TWI245603B - Isoflavone rich protein isolate and process for producing - Google Patents

Abstract

A process for providing an isoflavone rich protein isolate is provided, along with the isoflavone rich protein isolate produced thereby. A vegetable material containing protein and at least one isoflavone compound is extracted with an aqueous extractant having a neutral pH. The protein and isoflavones are extracted into the extractant, and the extractant containing the protein and isoflavones is separated from insoluble vegetable materials to form a protein extract. The pH of the protein extract is adjusted to about the isoelectric point of the protein to precipitate the protein. The extract containing the precipitated protein is cooled to a temperature of from 30 DEG F to 90 DEG F, and then the protein is separated from the extract. The cool separation temperatures unexpectedly significantly increase the concentration of isoflavones recovered in the separated protein, while the neutral extract pH inhibits loss of protein normally observed at cool or cold separation temperatures.

Description

1245603 V. Description of the invention (1) Background of the invention The present invention relates to isoflavone-enriched plant protein isolates and methods for preparing them. Isoflavones are found in a variety of legumes and oilseeds, including plant protein ingredients such as soybeans. These compounds, for the purpose of the present invention, include soy isoflavone glycosides, 6Π-0Ac soy isoflavone glycosides, 6 " -OMal soy isoflavone glycosides, Daugishuanghuanghuang 1, Sanjing Isoflavone, 6 " -〇Ac Sanjing Isoflavone, 6 "-OMal trihydroxy isoflavone glycosides, trihydroxy isoflavones, methoxydihydroxy isoflavone glycosides, 6 "-OMal trioxo isoflavone glycosides, methoxydihydroxy isoflavones, biocharni A and formononetin. As used herein, the tables "propylenediyl" and "Acn" represent πethylfluorenyl. The structures of these isoflavones are shown in Formulas 1 and 2 below.

Rv ^ V ° \

Compounds Ri r2 R, r4 Dihydroxy isoflavones OH H OH OH Soy isoflavones OH H H OH Methodihydroxy isoflavones OH och3 H OH Biochani A OH H OH och3 Methanone OH H H OCH,

O: \ 61 \ 61461.PTD Page 6 1245603 V. Description of the invention (2)

Compound r2 R; r4 Dihydroxy isoflavone glycoside Η H OH OH 6'0mal Dihydroxy isoflavone glycoside COCH2CH2H H OH OH 6 ”-OAc dihydroxy isoflavone glycoside COCH3 H OH OH Soy isoflavone glycoside Η HH OH 6'0mal soybean Isoflavone glycosides coch2co2h HH OH 6 ”-OAc Soy isoflavone glycosides COCH3 HH OH Methionol isoflavone glycosides OCH3 H OH 6” -OMal Methionone flavonoids coch3 OCH3 H OH It is understood in plant proteins such as soybeans The isoflavones contained in it can inhibit the growth of human cancer cells, such as breast cancer cells and prostaglandins, as described in the following article: π three-way isoflavones inhibit the growth of human breast cancer cells: independent of estrogen receptors and multidrugs "Resistance genes", Peterson and Barnes, Newsletter of Biochemical and Biophysical Research, Vol. 179, Issue 1, pp. 661-667, August 30, 1991; Trisoflavones and Bio-Chaney A inhibit human prostate Cancer cell growth, but non-epidermal growth factor tyrosine autophosphorylates π by Peterson and Bar

1245603 V. Description of the invention (3)

Nice, 22: 335-345 (1 9 93); " Soybean inhibits breast tumors in breast cancer models " 'by Barnes et al.' J | _ ^^ Proto- and carcinogenic '23, 23 9-253 ( 1 99 0). These isoflavones have also been found to reduce cardiovascular risk factors, such as reducing the amount of lipoproteins and low-density cholesterol induced by arteriosclerosis, and by increasing the endothelium-dependent vasodilation response. Yet another typical example is that these isoflavone compounds have been associated with the inherent bitterness of plant protein materials such as soybeans. In the commercial production of such proteinaceous materials, such as: mass isolates and protein concentrates, the focus is on removing these isoflavone compound cakes. T Plant protein isolates, such as soy protein isolates, are typical blood-transmitting types 1i protein-containing plants The raw materials are extracted with an aqueous alkaline extract solution / pH of white matter Ϊ from about 8 to about 11, and egg PH is extracted from insoluble plant matter, because the liquid I is preferably quite alkaline, often from about 9 to The raw material t of 10, protein is extremely soluble in higher alkaline extracts, resulting in self-vegetable plant material: $ protein extraction. Protein-containing extracts and insoluble proteins provide protein extracts. The extract is then recovered from the protein extract. Protein substances have self-electric points. Wide: From adjusting the pH of the extract to an appropriate protein with a suitable acid, the protein material of Γ is separated from the extract. Typically, the protein-killed eggs are separated at a temperature from about 12 ° to 15 ° C, because of the yield of sinking. For these temperatures, densely wrapped, enhanced recovery of protein material becomes loose and stained with Τ 之The temperature is avoided, because at these temperatures the self-extraction of Luoyou reduces the yield and commercial utility of the recovered protein. After the protein is scooped, the protein is thoroughly washed to remove residual sugar.

Page 8 1245603 V. Description of the invention (4), isoflavones, ash and other non-protein substances. When the plant material contains isoflavones, in addition to the protein, the isoflavones are dissolved together with the protein from the aqueous extract. Many isoflavones are still soluble in the extract after the protein components of the santin are separated from the extract. After the precipitated protein material is separated from the extract, the extract and the isoflavones dissolved in it are often discarded. Residual isoflavones left in the separated proteinaceous material are often removed by thorough washing of the proteinaceous material to confirm that the isoflavone-related flavor does not exist in the proteinaceous material. However, what is desired is to provide isoflavone-rich proteinaceous materials, and methods for preparing them, which are suitable for use in diets. Such isoflavone-rich protein materials, when used in diets, can provide the nutritional benefits of protein and the health benefits of isoflavones. SUMMARY OF THE INVENTION The present invention is a method for preparing isoflavone protein-rich material, and the isoflavone protein-rich material produced. The plant material containing protein and isoflavones is extracted with an aqueous extract with substantially neutral pH, and the extract is separated from the insoluble plant material to generate an isoflavone and protein-containing extract. The pH of the extract is adjusted to the isoelectric point of the grade protein to precipitate the protein substance containing isoflavones. The protein material and the extract are separated at a temperature of about 30 ° F to about 90 ° F, and washing of the separated protein material is avoided to generate an isoflavone-rich protein material. In another aspect, the present invention is a method similar to that described above, except that the separated proteinaceous material is washed with water. In a preferred embodiment, the separated protein material is washed with water at less than about 4 times the weight of the original plant material, and more preferably, less than about 2 times the weight of the original plant material. Better in another

1245603 V. Description of the invention (5) In the specific embodiment, the water used for cleaning and separating the protein material is about 30 ° to about 9 (TF temperature. In a preferred embodiment, the protein material prepared according to the present invention Isoflavones include at least one of the following: soy isoflavone glycosides, 6n-0Mal soy isoflavone glycosides, 6n-0AC soy isoflavone glycosides, soy isoflavones, trihydroxy isoflavone glycosides, 6n -OMal trihydroxy isoflavone glycosides, 6 ”-0AC trihydroxy isoflavone glycosides, trihydroxy isoflavones, methoxy dihydroxy isoflavone glycosides, 6n-0Mal methoxy dihydroxy isoflavone glycosides, trioxane isoflavones, biocharney A and methanone Its mixture.

With the method of the present invention, the isoflavone content of a protein material separated from a plant material containing protein and isoflavones is significantly higher than that of a protein material separated from such a plant material using a conventional protein separation method. First, avoid washing the separated protein material with water or using a limited amount of water to wash the separated protein material to increase the amount of isoflavones stored in the protein material. Compared to traditional methods, the separated protein material i is thoroughly washed. Secondly, the proteinaceous material separated and precipitated from the extract under cold or cold temperature did not unexpectedly significantly increase the amount of isoflavones trapped in the separated proteinaceous material. The inventors of the present invention have found that the cold or cold separation of the protein material at temperatures below 90 ° F greatly increases the amount of desired isoflavones recovered in the protein material. The separation of precipitated protein material under cold or cold temperatures has been avoided in traditional protein separation processes, so that no fluffy proteins are formed. The inventors have discovered that the formation of fluffy proteins at cold or cold separation temperatures below 90 ° F can be avoided by extraction of the primary plant raw material with a substantially neutral aqueous extract, rather than a medium or strong aqueous alkaline solution. Precipitated protein material formed from substantially neutral aqueous extracts

Page 10, 1245603 V. Description of the invention (6) Unexpectedly wrapped together in a tight substance, even at a cool or cold temperature of 90 ° F, so that the protein substance can be equivalent to the amount of protein protein recovered in traditional methods. Yield recovery, in which the recovered protein material is rich in isoflavones due to the low temperature separation process. Therefore, the use of neutral aqueous extracts allows the isoflavone content of the protein material to be increased, and the proteins are separated from the extracts at cool or cold temperatures without the formation of soft proteins typically at temperatures below 90 ° F. The resulting loss of protein yield. Description of the Preferred Embodiments Although the present invention will describe related soybean raw materials, and the method is particularly suitable for preparing isoflavone-rich protein isolates from soybean raw materials, this method can generally be applied to a variety of isoflavone-containing plant protein sources Preparation of protein isolates. Other vegetable protein sources containing isoflavones that can be used in the method of the present invention include, but are not limited to, one or more of the following plant materials: chickpea, peanut, marama bean, sword bean, off-sword bean, seaside bean, kalaw Beans, String Beans, Nuts, Beans, Kidney Beans, Beans, Cocoa Beans, Winged Beans, Jicama, Broad Beans, Potatoes, Golden Wheat Beans, Jump Beans, Florida Beans, African Locust Beans and Derivatives of Such Plant Materials Thing. The soybean starting material of the method of the present invention is a soybean raw material containing soybean protein and isoflavones, such as soybean cake, soybean flour and soybean fine powder. The isoflavone compounds contained in soybean starting materials typically include trihydroxy isoflavone glycosides, 6n -0Ma 1 trihydroxy isoflavone glycosides, 6 " -OAc trihydroxy isoflavone glycosides, trisomyrin isoflavones, soybean isoflavone glycosides , 6n -OMal soy isoflavone glycosides, 6n -OAc soy isoflavone glycosides, soy isoflavones, oxydihydroxy isoflavone glycosides, 6n -OMal methoxydihydroxy isoflavone glycosides and methoxydihydroxy isoflavones, shown above Formulas 1 and 2. From soybeans

O: \ 61 \ 61461.PTD Page 11 1245603 V. Description of the invention (7) ΐ ί ^ 2 You can / to modify it to adjust the content of isohuanghuang in the starting material ' The conjugate and isoflavone glucoside are converted into glucoside isoflavones by fermentation before conversion to the formula of Formula 1, and are converted in the formula I of the present invention using starting materials. The preferred starting material for the 4 agent or mechanical extraction of the present invention is soybean cake, from which the oil has been removed by the method of the invention, and it can be produced from soybean according to the traditional method. This protein and plant are described about soy cake starting materials, but other starting materials containing other soybeans, the white ones, can be used instead of the big cake. Extracted from the cake, it was extracted with an aqueous extract with substantially neutral pH, and it was determined as self-defining DH beta substance and isoxanthin. As used herein, the substantially neutral ρΗ sphere is in the range of about ρΗ 8. Use a substantially neutral PE extract to maximize the amount of protein recovered from the exudate during cold or warm separation of the protein material from the extract, and to form a mill. Optimally, the aqueous extract is soft protein. " .5. Typical alkali or acid reagents can be; liquid; === sexual extracts ... what you want ... including gas oxidation and so on: oxygen; weekly; calcium oxide, hydrochloric acid, sulfuric acid, acetic acid and phosphoric acid. The isoflavones desired by the lice and potassium lice are dissolved in the aqueous extract, and these compounds are mixed together in the aqueous extract during the wash period, and the weight ratio of the sexual extract is preferably controlled to Once the cake and water can dissolve more isoflavones and proteins. The egg and the degree can be carried out in a variety of ways, including the counterflow of soybean cake = quality and the weight ratio of the extraction solution of isoflavone to the soybean cake is about 5 ·· 1 to about 丨 2 · fL, which is preferably used for extraction. To re-extract the bean cake and provide protein * _____ which is the initial extraction solution, " /, page_ aqueous extraction solution. May page 1245603 V. Description of the invention (8) Alternatively, a two-step extraction process may be used, preferably where the second extraction of the extraction liquid in the first step in the weight ratio of the extraction liquid to the bean cake is about 3: It is completed at 1 to about 6: 1, so that the combined weight ratio of the extract solution and the bean cake in step 2 does not exceed the total weight ratio of the extract solution and the bean cake to be about 1 1: 1 to about 14: 1. Although not critical, extraction can be performed at a temperature of up to about 120 ° F, preferably at a temperature of about 90 ° F, for a period of about 5 minutes to about 60 minutes, preferably about 15 minutes .

The aqueous extract containing the proteinaceous material and isoflavones is then separated from the insoluble plant material. The extract can be separated from the insoluble plant material by conventional liquid / solid separation processes such as filtration or centrifugation. In a preferred embodiment, the protein / isoflavone extract is separated from the insoluble material by a centrifuge, and the extract is collected from the supernatant.

The pH of the aqueous protein extract containing isoflavone is adjusted to about the isoelectric point of the protein with edible acid to precipitate the protein substance containing isoflavone, so that the protein substance and other water-soluble substances extracted from plant raw materials, such as sugar Separated from ash and ash. The isoelectric point of soy protein is usually between about pp4.0 to about 5.0, and more specifically between about 4.4 to about 4.6 pH. The edible acid added to adjust the pH of the extract to about the isoelectric point of the protein may be any suitable edible acid, such as acetic acid, sulfuric acid, phosphoric acid, and hydrochloric acid. The acidic precipitation of proteins in the extract solution separates the extract into two layers, one layer is the precipitated protein clot, and the other layer is the aqueous extract. The protein clot is separated from the extract to form a protein isolate. Before separating the protein clot from the extract, the aqueous extract is cooled to a temperature below 90 ° F to significantly increase the concentration of isoflavones in the separated protein material.

Page 13 1245603 V. Description of Invention (9) Degree. The inventors of the present invention have discovered that the separation of protein clots from the extract under cool or cold conditions can unexpectedly significantly increase the concentration of isoflavones captured in the separated protein material. The concentration of isoflavones per unit of recovered protein material, and therefore the total amount of isoflavones in the recovered protein, increases as the temperature at which the protein clot and extract are separated separately decreases. Preferably, the protein clot separates from the extract at a temperature below about 90 ° F, typically from about 30 ° F to about 90 ° F, more preferably from about 40 ° F to About 80 ° F, and most preferably from about 50 ° F to about 700F. The quality of the white egg and the egg are separated from the sediment. The egg liquid is separated from the extract when the extractable liquid comes out of the extract and the water is extracted. After the egg is white, the egg is extracted. Extract cold PH from or in the cooling liquid and after the quality of the extraction of the temples, white acid, egg white, acid, ketones, yellow, white, and different degrees. Appropriate, the electricity is as regular as the experience. Quality, temperature and whiteness before whitening. The liquid extract is warmed and extracted from the cold, or the cold recipe is transferred to the system. From the previous time, the essentials must be at the temperature of the egg. However, when the cold temperature is changed with an opener, I want to think about it until the liquid can be extracted, but the cold is extracted. In the middle, the whole process is adjusted. Set or store cold liquid can be extracted in the middle of the extraction process excess 〇 small liquid in the extraction or extraction, but the liquid cooled out to be more white. After the cold egg has been filtered, the Chinese best liquid system is the most popular. The extraction is from the quality, such as the heart, and the solid separation is from the separated material. Dividing with yellow makes him strange. On, the machine can be divided into the center of the white egg of the heart and the heart of the white egg. Hair quality material Page 14 12456603 V. Description of the invention (10) It is also possible to increase the period of time before the separation of the isoflavones captured in the capture fluid, the typical geological material that is kept cool or separated at cold temperatures before separation Charge extraction induced by protein extraction, protein extraction and liquid extraction with separate production procedures and subsequent sinking, and protein precipitation obtained by extractable extraction liquid extraction during medium or strong alkaline extraction; House. The substance of the present invention can be dehydrated to replace the separated rich water to form a white substance-rich finish. Make sure that the separated protein is cooler or catches the isoflavone proteinaceous material when it is cooler, over time. Preferably, the protein substance is maintained at substantially neutrality, and preferably at about 1 / at less than about 90. Open from below 90 ° F. It would be better if tradition changed from being essentially neutral to being soft. White lambda is more likely because the protein is separated from the plutonium-rich protein monolayer separated by this suggested mechanism under the cold or cold temperature of the protein. Trucking Avoid measuring the amount of protein under cold separation. Protein material or self-extracted at cold temperature Increases a limited period of time> > Erlin's protein material Divides into the extract for an hour. At a temperature of ° F, it is divided into traditional aqueous extracts based on sexual aqueous extraction, cold or cold temperature, and extracted with medium-strength or strong aqueous extracts. The inventor believes that alkaline conditions are lighter in alkaline neutral extraction. Soft protein restriction. Isolate the protein of the isosal astragalus 1, or wash it and then remove it, and wash out the amount of the isoporus bream plus protein in the sinking eggs, and let it sink; keep it in the cold or cold as much as about 1 hour In the extract, at least 30 minutes, the amount may be unexpectedly yielded, and its isoflavones, such as the protein material of the lake above, are extracted from the extract, and the egg is not extracted at these temperatures. Degree, rejection, which leads to, however, not to avoid the formation of isoflavone-rich isoflavone-rich egg isoflavones protein, all substances are washed

1245603 5. In the description of the invention (11), it is better to reduce the degree of cleaning to substantially reduce the removal of isoflavones from proteins. Avoiding or reducing the separation of separated protein material can recover more than 2 times the isoflavones in the protein. Compared with the protein single ion formed in accordance with the traditional method of protein single ion formation, the protein material is in the self-extracted solution. After being separated, it was extensively cleaned.

If the proteinaceous material is washed, the washing is preferably limited to a single washing with a minimum amount of water. Preferably, if the protein material is to be washed, the washing should be a single washing, wherein the weight of the water washing liquid used is from 2 to 4 times the weight of the original plant starting material. Furthermore, it is preferred that the temperature of the water cleaning solution is cool or cold, preferably from about 30 ° F to about 90 ° F, to further reduce the amount of isoflavones removed in the cleaning solution. After separating the isoflavone-rich protein material from the extract and any washing of the protein material, the protein material can be dehydrated in a conventional manner. Preferably, the protein material is dehydrated by centrifugation or concentration, or a combination thereof. The dehydrated protein material can then be dried using conventional drying techniques, preferably spray-dried, to form a dry isoflavone-rich protein material.

The isoflavone-rich protein material formed by the method of the present invention contains at least 1 mg / g of isoflavones. If the isoflavone-rich protein material is not cleaned, the material preferably contains at least 2.8 mg / g isoflavones, and more preferably contains at least 4.2 mg / g isoflavones. If the isoflavone-rich proteinaceous material is washed, the material preferably contains at least 1.6 mg / g isoflavone, and more preferably at least 3.2 mg / g isoflavone. In a preferred embodiment, the isoflavone-rich protein material contains from about 2 mg / g isoflavone to about 40 mg / g isoflavone, and more preferably contains about 2.5 mg / g isoflavone to about 30 mg / Gram of isoflavones.

Page 16 1245603 V. Description of the invention (12) Isoflavone-rich protein substances can be incorporated into a variety of foods to provide the nutritional benefits of protein and the health benefits of isoflavones. For example, isoflavone-rich proteinaceous materials can be used in the following foods: meats, especially emulsified meats and textured meats; beverages such as nutritional beverages, sports drinks, protein-fortified beverages, fruit juices, milk, milk substitutes and weight loss beverages; cheese , Such as hard and soft cheeses, creamy roads and Qada milk networks; cold desserts such as ice cream, ice cream, low-fat frozen desserts, and non-dairy frozen desserts; curds; soups; puddings; baked products; salad dressings ; And dipping and spreading materials such as mayonnaise and flake food dipping. The above list of foods to which isoflavone-rich protein substances can be applied are examples, and a comprehensive list of foods that are not intended to be used for isoflavone-rich protein substances. The isoflavone-rich proteinaceous material can be incorporated into any special food, where the proteinaceous material is traditionally incorporated in a manner consistent with traditional methods of incorporating proteinaceous materials into special food types. The following non-limiting formulations illustrate dietary supplements that can be formed using isoflavone-enriched soy protein materials consistent with the method of the invention. The isoflavone-enriched soy protein material in the following formulations typically contains from about 1 to about 25 mg of isoflavone compounds of formulas 1 and 2 per gram of soybean protein. Formulations Formulation 1 Ready-to-drink beverages Ready-to-drink beverages can be formed from the following components: Composition Percentage, weight ratio Water 8 0-8 5 Isoflavone-rich isolated soy protein 10-15

Page 17 12456603 V. Description of the invention (13) Sucrose 5-8 Cocoa 0.1-1 Vitamins / Minerals 0. 1-1 Spices 0.1-1 Cellulose gum 0. 1-0.5. Ready-to-drink beverages can be 8 pans Served in portions, containing about 20 grams of isolated soy protein, including about 20 to about 500 mg of isoflavone compounds. Formulation 2 Powder Beverage Powder Beverage can be formed from the following ingredients: Percent composition, weight ratio rich in isoflavone_isolated soy protein 85-90 sucrose 8-15 maltodextrin 1-5 vitamins / minerals 0.5-2 Ah Spam 0-0.5 flavor 0-0. 5 3 0 g of powdered beverage formulation can be added to water to form a portion containing about 20 g of isolated soy protein, including about 20 to about 500 mg of isoflavone compounds.

Formulation 3 Food sticks Food sticks can be formed from the following components: Composition Composition percentage, weight ratio Isoflavone-enriched soy protein 20-30

Page 18 12456603 V. Description of the invention (14) Corn sugar poly 3 5-4 5 Rice syrup solids 7-14 Glycerin 1-5 Cocoa 2-7 Compound coating 15-25 Food bars can be supplied in 70 grams servings, including About 15 grams of soy protein, of which about 15 to about 375 mg isoflavone compound. Preparation 4 t Soy Sour Curd Soy Sour Curd can be formed from the following ingredients: Percent composition, weight ratio 65-75 5-15 3-8 1-5 0 · 3-1 1-3 0. 01-0.1 10 -20 Water-rich isoflavone-isolated soybean protein sucrose corn starch dextrin cellulose gum strain (sour curd) fruit vitamins / minerals 0.05-0.3 soy sour curd can be supplied in 170 grams servings, containing about 8 grams Soy protein with about 8 to about 200 mg of isoflavone compounds. The following examples are provided to illustrate the invention. In the examples, the term π trihydroxy isoflavone glycoside " is defined to include isoflavone trihydroxy isoflavones present in soy

O: \ 61 \ 61461.PTD Page 19 1245603 V. Description of the invention (15) All compounds of the flavonoid family, specifically, trisoflavone glycosides, trisoflavones, 6 " -OMal trisoflavone glycosides and 6 " -0Ac The total amount of trihydroxy isoflavone glycosides. " Soy isoflavone glycoside π is defined in the examples to include all compounds of the isoflavone soybean isoflavone family present in soybean, specifically soybean isoflavone glycoside, soybean isoflavone, 6 "-OMal soybean isoflavone glycoside and 6 Total π-0 Ac soy isoflavone glycosides. The examples are not certified to limit the scope of the invention. Example 1 is intended to illustrate the increased amount of isoflavones in the protein isolates produced by the method of the present invention. Traditional protein isolates are provided to show the recovery of isoflavones in the conventional method. 7。 100 defatted soybean cake was placed in the extraction tank, and extracted with 1000 pounds of water heated to 90 ° F, which was added enough calcium hydroxide to adjust the pH to 9.7. The weight ratio of water to bean cake is 10: 1. Bean cake was separated from the extract and re-extracted with 600 pounds of aqueous extract with a pH of 9.7 and a temperature of 90 ° F. The second extraction step provided a weight ratio of water to soybean cake of 6: 1. The bean cake was removed by centrifugation. And the 1st and 2nd extracts were combined and adjusted to ρ 5 4. 5 with hydrochloric acid to sink the protein clot in the hall. The coagulant from the acid sink is separated from the extract by centrifugation at 1 35 ° F, leaving the aqueous soy bean, and then washed with water at 7 times the weight of the original bean cake material to provide protein isolation. Thing. Protein single isolates, soy clears, waste soy cakes, and starting materials were analyzed for the content of π trihydroxy isoflavones " and " soybean isoflavone glycosides. The results are shown in Table 1 below, with the isoflavone concentration and also the isoflavone percentage recovery relative to the amount of isoflavone contained in the starting material.

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_ Most of the five and four Heming instructions (16) Table 1 Amount (mg / g dry weight) ° / c Trihydroxyiso3 Substances Trisomy isoflavone glycoside Soy isoflavone treasure protein single isolate 0.90 0.54 23 Douchi 3.24 3.30 75 Starting material 1.72 1.58-The above example clearly illustrates that in traditional methods the concentration is concentrated in soy white, which results in isoflavones in most commercial eggs. Example 2 The effect of separating protein on the amount of white protein extract at a relatively cool temperature from a plant protein material at a neutral pH extraction solution was determined, in which the protein separation was consistent with the traditional method of washing with a certain amount of water. Extraction of water with a temperature of 90 ° F for a period of 15 minutes, in which the protein, then the isoflavone protein in the protein is separated from the bean cake with a pH of 6. 8 and the ratio of water to bean cake is 6: 1. The bean cake was separated from the extract and re-extracted with a second volume of water at a temperature of 90 ° F, where the weight ratio of water to bean cake was 4: 1. The second extraction liquid and the insoluble soybean cake material were separated by centrifugation 'and combined with the first extraction liquid to form a combined extract. The combined extracts were adjusted to pH 4.5 with hydrochloric acid to precipitate protein clots. The resulting protein clot / extract slurry is cooled to 43 ° F. The protein clot was then separated from the extract at 45 ° F., leaving the aqueous soy protein. The separated protein clot was washed with 45 F water, in which the washing water was 8 times the weight of the original soybean cake material for protein isolation. Proteins isolated, soy and starting materials were analyzed for their contents of 'trihydroxy isoflavone π' and "soy isoflavone glycosides". The results are described in Table 2 below.

1245603 One ------ 5. Description of the invention (17)

Protein isolated tempeh starting material

Soy isoflavone glycoside 0.70 2.07 1.58 Bean isoflavone 31 ~ 54 22 14 As shown by the comparison with the single isolate in Example 丨, at a cool temperature, fish two: take the protein single isolate and want the isoflavone. Concentrations and percent yields were substantially increased compared to the concentration and recovery of Isoxan 1 in protein isolates prepared in accordance with conventional methods. Example 3 The protein was extracted from the plant protein material with a neutral pH extract, and then the effect of separating the protein from the extract at a relatively cool temperature on the placement of isopropanone in the separated protein was determined, in which the protein clot was separated for cleaning Avoided. 1000 grams of defatted soybean cake was extracted with 600 grams of water having a pH of 6.8 and a temperature of 90 ° F for 15 minutes. The soybean cake was separated from the extract and re-extracted with 400 grams of water having a temperature of 900 ° F. The second extraction liquid and the insoluble soybean cake material were separated by centrifugation and combined with the first extraction liquid. The combined extracts were adjusted to pH 4.5 with hydrochloric acid to precipitate protein clots. The resulting protein clot / extract slurry was cooled to 4 3 ° F. And kept at this temperature for 16 hours. The protein clot was then centrifuged at 10,000 rpm and 43 ° F to separate the clot from the extract, leaving the aqueous soy protein. The resulting protein clot was not washed. The protein clot, lysate, and starting materials were analyzed for the content of "trihydroxy isoflavone ridge and π soybean isoflavone ridge '. The results are shown in Table 3 below.

Page 22 12456603 V. Description of the invention (18) Material quality (mg / g protein single ions Douchi starting material Table 3 Rate glycoside isoflavone glycoside trihydroxyiso 5.49 2.25 4.29 5.22 4.20 4.54 60 25 Soy isoflavones 1 ---- 49 40 As shown by the comparison with the single-isolated substance in Example 1, the relative concentration and percentage of the desired isoflavones in the unwashed neutral-extracted protein clot separated by fish and beans at a cool temperature in the cold month The recovery rate is greatly increased compared to the concentration and recovery rate of the isoflavones in the egg prepared in accordance with the traditional method == single ions. Example 4. 9 Neutralization from plant protein material The pH extract was used to extract the protein, and then the effect of separating the protein from the extract on the amount of heterogeneous protein in the separated protein was determined at a relatively cool temperature, in which the separated protein clot was washed with a very small amount of water. The cake was extracted for 15 minutes with 600 g of water having a pH of 6.8 and a temperature of 90 F. The bean cake was separated from the extract and re-extracted with 600 g of water having a temperature of 90 ° F. The second extraction liquid Separated from insoluble soybean cake material by centrifugation and combined with the first extraction solution. Combined The pH of the effluent was adjusted to pH 4.5 with hydrochloric acid to kill the protein clot. The resulting protein clot / extract slurry was cooled to 4 3 ° F and maintained at this temperature for 16 hours. The protein clot was then Centrifuge at 10,000 rpm and 43 T for 15 minutes to separate the clot from the extract, leaving the aqueous soy protein. The resulting protein clot was washed with UF water. The water washing solution and the original bean cake were The weight ratio is 2: 1 (200 grams of water). The isolated protein isolates, tofu, and starting materials are analyzed for the content of "isoflavones in the three classics" and "soy isoflavone moss. The results are explained.于 表 4。 In Table 4 below.

Page 23, 1245603 V. Description of the invention (19) Table 4 Amount (mg / g dry weight)% Recovery Material 54 41 Douchi 2.59 4.81 31 49 Starting material 4.29 4.54-As shown by comparison with the single isolate from Example 1, clean the neutral extract protein isolate from the low-purity neutral isolate at low temperature under cool temperature. The relative concentration and percentage recovery of isoflavones are substantially increased compared to the concentration and recovery of isoflavones in protein isolates prepared in accordance with traditional methods. Example 5 White protein and isoflavones The effect of extracting proteins with a neutral pH aqueous extract from plant raw materials on the yield of recovered protein has been determined. Compared with white plant extracts, proteins are extracted with an aqueous alkaline extract. The extraction and separation efficiency of neutral and alkaline extracts were determined, and the content of isosum baicalensis was recovered at a traditional thermal separation temperature and at a effective force to recover the protein material σ. Comparison of heat retention at 100 lbs. The obtained defatted soybean cake was extracted with water with a pH of 6.8 and a temperature of 9 (P: F. The extraction was countercurrent extraction, in which the first part of water weighing 10.0 pounds was used to extract the soybean cake, and the weight A second portion of 60,000 pounds of water was used to re-extract the extracted soybean cake. The first and second portions were combined to form an extract containing protein and isoflavone 1. The extract was sampled, and The protein content of the sample was determined by Gelda analysis. The extract was then divided into 3 equal parts. The pH of the equal parts of the extract was

Page 24 1245603 V. Description of the invention (20) Hydrochloric acid was adjusted to 4.5 to precipitate the protein in equal parts of the extract. The temperature of the first aliquot was adjusted to 135 ° F, the temperature of the second aliquot was adjusted to 65 ° F, and the temperature of the third aliquot was adjusted to 43 ° F. The precipitated protein of each aliquot was then separated from the liquid layer of each aliquot by centrifugation and decantation of the supernatant. Each aliquot of the separated protein was washed with 4,000 pounds of water at 65 ° F and then dried. Aliquots of the recovered protein material were then weighed to determine the quality of the protein recovered by the extraction and separation method. The same procedure was performed on another 1000 pounds of defatted soybean cake, except that the extract was an aqueous alkaline (sodium hydroxide) extract having a pH of 9.7. The percentage of protein recovered from the extraction and separation of 3 equal parts of the neutral extract and 3 equal parts of the alkaline extract was calculated. The percentage of recovered protein is calculated as the percentage of protein recovered from the primary bean cake material and the percentage of protein recovered from the extract (as determined by Creta analysis). The results are shown in Table 5 below. Table 5 Extraction pH / separation temperature% protein recovered from soybean cake% protein recovered from extract pH 9.7-135 ° F 70 73 pH6.8-135 ° F 70 77 pH 9.7-65 ° F 60 63 pH 6.8-65 ° F 70 79 pH 9.7-43T 60 65 pH 6.8-43 ° F 69 75 Comparing protein recovery and alkali extraction at neutral pH at 65 ° F and 43 ° F The extraction rate of protein is shown in the figure.

Page 25 1245603 V. Description of the invention (21) The mass of protein recovered at open temperature is substantially greater than the mass of protein recovered from both primary bean cake material and protein extract under the same conditions using alkaline pH extraction solution. Of course, the foregoing are only the preferred specific embodiments of the present invention, and different variations and changes can be made without departing from the spirit and broader aspects of the scope of the patent application as described in the accompanying claims, which and the like The principle of patent law of quantity principle is interpreted consistently.

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Claims (1)

Compliance No. 88120439: Amendment VI. Patent Application Scope 1. A method for preparing isoflavone-rich protein materials, comprising: extracting a plant containing protein and at least one isoflavone with an aqueous extract having a pH of 6 to 8 Raw materials, and separating the extract and insoluble plant raw materials to generate an extract containing the isoflavones and the protein; adjusting the pH of the extract to the isoelectric point of the protein to precipitate the isoflavones containing at least one isoflavone Protein material; and separating the protein material from the extract at a temperature of 30 ° F to 90 ° F, and avoiding cleaning of the separated protein material. 2. The method according to item 1 of the patent application, wherein the aqueous extraction solution has a pH of 6.5 to 7.5. The method according to item 1 of the patent application, wherein the plant raw material is soybean material. 4. The method according to item 1 of the scope of patent application, wherein the plant raw material contains at least one kind of isoflavones selected from the group consisting of trihydroxy isoflavones, trihydroxy isoflavone glycosides, 6n-OMal trihydroxy isoflavone glycosides, 6n-OAc trihydroxy isoflavone glycoside, soybean isoflavone, soybean isoflavone glycoside, 6n-0Mal soybean isoflavone glycoside, 6 n-OAc soybean isoflavone glycoside, methoxydihydroxy isoflavone, methoxydihydroxy isoflavone glycoside, 6n-OMal oxydihydroxy isoflavone glycoside, formonetin, biochanin A, or a mixture thereof. 5. The method of claim 1 in which the protein material is separated from the extract at a temperature of 40 ° F to 80 ° F. 6. The method of claim 5 in which the protein material is separated from the extract at a temperature of 50 ° F to 70 ° F. 7. A method for preparing isoflavone-rich protein materials, including: O: \ 61 \ 61461-940728.ptc Page 28 12456603 _Case No. 88120439 July 44_ _ Charm_ VI. Application for patent scope Extraction of protein and at least 1 kind with aqueous extract with ρ Η of 6 to 8 Isoflavone plant raw materials, and separate the extract and insoluble plant raw materials to generate an extract containing the isoflavone and the protein; adjust the pH of the extract to the isoelectric point of the protein to precipitate at least 1 A kind of isoflavone protein substance; separating the protein substance from the extract at a temperature of 30 ° F to 90 ° F; and washing the separated substance with water at a temperature of 30 ° F to 90 ° F Protein material. 8. The method according to item 7 of the scope of patent application, wherein the protein material is washed with water less than 4 times the weight of the plant material. 9. The method according to item 7 of the scope of patent application, wherein the protein material is washed with water less than 2 times the weight of the plant material. 10. The method according to item 7 of the scope of patent application, wherein the water used to clean the protein material has a temperature of 50 ° F to 70 ° F. 1 1. The method according to item 7 of the patent application, wherein the aqueous extraction solution has a ρ Η of 6.5 to 7 · 5 2. The method according to item 7 of the patent application, wherein the plant material is a soybean material . 13. The method according to item 7 of the scope of patent application, wherein the plant raw material contains a group of at least one isoflavone in the following composition: trihydroxy isoflavone, trihydroxy isoflavone glycoside, 6n-0Mal trihydroxy isoflavone glycoside, 6n -0Ac trihydroxy isoflavone glycosides, soy isoflavones, soy isoflavone glycosides, 6n-0Mal soy isoflavone glycosides, 6n-OAc soy isoflavone glycosides, trioxo isoflavones, methoxydihydroxy isoflavone glycosides, 6n- OMal dioxin via isoflavone spine, methanone, biocharney A, or O: \ 61 \ 61461-940728.ptc Page 29 1245603 _ Case No. 88120439 Sixth, the scope of patent application is amended mixture. 14. The method according to item 7 of the patent application range, wherein the proteinaceous material is separated from the extract at a temperature of 40 ° F to 80T. 1 5. The method according to item 14 of the patent application range, wherein the proteinaceous material is separated from the extract at a temperature of 50 ° F to 70 ° F. O: \ 61 \ 61461-940728.ptc Page 30