Treatment of Koshu Must with Gelatin Hydrolysate.

47( J. Inst. Enol. Vitic. Yamanashi Univ. 17 47-54 1982 )Changes in General Constituents and Phenols
during Fermentation in Koshu Wine-Making
with Gelatin and Gelatin Hydrolysates *KOKI YOKOTSUKA, TOSHIHIDE MATSUDO,

and TADAE KUSHIDAThe Institute of Enology and Viticulture, Yamanashi University, Kofu 400.

Gelatin and gelatin hydrolysates of different molecular weights were added
to the must from Koshu grapes prior to the additon of wine yeast. The musts
or wines during fermentation were sampled several times over a month, on
which various physical and chemical analyses were performed. The addition of
the gelatin and gelatin hydrolysates affected the contents of various wine constituents, especially phenols, and the rate and extent of their changes. The
phenol-precipitation capacity of the gelatin hydrolysates of a molecular weight
more than 5,000 was similar to that of the gelatin of a molecular weight of

100,000.

Gelatin has been used as a fining agent
for long. It coagulates and precipitates
as a gelatin-tannin complex when added towine. This coagulation or precipitation isdue to a chemical combination of gelatin andtannin. The weight of tannin precipitatedvaries according to the sources and the isolation methods of gelatin!-7l Gelatins ofvarious kinds have been used up to the present and are commercially available now, but
no study of the use of hydrolysis productsof gelatins in wine-making has ever beenreported except for our previous study.8)
We showed, in the previous study, that theaddition of a gelatin hydrolysate of molecular weight of about 2,000 to Koshu must
reduced the phenol content of the resulting
wine appreciably while no change in thecontents of other wine constituents was ,observed.
• Chemical Studies on Coloring and Flavoring Substances
in Japanese Grapes and Wines (XVI)

In this paper, we used cattle-bone gelatin
and its hydrolysates of different molecularweights. They were added to the Koshu
must prior to the addition of wine yeast andchanges in various wine constituents during
fermentation were investigated.
Materials and Methods
Must and wine
Koshu grapes were obtained
from the Institute Vineyard in 1981. The must(about 2001) was obtained from the grapes witha Garolla crusher and a Vaslin-type press. Sulfur dioxide (30 g as potassium metabisulfite) was
added to the must and the must was allowed tosettle overnight. The sugar content of the must
was measured with a Atago refractometer and thereading was 14.5'. Sucrose was added to themust to give the reading of 24'.
To this, the culture solution (41) of wine yeast(Saccharomyces cerevisiae W-3) was added. Immediately after the addition of the yeast, gelatinor gelatin hydrolysate was added to the mixture
to give the final concentration of 500mg/l. The
gelatin and gelatin hydrolysates used were kindly

48

YOKOTSUKA, MATSUDO, and KUSHIDA

supplied by Nippi Inc. (Tokyo). The gelatin was
isolated from cattle bone by an alkali method.
The gelatin hydrolysates, PAlO and PA 100,
were obtained by acid hydrolysis of the above gelatin with dilute HCI, and the hydrolysates, PE 20
and PE 50, were prepared by enzymatic hydrolysis
with a protease, Miyapron (Teikoku Koso). According to Nippi, the number average molecularweights of the hydrolysates were about 1,000 for
PAlO, 2,000 for PE 20, 5,000 for PE 50, and10,000 for PA 100, and that of the gelatin was
about 100,000. Each of the hydrolysates or thegelatin was dissolved in water to give a concentration of 10% just before the addition to themust.
General analyses and determ ination of con·tents of total phenol and gelatin or gelatinhydrolysates
General analyses of the musts
and wines were carried out according to the methods in "Kokuzeicho Shoteibunsekihochukai "9) and"Methods for Analysis of Musts and Wines" .10)
All the samples were filtered through a membranefilter (Gelman, alpha-450) before analysis.
Total phenol content was determined by themethod of Singleton and RossLll)
The contents of the gelatin or gelatin hydrolysates left in soluble form in the must or wine
were determined as follows. An approriate volume of the must or wine made with the gelatin oreither of the gelatin hydrolysates was poured into
a test tube (14mm X 15cm) and evaporated to dryness. Two ml of 6 N HC] was added to the tube.
The tube was evacuated, sealed, and kept at 1l0·C
for 24h. After hydrolysis, analyses of amino acidcomposition were performed with an amino acidanalyzer (Kyowa Seimitsu, Tokyo). The contentsof all the amino acids found were summed up. At
the same time, the wine made without them was~ampled and the amino acid analysis was done as
described above. The content of the gelatin or
gelatin hydrolysates in the must or wine was estimated from the difference between the values
for the two samples.

Results and Discussion
Amounts of gelatin or gelatin hydroIysates to be added
In fining a white

wine, tannin is added first, followed bygelatin in order to prevent persistent cloudiness and overfining. Less than 100mg eachof tannin and gelatin are commonly used per
It of wine. In this experiment, however,
the gelatin or either of the gelatin hydrolysates of different molecular weights wasadded to the Koshu must in the high concentration (500mg/ l) prior to the addition of
the yeast while no tannin was used, since
we expected that the addition of larger
amounts of the gelatin or gelatin hydrolysates would present further distinctions of
the compositions among the wines made.
Specific gravity and extract
A considerable difference in specific gravity was
found among the wines sampled from day 5
to day 13 after the addition of the yeast.
During this period, the spec ific gravity of
the wines decreased with an increase in themolecular weights of the gelatin hydrolysates (or gelatin) added (F ig. 1).
The extract contents of all the wines, on
day 33, were about 2%. From day 5 to·day
33, the extract content of the wine madewithout the gelatin hydrolysates (or gelatin)
was larger than that of the wine with them.
As observed in the specific gravity, the extract' contents of the wines decreased as
the molecular weights of the gelatin hydrolysates (or gelatin) increased (F ig. 2).
Reducing sugar and ethanol
The ad·dition of the gelatin or gelatin hydrolysatesgave the significant effect on the fermentation rates of the musts. The fermentation
rate is indicated by the rates of the production of ethanol and the reduction of sugar.
In Figs. 3 and 4, the musts treated withthe gelatin or gelatin hydrolysates fermented
more rapidly than the must without them.
Among the wines sampled on day 5 and day
13, the content of the reduc ing sugar decreased and that of ethanol increased as
the molecular weights of the gelatin hydrolysates (or gelatin) increased.

Treatment of Koshu Must with Gelatin Hydrolysate.

49

1.100
o Control• Gelatint>. Gelatin hydrolysateo Gelatin hydr<llysatej.
Gelatin hydrolysate• Gelatin hydrolysate

PA 100
PE 50
PE 20
PA 10

....>.

'>

:!!

01
.~

:t::
u

~

1.000

(/)

0.980

o

5

10

20

15

25

30

35

Days after addition of wine yeast
Fig. 1.

Changes in specific gravity during fermentation.

ControlGelatint>. Gelatin hydrolysate PA 100
o Gelatin hydrolysate PE 50• Gelatin hydrolysate PE20
• Gelatin hydrolysate PA 10
o

•

20

E

--....
0
0

~

01

u

10

....:!!
)(

l.LJ

o

o

5

10

15

20

25

Days after addition of wine yeast
Fig. 2.

Changes in extract during fermentation.

30

35

YOKOTSUKA, MATSUDO, and KUSHIDA

50

o Control• Gelatin
6 Gelatin h¥drolysate
D Gelatin hydrolysate.. Gelatin hydrolysate• Gelatin hydrolysate--2

PA 100
PE 50
PE 20
PA 10

t

CI
:J
Ul

Cl

C

'u
:J
"C
QI

a::

5

10

15

20

25

30

35

Days after addition of wine yeast
Fig. 3.

Changes in reducing sugar during fermentation.

15

>

>-

o Control• Gelatin
6 Gelatin hydrolysate PA100o Gelatin hydrolysate PE50
to Gelatin hydrolysate PE20
• <klatin hydrolysate PAlO

5

5

10

15

20

25

30

35

Days after addition of wine yeast
Fig. 4.

Changes in ethanol during fermentation.

pH, total acid, and volatile acids
The maximum content of total acid and thelowest pH were obtained in the analyses of
the wines sampled on either day 2 or day 3(Figs. 5 and 6). The volatile acid contentsof all the wines had increased for 18 days
after the addition of the yeast (Fig. 7). No

significant differences in pH, total acid, andvolatile acids were found among the musts
and wines treated with either of various
gelatin hydrolysates or gelatin.
Total phenol
Figure 8 shows changes
in the total phenols of the various wines
during fermentation. The total phenol con·

Treatment of Koshu Must with Gelatin Hydrolysate.

51

...
2.0

:I:

o Control• GelatinlJ. Gelatin hydrolysateo Gelatin hydrolysate• Gelatin hydrolysate• Gelatin hydrolysate

Q.

1.0

PA 100
PE 50
PE 20
PA 10

OL-_-+---~---+---~----+----+------....-J

o

5

10

15

20

25

30

35

Days after addition of wine yeast
Fig. 5.

Changes in pH during fermentation.

o Control• GelatinlJ. Gelatin hydrolysate PA 100
o Gelatin hydrolysate PE 50• Gelatin hydrolysate PE 20• Gelatin hydrolysate PA 10

0.8

,-..

E 0.7
Q
0

01

'-"

"0

0.6

u

III

"iii
.....

~
0.5
0
0

5

10

15

20

25

30

35

Days after addition of wine yeast
Fig. 6.

Changes in total acid during fermentation.

tents of all the wines decreased abruptly
for 5 days after the addition of the yeast,and then decreased gradually from day 8 today 33. Most of the phenols lost seem tobe tannins because the monomeric and dimeric phenol fractions from grape seeds
were precipitated neither with the gelatin
nor gelatin hydrolysates, but the tannin frac-ztion was precipitated with either of them. 12)
The total phenol contents. of the wines madewith the gelatin or the gelatin hydrolysate
were smaller than that of the wine madewithout them when those of the wines sampled on the same day were compared. The
molecular weights of the gelatin hydrolysates (or gelatin) added showed significant

J¥OKOTSUKA, MATsuDa, and KUSHIDA

52

0,05

E 0,04

o
o

-rn

0,03

I II
"C

ControlGelatin6 Gelatin hydrolysate PA 100
o Gelatin hydrolysate PE 50• Gelatin hydrolysate PE 20• Gelatin hydrolySate PA 10o'uIII 0.02

~

.!!!
0

•

0.01

>

o

5

10

15

20

25

30

35

Days after addition of wine yeast
Fig. 7.

Changes in volatile acids during fermentation.

influence on the total phenol .contents.
Among the wines sampled on day 33, theloss of total phenol tended to increase as
the molecular weights of them increased.
Gelatin and gelatin hydrolysates
From 65% to 80% of the gelatin or gelatinhydrolysates added were precipitated for 2days after the addition of the yeast. It wasobserved on day 33 that 85% to 95% of themhad been precipitated. The amounts of thegelatin or gelatin hydrolysates left in eachwine hardly changed from day 8 to day 33
after the addition of the yeast (Fig. 9).
The amount of the total phenol in each wine
sampled on day 18 was similar to that on
day 33 as shown in Fig. 8. From theseControlGeratin
Geratin hydrolysate
Geratin hydrolysate
Geratin hydrolysate
Geratin hydrolysate

--

PA100
PE 50
PE 20
PA 10

01

E

100

o

' - - - - - < - - - - + - - - - _ + _ - - - - + - - - - - - l - - - - - - - ' - -_ _---'

o

5

10

15

20

25

Days after addition of wine yeast
Fig. 8.

Changes in total phenol during fermentation.

30

35

Treatment of Koshu Must with Gelatin Hydrolysate.

-

53

~

~ 500-..4-o Control• Gelatin

Q.I

A Gelatin
Cl Gelatinhydrolysate PA100
hydrolysate PE50.. Gelatin hydrolysate PE20
• Gelatin hydrolysate PA 10

1a
400
~
0

...

"U

>.J:.

300

c

:0::

III
~

OJ 200

...

\~

0

C

~

100

:;:::;

(!)

...M

~

III
~

0

0

5

--

~

•

'"
10

15

20

25

30

35

Days after addition of wine yeast
Fig. 9.

Changes in gelatin and gelatin hydrolysates added
during fermentation.

Table 1.

Loss of phenols by gelatin or gelatin hydrolysate.

Weight of gelatinor glatinhydrolysate
precipitated

Weight ofphenols lostfrom wine

Weight of phenols
lost by gelatin or
gelatin hydrolysate

(A) *

(B) *

[C = B- Control (187) ]o **187 ***

0

Control

Ratio
(C/A)

Gelatin

468 mg/l' wine

273

86

0.18

Gelatin hydrolysate
PA 100
PE 50
PE 20
PA 10

439
439
412
408

277
270
240
230

90
83
53
43

0.21
0.19
0.13
0.11

* These values were obtained by analyses of the wines sampled at 18 days
after the addition of the wine yeast.

** Each ge-1atin hydrolysate or gelatin was dissolved in the Koshu must to
give the final concentration of 500mg/l.

*** The total phenol content of the original mmit was 415mg/l (gallic acidequi valent) .

___~__-_------:....lJ

54

YOKOTSUKA. MATSUDO. and KUSHIDA

results, the ratio of the weight of the total
phenol lost to the weight of the gelatin or
gelatin hydrolysate precipitated was calculated from the data on the wines sampled
on day 18 in Figs. 8 and 9. The phenolprecipitation capacity of the gelatin hydrolysates PA 100 and PE 50 was very similar
to that of the gelatin as indicated by theratios in Table 1. However, the capacityof the gelatin hydrolysates PE 20 and PA
10 was smaller than that of the gelatin(about 1/2). Thus the loss of phenols was
not proportional to the molecular weights ofthe gelatin hydrolysates (or gelatin) added.
The major conceivable advantages of theuse of the gelatin hydrolysates which wereconfirmea in this experiment are removalof excessive tannins from must, rapid andhomogeneous solubilization of the hydrolysates, and no necessity of the use of tannins
for fining white wine.
Acknowledgements

References

1) Trunkel, H. : Biochem. Z., 26, 458 (1910).
2) Hartong, B. D. : Woch. Brauerei, 46, 11(1929).
3) Rudiger, M., Mayr, E. : Kolloid-Z., 47, 141(1929).
4) Page, R. O. : J Intern. Soc. Leather Trades
Chmists, 26, 71 (1942).
5) Kain, W. : FrUchteverwertung, 17, 10 (1967).
6) Kain, W. : FrUchteverwertung, 17, 109 (1967).
7) Kain, W. : Fruchteverwertung, 17, 201 (1967).
8) Yokotsuka, K., Matsudo, T., Kushida, T. : J.
Inst. Enol. Vitic. Yamanashi Univ., 16, 21(1981).
9) Kokuzeicho Shoteibunsekihochiikai, 3rd Ed.
(1974).
10) Amerine, M. A., Ough, C. S. : Methods forAnalysis of Musts and Wines, John Wiley& Sons, Inc., New York, Chichester, Bris·bane, and Toronto (1980).
ll) Singleton, V. L., Rossi, J. A., Jr. : Amer.
J. Enol. Vitic. 16, 144 (1965).
12) Yokotsuka, K., Singleton, V. L.: unpublished.
(Received August 31, 1982)

We thank Mr. S. Fujihara and Mr. H. Ushikawa
for their technical assistance.

s