JP2001008695A - A method for concentrating and recovering polyphenols and a polyphenol-rich yeast. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To selectively and efficiently inexpensively and efficiently concentrate and recover flavonols such as quercetin and resveratrol, which is a stillbene compound, present in wine and grape juice. It is an object of the present invention to develop a yeast containing resveratrol at a high concentration. SOLUTION: The present invention collects cells after fermentation of red wine and obtains the cells, and flavonols and stills adsorbed on yeast from the obtained cells using a hydrophilic polar solvent such as ethanol. It can be carried out by extracting a ben compound and obtaining a solution containing flavonol or resveratrol at a high concentration, or a solution with a higher concentration, or a powder. In addition, flavonols and resveratrol can be selectively adsorbed by adding and incubating commercially available yeasts such as baker's yeast to red grape juice and wine.
In particular, the invention can be carried out without going through a fermentation step. The present invention is characterized by selectively adsorbing the substance to yeast cells by contacting the yeast cells with fruit juice or wine containing flavonols or stilbene compounds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for efficiently concentrating and recovering polyphenols contained in grapes and wines, particularly quercetin and myricetin, which are flavonols, and resveratrol, which is a stillbene compound. It is intended to provide wine yeast contained in the concentration.

[0002]

BACKGROUND OF THE INVENTION Since red wine has been reported to unlock the key to the so-called "French paradox", consumption of red wine has increased since red wine is "healthy". Red wine is reported to have an effect on arteriosclerosis, heart disease, cancer, dementia, Alzheimer's disease, and the like, and it is said that the component exerting the effect is a polyphenol contained therein.

[0003] Polyphenol is a general term for those having a plurality of hydroxyl groups on the benzene ring, and among them, flavonols represented by quercetin are reported to be substances having high anticancer and antioxidant activities. In addition, resveratrol, which is also a kind of polyphenol, is a styrben compound, which has an anti-atherogenic effect by inhibiting the oxidation of LDL-cholesterol, which is a bad cholesterol, a thrombotic effect by inhibiting platelet aggregation, an anti-cancer effect, and phytoestrogens. It has attracted attention for its ability to prevent dementia and Alzheimer's disease.

[0004] As a method of taking polyphenols,
It is known to drink red wine and red grape juice. However, the concentrations of flavonols and resveratrol, which have high health effects, in wine or grape juice are relatively low. That is, the content of flavonols in grapes and wine is usually 20 to 50 mg / L [J. Oszmianski, CY Lee, A
m. J. Enol. Vitic., vol. 41, pp. 204-206 (1990),
PL Teissedre, EN Frankel, AW Waterhouse, H.
Peleg, JB German, J. Sci. Food Agric., Vol. 70, p
p. 55-61 (1996)]. In addition, the resveratrol content is even lower, and even at all stilbene compound concentrations, white wine
0.01 to 3.02 mg / L, 0.82 to 13.43 mg / L for red wine [M. Sat
o, Y. Suzuki, T. Okuda, K. Yokotsuka, Biosci. Biot
ech. Biochem., vol. 61, pp. 1800-1805 (1997)].

[0005]

2. Description of the Related Art Effective intake of flavonols and resveratrol requires a large intake of wine and juice. In the case of wine, alcohol harm is a problem. In the case of juice, excessive intake of sugar is required. There is a problem. If wine is concentrated by distillation under reduced pressure or the like, the problem of alcohol harm can be avoided, but the resulting concentrate is not suitable for eating and drinking because the amount of organic acids is too large. On the other hand, concentrating the juice does not solve the problem of excessive intake of sugar. Therefore, it has been desired to selectively increase the concentration of these effective polyphenols. Here, theoretically, an adsorption resin is used to adsorb substances with relatively low polarity,
It is conceivable that the solvent is eluted selectively using a solvent, but the construction of a resin tower and an adsorbent resin are required, and there is a problem that the cost is increased. Heretofore, there has been no known method for inexpensively selectively concentrating and recovering flavonols and resveratrol from wine, grape juice and the like.

[0006] On the other hand, the use of yeast as a dietary supplement has been practiced for a long time. However, yeast food containing a high concentration of polyphenols, especially flavonols and resveratrol among polyphenols, has been reported. There is no. As detailed above, flavonols such as quercetin and resveratrol, a stilbene compound, have various excellent effects such as anticancer properties and arteriosclerosis-preventing effects. Yeasts containing high concentrations of trolls have not yet been developed.

[0007]

SUMMARY OF THE INVENTION It is necessary to selectively and efficiently concentrate and recover flavonols such as quercetin and resveratrol which is a stillbene compound present in wine and grape juice at low cost. It is an object of the present invention to develop a yeast containing resveratrol at a high concentration.

[0008]

[Means for solving the problems] In the course of research on the effective utilization of wine yeast, we analyzed polyphenols contained in yeast cells after wine fermentation. Unexpectedly, quercetin and myricetin in polyphenols were surprisingly analyzed. Of flavonols and stilbene compounds such as resveratrol and its glycoside pie seed. As a result of further intensive studies, they have found that yeast can adsorb flavonols and stilbene compounds with high selectivity simply by adding it to grape juice or wine, and reached the present invention.

That is, the present invention relates to a method for recovering cells after fermentation of red wine and obtaining the cells, and for obtaining flavonols or the like adsorbed on yeast using a hydrophilic polar solvent such as ethanol from the obtained cells. Extracting stilbene compounds, a solution containing high concentrations of flavonols and stilbene compounds,
Alternatively, it can be carried out by obtaining a more concentrated solution or powder. Also, by adding and incubating a commercially available yeast such as baker's yeast to red grape juice or wine, the flavonols and the stilbene compound can be selectively adsorbed, so that the invention can be carried out without any particular fermentation step.
The present invention is characterized by selectively adsorbing the substance to yeast cells by contacting the yeast cells with fruit juice or wine containing flavonols or stilbene compounds.

Hereinafter, the present invention will be described in detail. The kind of fruit juice in the present invention is not particularly limited as long as it contains a target flavonol or a stilbene compound. As the fruit juice, red grape juice, apple juice, blueberry juice, Aronia juice, black currant juice and the like can be used, and preferably, red grape juice is used.

The type of wine used is not particularly limited. Usually, wine is fermented from grapes, but any wine obtained by fermenting fruit juice containing the above-mentioned flavonols and stilbene compounds can be used in the present invention. Preferably, red wine is used.

The method of contacting fruit juice and / or wine with yeast can utilize the fermentation process of wine brewing.
Alternatively, it can be carried out by using commercially available dried or yeast moist cells, adding it to fruit juice and / or wine, and then taking a short contact time. Commercially available yeast cells include baker's yeast (Oriental Yeast) and wine yeast for brewing (EC111).
8, Lallemand, etc.)
There is no particular limitation on the type. When the fermentation step is used, the conditions for contacting the fruit juice and / or wine with the yeast are 10 ° C to 30 ° C for 3 to 20 days, which are the conditions for wine fermentation. When yeast cells are added to juice and / or wine, the contact conditions between yeast and juice and / or wine should be 0-30 ° C, 30 ° C.
The contact time is 5 minutes to 20 ° C. for 5 hours, but the contact time is usually suitable.

When the yeast containing flavonols of the present invention is used after yeast fermentation of red wine, the yeast after fermentation of red wine is recovered by a cell separation operation usually performed for clarifying wine, such as centrifugation or filtration. Is done. The collected cells are appropriately dried in a drying step commonly used by those skilled in the art, such as vacuum drying, freeze drying, spray drying, and the like, to obtain a dry powder. Even when a commercially available yeast is added to and brought into contact with fruit juice and / or wine and flavonols or resveratrol is adsorbed on the yeast, the yeast can be collected and dried in the same manner as in the case of fermenting red wine yeast.

The amount of yeast used in the present invention to juice and / or wine is not particularly limited, but 0.1 to 10% (w / v) can be used based on the juice and / or wine. In the case of wine-fermented yeast, the amount of yeast added as an alcoholic beverage is extremely small, but the amount of yeast is usually about 0.5% (w / v) because the yeast grows during fermentation.

The desired flavonols and stilbene compounds can be extracted with a solvent from the recovered yeast that has come into contact with fruit juice and / or wine and adsorbed flavonols and stilbene compounds. The solvent used is not particularly limited as long as it is a solvent such as acetone, methanol, or ethanol that can extract flavonols and stilbene compounds from yeast cells. Considering the use for ethanol, ethanol is preferably used.

Extraction of flavonols and stilbene compounds from yeast cells may be carried out by conventional methods, and is not particularly limited. For example, it can be performed by bringing ethanol water into contact with yeast cells. The various conditions may be any as long as flavonols and stillbene compounds can be efficiently extracted.The ethanol concentration is 10% or more, the contact time with yeast is 10 minutes or more, and the extraction temperature is preferably in the range of 0 to 60 ° C. . Particularly preferably, the ethanol concentration is 20 to 80% (v /
v), the preferable contact time with the yeast is 30 minutes to 20 hours. In addition, the temperature at the time of extraction is 5-30
° C is selected.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0018]

EXPERIMENTAL EXAMPLE 1: An Example Utilizing a Wine Fermentation Process A red grape concentrated mast (produced in Spain) was diluted 4 times to prepare 100 L of juice having a sugar concentration of 20.2%.
rm CM, manufactured by Danster Ferment AG) at 1000 ppm.
Fermented at 0 ° C. 12 days of fermentation completely consumes sugar,
After the fermentation was completed, the cells were centrifuged (7000 rpm, 15 minutes) to separate the cells to obtain wet cells. The yield of wet cells was 2600 g. After freeze-drying the wet cells, dry yeast cells
g, 30 mL of 60% (v / v) ethanol was added, and the mixture was stirred with a stirrer at 20 ° C. for 20 hours to extract the polyphenol component adsorbed on the yeast. The obtained extract is centrifuged (3000 rpm, 1
0 minutes) to obtain a supernatant, which was subjected to 0.45 μm membrane filtration, and the total amount of polyphenols in the filtrate was determined by the Folin-Ciocalteu method in terms of gallicity. As a result, 3.54% (w / w) of polyphenol was contained in the red wine dry yeast. Since normal red wine contains 0.1-0.35% of total polyphenols, the total amount of polyphenols in red wine yeast was found to be more than 10 times the concentration of wine.

Further, the above filtrate was analyzed for high-performance liquid chromatography (HPLC) for stilbene compounds and flavonols. Detailed conditions for HPLC are described in the literature [M. Sato,
Y.Suzuki, T. Okuda, K. Yokotsuka, Biosci. Biotec
h. Biochem., vol. 61, pp. 1800-1805 (1997)]. Table 1 shows the analysis results.

Table 1 Contents of stilbene compounds and flavonol compounds in red wine yeast

[Table 1]

From Table 1, the dried yeast obtained by the above method contained resveratrol at 122.4 mg / kg and total stilbene compounds at 234.9 mg / kg. The resulting wine contained 3.1 mg / L of total stilbene compounds, which means that it contained approximately 80 times the stilbene compounds of the wine. In particular, resveratrol was contained at about 400 times the concentration of wine. On the other hand, for flavonols, the total amount of quercetin-3-glucoside, quercetin, myricetin, rutin, and kaempferol is 1498.7 m
g / kg was included. Since the wine obtained at this time contained 25.0 mg / L as the total flavonol concentration, it was found that the total flavonol concentration was also about 60 times that of the wine. Among them, the concentration of quercetin was about 250 times, and that of myricetin was about 200 times.

Example 2: Example in which yeast was added to fruit juice or wine and stirred The commercially available dried yeast was added to fruit juice or wine, and it was examined whether it was possible to selectively adsorb and concentrate flavonol compounds and stilbene compounds. Dried yeast (EC111) was added to 100 mL of grape juice diluted 4-fold from the concentrated red grape juice used in Example 1.
8, Lallemand) at 5 ° C or 20 ° C.
Stir for 5 or 20 hours. Similarly, 100 g of the red wine obtained in Example 1 was mixed with grape juice and dried yeast.
g was added and the grape juice was stirred. After stirring under each condition, the yeast was centrifuged (7000 rpm, 15 minutes) and washed once with 20 mL of distilled water. The yeast was dried in a freeze dryer. Table 2 shows the recovered amount of dried cells under each condition. The obtained dried yeast cells
0.3 g was collected, and 30 mL of 60% (v / v) ethanol was added.
The mixture was stirred with a stirrer for 20 hours to extract the polyphenol component adsorbed on the yeast. The obtained extract is centrifuged (3000 rp
m, 10 minutes) and the supernatant was analyzed by HPLC. Table 3 shows the analyzed stilbene compounds and flavonol compounds.
Table 4 shows the results of polyphenol adsorption by adding yeast to grape juice at 5 ° C, Table 4 shows the results of polyphenol adsorption by adding yeast to grape juice at 20 ° C, and Table 5 shows the results of polyphenol adsorption by adding yeast to wine at 5 ° C. Table 6 shows the results of adsorption of polyphenols by adding yeast to wine at 20 ° C.

Table 2 Changes in weight of recovered yeast in yeast addition test

[Table 2]

Table 3 Cell Adsorption Amount of Polyphenol by Adding Yeast to Grape Juice at 5 ° C.

[Table 3]

Table 4 Amount of adsorbed polyphenol cells by adding yeast to grape juice at 20 ° C

[Table 4]

Table 5 Cell adsorption amount of polyphenol by addition of yeast to wine at 5 ° C

[Table 5]

Table 6 Adsorption amount of polyphenols by adding yeast to wine at 20 ° C

[Table 6] Table 2 shows that the contact temperature between yeast and fruit juice or wine was 20
In the case of ℃, the amount of recovered cells was slightly larger than that in 5 ℃. This is probably due to the slight growth of yeast during contact. This tendency was remarkable when the fruit juice was contacted at 20 ° C for 20 hours.

As shown in Tables 3 and 4, the recovery rate (adsorption rate) of trans-resveratrol, which is the main substance, among the stilbene compounds contained in the original juice to the cells was 70 to 80%, which was extremely high. It can be seen that it is selectively adsorbed and collected on yeast. In addition, the recovery of quercetin and myricetin, which are the main substances among the flavonol compounds, in the cells was extremely high at 90% or more, indicating that they were very selectively and efficiently adsorbed to the cells and eluted with ethanol. From Tables 3 and 4, it was found that there was almost no difference between the adsorption reaction time at 5 ° C. and 5 hours or 20 hours, and the contact time of 5 hours was already sufficient. At 20 ° C, the amounts of stilbene and flavonol compounds adsorbed on yeast were slightly lower than at 5 ° C, and decreased with increasing reaction time. This was thought to be due to the yeast fermenting.
In other words, when the stilbene compound and the flavonol compound are adsorbed on the yeast from the fruit juice, the reaction should be carried out at a low temperature around 5 ° C. There was found.

As shown in Tables 5 and 6, the recovery rate (adsorption rate) of trans-resveratrol, which is the main substance, among the stilbene compounds contained in red wine to the cells was 43 to 60%. It can be seen that it is adsorbed and collected by yeast. Quercetin, which is the main substance in the flavonol compounds, was almost completely adsorbed on the cells and eluted with ethanol. From Tables 5 and 6, the adsorption reaction time is
In the case of 5 ° C, there was almost no difference between 5 hours and 20 hours, and it was found that a contact time of 5 hours was already sufficient. Also, 20
In the case of ° C, the amount of adsorption tended to be slightly larger in 5 hours than in 20 hours. The adsorption amount is higher at 5 ° C than at 20 ° C. When yeast is added to red wine to adsorb stilbene compounds and flavonol compounds, the reaction is preferably performed at a low temperature around 5 ° C, and the reaction time is 5 to 20 hours. It turned out to be almost the same. The total phenolic content of the dried yeast that has been in contact with wine at 5 ° C for 5 hours is 1.4%.
The total phenolic content of dried yeast in contact with wine for an hour is 1.
3%.

Example 3 In order to extract a stillbene compound and a flavonol compound from red wine yeast, the ethanol concentration of ethanol water used for the extraction was examined. 0.3 g of the dried red wine yeast obtained in Example 1 was weighed, and 0-99.5% ethanol water was added to make a total volume of 30 mL. After stirring with a stirrer at 20 ° C. for 20 hours, the mixture was centrifuged. 0.45 μm
After membrane filtration of, the extraction amounts of the stilbene compound and the flavonol compound were measured under the same HPLC conditions as in Example 1. Table 7 shows the results.

Since the extraction amount in Table 7 was obtained as a 1% bacterial cell solution, it was expressed in terms of 1 kg of bacterial cells. From Table 7, the extraction efficiency was 60% (v / v) with the highest extraction efficiency. 80
% Ethanol water showed a high extraction rate, but 99.5% ethanol did not extract at all. From Table 7, it was found that 40-80% ethanol water can be suitably used as the ethanol water extracted from the cells.

Table 7 Amount of extraction of stilbene compound and flavonol compound by ethanol water of each concentration

[Table 7] Example 4 Ethanol was directly added to the centrifuged cells obtained at the end of red wine fermentation, and 95.5% alcohol was added to 300 mL of the wet cells obtained in Example 1 to obtain extracts of the stilbene compound and the flavonol compound. Was added in various amounts, and practical extraction conditions were examined. After addition of the alcohol, the mixture was stirred at 20 ° C. overnight, centrifuged, and the supernatant was analyzed by HPLC to determine the concentrations of the stilbene compound and the flavonol compound in the obtained extract. Table 8 shows the results.

Table 8 Concentrations of stilbene compounds and flavonol compounds in extracts obtained from wet cells

[Table 8]

From Table 8, it can be seen that ethanol was added to the wet cells obtained after the fermentation to obtain an extract of the stilbene compound and the flavonol compound.
At the time of, the most concentrated extract was obtained. At this time, the total concentration of the stilbene compound and the flavonol compound was 7.7 times that of the wine. However, the ethanol concentration was 59.1% in the total amount of the total extracted stilbene compound and flavonol compound, and the result of Example 3 was reproduced. As described above, it was shown that a solution having a high concentration of the stilbene compound and the flavonol compound can be easily obtained by adding ethanol to the cells obtained in the wine production process after the fermentation of red wine. In order to obtain a higher concentration solution, a higher concentration solution can be prepared by a concentration step such as concentration under reduced pressure.

[0035]

Industrial Applicability According to the present invention, stilbene compounds and flavonol compounds having high antioxidant properties, anticancer properties, arteriosclerosis-preventing effects, and thrombosis-preventing effects are easily and selectively extracted from grape juice and wine. Is possible,
It has been shown that stilbene compounds and flavonol compounds can be efficiently recovered from cells after fermentation of red wine, which was conventionally discarded as waste. The obtained yeast cells can be used as health supplements, and the extract is expected to be widely applied to various alcoholic beverages and cosmetics.

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Claims (6)

[Claims] 1. A method for concentrating and recovering polyphenols, comprising contacting yeast with fruit juice and / or wine. 2. A method for concentrating and recovering stilbene compounds, comprising contacting yeast with fruit juice and / or wine. 3. A method for concentrating and recovering flavonols, which comprises contacting yeast with fruit juice and / or wine. 4. A yeast containing 5 g or more of total polyphenol in terms of gallic per 1 kg of dry weight. 5. A yeast containing at least 20 mg of a stillbene compound per 1 kg of dry weight. 6. The amount of flavonols per kg of dry weight is 5%.
A yeast containing at least 00 mg.