JP2007217367A - Method for preparing aglycon-type isoflavone from soy sauce cake - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply extracting and purifying an aglycon-type isoflavone from a soy sauce cake in which the obtained aglycon-type isoflavone is suitably useful as a food, a medicine and a health food material. <P>SOLUTION: The method for preparing an aglycon-type isoflavone comprises a process for bringing a soy sauce cake into contact with water to give a desalted soy sauce cake, a process for bringing the desalted soy sauce cake into contact with an alkali alcohol solution to give an extracted solution of an aglycon-type isoflavone, a process for keeping the extracted solution of an aglycon-type isoflavone at -50 to -5°C to precipitate impurities and a process for removing the impurities. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a technique for easily extracting and purifying aglycone type isoflavones, which are useful substances, from soy sauce koji discharged in the process of producing soy sauce.

  Isoflavone is a kind of polyphenol contained in soybean and the like, and is one of the nutrients attracting attention in recent years.

  Isoflavones prevent osteoporosis: Non-patent document 1 (Saito et al., J. Home Econ. Jpn. 54, 613 (2003)), lipid metabolism improving effect: Non-patent document 2 (Anthony et al., Journal of Nutrition, 126, 43-50 (1996)), inhibition of periodontal disease progression (Non-patent document 3 (Inagaki et al., Japanese Dental Preservation Journal 46,538 (2003)), cancer prevention effect (inhibition of growth of cultured cancer cells) Non-patent document 4 (Okura et al., Biochem. Biophys. Res. Commun., 157, 183 (1988)), suppression of experimental carcinogenesis: Non-patent document 5 (Sharm et al. J. Steroid Biochem. Mol). Biol., 43, 557 (1992)), suppression of oncogene expression: Non-Patent Document 6 (Z iller et al.Oncogene, has been reported to have 6,219 (1991)) or the like.

  Soybeans are rich in isoflavones, and the main ones are three types of aglycone isoflavones, daidzein, genistein, and glycitein, and their glycosides, malonyl glycosides, and acetyl glycosides.

  Most isoflavones contained in raw soybeans are present as glycosides, and the content of aglycone type isoflavones is low. In addition, it has been pointed out that the functionality of isoflavones is mainly observed in the aglycone type, not in glycosides.

  On the other hand, it is known that soy sauce cake obtained as a by-product in the brewing process of soy sauce contains a lot of aglycone type isoflavones. This is thought to be because the glycoside is converted to the aglycone type by the enzymatic action of microorganisms such as koji mold during soy sauce brewing. In addition, since aglycone type isoflavones are hardly soluble in water, they are contained in soy sauce cake at a higher concentration than soy sauce.

  A large amount of soy sauce cake is discharged during the production process of soy sauce, but only a part of it is used as livestock feed, and many are not used effectively and are handled as waste.

  Therefore, although soy sauce cake is suitable as a raw material for extracting aglycone type isoflavones, the high salt concentration of soy sauce cake has been a problem in use as a food material.

  On the other hand, in order to solve this problem, as an efficient extraction method, a first step of extracting useful substances using 1-propanol, 2-propanol, butyl alcohol, acetone, ether, etc., and water, ethanol, methanol A technique comprising a second step of desalting (Patent Document 1 (Patent No. 3567699)), extracting the heat-treated soy sauce cake with alkaline water, adsorbing the isoflavone in the extract to the adsorbent, A technique (Patent Document 2 (Japanese Patent Laid-Open No. 2002-003487)) that includes a step of elution with alkaline water, a step of adjusting the eluate to pH 6 or lower, and a step of collecting precipitates has been proposed.

However, in these conventional extraction methods, the organic solvent to be used is a solvent that is difficult to use in the final use of the extract as a food material, the adsorbent used is expensive, -There were problems such as discharge of alkaline waste liquid and low extraction efficiency.
Japanese Patent No. 3567699 (pages 1 to 10) JP 2002-003487 A (pages 1 to 5) Saito et al., J. Home Econ. Jpn. 54, 613 (2003)) Anthony et al. , Journal of Nutrition, 126, 43-50 (1996). Inagaki et al., Japanese Dental Conservation Journal 46,538 (2003) Okura et al. Biochem. Biophys. Res. Commun. , 157, 183 (1988) Sharm et al. J. et al. Steroid Biochem. Mol. Biol. , 43, 557 (1992) Zwiller et al. Oncogene, 6, 219 (1991)

  The present invention is intended to solve the above-mentioned problems, and is a method in which the obtained aglycone type isoflavone is suitable for use as a food material, a pharmaceutical material, etc. It is an object of the present invention to provide a method for easily extracting and purifying sucrose.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made contact with an alkaline alcohol solution after desalting by a water extraction treatment from a soy sauce cake in which a high salt concentration is a problem. Type isoflavones can be extracted with high efficiency, and by storing the extract at a low temperature, impurities such as lipids can be precipitated while the aglycone type isoflavones remain soluble. The inventors have found that a solution containing aglycone-type isoflavone having a high purity can be obtained, and based on this, the present invention has been completed.

  In a more specific case, after extracting the aglycone isoflavone from the soy sauce cake where high salt concentration becomes a problem by contacting the alkaline alcohol with the soy sauce cake desalted by the water extraction treatment at room temperature for a short time at room temperature, By storing only the impurities by storing the extract at a low temperature, the purity of the aglycone isoflavone in the extract can be increased.

In order to achieve the above object, the present invention provides, for example, the following means:
Item 1.
A method for preparing an aglycone type isoflavone,
A step of contacting the soy sauce cake with water to obtain a desalted soy sauce cake,
Contacting the desalted soy sauce cake with an alkaline alcohol solution to obtain an aglycone type isoflavone extract,
A method comprising a step of precipitating impurities by maintaining the aglycone type isoflavone extract at −50 ° C. to 5 ° C., and a step of removing the impurities.

Item 2.
Item 2. The preparation method according to Item 1, wherein the salt concentration of the desalted soy sauce cake is 0 to 1% by weight.

Item 3.
Item 2. The preparation method according to Item 1, wherein the alkaline alcohol solution is a 30 to 100 vol% alcohol aqueous solution containing 0.01 to 5M alkali.

Item 4.
Item 4. The preparation method according to Item 3, wherein the alkali is sodium hydroxide.

Item 5.
Item 2. The preparation method according to Item 1, wherein the alcohol is ethanol.

Item 6.
Item 2. The preparation method according to Item 1, wherein in the precipitation step, the aglycone type isoflavone extract is held at -50 to 5 ° C for 1 to 24 hours.

Item 7.
2. The preparation method according to item 1, further comprising the step of neutralizing the extract with an acid after removing the impurities.

  According to the method of the present invention, aglycone type isoflavones can be obtained from soy sauce cake with high extraction efficiency by an inexpensive and simple method. In the method of the present invention, since a solvent which is a problem when used as a food material, a pharmaceutical material or the like is not used, the obtained aglycone type isoflavone is very useful as a food material.

  Hereinafter, the present invention will be described in detail.

  In the present specification, “aglycone type isoflavone” refers to a non-sugar portion of glycoside isoflavone. Examples of aglycone type isoflavones include daidzein, genistein and glycitein. Aglycon type isoflavones are contained in a large amount in soy sauce cake.

  In the present specification, “soy sauce cake” is a by-product that remains after squeezing soy sauce from soy sauce raw materials such as soybeans in the soy sauce manufacturing process. Soy sauce cake is rich in aglycone type isoflavones and salt. The soybean used for the soy sauce raw material may be a soybean normally used in the field. Examples of soybeans used as soy sauce raw materials include whole soybeans, moulted soybeans and defatted soybeans. The soybean used for the soy sauce raw material is preferably defatted soybean. The soy sauce cake may be in a moist state just after squeezing soy sauce, or may be once dried.

  The method of the present invention includes a step of contacting a soy sauce cake with water to obtain a desalted soy sauce cake.

  The amount of water to be contacted with the soy sauce cake may be any amount as long as it can be efficiently desalted from the soy sauce cake, and can be appropriately selected. The amount of water can be, for example, about 2 times or more, preferably about 4 times or more the volume of soy sauce cake. Although there is no upper limit in particular in the quantity of water, For example, it may be about 20 times or less and about 10 times or less. Generally, soy sauce cake contains about 8% salt (particularly sodium chloride). When the soy sauce cake is brought into contact with water, the salt (particularly sodium chloride) contained in the soy sauce cake is dissolved in the water. On the other hand, since the aglycone type isoflavone hardly dissolves in water, it remains in the soy sauce cake. Therefore, the salt content is removed from the soy sauce cake by removing the moisture after bringing the soy sauce cake into contact with water.

  In the method of the present invention, it is important to desalinate before extracting the aglycone type isoflavones from the soy sauce cake. When the soy sauce cake is brought into direct contact with the alcohol solution without desalting, both the salt and the aglycone isoflavone are extracted into the alcohol solution, and then complicated and expensive to separate the salt and the aglycone isoflavone. Further processing is required.

  However, in the method of the present invention, by bringing the soy sauce cake into contact with water in advance, most of the salt contained in the soy sauce is extracted into water, while the aglycone isoflavones are hardly extracted into water. Therefore, by bringing the soy sauce cake into contact with water, the salt content and the aglycone type isoflavone can be separated, and the soy sauce cake can be desalted with almost no loss of the aglycon type isoflavone.

  As a desalting method, a batch method, a column method, or the like can be used as appropriate. For example, put soy sauce cake in a suitable container, add water, stir, then desalinate by filtration, etc. (batch method), fill the column with soy sauce cake, and pass water from the top to desalt A method (column method) or the like can be used. In the batch method, it is possible to further reduce the salt concentration of the soy sauce cake after the desalting treatment by repeating the same operation as necessary.

  The temperature at which the soy sauce cake is brought into contact with water can be any temperature as long as the salt is extracted from the soy sauce cake into the water. The temperature when the soy sauce cake and water are brought into contact with each other is preferably room temperature (about 25 ° C.), more preferably about 15 ° C. or more, and most preferably about 20 ° C. or more. The temperature at which the soy sauce cake is brought into contact with water is preferably about 50 ° C. or less, more preferably about 40 ° C. or less, and most preferably about 30 ° C. or less. If the temperature at which the soy sauce cake is brought into contact with water is too low, the desalination efficiency may be too low. If the temperature at the time of extraction is too high, the viscosity will increase and it will be difficult to filter. Separation may worsen.

  In the method of the present invention, a desalted soy sauce cake is obtained by contacting the soy sauce cake with water in this manner. The desalting treatment is preferably performed so that the salt concentration in the obtained desalted soy sauce cake is about 1/10 or less of the salt content in the soy sauce cake before the desalting treatment. The salt concentration in the desalted soy sauce cake is preferably 1% by weight or less, more preferably about 0.7% by weight or less, still more preferably about 0.5% by weight or less, and most preferably Is about 0.3% by weight or less. The salt concentration in the desalted soy sauce cake is preferably as low as possible, and when all is removed, it becomes 0% by weight. Therefore, the salt concentration in the desalted soy sauce cake is preferably 0% by weight to 0.3% by weight.

  The salt concentration in the desalted soy sauce cake is determined based on the weight of the soy sauce cake obtained after bringing the soy sauce cake and water into contact with each other as described above and then removing the water by a separation means such as filtration. The

  Next, the method of the present invention includes the step of bringing the desalted soy sauce cake into contact with an alkaline alcohol solution to obtain an aglycone type isoflavone extract.

  The alkaline alcohol liquid used in this step is a liquid containing an alkaline substance and alcohol. The alkaline alcohol solution is preferably an alkaline alcohol aqueous solution. That is, it contains an alkaline substance, alcohol and water. The alkaline alcohol solution can be prepared by any method known in the art. The alkaline alcohol liquid can be prepared, for example, by adding an alkali (or an aqueous solution thereof) to an alcohol or an aqueous alcohol solution. The alkaline alcohol aqueous solution may be prepared by adding an alkali to the alcohol aqueous solution, or may be prepared by adding an alkali to the alcohol and then mixing with water, or adding an alkali to the water and then the alcohol. And may be prepared by mixing with.

  Arbitrary alcohol can be used as alcohol. The alcohol is preferably an edible alcohol, more preferably ethanol or methanol, and most preferably ethanol.

  The alcohol liquid may be a mixture of alcohol and water, a pure alcohol, or a mixture of two or more alcohols. The alcohol liquid is preferably an aqueous alcohol solution. In the case of an aqueous alcohol solution, the mixing ratio of alcohol and water may be any ratio as long as the aglycone type isoflavone can be efficiently extracted, and can be appropriately selected. The volume% of alcohol in the aqueous alcohol solution is, for example, about 30% by volume or more, and preferably about 60% by volume or more. The volume% of alcohol in the aqueous alcohol solution is, for example, about 100% by volume or less, and preferably about 90% by volume or less. If the alcohol volume percentage is too low, extraction of the aglycone isoflavones may not be sufficient. If the alcohol volume percentage is too high, extraction of the aglycone isoflavones may not be sufficient.

  Any alkali can be used as long as it shows strong basicity. The alkali is preferably sodium hydroxide, potassium hydroxide, ammonia or sodium carbonate, more preferably sodium hydroxide or potassium hydroxide, most preferably sodium hydroxide.

  The concentration of alkali in the alkaline alcohol solution may be any concentration and can be selected as appropriate. The concentration of alkali in the alkaline alcohol solution is, for example, about 0.01M or more, preferably about 0.05M or more. The alkali concentration in the alkaline alcohol liquid is, for example, 5M or less, and preferably 0.3M or less. If the alkali concentration is too low, the extraction of isoflavones into the alcohol solution may not be promoted much. If the alkali concentration is too high, the neutralization treatment may be overburdened. “M” indicates the molar concentration and indicates the number of moles of alkali present in 1 liter of the alcohol liquid.

  The amount of the alkaline alcohol solution used for the extraction may be any amount as long as it can efficiently extract the aglycone type isoflavone, and can be appropriately selected. The amount (weight) of the alkaline alcohol solution is, for example, about 1 time or more, preferably about 2 times or more the weight of the soy sauce cake after the desalting treatment. The amount of the alkaline alcohol solution is not particularly limited, but is, for example, about 20 times or less, preferably about 10 times or less the weight of the soy sauce cake after the desalting treatment. If the amount of the alkaline alcohol solution is too small, the aglycone type isoflavone may not be sufficiently extracted. If the amount of the alkaline alcohol solution is too large, the neutralization treatment or the drying treatment may be excessively burdened.

  As an extraction method, a batch method, a column method, or the like can be used as appropriate. For example, a desalted soy sauce cake is put in a suitable container, an alkaline alcohol solution is added, and after stirring, a method of obtaining an extract by filtration or the like (batch method), the desalted soy sauce cake is packed in a column, The method (column method) etc. which obtain an extract by allowing an alkaline alcohol liquid to pass through from the upper part can be used. In the batch method, it is possible to increase the extraction amount of the aglycone type isoflavone by repeating the same operation as necessary.

  The extraction can be performed at any temperature as long as the alkaline alcohol liquid is liquid. The temperature at which the extraction is performed is preferably room temperature (about 25 ° C.), more preferably about 15 ° C. or more, and most preferably about 20 ° C. or more. The temperature at which the extraction is performed is preferably about 50 ° C. or less, more preferably about 40 ° C. or less, and most preferably about 30 ° C. or less. If the temperature at the time of extraction is too low, the extraction efficiency may be too low, and if the temperature at the time of extraction is too high, the volatilization of alcohol may become too intense.

  Thus, an aglycone type isoflavone extract is obtained by bringing the desalted soy sauce cake into contact with an alkaline alcohol solution.

  Next, the method of the present invention includes a step of precipitating impurities by maintaining the aglycone type isoflavone extract at −50 to 5 ° C. Preferably, the aglycone type isoflavone extract is held at −50 ° C. to 5 ° C. for 1 to 24 hours.

  The temperature at which the extract is cooled may be any temperature as long as the extract does not freeze and impurities precipitate, and can be appropriately selected. The temperature for cooling the extract is, for example, about 5 ° C. or lower, preferably about −20 ° C. or lower. The temperature which cools an extract is about -50 degreeC or more, for example. The “temperature at which the extract is cooled” means the temperature of the environment where the extract is placed to cool the extract, which substantially means the temperature of the extract.

  The time for cooling the extract may be any time as long as the extract is sufficiently cooled, and can be appropriately selected. The time for cooling the extract is, for example, about 1 hour or longer, preferably about 3 hours or longer. The time for cooling the extract is, for example, about 24 hours or less, preferably 15 hours or less. The “time for cooling the extract” means the time from when the extract begins to be placed in the cooling temperature condition to cool the extract until it ends, which is substantially the temperature of the extract. Means the time from when the cooling temperature is reached.

  When the extract is cooled in this manner, impurities are precipitated in the aglycone type isoflavone extract. When the desalted soy sauce cake is brought into contact with an alkaline alcohol solution, aglycone isoflavones are extracted from the soy sauce cake into the alkaline alcohol solution. At this time, not only the aglycone type isoflavone but also other impurities are extracted from the alkaline alcohol solution. One such impurity is lipid. Lipids are contained in soy sauce cake by about 10%, most of which are fatty acids. By cooling the extract, impurities such as fatty acids become insoluble precipitates and can be easily removed by separation means such as filtration. However, at this time, the aglycone type isoflavones remain soluble. Therefore, the purity of the aglycone isoflavone in the extract can be remarkably increased by cooling the extract to precipitate impurities and removing the precipitate.

  The method of the present invention includes the step of removing impurities (ie, precipitates) after the impurities are precipitated in the extract. Impurities can be removed by methods known in the art. Examples of methods for removing impurities include filtration, centrifugation, and decanting.

  The extract after removing the impurities can be pulverized by spray drying, freeze drying or the like after neutralizing with an acid. Any excipient can be used during spray drying.

  10kg of soy sauce cake (by volume 24L; water content 27% by weight; salt concentration 7.8% by weight), a by-product of soy sauce made from defatted soybeans, is pulverized, mixed with 50L of water, and filtered through filter paper To desalinate. This operation was performed twice in total to obtain 20 kg (volume: 19.2 L) of desalted soy sauce cake. The salt concentration of the obtained desalted soy sauce cake was about 0.3% by weight.

The salt concentration was calculated in terms of NaCl by measuring chloride ions (Cl ⁻ ) by ion chromatography.

  To this desalted soy sauce cake, 50 L of a 70% ethanol aqueous solution containing 0.1 M sodium hydroxide was added and stirred, and held at 25 ° C. for 15 minutes to extract aglycone isoflavones into this aqueous solution. Subsequently, by filtering this liquid, the soy sauce cake was removed to obtain 50 L of an extract. The concentration of aglycone type isoflavones was measured by HPLC. The concentrations of daidzein, genistein and glycitein were measured as aglycone type isoflavones. The total aglycone isoflavone concentration in the extract was as shown in Table 1 below.

  After performing desalting operation according to the same procedure as Example 1 using 10 kg of soy sauce cake, aglycone type isoflavone extraction with 50 L of 70% ethanol aqueous solution containing 0.1 M sodium hydroxide was performed twice, and aglycone type isoflavone contained 100 L of extract was obtained. The obtained extract was stored at −50 ° C., −20 ° C. or 5 ° C. overnight, and the precipitate was removed by filtration. After neutralizing the obtained filtrate with hydrochloric acid, 500 g of dextrin was added as an excipient and spray-dried to obtain an aglycone type isoflavone-containing powder.

  As a control, 100 L of the extract was stored overnight at room temperature (about 25 ° C.), and then the aglycone-type isoflavone-containing powder obtained in the same manner was used. The concentration of aglycone type isoflavones in this powder was measured by HPLC. The concentrations of daidzein, genistein and glycitein were measured as aglycone type isoflavones. The total concentration of aglycone type isoflavones in the obtained powder was as shown in Table 2 below.

この結果、冷却しないと沈澱が生じないが、冷却することによって沈澱が生じて不純物が除去され、得られるアグリコン型イソフラボン濃度が上昇することがわかった。

As a result, it was found that precipitation does not occur unless cooled, but precipitation occurs and impurities are removed by cooling, and the resulting aglycone isoflavone concentration increases.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, aglycone isoflavones, which are useful substances, can be easily extracted and purified from soy sauce cake, which is a by-product during soy sauce brewing and is a waste product. The obtained aglycone type isoflavones are suitable for use as materials for foods, pharmaceuticals, health foods and the like.

Claims (7)

A method for preparing an aglycone type isoflavone,
A step of contacting the soy sauce cake with water to obtain a desalted soy sauce cake,
Contacting the desalted soy sauce cake with an alkaline alcohol solution to obtain an aglycone type isoflavone extract,
A method comprising a step of precipitating impurities by maintaining the aglycone type isoflavone extract at −50 ° C. to 5 ° C., and a step of removing the impurities.   The preparation method according to claim 1, wherein the salt concentration of the desalted soy sauce cake is 0 to 1% by weight.   The preparation method according to claim 1, wherein the alkaline alcohol solution is a 30 to 100 vol% alcohol aqueous solution containing 0.01 to 5 M alkali.   The preparation method according to claim 3, wherein the alkali is sodium hydroxide.   The preparation method according to claim 1, wherein the alcohol is ethanol.   The preparation method according to claim 1, wherein in the precipitation step, the aglycone type isoflavone extract is held at −50 ° C. to 5 ° C. for 1 to 24 hours.   The preparation method according to claim 1, further comprising neutralizing the extract with an acid after removing the impurities.