JPH06113871A - Method for separating and recovering flavonoid compound and limonoid compound contained in citrus fruits - Google Patents

Abstract

PURPOSE:To obtain a method for separating and recovering a flavonoid and a limonoid compounds in which the flavonoid and limonoid compounds contained in citrus fruits can be separated and economically and advantageously recovered. CONSTITUTION:The objective method for separating and recovering a flavonoid and a limonoid compounds comprises the first step of adding a microorganism or a yeast to a marc of citrus fruits and subjecting impurities to the decomposing treatment and the second step of centrifuging the resultant treated substance obtained in the first step into a precipitated fraction containing the flavonoid compound and a supernatant fraction containing the limonoid compound.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to flavonoid compounds contained in citrus fruits having pharmacological activities such as stomach, antitussive, anti-inflammatory and hypotensive action, and anti-cancer activity, and physiological activity such as antifeedant activity against insects. The present invention relates to a method for efficiently separating and recovering a limonoid compound having

[0002]

2. Description of the Related Art Citrus fruits such as mandarin orange, summer mandarin orange, Hachisaku, and grapefruit are often squeezed into juice and used as beverages. Also occurs in large quantities. The squeezed lees that are produced in large quantities in processing factories may cause environmental pollution if they are discarded as they are. Therefore, they are usually used for livestock feed by heating and drying.

[0003]

However, in the squeezed lees, flavonoid compounds having pharmacological activities such as stomach, antitussive, anti-inflammatory and hypotensive action, anti-cancer activity, antifeedant activity against insects, etc. A limonoid compound having physiological activity is contained, and studies are being conducted to make effective use thereof.

[0004] Incidentally, as a technique for extracting and recovering flavonoid compounds from citrus juice residue, it is known to collect hesperidin by extracting alkali from raw juice residue and dried juice residue (dry skin). Since many impurities are contained in the citrus juice cake, the extraction effect of hesperidin is poor and it has not been put to industrial use. Further, limonoid compounds have been regarded as bitterness components of citrus fruit juice, and research has been advanced from the viewpoint of debittering, but no industrial extraction or recovery has been reported.

In view of such circumstances, it is an object of the present invention to provide a separation and recovery method capable of separating flavonoid compounds and limonoid compounds contained in citrus fruits and recovering them economically advantageously.

[0006]

The method for separating and recovering flavonoid compounds and limonoid compounds contained in citrus fruits according to the present invention, in order to achieve such an object,
The first step of adding microorganisms or enzymes to the citrus fruit juice cake to decompose contaminants, and the treated product obtained in the first step,
A second step of centrifuging into a precipitate fraction containing flavonoid compounds and a supernatant fraction containing limonoid compounds was performed.

In the above construction, the squeezed lees are not only primary squeezed lees (raw squeezed lees) but also secondary squeezed lees (squeezed lees obtained by pressing and dehydrating raw squeezed lees with lime). Is also included. In addition, in the first step, before adding the microorganisms or enzymes, an equal amount or more of water is added to the squeezed lees, and the pH is adjusted according to the enzymes and the microorganisms,
It is preferable that the squeezed lees are heated and sterilized by heating as above.

Examples of the enzyme used in the first step include pectinase, cellulase, amylase, protease and lipase. Aspergillus ni
ger , Aspergillus oryzae , As
pectinases originating from the genus Aspergillus (a type of filamentous fungus) such as pergillus pulverulentus, and genus Aspergillus such as Aspergillus niger or Trichoderma v
Cellulase originating from the genus Trichoderma (a type of filamentous fungus) such as iride is particularly preferable.

The temperature for decomposing contaminants by the enzyme in the first step is preferably about 40 to 50 ° C., and the amount of enzyme added is preferably about 0.02 to 0.2% by weight with respect to the raw material squeezed lees. The reaction time varies depending on the amount of enzyme added. For example, 0.02% is 2 days, and 0.2% is 16 hours or less.

Further, instead of using the enzyme agent in the first step, a microorganism producing the above enzyme can be used. As the microorganism used in the first step, Aspergill
us niger , Aspergillus oryz
ae , Aspergillus pulverulen
genus Aspergillus (a type of filamentous fungus) such as tus or Tr
The genus Trichoderma (a type of filamentous fungus) such as ichoderma viride can be mentioned.

It is preferable that the microorganism is previously cultivated in a liquid culture medium such as wort, koji or potato dextrose solution, and then added to the squeezed lees in the form of a bacterial solution.
The temperature for decomposing contaminants by microorganisms is preferably about 30 to 40 ° C., and the treatment is preferably performed for 3 to 5 days (static culture or shaking culture; shaking culture requires a shorter culture time).

When a microorganism is used in the first step, after culturing for a certain period of time and removing the cells in the culture solution,
It is preferable to perform centrifugation. In order to increase the recovery rate of the flavonoid compound, an equal amount or more of water is added to the precipitate fraction obtained in the second step, the mixture is stirred, and then centrifuged (washed), or the first step is completed. After that, before performing the centrifugation in the second step, the
It is preferable to collect only the fraction of 0 μm or less (remove the fraction of more than 150 μm).

The limonoid compound contained in the supernatant fraction obtained in the second step is adsorbed on a synthetic adsorption resin or an anion exchange resin to remove impurities such as sugars, and then a non-aqueous solvent such as methyl alcohol. After elution, the concentration can be easily increased by the vacuum concentration method. In this case, by performing the enzyme treatment, the amount of the supernatant increases, the absolute amount of the extracted limonoid compound increases, and the water-soluble polysaccharide has a low molecular weight, thereby increasing the purity of the limonoid compound. Has the advantage that

[0014]

According to the above construction, in the first step, the enzymes and the microorganisms are added to the squeezed meal to decompose the contaminants (pectin, cellulose, etc.) in the squeezed meal to form water-soluble substances such as sugars. After that, when it is centrifuged in the second step, it is separated into a precipitate fraction and a supernatant fraction.

The precipitate fraction thus obtained contains a large amount of flavonoid compounds, and the supernatant fraction contains a large amount of limonoid compounds as well as sugars.

[0016]

EXAMPLES The present invention will be described in detail below with reference to its examples.

[0017]

Example 1 In order to efficiently extract and recover a flavonoid compound, which is one of useful substances in citrus fruit juice cake,
An enzyme effective for decomposing and removing the contained impurities was selected. Equivalent amount of water is added to the secondary squeezed lees of Satsuma mandarin, pH is adjusted to 4.0 with dilute hydrochloric acid, and heat sterilization (85 ° C)
After that, the mixture was cooled (50 ° C.), and a commercial oxygen agent was added so that the ratio was 0.1% by weight. There were 48 kinds of oxygen agents used at this time, and pectinase, cellulase,
It is classified into amylase, protease, and lipase, and each is
H4.0, pH4.0, pH5.0, pH7.0, pH
It was adjusted to 7.0 and left to stand at 50 ° C. for 24 hours for enzyme reaction. Subsequently, centrifugation (10,000 rpm, 10 minutes)
The precipitation fraction was obtained.

After the precipitation fraction was dried, the solids retention, the concentration of hesperidin, which is one of the flavonoid compounds, and the hesperidin retention were determined and are shown in Table 1.

[0019]

[Table 1]

As shown in Table 1, it was shown that when pectinase or cellulase was used as the enzyme agent, the removal rate of contaminants was high, the yield of the obtained solids was small, and the concentration of hesperidin was high. In particular, the effect was large for the enzyme agents mainly containing pectinase and having cellulase and hemicellulase activities. Moreover, no loss of hesperidin was observed by any of the enzymes.

[0021]

Example 2 Mainly, an enzyme having pectinase activity and an enzyme having cellulase activity were mixed in equal amounts, and the same enzyme treatment as in Example 1 was performed. In other words, add an equal amount of water to Satsuma mandarin secondary juice and adjust the pH to 4.0 with dilute hydrochloric acid.
After heat sterilization (85 ° C.), the mixture was cooled (50 ° C.), and the mixed enzyme preparation was added at a ratio of 0.1% by weight.

After enzymatic reaction at 50 ° C. for 24 hours, 500 μm or more, 300-500 μm, 150-
The fractions of 300 μm and 150 μm or less were sieved, and a precipitate was obtained by centrifugation, and the precipitate was washed with 5 times the amount of water, and the precipitate fraction was recovered by centrifugation. The solid yield, hesperidin concentration, and hesperidin yield of the obtained precipitation fraction were measured and are shown in Table 2.

[0023]

[Table 2]

As shown in Table 2, by using the mixed enzyme, the removal rate of contaminants was increased and the solids yield was significantly reduced, whereby the concentration of hesperidin was increased. Especially, the hesperidin concentration of the fraction of 150 μm or less was 27.4%, and the untreated (hesperidin concentration 6.
4%) to about 4.3 times.

[0025]

Example 3 Filamentous fungi suspected of having pectinase and cellulase activities in a medium containing squeezed lees ( Aspergi
llus aculeatus, Aspergillu
sawamori , Aspergillus fumi
gasus , Aspergillus niger , A
spergillus usamii ) was cultured to decompose contaminants. That is, first, 1.5 times the amount of water was added to the Satsuma mandarin orange cake (secondary cake) to adjust the pH to 4.0, heat sterilize (85 ° C), and then cool to 30-40 ° C. Then, a bacterial solution (0.5 times the amount of squeezed lees) that had been previously cultured in a potato sucrose solution was added. Then, after culturing at 3O <0> C for 3 days (stationary culturing), the bacterial cells in the culture broth were removed, followed by centrifugation (10000 rpm, 1
A precipitation fraction was obtained by (0 minutes). The solid yield, hesperidin concentration, and hesperidin yield of the obtained precipitation fraction were measured and shown in Table 3.

[0026]

[Table 3]

As shown in Table 3, as a result of measuring the enzyme activity of the culture solution of each microbial cell, pectinase, cellulase and hemicellulase activities were observed in all cases, and the solid content residual rate was also reduced. In addition, no loss of hesperidin was observed during the microorganism culture, and as a result, it was possible to obtain a fraction having a high hesperidin concentration.

[0028]

[Example 4] For the purpose of recovering linomoid glycosides from Satsuma mandarin orange juice, an equal amount of water was added to the juice cake (1000 g), pH was adjusted to 4.0 with dilute hydrochloric acid, and heat sterilization was performed (85 ° C).
Then, the mixture was cooled (50 ° C), and a commercially available enzyme preparation having pectinase activity, cellulase activity, and hemicellulase activity (Cellulosin PE100 manufactured by Ueda Chemical Industry Co., Ltd.) was added at a ratio of 0.1% by weight, and the mixture was added at 50 ° C. The mixture was allowed to stand for 24 hours to carry out an enzymatic reaction. Centrifugation after enzyme treatment (10,000 rpm,
After 10 minutes, 1600 g of a supernatant fraction (supernatant solution) was obtained. Adsorb the obtained supernatant fraction to 150 ml of synthetic adsorption resin (Amberlite XAD-2 manufactured by Organo Corporation), remove impurities such as sugars with water (150 ml), and elute with methyl alcohol (450 ml). Then, a fraction containing a limonoid glycoside was obtained.

The concentration of limonoid glycosides in each of the squeezed lees, the enzyme-treated solution (supernatant fraction) and the methanol eluate was examined, and the results are shown in Table 4.

[0030]

[Table 4]

By removing methyl alcohol from the methanol eluate by a vacuum concentration method, the concentration of limonoid glycosides could be easily increased without affecting the component composition.

[0032]

The method for separating and recovering flavonoid compounds and limonoid compounds contained in citrus fruits according to the present invention is constituted as described above, and the flavonoid compounds or limonoid compounds contained in citrus fruits are contained in high concentrations. Can be separated into minutes.

The flavonoid compound or limonoid compound can be economically and advantageously recovered by concentrating the fraction containing the flavonoid compound or limonoid compound at a high concentration.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Yoshihiko Ozaki 1501-2 Tsubo, Shimotsu-cho, Seagrass-gun, Wakayama Prefecture (72) Inventor Kiyoshi Ifuku 3-7-84 Nishihama, Wakayama-shi

Claims (1)

[Claims] 1. A first step in which microorganisms or enzymes are added to squeezed lees of citrus fruits to decompose impurities, and the treated product obtained in the first step contains a precipitation fraction containing a flavonoid compound and a limonoid compound. A method for separating and collecting flavonoid compounds and limonoid compounds in citrus fruits, which comprises a second step of centrifuging with a supernatant fraction.