HK1053119B - Food product and process - Google Patents

Description

Food product and method of manufacture

Technical Field

The present invention relates generally to food and pharmaceutical formulations comprising one or more isoflavones. More particularly, the present invention relates to highly pure crystalline isoflavones, methods for their production, and the use of highly pure isoflavones in food processing, food products, and pharmaceutical preparations.

Background

Plant isoflavones and their metabolites have attracted considerable attention in the medical literature because these compounds produce some beneficial biological activity. Humans and other animals can benefit from the administration or intake of plant matter and plant extracts containing isoflavones. Biological benefits include estrogenic, antiestrogenic, antioxidant, anti-inflammatory, and anticancer effects. Other benefits of isoflavones include vascular compliance and function, treatment of osteoporosis, alteration of blood lipoprotein levels, reduction of the propensity to form thrombotic processes, and stabilization or reduction of menopausal symptoms. Biologically important isoflavones can be found almost exclusively in leguminous plants with the highest known amounts, as well as alfalfa, soybean, kudzu, and chickpea. There are four major plant isoflavones, represented by daidzein, genistein, and their corresponding methylated ethers, 7-hydroxy-4' -methoxyisoflavone and biochanin. It is believed that the amount of any or all of these isoflavones required to produce a beneficial health effect will generally be from 20 to 100mg per day.

All food products such as soy, chickpea, and lentils can provide the plant isoflavones. However, this approach is highly unreliable in order to achieve prescribed, predictable levels and ratios of isoflavones. Soybean meal is widely regarded as a very valuable source of plant isoflavones, typically having an isoflavone content of 100-300mg/100 g. However, since soybeans contain almost negligible levels of methylated isoflavones, 7-hydroxy-4' -methoxyisoflavone and biochanin, the isoflavone content is dominated by genistein and daidzein. Chickpeas and lentils generally contain all four of these isoflavones, but only about one-tenth the content of soy flour. Moreover, the type and content of isoflavones varies widely, depending on breeding background (variety and cultivation), plant age, environmental conditions and stress, storage conditions of the plant product, cooking and processing methods. Thus, it is very difficult, if not almost impossible, for the average person to obtain a predetermined amount of a particular isoflavone for a particular health benefit from the total diet. If not almost unpredictable, large amounts of food would be required to achieve the desired results due to the variability in isoflavone content and the low isoflavone content per gram of total food product. For these reasons, it is widely believed that it is necessary to extract isoflavones in a semi-pure form in order to provide them in the form of a health supplement in foods or in the form of a prescribed amount of a pharmaceutical preparation. In this way, the consumer can conveniently obtain a substantial amount of isoflavones.

There is another advantage to using isoflavones in a concentrated form. There is strong evidence that each of the four major estrogenic isoflavones in human foods (daidzein, genistein, 7-hydroxy-4' -methoxyisoflavone, and biochanin) have specific biological properties, and that selected health benefits can be obtained by administering specific isoflavones or mixtures thereof in specified doses. It is simply impossible for individuals seeking these benefits to obtain a diet containing a specified dose or ratio of isoflavones using a normal diet. Therefore, it would be desirable to be able to prepare isoflavone extracts having a defined or variable ratio of isoflavones.

In the scientific literature, there is a large body of reference to the application of phytoestrogens and isoflavones in foods and beverages. As mentioned previously, the concentration of isoflavones and the ratio of isoflavones in these additives vary and are astringent.

WO 00/64276(Chen et al) describes a water-in-oil spread comprising a calcium-and vitamin-containing phytoestrogen, an isoflavanone, which has a beneficial health-promoting effect. Spreads may also contain sterols and sterol esters, stanol and stanol esters, and soy protein.

The patent specification "health supplements containing phytoestrogens, analogues or metabolites thereof" (Kelly WO 93/23069) mentions that isoflavones can be extracted from plants of the family leguminosae etc. in the form of dry powders which can be conveniently formulated into food products such as bread, confectionery or beverages, or into pharmaceutical compositions such as tablets or capsules.

Crank et al, U.S. Pat. No. 5,858,449, describe isoflavone-enriched soy protein products and methods for their manufacture. The Crank product has a desirable flavor and can be included in infant formulas, nutritional drinks, milk replacers, bologna, imitation cheese spreads, yogurt, frozen desserts, and other dairy and meat-based products.

WO 98/08503 to Kelly and Joannou discloses the administration of isoflavone type compounds suitable for use in a variety of conditions. It can also be used as food additive for beverage and health chocolate candy.

U.S. patent 5,498,631 to Gorbach et al describes a method of treating menopause, premenstrual syndrome, or producing conditions that reduce estrogen levels by administering an effective amount of an isoflavonoid. The isoflavonoid can be consumed in the form of confectionery chocolate, cookies, cereals or beverages.

Gorbach, U.S. patent 5,733,926, describes a method for treating Alzheimer's disease or age-related cognitive loss, which includes specific isoflavonoids. Can be supplied in the form of candy, biscuit, cereal, or beverage.

U.S. Pat. No. 5,506,211 to Barnes et al describes the delivery of genistein/glucoside conjugates in various forms, including foods such as soy, in order to inhibit acid secretion by osteoclasts and reduce bone resorption.

U.S. patent 4,163,746 to Feuer et al describes 5-methylalkoxyisoflavones for use as weight gain enhancers. Such product compositions may be in liquid or solid form and may contain other beneficial additives such as vitamins and amino acids.

U.S. patent 5,654,011 to Jackson et al discloses a dietary supplement for increasing nutrition in perimenopausal (perimenopausal) women, which contains 8-less than 50mg of phytoestrogen, in combination with various vitamins and minerals. Such supplements may be supplied in the form of tablets, capsules, powders, gels or liquids, or dietetic sugar bars.

U.S. patent 5,464,619 to Kuznicki et al describes a composition, preferably in the form of a beverage, comprising solid green tea, flavonols, sodium and potassium ions, and carbohydrates.

Zilliken, U.S. patent 4,157,984, describes sweet fermented grain-based products for use as antioxidants in food compositions. Such products may be used alone or in combination with isoflavones or other compounds. Zilliken, U.S. patent 4,390,559, uses isoflavones as antioxidants to stabilize edible fats and oils.

U.S. patent 5,424,331 to Shylankevich discloses a material for the treatment and prevention of osteoporosis which includes one or more phytoestrogen compounds and other minerals. May be provided as a dietary supplement or in the form of a medicament.

Potter et al, U.S. patent 5,855,892, discloses the administration of daidzein in the form of a food supplement or a pharmaceutical that produces beneficial changes in cholesterol levels.

U.S. Pat. No. 5,569,459 to Shylankevich describes the use of phytoestrogen compounds to regulate estrogen secretion.

WO 9610341 to Schouten Industries discloses the use of soybean hypocotyls as a source of isoflavones. This material can be used in beverages, dairy products, various breads, health tea and other products. Schouten U.S. limited liability company currently produces a product isolated from soy, called soyfife, which can be included in a variety of food and diet beverages. Furthermore, Archer Daniels Midland (ADM) produces a food supplement called Novasoy which is rich in genistin and daidzin.

Internutria WO 9821947 describes a food or beverage containing phytoestrogen and melatonin to alleviate the symptoms of persistent nocturnal symptoms.

Many of the above references describe difficulties encountered in promoting the ingestion of isoflavone extracts and concentrates because of their often unpleasant taste and unpleasant mouthfeel caused by the nature and composition of the isoflavone extract, and because of the form in which they exist.

There are a number of methods commonly used to prepare isoflavone-rich extracts (see, e.g., BarnesS, Kirk M and Coward L, Soy foods for isoflavones and their conjugates: extraction conditions and HPLC-Mass Spectrometry analysis. (J Agrifood Food Chem)1994, 42: 2466-74.) the specific method depends in large part on whether the isoflavones are contained in the starting material in glycoside or aglycone form Extraction of isoflavones from plant materials such as kudzu or soy, or from by-products of the food industry such as soy syrup or soy whey, generally involves contacting the material with water, an organic solvent, or a mixture of water and an organic solvent. Any or all of these extraction methods suffer from the disadvantage that the extraction method is not selective. If aqueous media is used to extract the glycosides, hundreds of other water-soluble plant components are likewise extracted. Likewise, if the aglycone is extracted in an organic solvent phase, hundreds of other polar organic plant compounds such as saponins, sterols, and flavones also enter the solvent phase indiscriminately with the isoflavones. These various extraction processes generally produce a final product having an isoflavone content of 5-40%, but more typically about 10-20%. The presence of these high levels of non-isoflavone contaminants often results in an unpleasant taste to the final product, making the product unattractive as a food ingredient. In order to prepare a palatable mixture, masking agents such as sucrose must typically be employed. For pharmaceutical use, while the loose nature of the material tends to give the final formulation an unacceptably large volume, in the case of the material being in capsules and the like, this astringency is generally not a problem.

Several other steps, including the selective application of various organic solvents and chromatography, are recognized as suitable methods for increasing the purity of isoflavones. The disadvantages of these processes are that (a) they generally produce low yields of isoflavones, (b) the processes are expensive, and (c) for industrial scale, they are economically non-viable. Moreover, there is no mention of any process suitable for industrial scale, nor of any process that selectively preferentially separates specific isoflavones. For this reason, there is no mention in the literature of isolating a formulation containing isoflavones in the stated proportions.

There is therefore a need for isoflavone extracts and compositions which can be used as food additives and which have the following advantages: (a) having a sufficiently high purity to have a palatable or negligible taste, and (b) the mixture has the required structural characteristics and miscibility to allow them to be easily incorporated into a wide variety of food products. These properties also have significant industrial and practical advantages when applied to pharmaceutical formulations. There is another requirement for these isoflavone extracts and compositions to have a defined ratio of isoflavones.

Summary of The Invention

The present inventors have surprisingly found that it is possible to isolate the plant isoflavones in highly pure form in processable quantities and in the form of highly pure crystals. The purified isoflavones can be formulated into foods and health supplements having a suitable taste, texture and mouthfeel. Highly purified isoflavones can be conveniently added to common food products to facilitate the intake of these important isoflavones during the usual dietary process. Such as spreads, margarines, oils, sauces, breakfast cereals and the like, may contain an effective amount of the desired plant isoflavones without compromising the flavor and mouthfeel of the food product.

The process according to the invention generally comprises extracting the plant material containing isoflavones, such as alfalfa leaves and ground soybeans, with an aqueous organic solvent mixture, passing it through a fine size filter to remove large molecular weight compounds, optionally crystallizing the isoflavones by reducing the organic solvent phase by evaporation, and finally recovering the crystallized isoflavones. The resulting isoflavone product is most preferably in the alpha-crystalline form, which is highly pure, virtually colorless and odorless, and has a desirable taste, texture and mouthfeel.

According to a first aspect of the present invention, there is provided a process for producing an α -crystalline form of isoflavone, which comprises the steps of extracting isoflavone from isoflavone-containing plant material by contacting the isoflavone-containing plant material with an aqueous organic solvent mixture to obtain an extract solution; filtering the extraction solution to reduce the amount of plants with molecular weight greater than that of isoflavone; reducing the solvent in the filtrate to carry out isoflavone alpha-crystallization; recovering the alpha-crystalline isoflavones.

According to a second aspect of the present invention, there is provided a process for producing an α -crystalline form of isoflavone, which comprises the steps of extracting isoflavone from isoflavone-containing plant material by contacting the isoflavone-containing plant material with an aqueous organic solvent mixture to obtain an extract solution; filtering the extraction solution to reduce the amount of plants with molecular weight greater than that of isoflavone; reducing the solvent in the filtrate to carry out isoflavone alpha-crystallization; recovering the alpha-crystalline isoflavones; dissolving the recovered isoflavone crystals in an organic solvent; reducing the volume of the organic solvent gradually to perform selective alpha-crystallization of isoflavone; recovering the selectively crystallized alpha-crystalline isoflavones.

According to a third aspect of the present invention there is provided alpha-crystalline isoflavones prepared by the process of the first or second aspects of the present invention. The isoflavone content of the alpha-crystalline isoflavones is > 50%, more preferably > 65%. The selectively crystallized alpha-crystalline isoflavones have an isoflavone content of > 80%, more preferably > 90%.

According to a fourth aspect of the present invention there is provided an alpha-crystalline form of isoflavones. The alpha-crystalline form is colorless, odorless, and virtually tasteless when formulated into food products and preparations. The alpha-crystalline form of isoflavones, is generally substantially pure.

According to a fifth aspect of the present invention there is provided a food or pharmaceutical formulation comprising one or more highly pure isoflavones.

According to a sixth aspect of the present invention there is provided a process for the manufacture of a food product or pharmaceutical preparation comprising the step of admixing an alpha-crystalline form of isoflavone with one or more ingredients of said food product or pharmaceutical preparation. The alpha-crystalline form is substantially colorless, odorless, and virtually tasteless when formulated into said food or pharmaceutical preparation. Preferably, the alpha-crystalline isoflavones are ground or pulverized prior to formulation into said food and pharmaceutical preparations.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Drawings

FIG. 1 shows the use of 3^#High pressure liquid chromatogram of feed (alfalfa extract) for identification of isoflavone peak.

Figure 2 shows a high pressure liquid chromatogram with four different feeds.

Figure 3 shows chromatograms of various permeates or filtrates.

FIG. 4 shows the chromatogram of the filtrate using a one-stage (K1) and a two-stage (K1K1) ultrafiltration.

FIG. 5 shows a chromatogram of a retentate sample.

FIG. 6 shows an X-ray powder diffraction pattern of alpha-crystalline isoflavones.

Figure 7 shows the X-ray powder diffraction of a "crude" red alfalfa extract.

Figure 8 shows the X-ray powder diffraction of a "crude" red alfalfa extract.

Detailed Description

The isoflavones used in the methods and compositions of the present invention may be derived from all plant sources, synthetic or derived isoflavones, or isoflavone precursors and prodrugs. Readily available sources of isoflavones are derived from whole alfalfa plant material, with the isoflavones contained mostly in the leaves of this plant.

Suitable alfalfa includes red alfalfa (t.pratense), underground alfalfa (t.subteranean), or white alfalfa (t.depends). Other suitable plant materials containing isoflavones include other legumes such as kudzu, soy, and chickpea. The isoflavone content of the extract isolated therefrom can be controlled by selecting different types and amounts of plant material containing isoflavones. It is possible to use certain types of alfalfa, such as alfalfa having a high content of 7-hydroxy-4' -methoxyisoflavones, or to select another alfalfa having a different isoflavone base, such as biochanin.

In the case of leafy plant material such as alfalfa, the plant isoflavones are extracted by crushing or chopping the leaves, typically prior to the solvent extraction process. The isoflavones are preferably extracted in their aglycon form. After the leaves are pulverized, cut or ground, the plant body releases glucosidase contained in the plant, and isoflavone glycoside is subjected to enzymatic hydrolysis to produce corresponding aglycone. In the case of materials that are more inert, such as soy flour or waste stream materials of soy processing, the glycosidic bond can be broken using techniques known to those skilled in the art such as hydrolysis by exposure to heat and/or acid, or enzymatic treatment.

The ground or cut plant material is extracted with a solvent, typically water and an organic solvent, preferably an organic solvent miscible with water. The ratio of water to organic solvent may generally be from 1: 10 to 10: 1, and may for example comprise water and solvent in equal proportions. Any organic solvent or mixture of such solvents may be employed. The organic solvent may preferably be an organic solvent of C2-10, more preferably C1-4 (e.g., methanol, ethanol, isopropanol, butanol, succinaldehyde, propylene glycol, erythritol, chloroform, dichloromethane, trichloroethane, acetonitrile, ethylene glycol, ethyl acetate, methyl acetate, glycerol dihydroxyacetone, tetrahydrofuran, diethyl ether, or acetone), most preferably a C1-4 alcohol such as ethanol. Such extracts may be prepared by contacting the plant material with a water-solvent mixture by methods well known in the art. The mixture may also include enzymes that aid in breaking down the isoflavone glycosides into the aglycone form. This mixture can be vigorously stirred to produce an emulsion. The mixture may also be left to undergo enzymatic hydrolysis. The temperature of the mixture may, for example, be from ambient temperature to the boiling point of the solvent. The contact time may be from 1 hour to several weeks. The extract may be physically separated from the undissolved plant material by conventional methods such as filtration, centrifugation, or clarification, or other standard methods.

The inventors have surprisingly found that the aglycone isoflavones in the obtained solvent show a distinctive patterned behaviour in organic solvents, depending on the presence or absence of higher molecular weight compounds. It has now been found that when the resulting aqueous slurry is passed through a filter to remove particulate undissolved plant material as well as dissolved or suspended plant material having a molecular weight greater than that of isoflavones, isoflavones behave in a manner that is quite different from when the amount of higher molecular weight compounds present is not substantially reduced or removed at all. In particular, it has been found that in the absence of high molecular weight phytochemicals, the production of alpha-crystals of isoflavones can be induced. Without being bound by theory, it is believed that the desired alpha-crystalline isoflavones precipitated from the isoflavone containing organic solvent concentrate can be obtained in the filtration step.

The aqueous organic solvent mixture is forced through a fine-diameter physical separation barrier for filtration. This is typically an ultrafiltration device which includes a plastic or paper filter which blocks the passage of compounds having a molecular weight greater than about 500. This method removes most of the proteins, carbohydrates, lipids, oils, resins, and chlorophyll, leaving a generally colorless and transparent liquid that exits the device.

The aqueous organic solvent mixture obtained is then treated to reduce the amount of organic solvent in the mixture. This treatment is very easy to perform in a rotary evaporator. When the amount of organic solvent in the aqueous mixture reaches a critical point, there is noticeable precipitation of the alpha-crystalline form of the isoflavones. Precipitation typically occurs in water: the proportion of organic solvent is within a narrow range. At this point the evaporation can be stopped and the crystals collected by simple sedimentation or filtration. After drying in a drying oven or just at ambient temperature, the isoflavones typically have a purity of 65-75%, are relatively colorless and odorless, have only a slight astringent taste, and have a recovery of > 90% from the starting isoflavones, with the ratio of product isoflavones being similar to the starting mixture. Such isoflavone products can be considered as end products to be added to food or pharmaceutical preparations.

In addition, greater purification of the isoflavones in the final product, or isolation of selectively crystallized isoflavones, may be required. To obtain both results, further crystallization is required. For this purpose, the crystals of the first crystallization are dissolved in an organic solvent. The solvent is preferably a water-immiscible solvent, such as a C1-4 ester or ether. The solvent of choice is ethyl acetate. The solution was then fed to a rotary evaporator and the volume of solvent was gradually reduced. At the critical point of the process, a first crystallization occurs, followed by a second crystallization after a further reduction in the amount of solvent. The evaporation rate of the solvent is carefully controlled so that the first and second crystallization are easily separated. The first crystals were found to be predominantly 7-hydroxy-4 '-methoxyisoflavone and daidzein, containing about 8-12% biochanin and genistein, and the second crystals were found to be predominantly biochanin and genistein, containing 5-25% 7-hydroxy-4' -methoxyisoflavone and daidzein. Each crystal was collected and dried separately. In each case, the isoflavone content of the dry material was found to be 90-95%. Alternatively, the last two crystallization steps are not separated. The crystallization can be carried out as a process, and the crystals are recovered only after the second crystallization.

The crystals collected by filtration are air-dried and the crystals are pulverized into a fine powder by methods well known in the art, such as hammer milling or ball milling. Particularly when formulated into foods and pharmaceuticals, the resulting fine powder is almost colorless and odorless and practically tasteless. This makes the crystallized product particularly suitable for use as a food additive without masking the taste of the isoflavone product crystals. In addition, it has been found that the finely powdered alpha-crystalline isoflavones of the present invention blend well in a variety of food processing methods. The addition of isoflavone-rich crystals does not significantly increase the volume of food and pharmaceutical preparations and can be added to said preparations rapidly.

The use of membrane technology enables the present inventors to recover isoflavones in high yields and high purity while maintaining their physiological activity and function. Prior to the present invention, a general method for the isolation and recovery of isoflavones involved the evaporation of the organic solvent from an organic/aqueous solvent mixture, precipitation of the crude isoflavones, followed by recovery. The recovered isoflavones may be re-extracted into an organic solvent, washed with water to remove water soluble impurities, and re-precipitated to obtain a crude isoflavone extract. The present inventors have found that purification of the isoflavone extract from the initial isoflavone extract can be conveniently carried out using an ultrafiltration membrane. The use of F4 membranes is particularly effective in removing a significant amount of isoflavone impurities having a molecular weight greater than the molecular weight of the isoflavone itself.

The role of the ultrafiltration process is to purify the crude extract before recovering the solid by evaporation, followed by organic re-extraction, and precipitation in sequence. The removed impurities are typically natural biopolymer gums that have been found to interfere with process prediction and product quality control in downstream operations. Moreover, the gum component can significantly reduce the activity of the product, increase the colour (brown and green), co-precipitate with isoflavones, in particular dissolve in organic solvents.

The role of the ultrafilter is to prevent molecules larger than a certain size from passing through the filter. The most effective ultrafilter for the present invention is one that selects an ultrafilter with a smaller interception size than the biopolymer impurities, but larger than the valuable isoflavones. As can be seen from fig. 1-6, membrane F4 provided isoflavones with a very high purity and also at a much higher concentration than the other membranes tested.

The effect of ultrafiltration on product activity is shown in FIGS. 1-8 and is shown in Table 1 below. Table 1 includes isoflavone extracts (3) passed through various ultrafiltration membranes^#Feed) were analyzed, and the membranes were listed in order of decreasing membrane intercept size.

The results of "filtrate" showed that the isoflavone content (0.069. + -. 10.005%) and the ratio (0.85. + -. 0.06) of the first 6 filtrates were not changed at all, indicating that the isoflavones easily pass through the membrane. Membranes Y3 and F4 caused a reduction in isoflavone concentration because the size of the membrane interception was very close to the size of the isoflavone molecule. Even so, no change in the isoflavone ratio occurred with films Y3 and F4.

The data for "solid product" illustrates the composition of the solid recovered by precipitation from the filtrate. The main function of membrane filtration is to increase the activity of the product. The comparative example (no filtration) produced an activity of 47.1%, which was significantly exceeded with each membrane treatment. Both membranes Y3 and F4 produced activities that exceeded the target range of 40-70%.

The reason for the improved activity is that ultrafiltration removed some biopolymer impurities which were co-precipitated with isoflavones from 15% alcohol and co-extracted into ethyl acetate. The product activity of the conditioning film F4 (76.5% vs 85.2%) was modulated due to the slow passage of isoflavones compared to the unhindered passage of impurities, salt and sugar.

In the last column of table 1, the comparative example produced a ratio of 0.755, which is somewhat lower than the feed range because the residual solubility of biochanin in 15% alcohol is higher. Due to the effect of the solubility, the ratio of all film products except F4(0.85) was in a predetermined range of 0.69-0.81.

TABLE 1 analysis of ultrafiltration samples

3# feed	Filtrate		Product solids
					Film	Isoflavone%	Ratio of	Activity%	Ratio of
Is free of	0.074	0.81	47.3	0.73
					Is free of	0.065	0.91	47.0	0.78
K1	0.063	0.91	52.7	0.78
					K1	0.072	0.82	62.6	0.76
K1K1	0.068	0.82	64.1	0.77
					Om1	0.073	0.79	63.4	0.71
Y3	0.055	0.83	85.2	0.77
					F4	0.023	0.92	76.1	0.85

Table 1 above highlights the advantages of the ultrafiltration product compared to the microfiltration product. The microfiltration solid product is expected to be comparable to the comparative solid product described above without the use of a membrane, i.e. where the activity of the isoflavone extract is about 47% and the ratio is about 0.75. This indicates that microfiltration of the aqueous slurry of crude isoflavone extract is only a process improvement, whereas ultrafiltration of the crude extract results in an improvement in the product.

The effect of ultrafiltration is to remove flocculated impurities, which means that isoflavones can crystallize, preferably in their alpha-crystalline form, rather than being forced to sink by flocculation. Thus ultrafiltration significantly improves the throughput from the point of view of product activity without reducing the yield. The ultrafiltration step removes gum from the raw alfalfa extract and concentrates it to any purity desired by removing chlorophyll and biopolymers that cause flocculation and increase color. Importantly, the isoflavone extract after the ultrafiltration step, particularly after formulation into food products and preparations, is essentially colorless, odorless and virtually tasteless.

The crystalline isoflavone product of the present invention is significantly superior to previously known phytoestrogen extracts. For example, the water-in-oil spread described by Chen et al (WO 00/64276) comprises a phytoestrogen extract containing other botanical ingredients and impurities. In the crystalline isoflavones of the present invention, such contaminants as soy protein, sterols and esters of sterols, esters of stanol and stanol, plant gums, chlorophyll, and biopolymers are either absent or present in significantly reduced amounts. These contaminants generally impart undesirable color, astringency, and unpleasant mouthfeel to the food products containing them.

Therefore, the α -crystalline form of isoflavone products has been used as an important ingredient in foods such as spreads, margarines, cream products, oils, sauces, breakfast cereals, various breads, and beverages. The crystalline isoflavones can be incorporated into various types of food products by one skilled in the art using standard methods known. The existence of the isoflavone crystal product does not damage the flavor and mouthfeel of the food. Allowing the general public to be supplied with beneficial isoflavones on demand during a typical diet. The alpha-crystalline isoflavones of a particular food or pharmaceutical formulation are selected to obtain the benefits of ingestion of the plant isoflavones without the need to specifically target the diet to be supplemented with pills and capsules containing the isoflavone extract.

The invention is further illustrated and explained by the following examples. And these examples are not intended to limit the present invention.

EXAMPLE 1 alpha-crystalline form of isoflavones

Within 2 hours of harvesting, freshly harvested red alfalfa (100kg) was macerated and squeezed. The impregnated alfalfa was allowed to stand at ambient temperature for 6 hours to provide an enzymatic process to convert the isoflavones from their glycosides to the aglycone form. The impregnated alfalfa was then contacted with 1000 liters of 50% ethanol in water for 4 hours at room temperature with continuous stirring and agitation. The steep liquor is then separated from the remaining plant material by grating pressing or using a grooved drum.

The steep water then undergoes an ultrafiltration step, passing the water through a series of cartridges containing polyethylene filters. The primary filter cutoff was 1,000-10,000MW, and the secondary filter cutoff was 500-1000 MW. The liquid was forced through these filters at a pressure of about 2000 kPa.

The ultrafiltered maceration extract was distilled under reduced pressure to reduce the solvent from about 50% ethanol in water to about 10% ethanol. In the vicinity of this point in the evaporation process, the isoflavones crystallize in large quantities. The evaporation rate is reduced and the ethanol content of the solvent is further reduced by about 1-2%, at which point no more significant crystal formation is observed. At this point the distillation process was stopped, the solvent and crystal suspension were drained, the crystals and solvent were separated with a paper filter, air dried, and hammer milled to a fine powder. The isoflavone content of the product is determined by high pressure liquid chromatography.

The characteristics of this product are as follows:

physical appearance: light yellow to colorless, odorless and slightly astringent

Isoflavone content: purity of 78%

Chickpea extract 52%; 29% of 7-hydroxy-4' -methoxyisoflavone;

10% genistein; 9% daidzein

Example 2 margarine containing alpha-crystalline isoflavones

Margarine spreads are made from margarine fat comprising sunflower/canola oil and hardstock consisting of a mixture of fully hardened palm kernel oil and fully hardened palm oil optionally transesterified. Margarine fat is formulated into a margarine spread according to standard techniques known in the art, wherein the margarine fat is blended with the powdered alpha-crystalline isoflavone prepared according to example 1 along with other ingredients including salt, milk skin and whey powder, emulsifiers, pigments, flavors, vitamins, and water. The margarine spread was formulated so that the margarine spread contained 0.1% (w/w) of powdered isoflavone crystals. The spread comprising isoflavones shows no significant difference in taste, appearance, and mouthfeel from a comparable spread made without any isoflavone crystals. In contrast, red alfalfa extract, which was prepared without the ultrafiltration step, provided an unpleasant taste, color, and texture to the formulated spread containing such extract.

EXAMPLE 3 formulations of different isoflavone ratios

The alpha-crystalline isoflavone formulation prepared according to example 1 (1200g) was dissolved in ethyl acetate (50 liters). The solution was charged to a rotary evaporator, heated and vacuum applied. When the volume of ethyl acetate was reduced to about 30 liters, the first crystallization occurred. At this point the vacuum was released and the rotary evaporation was stopped and the crystals were allowed to settle. The crystals and solvent were removed, passed through a paper filter and the crystals were collected. The filtered solvent was then returned to the rotary evaporator, further heated and vacuum applied. When about 10 liters of ethyl acetate were further distilled off, a second crystallization occurred. These crystals were collected by filtration. The two batches were dried and ground to a powder.

The characteristics of both products are as follows:

first crystal product and second crystal product

Physical appearance: colorless, odorless, tasteless, colorless, odorless, and tasteless

Isoflavone content: 92 percent to 94 percent

7-hydroxy-4' -methoxyisoflavone: 82 percent to 20 percent

Chickpea extract: 11% to 75%

5 percent of daidzein and 1 percent of daidzein

Genistein 2% to 4%

Example 4X-ray powder diffraction data for alpha-crystalline isoflavones

The alpha-crystalline isoflavones isolated in example 1 were subjected to X-ray powder diffraction. Figure 6 shows an X-ray powder diffraction pattern. For comparison, the "crude" red clover extract was subjected to the same X-ray powder diffraction, the results of which are shown in fig. 7 and 8, where the noise had been removed. Currently, in Promensil (Novogen) production, crude red alfalfa extract is used. Fig. 7 and 8 show that the crude red alfalfa extract is an amorphous mixture of substances. In contrast, the α -crystalline isoflavones depicted in FIG. 6 have an exceptionally highly ordered crystal structure.

Example 5 analysis of molecular weight and isoflavones

General procedure

Samples were analyzed by size exclusion chromatography in 50% ethanol to illustrate the molecular size distribution of biomacromolecule impurities. The isoflavone profile was also analyzed simultaneously, as it coincidentally was partly similar.

The molecular weight range is indicative rather than absolute, as the alcohol has an effect on the calibration of the column and there are no closely related MW standards.

Four isoflavone cocktail Standard sensitization alfalfa extract samples (3)^#Feed) (fig. 1), isoflavone peaks were identified on the chromatogram. Isoflavones exhibit affinity for the column and are therefore eluted after their actual size of 250- & lton (daltons) (i.e. they occur at smaller LogMW values of 0-1.7).

Figure 2 shows that in a set of four different alfalfa extracts, there are many other minor components at Log MW 0-2.5 that may be closely related to isoflavones because they have similar affinities that make them pass through the end of a size exclusion column at Log MW 2.5. In the range of four different alfalfa extracts, minor impurities that absorb primarily UV occur at Log MW of 1.5-2.5.

Note that the isoflavone standard is not at the same weight as the peak position of isoflavones in alfalfa extract. Depending on the affinity, their elution is influenced by the type and amount of other components that compete for action or passage through the column.

The size range of the gum or biopolymer is about 1,000-. A sharp peak of green chlorophyll occurs near Log MW 3. Higher polymers with Log MW 3.5-4.0 appear yellow, brown, red brown, or black depending on concentration. The absorption of biopolymers is generally lower than that of small aromatic organic molecules, which means that the MW distribution shown here estimates the proportion of biopolymers present in the sample to be too low.

Microfiltration of crude isoflavone precipitate

The crude isoflavones precipitated from the 15% alcohol were recovered by microfiltration. The resulting thin suspension that is finally discharged is concentrated to a thick slurry that is still flowable. Concentration of the slurry aids in the purification of the isoflavone precipitate, removing water soluble components that remain in the filtrate.

In the long filtration experiments, at the highest pressure, the microfilter providing the greatest throughput was AF500, with a throughput of 68 L.m.at 100kPa^-2·h^-1To 45 L.m at 200kPa^-2·h^-1. The throughput drop at higher pressures is due to the formation of the filter cake and the compaction of the filter cake. In the appropriately selected low size range and with low feed pressure, the filtrate is completely transparent without any sign of haze. The recovery of isoflavones obtained by the microfiltration step was 95%. Performing secondary microfiltration, collecting the precipitate generated after the primary filtration, wherein the total recovery rate of the two-stage microfiltration isoflavone is 99%.

Ultrafiltration of isoflavones dissolved in 50% alcohol

The crude isoflavone extract was purified by ultrafiltration prior to sequential rotary evaporation-solid recovery-ethyl acetate re-extraction. It has been found that ultrafiltration removes polymeric impurities (i.e., gum removal) that interfere with process prediction and product quality in downstream operations. The gum component significantly reduces product activity, increases color (brown and green), co-precipitates with isoflavones in 15% alcohol, and is partially soluble in organic solvents. This part of the gum also causes objection as it is non-pharmaceutically effective as a pharmaceutical ingredient. The simplest and most desirable solution is to remove it.

The crude extract was analyzed by size exclusion chromatography to determine the size and mass distribution of the molecules, and the appropriate ultrafilter was selected using this information. The role of the ultrafilter is to prevent molecules larger than a certain size from passing through the filter, which is completely similar to the operation and biochemical functions of hemodialysis and kidney functions and of plant cell walls in preventing their release of genetic material.

The ultrafilter is distinguished by the size of the pores, which determine the size of the intercepted molecules, and the size of the pores is chosen to be smaller than the biopolymer but larger than the valuable small molecules. A dense membrane is adopted for carrying out the purification requirement,at 400kPa and 1-10 L.m^-2·h^-1The alfalfa extract is subjected to ultrafiltration under the conditions of (1). Membranes of this type, generally operated at a pressure of 1000-^-2·h^-1。

When all feeds were found to pass through a 50kD ultrafilter (fig. 3, membrane HZ20P, where P is the permeate or filtrate sample), chromatographic results with an upper MW limit of about 40kD (fig. 1 and 2) were experimentally confirmed. All permeation analyses in FIG. 3 were performed using different progressively denser membranes from the same feed (3)^#Feed) was performed. As shown in the series of curves in fig. 3, the removal of biopolymers increases as the membrane becomes denser.

It can be seen that membrane F4 enabled a significant reduction in chlorophyll. F4 was the only membrane that significantly affected the isoflavone material at LogMW of 1.8-2.5. Although the reduction in isoflavone concentration slows the mass transfer of isoflavones through the process, the enormous benefit in removing impurities is more important than this problem. The process K1K1 in FIG. 3 is 3^#The feed was treated in series by two-stage membrane K1. The improvement in biopolymer removal by one-stage (K1) and two-stage (K1K1) ultrafiltration is more clearly shown in FIG. 4. Isoflavones remain unaffected, but chlorophyll and biopolymers are significantly reduced. The purification effect obtained with K1K1 was superior to that obtained with Om1, even though Om1 was a denser membrane than K1 (this phenomenon is reasonable in basic principle for multi-stage membrane processes).

As for the films HZ20, GR61 and YW3, the performance of the debonding was insufficient.

One of the objectives of ultrafiltration is to improve the quality of the product. To demonstrate this, let 3^#The feed was ultrafiltered in 50% alcohol, (comparative example ═ no ultrafiltration). The filtrate was diluted with water to 15% alcohol and the isoflavone product was collected on a microfilter from which the isoflavones were re-extracted into 50% alcohol. Product analysis showed that in both cases of ultrafiltration, the product extract contained a higher concentration of isoflavones and a lower concentration of biopolymers than the control.

In addition, fig. 5 shows a retentate sample that was generated using ultrafiltration to reduce the initial volume of the feed by a factor of 28.8. The peak of the biopolymer is significantly enhanced compared to the feed sample of fig. 2, while the peak of the isoflavone remains unchanged. The isoflavone production lost to this retentate waste stream is proportional to the retentate volume and the feed volume. When the concentration factor was 28.8, the yield loss was 3.5%. Designing an even higher concentration factor or re-extraction (membrane filtration) retentate can further reduce this loss.

The reference to any prior art in this specification is not to be taken as an acknowledgment or any form of suggestion that prior art forms part of the common general knowledge in the field of endeavour.

It will be appreciated by persons skilled in the art that variations and modifications of the invention as described herein are possible, except as specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Claims (30)

1. A process for producing an alpha-crystalline form of isoflavones comprising the steps of:

(a) extracting isoflavones from the isoflavone-containing plant material by contacting the isoflavone-containing plant material with an aqueous organic solvent mixture to obtain an extract;

(b) ultrafiltering the extractive solution to reduce the amount of plant with molecular weight greater than that of isoflavone;

(c) reducing the solvent in the filtrate to cause alpha-crystallization of the isoflavone; and

(d) recovering the alpha-crystalline isoflavones.

2. The process of claim 1 wherein the aqueous organic solvent mixture has a ratio of water to organic solvent of from 1: 10 to 10: 1.

3. The method of claim 2, wherein the ratio of water to organic solvent is about 1: 1.

4. The method of claim 3, wherein the organic solvent is a C1-4 organic solvent selected from the group consisting of alcohols, chlorinated alkanes, glycols, alkyl esters, and alkyl ethers.

5. The method of claim 4, wherein the organic solvent is ethanol.

6. The method of claim 1 wherein the alpha-crystalline isoflavones have an isoflavone content of > 65%.

7. The process of any one of claims 1 to 6 wherein the alpha-crystalline form of isoflavone has an X-ray powder diffraction pattern substantially as shown in figure 6.

8. A process for producing an alpha-crystalline form of isoflavones comprising the steps of:

(a) extracting isoflavones from the isoflavone-containing plant material by contacting the isoflavone-containing plant material with an aqueous organic solvent mixture to obtain an extract;

(b) ultrafiltering the extractive solution to reduce the amount of plant with molecular weight greater than that of isoflavone;

(c) reducing the solvent in the filtrate to cause alpha-crystallization of the isoflavone;

(d) recovering the alpha-crystalline isoflavones;

(e) dissolving the isoflavone crystals recovered from step (d) in an organic solvent;

(f) gradually reducing the volume of the organic solvent to produce selective alpha-crystallization of the isoflavone; and

(g) isolating the selectively crystallized alpha-crystalline isoflavones.

9. The process of claim 8 wherein the water to organic solvent ratio of the aqueous organic solvent mixture in step (a) is from 1: 10 to 10: 1.

10. The method of claim 9, wherein the ratio of water to organic solvent is about 1: 1.

11. The method of claim 10, wherein the organic solvent is a C1-4 organic solvent selected from the group consisting of alcohols, chlorinated alkanes, glycols, alkyl esters, and alkyl ethers.

12. The method of claim 11, wherein the organic solvent is ethanol.

13. The process of claim 8 wherein the alpha-crystalline isoflavones have an isoflavone content of > 50%.

14. The method of claim 13 wherein the isoflavone content is > 65%.

15. The process of claim 8, wherein the organic solvent of step (e) is an ester or ether of C1-4.

16. The process of claim 15, wherein the organic solvent is ethyl acetate.

17. The method of claim 8 wherein the isoflavone content of the selectively crystallized α -crystalline isoflavones is > 80%.

18. The method of claim 17 wherein the isoflavone content of the selectively crystallized α -crystalline isoflavones is > 90%.

19. The method of claim 18 wherein the selectively crystallized α -crystalline isoflavones consist essentially of 7-hydroxy-4' -methoxyisoflavone and daidzein.

20. The method of claim 18 wherein the selectively crystallized α -crystalline isoflavones comprise predominantly biochanin and genistein.

21. The process of any one of claims 8 to 20, wherein the alpha-crystalline form of isoflavone has an X-ray powder diffraction pattern substantially as shown in figure 6.

22. An isoflavone in the form of an α -crystalline form, wherein the crystalline form has an X-ray powder diffraction pattern substantially as shown in figure 6.

23. The α -crystalline isoflavone of claim 22 wherein the α -crystalline form is substantially colorless, odorless, and virtually tasteless when formulated in food and pharmaceutical preparations.

24. The α -crystalline isoflavone of claim 23 wherein the α -crystalline form comprises greater than 50% isoflavones.

25. The α -crystalline isoflavone of claim 24 wherein the α -crystalline form comprises greater than 90% isoflavones.

26. The α -crystalline isoflavone of claim 25 wherein the α -crystalline form is substantially pure.

27. A food product or pharmaceutical formulation comprising the α -crystalline form of isoflavone of any of claims 22 to 26.

28. The food or pharmaceutical preparation according to claim 27, which is a spread, margarine, cream product, oil, sauce, breakfast cereal, various breads or beverages.

29. A process for preparing a food or pharmaceutical preparation comprising the step of admixing the α -crystalline form of isoflavone of claim 22 with one or more ingredients of said food or pharmaceutical preparation wherein the α -crystalline form is substantially colorless, odorless, and substantially tasteless when formulated in said food or pharmaceutical preparation.

30. The method of claim 29 wherein the α -crystalline isoflavones are ground or pulverized prior to being formulated in said food product or pharmaceutical preparation.