Europäisches Patentamt *EP000795553B1* (19) European Patent Office

Office européen des brevets (11) EP 0 795 553 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: C07D 311/36 of the grant of the patent: 09.01.2002 Bulletin 2002/02

(21) Application number: 97301595.1

(22) Date of filing: 11.03.1997

(54) Production of isoflavone enriched fractions from extracts Produktion von mit Isoflavonen angereicherten Fraktionen aus Sojabohnenextrakten Production de fractions enrichies en isoflavones à partir d’extraits de graines de soya

(84) Designated Contracting States: (56) References cited: DE FR GB NL EP-A- 0 348 781 WO-A-95/03816 WO-A-95/10512 US-A- 5 032 580 (30) Priority: 13.03.1996 US 614545 • DATABASE WPI Week 9247 Derwent (43) Date of publication of application: Publications Ltd., London, GB; AN 92-385398 17.09.1997 Bulletin 1997/38 XP002031911 & JP 04 283 518 A (KIKKOMAN CORP.) , 8 October 1992 (73) Proprietor: ARCHER DANIELS MIDLAND • PATENT ABSTRACTS OF JAPAN vol. 096, no. COMPANY 003, 29 March 1996 & JP 07 304655 A Decatur, Illinois 62526 (US) (KIKKOMAN CORP.), 21 November 1995, • DATABASE WPI Week 8626 Derwent (72) Inventors: Publications Ltd., London, GB; AN 86-166658 • Gugger, Eric T. XP002031912 & JP 61 100 524 A (JUNICHI Decatur, Illinois 62526 (US) IWAMURA K.K.) , 19 May 1986 • Dueppen, Daniel G. • DATABASE WPI Week 8426 Derwent Decatur, Illinois 62526 (US) Publications Ltd., London, GB; AN 84-161217 XP002031913 & JP 59 085 265 A (NAGANO-KEN (74) Representative: Bond, Bentley George et al KORI DOF) , 17 May 1984 Haseltine Lake & Co., Imperial House, 15-19 Kingsway London WC2B 6UD (GB)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 795 553 B1

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Description

[0001] The invention relates to process for separating isoflavone fractions from a vegetable starting material.

5 Background of the Invention

[0002] Isoflavones are a unique class of plant flavonoids that have a limited distribution in the plant kingdom and may be physically described as colorless, crystalline ketones. The most common and important dietary source of these isoflavones are which contain the following twelve isoflavone isomers: , , 6"-0-malonylgen- 10 istin, 6"-0-acetylgenistin; , , 6"-0-malonyldaidzin, 6"-0-acetyl genistin; , , 6"-0-malonylg- lycitin, 6"-0-acetylglycitin (Kudou, Agric. Biol. Chem. 1991, 55, 2227-2233). Ninety-seven to ninety-eight percent of the isoflavones are in the glycosylated form. [0003] Traditionally, individuals have been limited in their use of soy foods to increase their levels of dietary isofla- vones because the number and variety of soy foods available in the U.S. marketplace is limited. 15 [0004] The isoflavone, genistin, was first isolated from in 1931 by Walz (Justus Liebigs Ann. Chem 489, 118) and later confirmed in 1941 by Walter (J. Amer. Chem. Soc. 63, 3273). Patents have described the production of isoflavone enriched soy-protein products (WO 95/10512; WO 95/10529; WO 95/10530), genistin malonate and daidzin malonate (U.S. Patent 5,141,746), pharmaceutical-type compositions containing isoflavones (U.S. Patents 5,424,331; 4,883,788), and isolation and modification of isoflavones from (U.S. Patents 4,390,559; 4,366,248; 20 4,366,082; 4,264,509; 4,232,122; 4,157,984). [0005] JP-A-07304655 describes the preparation of soya milk, which can be used as a bathing agent, from soya bean material containing intra alia isoflavones. The content of isoflavones in the soya milk fraction is not disclosed. [0006] JP-A-4 283 518 relates to a process of obtaining isoflavones from soybeans by using an aqueous extraction at a high pH between 8 to 11. WO 95/10512 also discloses the separation of isoflavones by aqueous extraction at high 25 pH and subsequent enzyme treatment. JP-A-2 031 912 uses a high pH separation method and filtration for obtaining isoflavone fractions. [0007] However, the present patent relates to the manufacture of highly enriched isoflavone products containing either a wide-range of soy isoflavones or highly-purified genistin gained from an ethanol extract of defatted soybean flakes which can be directly used as a food supplement. 30 [0008] Since coronary heart disease (CHD) is a leading cause of death, especially in the United States and other industrialized nations, an elevated total and LDL cholesterol levels are important risk factors affecting human health. In humans, soy protein products appear to lower serum total cholesterol levels by. an average of about 9.3% and to lower low-density lipoprotein (LDL) cholesterol by an average of about 12.9% when consumed at an average intake level of 47 g soy protein per day (Anderson et al., NEJM, 333:276-282, 1995). 35 [0009] Isoflavones () are implicated as a class of compounds in soy protein products which is at least partly responsible for this cholesterol-lowering effect in animals (Setchell, in McLachlan JA, ed. Estrogens in the En- vironment II:69-85, 1985). In addition, studies with primates suggest that soy isoflavones may account for up to about 60-70% of the hypocholesterolemic properties of soy protein (Anthony et al., Circulation, 90:Suppl:I-235. (abstract), 1994; Anthony et al., J. Nutr., 125:Suppl 3S:803S-804S. (abstract), 1995; Anthony et al., Circulation, 91:925. (abstract), 40 1995). [0010] It has also been suggested that isoflavones have an ability to play a role in the prevention of certain. cancers. Japanese women who have consumed diets rich in isoflavones appear to have a very low incidence of breast cancer (Adlercreutz et al., J. Nutr. 125:757S-770S, 1995). Soy products have also been shown to decrease mammary tumor formation or to inhibit mammary tumor progression in rat breast cancer models (Barnes et al., Clin. Biol. Res. 347: 45 239-253; Hawrylewicz et al., J. Nutr. 121:1693-1698, 1991). Genistein has been shown to inhibit protein tyrosine kinase (Akiyama et al., J. Biol. Chem. 262:5592-5595, 1987), to inhibit angiogenesis (Fotsis et al., Proc. Natl. Acad. Sci. USA 90:2690-2694, 1993), and to induce differentiation in several malignant cell lines (Peterson, J. Nutr. 125:784S-789S, 1995), all of which may be important risk factors in cancer development. Genistein and glycitein (Biochanin A) also appear to inhibit the growth of androgen-dependent and independent prostatic cancer cells in vitro (Peterson and 50 Barnes, Prostate 22:335-345, 1993). Genistein may act as an antioxidant (Wei et al., Nutr. Cancer 20: 1-12, 1993).

Summary of the Invention

[0011] Accordingly, an object of this invention is to provide a isoflavone fractions from a vegetable starting material 55 for individuals to consume isoflavones either as a nutritional supplement or as an ingredient in more traditional types of food. [0012] In light of the potentially positive role of isoflavones and particularly genistein in the prevention of human disease, an object of this invention is to isolate purified forms of genistin, such as the glycone form of genistein, with

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or without subsequent recrystallization as a further purification step. [0013] Another object is to produce a product which is enriched in the entire range of soy isoflavones in order to provide isoflavone products for human diet supplementation or to enrich food products or food ingredients. This object is achieved by a method as claimed in claim 1. Preferred embodiments of the invention are disclosed in dependent 5 claims 2 to 24. [0014] In keeping with an aspect of the invention, selected isoflavones are extracted based on the differentials of the solubilities of isoflavones in aqueous solutions. Alcohol is removed from an extract of defatted soybean flakes by evaporation. Then, the remaining aqueous solution is subjected to ultrafiltration (UF) at an elevated temperature in order to separate soluble isoflavones from other and insoluble materials. The UF permeate containing the soluble 10 isoflavones may be treated in either one of two ways: 1) the permeate is treated with an adsorptive resin for enabling a recovery of a broad range of isoflavone isoforms while removing soluble sugars and salts; of 2) the permeate cools to promote a crystallization ot genistin. Then, genistin is isolated in a highly purified form by either centrifugation or filtration. The genistin may be further purified by a subsequent recrystallization from aqueous alcohol solutions.

15 Brief Description of Drawings

[0015] These and other objects of this invention will become more apparent from the following specification taken with the attached drawings, in which:

20 Fig. 1 is a graph showing the solubility of genistin in water vs. temperature; Fig. 2 is a graph showing the concentration of isoflavone in a UF permeate vs. temperature; and Fig. 3 is a process flow diagram showing the production of the inventive product.

Detailed Description of the Invention 25 [0016] This invention employs methods based on the differential of solubilities of isoflavones in aqueous solutions. Genistin is the least water soluble of the isoflavone glycosides, is insoluble in cold water, and is only slightly soluble in hot water (Fig. 1). [0017] In greater detail, Fig. 1 shows that the solubility of genistin is practically unchanged as the temperature in- 30 creases from 4°Cto50°C, but that the solubility increases rapidly as the temperature increases from 70° to 90°C. Therefore, if the manufacturing process is to recover genistin, the recovery step should be carried out at the high temperature end of the scale. [0018] All isoflavone glycosides other than genistin have higher solubilities in water and readily pass through an ultrafiltration membrane, along with other water soluble components. By increasing the temperature of the aqueous 35 solution prior to ultrafiltration, genistin and all other isoflavones can be separated from insoluble materials. The isofla- vones in the ultrafiltration permeate can be recovered by treating the solution with a resin, washing the resin with water to remove soluble sugars, and eluting the isoflavones with a mixture of ethanol and water. [0019] The starting material for the inventive processes is derived from an aqueous ethanol extract of hexanedetatted soybean flakes. The defatted soybean flakes are extracted with aqueous ethanol (approximately 60-80% ethanol by 40 volume) at temperatures in the range about 44°-63°C or 120-150°F. This aqueous ethanol extract is then subjected to a vacuum distillation in order to remove ethanol. The alcohol-stripped extract is also known as "soy molasses" or "soy solubles." [0020] Then the extract is adjusted within an appropriate temperature range (about 65-95°C) and subjected to ultra- filtration preferably by using a 10,000 molecular weight cut off (MWC0) membrane. However, the process is not limited 45 to this 10,000 cut-off membrane since any membrane which enables a filtration of the desired isoflavones may be used. The smallest cut-off membrane suitable for the inventive procedures should pass a molecular weight of 532, which provides a sufficient retention of insoluble material and passage of isoflavones. [0021] The effect of temperature on the concentration of two principle isoflavones, daidzin and genistin, in the UF permeate, is shown in Fig. 2. Cooler temperatures result in lower concentrations of genistin in the UF permeate. Daidzin 50 concentrations are much less affected by temperature. To achieve optimal concentrations of isoflavones in the UF permeate, ultrafiltration should be carried out at a temperature above 65°C. [0022] For example, Fig. 2 shows the differential between the concentration of daidzin and genistin in an aqueous solution permeate subjected to ultrafiltration. Ultrafiltration at 24°C produces a high concentration of daidzin and a low concentration of genistin. Therefore, if the manufacturing step is to recover daidzin and reject genistin, perhaps the 55 recovery should be carried out at the relatively low temperature of 24°C, although the exact temperature may be selected on a basis of how much genistin can be located in the permeate. On the other hand, if the manufacturing process is designed to recover both daidzin and genistin, perhaps it would be better to operate at the crossover point of about 78°C. For genistin, recovery should be carried out at a higher temperature.

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[0023] A flow diagram representing one example of a manufacturing processes is shown in Fig- 3. [0024] In greater detail, Fig. 3 shows at 20 that the preferred starting material is soy molasses which is subjected to ultrafiltration at 22. At 24, the retentate of the ultrafiltration is further processed, recycled, or otherwise used in another process. 5 [0025] If a batch type process is employed, the volume of the UF retentate fraction 24 is reduced during the ultrafil- tration process by about one-third to two-thirds of the original alcohol-stripped extract volume, or stated otherwise is up to 12-15% solids. The UF retentate may be diafiltered with about one to three retentate volumes of water, which has been previously adjusted to be within a temperature range of about 65-95°C in order to recover a greater percentage of isoflavones in the permeate. 10 [0026] With or without the diafiltered permeate, the ultrafiltration permeate at 26 contains a variety of isoflavones and is adjusted to an appropriate temperature (45-95°C). Then, it is treated with an adsorptive resin at 20 in either a batch or chromatography column type process, followed by washing the resin with water at 30. Next, the isoflavones are eluted at 32 with aqueous alcohol (20-100% by volume, at 25-85°C) as either a gradient or single percentage process. The resulting material is dried (not shown in Fig. 3), preferably by evaporation, in order to produce a product 15 which is approximately 30% isoflavones on a solids basis. The alcohol which is used at 32 may be ethanol, methanol, or isopropanol. The resin may be, but is not limited to, ethylvinylbenzene-divinylbenzene, styrene-divinylbenzene or polystyrene polymers, and may be either ionic or non-ionic. [0027] Alternatively or in addition, with or without a diafiltered permeate, the ultrafiltration permeate 26 is adjusted to an appropriate temperature (about 4-45°C) in order to promote genistin crystallization at 34. Highly purified genistin 20 crystals are then removed at 36 by a low-speed centrifugation or filtration and are finally washed with cold water (not shown in Fig. 3). The final product is between 70-90% pure genistin, measured on a dry basis. The genistin crystals can be further purified by recrystallization from aqueous alcohol solutions, such as aqueous ethanol, methanol, or isopropanol.

25 Examples

1) Ultrafiltration of Soy Solubles

[0028] Using a stainless steel steam-heated immersion coil, soy solubles (15.26 kg) were heated to a constant tem- 30 perature of about 80°C. The soy solubles were then passed through a model 92-HFK-131-UYU spiral wound, polysul- fone, 10,000 nominal molecular weight cut off ultrafiltration membrane (Koch Membrane Systems, Inc., St. Charles, IL) by using a parastaltic pump. Back pressure on the exit side of the membrane was adjusted by means of a hand- lightened clamp to provide a permeate flow of 70 mL/minute. Ultrafiltration was continued until 9.4 kg of permeate was collected leaving 4.8 kg of retentate. Isoflavone profiles of the various fractions are shown below: 35 Sample Weight %Solids Total Isoflavones Genistin Daidzin (kg) (g) (g) (g) Solubles 15.26 8.65 11.45 4.01 4.30 40 Retentate 4.8 11.5 4.63 1.75 1.67 Permeate 9.4 7.7 6.6 2.29 2.68

2) Diafiltration of UF Retentate

45 [0029] Ultrafiltration retentate (80°C initial temperature) was subjected to ultrafiltration as described in Example 1, except that 4.8 kg of tap water (25°C) was fed into the retentate at a feed rate which is the same as the permeate rate or flux of the permeate that was being produced. The retentate was then further ultrafiltered to a final weight of 1.93 kg. Isoflavone profiles of the various fractions is shown below:

50 Sample Weight %Solids Total Isoflavones Genistin Daidzin (kg) (g) (g) (g) Retentate 4.8 11.5 4.63 1.75 1.67 Diafilt. Permeate 7.25 4.28 2.12 0.72 0.96 55 Diafilt. Retentate 1.93 12.26 2.14 0.91 0.58

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3) Adsorption and Recovery of Isoflavones From a Resin

[0030] A glass liquid-chromatography column (2.54 cm i.d.) was slurry packed in 70% ethanol with Dow XUS 40323 divinylbenzene, ethylvinylbenzene copolymer resin. The resin was cleaned with an additional 500 mL of 70% wt ethanol 5 followed by 0.1% wt NaOH (500 mL) and water (500 mL). The resin was then back-flushed with water until the resin bed volume had expanded by about one half of its originally packed volume in order to partition the resin by size. The final packed volume was 100 mL. Fresh UF permeate (2000 mL or 20 column volumes) at an initial temperature of 60°C was fed through the resin bed at 6 column volumes/hour or 10 mL/minute. The resin bed was washed with 500 mL of water at 10 mL/minute to remove residual sugars and other impurities. Isoflavones were then eluted from the 10 resin with a linear gradient of 20-95% ethanol (500 mL total) at 10 mL/minute. Next, the entire ethanolic isoflavone containing fraction was vacuum dried to obtain a product with the following profile:

Sample Weight Total Isoflavones Genistin Daidzin (g) (g) (g) (g) 15 Column Product 6.56 2.2 0.92 0.83

4) Precipitation of Genistin From UF Permeate

20 [0031] A 7.69 kg volume of ultrafiltration permeate (85°C initial temperature) was allowed to cool gradually to an ambient temperature (22°C) during a 16 hour period, with constant stirring. The cooled permeate was then centrifuged at 900 x g for 10 minutes in order to pelletize the genistin precipitate. The supernatant was poured off. The white pellet was diluted with water (100 mL) and recentrifuged at 900 x g for 10 minutes in order to remove any residual supernatant. The white pellet was then vacuum dried to produce 1.02 g of a dried powder. The isoflavone composition of the dried 25 powder was as follows:

Sample Weight Total Isoflavones Genistin Daidzin (g) (g) (g) (g) 30 Genistin Precipitate 1.02 1.00 0.77 0.17

5) Recrystallization of Genistin From UF Permeate Precipitate

[0032] A volume of 80% ethanol (50 mL) was slowly added to 1 g of permeate precipitate while refluxing until the 35 precipitate dissolved. Then, the solution was filtered through Whatman 42 filter paper and slowly cooled (22°C) to produce fine yellow-white crystals. The crystals were harvested by centrifugation at 900 x G for 10 minutes. The su- pernatant was poured off. Next, the crystals were mixed with 50 mL of water (4°C) and recentrifuged to remove any residual supernatant. The water was then poured off and the crystals were vacuum dried to produce a product with the following isoflavone profile: 40

Sample Weight Total Isoflavones Genistin Daidzin (g) (g) (g) (g) Genistin Crystals 0.52 0.52 0.45 0.05 45

[0033] Those who are skilled in the art will readily perceive how to modify the invention. Therefore, the appended claims are to be construed to cover all equivalent structures which fall within the true scope and spirit of the invention.

50 Claims

1. A process for separating isoflavone fractions from an aqueous vegetable starting material, the process comprising the steps of:

55 (a) heating an aqueous vegetable starting material to a constant temperature selected on a basis of an aqueous solubility for given isoflavone fractions that are to be recovered; (b) passing the heated starting material of step (a) through an ultrafiltration membrane to obtain a retentate

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and a permeate, the membrane having a cutoff which passes the given isoflavone fractions; (c) adjusting back pressure in the retentate acting on the membrane of step (b) to adjust flow of the permeate while continuing the ultrafiltration; and (d) collecting isoflavone fractions from the permeate. 5 2. A process according to claim 1, wherein the starting material is soy molasses.

3. A process according to claim 2, wherein step (a) includes the further process step of making an aqueous ethanol extract of hexane-defatted soybean flakes at a temperature in the range from 44° to 63°C. 10 4. A process according to claim 2 or 3, wherein the ultrafiltration of step (b) is carried out at a temperature in the range from 65° to 95°C.

5. A process according to any one of claims 2 to 4, wherein the ultrafiltration membrane of step (b) has a nominal 15 molecular weight cut-off range of from 600 to 100,000.

6. A process according to any one of claims 2 to 4, wherein the ultrafiltration membrane of step (b) has a nominal molecular weight cut-off point of 10,000 nominal molecular weight.

20 7. A process according to any preceding claim, which includes the further step of adding water to the retentate of step (b) at a flow rate which is the same as the permeate rate of flux of the permeate, and the step of again passing the permeate with the added water through an ultrafiltration membrane.

8. A process according to any of claims 2 to 7, wherein the collection of isoflavones from the permeate, of step (d), 25 comprises the steps of:

(d1) treating the permeate with an adsorptive resin; (d2) washing the resin of step (d1) in water; and (d3) eluting adsorbed isoflavones from the washed resin of step (d2) with aqueous alcohol. 30 9. A process according to claim 8, wherein the resin of step (d1) is selected from ionic ethylvinylbenzene-divinylben- zene, non-ionic ethylvinylbenzene-divinylbenzene, ionic styrene-divinylbenzene, non-ionic styrenedivinylbenzene, ionic polystyrene polymers, and non-ionic polystyrene polymers; and the aqueous alcohol of step (d3) is selected from ethanol, methanol, and isopropanol. 35 10. A process according to claim 8, wherein step (d) includes the further step of:

(d4) drying the isoflavones eluted with aqueous alcohol in step (d3) to reduce the isoflavones to a solid basis.

40 11. A process according to claim 10, wherein the drying is effected by evaporating the aqueous alcohol eluate of step (d3) to reduce the isoflavones to a solid basis.

12. A process according to claim 8, wherein the step (d) includes the added steps of:

45 (e) packing a liquid-chromatography column with a slurry of ethanol and a polymeric resin; (f) back flushing the column with water until the resin expands by approximately fifty percent; (g) feeding the permeate of step (b) through the column; (h) eluting isoflavones from the resin by use of ethanol; and (i) drying the isoflavones eluted in step (h). 50 13. A process according to claim 12, wherein the slurry of step (e) is about 50-100% wt ethanol followed by about 1-5 column volumes of 0.1% wt. NaOH and 1-5 column volumes of water.

14. A process according to claim 12 or 13, wherein the temperature of the permeate during step (g) is in the range 55 from 45-95°C.

15. A process according to claim 12, 13 or 14, which includes an additional step, after step (g), of washing the resin with water to remove residual sugars and other impurities therefrom.

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16. A process according to any one of claims 12 to 15, wherein step (i) is a vacuum drying step.

17. A process according to claim 1 for separating isoflavones in an aqueous vegetable starting material, said process comprising the steps of: 5 (1) heating a vegetable starting material to an initial constant temperature in the order of about 65-95°C; (2) passing vegetable solubles produced in step (1) through an ultrafiltration membrane to provide a permeate; (3) gradually cooling the ultrafiltration permeate of step (2) from said initial hot temperature of about 85°Cto a cooled temperature of about 22°C while constantly stirring said permeate, 10 (4) centrifuging the cooled permeate of step (3) at about 900 x g for a period which is long enough to pelletize an isoflavones precipitate produced in step (4); (5) pouring off a supernatant; (6) diluting the precipitate with water; (7) recentrifuging the diluted precipitate of step (6) at about 900 x g to remove a residual supernatant; and 15 (8) drying the recentrifugated precipitate of step (7).

18. The process of claim 17 wherein said vegetable starting material is soy molasses.

19. The process of claim 18 and the further steps of adding ethanol to the washed precipitate of step (6) while refluxing 20 until the precipitate is dissolved, filtering the refluxed precipitate through a filter; and slowly cooling the filtered precipitate until fine crystals are formed.

20. The process of claim 19 and the added step of centrifuging said cooled and filtered precipitate to harvest the fine crystals, then pouring off the resulting supernatant, and finishing the process with said steps (7) and (8). 25 21. A process according to claim 1, comprising the steps of separating insoluble solids from isoflavones in a soy molasses starting material by using ultrafiltration, a permeate stream resulting from said ultrafiltration containing an isoflavone profile corresponding to a profile of soy in the soy molasses starting material, and controlling a temperature throughout said process to select an isoflavone from said profile in order to separate and recover said 30 selected isoflavone from said starting material.

22. A process according to claim 21, comprising the steps of ultrafiltering a feed stream of soy molasses starting material to isolate isoflavones in an ultrafiltration permeate, recovering the isoflavones by adsorption of the isofla- vones on a resin, washing said resin with water in order to remove residual-impurities from the adsorbed isofla- 35 vones, and eluting the washed isoflavones with aqueous alcohol.

23. A process according to claim 1 for recovering isoflavones from a starting material feed stream of soy molasses, said process comprising the steps of: heating said feed stream to a temperature which solubilizes genistin; ultra- filtering said heated feed stream to separate a purified genistin product in a permeate of said starting material, 40 cooling said permeate of said filtered feed stream to promote genistin crystallization, and separating genistin crys- tals by a centrifugation or filtration of said cooled permeate.

24. The process of claim 23 comprising the further steps of recrystallizing the genistin crystallization by forming a genistin precipitate from said crystallization of the ultrafiltration permeate, then making a heated aqueous alcohol 45 solution containing said precipitate, and next recrystallizing the precipitate from a heated aqueous alcohol solution by allowing the solution to cool.

Patentansprüche 50 1. Verfahren zum Separieren von Isoflavonfraktionen aus einem wasserhaltigen, pflanzlichen Ausgangsmaterial, wo- bei das Verfahren die Schritte umfaßt:

(a) Erhitzen eines wasserhaltigen, pflanzlichen Ausgangsmaterials auf eine konstante Temperatur, die auf 55 Basis der Wasserlöslichkeit für bestimmte Isoflavonfraktionen gewählt ist, die vorgesehen sind, wiedergewon- nen zu werden; (b) Leiten des erhitzten Ausgangsmaterials nach Schritt (a) durch eine Ultrafiltrationsmembran, um ein Re- tentat und ein Permeat zu erhalten, wobei die Membran ein Rückhaltevermögen aufweist, das die gegebenen

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Isoflavonfraktionen passieren läßt; (c) Einstellen eines Gegendrucks in dem auf die Membran nach Schritt (b) wirkenden Retentat, um den Durch- fluß des Permeats zu regulieren, während die Ultrafiltration fortgeführt wird; und (d) Sammeln der Isoflavonfraktionen aus dem Permeat. 5 2. Verfahren nach Anspruch 1, wobei das Ausgangsmaterial eine Sojamelasse ist.

3. Verfahren nach Anspruch 2, wobei Schritt (a) den weiteren Verfahrensschritt umfaßt, einen wasserhaltigen Etha- nolextrakt von hexanentfetteten Sojabohnenflocken bei einer Temperatur in dem Bereich von 44° bis 63°Czu 10 bilden.

4. Verfahren nach Anspruch 2 oder 3, wobei die Ultrafiltration nach Schritt (b) bei einer Temperatur in dem Bereich zwischen 65° und 95°C ausgeführt wird.

15 5. Verfahren nach einem der Ansprüche 2 bis 4, wobei die Ultrafiltrationsmembran nach Schritt (b) einen nominalen Abschneidebereich des Molekulargewichts von 600 bis 100,000 aufweist.

6. Verfahren nach einem der Ansprüche 2 bis 4, wobei die Ultrafiltrationsmembran nach Schritt (b) einen nominalen Abschneidepunkt des Molekulargewichts von 10,000 aufweist. 20 7. Verfahren nach einem der vorherigen Ansprüche, das umfaßt den weiteren Schritt, Wasser zu dem Retentat nach Schritt (b) bei einer Durchflußrate hinzuzufügen, die der Permeatrate oder dem Fluß des Permeats entspricht, und den Schritt, das Permeat wieder mit dem hinzugefügten Wasser durch eine Ultrafiltrationsmembran zu führen.

25 8. Verfahren nach einem der Ansprüche 2 bis 7, wobei das Sammeln der Isoflavone aus dem Permeat nach Schritt (d), die Schritte umfaßt:

(d1) Versetzen des Permeats mit einem adsorbierenden Harz; (d2) Waschen des Harzes nach Schritt (d1) in Wasser; und 30 (d3) Eluieren der adsorbierten Isoflavone aus dem gewaschenen Harz nach Schritt (d2) mit wasserhaltigem Alkohol.

9. Verfahren nach Anspruch 8, wobei das Harz nach Schritt (d1) gewählt ist aus ionischem Ethylvinylbenzol-Divinyl- benzol, nichtionischem Ethylvinylbenzol-Divinylbenzol, ionischem Styrol-Divinybenzol, nichtionischem Styrol-Di- 35 vinylbenzol, ionischen Polystyrolpolymeren und nichtionischen Polystyrolpolymeren; und wobei der wasserhaltige Alkohol nach Schritt (d3) gewählt ist aus Ethanol, Methanol und Isopropanol.

10. Verfahren nach Anspruch 8, wobei Schritt (d) den weiteren Schritt umfaßt:

40 (d4) Trocknen der eluierten Isoflavone mit wasserhaltigem Alkohol in Schritt (d3), um die Isoflavone zu einer Festkörperbasis zu reduzieren.

11. Verfahren nach Anspruch 10, wobei das Trocknen durch Verdampfen des wasserhaltigen Alkoholeluats nach Schritt (d3) bewirkt wird, um die Isoflavone zu einer Festkörperbasis zu reduzieren. 45 12. Verfahren nach Anspruch 8, wobei der Schritt (d) die zusätzlichen Schritte umfaßt:

(e) Füllen einer Flüssigchromatographiesäule mit einem Ethanolschlamm und einem Polymerharz; (f) Spülen der Säule mit Wasser, bis sich das Harz um ungefähr 50% ausdehnt; 50 (g) Führen des Permeats nach Schritt (b) durch die Säule; (h) Eluieren der Isoflavone aus dem Harz mittels Ethanol; und (i) Trocknen der in Schritt (h) eluierten Isoflavone.

13. Verfahren nach Anspruch 12, wobei der Schlamm nach Schritt (e) ungefähr 50 bis 100 Gew.%-iges Ethanol auf- 55 weist, gefolgt von ungefähr 1 bis 5 Säulennvolumen 0,1 Gew.-%-iges NaOH und 1 bis 5 Säulenvolumen Wasser.

14. Verfahren nach Anspruch 12 oder 13, wobei die Temperatur des Permeats während Schritt (g) in dem Bereich von 45 bis 95°C liegt.

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15. Verfahren nach Anspruch 12 oder 14, das einen zusätzlichen Schritt nach Schritt (g) aufweist, das Harz mit Wasser zu waschen, um Zukkerreste und andere Verunreinigungen von ihm zu entfernen.

16. Verfahren nach einem der Ansprüche 12 bis 15, wobei Schritt (i) ein Vakuumtrocknungsschritt ist. 5 17. Verfahren nach Anspruch 1 zum Separieren von Isoflavonen in einem wasserhaltigen, pflanzlichen Ausgangsma- terial, wobei das Verfahren die Schritte umfaßt:

(1) Erhitzen eines pflanzlichen Ausgangsmaterials auf eine konstante Ausgangstemperatur in der Größen- 10 ordnung von ungefähr 65 bis 95°C; (2) leiten des in Schritt (1) hergestellten pflanzlichen gelösten durch eine Ultrafiltrationsmembran, um ein Permeat zu bilden; (3) allmähliches Kühlen des Ultrafiltrationspermeats nach Schritt (2) von der heißen Ausgangstemperatur von ungefähr 85°C auf eine gekühlte Temperatur von ungefähr 22°C, während das Permeat konstant gerührt wird; 15 (4) Zentrifugieren des gekühlten Permeats nach Schritt (3) bei ungefähr 900 x g für eine Dauer, die lange genug ist, um ein in Schritt (4) gebildetes Isoflavon-Präzipitat zu pelletisieren; (5) Abgießen eines Überstands; (6) Verdünnen des Präzipitats mit Wasser; (7) erneutes Zentrifugieren des verdünnten Präzipitats nach Schritt (6) bei ungefähr 900 x g, um einen restli- 20 chen Überstand zu entfernen; und (8) Trocknen des erneut zentrifügierten Präzipitats nach Schritt (7).

18. Verfahren nach Anspruch 17, wobei das pflanzliche Ausgangsmaterial eine Sojamelasse ist.

25 19. Verfahren nach Anspruch 18 und den weiteren Schritten, Ethanol zu dem gewaschenen Präzipitat nach Schritt (6), während des Refluxierens hinzuzufügen, bis das Präzipitat aufgelöst ist, Filtrieren des refluxierten Präzipitats durch einen Filter; und langsames Abkühlen des gefilterten Präzipitats bis feine Kristalle gebildet werden.

20. Verfahren nach Anspruch 19 und dem zusätzlichen Schritt, das gekühlte und filtrierte Präzipitat zu zentrifügieren, 30 um feine Kristalle zu sammeln, dann Abgießen des resultierenden Überstands, und Beenden des Verfahrens mit den Schritten (7) und (8).

21. Verfahren nach Anspruch 1, umfassend die Schritte, unlösliche Feststoffe aus Isoflavonen in einem Sojamelasse- Ausgangsmaterial durch Verwendung einer Ultrafiltration zu separieren, wobei ein Permeatstrom aus der Ultrafil- 35 tration mit einem Isoflavon-Profil resultiert, das einem Sojaprofil in dem Sojamelasse-Ausgangsmaterial entspricht, und Regeln einer Temperaturwährend des Verfahrens, um Isoflavon aus dem Profil zuwählen, um das ausgewählte Isoflavon von dem Ausgangsmaterial zu trennen und wiederzugewinnen.

22. Verfahren nach Anspruch 21, umfassend die Schritte, einen Zustrom des Sojamelasse-Ausgangsmaterials zu 40 ultrafiltrieren, um Isoflavone in einem Ultrafiltrationspermeat zu isolieren, Wiedergewinnen der Isoflavone durch Adsorption der Isoflavone auf einem Harz, Waschen des Harzes mit Wasser, um restliche Verunreinigungen von den adsorbierten Isoflavonen zu entfernen, und Eluieren der gewaschenen Isoflavone mit wasserhaltigem Alkohol.

23. Verfahren nach Anspruch 1 zum Wiedergewinnen der Isoflavone aus einem Ausgangsmaterial-Zustrom der So- 45 jamelasse, wobei das Verfahren die Schritte umfaßt: Erhitzen des Zustroms auf eine Temperatur, die Genistin auflöst; Ultrafiltrieren des erhitzten Zustroms, um ein reines Genistin-Produkt in einem Permeat des Ausgangsmaterials abzuscheiden, Kühlen des Permeats des filtrierten Zustroms, um eine Genistin-Kristallisation zu fördern und Ab- scheiden der Genistin-Kristalle durch eine Zentrifugation oder Filtration des gekühlten Permeats. 50 24. Verfahren nach Anspruch 23, umfassend die weiteren Schritte, die Genistin-Kristallisation durch Bilden eines Ge- nistin-Präzipitats aus der Kristallisation des Ultrafiltrationspermeats zu rekristallieren, dann Bilden einer erhitzten, wasserhaltigen Alkohollösung mit dem Präzipitat, und als nächstes Rekristallisieren des Präzipitats aus einer erhitzten, wasserhaltigen Alkohollösung, indem es zugelassen wird, daß 55 sich die Lösung abkühlt.

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Revendications

1. Procédé pour la préparation de fractions d'isoflavones à partir d'une matière première végétale aqueuse, le procédé comprenant les étapes consistant : 5 (a) à chauffer une matière première végétale aqueuse à une température constante choisie sur une base d'une solubilité aqueuse pour des fractions d'isoflavones données qui doivent être récupérées ; (b) à faire passer la matière première chauffée de l'étape (a) à travers une membrane d'ultrafiltration pour obtenir un rétentat et un perméat, la membrane ayant une limite qui fait passer les fractions d'isoflavones 10 données ; (c) à ajuster la contre-pression dans le rétentat en agissant sur la membrane de l'étape (b) afin d'ajuster l'écoulement du perméat tout en continuant l'ultrafiltration ; et (d) à recueillir des fractions d'isoflavones à partir du perméat.

15 2. Procédé selon la revendication 1, dans lequel la matière première est constituée de molasses de soja.

3. Procédé selon la revendication 2, dans lequel l'étape (a) comprend l'autre étape du procédé consistant à fabriquer un extrait aqueux d'éthanol de flocons de graines de soja dégraissées dans de l'hexane à une température dans l'intervalle de 44° à 63°C. 20 4. Procédé selon la revendication 2 ou 3, dans lequel l'ultrafiltration de l'étape (b) est réalisée à une température dans l'intervalle de 65° à 95°C.

5. Procédé selon l'une quelconque des revendications 2 à 4, dans lequel la membrane d'ultrafiltration de l'étape (b) 25 présente un intervalle de limite de poids moléculaire nominal compris entre 600 et 100 000.

6. Procédé selon l'une quelconque des revendications 2 à 4, dans lequel la membrane d'ultrafiltration de l'étape (b) présente un point limite de poids moléculaire nominal de poids moléculaire nominal 10 000.

30 7. Procédé selon l'une quelconque des revendications précédentes, qui comprend l'étape supplémentaire consistant à ajouter de l'eau au rétentat de l'étape (b) à un débit qui est identique à la vitesse de perméat du flux du perméat et l'étape consistant à faire à nouveau passer le perméat avec l'eau ajoutée à travers une membrane d'ultrafiltration.

8. Procédé selon l'une quelconque des revendications 2 à 7, dans lequel le recueil des isoflavones à partir du perméat 35 de l'étape (d) comprend les étapes consistant :

(d1) à traiter le perméat avec une résine adsorbante ; (d2) à laver la résine de l'étape (d1) dans de l'eau ; et (d3) à éluer les isoflavones adsorbées de la résine lavée de l'étape (d2) avec de l'alcool aqueux. 40 9. Procédé selon la revendication 8, dans lequel la résine de l'étape (d1) est choisie parmi des polymères ioniques d'éthylvinylbenzène-divinylbenzène, non ioniques d'éthylvinylbenzène-divinylbenzène, ioniques de styrène-divi- nylbenzène, non ioniques de styrène-divinylbenzène, ioniques de polystyrène et des polymères non ioniques de polystyrène ; et l'alcool aqueux de l'étape (d3) est choisi parmi l'éthanol, le méthanol et l'isopropanol. 45 10. Procédé selon la revendication 8, dans lequel l'étape (d) comprend l'étape supplémentaire consistant:

(d4) à sécher les isoflavones éluées avec de l'alcool aqueux dans l'étape (d3) pour réduire les isoflavones en une base solide. 50 11. Procédé selon la revendication 10, dans lequel le séchage est réalisé par évaporation du produit d'élution avec de l'alcool aqueux de l'étape (d3) pour réduire les isoflavones en une base solide.

12. Procédé selon là revendication 8, dans lequel l'étape (d) comprend les étapes supplémentaires consistant : 55 (e) à garnir une colonne de chromatographie liquide avec une suspension d'éthanol et une résine polymère ; (f) à rincer la colonne avec de l'eau jusqu'à ce que la résine se dilate d'approximativement 50 % ; (g) à introduire le perméat de l'étape (b) à travers la colonne ;

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(h) à éluer les isoflavones de la résine en utilisant de l'éthanol ; et (i) à sécher les isoflavones éluées dans l'étape (h).

13. Procédé selon la revendication 12, dans lequel la suspension de l'étape (e) est d'environ 50-100 % en poids 5 d'éthanol suivie par environ 1-5 volumes de colonne de 0,1 % en poids de NaOH et 1-5 volumes de colonne d'eau.

14. Procédé selon la revendication 12 ou 13, dans lequel la température du perméat pendant l'étape (g) se trouve dans l'intervalle de 45-95°C.

10 15. Procédé selon la revendication 12, 13 ou 14, qui comprend une étape supplémentaire après l'étape (g) consistant à laver la résine avec de l'eau pour éliminer les sucres résiduels et d'autres impuretés de celle-ci.

16. Procédé selon l'une quelconque des revendications 12 à 15, dans lequel l'étape (i) est une étape de séchage sous vide. 15 17. Procédé selon la revendication 1 pour la séparation d'isoflavones dans une matière première végétale aqueuse, ledit procédé comprenant les étapes consistant :

(1) à chauffer une matière première végétale à une température constante initiale de l'ordre d'environ 65-95°C; 20 (2) à faire passer des matières végétales solubles produites dans l'étape (1) à travers une membrane d'ultra- filtration pour fournir un perméat ; (3) à refroidir progressivement le perméat de l'ultrafiltration de l'étape (2) de ladite température chaude initiale d'environ 85°C à une température refroidie d'environ 22°C tout en agitant constamment ledit perméat ; (4) à centrifuger le perméat refroidi de l'étape (3) à environ 900 x g sur une durée qui est assez longue pour 25 transformer un précipité d'isoflavones produit dans l'étape (4) en boulettes ; (5) à éliminer une matière surnageante en la versant ; (6) à diluer le précipité avec de l'eau ; (7) à recentrifuger le précipité dilué de l'étape (6) à environ 900 x g pour éliminer une matière surnageante résiduelle ; et 30 (8) à sécher le précipité recentrifugé de l'étape (7).

18. Procédé selon la revendication 17, dans lequel ladite matière première végétale est constituée de molasses de soja.

35 19. Procédé selon la revendication 18 et comprenant les autres étapes consistant à ajouter de l'éthanol au précipité lavé de l'étape (6) tout en mettant sous reflux jusqu'à ce que le précipité soit dissous, à filtrer le précipité mis sous reflux à travers un filtre ; et à refroidir lentement le précipité filtré jusqu'à ce que de fins cristaux soient formés.

20. Procédé selon la revendication 19 et comprenant l'étape supplémentaire consistant à centrifuger ledit précipité 40 refroidi et filtré pour recueillir les fins cristaux, à éliminer ensuite la matière surnageante résultante en la versant et à finir le procédé avec lesdites étapes (7) et (8).

21. Procédé selon la revendication 1 comprenant les étapes consistant à séparer des matières solides insolubles d'isoflavones dans un matière première de molasses de soja en utilisant l'ultrafiltration, un courant de perméat 45 résultant de ladite ultrafiltration contenant un profil d'isoflavone correspondant à un profil de soja dans la matière première des molasses de soja, et à régler une température tout au long dudit procédé pour choisir une isoflavone partir dudit profil afin de séparer et récupérer ladite isoflavone choisie à partir de ladite matière première.

22. Procédé selon la revendication 21 comprenant les étapes consistant à ultrafiltrer un courant d'alimentation de 50 matière première de molasses de soja pour isoler des isoflavones dans un perméat d'ultrafiltration, à récupérer les isoflavones par adsorption des isoflavones sur une résine, à laver ladite résine avec de l'eau afin d'éliminer des impuretés résiduelles des isoflavones adsorbées et à éluer les isoflavones lavées avec un alcool aqueux.

23. Procédé selon la revendication 1 pour la récupération d'isoflavones à partir d'un courant d'alimentation de matière 55 première de molasses de soja, ledit procédé comprenant les étapes consistant : à chauffer ledit courant d'alimen- tation à une température qui solubilise la génistine ; à ultrafiltrer ledit courant d'alimentation chauffé pour séparer un produit de génistine purifiée en un perméat de ladite matière première, à refroidir ledit perméat dudit courant d'alimentation filtré pour promouvoir la cristallisation de génistine et à séparer les cristaux de génistine par une

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centrifugation ou une filtration dudit perméat refroidi.

24. Procédé selon la revendication 23 comprenant les autres étapes consistant à recristalliser la cristallisation de génistine en formant un précipité de génistine à partir de ladite cristallisation du perméat de l'ultrafiltration, à cons- 5 tituer ensuite une solution aqueuse chauffée d'alcool contenant ledit précipité et à recristalliser ensuite le précipité à partir d'une solution aqueuse d'alcool chauffée en laissant la solution refroidir.

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