Method for manufacturing nuruk using three types of complex fungi and its use

Abstract

The present invention relates to novel fungi with excellent saccharifying power, such as Aspergillus oryzaeSU-Y (Deposit No.: KACC93367P), Lichtheimia ramosaKJ_WF (Deposit No.: KACC83070BP), and Aspergillus nigerJA_B (Deposit No.: KACC93366P), a composite starter culture using the three types of composite fungi, a rice cake starter culture using the same, and a method for manufacturing the same. It has no possibility of contamination by unwanted microorganisms or stability issues, possesses heat resistance, has a high spore count, and can be usefully used as a fermentation starter culture and fermentation agent for alcoholic beverages, vinegar, etc., by confirming the excellent activity of alpha-amylase (α-amylase), glucoamylase, and acidic carboxypeptidase involved in starch decomposition ability.

Description

Technology Field

[0001] The present invention relates to Aspergillus oryzae SU_Y, a novel fungus with excellent saccharification power isolated from traditional nuruk, Lichtheimia ramosa KJ_WF and Aspergillus niger JA_B, a complex starter culture using the three types of complex molds mentioned above, a rice cake starter culture using the same, a method of manufacturing the same, and uses thereof. It can be usefully utilized as a fermentation starter culture and fermentation agent that is safe and reduces risk factors for contamination by unwanted bacteria, thereby improving quality. Background Technology

[0003] Nuruk is used as a fermentation agent for brewing Korea's representative traditional alcoholic beverages, such as Makgeolli and Yakju. Since these are parallel fermentation beverages that use starchy raw materials, the saccharification of starch is essential during the brewing process; therefore, a fermentation agent in the form of enzyme-based koji (nuruk) produced by cultivating mold was used (So MH 1991). Nuruk has long served as a starch decomposer and alcohol fermentation agent in the production of Tak and Yakju. While it was primarily made from wheat, grains such as corn, barley, soybeans, oats, millet, and white rice were also used depending on the region (Lee et al 2002, Kim MJ 2002).

[0004] Nuruk is classified into traditional nuruk and improved nuruk depending on the manufacturing method. Traditional nuruk is produced by the proliferation of various microorganisms existing in the natural environment; alcohol is generated through saccharification and fermentation, and the resulting liquor has the unique advantage of having a distinctive taste and aroma. In the case of improved nuruk, to prevent contamination by unwanted bacteria and to produce liquor of uniform quality, Aspergillus ( Aspergillus ) and Rhizopus( RhizopusIt is produced by inoculating with fermentation starter cultures such as ). Due to the diverse manufacturing processes of traditional nuruk and the varying environmental fermentation microorganisms found in nature, there is an urgent need for standardized product production and quality standardization. Domestic breweries have been using imported starter cultures because using traditional nuruk resulted in low quality uniformity and prolonged manufacturing periods. As mentioned earlier, the traditional nuruk manufacturing process has the disadvantage of making it difficult to produce alcohol of standardized and uniform quality, as the flavor of the alcohol varies by season. Consequently, the competitiveness of Korean alcohol is low due to the poor quality of commercially available nuruk. To overcome this, research must be conducted to develop high-quality mass production technologies for nuruk suitable for the specific characteristics of traditional nuruk and its ingredients. This requires using domestic starter cultures instead of imported ones and conducting scientific research to improve nuruk quality, standardize products, and shorten manufacturing periods. Furthermore, it has been suggested that more systematic research and development regarding the enzymatic aspects of nuruk is necessary to resolve these issues (Heo et al., 2014).

[0005] Among the enzymes contained in nuruk, amylases refer to enzymes that hydrolyze starch and are the most widely used enzymes in the food industry to date. Based on their functional characteristics, they can be classified into alpha-amylase (α-amylase), beta-amylase (β-amylase), glucoamylase, and alpha-glucosidase (α-glucosidase). Alpha-amylase (α-amylase) is an enzyme involved in liquefying starch; it acts on α-1,4 bonds and breaks down starch into relatively large oligosaccharides. Beta-amylase (β-amylase) is a saccharifying enzyme involved in producing maltose by breaking down amylose from the ends of the starch. Glucoamylase is an enzyme involved in breaking down starch into glucose units starting from the non-reducing end, and plays a direct role in producing glucose from starch. Additionally, acidic carboxypeptidase is a proteolytic enzyme that assists in liquefaction and saccharification by participating in the release of alpha-amylase adsorbed to proteins.

[0006] Accordingly, the inventors isolated a novel mold with excellent saccharification power from traditional nuruk, and the novel mold Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF and Aspergillus niger The present invention was completed by using JA_B as a complex starter culture to produce rice cake nuruk and setting optimal fermentation conditions, and by confirming that the quality was improved with low possibility of contamination by unwanted bacteria and excellent saccharification power. The problem to be solved

[0008] The present invention relates to a novel mold with excellent saccharification power that is safe, heat-resistant, and free from the potential for contamination by unwanted microorganisms and stability issues associated with existing fermentation agents using a single strain. Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF and Aspergillus nigerJA_B, a complex starter culture using the three types of complex molds mentioned above, and the excellent spore count, alpha-amylase (α-amylase), glucoamylase, and acidic carboxypeptidase activities of the rice cake koji using the same, suggest that it can be used as a fermentation starter culture and fermentation agent for alcoholic beverages, vinegar, etc. means of solving the problem

[0010] In order to achieve the above objective,

[0011] The present invention relates to a novel mold with excellent saccharification power for the production of rice cake nuruk. Aspergillus oryzae SU_Y (Deposit No.: KACC93367P) is provided.

[0012] The present invention relates to a novel mold with excellent saccharification power for the production of rice cake nuruk. Lichtheimia ramosa Provides KJ_WF (Deposit No.: KACC83070BP).

[0013] The present invention relates to a novel mold with excellent saccharification power for the production of rice cake nuruk. Aspergillus niger Provides JA_B (Deposit No.: KACC93366P).

[0014] The present invention relates to a novel mold with excellent saccharification power for the production of rice cake nuruk. Aspergillus oryzae SU_Y(Deposit No.: KACC93367P ), Lichtheimia ramosa KJ_WF (Deposit No.: KACC83070BP) and Aspergillus niger Provides a complex starter culture for making rice cake nuruk mixed with JA_B (Deposit No.: KACC93366P).

[0015] The present invention provides a method for producing rice cake koji, comprising inoculating a starchy raw material with a complex starter culture for producing rice cake koji and fermenting it. Effects of the invention

[0017] The novel mold with excellent saccharification power of the present invention Aspergillus oryzae SU_Y(Deposit No.: KACC93367P), Lichtheimia ramosa KJ_WF and Aspergillus nigerJA_B, the complex starter culture using the three types of complex molds mentioned above and the rice cake koji using the same have no problem with contamination by unwanted bacteria or stability, and have heat resistance and high spore count, and excellent activity of alpha-amylase (α-amylase), glucoamylase, and acidic carboxypeptidase, so they can be usefully used as fermentation starter cultures and fermentation agents for alcoholic beverages, vinegar, etc. Brief explanation of the drawing

[0019] Figure 1 is a graph showing the change in spore count according to the inoculation ratio of a complex spawn of three fungi with excellent enzyme activity. Figure 2a is a graph showing the change in alpha-amylase (α-amylase) activity according to the inoculation ratio of three fungal strains with excellent enzyme activity. Figure 2b is a graph showing the change in glucoamylase activity according to the inoculation ratio of three strains of fungus with excellent enzyme activity. Figure 2c is a graph showing the change in carboxypeptidase activity according to the inoculation ratio of a complex inoculum of three fungi with excellent enzyme activity. Figure 3 is a schematic diagram showing the process of making rice cake koji using complex starter cultures. Specific details for implementing the invention

[0020] The present invention will be described in detail below.

[0021] According to a specific embodiment of the present invention, for the production of rice cake nuruk, two main ingredients and two auxiliary ingredients were selected from varieties that have a high amylose content, are easy to supply, and are preferably new varieties (Table 1).

[0022] Among the 21 commercially available fermentation agents collected, 5 agents with excellent enzyme activity were selected. After identifying and measuring the growth characteristics and enzyme activity of 15 isolated fungal strains and 2 additionally selected fungal strains (*), strains with excellent enzyme activity were selected, along with their potential for use in food manufacturing. Therefore, in this study Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus niger JA_B was selected (Table 2). The optimal growth temperature of the three selected fungi was generally 35℃, which is higher than the general optimal growth temperature of fungi (28℃), indicating heat resistance, and the optimal pH was found to be excellent at pH 5 (Tables 3 and 4).

[0023] Table 6 and Figure 1 show the results of measuring spore counts according to the culture period following treatment with the three strains used as complex spawn, based on inoculation ratios. Under most conditions, the highest spore count was observed on the 4th day of culture, followed by a tendency to gradually decrease or remain constant, while under the 16(0:1:2) condition, 3.2 x 10⁶ spores 8 It was measured as the highest value among all conditions in spore / mL (Table 6 and Fig. 1).

[0024] As a result of measuring the activities of three types of enzymes (α-amylase, glucoamylase, and carboxypeptidase), we intended to select the complex starter culture ratio conditions focusing on the activity of glucoamylase, which exhibits the most important saccharifying power in fermentation agents (nuruk). Among the mixing ratios Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus niger In the order of JA_B, glucoamylase activity was measured to be higher in the order of 16 (78.7 U / g) > 6 (59.0 U / g) > 10 (49.8 U / g) under conditions of 6 (0:1:1), 10 (1:1:2), and 16 (0:1:2), so the above three complex ratios were selected for the production of fermentation agents using complex starter cultures (Figs. 2a to 2c).

[0025] Next, the raw materials were crushed, and the main ingredients (Anjeunki, Deuraehyang) and auxiliary ingredients (Hogang, Dahyeon) were mixed in a 9:1 ratio, and the selected composite starter culture was inoculated according to each ratio. Tteoknuruk was prepared using 100 g of the composite raw materials for each. It was cultured at 35℃, the optimal culture temperature for the selected starter culture, and under conditions of 45% humidity; samples were taken at 24, 36, and 48 hours and used for quality characteristic analysis (Table 7).

[0026] As part of the investigation into the quality characteristics of the previously prepared rice cake nuruk according to the fermentation period, pH, acidity, and amino acid content were measured and compared (Table 8). The results of the acidity measurement showed a tendency to be inversely proportional to pH, and in most cases, the acidity was measured to be 2 or lower at the end of fermentation. Similarly, under conditions where the auxiliary ingredient Dahyun (D) was mixed, the acidity decreased sharply at 36 hours of fermentation and then rose again. In most other conditions, the acidity decreased as the fermentation time progressed. The amino acid content increased as the fermentation time progressed, but the difference was minimal. It can be confirmed that it was measured to be high when wheat (W) and Dahyun mung beans (D) were used (Table 8). The highest value was 20.4 mg / 100 g when fermented for 48 hours under condition 16 (complex spawn ratio) using wheat (W) and Dahyun mung beans (D), followed by a high value of 19.8 mg / 100 g when fermented for 48 hours under condition 6 (complex spawn ratio) using the same ingredients.

[0027] Glucoamylase activity, which indicates saccharification power, was measured to be high under all conditions when raw wheat (W) and mung bean Dahyun (D) were combined and used, regardless of the complex starter culture ratio. It was highest at 303.6 U / g when fermented for 48 hours under complex starter culture ratio condition 10 using raw materials W and D (wheat, mung bean), and when the same raw materials were used, it was found to be high at over 200 U / g under complex starter culture ratio conditions 6 and 16 when fermented for 48 hours. Based on these results, it was determined that the auxiliary raw material mung bean Dahyun (D) had a significant influence on saccharification power (Table 9).

[0028] α-amylase activity, which indicates liquefaction power, mostly increased as fermentation progressed, and it was confirmed that the increase was particularly high when raw wheat (W) and mung bean broccoli (D) were used in combination. Among them, when the highest complex spawn ratio of 10 (1:1:2) was inoculated, it was measured to be 118.2–103.3 U / g, which is higher than other conditions (Table 9).

[0029] The carboxypeptidase activity, which confirms protein degradation ability, showed the highest activity when rice (R) and Dahyeon mung bean (D) were used as the raw material mixture under most conditions. When raw materials R and D were used and fermented for 36 hours under condition 10 complex spawn ratio, the activity was measured to be high at 3933.6 U / g, and it was confirmed that the highest activity was observed when raw materials W and D were used and fermented for 48 hours under condition 10 complex spawn ratio (Table 9).

[0030] This is a complex spawn ratio of 10. Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF and Aspergillus niger It is suggested that rice cake nuruk produced using three types of mold in a 1:1:2 ratio can be used as an excellent fermentation starter and fermentation agent with improved quality and reduced risk factors of potential contamination by possessing high liquefaction and saccharification power.

[0031] The above fermentation can be carried out at 37°C to 40°C for 24 to 48 hours. In the production of a fermentation agent, it is important to sufficiently form spores of the inoculated mold, and by maintaining the above fermentation temperature and period, sufficient spores of the mold can be produced to produce a high-quality fermentation agent.

[0032] In a specific embodiment of the present invention, a method for producing rice cake nuruk is provided, comprising inoculating a prepared composite starter culture into a starchy raw material and fermenting it. The starchy raw material may be selected from the group consisting of grains such as rice, barley, wheat, mung beans, rye, oats, millet, sorghum, and buckwheat, tubers such as corn and sweet potatoes, and mixtures thereof.

[0033] The present invention will be described in detail below.

[0034] The present invention will be explained in detail below through examples and experimental examples.

[0035] However, the following examples and experimental examples are merely illustrative of the present invention in one aspect, and the present invention is not limited to the following examples and experimental examples.

[0037] <Example 1> Selection of Raw Materials for Manufacturing Rice Cake Nuruk

[0038] Wheat (Anjeunki) and rice (Deuraehyang) were collected or purchased as the main ingredients, and barley (Hogang) and mung beans (Dahyeon) were collected or purchased as auxiliary ingredients. As for the raw material conditions, mung beans (Dahyeon) were coarsely ground to a gap of 0.5 mm using a grain crusher, while the other raw materials were tested in their raw state (Table 1).

[0039]

[0040] (Table 1: List of collected raw materials for rice cake nuruk using complex starter cultures)

[0041] For the production of rice cake nuruk, raw materials with high amylose content, no supply difficulties, and preferably new varieties were selected first, and two main raw materials and two auxiliary raw materials were selected from varieties that have been studied infrequently in the past (Table 1).

[0043] <Example 2> Selection of Composite Starter Culture for Rice Cake Nuruk Production and Selection of Optimal Growth Conditions

[0044] Among the 21 commercially available fermenters collected, 5 fermenters with excellent enzyme activity were selected, and among a total of 17 mold strains, including molds isolated from the fermenters and an additional 2 mold strains from the Chungcheong region, 3 mold strains with excellent enzyme activity were selected based on their suitability for food manufacturing (Table 2).

[0045]

[0046] (Table 2: Analysis of enzyme activity of 17 selected fungal strains)

[0047] Among the 21 commercially available fermentation agents collected, 5 agents with excellent enzyme activity were selected. After identification, the growth characteristics and enzyme activity of 15 isolated fungal strains and 2 additionally selected fungal strains (*) were measured, resulting in the selection of strains with excellent enzyme activity and potential for food manufacturing use. Therefore, as a complex starter culture in this study Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus niger JA_B was selected (Table 2).

[0048] Add 5% (v / v) wheat bran to 50 mL of distilled water and sterilize in an autoclave at 121°C for 15 minutes, then 10 6 The three types of molds selected above, adjusted to spore / mL Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus niger After inoculating JA_B (1% (v / v)), the spore count was measured by incubating at different temperatures and pH levels. The spore count was determined by suspending 1 mL of wheat bran culture in 10 mL of Tween 80 solution (5%), adding 1 to 2 drops of methylene blue solution (1%), and microscopically examining the results using a hemocytometer (Tables 3 and 4).

[0049]

[0050] (Table 3: Growth characteristics of 3 selected fungi at different temperatures)

[0051]

[0052] (Table 4: Growth characteristics of 3 selected fungal strains by pH)

[0053] The above-mentioned selected fungus 3 weeks Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus nigerAs a result of verifying the growth characteristics by varying the growth conditions of JA_B by temperature and pH and measuring the number of spores after culture, it was found that the optimal growth temperature was generally 35℃, which is higher than the general optimal growth temperature of fungi (28℃), indicating heat resistance, and the optimal pH was 5, showing excellent activity (Tables 3 and 4). The three selected fungal strains have been deposited as patent strains at KACC.

[0055] <Example 3> Selection of Optimal Growth Conditions for Mixed Spawn According to Inoculation Ratio

[0056] Add 5% (v / v) wheat bran to 50 mL of distilled water and sterilize in an autoclave at 121°C for 15 minutes, then 10 6 The three strains of the selected fungi adjusted to spore / mL were inoculated at various ratios (1% (v / v)), and the spore count and enzyme activity were measured for different culture periods (0, 2, 4, 6, and 8 days). The spore count was determined by suspending 1 mL of wheat bran culture solution in 10 mL of Tween 80 solution (5%), adding 1 to 2 drops of methylene blue solution (1%), and microscopically examining the results using a hemacytometer (Tables 5, 6 and Fig. 1). Enzyme activity was analyzed using a Kikkoman enzyme activity test kit for brewing analysis; α-amylase, glucoamylase, and acidic protease activities were measured using the wheat bran culture solution and expressed as U / g values ​​(Figs. 2a to 2c).

[0057]

[0058] (Table 5: Setting of culture ratios for 3 selected fungal strains of complex spawn)

[0059]

[0060] (Table 6: Changes in spore count according to complex spawn inoculation ratio)

[0061] Table 6 and Figure 1 show the results of measuring spore counts according to the inoculation ratio and culture period for the three strains used as complex spawn. Under most conditions, the highest spore count was observed on the 4th day of culture, followed by a tendency to gradually decrease or remain constant. Additionally, under the 16 (0:1:2) condition, 3.2 x 10⁶ 8 It was measured as the highest value among all conditions in spore / mL (Table 6 and Fig. 1).

[0062] As a result of measuring the activities of three types of enzymes (α-amylase, glucoamylase, and carboxypeptidase), we intended to select the complex starter culture ratio conditions focusing on the activity of glucoamylase, which exhibits the most important saccharifying power in fermentation agents (nuruk). Among the mixing ratios Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF, Aspergillus niger In the order of JA_B, glucoamylase activity was measured to be higher in the order of 16 (78.7 U / g) > 6 (59.0 U / g) > 10 (49.8 U / g) under conditions of 6 (0:1:1), 10 (1:1:2), and 16 (0:1:2), so the above three complex ratios were selected for the production of fermentation agents using complex starter cultures (Figs. 2a to 2c).

[0064] <Example 4> Preparation of rice cake koji using complex starter culture

[0065] Based on the selected raw materials and starter cultures, rice cake nuruk under various conditions was produced through the process of crushing → mixing raw materials → inoculation with complex starter cultures → watering and molding → cultivation (Fig. 3). Wheat (Anjeunki) and rice (Deuraehyang), selected as main raw materials, and barley (Hogang), selected as auxiliary raw materials, were used in their polished state, while mung beans (Dahyeon), among the auxiliary raw materials, were used after coarsely crushing the surface to a 0.5mm gap using a grain crusher (Fig. 3).

[0066] Next, the main ingredients (Anjeunki, Deuraehyang) and auxiliary ingredients (Hogang, Dahyeon) were mixed in a 9:1 ratio, and the selected composite starter culture was inoculated according to each ratio. Tteoknuruk was prepared using 100 g of the composite ingredients for each. It was cultured at 35℃, the optimal culture temperature for the selected starter culture, and under conditions of 45% humidity; samples were taken at 24, 36, and 48 hours and used for quality characteristic analysis (Table 7).

[0067]

[0068]

[0069] (Table 7: Rice cake nuruk produced using complex starter cultures of different types)

[0071] <Example 5> Comparison of General Components of Prepared Rice Cake Nuruk According to Fermentation Period

[0072] The quality characteristics of the previously prepared rice cake nuruk according to the fermentation period were investigated by measuring and comparing pH, acidity, and amino acid content (Table 8). pH was measured using a pH meter after adding 10 times the amount of distilled water to 10 g of the sample, extracting, and filtering. Acidity was determined by adding 9 mL of distilled water to 1 mL of the sample filtrate and titrating with 0.1N NaOH, while amino acid content was determined by adding 9 mL of distilled water to 1 mL of the sample filtrate, liberating amino acids with a neutral formalin solution, and then titrating with 0.1N NaOH to convert the result to glycine.

[0073]

[0074]

[0075] (Table 8: Comparison of General Components of Complex Starter Culture Rice Cake Nuruk by Fermentation Period)

[0076] The acidity measurement results showed a tendency to be inversely proportional to pH, and in most cases, the acidity was measured to be 2 or lower at the end of fermentation. Similarly, under conditions where the auxiliary ingredient Dahyun (D) was mixed, the acidity decreased sharply at 36 hours of fermentation and then rose again. In most other conditions, the acidity decreased as the fermentation time progressed. The amino acid content increased as the fermentation time progressed, but the difference was minimal. It can be confirmed that it was measured to be high when wheat (W) and Dahyun mung beans (D) were used (Table 8). The highest value was 20.4 mg / 100 g when fermented for 48 hours under condition 16 (complex spawn ratio) using wheat (W) and Dahyun mung beans (D), followed by a high value of 19.8 mg / 100 g when fermented for 48 hours under condition 6 (complex spawn ratio) using the same ingredients.

[0078] <Example 6> Comparison of Enzyme Activity of Prepared Rice Cake Koji According to Fermentation Period

[0079] Enzyme activity was measured and compared by investigating the quality characteristics of the previously prepared rice cake nuruk according to the fermentation period. Enzyme activity was measured using a brewing analysis enzyme activity test kit (Kikkoman) to determine α-amylase, glucoamylase, and acidic carboxypeptidase activities, expressed as U / g values ​​(Table 9).

[0080]

[0081]

[0082] (Table 9: Comparison of Enzyme Activity of Complex Starter Culture Rice Cake Nuruk by Fermentation Period)

[0083] Glucoamylase activity, which indicates saccharification power, was measured to be high under all conditions when raw wheat (W) and mung bean dahyeon (D) were used in combination, regardless of the complex starter culture ratio. It was highest at 303.6 U / g when fermented for 48 hours under complex starter culture ratio condition 10 using raw materials W and D, and when the same raw materials were used, it was found to be high at over 200 U / g under complex starter culture ratio conditions 6 and 16 when fermented for 48 hours. Based on these results, it was determined that the auxiliary raw material mung bean dahyeon (D) had a significant influence on saccharification power (Table 9).

[0084] α-amylase activity, which indicates liquefaction power, mostly increased as fermentation progressed, and it was confirmed that the increase was particularly high when raw wheat (W) and mung bean broccoli (D) were used in combination. Among them, when the highest complex spawn ratio of 10 (1:1:2) was inoculated, it was measured to be 118.2–103.3 U / g, which is higher than other conditions (Table 9).

[0085] The carboxypeptidase activity, which confirms protein degradation ability, showed the highest activity under most conditions when rice (R) and mung bean (D) were used as the raw material mixture. When raw materials R and D (rice, mung bean) were fermented for 36 hours under condition 10 complex spawn ratio, the activity was measured to be high at 3933.6 U / g, and it was confirmed that the highest activity was observed when raw materials W and D (wheat, mung bean) were fermented for 48 hours under condition 10 complex spawn ratio (Table 9).

[0086] This is a complex spawn ratio of 10. Aspergillus oryzae SU_Y, Lichtheimia ramosa KJ_WF and Aspergillus nigerIt is suggested that rice cake nuruk produced using three types of molds in a ratio of 1:1:2% (v / v) can be used as an excellent fermentation starter and fermentation agent with improved quality and reduced risk factors of potential contamination by having the highest liquefaction and saccharification power.

[0089] Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC93367P Date of Deposit: 2021-10-07 Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC83070BP Date of Deposit: 2022-09-26 Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC93366P Date of Deposit: 2021-10-07

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 Novel mold with excellent saccharification ability Aspergillus oryzae SU-Y (Deposit No.: KACC93367P), Lichtheimia ramosa KJ_WF (Deposit No.: KACC83070BP) and Aspergillus niger Composite starter culture for making rice cake nuruk mixed with JA_B (Deposit No.: KACC93366P). Claim 8 delete Claim 9 A method for manufacturing rice cake koji comprising inoculating a starchy raw material with the complex starter culture for manufacturing rice cake koji of claim 7 and fermenting it. Claim 10 In claim 9, the rice cake nuruk is one having excellent alpha-amylase (α-amylase), glucoamylase, or acidic carboxypeptidase activity, Aspergillus oryzae SU-Y (Deposit No.: KACC93367P), Lichtheimia ramosa KJ_WF (Deposit No.: KACC83070BP) and Aspergillus niger A method for producing rice cake koji characterized by using a complex starter culture of JA_B (Deposit No.: KACC93366P). Claim 11 In Paragraph 7, the above Aspergillus oryzae SU-Y (accession number: KACC93367P) is a composite starter culture characterized by having the 18S rRNA nucleotide sequence of SEQ ID NO.

1. Claim 12 In Paragraph 7, the above Lichtheimia ramosa KJ_WF (Accession No.: KACC83070BP) is a complex starter culture characterized by having the 18S rRNA sequence of SEQ ID No.

2. Claim 13 In Paragraph 7, the above Aspergillus niger JA_B (accession number: KACC93366P) is a complex starter culture characterized by having the 18S rRNA nucleotide sequence of SEQ ID NO. 3.

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