- Email: [email protected]
Mycelial propagation and enzyme production in koji prepared with Aspergillus oryzae on various rice extrudates and steamed rice
JOURNAL. OF FERMENTATION AND BIOENGINEERING Vol. 79, No. 5, 509-512. 1995
Mycelial Propagation and Enzyme Production in Koji Prepared with Aspergillus oryzae on Various Rice Extrudates and Steamed Rice CHENG-CHUN
CHOU*
AND
JIN-HWEI
RWAN
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Received
11 July 1994/Accepted
24 January
1995
Rice flour with varied water content (11,15,20 and 25%) was extruded in a Brabender single-screw extruder at different barrel temperatures (190, 205 and 22OY). Growth and enzyme production during solid-state cultivation (koji making) of Aspergillus oryzae on the rice extrudate substrates and steamed rice were compared. The results revealed that the extent of mycelial propagation, the activities of amylase, neutral protease and acid protease, as well as the maltose-forming activity, varied according to the substrate used for koji making. Among the substrates tested, the greatest production of enzymes and growth of A. oryzae were found in koji prepared with a rice substrate extruded at 205°C and with an 11% water content. [Key words:
Aspergillus oryzae, koji,
rice extrudates,
Corresponding
propagation,
enzyme
production]
the extruded rice substrate, rice grain was first ground into flour (60 mesh). Various rice extrudates were prepared by using a single-screw extruder (Do-coder E330; Brabender Ohg, West Germany) under the following extrusion conditions: screw speed, 130 rpm; barrel temperature, 190, 205 or 220°C; feed moisture content 11, 1.5, 20 or 25%; feed rate 95 g/min; die diameter 6 mm. Steamed rice was prepared by soaking rice grain in water (1 : 1, w/w) overnight and steaming at 121°C for 30min after water was decanted. For the preparation of koji, various rice extrudates were first cut to a size similar to a steamed-rice granule. The moisture content of both the rice extrudates and steamed rice was adjusted to 50% and they were inoculated with 10% of the inoculum. They were then incubated in an automatic rice fermentor (Mini 15 PX; Yaegaki, Japan) in which the temperature and relative humidity were maintained at 30°C and greater than 90x, respectively, for 120 h. The mycelial mass in the koji was estimated by determining the amount of glucosamine, as described by Sakurai et al. (11). Dry weight was measured by weighing samples which had reached a constant weight after exposure to 105°C. Enzymes in the koji were extracted from log of koji with 100 ml of 1.O% NaCl solution by shaking for 2 h at 20°C and then filtering through Whatman no. 1 filter paper. The filtrate was used as the enzyme solution. A modification of the Wohlgemuth method (12) was used to determine a-amylase activity. One unit of (ramylase activity was defined as the amount of enzyme that catalyzed the hydrolysis of 10 mg soluble starch in 1 min under the assay conditions. To determine the maltose-forming activity, we also followed the modified Wohlgemuth method (12) and measured maltose in the reaction solution according to the DNS method (13). One unit of maltose-forming activity was defined as the amount of enzyme that liberated 1 /fg maltose per minute under the assay conditions. It is worth mentioning here is that cr-amylase, $-amylase, glucoamylase and n-glucosidase present in the assay enzyme solution may all contribute to the maltose-forming activity observed. A
Molds are involved in the preparation of most oriental fermented foods such as sake, soy sauce and miso. The starter of these fermented products, termed koji, is generally prepared by growing molds on steamed or cooked rice, soybeans, wheat bran or a mixture of these (1). In koji, molds serve as one of the main sources of hydrolytic enzymes such as amylases, proteases, lipases and many others to hydrolyse substrate components. Much effort is expended industrially to promote the rapid growth of molds and to increase enzyme activity in koji, which are regarded as essential both to reducing the processing duration and obtaining products of improved quality (1, 2). A high-temperature, short-duration food extruder may be regarded as a reactor for the chemical and physical modification of biological materials, including protein and starch (3-5). Extrusion processes have been applied in the food and feed industries for many years, but only a few studies on the pretreatment of the fermentation substrates have been reported (6-10; Imayasu, S. et al., Japanese patent, Showa 61-205479 (3), 1986). We thus conducted experiments to compare the growth and enzyme production by A. oryzae on various rice extrudates and steamed rice, which is the main substrate used to produce fermented foods such as sake, miso, tou-chiang and tien-chiu-niang, among others (1). Aspergillus oryzae no. 30428 obtained from the Food Industry Research and Development Institute, Shinchu, Taiwan and was used as the test organism. To prepare the inoculum, potato dextrose agar (Difco, Michigan, USA) in flasks were inoculated with one loopful of the activated test organism and incubated at 30°C for 4 d. Spores of the test organisms were harvested by flooding the surface of the agar with sterile distilled water containing 0.1% Tween 80. The spore suspension was adjusted with sterile distilled water to a concentration of cu. 106/ml and this served as the inoculum for koji making. Japonica rice (Taichung 189) with a polishing ratio of about 87%, was obtained in Taipei, Taiwan. To prepare *
mycelial
author.
509
510 TABLE
CHOU
J. FERMENT.BIOENG.,
AND RWAN
1. Maximum
propagation Max. mycelial propagation”
Substrate Steamed rice Rice extrudate 19O”C, 11% 19O”C, 15% 19O”C, 20% 19O”C, 25% 205°C. 11% 205”C, 15% 205°C. 20% 205”C, 25% 22O”C, 1 I,% 22O”C, 15% 22O”C, 20% 220°C. 25%
mycelial
HZ0 H,O H,O Hz0 Hz0 Hz0 HZ0 H,O Hz0 H,O H20 H,O
and enzyme
activities
during
Max. maltose-forming activitvb
of koji on steamed
the preparation
+Amvlase
58.9 (60)
40.0 (60)
21.0 19.9 18.6 17.3 21.6 21.1 20.6 18.6 19.9 20.3 18.1 16.1
76.8 60.0 55.2 49.2 94.2 75.6 72.0 50.4 75.6 72.0 61.2 39.6
53.0 42.0 36.0 31.5 69.0 65.0 47.5 40.0 63.0 54.0 45.0 41.0
(120) (120) (120) (120) (84) (96) (120) (96) (120) (120) (108) (120)
(120) (108) (120) (120) (72) (120) (108) (84) (120) (120) (120) (120)
Neutral
6.6 (96) 12.2 11.4 9.8 8.6 16.0 Il.8 10.8 10.0 11.6 10.0 9.0 7.2
rice extrudates
activity’
Acid urotease
17.1 (120)d (120) (120) (120) (120) (120) (120) (120) (120) (120) (120) (120) (120)
rice and various
Max. enzyme
urotease
4.2 (96)
(96) (96) (96) (96) (96) (96) (96) (96) (108) (96) (96) (96)
11.4 9.0 8.0 7.0 14.0
(96) (96) (96) (96) (96) 11.4(96) 10.4 (96) 10.0 (96) 11.8 (108) 10.2 (96) 9.2 (96) 7.4 (96)
a Data are presented as mg glucosamine/g dry matter. b Data are presented as units/g dry matter. c For tu-amylase, data are presented as units/g dry matter, and for acid and neutral protease as IO2units/g dry matter. d Numbers in parenthesis indicate the cultivation time (h) when the max. growth or max. enzyme activity was observed. modification of Anson’s method (12) sure acid and neutral protease activity
was used to meaat pHs 3.0 and 6.0, respectively. One unit of protease activity was defined as the amount of enzyme that liberated 1 1’8 of tyrosine in 1 min. Maximum mycelial propagation of A. oryzae expressed tivities vation
with an llfd feed moisture content, this time after 96 h of cultivation. Regardless of the barrel temperature during the extrusion process, rice extrudates obtained with a lower feed moisture content enabled A. oryzae to 80
by the glucosamine content and the maximum acof the various enzymes observed during the culti-
of A. oryzae on steamed rice and various rice extrudates are summarized in Table 1. Although the maximum mycelial propagation on rice extruded at 220°C and with a 25% feed moisture content, which had a glucosamine content of 16.1 mg/g dry matter, was less than that on steamed rice (17.1 mg/g dry matter), higher maximum growth of A. oryzae than that on steamed rice was observed on all the other rice extrudates. Among the extrudates examined, the test organism grew best on that obtained by extrusion at 205°C and with a feed moisture content of 1lyi. After cultivation for 120 h, a glucosamine content of 21.6 mg/g dry matter was observed in koji prepared with this rice extrudate. It is also interesting to note that extruding rice flour at 190°C or 205°C and decreasing the feed moisture content from 25 to 11% resulted in extrudates more suitable for the growth of A. oryzae. Maltose-forming and cr-amylase activities in steamed rice reached maxima of 58.9 and 40.0 units/g dry matter, respectively, after about 60 h of cultivation. In the rice extrudates, maximum maltose-forming and n-amylase activities varied with the extrusion conditions, but in all cases a longer cultivation duration was required than with steamed rice for these activities to reach maximum in the rice extrudates (Table 1). Some rice extrudates were found to give a higher yields of these enzymes than steamed rice did. Among all the substrates used for koji preparation, the rice extrudate prepared at 205°C with 11% moisture showed the highest maximum cu-amylase and maltose-forming activities of 69.0 and 94.2 units/g dry matter, respectively, after about 72-84 h of cultivation. When grown on rice extrudates, A. oryzae also produced higher maximum activities of acid and neutral protease than those produced in steamed rice, the highest being 1,600 and 1,400 units/g dry matter, respectively, which were also observed in rice extruded at 205°C and
r
C 0-M
P
Cuittvotion
time(h)
Cultivation
Cultsvation
time
(h)
time(h)
Cultivation
Cultivotlon
time(h)
time(h)
FIG. 1. Time courses of mycelial propagation and enzyme activity during the preparation of koji on steamed rice and rice extrudate. (A) Mycelial propagation; (B) maltose-forming activity; (C) namylase; (D) acid protease; (E) neutral protease. Symbols: 0, steamed rice; C), rice extrudate (205”C, 11% H,O).
NOTES
511
FIG. 2. Scanning electron micrographs of mycelial propagation on koji. (A) Rice extrudate (205, 11%moisture); (B) mycelial propagation on rice extrudate (205, 11% moisture); (C) mycelial propagation on steamed rice. For scanning electron microscopy, samples were first fractured after drying and mounted on specimen holders (copper-zinc alloy) with conductive adhesive. The samples were then coated with 200 gold metal in an IB-2 ion coater (Giko Engineering Co., Japan) and viewed on a Hitachi scanning electron microscope (Model S-550, Hitachi Co., Japan) with _ an accelerating voltage of 20 KV. exhibit greater more enzyme activity. Time courses of the mycelial propagation
of the test organism and enzyme activity in steamed rice and rice extrudate obtained by extrusion at 205°C and with a feed moisture of 11% are presented in Fig. 1. Although greater mycelial propagation was found in steamed rice than in the rice extrudate at 24 h cultivation, as the cultivation continued the rate of mycelial propagation in the extrudate became higher than that in steamed rice. After a 120-h cultivation period, maximum mycelial propagations of 21.6 and 17.1 mg glucosamine/g dry matter were found in the rice extrudate and steamed rice, respectively (Fig. 1A). As noted above, Figs. 1B and lC, show that maltose-forming and n-amylase activities in the extrudate reached their maxima after about 72-84 h, compared with about 60 h in steamed rice. Beyond this period, the activities of these enzymes remained rather stable in both substrates. A similiar trend in the changes of acid and neutral protrase activities in steamed rice and the rice extrudate was observed during the entire cultivation period (Figs. 1D and 1E). The activities of these enzymes increased with cultivation time and reached their maxima at about 96 h. Further extension of the cultivation time resulted in a decline of the enzyme activities in both steamed rice and the extrudate. The protease activity of the rice extrudate was significantly higher than that in steamed rice. High temperature, high shear force and high pressure in the extrusion process cause physical and chemical modifications of food components (3, 4, 14, 15). Mercier and Feillet (4) showed that starch can be liquified by extrusion cooking at low moisture levels, Linko et al. (9) indicated that extrusion-liquified barley starch can be saccharified rapidly by glucoamylase without any prior treatment with a-amylase, while Chay et al. (6) demontrated the adequacy of using extrusion-hydrolyzed starch and flours as fermentation substrates for ethanol production by yeasts. On the other hand, Okamoto et al. (10) reported that sake obtained from extruder-cooked rice contained higher amounts of aroma components than that prepared with steamed rice. We investigated some physicochemical properties of the tested extruded rice as
affected by the feed moisture and barrel temperature. The degree of gelatinization (DG), and the contents of water-soluble carbohydrate (WSC) and water-soluble protein (WSP) of the extruded rice obtained were higher than those of steamed rice. Among the extrudates, rice extrudate (205”C, 11% moisture) exhibited the highest DG, WSC and WSP of about lOO.OO%, 37.75% and ll.SO%, respectively, compared to 87.00%, 6.50% and 4.70% found in steamed rice (16). Additionally, the rice extrudates were more susceptible to the activities of aand P-amylase than steamed rice (16) and became porous in structure (Fig. 2A), thus providing more suitable conditions for the growth of molds than steamed rice. As shown in Fig. 2B, an abundant mycelial growth was observed in the pores of the rice extrudate, whereas mycelial propagation was limited on the surface of the steamed rice (Fig. 2C). Therefore, chemical and physical changes in rice extrudates caused by the extrusion may account for the improved growth and greater enzyme production by A. oryzae observed in this study. This observation is consistent with the report of Ota et al. (17), which showed that koji made from puffed rice has higher acid protease and acid carboxypeptidase activities than that made from ordinary steamed rice. A similiar phenomenon was reported by Fujita et al. (7) and Imayasu et al. (Japanese patent, Showa 61-205479 (3), 1986), who showed that extruding a mixture of grains for koji making resulted in higher enzymes activities. Fujita et al. (7) also indicated that during further moromi mashing, the nitrogen and sugar utilization rate was increased when the koji was prepared with an extruded grain mixture. Based on the data obtained from this study along with the high susceptibility of extrudates to enzyme activities (16), we conclude that rice pretreated with an appropriate extrusion process becomes a more suitable substrate for microbial growth and enzyme production. An extrusion process at a high temperature for a brief duration may beneficially replace the protracted and tedious cooking processes that are currently employed for the pretreatment of rice or other grain substrates in the fermentation industries. Instead of steamed rice, a rice extrudate may be a useful alternative sub-
512
CHOU
J. FERMENT.BIOENG.,
AND RWAN
strate for the fermentation of sake, miso, vinegar, etc., thereby shortening the entire fermentation period, increasing the fermentation yield and to reducing the cost. This research was supported Development Council (ROC).
by a grant
from
the Agricultural
REFERENCES 1. Su, Y. C.: Traditional fermented food in Taiwan, p. 15-45. In Proceedings of the fermented foods. Food Industry Research and Development Institute, Taiwan, Republic of China (1980). changes occurring during the fer2. Whitaker, J. R.: Biochemical mentation of high-protein foods. Food Technol., 32, 175-180 (1978). CRC Crit. Rev. Food Sci. 3. Harper, J. M.: Food extrusion. Nutr., 11. 155-215 (1978). of carbohydrate com4. Mercier, C. and Feillet, P.: Modification ponents by extrusion cooking of cereal products. Cereal Chem., 52, 283-297 (1975). 5. Gomez, M. H. and Aguilera, J. M.: Changes in the starch fraction during extrusion cooking of corn flour. J. Food Sci., 48, 378-3812 (1983). 6. Chay, P. B., Chouvel, H., Cheftel, J. C., Ghommidh, C., and Navarro, J. M.: Extrusion-hydrolyzed starch and flour as fermentation substrates for ethanol production. Lebnsm-Wiss. u.Technol., 17, 257-267 (1984). A., Watanabe, Y., Kishi, K., Ogawa, G., and 7. Fujita, Yoshizaki, T.: Evaluation of the coextruding process of wheat bran, flour and soybean meal for making soy sauce koji.
Nippon Shokuhin Kogyo Gakkaishi, 27, 483-488 (1980). 8. Motoi, H., Watanabe, Y., Kishi, K., and Yoshizaki, T.: The studies on the soy sauce fermentation by reducing koji amounts using co-extruded wheat and soybean flour (part 2). J. Jpn. Soy Sauce Res. Inst., 8, 254-259 (1982). S., and Linco, Y.Y.: Extrusion cooking 9. Linco, P., Hakulin, of barley starch for the production of glucose syrup and ethanol. J. Cereal Sci., 1, 275-284 (1983). T., Kobayashi, Y., Ono, A., Takahashi, Y., and 10. Okamoto, Tashizo, M.: Use of twin screw extruder cooked rice in sake brewine. J. Brew. Sot. Jauan, 85, 506-513 (1990). 11. Sakurai, Y., Lee, T. H., and. Shiota, H.: On the convenient method for glucosamine estimation in koji. Agr. Biol. Chem.. 41, 619-624 (1977). University: Experimental agricultural chemistry. I. 12. Tokyo Asakura Shokuden, Tokyo (1962). acid reagent for determi13. Miller, G. L.: Use of dinitrosalicyclic nation of reducing sugar. Anal. Chem., 31, 426-428 (1959). J.. van de Voort, F. R., and Stanley, D. W.: 14. Owusu-Ansha. Physicochemical changes in corn starch as function extrusion variables. Cereal Chem., 60, 319-324 (1983). R. and Hanna, M. A.: Relationship between 15. Chinnaswamy, amylose content and extrusion expansion properties of corn starches. Cereal Chem., 65, 138-143 (1988). 16. Rwan, J. H., Chou, C. C., and Lii, C. Y.: Effect of feed moisture and barrel temperature on some physicochemical properties of extruded rice. Food Sci., 20, 441-449 (1993). 17. Ota, T., Kuwahara, K., Obata, T., Imamura, T., Hara, S., and Yoshizawa, K.: Brewing of sake using polished (r-rice made from preboiled rice grain. J. Brew. Sot. Japan, 81, 329332 (1986).
Mycelial Propagation and Enzyme Production in Koji Prepared with Aspergillus oryzae on Various Rice Extrudates and Steamed Rice CHENG-CHUN
CHOU*
AND
JIN-HWEI
RWAN
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Received
11 July 1994/Accepted
24 January
1995
Rice flour with varied water content (11,15,20 and 25%) was extruded in a Brabender single-screw extruder at different barrel temperatures (190, 205 and 22OY). Growth and enzyme production during solid-state cultivation (koji making) of Aspergillus oryzae on the rice extrudate substrates and steamed rice were compared. The results revealed that the extent of mycelial propagation, the activities of amylase, neutral protease and acid protease, as well as the maltose-forming activity, varied according to the substrate used for koji making. Among the substrates tested, the greatest production of enzymes and growth of A. oryzae were found in koji prepared with a rice substrate extruded at 205°C and with an 11% water content. [Key words:
Aspergillus oryzae, koji,
rice extrudates,
Corresponding
propagation,
enzyme
production]
the extruded rice substrate, rice grain was first ground into flour (60 mesh). Various rice extrudates were prepared by using a single-screw extruder (Do-coder E330; Brabender Ohg, West Germany) under the following extrusion conditions: screw speed, 130 rpm; barrel temperature, 190, 205 or 220°C; feed moisture content 11, 1.5, 20 or 25%; feed rate 95 g/min; die diameter 6 mm. Steamed rice was prepared by soaking rice grain in water (1 : 1, w/w) overnight and steaming at 121°C for 30min after water was decanted. For the preparation of koji, various rice extrudates were first cut to a size similar to a steamed-rice granule. The moisture content of both the rice extrudates and steamed rice was adjusted to 50% and they were inoculated with 10% of the inoculum. They were then incubated in an automatic rice fermentor (Mini 15 PX; Yaegaki, Japan) in which the temperature and relative humidity were maintained at 30°C and greater than 90x, respectively, for 120 h. The mycelial mass in the koji was estimated by determining the amount of glucosamine, as described by Sakurai et al. (11). Dry weight was measured by weighing samples which had reached a constant weight after exposure to 105°C. Enzymes in the koji were extracted from log of koji with 100 ml of 1.O% NaCl solution by shaking for 2 h at 20°C and then filtering through Whatman no. 1 filter paper. The filtrate was used as the enzyme solution. A modification of the Wohlgemuth method (12) was used to determine a-amylase activity. One unit of (ramylase activity was defined as the amount of enzyme that catalyzed the hydrolysis of 10 mg soluble starch in 1 min under the assay conditions. To determine the maltose-forming activity, we also followed the modified Wohlgemuth method (12) and measured maltose in the reaction solution according to the DNS method (13). One unit of maltose-forming activity was defined as the amount of enzyme that liberated 1 /fg maltose per minute under the assay conditions. It is worth mentioning here is that cr-amylase, $-amylase, glucoamylase and n-glucosidase present in the assay enzyme solution may all contribute to the maltose-forming activity observed. A
Molds are involved in the preparation of most oriental fermented foods such as sake, soy sauce and miso. The starter of these fermented products, termed koji, is generally prepared by growing molds on steamed or cooked rice, soybeans, wheat bran or a mixture of these (1). In koji, molds serve as one of the main sources of hydrolytic enzymes such as amylases, proteases, lipases and many others to hydrolyse substrate components. Much effort is expended industrially to promote the rapid growth of molds and to increase enzyme activity in koji, which are regarded as essential both to reducing the processing duration and obtaining products of improved quality (1, 2). A high-temperature, short-duration food extruder may be regarded as a reactor for the chemical and physical modification of biological materials, including protein and starch (3-5). Extrusion processes have been applied in the food and feed industries for many years, but only a few studies on the pretreatment of the fermentation substrates have been reported (6-10; Imayasu, S. et al., Japanese patent, Showa 61-205479 (3), 1986). We thus conducted experiments to compare the growth and enzyme production by A. oryzae on various rice extrudates and steamed rice, which is the main substrate used to produce fermented foods such as sake, miso, tou-chiang and tien-chiu-niang, among others (1). Aspergillus oryzae no. 30428 obtained from the Food Industry Research and Development Institute, Shinchu, Taiwan and was used as the test organism. To prepare the inoculum, potato dextrose agar (Difco, Michigan, USA) in flasks were inoculated with one loopful of the activated test organism and incubated at 30°C for 4 d. Spores of the test organisms were harvested by flooding the surface of the agar with sterile distilled water containing 0.1% Tween 80. The spore suspension was adjusted with sterile distilled water to a concentration of cu. 106/ml and this served as the inoculum for koji making. Japonica rice (Taichung 189) with a polishing ratio of about 87%, was obtained in Taipei, Taiwan. To prepare *
mycelial
author.
509
510 TABLE
CHOU
J. FERMENT.BIOENG.,
AND RWAN
1. Maximum
propagation Max. mycelial propagation”
Substrate Steamed rice Rice extrudate 19O”C, 11% 19O”C, 15% 19O”C, 20% 19O”C, 25% 205°C. 11% 205”C, 15% 205°C. 20% 205”C, 25% 22O”C, 1 I,% 22O”C, 15% 22O”C, 20% 220°C. 25%
mycelial
HZ0 H,O H,O Hz0 Hz0 Hz0 HZ0 H,O Hz0 H,O H20 H,O
and enzyme
activities
during
Max. maltose-forming activitvb
of koji on steamed
the preparation
+Amvlase
58.9 (60)
40.0 (60)
21.0 19.9 18.6 17.3 21.6 21.1 20.6 18.6 19.9 20.3 18.1 16.1
76.8 60.0 55.2 49.2 94.2 75.6 72.0 50.4 75.6 72.0 61.2 39.6
53.0 42.0 36.0 31.5 69.0 65.0 47.5 40.0 63.0 54.0 45.0 41.0
(120) (120) (120) (120) (84) (96) (120) (96) (120) (120) (108) (120)
(120) (108) (120) (120) (72) (120) (108) (84) (120) (120) (120) (120)
Neutral
6.6 (96) 12.2 11.4 9.8 8.6 16.0 Il.8 10.8 10.0 11.6 10.0 9.0 7.2
rice extrudates
activity’
Acid urotease
17.1 (120)d (120) (120) (120) (120) (120) (120) (120) (120) (120) (120) (120) (120)
rice and various
Max. enzyme
urotease
4.2 (96)
(96) (96) (96) (96) (96) (96) (96) (96) (108) (96) (96) (96)
11.4 9.0 8.0 7.0 14.0
(96) (96) (96) (96) (96) 11.4(96) 10.4 (96) 10.0 (96) 11.8 (108) 10.2 (96) 9.2 (96) 7.4 (96)
a Data are presented as mg glucosamine/g dry matter. b Data are presented as units/g dry matter. c For tu-amylase, data are presented as units/g dry matter, and for acid and neutral protease as IO2units/g dry matter. d Numbers in parenthesis indicate the cultivation time (h) when the max. growth or max. enzyme activity was observed. modification of Anson’s method (12) sure acid and neutral protease activity
was used to meaat pHs 3.0 and 6.0, respectively. One unit of protease activity was defined as the amount of enzyme that liberated 1 1’8 of tyrosine in 1 min. Maximum mycelial propagation of A. oryzae expressed tivities vation
with an llfd feed moisture content, this time after 96 h of cultivation. Regardless of the barrel temperature during the extrusion process, rice extrudates obtained with a lower feed moisture content enabled A. oryzae to 80
by the glucosamine content and the maximum acof the various enzymes observed during the culti-
of A. oryzae on steamed rice and various rice extrudates are summarized in Table 1. Although the maximum mycelial propagation on rice extruded at 220°C and with a 25% feed moisture content, which had a glucosamine content of 16.1 mg/g dry matter, was less than that on steamed rice (17.1 mg/g dry matter), higher maximum growth of A. oryzae than that on steamed rice was observed on all the other rice extrudates. Among the extrudates examined, the test organism grew best on that obtained by extrusion at 205°C and with a feed moisture content of 1lyi. After cultivation for 120 h, a glucosamine content of 21.6 mg/g dry matter was observed in koji prepared with this rice extrudate. It is also interesting to note that extruding rice flour at 190°C or 205°C and decreasing the feed moisture content from 25 to 11% resulted in extrudates more suitable for the growth of A. oryzae. Maltose-forming and cr-amylase activities in steamed rice reached maxima of 58.9 and 40.0 units/g dry matter, respectively, after about 60 h of cultivation. In the rice extrudates, maximum maltose-forming and n-amylase activities varied with the extrusion conditions, but in all cases a longer cultivation duration was required than with steamed rice for these activities to reach maximum in the rice extrudates (Table 1). Some rice extrudates were found to give a higher yields of these enzymes than steamed rice did. Among all the substrates used for koji preparation, the rice extrudate prepared at 205°C with 11% moisture showed the highest maximum cu-amylase and maltose-forming activities of 69.0 and 94.2 units/g dry matter, respectively, after about 72-84 h of cultivation. When grown on rice extrudates, A. oryzae also produced higher maximum activities of acid and neutral protease than those produced in steamed rice, the highest being 1,600 and 1,400 units/g dry matter, respectively, which were also observed in rice extruded at 205°C and
r
C 0-M
P
Cuittvotion
time(h)
Cultivation
Cultsvation
time
(h)
time(h)
Cultivation
Cultivotlon
time(h)
time(h)
FIG. 1. Time courses of mycelial propagation and enzyme activity during the preparation of koji on steamed rice and rice extrudate. (A) Mycelial propagation; (B) maltose-forming activity; (C) namylase; (D) acid protease; (E) neutral protease. Symbols: 0, steamed rice; C), rice extrudate (205”C, 11% H,O).
NOTES
511
FIG. 2. Scanning electron micrographs of mycelial propagation on koji. (A) Rice extrudate (205, 11%moisture); (B) mycelial propagation on rice extrudate (205, 11% moisture); (C) mycelial propagation on steamed rice. For scanning electron microscopy, samples were first fractured after drying and mounted on specimen holders (copper-zinc alloy) with conductive adhesive. The samples were then coated with 200 gold metal in an IB-2 ion coater (Giko Engineering Co., Japan) and viewed on a Hitachi scanning electron microscope (Model S-550, Hitachi Co., Japan) with _ an accelerating voltage of 20 KV. exhibit greater more enzyme activity. Time courses of the mycelial propagation
of the test organism and enzyme activity in steamed rice and rice extrudate obtained by extrusion at 205°C and with a feed moisture of 11% are presented in Fig. 1. Although greater mycelial propagation was found in steamed rice than in the rice extrudate at 24 h cultivation, as the cultivation continued the rate of mycelial propagation in the extrudate became higher than that in steamed rice. After a 120-h cultivation period, maximum mycelial propagations of 21.6 and 17.1 mg glucosamine/g dry matter were found in the rice extrudate and steamed rice, respectively (Fig. 1A). As noted above, Figs. 1B and lC, show that maltose-forming and n-amylase activities in the extrudate reached their maxima after about 72-84 h, compared with about 60 h in steamed rice. Beyond this period, the activities of these enzymes remained rather stable in both substrates. A similiar trend in the changes of acid and neutral protrase activities in steamed rice and the rice extrudate was observed during the entire cultivation period (Figs. 1D and 1E). The activities of these enzymes increased with cultivation time and reached their maxima at about 96 h. Further extension of the cultivation time resulted in a decline of the enzyme activities in both steamed rice and the extrudate. The protease activity of the rice extrudate was significantly higher than that in steamed rice. High temperature, high shear force and high pressure in the extrusion process cause physical and chemical modifications of food components (3, 4, 14, 15). Mercier and Feillet (4) showed that starch can be liquified by extrusion cooking at low moisture levels, Linko et al. (9) indicated that extrusion-liquified barley starch can be saccharified rapidly by glucoamylase without any prior treatment with a-amylase, while Chay et al. (6) demontrated the adequacy of using extrusion-hydrolyzed starch and flours as fermentation substrates for ethanol production by yeasts. On the other hand, Okamoto et al. (10) reported that sake obtained from extruder-cooked rice contained higher amounts of aroma components than that prepared with steamed rice. We investigated some physicochemical properties of the tested extruded rice as
affected by the feed moisture and barrel temperature. The degree of gelatinization (DG), and the contents of water-soluble carbohydrate (WSC) and water-soluble protein (WSP) of the extruded rice obtained were higher than those of steamed rice. Among the extrudates, rice extrudate (205”C, 11% moisture) exhibited the highest DG, WSC and WSP of about lOO.OO%, 37.75% and ll.SO%, respectively, compared to 87.00%, 6.50% and 4.70% found in steamed rice (16). Additionally, the rice extrudates were more susceptible to the activities of aand P-amylase than steamed rice (16) and became porous in structure (Fig. 2A), thus providing more suitable conditions for the growth of molds than steamed rice. As shown in Fig. 2B, an abundant mycelial growth was observed in the pores of the rice extrudate, whereas mycelial propagation was limited on the surface of the steamed rice (Fig. 2C). Therefore, chemical and physical changes in rice extrudates caused by the extrusion may account for the improved growth and greater enzyme production by A. oryzae observed in this study. This observation is consistent with the report of Ota et al. (17), which showed that koji made from puffed rice has higher acid protease and acid carboxypeptidase activities than that made from ordinary steamed rice. A similiar phenomenon was reported by Fujita et al. (7) and Imayasu et al. (Japanese patent, Showa 61-205479 (3), 1986), who showed that extruding a mixture of grains for koji making resulted in higher enzymes activities. Fujita et al. (7) also indicated that during further moromi mashing, the nitrogen and sugar utilization rate was increased when the koji was prepared with an extruded grain mixture. Based on the data obtained from this study along with the high susceptibility of extrudates to enzyme activities (16), we conclude that rice pretreated with an appropriate extrusion process becomes a more suitable substrate for microbial growth and enzyme production. An extrusion process at a high temperature for a brief duration may beneficially replace the protracted and tedious cooking processes that are currently employed for the pretreatment of rice or other grain substrates in the fermentation industries. Instead of steamed rice, a rice extrudate may be a useful alternative sub-
512
CHOU
J. FERMENT.BIOENG.,
AND RWAN
strate for the fermentation of sake, miso, vinegar, etc., thereby shortening the entire fermentation period, increasing the fermentation yield and to reducing the cost. This research was supported Development Council (ROC).
by a grant
from
the Agricultural
REFERENCES 1. Su, Y. C.: Traditional fermented food in Taiwan, p. 15-45. In Proceedings of the fermented foods. Food Industry Research and Development Institute, Taiwan, Republic of China (1980). changes occurring during the fer2. Whitaker, J. R.: Biochemical mentation of high-protein foods. Food Technol., 32, 175-180 (1978). CRC Crit. Rev. Food Sci. 3. Harper, J. M.: Food extrusion. Nutr., 11. 155-215 (1978). of carbohydrate com4. Mercier, C. and Feillet, P.: Modification ponents by extrusion cooking of cereal products. Cereal Chem., 52, 283-297 (1975). 5. Gomez, M. H. and Aguilera, J. M.: Changes in the starch fraction during extrusion cooking of corn flour. J. Food Sci., 48, 378-3812 (1983). 6. Chay, P. B., Chouvel, H., Cheftel, J. C., Ghommidh, C., and Navarro, J. M.: Extrusion-hydrolyzed starch and flour as fermentation substrates for ethanol production. Lebnsm-Wiss. u.Technol., 17, 257-267 (1984). A., Watanabe, Y., Kishi, K., Ogawa, G., and 7. Fujita, Yoshizaki, T.: Evaluation of the coextruding process of wheat bran, flour and soybean meal for making soy sauce koji.
Nippon Shokuhin Kogyo Gakkaishi, 27, 483-488 (1980). 8. Motoi, H., Watanabe, Y., Kishi, K., and Yoshizaki, T.: The studies on the soy sauce fermentation by reducing koji amounts using co-extruded wheat and soybean flour (part 2). J. Jpn. Soy Sauce Res. Inst., 8, 254-259 (1982). S., and Linco, Y.Y.: Extrusion cooking 9. Linco, P., Hakulin, of barley starch for the production of glucose syrup and ethanol. J. Cereal Sci., 1, 275-284 (1983). T., Kobayashi, Y., Ono, A., Takahashi, Y., and 10. Okamoto, Tashizo, M.: Use of twin screw extruder cooked rice in sake brewine. J. Brew. Sot. Jauan, 85, 506-513 (1990). 11. Sakurai, Y., Lee, T. H., and. Shiota, H.: On the convenient method for glucosamine estimation in koji. Agr. Biol. Chem.. 41, 619-624 (1977). University: Experimental agricultural chemistry. I. 12. Tokyo Asakura Shokuden, Tokyo (1962). acid reagent for determi13. Miller, G. L.: Use of dinitrosalicyclic nation of reducing sugar. Anal. Chem., 31, 426-428 (1959). J.. van de Voort, F. R., and Stanley, D. W.: 14. Owusu-Ansha. Physicochemical changes in corn starch as function extrusion variables. Cereal Chem., 60, 319-324 (1983). R. and Hanna, M. A.: Relationship between 15. Chinnaswamy, amylose content and extrusion expansion properties of corn starches. Cereal Chem., 65, 138-143 (1988). 16. Rwan, J. H., Chou, C. C., and Lii, C. Y.: Effect of feed moisture and barrel temperature on some physicochemical properties of extruded rice. Food Sci., 20, 441-449 (1993). 17. Ota, T., Kuwahara, K., Obata, T., Imamura, T., Hara, S., and Yoshizawa, K.: Brewing of sake using polished (r-rice made from preboiled rice grain. J. Brew. Sot. Japan, 81, 329332 (1986).