodiment, a method for analyzing pongamia compositions is provided, comprising: mixing the pongamia composition with an alkyl alkanoate solvent to form an extraction mixture; irradiating the extraction mixture with microwave radiation to obtain an irradiated mixture; separating the irradiated mixture into the extracted pongamia composition and the alkyl alkanoate extract; and measuring the concentration of carangin in the alkyl alkanoate extract.

In some embodiments, the pongamia composition includes pongamia seeds. In other embodiments, the pongamia composition comprises a defatted pongamia seed cake. In some embodiments, the pongamia composition comprises pongamia seeds and/or defatted pongamia seed cake.

In embodiments of the foregoing in which the pongamia composition is a defatted pongamia seed cake, the defatted pongamia seed cake is obtained by mechanical extraction. In some embodiments, the defatted pongamia seed cake is obtained by mechanical extraction of the pongamia seeds. In other embodiments, the defatted pongamia seed cake is obtained by mechanical extraction of the pongamia seed cake. In some embodiments, the defatted pongamia seed cake is obtained by mechanical extraction using a screw press. In other embodiments where the pongamia seed cake composition comprises a defatted pongamia seed cake, the defatted pongamia seed cake is obtained by solvent extraction of the pongamia seed or pongamia seed cake. In some embodiments, a defatted pongamia seed cake is prepared by solvent extraction of the pongamia seed cake with an alkyl alkanoate solvent containing at least one alkyl alkanoate, such as ethyl acetate. In yet other embodiments, where the pongamia composition comprises defatted pongamia seed cake, the defatted pongamia seed cake is obtained by mechanical extraction, solvent extraction, or a combination thereof.

In some embodiments of the above method, the pongamia composition is mixed with an alkyl alkanoate solvent. In some embodiments, the alkyl alkanoate solvent is a solvent containing at least one alkyl alkanoate. In some embodiments, the solvent contains one alkyl alkanoate. In other embodiments, the solvent contains a mixture of alkyl alkanoates. The alkyl alkanoate solvent may contain only alkyl alkanoate(s) or, alternatively, may contain one or more additional co-solvents that are not alkyl alkanoates. In some embodiments, the pongamia composition is mixed with an alkyl alkanoate solvent containing at least one alkyl alkanoate. In some embodiments, the alkyl alkanoate solvent contains at least one alkyl alkanoate and one or more co-solvents that are not alkyl alkanoates. In other embodiments, the alkyl alkanoate solvent contains at least one alkyl alkanoate, but does not contain any co-solvents that are not alkyl alkanoates. In some embodiments, "alkylalkanoate" includes at least one ester group in which the hydrogen atom of the carboxylic acid group is replaced by an alkyl group. In some embodiments, the alkyl alkanoate includes one ester group in which the hydrogen atom of the carboxylic acid group is replaced by an alkyl group.

In some embodiments of the solvent, the alkyl in the alkyl alkanoate is methyl, ethyl, propyl, or butyl. In other embodiments, the solvent includes methyl alkanoate, ethyl alkanoate, propyl alkanoate, or butyl alkanoate, or any combination thereof. In some embodiments, the solvent includes ethyl alkanoate. In some embodiments, the alkanoate is ethanoate, propionate, butanoate, or pentanoate. In some embodiments, the solvent includes an alkyl ethanoate, an alkyl propionate, an alkyl butanoate, an alkyl pentanoate, or any combination thereof. In some embodiments, the solvent includes an alkyl ethanoate. In some embodiments, the implementation of the solvent includes ethyl acetate. In other embodiments, the solvent is ethyl acetate.

In some embodiments, the alkyl alkanoate solvent comprises an alkyl alkanoate selected from the group consisting of methyl methanoate, methyl ethanoate, methyl propanoate, methyl butanoate, methyl pentanoate, ethyl methanoate, ethyl ethanoate, ethyl propanoate, ethyl butanoate, ethyl pentanoate, propyl methanoate, propyl ethanoate, propyl propanoate, propyl butanoate, propyl pentanoate, butyl methanoate, butyl ethanoate, butyl propanoate, butylbutanoate and butylpentanoate; and any combinations thereof. In some embodiments, the alkyl alkanoate solvent comprises an alkyl alkanoate selected from the group consisting of methyl ethanoate, methyl propanoate, methyl butanoate, ethyl methanoate, ethyl ethanoate, ethyl propanoate, ethyl butanoate, propyl methanoate, propyl ethanoate, propyl propanoate, propyl butanoate, butyl methanoate, butyl ethanoate, butyl propanoate, and butyl butanoate; and any combinations thereof.

In addition, it should be noted that the chemical names used here in accordance with the International Union of Pure and Applied Chemistry (IUPAC) nomenclature standards may also be referred to by their respective common names, e.g., acetate for ethanoate, propionate for propanoate, butyrate for butanoate, valerate for pentanoate and so on. Thus, alkyl ethanoate may also be referred to as an acetate ester.

In other embodiments, the method includes mixing the pongamia composition with a solvent containing at least one alkyl alkanoate of formula (I):

Where:

R ¹ is C ₁ -C ₄ alkyl; And

R ² is hydrogen or C ₁ -C ₄ alkyl.

In some embodiments, R ¹ is C ₁ -C ₄ alkyl. In other embodiments, R ² is hydrogen or C ₁ -C ₄ alkyl. In some embodiments, R ¹ and R ² are independently C ₁ -C ₄ alkyl. In some other embodiments, R ¹ is C ₁ -C ₄ alkyl and R ² is hydrogen.

In some embodiments, where R ¹ is C ₁ -C ₄ alkyl, R ¹ is CH ₃ -, CH ₃ CH ₂ -, CH ₃ CH ₂ CH ₂ -, (CH ₃ ) ₂ CH-, CH ₃ CH ₂ CH ₂ CH ₂ -, CH ₃ CH ₂ (CH ₃ )CH-, (CH ₃ ) ₂ CHCH ₂ - or (CH ₃ ) ₃ C-. In some embodiments, R ¹ is CH ₃ CH ₂ -. In other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ CH ₂ -. In yet other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ -.

In some embodiments, R ² is hydrogen. In other embodiments, R ² is C ₁ -C ₄ alkyl. In some embodiments, where R ² is C ₁ -C ₄ alkyl, R ² is CH ₃ -, CH ₃ CH ₂ -, CH ₃ CH ₂ CH ₂ -, (CH ₃ ) ₂ CH-, CH ₃ CH ₂ CH ₂ CH ₂ -, CH ₃ CH ₂ (CH ₃ )CH-, (CH ₃ ) ₂ CHCH ₂ - or (CH ₃ ) ₃ C-. In some embodiments, R ² is hydrogen, CH ₃ -, CH ₃ CH ₂ - or CH ₃ CH ₂ CH ₂ -.

In still other embodiments, R ¹ is CH ₃ CH ₂ - and R ² is CH ₃ -. In some embodiments, R ¹ is CH ₃ CH ₂ - or CH ₃ CH ₂ CH ₂ CH ₂ - and R ² is hydrogen. In other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ - and R ² is CH ₃ CH ₂ CH ₂ - or CH ₃ CH ₂ CH ₂ CH ₂ -.

In other embodiments, R ¹ is C ₁ -C ₃ alkyl. In still other embodiments, R ¹ is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is C ₂ -C ₄ alkyl. In some embodiments, R ¹ is ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or t-butyl. In other embodiments, R ² is hydrogen or C ₁ -C ₃ alkyl. In some embodiments, R ² is hydrogen, methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ² is methyl. In still other embodiments, R ¹ is ethyl and R ² is methyl.

The analytical method of the present disclosure uses the alkyl alkanoate solvents described herein in combination with microwave irradiation to provide high extraction efficiency for carangin and pongamol, thereby giving more accurate measurements of carangin concentrations than other analytical methods based, for example, on methanol or hexane extraction. . In some embodiments, the alkyl alkanoate solvents used in the analytical methods described here exclude any co-solvents. In some embodiments of the above assay method, the alkyl alkanoate solvent that is mixed with the pongamia composition is free of alcohol, alkane, ketone, ether, and/or aromatic hydrocarbon. In some embodiments, the solvent does not contain methanol, ethanol, propanol, hexane, methyl tert-butyl ether, diethyl ether, toluene, benzene, or acetone. In other embodiments, the alkyl alkanoate solvent does not contain a diketone or diester, such as succinates, sebacates, glutarates, or malonates.

However, it should be noted that in some embodiments, the alkyl alkanoate solvent may contain trace amounts or residual concentrations of the excluded solvents described above. These additional trace amounts of solvent may, for example, be incorporated into the alkyl alkanoate solvent by standard chemical manufacturing or processing procedures. Residual concentrations of solvents, such as methanol or hexane, can be maintained below a certain threshold of total impurities in the alkyl alkanoate solvent, which is considered acceptable for standard analytical measurements, so as not to significantly affect the performance of the analytical method described here. For example, in some embodiments, the alkyl alkanoate solvent includes one or more co-solvents that are not alkyl alkanoate solvents, wherein the total concentration of the one or more co-solvents is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. solvent.

In some embodiments, the method includes mixing the pongamia composition and an alkyl alkanoate solvent to form an extraction mixture. In some embodiments, mixing the pongamia composition and the alkyl alkanoate solvent may include mixing, agitating or agitating the pongamia composition and the alkyl alkanoate solvent together to form an extraction mixture. In other embodiments, mixing the pongamia composition and the alkyl alkanoate solvent may include heating the pongamia composition and the alkyl alkanoate solvent to form an extraction mixture. It should be noted that the pongamia composition and the alkyl alkanoate solvent can also be separately mixed or shaken or heated prior to mixing. It should also be noted that the methods of the present disclosure also involve changing other parameters that may be relevant to the mixing step, including, for example, the length of time that the pongamia composition and the alkyl alkanoate solvent are mixed, the temperature and/or pressure at which they are mixed, the ratio of the pongamia composition to the alkyl alkanoate solvent; and other physical properties of the pongamia composition, such as particle size distribution.

In some embodiments, the implementation of the extraction mixture is irradiated to obtain an irradiated mixture. In some embodiments, the implementation of the extraction mixture is irradiated with microwave radiation to obtain an irradiated mixture. In some embodiments, the implementation of the extraction mixture is irradiated using a microwave extractor. In other embodiments, the present disclosure also provides for changes to parameters that may be related to the irradiation step, including, for example, the length of time, temperature, pressure, and microwave frequency at which the extraction mixture is irradiated.

It should be noted that the analytical method of the present disclosure, by combining an alkyl alkanoate solvent and microwave radiation, does not require certain technical means that are commonly used in existing analytical methods, such as Soxhlet extraction, pre-soaking seeds or pongamia seed cake in sodium hydroxide, or pre-treatment seeds or pongamia seed cake with subcritical water/steam. For example, in some embodiments, the present disclosure provides an analysis method where the method does not include Soxhlet extraction. In other embodiments, the assay method does not include soaking pongamia seeds or seed cake in a base (eg, hydroxide solution).

In some embodiments, the irradiated mixture is separated into a solid component and a liquid component. The solid component is referred to here as the extracted pongamia composition and the liquid component is referred to as the solvent extract (or alternatively the alkyl alkanoate extract). The irradiated mixture can be separated into the extracted pongamia composition and the solvent extract by any suitable means known in the art for separating solid and liquid phases. For example, in some embodiments, the irradiated mixture is separated by centrifugation. In some embodiments, the irradiated mixture is separated by decanting. In other embodiments, the irradiated mixture is separated by filtration.

In some embodiments, the extracted pongamia composition contains solid material and/or intrinsic chemical components that were originally present in the pongamia composition in combination with the alkyl alkanoate solvent, but which were insoluble in the alkyl alkanoate solvent and thus were not separated into the liquid phase of the extract.

In some embodiments, the solvent extract contains an alkyl alkanoate solvent (including the alkyl alkanoate and any co-solvents) and certain chemical components that are intrinsic to pongamia and that have been extracted from the pongamia composition into the alkyl alkanoate solvent. In some embodiments, the extract contains furanoflavonoids. Furanoflavonoids can be further identified by subclasses including, for example, flavones, flavonols (eg carangin) and dibenzoylmethanes (eg pongamol). In some embodiments, the extract contains carangin. In other embodiments, the extract contains pongamol. In some embodiments, the extract contains carangin and other furanoflavonoids. In some embodiments, the extract contains at least one or more furanoflavonoids selected from the group consisting of carangin, pongamol, lanceolatin, kanjon, pongaglabron, pongaglabol, ovalifolin, sanaganone, pinnathine, gamatine, pongon, glabon, caranjonol, pongkapin, pachycarin, pongagaglabol methyl ester, isopongagaglabol, methoxyisopongaglabol, pongol methyl ester, millettocalixin, 6-methoxyisopongaglabol, pongamoside A, pongamoside B, ponganone XI, pongamoside C, glabra I, ovalitenon, ponganone IX and pongarotene.

In some embodiments, after separating the irradiated mixture into the extracted pongamia composition and the solvent extract, the method further comprises analyzing the solvent extract. As described herein, the step of analyzing the solvent extract includes measuring the concentrations of certain chemical constituents in the solvent extract, which serve as proxy measurements for the concentrations of such chemical constituents originally present in the pongamia composition. In some embodiments, the method includes measuring individual concentrations of one or more furanoflavonoids in the solvent extract. In some embodiments, the method includes measuring the concentration of carangin in the solvent extract. In other embodiments, the method includes measuring the concentration of pongamol in the solvent extract.

Measurement of the concentrations of carangin, pongamol and other furanoflavonoids in the solvent extract can be performed using separation and detection analytical methods known in the art. In some embodiments, the concentrations of carangin, pongamol, and other furanoflavonoids are determined by high performance liquid chromatography (HPLC). In other embodiments, the concentrations of carangin, pongamol, and other furanoflavonoids are determined by HPLC-mass spectrometry (HPLC-MS). In some embodiments, the concentrations of carangin, pongamol, and other furanoflavonoids are determined by HPLC tandem mass spectrometry (HPLC-MS/MS). In some embodiments, the concentrations of carangin, pongamol, and other furanoflavonoids are determined by HPLC-ultraviolet-visible spectroscopy (HPLC-UV-visible).

In some embodiments, the assay method described herein may be referred to as "alkyl alkanoate solvent microwave extraction analytical method." In some embodiments in which a specific alkyl alkanoate is used in an alkyl alkanoate solvent, the extraction may be specifically referred to as the specific alkyl alkanoate used. For example, in some embodiments of the above methods in which the alkyl alkanoate solvent includes ethyl acetate, the assay method may be referred to as "ethyl acetate microwave analytical method".

It should be noted that reference to "alkyl alkanoate solvent microwave analytical method" includes embodiments in which the alkyl alkanoate solvent contains at least one alkyl alkanoate solvent and optionally one or more co-solvents that are not alkyl alkanoates. For example, "ethyl acetate microwave extraction analytical method" may refer to the use of an alkyl alkanoate solvent containing ethyl acetate and optionally one or more co-solvents.

Methods for preparing pongamia compositions

As described above, previous attempts to develop improved methods for preparing pongamia compositions having low concentrations of residual oil, caranjin, and pongamol have previously been hampered by the unreliability and unreliability of existing analytical methods for determining such concentrations. However, the development of improved methods for preparing pongamia compositions having low concentrations of carangin is now possible due to the analytical method described above, which provides a more accurate and reliable measurement of the concentration of carangin. Thus, the present disclosure provides more efficient methods for removing carangin and other furanoflavonoids from pongamia seeds and seed cake, including methods for preparing pongamia compositions, described below, in which the pongamia composition has a low concentration of carangin. More specifically, the present disclosure provides methods for preparing pongamia compositions wherein the pongamia composition contains carangin and has a concentration of carangin less than or equal to 100 ppm.

In one aspect, methods are provided herein for preparing pongamia compositions having low concentrations of carangin as determined by the alkyl alkanoate solvent microwave extraction analytical methods described above. In some embodiments, methods are provided herein for preparing pongamia compositions wherein the pongamia compositions have carangin concentrations of less than or equal to 100 ppm as determined by the alkyl alkanoate solvent microwave extraction analytical methods described above. In other embodiments, methods for preparing pongamia compositions are provided herein, wherein the pongamia compositions contain 20% less carangin, as determined by the alkyl alkanoate solvent microwave extraction analytical methods described above, compared to the original or first pongamia compositions from which they were derived.

In one aspect, provided herein is a process for preparing pongamia compositions, wherein the method comprises mixing a first pongamia composition with an alkyl alkanoate solvent to form an extraction mixture and separating the extraction mixture to form a miscella and a second pongamia composition, wherein the second pongamia composition has (i) a carangin concentration that is less than 20% of the concentration of carangin in the first pongamia composition, or (ii) a concentration of carangin less than or equal to 100 ppm.

With reference to FIG. 2, process 200 is an exemplary process for preparing a pongamia composition. At step 202, a first pongamia composition is provided. The first pongamia composition is mixed with the alkyl alkanoate solvent in step 204, thus obtaining an extraction mixture. The extraction mixture is separated at 206 to obtain a second pongamia and miscella composition.

It should be understood that in other embodiments, process 200 may include additional processing steps. In still other embodiments, certain steps of process 200 may be omitted.

In one embodiment, a process for preparing pongamia compositions is provided, comprising: providing a first pongamia composition; mixing the first pongamia composition with a solvent containing at least one alkyl alkanoate to obtain an extraction mixture; and separating the extraction mixture into a miscella and a second pongamia composition. In some embodiments, the second pongamia composition has (i) a concentration of carangin that is less than 20% of the concentration of carangin in the first pongamia composition, or (ii) a concentration of carangin that is less than or equal to 100 ppm.

In some embodiments, the first pongamia composition is derived from plant material derived from the pongamia tree or plant (also known as "Cytisus pinnatus", "Dalbergia arborea", "Derris indica", "Galedupa pungum", "karanj", "Millettia pinnata" , "pongam", "pongamia", "Pongamia glabra", "Pterocarpus flavus", "Pongamia pinnata" and "Robinia mitis", "Indian beech" and "mempari").

In some embodiments, the first pongamia composition is a defatted pongamia seed cake. The defatted pongamia seed cake can be described in terms of the previous processing that results in the defatted pongamia seed cake. For example, in some embodiments, the first composition of pongamia is a defatted pongamia seed cake, wherein the defatted pongamia seed cake is obtained by mechanical extraction. In other embodiments, the first pongamia composition is a defatted pongamia seed cake obtained by mechanical extraction of pongamia seeds or pongamia seed cake. In some embodiments, the defatted pongamia seed cake is obtained by mechanical extraction using a screw press. It should be noted that one or more cycles of mechanical extraction may be applied to either the seeds and/or the pongamia seed cake to produce a defatted pongamia seed cake as the first pongamia composition. In some embodiments, the first pongamia composition is not a pongamia oilseed or oilseed. In other embodiments, the first pongamia composition is not a defatted pongamia seed cake obtained by solvent extraction.

The first pongamia composition can be further characterized by other parameters including, for example, carangin concentration, oil content, moisture content, and particle size distribution, which may be particularly advantageous for extracting carangin and pongamol from the first pongamia composition. For example, in some embodiments, the implementation of the first composition of pongamia has a concentration of carangin, at least 200 ppm. In other embodiments, the implementation of the first composition of pongamia has a concentration of carangin, at least 500 ppm. In some embodiments, the first pongamia composition contains 8-40% oil by weight, 10-35% oil by weight, or 8-30% oil by weight. In some embodiments, the implementation of the first composition of pongamia contains 8-30% oil by weight.

In some embodiments of the foregoing method, obtaining the first pongamia composition may further include any steps of obtaining the first pongamia composition. For example, in some embodiments, the method includes providing pongamia oilseeds and subjecting the pongamia oilseeds to mechanical extraction to produce a defatted pongamia seed cake as the first pongamia composition. In some embodiments, the method includes mechanically pressing pongamia oilseeds to produce a defatted pongamia seed cake as the first pongamia composition. In other embodiments, the method may include obtaining a defatted pongamia seed cake and subjecting the defatted pongamia seed cake to mechanical extraction to obtain a first pongamia composition having the desired oil content and/or carangin concentration. In still other embodiments, the implementation of the method may include providing the first composition of pongamia, as described here, and further minimizing the oil content in the first composition of pongamia. In yet other embodiments, the method may include providing defatted pongamia seeds and further crushing the defatted pongamia seed cake to obtain a first pongamia composition having the desired particle size distribution.

In some embodiments of the above methods, the first pongamia composition can be mixed with any of the described solvents used in analytical methods. For example, in some embodiments, the solvent is an alkyl alkanoate solvent. In some embodiments, the alkyl alkanoate solvent may contain only alkyl alkanoates or, alternatively, may contain one or more additional co-solvents that are not alkyl alkanoates. In some embodiments, the solvent contains at least one alkyl alkanoate and one or more co-solvents that are not alkyl alkanoates. In other embodiments, the solvent contains at least one alkyl alkanoate, but does not contain any co-solvents that are not alkyl alkanoates. In some embodiments, the solvent is an alkyl alkanoate.

In some embodiments, the alkyl in the alkyl alkanoate is methyl, ethyl, propyl, or butyl. In other embodiments, the solvent includes methyl alkanoate, ethyl alkanoate, propyl alkanoate, or butyl alkanoate, or any combinations thereof. In some embodiments, the solvent includes ethyl alkanoate. In some embodiments, the alkanoate is methanoate, ethanoate, propionate, butanoate, or pentanoate. In other embodiments, the solvent includes alkyl methanoate, alkyl ethanoate, alkyl propionate, alkyl butanoate, alkyl pentanoate, or any combination thereof. In some embodiments, the solvent includes an alkyl ethanoate. In some embodiments, the implementation of the solvent includes ethyl acetate. In other embodiments, the solvent is ethyl acetate.

In some embodiments, the solvent comprises an alkyl alkanoate solvent selected from the group consisting of methyl methanoate, methyl ethanoate, methyl propanoate, methyl butanoate, methyl pentanoate, ethyl methanoate, ethyl ethanoate, ethyl propanoate, ethyl butanoate, ethyl pentanoate, propyl methanoate, propyl ethanoate, propyl propanoate, propyl butanoate, propyl p entanoate, butylmethanoate, butylethanoate, butylpropanoate , butylbutanoate and butylpentanoate and any combinations thereof. In some embodiments, the solvent comprises an alkyl alkanoate solvent selected from the group consisting of methyl ethanoate, methyl propanoate, methyl butanoate, ethyl methanoate, ethyl ethanoate, ethyl propanoate, ethyl butanoate, propyl methanoate, propyl ethanoate, propyl propanoate, propyl butanoate, butyl methanoate, butyl ethanoate, butyl propanoate, and butyl butyl anoate and any combination thereof.

In other embodiments, the method includes mixing the first pongamia composition with an alkyl alkanoate solvent containing at least one alkyl alkanoate of formula (I):

Where:

R ¹ is C ₁ -C ₄ alkyl; And

R ² is hydrogen or C ₁ -C ₄ alkyl.

In some embodiments, R ¹ is C ₁ -C ₄ alkyl. In other embodiments, R ² is hydrogen or C ₁ -C ₄ alkyl. In some embodiments, R ¹ and R ² are independently C ₁ -C ₄ alkyl. In some other embodiments, R ¹ is C ₁ -C ₄ alkyl and R ² is hydrogen.

In some embodiments, where R ¹ is C ₁ -C ₄ alkyl, R ¹ is CH ₃ -, CH ₃ CH ₂ -, CH ₃ CH ₂ CH ₂ -, (CH ₃ ) ₂ CH-, CH ₃ CH ₂ CH ₂ CH ₂ -, CH ₃ CH ₂ (CH ₃ )CH-, (CH ₃ ) ₂ CHCH ₂ - or (CH ₃ ) ₃ C-. In some embodiments, R ¹ is CH ₃ CH ₂ -. In other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ CH ₂ -. In yet other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ -.

In some embodiments, R ² is hydrogen. In other embodiments, R ² is C ₁ -C ₄ alkyl. In some embodiments, where R ² is C ₁ -C ₄ alkyl, R ² is CH ₃ -, CH ₃ CH ₂ -, CH ₃ CH ₂ CH ₂ -, (CH ₃ ) ₂ CH-, CH ₃ CH ₂ CH ₂ CH ₂ -, CH ₃ CH ₂ (CH ₃ )CH-, (CH ₃ ) ₂ CHCH ₂ - or (CH ₃ ) ₃ C-. In some embodiments, R ² is hydrogen, CH ₃ -, CH ₃ CH ₂ - or CH ₃ CH ₂ CH ₂ -.

In still other embodiments, R ¹ is CH ₃ CH ₂ - and R ² is CH ₃ -. In some embodiments, R ¹ is CH ₃ CH ₂ - or CH ₃ CH ₂ CH ₂ CH ₂ - and R ² is hydrogen. In other embodiments, R ¹ is CH ₃ CH ₂ CH ₂ - and R ² is CH ₃ CH ₂ CH ₂ - or CH ₃ CH ₂ CH ₂ CH ₂ -.

In other embodiments, R ¹ is C ₁ -C ₃ alkyl. In still other embodiments, R ¹ is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is C ₂ -C ₄ alkyl. In some embodiments, R ¹ is ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or t-butyl. In other embodiments, R ² is hydrogen or C ₁ -C ₃ alkyl. In some embodiments, R ² is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R ² is methyl. In still other embodiments, R ¹ is ethyl and R ² is methyl. In other embodiments, R ² is hydrogen, ethyl, or n-propyl. In other embodiments, R ¹ is ethyl, n-propyl, or n-butyl, and R ² is hydrogen, methyl, ethyl, or n-propyl. In some embodiments, R ² is methyl. In still other embodiments, R ¹ is ethyl and R ² is methyl.

In some embodiments, the alkyl alkanoate solvent is produced in situ . For example, an alkyl alkanoate can be prepared by mixing the appropriate alcohol with the appropriate carboxylic acid. In some embodiments, an alkyl alkanoate of formula (I) is prepared in situ by mixing an alcohol R ¹ -OH with a carboxylic acid R ² -COOH, where R ¹ and R ² are as defined above. In some embodiments, where the alkyl alkanoate is ethyl acetate, the ethyl acetate is produced in situ by mixing ethanol with acetic acid. In some embodiments, the alkyl alkanoate is prepared in situ prior to mixing the alkyl alkanoate solvent with the first pongamia composition. In other embodiments, the alkyl alkanoate is produced in situ with the first pongamia composition. For example, in some embodiments where the method includes mixing the first pongamia composition with a solvent containing ethyl acetate and the ethyl acetate is produced in situ , the method includes mixing the first pongamia composition with ethanol and acetic acid.

In some embodiments, the solvent may contain one or more co-solvents that are not alkyl alkanoates. However, in some embodiments, the solvent does not include any co-solvents. For example, in some embodiments, the alkyl alkanoate solvent does not contain an alkane, a ketone, an ether, and/or an aromatic hydrocarbon. In some embodiments, the alkyl alkanoate solvent does not contain hexane, methyl tert-butyl ether, diethyl ether, toluene, benzene, and/or acetone. In other embodiments, the alkyl alkanoate solvent does not contain a diketone and/or diester, such as succinates, sebacates, glutarates, or malonates.

In some embodiments, the implementation of the first composition of pongamia and the solvent are mixed to obtain an extraction mixture. In some embodiments, mixing the first pongamia composition and the solvent comprises mixing the first pongamia composition and the solvent in an extractor to form an extraction mixture. In some embodiments, the mixing step includes mixing, agitating, or agitating the extraction mixture in an extractor. In some embodiments, mixing the first pongamia composition and solvent to form an extraction mixture comprises heating the first pongamia composition and solvent to form an extraction mixture. In other embodiments, the method further comprises heating the extraction mixture. It should be noted that the above methods may include changes to other parameters that may be related to the mixing step, including, for example, the length of time the extraction mixture is in the extractor, the temperature and pressure of the extractor, the speed of the extractor chain, the particle size distribution of the first pongamia composition, the ratio of the first the composition of pongamia and the alkylalkanoate solvent; and the feed rate of the composition of pongamia and the alkylalkanoate solvent to the extractor.

In some embodiments, the implementation of the method may further include irradiating the extraction mixture with microwave radiation. In some embodiments, the extraction mixture is irradiated with microwave radiation after the mixing step and before the separation step. The present disclosure also provides for changes to parameters that may be related to the irradiation step, including, for example, the length of time, temperature, pressure, and frequency of microwave irradiation at which the extraction mixture is irradiated.

In some embodiments, the extraction mixture is separated into a miscella and a second pongamia composition. The miscella predominantly contains the liquid fraction of the extraction mixture (oil, alkyl alkanoate solvent and any soluble components), while the second pongamia composition is largely composed of the residual insoluble solid material or meal that remains from the first pongamia composition. The step of separating the extraction mixture into a miscella and a second pongamia composition may include any suitable methods known in the art for separating solid and liquid phases. In some embodiments, the extraction mixture is separated by filtration. In other embodiments, the extraction mixture is separated by decanting.

In some embodiments, the miscella contains a mixture of extracted oil, carangin, other furanoflavonoids, and an alkyl alkanoate solvent (including the alkyl alkanoate and any co-solvents). In other embodiments, the miscella has a concentration of carangin equal to or greater than 4000 ppm. In some embodiments, the miscella has a concentration of carangin equal to or greater than 4000 ppm as measured by the method described above. In some embodiments, the miscella can be characterized by oil content, water content, moisture content, solids content, or other characteristics known in the art.

In some embodiments, the second pongamia composition has a carangin concentration of less than or equal to 100 ppm. In other embodiments, the second pongamia composition has a concentration of carangin that is less than 20% of the concentration of carangin in the first pongamia composition. In still other embodiments, the second pongamia composition has a carangin concentration of less than or equal to 100 ppm, as determined by the alkyl alkanoate solvent microwave extraction analytical method described herein. In still other embodiments, the second pongamia composition has a concentration of carangin that is less than 20% of the concentration of carangin in the first pongamia composition, as determined by the alkyl alkanoate solvent microwave extraction analytical method described herein.

Due to the effectiveness of the methods of preparation described here, it should be noted that the second composition of pongamia has a concentration of carangin less than or equal to 100 ppm. In some embodiments, the second pongamia composition may have concentrations of carangin and/or pongamol of exercise. In some embodiments, the second pongamia compositions may have carangin and/or pongamol concentrations of less than 100 ppm, which are not detectable by conventional hexane and methanol based analytical methods. In other embodiments, the second pongamia composition may have trace concentrations of carangin and/or pongamol on the order of parts per billion (ppb) or parts per trillion (ppt). In circumstances where trace concentrations are present, the detection of carangin and pongamol by the alkyl alkanoate solvent microwave analytical methods described herein may be limited by the detection limits of the liquid chromatography methods and materials used. In some embodiments, trace amounts of carangin and/or pongamol may not be detected by the alkyl alkanoate solvent microwave extraction analytical methods described herein.

As described above, the first pongamia composition can be obtained from plant material derived from a pongamia tree or plant. Accordingly, in some embodiments, a second pongamia composition obtained from a first pongamia composition by the methods described herein can also be characterized as derived from plant material derived from the pongamia tree or plant (also known as "Cytisus pinnatus", "Dalbergia arborea", "Derris indica", "Galedupa pungum", "karanj", "Millettia pinnata", "pongam", "pongamia", "Pongamia glabra", "Pterocarpus flavus", "Pongamia pinnata" and "Robinia mitis", "Indian beech" and "mempari").

As also described above, the second pongamia composition largely consists of the residual insoluble solid material or meal that remains from the first pongamia composition after extraction with an alkyl alkanoate solvent and solid-liquid separation to remove the miscella. In some embodiments, the second pongamia composition is a meal. The resulting second pongamia compositions having a carangin concentration of less than or equal to 100 ppm, as described herein, may additionally contain any number of components such as carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones, which are originally present in the first composition of pongamia. For example, in some embodiments where the second pongamia composition contains protein, the second pongamia composition contains at least 30% protein by dry weight. In some embodiments, the implementation of the second composition of pongamia contains 30-50% protein or 30-40% protein by dry weight. In other embodiments, where the second pongamia composition contains carbohydrates, the second pongamia composition contains at least 40% carbohydrates by dry weight. In some embodiments, the second pongamia composition contains 40-70% carbohydrates, 50-70% carbohydrates, or 50-60% carbohydrates by dry weight.

Additional components may be present in the second pongamia composition as a percentage by weight of the total composition, reflecting the non-destructive techniques applied to the first pongamia compositions. That is, the methods of the present disclosure may be particularly suitable for removing carangin and pongamol while maintaining or maintaining the concentrations of nutrient components such as carbohydrates, proteins, fibers, ash, or any combination thereof, compared to the concentrations present in the first pongamia composition from which the a pongamia composition having a concentration of carangin less than or equal to 100 ppm. In other embodiments, the methods described herein can result in a marked increase in the concentration of these additional components by removing residual oil during extraction with the alkyl alkanoate solvent and subsequently reducing the overall weight of the second pongamia composition.

In some embodiments, the second pongamia composition, having a concentration of carangin less than or equal to 100 ppm, contains at least one component selected from the group consisting of carbohydrates, proteins, fibers, ash, and any combinations thereof, in a weight percent of the second pongamia composition, which is at least 90% by weight of the corresponding component present in the first pongamia composition. In some embodiments, the second pongamia composition, having a concentration of carangin less than or equal to 100 ppm, contains at least one component selected from the group consisting of carbohydrates, proteins, fibers, ash, and any combinations thereof, in a weight percent of the second pongamia composition, which is 90-125% by weight of the corresponding component present in the first pongamia composition.

In some embodiments, the second pongamia composition contains carbohydrates in a weight percent of the second pongamia composition that is 90-125% of the weight percent of carbohydrates present in the first pongamia composition. In other embodiments, the second pongamia composition contains proteins in a weight percent of the second pongamia composition that is 90-125% of the weight percent of proteins present in the first pongamia composition. In still other embodiments, the second pongamia composition contains fibers in a weight percent of the second pongamia composition that is 90-150% by weight of the fibers present in the first pongamia composition. In other embodiments, the second pongamia composition contains ash in a weight percent of the second pongamia composition that is 90-125% by weight of the ash present in the first pongamia composition.

In some embodiments, the concentration of other components in the second pongamia composition may be slightly reduced compared to the first pongamia composition. In some embodiments, the second pongamia composition contains trypsin inhibitors in a weight percent of the second pongamia composition that is 60-90% by weight of the trypsin inhibitors present in the first pongamia composition. In other embodiments, the second pongamia composition contains chalcones and/or other furanoflavonoids in a weight percent of the second pongamia composition that is less than 100% of the weight percent of chalcones and/or other furanoflavonoids present in the first pongamia composition.

In other embodiments, the total protein content of pongamia compositions can be further characterized by an amino acid profile. The amino acid profile may include characterization of pongamia compositions based on the amounts of individual amino acids present, the amounts of various combinations of different amino acids present, or the total amount of amino acids present. In some embodiments, the second pongamia composition has a total amino acid content of at least 20% by weight of the composition. In other embodiments, the second pongamia composition has a total amino acid content of 20-30% by weight of the composition. In other embodiments, the second pongamia composition has a total amino acid content that is at least 90% of the total amino acid content present in the first pongamia composition. In some embodiments, the second pongamia composition has a total amino acid content in the weight percent of the second pongamia composition that is 90-125% by weight of the total amino acids present in the first pongamia composition.

It should be noted that due to the nature of the extraction method described herein, which includes mixing the first pongamia composition with a solvent, the second pongamia composition may contain residual solvent concentrations. For example, the second pongamia composition may contain residual concentrations of the particular alkyl alkanoate(s) and any co-solvents in the alkyl alkanoate solvent used, even after separation of the miscella from the second pongamia composition. Thus, in some embodiments, the second pongamia composition contains an alkyl alkanoate solvent. In some embodiments, the second pongamia composition has an alkyl alkanoate solvent concentration of less than 100,000 ppm. In other embodiments, where the alkyl alkanoate solvent mixed with the first pongamia composition contains ethyl acetate, the second pongamia composition contains ethyl acetate. In some embodiments, where the pongamia composition contains ethyl acetate, the pongamia composition has an ethyl acetate concentration of less than 100,000 ppm.

The method of the present disclosure may further include a dry heating or roasting step to desolventize, i.e. to reduce the concentration of residual alkyl alkanoate solvent in the second pongamia composition. Therefore, in some embodiments, the method further comprises toasting the second pongamia composition to produce a toasted pongamia composition. In some embodiments, after toasting the second pongamia composition, the toasted pongamia composition contains an alkylalkanoate solvent and has an alkylalkanoate solvent concentration of less than or equal to 5000 ppm. In some embodiments, the toasted pongamia composition has an alkylalkanoate solvent concentration of 0 ppm to 5000 ppm, 0 ppm to 1000 ppm, 1000 to 3000 ppm, or 3000 to 5000 ppm. In still other embodiments, where the alkyl alkanoate solvent mixed with the first pongamia composition contains ethyl acetate and the second pongamia composition is toasted, the toasted pongamia composition contains ethyl acetate and has an ethyl acetate concentration of less than or equal to 5000 ppm. In some embodiments, where the toasted pongamia composition contains ethyl acetate, the toasted pongamia composition has an ethyl acetate concentration of 0 ppm to 5000 ppm, 0 ppm to 1000 ppm, 1000 ppm to 3000 ppm, or 3000 ppm to 5000 ppm of ethyl acetate.

Pongamia compositions

As previously mentioned, pongamia formulations having low concentrations of carangin and other antinutrients are required for later use. However, prior to the development of the above methods for analyzing pongamia compositions, residual carangin concentrations in treated pongamia compositions were difficult to assess accurately and reliably, making the preparation of pongamia compositions having low concentrations of carangin just as difficult to achieve. The alkylalkanoate solvent microwave extraction analytical methods of the present invention make it possible to obtain and test pongamia compositions having low concentrations of carangin. Here are the compositions of pongamia, in which the concentration of carangin is less than or equal to 100 ppm. Also provided herein are compositions of pongamia in which the concentration of carangin is less than or equal to 100 ppm, obtained by the methods described here and/or determined by the analytical methods described here for microwave extraction with an alkyl alkanoate solvent.

In one aspect, there is provided a pongamia composition comprising carangin, wherein the pongamia composition has a concentration of carangin less than or equal to 100 ppm, less than or equal to 90 ppm, less than or equal to 80 ppm, less than or equal to 70 ppm, less than or equal to 60 ppm, less than or equal to equal to 50 ppm, less than or equal to 40 ppm, less than or equal to 30 ppm, less than or equal to 20 ppm, or less than or equal to 10 ppm. In some embodiments, the pongamia composition has a carangin concentration of less than or equal to 100 ppm.

In another aspect, there is provided a pongamia composition comprising pongamol, wherein the pongamia composition has a pongamol concentration of less than or equal to 100 ppm, less than or equal to 90 ppm, less than or equal to 80 ppm, less than or equal to 70 ppm, less than or equal to 60 ppm, less than or equal to equal to 50 ppm, less than or equal to 40 ppm, less than or equal to 30 ppm, less than or equal to 20 ppm, or less than or equal to 10 ppm. In some embodiments, the pongamia composition has a pongamol concentration of less than or equal to 100 ppm.

In some embodiments, the pongamia composition is derived from plant material derived from the pongamia tree or plant (also known as "Cytisus pinnatus", "Dalbergia arborea", "Derris indica", "Galedupa pungum", "karanj", "Millettia pinnata", "pongam", "pongamia", "Pongamia glabra", "Pterocarpus flavus", "Pongamia pinnata" and "Robinia mitis", "Indian beech" and "mempari").

In some embodiments, a pongamia composition is provided herein, wherein the composition is or may be obtained by solvent extraction of defatted pongamia seed cake using an alkyl alkanoate solvent. In some embodiments, a pongamia composition obtained by microwave extraction with an alkyl alkanoate solvent is provided herein. In some embodiments, the pongamia composition is obtained by microwave extraction with an alkylalkanoate solvent from the defatted pongamia seed cake.

In another aspect, a pongamia composition is provided, comprising: karangin; and at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones.

In some embodiments, the pongamia composition has a carangin concentration of less than or equal to 100 ppm. In other embodiments, the pongamia composition has a carangin concentration of less than or equal to 100 ppm as determined by the alkyl alkanoate solvent microwave analytical method described above. In other embodiments, there is provided a pongamia composition comprising carangin, wherein the pongamia composition has a carangin concentration of less than or equal to 100 ppm, and wherein the carangin concentration is determined by treating the pongamia composition with an alkyl alkanoate solvent under microwave radiation.

In some embodiments, the pongamia composition comprises carangin and at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. In other embodiments, the pongamia composition contains carbohydrates and proteins. In some embodiments, the pongamia composition contains tannins and trypsin inhibitors. In some embodiments, the pongamia composition contains fibers and ash. In other embodiments, the pongamia composition contains other furanoflavonoids and chalcones. In some embodiments, the pongamia composition contains carbohydrates and fibers. In some embodiments, the pongamia composition contains carbohydrates and ash.

In some embodiments, a pongamia composition having a low concentration of karanjin may be prepared or prepared using the preparation methods described herein. In still other embodiments, the pongamia composition having a carangin concentration of less than or equal to 100 ppm is the second pongamia composition obtained by the method for preparing pongamia compositions as described herein. In some embodiments, the pongamia composition is prepared from a first pongamia composition having a concentration of carangin of at least 200 ppm. In other embodiments, the pongamia composition is prepared from a first pongamia composition having a concentration of carangin of at least 500 ppm.

As noted above, it should be understood that pongamia compositions having a concentration of carangin and/or pongamol less than or equal to 100 ppm as described herein may contain additional components (carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones), if present, as a weight percentage of the composition as a whole, reflecting the non-destructive method for preparing pongamia compositions as described here. For example, in some embodiments where the pongamia composition contains protein, the pongamia composition contains at least 30% protein by dry weight. In some embodiments, the pongamia composition contains 30-50% protein or 30-40% protein by dry weight. In other embodiments, where the pongamia composition contains carbohydrates, the pongamia composition contains at least 40% carbohydrates by dry weight. In some embodiments, the pongamia composition contains 40% to 70% carbohydrates, 50% to 70% carbohydrates, or 50% to 60% carbohydrates by dry weight.

In still other embodiments, the implementation of the total protein content in the compositions of pongamia can be further characterized by an amino acid profile. An amino acid profile may include characterization of pongamia compositions based on the amounts of individual amino acids present, the number of different combinations of different amino acids present, or the total amount of amino acids present. In some embodiments, the pongamia composition has a total amino acid content of at least 20% by weight of the composition. In other embodiments, the pongamia composition has a total amino acid content of 20-30% by weight of the composition.

Furthermore, it should be understood that pongamia compositions having low concentrations of carangin as described herein and obtained by the alkyl alkanoate extraction methods described herein may still contain residual pongamia oil and an alkyl alkanoate solvent.

In some embodiments, the pongamia composition contains an oil. In some embodiments, the pongamia composition contains less than 5% oil by dry weight. In some embodiments, the pongamia composition contains 1% to 5% oil by dry weight.

In some embodiments, the pongamia composition further comprises an alkyl alkanoate solvent. In other embodiments, the pongamia composition has an alkyl alkanoate solvent concentration of less than or equal to 100,000 ppm. In other embodiments, the pongamia composition has an alkyl alkanoate solvent concentration of less than or equal to 5000 ppm. In some embodiments, the pongamia composition has an alkyl alkanoate solvent concentration of 0 to 5000 ppm, 0 to 1000 ppm, 1000 to 3000 ppm, or 3000 to 5000 ppm.

In some embodiments, where the pongamia composition comprises an alkyl alkanoate solvent and the alkyl alkanoate solvent comprises ethyl acetate, the pongamia composition comprises ethyl acetate. In some embodiments, where the pongamia composition comprises ethyl acetate, the pongamia composition has an ethyl acetate concentration of less than or equal to 100,000 ppm. In yet other embodiments, the pongamia composition has an ethyl acetate concentration of less than or equal to 5000 ppm. In some embodiments, where the pongamia composition comprises ethyl acetate, the pongamia composition has an ethyl acetate concentration of 0 to 5000 ppm, 0 to 1000 ppm, 1000 to 3000 ppm, or 3000 to 5000 ppm of ethyl acetate.

As described herein, methods for preparing pongamia compositions can result in pongamia compositions having extremely low concentrations of carangin and/or pongamol. In some embodiments, the pongamia composition has a concentration of carangin and/or a concentration of pongamol on the order of single digit parts per million or fractional amounts thereof. In some embodiments, pongamia compositions may have carangin and/or pongamol concentrations of less than 100 ppm, which are not detectable by conventional hexane and methanol based analytical methods. In other embodiments, the pongamia composition may have trace concentrations of carangin and/or pongamol on the order of parts per billion (ppb) or parts per trillion (ppt). In some embodiments, the pongamia compositions described herein may contain trace amounts of carangin and/or pongamol that are not detectable by the alkyl alkanoate solvent microwave extraction analytical methods described here.

Accordingly, in some embodiments, where the pongamia composition has a concentration of carangin that is detectable by the alkyl alkanoate solvent microwave extraction analytical methods described herein, the pongamia composition can be characterized by other components present in the composition, including carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors , other furanoflavonoids, chalcones, alkyl alkanoate solvent(s) or amino acid content, or any combination thereof.

In some embodiments, a pongamia composition is provided herein comprising at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones, wherein the pongamia composition has Karanjin concentration less than or equal to 100 ppm. In some embodiments of the above, the pongamia composition has an undetectable concentration of carangin, as determined by the alkyl alkanoate solvent microwave extraction analytical method described herein. In some embodiments, the pongamia composition contains (i) 30-50% protein by dry weight; (ii) 40-70% carbohydrates; (iii) a total amino acid content of 20-30% by dry weight; or any combination of them.

The use of pongamia compositions

Pongamia compositions having low concentrations of carangin and pongamol as described above and prepared by the methods described above may be particularly useful as a feed additive or main feed in ruminant feed compositions such as cattle feed compositions. The non-destructive methods for preparing pongamia compositions described here result in the effective removal of the anti-nutritional components of carangin and pongamol without reducing the amount of other components, including macronutrients (such as protein and carbohydrates), which are very important for achieving acceptable feed efficiency. The pongamia compositions can be used alone in a ruminant feed formulation or in combination with a non-pongamia base feed to form a mixed ruminant feed composition.

As described herein, the term "ruminant" should be understood to include any wild or domesticated ungulate mammal having a multi-chambered stomach (including a rumen) adapted to digest plant matter. Suitable ruminants may include, but are not limited to, cattle, yaks, buffalo, goats, sheep, deer, gazelles, and antelope. In some embodiments, the implementation of the cattle is a beef cattle.

In one aspect, ruminant feed compositions are provided comprising pongamia compositions having low concentrations of carangin as described above. In some embodiments, ruminant feed compositions comprise a pongamia composition having a carangin concentration of less than or equal to 100 ppm. In other embodiments, ruminant food compositions comprise a staple food and a pongamia composition, wherein the pongamia composition has a carangin concentration of less than 100 ppm, as described herein.

In some embodiments, cattle feed compositions are provided herein containing pongamia compositions having low concentrations of carangin, as described above. In some embodiments, the cattle feed compositions comprise a pongamia composition having a carangin concentration of less than or equal to 100 ppm. In other embodiments, the cattle feed compositions comprise a staple feed and a pongamia composition, wherein the pongamia composition has a carangin concentration of less than 100 ppm, as described herein.

In some embodiments, a ruminant feed composition (including, for example, a bovine feed composition) is provided herein comprising any of the pongamia compositions described herein. In one embodiment, a ruminant feed composition (including, for example, a bovine feed composition) is provided herein comprising: a base feed and any of the pongamia compositions described herein.

In some embodiments of the foregoing, the pongamia composition contains: karangin; and at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones, and the pongamia composition has a carangin concentration of less than or equal to 100 ppm. In other embodiments, the pongamia composition contains: at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones, and the pongamia composition has a concentration of carangin less than or equal to 100ppm.

The pongamia compositions described herein, due to their low concentrations of carangin, can be used in ruminant feed compositions, such as cattle feed compositions, in greater amounts than previously used, and with less anti-nutritional or long-term pathological effects than previously observed. Thus, in some embodiments, the ruminant or bovine feed composition contains at least 30% by weight or at least 40% by weight of the pongamia composition, wherein the pongamia composition has a carangin concentration of less than or equal to 100 ppm. In some embodiments, the pongamia composition has a carangin concentration of less than or equal to 100 ppm, where the carangin concentration is determined by treating the pongamia composition with an alkyl alkanoate solvent under microwave radiation. In other embodiments, the pongamia composition has a concentration of carangin less than or equal to 100 ppm, where the concentration of carangin is determined by an analytical method of microwave extraction with an alkyl alkanoate solvent.

In some embodiments, a ruminant feed composition (including, for example, a bovine feed composition) is included in the main feed. A suitable staple feed for the ruminant feed compositions described herein can be any non-pongamia derived feedstock known in the art as forage or feed, including, for example, hay, straw, silage, grain, legumes, food scraps, and forage. In some embodiments, the staple food may include one or more foods selected from the group consisting of wheat food, corn food, barley food, oatmeal, soybean meal, sunflower meal, peanut meal, peanut meal, and hay. In some embodiments, the base food includes wheat food, corn food, soy flour, or any combination thereof. Due to the low concentrations of carangin in the pongamia compositions described herein, pongamia compositions can be mixed with basic feeds to produce ruminant feed compositions containing high proportions of pongamia-derived feeds. Thus, the amount of basic food in the animal compositions of the present invention can be reduced. In other embodiments, the implementation of the feed composition for ruminants (including, for example, feed composition for cattle) contains less than 60% or less than 70% by weight of the main feed.

It should be noted that the ruminant feed compositions described herein (including, for example, cattle feed compositions) may contain additional feed additives known in the art, including, for example, antibiotics and other veterinary agents, growth hormones, vitamins, minerals. substances or biologically active additives, flavor enhancers, processing aids and the like.

In yet another aspect, the present disclosure provides methods for feeding a ruminant, such as a cow, comprising providing pongamia compositions or ruminant feed compositions, as described herein, to the ruminant. In some embodiments, the present disclosure provides a method of feeding a ruminant, comprising providing the ruminant with a pongamia composition having a carangin concentration of less than or equal to 100 ppm, as determined by an alkyl alkanoate solvent microwave extraction analytical method. In other embodiments, provided herein is a method of feeding a ruminant, comprising providing the ruminant with a bovine feed composition, wherein the ruminant feed composition comprises a pongamia composition having a carangin concentration of less than or equal to 100 ppm, and wherein the concentration is determined by processing the pongamia composition. any of the alkylalkanoate solvents described here under the influence of microwave radiation.

In some embodiments, the ruminant is a cow. In some embodiments, the cow is a beef cow. In some embodiments, the present disclosure provides methods for feeding a cow, comprising providing the cow with pongamia compositions or ruminant feed compositions, as described herein. In some embodiments, the present disclosure provides a method of feeding a cow, comprising providing the cow with a pongamia composition having a carangin concentration of less than or equal to 100 ppm, as determined by an alkyl alkanoate solvent microwave extraction analytical method. In other embodiments, a method of feeding a cow is provided herein, comprising providing the cow with a bovine feed composition, wherein the bovine feed composition comprises a pongamia composition having a carangin concentration of less than or equal to 100 ppm, and wherein the carangin concentration is determined by treating the pongamia composition. any of the alkyl alkanoate solvents described here under the influence of microwave radiation.

For the ruminant or bovine feeding methods described herein, the ruminant feed compositions (including, for example, bovine feed compositions) can be provided in various forms suitable for the ruminant or bovine. In some embodiments, the ruminant feed composition is provided as a ground meal, kibble, liquid feed, or mash. For example, in some embodiments, the ruminant feed composition may be provided as a ground meal or kibble.

Notably, the non-destructive processing methods described here provide pongamia compositions that not only have low concentrations of anti-nutrients such as caranjin and pongamol, but can also retain or maintain comparable concentrations of nutrients and nearby compounds present in the original pongamia compositions from which the compositions are derived. pongamia with low concentrations of carangin. Thus, it should also be noted that pongamia compositions prepared by the methods described herein may also have specific concentrations of nutrients or proximate compounds (ashes, moisture, proteins, fats, carbohydrates, minerals, vitamins) that are particularly suited to meet nutritional requirements. nutrients of ruminants (such as cattle) to be fed, in particular with regard to feed efficiency.

In other aspects, an article of manufacture is provided, such as a container containing pongamia compositions as described herein, or a food containing pongamia compositions as described herein; and a label containing instructions for use of such pongamia or food compositions.

In still other aspects, a kit is provided that includes pongamia compositions as described herein or a food containing pongamia compositions as described herein; and a package insert containing instructions for use of such pongamia or food compositions.

EMBODIMENTS LISTED

The following embodiments below represent some aspects of the invention.

1. Method, including:

mixing the pongamia composition with an alkyl alkanoate solvent to obtain an extraction mixture;

irradiating the extraction mixture with microwave radiation to obtain an irradiated mixture;

separating the irradiated mixture into the extracted pongamia composition and the alkyl alkanoate extract; And

measuring the concentration of carangin in the alkyl alkanoate extract.

2. The method of embodiment 1 wherein the alkyl alkanoate solvent comprises an alkyl alkanoate selected from the group consisting of methyl methanoate, methyl ethanoate, methyl propanoate, methyl butanoate, methyl pentanoate, ethyl methanoate, ethyl ethanoate, ethyl propanoate, ethyl butanoate, ethyl pentanoate, propyl methanoate, propyl ethanoate, propyl propanoate , propylbutanoate, propylpentanoate, butylmethanoate, butylethanoate, butylpropanoate, butylbutanoate and butylpentanoate, and any combinations thereof.

3. The method of embodiment 1 or 2 wherein the alkyl alkanoate solvent comprises ethyl acetate.

4. The method of one of embodiments 1-3, wherein the pongamia composition is a defatted pongamia seed cake.

5. The method of one of embodiments 1-4, wherein the pongamia composition is obtained by mechanical extraction, solvent extraction, or a combination thereof.

6. The method of one of embodiments 1-5, wherein measuring the concentration of caranjin in the alkyl alkanoate extract comprises determining the concentration of caranjin by high performance liquid chromatography (HPLC).

7. Method, including:

providing the first composition of pongamia,

mixing the first composition of pongamia with an alkyl alkanoate solvent to obtain an extraction mixture; And

separating the extraction mixture into a miscella and a second pongamia composition, wherein the second pongamia composition has (i) a concentration of carangin that is less than 20% of the concentration of carangin in the first pongamia composition, or (ii) a concentration of carangin that is less than or equal to 100 ppm.

8. The method of Embodiment 7, wherein the second pongamia composition has a carangin concentration of less than or equal to 100 ppm as determined by the method of one of Embodiments 1-5.

9. The method of embodiment 7 or 8 wherein the first pongamia composition is a defatted pongamia seed cake.

10. The method of embodiment 9 wherein the first pongamia composition is a defatted pongamia seed cake obtained by mechanical extraction.

11. The method of embodiment 9 or 10 wherein the first pongamia composition is not a defatted pongamia seed cake obtained by solvent extraction.

12. The method of one of embodiments 7-11, wherein the first pongamia composition has a concentration of carangin of at least 200 ppm.

13. The method of one of embodiments 7-12, wherein the first pongamia composition contains 8-30% oil by weight.

14. The method of one of embodiments 7-13, wherein the miscella has a concentration of carangin greater than or equal to about 4000 ppm.

15. The method of one of embodiments 7-14, wherein the second pongamia composition has a pongamol concentration of less than or equal to 100 ppm.

16. The method of one of embodiments 7-15, wherein the alkyl alkanoate solvent comprises ethyl acetate.

17. The method of one of embodiments 7-16, wherein the method further comprises irradiating the extraction mixture with microwave radiation.

18. Composition of pongamia, containing:

karangin; And

at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones,

wherein the pongamia composition has a concentration of carangin less than or equal to 100 ppm.

19. The pongamia composition of embodiment 18, wherein the pongamia composition has a carangin concentration of less than or equal to 100 ppm as determined by the method of one of embodiments 1-6.

20. The pongamia composition of embodiment 18 or 19, wherein the pongamia composition further comprises pongamol.

21. The pongamia composition of one of embodiments 18-20, wherein the pongamia composition has a pongamol concentration of less than or equal to 100 ppm.

22. A pongamia composition according to one of embodiments 18-21 further comprising an alkyl alkanoate solvent.

23. The pongamia composition of embodiment 22, wherein the pongamia composition has an alkyl alkanoate solvent concentration of less than 100,000 ppm.

24. The pongamia composition of embodiment 22 or 23, wherein the pongamia seed meal has an alkyl alkanoate solvent concentration of less than 5000 ppm.

25. The pongamia composition of one of embodiments 22-24, wherein the alkyl alkanoate solvent comprises ethyl acetate.

26. The pongamia composition of one of embodiments 18-25, wherein the pongamia composition contains less than 5% oil by dry weight.

27. The pongamia compositions of one of embodiments 18-26, wherein the pongamia composition contains 1% to 5% oil by dry weight.

28. The pongamia composition of one of embodiments 18-27, wherein the pongamia composition contains at least 30% protein by dry weight.

29. A pongamia composition according to one of embodiments 18-28, wherein the pongamia composition contains 30-40% protein by dry weight.

30. The pongamia composition of one of embodiments 18-29, wherein the pongamia composition has a total amino acid content of at least 20% by weight.

31. The pongamia composition of one of embodiments 18-30, wherein the pongamia composition has a total amino acid content of 20-30% by weight.

32. The pongamia composition of one of embodiments 18-31, wherein the pongamia composition contains at least 40% carbohydrate by weight.

33. The pongamia composition of one of embodiments 18-32, wherein the pongamia composition contains 50-70% carbohydrates by weight.

34. The pongamia composition of one of embodiments 18-33, wherein the pongamia composition is derived from an original pongamia composition having a concentration of carangin of at least 200 ppm.

35. The pongamia composition of one of embodiments 18-34, wherein the pongamia composition is obtained by solvent extraction of defatted pongamia seed cake using an alkyl alkanoate solvent.

36. The pongamia composition of one of embodiments 18-35, wherein the pongamia composition is obtained by solvent extraction of defatted pongamia seed cake using an alkyl alkanoate solvent and microwave radiation.

37. Pongamia composition obtained or obtained by the method according to one of embodiments 1-17.

38. Pongamia composition containing:

at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones,

wherein the pongamia composition has a concentration of carangin less than or equal to 100 ppm.

39. Pongamia composition containing:

at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones,

wherein the pongamia composition has a carangin concentration of less than or equal to 100 ppm, and wherein the pongamia composition has a pongamol concentration of less than or equal to 100 ppm.

40. Pongamia composition containing:

caranjin, or pongamol, or a combination of caranjin and pongamol;

at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones,

wherein if carangin is present, the pongamia composition has a concentration of carangin less than or equal to 100 ppm,

wherein if pongamol is present, the pongamia composition has a pongamol concentration of less than or equal to 100 ppm.

41. Pongamia composition containing:

karangin; And

at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids and chalcones,

wherein the pongamia composition has a caranjin concentration of less than or equal to 100 ppm, and wherein the caranjin concentration is determined by treating the pongamia composition with an alkyl alkanoate solvent under microwave radiation.

42. A pongamia composition according to one of embodiments 18-41, wherein the pongamia composition is derived from plant material derived from a pongamia tree or plant.

43. The pongamia composition of one of embodiments 18-42, wherein the pongamia composition is a meal.

44. Feed composition, including:

a pongamia composition according to one of embodiments 18-43; And

main feed for cattle.

45. Feed composition, including:

a pongamia composition according to one of embodiments 18-43; And

main feed.

46. The food composition of embodiment 44 or 45, wherein the food composition comprises at least 30% by weight or at least 40% by weight of the pongamia composition.

47. A food composition according to one of embodiments 44-46, wherein the food composition comprises less than 60% by weight or less than 70% by weight of the main food.

48. A food composition according to one of embodiments 44-47, wherein the food composition is a kibble.

49. A method of feeding a ruminant, comprising providing the ruminant with the pongamia compositions of one of embodiments 18-43 or the feed composition of one of embodiments 44-48.

50. The method of claim 49, wherein the ruminant is selected from the group consisting of cow, yak, buffalo, goat, sheep, deer, gazelle, and antelope.

51. The method of embodiment 50 wherein the ruminant is a cow.

EXAMPLES

The present disclosed subject matter will be better understood with reference to the following examples, which are given by way of example of the invention and not by way of limitation.

EXAMPLE A: ANALYTICAL METHODS

EXAMPLE A1: COMPARATIVE METHANOL BASED EXTRACTION METHODS

The following example describes experiments comparing the extraction of carangin and pongamol from pongamia seed cake using methanol as solvent.

Homogenization of the extraction of caranjin and pongamol. 0.5 g of pongamia seed cake was placed in a 50 ml polypropylene centrifuge tube with 5 ml of methanol in a final ratio of 10:1 (solvent:solids). The samples were then placed in a plant/tissue homogenizer for shaking for 2 minutes at 1500 rpm. The samples were then centrifuged for 5 minutes at 3000 rpm to separate the solvent from the solids, and the supernatant was decanted into a clean 50 ml polypropylene tube. The extraction process was repeated 5 times in order to improve the extraction of carangin and pongamol.

Extraction homogenization with soaking in NaOH. Prior to extraction homogenization, 0.5 g of pongamia seed cake was placed in a 50 ml polypropylene centrifuge tube with 1 ml of 2% NaOH. The mixture was then incubated for 24 hours. After incubation, 10 ml of water was added and the tubes were placed on a mechanical shaker at high speed for 10 minutes. The tubes were then centrifuged at 3000 rpm to precipitate the solid material. The water was kept for analysis and the washing process was repeated once more to ensure removal of the NaOH. After incubation with NaOH, the samples were extracted with methanol as described above.

Homogenization of extraction with soaking in methanol. Prior to extraction homogenization, 0.5 g of pongamia seed cake was placed in a 50 ml polypropylene centrifuge tube with 25 ml of methanol in a final ratio of 50:1 (solvent:solids). The mixture was then incubated for 24, 48, 72 or 96 hours at room temperature. After incubation, the samples were placed in a plant/tissue homogenizer for shaking for 2 minutes at 1500 rpm. After homogenization, the samples were centrifuged for 5 minutes at 3000 rpm to separate the solvent from solid particles, and the supernatant was decanted into a clean 50 ml polypropylene tube. Homogenization was repeated 5 times in order to improve the extraction of carangin and pongamol.

Homogenization extraction with filtration. 0.5 g of pongamia seed cake was placed in a 50 ml polypropylene centrifuge tube with 25 ml of methanol in a final ratio of 50:1 (solvent:solids). The samples were then placed in a plant/tissue homogenizer for shaking for 2 minutes at 1500 rpm. The samples were then filtered to separate solids from methanol. The extraction process was repeated 5 times in order to improve the extraction of carangin and pongamol.

Soxhlet extraction of caracine and pongamol. 0.5 g of pongamia seed cake was placed in an extraction thimble with 125 ml of methanol. Extractions were carried out for 24 or 48 hours, after which the Soxhlet extract was transferred into a clean polypropylene tube.

Standard solutions for HPLC. Commercially available caranjin and pongamol were mixed with methanol to give the following HPLC standard solutions: 0.05, 0.1, 0.2, 0.5, 1.0, 5.0 and 20.0 µg/mL.

Instruments for HPLC. HPLC analysis was performed with a mobile phase consisting of: solvent A (0.1% formic acid in HPLC water) and solvent B (0.1% formic acid in acetonitrile). The injection volume was 2 μl and the flow rate was 0.75 ml/min. The column was a HPLC C18 5 µm, 50×2 mm column. All HPLC analyzes were performed in negative ion mode. MS parameters were as follows: 30 psi gas curtain; impact gas 4 psi; drying gas (GS1) 50 psi; drying gas (GS2) 50 psi; ion spray voltage 5000; temperature 500°C; declustering potential (DP) 51 V; input potential 10 V; collision energy (CE) 60 eV for karanjin and 30 eV for pongamol.

MS/MS quantitation of caranjin and pongamol in the extract. Multiple reaction monitoring (MMR) of ionic transitions was monitored for both Karanjin and Pongamol. The concentrations of carangin and pongamol present in the extracted samples were calculated using Analyst software version 1.6.3. Briefly, the peak area of carangin and pongamol in the extracted samples were compared with the peak area of the calibration standards to determine the ppm of carangin and pongamol.

Extraction of caranjin with methanol and extraction of pongamol from pongamia seed cake. Table 1 lists the concentrations of carangin and pongamol that can be extracted from pongamia seed cake (FIGS. 3A and 3B) by the homogenization extraction method using methanol as the extraction solvent (solvent:solids ratio 10:1). The amounts of carangin and pongamol obtained in each of the five consecutive extractions were summed to provide a determination of the total amount of caranjin extracted and the total amount of pongamol extracted, respectively, as shown in Table 1.

Table 1.

Sample number Raw material Total carangin extracted (ppm) Total pongamol extracted (ppm) 1.1 Seed cake 4154 318

Evaluation of the effect of 24-hour soaking in NaOH on the extraction of carangin and pongamol. The amounts of carangin and pongamol obtained from each of the five successive extractions were added together to obtain a measurement result of total extracted carangin and total extracted pongamol, respectively, as shown in Table 2. The table shows that the treatment of pongamia seed cake with NaOH before homogenizing the extraction with methanol (at a solvent:solids ratio of 10:1) reduced the total concentration of extracted and residual carangin and pongamol compared to extraction without treatment (FIGS. 3A and 3B).

Table 2.

Sample number Raw material Total carangin extracted (ppm) Total pongamol extracted (ppm) 1.1 Extraction without NaOH (seed cake) 4154 318 2.1 Extraction with NaOH (seed cake) 2219 15

Determination of the concentration of carangin and pongamol in the NaOH wash water. Table 3 shows that carangin and pongamol leach into the wash water after soaking in NaOH. However, the concentration of carangin and pongamol in the wash water is insufficient to explain the decrease in the concentration of carangin and pongamol isolated after treatment with NaOH.

Table 3

Sample number Raw material Carangin extracted from wash water (ppm) Pongamol extracted from wash water (ppm) 3.1 NaOH wash with water 463 112

Evaluation of the effect of soaking in methanol on the extraction of carangin. The amounts of carangin and pongamol obtained from each of the five successive extractions were added together to obtain the measurement result of total extracted carangin and total extracted pongamol, respectively, as shown in Table 4. Table 4 demonstrates that soaking pongamia seed cake in methanol prior to extraction for any period of time reduced the extraction of carangin compared with extraction without soaking in methanol (at a ratio of solvent:solids 50:1) (figa and 3B).

Table 4

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 4.1 No soaking 4580 329 4.2 24 hour soak in methanol 4117 263 4.3 48 hour soak in methanol 4331 284 4.4 72 hour soak in methanol 3999 439 4.5 96 hour soak in methanol 4145 223

Evaluation of carangin and pongamol recovery using filtration rather than centrifugation after homogenizing the extraction with methanol. The amounts of carangin and pongamol obtained from each of the five successive extractions (at a solvent:solids ratio of 50:1) were added together to obtain the measurement result for total carangin extracted and total pongamol extracted, respectively, as shown in Table 5. Table 5 demonstrates that homogenization of extraction with methanol with filtration allows the extraction of similar concentrations of carangin and pongamol from pongamia seed cake compared to homogenization of extraction with methanol with centrifugation (FIGS. 3A and 3B).

Table 5

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 4.1 centrifugation 4580 329 5.1 Filtration 5240 302

Soxhlet extraction of caracine and pongamol. Table 6 shows a comparison of the homogenization of the methanol extraction (solvent:solids ratio 50:1) and the methanol Soxhlet extraction. The total amount of carangin and pongamol extracted for methanol treatment homogenization is the sum of carangin and pongamol obtained from each of the five consecutive extractions; the total amount of carangin and pongamol extracted from the Soxhlet treatment is the amounts of carangin and pongamol obtained in one Soxhlet cycle during the specified time period. The values determined for the 24-hour and 48-hour Soxhlet extraction with methanol were taken as the average of two separate experiments, respectively. Table 6 shows that the 24- and 48-hour methanol Soxhlet extraction released more carangin and pongamol than the methanol extraction homogenization method (FIGS. 3A and 3B).

Table 6

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 4.1 Homogenization with methanol 4580 329 6.1 Methanol Soxhlet (24 hours) 5670 521 6.2 Methanol Soxhlet (48 hours) 5933 508

EXAMPLE A2: SOLVENT DEPENDENCE OF EXTRACTION METHODS

The following example describes experimental attempts to evaluate the ability of various solvents to extract caranjin and pongamol from pongamia seed cake.

Homogenization of the extraction of caranjin and pongamol. In this example, the extraction homogenization was carried out in the same way as in example A1. However, the solvents tested included ethanol, hexane, methyl tert-butyl ether (MTBE), toluene, diethyl ether, ethyl acetate, and acetone, all in a 50:1 solvent:solids ratio. Finally, experiments were performed to evaluate the effect of homogenization time on ethyl acetate solvent extraction comparing 10 minutes (2 min/cycle, 5 cycles) and 50 minutes (10 min/cycle, 5 cycles) homogenization.

Soxhlet extraction of caracine and pongamol. In this example, the Soxhlet extraction was carried out in the same way as in example A1. However, the solvents tested were methanol, MTBE and ethyl acetate. In addition, the reaction times tested for ethyl acetate were 6, 24, 48, 72 and 96 hours.

Evaluation of the extraction efficiency of carangin and pongamol with various solvents using extraction homogenization. The amounts of carangin and pongamol obtained from each of the five successive extractions were added together to give a measurement result of total carangin extracted and total pongamol extracted, respectively, as shown in Table 7. Table 7 demonstrates that ethyl acetate extracted more carangin and pongamol than any other solvent tested using the homogenization method (FIGS. 3A, 3B, 4A and 4B).

Table 7

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 4.1 methanol 4580 329 7.1 Ethanol 3879 268 7.2 Hexane 4341 323 7.3 MTBE 5503 329 7.4 Toluene 6307 325 7.5 diethyl ether 7465 379 7.6 ethyl acetate 8373 471 7.7 Acetone 4812 292

Evaluation of the effect of increasing the homogenization time on the extraction of carangin and pongamol with ethyl acetate. Table 8 compares extraction homogenization using ethyl acetate as solvent with a total 10 minute extraction period and a total 50 minute extraction period. Table 8 demonstrates that increasing the total homogenization time from 10 to 50 minutes reduces the efficiency of extraction of carangin and pongamol with ethyl acetate (FIGS. 4A and 4B).

Table 8

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 7.6 10 minutes 8373 471 7.6(b) 50 minutes 4011 317

Evaluation of the efficiency of extraction of carangin and pongamol with various solvents using Soxhlet extraction. Table 9 shows the total amount of carangin and the total amount of pongamol extracted in single Soxhlet cycles using various solvents for various lengths of time. Table 9 shows that ethyl acetate extracted more carangin and pongamol than any other solvent tested using the Soxhlet extraction method (FIGS. 3A, 3B, 4A and 4B). In addition, Table 9 demonstrates that a shorter Soxhlet extraction time was more effective than a longer time for carangin isolation. However, a longer Soxhlet extraction time was generally more effective than a shorter time to isolate pongamol.

Table 9

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 6.1 Methanol/Soxhlet 24 h 5670 521 6.2 Methanol/Soxhlet 48 h 5993 508 8.1 Methanol/Soxhlet 6 h 5920 512 8.2 Methanol/Soxhlet 72 h 4158 591 8.3 Methanol/Soxhlet 96 h 3912 333 8.4 MTBE/Soxhlet 6 h 5240 544 8.5 MTBE/Soxhlet 24h 5560 520 8.6 MTBE/Soxhlet 48 h 4640 508 8.7 MTBE/Soxhlet 72 h 3876 418 8.8 MTBE/Soxhlet 96 h 4080 424 8.9 Ethyl acetate/Soxhlet 6 h 7747 641 8.10 Ethyl acetate/Soxhlet 24 h 7626 632 8.11 Ethyl acetate/Soxhlet 48 h 7386 713 8.12 Ethyl acetate/Soxhlet 72 h 7306 721 8.13 Ethyl acetate/Soxhlet 96 h 7480 755

EXAMPLE A3: MICROWAVE EXTRACTION (ME) METHOD

The following example describes experimental attempts to develop an ethyl acetate ME method for Karanjin and Pongamol.

Preparation of ionic liquid. An ionic liquid was prepared by adding 40.1 g of 1-butyl-3-methylimidazolium bromide and 75 ml of 0.8 N HCl to a glass bottle. The mixture was then shaken to dissolve the solids.

Microwave extraction of carangin and pongamol. 0.5 g pongamia seed cake was added to a microwave extraction tube. Then, 15 ml of ethyl acetate or ionic liquid was added to the sample tubes and shaken to mix. Next, the samples were extracted using a microwave extractor under the following conditions: 1) the rate of temperature change over 15 minutes to 70°C, 2) exposure at 70°C for 10 minutes. After cooling, the supernatant was filtered using filter paper on a Buchner funnel under vacuum.

Evaluation of the efficiency of microwave extraction of carangin and pongamol with ethyl acetate. Table 10 shows that more than 9600 ppm carangin and 790 ppm pongamol were extracted from the seed cake with ME ethyl acetate (also FIGS. 5A and 5B).

Table 10

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 9.1 ME/ethyl acetate 9675 792 9.2 ME/ionic liquid 979.5 481.5

Summary of seed cake extraction. Table 11 presents a summary of the extraction data for karagin and pongamol, detailed in examples A1-A3. 6A and 6B are histograms of the relative efficiency of each extraction treatment, as measured by the total amount of carangin and pongamol extracted in ppm.

Table 11

Sample number Treatment Total carangin extracted (ppm) Total pongamol extracted (ppm) 1.1 Methanol (homogenization-10:1) 4154 318 2.1 Methanol (homogenization - 10:1 with NaOH soak) 2219 15 4.1 Methanol (homogenization-50:1) 4580 329 4.2 Methanol (homogenization - 50:1 with soaking in methanol for 24 hours) 4117 263 4.3 Methanol (homogenization - 50:1 with soaking in methanol for 48 hours) 4331 284 4.4 Methanol (homogenization - 50:1 with soaking in methanol for 72 hours) 3999 439 4.5 Methanol (homogenization - 50:1 with soaking in methanol for 96 hours) 4145 223 5.1 Methanol (homogenization-10:1 with filtration) 5240 302 6.1 Methanol (Soxhlet 24h) 5670 521 6.2 Methanol (Soxhlet 48 h) 5933 508 7.1 Ethanol (homogenization-50:1) 3879 268 7.2 n-hexane (homogenization-50:1) 4341 323 7.3 MTBE (homogenization-50:1) 5503 329 7.4 Toluene (homogenization-50:1) 6307 325 7.5 Diethyl ether (homogenization - 50:1) 7465 379 7.6 Ethyl acetate (homogenization-50:1) 8373 471 7.6(b) Ethyl acetate (homogenization - 50:1, 50 minutes) 4011 317 7.7 Acetone (homogenization - 50:1) 4812 292 8.1 Methanol (Soxhlet 6 h) 5920 512 8.2 Methanol (Soxhlet 72 h) 4158 591 8.3 Methanol (Soxhlet 96 h) 3912 333 8.4 MTBE (Soxhlet 6 h) 5240 544 8.5 MTBE (Soxhlet 24h) 5560 520 8.6 MTBE (Soxhlet 48h) 4640 508 8.7 MTBE (Soxhlet 72h) 3876 418 8.8 MTBE (Soxhlet 96h) 4080 424 8.9 Ethyl acetate (Soxhlet 6 h) 7747 641 8.10 Ethyl acetate (Soxhlet 24h) 7626 632 8.11 Ethyl acetate (Soxhlet 48 h) 7386 713 8.12 Ethyl acetate (Soxhlet 72 h) 7306 721 8.13 Ethyl acetate (Soxhlet 96 h) 7480 755 9.1 Ethyl acetate (ME) 9675 792 9.2 Ionic liquid (ME) 979.5 481.5

EXAMPLE A4: MICROWAVE EXTRACTION (ME) METHOD WITH ALKYL ALKANOATES

The following examples describe experimental attempts to evaluate the ability of various alkyl alkanoate solvents to extract carangin and pongamol from seed cake under microwave extraction conditions.

The Pongamia seed cake samples used in Parts I-V below were of the same origin and date of preparation as the Pongamia seed cake used in Examples A1-A3 above. Pongamia seed cake was stored for 18 months (at -20 degrees Celsius) at the start of Example A4.

Various alkyl alkanoate solvents were evaluated for their ability to extract carangin and pongamol under microwave extraction conditions.

The partially defatted pongamia meal was homogenized using dry ice. Samples were kept frozen and dry ice allowed sublimation. 0.5 g +/- 0.02 g of pongamia meal was placed in separate microwave extraction tubes, and 15.0 ml of each solvent (solvent:solids (v/w) ratio 30:1) was added to the appropriate tube for microwave oven, which was then covered with a lid and shaken. The extraction was carried out using a MARS6 microwave extractor under the following conditions: 1) the rate of temperature change over 15 min to 70°C; 2) exposure at 70°C for 10 minutes. After the supernatant was cooled to room temperature, the extract was filtered through Whatman GF/F filter paper on a Buchner funnel under vacuum, and the extract was poured into pre-labeled 50 ml centrifuge tubes.

Analysis. All sample extracts were diluted 10-fold and 100-fold for LC-MS/MS analysis or directly using LC-MS/MS vials or volumetric flasks. (10-fold dilution: 100 µl of sample extract was added to 900 µl of the appropriate solvent and shaken; 100-fold dilution: 10 µl of the sample extract was added to 990 µl of the appropriate solvent and shaken.) LC-MS/MS assay parameters were identical to those described in example A1 above.

Evaluation of the efficiency of microwave extraction of carangin and pongamol using various alkyl alkanoate solvents. Table 12 lists the total amounts of carangin and pongamol extracted from seed cake samples using the listed solvents under microwave extraction conditions (see also FIGS. 7A and 7B).

Table 12

Solvent Total carangin extracted (ppm) Total pongamol extracted (ppm) Butyl acetate 4989 473 Butyl butyrate 4315 425 Butyl propanoate 4939 417 ethyl acetate 4338 446 ethyl butyrate 4222 389 Ethyl propanoate 4248 375 Methyl acetate 4003 398 Methyl butyrate 4359 399 Methyl propanoate 4435 421 propyl acetate 4952 464 propyl butyrate 4997 486 propyl formate 4342 449 Propylopropanoate 5119 473

Example B Large-Scale Extraction Techniques

This example details experimental attempts to increase the extraction of carangin and pongamol to commercially viable levels.

Part I - Machining

Thermal extrusion method and screw press method. Pongamia seed cake samples were separately subjected to the thermal extrusion method (“Seed Conditioning”) and the screw press method (“Screw Press”, first (1st) or second (2nd) pressing cycle) to remove oil, carangin and pongamol from the cake pongamia seeds.

Evaluation of the efficiency of extraction of karaginin and pongamol by mechanical processing. Pongamia seed cake samples were analyzed by ethyl acetate microwave extraction (according to the protocol of example A3 above) to determine the amounts of carangin and pongamol remaining in the treated pongamia seed cake after treatment with seed conditioning treatments and screw presses. Tables 13 and 14 demonstrate that both seed conditioning and screw press mechanical processing methods can be used to extract caranjin and pongamol from pongamia seed cake on a commercial scale (FIGS. 8A and 8B).

Table 13

Extraction process Residual carangin remaining in seed cake (ppm) Residual pongamol remaining in seed cake (ppm) cold pressing 11040 1645.5 Extruded (160°C) 9525 1099.5 Extruded (175°C) 6705 723

Table 14

Extraction process Residual carangin remaining in seed cake (ppm) Residual pongamol remaining in seed cake (ppm) Seed conditioning 11490 967.5 Screw press (1st pressing) 4485 303 Screw press (2nd pressing) 4185 322.05

Part II - Solvent Extraction

Extraction of carangin and pongamol with one solvent. Pongamia seed cake was introduced into the immersion extractor using a volumetric dispenser. The feed rate was controlled so that each blade section of the extractor was about 50% full. Table 15 lists specific extraction parameters for each solvent.

Table 15

Solvent extraction (pre-machining) Duration (min) Chain speed
(in/min) Solvent: feed Feeding speed (kg/min) Dissolution rate (ml/min) Hexane (cold pressing) 60 2.65 5:1 0.06 455 Methanol (cold pressing) 180 0.88 7:1 0.02 179 Ethyl acetate (cold pressed) 180 0.88 7:1 0.013 101 Ethyl acetate (screw press, 2nd pressing) 180 1.79 5:1 0.11 580

Double solvent extraction of carangin and pongamol. After the primary extraction with hexane, detailed above, pongamia seed cake was collected and introduced into the immersion extractor using a volumetric dispenser. The feed rate was adjusted so that each section of the extractor vanes was about 50% full. Table 16 lists specific extraction parameters for each solvent.

Table 16

Combination of extraction solvents Duration (min) Chain speed
(in/min) Solvent: feed Feeding speed (kg/min) Dissolution rate (ml/min) 1°: Hexane;
2°: Methanol (cold pressing) 120 1.32 5:1 0.014 88 1°: Hexane;
2°: Ethyl acetate (cold pressed) 120 1.32 5:1 0.014 78

Evaluation of large-scale solvent extraction of carangin and pongamol. Each of the samples obtained from the combination of mechanical and solvent extraction methods described in Tables 15 and 16 was analyzed by ethyl acetate microwave extraction (according to the protocol in Example A3 above) to determine the amounts of residual carangin and residual pongamol remaining in pongamia seed cake after extraction treatment. Table 17A demonstrates that ethyl acetate, when used in a single extraction, regardless of the prior mechanical extraction method, was the most effective solvent for removing carangin and pongamol from pongamia seed cake (FIGS. 9A and 9B).

Table 17A.

Extraction solvent(s) Residual carangin remaining in seed cake (ppm) Residual pongamol remaining in seed cake (ppm) Hexane (cold pressing) 909 64.05 Methanol (cold pressing) 290.7 32.97 Ethyl acetate (cold pressed) 148.05 18.15 Hexane; Methanol (cold pressing) 164.55 20.205 Hexane; Ethyl acetate (cold pressed) 191.7 21.21 Ethyl acetate (screw press, 2nd pressing) 1.5 2.8

Screw press treated (2nd pressing), ethyl acetate extracted seed cake was subsequently analyzed a second time for carangin and pongamol concentrations according to the same protocol used previously and the conditions of Example A3 to confirm the original measurement in Table 17A. The results of the second cycle were slightly higher than the initial measurement. The first measurement (cycle #1, such as in Table 17A above), the second measurement (cycle #2), and the average of the two measurements (“Average”) are shown in Table 17B below.

Table 17B.

Extraction solvent(s) Cycle No. Residual carangin remaining in seed cake (ppm) Residual pongamol remaining in seed cake (ppm) Ethyl acetate (screw press, 2nd pressing) 1 1.5 2.8 2 7.5 8.2 Average value 4.5 5.5

Part III - Compositional profile of the extracted seed cake

Following the extraction of the pongamia seed cake in Part II above, the original pongamia seed cake samples in Part I and the solvent extracted pongamia seed cake samples in Part II were analyzed to determine the effect, if any, of mechanical pressing and solvent extraction on the compositional profile of the seed cake. The total protein, total carbohydrate, and amino acid profiles of the mechanically pressed seed cake and the mechanically compressed, ethyl acetate-extracted seed cake were determined.

The content of amino acids in the pongamia seed cake was determined by various methods depending on the name of the amino acid to be quantified. For example, the measurement of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, tyrosine, and valine was performed by subjecting samples of Pongamia seed cake to acid hydrolysis in 6 N HCl at 110°C in within 24 hours followed by quantitative determination by ion-exchange chromatography with post-column ninhydrin reaction and detection in the UV/visible region (modified reference method AOAC 982.30). The measurement of tryptophan in the seed cake samples was performed by alkaline cooking of the seed cake using lithium hydroxide at 110° C. for 22 hours, followed by quantification by reverse phase chromatography with fluorescence detection (AOAC reference method 998.15). The amounts of cysteine and methionine were measured by treating seed cake samples with performic acid oxidation to convert cysteine to cysteic acid and methionine to methionine sulfone. The oxidized sample was then hydrolyzed to release cysteic acid and methionine sulfone from the protein, followed by quantification of the released cysteic acid and methionine sulfone by ion exchange chromatography (modified reference method AOAC 994.12).

The average amino acid profile of pongamia meal from two separate cycles is presented below as amino acid content after mechanical pressing but before solvent extraction (screw press, 2nd pressing, “before processing”) and after both mechanical pressing and extraction with ethyl acetate solvent (extracted with ethyl acetate after screw press, 2 pressing, "after processing"), shown in table 18 below. The amino acid profile is expressed as a percentage by weight (% w/w) of the absolute amino acid content of the meal and as a percentage of the total amino acid content.

Table 18

Before processing After processing absolute content of amino acids (%) % total amino acids absolute content of amino acids (%) % total amino acids Alanine 0.93 4.28 1.11 4.57 Arginine 1.27 5.85 1.30 5.36 Aspartic acid 2.54 11.69 3.03 12.44 Cysteine 0.44 2.0 0.45 1.85 Glutamic acid 3.35 15.42 3.93 16.16 Glycine 0.99 4.56 1.17 4.82 Histidine 0.59 2.72 0.63 2.58 Isoleucine 0.85 3.91 1.02 4.18 Leucine 1.94 8.93 2.28 9.37 Lysine 1.79 8.24 1.35 5.57 Methionine 0.24 1.10 0.25 1.02 Phenylalanine 1.31 6.03 1.50 6.15 Proline 1.14 5.25 1.29 5.32 Serene 1.23 5.66 1.45 5.94 Threonine 0.81 3.73 0.97 3.97 tryptophan 0.39 1.80 0.44 1.81 Tyrosine 0.78 3.59 0.87 3.57 Valine 1.13 5.20 1.30 5.34 Total 21.72 100 24.33 100

As shown in Table 18 above, the relative amino acid content and profiles of the seed cake samples were predominantly maintained after extraction with ethyl acetate solvent to remove carangin and pongamol. The solvent-extracted seed cake generally showed a slightly higher concentration of amino acids than the original seed cake, except for the lysine content, which was reduced. The higher concentration of amino acids in the solvent-extracted meal can be partly explained by the removal of pongamia oil during solvent extraction and hence the reduction in overall weight of the seed cake.

Total proteins and carbohydrates were also measured in seed cake samples before and after extraction with ethyl acetate solvent. The total protein content was determined by placing pongamia seed cake samples in the combustion chamber of a protein analyzer, measuring the total nitrogen content of the combustion gas, and calculating the protein from the observed nitrogen content (protein content=6.25×nitrogen content).

The total carbohydrate content was calculated as the remaining percentage in the pongamia seed cake (100%) minus the sum of the total ash content (%), total protein content (%), total moisture content (%) and total fat (%). The total ash content was determined by placing samples of seed cake (2 g) in a crucible, staining the samples in an oven, ashing the samples in a muffle furnace at 600°C, and measuring the ash weight (reference method AOAC 942.05). The total moisture content was determined by heating a portion of the sample at 130° C. for 2 hours in a forced ventilation oven and determining the difference in weight of the sample, the difference in % being calculated as the moisture content (reference method AOCS BA 2A-38). The total fat content was determined by solvent extraction under heating in a flask under reflux with petroleum ether (modified reference method AOCS BA3-38).

Table 19 shows the average total protein content and the average total carbohydrate content (in weight percent of the total sample weight) for two cycles after mechanical pressing (screw press, 2nd pressing, "before processing"), but before solvent extraction, and after both mechanical pressing. pressing, and extraction with ethyl acetate solvent (extracted with ethyl acetate after screw press, 2 pressing, "after processing").

Table 19

Before processing After processing Total protein % 30.64 35.71 Total Carbs % 48.35 58.24

The solvent extraction process resulted in no loss of protein or carbohydrate content in the pongamia seed cake. Similar to the amino acid profiles presented above, samples of solvent-extracted seed cake showed slightly higher concentrations of total protein and total carbohydrates than the original screw press processed seed cake.

EXAMPLE C: RUMINANT FEED COMPOSITIONS

The following examples detail experimental attempts to evaluate the effectiveness of incorporating ethyl acetate-extracted pongamia meal into the diet of cattle.

EXAMPLE C1: PONGAMIA ADDITIVE STUDY

The example below describes a study comparing the use of ethyl acetate-extracted pongamia seed cake and soybean meal as separate supplemental protein sources with low-quality bovine diets.

Thirteen steers were used in a fully randomized study and were fed one of three diets, including poor quality hay (5.0% crude protein) as control (CON (n=4), a diet based on corn and dry distiller grains supplemented with soybean meal (SBM) (n=4) and a distiller's dry grain diet supplemented with ethyl acetate-extracted pongamia seed cake (PSC) (n=5).The pongamia seed cake used in this study was screw press processed, 2 pressing, ethyl acetate-extracted pongamia meal prepared in Example B, Part II.

Table 20 shows the composition of the three study diet groups - hay (control), soybean meal (SBM), and solvent-extracted pongamia seed cake (PSC) used in the study.

Table 20

Hay (control, CON) Soybean Meal (SBM) Pongamia Seed Cake (PSC) Additive composition, g/kg SBM - 420 0 Cornflower - 380 0 Dry Grain Distiller - 200 600 PSC - 0 400

Table 21 shows the chemical composition of hay, soybean meal and pongamia seed cake.

Hay, SBM and PSC were dried in a forced ventilation oven for 96 hours at 55° C. and allowed to air to determine the partial dry matter content (DM). Hay and additives were mixed throughout the day on an equal weight basis, then ground through a 1 mm sieve using a Wiley mill and dried at 105°C for DM determination. Organic matter (OM) was defined as the dry weight loss when burned in a muffle furnace for 8 hours at 450°C. Nitrogen was measured using an Elementar nitrogen analyzer (Elementar, Hanua, Germany) and crude protein (CP) was calculated as N×6.25. Analysis of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was performed sequentially using an Ankom fiber analyzer with amylase.

The content of carangin and pongamol in pongamia seed cake was determined by the microwave solvent extraction method using ethyl acetate described in Example A3 and Example A4.

Table 21

Index Hay (control, CON) Soybean Meal (SBM) Pongamia Seed Cake (PSC) Chemical composition, g/kg dry matter Organic Matter (OM) 927 950 947 Neutral Detergent Fiber (NDF) 734 175 372 Acid Detergent Fiber (ADF) 436 54 137 Crude protein (CP) 50 297 312 Caranjin, ppm - - 4.5 Pongamol, ppm - - 5.5

The experimental bulls were fed a specially defined diet for a total of 21 days, including 13 days for adaptation and 8 days for sampling. Determination of dry matter intake and digestibility was facilitated by total stool collection using fecal bags for six days.

Statistical analysis. Dry matter intake and digestibility were analyzed using the MIXED procedure in SAS 9.2 (SAS Inst. Inc., Cary, NC). The model parameters included treatment and period using a steer as a random effect. The model parameters were: treatment, period, hour and hour×treatment, with bull and treatment×period×steer included as random parameters. The recurring parameter was an hour with treatment×bull as the subject. Processing averages were calculated using the LSMEANS option. Table 22 shows the results of the statistical analysis for the three treatment groups.

Table 22.

Index Treatment CON SBM PSC SEM p-value CON versus SBM CON versus PSC SBM versus PSC Observation number 4 4 5 Dry matter intake, % of body weight Fodder 1.48 1.95 1.83 0.092 0.01 <0.01 0.02 0.36 Additive 0.00 0.15 0.16 0.002 <0.01 <0.01 <0.01 0.26 Total 1.48 2.10 1.99 0.093 <0.01 <0.01 <0.01 0.37 digestible 0.76 1.06 1.16 0.049 <0.01 <0.01 <0.01 0.14 Digestibility, % 51.4 55.2 53.6 1.99 0.43 0.21 0.45 0.55

CON=control; SBM=soybean meal; PSC=pongamia seed cake

As shown in Table 22, feed dry matter intake for both soybean meal and pongamia seed cake diets was greater than for the hay control diet. Digestible dry matter intake for diets containing soybean meal and pongamia seed cake was also greater than for the hay control diet. No differences were observed in the digestibility of the diets. No statistically significant differences were observed between the soybean meal test group and the pongamia seed cake test group.

EXAMPLE C2: COMPARATIVE FORAGE-BASED CATTLE FEED DIET WITH A PONGAMIA PROTEIN SUPPLEMENT OR A MARKETLY AVAILABLE PROTEIN SUPPLEMENT

The present example details a comparative study in which experimental cattle were fed one of three diets as shown in Table 23, including forage with pongamia protein supplement (Group A), forage only (Group B) and forage with commercially available protein. additive (group C).

The pongamia seed cake used in this study was the screw-pressed, 2-press, ethyl acetate-extracted pongamia meal prepared in Example B, Part II above. Pongamia seed cake was mixed with distiller's dry grains (DDGS) at a ratio of 30:70 by weight to form the pongamia protein supplement. Table 24 shows the concentrations of caranjin and pongamol in the pongamia composition used in this study, as determined by the ethyl acetate solvent microwave analytical method described in Example A4 above. Sweet Pro CattleKandi protein supplement was used as the commercially available supplement for Group C cattle for the first 30 days of the study and was replaced with SweetPro 16 for the remainder of the study.

Table 23

Group designation Experimental group Description Group A Experimental Processing Group Forage + protein supplement pongamia Group B Negative control group Forage only Group C Positive control group Forage + commercially available protein supplement

Table 24

Meal type Karanjin (ppm) Pongamol (ppm) Meal extracted with ethyl acetate 4.5 5.5

Three Black Angus × Wagyu beef cows (neutered males, aged 22-24 months) were used for each test group (nine bulls in total) and kept in 3-5 acre pens designated for each test group. In each pen, drinking water and access to pen food were provided ad libitum throughout the study. The test treatment group (Group A) received 1 kg for each pongamia protein supplement in a common feeder available to all three animals in the test treatment pen. Cattle in the positive control group (Group C) received a standard protein supplement diet based on SweetPro tub. No protein supplements were given to cattle in group B.

Experimental cattle were monitored daily and subjected to qualitative assessment to confirm the consumption of at least the entire daily diet of pongamia protein supplement and to confirm the absence of adverse health effects throughout the study. Experimental cattle from all three groups were weighed approximately every thirty days to monitor weight gain. Recorded weight gain for each of the test cattle and for the treatment groups is shown in Table 25.

Table 25

Group Tag No. Weight in pounds Average daily gain by groups Day 1 Day 33 Day 62 Day 93 A 1 901 975 1070 1155 2 901 1125 1155 1240 3 901 1030 1115 1180 Average value 901 1043 1113 1192 Delta 142 70 78 3.2 IN 4 911 910 965 1085 5 911 895 975 1100 6 911 1085 1175 1230 Average value 911 963 1038 1138 Delta 52 75 100 2.5 WITH 7 878 1040 1120 1190 8 878 1005 1055 1135 9 878 995 1040 1145 Average value 878 1013 1072 1157 Delta 135 58 85 3.1

As shown in Table 25, a bovine diet incorporating a protein supplement derived from pongamia meal resulted in increased weight gain over a forage-only diet and showed comparable average weight gain in test calves compared to a diet including commercially available protein supplement.

Claims (21)

1. Pongamia composition, which is a meal, containing: carangin at a concentration less than or equal to 100 ppm (ppm); And at least one or more components selected from the group consisting of carbohydrates, proteins, fibers, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. 2. A pongamia composition according to claim 1, wherein the pongamia composition further comprises pongamol. 3. A pongamia composition according to claim 2, wherein the pongamia composition has a pongamil concentration of less than or equal to 100 ppm. 4. Pongamia composition according to claim 2 or 3, additionally containing an alkylalkanoate solvent with a concentration of less than 5000 ppm, while the alkylalkanoate solvent optionally includes ethyl acetate. 5. The composition of pongamia according to any one of paragraphs. 1-4, where the composition contains less than 5% oil by dry weight, and/or at least 30% protein by dry weight, and/or 50-70% carbohydrates by weight, and/or the composition has a total amino acid content of 20-30 % by weight. 6. The composition of pongamia according to any one of paragraphs. 1-5, wherein the pongamia composition is obtained from defatted pongamia seed cake having a carangin concentration of at least 200 ppm. 7. Feed composition containing: at least 30% by weight of the pongamia composition according to any one of paragraphs. 1-6; And main feed. 8. A method of obtaining a composition of pongamia according to any one of paragraphs. 1-6, including: mechanical extraction of pongamia oilseeds to obtain a defatted pongamia seed cake, wherein the defatted pongamia seed cake contains 8-30% oil by weight; mixing the defatted pongamia seed cake with an alkyl alkanoate solvent to obtain an extraction mixture in which the alkyl alkanoate solvent optionally includes ethyl acetate; measuring the concentration of carangin in the alkylalkanoate extract; And separation of the extraction mixture into miscella and pongamia composition, wherein the pongamia composition is a meal having (i) a caranjin concentration that is less than 20% of the caranjin concentration in the defatted pongamia seed cake, or (ii) a caranjin concentration that is less than or equal to 100 ppm. 9. The method of claim 8, wherein the pongamia composition has a concentration of carangin less than or equal to 100 ppm. 10. The method according to claim 8 or 9, wherein the defatted pongamia seed cake has a carangin concentration of at least 200 ppm, and/or the miscella has a carangin concentration greater than or equal to about 4000 ppm, and/or the pongamia composition has a pongamol concentration of less than 100 ppm. 11. A method of feeding a ruminant, comprising providing the ruminant with a composition according to any one of paragraphs. 1-6 or the feed composition according to claim 7, wherein the ruminant is optionally a cow.

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