Koshu grape pectins.

59( J. Inst. Enol. Vitic. Yamanashi Univ. 17 59-63 1982 )NoteKoshu Grape Pectins : Isolation, ChemicalComposition, and Precipitation *KOKI YOKOTSUKA, KAzuo ITo, KAzuHIKo NOZAKI,and T ADAE K USHIDAThe Institute of Enology and Viticulture, Yamanashi University,Kofu 400.

Crude pectins were extracted from Koshu grapes with hot water. Thepectins were· precipitated by ethanol and purified by DEAE-Sephadex A- 50
chromatography. The purified pectin contains galacturonic acid (92%), galactose,arabinose, xylose, and rhamnose as polysaccharide components. The degree ofesterification was 70%. Various factors influencing the precipitation of thepectin in musts or wines were investigated. The pectin was effectively precipitated at low temperatures, at pH below 3.0 and at more than 7% ethanol
concentration. The pectin was also precipitated at the low concentrationsof metal ions such as Cu,++ Ca,++ Fe,++ and K,+ and Koshu must proteins.

Pectins are one of the colloidal constituents of musts ~nd wines. The presenceof a large amount of pectins sometimes
makes clarification of musts and filtrationof wines difficult. Therefore, pectic enzymes have been used for the increase of
the yield of free-run juices from musts, orfor clarification or filtration of juices orwines. However, the use of the enzymes
increases the galacturonic acid and methyl
alcohol contents in wines.
Most of must pectins are hydrolyzed bynaturally occurring pectic enzymes and precipitated by the alcohol produced during
fermentation, hence the concentration ofpectins in wines is smaller than that inmusts. Pectins and their hydrolysis products act as protective colloids to prevent* Chemical Studies on Coloring and Flavoring
Substances in Japanese Grapes and Wines(XV).

7

the precipitation of suspended materials.
They may also be bound to various wine
components to form haze or sediment. However, the details are still not completely
elucidated.
This report describes the isolation ofpectins from Koshu grapes, their chemicalcomposition, and factors influencing the pre·cipitation of them.
Koshu grapes were crushed and pressedwith a Garolla crusher and a Vaslin-type
press. To the pomace (1 kg) left in thepress, water (2l) was added and boiled for30min. The pH of the extract was 4.0 to4.5. The extract was filtered through a
Toyo filter No. 101. The filtrate was cooled, concentrated to one-third of its original
volume. After the concentrate had beenadjusted to pH 1.0 with Hel, 3 volumes ofethanol were added to the concentrate. The
precipitate formed was collected by filtra-I.

YOKOTSUKA, ITO. NOZAKL and KUSHIDA

60-Table 1. General composition of the crude and purified pectins from Koshu grapes.

Component

Crude pectin%

Purified pectin%

Polysaccharide

79.497.0Phenol

14.5

0.8

Ash

2.5

1.8

Others3.6

0.4

Table 2. Composition of polysaccharide components of the purified pectin.
Components%

Galacturonic acid95.4

Arabinose

trace

Galactose

1.8Rhamnose

2.8

Xylosetrace

tion through a Toyo filter No.2, washedwith ethanol and acetone, and dried. The
yield of the crude pectin was about 4g.
The pectin contents of the must from theKoshu grapes and the wine were about 0.3
g/ t and O.06g/ t, respectively when determined by the carbazole-H2S04 method.n
The crude pectin was dissolved in a small
amount of water. Insoluble substances wereremoved by centrifugation. The supernatant was applied to a column of DEAE·Sephadex A-50 (2.3 X 25cm) equilibrated
with 1/100 M phosphate buffer (pH 6.0).
The column was washed with the pH 6.0
buffer (350 mt). Pectins were eluted with
0.1 N HCl. The yield was l.lg. This

pectin fraction was used as the purified
pectin fraction in the subsequent experiments.
Tables 1 and 2 show the compositions of
the pectins. Polysaccharides and total phenols were determined by the methods ofHodge and Hofreiter,2) and Singleton andRossi,3) respectively. Ash content was determined by the method in "Office International de Vigne et du Vin."4) The composition of the cations in the ash was analyzed
with a Hitachi atomic absorption spectrometer (Model 170-30). The concentrations of cations were 16.2mg/g for Na; 0.33
mg for Fe;-+ 0.16mg for Mg;-+ 0.10mg forCu;-+ 0.094mg for K; and 0.082mg for Ca:-+Koshu grape pectins.

The pectin was hydrolyzed in 0.2 N H2S04
at 100·e for 6h. The hydrolysate wasneutralized with barium carbonate. The
precipitate formed was removed by centrifugation. The supernatant was passed
through a column of Amberlite IR-120(1.0X 10cm). To the eluate, an equal volumeof acetone was added, and the mixture wasfiltered through a Toyo No.5 c filter. The
filtrate was evaported to a syrup.
This
was used as a sample for thin-layer chromatography. Ascending thin-layer chromatography was carried out at room temperature
with a solvent system of ethyl acetate:pyridine: acetic acid: water (5 : 5 : 1 : 3,v/v). Avicel cellulose AF (Asahi Kasei,109) was vigorously shaken with 35ml ofwater. The slurry produced was poured
onto a glass plate (20 X20cm). After airdrying, the plate was heated at 60· to 80·efor 20 min. After the solvent front hadreached within about 7 cm of the upper edgeof the plate, the plate was removed from achamber and dried under a slow air current.
The dried plate was sprayed with the diphenylamine-aniline-phosphoric acid reagent:)and heated at 80·e for 4min. The plate
was scanned zigzag at 400nm for arabinose,
xylose, and rhamnose, 440nm for galacturonic acid, and 640 nm for galactose .w ith aShimadzu microdensitometer (Model es900). The Rf values of galacturonic acid,galactose, arabinose, xylose, and rhamnosewere 0.19, 0.51, 0.56, 0.73, and 0.84, respectively. Galacturonic acid constituted 95.4%of the total polysaccharide (Table 2).
Degree of esterification and intrinsic viscosity were examined bythe methods of Geeet al~j) and Owens et al.~) respectively. The
degree of the esterification of the pectin
was 70%. The degree of the polymerization
seemed to be low, because the intrinsicviscosity was low (0.6 dl/g).
The pectin was dissolved in water to givep61

a concentration of 0.2%. The pectin solution(2 ml) was mixed with 0.1 M potassium
tartrate buffer (2ml) with different pH (pH2.5 to 4.5). The mixture was allowed tostand at room temperature for 48h and centrifuged at 30,000 rpm for 30min. The pectin content in the supernatant was measured
by the carbazole-H2S04 method. About 14% of the amount of the pectin added at pH
2.5 and 3% of it at pH 3.0 were precipitated,but the pectin was not precipitated at pHlarger than 3.0 (Fig. la).

~::~
2.5

P2Z!
3.00

3.5

4D

4.53040pH;;-~

"QI~

:§.
uQIL-a.

.£UQIa.

::~0f22l
1020Temperature(OC)]~')
=~:J5

7.5

10

12.5

15

Concn. of ethanol (0/0)

Fig. 1. Effects of pH, temperature, and ethanolon the precipitation of the pectin.

The pectin solution (2ml) was mixed
with the pH 3.0 buffer (2ml) and allowed
to stand at various temperatures for 48h,but no pectin was precipitated between 20and 40·e (Fig. Ib).
The pectin solution was mixed with thepH 3.0 buffer containing various concentrations of ethanol. The other experimental•YOKOTSUKA. ITO. NOZAKI, and KUSHIDA

62

1 2 3100~~I/'~0~

0-~'0CII~'u'0.

...G>

23~50~

CL.5
ti

3CIIa.

30

11 2:,~ 1 2 3K+

Na+

2 3

Ca++

Mg++

Vl~
Fe++

Cu+·

Fig. 2. Effects of various cations on the precipitation of the pectin.
The concentration of each cation was 5mM (1),10mM (2), and 15mM (3).

conditions were the same as describedabove. Although the amount of the precipitated pectin increased with an increase in
the ethanol concentration, only 5% of thetotal pectin added was precipitated evenwhen the concentration of ethanol was 15%(Fig. 1c). On the contrary, a large amountof pectin precipitate was produced by theaddition of low concentrations of some cations (Fig. 2) . The pectin solution (2ml)
was mixed with the pH 3.0 buffer containing
various concentrations of a cation salt suchas potassium tartrate, sodium tartrate, calcium tartrate, magnesium sulfate, copper
sulfate, or iron sulfate. The concentrationof each cation was from 5 mM to 15 mM.
The cations such as Cu;-r Ca;-r and Fe;-r
combined with the pectin and insoluble complexes occurred. However, the concentrations of these cations in the Koshu must orwine were usually below 0.1 mM. Therefore, the formation of precipitate due tobinding of the pectin to these cations seemed
improbable to occur. The concentration ofK+ in the wine was about 25mM. The addition of K+ at the concentration of 15 to50mM produced the precipitate corresponding to 9 to 11% of the original amount ofthe pectin.
The Koshu must (60 l) was dializedagainst running water for 24h. The dialyzate(69.5l) was centrifuged at 8,000 rpm for
30min. To the supernatant, DEAE-Sepha·dex A-50 gels (69.5g as dry weight) equilibrated with 1/100 M phosphate buffer (pH6.5) were added. The mixture was adjusted
to pH 6.5 with dilute NaOH and stirred at4°C for 24 h.
The mixture was filteredthrough a Toyo filter No.2 with a Biichner
funnel. The gels collected were washedwith 5l of the pH 6.5 phosphate buffer containing 2 M NaCl. The eluate was dialyzed
against deionized water overnight. To thedialyzate, DEAE-Sephadex A-50 gels (8gas dry weight) were added and stirred. The
mixture was packed into a glass column, andthe column was eluted with the pH 6.5 buffer
containing 2 M NaCl. A protein concentration was measured at 280nm. A portionof proteins was collected and dialyzed threetimes against a large amount of water. The
dialyzate was lyophilized. The yield of theKoshu grape pectins.

must protein was 630mg by weight. All
these procedures were carried out at 4·e.

The protein concentration of the lyophilisate was estimated from the sum of allamino acids found by amino acid analysis
after hydrolysis. Amino acid analysis wascarried out according to the method described previously.8) The amino acid compositionof the must protein was as follows: asparticacid, 15.7% : threonine, 11.4% : serine, 8.7% : glutamic acid, 8.2% ; proline, 5.6% ;glycine, 12.7% ; alanine, 9.9% ; valine, 4.9% ;isoleucine, 2.3%, leucine, 4.7% ; tyrosine,2.5% ; phenylalanine, 6.5% ; histidine, 1.6% ;lysine, 3.0% ; and arginine, 2.2% (mole %) .
The amino acid composition was similar tothat of the must protein obtained by precipitation with trichloroacetic acid.8) The pectin(0.5 g) was dissolved in 100mI of water.
The must protein was dissolved in 1/100 M
potassium tartrate buffer, pH 3.0, to givethe final concentrations of 20 J.l.g/mi to 100

15~c52040Mus

6080100protein (}-Ig)

Fig. 3. Effects of Koshu must protein on theprecipitation of the pectin.

763The protein solution (0.5ml) wasmixed with the mixture (3.5ml) of the pectin solution (0.8mt) and the pH 3.0 buffer(2.7 ml) . The sample solution was stored
at room temperature for 48h, and centrifuged at 40,OOOrpm for 30min. The amountof the pectin precipitate increased with anincrease in the amount of the protein (Fig.
3) .
Thus, the pectin was bound to the must
protein to produce the insoluble complexes,
but the phenols isolated from Koshu wineby the extraction method 9) and bovine serum
albumin (fraction V, Sigma) gave no effect on the precipitation of the pectin.
J.l.g/mi.

References

1) Bitter, T., Muir, H. M. : Anal. Bioehem., 4,330 (1962).
2) Hodge, J. E., Hofreiter, B. T. : Methods in
Carbohydrate Chemistry (Whistler, R. L.,
Wolfrom, M. L.), Vo!.l. 388, Academic Press,
New York and London (1962).
3) Singleton, V. L., Rossi, J. A., Jr. : Amer. J.
Enol. Vitie. 16, 144 (1965).
4) Reeeuil des Methodes internationales d'Analyse des Vins, 5th ed., Office International deVigne et du Vin, Paris, 1978.
5) Bailey, R. W., Bourne, E. J. : J. Chromatogr.,
4, 206 (1960).
6) Gee, M., Reeve, R. M., McCready, R. M. : J.
A gr. Food Chem., 7, 34 (1959).
7) Owens, H. S., McCready, R. M., Shepherd, A.
D., Schultz, E. L., Swenson, H. A., Miers, J.
C., Ehrlandsen, R. F., Maclay, W. D. : U. S.
Dept. A gr., Bur. A gr. Ind. Chem., AIC 340(1952).
8) Yokotsuka, K., Yoshii, M., Aihara, T., Kushida,T. : J. Fe1·ment. Tee/mol., 55, 510 (1977).
9) Yokotsu 'a, K., Takahashi Y, Yoshii, M., KushidaT. : J. Ferment. Technol., 56, 122 (1978).
(Received August 31, 1982)