Process for the preparation of proanthocyanidins

The present invention aims at enhancing purity of the proanthocyanidin extracts by acting yeast to the proanthocyanidin extracts for fermenting them to thereby assimilate the impurities such as saccharides with yeast to reduce them.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for preparing proanthocyanidins 
in a high yield through a yeast fermentation of proanthocyanidin extracts. 
2. Description of the Related Art 
Proanthocyanidins are a group of compounds comprising, as constitutional 
units, condensed type tannin existing in various plants, namely 
flavan-3-ols and flavan-3,4-diols, which are bonded by condensation or 
polymerization. Since these compounds are transformed into anthocyanidins 
such as cyanidin, delphinidin and pelargonidin by an acid treatment, they 
are called by the above name. The compounds include proanthocyanidins such 
as higher molecular procyanidin, prodelphinidin and propelargonidin, and 
their stereoisomers, which are dimers, trimers, tetramers, or decamers of 
said constitutional units. 
The present inventors had discovered that these proanthocyanidins had a 
strong antioxidative activity, and filed a patent application on this 
discovery (Patent Kokoku No. 3-7232). 
Proanthocyanidins can be obtained by extracting from various species of 
plants with water or an organic solvent such as ethanol or acetone, but 
analyses of the extracts confirmed presence of other substances than the 
preferred proanthocyanidins, such as crude proteins, carbohydrates and 
ash. Therefore, if an extract solution is directly concentrated and made 
into powder, there would remain a substantial amount of said substances as 
impurities in the product, which may cause a decreased commercial value of 
the product. 
Analysis of carbohydrates of a proanthocyanidin extract from grape seeds 
showed that the main constituents of the extract were fructose and 
glucose. Considering that it should be possible to enhance purity of 
proanthocyanidins by removing these saccharides from the extract, the 
present inventors have pursued studies in this line and found that the 
desired effect could be obtained by subjecting the proanthocyanidin 
extract to a fermentation treatment with yeast. It was further found that 
heavy metals contained in the extract could be also assimilated by this 
treatment and the purifying effects could be obtained by reduction of 
heavy metal content. 
The present invention has been attained on the basis of the above finding. 
SUMMARY OF THE INVENTION 
The present invention provides a process for preparing proanthocyanidins 
which comprises adding yeast to a proanthocyanidin extract to cause its 
fermentation, and subjecting the fermented extract to solid-liquid 
separation. 
A detailed explanation of the present invention is given below. 
The proanthocyanidin extracts which fall within the conception of the 
present invention include the extract liquids containing proanthocyanidins 
extracted from various plant sources such as grape seeds, grape skins 
cranberries, apples, adzuki beans, and the barks of matsu (pine), Japanese 
ceder and Japanese cypress with water or an organic solvent, concentrated 
liquid thereof, and aqueous solutions formed by redissolving the dried and 
powdered concentrate in water. These solutions contain proanthocyanidins 
in amount of at least 10% calculated on dry basis. 
In case extraction has been conducted using water or hydrous ethanol, the 
extract may be used directly in the form as it is or after concentration, 
but in case extraction has been carried out with acetone and the like, it 
needs to remove acetone or such by concentration. 
As the yeast to be added to the proanthocyanidin extract for fermentation, 
there can be used, for instance, those belonging to the genera 
Saccharomyces and Zygosaccharomyces, which include commercially available 
wine yeast, sake yeast and baker's yeast. 
The yeast may be added and acted at a temperature and pH optimal for the 
action of the yeast used; for instance, the yeast may be added by 
adjusting the temperature of the proanthocyanidin extract to 
5.degree.-35.degree. C. and its pH to 3-6. The fermentation time is 12 
hours or more, preferably 24 hours or more. 
The amount of yeast added is 10.sup.4 -10.sup.8 cells, preferably 10.sup.5 
-10.sup.7 cells, per ml of the extract. 
As a consequence of the above treatment, the saccharides in the 
proanthocyanidin extract are utilized by yeast to give an alcohol, so that 
the proanthocyanidin products obtained by concentrating and drying the 
extract after yeast fermentation have a lower amount of impurities and 
have a high proanthocyanidin content. 
In case yeast fermentation is insufficient due to the lack of saccharide in 
the extract, saccharide may be added to the extract. In this way, the 
impurities such as heavy metals in the extract can be reduced through 
assimilation. 
Of course, these products may be subjected to additional purification. In 
this case, since the substantial part of the impurities has already been 
removed, purification can be performed in a very simple way. 
The present invention is further specifically illustrated by the following 
examples. The quantitative determination of proanthocyanidins was carried 
out according to R. Jambunathan et al method (J. Agric. Food Chem., 34, 
425-429, 1986): A prothocyanidin-containing sample is heated in the 
presence of dilute hydrochloric acid to redden proanthocyanidin, and it is 
quantified from the measurement of absorbence at 550 nm and calibration 
curve drawn up using as standard a procyanidin tetramer separated and 
purified from cider according to A.G.H. Lea method (J. Sci. Food Agric., 
34, 471-477, 1978). 
Heavy metals were quantified by colorimetry using sodium sulfide, and the 
amount was calculated on the lead basis. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS

EXAMPLE 1 
One kg of seeds of grape (species: Koshu) were extracted in 2 liters of 20% 
ethanol with stirring at 25.degree. C. for one week followed by 
solid-liquid separation and addition of water to obtain 4 liters of a 
clear solution (alcohol concentration: 9%). 
This clear solution was divided into two equal portions, and yeast (powdery 
wine yeast produced by Lallemand Inc. EC1118) was added to one portion to 
a concentration of 300 ppm to carry out stationary fermentation at 
25.degree. C. for 7 days, after which the solution was subjected to 
solid-liquid separation according to conventional method to obtain about 2 
liters of clear solution. 
This clear solution was concentrated and freeze-dried to produce a 
proanthocyanidin powder. 
The other portion (2 liters) of the clear solution was used as control, and 
it was concentrated and freeze-dried with no yeast added thereto to obtain 
a proanthocyanidin powder. 
The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Present 
invention Control 
______________________________________ 
Yield of powdery 6.65 g 9.50 g 
product 
Proanthocyanidin 51.0% 35.7% 
content in product 
Heavy metal content in 
2 ppm 19 ppm 
product 
______________________________________ 
EXAMPLE 2 
Two kg of seeds of grape (species: Chardonnay) were extracted with 
90.degree. C. hot water for 2 hours, and after solid-liquid separation, 
the solution was concentrated under reduced pressure to obtain 1,200 ml of 
a concentrated solution with 10% solid content. This solution was divided 
into two equal portions, and sake yeast (Japan Brewage Association Yeast 
#7) was added to one portion in an amount of 1.times.10.sup.7 cells per ml 
of the concentrated solution to carry out a fermentation at 20.degree. C. 
for 2 days, followed by the same treatments as in Example 1 to obtain a 
proanthocyanidin powder. The other portion of the solution, used as 
control, was concentrated and freeze-dried without adding yeast to obtain 
a powdery product. The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Present 
invention Control 
______________________________________ 
Yield of powdery 58.5 g 89.6 g 
product 
Proanthocyanidin 59.0% 32.0% 
content in product 
Heavy metal content in 
2 ppm 15 ppm 
product 
______________________________________ 
EXAMPLE 3 
100 g of a powdery grape seed extract (proanthocyanidin content in the 
powder: 41%) was dissolved in 2 liters of a 10% ethanol solution, to which 
600 ppm of powdery wine yeast (Lalvin KI by Lallemand Inc.) was added to 
carry out a fermentation at 25.degree. C. for 7 days, followed by 
solid-liquid separation, concentration and freeze-drying in the same way 
as in Example 1 to obtain 101 g of a powdery product. The proanthocyanidin 
content in this product was determined to be 61%, which represents about 
50% improvement of the degree of purification. 
EXAMPLE 4 
100 g of powdery grape seed extract (proanthocyanidin content in the 
powder: 38%) was dissolved in one liter of water, to which powdery wine 
yeast (Lalvin L2226 by Lallemand Inc.) was added in an amount of 300 ppm 
to carry out a fermentation at 30.degree. C. for 5 days, followed by 
solid-liquid separation, concentration and freeze-drying according to 
Example 1 to obtain 100 g of a powdery product. Analysis determined the 
proanthocyanidin content in the product was 54%, corresponding to about 
42% improvement of the degree of purification. 
According to the present invention, it is possible to enhance purity of 
proanthocyanidin preparations and to reduce heavy metals and other 
impurities therein by yeast fermentation to the proanthocyanidin extracts 
obtained from various plant sources.