SAPONIN CONTAINING EXTRACTS PREPARED FROM HESPERALOE USEFUL IN THE TREATMENT OF NON-HUMAN ANIMALS
Disclosed are animal feed compositions comprising a saponin extracted from a non-woody plant of the genus Hesperaloe. The saponin containing extracts, which generally constitute the water soluble fraction, are useful as additives to basal animal feed to reduce the production of ammonia. In certain embodiments, saponin containing compositions of the present invention may inhibit proteolytic microorganisms, reducing ammonia and improving animal welfare and animal performance. In other embodiments, administration of saponin containing compositions of the present invention to non-human animal inhibits the release of gaseous ammonia through the inhibition urease. The compositions of the present invention may be administered to non-human animals, particularly poultry, to reduce ammonia emissions. In certain embodiment, the animal feed composition comprises a basal animal feed and an extract from a non-woody plant of the genus Hesperaloe, the extract comprising 25(27)-dehydrofucreastatin, 5(6), 25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside C, furcreastatin, yuccaloiside, or a mixture thereof.
Plants produce a vast and diverse assortment of organic compounds, the great majority of which do not appear to participate directly in their growth and development. These substances, traditionally referred to as secondary metabolites or plant natural products, often are distributed among limited taxonomic groups within the plant kingdom. The functions of secondary metabolites remain largely unknown, although a number of compounds have been associated with attributes useful to the plants e.g. protection against herbivores and protection against microbial infection, as attractants for pollinators and seed-dispersing animals, and as compounds that influence competition among plant species (allelochemicals). There is a growing interest in plant natural products, since these products often have a wide range of applications in different kinds of industries, including pharmaceutical industries, cosmetic industries, food industries, detergent industries, and the like.
A particular group of plant secondary metabolites of interest are saponins. Saponins are glycosylated compounds classified as either triterpenoids, steroids, or steroidal glycoalkaloids. Saponins consist of one or two sugar moieties which are coupled to the aglycon (mono- and bisdesmosides, respectively). Saponins can be hydrolyzed to sapogenins and sugar moieties by acid hydrolysis or enzymatic methods. Saponins are generally water soluble high molecular weight compounds with molecular weights ranging from 600 to more than 2,000 daltons.
The asymmetric distribution of their hydrophobic (aglycone) and hydrophilic (sugar) moieties confers an amphipathic character to these compounds which are largely responsible for their detergent-like properties. The ability of lowering surface tension make saponins potentially well suited for use in the cosmetic and in the detergent industries.
Saponins also have the ability of forming insoluble complexes with cholesterol, which makes some of them suitable for use in the pharmaceutical industry as cholesterol lowering agents. Other saponins are associated with formation of immunostimulating complexes that are useful in vaccine strategies.
Currently, a major limitation to the broad exploitation of saponins is the fact that commercially available saponins are relatively expensive. The expenses is due in large part to the limited number of plant extracts having significant amounts of saponins. Currently, commercially available plant extracts containing saponins include Saponaria officinalis, Quillaia bark and stem, Castanea sativa seeds, and extracts of various Yucca species.
Plant extracts containing saponins are thus of general interest within a wide range of different industries. There is therefore a growing need in the art for alternative sources of saponin extracts and these plant sources should preferably be cheap, easy to obtain, and preferably the saponin content should be relatively high.
SUMMARYThe present inventors have now discovered that water soluble solids extracted from non-woody plants of the genus Hesperaloe comprise one or more saponins that may be useful as additives to basal animal feed to reduce the production of ammonia. In certain embodiments, saponin containing compositions of the present invention may inhibit proteolytic microorganisms, reducing ammonia and improving animal welfare and animal performance. In other embodiments, administration of saponin containing compositions of the present invention to non-human animal inhibits the release of gaseous ammonia through the inhibition urease, which catalyze hydrolysis of urea into carbon dioxide and ammonia resulting in the release of gaseous ammonia.
Accordingly, in one embodiment, the present invention provides novel pharmaceutical, dietary supplements and food ingredient compositions that are safe for non-human animal consumption, reduce ammonia emissions, have beneficial health effects, and can enhance the growth animals treated therewith.
In other embodiments, the present invention provides novel pharmaceutical, dietary supplements and food ingredient compositions comprising at least one component selected from the extract(s), fraction(s), active compound(s) and phytochemical(s) or mixtures thereof derived from non-woody plants of the genus Hesperaloe including, for example, Hesperaloe funifera, Hesperaloe nocturne, Hesperaloe parviflora, and Hesperaloe chiangii. The novel pharmaceutical, dietary supplements and food ingredient compositions may optionally contain one or more of pharmaceutically and dietetically acceptable phytochemical actives, diluents, vehicles, carriers and actives or mixtures thereof.
In a particularly preferred embodiment, the present invention provides an animal feed additive comprising water soluble solids derived from non-woody plants of the genus Hesperaloe. Preferably the water soluble solids comprise at least about 2 wt % saponin, more preferably at least about 5 wt % saponin and still more preferably at least about 10 wt % saponin, such as from about 2 to about 30 wt % saponin, such as from about 5 to about 25 wt % saponin, wherein the weight percentages are based upon the total bone dry weight of the water soluble solids. In a particularly preferred embodiment, the present invention provides an animal feed composition comprising a basal animal feed and a mixture of saponins derived from non-woody plants of the genus Hesperaloe. In certain embodiments the mixture of saponins may comprise one or more of 25(27)-dehydrofucreastatin (
In other embodiments the present invention provides methods of treating, controlling or preventing of one or more conditions in non-human animals, especially birds and more particularly poultry by administering thereto a pharmaceutical, dietary supplement or food ingredient composition comprising at least one component selected from the extract(s), fraction(s), active compound(s) and phytochemical(s), or mixtures thereof, derived from non-woody plants of the genus Hesperaloe. For example, saponin containing compositions derived from Hesperaloe biomass may be administered to non-human animals to reduce environmental ammonia and odor. In a particularly preferred embodiment, the saponin compositions of the present invention may be administered orally to poultry to reduce environmental ammonia and odor.
In another embodiment the present invention provides an animal feed composition comprising basal animal feed; and a water soluble composition extracted from a non-woody plant of the genus Hesperaloe and comprising at least one saponin. In a particularly preferred embodiment total amount of saponin, based upon the total mass of feed, ranges from about 1.0 to about 30.0 grams per 100 kilograms of feed.
As used herein the term “biomass” generally refers to whole plants and plant organs (i.e., leaves, stems, flowers, roots, etc.) of the genus Hesperaloe including, for example, Hesperaloe funifera, Hesperaloe nocturne, Hesperaloe parviflora, and Hesperaloe chiangii.
As used herein the term “bagasse” generally refers to biomass that has been cut to size and then subjected to high pressure so that the resulting material has less liquid than the biomass from which it is derived. High pressure can be achieved by using compression pressure, such as that provided by machines such one or more opposed counter-rotating rolls, a mechanical press, a screw press as well as by direct hydraulic pressure and other processes to apply pressure to the biomass and remove intercellular and intracellular liquid.
As used herein the term “milling” generally refers to the application of sufficient pressure to force the intercellular and intracellular liquid from the biomass.
As used herein the term “saponin” generally refers to glycosides comprising a sugar component referred to as a glycone and a non-sugar component referred to as an aglycone. Depending on the structure of the aglycone the saponin may be classified as a triterpenoid saponin, illustrated in
As used herein the term “water soluble solids” generally refers to dry matter which remains after the extract has been centrifuged, filtered and all water is evaporated. The procedure for measuring water soluble solids of a biomass extract of the present invention is described in detail in the Test Methods section below. Water soluble solids may be expressed on a percentage basis relative to the mass of bone dry biomass.
As used herein the term “water insoluble solids” generally refer to the fraction of extract that is removed by centrifugation and filtration in the course of measuring water soluble solids, as described in the Test Methods section below.
As used herein, “reducing ammonia emissions” from a non-human animal treated with saponin containing composition of the present invention refers to reducing emitted ammonia gas relative to an untreated non-human animal. In some embodiments, the reduction is from about 10 to about 30% in ammonia emissions when compared to an untreated animal. In some embodiments, the reduction is from about 15 to about 25% in ammonia emissions when emissions are standardized to animal live weight. In some embodiments, the reduction of ammonia emissions from the animal does not significantly negatively affect the animal, such as, for example, lowering body weight, or decreasing eating quality.
As used herein, “reducing carbon dioxide emissions” from a non-human animal treated with saponin containing composition of the present invention refers to reducing emitted carbon dioxide gas relative to an untreated animal. In some embodiments, the reduction is about 5% in carbon dioxide emissions when compared to an untreated animal when carbon dioxide emissions are standardized to animal live weight.
The term “effective amount”, in the context of administration, refers to the quantity of saponin administered to a non-human animal in need of treated, which amount is sufficient to reduce ammonia or carbon dioxide emissions from the animal as compared to an untreated animal.
DETAILED DESCRIPTIONThe present invention relates to novel pharmaceutical, dietary supplements and food ingredient compositions comprising at least one component selected from the extract(s), fraction(s), active compound(s) and phytochemical(s) or mixtures thereof derived from non-woody plants of the genus Hesperaloe including, for example, Hesperaloe funifera, Hesperaloe nocturne, Hesperaloe parviflora, and Hesperaloe chiangii, optionally containing one or more of pharmaceutically and dietetically acceptable phytochemical actives, diluents, vehicles, carriers and actives or mixtures thereof. In a particularly preferred embodiment, the present invention provides an animal feed additive comprising water soluble solids derived from non-woody plants of the genus Hesperaloe.
The novel saponin containing pharmaceutical, dietary supplements and food ingredient compositions may be administered to a non-human animal to reduce gas emissions, particularly ammonia and carbon dioxide. The compositions of the present invention are particularly well suited for the treatment of non-human-animals including, for example, bovine, fowl, porcine, ovine, and equine species. By way of example, the methods and compositions of the invention can be used for the treatment of cattle, chickens, turkeys, ducks, quail, geese, pigs, and sheep. In a particularly preferred embodiment, the methods and compositions of the present invention can be used for the treatment of poultry and more particularly may be administered to poultry to reduce gas emissions.
In certain embodiments, the pharmaceutical, dietary supplements and food ingredient compositions comprise a mixture of saponins. In this manner the compositions of the present invention may comprise least 2 wt %, based upon the bone dry weight of the composition, saponins as measured by the total saponin assay set forth in the Test Methods section below. In a particular embodiment, the saponin containing composition used in accordance with the invention comprises at least about 2 wt % saponin, more preferably at least about 5 wt % saponin and still more preferably at least about 10 wt % saponin, such as from about 2 to about 30 wt % saponin, such as from about 10 to about 25 wt %. It is believed that the effects of the composition are related to the total amount of saponins present. Thus, one of skill in the art will appreciate that if a certain amount of saponins is desired it can be achieved either through varying the volume of a certain concentration composition administered, varying the concentration of a certain volume of a composition, or both.
Saponins useful in formulating the pharmaceutical, dietary supplements and food ingredient compositions of the present invention are preferably extracted from non-woody plants of the genus Hesperaloe. Saponins derived from Hesperaloe may have at least one of the following aglycones or genins: kammogenin, manogenin, gentrogenin, hecogenin, tigogenin, sarsapogenin, chlorogenin and gitogenin or their corresponding isomer or oxidized or reduced forms with at least one of the following glycosidic moieties (in the form of acid or salt): glucose, xylose, rhamnose, arabinose, or galactose. In other embodiments the steroidal saponins may comprise agamenoside, agaveside, agavoside, magueyside, agavasaponi, cantalasaponin, sisalsaponin, gabrittonoside, dongnoside or amolonin, or other steroidal saponins.
Extractives may be recovered from non-woody plants of the genus Hesperaloe by extracting biomass, particularly the leaves and more particularly the leaves above the crown of the plant, with at least one solvent selected from the group consisting of water, methanol, ethanol, butanol, and isopropanol and mixtures thereof. For example, in one embodiment, the process comprises contacting biomass with an extractant solution comprising water and separating the water soluble fraction from the insoluble biomass fraction. In other embodiments the extractant solution may comprise, in addition to water, a surfactant, a solvent and optionally extract-bearing juice. The extract-bearing juice can come from, for example, an earlier extraction step or an earlier milling step.
A simple water extraction of Hesperaloe biomass may yield a crude aqueous extract comprising saccharides, polysaccharides, inorganic salts, saponins and sapogenins. A crude extract may also be produced using methanol as a solvent, or a mixture of methanol and water, to extract biomass, which may have been previously extracted with acetone or diethyl ether to remove lipids and pigments. In other instances, the biomass may be extracted with a 4:1 ethanol-water solvent, followed by subsequent defatting of the extract with a non-polar solvent such as hexane. In certain instances, the defatted extract may be subjected to further treatment to isolate specific water soluble components, such as saponins, which may be purified from the defatted extract by mixing with butanol and separating the butanol phase to yield a mixture of saponins that are substantially free from proteins and free saccharides and polysaccharides.
Hot aqueous extractants can also be used. For example, in one embodiment water soluble solids may be extracted from Hesperaloe biomass, particularly the leaves, by extracting the biomass with hot aqueous ethanol or isopropanol (75 to 95% by weight alcohol). The aqueous alcohol extraction fluid may then be filtered and concentrated, and the fat-soluble material may be removed by mixing the extraction fluid with a non-polar solvent such as hexane. A substantially pure saponin composition may then be prepared by further extracting defatted extract with a polar solvent such as butanol.
For the purpose of preparing the compositions of the present invention, and for use in the present method, a simple aqueous extract may be preferred, although other extraction methods are within the scope of the present invention. In a particularly preferred embodiment, Hesperaloe biomass may be cut to size, pressed, and extracted with an aqueous solvent to remove water soluble extracts such as inorganic salts, saccharides, polysaccharides, organic acids and saponins. The water soluble extracts are collected and may be concentrated by techniques well known in the art such as, for example, evaporation, spray-drying, drum drying and the like. The extract may be concentrated until it has a solids content of about 20 to about 100% solids by weight, such as from about 20 to about 95% solids by weight, such as from about 20 to about 80% solids by weight.
In a particularly preferred embodiment water soluble extracts are concentrated by spray drying by feeding the extract solution to atomizing equipment. Suitable atomizing equipment includes, but is not limited to, a rotary wheel atomizer, a pressure nozzle atomizer, and a dual fluid nozzle atomizer. Rotary wheel, pressure nozzle and dual fluid nozzle atomizers are known to those of ordinary skill in the art and include those in spray dryers commercially available from a variety of sources, such as GEA Process Engineering.
In certain embodiments biomass may be milled to separate the bagasse and water soluble solids using a roll, screw, and other forms of presses. In certain preferred embodiments biomass is passed between one or more nips of opposed counter-rotating rolls to maximize the mechanical removal of juice. The bagasse can then be contacted with the juice in a subsequent milling step, as will be described more fully below. In certain instances, the biomass may be cut to size and cleaned prior to milling. Cutting and cleaning may be carried out using well known methods in the art. In a particularly preferred embodiment, the biomass is cleaned to remove debris such as dirt without the use of water or other solvents.
In other embodiments the water soluble solids may be recovered from biomass by diffusion. In diffusion, the biomass brought into contact with the liquid to extract the liquid components. Usually, the biomass is prepared by first cutting, but not shearing or crushing so as to minimize the damage to fibers and avoid the creation of an excessive amount of fines. The prepared biomass is then washed repeatedly, usually using a solvent, to extract the liquid contained in the biomass. The solvent can be any of the foregoing solvents. An exemplary treatment solvent is water, particularly hot water such as water heated to a temperature from about 40 to about 90° C. The solvent can be circulated and reused so that the solvent used for a first extraction is reused as a solvent to extract subsequent prepared biomass.
Various types of diffusers are known in the art and can be adapted for use with biomass as described herein. Suitable diffusers include a ring diffuser, a tower diffuser, or a drum diffuser. Exemplary diffusion systems are discussed, for example, in U.S. Pat. Nos. 4,182,632, 4,751,060, 5,885,539 and 6,193,805 the contents of which are hereby incorporated in a manner consistent with the present disclosure. Numerous other diffusion methods and devices for the diffusion method are known and can be adapted for use in the methods described herein. One such diffuser is the continuous-loop, counter-current, shallow-bed Crown Model III Percolation Extractor, commercially available from Crown Iron Works, Blaine, Minn.
The biomass, cut or uncut, may be extracted by any suitable extraction process as discussed above. In a particularly preferred embodiment, the solvent used for extraction comprises water. One of skill in the art will recognize the ratio of extraction solvent to biomass will vary based on the solvent, the amount of biomass to be extracted and the extraction procedure. In certain preferred embodiments, the extraction solvent is water and the ratio of extraction solvent to biomass, on the basis of liters of extraction solvent to kilogram of bone-dry biomass, is from about 1:5 to about 1:100, such as from about 1:5 to about 1:50 and more preferably from about 1:5 to about 1:20.
The pH of the extraction solvent can be between about pH 5.0 and 8.0, such as, for example, between about pH 6.0 and about pH 8.0, between about pH 6.5 and about pH 7.5. In a particular embodiment, the extraction solvent is water having a pH between about pH 6.5 and about pH 7.5. In those embodiments where extraction includes imbibition with a crude juice, the imbibition fluid may have a pH from about 4.0 to about 5.0.
The extraction may be carried out at temperatures between about 25 and about 90° C., such as, for example, between about 30 and about 80° C., between about 35 and about 75° C., between about 40 and about 70° C., between about 45 and about 65° C. or between about 50 and about 60° C.
In embodiments where the extraction process is a batch extraction process, the duration of extraction may range from about 0.25 to about 24 hours, such as, for example, from about 0.5 to about 2 hours, from about 1 to about 8 hours, or from about 1 to about 6 hours.
In embodiments where the extraction process is a continuous process, the duration of extraction may range from about 0.25 to about 5 hours, such as, for example, from about 0.5 to about 3 hours.
After extraction the water insoluble biomass material may be separated from the water soluble solids by filtration to provide a filtrate containing inorganic salts, saccharides, polysaccharides, organic acids and saponins (referred to herein as the “first filtrate”). Separation can be achieved by any suitable means including, but not limited to, gravity filtration, a plate-and-frame filter press, cross flow filters, screen filters, Nutsche filters, belt filters, ceramic filters, membrane filters, microfilters, nanofilters, ultrafilters or centrifugation. Optionally various filtration aids such as diatomaceous earth, bentonite, zeolite, and the like, may also be used in this process.
After separation, the pH of the first filtrate may be adjusted to remove additional impurities. In one embodiment, the pH of the first filtrate can be adjusted to between about 8.5 and about 10.0 by treatment with a base, such as, for example, calcium oxide or hydroxide (about 1.0% from the volume of filtrate) with slow agitation.
In other instances the first filtrate may be concentrated or subjected to further processing to isolate a particular mixture of saponins or individual saponins. In other instances, the clarified juice may be further purified to remove saccharides, polysaccharides, and organic acids to yield composition comprising saponins and being substantially free from remove saccharides, polysaccharides, and organic acids. The foregoing impurities may be removed by mixing the juice or first filtrate with a water-immiscible polar solvent such as alcohols having from 4 to 6 carbon atoms, such as butyl, amyl, hexyl and cyclohexyl alcohols. The solvent phase containing the saponin may be subjected to further treatment to separate the saponin from the alcohol phase. This can be accomplished in various ways including, for example, by cooling, by dehydrating the solvent extract, or by adding an organic solvent which is miscible with the alcohol solvent but in which the saponin is insoluble. Suitable precipitating solvents include, for example, diethyl ether, petroleum ether, acetone, and chloroform.
In a particularly preferred embodiments, the saponin is separated from the alcohol by flash evaporation. Flash evaporation is a technique known in preparative chemistry for the rapid removal of a volatile component from a liquid mixture. The volatile liquid is removed from solution by rapid conversion to a vapor phase by creating a thin film of the solution over a large surface area under reduced pressure often accompanied by an increase of temperature of the solution above ambient but less than the boiling point of the solution at atmospheric pressure. The actual thickness of the film and the area over which it is applied is chosen to provide optimum evaporation and ease of use, but evaporation may be substantially instantaneous (hence the name “flash” evaporation). Flash evaporation avoids the prolonged use of high temperatures that may degrade the intended product and has the ability to remove almost all of the alcohol component (which makes the remaining solution suitable for the preferred practice of spray drying employed in the next step. The alcohol may be recovered from this step and reused in the extraction process.
The saponin content of the alcohol extract can be further increased by passage over an ultrafiltration membrane without significant alteration to or loss of the saponin composition. This concentrated saponin fraction where the saponin content is in the range of 85-90%, can then be further purified in a liquid state or reduced to a dry state. Individual saponins may be recovered by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC. Alternatively, the alcohol extract containing saponins can be fractionated directly by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC.
In still other embodiments saponins may be purified from juice prepared according to the present invention comprises the steps of mixing the juice with a salt and a solvent to form a first solution. The solvent may comprise one or more solvents selected from acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme, 1,2-dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide, hexamethylphosphorous triamide, hexane, methanol, methyl-t-butyl ether, methylene chloride, N-methyl-2-pyrrolidinone, pentane, perchloroethylene, petroleum ether, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, triethylamine, trifluorotoluene, water, xylene, or any combination of the forgoing. In some embodiments the solvent is water. The salt may be selected from an alkali metal salt, an alkaline earth salt, a transition metal salt, an ammonium salt, or combinations of the forgoing. In certain preferred embodiment the salt added to the plant extract to form the solution is an alkaline earth metal salt. In particularly preferred embodiments the salt is calcium chloride (CaCl2)), magnesium chloride (MgCl2), or a mixture thereof.
The pH of the first solution is generally adjusted to a pH from about 6.0 to about 9.0, such as from about 6.0 to about 8.0, such as from about 6.0 to about 7.0. At least one phosphate may then be added to the first solution to form an ion-polysaccharides complex precipitate. Useful phosphates include, for example, sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), or sodium hydrogen bisphosphate (Na2H2PO7).
The precipitated ion-polysaccharides complex may be removed by filtration to yield a second solution, which may be further clarified to produce an extract of purified saponins. Optionally, the extract can be concentrated by any filtration technique known in the prior art. Preferably, the concentration of the extract of purified saponins is carried out by nanofiltration, ultrafiltration and diafiltration, or any combination of these techniques. In some embodiments, the saponin extract is substantially free of proteins. In some embodiments, the saponin extract is substantially free of polysaccharides. In some embodiments, the saponin extract is substantially free of phenolic compounds.
The amount of water soluble solids recovered from biomass may vary depending on the extraction efficiency, however, in certain instances from about 100 to about 400 grams of water soluble solids may be extracted per kilogram of bone dry biomass, such as from about 120 to about 350 grams per kilogram, such as from about 150 to about 300 grams per kilogram. Of the extracted water soluble solids, the total saponins may comprise at least about 5 wt %, such as at least 10 wt %, such as at least 20 wt %, such as from about 5 to about 40 wt %, such as from about 10 to about 30 wt %, based upon the bone dry weight of the water soluble solids. In certain instances the amount of total saponins that may be extracted from biomass may range from about 10 to about 400 grams per bone dry kilogram of biomass, such as from about 20 to about 300 grams, such as from about 25 to about 200 grams, such as from about 10 to about 100 grams. In certain instances, the amounts of materials (on bone dry grams per kilogram of bone dry biomass) removed from the biomass during the extraction process may range as set forth in Table 1, below.
The total amount of saponins that may be extracted from Hesperaloe biomass according to the present invention may range from about 10 to about 100 grams per bone dry kilogram of biomass, such as from about 20 to about 80 grams, such as from about 25 to about 75 grams. The saponins may be provided as part of a crude juice, as part of a dried water soluble solids compositions, as a partially purified compositions or as a substantially pure composition comprising a mixture of saponins.
In certain embodiments saponins extracted from Hesperaloe biomass may comprise one or more saponins selected from 25(27)-dehydrofucreastatin (
Compositions useful in the present invention may be prepared by blending an aqueous extract from Hesperaloe biomass with one or more polyhydroxy alcohols including glycerol, propylene glycol, polyalkylene glycol such as polyethylene glycol and polypropylene glycol, and polyglycerol. Preferred polyhydroxy alcohols have less than about eight carbon atoms. Glycerol and propylene glycol are particularly preferred polyhydroxy alcohols.
The composition may also comprise saccharides, which may be present in the aqueous extract or may be added after extraction during formulation. Saccharides useful in compositions of the present invention include monosaccharides such as glucose, disaccharides such as sucrose and polysaccharides such as starch.
In still other embodiments compositions in accordance with embodiments of the invention can include various other additives known in the art to have benefits for the maintenance and well-being of non-human animals. By way of example, compositions can also include components such as Vitamin E, Vitamin A Propionate, Vitamin A Palmitate, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12, D-Activated Animal Sterol (source of Vitamin D3), yeast components, dried egg solids, dried casein, and dried whey.
Saponin containing compositions of the present invention may be in liquid or dry forms. By way of example, a saponin containing Hesperaloe extract may be dried into a powder form. In this form, the saponin containing composition may be administered to an animal as a pill or bolus or mixed in with other components such as a feed ration. For example, dry powder formulations of saponin containing compositions may be added to the feed ration via a micro-ingredient machine or added to a feed mix truck and mixed thoroughly to assure even distribution in the feed. Saponin containing Hesperaloe extract may also be in liquid form with an amount of a carrier liquid such as water. In this form, the saponin containing composition may be administered to an animal as a liquid drench.
Saponin containing compositions of the present invention may be administered to non-human animals in need there of as a single dose, as multiple doses as part of a feeding regiment. For example, a non-human animal may receive an initial dose and then receive subsequent maintenance doses in lesser amounts. A non-human animal may receive multiple doses of a saponin containing composition in one day or may receive multiple doses over multiple days.
In certain embodiments, non-human animals, especially birds and more particularly poultry, can be treated with a saponin containing composition in an amount that is effective to improve the feeding characteristics of an animal and/or improve the production characteristics of an animal in comparison to an untreated control animal. Production characteristics can include carcass quality grades, yield grades, average daily gain, and the like. In an embodiment, when administered as a feed additive to poultry, the amount of saponins per 100 kilograms of feed is preferably at least about 1 g/100 kg, such as from about 1 to about 50 g/100 kg, such as from about 2 to about 40 g/100 kg.
In particularly preferred embodiment the administration of saponins to a non-human animal in need thereof is carried out by including the saponins in an animal feed. The basal animal feed may be a dry feed, or a liquid feed and the compositions of the present invention may be formulated as a liquid, a slurry, a dry powder, a dry granular mix, a paste, a pellet, or a block. Suitable feeds may be prepared by applying saponins containing extracts, such as my mixing or spraying, with suitable basal animal feeds commonly employed in the feeding of animals. Typical basal animal feeds useful in the present invention may comprise one or more of corn meal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybean mill run, cottonseed oil meal, bone meal, ground corn, corncob meal, wheat middlings, limestone, dicalcium phosphate, sodium chloride, urea, distillers dried grain, vitamin and/or mineral mixes, cane molasses or other liquid carriers and the like. Such basal animal feeds promote a uniform distribution and administration of the saponins.
In still other embodiments saponin containing extracts of Hesperaloe may be formulated as urease inhibiting compositions. The as urease inhibiting compositions of the present invention may comprise an amount of Hesperaloe extract sufficient to provide urease inhibiting activity. In certain instances, when administered to inhibit urease, the total saponins in the composition is at least about 1 wt %, and more preferably at least about 2 wt %, and still more preferably at least about 5 wt %, based on the total weight of the composition. In certain preferred embodiments the urease inhibiting compositions of the present invention comprise one or more saponins extracted from Hesperaloe where the saponins include 25(27)-dehydrofucreastatin (
Urease inhibiting compositions may be provided as animal feed, thereby simplifying dosing. The compositions may include a saponin composition extracted from Hesperaloe and an animal feedstuff, such as those discussed above, and may be formulated in various ways. For example urease inhibiting compositions can be formulated as a liquid, slurry, dry powder, dry granular mix, paste, pellets, block, or the like. Compositions may be administered to an animal as a pill, a bolus, or a liquid drench. In certain instances urease may be inhibited, and ammonia emissions reduced, by administering an effective amount of saponin derived from Hesperaloe along with the animal's feed ration. For example, in some embodiments, a composition containing saponin derived from Hesperaloe, such as a liquid composition, can be mixed in with an animal's water.
Test Methods Water Soluble SolidsTotal biomass water soluble solids may be determined using an Accelerated Solvent Extraction system (ASE) such as a Dionex™ ASE™ 350 (Thermo Fisher Scientific, Waltham, Mass.). Approximately 10 grams of harvested biomass is dried to a constant weight in an oven, typically 4 hours at 125° C. After drying 1.5-2.0 grams of the bone dry biomass is accurately weighed and the weight (Wb) recorded to the nearest 0.001 gram. Using water as the solvent, biomass is extracted using the conditions set forth in the table below. The ratio of biomass to solvent is generally 21:1 and five consecutive water extraction cycles are performed. At the end of each extraction cycle, the liquid phase is collected, dried under vacuum at approximately 40° C. and the weight of the dried material (Wi) is recorded to the nearest 0.001 g. The total weight of water soluble solids (We) is calculated by summing the weight of solids recovered from each extraction cycle (Wi). Total water soluble solids as a percentage of bone dry biomass is then determined using the following equation: Water Soluble Solids (wt %)=We/Wb*100.
The total water soluble solids in biomass extract may be determined by withdrawing an appropriate aliquot, typically about 10-50 ml, transferring to clean, pre-weighed centrifuge tube. The tube is weighed to the nearest 0.001 gram and centrifuged at 7000 rpm for 20 minutes. The weight of extract (W1) is calculated. The supernatant was then transferred to clean, pre-weighed beaker (D0), and weighed. The beaker containing the sample is then placed at 140° C. in a hot air oven for overnight drying. The beaker is removed from the oven and desiccated to cool to room temperature then weighed to the nearest 0.001 gram (D1). The weight percentage of soluble solids, based upon the weight of the extract, is determined using the formula below:
D1=mass of empty beaker+dried soluble solids, D0=mass of empty beaker, W1=initial bone dry mass of biomass.
Total SaponinsTotal saponins were measured generally as described in Makkar, Harinder P. S., Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites, chapter 17, pp 93-100. A standard saponin solution was prepared by weighing 10 mg of diosgenin (MilliporeSigma >93%), dissolving in 16 mL of methanol and adding 4 mL of distilled water. The solution was mixed thoroughly to yield a 0.5 mg/mL diosgenin solution in 80% methanol solvent. The standard was used to produce a calibration curve by transferring various amounts of the standard (0, 10, 20, 40, 60, 80, and 100 μL) into 13-mm glass test tubes. A solution of 80% aqueous methanol was added to a total volume of 100 μL.
Prior to testing samples of biomass extract were adjusted to about 0.5 wt % total solids by dilution with water to ensure absorbency result fell along the saponin standard calibration curve range. Samples of diluted extract (20-μL) were pipetted into 13-mm glass test tubes and the volume was brought up to 100 μL with 80 μL methanol. Each sample was tested in triplicate.
To each sample 100 μL of vanillin reagent (prepared by dissolving 800 mg of vanillin in 10 mL of 99.5% ethanol (analytical grade)) and then 1.0 mL of 72% (v/v) sulfuric acid (72% (v/v) sulfuric acid prepared by adding 72 mL of sulfuric acid (analytical grade, 95%, w/w) to 28 mL of distilled water) were added. Solutions were mixed well and heated at 60° C. for 10 minutes. Samples were then cooled in an ice bath and 1 mL of solution was transferred into respective cuvette and absorbance at 544 nm was read. The total mass of saponins in the sample may be calculated based upon the standard absorbency curve as follows:
Saponin (μg)=[Slope]×Measured Absorbency−[Intercept]
The inventive extract was prepared by forage harvesting mature Hesperaloe funifera leaves above the crown, cutting the leaves into pieces ranging from about 0.50 to about 8.0 cm and pressing the cut biomass using a sugar cane tandem press, each mill of the tandem press having 3-rollers. The biomass was passed through the tandem mill three times. Imbibition water was added prior to first mill in the tandem. The crude juice was collected and passed through 25 mm filter and heated to boiling in a flat pan evaporator (Leader Evaporator Company, Swanton, Vt.) to concentrate the extract to 29% solids. The water-soluble solids comprised 21 wt % total saponins, based upon the bone dry weight of water soluble solids. The concentrated juice was diluted with water to provide a composition comprising 5 wt % total saponins.
For comparison a composition containing 5 wt % total saponins extracted from Yucca was prepared. The Yucca derived saponin are marketed under the tradename FOAMATION™ (commercially available from Ingredion, Westchester, Ill.) and were provided as 50 wt % total saponins. FOAMATION™ was diluted with water to provide a composition comprising 5 wt % saponins.
To test the efficacy of urease inhibitors, 2.0 mL of a putative inhibitor and 2.0 mL Jack Bean urease (diluted to 10 mg/mL) (Sigma Diagnostics, St. Louis, Mo., Sigma U-4002) were added to a cuvette. To this mixture, 18 mL of urine is added to the cuvette and the mixture is incubated for 2 hours. The liberation of ammonia was determined using Gastec's standard ammonia (NH3) detector tube system (Model 1M 003MJ1, supplied by Gastec Corporation, Kanagawa, Japan). The reduction in ammonia achieved with the inventive compositions is shown in Table 2, below.
First Embodiment: An animal feed composition comprising a basal animal feed; and a water-soluble extract of a non-woody plant of the genus Hesperaloe comprising at least one saponin.
Second Embodiment: The animal feed composition of the first embodiment wherein the composition is a liquid, a slurry, a dry powder, a dry granular mix, a paste, or a solid.
Third Embodiment: The animal feed composition of the first or the second embodiment wherein the basal animal feed comprises one or more of corn meal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybean mill run, cottonseed oil meal, bone meal, ground corn, corncob meal, wheat middlings, limestone, dicalcium phosphate, sodium chloride, urea or distillers dried grain.
Fourth Embodiment: The animal feed composition of any one of the first through third embodiments wherein the basal animal feed comprises water and is a liquid.
Fifth Embodiment: The animal feed composition of any one of the first through fourth embodiments wherein the total amount of saponin ranges from about 1 to about 100 g per 100 kg of animal feed.
Sixth Embodiment: The animal feed composition of any one of the first through fifth embodiments wherein the at least one saponin consists of kammogenin, manogenin, gentrogenin, hecogenin, tigogenin, sarsapogenin, chlorogenin and gitogenin or their corresponding isomer or oxidized or reduced forms and at least one glycosidic moiety selected from glucose, xylose, rhamnose, arabinose, or galactose.
Seventh Embodiment: The animal feed composition of any one of the first through sixth embodiments wherein the at least one saponin is 25(27)-dehydrofucreastatin (
Eighth Embodiment: The animal feed composition of any one of the first through seventh embodiments wherein the mass of the water-soluble solids ranges from about 100 to about 1,000 g per kilogram of basal animal feed.
Ninth Embodiment: The animal feed composition any one of the first through eighth embodiments wherein the water-soluble solids comprise from about 10 to about 25 wt % saponin.
Tenth Embodiment: Administering to a non-human animal the animal feed composition of any one of embodiments one through nine to reduce environmental ammonia and odor, to provide a hypocholesterolemic effect, reduce inflammation, provide an anti-protozoal effect, control parasitic nematodes, promote weight gain or improve feed conversion efficiency.
Eleventh Embodiment: Administered orally to poultry to reduce environmental ammonia and odor the animal feed composition of any one of embodiments one through nine.
Twelfth Embodiment: A urease inhibitor comprising an extract of a non-woody plant of the genus Hesperaloe comprising at least one saponin.
Thirteenth Embodiment: A proteolytic microorganism inhibit comprising an extract of a non-woody plant of the genus Hesperaloe comprising at least one saponin.
Claims
1. An animal feed composition for reducing gaseous emissions, the feed comprising:
- a. a basal animal feed; and
- b. an extract from a non-woody plant of the genus Hesperaloe, the extract comprising 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside C, furcreastatin, yuccaloiside, or a mixture thereof.
2. The animal feed composition of claim 1 comprising from about 1 to about 100 g of total saponins per 100 kg of basal animal feed.
3. The animal feed composition of claim 1 wherein extract solids further comprise saccharides, proteins, lipids, or a mixture thereof.
4. The animal feed composition of claim 1 wherein the extract substantially free from saccharides, proteins, and lipids.
5. The animal feed composition of claim 1 wherein the extract comprises at least about 90% water soluble solids.
6. The animal feed composition of claim 1 wherein the extract comprises less than about 2% water insoluble solids.
7. The animal feed composition of claim 1 wherein the extract comprises 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside, 5(6)-disdehydroyuccaloiside, furcreastatin and yuccaloiside.
8. The animal feed composition of claim 1 wherein the non-woody plant is selected from Hesperaloe funifera, Hesperaloe nocturne, Hesperaloe parviflora and Hesperaloe chiangii,
9. The animal feed composition of claim 1 comprising from about 100 to about 1,000 g water soluble solids per kilogram of basal animal feed.
10. The animal feed composition of claim 1 wherein the extract comprise from about 5 to about 25 wt % saponin.
11. The animal feed composition of claim 10 wherein the saponins comprise a mixture of 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside, furcreastatin and yuccaloiside C.
12. A method of reducing gaseous emissions from a non-human animal comprising administering thereto a feed composition comprising a basal animal feed and an extract from a non-woody plant of the genus Hesperaloe, the extract comprising at least one saponin.
13. The method of claim 12 wherein the total amount of saponin ranges from about 1 to about 100 g per 100 kg of animal feed.
14. The method of claim 12 wherein the extract further comprise saccharides, proteins, and lipids.
15. The method of claim 12 wherein the extract is substantially free from saccharides, proteins, and lipids.
16. The method of claim 12 wherein the at least one saponin is selected from 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside, furcreastatin and yuccaloiside C.
17. The method of claim 12 wherein the extract comprise a mixture of 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloiside C, 5(6)-disdehydroyuccaloiside, furcreastatin and yuccaloiside C.
18. The method of claim 12 wherein the feed composition comprises from about 100 to about 1,000 g water soluble solids per kilogram of basal animal feed.
19. The method of claim 12 wherein the extract comprises from about 10 to about 25 wt % saponin.
20. The method of claim 12 wherein the gas emissions are ammonia or carbon dioxide.
21. The method of claim 12 wherein the non-human is a bovine, a fowl, a porcine, an ovine or an equine species.
22. The method of claim 12 wherein the feed composition is administered orally.
23. The method of claim 12 wherein the feed composition is a liquid, a slurry, a dry powder, a dry granular mix, a paste, or a solid.
24. The method of claim 12 wherein the basal animal feed comprises one or more of corn meal, corncob grits, soybean meal, alfalfa meal, rice hulls, soybean mill run, cottonseed oil meal, bone meal, ground corn, corncob meal, wheat middlings, limestone, dicalcium phosphate, sodium chloride, urea or distillers dried grain.
25. The method of claim 12 wherein the basal animal feed comprises water and is a liquid.
Type: Application
Filed: Jun 21, 2021
Publication Date: Aug 24, 2023
Inventors: Ning Wei (Roswell, GA), Thomas G. Shannon (Neenah, WI)
Application Number: 18/011,341
