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Ethanol fermentation of aromatic red rice without cooking, studies on red rice wine brewing (part 1)
JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 70, No. 5, 326-328. 1990
Ethanol Fermentation of Aromatic Red Rice without Cooking, Studies on Red Rice Wine Brewing (Part 1) S E I N O S U K E U E D A , 1. T A T S U R O UEKI, 1 R I I C H I R O O H B A , l YUJI T E R A M O T O , l AND K I Y O S H I Y O S H I Z A W A 2
Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, j and Faculty of Brewing Science, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagayaku, Tokyo 156,2 Japan Received 27 June 1990/Accepted 20 August 1990 Using aromatic red rice (Oryza sativa vat. Indica, Tapol), which contained anthocyanin pigments, as a raw material, fermentation without cooking was done at pH 3.5 and 30°C to produce a novel type of alcoholic beverage designated as aromatic red rice wine. The final ethanol concentration achieved during fermentation was 9.3% by volume. Aromatic red rice wine was rich in a fruity aroma and had a characteristic sour taste by gas chromatographic analysis and organoleptic test. Fermentation without cooking was effective for conservation of the red pigment and aroma components of aromatic red rice. The uncooked ethanol fermentation of aromatic red rice in this work has been proven by experiment to be suitable for economical production of a novel type of ros6-wine-like alcoholic beverage.
As fermentation technology and the brewing industry progress, many kinds o f alcoholic beverages have been produced year by year to catch the p o p u l a r fancy. In Japan, red rice (1-3), which contained a red pigment in its bran, has been used for production o f an ancient alcoholic beverage and traditional c o o k e d rice, Sekihan, for celebrations, but red rice has not been used by commercial fermentation industries. Recent extensive studies (4-6) on ethanol fermentation from u n c o o k e d starchy raw materials have shown that the uncooked fermentation could be effective to economically produce ethanol since it does not require as much energy. This paper investigates the application o f aromatic red rice to uncooked ethanol fermentation system to produce an economically novel type o f rice wine rich in a r o m a and fit for the public taste. F o r comparison, cooked and uncooked fermentation o f various rices are also described.
Erlenmeyer flask with a gas trap. The p H of the initial broth was adjusted to p H 3 . 5 with I N HC1 and I N N a O H . Fermentation was done at 30°C. The weight decrease o f the whole Erlenmeyer flask by the a m o u n t of CO2 gas evolved was weighed every 24 h. Runs 1,2, 3, and 4 contained aromatic red rice, red rice, glutinous rice, and nonglutinous rice as raw material, respectively. Runs 5, 6, 7, and 8 contained cooked aromatic red rice, cooked red rice, cooked glutinous rice, and cooked nonglutinous rice, respectively. Ethanol measurement Ethanol was measured as previously described (5, 6) by a gas c h r o m a t o g r a p h i c method using a Shimadzu model GC-14A gas c h r o m a t o g r a p h . The distillation methods (7) were also used for ethanol measurement for the final fermented broth. General analytical methods Samples of fermented broth were analyzed as previously described (5, 6). Starch in the feed and in the residue of fermented broth was hydrolyzed by 1 N HC1 in a boiling water bath for 3 h, and the reducing sugar in hydrolysate was measured by the methods of Nelson (8) and Somogyi (9). Acidity was measured by titrating 10ml of fermented broth with 0.1 N N a O H . Acidity was defined as the a m o u n t of N a O H solution used for the titration. A r o m a analysis o f fermented broth was done by gas chromatographically (10-13). Organoleptic tests were also done to evaluate the various fermented broths.
MATERIALS AND METHODS Starchy material A r o m a t i c red rice (Oryza sativa vat. Indica, Tapol), red rice (Oryza sativa vat. Indica), glutinous rice (Oryza sativa var. Japonica), and nonglutinous rice (Oryza sativa var. Japonica) was kindly d o n a t e d by the K u m a m o t o Agricultural Research Institute. All rices were unpolished and ground to 2-3 mm mesh by an electric grinder (Kansai Toki model " H i k i k k o " K-80) and used for fermentation tests. E n z y m e preparation and yeast Glucoamylase AN-2 (a glucoamylase p r e p a r a t i o n p r o d u c e d by Aspergillus niger, Shinnihon Kagaku Kogyo Co. Ltd., Anjo) was used as the saccharifying enzyme. Compressed baker's yeast (Kyowa H a k k o Kogyo Co. Ltd., Tokyo) was used for ethanol fermentation. Procedure for ethanol fermentation (5, 6) Thirty grams o f various raw ground unpolished rices, 0.2 g o f glucoamylase AN-2, 3 g o f compressed baker's yeast, and 100ml o f tap water were dispensed into a 300-ml
RESULTS Fermentation o f various kinds of uncooked rice
We
have examined the influence o f important factors such as initial pH, inoculum, and a m o u n t o f saccharifying enzyme. Acidic conditions, initially p H 3.5, and an excess of yeast were inevitable for prevention of contamination. Figure 1 shows the changes in the cumulative a m o u n t of carbon dioxide generated from broth for runs 1, 2, 3, and 4. Each fermentation was continued until the generation of carbon dioxide from broth ceased. C o m p a r i n g with the experiment, the fermentation rate o f run 1 was slower than
* Corresponding author. 326
VoL 70, 1990
PRODUCTION OF AROMATIC RED RICE WINE
10.0
327
0
cn v v
5.0
O ¢..9
00-
I I
I
I
I
2 T i me
3
4
(d) FIG. I. Courses of fermentation of mashes used rices without cooking. Symbols: o, unpolished aromatic red rice; A, unpolished
[
I
I
1
I
O0
1
2
3
/4
T i me
(d)
FIG. 2. Coursesof fermentation of mashes used rices with cooking. Symbols: O, unpolished aromatic red rice; A, unpolished red rice; [], unpolished glutinous rice; 0, unpolished nonglutinous rice. Experimental details are described in the text.
red rice; •, unpolished glutinous rice; 0 , unpolished nonglutinous rice. Experimental details are described in the text.
that of runs 2, 3, and 4. The a m o u n t of carbon dioxide evolved in run 1 was 9.7 g and that of runs 2, 3, and 4 was 9.9 g. The ratio of glucose c o n s u m p t i o n in run 1 was 9 2 ~ and that of runs 2, 3, and 4 was 94%. The acidity of the fermented broth in runs 1, and 2 was 9.3 and that of runs 3, and 4 was 8.2 (Table 1). Fermentation of various kinds of cooked rice Figure 2 shows the change in the cumulative a m o u n t of carbon dioxide generated from broth for runs 5, 6, 7, and 8. The a m o u n t of carbon dioxide evolved in run 5 was 9.8 g and that of runs 6, 7, and 8 was about 10 g. The ratio of glucose c o n s u m p t i o n in runs 5, 6, 7, and 8 was about 960/00. The acidity of the fermented broth in runs 5, and 6 was about 8.0 and that of runs 7, and 8 was 7.3 (Table 1). Characteristics of the various rice wines Comparing the aromatic c o m p o n e n t of the various rice wines, the a m o u n t s of isobutyl alcohol and isoamyl alcohol in the broth from uncooked fermentations were 3.5-4.0 and 1.21.6 times higher than that from cooked fermentations (Table 2). The a m o u n t of ethyl acetate in the broth from uncooked fermentation was also higher than that from traditional cooked fermentation. By the organoleptic tests, the rice wines produced by uncooked fermentation were much better than the rice wines produced by cooked fermentation. Especially, the aromatic red rice wine produced by uncooked fermentation was rich in fruity a r o m a
and had a ros~-wine-like taste by the organoleptic test. DISCUSSION Under optimal conditions (initial pH 3.5, 30°C), a novel type of ros~-wine-like alcoholic beverage was produced within 4 d by uncooked fermentation of aromatic red rice. The final ethanol concentration achieved during uncooked fermentation was 9.3%0 by volume. Aromatic red rice wine produced by uncooked fermentation had a characteristic ruby color, fruity aroma, and sour taste. Here, we propose an economical application of aromatic red rice and a possibility of developing original alcoholic beverages with ruby color and fruity fragrance. Maekawa and Shinke (1) also reported sake brewing using red rice. Comparing with the above case, the fermentation procedure in this work was relatively economical because it reduced the cooking process energy. Moreover, the red color of the fermented broth was vivid in this case. Volatile esters and longer-chain alcohols were found a b u n d a n t l y in the aromatic red rice wine produced by uncooked fermentation. On the contrary, broth of the cooked rice fermentation contained less of such aromatic compounds. In comparing the various rice wines, the process of uncooked fermentation was essential for conservation
TABLE 1. Characteristics of the various rice wines Runs Cooking Volume of filtrate (ml) Weight of residue (g) CO2 output (g) Total glucose of feed (g) Total glucose of residue (g) Consumed glucose (g) Consumption of glucose (%) Acidity (ml)
1 95 21.3 9.7 19.4 1.6 17.8 92 9.2
5 + 93 19.9 9.8 19.4 0.8 18.6 96 8.1
2 96 20.3 9.9 18.6 1.2 17.4 94 9.3
6 + 92 19.3 10.1 18.6 0.9 17.7 95 7.9
3 94 19.3 9.9 19.4 1.2 18.2 94 8.0
7 + 93 19.2 10.2 19.4 0.8 18.6 96 7.2
4 97 19.8 9.9 19.3 1.2 17.9 94 8.4
8 + 96 19.0 10.1 19.3 0.8 18.5 95 7.4
328
UEDA ET AL.
J. FERMENT. BIOENG., TABLE 2.
Runs Cooking
1 -
Ethyl alcohol ( ~ ) Isobutyl alcohol (ppm) Isoamyl alcohol (ppm) Acetaldehyde (ppm) Isovaleraldehyde (ppm) Ethyl acetate (ppm) Acetic acid (ppm) Organoleptic test a
9.3 230 250 1100 2.2 370 150 -+
Flavor components of the various rice wines 5 ~-
2
6 +
9.5 9.4 97 220 200 230 1170 1100 1.4 2.6 200 300 48 190 . . . . .
9.5 130 220 1100 1.4 200 51 ~
3 9.4 200 210 1090 3.4 340 170 ~
7 +
4
8 +
9.6 140 200 980 2.7 170 53 +
9.5 210 220 1100 3.4 350
9.6 120 210 1060 4.4 170 48
190
'~ Organoleptic test. +b+, Excellent; ~ , fairly; ~, positive; + , slightly.
o f the sweet smell in a r o m a t i c red rice. A r o m a t i c r e d rice w h i c h c o n t a i n e d a c h a r a c t e r i s t i c r e d p i g m e n t a n d a specific a r o m a h a d n o t b e e n p r o p e r l y used in t h e f o o d i n d u s t r y . N o w a d a y s , p u b l i c t a s t e values q u a l i t y a n d n o v e l t y . A r o m a t i c red rice is a n excellent m a t e r i a l t h a t provides an original alcoholic beverage. P r e c i s e a r o m a a n a l y s i s o f a r o m a t i c red rice w i n e a n d t h e m e c h a n i s m o f a r o m a f o r m a t i o n will be r e p o r t e d in o u r next paper.
4. Yamasaki, I. and Ueda, S.: Action of black-koji-amylase on raw starch I. J. Agric. Chem. Soc. Jpn., 24, 181-185 (1951).
5. Fujio, Y., Suyanadona, P., Attasampunna, P., and Ueda, S.: 6.
7. 8.
ACKNOWLEDGMENT The authors are grateful to the Kumamoto Agricultural Research Institute for generously providing various rices and Shinnihon Kagaku Kogyo Co. Ltd. for generously providing the glucoamylase preparations. The authors also thank Bishonen Co. Ltd. for his support during the study.
REFERENCES 1. Maekawa, K. and Shinke, R.: Properties of red rice pigments, and sake brewing by using red rice as raw material. J. Brew. Soc. Japan, 84, 787-793 (1989).
2. Takahashi, K., Sugimoto, T., Miura, T., Wasizu, Y., and Yoshizawa, K.: Isolation and identification of red rice pigment. J. Brew. Soc. Japan, 84, 807-812 (1989). 3. Yokoo, M.: Kome. Seibutsu to Kagaku, 28, 49-55 (1990).
9.
Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking. Biotechnol. Bioeng., 26, 315-319 (1984). Fujio, Y., Ogata, M., and Ueda, S.: Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas ci~'culation type fermentoe Biotechnol. Bioeng., 27, 1270-1273 (1985). Honda, N.: Alcohol handbook, 5th ed. Japanese Assoc. Indust. Ferment., Japan, Tokyo (1978). Nelson, N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375-380 (1944). Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952).
10. Yajima, I., Yanai, T., Nakamura, M., Sakakibara, H., and Hayashi, K.: Volatile flavor components of cooked kaorimai. Agric. Biol. Chem., 43, 2425-2429 (1979). l l. Ishikawa, T. and Yoshizawa, K.: Seishukobo niyoru koki ester no seisei to shibosan, p. 103-115. In Akiyama, Y. (ed.), Kobo no riyo to kaihatsu. Gakkai Shuppan Center, Tokyo (1979).
12. Iwata, H., Nakajima, N., Mizuno, A., Izumi, T., Ikeda, E., Kumagai, C., Umeda, N., and Tanaka, T.: Comparison of aromatic compounds in sake making using Kyokai no. 9 and no. l0 yeast. J. Brew. Soc. Japan, 83, 411-415 (1988). 13. Supriant, Ohba, R., Koga, T., and Ueda, S.: Liquefaction of glutinous rice and aroma formation in tap6 preparation by ragi. J. Ferment. Bioeng., 67, 249-252 (1989).
Ethanol Fermentation of Aromatic Red Rice without Cooking, Studies on Red Rice Wine Brewing (Part 1) S E I N O S U K E U E D A , 1. T A T S U R O UEKI, 1 R I I C H I R O O H B A , l YUJI T E R A M O T O , l AND K I Y O S H I Y O S H I Z A W A 2
Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, j and Faculty of Brewing Science, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagayaku, Tokyo 156,2 Japan Received 27 June 1990/Accepted 20 August 1990 Using aromatic red rice (Oryza sativa vat. Indica, Tapol), which contained anthocyanin pigments, as a raw material, fermentation without cooking was done at pH 3.5 and 30°C to produce a novel type of alcoholic beverage designated as aromatic red rice wine. The final ethanol concentration achieved during fermentation was 9.3% by volume. Aromatic red rice wine was rich in a fruity aroma and had a characteristic sour taste by gas chromatographic analysis and organoleptic test. Fermentation without cooking was effective for conservation of the red pigment and aroma components of aromatic red rice. The uncooked ethanol fermentation of aromatic red rice in this work has been proven by experiment to be suitable for economical production of a novel type of ros6-wine-like alcoholic beverage.
As fermentation technology and the brewing industry progress, many kinds o f alcoholic beverages have been produced year by year to catch the p o p u l a r fancy. In Japan, red rice (1-3), which contained a red pigment in its bran, has been used for production o f an ancient alcoholic beverage and traditional c o o k e d rice, Sekihan, for celebrations, but red rice has not been used by commercial fermentation industries. Recent extensive studies (4-6) on ethanol fermentation from u n c o o k e d starchy raw materials have shown that the uncooked fermentation could be effective to economically produce ethanol since it does not require as much energy. This paper investigates the application o f aromatic red rice to uncooked ethanol fermentation system to produce an economically novel type o f rice wine rich in a r o m a and fit for the public taste. F o r comparison, cooked and uncooked fermentation o f various rices are also described.
Erlenmeyer flask with a gas trap. The p H of the initial broth was adjusted to p H 3 . 5 with I N HC1 and I N N a O H . Fermentation was done at 30°C. The weight decrease o f the whole Erlenmeyer flask by the a m o u n t of CO2 gas evolved was weighed every 24 h. Runs 1,2, 3, and 4 contained aromatic red rice, red rice, glutinous rice, and nonglutinous rice as raw material, respectively. Runs 5, 6, 7, and 8 contained cooked aromatic red rice, cooked red rice, cooked glutinous rice, and cooked nonglutinous rice, respectively. Ethanol measurement Ethanol was measured as previously described (5, 6) by a gas c h r o m a t o g r a p h i c method using a Shimadzu model GC-14A gas c h r o m a t o g r a p h . The distillation methods (7) were also used for ethanol measurement for the final fermented broth. General analytical methods Samples of fermented broth were analyzed as previously described (5, 6). Starch in the feed and in the residue of fermented broth was hydrolyzed by 1 N HC1 in a boiling water bath for 3 h, and the reducing sugar in hydrolysate was measured by the methods of Nelson (8) and Somogyi (9). Acidity was measured by titrating 10ml of fermented broth with 0.1 N N a O H . Acidity was defined as the a m o u n t of N a O H solution used for the titration. A r o m a analysis o f fermented broth was done by gas chromatographically (10-13). Organoleptic tests were also done to evaluate the various fermented broths.
MATERIALS AND METHODS Starchy material A r o m a t i c red rice (Oryza sativa vat. Indica, Tapol), red rice (Oryza sativa vat. Indica), glutinous rice (Oryza sativa var. Japonica), and nonglutinous rice (Oryza sativa var. Japonica) was kindly d o n a t e d by the K u m a m o t o Agricultural Research Institute. All rices were unpolished and ground to 2-3 mm mesh by an electric grinder (Kansai Toki model " H i k i k k o " K-80) and used for fermentation tests. E n z y m e preparation and yeast Glucoamylase AN-2 (a glucoamylase p r e p a r a t i o n p r o d u c e d by Aspergillus niger, Shinnihon Kagaku Kogyo Co. Ltd., Anjo) was used as the saccharifying enzyme. Compressed baker's yeast (Kyowa H a k k o Kogyo Co. Ltd., Tokyo) was used for ethanol fermentation. Procedure for ethanol fermentation (5, 6) Thirty grams o f various raw ground unpolished rices, 0.2 g o f glucoamylase AN-2, 3 g o f compressed baker's yeast, and 100ml o f tap water were dispensed into a 300-ml
RESULTS Fermentation o f various kinds of uncooked rice
We
have examined the influence o f important factors such as initial pH, inoculum, and a m o u n t o f saccharifying enzyme. Acidic conditions, initially p H 3.5, and an excess of yeast were inevitable for prevention of contamination. Figure 1 shows the changes in the cumulative a m o u n t of carbon dioxide generated from broth for runs 1, 2, 3, and 4. Each fermentation was continued until the generation of carbon dioxide from broth ceased. C o m p a r i n g with the experiment, the fermentation rate o f run 1 was slower than
* Corresponding author. 326
VoL 70, 1990
PRODUCTION OF AROMATIC RED RICE WINE
10.0
327
0
cn v v
5.0
O ¢..9
00-
I I
I
I
I
2 T i me
3
4
(d) FIG. I. Courses of fermentation of mashes used rices without cooking. Symbols: o, unpolished aromatic red rice; A, unpolished
[
I
I
1
I
O0
1
2
3
/4
T i me
(d)
FIG. 2. Coursesof fermentation of mashes used rices with cooking. Symbols: O, unpolished aromatic red rice; A, unpolished red rice; [], unpolished glutinous rice; 0, unpolished nonglutinous rice. Experimental details are described in the text.
red rice; •, unpolished glutinous rice; 0 , unpolished nonglutinous rice. Experimental details are described in the text.
that of runs 2, 3, and 4. The a m o u n t of carbon dioxide evolved in run 1 was 9.7 g and that of runs 2, 3, and 4 was 9.9 g. The ratio of glucose c o n s u m p t i o n in run 1 was 9 2 ~ and that of runs 2, 3, and 4 was 94%. The acidity of the fermented broth in runs 1, and 2 was 9.3 and that of runs 3, and 4 was 8.2 (Table 1). Fermentation of various kinds of cooked rice Figure 2 shows the change in the cumulative a m o u n t of carbon dioxide generated from broth for runs 5, 6, 7, and 8. The a m o u n t of carbon dioxide evolved in run 5 was 9.8 g and that of runs 6, 7, and 8 was about 10 g. The ratio of glucose c o n s u m p t i o n in runs 5, 6, 7, and 8 was about 960/00. The acidity of the fermented broth in runs 5, and 6 was about 8.0 and that of runs 7, and 8 was 7.3 (Table 1). Characteristics of the various rice wines Comparing the aromatic c o m p o n e n t of the various rice wines, the a m o u n t s of isobutyl alcohol and isoamyl alcohol in the broth from uncooked fermentations were 3.5-4.0 and 1.21.6 times higher than that from cooked fermentations (Table 2). The a m o u n t of ethyl acetate in the broth from uncooked fermentation was also higher than that from traditional cooked fermentation. By the organoleptic tests, the rice wines produced by uncooked fermentation were much better than the rice wines produced by cooked fermentation. Especially, the aromatic red rice wine produced by uncooked fermentation was rich in fruity a r o m a
and had a ros~-wine-like taste by the organoleptic test. DISCUSSION Under optimal conditions (initial pH 3.5, 30°C), a novel type of ros~-wine-like alcoholic beverage was produced within 4 d by uncooked fermentation of aromatic red rice. The final ethanol concentration achieved during uncooked fermentation was 9.3%0 by volume. Aromatic red rice wine produced by uncooked fermentation had a characteristic ruby color, fruity aroma, and sour taste. Here, we propose an economical application of aromatic red rice and a possibility of developing original alcoholic beverages with ruby color and fruity fragrance. Maekawa and Shinke (1) also reported sake brewing using red rice. Comparing with the above case, the fermentation procedure in this work was relatively economical because it reduced the cooking process energy. Moreover, the red color of the fermented broth was vivid in this case. Volatile esters and longer-chain alcohols were found a b u n d a n t l y in the aromatic red rice wine produced by uncooked fermentation. On the contrary, broth of the cooked rice fermentation contained less of such aromatic compounds. In comparing the various rice wines, the process of uncooked fermentation was essential for conservation
TABLE 1. Characteristics of the various rice wines Runs Cooking Volume of filtrate (ml) Weight of residue (g) CO2 output (g) Total glucose of feed (g) Total glucose of residue (g) Consumed glucose (g) Consumption of glucose (%) Acidity (ml)
1 95 21.3 9.7 19.4 1.6 17.8 92 9.2
5 + 93 19.9 9.8 19.4 0.8 18.6 96 8.1
2 96 20.3 9.9 18.6 1.2 17.4 94 9.3
6 + 92 19.3 10.1 18.6 0.9 17.7 95 7.9
3 94 19.3 9.9 19.4 1.2 18.2 94 8.0
7 + 93 19.2 10.2 19.4 0.8 18.6 96 7.2
4 97 19.8 9.9 19.3 1.2 17.9 94 8.4
8 + 96 19.0 10.1 19.3 0.8 18.5 95 7.4
328
UEDA ET AL.
J. FERMENT. BIOENG., TABLE 2.
Runs Cooking
1 -
Ethyl alcohol ( ~ ) Isobutyl alcohol (ppm) Isoamyl alcohol (ppm) Acetaldehyde (ppm) Isovaleraldehyde (ppm) Ethyl acetate (ppm) Acetic acid (ppm) Organoleptic test a
9.3 230 250 1100 2.2 370 150 -+
Flavor components of the various rice wines 5 ~-
2
6 +
9.5 9.4 97 220 200 230 1170 1100 1.4 2.6 200 300 48 190 . . . . .
9.5 130 220 1100 1.4 200 51 ~
3 9.4 200 210 1090 3.4 340 170 ~
7 +
4
8 +
9.6 140 200 980 2.7 170 53 +
9.5 210 220 1100 3.4 350
9.6 120 210 1060 4.4 170 48
190
'~ Organoleptic test. +b+, Excellent; ~ , fairly; ~, positive; + , slightly.
o f the sweet smell in a r o m a t i c red rice. A r o m a t i c r e d rice w h i c h c o n t a i n e d a c h a r a c t e r i s t i c r e d p i g m e n t a n d a specific a r o m a h a d n o t b e e n p r o p e r l y used in t h e f o o d i n d u s t r y . N o w a d a y s , p u b l i c t a s t e values q u a l i t y a n d n o v e l t y . A r o m a t i c red rice is a n excellent m a t e r i a l t h a t provides an original alcoholic beverage. P r e c i s e a r o m a a n a l y s i s o f a r o m a t i c red rice w i n e a n d t h e m e c h a n i s m o f a r o m a f o r m a t i o n will be r e p o r t e d in o u r next paper.
4. Yamasaki, I. and Ueda, S.: Action of black-koji-amylase on raw starch I. J. Agric. Chem. Soc. Jpn., 24, 181-185 (1951).
5. Fujio, Y., Suyanadona, P., Attasampunna, P., and Ueda, S.: 6.
7. 8.
ACKNOWLEDGMENT The authors are grateful to the Kumamoto Agricultural Research Institute for generously providing various rices and Shinnihon Kagaku Kogyo Co. Ltd. for generously providing the glucoamylase preparations. The authors also thank Bishonen Co. Ltd. for his support during the study.
REFERENCES 1. Maekawa, K. and Shinke, R.: Properties of red rice pigments, and sake brewing by using red rice as raw material. J. Brew. Soc. Japan, 84, 787-793 (1989).
2. Takahashi, K., Sugimoto, T., Miura, T., Wasizu, Y., and Yoshizawa, K.: Isolation and identification of red rice pigment. J. Brew. Soc. Japan, 84, 807-812 (1989). 3. Yokoo, M.: Kome. Seibutsu to Kagaku, 28, 49-55 (1990).
9.
Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking. Biotechnol. Bioeng., 26, 315-319 (1984). Fujio, Y., Ogata, M., and Ueda, S.: Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas ci~'culation type fermentoe Biotechnol. Bioeng., 27, 1270-1273 (1985). Honda, N.: Alcohol handbook, 5th ed. Japanese Assoc. Indust. Ferment., Japan, Tokyo (1978). Nelson, N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375-380 (1944). Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952).
10. Yajima, I., Yanai, T., Nakamura, M., Sakakibara, H., and Hayashi, K.: Volatile flavor components of cooked kaorimai. Agric. Biol. Chem., 43, 2425-2429 (1979). l l. Ishikawa, T. and Yoshizawa, K.: Seishukobo niyoru koki ester no seisei to shibosan, p. 103-115. In Akiyama, Y. (ed.), Kobo no riyo to kaihatsu. Gakkai Shuppan Center, Tokyo (1979).
12. Iwata, H., Nakajima, N., Mizuno, A., Izumi, T., Ikeda, E., Kumagai, C., Umeda, N., and Tanaka, T.: Comparison of aromatic compounds in sake making using Kyokai no. 9 and no. l0 yeast. J. Brew. Soc. Japan, 83, 411-415 (1988). 13. Supriant, Ohba, R., Koga, T., and Ueda, S.: Liquefaction of glutinous rice and aroma formation in tap6 preparation by ragi. J. Ferment. Bioeng., 67, 249-252 (1989).