AU667100B2 - Method of sake making from non-cooked rice grain - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Mlzoknrnl Sake Brewing Co., Ltd.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Method of sake making from non-cooked rice grain The following statement is a full description of this invention, including the best method of performing it known to me/us:- 44 *444 4 40*444 0 444G*4 4 *4*o 0 4004 4. 04 *0t4 44 4* 4 44 0 4 4 0044 Is..

.4 I I. 0 1A- FIELD OF THE INVENTION The present invention relates to a method of sake making, and more specifically to a method of sake making from non-cooked rice grain.

BACKGROUND OF THE INVENTION Sake is essentially a beverage having an alcohol content of not lower than 15% by volume, obtained by fermentation of cooked rice, sake koji prepared by using e Aspergillus oryzae and seeding yeast mash, wherein the major starting material rice is cooked or otherwise heated and then fermented in the presence of sake koji and seeding yeast mash. This is because cooking the starting a a material rice is very effective in facilitating rice saccharification by the enzyme of koji, as well as in .'20 killing undesirable contaminant bacteria. Supported by e much experience, the practice of cooking the starting material rice has long been common in sake making.

However, among the processes for sake production, the cooking of the starting material rice and koji making processes require much labor and energy. Accordingly, there is urgent need for saving labor and energy in the sake making industry. Another problem has been pointed 2 out that in sake making, particularly in the koji making process, it is almost technically impossible to perfectly eliminate contamination of undesirable bacteria. However, when the starting material rice is charged at least under low temperature and acidic conditions, fermentation is carried out at a rate such that the alcohol concentration exceeds 3.5% after 24 hours. Almost no proliferation of contaminant bacteria occurs. This is because the growth of contaminant bacteria is inhibited or they are killed under such conditions in their lag phase and subsequent initial logarithmic growth phase. It is generally observed, however, that such bacterial contamination and proliferation in sake making are likely to occur during koji making and during charge of cooked rice and koji, and 15 to occur when the rate of alcohol production is low due to

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unsatisfactory conditions at the beginning of fermentation. It would be, therefore, possible to prevent bacterial contamination and proliferation by increasing 0* the rate of alcohol production.

In sake making, koji produced with Aspergillus oryzae is normally used. The starch-decomposing enzyme "amylase" system used in this case mainly comprises a-amylase whose f maltose productivity is relatively high, and small activities of glucoamylase and a-glucosidase both of which produce glucose. In this amylase system, the major enzyme a-amylase does not act on non-cooked rice, while it acts on cooked rice to convert its gelatinized starch to 3 dextrin, then oligosaccharides and finally fermentable maltose and glucose. Therefore, a problem remains unsolved of considerable time requirement in the production and accumulation of the fermentable sugars when cooked rice and a koji produced with Aspergillus oryzae are charged. In addition, the rate of alcohol fermentation from maltose, which accounts for the majority of the resulting fermentable sugars, is considerably lower than that from glucose. In other words, in the conventional method for sake making, it is not until 12 to 14 hours after charging when the reducing sugar content in moromi (mash) exceeds based on glucose content, and fermentation occurs. Moreover, the alcohol concentration obtained 24 hours after mashing is generally at most around For this reason, in the conventional method for sake making, contaminant bacterial proliferation cannot be inhibited perfectly, as stated above.

On the other hand, in China, maotai-chu and go.

Fheng-chu, both distilled alcoholic beverages, have traditionally been produced. The major starting material for fermentation is sorghum, which is saccharified with the glucoamylase produced by a filamentous fungus of the genus Rhizopus inoculated after the starting material is allowed to simply absorb water without cooking. Since the glucoamylase derived from the genus Rhizopus is capable of producing glucose directly from starch, it easily saccharifies non-cooked cereal starch to glucose. It I -4appears that the Chinese empirically discovered this fact and has applied it to the production of the above-mentioned distilled alcoholic beverages.

A few study reports have been presented of direct alcohol fermentation from non-cooked polished rice with different purposes. However, none of these reports referred to sake making, though alcohol producing effects were noted. The commercial enzymes so far used derived from a fungus of the genus Aspergillus or Rhizopuswere very low in the activity and purity, so that the enzyme preparation must be used at high amounts. Furthermore, they contained proteinase which unfavourably affects the quality of sake.

SUMMARY OF THE INVENTION The present invention is directed to providing a method for sake making, wherein labor, energy and natural resources are saved, while retaining the essential 15 quality of sake, and wherein bacterial contamination and proliferation can be 'e prevented by increasing the rate of alcohol production.

It has been found that the use of purified glucoamylase derived from a fungus of the genus Rhizopus makes saccharification possible with an amount of enzyme which is within the legally acceptable range for sake making in Japan, and makes it possible to brew sake directly from the starting material polished rice under the addition of koji in a minimum amount sufficient to provide sake acceptable in aspects such as body.

According to a first aspect of the present invention there is provided a method of sake making comprising the steps of: 25 charging non-cooked polished rice and water with or without an organic acid to a fermenter; adding a purified glucoamylase containing substantially no proteinase and a yeast seed prepared with koji extract thereto; and fermenting them.

Further, according to a second aspect of the present invention there is provided a method of sake making comprising the steps of: charging non-cooked polished rice, water with or without an organic acid and 951205,p:\operrmh,40117-93.339,4 a koji to a fermenter; adding a purified glucoamylase containing substantiaily no proteinase and a yeast seed prepared with koji extract thereto; and fermenting them.

BRIEF DESCRIPTION OF THE DRAWING The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawing which is given byway of illustration only, and thus is not limitative of the present invention, and wherein: Figure 1 is a graph showing time courses of carbon dioxide generation during fermentation of non-cooked polished rice in the presence of purified glucoamylase and a yeast seed in Example 1.

0. 0 a 0 *o 951205,p:\aperrnfih4O117-93.339,5 6 DETAILED DESCRIPTION OF THE INVENTION Unlike the conventional method for sake making, the method of sake making of the present invention is characterized in that the starting material rice is charged without cooking and the amount of koji added is minimized or omitted, and in some cases, the koji is used only to cultivate seeding mash. Preferably, purified glucoamylase is used at 0.02 to 0.1% by weight, based on the non-cooked polished rice, and the purified glucoamylase is preferably derived from Rhizopus delemer or Rhizopus niveus.

In the conventional method for sake making, the starting material rice is necessary to be polished to high S degrees to minimize the content of rice-derived protein or 1..15 decomposition products thereof, since the presence of amino acids at concentrations exceeding a given level in the starting material rice results in production of much fusel oil or production of bitter and other undesirable substances. Specifically, to obtain a starting material rice of reduced protein content by polishing, about 30 to 0 by weight of the husked rice is removed as bran.

Also, the conventional method for sake making requires o koji addition, wherein the koji must be managed under culturing conditions which minimize its proteolytic activity. On the other hand, in sake making according to the present invention, proteinase-free glucoamylase is used in place of koji. Therefore, the starting material 7 rice need not be polished to high degrees; as shown in column C of Table 1, even a degree of polishing of (commonly used for ordinary rice cooking) is acceptable for charging. In the brewing method of the present invention, if necessary, a minimum sufficient .mount of koji may be charged as a starting material fo sake. If koji is added, its amount is normally not more than preferably not more than 10%, and more preferably not more than based on the amount of starting material rice charged. The fermentation being supplied with koji in an amount of more than 15% usually results in fermentation of undesirable order and taste. The sake making by the present methods can be achieved without using koji.

However, the addition of small quantities of koji brings a "015 great growth of yeast, and thus it is recommended to e culture the yeast as the yeast seed mush using koji extract which is now available to obtain commercially.

Although commercially available glucoamylase may be used for the present invention, the use of a commercial product as such slightly affects the flavor of sake,

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o" ~because it contains other enzymes, particularly proteinase and lipase, as impurities. The glucoamylase used for the present invention is therefore preferably a, purified glucoamylase which is free of such undesirable enzymes and from which colored substances have been removed as much as possible. To prepare purified glucoamylase from commercially available glucoamylase, the method of 8 Fukumoto et al. [Hisaichiro Fukumoto, Daisuke Tsuru, Takehiko Yamamoto, Agr. Biol. Chem., 31, 710-717 (1967)], for instance, is used. Specifically, the chromatography on a column of strong anion exchanger such as Duolite A- 102D (manufactured by Diamond Shamrock Corp.) is effective, in which proteinase is adsorbed on the column which glucoamylase passed through down the column with no loss of activity. In the present specification, "purified glucoamylase containing substantially no proteinase" is defined as glucoamylase which is free of proteinase activity or glucoamylase containing proteinase as impurities whose proteinase content is such that the quality of sake is not affected.

"I When no commercial product is used, glucoamylase can 0*#Qoe be obtained by cultivating a glucoamylase-producing fungus and extracting and purifying it from the resulting culture. Glucoamylase-producing fungi which can be used for this purpose include filamentous fungi of the genus Rhizopus. The glucoamylase derived from these fungi exhibits strong saccharifying and decomposing activity on a raw cereal starch, with high decomposition limit. In addition, its application to food processing is a internationally accepted, and proteinase removal is easy.

With these features, said glucoamylase is most appropriate for the object of the present invention. Such filamentous fungi of the genus Rhizopus include, but are not limited to, Rhizopus delemer and Rhizopus niveus, which are 9 preferable glucoamylase-producing fungi.

On the other hand, the glucoamylase obtained from Aspergillus niger or a related fungus is thought to surpass the above glucoamylase in heat resistance and acid tolerance at relatively high temperatures. However, it is understood that the glucoamylase derived from the genus Rhizopus is preferred, considering the fact that 1) at low temperatures, it is very stable even on the acidic side 18 0 C, pH 2.8 to its enzyme stability under these conditions is almost equivalent to that at pH 4.8 to and it is therefore capable of almost completely saccharifying and decomposing cereal starch to glucose, provided that reaction time is sufficient, and that 2) the glucoamylase obtained from Aspergillus niger or a related fungus contains not only proteinase but also **4 carboxypeptidase, which impurity enzymes are not always easy to remove.

4 4 Of course, the fact that a trace amount of a-amylase contributes to the complete saccharification of raw starch 4W 20 to glucose has already been shown by the inventors .64.0 [Takehiko Yamamoto, Izumi Miyahara, Kyohei Mizokami, Denpun Kagaku, 37, 129-136 (1990)]. The glucoamylase used o for the present invention may therefore contain a-amylase derived from the same fungus.

For preparing glucoamylase from a filamentous fungus of the genus Rhizopus, which glucoamylase passes through anion exchangers because its isoelectric point is around rr 10 an extract from cells of a filamentous fungus of the genus Rhizop,1- or a culture of a filamentous fungus of the genus Rhizopus is passed through a column of a strong anion exchanger such as Duolite A2 or Duolite AlOaD (manufactured by Diamond Shamrock Corp.), previously buffered to pH 5.2 to 5.4, to adsorb and remove proteinact', together with co-existing acid dyes, whereby the glucoamylase is recovered in the colorless effluent without loss of glucoamylase activity (the above-mentioned reference). After desalting, the effluent may be appropriately concentrated and then lyophilized or spray-dried to yield highly purifie3 glucoamylase.

Seeding mash is prepared by cultivating a brewing yeast in two ways, wherein the medium used to cultivate 15 the yeast is koji suspension or koji extract previously 0**0 0:00 prepared from the koji suspension. In this cultivation, a lactic acid bacterium may be proliferated at the same time to produce an appropriate amount of lactic acid for prevention of contamination with other bacteria, or an appropriate amount of lactic acid may be added to the medium for yeast cultivation to obtain seeding mash.

To completely ferment polished rice by means of 0* purified glucoamylase according to the brewing method of the present invention, non-cooked polished rice, water with or without lactic acid (hereinafter also referred to as charged water), and where necessary a minimum sufficient amount of koji are charged to a fermenter, and 11 purified glucoamylase containing substantially no proteinase and a yeast seed prepared with koji extract (horeinafter also referred to as yeast seeding mash) are added theretc, followed by fermentation to brew sake.

Specifically, polished rice for sake making is first washed with water, and after the water is thoroughly removed, charged water supplemented with an organic acid such as lactic acid, malic acid and citric acid, for example, about 0.04 to 0.2% lactic acid or about 0.04 to 0.15% malic acid, to sterilize the charged water and charged polished rice, is added to the washed rice, and the mixture is kept standing at 6 to 20 0 C, preferably about 15°C, with a lid, for at least 2 to 3 hours, or overnight if possible (16 hours). Although the pH is 1...15 about 2.7 immediately after the charged water is added to the washed rice, the pH rises during the period when the mixture is being kept standing, reaching 3.2 or higher.

Upon reach of this pH level, purified glucoamylase and seeding mash are added, followed by sake fermentation at a s.

temperature of 6 to 20 0 C, preferably about 15 0 C, as above 4 4 ses under conventional brewing condition management in which the fermenting mash is stirred occasionally(temperature control, stirring, etc.). Here, the amount of purified glucoamylase used is normally 0.02 to 0.1% by weight, preferably 0.05 to 0.1% by weight, based on the non-cooked polished rice. When the amount of the enzyme to be added is over the level of a rapid fermentation occurs 12 which spoils the quality of sake. The use too small amounts of enzyme takes a long time for fermentation. The amount of yeast seeding mash used is set in accordance with conventional sake making. Specifically, it is used in an amount of 3 to 7% by volume against total fermenting mash.

For example, when a fermentation temperature of about 0 C and a fermentation period of 15 to 25 days are used in the brewing method of the present invention, the fermented alcoholic beverage obtained is comparable to the sake obtained by the conventional brewing method with respect to both !cceptability and chemical composition, provided that the above conditions are used for at least charging.

The method for sake making based on the concurrent 06** process of saccharification of rice with enzyme and fermentation of the produced sugar with yeast acccrding to the present invention uses non-cooked polished rice.

Needless to say, the method using purified glucoamylase according to the present invention can also be applied to cooked polished rice, and the minimum sufficient amount of enzyme used in this case is much lower than that used in the case of non-cooked polished rice.

The fermented alcoholic beverage obtained according to the present invention is analytically comparable to those obtained by the conventional method, surpassing fermented alcoholic beverages obtained by the conventional 13 brewing method in sensory evaluation. Also, sake making according to the present invention is highly effective in saving labor, energy and natural resources. According to the brewing method of the present invention, it is possible to prevent bacterial contamination and proliferation because the rate of alcohol production is high.

EXAMPLES

The present invention is hereinafter described in r.ore detail by means of the following working examples, which are not to be construed as limitative.

Example 1 To 200 g of polished rice in a jar fermenter having an inside volume of about 1.5 liter, about 1 liter of well water was added. After the rice was washed by vigorously shaking the jar up and down, the jar was kept standing for several minutes, and the washing was removed by 0* 0 decantation. After this operation was repeated five 20 cycles, a gauze was applied to the jar's mouth and the jar was inverted to thoroughly drain the water. The jar was then returned to the normal position, 300 ml of well water e containing 0.15% lactic acid was added, and the jar was kept standing at 18*C. Three hours later, the supernatant's pH, initially about 2.8, became 3.4 to At this time, purified glucoamylase and a yeast seed prepared with koji extract were added, and a U-shaped tube 14 containing an appropriate amount of sulfuric acid was attached to the jar fermenter, followed by fermentation at 18°C. The purified glucoamylase used was a commercial product "Super Sumizyme" (produced from a Rhizopus fungus by Shinnihon Kagaku Kogyo Co., Ltd.; 24.2 U/mg). Here, 1 unit means 1 p mole as glucose/minute. The yeast used was grown aerobically in 7% koji extract at 30 0 C overnight.

The yeast cells were collected by centrifugation and inoculated in a ratio of 107 cells per ml mash to be fermented.

During fermentation, a loss of fermentation broth weight (total weight (jar fermenter tare sulfuric acid weight)) was determined; the weight loss was attributed to generation of carbon dioxide. Figure 1 shows time courses a 15 of fermentation broth weight loss. Table 1 gives 0ee analytical values for the fermentation broth after 17 days of fermentation.

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S. e 0* S S S S S S S a S r 55 Table 1 Direct Alcohol Fermentation (18 with'Non-Cooked Rice by Enzymatic Method and Analytical Results of Fermentation Liquid A B C Degree of polishing of rice 70 70 Weight of non-cooked rice g 200 200 200 Charged Activity of enzyme added U(wt,g) 8000(0.4g) 4000(0.2g) 4000(0.2g) Materials Yeast (Kyokai 7) g 1 1 1 (2.3 10 9 cells/g) 0.15% Lactic acid ml 350 350 350 Initial total weight g 550 550 550 Final total weight g 480 484 480 Amount of CO 2 generated g 70 66 Yield of fermentation 85 81 After 17 days Alcohol concentration 18.0 17.5 18.0 Fermentation Sake-cake (dry weight) g 38.2 34.6 35.2 Total acids ml 4.3 4.8 5.3 Amino acids ml 1.5 1.1 0.8 Sake meter -10 -3 +17 pH 4.1 4.0 3.8 16 In Table 1, figures for fermentation yield are expressed on the basis of the amount of alcohol produced as estimated in accordance with Gay-Lussac's law, on the assumption that all weight loss was due to generation of carbon dioxide. Figures for the amount of enzyme added are expressed in weight percent ratio to the amount of charged starting material rice (pre-washing weight). The glucoamylase used had an activity of 20 units per mg.

Here, 1 unit of saccharification activity is defined as the activity resulting in the production of 1 p mol of glucose per minute at pH 5.2 and 40 0 C using a 1% gelatinized corn starch solution as a substrate. Assay for reducing sugars was conducted by the Shaffer-Somogyi method Biol. Chem., 100, 695 (1933)].

S..15 As seen in Figure 1, alcohol fermentation of the 0 washed polished rice began rapidly upon addition of purified glucoamylase derived from Rhizopus niveus and a yeast. When 0.4 g of the enzyme was added under the conditions of Table 1, fermentation had proceeded to not lower than 90% of the theoretical value 7 days later.

Even when 0.2 g of the enzyme was added, fermentation had 0: been nearly completed 17 days later. These data are **So 4'4 almost the same as those obtained with commercially available refined sake without additives. Also, the purified glucoamylase used is understood to be an enzyme preparation possessing a saccharifying activity sufficient to almost complete the fermentation of charged non-cooked 17 rice within 3 weeks in an amount of not more than 0.1% by weight to polished rice. Also, as shown in Table 1, the obtained fermented alcoholic beverage is deemed to be almost equivalent to commercially available refined sake without additives in general analytical values.

Example 2 The brewing method of the present invention was carried out by three-step charging, which has long been recognized as a good practice. Table 2 shows the amounts of charged materials by stage. The results are shown in Table 3.

Despite proteinase removal from Rhizopus niveus glucoamylase appropriate to the object of the present :SO"O! invention by chromatography with anion exchanger, a trace amount of a-amylase remained in the purified glucoamylase as impurities. Experiments using this glucoamylase revealed that when added at 0.1% by weight to 100% by 0 weight of polished rice, the enzyme is capable of 5* 0 sufficiently saccharifying the non-cooked rice to glucose within the ordinary period of sake making, about 3 5 eel weeks, and that the saccharification of the non-cooked rice by the enzyme is markedly accelerated by the presence of yeast, while the lactic acid added for sterilization is esterified during alcohol fermentation, resulting in a moromi pH of about 4.0 at the time of completion of fermentation.

0 4*0 0 4 4 00 4 0* .0 0 a aaa 0- a *a *0 0* 0 0 *0 0 a a a 0* 4 0 0 *40 *a4 a Table 2 Sake Making with Non-Cooked Rice by- the Three-step Charging Me~thod Formula for Three-Step Charging lst-step 2nd-step 3rd-step of Non-Cooked Rice charging charging charging Polished rice g 230 270 500 Charged water ml 500 630 870 Yeast (Kyokai 7) g Number of yeast cells cells/mi 2i3xl0 7 3.6 x 10ll 4.1 x10 8 Enzyme U 6000(0.3g) 6000(0.3g) 800fl(0.4g) 75%1 Lactic acid ml Incubation temperature 15'C 48 hours 10 0 C 24 hours 18-C

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e l go c Table 3 Analytical Results Sample of Commercial Refined Sake Without Additives Amount of non-cooked rice used Total weight Final weight Fermentation time Amount of CO 2 generated Amount of sake obtained Amount of sake-cake (dry weight) Alcohol concentration Total acids Amino acids Sake meter Amount of ;otal nitrogen m g g g day g ml g ml ml g/ml 1 0 0 0 1000 2 9 4 0 2564 2 5 6 4 18 days after 3 7 6 2 2 6 0 1 3 9 2260 139 1 7. 2 2. 1 0. 7 5 5. 3 4. 0 the 3rd step 1 1 2.

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20 As seen in Table 3, the alcohol concentration' amounted to 17.2% after 18 days fermentation at around to 18 0 C. Also, the general analytical values obtained were comparable to those from commercially available refined sake without additives; in particular, sugar consumption was good, with a nihonshudo (1/10 Baum6 degree: sake meter) of as high as The amino acid content (amino group content) was low, though the total nitrogen content was high, about 2.1 times that of the commercial sake sample. To explain this, it is assumed that the proteins in the charged rice grains had not been denatured, and prolamine, one of its constituent proteins, eluted with the rise in alcohol concentration. In this .regard, some remains to be studied, but it is judged that the protein can mostly be removed by activated charcoal treatment and poses no problem of bad taste.

In comparison with fermented alcoholic beverages obtained by the conventional brewing method, the fermented alcoholic beverage obtained by the brewing method of the present invention is characterized in that the exhalation from the drinker who has taken a certain amount of alcoholic beverage has little or no sake odor but a light fruity fragrance, though it is difficult to show objective sensory differences therebetween. This may be because production of odorous substances due to the physiological and biochemical actions of koji and koji mould is suppressed, while yeast-produced alcohol and trace amounts 21 of other volatile substances, and secondary volatile substances, such as esters produced chemically or biochemically during fermentation, are produced in the moromi, since the starting material rice is charged after only washing in the brewing method of the present invention.

Example 3 Using 200kg of non-cooked rice, sake making was carried out by three-step charging in the same manner as in Example 2. The yeast seed, Kyokai 7, produced by Takarashuzo Co., Ltd. was added at the 1st step charging.

"Super Sumizyme" (produced from a Rhizopus fungus by Shinnihon Kagaku Kogyo Co., Ltd.; 24.2 U/mg) was added in an amount of 60g at the 1st step charging, 64g at the 2nd ".15 step charging and 76g at the 3rd step charging, respectively. Koji being treated at 60 0 C for 1 hour was added to a final concentration of 7% at the 1st step 0 0 charging. The charging was carried out with 0.15% of lactic acid and 0.0825% of malic acid, respectively. The charging formula is shown in Table 4.

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a a Table 4 Formula for Three-Step Charging in Large Scale 1st-step 2nd-step 3rd-step Total charging charging charging Temperature 16 12 12 Total rice (kg) 46 54 100 200 Water charged 87 110 153 350 Yeast 240 0 0 240 Enzyme 60 64 76 200 k'.

23 The analytical results at charging are shown in Table The analytical results of sake obtained are shown in Table 6.

As seen in Table 6, the sake thus obtained was evaluated to be a higher class in the quality, especially in the taste and flavor, though the analytical data were almost similar to those of sake in marketing.

a a a a S sea C. a a. a a a a a S Ca a a a a a a Table 5 Analytical Results at Charging Lactic acid (0.15%) Malica acid (0.0825%) At 1 st-step charging 77 Koji M(7) Acids (m)0.8 0.7 Amino acids (m)0.65 0.65 Number ofLc yeast cells X10 6 /Ml) 9.7 5.8 During 1st-step charging Acids (ml) 2.3 2.2 Amino acids (ml) 0.90 0.95 Number of yeast cells (x 10 8 1.0 0.9 Just before 2nd-step charging Acids (ml) 2.5 3.1 Amino acids (ml) 0.4.0 0.50 a.. a a a .aa a baa 09 a a. a a a Table 5 (continued) Lactic acid Malic acid 110.15%) (0.0825%) Imediately after 2nd-st,: p charging Acids (ml) 1.3 Amino acids (ml) 0.20 0.20 Number of yeast cells (X 10 7 /ml) 3.5 5.3 Just before 3rd-step charging Acids (ml) 2.2 2.3 Amino acids (ml) 0.10 0.05 Imediately after 3rd-step charging Acids (Ml) 1.2 Amino acids (Ml) 0.05 0.05 Number of yeast cells (XlO

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7/ml) 4.o 6.1 26 Table 6 Analytical Results of Sake Obtained

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sees 69 at, 0 *0O *600 at** 46.0 &0S 5 Acid added Analytical results of Sake Lactic acid Malic acid (0.0825%) Total acids (ml) 3.05 3.00 Amino acids (ml) 0.80 0.80 Sake meter +20.0 24.0 Alcohol concentration 17.0 17.2 Sake-cake (dry weight) 43.69 40.63 Conditions: 200kg of 70% polished rice, temperature of 16 °C, 17kg of koji, 350kg of water charged, 200g of Super Sumizyme, 240g of yeast, and fermentation time of 28 days 27 The present invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

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Claims (8)

1. A method of sake making comprising the steps of: charging non-cooked polished rice and water with or without an organic acid to a fermenter; adding a purified glucoamylase containing substantially no proteinase and a yeast seed prepared with koji extract thereto; and fermenting them.

2. A method of sake making comprising the steps of: charging non-cooked polished rice, water with or without an organic acid and a koji to a fermenter; adding a purified glucoamylase containing substantially no proteinase and a yeast seed prepared with koji extract thereto; and 15 fermenting them.

3. The method according to claim 1 or claim 2, wherein said purified glucoamylase is added in an amount of 0.02 to 0.1% by weight, based on said non- cooked polished ric,

4. The method according to any one of claims 1 to 3, wherein said purified glucoamylase is derived from Rhizopus delemeror Rhizopus niveus

5. The method according to any one of claims 1 to 4, wherein said organic acid 25 is at least one kind selected from the group consisting of lactic acid, malic acid and citric acid.

6. The method according to any one of claims 2 to 5, wherein said koji is charged in an amount of not more than 15% by weight, based on said non-cooked polished rice. 951208,p:\oper\nh,40117-93.339;28 I 29

7. A method of making sake substantially as hereinbefore described, with reference to the drawings and/or Examples.

8. Sake when made by the method claimed in any one of claims 1 to 7. DATED this 5th day of December, 1995 Mizokami Sake Brewing Co., Ltd. by DAVIES COLLISON CAVE Patent Attorneys for the Applicant 6 'SS *6* 6 @64* 6*e6 ~6 *6 6 6 6*6 6 66*6 6 6* 0 0 6 *6 6 0 6 6 66* 6 *6.606 6 951205,p:\oper\nnb,40117-93.339,29 ABSTRACT OF THE DISCLOSURE The present invention is directed to a method of sake making having the step of fermentation of non-cooked polished rice, water supplemented with or without an organic acid, and where necessary, a minimum sufficient amount of koji, as starting materials, in the presence of purified glucoamylase containing substantially no proteinase and a yeast seed prepared with koji extract. The fermented alcoholic beverage obtained according to the present invention is analytically comparable to those obtained by the conventional method, surpassing fermented alcoholic beverages obtained by the conventional brewing method in sensory evaluation. Also, the sake making is highly effective in saving labor, energy and natural resources, and it is possible to prevent bacterial contamination and proliferation because the rate of *t alcohol production is high. B*.