APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1979, p.

892-896
0099-2240/79/05-0892/05$02.00/0

Vol. 37, No. 5

Production of Higher Alcohols During Indonesian Tape Ketan

Fermentationt
T. C. CRONK,4 L. R. MATTICK, K. H. STEINKRAUS,* AND L. R. HACKLER
New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456

Received for publication 26 February 1979

Tape ketan is a traditional Indonesian fermented food prepared from glutinous rice which
has been steamed, inoculated, and allowed to
ferment for 24 to 48 h or longer at ambient
temperatures (25 to 30C). The inoculum,
known as ragi, consists of dry circular cakes
prepared locally from rice flour and distinctive
spices and contains the essential microflora. The
fermentation is dependent on at least one amylolytic filamentous fungus and one or more
yeasts. The fermented product is a partially
liquified, yet cohesive mass having a sweet-sour
and mildly alcoholic taste. It is consumed without further processing as a dessert or snack item
(4, 5, 7, 10, 12).
In a previous paper (4), we reported results
describing the fundamental biochemical changes
occurring during the tape ketan fermentation. A
filamentous mold, Amylomyces rouxii Calmette
(6), and eight yeasts, including species of Endomycopsis, Candida, and Hansenula, each previously isolated from ragi (tap6 inoculum), were
studied. The highest quality tape was prepared
t Journal Paper no. 3204 from the New York State Argicultural Experiment Station.
: Present address: The Pillsbury Co., Minneapolis, MN
55414.

from rice fermented for 36 to 48 h at 30C using

a combination of A. rouxii and Endomycopsis
burtonii. The growth of the various yeasts was
shown to be dependent on the amylolytic capacity of the mold, and these yeasts were observed
to be primarily responsible for the characteristic
tape ketan flavor. Ethanol production during the
initial phase of fermentation was shown to be
due primarily to the mold A. rouxii.
Typically associated as secondary products
with alcoholic fermentations are the higher-molecular-weight alcohols, commonly referred to as
fusel oils. Fusel oils is a collective term used to
describe that distillate fraction which generally
includes n-propanol (1-propanol), isobutanol (2methyl-1-propanol) and active amyl (2-methyl1-butanol) and isoamyl (3-methyl-1-butanol) alcohols. Each of these alcohols has distinct organoleptic qualities. Both amyl alcohols are generally considered unattractive in odor when concentrated; however, they tend to contribute favorably to the aroma of a fermented beverage
when present at appropriate concentrations (R.
W. Plank, Abstr. Annu. Meet. Am. Soc. Enol.
1970).
This study was conducted to further characterize the tape fermentation and to determine

892

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A study was made of the higher alcohols (fusel oils) produced during the
Indonesian tape ketan fermentation using Amylomyces rouxii as the principal
mold, alone or in combination with yeasts belonging to genera commonly found
in the tape ketan fermentation (Endomycopsis, Candida, and Hansenula). Total
fusel oils increased with length of fermentation. Fusel oils detected in the product
distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol
was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts.
A. rouxii alone produced nearly the same quantity of fusel oils (total production,
275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total
production, 292 mg/liter at 192 h). A. rouxii and Endomycopsisfibuliger produced
fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in
combination with Candida yeasts produced somewhat more fusel oils, ranging
from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula
yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During
the first 36 h, production of fusel oils was higher at 30 and 35C than at 25C. At
48 h fusel oil production was slightly higher at 30C than at 35C. Beyond 48 h,
production of fusel oils was higher at 25C. A. rouxii in combination with
Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl
acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192
h.

VOL. 37, 1979

ALCOHOLS IN TAPE KETAN FERMENTATION

893

qualitatively and quantitatively the higher al- Ominiscribe strip chart recorder (1 mV; 1 inch [ca.
cohols which contribute to the unique flavor of 2.54 cm]/min).
Three standard solutions containing varying contap6 ketan.
centrations of n-propanol, isobutanol and isoamyl and
MATERLALS AND METHODS
active amyl alcohols were prepared. To each solution
was added a constant amount of 3-pentanol as the
iiternal standard.
The response factor for each alcohol in each standard was calculated from the peak area ratio and the
concentration ratios of the alcohol and internal standard based on the average of three determinations.
The concentration of the alcohols was determined
according to the following equation: Concentration of
alcohol = response factor x concentration of internal
standard x peak area ratio x dilution factor. Retention
times were 4.91, 5.43, 9.90, and 10.26 min for isobutanol, 3-pentanol, active amyl alcohol, and isoamyl
alcohol, respectively. Ethyl acetate was also determined quantitatively as part of the fusel oil alcohol
chromatogram (retention time, 1.21 min).

RESULTS AND DISCUSSION

The fusel oil alcohols and the quantities of
each produced in the fermentation of rice by
various yeasts, each in combination with A.
rouxii, are presented in Table 1. No n-propanol
was detected in this study. Concentrations of
TABLE 1. Fusel oil alcohols produced by A. rouxii
in combination with various yeasts on rice
fermented at 30C

Organisms

Alcohol

Isobutanol
Active amyl
buliger
Isoamyl
Total
Isobutanol
A. rouxii + C.
Active amyl
lactosa
Isoamyl
Total
Isobutanol
A. rouxii + C.
Active amyl
melinii
Isoamyl
Total
Isobutanol
A. rouxii + CanActive amyl
dida parapsilosis
Isoamyl
Total
A. rouxii + H. an- Isobutanol
Active amyl
omala
Isoamyl
Total
A. rouxii + Han- Isobutanol
Active amyl
senula maIsoamyl
langa
Total
+
H.
Isobutanol
A. rouxii
Active amyl
subpelliculosa
Isoamyl
A. rouxii + E. fi-

Total

Amt of alcohol (mg/

liter) in product distillate after the following fermentation
times (h):a
36
13
13
47
72
47
17
87
151
38
9
40
87
36
7
39
82
25
4
21
50
20

_b
37
57
21
-

17
38

aEach value is a mean of three analyses.

b-, Detectable but not measurable.

48
41
27

119
186
116
30
109
255
104
26
106
324
116
23
99
238
48
10
50
109
57
8
62
128
56
14
46
116

96
135
65
261
461
213
48
173
434
229
60
203
492
230
45
183
459
67
18

192
175
86
298
558
280
78
241
600
280
86
253
618
293
66
232
590
65
16

77
163
112
25
143
280
80
38
80
198

63
143
100
23
126
248

84
24
60
168

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Microorganisms and substrate. The microorganisms, their sources, and the techniques employed for
culture maintenance and inoculum preparation used
in this study have been described previously (4). The
fermentation substrate consisted of hydrated, polished
white rice (Carolina Brand). A total of 40 g of rice and
100 ml of water were added to a circular Nalgene
specimen dish (105 by 42 mm). Dishes were covered,
steamed for 15 min, and subsequently autoclaved for
15 min at 1210C.
Sample preparation. A sample consisted of the
entire contents of one dish. At each sample point, a
dish was removed from the incubator, tightly covered,
and quickly frozen at -20C until the analysis could
be performed. Before analysis, samples were briefly
thawed at room temperature, quantitatively diluted
approximately 3:1 with distilled water and comminuted with an Omnimixer. By using a micro-Kjeldahl
distillation apparatus, 50 ml of the comminuted fermented product was distilled, and an equal volume of
the distillate was collected over an interval of approximately 20 min. Samples were further prepared for
analysis by the addition of 0.1 ml of 3-pentanol (12.11
mg/ml) as an internal standard to each 10-ml sample
of the steam distillate.
Gas chromatographic determination. Fusel oil
alcohols were determined qualitatively and quantitatively by gas chromatography, using the procedure
(method 1) of Kahn and Blessinger (8). The gas chromatographic instrument used was a Carle model 9000
equipped with a flame ionization detector. A coiled
copper column (10 feet [ca. 3.05 m] by 'k8-inch [ca 3.2mm] OD, with a wall thickness of 0.031 inch [ca. 0.79
mm]) was fitted into the injection port to allow oncolunm injection.
The column packing was 2.0% glycerol-2% 1,2,6hexanetriol (wt/wt) on Gas Chrom R 100/120 mesh.
The column was prepared by dissolving 0.2 g each of
glycerol and 1,2,6-hexanetriol in a small volume of
methyl alcohol. The alcoholic solution of the two
liquid phases was added to an evaporating dish containing 9.6 g of Gas Chrom R which had been previously moistened with methyl alcohol. After thorough
mixing, the methyl alcohol was slowly boiled off by
gently heating the mixture in a steam bath with continuous stirring until complete removal of the solvent
occurred. After packing, the column was conditioned
by heating overnight at 78C with the detector end
disconnected but maintaining carrier gas flow. The
column oven was maintained at 78C, with the injector
port and detector approximately 1C higher.
The carrier gas (nitrogen) was maintained at a flow
rate of 25 ml/min. The combustion gases were hydrogen and air maintained at 25 and 300 ml/min, respectively. A 2- to 3-p1 sample was injected into the instrument with two no. 701 Hamilton syringes. The instrument sensitivity was maintained at 5 x 10`0 AFS.
A Hewlett-Packard model 3370A integrator connected in series to the gas chromatographic instrument
was used to determine peak areas. A visual record of
the chromatogram was achieved by using a Houston

894

APPL. ENVIRON. MICROBIOL.

CRONK ET AL.

burtonii were influenced by temperature (Tables 2 and 3). Quantities of the higher alcohols
were similar for rice fermented by mold and by
mold plus yeast when compared at the same
temperature. In both fermentations, the concentration of the fusel oil alcohols increased with
fermentation time at 25C. At 30 and 350C, the
levels reached a maximum at 96 h and decreased
thereafter.
During the first 36 h of fermentation, the
concentration of fusel oil alcohols increased with
increasing temperature for rice fermented both
by mold and by mold plus yeast. At 48 h the
concentrations increased with an increase in
temperature from 25 to 300C, but decreased with
a further increase to 350C. At both 96 and 144 h
there was a consistent decrease in concentrations with increasing temperature.
The concentrations of the higher alcohols produced by the fermentation of rice with A. rouxii
alone at 300C (Table 2) tended to be higher than
the concentrations produced by Hansenula species plus A. rouxii and lower than those produced by all other yeasts plus A. rouxii (Table
1). The concentrations of fusel oil alcohols in
rice femented by Endomycopsis burtonii plus A.
rouxii at 30C (Table 3) were generally higher
compared with the concentrations for the Hansenula species and lower compared with Endomycopsis fibuliger and Candida species.
The highest concentration of the fusel oil alcohols in tap6 ketan fermented for 48 h was
produced at 300C, which corresponds to the
highest concentration of ethanol, 3.0%, produced
under the same conditions (4). An incubation
temperature of 350C appears to lower the level
TABLE 2. Influence of temperature on fusel oil
alcohols produced by A. rouxii on rice
Amt of alcohol (mg/liter) in
Temp

Alcohol

(OC)

product distillate after the

following fermentation times
h:
36

25

Isobutanol
Active amyl
Isoamyl
Total
Isobutanol
Active amyl
Isoamyl
Total
Isobutanol
Active amyl

_b

48

96

92 139
7
20
54 102
152 262
30
62 109 132
11
22
28
68 102
90 188 256
35
61
91
90
4
15
20
40
Isoamyl
73
82
Total
106 177 192
a Each value is a mean of triplicate analyses.
b-, Detectable but not measurable.

192

146
27
102
275
132
21
96
249
78
18
76
170

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total fusel oil alcohols at 36 h ranged from 38

mg/liter, produced by Hansenula subpelliculosa plus A. rouxii, to 151 mg/liter, produced by
Candida lactosa plus A. rouxii. At 48 h, the
maximum concentration (324 mg/liter) was produced by Candida melinii, and the minimum
concentration (109 mg/liter) was produced by
Hansenula anomala, both in combination with
A. rouxii. These same combinations of organisms at subsequent intervals continued to produce the highest and lowest concentrations of
fusel oil alcohols, respectively.
With each yeast (in combination with A.
rouxii), except the Hansenula species, there was
a progressive increase with time in the total fusel
oil alcohol concentrations. In rice fermented
with the Hansenula yeasts, the maximum concentrations of fusel oil alcohols were observed at
96 h. By 192 h, these concentrations decreased,
although higher concentrations may have been
reached before or after 96 h. Of all yeasts studied
in combination with A. rouxii, the Hansenula
species produced the lowest concentrations of
fusel oil alcohols at each time interval examined.
At 48 h in typical tape ketan, the total quantities of fusel oil alcohols produced, except with
C. melinii plus A. rouxii, were comparable to or
less than the quantities in grape wines, which
range from 200 to 300 mg/liter (3). The level of
fusel oil alcohols never exceeded 300 mg/liter
for any of the rice fermented by Hansenula
species plus A. rouxii. For all other yeasts, the
level of these alcohols exceeded 400 mg/liter by
96 h and 500 mg/liter by 192 h.
By 96 h the fermented rice may still be consumed as tape ketan, but more commonly the
liquid portion would be separated from the solid
and used for the production of brem or arak.
Brem is consumed as a solid "candy" prepared
by sundrying; arak constitutes an alcoholic beverage prepared through distillation. Consumed
as either of these products, the level of fusel oil
alcohols would be appreciably higher than the
values presented in Table 1.
Of the individual fusel oil alcohols, isobutanol
and isoamyl alcohol were produced in the greatest amounts. Within a particular fermentation,
there was also a tendency for either isobutanol
or isoamyl alcohol to predominate throughout
the fermentation. Therefore, in addition to the
total concentration of fusel oil alcohols, the species of yeast also influenced the amount of an
individual alcohol produced. Similar observations were reported by Webb and Kepner (11)
and Rankine (9).
The total and individual quantities of fusel oil
alcohols produced in the fermentation of rice by
A. rouxii separately and in combination with E.

ALCOHOLS IN TAPE KETAN FERMENTATION

VOL. 37, 1979

895

Each value is a mean of triplicate analyses.

organisms.

Esters, particularly ethyl acetate, are important constituents of fermented foods and beverages. Ethyl acetate at a level below 200 mg/liter
tends to impart a desirable odor in wines, but
higher concentrations tend to be associated with
spoilage (2). Total esters (as ethyl acetate) of
TABLE 3. Influence of temperature on fusel oil
alcohols produced by A. rouxii in combination with
E. burtonii on rice
Amt of alcohol (mg/liter) in
product distillate after the folTemp

Alcohol

lowing

fermentation

times

(h):a

(00)

36

48

96

90 131
7
19
61 116
Isoamnyl
158 266
Total
87 128
42
Isobutanol
30
17
9
Active amyl
66 115
28
Isoamyl
69 162 260
Total
87 143
49
35
Isobutanol
16
9
Active amyl
94
32
60
Isoamyl
81 156 253
Total
aEach value is a mean of triplicate analyses.
b-, Detectable but not measurable.

25

Isobutanol
Active amyl

_b
-

192

130
21
141
292
126
17
104
247
94
11
75
179

wines analyzed by Amerine (1) ranged from

about 200 to 400 mg/liter.
Although a higher concentration of ethyl acetate was produced at 36 h by H. anomala than
by H. subpelliculosa, the levels were comparable at each interval thereafter. After 36 h of
fermentation, the odor of ethyl acetate was exceedingly strong and would be considered objectionable. A substantial decrease in the concentration of ethyl acetate occurred beyond 96 h.
At 192 h the fermented rice which would be used
for the production of brem or arak contained a
level of ethyl acetate within the range for various
wines (1).
The results of this study demonstrate that
appreciable quantities of isobutanol and active
amyl and isoamyl alcohols are produced during
the fermentation of rice by A. rouxii, separately
or in combination with various yeasts. The total
quantity of the fusel oil alcohols, as well as the
quantities of individual alcohols, were influenced
by the temperature and the species of yeast.
Ethyl acetate was also shown to be produced in
high concentrations by two species of Hansenula.
LITERATURE CITED
1. Amerine, M. A. 1954. Composition of wines. I. Organic
constituents. Food Res. 5:353-510.
2. Amerine, M. A., H. W. Berg, and W. V. Cruess. 1967.

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of fusel oil alcohols in products such as brem TABLE 4. Influence of temperature on the ratio of
active amyl to isoamyl alcohol produced by A.
and arak where the fermentation continues berouxii alone and in combination with E. burtonii on
yond'48 h.
rice,
Temperature influenced the ratio of active
Ratio of active amyl to isoamyl to isoamyl alcohol produced by A. rouxii
separately and in combination with E. burtonii
Temp amyl alcohol after the following fermentation times (h):
Organism(s)
(OC)
(Table 4). For rice fermented both by mold and
by mold plus yeast, the ratios increased as the
192
96
48
36
fermentation progressed at 25 and 300C, indica- A. rouxii
0.130 0.196 0.265
25
tive of a higher rate of active amyl alcohol
0.162 0.215 0.219
30
At
alcohol.
production compared with isoamyl
0.100 0.205 0.244 0.237
35
48 h the ratios for both fermentations increased A. rouxii + E.
0.115 0.164 0.149
25
with an increase in temperature. The ratio of
0.136 0.148 0.163
30
burtonii
0.150 0.170 0.147
35
active amyl to isoamyl alcohol for tape ketan
fermented for 48 h at 300C by A. rouxii alone
and in combination with all yeasts studied
TABLE 5. Quantities of ethyl acetate produced in
ranged from 0.129 to 0.304. These extremes, both rice fermented by A. rouxii in combination with H.
anomala and H. subpelliculosa at 30C
produced by species of Hansenula (each in combination with A. rouxii), indicate the influence
Amt of ethyl acetate (mg/liwhich the species of yeast may have on the taste
ter) in product distillate
after the following fermentaand odor of the fermented product.
tion times (h):a
Orgamsms5
The presence of ethyl acetate was detected
and
H.
in
H.
anomala
by
fermented
rice
only
96
192
36
48
subpelliculosa, each in combination with A. A. rouxii + H. anom369
859
199
543
rouxii (Table 5). These qualitative findings by
ala
gas chromatography are in accord with the easily
145 537 905 354
A. rouxii + H. subpelrecognized, strong odor of ethyl acetate that was
liculosa
present only in the rice fermented by these two

896

3.
4.
5.
6.

7.
8.

APPL. ENVIRON. MICROBIOL.

CRONK ET AL.

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10.
11.
12.

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fermented foods of Indonesia. Ann. Bogor. 4:187-204.
Webb, A. D., and R. E. Kepner. 1961. Fusel oil analysis
by means of gas-liquid partition chromatography. Am.
J. Enol. Vitic. 12:51-59.
Went, F. A. F. C., and H. C. Prinsen Geerligs. 1895.
Beobachtungen uber die Hefearten und Zuckerbildenden Pilze der Arackfabrikation. Verh. K. Akad. Wet.
Amsterdam Afd. Natunrkd. Reeks 2 4:3-31.

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