27

Antibacterial Substance from Grapes and Wines(J. lnsti. Enol. Viticul. Yamanashi Univ. 16

27-33

1981)

Fractionation and Properties of Antibacterial
Substance from Grapes and Wines*
KOKI YOKOTSUKA, TOSHIHIDE MATSUDO, and TADAE KUSHIDAThe Institute of Enology and Viticulture, Yamanashi University, Kofu 400
An antibacterial substance was obtained from various grape juices and wines by
Dowex 50-X2 chromatography followed by treatmen't with charcoal. The substance had
distinct antibacterial activity against many bacteria, but the degre~ of the ant~bac­IIbterial activity varied with the strains of bacteria.

The activity was considerably

affected by other wine components, particularly organic acids.

However, heating of

the substance at various pH resulted in no loss of the antibacterial activity.

It

was found that the antibacterial substance was not, in fact, the materials such as
alcohols, phenol compounds, S02' organic acids, amino acids, peptides, and metal salts.

The antibacterial properties of grape juices

ent broth-agar medium seeded with the bacteria

and wines have commonly been known for long.1 ,2)tested were poured into Petri-dishes, and small

The properties seem to have been attributed

filter paper discs

to phenolic compounds.3 ,4) Few studies, however,

0,7 mm in thickness) soaked in samples

have been reported on the antibacterial activity

placed on the surface of the agar.

of wine components other thancom-

was carried out at 30°C for 18 hr. The diameters

pounds. This report describes the isolation andof clear inhibitory zones occurred around thefractionationofdiscs were measured.

non-phenolic

substance

phenolic

antibacterial

activity

obtainedofa(Toyo, 8 mm indiameter,were

Incubationfrom grape

juices and wines, and its properties.

Results and Discussion
Grape juice or wine (2 £) was diluted

Materials and Methods
Grape juices and wines

twicewith water. The sample was applied to a column

The grape varieties

of Dowex 50-X2 (H+type, 2.2 X 52,,6 cm) and theused were Koshu, Delaware, Neomuscat, Muscat

column was washed with water.

Bailey A, and Cabernet Sauvignon, grown in thefraction was eluted with 2 £ of 10 % pyridine-

Institute Vineyard in 1975-1980.

acetic acid buffer

Three whitewines and two red wines were made fromabove grapes in the usual

theway at the Insti-

tute Experimental Winery,(pH 5,6).

The

adsorbed

The eluate wasdried in vacuo and the residue obtained
dissolved in water,

was

The antibacterial activity

in the sample solution was inve,stigated by the

Culture medium for growth of bacteria

Glu-

paper

disc

methodusing

B. subtills,

E.

coli,

cose nutrient broth (pH 6.6) was used as growthP. aeruginosa, and S. aureus. All of the sample

medium for bacteria. The bacteria used in thissolutions from the grape juices and wines used

experiment were listed in Table 4.

showed clear inhibitory zones against

Assay of antibacterial activity

Glucose nutri-

the bacteria.

all

of

However, it seems plausible to* Chemical Studies on Coloring and Flavoring Substances in Japanese Grapes and Wines (XIII).

YOKOTSUKA· MATSUDO and KUSHIDA

28

confirm that the inhibitory zones occurred bysubstances such as pyridine or acetic acid used

and the column. was successively washed withas eluting solution, pyridinium acetate, or theacids and peptides.

eluate from Dowex 50 resin,which werewater,

notCharcoal (50 f.lg to 350 flg) was added tom~potentially present in grape or wine. To confirm
the above statement, a model wine

(pH 3.0),

4

the aqueous solution including the an·tibacterial

activity,

which was obtained fromof

80 m~ of juice or wine, to remove

was applied to the columnofcompounds. The mixture was vigorously stirred

phenolic

Dowex 50-X2 instead of the juice or wine. The

and filtered through a Toyo No. 5c filter paper.

eluate from the column with 10 % pyridine-

The charcoal on the filter was washed with 4a"cetic acid buffer(pH 5.6)

was obtained

the same procedure as stated above,
evaporated to dryness.

m~byand wasAntibacterial activityof water and the two filtrates were combined.

Absorbance at

400 nm,phenolantibacterial

activity ofthecontent,
filtrateandwereof the residue was examined, but no activity

examined. The absorbance became about 0 by theThis shows that the antibacte·

addition of 300 mg of charcoal, and no phenol

was obtained.

rial activity was due to grape juice or wine

content was observed. However, no antibacterial

components.

activity was lost.

When 4 N NH 4 0H, 0.1 N NaOH, 0.2 M Ba(OH) 2' and mixtures of these solutions andorganic solvents were used as eluting

The filtrate was lyophilized

and this was used as crude antibacterialsomestance. The yield of

solution

the substance was 135

Table 1 shows the antibacterial

activity

5.6), the residues of the eluates did not show any

the substance against the four bacteria.

activity.

substance had higher activity against

The concentrates of the6.6) also showed no activity.
ings,

the antibacterial

wines (pH

From these find-activitywaselutedB.

subtilis,P.

andagainst S. aureus.
stance obtained

wine was applied to a column of Dowex 50-X2

than that from the wines.

from theactivityofThe

E. co Ii,

aeruginosa than it
Thewith the pH 5.6 buffer after grape juice or

Table

sub-

mg from 1 ~ of Koshu wine.

instead of 10 % pyridine-acetic acid buffer (pHdidof the sub-juices washigher1.
Antibacterial activities of the crude substances obtained from various
grape juices and wines.

Grape juices
or wines

Diameters of inhibitory zones against
B. subtilis
E. coliP. aeruginosa

Grape juices
Koshu
Neomuscat
Muscat Bailey A
Wines
Koshu
Neomuscat
Muscat Bailey A
DelawareCabernet Sauvignon

7ofand 2 %

consisting of 14 % of ethanol
tartaric acid,

4N NH 4 0H, and water to remove aminoS.

~ureus

(mm)

23.3
20.1
22.7

24.7
21. 7
26.0

21.3
18.5
20.8

20.1
10.4
11.1

14.7
17.5
11.5
10.5
15.2

18.7
24.7
17.7
18.0
21.0

15.8
18.2
14.2
14.2
17.3

16.4
10.5
10.4
10.4
10.3

t

29

Antibacterial Substance from Grapes and Wines
The crude antibacterial substance (270 mg)

from No. 7 to No. 11, and the fractions of No.

from 2 £ of Koshu wine was dissolved in water.

25 and No. 26.

The sample solution was applied to a column ofgainstDowex 50-X2 (H+type, 2.2 X 52.6 em) and theagainst the other three bacteria.

column was eluted with 2 R, of 10 % pyridine-

No. 5

acetica flow rate

11 (F 2), and the fractions of No. 25 and No.

of 60 m.e!hr. Fractions of 50 mt were collected.

26 (F 3) were collected, concentrated by rotary

The substance contained a large amount of Na+

evaporation,

and K,+ and a small amount of Zn.+ + The concen-

F 3 were rechromatographed on Dowex 50-X2. The

trations

columnacidofbuffer (pH 5.6) atthese

cations

weredetermined

The degree of the activity a·P. aeruginosa was smaller than those(F 1),

The fraction

the fractions from No. 7 to No.

and lyophilized.

sizes

and

fraction

F I,

F 2,

volumesandcollected

were 1.8 X 7.9 em and 5 m£ for F I, 2.2 X 44.7.,-,

em and 40 mt for F 2, and 1.5 X 5.7 em and 2.5

K+10,000

,

.

,
,

I
I

Na+,'

~

•
I

m£ for F 3. Each sample was dissolved in water

I
I

, ,

,

..

~

z: e

5,000

o ""
Po

. "

with

% pyridine-acetic acid buffer (pH

1. 6

3.1).

Stepwise elution was carried out

the pH 3.1 buffer and 10

F2

... N
0

cation, and applied to ea:ch column equilibrated

\

C

g +
:.:; :><:

I,:3" ++'o" ...

to give a final concentration of 0.1 M of eachwith% pyridine-acetic

20
a) Fl
F3

-

I -H- II - H - -

m~

Ul

'""o

F2

20

2,000

F3

N

.I

e

20

""""
+

F3

b) F2
N

~+-

" 2,000

~

...

0

F3

20

'"
is'"

E

10

+-

,/

:><:

c) F3

.

20

30 +

Fraction numbers (50 m£)

~

"

,/

b

d) F2

"'"

o

spectrophotometer.

<.)

tivity.

Figure 1 shows the elution
andantibactrialac-

Na+ was eluted in the fractions

from

No. 5 to No. 11, K+ in the fractions from No.
8 to No. 17, and Zn ++ in the fractions from No.
23 to No. 28. The antibacterial activity in eachh-action was examined. The activity

was

~~

VDI -H- IX -lob-served in the fraction of No.5, the fractions

2,000

.~
co

with a Hitachi, Model 170-30, atomic absorption
the cations

vn

Z

Fig. 1. Fractionation of the crude antibacterial
substance on Dowex 50- X2 chromatography.

patterns of

V ~~Vl ~

it

N

...

buffer

l

~Na+

+

Ul

pH 5.6bufferpH 3.1~Zn ++"""x ~+XI"

"o

K+
2,000
pH 5.6buffer

,/

o

o

10

40
30
20
Fraction numbers

J

50

Fig. 2. Fractionation of the antibacterial fractions on Dowex 50-X2 (a-c) and Dowex50-X8(d) chromatographies.

YOKOTSUKA· MATSUDO and KUSHIDA

30

acid buffer (pH 5.5). Fractions indicatedI to IX in Fig.

2 were collectedbymeeluting buffer. Fractions of 10

separately

lected.

InFig.

2-(d),

thewere col-antibacterial' ac·

and antibacterial activity of thefractions wastivity (fraction X)

investigated.

observed infrom each other on this chromatography.

The activity

wasthe fractions I, V, and IX (Fig. 2-(a)-(c)).

Thus,

the

and K+ could be separated

crude

antibacterial

substance

least

thtee different

activities,

The fraction F 2 was also rechromatograped oncontained at

Dowex 50-X8. The column size and the flow rate

and two of these were metal

were 2.2 X 26.3 cm and 60 m.e/hr, respectively,

2 shows the activities of the fractions obtain-

with

10

% pyridine-acetic acid (pH 5.6) as

ion-free. Tableed.

Table 2.
Antibacterial activities of the fractions obtained by
Dowex 50-X2 and Dowex 50-X8 chromatographies.
Fractions

*
B.

I
II
III
N
V

Diameters of inhibitory zones against
subtilis
E. coliP. aeruginosa
(mm)
16

11
0

0

0
0
14
0
0
0
20
14

VI
VII
Vill
IX
X
XI

12

13

o
o
o

o
o
o

14

25

o
o
o

o
o
o

10

11

0

0
10
0
0
0
13

S. aureus0* See Fig. 2.
Figure 3 represents the relationship between

through a column of Dowex 50-X2 (H+ type, 3X28

the amounts of the crude. antibacterial substance

em),

and

the diametersagainstthefour

The non-adsorbed fractionwaspassedinhibitoryzones

through a column of Amberlite IR-4B (OH-type,bacteria graphically.

The

3 X 28 cm). The fraction not adsorbed on this

of

the

range of linearlity was between at least50

Ilg and 400 Ilg of the antibacteri&l substance.

column was designated as a neutral

fraction(1.5 .e). The adsorbed fraction was eluted with

The relative activities in Tables 3 and 4 were

1 .e of 2 N NH 4 0H. The elua,te was evaporated toobtained using these standard curves.

dryness at 40·C, and to the residue, 500 m.e

As stated earlier, the Koshu wine concentratewater was added.

The solution

was

of

passeddid not show antibacterial activity. We assumed

through a column of Amberlite IR-120 (H+ type,that the antibactrial substance was

3 X 28 cm) to remove NH a and the column wassome components in the wine,

bound

to

and the complexes

formed did not have any activity.

To' elucidate

washed with water.

The non-adsorbed fraction(1.5 .e) was obtained and was designated as an

this assumption, the following experiments wereacidic fraction.

The crude antibacterial

frac-carried out.

tion

was dissolved in 300 m.eofKoshu wine (I.e) was

passed(13.5 mg)«

31

Antibacterial Substance from Grapes and Wines
the

B.
E.
P. aeruginosa

.,"'

"o
N

neutralfractionortheacidicfraction.

Each fraction was adjusted to pH 3.0 and wasallowed to stand for 1 hr at room temperature.
After concentration todryness,

1.25

water was added to the residue.

meof

The solution

was adjusted to pH 6.6 and was subjected toassay of the antibacterial activity.
In Table

3, the activity of the crude anti·....obacterial substance decreased considerably or"'.... 10completely by the addition ofsi5"tion or the acidic fraction. The neutral fraction

2.,

contained
O'----o----+----+--~---o-o_ _____.J

25
50
100 200 400
Concentration of crude
antibacterial substance (mg/me)

Fig. 3. Standard curves of the crude antibacterial substance for four bacteria.

glucose,andthe neutral frac-theacidicoforganic acids and glucose on the activity

were examined.
erablyThe activity decreased consid-

by the addition of some organic acids,

but increased slightly by glucose. The additionof metal salts gave no effect.

Table 3
Effects of wine components on antibacterial activity of the crude
substance.
Wine components
added

B.

None
Neutral fraction I)
(a)

(b)
(c)
(d)
(e)

Acidic fraction 1) (a)-(elAcetic acid (0.16g) 3)
Lactic acid (0.02g)
Malic acid (l.46g)
Tartaric acid (0.67g)
Citric acid (0.02g)
Glucose (0.18g)(0.35g)(0.53g)(1. 75 g)
NaCI (LOg)KCl(1.3g)
1)

2)

subtilis

fractioncontained organic acids. Therefore, the effects

Relative activity 2)
E. coliP. aeruginosa

S. aureus

100

100

100

100

100
86
86
73
86
0
60
30
21
21
17

36
36
36
42
42
0
82
18
18
18
30

68
55
61
55
68
0

108
92
100
92
85
0
59
72
53
53

77

27
52
52
25
113
106
114
114
105
100

72

The crude antibacterial substance (l3.5mg) from Koshu wine was added to300me of the neutral fraction or the acidic fraction from Koshu wine, and the
mixture was adjusted to pH 2.0 (a), pH 4.0 (bl, pH 6.0 (c), pH 8.0 (d), or
pH 10.0 (el. The mixture was allowed to stand for I hr at room temperature,then adjusted to pH 6.6.
Relative activity was obtained by use of the standard curves in Fig. 3.

32

YOKOTSUKA· MATSUDO and KUSffiDA
3)

The values in Parentheses are the amounts of the organic acids added. The
composition of organic acids in Koshu wine was analyzed by the methods ofYamashita et aJ. 5,6) Each organic acid was added to the aqueous solution of
the crude antibacterial substance on the basis of the contents of organic acids
which had been determined above.

Table 4.
Minimum inhibitory concentration of crude antibacterial substanceagainst various bacteria.
Minimum
inhibitory
concentration(mg/mt)Bacteria tested
Ba c illus brevis JAM 1031
Bllc illus cereus JAM 1029

Bacillus cereus JAM1190
var. mycoides
Bacillus circulans JAMl.140
Ba cillus firmus JAM 1188
Bacillus licheniformis JAM 11054
Bacillus macerans JAM1243
Bacillus megaterium JAM1166
Bacillus polymyxa JAM 1210
Bacillus sphaericus JAM 1244
Bacillus subtilis JAM 1064
Bacterium ammoniagenes JAMl641

1)
2)

Table

1.81)
3.2
1.7
3.2(1.7) z)
3.2(p)
3.2
0.8(0.2)
1.7
1.7
1. 7(0.8)
3.2
1.7(0.8)

Bacteria tested

Es cherichia coli JAM 1239
1. 7(0.8)
Eschericia coli (M) Cost &1. 7(0.8)
Chalm NJHJ JC-l
Proteus mirabilis OM-1
1. 7(0.8)
Pseudomonas aeruginosa JAMI095 0.8
Pseudomonas decunhae JAM 1048
0.8
Pseudomonas diminuta JAM 1513
0.8(0.3)
0.8
Ps eudomona s ova lis JAM 1002
Pseudomonas putrefaciens AJ2065 0.3
Pseudomonas putrefaciens AJ2261 0.8
Pseudomonas rubescens NRRL1551 0.5(0.3)
Pseudomonas taetrolens JAM 1653 0.8(0.5)
3.2(0.8)
Staphylococcus aureus JAM 1011

Bacteriocidal action
Bacteriostatic action

4 shows the minimum inhibitory con·dissolved in water at the concentration of 1.35 g/t

centration of the crude antibacterial substanceand adjusted to pH 2.0-10.0 with dilute

HCI

against various bacteria. The substance showedor NaOH. The samples were allowed to

standbacteriocidal action against all of the bacteria

in boiling water for 1 hr,cooled in runningtested above the concentration of 3.2 mg/mt,

water, adjusted to pH 6.6, and then subjected

and bacteriostatic action against B. maceransat the concentration of 0.2 mg/mt and against

to assay of

P. diminuta and P.

above treatment. However, most of the activity

rubescens attration of 0.3 mg/IDt.
bacteriocidal

actionHere,
asthe concen-

we defined thebeing in a stateofno bacterial growth for one week after inoculation.

The bacteriostatic action was defined

activity

the antibacterial

never

was lost

afterchangedactivity.

The

before and after

the aqueous solution of

the
the

substance was stored at 4'C for several months.
From these results,

the antibacterialsub·stance was not the materials such as alcohols,

as being in a state of no growth for one day

phenol compounds, SOz' organic acids, and metal

after inoculation and good growth after that.

salts, which have been known as antibacterial

The effects of pH and temperature on
ac ti v i ty

andstability

substance were examined.

I

Minimum
inhibitory
concentration(mg/mt)

of

the

the

antibacterial

The substance wassubstance.

The substance present ingrapesand wines was the inactive form which was boundto some grape or wine components.

33

Antibacterial Substance from G rapes and Wines
Research, Suppl. 1, Academic Press,

Acknowledgement

We thank Mr. T. Yajima, Mr. M. Nakamura, Miss
Y. Kanai, Miss A. Higuchi, and Miss S. Uesugi
for their technicalassistance,and MissAmino for reading an earlier draft

of

Y.
this

3) Powers, J. J., Somaatmadja, D., Pratt, D. E.,
Hamdv, M. K. : Food Technol., 14, 626 (1960)
4) Pratt,' D. E., Powers, J. J.,

Somaatmadja,

D. : Food Research, 25, 26 (1960)

manuscript.

5) Yamashita,
References

1) Masquelier, J.: The Pharmacology of Plant

2)

New

York (1969)

I., Tamura, T., Yoshikawa,S.,

Suzuki, S.: Japan Analyst, 22, 1334 (1973)
6) Yamashita,

1.,

Tamura, T., Yoshikawa, S.,

Phenolics, ed. by J. W. Fairbairn, Academic

Shimamoto, T., Matsumoto, A.: Nippon Nog·

Press, New York, p 123 (1959)

eikagaku Kaishi, 48, 151 (1974)

Sinl(leton, V. L., Esau, P. : Advances in Food