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THE KEY PRODUCT IN JAPANESE ALCOHOLIC BEVERAGES AND FERMENTED FOODS
Tokyo University of Agriculture, Sedagaya-ku, Tokyo, Japan
ABSTRACT Koji is a culture of koji mold, Aspergilhs grown on cereal substrates. Koji making process is one of the most typical solid state cultivation. In Japan, both alcoholic beverages and fermented foods are made with koji, although different strains and substrates are used depending on the final product. i t is the most important role of koji to supply various enzymes. These enzymes generally decompose complex compounds, including carbohydrates, proteins and fats, into smaller molecules. In addition, koji is an important contributor of flavor, aroma and color materials to the final products. In submerged culture, none of such substances are produced in them. Accordingly, attempts to use a submerged culture broth as a substitutes for koji were unsuccessful. It is worth noting that no aflatoxin-producing strains are found among industrial strains of koji mold. Conventionally, koji is prepared by manpower using small wooden trays. This method, however, is very trouble some and needs much labor. Therefore, in order to save labor, automatic methods have been devised successfuily for commercial application. In koji preparation, enzyme production of koji mold is much influenced by temperature. Being cultured at a relatively high temperature, e.g. 45OC. it produce amylase in greater amount. On the contrary, at a relatively low temperature, e.g. 3s0C, it produces protease more abundantly. Improvement of strains by mutation was also carried out. To prevent excess color development of snake, a mutant strain of Aspergillus oryzae lacking deferriferrichrysin productivity was selected by UV irradiation of conidia. As for A. oryzae used in soy sauce production, a strain having higher productivity of protease than ordinary strains was made by UV irradiation followed by protoplast fusion.
Koji is a culture of koji mold, Aspergillus grown on cereal substrates, such as rice, barley or soybean. Koji making process is one of the typical solid state cultivation. i n many Asian countries, Aspergillus are used to produce seasoning agents, such as soy sauce. To produce alcoholic beverages, however, molds other than Aspergillus generally used (Table 1). In Japan, both alcoholic beverages and fermented foods are produced using koji, although different strains and substrates are used depending on the final products (Table 2).
Though there are several species being industrially used in the genus Aspergillus, the most important one is A. oryzae. This mold produces many kinds of enzymes, among which amylase and protease are the most important.
such as shoyu and miso. soy bean soy bean + toasted and ground wheat Note spirits spirits made in Okinawa soy paste soy sauce It is worth noting that no aflatoxin producing strains are found among industrial strains of k o j i mold. Names given in various countries to a starter used to manufacture certain food product. Table 1. Because almost all of the Japanese indigenous alcoholic beverages and fermented foods are made using k o j i molds as enzyme sources. Table 2. Name Sake Shochu A wamori Miso Sho yu Microorganism A. Koji used in Japanese indigenous beverages and fermented foods. kawachii A. Name Country Philippines Indonesia Malaysia Thailand China Korea Nepal Microorganism" Rhizopus Absidia Mucor A m ylom yces Endornycopsis Sacchromyces Candida Ragi Ragi Luk-pang Chuzu Nurook Murcha * Microorganisms listed here were isolated from these starters by many workers. . flavus had become a serious problem for Japanese. oryzae A. soyae Substrate white rice rice broken rice rice. awamori A. and none of kojimold has been found t o produce them. oryzae. One strain may produce more amylase and less protease and vice versa. oryzae A. barley. oryzae A. Therefore. The k o j i molds have long been considered t o belong t o Aspergillus oryzae and/or Aspergillus flavus by many investigators. the Japaneses have extensively looked at the productivity of aflatoxins by A. outbreak of aflatoxin being produced by A.Aspergillus oryzae includes many strains in which there are slight graded variations in morphological and physiological properties from strain t o strain. Strains producing much amylase are used for sake brewing and others producing much protease are used for making fermented foods.
32 for miso. 1 97 1 ). Number of industrial strains of AspergiNus examined for their aflatoxin productivity by Murakami and Yokotsuka. Tane-koji is a koji which has a great deal of spore and yellow-greenish color. Later. These are dried t o 10% moisture content in the sun or by fire. by a multivariate statistical anaiysis. None of koji is made with spontaneous fermentation. and cultivated at about 30° C. (Table 3). The cultivation is finished after 5-6 days when the materials became abundunt with spores. they preserved it in a form of tane-koji for a long time. So. oryzae is one of the oldest horticultural plant in the world. Once they got such a strain as they had expected. Table 3. and made by special makers in Japan. tane-koji is produced and distributed t o sake brewers or food factories by some ten or so makers. 44 for shoyu. tane-koji makers have been isolating a strain of A. A t present. The steamed brown rice is mixed with wood ashes and inoculated with spores of some specific molds which have been prepared i n advance. (Murakami. then packed. though there were some strains producing compounds that were very similar t o those of aflatoxins i n fluorescence spectra and Rf values in thin layer chromatograms. oryzae which has superior quality. (19 6 7 ) likewise studied the aflatoxin production in 7 3 industrial strains of Aspergillus and found all of them t o be negative. tane-koji is used as inoculum. (1 967. 16 for spirits and 22 wild strains) for the production of aflatoxin after cultivation in agar slants and in a liquid medium by shaking. A n objective of using ashes is t o give molds some nutrients and t o . It is reasonable t o say that A. oryzae group and definitely discriminated from the A. neither alcoholic beverages nor fermented foods are made with wild strains of A. Investigator Strains For sake For rniso For shoyu For spirits Total Murakami Yokotsuka TANE-KOJI (SEED KOJI) To make koji. 1 9 6 8 ) investigated 214 industrial strains of Aspergillus ( 100 for sake. the tane-kojimay contain about 4 x 1O9 spores per g. No aflatoxin producers was found. oryzae. For the past millenium. flavus group producing aflatoxin. in Japan.Murakami et a/. the koji mold were proved t o belong t o the A. Tane-koji is made of brown rice. Yokotsuka et a/.
1 9 5 6 ) (Table 41.1 %.prevent the media from bacterial contamination by giving a high pH t o the media. After the steamed rice has been cooled t o about 3 s 0 C . After 10. proteins and fats. it is the most important role of k o j i to supply various enzymes. the greater the activities of amylase (Suzuki eta/. the k o j i mass should be stirred periodically t o maintain uniform temperature. As mold growth continuesly. moisture and airation. the k o j i making process usually is finished. Cultural conditions influence the formation of enzymes. the k o j i mass in the trays is mixed by hand. A t 20 hours. Forty four hours after the inoculation. a t 6-8 hour intervals. in which temperature (28OC) and humidity are controlled at suitable level for the growth of mold. Acid protease Alkaline protease * Numerical value shows relative activity of enzymes. oryzae strains. none of such substances are produced. including carbohydrates. 1957) (Table 5). Accordingly. aroma and cotor materials t o the final products. at this stage the developing k o j i is distributed in small shallow wooden trays t o about 5 c m in depth. the temperature of the k o j i mass could rise t o 40°C or higher. Temp. a slight mycerial growth is apparent. kojiis made with small wooden trays b y manpower. it is transferred into the cultivation room. Then the rice is mixed thoroughly and heaped on a wooden bed. In general. and lower temperatures favor the development of protease activities (Suzuki eta/. Kojihas a pleasant smell. In addition. ConventionalKoji Making Process Conventionally. Effect of cultivation temperature on protease activity of rice koji. Tane-koji contains at least t w o or three different strains of Aspergillus oryzae which have been mixed by each maker secretly as his particular product. attempts t o use a submerged culture broth as a substitutes for koji were unsuccessful yet. into smaller molecules.1 2 hours. the higher the cultivation temperatures. and is quite sweet in taste. Thereafter. In submerged culture broth. the rice is mixed again and heaped. 84 . These enzymes generally decompose complex compound. Tane-koji is sprayed on t o rice a t a rate of 0. rice grain is completely covered w i t h white mycelium of the inoculated A. Table 4. The Role of Koji Undoubtedly. and the trays are stacked on a shelf. A t this stage. Therefore. k o j i is an impoiiant contributor of flavor.
(1 967) found ferrichrysin. A. because of excess color development of produced sake. RECENT PROGRESS OF KOJl MAKING TECHNOLOGY Recent progress of koji making technology goes toward t w o directions. Based on the above described reason. 01 -Amylase Glucoamylase 32OC C 43 O 100" 140" 100" 132" " Numerical value shows relative activity of enzyme. in sak8. of DFC" (mglkg of koji) * DFC: Deferriferrichrysin.1 1 (mutant) RIB 647 (parent) RIB 647-88 (mutant) RIB 326 (parent) RIB 326-77 (mutant) Conc.Table 5 . One is the improvement of used strains. and the other is the development of koji making machine. Deferriferrichrysin productivity of Aspergillus oryzae. oryzae lacking deferriferrichrysin productivity. Effect of cultivation temperature on amylase activity of rice koji. but it is difficult t o decrease iron in water t o this level. it has been well known that iron containing water is not suitable for sake brewing. another trial was attempted t o get a mutant strain of A. oryzae used in rice-koji making produces deferriferrichrysin. Temp. Strain RIB 203 (parent) RIB 203-27 (mutant) RIB 1 5 5 (parent) RIB 1 55.02 ppm of iron. To prevent sake from excess color development. DeferriferrichrysinNon-ProducingMutants o Aspergillus oryzae f For the past 100 years. Table 6. it was concluded that the brewing water for sake should not contain more than 0. red colorant containing iron. but its mechanism was not elucidated before Tadenuma eta/. which chelates ferric ion t o form ferrichrysin. .
8 5 3 6 No.76 cys- n.2 7 now put into commercial use. Rice-kojimade with one of these 6 strains (RIB 2 0 3 2 7 ) contains no detectable amount of deferriferrichrysin and shows equal amylase activity t o that of parent strain. Phenotype Color green yellow green white Requirement Growth' (mm) Protease ( x 1o2 unitlg) 4. Strain No. No. ( 1 9 7 4 ) selected 6 mutant strains having lower deferriferrichrysin productivity than their parent strains. they derived new mutant strains having nutritional marker using UV irradiation (Table 7).5 (x I unitlg) o2 9. 13. 13 ac7 No. 8 5 3 6 was nicotinic acidless and had white conidia. $536. 1 3 using UV irradiation before. which was derived from strain No.a. The mutant strain RIB 2 0 3 . 13 was cysteinless and had yellow conidia. Breeding of Aspergillus oryzae by Protoplast Fusion For Soy Sauce Production A. Hara eta/. 1 3 Growth (mm) 76. oryzae var.2 * Growth was indicated by measuring the diameter of colonies of 5 days culture on koji extract agar. 13 and exhibited faster growth than strain No.30 4.- 78.5 78. Properties of green strains.9 49.0 49. produces $-times more protease than strain No. ( 1 9 8 3 ) carried out by protoplast fusion t o breed a new strain which produced more protease than strain No. Table 8.5 50. 13 for soy sauce production exhibits fast growth. Strain No. g 201 g 344 No.85 33.38 4.68 9. Mutant strain No.38 32. 8 5 3 6 ac14 Properties of used strains. 8 5 3 6 . Strain ac7 derived from strain No. both parent and its mutant strains. Strain ac14 derived from strain No. but exhibits slow growth.1 76. Furuya et a/. Table 6 shows deferriferrichrysin productivity of Aspergillus oryzae. No.By UV irradiation of conidia on the surface of Czapek's medium. Table 7.14 33.5 78. A t first.30 Protease .
they fused effectively and formed heterocaryons. Drain Eliminator Shower Damper Air Filter / Thermometer Koji Fig.Protoplasts of t w o mutant strains were obtained'using snail gut juice. fast growth.3-times more protease than strain No. 2 shows the fully automatic k o j i making machine. T w o of the green strains derived from the heterocaryon showed high stability. 13 (Table 8). however. . The fusion frequency was 1 %. 1 . charge of the steamed rice and discharge of kojiare carried out by manpower. Probes buried in the koji monitor the temperature and humidity of the air blast to maintain conditions in the k o j i optimal for mold growth and enzyme production. Semi automatic koji making machine. Fig. producing 2. automatic kojimaking machine have been devised successfully for commercial application. In this machine. which has t w o cultivation chambers. many fermentation factories are so large that they usually produce five or more k/ of sake or shoyu a day. The bottom of the bed is perforated and a steady stream of forced and filtered air blow through k o j i mass. When the protoplasts of the t w o strains were treated with a solution containing polyethylene glycol. and make one or more ton of kojia day. Fig. Development of Koji Making Machine In Japan. 1 shows the semi automatic k o j i making machine having a fixed bed. upper and lower. Since the conventional k o j i making process is very troublesome and needs much labor. and each chamber has the rotary bed.
M. 13. M.. Y. AND MURAKAMI. OBA.S. Y..AND HIROSHIMA. 4 1. charge of the steamed rice. ( 1 968).Upper Screw Cultivation Stirring for outlet Chamber Apparatus Air Conditioner /Control Panel Screw for Outlet Rotary Bed ' Lower Cultivation Chamber (By courtesy of NAGATA BREWlNG MACHINE Co. S.. MURAKAMI. UCHIDA. AND SATO. J. H. Appl.M. S.. REFERENCES FURUYA. Gen. 477. (1983). KIKUCHI.. ISHIGAKI. SUZUKI.(1967). TADENUMA. HASEGAWA.. MURAKAMI.. 1482. NUNOKAWA.J. JAPAN) Fig. SASAKI. 32. Microbiol.. I n this fully automated machine. The diameter of the largest machine is ten meter and has the capacity of producing 10 ton of koji a day. 97. G. JozokyokaiZasshi. MURAKAMI. T. Y. (1967). (197 1). HONGO. Kaishi. M. ASAO. Biol. S. 306. K. 52. H. S. T. TAKASE. Appl. A.. K. The steamed rice is charged into the upper chamber. AND YOSHINO. TAKASE. 52. ITO. H. T. Microbiol. AND KUWABARA. AND SEKIZAWA... ( 1967).. Microbioi. Nippon Nogeikagaku T. K. Gen.. SAGAMIHARA. 14. HARA. (1957).. 2. Gen.. Agr. Y.. HakkokogakuZasshi. 1 54. TANAKA. koji mass are transferred into the lower chamber.. 28 1. NUNOKAWA. M. YOKOTSUKA. J. T. AND NOBUHARA. K. 1 7. Fully automatic koji making machine. H. After 20 hours cultivation. stirring and discharge of koji are carried out mechanically.. 9 14. (1974).. and cultivated for 24 hours with stirring at intervats. 323. M. . Nippon Nogeikagaku Kaishi. Nippon N. inoculation. Nippon S. (1956). KOEAYASHI.. AND ISHI. SUGAMA. H. 3 1. Nippon JozokyokaiZasshi. Appl. Chern. 57. 5 1. SUZUKI. 1 .
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