Utilization of Gelatin Hydrolysate in White Wine-Making(J. Insti. Enol. Viticul. Yamanashi Univ.

1621-25211981)

Utilization of Gelatin Hydrolysate InWhite Wine-Making*
KOKI YOKOTSUKA, TOSHIHIDE MATSUDO. and TADAE KUSHIDAThe Institute of Enology and Viticulture, Yamanashi University, Kofu 400
Koshu white wine was made from the musts treated with a gelatin hydrolysate prior
to the addition of yeast.

Analyses and sensory evaluation showed that the gelatinhydrolysate was a possible material for sulfur dioxide to prevent browning, otheroxidative reactions, and formation of cloudiness. The phenol content in that wine,
particularly the tannin content, was smaller than that in the wine, made from themusts treated with sulfur dioxide. The peptide content, however, was similar to thatin the wine made from the musts treated with sulfur dioxide.

There was no appreciable

difference in their chemical properties other than the phenol content between thewine made from the musts treated with the gelatin hydrolysate and that made from themust treated withsulfur dioxide.

Sulfur dioxide has been used in must and wine

colloidal

cloud caused by excessive tannin inThewine.

Recently,powers are known as to decrease its combination

used

for fining ofwith aldehydes,

poor solubility in water, and the presence

for its antiseptic and antioxidant powers.

sugars, or other constituents

dioxideare not active

gelatinwhite wine,ishardlysince it hasofofcontaminants in it may prod1,1ce off-flavor or

as is the free

off-odor in wine, and overfining and the sec·in must and wine since the bound forms
sulfurhowever,form. The odor of free sulfur dioxide, however,ondaryis pungent and it has a negative quality,henceby addition of gelatin.

formation of cloudiness may be caused

We treated Koshu must with a gelatin hydro-

many researchers have searched for a substitutecontrolling undesirable

prior to the addition of wine yeast
order
to remove excessive tannin, and toin
search for a possible substitute for sulfur

microorganisms and does not bind acetoaldehyde

dioxide, or to use lower level of dose of sulfur

asdioxide.

for sulfur
isdioxide.

In case that ascorbic acidused as one of thevery

littlesulfur

effectdioxideonantioxidants,

does.

ithas

Furthermore, it haslysate

The results of this experiment show

been well known that a butter-like and age·that a gelatin hydrolysate may be a substituteranium-likefor sulfur dioxide, therefore,

off-odors occured inwine

withexcessive use of ascorbic acid.

Therefore, atpresent there is no desirable

substitute forthis work gives

a new method for wine-making with the additionofthe gelatin hydrolysate to musts prior tofermentation.

sulfur dioxide.
Gelatin has been commonly used as a fining
agent

forwine, especially forredwine tostabilize and clarify a wine with an unstable

Materials and Methods
Must and wine

Koshu grapes were obtained* Chemical Studies on Coloring and Flavoring Substances in Japanese Grapes and Wines(XII).

YOKOTSUKA· MATSUDO and KUSHIDA

22

from the Institute Vineyard in 1979. The Koshu(Corona UT-l1)

must was obtained with a Garolla crusher andAnalyses of phenolic compounds were carried

About 65 ta Vaslin-type press.

was obtained from 100 kg ofgelatinhydrolysateorsulfurof the must

the grapes.

Adioxide(asand wasexpressed

as unit.

out as follows. The wine samples were adjusted
to pH 2.0 with dilute hydrochloric acid.

To5 mt of the wine in a test tube with a glass

potassium metabisulfite) was added to the must

stopperin various concentrations.

saturated with distilled water and the mixture

Then, wine yeast

was added an equalvolume of ether(Saccharomyces cerevisiae OC-2) was immediately·was shaken, then allowed to stand.

added to the musts treated with the above twophase was removed and the lower phase wasmaterials.

extracted twice more in the same way.

Racking was done twice at a monthand six months after the addition of theyeast,

then the wines were bottled and stored at 15'C
for one year.

General analyses of the wines

were carried out according to the methods inphase

The gelatin hydrolysate
supplied by(PE 20)

NippiInc.

usedwasremaining

aftertheTheextraction

with ether was extracted three times with anequalvolume of butanol-2 saturated

with di·stilled water as described above. The combinedupper"Jozo Bunsekiho:'l)kindlylower

The upper

phases

werevolume of benzene.

extracted

withanequal

The aqueous phase was(Tokyo).

dried by rotary evaporation in vacuobyat 30'C.

enzymatic

To the residue was added 1 mt of 0.1 N HC1,hydrolysis with a protease, Miyapron (Teikokuand the mixture was heated in boiling waterKoso, Tokyo).

for 20 min.

Thehydrolysate

Aminoacids,wasobtained

peptides,and nonvolatile acids

cted

The resulting solution was extra·

three times with 1

meof ether.

TheThe wines were centrifuged at 40,000 rpm for

combined ether extracts and the aqueous layer

30 min at 15T. The supernatant waswere dried.

rated

under

reduced pressurelyophilized.
boththeatconcent40'C

andThe con.tents of amino acids inlyophilizedmaterials and their acid

Each residue was dissolved in 5

mt of water and their phenolic contents weredetermined
method?}bythe

modified

Folin-Ciocalteu

The ether-extractable phenolics werehydrolysates (hydrolyzed in 6 N He1 for 24 hr

non-tannin phenolics and most of the phenolics

at 110'C) were determined with a JEOL

in the aqueous phase were tannin.3)

Model

JLC-6AH amino acid analyzer. The amino acids
constituting

thetotal

peptides·wereestimatedfrom the differences between the values

thustreateddetermined.
Nonvolatileacidswereanalyzed by

highperformance liquid chromatography. The samples
wereappliedtoacolumn (0.8 X 50 cm)

with apH 1.9of

withsulfurdioxide(150theanda combined dose of sulfur dioxide (75 ppm) andthe gelatin hydrolysate (75 ppm).

The number

pre-treatment,

aqueous solution

about 2,000 according to the personal communica-(adjusted with perchloric acid).

The column

was eluted with the same solution ataflow

tion with Nippi. The hydrolysate was able to beeasily dissolved in water without heating andwas colorless. Various Koshu wines made fromrate of 1.0 mt/min.
Browning,ppm),

gelatin hydrolysate (l00 ppm to 750 ppm),

average molecular weight of the hydrolysate was

Shimadzu gel SCR 101 without
equilibrated

Results and Discussion
Koshu white wines were made from the musts

turbidity,andconcentration

ofthemusts

treatedwith

theabovematerials

browning was

were not much different from one another inmeasured by optical density at 400 nm. Degree

specific gravity, and contents of extract andof turbidity was determined with a turbidmeter

ethanol (Table 1).

phenolic compounds

Degreeof1

Utilization of Gelatin Hydrolysate in White Wine-Making
Table 1.

Specific gravity and contents of extract and ethanol.

Wines

Sulfur dioxide
150 ppm
Gelatin hydrolysate 100 ppm
150 ppm
500 ppm
750 ppm
Sulfur dioxide 75 ppm +gelatin hydrolysate 75 ppm

Table 2.

23

Specificgravity

Extract

Ethanol

0.99000.99000.99000.99000.9900

2.00%
2.06
2.13
2.06
2.24

13.2
13.4
13.6
13.4
14.0

0.99002.08

13.5

Contents of free amino acids and nonvolatile acids.
Free

Nonvolatile acids

amino

Wines

Sulfur dioxide
150 ppm
Gelatin hydrolysate 100 ppm
150 ppm
500 ppm
750 ppm
Sulfur dioxide 75 ppm +gelatin hydrolysate 75 ppm

acids
mg/.e

Citricacid

Tartaric
acid g/.e

Malic
acid

Aceticacid

867
791804
880
1054

0.68
0.66
0.40
0.70
0.51

2.39
2.42
2.33
2.47
2.60

1.83
1.47
1.25
1.32
1.48

0.29
0.51
0.50
0.61
0.49

8114

0.61

2.37

1.32

0.97

The proteins present in the Koshu wine were

75 ppm of the hydrolysate and 75 ppm of sulfur

0.1 to 0.4 mg/.e.4) The values were considerably

dioxide were 113.4 mg, 101.5 mg, and 112.1 mg,

small compared with those ofthe totalfreerespectively (Table 3).

These values wereamino acids and the peptides (989 mg a~d 137

similar to thethemg, respectively) in the wine.5) Therefore, thesulfur

dioxide.

Thewine samples were not deproteinized whenacids

constituting

thedetermined the contents offree

wewinewithonlycompositions

of

amino

peptidesthe wines

inother than the two wines with 500 ppm and 750

amino acid contents in the wines

tion of 100 ppm or 150 ppm were removed asppm of the hydrolysate added at the concentra-

with the hydrolysate were notmuch different

from those of the wine with only sulfur dioxidewith the exception of those in the wine with750 ppm of the hydrolysate.
composi tions ofble 2.

inamino acids

and peptides.
The freevalue

precipitate or assimilated by yeast as nitrogensource during fermentation and aging.
The addition of

the hydrolysate at the con-

The contents andcentration of 100 ppm or 150 ppm decreased

nonvolatile acids show in Ta-

the degree of browning, and it did not affectIn each nonvolatile acid, thecontents

in the six wine samples were rather similar toone another.
The peptide contents in the wines with 100ppm and 150 ppm of the hydrolysate, and bothturbidity formation.

It appears thatturbidityand browning are primarily affected by phenol
contents

and

their

phenols are theturbidityoxidation,namely,

thesubstrate forbrowningandformation.

Thephenolcontents,

24

YOKOTSUKA . MATSUDO and KUSHIDA

Table 3.

Compositions of amino acids constituting peptides.
Wines
Sulfur dioxide Gelatin
GelatinGelatinGelatin·
Sulfur150 ppm hydrolysate hydrolysate hydrolysate hydrolysate dioxide 75 ppm
100 ppm
150 ppm
500 ppm
750 ppm+Gelatin 75 ppm
hydrolysate(mg/ e)

Asp
Thr + SerGluProGlyAlaValMet
lie
Leu
Tyr
Phe
His 1)
Lys
Arg

20.2
4.3
6.9
trace
9.0
7.3
9.3
1.9
6.7
7.5
3.4
3.5
10.5
10.8
5.1

24.2
4.1
9.9
trace
7.3
3.0
13.2
trace
6.6
5.4
7.4
7.4
13.3
10.4
6.4

21.0
1.3
7.9
trace
9.7
7.8
9.0
1.6
5.9
5.6
5.1
5.1
14.5
6.0
6.1

49.9
9.5
17.1
trace
39.7
16.3
18.4
5.2
12.9
12.6
9.1
9.1
15.0
18.0
13.6

51.1
4.9
19.1
trace
25.7
30.5
19.7
4.6
14.7
16.0
15.0
15.0
18.7
22.5
14.328.2
3.9
11.2
trace
6.5
6.4
8.5
trace
6.3
7.0
5.1
5.1
9.4
10.1
6.8

Total

106.4

113.4

101.5

240.7

256.8

112.1

1)

These values include histidine and at least too unknown ninhydrin-positive compounds.

Tadle 4.
Browning, turbidity, and contents of ether-extractablenontannin phenolics and butanol-2-extractable tannins.
Absorbanceat 400 nm

Turbidity(units)Wines

Sulfur dioxide
150 ppm
Gelatin hydrolysate 100 ppm
150 ppm
500 ppm
750 ppm
Sulfur dioxide 75 ppm +gelatin hydrolysate 75 ppm
1)
2)

Etherextractablenon-tannin
phenolics(mg/e) 1)

Butanol-2extractabletannins(mg/ e)

0.145
0.105
0.115
0.130
0.145

0.22
0.17
0.24
0.36
0.46

38.0
23.5
33.0
31.5
39.0

102.0
61.5
72.0
73.5
81.0

0.100

0.05

37.5

75.02)

as gallic acid equivalent.
as catechin equivalent.

«25

Utilization of Glatin Hydrolysate in White Wine-Making
especiallythetannin

contents

inthe wines

odor was the one obtained with use of the mustwith the hydrolysate were smaller than thosetreated with both sulfur dioxide and the hy·

in the wine with only sulfur dioxide (Table 4).

drolysate.

The hydrolysate was found to bind with ease to
many phenols such as catechin,Acknowl edgement

dimeric phenols,and tannins, but its binding ability to the tann·We thank Mr. S.

in was poorer than that of the unhydrolyzed

assistance and Miss Y.

gelatin.6l

earlier draft of this manuscript.

Therefore, there seemed to be little

danger of overfining even if a large amountthe hydrolysate was used.

ofbinding ofIn fact, theAmino for reading anThis study was supported, in part, by

the hydrolysate to the tannins inaging seemed to occur

resultinginremoval

References

1) Kokuzeicho Shoteibunsekihochukai,

The wines made from the musts with the hy·2) Singleton,drolysate (100 ppm or 150 ppm) were slightly

Eno /.

brownish but not cloudy. The sensory impres·3) Yokotsuka,

sion of the aftllrtaste of the wines wasthey had noolfactory

fresh,characteristicofJ.

L., Rossi,V.
K.,

Ferment.

4) Yokotsuka,Ferment.

were perceived. These sensory impressions are5) Yokotsuka,

Shinkai,K., Kato,

K., Aihara,

Kushida, T.: J. Ferment.
(1975)

slight bitterness.

T.

J.

T., Umehara,Y.,

A., Kushida,Techno/., 56, 606 (1978)

the hydrolysate of 500 ppm and 750 ppm had aaS., Kushida, T.

Technol., 58, 107 (1980)

common in Koshu wine. However, the wines with

Of the wines made, the best wine in taste andJ. A.: Amer. J.

Viticult., 16, 144 (1965)

bouquet and aroma. No off-flavors or off-odors

aldehyde-odor and3rd. Ed.,

p. 52, Nippon J ozo Kyokai, Tokyo (1974)

of a limited amount of tannins.

littlethescience fund granted by Nippi Inc.

the must or the wines during fermentation andbut

Amemiya for his technicalTechnol., 53, 6316) Yokotsuka, K., Singleton, V. L., unpublished