JPH0352447B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing soybean saponin using as a starting material soy sauce lees obtained as a by-product in the soy sauce production process.

Generally, saponins are used as emulsifiers and dispersants in cosmetics and health drugs, and also in the food industry. Recently, saponin crude drugs have been reconsidered in the pharmaceutical field, and their anti-inflammatory, anti-ulcer, and anti-allergic effects have also come to be utilized.

Soybean saponin has long been known as a low-toxic saponin with the weakest hemolytic effect, which is a biological property of saponins, but the separation process from soybeans is complicated and economically disadvantageous, making it difficult to use in practical applications. It is considered a drawback.

The present inventors have discovered that this soybean saponin can be industrially advantageously produced using soy sauce lees, which is a by-product in the soy sauce manufacturing process, as a starting material.

The present invention involves adding water and/or lower alcohol to soy sauce lees or defatted soy sauce lees obtained by degreasing this with an organic solvent, and extracting the resulting extracted portion.
Production of soybean saponin, characterized in that soybean saponin is separated by applying at least one separation method selected from partition extraction with an organic solvent, adsorption/desorption with a nonionic adsorption resin, fractional precipitation, and chromatography. It is the law.

The soy sauce lees used as the starting material of the present invention is
Soy sauce lees, which is a by-product in the soy sauce production process and is a by-product in the production of various soy sauces and soy sauce-like seasonings, can be used. Since soy sauce lees contains a large amount of salt, it has been difficult to use or dispose of it, but according to the present invention, it can be turned into an industrial product with high utility value.

Since soy sauce lees usually contains lipids such as soy sauce oil, it is preferably used as defatted soy sauce lees obtained by previously defatting the soy sauce lees in a conventional manner using an organic solvent. Examples of solvents for degreasing include chloroform and other halogenated hydrocarbons, ethers such as ethyl ether and isopropyl ether, alcohols such as methanol and ethanol, petroleum ether, hydrocarbons such as benzene, acetone and other ketones, Ethyl acetate and other esters or mixtures of these solvents can be used.

According to the present invention, soy sauce lees or defatted soy sauce lees is extracted with water and/or lower alcohol. In this case, extraction may be performed using water alone, but extraction using an aqueous lower alcohol or lower alcohol is more preferable in terms of extraction efficiency. As the lower alcohol, for example, methanol, ethanol, n- and isopropanol, n-butanol, etc. are used, but methanol or aqueous ethanol is particularly preferred from the viewpoint of the yield of soybean saponin. Extraction is carried out in a conventional manner using a batch-type or continuous-type extraction device at room temperature or under heating using the above-mentioned solvent in an amount of 5 to 20 times the weight of the soy sauce lees or defatted soy sauce lees.

Next, one or two of the above-mentioned separation means for separating soybean saponin are added to the extract thus obtained.
Apply in combination of more than one species. Prior to this separation process, the extract is clarified, for example, by filtration, centrifugation, or other means, and unless it is directly subjected to adsorption and desorption using a nonionic adsorption resin, it is usually concentrated under reduced pressure to remove the solvent. Collect.

In order to separate soybean saponin from the extract obtained as a remainder after the above-mentioned solvent recovery, a partition extraction method using an organic solvent can be first applied. This method utilizes the difference in solubility of soybean saponin and other substances in organic solvents, and includes shaking extraction, one-stage distribution methods such as continuous perforation, and multi-stage distribution methods that combine these methods. can give.

Organic solvents used in this partition extraction method include alcohols such as methanol and ethanol, acetone and other ketones, ethers such as ethyl ether, ethyl acetate and other esters, chloroform and other halogenated hydrocarbons, petroleum It is a mutually immiscible solvent system of two or more selected from hydrocarbons such as ether, and water is also used. Examples of preferred solvent systems are water and n-butanol;
These are the upper and lower phases of a mixed system of chloroform, methanol, and water in various proportions that separate into two phases.

As another method for separating soybean saponin, adsorption/desorption using a nonionic adsorption resin can also be used.
As the nonionic adsorption resin, for example, styrene-based adsorption resins such as Serverchrome XAD-2 (manufactured by Server Co., Ltd.), Amberlite XAD-2 (manufactured by Rohm and Haas Co., Ltd.), etc. are used. water or generally 30%
In the case of an aqueous lower alcohol extract of less than V/V, soybean saponin can be selectively adsorbed to the adsorption resin as it is. When extracting with a higher concentration of aqueous lower alcohol or pure lower alcohol, the concentration of lower alcohol in the extract is generally reduced to 30% V/V by concentrating under reduced pressure to recover the lower alcohol or adding water. Adsorption is performed after reducing the amount to less than Then, the soybean saponin adsorbed on the adsorption resin can be desorbed using a lower alcohol or a highly concentrated aqueous lower alcohol.

Note that if a relatively large amount of lipids other than soybean saponin coexists in the extract or its concentrate in a suspended state, the selective adsorption and desorption of soybean saponin by the adsorption resin will be hindered, so static separation must be performed in advance. Alternatively, good results can be obtained by removing this by filtration using a supernatant such as diatomaceous earth.

Similar to the partition extraction method described above, the fractional precipitation method can also be advantageously applied to the separation of soybean saponin, as it utilizes the difference in solubility in organic solvents between soybean saponin and other substances. That is, raw materials containing soybean saponin and other substances are dissolved in a solvent in which they can be dissolved, and this solution is gradually added to a solvent in which one of them is more difficult to dissolve. The final fraction containing the precipitated soybean saponin is collected and the fraction containing other substances is removed. This operation may be repeated with the same solvent system, but it is also possible to select different solvent systems and combine them to make the separation of soybean saponins more effective.

The solvent system for this method is preferably a mutually miscible solvent system consisting of two or more solvents selected from among the solvents listed in the above-mentioned partition extraction method. Examples include combinations of methanol and ethyl acetate, methanol or highly concentrated aqueous ethanol and acetone.

Chromatography is also an effective means of separating soybean saponins. In this case, the treated starting materials, which are mixtures of soybean saponin and other substances, are mixed with organic solvents or mixed solvents in which they are soluble, such as methanol alone, methanol and water, chloroform,
Fillers that are soluble in a mixed solvent of methanol and water and capable of adsorbing or distributing soybean saponin and other substances, such as silica gel, chemically bonded silica gel,
It is passed through a column filled with granular activated carbon, alumina, etc. in advance to adsorb soybean saponin or other substances.

When soybean saponin is adsorbed, the soybean saponin is further eluted by developing with the same series of organic solvents with different mixing ratios or with a different series of organic solvents that easily elute soybean saponin. When substances other than soybean saponin are adsorbed, soybean saponin is obtained as a permeate. The soybean saponin in these solutions can be collected by recovering the organic solvent by concentration under reduced pressure. In addition, reverse-phase chemically bonded silica gel, such as Micro Bonder Pack
C8 or C18 (manufactured by Waters) and similar fillers are suitable for purification of the target soybean saponin.

Furthermore, good results can be obtained by appropriately combining the above separation means.

The soybean saponin thus separated may be used as a product as it is, but if necessary, it can be further recrystallized using an aqueous lower alcohol such as aqueous methanol, aqueous ethanol, etc. to obtain highly purified soybean saponin. .

The present invention has extremely great industrial value in that useful soybean saponin can be provided at low cost from soy sauce lees, which has low utility value.

Example 1 Koikuchi brewed soy sauce lees was crushed into powder,
400 g of the mixture was placed in a 2-volume round bottom flask, 1 portion of ethyl acetate was added thereto, and the mixture was heated under reflux for 5 hours.
Then, the ethyl acetate extract is removed by filtration, and the residue is subjected to the same ethyl acetate extraction five times in total.

The defatted soy sauce lees obtained in this way was taken into a 2-volume round bottom flask, and 1 volume of methanol was added to it.
Heat to reflux for an hour. Then, the methanol extract is collected by filtration, and the residue is subjected to the same methanol extraction three times in total. These methanol extracts are combined and the solvent is distilled off under reduced pressure to obtain 65 g of methanol extract.

65 g of this methanol extracted extract is distributed between 300 ml of n-butanol and 300 ml of water, and the solvent transferred to n-butanol is distilled off to obtain 19 g of extract.

Dissolve 19 g of this extract in 30 ml of methanol, add dropwise to 1.5 mL of ethyl acetate, and collect the resulting precipitate by centrifugation to obtain 6 g of precipitate.

Dissolve 6g of this precipitate in 300ml of methanol,
Add 20g of activated carbon and stir for 12 hours. Then, the activated carbon was removed by filtration, and the liquid was concentrated under reduced pressure.
2 g of crude soybean saponin fraction is obtained.

Dissolve 2 g of the crude soybean saponin fraction in 500 ml of methanol, put it in a 1 volume round bottom flask, and add 5
After adding 20 ml of % caustic potash methanol solution and heating under reflux for 1 hour, the mixture was neutralized with a strongly acidic ion exchange resin. Then, the resin is removed by filtration, and the solvent is distilled off from the liquid under reduced pressure. 1.8 g of the residue thus obtained was subjected to reverse phase silica gel column chromatography [Carrier: Bondapak C18 (manufactured by Waters)]
100g, solvent: methanol-water (1:2-1:
1)] yields 1.5g of soybean saponin.

This soybean saponin was analyzed by thin layer chromatography using silica gel [pre-coated silica gel].
60F254, layer thickness 0.25 mm (manufactured by Merck), developing solvent: chloroform-methanol-water (65:35:10)
When sprayed with 50% sulfuric acid solution and heated to 90-95℃ for 5 minutes, spots with an Rf value of 0.26,
For all of 0.30, 0.34 and 0.37, reddish-purple spots characteristic of soybean saponin are obtained. For example, Journal of the American Oil Chemistry Society, Vol. 47, No. 3, pp. 86-90 (1970)
This result agrees well with the thin layer chromatography results for soybean saponin described in 2009, confirming that the saponin obtained as described above is soybean saponin.

Example 2 Koikuchi brewed soy sauce lees is crushed into flakes, 2 kg of which is put into a 20-volume enamel tank, 12 parts of 80% V/V aqueous ethanol is added thereto, the lid is closed, and immersion extraction is carried out overnight at room temperature. This extract is collected by a conventional decanting method, and the residue is subjected to a total of two similar aqueous ethanol extractions. The aqueous ethanol extracts are combined and filtered, and the resulting clear liquid is concentrated under reduced pressure using a flash evaporator until the total volume is approximately 1.2.

Pour the concentrated solution into a liquid chromatography column with a diameter of 60 cm and a height of 50 cm, and leave it overnight. At that time, the concentrate is 2
Since it is separated into layers, the lower layer is taken out from the bottom of the column and filtered through diatomaceous earth as an auxiliary agent to remove some remaining lipids, yielding a clear liquid. The solvent is distilled off from this clear liquid using a flash evaporator under reduced pressure to obtain 230 g of extract.

Add 1 part methanol to 230g of this extract, and add approximately
Heat to 70℃ to dissolve as much of the extract as possible, then cool and leave. The formed precipitate was removed by filtration, and the liquid was similarly concentrated to dryness under reduced pressure to obtain 160 g of methanol-soluble extract. This extract
Distribute 160 g with 400 ml of n-butanol and 600 ml of water,
When the solvent in the portion transferred to n-butanol is distilled off, 42 g of extract is obtained.

Dissolve 42 g of this extract in 60 ml of methanol, add dropwise to 1.2 g of acetone, and collect the resulting precipitate by centrifugation (yield: 24 g). 24g of this sediment
Dissolve in 1.2 methanol and add 40 g of activated carbon to this.
Add and stir for 12 hours. Activated carbon is removed from this solution by filtration, and the solvent is distilled off from the solution.
10 g of crude soybean saponin fraction is obtained.

When 10 g of this crude soybean saponin fraction was further purified in the same manner as in Example 1, the desired soybean saponin was obtained.
7.2g obtained.

Example 3 200 ml of water was added to the lower layer 1 of the concentrated separation liquid of the aqueous ethanol extract obtained by processing 2 kg of Usukuchijozo soy sauce lees by the method of Example 2, and the alcohol concentration was adjusted to 30% V/V. After adjustment, the suspended matter is removed by filtration using diatomaceous earth, and the clear liquid has a concentration of 1.2
can get.

This clarified liquid 1.2 was passed through a column (diameter 6 cm x height 50 cm) of styrene adsorption resin Amberlite XAD-2 (manufactured by Rohm and Haas) at a flow rate of SV = 1, and 400 ml of 30% V/V aqueous ethanol After washing, the adsorption area is eluted with 1 part of methanol, and the solvent of the eluate is distilled off under reduced pressure to obtain 40 g of extract.

Dissolve 40 g of this extract in 60 ml of methanol, add dropwise to 1.2 mL of acetone, and collect the resulting precipitate by centrifugation to obtain 20 g of the precipitate. Thereafter, activated carbon treatment was performed in the same manner as in Example 2, and further purification treatment was performed in the same manner as in Example 1, to obtain 7.4 g of the desired soybean saponin.

Claims (1)

[Claims] Soy sauce lees or defatted soy sauce lees obtained by degreasing this with an organic solvent is extracted by adding water and/or lower alcohol, and the resulting extraction part is subjected to partition extraction with an organic solvent and nonionic adsorption resin. A method for producing soybean saponin, which comprises separating soybean saponin by applying at least one type of separation means selected from adsorption/desorption, fractional precipitation, and chromatography.