HK1162127B - Oil body associated protein compositions and methods as well as use thereof for reducing the risk of cardiovascular disease - Google Patents

Description

Compositions and methods for oil body associated proteins for reducing the risk of cardiovascular disease and uses thereof

the present application is a divisional application of an invention patent application having an application date of 2003, month 4 and day 17 and an application number of 038140152, and having the same name as the present invention.

This application claims priority from U.S. provisional patent application serial No. 60/373,460, filed on 18/4/2002, the disclosure of which is hereby specifically incorporated by reference in its entirety.

Technical Field

The present invention relates to novel compositions, polypeptides and methods for reducing cholesterol levels and reducing the risk of cardiovascular disease. More particularly, the present invention relates to novel compositions for the prevention and treatment of elevated cholesterol and cardiovascular diseases, including oil body associated proteins, and uses thereof.

Background

Cardiovascular disease is a major cause of morbidity and mortality in the human population. Especially in the united states and western european countries. Many etiological factors have been implicated in the development of cardiovascular disease. Some of these factors include genetic predisposition to the disease, lifestyle factors such as smoking and diet, age, sex, hypertension and hyperlipidemia, including hypercholesterolemia. Many factors, particularly hyperlipidemia and hypercholesterolemia, contribute to the development of atherosclerosis, the underlying cause of cardiovascular disease.

High blood cholesterol concentrations are a major risk factor for cardiovascular disease in humans. Elevated low density lipoprotein cholesterol ("LDL") and total cholesterol are directly associated with an increased risk of coronary heart disease. (Anderson et al, 1987). Although high levels of total cholesterol and LDL are risk factors in the development of atherosclerosis and vascular disease, high density lipoprotein cholesterol ("HDL") has recently been considered an additional risk factor for the development of these conditions. Several clinical trials support the theory of the protective role of HDL against atherosclerosis. One such study has demonstrated that in women, every 1-mg/dl increase in HDL in the blood reduces the risk of coronary vascular disease by 3%. (Gordon et al, 1989).

Studies have shown that dietary changes can reduce cholesterol in humans. In this context, special studies have shown that the quality and quantity of ingested protein greatly influences serum cholesterol levels. (Carol and Hamilton, 1975; Nagaoka et al, 1992; Potter, 1995). The intake of vegetable protein material instead of animal proteins in the daily diet is associated with a lower risk of cardiovascular disease, which may be reflected in a reduction of serum cholesterol levels. In particular, soy protein, a vegetable protein, has been shown to reduce serum cholesterol levels relative to the animal protein casein (Nagaoka et al, 1999). More recent analysis of changes in soy protein intake on the effects of serum lipids in humans has shown that dietary soy protein is significantly associated with a decrease in total and LDL serum concentrations in humans, and does not significantly affect HDL-cholesterol concentrations (Anderson et al, 1995).

One factor responsible for the cholesterol lowering role of soy protein is the high molecular weight fraction (HMF). HMF constitutes the nondigestible fraction of soy protein that remains intact after proteolytic digestion. This fraction therefore consists of many different peptides or peptide fragments. HMF of soy protein has in fact been shown to significantly reduce serum cholesterol in animal and human studies. It is believed that the non-digestible HMF prevents cholesterol uptake by preventing passive uptake of cholesterol through the brush membrane or by preventing protein-mediated cholesterol uptake. In contrast, low molecular weight fractions, digestible fractions of soy protein, have in fact been shown to increase serum cholesterol (Sugano et al, 1988).

Although the potential therapeutic value of soy protein and HMF in particular is as a cholesterol-lowering factor, no specific component has been identified that is responsible for its non-digestive and hypocholeretic activities. There remains a need, therefore, for an effort to identify these active ingredients to provide therapeutic agents that more effectively reduce cholesterol and other risk factors associated with cardiovascular disease.

Summary of The Invention

In one aspect, the present invention provides a method of preparing a food product comprising the steps of: (a) obtaining the selected food product; and (b) adding the isolated oil body associated protein to the food product, wherein consumption of an effective amount of the food product reduces serum cholesterol in a subject in need of the food product. In certain embodiments, the method further comprises adding at least one compound selected from the group consisting of saponins that are substantially resistant to digestion, phytoestrogens, phospholipids and carbohydrates. The oil body associated protein may include lipoproteins and/or oleosins. In one embodiment, the food product is soy-based. The composition may lack or include oil body associated bulk protein prior to the step of adding. Examples of food products include, but are not particularly limited to, soy flour, soy grain, soy meal, soy flakes, soy milk powder, soy protein concentrate, soy protein isolate, and isolated soy polypeptide. In certain embodiments of the invention, the soy protein isolate is a high molecular weight fraction of the soy material treated with a protease. In a further embodiment, the isolated soy polypeptide comprises β -conglycinin, or a fragment thereof, and/or is glycinin, or a fragment thereof.

In another aspect, the present invention provides a composition for treating or preventing hypercholesterolemia, comprising: (a) glycinin and/or beta-conglycinin or fragments thereof; in one embodiment, the glycinin or beta-conglycinin is at least partially hydrolyzed by an enzyme or mixture of enzymes. In a further embodiment, the composition comprises glycinin or fragments thereof and purified oil body associated protein, while in another embodiment comprises β -conglycinin or fragments thereof and purified oil body associated protein. In certain embodiments of the invention, the composition may comprise from about 1% to about 5%, from about 5% to about 10%, greater than about 10%, or from about 30% to about 50%, by weight, of the oil body associated protein.

The compositions provided herein may further comprise at least one additional compound, including saponins that are substantially resistant to digestion, phytoestrogens, phospholipids and carbohydrates. Examples of phytoestrogens include isoflavones. Examples of isoflavones include genistein, bipartite zein (diadzein), equol, biochanin a, formononetin, and their respective naturally occurring glycosides and glycoside conjugates. Examples of carbohydrates include high amylose starch, fructooligosaccharides and soy cotyledon fiber. In one embodiment of the invention, the phospholipid is selected from the group consisting of lecithin, lysolecithin and lecithin having an improved fatty acid component. In a further embodiment, the saponin is selected from the group consisting of soybean saponin a, saponin B, saponin E, sapogenol a, sapogenol B and sapogenol E. The oil body associated protein may comprise lipoprotein, including mammalian lipoprotein, egg yolk lipoprotein, or fat globule membrane protein. The oil body associated protein can also be an oleosin, including the low molecular weight fraction of oleosin.

In one embodiment of the invention, the oil body associated protein comprises a polypeptide fragment comprising an amphipathic sequence. Wherein the composition comprises glycinin, and may comprise the basic subunits of glycinin, including the B-1B subunit. The compositions may also include β -conglycinin, including the α' subunit or fragment thereof. The composition may be further defined as comprising greater than 40% beta-conglycinin, or fragment thereof. The composition may be further defined as comprising one or more polypeptide sequences selected from the group consisting of SEQ ID NOs: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

in another aspect, the present invention provides a method for treating or preventing hypercholesterolemia, comprising the steps of: (a) adding oil body associated protein to the selected food product; and (b) providing the food product to a subject in need thereof in an amount sufficient to treat or prevent hypercholesterolemia. The method may further comprise adding to the food product at least one compound selected from the group consisting of saponins that are substantially resistant to digestion, phytoestrogens, phospholipids and carbohydrates. In one embodiment, the oil body associated protein comprises a lipoprotein, such as oleosin. In another embodiment, the food product is soy-based. Examples of food products include soy flour, soy granules, soy meal, soy flakes, soy milk powder, soy protein concentrate, soy protein isolate, and isolated soy polypeptide. The food product may lack or include oil body associated bulk protein prior to the step of adding. The soy protein isolate may include a high molecular weight fraction of the soy material treated with the protease. In certain embodiments of the invention, the isolated soy polypeptide comprises β -conglycinin, or a fragment thereof, and/or is glycinin, or a fragment thereof.

In another aspect, the present invention provides a method for treating or preventing hypercholesterolemia comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a purified oil body associated protein. The pharmaceutical compositions may be administered in any manner, including as a pill or capsule or as a nutritional supplement. In this method, cardiovascular disease may be prevented by reducing the total serum and/or liver cholesterol or triglyceride concentration. Serum cholesterol concentrations can be decreased by decreasing the concentration of low density lipoproteins, as well as increasing the concentration of high density lipoproteins.

In another aspect, there is provided a polypeptide having the sequence of SEQ ID NO: 1, or a polypeptide having an amino acid sequence with at least 95% sequence homology thereto and having the same biological activity as same.

Brief Description of Drawings

These and other details, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

fig. 1 depicts the sequence of oleosin protein P24 oleosin isoform a from soybean (P89), accession number P29530, and corresponding to SEQ ID NO: 12.

fig. 2 depicts the sequence of the oleosin protein P24 oleosin isoform B (P91) from soybean, accession number P29531, and corresponding to SEQ ID NO: 13.

fig. 3 depicts the amino acid sequence (accession No. AAA68066) of the oil body associated protein 17kDa oleosin (oleo 17) from maize (Zea mays) and corresponds to SEQ ID NO: 14.

fig. 4 depicts the amino acid sequence (accession No. AAA68065) of the oil body associated protein 16kDa oleosin (oleo 16) from maize (zea) and corresponds to SEQ ID NO: 15.

FIG. 5 depicts the amino acid sequence (accession AAA67699) of oleosin KD18(KD 18; L2) from maize (Zea) and corresponds to SEQ ID NO: 16.

fig. 6 depicts the amino acid sequence of sunflower oleosin (accession number CAA55348) and corresponds to SEQ ID NO: 17.

fig. 7 depicts 5 polypeptides comprising HMF from a soy protein isolate called OBAP (+) separated by polyacrylamide gel electrophoresis of gel bands.

FIG. 8 depicts oil body associated proteins (P34 and oleosin) purified and isolated on polyacrylamide gels.

Fig. 9 depicts a gel comprising HMF from pepsin digested soy protein isolate produced from soybeans lacking G1 glycinin.

FIG. 10 depicts a graph illustrating dose-dependent inhibition of cholesterol uptake in Caco-2 cells by HMF isolated from OBAP (+) in comparison to OBAP (-) soy protein isolate.

Figure 11 depicts a graph illustrating the difference between cholesterol absorption in the presence of sitostanol and that in the presence of glycinin-null and α/α' β -conglycinin-null HMF, demonstrating a mechanistic difference.

The gel depicted in fig. 12 illustrates the presence of oleosin in each sample.

FIG. 13 depicts a gel illustrating the presence or absence of oleosins in the indicated samples.

Detailed Description

The present invention overcomes the limitations of the prior art by identifying compositions that have cardiovascular health benefits. In particular, one aspect of the invention relates to the discovery of oil body associated proteins capable of lowering cholesterol levels. Meanwhile, plant components, such as soy protein, are known to have hypocholesterolemic activity, but the particular component responsible for this activity has not been characterized previously. Thus, the present invention relates to the discovery that oil body associated proteins, including oleosins and egg yolk lipoproteins, are capable of conferring cholesterol lowering characteristic capabilities to plant proteins. As such, the present invention relates to synergistic combinations of these specific ingredients with plant materials such as soy food. Without being bound to any particular theory, it is believed that the oil body associated proteins prevent digestion of the physiologically active peptides present in the soy material and thereby synergistically increase the hypocholesterolemic activity of the composition. In addition, the present invention relates to a synergistic combination of soy material binding ingredients and an additive ingredient selected from the group consisting of saponins, phytoestrogens, phospholipids, and carbohydrates that are substantially resistant to digestion, or any combination thereof, to further increase the hypocholesterolemic activity of the combination. The invention also includes the therapeutic use of these peptides, alone or in combination with other compounds, for lowering the total cholesterol concentration in the blood, and in particular for lowering the LDL-cholesterol concentration, inhibiting the development of cardiovascular diseases.

Oil bodies are small, spherical subcellular organelles that encapsulate stored triacylglycerides, and are an energy reserve used by many plants. Although they are found particularly abundant in seeds of oilseeds in most plants and in different tissues, they typically range in size from 1 micron to several microns in diameter. Oil bodies are generally composed of triacylglycerides and surrounding lipoproteins and proteins. "oil body associated proteins" include any and all of these proteins and lipoproteins physically associated with oil bodies. In plants, the major oil body associated protein is oleosin. Oleosins have been cloned and sequenced from a number of plant sources, including corn, rapeseed, carrot, and cotton. Oleosins from different species are generally highly conserved.

I. Abbreviations and Definitions

To facilitate an understanding of the invention, a number of terms and abbreviations used herein are defined as follows:

HMF ═ high molecular weight fraction

As used herein, "high molecular weight fraction" refers to the fraction of the retained plant protein isolate after hydrolysis or chemical digestion of the isolate, which can be separated by centrifugation at pH 6-7 at 4,000-10,000 Xg for 15-20 minutes.

As used herein, "additional compounds" generally refers to a single compound or a group of compounds added to the compositions of the present invention. These compounds are selected from the group consisting of saponins, phytoestrogens, phospholipids and carbohydrates.

As used herein, the term "amino acid" is used in a broad sense and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules comprising amino acid moieties. Those skilled in the art will recognize that, in view of this broad definition, amino acids referred to herein include, for example, naturally occurring proteinaceous L-amino acids; a D-amino acid; chemically modified amino acids, such as amino acid analogs and derivatives; as used herein, the term "proteinaceous" indicates that an amino acid may be integrated into a peptide, polypeptide, or protein in a cell by a metabolic pathway.

As used herein, the term "pharmaceutically acceptable salt" includes salts commonly used to form alkali metal salts and addition salts of the free acid or free base. The nature of the salt is not critical if it is pharmaceutically acceptable.

As used herein, "secretory sequence" or "signal peptide" or "signal sequence" refers to a sequence that directs the re-synthesis of a secreted or membrane protein to and through the membrane of the endoplasmic reticulum, or across the bacterial inner membrane from the cytoplasm to the periplasm, or from the mitochondrial matrix to the internal space, or from the chloroplast matrix into the cysts. Fusion of such sequences to genes to be expressed in a heterologous host ensures that the recombinant protein is secreted from the host cell.

As used herein, "polypeptide" and "oligopeptide" are used interchangeably and refer to polymers of at least 2 amino acids linked together by peptide bonds.

As used herein, "sequence" refers to a linear sequence in which monomers are present in a polymer, e.g., the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

As used herein, "ground whole soybeans" include, for example, soybean material formed from flaked or ground whole soybeans, including the hulls and embryos of soybeans. The comminuted whole soybean material may comprise inherent fat of the soybean or may be defatted.

As used herein, "soy flour" includes, for example, on a moisture free basis, a soy material containing less than 65% by weight soy protein content, consisting of dehulled soybeans and having an average particle size of 150 microns or less. The term "soy flour" may include, for example, soymilk powder. The soy flour may contain inherent fat of the soy or may be defatted.

As used herein, "soy particulates" include, for example, a soy material comprising less than 65% by weight soy protein content on a moisture free basis, consisting of dehulled soybeans and having an average particle size of 150 microns to 1000 microns. The soy particles may contain fats inherent to soy or may be defatted.

As used herein, "soybean meal" includes, for example, a soybean material comprising less than 65% by weight of soybean protein content on a moisture-free basis, consisting of dehulled soybeans, which are not within the definition of soybean fines or soybean granules. The soybean meal may contain inherent fat of the soybeans or may be defatted.

As used herein, "soy flakes" include, for example, a flaked soy material formed from flaked, dehulled soybeans comprising less than 65% by weight soy protein content on a moisture free basis. The soy flakes may contain inherent fat from the soy or may be defatted.

As used herein, "soy protein concentrate" includes, for example, soy protein material prepared from high quality whole, clean dehulled soybean seeds. Soy protein concentrates are prepared by removing a substantial portion of the oil and water soluble non-protein components and typically contain not less than 65% protein on a moisture free basis. In another embodiment, "soy protein concentrate" may also be construed to additionally include a mixture of soy protein and phospholipids, wherein the total protein on a moisture free basis is between about 65 to about 90% protein. Generally, this is prepared from dehulled defatted soybeans by 3 basic steps: acid soaking (about pH4.5), extracting with alcohol (about 55-80%), and heat-denaturing the protein with heat before extraction with water. Soy protein concentrates of low water solubility (aqueous alcohol extraction) are subjected to heating (steam injection or air jet cooking) and mechanical processing (homogenization) to enhance solubility and functionality. When referring to a mixture of soy protein and phospholipids, wherein the total protein on a moisture free basis is 65-90% protein, such materials may be prepared by combining soy protein isolate and phospholipids or by fractionating phospholipids from soy: protein complexes (i.e., oil body proteins and associated phospholipids). The term "soy protein concentrate" may include, for example, soymilk powder.

As used herein, "soy protein extract" includes, for example, soy protein concentrates or isolates that are enriched in certain soy proteins. The fractionated soy protein material from the intact soy protein material may be prepared, for example, from soy protein ingredient waste or whey streams (alcoholic or acidic water extraction step).

As used herein, "soy protein isolate" includes, for example, soy protein material, which is the major protein fraction of soybeans prepared from dehulled soybeans by removing most of the non-protein compounds, preferably containing no less than 90% protein on a moisture free basis. In another embodiment, "soy protein isolate" may also be construed to additionally include certain types of soy proteins such as, for example, β -conglycinin, glycinin, and oleosin, or alternatively lack certain types of soy proteins, such as soy protein material enriched in oleosin. Extracting protein from unheated defatted soybean flakes, typically with water or mild alkali, at a pH of 8-9, and then removing insoluble fibrous residues by centrifugation; adjusting the resulting extract to a pH of 4.5, wherein the majority of the protein precipitate is a clot; the coagulum is separated from the soluble oligosaccharides by centrifugation, followed by multiple washes, neutralization with sodium or potassium hydroxide (to make it more soluble and functional), heat treatment (e.g. using jet-cooking) and spray drying. The addition of a protease prior to the heat treatment step may also serve to partially hydrolyze the protein and increase the solubility of the soy protein isolate.

As used herein, "peptide" and "protein" are used interchangeably and refer to a compound consisting of two or more amino acids joined by peptide bonds.

As used herein, "recombinant protein" refers to a protein, whether comprising a native or mutated primary amino acid sequence, that is obtained by expressing a gene carrying a recombinant DNA molecule in a cell other than the cell in which the gene and/or protein is naturally occurring. In other words, the gene is heterologous to the host in which it is expressed. It should be noted that any variation of a gene, including the addition of a polynucleotide encoding an affinity purified portion of the gene, renders the gene unnatural for the purpose of this definition, and thus the gene does not exist "naturally" in any cell.

As used herein, "target sequence" refers to within the context of a protein or peptide, and "target sequence" refers to a nucleotide sequence that encodes an amino acid sequence, the presence of which results in the targeting of the protein to a particular destination within a cell.

The phrase "therapeutically-effective" indicates that the agent is capable of preventing or ameliorating the severity of the disease while avoiding the adverse side effects typically associated with alternative therapies. It is understood that the phrase "therapeutically-effective" is equivalent to the phrase "effective for treatment or prevention" and is intended to define, for example, the amount of the composition used in the methods of the invention that achieves the goal of reducing the risk of cardiovascular disease or preventing the disease in question, while avoiding the adverse side effects typically associated with alternative therapies.

The polypeptides of the invention

Applicants have identified sequences for polypeptides that isolate HMF from soybean material, more specifically from soybean material having hypocholesterolemic activity. These sequences encode peptides derived from glycinin or beta-conglycinin. The proteins glycinin and beta-conglycinin are seed storage proteins. Glycinin has an approximate molecular weight of 320 kilodaltons ("kDa") and consists of 6 subunits, each consisting of acidic and basic subunits. In addition, β -conglycinin has an approximate molecular weight of 150kDa and is composed of 3 different subunits (α, α', and β) in different proportions. Glycinin and beta-conglycinin type proteins are highly conserved among different plant species.

Thus, in one aspect of the invention, one to several isolated polypeptides or polypeptide fragments from a glycinin protein are included. In one embodiment, the sequence of the isolated polypeptide is set forth in seq id NO: provided in 1 and corresponding to VFDGELQEGRVLIVPQNFVVAARSQSDNFEYVSFK.

In yet another aspect of the invention, one to several isolated polypeptides or polypeptide fragments from a beta-conglycinin protein are provided. In one embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 2 and corresponds to LRMITLAIPVNKPGRFESFFL. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 3, and corresponds to IFVIPAGYPVVVNATSHLNFFAIGI. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 4, and corresponding to lqesviteiskk. in another embodiment, the sequence of the isolated polypeptide is set forth in SEQ ID NO: 5 and corresponds to QQQEEQPLEVRK. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 6, and corresponds to NQYGHVR. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 7 and corresponds to AIVILVINEGDANIELVGL. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 8, and corresponds to NILEASYDTKFEEINK. In another embodiment, the sequence of the isolated polypeptide is as set forth in SEQ ID NO: 11 and corresponds to IFVIPAGYPVVVNATSDLNFFAFGI.

Applicants have also identified sequences of oleosins that demonstrate hypocholesterolemic activity. Oleosins are found primarily in the membrane components of vegetable oil bodies. Oleosin proteins consist of 3 domains; the 2-terminal, N-and C-terminal ends of the protein are essentially hydrophilic and remain on the surface of the oil body exposed to the cellular fluids, while the highly hydrophobic central core of oleosin is firmly anchored within the membrane and triacylglycerides. Oleosins also contain an amphipathic α -helical domain at or near the C-terminus. Oleosins from different species represent a small family of proteins that display a considerable conservation of amino acid sequence, particularly in the central region of the protein. Small numbers of different isoforms may be present within individual species.

Thus, in another aspect of the invention, one to several isolated polypeptides or polypeptide fragments from oleosin proteins are included. In one embodiment, the oleosin is isoform A P24 from soybean (P89) (accession number P29530; SEQ ID NO: 12), which corresponds to FIG. 1. In another embodiment, the oleosin is isoform B P24 from soybean (P91) (accession number P29531; SEQ ID NO: 13), which corresponds to FIG. 2. In another embodiment, the oleosin is the oil body associated protein 17kDa oleosin (oleo 17) from maize (Zea) (accession No. AAA 68066; SEQ ID NO: 14), which corresponds to FIG. 3. In another embodiment, the oleosin is the oil body protein 16kDa oleosin (oleo 16) from maize (Zea) (accession No. AAA 68065; SEQ ID NO: 15), which corresponds to FIG. 4. In another embodiment, the oleosin is oleosin KD18(KD 18; L2) (accession number AAA 67699; SEQ ID NO: 16) from maize (Zea), which corresponds to FIG. 5. In another embodiment, the oleosin is sunflower oleosin (sunflower) (accession number CAA 55348; SEQ ID NO: 17), which corresponds to FIG. 6. In a further embodiment, the isolated oleosin polypeptide has a sequence set forth in SEQ ID NO: 9 and corresponds to VKFITAATIGITLLLL. In another embodiment, the isolated oleosin polypeptide has a sequence set forth in SEQ ID NO: 10 and corresponds to YETNSSLNNPPSR.

The invention also includes compositions related to oleosins and SEQ ID NOs: 12. 13, 14, 15, 16, 17, preferably 95%, more preferably 97%, and more preferably 99% sequence homology. A further embodiment of the invention provides a polypeptide which is substantially identical to SEQ ID NO: 1-11, and preferably 95%, more preferably 97%, and more preferably 99%.

"homology," as is well understood in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, "homology" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between such strings of sequence symbols. "homology" can be readily calculated by known methods, including but not limited to those described in comparative Molecular Biology, 1988; biocomputing: information and Genome Projects, 1993; computer Analysis of Sequence Data, part I, 1994; sequence analysis in Molecular Biology, 1987; sequence analyst primer, 1991; and the method described by Carillo and Lipman, 1988. The method of determining homology is designed to give the maximum match between the sequences to be tested. Furthermore, methods for determining homology are compiled into publicly available programs. Computer programs that can be used to determine identity/homology between 2 sequences include, but are not limited to, GCG (Devereux et al, 1984); a set of 5 BLAST programs, 3 designed for nucleotide sequence queries (BLASTN, BLASTX and TBLASTX) and 2 designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, 1994; Birren et al, 1997). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual; Altschul et al, 1990). Homology can also be determined using the well-known Smith Waterman algorithm.

The invention also relates to isolated proteins. As used herein, the term protein includes fragments, analogs and derivatives of glycinin, beta-conglycinin or oleosin proteins. The protein of the present invention may be a natural protein, a recombinant protein or a synthetic protein or polypeptide.

One of ordinary skill in the art will recognize that altering the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent or possibly improved sub-second generation peptides, etc., that exhibit equivalent or higher functional properties when compared to the original amino acid sequence. The present invention therefore includes such altered amino acid sequences. Alterations may include amino acid insertions, deletions, substitutions, truncations, fusions, recombination of subunit sequences, and the like, provided that the peptide sequence produced by such alterations has substantially the same functional properties as the naturally occurring counterpart disclosed herein. Biological activity or function can be determined, for example, by the ability of the protein to reduce total serum cholesterol as demonstrated in the examples below.

One factor that may be considered in making such changes is the hydropathic index of amino acids. The importance of the hydrophilic amino acid index in conferring protein interactions on biological functions has been discussed by Kyte and Doolittle (1982). It is well recognized that the relatively hydrophilic nature of amino acids contributes to the secondary structure of the synthesized protein. This in turn affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

The hydropathic index of each amino acid, in terms of its hydrophobic and charge properties, has been assigned as follows: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid/glutamate/aspartic acid/asparaginic acid (-3.5); lysine (-3.9); and arginine (-4.5).

As is known in the art, certain amino acids in a peptide or protein may be substituted for other amino acids having a similar hydrophilicity index or score, and result in a synthetic peptide or protein having similar biological activity, i.e., still maintaining biological function. In making such a change, it is preferable that amino acids having a hydrophilicity index within. + -. 2 be substituted for each other. More preferably the substitution is an amino acid wherein the amino acid hydrophilicity index is within ± 1. Most preferred substitutions are those in which the amino acid hydrophilicity index is within ± 0.5.

Similar amino acids may also be substituted for hydrophilicity. U.S. Pat. No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein depends on the hydrophilicity of its adjacent amino acids, which is related to the biological properties of the protein. The following hydrophilicity values have been assigned to the amino acids: arginine/lysine (+ 3.0); aspartate/glutamate (+3.0 ± 1); serine (+ 0.3); asparagine/glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus, one amino acid in a peptide, polypeptide or protein can be replaced by another amino acid with a similar hydrophilicity score and still produce a synthetic protein with similar biological activity, i.e., still maintaining the correct biological function. In making such changes, amino acids having a hydropathic index within. + -. 2 are preferably substituted for each other, amino acids having a hydropathic index within. + -. 1 are more preferred, and amino acids having a hydropathic index within. + -. 0.5 are most preferred.

As discussed above, the substitution of amino acids in the peptides of the invention may depend on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. In order to produce conservative amino acid changes that result in silent changes within the peptides provided, illustrative substitutions that take into account various of the above characteristics may be selected from other members of the class to which the naturally occurring amino acids belong. Amino acids can be divided into four groups: (1) an acidic amino acid; (2) a basic amino acid; (3) a neutral polar amino acid; and (4) a neutral nonpolar amino acid. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids, such as arginine, histidine and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that variations that are not expected to be beneficial may also be useful if they can result in the production of functional sequences.

The term protein also includes forms of protein to which one or more substituent groups have been added. Substitution is an atom or group of atoms that is introduced into a molecule by replacement of another atom or group of atoms. Such groups include, but are not limited to, lipids, phosphate groups, sugars, and carbohydrates. Thus, the term protein includes, for example, lipoproteins, glycoproteins, phosphoproteins, and phospholipoproteins.

Compositions and methods of the invention

Another aspect of the invention provides a composition for treating or preventing cardiovascular disease. The composition in one embodiment comprises a plant-based food product to which the isolated oil body associated protein has been added. In one embodiment of the invention, the food product is soy-based. In another embodiment, the composition comprises isolated soy material, isolated oil body associated protein, and at least one additional compound selected from the group consisting of saponins that are substantially resistant to digestion, phytoestrogens, phospholipids, and carbohydrates. In certain embodiments of the invention, the isolated oil body associated protein may be a purified fraction isolated from a plant protein. For example, the isolated oil body associated protein may be enriched by 1, 10, 100, 200, 1000 or more fold relative to the native plant protein. In certain embodiments of the invention, the isolated oil body associated protein may be enriched so as to be enriched by a factor of 1, 5, 10, 50, or more than 100 relative to, for example, a purification fraction such as HMF oil body associated protein.

The isolated soy material used in the present invention may be prepared and isolated from any soybean plant to the extent that the isolated soy material contains the components necessary for the isolated soy material to exhibit the desired hypocholesterolemic activity. Isolated soy materials may include, for example, ground whole soybeans, soybean fines, soymilk powder, soybean grains, soybean meal, soybean flakes, soybean concentrate, soybean protein isolate, and soybean protein extract. In one embodiment, the soy material is soy concentrate. In another embodiment, the soy material is an isolate of soy protein. In a further embodiment, the soy material is an extract of soy protein. Any method known in the art, including the methods detailed herein, can be used to isolate a particular soybean material.

One aspect of the present invention provides isolated soy materials enriched in certain soy proteins or peptides, such as beta-conglycinin, glycinin, and oleosins. These soy materials can be prepared from isolates, concentrates, or from waste streams of isolate and concentrate products. They may also be prepared from soybeans with improved protein composition, such as soybeans with twice the normal level of β -conglycinin and low levels of glycinin (such as described in U.S. Pat. No. 6,171,640).

Food products are well known to those skilled in the art. In the case of soybeans, common food uses include, but are not limited to, products such as seeds, bean sprouts, baked soybeans, full fat soybean fines for various baked products, baked soybeans for cakes, soy nut butters, soy coffees, and other soy derivatives of oriental foods. Soy protein products (e.g., meal) can be divided into soy flour concentrates and isolates, which are useful for both food/feed and industrial applications. Soy flour and particles are often used in the manufacture of meat supplements and the like, pet food, bakery ingredients, and other food products. Food products prepared from the soybean fine powder and isolate include baby food, confectionery products, cereals, food beverages, noodles, baking powder, beer, malt liquor, etc.

In certain aspects of the invention, an oil body associated protein, such as an oleosin, is added to an isolated soy material that lacks, or contains low amounts of, certain classes of soy proteins, such as oleosins, to improve or restore the low cholesterol properties of the soy protein or soy protein composition. In further embodiments of the invention, oil body associated protein is added to a final concentration of about 0.5%, 1%, 3%, 5%, 10%, 20% or more by weight, including all intermediate ranges within these concentrations. Depending on the particular application, soy materials without oleosins or with reduced amounts of oleosins may be highly beneficial. For example, oleosins may be considered undesirable because they limit flavor, resulting in a soy food product with relatively poor flavor quality. In addition, oleosins are also poorly soluble and result in a larger particle fraction when the defatted soy milk is dispersed in water. In one embodiment, oleosin may be removed by allowing the large particle fraction comprising oleosin to settle in a tank or by using an ultrafiltration membrane. Alternatively, oleosins can be precipitated from soy protein isolates in the presence of sodium sulfate and calcium chloride at pH 2.8 (Samoto et al, 1998).

The present invention also includes compositions comprising an isolated soy material and an isolated oil body associated protein, wherein the isolated soy material is the high molecular weight fraction ("HMF") of a soy protein isolate. The HMF used in any of the compositions of the present invention may be prepared and isolated from any plant protein isolate in which it naturally occurs to the extent that the fraction has the desired hypocholesterolemic activity. In one embodiment, the HMF is prepared from a soy protein source. Typical soy materials from which HMF can be prepared include soy, dehulled soy, soy meal, soy flour, soy granules, soy milk powder, soy flakes (full and defatted), soy syrup, soy protein concentrate, soy whey protein, and soy protein isolate. In a preferred embodiment, the HMF is prepared from soy protein isolate.

To prepare HMF, the selected vegetable protein isolate is subjected to hydrolytic or chemical digestion. Typical reagents for this digestion process include pepsin or microbial proteases. Typically, for example, soy protein is incubated with pepsin (0.2% NaCl in the aqueous phase, 38 ℃, pH 1.1), 0.5-5% of the soy protein isolate, for 13-17hrs, heat treated at 90 ℃ for 20 minutes to inactivate the pepsin, cooled on ice, and treated with Na₂CO₃The pH was adjusted to 6-7 and centrifuged at 4,500g for 20 minutes. The precipitated particles can then be washed 3 times with water and identified. Other methods of using pepsin are known in the art.

The microbial protease may, for example, be Sumizyme FP (Aspergillus niger protease, enzyme activity 48800U/gm, Shin Nippon Kagaku KabushikiKaisha) and incubated with soybean protein at 60 ℃ for 5hrs. Then centrifuged at 10,000 Xg for 10 minutes. The precipitate was collected and identified. Additional methods are known in the art. See, e.g., Hori et al (1999).

HMF is then isolated from the digested plant protein isolate by any means generally known in the art. In one embodiment, the HMF is isolated by centrifugation. In general, the separation step may be performed, for example, by drying (e.g., freeze drying) the centrifuged fraction of the aqueous suspension of soy protein isolate treated with microbial protease or pepsin (protease is 0.5-6% of the total protein) and incubating the fraction at 30-70 ℃ for 1-20 hours. Additional methods are known in the art. See, Nagako et al, (1999).

Furthermore, any peptide isolated from HMF may be used in the composition of the invention to the extent that the peptide has the desired hypocholesterolemic activity. Generally, however, the peptides used in the compositions are at least 10 amino acid residues in length, more generally from about 10 to about 100 amino acid residues in length, and most generally from about 30-80 amino acid residues in length. Generally, peptides are substantially hydrophobic in nature, having a hydrophobic amino acid composition of about more than 30 weight percent to preferably about more than 35 weight percent. Furthermore, the peptides used may have 0, 1 or more regions of amphiphilicity. In addition, the peptides used may have a hydrophobic surface or a hydrophobic region, which is not due to a series of hydrophobic amino acids, but rather to the helical structure of the peptide.

Another embodiment of the present invention provides a composition comprising an isolated soy polypeptide isolated from soy material, wherein the soy polypeptide comprises beta-conglycinin and glycinin. The structure of β -conglycinin and glycinin are described in detail above, and details regarding their structure are provided by Utsumi et al (1997). Applicants are not bound by a single theory and believe that these peptides have hypocholesterolemic activity because they are free from digestion and are absorbed into the bloodstream or bind bile acids. It is also beneficial, although not required, that amphiphilic α -helical regions are present within β -conglycinin. Without being limited to applying any single theory, it is believed that the presence of the amphipathic α -helical region is beneficial because it forms a hydrophobic surface that facilitates interaction with various important receptor sites that confer hypocholesterolemic activity.

In addition, and without the applicants being bound to any single theory, the identified polypeptides of the invention may also serve as a nitrogen source for beneficial bacteria in the colon. These bacteria can therefore produce short chain fatty acids, such as propionic acid, which positively affects lipid metabolism. Short chain fatty acids inhibit fatty acid synthesis in the liver, reduce the rate of triglyceride secretion and reduce hepatic cholesterol synthesis. One aspect of the invention is the use of soy protein polypeptides to promote the production of short chain fatty acids in the colon and to reduce serum cholesterol and triglycerides. The effectiveness of undigested soy polypeptides to promote the growth of beneficial bacteria in the colon may be most pronounced in the presence of undigested carbohydrates, such as high amylose corn starch, which provides an important fuel for the microbiota of the human colon. Thus another embodiment of the invention is the combination of soy polypeptide ingredient with an undigested carbohydrate source to optimize the beneficial effects of the polypeptide on serum cholesterol and triglycerides.

Thus in one aspect of the invention, compositions are provided having specific polypeptides isolated from beta conglycinin. In general, these polypeptide sequences correspond to SEQ ID NOs: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof. These sequences can be isolated from β -conglycinin by a method comprising, for example: (1) enzymatic hydrolysis of soy material followed by separation of high molecular weight fractions, e.g., by centrifugation, or (2) enzymatic digestion of soy material followed by further purification by ultrafiltration membranes, ion exchange resin columns, and gel filtration column chromatography, gives peptides in the molecular weight range of about 200 to about 5,000 kilodaltons (see, e.g., the methods detailed in Yamauchi and Suetsuna (1993)). The peptides can be further fractionated by using ion exchange chromatography (Chen et al, 1995). Alternatively, rather than isolating individual sequences from a particular β -conglycinin subunit, it is also possible to utilize soybean with twice the normal level of β -conglycinin and isolate sequences from only soybean that are isolated as β -conglycinin. Likewise, it is also possible to use the same germplasm in which only a specific β -conglycinin subunit is expressed to obtain a natural preparation of that subunit. The active peptides are then produced in the intestine by consumption of the natural product by pepsin and pancreatin hydrolysis, or are obtained during ingredient manufacture using microbial enzymes. Thus, in another embodiment of the invention, there is provided a composition comprising a crude preparation of a particular subunit of β -conglycinin in combination with an oleosin preparation.

Another aspect of the invention provides compositions having specific polypeptides isolated from glycinin. In one embodiment the composition comprises a polypeptide isolated from the basic subunit of glycinin. In another embodiment, the composition comprises a polypeptide isolated from the B-1B subunit of glycinin. In a preferred embodiment, the composition comprises a peptide isolated from glycinin corresponding to seq id NO: 1. Glycinin is generally isolated from the protein source using separation methods well known in the art. Examples of such separation methods are methods for separating other protein isolates as listed above.

The compositions of the present invention also generally comprise an oil body associated protein. As used herein, the term "oil body associated protein" includes any protein, lipoprotein, or peptide that is physically associated with an oil body structure or intracellular lipid particle. In general, most eukaryotic cells from, for example, plant, mammalian, non-mammalian cell, algae, and yeast species contain intracellular lipid particles. These particles are called liposomes, lipid droplets or, depending on the species, oil bodies, oleosomes or spherosomes, in particular in plants. Lipid particles of eukaryotic cells consist of a highly hydrophobic core of neutral lipids, mainly triglycerides and/or sterol esters, surrounded by a phospholipid monolayer and a small amount of embedded proteins. A typical composition of oil bodies isolated from corn is triacylglycerols (95%), diacylglycerols (4%), phospholipids (0.9%) and proteins (1.4%).

Typical oil body associated proteins that may be used in the compositions of the present invention include oil body proteins, such as oleosins in the form of apoproteins, or lipoproteins, and 34-kD soybean seed storage vacuolar proteins associated with oil bodies. Accordingly, one aspect of the present invention provides a composition comprising isolated soy material and a peptide isolated from an oil body associated protein, wherein the peptide is oleosin or a peptide fragment from oleosin. The oleosin may be represented by P24 oleosin isoform a (P89), sequence accession number P29530, as shown in SEQ ID NO: 12 and corresponds to figure 1; p24 oleosin isoform B (P91), accession number P29531, as set forth in seq id NO: 13 and corresponds to figure 2; oil body associated protein 17kDa oleosin (oleo 17), accession No. AAA68066, as set forth in SEQ ID NO: 14, and corresponds to fig. 3; oleosin 16kDa oleosin (oleo 16), accession No. AAA68065, as set forth in SEQ ID NO: 15 and corresponds to fig. 4; oleosin KD18(KD 18; L2) accession number AAA67699 as provided by SEQ ID NO 16 and corresponds to FIG. 5; and sunflower oleosin, accession number CAA55348, as set forth in SEQ ID NO: 17 and corresponds to figure 6. More generally, the peptides used in the compositions are derived from low molecular weight oleaginous proteins having a molecular weight of approximately 17 kilodaltons. In certain embodiments, the peptide is from an oleosin and corresponds to SEQ ID NO: 9 or SEQ ID NO: 10.

The oleosin peptides used in the compositions of the invention may be isolated from intact oil bodies. Thus, intact oil bodies can be isolated from seeds that are a source of oil body associated proteins. In WO 00/30602, for example, a list of methods and seed types that can be used is provided. Methods for preparing oil bodies are also described in Japanese patent application laid-open No. 2002-101820. The oil can be extracted from the oil bodies with diethyl ether, leaving interfacial substances (oleosins and phospholipids) in the aqueous fraction. The phospholipids can be extracted with chloroform/methanol (2: 1, vol/vol) or other suitable organic solvent. In particular, oleosins are isolated as aggregated fractions (Tzen and Huang 1992). In addition, if a high pH extraction method is present, it can be used to remove P34 protein. The P34 protein has an isoelectric pH below 6.5 and therefore, can be solubilized at high pH, where oleosins have their own isoelectric pH and precipitate.

Oleosins can also be isolated from whole soybeans that are soaked in water or from defatted soybean flour. Oleosins can be isolated from a protein source using isolation methods well known in the art. Examples of such separation methods are the methods used to separate oleosins from defatted soy flour, as outlined above. Another example is the isolation of oleosins from intact soybeans (JP 2002-. Additional sources of oleosins include plant cells, fungal cells, yeast cells (Leber et al, 1994), bacterial cells (Pieper-F üst et al, 1994), and algal cells (Rossler, 1988).

In a preferred embodiment of the invention, the oleosins are obtained from plant cells, including cells from pollen, spores, seeds, and vegetative plant organs, in which oleosins or oil body-like organelles are present (Huang, 1992). More preferably, the oleosins of the invention are obtained from plant seeds, and most preferably the plant species comprises: rapeseed (brassica), soybean (Glycine max), sunflower (Helianthus annuus), oil palm (Elaeis guineeis), cotton seed (Gossypium spp), groundnut (arachis nigroaa), coconut (Cocus nucifera), castor (Ricinus cummunis), safflower (Carthamus tinctorius), mustard (brassica and white mustard alba), coriander (coriandem sativum), squarch (Cucurbita maxima), linested/flax (Linum usitatissimum), brazil nut (berthileaexcel), jojoba (Simmondsia chinensis), maize (Zea mays), Crambe abyssi (Crambe persica), and caterpillars (erucia sativa).

Another aspect of the invention includes a composition comprising an isolated soy material and peptides isolated from oil body associated proteins in which the peptides are lipoproteins lipoprotein is a non-covalent, non-stoichiometric particulate complex of neutral lipids, phospholipids and proteins found in animal and plant cells. In one embodiment, the lipoprotein is a mammalian lipoprotein. In another embodiment, the lipoprotein is egg yolk lipoprotein (for a review of egg yolk structure, see examples, Bringe (1997), the contents of which are hereby incorporated by reference). In another embodiment, the lipoprotein comprises a membrane protein of a lipoglobulin.

Lipoproteins can be isolated from the oil body associated protein source using methods well known in the art examples of such isolation methods are those listed above for the isolation of oil body proteins or oleosins from defatted soy flour. In addition, egg yolk lipoproteins can be isolated from the oil body associated protein source using separation methods well known in the art.

As an example of these separation methods, egg yolk lipoproteins can be separated by extracting triglycerides and cholesterol using carbon dioxide in a supercritical state (see example, Bringe and Cheng, 1995). A further aspect of the invention provides a composition comprising a soy material in combination with an oil body associated protein and an additional compound. The additional compound may include any compound that therapeutically enhances the composition. Generally, however, the additional compound is selected from the group consisting of saponins which are substantially resistant to digestion, phytoestrogens, phospholipids and carbohydrates. In general, without being bound by any particular theory, it is believed that these additional compounds substantially enhance the hypocholesterolemic activity of the composition by preventing digestion of the cholesterol-lowering polypeptides in the isolated soy material. Because of these properties, the additional compounds of the present invention increase the therapeutic capacity of the composition. Thus in a preferred embodiment, the compositions of the present invention can have any combination of the specific additional ingredients identified above in combination with the isolated soy material and the isolated oil body associated protein.

Thus in one embodiment, the composition of the invention may comprise one to several saponins as additional compounds. Saponins may be isolated from their naturally occurring plant sources by any known method, for example the method of Gurfinkel et al (2002), or may be synthetically prepared by any known method. Saponins are suitable for use in the present invention to the extent that the selected compound enhances the properties of the composition for use as a hypocholesterolemic agent. Typically, however, the saponin used is isolated from legumes of plants such as alfalfa or soybean, or from the seeds of oats or other plants. More generally, the saponin is isolated from soybean seeds, and in particular from soybean embryos. Sources of saponins include, for example, soy, quillaja, alfalfa, and quillaja soapbark. Soybean saponins include, for example, saponin A, B, E, and sapogenol A, B and E.

Furthermore, the composition of the invention may comprise one to several phytoestrogens as additional compounds. Phytoestrogens can be isolated from their naturally occurring plant sources by any known method, such as those detailed in U.S. Pat. No. 5,855,892 or WO 00/30663, or can be synthetically prepared by any known method. Any phytoestrogen is suitable for use in the present invention to the extent that the selected compound enhances the properties of the composition for use as a hypocholesterolemic agent. Typically, the phytoestrogen used in the composition is an isoflavone. More generally, isoflavones are genistein, daidzein (including its metabolites o-desmethylangolenin, dihydroclaidzenin and equol), biochanin A, formononetin and their respective glycosides and glycoside conjugates naturally occurring in soy or clover.

The compositions of the present invention may also include one or more phospholipids as additional compounds. Phospholipids may be derived from a variety of sources, but are generally isolated from, for example, seeds, and more generally from oilseeds of the soybean plant. In addition, phospholipids having improved fatty acid composition can be used. The phospholipid may be an enzyme modified soybean phospholipid. Enzymatically modified phospholipids can be prepared, for example, from soybean phospholipids (SLP; eulecithin, Mie, Japan) by treatment with phospholipase A2(Novo industry, Bagsvaerd, Denmark) (Nagaoka, et al, 1999). In addition, phospholipids having improved fatty acid composition can be obtained from plants or plant seeds that have been genetically altered to produce phospholipids having improved fatty acid composition. An example of a phospholipid having an improved fatty acid composition is lecithin having an improved fatty acid composition. Other methods known in the art may also be used to modify the fatty acid content of the phospholipid.

In addition, the compositions of the present invention may also include one to several carbohydrates. Any carbohydrate may be used in the composition of the invention to the extent that the selected compound enhances the properties of the composition for use as a hypocholesterolemic agent. Generally, however, the carbohydrate used is a carbohydrate that is substantially resistant to digestion. As used herein, "substantially resistant to digestion" when used to describe carbohydrates is defined in the art to generally mean, for example, that the carbohydrate is greater than about 70% resistant to digestion, preferably greater than about 80% resistant to digestion, and more preferably greater than about 90% resistant to digestion. Generally, carbohydrates rich in amylose or fiber are particularly suitable for use in the composition. In one embodiment, for example, the carbohydrate used in the composition is high amylose starch, fructo-oligosaccharides or soy cotyledon fiber. Further embodiments are, for example, physically inaccessible starches (partially milled grains and seeds), resistant granules (raw potatoes, unripe bananas, some legumes and high amylose starches), retrograded starches (cooked and cooled potatoes, breads and corn chips), and chemically modified starches (etherified, esterified or cross-linked starches (for processing foods)) (Topping and Clifton, 2001).

Any combination of isolated soy material and isolated oil body associated protein, in the presence or absence of at least one additional compound, can be combined to form the compositions of the present invention. Table 1 below, for example, illustrates a number of general formulations for different embodiments of the compositions.

TABLE 1 formulation of the compositions

In one embodiment of the invention, the composition has an isolated soy material content of not less than 50% by weight of the composition and an oil body associated protein content of not less than 0.5% by weight of the composition. More preferably, however, the composition has an isolated soy material content of not less than about 70 to about 90% by weight of the composition and an oil body associated protein content of about 1 to about 5% by weight of the composition. When present, additional compounds such as isoflavone or saponin compounds typically comprise not less than 10mg per 100g of the composition, and may further comprise about 30-300mg of additional compound per 100g of the composition. Further, when present, additional compounds such as substantially digestive resistant phospholipids or carbohydrates typically make up no less than 2% by weight of the composition and may further make up about 10-50% by weight of the composition.

The compositions of the present invention may be administered to a mammal as an agent for the prevention or treatment of atherosclerosis and vascular diseases. More specifically, the compositions of the present invention can be administered to a mammal, preferably a human, to reduce total serum cholesterol concentration, reduce low density lipoprotein concentration, increase high density lipoprotein concentration, reduce cholesterol concentration in the liver, and reduce serum triglyceride concentration. In general, without being bound to any particular theory, it is believed that the compositions of the present invention exert their hypocholesterolemic activity by preventing the absorption of cholesterol, substantially inhibiting the reabsorption of bile acids and/or being useful as a source of nitrogen for bacteria in the colon of mammals.

Pharmaceutical compositions and administration

Any of the compositions of the present invention may be formulated as a pharmaceutical or nutraceutical composition. Such compositions may be administered orally, parenterally, by inhalation spray, rectally, intradermally, transdermally or topically, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles as desired. Topical administration may also include the use of transdermal administration, such as transdermal patches or iontophoretic devices. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques. Dosage forms of drugs are discussed, for example, in Remington's pharmaceutical Sciences, (1975), and Liberman and Lachman (1980).

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable carriers and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Dimethylacetamide, surfactants including ionic and nonionic detergents, and polyethylene glycol can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.

Suppositories for rectal administration of the compounds discussed herein can be prepared by mixing the active agent with a suitable non-irritating excipient, such as cocoa butter, synthetic mono-, di-or triglycerides, fatty acids or polyethylene glycols, which are solid at ordinary temperatures but liquid at the rectal temperature and therefore will melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules. In such solid dosage forms, the compounds of the invention are generally admixed with one or more adjuvants appropriate to the indicated route of administration. If administered orally, the compounds may be mixed with lactose, sucrose, starch, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then compressed into tablets or encapsulated for convenient administration. In the case of capsules, tablets and pills, the dosage form may also include buffering agents such as sodium or calcium citrate or magnesium or bicarbonate. Tablets and pills may additionally be prepared with an enteric coating.

For therapeutic purposes, the dosage form for parenteral administration may be in the form of an aqueous or anhydrous isotonic sterile injectable solution or suspension. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the mentioned carriers or diluents for oral dosage forms. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical arts.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, as well as sweetening, flavoring and perfuming agents.

The amount of therapeutically active compound administered and the dosage regimen for treating a cardiovascular disease condition using the compounds and/or compositions of the invention depends on a variety of factors including the age, weight, sex and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound used and therefore may vary widely. A generally acceptable and effective daily dosage may be from about 0.1 to about 6000mg/Kg body weight per day, more typically from about 100 to about 2500mg/Kg per day, and most preferably from about 200 to about 1200mg/Kg per day.

The above detailed description is provided to assist those skilled in the art in carrying out the invention. Even so, this detailed description should not be construed as unduly limiting this invention as variations and changes in the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit or scope of the discovery.

All publications, patents, patent applications, and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent application, or other reference were specifically and individually indicated to be incorporated by reference.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

V. examples

Example 1

Identification of HMF from soy protein

The polypeptide present in the soy protein HMF has potent cholesterol-lowering properties (see example 2). Studies were conducted to identify the origin and partial sequence of these polypeptides. The method used was as follows:

A. polyacrylamide gel electrophoresis.

The polyacrylamide gel electrophoresis method is performed according to methods known in the art. The present invention uses several different methods, which proceed as follows:

Tris-Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

A sample of soy protein for analysis was prepared by freezing whole or ground soybeans, pulverizing in a mortar (pulverization not necessary for soy protein isolate), and extracting the protein with 0.03M Tris, 0.01M 2-mercaptoethanol at pH 8.0 for 1hr at room temperature. A4 mg/ml SDS-solubilized solution of these proteins (0.0625M Tris, 2.3% SDS, 5% beta-mercaptoethanol, 10% glycerol, pH6.8, trace bromophenol blue as tracer dye) was prepared. The sample was heated at 70 ℃ for 10 minutes, cooled for 5 minutes, and then centrifuged to precipitate insoluble material. mu.L (20. mu.g) of each sample supernatant was loaded onto 10-20% total acrylamide gels (as described by Laemmli (1970)) and separated by electrophoresis at 15-30mA (constant current) or 60-100 volts (constant voltage) per gel. The electrophoresis was terminated when the tracer dye was within 2mm of the bottom of the gel. SYPRO orange may replace Coomassie after method 2 in Malone et al, (2001).

C. Analysis of SDS-PAGE by NuPAGE gel electrophoresis

Samples were prepared using 4 x NuPAGE LDS sample buffer (NOVEX catalog No. NP0003, prepared as detailed in the Tris glycine SDS-PAGE subsection with a sample volume of 1/4 and a sample volume of 1/10 mM dithiothreitol.4 μ L (16 μ g) of each sample supernatant was loaded onto NOVEX 4-12% acrylamide Bis-Tris gels.novex Xcel12 mini gel wells were filled with NuPAGE MES running buffer (50mM MES, 50mM Tris, 3.5mM SDS, 1.025mM EDTA, pH 7.7) and proteins were separated by running at 200 volts (constant pressure) until the tracer dye reached the slit at the bottom of the gel.

D. 2-D PAGE of Soy protein

Soybean protein was extracted as detailed in the Tris-glycine SDS-PAGE section. Samples were supplemented to contain 8M urea, 2% CHAPS, 0.35% dithiothreitol, 0.2% ampholyte and 15% isopropanol to a final volume of 450. mu.L containing 0.6-1.05mg total protein. A18 cm strip of pH 3-10 Immobilized PH Gradient (IPG) gel dried strip was allowed to swell with 430. mu.L of this solution for an additional 24-30 hours (the strip was covered with mineral oil during the re-swelling). Using the water-soaked electrode strips, the IPG strips (covered with mineral oil) were focused 50,000-70,000V-hours using the following voltage ramping method. Starting at 100 volts (v) for 1hr, then at 200v 1hr, then at 400v 2hrs, at 400-. Each IPG strip was soaked in 1.5mL of sample equilibration solution (62.5mM Tris, 2.3% SDS, 5% 2-mercaptoethanol, pH6.8, and a trace of bromophenol blue as a tracer dye) for 3.5 minutes. The gel strips were drained and then each placed on a 10-20% acrylamide Tris glycine gel, which was blocked in situ using hot 1% agarose in equilibration solution. A second dimension of gel electrophoresis was performed and stained as detailed in the Tris-glycine SDS-PAGE section.

E. In situ pancreatic enzyme digestion of proteins in acrylamide gels

The gel bands or spots were cut out and placed in 1500 μ l siliconized microcentrifuge tubes. The dye was removed by washing twice with 50% methanol (30 minutes for each wash). The gel fragment was equilibrated in 50% acetonitrile (in 200mM ammonium acetate pH 8.0) for 15 min. The washing was repeated twice. 100% acetonitrile washing 15 minutes, then in Speedvac evaporation to dryness, using 20 u g/mL sequencing grade improved trypsin (Promega catalog number V5111) in 200mM ammonium acetate pH-8.0 10% acetonitrile, at 37 degrees C trypsin action 16-20 hrs. The peptides were extracted with 50% acetonitrile, 0.1% trifluoroacetic acid (TFA) with nutor agitation for 20 minutes. Extraction was repeated with 80% acetonitrile 0.1% TFA in ammonium acetate for 20 min. The final extraction was repeated for 30 minutes. All supernatants were combined and the extracts were dried in a Speed-vac.

F. The bands are trypsinized to provide a trypsinized polypeptide of the polypeptide present in a given band.

To provide a tryptized polypeptide of the polypeptide present in a given band, the tryptic digestion of the band was performed in the gel according to the following method:

1) the gel spots were cut such that the largest dimension of any fragment was less than 1 mm. The gel block was placed in 1500 μ l siliconized microcentrifuge tubes.

2) The gel block (200. mu.l per tube) was washed 2 more times (30 minutes each) with 50% methanol to remove the stain. Coomassie stained gels may require additional washing times to remove the stain. The gel pieces can be stored in this solution. The tube was agitated using Nutator.

3) The gel blocks were washed twice in 200 μ l 50% acetonitrile (in 200mM ammonium acetate, pH 8.0-adjusted with NH 40H), for 15 minutes each, and then once for another 30 minutes. Nutator was used for agitation.

4) Wash with 100% acetonitrile for 15 minutes.

5) The solution was removed from the gel block and evaporated to dryness in Speedvac (15 minutes).

6) A stock solution of trypsin was prepared by adding 1ml of 10% acetonitrile (in 200mM NH40Ac, pH 7.8-8.3-adjusted with NH 40H) to a vial of 20. mu.g trypsin. (Promega sequencing grade modified trypsin-catalog No. V5111 was used).

7) The proteins were digested by adding a trypsin solution just enough to cover the gel mass in each tube. Generally, 20. mu.l is sufficient.

8) The gel blocks were incubated at 37 ℃ for 16-20 hours.

9) The tube was cooled to room temperature and microcentrifuged to allow the water to reach the bottom.

10) The peptide was extracted with 200. mu.l of 50% acetonitrile, 0.1% trifluoroacetic acid (TFA) with nutor agitation for 20 minutes.

11) The supernatant of each tube was retained separately and then agitated with Nutator using 200 μ l of 80% acetonitrile, 0.1% TFA and extracted for 15 minutes.

12) The supernatant was retained, added to each tube previously retained, and then agitated on Nutator using 200 μ 180% acetonitrile, 0.1% TFA, for a final extraction of 30 minutes.

13) The supernatant was retained and the previously retained tubes were added. The extract was dried in a Speed-vac.

MALDI-TOF analysis

The trypsinized polypeptides from the bands are ionized using laser desorption (for MALDI) or electrospray (for LC/MS and LC/MS) to generate a mass spectrum. Measuring the mass of the peptide using MALDI as a mixture; samples were prepared for MALDI analysis using a slightly modified published method (Shevchenko et al, 1996). Samples were recovered by adding 10 μ l of 0.1% TFA to each tube containing the dried peptide. The matrix was prepared by dissolving 10mg/ml of nitrocellulose and 20mg/ml of alpha-cyano-4-hydroxycinnamic acid in a 1: 1 (v: v) mixture of acetone and isopropanol. The matrix was spotted by delivering 0.5. mu.l of the matrix solution onto a MALDI plate. Similarly, 1. mu.l of sample from each sample was placed on a spotted substrate. The sample was allowed to dry on the matrix for approximately 10 minutes to ensure that the peptide bound to the nitrocellulose. Finally, 3 aqueous washes were performed by precipitating 5 μ l of 5% formic acid onto each spot and immediately removing the solution using a vacuum tube. The MALDI template is then moved to a mass spectrometer for MALDI mass spectrometry analysis. Data were collected in reflectance mode using a Voyager DE-STR (Perseptive biosystems, Framingham, Mass.). The spectra were calibrated internally using known trypsin autolysis peaks. A list of peaks of mass of tryptic peptides was generated and searched against the NCBI non-redundant protein sequence database using the MS-Fit search tool to identify the protein (Clauser et al, (1999).

Sometimes, the match is "ambiguous" and needs to be confirmed, or the data is a weak or no significant match at all. In this method, the digested mixture is injected onto a reversed phase column so that the peptides are separated and introduced into a mass spectrometer (in this case an electrospray tandem mass spectrometer) one at a time (or at least one pair coupled at a time). The mass spectrometer measures the mass of the peptide, separates the largest amount of material by filtering out everything else, and sends the peptide to a collision cell where it collides with argon. Peptides tend to break into fragments near the amide bond, so the fragments can exhibit amino acid sequences. The lysis data may be sent to a database search tool that matches the data to a protein sequence database. It uses the partial sequence and initial mass of the tryptic peptide to obtain the match. The MS/MS data for a single tryptic peptide can be used to identify proteins with high confidence.

H. Nano HPLC

The tryptic fragments were separated from the purified proteins (by electrophoretic purification) using nano-HPLC, so that only one (or some) peptide was introduced into the electrospray tandem mass spectrometer at a time. Samples were injected into a CapLC (Waters, Milord, Mass.) system equipped with an autosampler, gradient, and auxiliary pump 5 microliters were injected in "microliter pickup" mode and passed through a 300 μm by 5mm C₁₈The cut-off column (LC Packings, San Francisco, Calif.) was desalted instantaneously. Samples were desalted at high flow rate (30. mu.L/min) for 3 minutes. Magic C at 100 μm × 150mm before introduction into the mass spectrometer₁₈Peptides were isolated on columns (Michrom BioResources, Auburn, Calif.). A reverse phase gradient of generally low to high organic is used for more than about 30 minutes. Mobile phase a was 0.1% formic acid and B was 100% acetonitrile, 0.1% formic acid. The flow rate also increases linearly with the organic flow phase. The system uses a small split, resulting in a column flow rate of about 300-.

MS/MS analysis and data processing

For LC/MS there is a separation step (reverse phase LC) so that the peptides of trypsin are introduced into the mass spectrometer one at a time (theoretically). It is called MS/MS, or tandem MS, because the mass of a peptide is measured first and the mass of a fragment of the peptide is measured second. The tryptic peptides were fragmented by collision with a steady state gas (Ar) and the mass of the fragments was measured. This may often give at least part of the peptide sequence. Data dependent MS/MS studies were performed on a Q-Tof mass spectrometer (Micromass, Beverly, Mass.). The inlet is a modified nano-jet source (New Objective, cambridge, MA) designed to hold the millionths. The collision energy for CAD is determined from the mass and charge state of the peptide. Data was processed by ProteinLynx version 3.4(Micromass, Beverly, Mass.) to generate a Peak List file. Data were searched against NCBI non-redundant protein sequence databases using the search engine MASCOT (Matrix Science, london, uk). The search engine reports the top 20 records. Sequence data that does not match any entry in the database is searched against the NCBI's dbEST database as well as against internally generated sequence databases.

J. Study 1

HMF from soy protein isolate referred to as OBAP-rich was separated using polyacrylamide gel electrophoresis. The 5 bands or gel regions containing the polypeptide were trypsinized and the amino acid sequence was analyzed using mass spectrometry (fig. 7). The origin of the band was identified as shown in Table 2:

table 2.

Simultaneous alignment of the glycinin sequences to determine residues 401-435(VFDGELQEGRVLIVPQNFVVAARSQSDNFEYVSFK) (SEQ ID NO: 1) of the basic subunit of G1 glycinin (72296, AlaBx precursor) (SEQ ID NO: 1):

a VFDGELQEGR and SQSDNFEYVSFK

B is the same as 2 of a, plus: VLIVPQNFVVAAR

K. Study 2

The above putative oleosin sequences do not match known oleosin sequences. The sequence of the low molecular weight soybean oleosin (-18 kDa) is not known. The purpose of the following study was to determine whether the above "putative oleosin" sequence was derived from a 18kDa soybean oleosin. Oil body associated proteins (p34 protein and oleosin) were purified and isolated on polyacrylamide gels (FIG. 8). The band was trypsinized and analyzed by MS.

The presence of a tryptic peptide released from the amphiphilic N-terminal region immediately to the right of the oleosin, next to the hydrophobic domain, was confirmed in the 12kDa band isolated from HMF. The average mass per amino acid was 111.1 daltons. Thus, the number of amino acids in the 12kDa peptide is about 108 amino acids. Therefore, the peptides found in HMF must also comprise a portion of the hydrophobic domain of oleosins. Sequence YETNNSSLNNPPSR (SEQ ID NO: 10) shows residues 33-45 of the putative oleosin, which falls just before the beginning of the hydrophobic core region of the protein.

As a result: it was confirmed that sequence YETNSSLNNPPSR (SEQ ID NO: 10) was found in a low molecular weight soybean oleosin.

Table 3.

L. study 3

The HMF characterized in study 1 consisted of a soy protein fraction with small β -conglycinin. This helps identify the glycinin subunit present in HMF. The following study was performed to determine what, if any, β -conglycinin sequences could be identified in HMF. Soy protein isolate is produced using soybeans lacking G1 glycinin and the isolate is digested with pepsin to produce HMF. HMF was separated using polyacrylamide gel electrophoresis (fig. 9) and the band was trypsinized and characterized using MS.

As a result: the sequence of the polypeptide from beta-conglycinin is determined.

Table 4.

Strip tape	Protein identification from MALDI
		A1	Beta subunit of beta-conglycinin
A2	Same as A1
		A3	Alpha or alpha-major subunit of beta-conglycinin
A4	Peptide identical to A3
		A5	Peptide identical to A3
A6	Peptide identical to A3

Precursor α -primary β -conglycinin (121286): QNPSHNKCLR (SEQ ID NO: 18), and the sequence in SEQ 4191814.

α -major subunit of β -conglycinin (4191814): NQYGHVR (SEQ ID NO: 6), and the sequence in 7442025 below.

α subunit of β -conglycinin (7442025):

NILEASYDTKFEEINK(SEQ ID NO：8)，LQESVIVEI SKK(SEQ ID NO：4)，QQQEEQPLEVRK(SEQ ID NO：5)

example 2

Effect of HMF on Cholesterol uptake

The Caco-2 cell line is derived from human colorectal cancer and is commonly used to study the physiology of intestinal epithelial cells. These cells have been used to study cholesterol, glucose, amino acids, vitamins, fatty acids, bile acids and drug transport processes (Hidalgo et al, 1989); artursson, 1990; homan and Hamelehle, 1998). These cells express lipid and sterol metabolizing enzymes and regulate transporters similar to intestinal intracellular transporters (Levy et al, 1995). Furthermore, they are known to express SR-B1, a recently identified protein that may play a role in cholesterol absorption (Werder et al, 2001) and a number of proteins in the ATP-binding cassette transport family, also play a role in the regulatory network of cholesterol uptake by intestinal epithelial cells (Taipalensu et al, 2001).

Soy protein is the source of HMF. Briefly, soy protein was incubated with pepsin for 17 hours (0.2% NaCl in aqueous phase, 38 ℃, pH 1.1, pepsin 5% of soy protein isolate), heat treated at 90 ℃ for 20 minutes to inactivate pepsin, cooled on ice, and treated with 0.2M Na₂CO₃The pH was adjusted to 6.21 and centrifuged at 4,500g for 20 minutes. The precipitated particles were washed 3 times with water and identified as HMF.

A. Culture of Caco-2 cells for cholesterol absorption assays

1. 96-well Costar solid blank tissue-culture treatment plates (Costar #3917) were pre-coated with rat tail collagen as follows:

a) a solution of 20. mu.g/ml of rat tail collagen was prepared in 0.02N acetic acid (Becton Dickinson/collagen Biomedical catalog No. 40236):

0.02N acetic acid: 11.5ml of 17.4N acetic acid/10 m sterile H were added₂O60. mu.l of collagen (3.32mg/ml) was added per 10ml of 0.02N acetic acid

b) For 96-well plates, 250 μ l of collagen solution/well was added and the cover sheet was allowed to stand overnight at room temperature

c) Wells were rinsed with 1X 250. mu.l DMEM, then 100ml DMEM

2. By 10ml Ca-free²⁺And Mgt²⁺Dulbeco's phosphate buffered saline (applied to cells with passage numbers between 41 and 60) rinsed Caco-2 cells grown to 70-80% full in 2T-150 flasks (Costart # 430825).

3. 6ml of 0.25% trypsin/EDTA solution was added to each flask and incubated for 5-10 minutes at 37 ℃.

4. Cells were washed off the T-150 flask using a 10ml pipette and cell clumps were broken up. Transfer cell suspension to 50ml tubes.

5. To the cell suspension, 12ml of complete medium DMEM with 10% fetal bovine serum, 1 × non-essential amino acids, 50mg/ml gentamicin was added, mixed and the cell particles were pelleted by centrifugation at 2000rpm for 5 minutes (Sorvall RT 7).

6. The medium was removed and replaced with 20ml of complete medium the cells were dispersed using a 10ml pipette. [2 XT 150 flasks-80% overgrown-10X 10⁶Cells]。

7. Cells were seeded into 96-well collagen-coated microtiter plates at a density of 3200 cells/100 μ l per well.

8. Cells were cultured with complete medium every other day. Cells will express surface receptors necessary for cholesterol absorption 13 days after seeding.

B. Cholesterol absorption assay

Uptake of micellar cholesterol by Caco-2 cells was performed by an improved method reported by Field et al (1991). Our method is described below:

1. the compounds/peptides to be tested that inhibit cholesterol absorption in Caco-2 cells were dissolved in DMSO as stock solutions from which dilutions of the test compounds were prepared in pentanol. The diluted compound used in the Caco-2 cholesterol absorption inhibition assay should be 10 times higher than the final dilution to be assayed, i.e., a 2mM solution in the aspiration well will eventually yield 200 μ M for analysis. Sitostanol was used as a control cholesterol absorption inhibitor with maximum inhibition at 200mM (0% cholesterol absorption modulation). The solvent was used only for 100% cholesterol absorption (high dpm).

2. Dilutions of control and test compound/peptide were pipetted in triplicate into a polypropylene shallow well microtiter plate (Sigma M-4029).

3. The plates were dried in a GeneVac instrument at a temperature of 35 ℃ to 45 ℃ overnight. The plates were checked for complete drying before the next step.

4. Prepared as follows in a Trace-Clean amber vial with septum-lined lid (VWR catalog No. 15900-036)³H-cholesterol micelle mixture:

to 1.0ml of 10 × stock solution micelle solution (50mM taurocholate, 1mM oleic acid, 1mM cholesterol), 37.5 μ l was added³H-cholesterol (NEN catalogue No. NET-725) (0.75 nmol cholesterol) 37.5 μ Ci³H-cholesterol (solution containing 0.075% cholesterol as radiolabel ═ tracer). For each microtiter plate 1.8ml was prepared for the excess.

5. 15. mu.l/well of diluent CH₃MeOH (1: 1) was pipetted into all the wells of the dried plate to solvate the dried residue. A polypropylene bath was used for this purpose and the bath was kept on ice to minimize evaporation of the solvent.

6. For the same plate, 15. mu.l of each well was added using a polypropylene solution bath on ice³The H-cholesterol mixture may comprise a radiolabeled solution.

7. The dried plates were flushed with nitrogen in a preheated 37 ℃ evaporation chamber (VWR) for approximately 20-30 minutes.

8. The dried wells were rinsed with 50. mu.l/well of diethyl ether and re-dried in an evaporation chamber. The ether rinse was repeated more than once and dried.

9. The plate was placed in a vacuum desiccator overnight.

10. 150 μ l of room temperature Hank's Balanced Salt Solution (HBSS) (Sigma H-8264) was pipetted into each dry well using a Quadra 96 manipulator. The final concentrations of micellar lipid were as follows: 5mM taurocholate, 100. mu.M oleic acid, 100. mu.M cholesterol and 3.75. mu. Ci/ml³H-cholesterol (or approximately 5. mu.M).

11. The plates were sealed with an adhesive sealing tape (Packard TopSeal-a adhesive tape or Sigma mylar tape T-2162) and shaken at room temperature on a Labline plate shaker, model 4625, (set ═ 6) for at least 30 minutes to dissolve the micelles.

12. Caco-2 cells (as described above) seeded on 96-well plates were washed 5 times with HBSS using an automatic cell washing machine. Just before step 13. Any excess liquid was removed by beating the plate on a paper towel before the next addition of micelles.

13. 100 μ l of the solubilized micelles were transferred to appropriately labeled Caco-2 cell plates using a Quadra 96 manipulator apparatus. The plate was returned to the 37 ℃ incubator for 4 hr.

14. Cells were washed 5 times with 1mM taurocholate ((1mM TC) in cold HBSS using an automated cell washing machine.

15. 200. mu.l/well of scintillation cocktail (Packard MicroScint 40, Cat. 6013641) was pipetted into the cell plate using a Quadra 96 manipulator.

16. The plate (Packard TopSeal-S heat-seal film) was sealed with heat-seal tape and shaken (set at least 6) for at least 20 minutes to mix the luminophore and cell sample.

17. Plates were kept overnight in the dark.

18. Dpm was calculated using a Packard TopCount NXT instrument.

19. The results were calculated as% of control (no inhibitor) as follows:

soybean HMF from various sources showed dose-dependent inhibition of cholesterol uptake by Caco-2 cells (see, e.g., fig. 10). In addition to the beta-conglycinin assay (4.7mg/mL), the concentration of HMF required to reduce 50% cholesterol uptake was between 1.2-2.9 mg/mL.

Example 3

Molecular pharmacological characterization of the mechanism of action of HMF.

To determine whether the test compound/peptide influences³Solubility of H-cholesterol in the preparation of micellar solutions for cholesterol absorption assays (see method of example 2), the following assays were performed:

1. micelles were prepared up to and including step #11 as described in the method of example 2.

2. Transfer 20 μ l of the solubilized micelles to an opaque Costar plate (Costar #3917) using a Quadra 96 manipulator.

3. 200 μ l/well of scintillation cocktail (PackardMicroScint 40, Cat. No. 6013641) was pipetted into the wells of a microtiter plate using a Quadra 96 manipulator.

4. The plate was sealed with a heat seal tape (Packard TopSeal-S heat seal film) and shaken (set at least 6) for at least 20 minutes to mix the luminophore and micelle samples.

5. Plates were kept overnight in the dark.

6. Dpm was calculated using a Packard TopCount NXT instrument.

7. The results were calculated as% of control (no inhibitor) as follows:

8. if the compound/peptide to be tested is substituted for that from the micelle³H-cholesterol, for higher concentrations of the test compound/peptide, a decrease in dpm of the test compound/peptide will be observed. Otherwise, dpms in the solubilized micelles will remain fairly constant over the dilution range of the tested compounds/peptides to be tested.

FIG. 11 illustrates this in contrast to the primary mechanism of cholesterol inhibition by sitostanol.

Example 4

Characterization of soy protein HMF

The yields of HMF from the soy protein isolate fraction with low amounts of oil body associated protein (OBAP (-), the fraction enriched in oil body associated protein (OBAP (+)), and the control were compared. All of these were made from the same soybean (variety a 2247).

Briefly, the soy protein fraction was incubated with pepsin (0.2% NaCl in the aqueous phase, 38 ℃, pH 1.4, pepsin 5% of the soy protein isolate) for 17 hours, heat treated at 90 ℃ for 20 minutes to inactivate the pepsin, cooled on ice, and treated with 0.2M Na₂CO₃The pH was adjusted to 6.21 and centrifuged at 4,500g for 20 minutes. The precipitated particles were washed 2 times with water and freeze-dried. Comparing the weight of the dried precipitated particles to the weight of the original amount of soy protein isolate used to determine the HMF yield analysis [ (last weight/starting weight). times.100 ×]. Both the OBAP (+) and control soy protein samples produced HMF. However, the OBAP (-) fraction did not produce any HMF, indicating that oleosins and or oleosin-related ingredients (e.g. saponins, phospholipids) control the digestibility of soy protein.

The following is a method for fractionating soy protein into Soy Protein Isolate (SPI):

A. the method comprises the following steps: OBAP (-)

1.1 kg of defatted soy flakes (from Cargill) was added to 15kg of deionized water. The pH was adjusted to 7.5 with 1N NaOH. Mix at room temperature for 1 hour.

2. The mixture was centrifuged at 10,000g for 10 minutes and the supernatant was collected.

3. Mixing Na₂SO₄And CaCl₂The supernatant was added at a concentration of 30mM, respectively.

4. The pH of the supernatant from step 3 was adjusted to pH 2.8 with 2N HCl. The mixture was centrifuged at 10,000g for 10 minutes. The supernatant was collected.

5. The supernatant (from step 4) was diluted with 4 times deionized water (e.g., from 1L to 4L). The pH was adjusted to 4.5 with 2N NaOH. Centrifuge at 10,000g for 10 minutes. And collecting the precipitate.

6. The precipitate from step 5 was redissolved in deionized water and the pH was adjusted to 7.5 with 2N NaOH.

7. The neutralized protein mixture was spray dried at an inlet temperature of 200 ℃ and an outlet temperature of 90-95 ℃.

B. The method 2 comprises the following steps: OBAP (+)

1.1 kg of defatted soy flakes (from Cargill) was added to 15kg of deionized water. The pH was adjusted to 7.5 with 1N NaOH. Mix at room temperature for 1 hour.

2. The mixture was centrifuged at 10,0008 for 10 minutes and the supernatant was collected.

3. Mixing Na₂SO₄And CaCl₂The supernatant was added at a concentration of 30mM, respectively.

4. The pH of the supernatant from step 3 was adjusted to pH 2.8 with 2N HCl. The mixture was centrifuged for 10 minutes at 10,0008. The precipitate was collected.

5. The precipitate from step 4 was redissolved in deionized water and the pH was adjusted to 7.5 with 2N NaOH.

6. The neutralized protein mixture was spray dried at an inlet temperature of 200 ℃ and an outlet temperature of 90-95 ℃.

C. The method 3 comprises the following steps: control

1.1 kg of defatted soy flakes (from Cargill) was added to 15kg of deionized water. The pH was adjusted to 7.5 with 1N NaOH. Mix at room temperature for 1 hour.

2. The mixture was centrifuged at 10,000g for 10 minutes and the supernatant was collected.

3. The supernatant (from step 2) was diluted with 4 times deionized water (e.g., from 1L to 4L). The pH was adjusted to 4.5 with 2N HCl. Centrifuge at 10,000g for 10 minutes. And collecting the precipitate.

4. The precipitate from step 5 was redissolved in deionized water and the pH was adjusted to 7.5 with 2N NaOH.

5. The neutralized protein mixture was spray dried at an inlet temperature of 200 ℃ and an outlet temperature of 90-95 ℃.

In one study, the total protein content of the samples was OBAP (-) (94.8%), OBAP (+) (91.8%) and control (68%) (Official Methods of Analysis, AOAC, 16 th edition, 1995, 990.02, Locator # 4.2.08). The yield of HMF from each fraction was determined. The results demonstrate that no HMF was produced from the OBAP (-) samples (see table 5). This study indicates the importance of OBAP and/or the importance of phospholipids, isoflavones and saponins, associated with obtaining this fraction in HMF, although oil body associated proteins do not make up the majority of HMF.

In the same study, the trypsin inhibitor activity of OBAP (-), OBAP (+) and control soy protein isolates were 24, 31.5 and 47.5TIU/mg, respectively (using standard AACC method, 1995, 9 th edition, method 71-10). These activities are not related to the yield of HMF of the sample; thus, the possibility of the lack of trypsin inhibitor activity in the OBAP (-) isolate causing a lack of HMF formation is eliminated.

The chymotrypsin inhibitor activity of the soy protein samples was not correlated with HMF production of the samples (table 5) (AACC, 10 th edition, methods 22-40).

TABLE 5 production of chymotrypsin inhibitory units and HMF per mg of sample.

	CTIU/mg	HMF yield M
			Glycinin	2.0	5
Beta-conglycinin	2.3	14
			OBAP(-)	5.2	0
BC-invalid	8.8	8
			Intermediate product	9.2	24
OBAP(+)	24.5	8
			Glycinin-null	39.2	19

Other soy fractions (glycinin, beta-conglycinin, intermediates) were also prepared at pilot plant at Iowa State university (15 kg experimental plant method # 2; Wu et al, 1999). The values of oleosins in these samples were also determined by Western blotting using oleosin antibodies (see figure 12). Furthermore, it was demonstrated that samples with higher amounts of oleosin produced higher yields of HMF (table 6).

Samples of pepsin digested SPI (OBAP and control), undigested SPI (control, OBAP (-), OBAP (+), oil body protein preparation and ground soybean samples of control soybean IA-2032 were resolved on an 18% Tris/glycine gel and transferred to PVDF membranes. These blots were probed with antisera to oleosin (fig. 13). The antiserum was developed in rabbits using full-length oleosins overexpressed and purified in E.coli as described by QIAexpressinst (QIAGEN, Valencia, Calif.). These blots indicate that samples with higher amounts of oleosin produced higher yields of HMF (table 6).

Example 5

Effect of ethanol extraction on HMF yield

To observe the extent of high yields achieved with intermediate fractions from phospholipids, saponins and isoflavones, the components were extracted from the fraction using 70% ethanol and the fraction was tested repeatedly for HMF production. The result was a low yield of 50%. Adding the extracted fractions to the low yield fractions (glycinin) helps to improve the HMF yield (80%) of those fractions. These results (see table 6) indicate that the ability of HMF polypeptides to resist protease digestion may depend in part on the presence of alcohol extractable components, such as saponins, isoflavones, and phospholipids. The results produced by the above examples are also included in the summary together with Table 6. the conclusion from this summary is that oleosins, beta-conglycinins, alcohol extractable materials (phospholipids, saponins, isoflavones) and basic glycinin subunits contribute to high yields of HMF, which may function as cholesterol lowering substances. The most important components are lipoproteins (oleosins and related phospholipids). It is contemplated that the cholesterol-lowering properties of a soy protein component containing β -conglycinin and glycinin may be enhanced by the addition of vegetable lipoproteins (e.g., oleosin-related phospholipids) or other sources of lipoproteins (e.g., egg yolk lipoproteins).

Table 6: comparison of Source Material and HMF yields

*************

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain factors which are both chemically and physiologically related may be substituted for the factors described herein while the same or similar results would be achieved. It will be apparent to those skilled in the art that all such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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Claims (26)

1. A method of preparing a food product comprising the steps of:

(a) obtaining a selected food product comprising glycinin and/or beta-conglycinin; and

(b) adding to the food product an isolated oil body associated protein and a phospholipid, wherein the oil body associated protein is an oleosin and has a final concentration of at least 5% by weight and the phospholipid has a final concentration of at least 2% by weight.

2. The method of claim 1, further comprising adding at least one compound selected from the group consisting of saponins that are substantially resistant to digestion, phytoestrogens, and carbohydrates.

3. The method of claim 1 wherein the food product is soy-based.

4. The method of claim 1 wherein the food product lacks oil body associated bulk protein prior to the step of adding.

5. The method of claim 1 wherein the food product comprises oil body associated proteins prior to the step of adding.

6. The method of claim 3 wherein the food product is selected from the group consisting of soy flour, soy grain, soy meal, soy flakes, soy milk powder, soy protein concentrate, soy protein isolate, and isolated soy polypeptide.

7. The method of claim 6 wherein the soy protein isolate is a high molecular weight fraction of the soy material treated with a protease.

8. The method of claim 6, wherein the isolated soy polypeptide comprises β -conglycinin, or a fragment thereof.

9. The method of claim 6, wherein the isolated soy polypeptide is glycinin or a fragment thereof.

10. A composition, comprising:

(a) glycinin and/or beta-conglycinin or fragments thereof,

(b) an oil body associated protein, and

(c) a phospholipid;

wherein the oil body associated protein is an oleosin and has a final concentration of at least 5% by weight and a final concentration of phospholipids of at least 2% by weight.

11. The composition of claim 10 wherein glycinin or β -conglycinin is at least partially hydrolyzed by an enzyme or mixture of enzymes.

12. The composition of claim 10, defined as comprising glycinin or fragments thereof and purified oil body associated protein.

13. The composition of claim 10, defined as comprising β -conglycinin, or a fragment thereof, and a purified oil body associated protein.

14. The composition of claim 10, further comprising at least one additional compound, wherein the additional compound is selected from the group consisting of saponins that are substantially resistant to digestion, phytoestrogens, and carbohydrates.

15. The composition of claim 14, wherein the phytoestrogen comprises an isoflavone.

16. The composition of claim 15, wherein the isoflavones are selected from the group consisting of genistein, bipartite zein, equol, biochanin a, formononetin, and naturally occurring glycosides and glycoside conjugates thereof, respectively.

17. The composition of claim 14 wherein the carbohydrate is selected from the group consisting of high amylose starch, fructooligosaccharides, and soy cotyledon fiber.

18. The composition of claim 10, wherein the phospholipid is selected from the group consisting of lecithin, lysolecithin, and lecithin with improved fatty acid composition.

19. The composition of claim 14, wherein the saponin is selected from the group consisting of soybean saponin a, saponin B, saponin E, sapogenol a, sapogenol B, and sapogenol E.

20. The composition of claim 14, wherein the isolated oil body associated protein is a low molecular weight fraction of an oleosin.

21. The composition of claim 14, wherein the oil body associated protein comprises a polypeptide fragment comprising an amphipathic sequence.

22. The composition of claim 10 wherein the glycinin is the basic subunit of glycinin.

23. The composition of claim 22, wherein said glycinin base subunit is a B-Ib subunit.

24. The composition of claim 10, wherein the β -conglycinin protein is the α' subunit or fragment thereof.

25. The composition of claim 10, further defined as comprising greater than 40% β -conglycinin, or fragment thereof.

26. The composition of claim 10, further defined as comprising one or more polypeptide sequences selected from the group consisting of SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: 11.