JP5804471B2 - Method for producing sudachi polyphenol from sudachi (squeezed residue) - Google Patents

Description

  The present invention relates to a method for producing sudachi polyphenol effective for prevention and / or treatment of metabolic syndrome from the juice residue of sudachi.

Citrus contains many polyphenols (polymethoxyflavonoids), and these polyphenols are known to have the effect of preventing and improving lifestyle-related diseases such as diabetes and hyperlipidemia. (Patent Documents 1 and 2, Non-Patent Document 2).
In addition, Sudachi contains a large amount of Sudachi polyphenols (Sudachitin, demethoxysdachitin and glycosides thereof) having a unique structure unique to Sudachi. For example, the dried peel of sudachi (here, the skin covering the surface of the fruit) contains about 0.1% by mass of sudachitin, and at the same time, about 6 times the amount of sudachitin glycoside. (Patent document 3).
Thus, many attempts have been made so far to isolate and recover sudachi polyphenols from dried pericarp and squeezed residue and to make effective use of them. For example, it has been reported that sudachitin can be efficiently extracted by extracting sudachitin from a juice residue or hydrolyzing sudachitin glycoside with hydrochloric acid while irradiating with microwaves (Patent Document 3). Furthermore, it has been reported that when sake yeast is added to and reacted with a mixture of sudachitin and sudachitin glycoside extracted from the dried pericarp of sudachi, sudachitin glycoside is hydrolyzed to produce sudachitin (non-) Patent Document 1).

  However, since the above method requires microwave irradiation, it is necessary to scale up and isolate and purify Sudachi polyphenol on an industrial scale, which requires a special device and is expensive in terms of cost. It was difficult to synthesize. Therefore, there has been a demand for a simple and inexpensive manufacturing method on a large scale.

JP 2007-77139 A WO2002 / 0875567 JP 2008-208064 A

Ministry of Agriculture, Forestry and Fisheries Food Industry Green Project Technology Demonstration Model Project, 2009, Business Results 17 Subject Name “Demonstration of Separation Technology from Citrus Skin” Kinki Chugoku-Shikoku Agricultural Research Center Research Report No. 5, pp. 19-84, 2005 “Research on functional components of citrus fruits and their effective use”

  An object of the present invention is to provide a method for efficiently producing a large amount of sudachi polyphenol from the juice residue of sudachi.

  The present inventors have intensively studied a method for efficiently producing a large amount of sudachi polyphenol from a juice residue of sudachi, but a commercially available enzyme for food processing using a juice residue (dry powder) of sudachi has been studied. By making it act, it discovered that water-soluble sudachi polyphenol (a glycoside is included) can be isolated and purified more efficiently, and completed this invention.

The gist of the present invention is as follows.
(1) A method for producing a sudachi polyphenol from a sudachi juice residue or a sudachi peel,
Select one or more of food processing enzymes, Magnax JW-2, Cochlase SS, Denapsin 10P, Gluc S, Dena Team AP, Gluc 100, Gluco Team # 20000, Cellulosin T3,
In the enzyme aqueous solution, add Sudachi juice residue or Sudachi peel and stir at the appropriate temperature of the enzyme.
After further heating and stirring,
A method for producing sudachi polyphenol, comprising separating a supernatant of a reaction solution and then isolating and purifying the supernatant.
(2) The method for producing sudachi polyphenol according to (1) above, wherein the appropriate temperature of the enzyme is 35 ° C.
(3) The manufacturing method of sudachi polyphenol as described in said (1) or (2) whose said heating stirring is 50 degreeC.
(4) The method for producing sudachi polyphenol according to any one of (1) to (3), wherein the food processing enzyme contains Magnax JW-2.
(5) The method for producing sudachi polyphenol according to any one of (1) to (4), wherein the juice residue or the peel is in a dry powder form.

  The method for producing sudachi polyphenol of the present invention is a method of more efficiently extracting sudachi polyphenol and its glycoside contained in sudachi juice residue or sudachi peel, from the juice residue, etc. It is possible to achieve the intended purpose only by adding a general-purpose food processing enzyme without requiring additional means such as irradiation. Thus, the production method of the present invention is easy to operate, and industrial mass production of water-soluble Sudachi polyphenols has become possible. This has made it possible to develop foods or apply therapeutics to prevent diabetic diseases and metabolic syndrome using water-soluble sudachi polyphenols.

It is a figure of the HPLC analysis chart which performed the fixed_quantity | quantitative_assay of the demethoxysudachitin and sudachitin in the reaction liquid supernatant obtained by carrying out the enzyme treatment (Denapsin 10P) of the juice residue of Sudachi. It is a figure of the HPLC analysis chart of the supernatant liquid of an enzyme blank instead of the analysis chart in the reaction liquid supernatant processed with the enzyme like FIG.

The “sudachi juice residue or sudachi peel” of the present invention is a residue produced as a by-product when sudachi juice is squeezed from sudachi, and is obtained by treating this residue with an inner / outer skin separator (slicer). Says the pericarp. The juice residue is desirably dried and then pulverized to be used as a dry powder Sudachi powder.
The “food processing enzyme” of the present invention is selected from one or more of Magnax JW-2, Cochlase SS, Denapsin 10P, Gluc S, Denateam AP, Gluc 100, Glucozyme # 20000, and Cellulosin T3. It is what you use. That is, Magnax JW-2 is a glucoamylase manufactured by Nitto Kasei Kogyo. Coclase SS is a proteolytic enzyme agent manufactured by Mitsubishi Chemical Foods, and Denapsin 10P is a proteolytic enzyme agent consisting of an acidic protease manufactured by Nagase ChemteX. Gluc S and Gluc 100 are saccharifying enzyme agents consisting of α-amylase and glucoamylase manufactured by Amano Pharmaceutical. Denateam AP is a neutral protease proteolytic enzyme agent manufactured by Nagase ChemteX, and Glucoteam # 20000 is a glucoamylase saccharifying enzyme agent manufactured by Nagase Chemtex. Cellulosin T3 is a saccharifying enzyme agent comprising cellulase manufactured by Hibiai.

The “sudachi polyphenol” of the present invention refers to polymethoxyflavonoids and their glycosides contained in the static. Examples of polymethoxyflavonoids contained in the statie include sudachitin, demethoxysdachitin, nobiletin, and tangeretin. As shown in Patent Document 3, Sudachi contains a relatively large amount of sudachitin, demethoxysdachitin and their glycosides.
The water-soluble sudachi polyphenol is polymethoxyflavonoids and their glycosides contained in statis which are easily dissolved in water.
The “appropriate temperature of the enzyme” of the present invention varies slightly depending on the enzyme used, but a range of about 35 to 50 ° C. is used in consideration of the thermal stability of each enzyme. For example, the range of 40-50 degreeC is used for the degrading enzyme regarding 1.1 (4) alpha-glucan such as amylase. The range of 35-40 degreeC is used for the degrading enzyme regarding 1.3 (4) beta-glucan such as cellulase. A proteolytic enzyme such as protease is used in the range of 35 to 45 ° C. More preferable temperature for the enzyme reaction includes about 55 ° C.
The time for the enzyme reaction is not particularly limited, and can be appropriately adjusted in view of the degree of progress of the enzyme reaction.

The “heating and stirring” of the present invention refers to heating and stirring performed to accelerate and complete the enzymatic decomposition reaction. Here, the temperature rise means to further increase the reaction temperature, and it is preferable to stir at 40 to 50 ° C. More preferably, a temperature of about 50 ° C. can be mentioned.
The superheated stirring time can be appropriately adjusted while checking the content of sudachi polyphenol liberated from the juice residue or the like, and is not particularly limited.
The “reaction supernatant” of the present invention refers to a supernatant obtained by allowing the reaction solution to stand or be centrifuged. The supernatant may be further filtered with a membrane filter or the like.
The “isolation / purification” of the present invention refers to a conventional means described in a document such as a laboratory chemistry course. For example, it can be isolated by a conventional isolation and purification method such as column chromatography.

EXAMPLES Next, although an Example is given and this invention is further demonstrated, this invention is not limited to these.
(Example 1) Reaction of Sudachi Juice Residue with Various Food Processing Enzymes Sudachi Juice Residue was dried and then pulverized to obtain Sudachi Powder An attempt was made to solubilize functional ingredients by allowing food processing enzyme agents to act on the powder. That is, 0.5 g of sudachi powder was taken into a 15 ml centrifuge tube, 10 ml of 0.5% aqueous solution for food processing enzyme was added, reacted at 35 ° C. for 6 hours, then at 50 ° C. for 15 hours, and then in a boiling water bath Treated for minutes. After centrifugation of these reaction solutions, the amount of polyphenol (including glycoside) as a solubilized product by enzyme treatment was analyzed by the method of Test Example 1 for the solution obtained by filtering the supernatant with a membrane filter (φ0.45 μm).

(Test Example 1) Quantification of sudachi polyphenol (1) Polyphenol measurement method Polyphenol was quantified by the Folin-Denis method. That is, 1 ml of the sample solution and 0.5 ml of 1N phenol reagent (forein thiocult reagent) and 5 ml of 0.4 M Na ₂ CO ₃ were mixed in a test tube and reacted at 30 ° C. for 30 minutes. After the reaction solution was cooled to room temperature, the absorbance at 660 nm was measured. Gallic acid was used for the preparation of a calibration curve for quantification, and the amount of polyphenol in the sample was represented by a considerable amount of gallic acid.
(2) Measurement result of polyphenol amount Table 1 shows the amount of polyphenol in each enzyme-treated solution obtained in Example 1. When treated under the same conditions without adding enzyme (no enzyme), that is, the amount of polyphenol per 1 g of Sudachi powder solubilized by water extraction is 37.1 mg, and increases and decreases with various enzyme treatments was there. The results are shown in descending order of polyphenol recovery.

(3) Quantification of demethoxysudatin and sudachitin in polyphenol Hitachi high-performance liquid chromatograph system was used for quantitative analysis of demethoxysdachitin and sudachitin. Analysis conditions were set as follows.
[Column] Wakosil-II 5C18RS (4.6 mm ID × 250 mm)
[Mobile Phase] A: NaH ₂ PO ₄ (pH 2.3), B: CH ₃ CN
Linear gradient from A (88%) / B (12%) to A (40%) / B (60%) over 40 minutes from the start of analysis, A (40%) / B (60%) from 40 to 50 minutes Analyze with.
[Column temperature] 40 ° C
[Flow rate] 1.0 ml / min
[Detection] 325nm
Quantification of demethoxysudacitin and sudachitin in each enzyme treatment solution obtained in Example 1 was performed. The results are also shown in Table 1, and an analysis example of sudachi polyphenol is shown in FIG.

From Table 1 above, in order to efficiently recover and isolate sudachi polyphenol, sudachi with the enzymes of Magnax JW-2, Cochlase SS, Denapsin 10P, Gluc S, Denateam AP, Gluc 100, Glucozyme # 20000, and Cellulosin T3. It turns out that it is necessary to react the powder.
In addition, among the sudachi polyphenols, in order to efficiently recover sudachitin, denapsin 10P was the best, and it was shown that the recovery efficiency was about 2.1 times that of the case without enzyme. Further, as shown in Table 1, it was found that the sensitivity to the enzyme was different between demethoxysudatin and sudachitin. In the case of sudachitin, denapsin 10P had the highest recovery efficiency, but in the case of demethoxysdachitin, it was gluc S.

  The method for producing sudachi polyphenol of the present invention does not require the installation of a microwave irradiation device, etc., and can be easily and more efficiently recovered and isolated by using a commercially available food processing enzyme. . Therefore, it becomes possible to effectively use the juice residue of Sudachi, which was often disposed of as compost or industrial waste, and is likely to be used for the prevention and treatment of diseases such as diabetes and lifestyle-related diseases. Sudachi polyphenol can be mass-produced.

Claims (5)

A method for producing sudachi polyphenol containing sudachitin and demethoxysdachitin from the juice residue of sudachi or sudachi peel,
Select one or more of food processing enzymes, Magnax JW-2, Cochlase SS, Denapsin 10P, Gluc S, Dena Team AP, Gluc 100, Gluco Team # 20000, Cellulosin T3,
In the enzyme aqueous solution, add Sudachi juice residue or Sudachi peel and stir at the appropriate temperature of the enzyme.
After further heating and stirring,
A method for producing sudachi polyphenol, comprising separating a supernatant of a reaction solution and then isolating and purifying the supernatant.   The method for producing sudachi polyphenol according to claim 1, wherein the appropriate temperature of the enzyme is 35 ° C.   The method for producing sudachi polyphenol according to claim 1 or 2, wherein the heating and stirring is 50 ° C.   The method for producing sudachi polyphenol according to any one of claims 1 to 3, wherein the food processing enzyme comprises Magnax JW-2.   The method for producing sudachi polyphenol according to any one of claims 1 to 4, wherein the squeezed residue or peel is in a dry powder form.

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