Pongamia compositions, methods for their preparation and analysis, and their uses

Abstract

To provide pongamia compositions having low concentrations of residual oil, karanjin and pongamol, to provide methods of producing such pongamia compositions, and to provide more accurate methods for analyzing pongamia compositions produced by various common processing methods.SOLUTION: Provided is a method of producing a composition comprising: producing a deoiled pongamia seedcake by mechanical extraction of pongamia oilseeds, the deoiled pongamia seedcake containing 8 to 30 wt.% of oil; preparing an extraction mixture by mixing the deoiled pongamia seedcake with an alkyl alkanoate solvent; and separating the extraction mixture into a miscella and a pongamia composition, the pongamia composition being a meal having (i) a karanjin concentration that is less than 20% of the karanjin concentration in the deoiled pongamia seedcake, or (ii) a karanjin concentration that is 100 ppm or less.SELECTED DRAWING: Figure 9A

Description

Detailed Description of the Invention

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 741,351, filed October 4, 2018, the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] [Technical Field] This disclosure relates generally to products of pongamia oilseeds, and more specifically to pongamia compositions having low concentrations of residual pongamia oil, karanjin, and pongamol, and methods for preparing and using such pongamia compositions. This disclosure also relates to methods for analyzing pongamia compositions prepared by the methods described herein and by other processing methods known in the art. This disclosure also relates to the use of pongamia compositions as feed for cattle and other ruminant animals.

[0003] 〔background〕 Pongamia seed cake is a by-product of oil extraction from pongamia oilseeds and offers a potential renewable protein source for use in food products. However, crude pongamia seed cake contains residual oil and inherent chemical components, such as karangin and pongamol. It is desirable to reduce the amount of karangin and pongamol in the seed cake so that it can be used as a suitable food source. Karangin and pongamol, in particular, have been identified as economically valuable for their own merits. As a result, various processes have been explored for extracting high-purity karangin and pongamol from crude pongamia seed cake. However, existing methods often result in incomplete removal of residual oil, karangin, and pongamol from pongamia seed cake, thus preventing downstream uses of the seed cake itself.

[0004] Currently, there is a need for pongamia compositions having low levels of residual oil, karanjin and pongamol, as well as improved methods for more complete and thorough extraction of karanjin and pongamol from crude pongamia seed cake.

[0005] Furthermore, a major obstacle to the development of improved extraction methods has been the lack of a standardized method for assessing the levels of karanjin and pongamol remaining in the seed cake after such processing. Although many methods currently exist for quantifying residual karanjin and pongamol in pongamia seed cake after extraction processing, these methods are often inaccurate and / or unclear.

[0006] Most analytical methods assess the concentrations of karanjin and pongamol in pongamia compositions by measuring the concentrations of these chemical components in the corresponding methanol or hexane solvent extracts, thereby alternatively assessing their concentrations in pongamia compositions. However, these methods rely on the efficiency of methanol or hexane extraction, which varies depending on the nature of the material being analyzed and its previous processing history, resulting in inaccurate values ​​reported for karanjin and other compounds in pongamia seed cake. Furthermore, different analytical methods report different concentrations of oil, karanjin, and pongamol for the same pongamia seed cake sample, and these analytical methods often do not provide an internally consistent reference scale across different processing methods. As a result, meaningful comparison of different processing methods based on existing analytical methods has been difficult in the art.

[0007] Therefore, there is a need for pongamia compositions having low concentrations of residual oil, karanjin, and pongamol, and alternative methods for producing such pongamia compositions, as well as more accurate methods for analyzing pongamia compositions produced by various common processing methods.

[0008] 〔overview〕 In one aspect, provided herein is a method comprising: mixing a pongamia composition with an alkyl alkanoate solvent to prepare an extraction mixture; irradiating the extraction mixture with microwave radiation to prepare an irradiated mixture; separating the irradiated mixture into an extracted pongamia composition and an alkyl alkanoate extract; and determining the concentration of karanjin in the alkyl alkanoate extract.

[0009] In another aspect, provided herein is a method comprising: preparing a first pongamia composition, wherein the first pongamia composition is a de-oiled pongamia seed cake obtained by mechanical extraction and comprises 8-30% oil by weight; preparing an extraction mixture by mixing the first pongamia composition with an alkyl alkanoate solvent; and separating the extraction mixture into miscella and a second pongamia composition, wherein the second pongamia composition has a karangin concentration that is (i) less than 20% of the karangin concentration in the first pongamia composition, or (ii) less than or equal to 100 ppm.

[0010] In yet another aspect, provided herein is a pongamia composition comprising karanjin and at least one or more components selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. In some variations, the pongamia composition has a karanjin concentration of 100 ppm or less. In certain variations, the karanjin concentration is measured by treating the pongamia composition with an alkyl alkanoate solvent under microwave irradiation.

[0011] In certain aspects, provided are pongamia compositions produced according to the methods described herein. In another aspect, provided herein are feed compositions comprising any of the pongamia compositions described herein.

[0012] In yet another aspect, there is provided a method of feeding a ruminant comprising providing said ruminant with a Pongamia composition described herein or any of the feed compositions described herein.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS The present application can be understood by reference to the following description taken in conjunction with the accompanying drawings.

[0014] FIG. 1 shows an exemplary process for analyzing Pongamia composition.

[0015] FIG. 2 shows an exemplary process for preparing a pongamia composition having a karanjin concentration of 100 ppm or less.

[0016] Figures 3A and 3B show bar graphs comparing the total concentrations (adjusted to ppm, relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various methanol-based extraction methods.

[0017] Figures 4A and 4B show bar graphs comparing the total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various solvents (methyl tert-butyl ether, ethanol, hexane, toluene, and ethyl acetate) in combination with various extraction methods.

[0018] Figures 5A and 5B show bar graphs comparing the total concentrations (in ppm, adjusted for the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using microwave-assisted extraction with ethyl acetate or ionic liquid as the solvent.

[0019] Figures 6A and 6B show a comparison of the total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using the various methods and solvents shown in Figures 3A-5B.

[0020] 7A-7B show bars for the observed total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various alkyl alkanoate solvents in combination with microwave-assisted solvent extraction.

[0021] 8A and 8B show bar graphs comparing the residual concentrations of karanjin and pongamol (adjusted to ppm relative to the amount of starting material) in pongamia seed cakes subjected to various mechanical treatments, as determined by analysis of microwave-assisted ethyl acetate extractions.

[0022] 9A and 9B show bar graphs comparing the residual concentrations (ppm, adjusted for the amount of starting material) of karanjin and pongamol in pongamia seed cakes subjected to various mechanical treatments in combination with solvent extraction processes, as determined by analysis of microwave-assisted ethyl acetate extractions.

[0023] Detailed Description The following description describes example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but instead is provided as a description of example embodiments.

[0024] The following description relates to pongamia compositions having low concentrations of karanjin and pongamol, and methods for preparing and using pongamia compositions having low concentrations of karanjin, as well as methods for analyzing pongamia compositions.

[0025] [Method for analyzing Pongamia compositions] In some aspects, provided herein are methods for analyzing pongamia compositions. In some embodiments, provided herein are methods for measuring the concentrations of karanjin and pongamol in pongamia.

[0026] Oilseeds collected from pongamia (also known as Cytisus pinnatus, Dalbergia arbore, Derris indica, Galedupa pungum, karanj, Millettia pinnata, 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, emerging interest in non-petroleum-based fuel sources has led to the use of pongamia oil as a feedstock for producing biodiesel in many parts of the world.

[0027] Deoiled pongamia seed cake, remaining after oil extraction from pongamia oilseeds, has long been recognized as a potentially sustainable source of protein that can be used as a nutritional supplement. However, deoiled pongamia seed cake contains high concentrations of karanjin and pongamol. These high concentrations have generally prevented the use of seed cake in foods without adverse health effects. These compounds can render the seed cake inedible and potentially harmful to humans and animals. Previous attempts to develop edible pongamia compositions have been unsuccessful, in part, due to the fact that a consistent, maximum acceptable threshold for the concentration of karanjin and other antinutritional compounds intended for consumption has not yet been established. Furthermore, existing methods for analyzing pongamia compositions are inaccurate and unreliable, making determining the maximum acceptable karanjin concentration, let alone assessing the concentration of karanjin present in pongamia compositions, a formidable challenge. Therefore, there remains a need for more accurate methods for measuring the levels of karanjin and other antinutritional compounds present in pongamia compositions.

[0028] The present disclosure addresses this need by providing methods for analyzing pongamia compositions, i.e., methods for measuring the concentration of karanjin and other compounds inherent in pongamia oil seeds with greater accuracy and specificity than existing methods. Specifically, in some aspects, the present disclosure provides microwave-assisted solvent extraction analytical methods for determining the concentration of karanjin and pongamol in pongamia compositions. Solvents suitable for use in such methods are described herein and can include solvents comprising alkyl alkanoates.

[0029] Surprisingly, the analytical method of the present disclosure not only provides a more accurate measurement of karanjin and pongamol in pongamia compositions than previously existing methods, but also has been found to significantly underreport the concentration of residual karanjin in processed pongamia compositions. The use of an alkyl alkanoate solvent, including at least one alkyl alkanoate, in combination with microwave radiation leads to improved extraction efficiency of karanjin and pongamol from pongamia compositions and, consequently, improved quantification of residual karanjin and pongamol remaining in the processed pongamia compositions. Thus, the analytical method described herein provides a broadly applicable yet reliable means of detecting and quantifying the presence of karanjin and pongamol in various pongamia-derived compositions at concentrations lower than those detectable by conventional hexane- and methanol-based methods.

[0030] In one aspect, provided herein is a method for analyzing a pongamia composition, the method comprising: mixing the pongamia composition with an alkyl alkanoate solvent to prepare an extraction mixture; irradiating the extraction mixture with microwave radiation to prepare an irradiated mixture; separating the irradiated mixture into an extracted pongamia composition and an alkyl alkanoate extract; and measuring the concentrations of karanjin and pongamol in the alkyl alkanoate extract and their corresponding concentrations in the pongamia composition by surrogates. In another aspect, provided herein is a method for measuring the concentrations of karanjin and pongamol in a pongamia composition, the method comprising treating the pongamia composition with an alkyl alkanoate solvent under microwave irradiation.

[0031] Referring to Figure 1, process 100 is an exemplary process for analyzing a pongamia composition. In step 102, a pongamia composition is prepared. The pongamia composition is mixed with an alkyl alkanoate solvent in step 104, thereby providing an extraction mixture. The extraction mixture includes the pongamia composition and the alkyl alkanoate solvent. The extraction mixture is irradiated with microwave radiation in step 106 to provide an irradiated mixture. The irradiated mixture is separated in step 108 to produce an extracted pongamia composition and an alkyl alkanoate extract. In step 110, the alkyl alkanoate extract is analyzed.

[0032] It should be understood that in other variations, process 100 may include additional processing steps. In still other variations, certain steps of process 100 may be omitted.

[0033] In one variation, a method for analyzing a pongamia composition is provided, the method comprising: combining the pongamia composition with an alkyl alkanoate solvent to prepare an extraction mixture; irradiating the extraction mixture with microwave radiation to prepare an irradiated mixture; separating the irradiated mixture into the extracted pongamia composition and an alkyl alkanoate extract; and determining the concentration of karanjin in the alkyl alkanoate extract.

[0034] In some embodiments, the pongamia composition comprises pongamia seeds. In other embodiments, the pongamia composition comprises de-oiled pongamia seed cake. In particular embodiments, the pongamia composition comprises pongamia seeds and / or de-oiled pongamia seed cake.

[0035] In the foregoing variations in which the pongamia composition is a de-oiled pongamia seed cake, the de-oiled pongamia seed cake is obtained by mechanical extraction. In certain embodiments, the de-oiled pongamia seed cake is obtained by mechanical extraction of pongamia seeds. In other embodiments, the de-oiled pongamia seed cake is obtained by mechanical extraction of pongamia seed cake. In certain embodiments, the de-oiled pongamia seed cake is obtained by mechanical extraction using an expeller press. In other embodiments in which the pongamia composition comprises a de-oiled pongamia seed cake, the de-oiled pongamia seed cake is obtained by solvent extraction of pongamia seeds or pongamia seed cake. In certain embodiments, the de-oiled pongamia seed cake is obtained by solvent extraction of 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 de-oiled pongamia seed cake, the de-oiled pongamia seed cake is obtained by mechanical extraction, solvent extraction, or a combination thereof.

[0036] In some embodiments of the aforementioned method, the pongamia composition is mixed with an alkyl alkanoate solvent. In some variations, the alkyl alkanoate solvent is a solvent containing at least one alkyl alkanoate. In certain variations, the solvent contains one alkyl alkanoate. In other variations, the solvent contains a mixture of alkyl alkanoates. The alkyl alkanoate solvent may contain only alkyl alkanoates, or it 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 certain 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 variations, the "alkyl alkanoate" contains at least one ester group in which a hydrogen atom of the carboxylic acid group is replaced by an alkyl group. In certain variations, the alkyl alkanoate contains one ester group in which a hydrogen atom of the carboxylic acid group is replaced by an alkyl group.

[0037] In some embodiments of the solvent, the alkyl of the alkyl alkanoate is methyl, ethyl, propyl, or butyl. In other embodiments, the solvent comprises methyl alkanoate, ethyl alkanoate, propyl alkanoate, or butyl alkanoate, or any combination thereof. In certain embodiments, the solvent comprises ethyl alkanoate. In some embodiments, the alkanoic acid is ethanoic acid, propanoic acid, butanoic acid, or pentanoic acid. In certain embodiments, the solvent comprises alkyl ethanoate, alkyl propanoate, alkyl butanoate, alkyl pentanoate, or any combination thereof. In certain embodiments, the solvent comprises alkyl ethanoate. In certain embodiments, the solvent comprises ethyl acetate. In other embodiments, the solvent is ethyl acetate.

[0038] 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, butyl butanoate, and butyl pentanoate, and any combination thereof. In certain 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 combination thereof.

[0039] It should also be recognized that chemical names used herein in accordance with the International Union of Pure and Applied Chemistry (IUPAC) nomenclature standard may also be referred to by their corresponding common names, e.g., acetate for ethanoate, propionate for propanoate, butyrate for butanoate, valerate for pentanoate, etc. As such, alkyl ethanoates may also be referred to as acetates.

[0040] In other embodiments, the method comprises combining the pongamia composition with a solvent comprising at least one alkyl alkanoate of formula (I), [ka] During the ceremony, R 1 is C1-C4 alkyl, R 2 is hydrogen or C1-C4 alkyl.

[0041] In some embodiments, R 1 is C1-C4 alkyl, and R 2 is hydrogen or C1-C4 alkyl. In certain embodiments, R 1 and R 2 are independently C1-C4 alkyl. In certain other embodiments, R 1 is C1-C4 alkyl, and R 2 is hydrogen.

[0042] R 1 In some embodiments, where R is C-C alkyl, 1 is CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2(CH3)CH-, (CH3)2CHCH2-, or (CH3)3C-. 1 is CH3CH2-. In another embodiment, R 1 is CH3CH2CH2CH2-. In yet another embodiment, R 1 is CH3CH2CH2-.

[0043] In some embodiments, R 2 is hydrogen. In other embodiments, R 2 is C1-C4 alkyl. R 2 In some embodiments, where R is C-C alkyl, 2 is CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2(CH3)CH-, (CH3)2CHCH2-, or (CH3)3C-. 2 is hydrogen, CH3-, CH3CH2-, or CH3CH2CH2-.

[0044] In yet another embodiment, R 1 is CH3CH2 and R 2 is CH3-. In some embodiments, R 1 is CH3CH2- or CH3CH2CH2CH2-, and R 2 is hydrogen. In other embodiments, R1 is CH3CH2CH2- and R 2 is CH3CH2CH2- or CH3CH2CH2CH2-.

[0045] In other embodiments, R 1 is C1-C3 alkyl. In yet another embodiment, R 1 is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R 1 is ethyl. In some embodiments, R 1 is C2-C4 alkyl. In certain embodiments, R 1 is ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. 2 is hydrogen or C1-C3 alkyl. In certain embodiments, R 2 is hydrogen, methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R 2 is methyl. In yet another embodiment, R 1 is ethyl, and R 2 is methyl.

[0046] The analytical method of the present disclosure uses the alkyl alkanoate solvent described herein in combination with microwave radiation to provide high extraction efficiency for karanjin and pongamol. This results in a more accurate measurement of karanjin concentration than other analytical methods based on, for example, methanol or hexane extraction. In some embodiments, the alkyl alkanoate solvent used in the analytical method described herein excludes certain co-solvents. In some embodiments of the aforementioned analytical method, the alkyl alkanoate solvent mixed with the pongamia composition does not contain alcohols, alkanes, ketones, ethers, and / or aromatic hydrocarbons. In certain embodiments, the solvent does not contain methanol, ethanol, propanol, hexane, methyl tert-butyl ether, diethyl ether, toluene, benzene, or acetone. In still other embodiments, the alkyl alkanoate solvent does not contain diketones or diesters, such as succinates, sebacates, glutarates, or malonates.

[0047] However, it should be recognized that in some variations, the alkyl alkanoate solvent may contain trace amounts or residual levels of the excluded solvents disclosed above. These traces of additional solvents may be introduced into the alkyl alkanoate solvent, for example, by standard chemical manufacturing or handling procedures. Residual levels of solvents such as methanol or hexane may be maintained below a certain threshold considered acceptable for standard analytical measurements of total impurities in the alkyl alkanoate solvent, so that the effectiveness of the analytical methods described herein is not significantly affected. For example, in some embodiments, the alkyl alkanoate solvent contains one or more additional solvents that are not alkyl alkanoate solvents, and the total concentration of the one or more additional solvents is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the solvent.

[0048] In some embodiments, the method includes mixing the pongamia composition with an alkyl alkanoate solvent to provide an extraction mixture. In certain embodiments, mixing the pongamia composition with the alkyl alkanoate solvent can include mixing, stirring, or turbulently stirring the pongamia composition and the alkyl alkanoate solvent together to prepare the extraction mixture. In other embodiments, mixing the pongamia composition with the alkyl alkanoate solvent can include heating the pongamia composition and the alkyl alkanoate solvent to prepare the extraction mixture. It should also be appreciated that the pongamia composition and the alkyl alkanoate solvent may be individually stirred, turbulently stirred, or heated before mixing. It should also be appreciated that the methods of the present disclosure provide for variation of other parameters that may be part of the mixing process, including, for example, the duration for which 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 and the alkyl alkanoate solvent to be mixed, and other physical characteristics of the pongamia composition, such as particle size distribution.

[0049] In some embodiments, the extraction mixture is irradiated to provide an irradiated mixture. In certain embodiments, the extraction mixture is irradiated with microwave radiation to provide the irradiated mixture. In some embodiments, the extraction mixture is irradiated using a microwave extractor. In other embodiments, the present disclosure also provides variations in parameters that may be associated with the irradiating step, including, for example, the duration, temperature, pressure, and frequency of microwave radiation with which the extraction mixture is irradiated.

[0050] It should be noted that, due to the effectiveness of the combination of alkyl alkanoate solvent and microwave radiation, the analytical method of the present disclosure does not require specific techniques commonly used in existing analytical methods, such as Soxhlet extraction, pre-soaking of pongamia seeds or seed cake with sodium hydroxide, or pre-treatment of pongamia seeds or seed cake with subcritical water / steam. For example, in some embodiments, the present disclosure provides an analytical method that does not include Soxhlet extraction. In other embodiments, the analytical method does not include soaking pongamia seeds or seed cake in a base (e.g., hydroxide solution).

[0051] In some embodiments, the irradiated mixture is separated into a solid component and a liquid component. The solid component is referred to herein 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 method known in the art for solid-liquid separation. For example, in certain embodiments, the irradiated mixture is separated by centrifugation. In some embodiments, the irradiated mixture is separated by decantation. In other embodiments, the irradiated mixture is separated by filtration.

[0052] In some embodiments, the extracted pongamia composition includes any solid materials and / or inherent chemical components originally present in the pongamia composition mixed with the alkyl alkanoate solvent, but which were insoluble in the alkyl alkanoate solvent and therefore did not partition into the liquid phase of the extract.

[0053] In some variations, the solvent extract contains an alkyl alkanoate solvent (including an alkyl alkanoate and optional co-solvents) and specific chemical components unique to Pongamia. The specific chemical components are extracted from the Pongamia composition into the alkyl alkanoate solvent. In some embodiments, the extract contains furanoflavonoids. Furanoflavonoids can be further identified by sub-classes, including, for example, flavones, flavonols (e.g., karanjin), and dibenzoylmethanes (e.g., pongamol). In certain embodiments, the extract contains karanjin. In other embodiments, the extract contains pongamol. In some embodiments, the extract contains karanjin and other furanoflavonoids. In some embodiments, the extract comprises karanjin, pongamol, lanceolatin, canjong, pongaglabron, pongaglabol, obalifolin, sanaganone, pinnatin, gamatin, pongon, glabone, karanjonol, pongapin, pachycarin, pongaglabol methyl ether, isopongaglabol, methoxyisopongaglabol, pongol methyl ether, millettocalyxin, 6-methoxyisopongaglabol, pongamoside A, pongamoside B, ponganone XI, pongamoshi The compound contains at least one or more furanoflavonoids selected from the group consisting of flavonoid C, glabra I, obalitenone, ponganone IX, and pongarotene.

[0054] In some embodiments, following separation of the irradiated mixture into the extracted pongamia composition and the solvent extract, the method further comprises analyzing the solvent extract. As described herein, analyzing the solvent extract comprises measuring the concentration of specific chemical components in the solvent extract, which serves as a surrogate measure of the concentration of those chemical components originally present in the pongamia composition. In some embodiments, the method comprises measuring the individual concentrations of one or more furanoflavonoids in the solvent extract. In certain embodiments, the method comprises measuring the concentration of karanjin in the solvent extract. In other embodiments, the method comprises measuring the concentration of pongamol in the solvent extract.

[0055] The concentration of karanjin, pongamol, and other furanoflavonoids in the solvent extract can be measured using analytical separation and detection techniques known in the art. In some embodiments, the concentration of karanjin, pongamol, and other furanoflavonoids is measured by high-performance liquid chromatography (HPLC). In other embodiments, the concentration of karanjin, pongamol, and other furanoflavonoids is measured by HPLC-mass spectrometry (HPLC-MS). In certain embodiments, the concentration of karanjin, pongamol, and other furanoflavonoids is measured by HPLC-tandem mass spectrometry (HPLC-MS / MS). In some embodiments, the concentration of karanjin, pongamol, and other furanoflavonoids is measured by HPLC-ultraviolet-visible spectrophotometry (HPLC-UV-vis).

[0056] In some variations, the analytical methods described herein may be referred to as "microwave-assisted alkyl alkanoate solvent extraction analytical methods." In certain embodiments where a specific alkyl alkanoate is used as the alkyl alkanoate solvent, the extraction may be more specifically referred to by the specific alkyl alkanoate used. For example, in certain embodiments of the aforementioned methods where the alkyl alkanoate solvent comprises ethyl acetate, the analytical method may be referred to as "microwave-assisted ethyl acetate extraction analytical methods."

[0057] It should be recognized that references to "microwave-assisted alkyl alkanoate solvent extraction analysis" include embodiments in which the alkyl alkanoate solvent comprises at least one alkyl alkanoate solvent and, optionally, one or more co-solvents that are not alkyl alkanoates. For example, "microwave-assisted ethyl acetate extraction analysis" can refer to the use of an alkyl alkanoate solvent comprising ethyl acetate and, optionally, one or more co-solvents.

[0058] [Method for preparing Pongamia composition] As described above, previous efforts to develop improved methods for preparing pongamia compositions with low concentrations of residual oil, karangin, and pongamol have previously been hampered by the unreliability and inconsistency of existing analytical methods for measuring such concentrations. However, the development of improved methods for preparing pongamia compositions with low concentrations of karangin is now possible thanks to the analytical methods described above. Such analytical methods provide greater accuracy and reliability for measuring karangin concentration. Thus, the present disclosure provides a more efficient method for removing karangin and other furanoflavonoids from pongamia seeds and seed cakes. The present disclosure includes methods for preparing pongamia compositions with low karangin concentrations, as described below. More specifically, the present disclosure provides methods for preparing pongamia compositions containing karangin and having a karangin concentration of 100 ppm or less.

[0059] In one aspect, provided herein is a method for preparing a pongamia composition having a low karanjin concentration, as measured by the microwave-assisted alkyl alkanoate solvent extraction analysis method described above. In some embodiments, provided herein is a method for preparing a pongamia composition having a karanjin concentration of 100 ppm or less, as measured by the microwave-assisted alkyl alkanoate solvent extraction analysis method described above. In other embodiments, provided herein is a method for preparing a pongamia composition having less than 20% less karanjin, as measured by the microwave-assisted alkyl alkanoate solvent extraction analysis method described above, compared to the initial or first pongamia composition obtained.

[0060] In one aspect, provided herein is a method for preparing a pongamia composition, the method comprising combining a first pongamia composition with an alkyl alkanoate solvent to provide an extraction mixture; and separating the extraction mixture to provide a miscella and a second pongamia composition, wherein the second pongamia composition has (i) a karanjin concentration that is less than 20% of the karanjin concentration in the first pongamia composition, or (ii) a karanjin concentration of 100 ppm or less.

[0061] Referring to Figure 2, process 200 is an exemplary process for preparing a pongamia composition. In step 202, a first pongamia composition is prepared. The first pongamia composition is mixed with an alkyl alkanoate solvent in step 204, thereby preparing an extraction mixture. The extraction mixture is separated in step 206 to produce a second pongamia composition and miscella.

[0062] It should be understood that in other variations, process 200 may include additional processing steps. In still other variations, certain steps of process 200 may be omitted.

[0063] In one variation, a method for preparing a pongamia composition is provided, comprising: preparing a first pongamia composition; mixing the first pongamia composition with a solvent comprising at least one alkyl alkanoate to prepare an extraction mixture; and separating the extraction mixture into a miscella and a second pongamia composition. In certain variations, the second pongamia composition has (i) a karanjin concentration that is less than 20% of the karanjin concentration in the first pongamia composition, or (ii) a karanjin concentration of 100 ppm or less.

[0064] In some embodiments, the first pongamia composition is obtained 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").

[0065] In some embodiments, the first pongamia composition is a de-oiled pongamia seed cake. The de-oiled pongamia seed cake can be described as a result of the aforementioned treatments to obtain the de-oiled pongamia seed cake. For example, in some embodiments, the first pongamia composition is a de-oiled pongamia seed cake, and the de-oiled pongamia seed cake is obtained by mechanical extraction. In other embodiments, the first pongamia composition is a de-oiled pongamia seed cake obtained by mechanical extraction of pongamia seeds or pongamia seed cake. In certain embodiments, the de-oiled pongamia seed cake is obtained by mechanical extraction using expeller pressing. It should be appreciated that one or more iterations of mechanical extraction may be applied to either the pongamia seeds and / or seed cake to provide the de-oiled pongamia seed cake as the first pongamia composition. In some embodiments, the first pongamia composition is a pongamia oilseed or is not an oilseed. In other embodiments, the first pongamia composition is not a de-oiled pongamia seed cake obtained by solvent extraction.

[0066] The first pongamia composition may be further defined by other attributes, including, for example, its karanjin concentration, oil content, moisture content, and particle size distribution. These attributes may be particularly advantageous for the extraction of karanjin and pongamol from the first pongamia composition. For example, in some embodiments, the first pongamia composition has a karanjin concentration of at least 200 ppm. In other embodiments, the first pongamia composition has a karanjin concentration of 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 certain embodiments, the first pongamia composition contains 8-30% oil by weight.

[0067] In some embodiments of the aforementioned methods, preparing a first pongamia composition may further include any optional steps for producing the first pongamia composition. For example, in some embodiments, the method includes preparing pongamia oil seeds and subjecting the pongamia oil seeds to mechanical extraction to prepare a de-oiled pongamia seed cake as the first pongamia composition. In certain embodiments, the method includes mechanically pressing the pongamia oil seeds to prepare a de-oiled pongamia seed cake as the first pongamia composition. In other embodiments, the method may include preparing a de-oiled seed cake and subjecting the de-oiled pongamia seed cake to mechanical extraction to prepare a first pongamia composition having a desired oil content and / or karanjin concentration. In still other embodiments, the method may include preparing a first pongamia composition as described herein and further minimizing the oil content of the first pongamia composition. In yet another embodiment, the method may include providing a de-oiled pongamia seed cake and further disintegrating the de-oiled pongamia seed cake to provide a first pongamia composition having a desired particle size distribution.

[0068] In some embodiments of the aforementioned method, the first Pongamia composition can be mixed with any of the solvents described for use in the analytical method. For example, in some variations, the solvent is an alkyl alkanoate solvent. In certain variations, the alkyl alkanoate solvent can contain only alkyl alkanoates, or alternatively, can contain one or more additional co-solvents that are not alkyl alkanoates. In certain embodiments, the solvent contains at least one alkyl alkanoate and one or more additional 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 certain embodiments, the solvent is an alkyl alkanoate.

[0069] In some embodiments, the alkyl of the alkyl alkanoate is methyl, ethyl, propyl, or butyl. In other embodiments, the solvent comprises methyl alkanoate, ethyl alkanoate, propyl alkanoate, or butyl alkanoate, or any combination thereof. In certain embodiments, the solvent comprises ethyl alkanoate. In some embodiments, the alkanoic acid of the alkyl alkanoate is methanoic acid, ethanoic acid, propanoic acid, butanoic acid, or pentanoic acid. In other embodiments, the solvent comprises alkyl methanoate, alkyl ethanoate, alkyl propanoate, alkyl butanoate, alkyl pentanoate, or any combination thereof. In certain embodiments, the solvent comprises alkyl ethanoate. In certain embodiments, the solvent comprises ethyl acetate. In other embodiments, the solvent is ethyl acetate.

[0070] 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 pentanoate, butyl methanoate, butyl ethanoate, butyl propanoate, butyl butanoate, and butyl pentanoate, and any combination thereof. In certain 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 butanoate, and any combination thereof.

[0071] In other embodiments, the method comprises combining a first pongamia composition with an alkyl alkanoate solvent comprising at least one alkyl alkanoate of formula (I), [ka] During the ceremony, R 1 is C1-C4 alkyl, R 2 is hydrogen or C1-C4 alkyl.

[0072] In some embodiments, R 1 is C1-C4 alkyl. In other embodiments, R 2 is hydrogen or C1-C4 alkyl. In certain embodiments, R 1 and R 2 are independently C1-C4 alkyl. In certain other embodiments, R 1 is C1-C4 alkyl, and R 2 is hydrogen.

[0073] R1 In some embodiments, where R is C-C alkyl, 1 is CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2(CH3)CH-, (CH3)2CHCH2-, or (CH3)3C-. 1 is CH3CH2-. In another embodiment, R 1 is CH3CH2CH2CH2-. In yet another embodiment, R 1 is CH3CH2CH2-.

[0074] In some embodiments, R 2 is hydrogen. In other embodiments, R 2 is C1-C4 alkyl. R 2 In certain embodiments, where R is C1-C4 alkyl, 2 is CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2(CH3)CH-, (CH3)2CHCH2-, or (CH3)3C-. 2 is hydrogen, CH3-, CH3CH2-, or CH3CH2CH2-.

[0075] In yet another embodiment, R 1 is CH3CH2- and R 2 is CH3-. In some embodiments, R 1 is CH3CH2- or CH3CH2CH2CH2-, and R 2 is hydrogen. In other embodiments, R 1 is CH3CH2CH2- and R 2 is CH3CH2CH2- or CH3CH2CH2CH2-.

[0076] In other embodiments, R 1 is C1-C3 alkyl. In yet other embodiments, R 1 is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R 1is ethyl. In some embodiments, R 1 is C2-C4 alkyl. In certain embodiments, R 1 is ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. 2 is hydrogen or C1-C3 alkyl. In certain embodiments, R 2 is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R 2 is methyl. In yet another embodiment, R 1 is ethyl, and R 2 is methyl. In yet another embodiment, R 2 is hydrogen, ethyl, or n-propyl. 1 is ethyl, n-propyl, or n-butyl, and R 2 is hydrogen, methyl, ethyl, or n-propyl. In certain embodiments, R 2 is methyl. In yet another embodiment, R 1 is ethyl, and R 2 is methyl.

[0077] In some embodiments, the alkyl alkanoate solvent is prepared in situ. For example, the alkyl alkanoate can be prepared by mixing the corresponding alcohol with the corresponding carboxylic acid. In some embodiments, the alkyl alkanoate of formula (I) is prepared by mixing the alcohol R 1 -OH to carboxylic acid R 2 -COOH, where R 1 and R 2is as defined above. In certain embodiments where the alkyl alkanoate is ethyl acetate, the ethyl acetate is prepared in situ by mixing ethanol with acetic acid. In some embodiments, the alkyl alkanoate is prepared in situ before the alkyl alkanoate solvent is mixed with the first pongamia composition. In other embodiments, the alkyl alkanoate is prepared in situ using the first pongamia composition. For example, in some embodiments, the method comprises mixing the first pongamia composition with a solvent comprising ethyl acetate, where the ethyl acetate is prepared in situ, and the method comprises mixing the first pongamia composition with ethanol and acetic acid.

[0078] In some variations, the solvent may contain one or more co-solvents that are not alkyl alkanoates. However, in some embodiments, the solvent excludes certain co-solvents. For example, in some variations, the alkyl alkanoate solvent does not contain alkanes, ketones, ethers, and / or aromatic hydrocarbons. In certain embodiments, the alkyl alkanoate solvent does not contain hexane, methyl tert-butyl ether, diethyl ether, toluene, benzene, and / or acetone. In still other embodiments, the alkyl alkanoate solvent does not contain diketones and / or diesters, such as succinates, sebacates, glutarates, or malonates.

[0079] In some embodiments, the first pongamia composition and the solvent are mixed to prepare an extraction mixture. In certain embodiments, mixing the first pongamia composition and the solvent comprises mixing the first pongamia composition and the solvent in an extractor to prepare an extraction mixture. In certain embodiments, the mixing step comprises mixing, stirring, or turbulently stirring the extraction mixture in the extractor. In some embodiments, mixing the first pongamia composition and the solvent to provide an extraction mixture comprises heating the first pongamia composition and the solvent to prepare an extraction mixture. In still other embodiments, the method further comprises heating the extraction mixture. It should be noted that the above-described methods may include variations in other parameters that may be part of the mixing step, including, for example, the residence time of the extraction mixture in the extractor, the extractor temperature and pressure, the speed of the extractor chain, the particle size distribution of the first pongamia composition, the ratio of the first pongamia composition to the alkyl alkanoate solvent, and the feed rates of the pongamia composition and the alkyl alkanoate solvent to the extractor.

[0080] In some embodiments, the method may further include irradiating the extraction mixture with microwave radiation. In certain embodiments, the extraction mixture is irradiated with microwave radiation after the mixing step and before the separation step. The present disclosure also provides variations in parameters that may be associated with the irradiating step, including, for example, the duration, temperature, pressure, and frequency of microwave radiation to which the extraction mixture is irradiated.

[0081] In some embodiments, the extraction mixture is separated into a miscella and a second pongamia composition. The miscella contains primarily the liquid fraction of the extraction mixture (oil, alkyl alkanoate solvent, and any soluble compounds), while the second pongamia composition is composed largely of residual insoluble solid material or meal remaining from the first pongamia composition. Separating the extraction mixture into a miscella and a second pongamia composition can include any suitable method known in the art for solid-liquid separation. In certain embodiments, the extraction mixture is separated by filtration. In other embodiments, the extraction mixture is separated by decantation.

[0082] In some embodiments, the miscella comprises a mixture of extracted oil, karanjin, other furanoflavonoids, and an alkyl alkanoate solvent (including the alkyl alkanoate and any co-solvents). In other embodiments, the miscella has a karanjin concentration of 4000 ppm or more. In certain embodiments, the miscella has a karanjin concentration of 4000 ppm or more as measured by the above method. In certain embodiments, the miscella can be characterized by oil content, water content, moisture content, solids content, or other properties known in the art.

[0083] In some embodiments, the second pongamia composition has a karanjin concentration of 100 ppm or less. In other embodiments, the second pongamia composition has a karanjin concentration that is less than 20% of the karanjin concentration in the first pongamia composition. In still other embodiments, the second pongamia composition has a karanjin concentration of 100 ppm or less, as measured by the microwave-assisted alkyl alkanoate solvent extraction analytical method described herein. In still other embodiments, the second pongamia composition has a karanjin concentration that is less than 20% of the karanjin concentration in the first pongamia composition, as measured by the microwave-assisted alkyl alkanoate solvent extraction analytical method described herein.

[0084] It should be appreciated that, thanks to the preparation method described herein, the second pongamia composition has a karanjin concentration of 100 ppm or less. In some embodiments, the second pongamia composition may have a karanjin and / or pongamol concentration in the order of parts per million, or fractions thereof. In some embodiments, the second pongamia composition may have a karanjin and / or pongamol concentration of less than 100 ppm, which is undetectable by conventional hexane and methanol-based analytical methods. In yet further embodiments, the second pongamia composition may have a trace concentration of karanjin and / or pongamol in the order of parts per billion (ppb) or parts per trillion (ppt). In situations where trace concentrations exist, the detection of karanjin and pongamol using the microwave-assisted alkyl alkanoate solvent extraction analytical method described herein may be limited by the detection limits of the liquid chromatography technique and the materials used. In some embodiments, trace amounts of karanjin and / or pongamol may be undetectable by the alkyl alkanoate-based microwave-assisted solvent extraction analytical methods described herein.

[0085] As noted above, the first pongamia composition can be obtained from plant material derived from a pongamia tree or plant. Thus, in some embodiments, the second pongamia composition obtained from the first pongamia composition by the methods described herein can be characterized as being obtained from plant material derived from a pongamia tree or plant (such 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).

[0086] As further described above, the second pongamia composition is composed largely of the residual insoluble solid material or meal remaining from the first pongamia composition following extraction with an alkyl alkanoate solvent and solid-liquid separation to remove miscella. In some embodiments, the second pongamia composition is meal. The resulting second pongamia composition, having a karanjin concentration of 100 ppm or less as described herein, may further contain any number of components originally present in the first pongamia composition, such as carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. For example, in some embodiments in which the second pongamia composition contains protein, the second pongamia composition contains at least 30% protein by dry weight. In certain embodiments, the second pongamia composition contains 30-50% protein, or 30-40% protein by dry weight. In other embodiments in which the second pongamia composition contains carbohydrates, the second pongamia composition contains at least 40% carbohydrates by dry weight. In certain embodiments, the second pongamia composition comprises 40-70% carbohydrates, 50-70% carbohydrates, or 50-60% carbohydrates by dry weight.

[0087] The additional components may be present in the second pongamia composition in a weight percentage of the total composition, reflecting the non-destructive method applied to the first pongamia composition. That is, the method of the present disclosure may be particularly suitable for removing karanjin and pongamol while maintaining or preserving the levels of nutritional components, such as carbohydrates, protein, fiber, ash, or any combination thereof, compared to the levels present in the first pongamia composition, resulting in a pongamia composition having a karanjin concentration of 100 ppm or less. In yet a further embodiment, the method described herein may result in a significant increase in the concentration of these additional components due to the removal of residual oil during extraction with the alkyl alkanoate solvent and the resulting reduction in the total weight of the second pongamia composition.

[0088] In some embodiments, the second pongamia composition having a karanjin concentration of 100 ppm or less comprises at least one component selected from the group consisting of carbohydrates, protein, fiber, ash, and any combination thereof, in a weight percentage of the second pongamia composition that is at least 90% of the weight percentage of the corresponding component present in the first pongamia composition. In certain embodiments, the second pongamia composition having a karanjin concentration of 100 ppm or less comprises at least one component selected from the group consisting of carbohydrates, protein, fiber, ash, and any combination thereof, in a weight percentage of the second pongamia composition that is 90-125% of the weight percentage of the corresponding component present in the first pongamia composition.

[0089] In some embodiments, the second pongamia composition comprises carbohydrates at a weight percentage of the second pongamia composition that is 90-125% of the weight percentage of carbohydrates present in the first pongamia composition. In other embodiments, the second pongamia composition comprises protein at a weight percentage of the second pongamia composition that is 90-125% of the weight percentage of protein present in the first pongamia composition. In still other embodiments, the second pongamia composition comprises fiber at a weight percentage of the second pongamia composition that is 90-150% of the weight percentage of fiber present in the first pongamia composition. In still other embodiments, the second pongamia composition comprises ash at a weight percentage of the second pongamia composition that is 90-125% of the weight percentage of ash present in the first pongamia composition.

[0090] In certain 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 a trypsin inhibitor at a weight percentage of the second Pongamia composition that is 60-90% of the weight percentage of the trypsin inhibitor present in the first Pongamia composition. In other embodiments, the second Pongamia composition contains chalcones and / or other furanoflavonoids at a weight percentage of the second Pongamia composition that is less than 100% of the weight percentage of chalcones and / or other furanoflavonoids present in the first Pongamia composition.

[0091] In yet further embodiments, the total protein content of the Pongamia composition may be further characterized by an amino acid profile. The amino acid profile may include characterizing the Pongamia composition based on the amount of individual amino acids present, the amount 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 still other embodiments, the second Pongamia composition has a total amino acid content that is at least 90% of the total amino acid content of the first Pongamia composition. In certain embodiments, the second Pongamia composition has a total amino acid content of 90-125% by weight of the total amino acid content of the second Pongamia composition.

[0092] It should be recognized that due to the nature of the extraction methods described herein, which involve mixing a first pongamia composition with a solvent, the second pongamia composition may contain residual levels of the solvent. For example, even after separating the miscella from the second pongamia composition, the second pongamia composition may contain residual levels of the specific alkyl alkanoate and any co-solvents in the alkyl alkanoate solvent used. Thus, in some embodiments, the second pongamia composition contains an alkyl alkanoate solvent. In certain embodiments, the second pongamia composition has an alkyl alkanoate solvent concentration of less than 100,000 ppm. In other embodiments, in which the alkyl alkanoate solvent combined with the first pongamia composition contains ethyl acetate, the second pongamia composition contains ethyl acetate. In certain embodiments in which the pongamia composition contains ethyl acetate, the pongamia composition has an ethyl acetate concentration of less than 100,000 ppm.

[0093] The disclosed methods may further include a dry heating or toasting step to desolventize, i.e., reduce the level of residual alkyl alkanoate solvent in the second pongamia composition. Thus, in some embodiments, the method further includes baking the second pongamia composition to prepare a baked pongamia composition. In some embodiments, after baking the second pongamia composition, the baked pongamia composition contains an alkyl alkanoate solvent and has an alkyl alkanoate solvent concentration of 5,000 ppm or less. In certain embodiments, the baked pongamia composition has an alkyl alkanoate solvent concentration of 0 ppm to 5,000 ppm, 0 ppm to 1,000 ppm, 1,000 ppm to 3,000 ppm, or 3,000 ppm to 5,000 ppm. In still other embodiments, the alkyl alkanoate solvent mixed with the first pongamia composition contains ethyl acetate, and the second pongamia composition is baked. The baked pongamia composition comprises ethyl acetate and has an ethyl acetate concentration of 5,000 ppm or less. In certain embodiments, the baked pongamia composition comprises ethyl acetate. The baked pongamia composition has an ethyl acetate concentration of 0 ppm to 5,000 ppm, 0 ppm to 1,000 ppm, 1,000 ppm to 3,000 ppm, or 3,000 ppm to 5,000 ppm.

[0094] [Pongamia composition] As mentioned above, pongamia compositions with low concentrations of karanjin and other antinutrients are desirable for downstream use. However, prior to the development of the above-described method for analyzing pongamia compositions, the residual karanjin concentration in processed pongamia compositions was difficult to accurately and consistently assess, and thus, the preparation of pongamia compositions with low karanjin concentrations was similarly difficult to achieve. The microwave-assisted alkyl alkanoate solvent extraction analytical method disclosed herein has made it possible to prepare and verify pongamia compositions with low karanjin concentrations. The present invention provides pongamia compositions with karanjin concentrations of 100 ppm or less. Also disclosed herein are pongamia compositions with karanjin concentrations of 100 ppm or less, as prepared by the methods described herein and / or as measured by the microwave-assisted alkyl alkanoate solvent extraction analytical method described herein.

[0095] In one aspect, the present disclosure provides a pongamia composition comprising karanjin, wherein the pongamia composition has a karanjin concentration of 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 60 ppm or less, 50 ppm or less, 40 ppm or less, 30 ppm or less, 20 ppm or less, or 10 ppm or less. In certain embodiments, the pongamia composition has a karanjin concentration of 100 ppm or less.

[0096] In another aspect, the present specification provides a pongamia composition comprising pongamol, wherein the pongamia composition has a pongamol concentration of 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 60 ppm or less, 50 ppm or less, 40 ppm or less, 30 ppm or less, 20 ppm or less, or 10 ppm or less. In certain embodiments, the pongamia composition has a pongamol concentration of 100 ppm or less.

[0097] In some embodiments, the pongamia composition is obtained 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").

[0098] In some embodiments, provided herein are pongamia compositions obtained or obtainable by solvent extraction of de-oiled pongamia seed cake with an alkyl alkanoate solvent. In some embodiments, provided herein are pongamia compositions prepared by microwave-assisted alkyl alkanoate solvent extraction. In certain embodiments, the pongamia composition is prepared by microwave-assisted alkyl alkanoate solvent extraction of de-oiled pongamia seed cake.

[0099] In another aspect, a pongamia composition is provided comprising karanjin and at least one or more components selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones.

[0100] In some embodiments, the pongamia composition has a karangin concentration of 100 ppm or less. In other embodiments, the pongamia composition has a karangin concentration of 100 ppm or less, as measured by the microwave-assisted alkyl alkanoate solvent extraction analytical method described above. In yet other embodiments, provided herein are pongamia compositions containing karangin, wherein the pongamia composition has a karangin concentration of 100 ppm or less, the karangin concentration being measured by treating the pongamia composition with an alkyl alkanoate solvent under microwave irradiation.

[0101] In some embodiments, the pongamia composition comprises karanjin and at least one or more components selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones. In other embodiments, the pongamia composition comprises carbohydrates and protein. In certain embodiments, the pongamia composition comprises tannins and trypsin inhibitors. In some embodiments, the pongamia composition comprises fiber and ash. In other embodiments, the pongamia composition comprises other furanoflavonoids and chalcones. In certain embodiments, the pongamia composition comprises carbohydrates and fiber. In some embodiments, the pongamia composition comprises carbohydrates and ash.

[0102] In some embodiments, the pongamia composition having a low karanjin concentration may be prepared or obtained by the preparation method described herein. In still other embodiments, the pongamia composition having a karanjin concentration of 100 ppm or less is a second pongamia composition obtained by the method for preparing a pongamia composition described herein. In some embodiments, the pongamia composition is obtained from a first pongamia composition having a karanjin concentration of at least 200 ppm. In other embodiments, the pongamia composition is obtained from a first pongamia composition having a karanjin concentration of at least 500 ppm.

[0103] As noted above, it should be recognized that pongamia compositions having karanjin and / or pongamol concentrations of 100 ppm or less as described herein may contain additional components (carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones) by weight of the total composition, if present, reflecting the non-destructive method of preparing the pongamia compositions described herein. For example, in some embodiments in which the pongamia composition contains protein, the pongamia composition contains at least 30% protein by dry weight. In certain embodiments, the pongamia composition contains 30-50% protein or 30-40% protein by dry weight. In other embodiments in which the pongamia composition contains carbohydrates, the pongamia composition contains at least 40% carbohydrates by dry weight. In certain embodiments, the pongamia composition contains 40-70% carbohydrates, 50-70% carbohydrates, or 50-60% carbohydrates by dry weight.

[0104] In yet further embodiments, the total protein content of the Pongamia composition may be further characterized by an amino acid profile. The amino acid profile may include characterizing the Pongamia composition based on the amount of individual amino acids present, the amount of various 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.

[0105] Additionally, it should be recognized that pongamia compositions having low karanjin concentrations described herein and prepared by the alkyl alkanoate-based extraction methods described herein may still contain residual pongamia oil and alkyl alkanoate solvent.

[0106] In some embodiments, the pongamia composition comprises oil. In certain embodiments, the pongamia composition comprises less than 5% oil by dry weight. In certain embodiments, the pongamia composition comprises 1% to 5% oil by dry weight.

[0107] 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 100,000 ppm or less. In still other embodiments, the pongamia composition has an alkyl alkanoate solvent concentration of 5,000 ppm or less. In specific embodiments, the pongamia composition has an alkyl alkanoate solvent concentration of 0 ppm to 5,000 ppm, 0 ppm to 1,000 ppm, 1,000 ppm to 3,000 ppm, or 3,000 ppm to 5,000 ppm.

[0108] In some embodiments, the Pongamia composition comprises an alkyl alkanoate solvent, and the alkyl alkanoate solvent comprises ethyl acetate. In certain embodiments, the Pongamia composition comprises ethyl acetate. In certain embodiments, the Pongamia composition comprises ethyl acetate at a concentration of 100,000 ppm or less. In still other embodiments, the Pongamia composition comprises 5,000 ppm or less. In certain embodiments, the Pongamia composition comprises ethyl acetate at a concentration of 0 ppm to 5,000 ppm, 0 ppm to 1,000 ppm, 1,000 ppm to 3,000 ppm, or 3,000 ppm to 5,000 ppm.

[0109] As described herein, the methods for preparing pongamia compositions may result in pongamia compositions having extremely low karanjin and / or pongamol concentrations. In some embodiments, the pongamia compositions have karanjin and / or pongamol concentrations in the parts per million order, or fractions thereof. In some embodiments, the pongamia compositions may have karanjin and / or pongamol concentrations of less than 100 ppm. These concentrations are undetectable by conventional hexane- and methanol-based analytical methods. In yet further embodiments, the pongamia compositions may have trace karanjin and / or pongamol concentrations in the parts per billion (ppb) or parts per trillion (ppt) order. In some embodiments, the pongamia compositions described herein may contain trace amounts of karanjin and / or pongamol that are undetectable by the alkyl alkanoate-based microwave-assisted solvent extraction analytical methods described herein.

[0110] Thus, in some embodiments in which a pongamia composition has a concentration of karanjin that is undetectable by the alkyl alkanoate-based microwave-assisted solvent extraction analytical methods described herein, the pongamia composition may be characterized by other components present in the composition, including carbohydrate, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, chalcones, alkyl alkanoate solvents, or amino acid content, or any combination thereof.

[0111] In some embodiments, a pongamia composition is provided that includes at least one component selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones, and the pongamia composition has a karanjin concentration of 100 ppm or less. In certain of the foregoing embodiments, the pongamia composition has an undetectable karanjin concentration as measured by the microwave-assisted alkyl alkanoate solvent extraction analytical method described herein. In some embodiments, the pongamia composition includes (i) 30-50% protein by dry weight; (ii) 40-70% carbohydrate; (iii) 20-30% total amino acid content by weight; or any combination thereof.

[0112] Use of Pongamia Composition Pongamia compositions having low concentrations of karanjin and pongamol and prepared by the above-described methods can be particularly useful as nutritional supplements or primary feeds in ruminant feed compositions, such as cattle feed compositions. The non-destructive method for preparing pongamia compositions described herein results in the successful removal of the anti-nutritional components karanjin and pongamol without reducing the amounts of other components, including macronutrients (e.g., protein and carbohydrates), which are crucial for achieving acceptable feed conversion efficiencies. The pongamia compositions can be used alone in ruminant feed compositions or in combination with non-pongamia-derived base feeds to provide complex ruminant feed compositions.

[0113] As used herein, the term "ruminant" should be understood to include any wild or domesticated hoofed mammal having a chambered stomach (including a rumen) adapted for the digestion of plant material. Suitable ruminants may include, but are not limited to, cattle, yaks, buffalo, goats, sheep, deer, gazelles, and antelopes. In certain embodiments, the cattle are beef cattle.

[0114] In one aspect, provided herein is a ruminant feed composition comprising a pongamia composition having a low karanjin concentration as described above. In some embodiments, the ruminant feed composition comprises a pongamia composition having a karanjin concentration of 100 ppm or less. In other embodiments, the ruminant feed composition comprises a base feed and a pongamia composition, wherein the pongamia composition has a karanjin concentration of less than 100 ppm as described herein.

[0115] In some embodiments, provided herein is a cattle feed composition comprising a pongamia composition having a low karanjin concentration as described above. In some embodiments, the cattle feed composition comprises a pongamia composition having a karanjin concentration of 100 ppm or less. In other embodiments, the cattle feed composition comprises a base feed and a pongamia composition, wherein the pongamia composition has a karanjin concentration of less than 100 ppm as described herein.

[0116] In some embodiments, provided herein are ruminant feed compositions (including, for example, bovine feed compositions) comprising any of the pongamia compositions described herein. In one embodiment, provided herein are ruminant feed compositions (including, for example, bovine feed compositions) comprising a base feed and any of the pongamia compositions described herein.

[0117] In some variations of the foregoing, the pongamia composition comprises karanjin and at least one or more components selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones, and the pongamia composition has a karanjin concentration of 100 ppm or less. In other variations, the pongamia composition comprises at least one or more components selected from the group consisting of carbohydrates, protein, fiber, ash, tannins, trypsin inhibitors, other furanoflavonoids, and chalcones, and the pongamia composition has a karanjin concentration of 100 ppm or less.

[0118] Due to their low concentrations of karangin, the pongamia compositions described herein can be utilized in ruminant feed compositions, such as cattle feed compositions, in larger amounts than previously used and with fewer anti-nutritional or long-term pathological effects than previously observed. Thus, in some embodiments, a ruminant feed composition or cattle feed composition comprises at least 30% or at least 40% by weight of a pongamia composition, and the pongamia composition has a karangin concentration of 100 ppm or less. In some embodiments, the pongamia composition has a karangin concentration of 100 ppm or less, measured by treating the pongamia composition with an alkyl alkanoate solvent under microwave irradiation. In other embodiments, the pongamia composition has a karangin concentration of 100 ppm or less, measured by microwave-assisted alkyl alkanoate solvent extraction analysis.

[0119] In some embodiments, ruminant feed compositions (including, e.g., cattle feed compositions) comprise a base feed. Suitable base feeds for the ruminant feed compositions described herein can be any non-pongamia-derived feedstock known in the art for use as forage or livestock feed, including, for example, hay, straw, silage, grains, legumes, food waste, and food processing by-products. In certain embodiments, the base feed may comprise one or more feeds selected from the group consisting of wheat feed, corn feed, barley feed, oat feed, soybean meal, cottonseed meal, safflower seed meal, sunflower seed meal, peanut meal, groundnut meal, and hay. In certain embodiments, the base feed comprises wheat feed, corn feed, soybean meal, or any combination thereof. Due to the low concentration of karanjin in the pongamia compositions described herein, the pongamia composition can be combined with the base feed to produce a ruminant feed composition containing a large proportion of pongamia-derived feed. Thus, the amount of base feed in the animal compositions of the present disclosure can be reduced. In other embodiments, ruminant feed compositions (including, for example, cattle feed compositions) comprise less than 60% or less than 70% by weight of base feed.

[0120] It should be appreciated that the ruminant feed compositions (including, e.g., bovine feed compositions) described herein may contain additional feed additives known in the art, including, for example, antibiotics and other veterinary drugs, growth hormones, vitamins, mineral or nutritional supplements, palatability enhancers, processing additives, and the like.

[0121] In yet another aspect, the present disclosure provides a method for feeding a ruminant, such as a cow, comprising providing the ruminant with a pongamia composition or a ruminant feed composition described herein. In certain embodiments, the present disclosure provides a method for feeding a ruminant, comprising providing the ruminant with a pongamia composition having a karanjin concentration of 100 ppm or less, as measured by microwave-assisted alkyl alkanoate solvent extraction analysis. In other embodiments, a method for feeding a ruminant, comprising providing a cow feed composition to the ruminant, wherein the ruminant feed composition comprises a pongamia composition having a karanjin concentration of 100 ppm or less, and the karanjin concentration is measured by treating the pongamia composition with any of the alkyl alkanoate solvents described herein under microwave irradiation.

[0122] In some embodiments, the ruminant is a cow. In particular embodiments, the cow is a beef cow. In certain variations, the present disclosure provides a method of feeding a cow, comprising providing the cow with a pongamia composition or a ruminant feed composition as described herein. In certain embodiments, the present disclosure provides a method of feeding a cow, comprising providing the cow with a pongamia composition having a karanjin concentration of 100 ppm or less, as measured by microwave-assisted alkyl alkanoate solvent extraction analysis. In other embodiments, a method of feeding a cow with a cattle feed composition is provided herein, comprising providing the cattle with a pongamia composition having a karanjin concentration of 100 ppm or less, as measured by microwave irradiation with any of the alkyl alkanoate solvents described herein.

[0123] For the methods of feeding ruminants or cattle described herein, the ruminant feed composition (including, for example, cattle feed composition) can be provided in a variety of forms suitable for ruminants or cattle. In some embodiments, the ruminant feed composition is provided as a powdered meal, a pelleted feed, a liquid feed, or a mashed feed. For example, in some embodiments, the ruminant feed composition may be provided as a powdered meal or a pelleted feed.

[0124] In particular, the non-destructive processing methods described herein not only provide pongamia compositions having low levels of anti-nutrients, such as karanjin and pongamol, but also preserve or maintain comparable levels of nutrients and proxies present in the original pongamia composition resulting in a pongamia composition having a reduced karanjin concentration. Accordingly, it should also be appreciated that the pongamia compositions prepared by the methods described herein may also have specific levels of nutrients or proxies (ash, moisture, protein, fat, carbohydrates, minerals, vitamins) that are particularly suited to the nutritional requirements of ruminant animals (e.g., cattle) being fed the feed, particularly with regard to feed conversion efficiency.

[0125] In other aspects, there is provided an article of manufacture, such as a container comprising a pongamia composition as described herein, or a feed comprising a pongamia composition as described herein, and a label comprising instructions for use of such pongamia composition or feed.

[0126] In yet another aspect, there is provided a kit comprising a Pongamia composition as described herein, or a feed comprising a Pongamia composition as described herein, and a package insert containing instructions for use of such Pongamia composition or feed.

[0127] Enumerated Embodiments The embodiments listed below are representative of some aspects of the present invention. (1) mixing a pongamia composition with an alkyl alkanoate solvent to prepare an extraction mixture; irradiating the extraction mixture with microwave radiation to provide an irradiated mixture; separating the irradiated mixture into an extracted pongamia composition and an alkyl alkanoate extract, and determining the concentration of karandin in the alkyl alkanoate extract. (2) The method of (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, propyl butanoate, propyl pentanoate, butyl methanoate, butyl ethanoate, butyl propanoate, butyl butanoate, and butyl pentanoate, and any combination thereof. (3) The method of any one of (1) to (2), wherein the alkyl alkanoate solvent comprises ethyl acetate. (4) The method of any one of (1) to (3), wherein the pongamia composition is a deoiled pongamia seed cake. (5) The method according to any one of (1) to (4), wherein the Pongamia composition is obtained by mechanical extraction, solvent extraction, or a combination thereof. (6) The method of any one of embodiments (1) to (5), wherein measuring the karanjin concentration in the alkyl alkanoate extract comprises measuring the karanjin concentration by high performance liquid chromatography (HPLC). (7) providing a first pongamia composition; preparing an extraction mixture by combining the first pongamia composition with an alkyl alkanoate solvent; separating the extract mixture into miscella and a second pongamia composition; wherein the second pongamia composition has (i) a karanjin concentration that is less than 20% of the karanjin concentration in the first pongamia composition, or (ii) a karanjin concentration that is 100 ppm or less. (8) The method of embodiment 7, wherein the second pongamia composition has a karanjin concentration of 100 ppm or less when measured by the method of any one of embodiments 1 to 5. (9) The method of any one of (7) to (8), wherein the first pongamia composition is a cake of deoiled pongamia seeds. 10. The method of claim 9, wherein the first pongamia composition is a de-oiled pongamia seed cake obtained by mechanical extraction. (11) The method of any one of (9) to (10), wherein the first pongamia composition is not a deoiled pongamia seed cake obtained by solvent extraction. (12) The method of any one of (7) to (11), wherein the first pongamia composition has a karanjin concentration of at least 200 ppm. (13) The method of any one of (7) to (12), wherein the first pongamia composition comprises 8 to 30 wt. % oil. (14) The method of any one of (7) to (13), wherein the miscella has a karanjin concentration of about 4,000 ppm or more. (15) The method according to any one of embodiments (7) to (14), wherein the second pongamia composition has a concentration of pongamol of 100 ppm or less. (16) The method of any one of (7) to (15), wherein the alkyl alkanoate solvent comprises ethyl acetate. (17) The method according to any one of embodiments (7) to (16), wherein the method further comprises irradiating the extraction mixture with microwave radiation. (18) The pongamia composition is Karanjin 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 karanjin concentration of 100 ppm or less. (19) The Pongamia composition according to embodiment (18), wherein the Pongamia composition has a karanjin concentration of 100 ppm or less when measured by the method of any one of embodiments (1) to (6). (20) The Pongamia composition according to embodiment (18) or (19), wherein the Pongamia composition further comprises pongamol. (21) The Pongamia composition according to any one of embodiments (18) to (20), wherein the Pongamia composition has a concentration of pongamol of 100 ppm or less. (22) The Pongamia composition according to any one of embodiments (18) to (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 5,000 ppm. (25) The Pongamia composition of any one of embodiments (22) to (24), wherein the alkyl alkanoate solvent comprises ethyl acetate. (26) The Pongamia composition according to any one of embodiments (18) to (25), wherein the Pongamia composition comprises less than 5% oil by dry weight. (27) The Pongamia composition according to any one of embodiments (18) to (26), wherein the Pongamia composition comprises 1% to 5% oil by dry weight. (28) The Pongamia composition according to any one of embodiments (18) to (27), wherein the Pongamia composition comprises at least 30% protein by dry weight. (29) The Pongamia composition according to any one of embodiments (18) to (28), wherein the Pongamia composition comprises 30 to 40% protein by dry weight. (30) The Pongamia composition according to any one of embodiments (18) to (29), wherein the Pongamia composition has a total amino acid content of at least 20% by weight. (31) The Pongamia composition according to any one of embodiments (18) to (30), wherein the Pongamia composition has a total amino acid content of 20 to 30% by weight. (32) The Pongamia composition according to any one of embodiments (18) to (31), wherein the Pongamia composition comprises at least 40% by weight of carbohydrates. (33) The Pongamia composition according to any one of embodiments (18) to (32), wherein the Pongamia composition has a total amino acid content of carbohydrates of 50 to 70% by weight. (34) The Pongamia composition according to any one of embodiments (18) to (33), wherein the Pongamia composition is obtained from an initial Pongamia composition having a karanjin concentration of at least 200 ppm. (35) The Pongamia composition according to any one of embodiments (18) to (34), wherein the Pongamia composition is obtained by solvent extraction of deoiled Pongamia seed cake with an alkyl alkanoate solvent. (36) The Pongamia composition according to any one of embodiments (18) to (35), wherein the Pongamia composition is obtained by solvent extraction of deoiled Pongamia seed cake with an alkyl alkanoate solvent and microwave irradiation. (37) A Pongamia composition obtained or obtainable by the method according to any one of embodiments (1) to (17). (38) The pongamia composition is 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 karanjin concentration of 100 ppm or less. (39) The pongamia composition is 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 karanjin concentration of 100 ppm or less; wherein the pongamia composition has a pongamol concentration of 100 ppm or less. (40) The pongamia composition is Karanjin, or pongamol, or a mixture of karanjin 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, when karancin is present, the pongamia composition has a karancin concentration of 100 ppm or less; wherein, when pongamol is present, the pongamia composition has a pongamol concentration of 100 ppm or less. (41) The pongamia composition is Karanjin 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 karanjin concentration of 100 ppm or less, the karanjin concentration being measured by treating the pongamia composition with an alkyl alkanoate solvent under microwave irradiation. (42) The Pongamia composition according to any one of embodiments (18) to (41), wherein the Pongamia composition is obtained from plant material derived from a Pongamia tree or plant. (43) The Pongamia composition according to any one of embodiments (18) to (42), wherein the Pongamia composition is meal. (44) A feed composition comprising: A Pongamia composition according to any one of embodiments (18) to (43), a base cattle feed; and a feed composition comprising: (45) A feed composition comprising: A pongamia composition according to any one of embodiments 18 to 43, A feed composition comprising: a base feed; (46) The feed composition according to embodiment (44) or (45), wherein the feed composition comprises at least 30% by weight or at least 40% by weight of the Pongamia composition. (47) The feed composition according to any one of embodiments (44) to (46), wherein the feed composition comprises less than 60% or less than 70% by weight of the base feed. (48) The feed composition according to any one of embodiments (44) to (47), wherein the feed composition is a pelleted feed. (49) A method of feeding a ruminant, comprising providing the ruminant with a Pongamia composition according to any one of embodiments (18) to (43) or a feed composition according to any one of embodiments (44) to (48). 50. The method of claim 49, wherein the ruminant is selected from the group consisting of cattle, yaks, buffalo, goats, sheep, deer, gazelles, and antelopes. 51. The method of embodiment 50, wherein the ruminant is a cow.

[0128] [Example] The subject matter of the present disclosure will be better understood by reference to the following examples, which are offered by way of illustration of the invention and not by way of limitation.

[0129] Example A: Analytical Methods Example A1: Comparison of Methanol-Based Extracts The following example describes an experiment comparing the extraction of karanjin and pongamol from pongamia seed cake using methanol as the solvent.

[0130] (Homogenized Extraction of Karanjin and Pongamol) 0.5 g of pongamia seed cake was placed in a 50 mL polypropylene centrifuge tube containing 5 mL of methanol for a final ratio of 10:1 (solvent:solid). The sample was then placed in a plant / tissue homogenizer and shaken at 1500 rpm for 2 minutes. The sample was then centrifuged at 3000 rpm for 5 minutes to separate the solvent from the solids, and the supernatant was poured into a clean 50 mL polypropylene tube. The extraction process was repeated five times to improve the extraction of karanjin and pongamol.

[0131] (Homogenized extraction by soaking in NaOH) Prior to homogenization extraction, 0.5 g of pongamia seed cake was placed in a 50 mL polypropylene centrifuge tube containing 1 mL of 2% NaOH. The mixture was then incubated for 24 hours. After incubation, 10 mL of water was added, and the tube was placed on a mechanical shaker and shaken on high for 10 minutes. The tube was then centrifuged at 3000 rpm to pellet the solid material. The water was saved for analysis, and the washing process was repeated once more to ensure removal of the NaOH. After incubation with NaOH, the sample was extracted with methanol as described above.

[0132] (Homogenized extraction by methanol immersion) Prior to homogenization, 0.5 g of pongamia seed cake was placed in a 50 mL polypropylene centrifuge tube containing 25 mL of methanol to a final ratio of 50:1 (solvent:solid). The mixture was then incubated at room temperature for 24, 48, 72, or 96 hours. After incubation, the sample was placed in a plant / tissue homogenizer and shaken at 1500 rpm for 2 minutes. After homogenization, the sample was centrifuged at 3000 rpm for 5 minutes to separate the solvent from the solids, and the supernatant was poured into a clean 50 mL polypropylene tube. Homogenization was repeated five times to improve the extraction of karanjin and pongamol.

[0133] (Homogenized extraction by filtration) 0.5 g of pongamia seed cake was placed in a 50 mL polypropylene centrifuge tube containing 25 mL of methanol for a final ratio of 50:1 (solvent:solid). The sample was then placed in a plant / tissue homogenizer and shaken at 1500 rpm for 2 minutes. The sample was then filtered to separate the solids from the methanol. The extraction process was repeated five times to improve karanjin and pongamol extraction.

[0134] (Soxhlet extraction of Karanjin and Pongamol) 0.5 g of Pongamia seed cake was placed in an extraction thimble containing 125 mL of methanol. Extraction was allowed to proceed for 24 or 48 hours, at which point the Soxhlet extract was transferred to a clean polypropylene tube.

[0135] (HPLC standard solution) Commercially available karanjin and pongamol were mixed with methanol to produce the following HPLC standards: 0.05, 0.1, 0.2, 0.5, 1.0, 5.0, and 20.0 μg / mL.

[0136] (HPLC equipment) HPLC analysis was performed using 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. A C18 5 μm, 50 × 2 mm HPLC column was used. All HPLC analyses were performed in negative ion mode. MS parameters were: curtain gas 30 psi, collision gas 4 psi, nebulizer gas (GS1) 50 psi, drying gas (GS2) 50 psi, ion spray voltage 5000, temperature 500 °C, declustering potential (DP) 51 V, entrance potential 10 V, and collision energy (CE) 60 eV for karanjin and 30 eV for pongamol.

[0137] (MS / MS quantification of karanjin and pongamol in extracts) Multiple reaction monitoring (MRM) ion transitions were monitored for both karanjin and pongamol. The levels of karanjin and pongamol present in the extracted samples were calculated using Analyst version 1.6.3. Briefly, the peak areas of karanjin and pongamol in the extracted samples were compared with the peak areas of calibration standards to determine the parts per million of karanjin and pongamol.

[0138] (Methanol extraction of karanjin and pongamol from pongamia seed cake) Table 1 shows the levels of karangin and pongamol that can be extracted from pongamia seed cake (Figures 3A and 3B) under homogenized extraction using methanol (solvent:solid ratio of 10:1) as the extraction solvent. The amounts of karangin and pongamol obtained from each of the five consecutive extractions were added together to obtain the measurements of total extracted karangin and total extracted pongamol, respectively, as shown in Table 1.

[0139] [Table 1] (Evaluation of the effect of 24-hour NaOH soaking on the extraction of karanjin and pongamol) The amounts of karanjin and pongamol obtained from each of the five consecutive extractions were combined and added to obtain measurements of the total amount of karanjin extracted and the total amount of pongamol extracted, respectively, as shown in Table 2. Table 2 showed that when pongamia seed cake was treated with NaOH prior to methanol homogenization extraction (solvent:solid ratio of 10:1), the total extracted and residual karanjin and pongamol levels were reduced compared to the untreated extraction (Figures 3A and 3B).

[0140] [Table 2] (Measurement of Karanjin and Pongamol concentrations in NaOH wash water) Table 3 shows that karanjin and pongamol leached into the wash water after NaOH immersion. However, the levels of karanjin and pongamol in the wash water were not sufficient to explain the reduced levels of karanjin and pongamol isolated after NaOH treatment.

[0141] [Table 3] (Evaluation of the effect of methanol soaking on karanjin extraction) The amounts of karanjin and pongamol obtained from each of the five consecutive extractions were combined and added to obtain measurements of the total amount of karanjin extracted and the total amount of pongamol extracted, respectively, as shown in Table 4. Table 4 showed that soaking the pongamia seed cake in methanol for any length of time before extraction reduced the recovery of karanjin compared to extraction without methanol soaking (solvent:solid ratio of 50:1) (Figures 3A and 3B).

[0142] [Table 4] (Evaluation of the recovery of karanjin and pongamol using filtration rather than centrifugation after methanol homogenization extraction) The amounts of karangin and pongamol obtained from each of the five consecutive extractions (solvent:solid ratio of 50:1) were combined and added to obtain the measurements of total karangin extracted and total pongamol extracted, respectively, as shown in Table 5. Table 5 demonstrated that methanol homogenization extraction by filtration could recover similar levels of karangin and pongamol from pongamia seed cake compared to methanol homogenization extraction by centrifugation (Figures 3A and 3B).

[0143] [Table 5] (Soxhlet extraction of Karanjin and Pongamol) Table 6 shows a comparison of methanol homogenization extraction (50:1 solvent:solid ratio) and methanol Soxhlet extraction. The total karangin and pongamol extracted by the methanol homogenization process is the sum of karangin and pongamol obtained from each of five consecutive extractions, while the total amount of karangin and pongamol extracted by the Soxhlet process is the amount of karangin and pongamol obtained from a single Soxhlet run for the indicated time period. The values ​​measured in the 24-hour and 48-hour methanol Soxhlet extractions were each the average of two separate runs. Table 6 shows that the 24-hour and 48-hour methanol Soxhlet extractions isolated more karangin and pongamol than the methanol homogenization extraction technique (Figures 3A and 3B).

[0144] [Table 6] Example A2: Solvent dependency of extraction method The following examples describe experimental efforts to evaluate the ability of various solvents to extract karanjin and pongamol from pongamia seed cake.

[0145] (Homogenized Extraction of Karanjin and Pongamol) In this example, homogenization extraction was performed similarly to Example A1. However, the solvents tested included ethanol, hexane, methyl tert-butyl ether (MTBE), toluene, diethyl ether, ethyl acetate, and acetone, all at a solvent:solid ratio of 50:1. Finally, experiments were conducted to evaluate the effect of homogenization time on ethyl acetate solvent extraction, comparing 10 minutes (2 minutes per cycle, 5 cycles) and 50 minutes (10 minutes per cycle, 5 cycles) of homogenization.

[0146] (Soxhlet extraction of Karanjin and Pongamol) In this example, Soxhlet extraction was performed similarly to Example A1. However, the solvents tested were methanol, MTBE, and ethyl acetate. Furthermore, the reaction times tested for ethyl acetate included 6 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours.

[0147] (Evaluation of Karanjin and Pongamol Extraction Efficiency with Different Solvents Using Homogenized Extraction) The amounts of karanjin and pongamol obtained from each of the five consecutive extractions were combined and added to obtain the measurements of total karanjin extracted and total pongamol extracted, respectively, as shown in Table 7. Table 7 shows that ethyl acetate extracted more karanjin and pongamol than the other solvents tested using the homogenization technique (Figures 3A, 3B, 4A, and 4B).

[0148] [Table 7] (Evaluation of the effect of extended homogenization time on the extraction of karanjin and pongamol with ethyl acetate) Table 8 shows a comparison of homogenization extraction for a total of 10 minutes using ethyl acetate as the solvent with homogenization extraction for a total of 50 minutes using ethyl acetate as the solvent. Table 8 demonstrates that increasing the total homogenization time from 10 to 50 minutes decreases the ethyl acetate extraction efficiency of karanjin and pongamol (Figures 4A and 4B).

[0149] [Table 8] (Evaluation of extraction efficiency of karanjin and pongamol with different solvents using Soxhlet extraction) Table 9 shows the total karanjin and total pongamol extracted from a single Soxhlet experiment using various solvents at different times. Table 9 shows that ethyl acetate extracted more karanjin and pongamol than the other solvents tested using the Soxhlet extraction technique (Figures 3A, 3B, 4A, and 4B). Furthermore, Table 9 shows that shorter Soxhlet extraction times were more efficient at isolating karanjin than longer extraction times. However, in general, longer Soxhlet extraction times were more efficient at isolating pongamol than shorter extraction times.

[0150] [Table 9] Example A3: Microwave-assisted extraction (MAE) The following examples describe experimental efforts to develop an ethyl acetate MAE process to karanjin and pongamol.

[0151] (Preparation of ionic liquid) The 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, and then vortexing the mixture to dissolve the solids.

[0152] (Microwave-assisted extraction of Karanjin and Pongamol) 0.5 g of pongamia seed cake was added to a microwave extraction tube. 15 mL of either ethyl acetate or ionic liquid was then added to the sample tube and vortexed to mix. The sample was then extracted using a microwave extractor under the following conditions: 1) ramp to 70°C for 15 minutes, 2) hold at 70°C for 10 minutes. Once cooled, the supernatant was filtered under vacuum using filter paper in a Buchner funnel.

[0153] (Evaluation of the efficiency of microwave assisted ethyl acetate extraction of karanjin and pongamol) Table 10 shows that ethyl acetate MAE extracted over 9600 ppm of karanjin and over 790 ppm of pongamol from pongamia seed cake (see also Figures 5A and 5B).

[0154] [Table 10] (Seed cake extraction summary) Table 11 provides a summary of the karanjin and pongamol extraction data detailed in Examples A1-A3. Figures 6A and 6B show bar graphs of the relative efficiency of each extraction treatment as measured in ppm for total karanjin and total pongamol extracted.

[0155] [Table 11] Example A4: Microwave-assisted extraction (MAE) using alkyl alkanoates The following example describes experimental efforts to evaluate the ability of various alkyl alkanoate solvents to extract karanjin and pongamol from pongamia seed cake under microwave-assisted extraction.

[0156] The pongamia seed cake samples used in Parts I-V below were of the same origin and preparation date as the seed cake used in Examples A1-A3 above. The pongamia seed cake had been stored for 18 months (at -20°C) at the start of Example A4.

[0157] The extraction ability of various alkyl alkanoate solvents for karanjin and pongamol under microwave-assisted extraction conditions was evaluated.

[0158] Partially defatted pongamia meal was homogenized using dry ice. Samples were stored frozen and the dry ice was allowed to sublimate. 0.5 g + / - 0.02 g of pongamia meal was placed in separate microwave extraction tubes, and 15.0 mL of each solvent (30:1 solvent:solid (v / w) ratio) was added to the corresponding microwave tube. The microwave tubes were then capped and vortexed. Extraction was performed using a MARS6 microwave extractor under the following conditions: 1) ramp to 70°C for 15 minutes, 2) hold at 70°C for 10 minutes. After cooling the supernatant to room temperature, the extract was filtered under vacuum through Whatman GF / F filter paper in a Buchner funnel, and the extract was poured into pre-labeled 50 mL centrifuge tubes.

[0159] (analysis) All sample extracts were diluted 10-fold and 100-fold for LCMS / MS analysis using LCMS / MS vials or volumetric flasks directly. (10-fold dilution: 100 μL of sample extract was added to 900 μL of the appropriate solvent and vortexed. 100-fold dilution: 10 μL of sample extract was added to 990 μL of the appropriate solvent and vortexed.) The parameters for LCMS / MS analysis were identical to those described in Example A1 above.

[0160] Evaluation of microwave-assisted extraction efficiency of karanjin and pongamol using various alkyl alkanoate solvents Table 12 shows the total amount of karanjin and total amount of pongamol extracted from pongamia seed cake samples using the listed solvents under microwave-assisted extraction conditions (see also Figures 7A and 7B).

[0161] [Table 12] Example B: Large-scale extraction method This example details the experimental efforts to scale up the extraction of karanjin and pongamol to commercially viable levels.

[0162] Part I - Mechanical Processing (Hot extrusion method and continuous press extraction method) Pongamia seed cake samples were separately subjected to hot extrusion process ("seed conditioning") and expeller pressing process ("expeller pressing", one (1st) or two (2nd) pressings) to remove oil, karanjin and pongamol from the pongamia seed cake.

[0163] (Evaluation of the extraction efficiency of karanjin and pongamol by mechanical processing) Pongamia seed cake samples were analyzed by microwave-assisted ethyl acetate solvent extraction (following the protocol in Example A3 above) to determine the amount of karanjin and pongamol remaining in the processed pongamia seed cake after treatment by the seed conditioning and expeller-press processing methods. Tables 13 and 14 demonstrate that both seed conditioning and expeller-press mechanical processing methods can be used to extract karanjin and pongamol from pongamia seed cake on a commercial scale (Figures 8A and 8B).

[0164] [Table 13] [Table 14] Part II - Solvent Extraction (Single solvent extraction of karanjin and pongamol) Pongamia seed cake was introduced into the immersion extractors using a metering feeder. The feed rate was adjusted so that the paddle section of each extractor was approximately 50% full. Table 15 shows the specific extraction settings for each solvent.

[0165] [Table 15] (Dual solvent extraction of Karanjin and Pongamol) Following the primary extraction with hexane detailed above, the pongamia seed cake was collected and introduced into the immersion extractors using a metered feeder. The feed rate was adjusted so that the paddle section of each extractor was approximately 50% full. Table 16 shows the specific extraction settings for each solvent.

[0166] [Table 16] (Evaluation of large-scale solvent extraction of karanjin and pongamol) Each sample produced from the combined mechanical and solvent extraction methods outlined in Tables 15 and 16 was analyzed by microwave-assisted ethyl acetate extraction (following the protocol in Example A3 above) to determine the amount of residual karanjin and pongamol remaining in the pongamia seed cake after the extraction procedure. Table 17A shows that a single extraction with ethyl acetate is the most efficient solvent for removing karanjin and pongamol from pongamia seed cake, regardless of the conventional mechanical extraction method (Figures 9A and 9B).

[0167] [Table 17A] The expeller-pressed (second press), ethyl acetate-extracted seed cake was analyzed a second time at a later date for karanjin and pongamol concentrations under the conditions previously used in Example A3, confirming the initial determinations in Table 17A. The results of the second experiment were observed to be slightly higher than the initial determinations. The results of the first experiment (Experiment No. 1, same as Table 17A above), the second experiment (Experiment No. 2), and the average of the two determinations ("Average") are shown below in Table 17B.

[0168] [Table 17B] Part III - Compositional profile of extracted seed cake Following the extraction of the pongamia seed cake in Part II above, the starting pongamia seed cake samples in Part I and the solvent-extracted pongamia seed cake samples in Part II were evaluated to determine the effect, if any, of mechanical processing and solvent extraction on the compositional profile of the seed cake. The total protein, total carbohydrate, and amino acid profiles of the mechanically pressed pongamia seed cake and the mechanically pressed ethyl acetate-extracted seed cake were measured.

[0169] The amino acid content in pongamia seed cake was measured by various methods depending on the identity of the amino acid being quantified. For example, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, tyrosine, and valine were measured by acid hydrolysis of pongamia seed cake samples in 6N HCl for 24 hours at 110°C, followed by quantification by ion-exchange chromatography with ninhydrin post-column reaction and UV / vis detection (AOAC 982.30 reference method, modified). Tryptophan in seed cake samples was measured by alkaline digestion of seed cake with lithium hydroxide for 22 hours at 110°C, followed by quantification by reverse-phase chromatography with fluorescence detection (AOAC 998.15 reference method). The amounts of cysteine ​​and methionine were measured by oxidizing seed cake samples with performic acid to convert cysteine ​​to cysteic acid and methionine to methionine sulfone. The oxidized samples were 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 (AOAC 994.12 reference method, modified).

[0170] The average amino acid profile of pongamia meal from two separate experiments is shown below in Table 18 as the amino acid content after mechanical expression but before solvent extraction (expeller expression, second expression, "pre-processing") and after both mechanical expression and solvent extraction with ethyl acetate (expeller expression, second expression, "post-processing") The amino acid profile is expressed as the weight percentage (% w / w) of the relative amino acid content of the meal and as a percentage of the total amino acid content.

[0171] [Table 18] As shown in Table 18 above, the relative amino acid content and profile of the seed cake samples were largely preserved after ethyl acetate solvent extraction to remove karanjin and pongamol. The solvent-extracted seed cake generally exhibited slightly higher amino acid concentrations than the starting seed cake, except for a decrease in lysine content. The higher concentrations of amino acids in the solvent-extracted meal may be due in part to the removal of pongamia oil during solvent extraction and, therefore, a decrease in the total weight of the seed cake.

[0172] Total protein and carbohydrates were also measured in seed cake samples before and after ethyl acetate solvent extraction. 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 gas produced by combustion, and calculating protein from the observed nitrogen content (protein content = 6.25 × nitrogen content).

[0173] Total carbohydrate content was calculated as the percentage of the remaining pongamia seed cake (100%) minus the sum of the total ash content (%), total protein content (%), total moisture content (%), and total lipid content (%). Total ash content was measured by placing a seed cake sample (2 g) in a crucible, staining the sample in an oven, ashing the sample in a muffle furnace at 600 °C, and weighing the ash (AOAC 942.05 reference method). Total moisture content was determined by heating a weighed sample in a forced draft oven for 2 hours at 130 °C and determining the difference in sample weight for the calculated percentage difference in moisture content (AOCS BA 2A-38 reference method). Total lipid content was determined by solvent extraction with petroleum ether under reflux (AOCS BA3-38 reference method, modified).

[0174] Table 19 shows the average total protein and average total carbohydrate content (as a weight percent of the total sample weight) for two experiments after mechanical expression but before solvent extraction (expeller press, second press, "pre-processing") and after both mechanical expression and solvent extraction with ethyl acetate (expeller press followed by ethyl acetate extraction, second press, "post-processing").

[0175] [Table 19] The solvent extraction process did not result in a loss of protein or carbohydrate content in the Pongamia seed cake. Similar to the amino acid profiles described above, the solvent-extracted seed cake samples showed slightly higher concentrations of total protein and total carbohydrate than the expeller-pressed starting seed cake.

[0176] Example C: Ruminant Feed Composition The following example details experimental efforts to evaluate the feasibility of administering ethyl acetate extracted pongamia meal to cattle.

[0177] Example C1: Pongamia supplement test The following example describes a study comparing the use of ethyl acetate extracted pongamia seed cake with the use of soybean meal as a separate supplemental protein source in a low quality forage diet in cattle.

[0178] Thirteen steers were utilized in a fully randomized study and fed one of three diets, including a low-quality hay (5.0% crude protein) control (CON (n = 4), a corn and distillers dried grains-based diet (SBM) supplemented with soybean meal (n = 4), and a distillers dried grains-based diet supplemented with ethyl acetate-extracted pongamia seed cake (PSC) (n = 5). The pongamia seed cake used in this study was expeller-pressed, second-press, ethyl acetate-extracted pongamia meal prepared in Example B, Part II.

[0179] Table 20 shows the composition of the three test diet groups utilized in the study: hay (control), soybean meal (SBM), and solvent extracted pongamia seed cake (PSC).

[0180] [Table 20] Table 21 shows the chemical composition of hay, soybean meal and pongamia seed cake.

[0181] Hay, SBM, and PSC were dried in a forced-air oven at 55°C for 96 hours and air-equilibrated for partial dry matter (DM) determination. Hay and supplements were pooled on an equal weight basis for 1 day, then ground through a 1 mm screen using a Wiley mill and dried at 105°C to determine DM. Organic matter (OM) was measured as the loss in dry weight upon combustion in a muffle furnace at 450°C for 8 hours. Nitrogen was measured using an Elementar rapid N cube (Elementar, Hanua, Germany), and crude protein (CP) was calculated as N x 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) analyses were performed consecutively using an Ankom fiber analyzer with amylase.

[0182] Karanjin and pongamol contents in pongamia seed cake were determined by microwave-assisted solvent extraction method using ethyl acetate as described in Example A3 and Example A4.

[0183] [Table 21] Test steers were fed the designated diets for a total duration of 21 days, including 13 days for adaptation and 8 days for sample collection. Measurement of dry matter intake and digestibility was facilitated by 6 days of total fecal collection via fecal bags.

[0184] (statistical analysis) Dry matter intake and digestibility were analyzed using the MIXED procedure in SAS 9.2 (SAS InstInc., Cary, NC). Model terms included treatment and period, with steer as a random effect. Model terms were treatment, period, time, and time x treatment, with steer and treatment x period x steer included as random terms. The replicate period was 1 hour, with treatment x steer. Treatment means were calculated using the LSMEANS option. Table 22 shows the results of the statistical analysis across the three treatment groups.

[0185] [Table 22] As shown in Table 22, feed dry matter intake for both the soybean meal and pongamia seed cake containing diets was greater than the hay control diet. Digestible dry matter intake for the soybean meal and pongamia seed cake containing diets was also greater than the hay control diet. No differences in feed digestibility were observed. No significant differences were observed between the soybean meal test group and the pongamia seed cake test group.

[0186] Example C2: Comparison of cattle fed forage-based diets with a pongamia-based protein supplement or a commercial protein supplement This example details a comparative study in which test cattle were fed one of three diets as shown in Table 23, including a diet containing a Pongamia protein supplement (Group A), a diet only (Group B), and a diet containing a commercial protein supplement (Group C).

[0187] The pongamia seed cake used in this study was the expeller-pressed, second-pressed, ethyl acetate-extracted pongamia meal prepared in Example B, Part II above. The pongamia seed cake was mixed with distillers' dried grains with solubles (DDGS) in a 30:70 weight ratio to prepare a pongamia protein supplement. Table 24 shows the karanjin and pongamol concentrations of the pongamia compositions used in this study, as determined by the microwave-assisted solvent extraction analytical method with ethyl acetate described in Example A4 above. Sweet Pro CattleKandi protein supplement was used as the commercial supplement for Group C cattle for the first 30 days of the study, and was replaced with Sweet Pro 16 supplement for the remainder of the study.

[0188] [Table 23] [Table 24] Each test group (nine steers total) consisted of Black Angus x Wagyu beef cattle (male castrates, 22-24 months old) housed in a 3-5 acre pen of the size designated for each test group. Drinking water and access to feed within each pen were provided ad libitum throughout the study. Test treatment groups (Group A) were each administered 1 kg of a Pongamia-based protein supplement in a common sampling tank accessible to all three cattle in the test treatment pen. Cattle in the positive control group (Group C) were given a standard SweetPro tank-based protein supplement formula. Cattle in Group B received no protein supplement.

[0189] Test cattle were monitored and received at least the full amount of the Pongamia-based protein supplement per day, and daily qualitative assessments were performed to ensure no negative health effects occurred throughout the study. Test cattle in all three groups were weighed approximately every 30 days to monitor weight gain. Weight gain recorded for each test cattle and treatment group is shown in Table 25.

[0190] [Table 25] As shown in Table 25, cattle diets containing a pongamia meal-derived protein supplement provided improved weight gain compared to the forage-only diet and showed comparable mean weight gain for the test steers compared to diets containing a commercial protein supplement. [Brief explanation of the drawings]

[0191] [Figure 1] FIG. 1 shows an exemplary process for analyzing Pongamia composition. [Figure 2] FIG. 2 shows an exemplary process for preparing a pongamia composition having a concentration of karanjin that is 100 ppm or less. [Figure 3A]Figure 3A shows a bar graph comparing the total concentrations (adjusted to ppm, relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various methanol-based extraction methods. [Figure 3B] Figure 3B shows a bar graph comparing the total concentrations (adjusted to ppm, relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various methanol-based extraction methods. [Figure 4A] Figure 4A shows a bar graph comparing the total concentrations (adjusted to ppm, relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various solvents (methyl tert-butyl ether, ethanol, hexane, toluene, and ethyl acetate) in combination with various extraction methods. [Figure 4B] Figure 4B shows a bar graph comparing the total concentrations (adjusted to ppm, relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various solvents (methyl tert-butyl ether, ethanol, hexane, toluene, and ethyl acetate) in combination with various extraction methods. [Figure 5A] Figure 5A shows a bar graph comparing the total concentrations (in ppm, adjusted for the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using microwave-assisted extraction with ethyl acetate or ionic liquid as the solvent. [Figure 5B] Figure 5B shows a bar graph comparing the total concentrations (in ppm, adjusted for the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using microwave-assisted extraction with ethyl acetate or ionic liquid as the solvent. [Figure 6A] Figure 6A shows a comparison of the total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using the various methods and solvents shown in Figures 3A-5B. [Figure 6B] Figure 6B shows a comparison of the total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using the various methods and solvents shown in Figures 3A-5B. [Figure 7A] FIG. 7A shows the observed total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various alkyl alkanoate solvents in combination with microwave-assisted solvent extraction. [Figure 7B] FIG. 7B shows the observed total concentrations (adjusted to ppm relative to the amount of starting material) of karanjin and pongamol extracted from deoiled pongamia seed cake using various alkyl alkanoate solvents in combination with microwave-assisted solvent extraction. [Figure 8A] Figure 8A shows a bar graph comparing the residual concentrations of karanjin and pongamol (adjusted to ppm relative to the amount of starting material) in pongamia seed cakes subjected to various mechanical treatments, as determined by analysis of microwave-assisted ethyl acetate extraction. [Figure 8B] Figure 8B shows a bar graph comparing the residual concentrations of karanjin and pongamol (adjusted to ppm relative to the amount of starting material) in pongamia seed cakes subjected to various mechanical treatments, as determined by analysis of microwave-assisted ethyl acetate extraction. [Figure 9A] Figure 9A shows a bar graph comparing the residual concentrations (ppm, adjusted for the amount of starting material) of karanjin and pongamol in pongamia seed cakes subjected to various mechanical treatments combined with solvent extraction treatments, as determined by analysis of microwave-assisted ethyl acetate extractions. [Figure 9B]Figure 9B shows a bar graph comparing the residual concentrations (ppm, adjusted for the amount of starting material) of karanjin and pongamol in pongamia seed cakes subjected to various mechanical treatments combined with solvent extraction treatments, as determined by analysis of microwave-assisted ethyl acetate extractions.

Claims

1. producing de-oiled pongamia seed cake by mechanically extracting pongamia oilseeds using expeller pressing; wherein the de-oiled pongamia seed cake contains 8 to 30% by weight of oil; preparing an extraction mixture by mixing the de-oiled pongamia seed cake with ethyl acetate; separating the extract mixture into miscella and a pongamia composition; wherein the pongamia composition is a meal having a karanjin concentration of 100 ppm or less.

2. 10. The method of claim 1, wherein the pongamia composition has a karanjin concentration of 100 ppm or less as measured by the following analytical method: mixing the Pongamia composition with ethyl acetate to prepare an extraction mixture; irradiating the extraction mixture with microwave radiation to provide an irradiated mixture; separating the irradiated mixture into an extracted pongamia composition and an ethyl acetate extract, and determining the concentration of karanjin in the ethyl acetate extract.

3. 3. The method according to claim 1 or 2, wherein the de-oiled pongamia seed cake has a karanjin concentration of at least 200 ppm.

4. 4. The method according to any one of claims 1 to 3, wherein the miscella has a karanjin concentration of about 4000 ppm or more.

5. 5. The method of any one of claims 1 to 4, wherein the pongamia composition has a pongamol concentration of less than 100 ppm.

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