A process for producing sake based on multi-strain mixed fermentation and its flavor

Abstract

This invention discloses a process for producing sake based on multi-strain mixed fermentation and its flavor profile, belonging to the field of sake brewing technology. This invention provides a method using japonica rice from Northeast China as raw material, employing multi-strain, sequential low-temperature fermentation to regulate ester, acid, and alcohol production during the sake fermentation process. This results in a sake product with flavor compounds at levels and proportions comparable to Japanese sake. The strains include *Rhizopus oryzae*, *Rhizopus chinensis*, *Issaccharomyces orientalis*, and *Saccharomyces cerevisiae*. Compared to existing technologies, the microbial combination, inoculation amount, and inoculation sequence of this invention, combined with synergistic saccharification and fermentation, achieves a ratio of isoamyl acetate, ethyl hexanoate, ethyl lactate, and ethyl acetate, as well as a total ester content, reaching the levels of typical Japanese sake.

Description

Technical Field

[0001] This invention discloses a process for producing sake based on multi-strain mixed fermentation and its flavor, belonging to the field of sake brewing technology. Background Technology

[0002] With increasing health awareness among the Chinese people, the drinking habits of the new generation of consumers are evolving, with the consumption of low-alcohol, nutrient-rich non-distilled spirits rising year by year. Sake, as a low-alcohol beverage, is highly sought after by young consumers for its refreshing and natural style, gaining increasing attention and popularity in China, and achieving a growing market share. Sake is also becoming increasingly popular in some Asian countries. Currently, the taste and flavor of sake produced in China differ from typical Japanese sake; therefore, the vast majority of Japanese sake sold in China is imported directly from Japan.

[0003] Japanese sake is a non-distilled spirit made from water, rice, and rice koji (using Aspergillus oryzae as the starter culture) fermented with sake yeast. It is Japan's national sake, with an alcohol content typically between 13% and 20% vol. It has a light, clear, and transparent taste, a pale yellow or colorless color, and distinct fruity and floral aromas.

[0004] The outer layer of rice, the raw material for Japanese sake, contains a large amount of protein, ash, fat, and various vitamins and minerals. These nutrients cause vigorous fermentation by sake yeast, and excessive protein degradation products such as amino acids can also cause off-flavors in the sake. To eliminate the adverse effects of these substances on the fermentation process, the taste, and color of the sake, the outer layer of rice used for sake brewing is milled to remove the surface fat and protein. Generally speaking, the more of the rice is milled away, the cleaner the taste, the more fragrant the aroma, and the higher the quality of the sake. Sake with specific names in Japan is classified according to the rice polishing rate. For example, Honjozo sake has a rice polishing rate of less than 70%, Ginjo and Junmai Ginjo sake have a rice polishing rate of less than 60%, and Junmai Daiginjo sake, the highest grade of sake, requires rice with a polishing rate of less than 50%.

[0005] Traditional Chinese rice wine does not emphasize rice polishing as much as sake. The rice used in Chinese rice wine brewing, specifically the first and second grade rice, has a polishing degree of around 90%, which is the same as the polishing rate of edible rice. About 10% of the surface of the brown rice is removed as bran, and its protein content is relatively high, about 6-8% (w / w).

[0006] The main functional microorganisms used in Japanese sake brewing are Aspergillus oryzae and sake yeast. Sake yeast is the key microorganism in sake fermentation, playing a decisive role in the aroma characteristics of sake. The various alcohols, esters, amino acids, and organic acids produced by its metabolism interact to determine the final aroma characteristics of sake. Alcohols and esters in sake impart fruity and floral aromas, giving consumers a pleasant aftertaste. Esters are the main components of the aroma in Japanese ginjo sake, primarily including ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl lactate. Isoamyl acetate has a banana flavor, and ethyl hexanoate has an apple aroma; these are the most important aroma compounds in high-end ginjo sake.

[0007] Currently, domestic research institutions and production enterprises have conducted numerous trials on producing Japanese sake in China. Chinese invention patent CN106434399A provides a mixed yeast starter for glutinous rice wine and a method for preparing the mixed yeast starter, including using Rhizopus oryzae liquid, Saccharomyces cerevisiae liquid, Rhizopus chinensis liquid, and Rhizopus nigricans liquid; the resulting glutinous rice wine has stable flavor and high safety performance. Chinese invention patent CN116622462A provides a flavored sake and its preparation method, using Tonghua fragrant rice as raw material and incorporating ingredients such as peach, plum, hawthorn, and peach to prepare sake with different single and mixed flavors. Chinese invention patent CN112522050A relates to a sake brewing method, using rice as raw material, Rhizopus oryzae and Saccharomyces cerevisiae as fermentation strains, and adding medicinal and edible ingredients such as licorice, dried tangerine peel, dried rose petals, and stevia.

[0008] Chinese invention patent CN110551592A discloses a method for brewing sake using brown rice as raw material. The method uses ordinary brown rice and sweet sake koji as raw materials. At a low temperature of 6-10℃, the yeast activity in the sweet sake koji is inhibited, and saccharifying bacteria are used to complete the liquefaction and saccharification of rice starch. The fat and protein are pressed and separated, and then yeast is introduced for fermentation to obtain Japanese sake with a pure taste, clear body, and total acidity, amino acid nitrogen and various sensory indicators that conform to the Daiginjo style. However, the text does not provide specific data on sensory and physicochemical indicators. Chinese invention patent CN110157570A discloses a brewing process for Chinese sake. This invention improves upon existing brewing techniques for Japanese sake and Chinese yellow wine. The process involves rice milling, washing, soaking, steaming, cooling, fermentation in tanks (initial, second, and third fermentation), pressing, initial filtration, initial sterilization, blending, decolorization, secondary filtration, freezing, tertiary filtration, bottling, and secondary sterilization. The resulting sake has a delicate aroma, refreshing taste, and harmonious balance of sweet and sour flavors. Chinese invention patent CN111019789A discloses a method for brewing Japanese sake using *Corynebacterium tumefaciens*. Its main purpose is to reduce the higher alcohol content of the sake by inoculating it with *Corynebacterium tumefaciens*, but it does not compare its flavor with that of Japanese sake, particularly in terms of ester content. Zhang Ruiting (Zhang Ruiting, Research on Fermentation Process Conditions and Antioxidant and Antibacterial Properties of Glutinous Rice Sake, Master's Thesis, Yanbian University, 2021) studied the effects of glutinous rice polishing rate, fermentation temperature, and yeast species on the composition and flavor of sake and its antibacterial activity. The conclusion was that using a fermentation temperature of 15℃, a raw material polishing rate of 70%, and F5 yeast strain resulted in glutinous rice sake with a unique taste, aroma, and antibacterial activity. Li Li (Li Li, Huang Yuanyuan, Tang Weizu. Research on Sake Brewing with Different Yeasts, Brewing Technology, 2021, 7: 54-58) studied the physicochemical indicators, sensory evaluation, volatile flavor compounds, and organic acid content of sake brewed with different yeasts, concluding that yeast strain No. 2 is an excellent strain for sake brewing.

[0009] In alcoholic beverage brewing, the main method for increasing the ester content of alcoholic beverages is the use of aroma-producing yeasts. Common aroma-producing yeasts include *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Kluyveromyces martensii*, *Issa mesasura*, and *Wickhamia lanceolata*, among others. There are already literature reports and patents on this topic. Yang Zilin et al. (Yang Zilin, Wu Shihua, Huang Cuiji, Yi Yi. Study on the improvement of glutinous rice wine flavor by mixed liquid fermentation of *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae*. *Journal of Guangxi University of Science and Technology*, 2016, 3:95-100) investigated how to improve the flavor of glutinous rice wine fermented by single-strain *Saccharomyces cerevisiae*. They mixed *Saccharomyces cerevisiae* with *Saccharomyces cerevisiae* for fermentation. This increased the total acid and amino acid nitrogen content in the glutinous rice wine, decreased the content of methanol, isobutanol, and isoamyl alcohol, and increased the lower ester content to 3.47 times that of single-strain fermentation, thus improving the flavor and taste of the glutinous rice wine. Zheng Chaoqun et al. (Zheng Chaoqun, Chen Chen, Jiang Yujian, Zhang Lei, Xie Guangfa, Study on the fermentation process of rice wine with Saccharomyces cerevisiae and Hansenula anomala, Chinese Journal of Food Science) used Saccharomyces cerevisiae and Hansenula anomala as fermentation strains for rice wine. The volatile ester content of rice wine fermented by sequential mixing was 1.66±0.028 g / L, which was significantly higher than that of rice wine fermented by simultaneous mixing. However, they only measured the total ester content and did not study the types, proportions, or absolute contents of esters. Liu Meng et al. (Liu Meng, Miao Lihong, Liu Pulin, et al. Effect of mixed fermentation of Kluyveromyces martensii and Saccharomyces cerevisiae on the flavor of liquid rice wine [J]. Food and Fermentation Industries, 2021, 47(9):160~167) used glutinous rice saccharification liquid as fermentation substrate and studied the effects of Kluyveromyces martensii at different fermentation temperatures, different aeration conditions, and mixed fermentation with Saccharomyces cerevisiae on the flavor substances and sensory properties of rice wine. The results showed that compared with fermentation at 37℃, fermentation at 28℃ significantly reduced the content of higher alcohols such as isoamyl alcohol and significantly increased the content of organic acids such as acetic acid. The ethyl acetate content produced by shaker fermentation was 9.66 times that of static fermentation. There was no significant difference in alcohol content between 1:1 mixed fermentation of *Kluyveromyces martensii* and *Saccharomyces cerevisiae* and fermentation with *Saccharomyces cerevisiae* alone, but the ethyl acetate and acetic acid contents of the mixed-fermented rice wine samples were 2.46 and 1.31 times higher than those of single-fermentation samples, respectively. The total amount of higher alcohols (n-propanol, isobutanol, isoamyl alcohol, and β-phenylethanol) was 81.9% of that in samples fermented with single-fermentation samples. Chinese invention patents CN102586125A and CN102102084A disclose a new high-ester-producing *Isais oryzae* strain, a brewing agent composition containing this strain, and the application of the high-ester-producing *Isais oryzae* strain and the brewing agent composition in baijiu brewing. Baijiu fermented with this strain exhibits a unique flavor and significantly increased ethyl acetate content, but this strain does not produce ethyl hexanoate or isoamyl acetate.Chinese invention patent CN111534407A provides a method for producing ginkgo rice wine through co-fermentation of *Anomala sacchariformis* and *Saccharomyces cerevisiae*. The method involves saccharifying glutinous rice and ginkgo separately, adding 0-80% of ginkgo enzymatic hydrolysate to the glutinous rice saccharification solution, inoculating *Anomala sacchariformis* and *Saccharomyces cerevisiae* at a ratio of 1-1000:1, fermenting at 24-36℃ for 48-96 hours, centrifuging, and obtaining the supernatant as ginkgo rice wine.

[0010] The main problems currently existing in the production of Japanese sake in China

[0011] Sake produced in China differs from sake from that produced in Japan in terms of the types and proportions of esters, alcohols, and acids, particularly in the content of several typical esters in ginjo sake (ethyl acetate, isoamyl acetate, ethyl hexanoate, and ethyl lactate, etc.). The reasons for this discrepancy may be the following factors:

[0012] (1) Lack of rice specifically for sake brewing. In the traditional Japanese koji-making process, rice is usually required to be large-grained, soft (i.e., with strong water absorption, soft inside and hard outside and elastic, easy to grow Aspergillus oryzae, and good solubility in mash), high white core ratio, low protein and fat content, and high starch content.

[0013] If the production requirements of Japanese sake are strictly followed, the rice polishing rate should generally be below 75%, and the rice needs to be milled and polished for a long time. Chinese edible rice has a very high risk of breaking during this process, resulting in high production costs and difficulties in subsequent steaming, fermentation and quality control. The time and energy consumption costs are also higher than those of yellow wine and rice wine.

[0014] (2) Lack of Aspergillus oryzae specifically for sake. Different microorganisms secrete different types and activities of extracellular enzymes, resulting in variations in the saccharifying enzyme activity, liquefying enzyme activity, and protease activity of the koji used in sake brewing. Amino acids are derived from proteins through the action of proteases. Different microorganisms secrete proteases that degrade rice protein at different cleavage sites, potentially leading to differences in the types and amounts of amino acids and peptides produced. Different amino acids contribute to different flavors, thus affecting the taste of sake.

[0015] (3) Lack of sake-specific yeast. Yeast is the most important factor affecting the content of flavor compounds in sake. Among them, esters are important aroma compounds in ginjo sake. Ethyl acetate has the aroma of apples and pears, ethyl hexanoate has the aroma of apples and peaches, and isoamyl acetate has the aroma of honeydew melons and bananas. At present, various yeasts have been used to produce sake in China. However, as mentioned in the background technology section above, the total amount of esters and the ratio of ethyl hexanoate, isoamyl acetate, ethyl lactate and ethyl acetate in sake produced by yeast fermentation are still unsatisfactory.

[0016] To address the aforementioned technical deficiencies in existing technologies, it is necessary to find one or more microorganisms (including yeast and mold) and a matching brewing process to produce Japanese sake with a typical taste and flavor, particularly in the proportions of esters such as ethyl acetate, isoamyl acetate, ethyl hexanoate, and ethyl lactate, as well as the total ester content. Summary of the Invention

[0017] To address the aforementioned problems in the prior art, this invention provides a Japanese sake brewing process that uses microbial technology to naturally generate flavor substances. Specifically, by controlling process parameters such as the composition and inoculation amount of different microorganisms, fermentation temperature, and inoculation sequence, the flavor substances in the sake, including esters, alcohols, organic acids, and amino acids, are adjusted to ensure that the flavor substances in the produced sake product have the same level and composition ratio as those in Japanese sake.

[0018] This invention provides a method for producing sake based on multi-strain mixed fermentation, wherein the multi-strain includes Rhizopus chinensis, Rhizopus oryzae, Issatchenkia orientalisi, and Saccharomyces cerevisiae. The production method includes the following steps:

[0019] (1) Steamed rice;

[0020] (2) Cultivation of bacteria: After steaming the rice, add rice koji for fermentation to obtain fermented rice; the rice koji is made by mixing pure Rhizopus hualiensis rice koji and pure Rhizopus oryzae rice koji in a ratio of 1:1.0 to 1:1.5.

[0021] The pure Rhizopus oryzae rice koji and pure Rhizopus huassifolia rice koji are obtained by inoculating Rhizopus oryzae and Rhizopus huassifolia into steamed rice and fermenting them, respectively. The Rhizopus oryzae rice koji has a saccharification power ≥1900 U / g koji, an acidic protease activity ≥28 U / g koji, and a liquefaction power ≥190 U / g koji. The Rhizopus huassifolia rice koji has an ester synthesis lipase activity ≥25 U / g koji, an acidic protease activity ≥8 U / g koji, a liquefaction power ≥45 U / g koji, and a saccharifying enzyme activity ≥200 U / g koji.

[0022] (3) Primary fermentation:

[0023] a. Inoculating with yeast:

[0024] Add Oriental Isaac yeast liquid and brewing water to the rice after fermentation with cultured bacteria, control the temperature at 18-25℃, and ferment for 24-26 hours;

[0025] b. Inoculate with brewer's yeast:

[0026] After step a is completed, pump brewer's yeast liquid into the fermentation tank, control the temperature at 18-25℃, and ferment for 3-5 days;

[0027] (4) Post-fermentation: After the main fermentation is completed, the temperature is controlled at 8-10℃ and the post-fermentation lasts for 30-40 days;

[0028] (5) After the post-fermentation is completed, the product sake is obtained by pressing, clarifying, sterilizing, aging, fine filtering, bottling, and sterilizing.

[0029] In one embodiment of the present invention, in step (2), the Rhizopus oryzae includes, but is not limited to: Rhizopus oryzae CICC41203, Rhizopus oryzae CICC40865, Rhizopus oryzae CICC 41440, Rhizopus oryzae CICC 3083, and Rhizopus oryzae CICC 40469. These strains all have a strong ability to hydrolyze starch and protein.

[0030] In one embodiment of the present invention, in step (2), the Rhizopus chinensis is CICC41505; this strain has high ester synthesis lipase activity, which is beneficial to increasing the content of ester substances in sake fermentation.

[0031] In one embodiment of the present invention, in step (2), the preparation method of Rhizopus oryzae and Rhizopus oryzae rice koji is as follows: activated Rhizopus oryzae spores and Rhizopus oryzae spores are inoculated into rice, mixed, cultured at 30-35℃ for 48-72h, and dried at 40-45℃ for 8-12h to obtain koji seeds; after steaming the japonica rice, it is cooled to 33-38℃, and 0.5-1.0% of the above koji seeds are inoculated into it, cultured at 30-35℃ and 80-90% humidity for 48-72h, cultured at 35-42℃ and 70-80% humidity for 18-24h, and finally dried at 40-45℃ for 8-12h to obtain Rhizopus oryzae rice koji and Rhizopus oryzae rice koji.

[0032] In one embodiment of the present invention, the pure Rhizopus oryzae koji is obtained by the following steps:

[0033] (1) Inoculate Rhizopus oryzae onto rice koji juice culture medium plates for activation. The culture temperature is 30-35℃ and the activation time is 72-96h.

[0034] (2) Wash the activated Rhizopus oryzae spores with sterile water and inoculate them into steamed rice. Shake well and incubate at 30-35℃ for 48-72 hours. Dry at 40℃ for 8-12 hours to obtain the starter culture.

[0035] (3) After the japonica rice is steamed and cooled to 35°C, 0.5-1.0% of the above-mentioned starter culture is inoculated and cultured at 30-35°C and 80-90% humidity for 48-72 hours. Then, it is cultured at 35-42°C and 70-80% humidity for another 18-24 hours. Finally, it is dried at 40-45°C for 8-12 hours to obtain rice starter culture.

[0036] The saccharification power and acidic protease activity of koji are relatively high. The liquefaction power of Rhizopus oryzae is ≥190U / g, the saccharification power is ≥1900U / g, and the acidic protease activity is ≥28U / g.

[0037] In one embodiment of the present invention, the rice koji juice culture medium is prepared by the following steps: 20 kg of crushed pure Rhizopus oryzae rice koji is added to every 100 kg of cooked rice, and brewing water is added in amounts three times the total weight of the cooked rice and koji. The mixture is saccharified at 62°C for 3–5 hours, filtered to obtain the juice, and the solid content is adjusted to 8–10% (w / w). The pH is adjusted to 5.5 with lactic acid, and the mixture is sterilized at 121°C for 15 minutes. The rice koji juice agar plate is prepared by adding 2% agar to the rice koji juice liquid culture medium, sterilizing under the same conditions, pouring the plates under aseptic conditions, and allowing them to solidify.

[0038] In one embodiment of the present invention, the raw materials and process conditions used in the pure Rhizopus oryzae koji are the same as those in Rhizopus oryzae koji, and the Rhizopus oryzae koji ester synthesis lipase activity is ≥25U / gkoji, the acid protease activity is ≥8U / gkoji, the liquefaction power is ≥45U / gkoji, and the saccharifying enzyme activity is ≥200U / gkoji.

[0039] In one embodiment of the present invention, in step (2), the amount of rice koji added is 8-10 kg / 100 kg of raw rice; before adding the rice koji, sterile water of 3-5 times the weight of the rice koji is added to mix the rice koji.

[0040] In one embodiment of the present invention, in step (2), the fermentation conditions are: cultured at 28-30°C for 36-48 hours, and the sugar content of the mash is 40-44% (w / w).

[0041] In one embodiment of the present invention, in step (2), in step (3), step a is as follows: 8-10 kg of *Isabella orientalis* seed liquid per 100 kg of raw rice is pumped into the fermenter, and brewing water is added at a total weight of 1.8-2.2 times the weight of the raw rice. The mixture is then thoroughly mixed with sterile air, and the temperature is controlled at 18-25°C. The initial density of *Isabella orientalis* is 0.5-1.5 × 10⁻⁶. 7 CFU / mL, sugar content controlled at 20-24% (w / w), fermented with sterile air every 12 hours, and temperature controlled.

[0042] In one embodiment of the present invention, in step (2) and step (3), 8-10 kg of brewer's yeast seed liquid per 100 kg of raw rice is pumped into the fermenter, and the brewer's yeast density after inoculation is 0.5-1.5 × 10⁻⁶. 7 CFU / mL, with sterile air rake applied every 12 hours for temperature-controlled fermentation.

[0043] In one embodiment of the present invention, in step (2) and step (3), the Saccharomyces cerevisiae includes, but is not limited to: Saccharomyces cerevisiae CICC 31693, Saccharomyces cerevisiae CICC 1273, Saccharomyces cerevisiae CICC 31129, Saccharomyces cerevisiae CICC 32163, and Saccharomyces cerevisiae CICC 31431.

[0044] In one embodiment of the present invention, in step (2), the brewing yeast includes, but is not limited to: brewing yeast CICC 1299, brewing yeast CICC 1009, brewing yeast CICC 1206, brewing yeast CICC 1442, brewing yeast CICC31324, brewing yeast CICC 32937, and brewing yeast CICC 33487.

[0045] In one embodiment of the present invention, step (2) involves the following steps in the preparation of the *Issa mesasura* spore liquid and the *Saccharomyces cerevisiae* spore liquid:

[0046] (1) Inoculate the Oriental Isaacs yeast or Saccharomyces cerevisiae strains into rice koji juice medium and culture them at 25-28℃ for 36-48h to obtain seed liquid; preferably, the seed liquid can be obtained by gradually expanding the seed liquid to meet the inoculation requirements of the yeast tank;

[0047] (2) After steaming the rice, add water, the pure Rhizopus oryzae koji as described in claim 1, and saccharifying enzyme, and keep warm at 60-65℃ for 3-5 hours for saccharification. Add brewing water to adjust the sugar content of the system to 10-12% (w / w), and then cool after sterilization.

[0048] (3) After cooling, the seed liquid obtained in step (1) is inoculated into the system obtained in step (2), and fermented at 28-30℃ to prepare the Oriental Isaac yeast liquid and the brewing yeast liquid respectively.

[0049] In one embodiment of the present invention, the yeast culture is prepared by the following steps:

[0050] (1) Inoculate the Saccharomyces orientalis or Saccharomyces cerevisiae strains preserved on test tube slant onto rice koji juice agar plates and activate for 48 h at 30 °C. Pick the activated yeast colonies with good morphology and transfer them to rice koji juice liquid agar, and incubate at 20 °C for 24 h.

[0051] (2) Yeast seed culture:

[0052] a. Under aseptic conditions, inoculate one loop of the slant culture into a test tube containing 10 mL of rice koji juice and incubate at 25°C for 24 h.

[0053] b. Shake the activated test tube culture medium well, and under aseptic conditions, add 1 mL to each test tube containing 10 mL of rice koji juice culture medium, and incubate at 25°C for 24 h.

[0054] c. Transfer 10 mL of yeast culture medium to a 150 mL Erlenmeyer flask containing 50 mL of rice koji juice, and incubate at 25 °C for 24 hours.

[0055] d. Transfer 50 mL of yeast culture medium to a 1000 mL Erlenmeyer flask containing 500 mL of rice koji juice, and incubate at 25 °C for 24 hours;

[0056] e. Transfer 500 mL of yeast culture medium to a 5000 mL Erlenmeyer flask containing 3000 mL of rice koji juice, and incubate at 25°C for 24 hours;

[0057] f. Transfer 3000 mL of yeast culture medium to a Karl flask containing 25 L of rice koji juice and incubate at 25 °C for 24 h to 48 h.

[0058] g. Transfer the yeast culture medium in the Karl arsenic tank to a seed tank containing 300L of rice koji juice and incubate at 25°C for 24h to 48h.

[0059] (3) After soaking glutinous rice for 24 hours, steam the rice until it is cooked but not mushy and has no raw center. In the yeast starter tank, add 280-300 kg of water, 4-5 kg ​​of pure Rhizopus oryzae koji, and 50 U / g rice saccharifying enzyme per 100 kg of glutinous rice. Saccharify at 58-62℃ for 4-5 hours, stirring once every hour. Add brewing water to adjust the sugar content to 12% (w / w). Sterilize at 121℃ for 15 minutes, cool to 30℃, and inoculate with 10% (v / v) of *Issa mesasura* or *Saccharomyces cerevisiae* seed culture. Incubate at 28-30℃ for 40-48 hours until the yeast cell concentration reaches 1.0-2.0 × 10⁻⁶. 8 CFU / mL.

[0060] In one embodiment of the present invention, the produced Japanese sake has an alcohol content of 15-16% vol, a total acid content of 2.2-3.5 g / L, a total ester content of 1.2-3.5 g / L, a total sugar content of 25-50 g / L, an ethyl acetate content of 30-120 mg / L, an isoamyl acetate content of 3-15 g / L, a β-phenylethanol content of 70-84 mg / L, and an ethyl hexanoate content of 3-10 g / L. The sake is pure, clear in color, and has a light, refreshing aroma of banana, pear, and apple. On the palate, it has a sweet taste of banana, peach, and apple, a subtle aroma of steamed rice, and a slight umami flavor.

[0061] In a preferred embodiment of the present invention, the produced Japanese sake has an alcohol content of 15.4% vol, a total acid content of 2.35 g / L, a total sugar content of 32.5 g / L, an ethyl acetate content of 45.7 mg / L, an isoamyl acetate content of 15.5 mg / L, a β-phenylethanol content of 46.2 mg / L, and an ethyl hexanoate content of 8.2 mg / L.

[0062] In one embodiment of the present invention, the brewing process of the Japanese sake includes the following steps:

[0063] (1) Raw material pretreatment: Japonica rice from Northeast China is used. After the rice is dehulled, it is milled and polished twice using a rice milling machine. The rice milling rate is controlled at 80-85%. The germ is completely removed, and the protein and fat on the surface of the rice are partially removed.

[0064] (2) Soaking rice: The raw rice is transported to the soaking tank, water is added, the rice-to-water ratio is 1:1.2, the foam is removed, and the rice is soaked at room temperature for 16-24 hours.

[0065] (3) Steaming rice: The soaked rice is conveyed from the bottom of the soaking tank to the rice steamer via a conveyor belt for steaming. The steam pressure of the rice steamer is 0.8-1.0 MPa, the steam temperature is 110-120℃, and the steaming time is 15-30 minutes.

[0066] The steamed rice is cooled to 20℃~25℃ using a fan. The cooked rice is distinct grains, firm on the outside and soft on the inside, with no white center, loose and not mushy, cooked but not mushy.

[0067] (4) Cultivation: The rice is transported to a mechanized stainless steel sake fermentation tank. During the transport, rice koji is added at a ratio of 8-10 kg / 100 kg of polished rice and mechanically mixed. The mixture is then incubated at 28-30°C for 36-48 hours, and the sugar content of the mash is 40-44% (w / w). Preferably, the rice koji is made by mixing pure Rhizopus oryzae rice koji and pure Rhizopus oryzae rice koji in a ratio of 1:1.0 to 1:1.5. Before adding the rice koji, sterile water with a weight of 3-5 times the rice koji is added to mix the rice koji thoroughly.

[0068] (5) Primary fermentation: Sake is obtained by inoculating and fermenting Oriental Isaac yeast and brewing yeast in sequence.

[0069] a. Inoculation with *Issa mesapiens*: Pump 8-10 kg of *Issa mesapiens* seed liquid per 100 kg of raw rice into the fermenter. Add 1.8-2.2 times the total weight of the rice to brewing water. Mix thoroughly with sterile air. Control the temperature at 18-25℃. The initial density of *Issa mesapiens* should be 0.5-1.5 × 10⁻⁶. 7 CFU / mL, sugar content controlled at 20-24% (w / w), stir with sterile air every 12 hours, and ferment at controlled temperature for 24-26 hours;

[0070] b. Inoculation with brewer's yeast: After 24-26 hours of temperature-controlled fermentation, pump 8-10 kg of brewer's yeast seed liquid per 100 kg of raw rice into the fermenter. The brewer's yeast density after inoculation should be 0.5-1.5 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation at 18-25℃ for 3-5 days;

[0071] (6) Post-fermentation: When the alcohol content in the fermentation liquid is ≥10%vol, the temperature of the fermentation tank is lowered to 8-10℃ at a rate of 2℃ / h, and the post-fermentation lasts for 30-40 days. Through the low-temperature fermentation process, the fermentation process is always stable and slow, which can effectively reduce the content of higher alcohols in rice wine.

[0072] (7) Coarse pressing: The wine is pressed using an air-membrane plate and frame filter press. Before pressing, the temperature of the fermented mash is controlled at 2-8℃, and after pressing, the temperature of the wine is controlled at 4-10℃.

[0073] (8) Clarification: Let stand in the clarification tank for 2 days;

[0074] (9) Sterilization: Sterilize raw wine at 100-121℃ in an instant sterilization system for 5-30 seconds;

[0075] (10) Aging: Sake is obtained by storing it in large tanks at 10°C for 3 to 6 months.

[0076] (11) Freezing and settling: The wine after aging is cooled to -2°C through a plate heat exchanger and pumped into a settling tank for settling for 2 days.

[0077] (12) The wine is filtered through a freezing membrane at a temperature of -3 to 5°C;

[0078] (13) Fine filtration: The wine is finely filtered using a nanofiltration membrane at a temperature of -2℃;

[0079] (14) Filling;

[0080] (15) Tunnel heat sterilization: 60-65℃ constant temperature for 25-30 minutes to obtain the finished product.

[0081] In one embodiment of the present invention, after freezing and filtration, the raw wine is sterilized in an instantaneous sterilization system at 121°C for 5-30 seconds. After 3-6 months, the wine is filtered through a frozen membrane and then through a nanofiltration membrane for precision filtration and packaging to obtain the finished sake.

[0082] In one embodiment of the present invention, the brewing water is characterized by being treated by a series of processes, including quartz sand filtration to remove impurities, coconut shell activated carbon filtration to remove odors, reverse osmosis filtration to remove ions, and fine filtration.

[0083] In one embodiment of the present invention, the time interval between milling and use of the raw rice is less than 7 days, the protein content is 4.0-4.5% (w / w), the fat content is 0.10-0.15% (w / w), the fatty acid value is ≤10mg KOH / 100g rice, and the freshness color reaction is light green to green.

[0084] In one embodiment of the present invention, the multi-strain includes Rhizopus chinensis, Rhizopus oryzae, Issatchenkia orientalisi, and Saccharomyces cerevisiae.

[0085] In one embodiment of the present invention, the *Issatchenkia orientalisi* was purchased from the China Industrial Microbial Culture Collection Center, with the numbers CICC31693, CICC 1273, CICC 31129, CICC 32163 or CICC 31431, and its taxonomic name is *Issatchenkia orientalisi*.

[0086] In one embodiment of the present invention, the brewing yeast was purchased from the China Industrial Microbial Culture Collection Center, with the numbers CICC1299, CICC 1009, CICC 1206, CICC 1442, CICC 31324, CICC 32937 or CICC33487, and its taxonomic name is Saccharomyces cerevisiae.

[0087] In one embodiment of the present invention, the Rhizopus oryzae was purchased from the China Industrial Microbial Culture Collection Center, with the numbers CICC41203, CICC 40865, CICC 41440, CICC 3083 or CICC 40469, and its taxonomic name is Rhizopus oryzae.

[0088] In one embodiment of the present invention, the Rhizopus chinensis was purchased from the China Industrial Microbial Culture Collection Center, number: CICC41505, and its taxonomic name is: Rhizopus chinensis.

[0089] In one embodiment of the present invention, the inoculum size of *Issa mesasura* is 0.5–1.5 × 10⁻⁶. 7 CFU / mL, with Saccharomyces cerevisiae at 0.5–1.5 × 10⁻⁶ CFU / mL. 7 CFU / mL, preferably 0.8–1.2 × 10⁻⁶. 7 CFU / mL and 0.8–1.2 × 10 7 CFU / mL, further preferably 1.0 × 10⁻⁶ 7 CFU / mL and 1.0×10 7 CFU / mL.

[0090] Another aspect of the present invention provides a method for increasing the flavor substances in sake or controlling the content of esters in sake, wherein the method involves inoculating Rhizopus schlegelii and Isaac's yeast, respectively, during the fermentation process of sake brewing.

[0091] In one embodiment of the present invention, the esters include isoamyl acetate, ethyl hexanoate, ethyl lactate, and ethyl acetate. Compared with the prior art, by employing the microbial combination, corresponding inoculation amount, and inoculation sequence of the present invention, synergistic saccharification and fermentation are achieved, and the ratio of isoamyl acetate, ethyl hexanoate, ethyl lactate, and ethyl acetate, as well as the total amount of esters, can reach the level of typical Japanese sake.

[0092] In one embodiment of the present invention, the fermentation process is as follows: steamed rice is mixed with rice koji containing Rhizopus huassifolia and Rhizopus oryzae for inoculation. After 24 hours, it is inoculated with Isaac's Oriental yeast for ester production. After 48 hours, it is inoculated with Saccharomyces cerevisiae for alcohol production. This inoculation sequence is used to increase the ester content of sake.

[0093] In one embodiment of the present invention, the method includes the following steps:

[0094] (1) Raw material pretreatment: Japonica rice from Northeast China is used. After the rice is dehulled, it is milled and polished twice using a rice milling machine. The rice milling rate is controlled at 80-85%. The germ is completely removed, and the protein and fat on the surface of the rice are partially removed.

[0095] (2) Soaking rice: Transfer the raw rice to the soaking tank, add water, the rice-to-water ratio is 1:1.2, remove the foam, and soak at room temperature for 16-24 hours.

[0096] (3) Steaming rice: The soaked rice is conveyed from the bottom of the soaking tank to the rice steamer via a conveyor belt for steaming. The steam pressure of the rice steamer is 0.8-1.0 MPa, the steam temperature is 110-120℃, and the steaming time is 15-30 minutes.

[0097] The steamed rice is cooled to 20℃~25℃ using a fan. The cooked rice is distinct grains, firm on the outside and soft on the inside, with no white center, loose and not mushy, cooked but not mushy.

[0098] (4) Cultivation: The rice is transported to a mechanized stainless steel sake fermentation tank. During the transport, rice koji is added at a ratio of 8-10 kg / 100 kg of polished rice and mechanically mixed. The mixture is then incubated at 28-30°C for 36-48 hours, and the sugar content of the mash is 40-44% (w / w). Preferably, the rice koji is made by mixing pure Rhizopus oryzae rice koji and pure Rhizopus oryzae rice koji in a ratio of 1:1.0 to 1:1.5. Before adding the rice koji, sterile water with a weight of 3-5 times the rice koji is added to mix the rice koji thoroughly.

[0099] (5) Primary fermentation: Sake is obtained by inoculating and fermenting Oriental Isaac yeast and brewing yeast in sequence.

[0100] a. Inoculation with *Issa mesapiens*: Pump 8-10 kg of *Issa mesapiens* seed liquid per 100 kg of raw rice into the fermenter. Add 1.8-2.2 times the total weight of the rice and brewing water. Mix thoroughly with sterile air. Control the temperature at 18-25℃. The initial density of *Issa mesapiens* should be 0.5-1.5 × 10⁻⁶. 7 CFU / mL, sugar content controlled at 20-24% (w / w), stir with sterile air every 12 hours, and ferment at controlled temperature for 24-26 hours;

[0101] b. Inoculation with Saccharomyces cerevisiae: After 24–26 hours of temperature-controlled fermentation, pump 8–10 kg of Saccharomyces cerevisiae seed liquid per 100 kg of raw rice into the fermenter. The Saccharomyces cerevisiae density after inoculation should be 0.5–1.5 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation at 18-25℃ for 3-5 days;

[0102] (6) Post-fermentation: When the alcohol content in the fermentation liquid is ≥10%vol, the temperature of the fermentation tank is lowered to 8-10℃ at a rate of 2℃ / h, and the post-fermentation lasts for 30-40 days. Through the low-temperature fermentation process, the fermentation process is always stable and slow, which can effectively reduce the content of higher alcohols in rice wine.

[0103] (7) Coarse pressing: The wine is pressed using an air-membrane plate and frame filter press. Before pressing, the temperature of the fermented mash is controlled at 2-8℃, and after pressing, the temperature of the wine is controlled at 4-10℃.

[0104] (8) Clarification: Let stand in the clarification tank for 2 days;

[0105] (9) Sterilization: Sterilize raw wine at 100-121℃ in an instant sterilization system for 5-30 seconds;

[0106] (10) Aging: Sake is obtained by storing it in large tanks at 10°C for 3 to 6 months.

[0107] (11) Freezing and settling: The wine after aging is cooled to -2°C through a plate heat exchanger and pumped into a settling tank for settling for 2 days.

[0108] (12) The wine is filtered through a freezing membrane at a temperature of -3 to 5°C;

[0109] (13) Fine filtration: The wine is finely filtered using a nanofiltration membrane at a temperature of -2℃;

[0110] (14) Filling;

[0111] (15) Tunnel heat sterilization: 60-65℃ constant temperature for 25-30 minutes to obtain the finished product.

[0112] The present invention also provides the application of the above method in improving the flavor substances in sake or in controlling the content of esters in sake, wherein the flavor substances include, but are not limited to, ethyl acetate content, isoamyl acetate content, β-phenylethanol content, and ethyl hexanoate content.

[0113] Beneficial effects

[0114] (1) A new method for producing Japanese sake with typical taste and flavor can be achieved by using the compound microbial agent composed of Rhizopus oryzae, Rhizopus huassifolius, Isaac's yeast of the Orient and Saccharomyces cerevisiae provided by the present invention and the corresponding process conditions, providing a solution for the domestic production of Japanese sake.

[0115] (2) The present invention uses a mixed rice koji composed of Rhizopus oryzae and Rhizopus oryzae, and uses rice with a polishing rate of 80-85% to produce Japanese sake, which saves raw materials and cumbersome rice milling operations and reduces production costs.

[0116] The ratio of *Rhizopus oryzae* to *Rhizopus schlegelii* is 1:1.0 to 1:1.5. This invention emphasizes the complementary advantages achieved by using *Rhizopus oryzae* and *Rhizopus schlegelii* in combination. *Rhizopus oryzae* rice koji contains strong acidic protease, saccharification power, and liquefaction power, while *Rhizopus schlegelii* has strong lipase and ester synthase activity, but lower saccharification power, liquefaction power, and acidic protease activity. Therefore, a combination of pure *Rhizopus oryzae* and pure *Rhizopus schlegelii* is used to provide liquefying and saccharifying enzymes for starch conversion to alcohol, and acidic protease for protein conversion to amino acids. The mixed rice koji of this invention, composed of *Rhizopus oryzae* and *Rhizopus schlegelii*, provides amylase, protease, lipase, and ester synthase as enzyme sources for the mash. The enzyme system of the strains is very rich. Through the action of these various enzymes, the raw materials are fully utilized, converting starch, protein, and fat in the raw materials into nutrients such as sugars, amino acids, and fatty acids. *Issa mesasura* utilizes these components to generate esters and other components during low-temperature fermentation. The proportion and content of these components determine the style and quality of the sake. The main reason why this invention can use rice with a polishing rate of 80-85% to produce Japanese sake is that the remaining protein and fat in the polished rice are fully utilized and transformed into flavor components of sake.

[0117] (3) The selection of Rhizopus chinensis fully utilizes its advantages of producing certain flavor substances and having strong activity of lipase and ester synthase. During the cultivation of Rhizopus chinensis, it provides sake with complex aroma substances and their precursors. The lipase secreted by Rhizopus chinensis can decompose the residual fat in the raw rice, providing a material basis for the subsequent growth of yeast and the formation of esters. This not only makes full use of the raw materials but also effectively reduces production costs. The membrane-bound ester synthase secreted by Rhizopus chinensis plays a role in the synthesis of esters such as ethyl hexanoate and isoamyl acetate in sake during low-temperature fermentation, which helps to increase the fullness of sake and the typicality of ginjo aroma.

[0118] (4) The ester synthesis ability of Oriental Isaac yeast is strong, especially the synthesis ability of isoamyl acetate. After culturing the bacteria and then inoculating Oriental Isaac yeast for fermentation, it plays an important role in the synthesis of esters in sake.

[0119] The sequential fermentation using Oriental Isaac yeast and brewing yeast maximizes the aroma-producing characteristics of Oriental Isaac yeast, significantly increases the content of isoamyl acetate and ethyl hexanoate, and makes a significant contribution to the production of flavor compounds. This makes the flavor of the sake close to that of sake made with sake-specific Aspergillus oryzae, yeast, rice, and 70% rice polishing, resulting in Japanese sake with typical flavor, gorgeous fruit aroma, and elegant and mellow taste.

[0120] (5) It highlights the advantages of microbial cultivation and low-temperature fermentation.

[0121] In the early stages of fermentation, bacteria are first cultivated, with Rhizopus oryzae and Rhizopus huassifolia multiplying and expanding to form highly active ester synthases and abundant nutrients, creating conditions for low-temperature controlled fermentation and providing sufficient carbon and nitrogen sources for subsequent yeast reproduction and flavor compound production. Then, Saccharomyces orientalis and Saccharomyces cerevisiae are inoculated sequentially for low-temperature fermentation. Low-temperature fermentation can increase the activity of ester synthases, increase the ester content of sake, make the taste cleaner, less bitter, and more fruity.

[0122] Through the cultivation of microorganisms, the amylase, protease, and lipase secreted by *Rhizopus oryzae* and *Rhizopus huassifolius* produce abundant nutrients, making low-temperature fermentation of sake possible. Low-temperature fermentation, in turn, creates favorable environmental conditions for the formation of esters in sake. Low temperatures are more conducive to the function of ester synthases, playing a crucial role in the formation of sake esters. At the same time, the formation of sake esters requires a sufficiently long time; that is, if the low-temperature fermentation time is too short, the typical characteristics of sake esters will be poor. Therefore, low-temperature fermentation is a vital step in the formation of sake's aroma components.

[0123] (6) The process of first cultivating bacteria and then sequentially inoculating yeast for fermentation reduces the content of bitter amino acids and bitter peptides in the product.

[0124] During the cultivation of bacteria, the protease secreted by Rhizopus oryzae preferentially acts on the protein in the rice, producing less bitter substances. Compared with the process of adding rice koji and wheat koji to the jar at the same time, the dry rice wine has a more harmonious taste.

[0125] By comprehensively employing the above-mentioned technological measures in production, a high-quality Japanese sake product with an elegant aroma, long-lasting aftertaste, and prominent fruity fragrance was obtained. Attached Figure Description

[0126] Figure 1 This invention provides an analytical chromatogram of amino acid content in sake.

[0127] Figure 2 This invention provides an analytical chromatogram of organic acid content in sake.

[0128] Figure 3 The present invention provides an analytical chromatogram of flavor compounds (alcohol and ester content) in sake. Detailed Implementation

[0129] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0130] The Rhizopus oryzae CICC 41203, Rhizopus huassifolius CICC 41505, Isaac's orientalis CICC 31693, and Saccharomyces cerevisiae CICC 1299 involved in the following examples were purchased from the China Industrial Microbial Culture Collection Center, and the saccharifying enzyme involved was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0131] The brewing water used in the following examples was provided by the winery, and the specific parameters are shown in Table 1:

[0132] Table 1: Indicators of Brewing Water

[0133]

[0134]

[0135] The detection methods used in the following embodiments:

[0136] (1) The alcohol content, total sugar, total acid and volatile esters were tested according to the method in GB / T13662-2018 Yellow Rice Wine.

[0137] (2) The count of yeast and lactobacillus in rice wine was conducted in accordance with the national standard GB4789.15-2016; the determination of alcohol content and total acidity was conducted in accordance with the national standard GB / T13662-2018.

[0138] (3) Analysis of amino acid profiles

[0139] The OPA-FMOC pre-column derivatization method was employed. A high-performance liquid chromatography (HPLC) system equipped with a UV detector was used. The column model was ODS Hypersil, 250 mm × 4.6 mm, 5 μm; column temperature: 40 °C; mobile phase: 20 mmol / L sodium acetate and methanol:acetonitrile = 1:2 (v:v) mixture; flow rate: 1 mL / min; detection wavelength: 210 nm. The sample was filtered through a 0.45 μm filter membrane, and the injection volume was 10 μL.

[0140] (4) Analysis of organic acid spectra

[0141] Organic acid analysis was performed using a high-performance liquid chromatography (HPLC) system equipped with a UV detector. The chromatographic column was a Waters Atlantis C18, 250 mm × 4.5 mm, 5 μm; column temperature: 30 °C; mobile phase: 0.05 mmol / L H₃PO₄: methanol = 95:5 (v:v); flow rate: 0.8 mL / min; detection wavelength: 210 nm. Samples were filtered through a 0.45 μm filter membrane, and the injection volume was 10 μL.

[0142] (5) Analysis of sugar profile

[0143] Sugar analysis was performed using a high-performance liquid chromatograph equipped with a refractive index detector. The chromatographic column was a Waters Sugarpak1, 300 mm × 6.5 mm, 5 μm; the column temperature was 85 °C; the flow rate was 0.4 mL / min; and the eluent was pure water. The sample was filtered through a 0.45 μm filter membrane, and the injection volume was 10 μL.

[0144] (6) Determination of alcohol and ester content

[0145] HS-SPME conditions: A 50μm / 30μm CAR / DVB / PDMS SPME fiber extraction head was used for the extraction of flavor compounds; 8 mL of wine sample and 10 μL of internal standard 2-octanol (final concentration: 50 μg / L) were accurately transferred into a 20 mL extraction bottle and extracted at 50℃ for 30 min.

[0146] GC-MS conditions: A DB-Wax column (30 m × 0.5 mm × 0.5 μm) was used, with helium as the carrier gas at a flow rate of 0.8 mL / min. The sample was introduced into the detector along with the carrier gas. The injection port temperature and detector temperature were 250 °C, and the ion source temperature was 200 °C. MS conditions: EI was used as the ionization source, with an electron energy of 70 eV and a scan range of 32.0–350.0 m·z. -1 Temperature program: Initial temperature 40℃, hold for 4 min, increase to 90℃ at 5℃ / min, increase to 250℃ at 105℃ / min, hold for 7 min.

[0147] Example 1: Preparation of pure Rhizopus oryzae koji and Rhizopus wort koji

[0148] The specific steps are as follows:

[0149] (1) Inoculate Rhizopus oryzae CICC41203 or Rhizopus oryzae CICC41505 into rice koji juice plates for activation. The culture temperature is 33℃ and the activation time is 86h.

[0150] Rice koji juice plate: Add 300g of water and 100U / g of rice saccharifying enzyme to 100g of rice, keep warm at 60℃ for 5 hours, stirring once every hour, add brewing water to adjust the sugar content of the system to 12% (w / w), add 2g / L of agar, and sterilize at 121℃ for 15 minutes.

[0151] (2) The activated Rhizopus oryzae CICC41203 or Rhizopus oryzae CICC41505 spores were inoculated into steamed rice, shaken thoroughly, cultured at 35℃ for 60h, and dried at 40℃ for 10h to obtain Rhizopus oryzae starter and Rhizopus oryzae starter, respectively.

[0152] (3) After steaming the japonica rice and cooling it to 35℃, 0.8% (w / w) of the above-mentioned koji was inoculated into the rice. The rice was then cultured at 33℃ and 85% humidity for 15 hours, followed by 21 hours at 38℃ and 75% humidity. Finally, the rice was dried at 42℃ for 10 hours to prepare Rhizopus oryzae koji and Rhizopus huassifolia koji, with mycotoxin concentrations of 2.1 × 10⁻⁶ and 2.1 × 10⁻⁶, respectively. 8 CFU / g, 3.6×10 8 CFU / g song.

[0153] The results showed that the liquefaction power of Rhizopus oryzae was 212 U / g, the saccharification power was 1906 U / g, and the acidic protease activity was 29 U / g.

[0154] The activity of the lipase synthase bound to the mycelium of Rhizopus was 26 U / g, the liquefaction power was 49 U / g, the saccharification power was 206 U / g, and the acid protease activity was 8.6 U / g.

[0155] Example 2: Preparation of pure culture of *Isapoma orientalis* and *Saccharomyces cerevisiae*

[0156] The specific steps are as follows:

[0157] (1) The Saccharomyces davidiana CICC31693 or Saccharomyces cerevisiae CICC1299 strain preserved on test tube slant was inoculated onto a rice koji juice culture medium plate and activated for 48 h. The rice koji juice culture medium plate was the same as in Example 1, and the activation temperature was 30 °C.

[0158] Activated Isaac's yeast or Saccharomyces cerevisiae strains with good colony morphology were selected and cultured in rice koji liquid medium at 20℃ for 24 h to prepare Isaac's yeast culture medium and Saccharomyces cerevisiae culture medium, respectively.

[0159] Rice koji liquid culture medium: 20 kg of crushed pure Rhizopus oryzae rice koji obtained from Example 1 was added to every 100 kg of cooked rice. Brewing water was added in a ratio of 3 times the total weight of cooked rice and rice koji. After saccharification at 62°C for 4 h, the juice was filtered and the solid content in the liquid was adjusted to 10% (w / w). The pH was adjusted to 5.5 with lactic acid and sterilized at 121°C for 15 min.

[0160] Rice koji juice agar plates are made by adding 2% agar to rice koji juice liquid culture medium, sterilizing under the same conditions, pouring the plates under aseptic conditions, and allowing them to solidify.

[0161] (2) Seed culture of *Isapioca orientalis* CICC31693 or *Saccharomyces cerevisiae* CICC1299:

[0162] 12% (w / w) rice koji juice culture medium: The pure Rhizopus oryzae rice koji cultured in Example 1 was crushed, 3 times the amount of brewing water was added, saccharified at 60°C for 3 hours, filtered to obtain juice, water was added to adjust the solids concentration of the filtrate to 12% (w / w), the pH value was adjusted to 5.5 with lactic acid, and sterilized at 121°C for 15 minutes.

[0163] The preferred and specific method for expanding the culture is as follows:

[0164] a. Under aseptic conditions, add one loopful of the slant culture to a test tube containing 10 mL of the rice koji liquid culture medium described in step (1) and incubate at 25°C for 24 hours.

[0165] b. Shake the activated test tube culture medium well, and under aseptic conditions, add 1 mL to each test tube containing 10 mL of the above rice koji juice liquid culture medium, and incubate at 25°C for 24 hours.

[0166] c. Transfer 10 mL of yeast culture medium to a 150 mL Erlenmeyer flask containing 50 mL of the above rice koji juice liquid culture medium, and incubate at 25 °C for 24 hours.

[0167] d. Transfer 50 mL of yeast culture medium to a 1000 mL Erlenmeyer flask containing 500 mL of the above rice koji juice liquid culture medium, and incubate at 25°C for 24 hours;

[0168] e. Transfer 500 mL of yeast culture medium to a 5000 mL Erlenmeyer flask containing 3000 mL of the above rice koji juice liquid culture medium, and incubate at 25°C for 24 hours.

[0169] f. Transfer 3000 mL of yeast culture medium to a Karl flask containing 25 L of the above rice koji liquid culture medium, and incubate at 25°C for 24 to 48 hours.

[0170] g. Transfer the yeast culture medium in the Karl argutor tank to a seed tank containing 300L of the above-mentioned rice koji liquid culture medium, and incubate at 25°C for 24 to 48 hours.

[0171] (3) After soaking glutinous rice for 24 hours, the rice is steamed. The rice is cooked but not mushy and has no raw center. In the yeast tank, 300 kg of water, 5 kg of pure Rhizopus oryzae rice koji prepared in Example 1 and 50 U / g rice saccharifying enzyme (purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.) are added to 100 kg of glutinous rice. The mixture is kept at 60°C for 5 hours, stirred once every hour, and brewing water is added to adjust the sugar content of the system to 12% (w / w). The mixture is sterilized at 121°C for 15 minutes and cooled to 30°C.

[0172] The 10% (v / v) of *Isabella orientalis* seed culture or *Saccharomyces cerevisiae* seed culture prepared in step (2) was added to the system after adjusting the sugar content, and cultured at 29°C for 44 hours to prepare *Isabella orientalis* culture and *Saccharomyces cerevisiae* culture, respectively.

[0173] The results showed that the density of *Isaporphyromonas orientalis* in the *Isaporphyromonas orientalis* culture was 1.8 × 10⁻⁶. 8 CFU / mL, the yeast density of *Saccharomyces cerevisiae* in the culture was 1.6 × 10⁻⁶. 8 CFU / mL.

[0174] In addition, the preparation methods of the above-mentioned *Saccharomyces cerevisiae* CICC31025, *Saccharomyces cerevisiae* CICC31194, *Saccharomyces cerevisiae* CICC1728, and *Pichia pastoris* CICC33440 are the same as those in steps (1) to (3).

[0175] Example 3: Mechanized Large-Tank Sake Brewing Process

[0176] The specific steps are as follows:

[0177] 1. Sake brewing

[0178] (1) Soaking rice

[0179] Raw rice pretreatment: Japonica rice from Northeast China (85% milled rice rate) is used. The raw rice is milled twice to achieve a milled rice rate of 85%.

[0180] Soaking rice: The processed raw rice is transferred to a soaking tank, water is added, the rice-to-water ratio is 1:1.2, the scum is removed, and the rice is soaked at room temperature for 24 hours.

[0181] (2) Steamed rice:

[0182] After soaking, the raw rice is conveyed from the bottom of the soaking tank to the rice steamer via a conveyor belt for cooking. The steam pressure of the rice steamer is 0.9 MPa, the steam temperature is 115℃, and the steaming time is 22 minutes.

[0183] The steamed rice is cooled to 23°C using a fan. The cooked rice is distinct grains, firm on the outside and soft on the inside, with no white center, loose and not mushy, cooked but not mushy.

[0184] (3) Culture:

[0185] 1) Processing of rice koji

[0186] The pure Rhizopus oryzae rice koji and pure Rhizopus oryzae rice koji prepared in Example 1 were mixed in a mass ratio of 1:1, and then sterile water was added in a mass ratio of 4 times the total weight of the rice koji. The rice koji was thoroughly mixed and placed in a mechanized stainless steel sake fermentation tank.

[0187] 2) The steamed rice obtained in step (2) is sent into a mechanized stainless steel sake fermentation tank. During the transportation process, 9 kg of rice koji obtained in step 1) is added for every 100 kg of raw rice. The mixture is mechanically mixed and incubated at 29°C for 36 hours. After the incubation is completed, the sugar content of the mash is 42% (w / w).

[0188] (4) Primary fermentation:

[0189] a. Inoculating with yeast:

[0190] After the incubation in step (3) is completed, pump 9 kg of *Isabella orientalis* culture (prepared in Example 2) per 100 kg of raw rice into the fermenter, and add brewing water at a rate of 2.0 times the total weight of the raw rice. Mix thoroughly with sterile air, maintain the temperature at 21°C, and set the initial density of *Isabella orientalis* at 1.0 × 10⁻⁶. 7 The sugar content in the fermenter was controlled at 22% (w / w) with CFU / mL. The fermenter was stirred with sterile air every 12 hours and fermented at a controlled temperature for 25 hours.

[0191] b. Inoculate with brewer's yeast:

[0192] After fermenting with *Issa mesasura* broth for 25 hours, 10 kg of *Saccharomyces cerevisiae* broth (prepared in Example 2) per 100 kg of raw rice was pumped into the fermenter. The density of *Saccharomyces cerevisiae* after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21℃ (alcohol content in fermentation broth ≥10% vol).

[0193] (5) Post-fermentation: After the main fermentation is completed, the temperature of the fermentation tank is reduced to 9℃ at a rate of 2℃ / h, and the post-fermentation is carried out for 35 days at 9℃.

[0194] (6) Coarse pressing: The wine is pressed using a gas membrane plate and frame filter press. Before pressing, the temperature of the fermented mash is controlled at 2-8℃, and after pressing, the temperature of the wine is controlled at 6℃.

[0195] (7) Clarification: Let stand in the clarification tank for 2 days.

[0196] (8) Sterilization: Sterilize raw wine at 121℃ in an instant sterilization system for 20 seconds.

[0197] (9) Aging: The sake is obtained by storing it in a large tank at 10°C for 4 months.

[0198] (10) Freezing and settling: The wine after aging is cooled to -2°C through a plate heat exchanger and pumped into a settling tank for settling for 2 days.

[0199] (11) The wine is filtered through a freezing membrane at a temperature of 1°C.

[0200] (12) Fine filtration: The wine is finely filtered using a nanofiltration membrane at a temperature of -2℃.

[0201] (13) Filling.

[0202] (14) Tunnel-type heat sterilization: 63℃ constant temperature for 27 min to obtain the finished product.

[0203] 2. Specifications of brewed sake

[0204] (1) The conventional physicochemical properties of sake are shown in Table 2.

[0205] Table 2. Routine Physicochemical Indicators of Sake

[0206]

[0207] The results showed that the sake obtained by the method of the present invention was pure, clear in color, and had a light and refreshing aroma of banana, pear and apple. On the palate, it had a sweet aroma of banana, peach and apple, a light aroma of steamed rice and a little bit of umami.

[0208] (2) The flavor compounds of the sake in this embodiment are shown in Table 3.

[0209] Table 3. Alcohol and ester content of sake (mg / L)

[0210]

[0211] The results show that the sake prepared by the method of the present invention contains the main aroma components of Japanese sake: ethyl acetate, ethyl butyrate, isoamyl acetate, and ethyl hexanoate. Isoamyl acetate has a banana aroma, and ethyl hexanoate has an apple aroma. These are the most important aroma substances in high-end Japanese sake. The content of isoamyl acetate in the sake of the present invention also reaches the upper limit of the content in Japanese sake. Therefore, the sake prepared by the method of the present invention is rich in aroma substances and has the typical aroma characteristics of Japanese sake.

[0212] (3) The free amino acids of the sake in this embodiment are shown in Table 4. The amino acid data of Japanese sake in the table are referenced from (Wang Shuying, Xu Yan; Study and analysis of amino acid composition and formation of Chinese yellow wine and Japanese sake [J]. Brewing, 1997(6):10-11.).

[0213] Table 4. Free amino acid profile of sake

[0214]

[0215]

[0216] As can be seen from the comparison of amino acid data between the sake of this embodiment and Japanese sake in Table 4, although the rice polishing rate of the raw rice used in this invention is only 80-85%, which is much higher than that of Japanese sake (<70%), the overall amino acid content of the sake of this invention is lower than that of Japanese sake by adopting the multi-strain mixed fermentation and cultivation process of this invention. In particular, the bitter and astringent amino acids are significantly lower than those of Japanese sake.

[0217] (4) The organic acid content of the sake in this embodiment is shown in Table 5.

[0218] Table 5 Organic Acid Profile of Sake

[0219]

[0220]

[0221] The results showed that the content of organic acids in the sake prepared by the method of the present invention was not significantly different from that in Japanese sake.

[0222] Example 4:

[0223] The specific implementation method is the same as in Examples 1-3, except that the Oriental Isaac yeast is adjusted to Oriental Isaac yeast CICC1273, Oriental Isaac yeast CICC 31129, Oriental Isaac yeast CICC 32163 or Oriental Isaac yeast CICC 31431. Sake is prepared according to the method in Examples 1-3. The results show that the flavor substances of the obtained sake, such as the ratio of isoamyl acetate, ethyl hexanoate, ethyl lactate and ethyl acetate and the total amount of esters, can reach the level of typical Japanese sake.

[0224] The specific implementation method is the same as in Examples 1-3, except that the brewing yeast is adjusted to brewing yeast CICC 1009, brewing yeast CICC 1206, brewing yeast CICC 1442, brewing yeast CICC 31324, brewing yeast CICC 32937, and brewing yeast CICC 33487. Sake is prepared according to the method in Examples 1-3. The results show that the flavor substances of the obtained sake, such as the ratio of isoamyl acetate, ethyl hexanoate, ethyl lactate, and ethyl acetate, and the total amount of esters, can reach the level of typical Japanese sake.

[0225] The specific implementation method is the same as in Examples 1-3, except that the Rhizopus oryzae is adjusted to Rhizopus oryzae CICC40865, Rhizopus oryzae CICC 41440, Rhizopus oryzae CICC 3083, and Rhizopus oryzae CICC 40469. Sake is prepared according to the method in Examples 1-3. The results show that the flavor substances of the obtained sake, such as the ratio of isoamyl acetate, ethyl hexanoate, ethyl lactate, and ethyl acetate, and the total amount of esters, can reach the level of typical Japanese sake.

[0226] Comparative example:

[0227] To better illustrate the advantages of the Japanese sake brewed by this invention, comparative examples 1 to 5 were also prepared. In these comparative examples, the Kluyveromyces CICC33440, Saccharomyces cerevisiae CICC31025, Saccharomyces cerevisiae CICC31194, and Saccharomyces cerevisiae CICC1728 used were all purchased from the China Industrial Microbial Culture Collection Center. They are all aroma-producing and ester-producing yeasts currently used in alcoholic beverage brewing. Saccharomyces cerevisiae is an aroma-producing yeast widely used in the production of rice wine and huangjiu (yellow wine), while Kluyveromyces, Saccharomyces cerevisiae, and Saccharomyces cerevisiae are aroma-producing yeasts commonly used in wine and other fruit wines.

[0228] The specific details of the comparison are as follows:

[0229] Comparative Example 1

[0230] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, only brewing yeast is inoculated, and not Oriental Isaac yeast, that is:

[0231] (4) Primary fermentation: 10 kg of brewer's yeast culture (prepared in Example 2) per 100 kg of raw rice was pumped into the fermenter after the inoculation in step (3). The brewer's yeast density after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21℃.

[0232] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0233] Comparative Example 2

[0234] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, only pure Rhizopus oryzae koji is added, and Rhizopus oryzae koji is not added. The amount of pure Rhizopus oryzae koji added is the sum of Rhizopus oryzae koji and Rhizopus oryzae koji in Example 3, that is:

[0235] Step (3) is adjusted to:

[0236] The steamed rice obtained in step (2) was sent into a mechanized stainless steel fermentation tank. During the transportation process, 9 kg of pure Rhizopus oryzae rice koji prepared in Example 1 was added based on 100 kg of raw rice. The mixture was mechanically mixed and cultured at 29°C for 36 h. After the culture was completed, the sugar content of the mash was found to be 42% (w / w).

[0237] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0238] Comparative Example 3

[0239] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, rice, rice koji, Oriental Isaac yeast, brewing yeast, and brewing water are added to the tank simultaneously, without the sequential fermentation process of first inoculating Oriental Isaac yeast and then inoculating brewing yeast. That is, step (3) of inoculating the bacteria is omitted, and the main fermentation is carried out after steaming the rice. Specifically:

[0240] (3) Primary fermentation:

[0241] The steamed rice obtained in step (2) was sent into a mechanized stainless steel sake fermentation tank, and 9 kg / 100 kg of raw rice of Oriental Isaac yeast liquid (prepared in Example 2), 10 kg / 100 kg of raw rice of brewing yeast liquid (prepared in Example 2), brewing water (2.0 times the total weight of the rice) and 9 kg / 100 kg of raw rice koji (the pure Rhizopus oryzae koji and pure Rhizopus oryzae koji prepared in Example 1 were mixed in a mass ratio of 1:1, and then sterile water was added at 4 times the total weight of the koji to mix the koji thoroughly). The mixture was mixed evenly and fermented at 21°C for 5 days.

[0242] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0243] Comparative Example 4

[0244] The specific implementation method is the same as in Examples 1-3, except that the sake is brewed using mechanized large-tank fermentation at the conventional fermentation temperature for rice wine (main fermentation temperature 30℃, secondary fermentation temperature 15℃).

[0245] Adjust the primary fermentation conditions in step (4) as follows:

[0246] After inoculating with brewer's yeast, the temperature was controlled at 30℃, and fermentation continued for 4 days.

[0247] At the same time, the fermentation conditions for the post-fermentation in step (5) are adjusted as follows:

[0248] After the primary fermentation is completed, the temperature of the fermentation tank is reduced to 15℃ at a rate of 2℃ / h, and then the secondary fermentation is carried out for 35 days at 15℃.

[0249] The brewing process for other sake is the same as in Example 3.

[0250] Comparative Example 5

[0251] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, the inoculated *Issa mesasura* is replaced with *Kluyveromyces pubescens* CICC33440, that is, the primary fermentation in step (4) is:

[0252] a. Inoculating with yeast:

[0253] After the inoculum cultivation in step (3) is completed, pump 9 kg of Kluyveromyces CICC 33440 bacterial culture per 100 kg of raw rice (prepared in Example 2) into the fermenter, and add brewing water at 2.0 times the total weight of the rice. Mix thoroughly with sterile air, control the temperature at 21°C, and maintain the initial density of Kluyveromyces CICC 33440 at 1.0 × 10⁻⁶. 7 With CFU / mL, the sugar content in the fermenter was controlled at 22% (w / w). The fermenter was stirred with sterile air every 12 hours and fermented at controlled temperature for 25 hours.

[0254] b. Inoculate with brewer's yeast:

[0255] After 25 hours of fermentation with Kluyveromyces oryzae, 10 kg of Saccharomyces cerevisiae broth (prepared in Example 2) per 100 kg of raw rice was pumped into the fermenter. The Saccharomyces cerevisiae density after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21°C (alcohol content in fermentation broth ≥10% vol).

[0256] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0257] Comparative Example 6

[0258] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, the inoculated *Issa mesasura* is replaced with *Cytomyces commune* CICC31025, that is, the primary fermentation in step (4) is:

[0259] a. Inoculating with yeast:

[0260] After the incubation in step (3) is completed, pump 9 kg of CICC 31025 yeast culture solution per 100 kg of raw rice (prepared in Example 2) into the fermenter, and add brewing water at 2.0 times the total weight of the rice. Mix thoroughly with sterile air, control the temperature at 21°C, and maintain the initial density of CICC 31025 yeast at 1.0 × 10⁻⁶. 7 The sugar content in the fermenter was controlled at 22% (w / w) with CFU / mL. The fermenter was stirred with sterile air every 12 hours and fermented at a controlled temperature for 25 hours.

[0261] b. Inoculate with brewer's yeast:

[0262] After fermenting with *Saccharomyces cerevisiae* for 25 hours, 10 kg of *Saccharomyces cerevisiae* broth (prepared in Example 2) per 100 kg of raw rice was pumped into the fermenter. The density of *Saccharomyces cerevisiae* after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21℃ (alcohol content in fermentation broth ≥10% vol).

[0263] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0264] Comparative Example 7

[0265] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, the inoculated *Issa mesasura* is replaced with *Wickhamia lanceolata* CICC31194, that is, the primary fermentation in step (4) is:

[0266] a. Inoculating with yeast:

[0267] After the inoculum cultivation in step (3) is completed, pump 9 kg of abnormal Wickham yeast CICC 31194 culture solution per 100 kg of raw rice (prepared in Example 2) into the fermenter, and add brewing water at 2.0 times the total weight of the rice. Mix thoroughly with sterile air, control the temperature at 21°C, and maintain the initial density of abnormal Wickham yeast CICC 31194 at 1.0 × 10⁻⁶. 7 With CFU / mL, the sugar content in the fermenter was controlled at 22% (w / w). The fermenter was stirred with sterile air every 12 hours and fermented at controlled temperature for 25 hours.

[0268] b. Inoculate with brewer's yeast:

[0269] After fermenting for 25 hours with abnormal Wickham yeast, 10 kg of Saccharomyces cerevisiae (prepared in Example 2) per 100 kg of raw rice was pumped into the fermenter. The Saccharomyces cerevisiae density after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21℃ (alcohol content in fermentation broth ≥10% vol).

[0270] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0271] Comparative Example 8

[0272] The specific implementation method is the same as in Examples 1-3, except that during sake brewing, the inoculated *Issa mesasura* is replaced with *Hansenula polymorpha* CICC1728, that is, the primary fermentation in step (4) is:

[0273] a. Inoculating with yeast:

[0274] After the inoculum cultivation in step (3) is completed, pump 9 kg of abnormal Hansenula polymorpha CICC 1728 bacterial culture per 100 kg of raw rice (prepared in Example 2) into the fermenter, and add brewing water at a rate of 2.0 times the total weight of the rice. Mix thoroughly with sterile air, control the temperature at 21°C, and maintain the initial density of abnormal Hansenula polymorpha CICC 1728 at 1.0 × 10⁻⁶. 7The sugar content in the fermenter was controlled at 22% (w / w) with CFU / mL. The fermenter was stirred with sterile air every 12 hours and fermented at controlled temperature for 25 hours.

[0275] b. Inoculate with brewer's yeast:

[0276] After fermenting with abnormal Hansenula polymorpha for 25 hours, 10 kg of Saccharomyces cerevisiae culture per 100 kg of raw rice was pumped into the fermenter. The Saccharomyces cerevisiae density after inoculation was 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, and continue fermentation for 4 days at 21℃ (alcohol content in fermentation broth ≥10% vol).

[0277] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0278] Comparative Example 9

[0279] The specific implementation method is the same as in Examples 1-3, except that the yeast inoculation order is adjusted during sake brewing, that is, the primary fermentation in step (4) is:

[0280] a. Inoculate with brewer's yeast:

[0281] After the inoculation in step (3) is completed, pump 10 kg of brewer's yeast culture (prepared in Example 2) per 100 kg of raw rice into the fermenter. The brewer's yeast density after inoculation is 1.0 × 10⁻⁶. 7 CFU / mL, rake with sterile air every 12 hours, ferment at 21℃ for 25 hours.

[0282] b. Inoculation with *Isapia orientalis*:

[0283] After fermentation for 25 hours using brewing yeast, 9 kg of *Isabella orientalis* culture (prepared in Example 2) per 100 kg of raw rice was pumped into the fermentation tank, along with brewing water at 2.0 times the total weight of the rice. The mixture was then thoroughly mixed with sterile air, and the temperature was controlled at 21°C. The initial density of *Isabella orientalis* was 1.0 × 10⁻⁶. 7 CFU / mL, the sugar content in the fermenter was controlled at 22% (w / w), and the fermenter was stirred with sterile air every 12 hours. Fermentation was carried out at 21°C for 4 days (the alcohol content in the fermentation broth was ≥10% vol).

[0284] Other brewing process parameters are the same as those for the sake brewing process in Example 3.

[0285] Analysis results of the physicochemical indicators of the comparative finished wine:

[0286] Table 6. Conventional physicochemical properties of sake in each comparative example.

[0287]

[0288] The results show:

[0289] Comparative Example 1: Sake fermented with brewer's yeast had a higher content of isoamyl alcohol, but the content of esters such as ethyl hexanoate and isoamyl acetate was the lowest, and the aroma was weaker.

[0290] The results of Comparative Example 2 show that *Rhizopus oryzae* has a significant impact on the ethyl hexanoate content in sake. In Comparative Example 2, the ethyl hexanoate content decreased significantly without the addition of *Rhizopus oryzae* rice koji. *Rhizopus oryzae* secretes highly active ester synthases, which enhance esterification capabilities, thereby increasing the ethyl hexanoate content in sake. Ethyl hexanoate is the most important lipid component in the aroma of Japanese sake ginjo and has a significant impact on sake quality.

[0291] In Comparative Example 3, sake inoculated simultaneously with *Rhizopus oryzae*, *Rhizopus chinensis*, *Issa mesosoma orientalis*, and *Saccharomyces cerevisiae* resulted in sake with low ester content and high total acid content, indicating antagonistic effects between different microorganisms. Sequential fermentation using *Issa mesosoma orientalis* and *Saccharomyces cerevisiae* maximizes the aroma-producing characteristics of *Issa mesososoma orientalis*, significantly increasing the content of ethyl acetate, isoamyl acetate, and ethyl hexanoate, making a significant contribution to the production of flavor compounds. This results in a sake flavor close to that of sake produced using a process employing specific *Aspergillus oryzae*, specific yeast, specific rice, and 70% rice polishing, giving the produced Japanese sake a typical flavor profile, a gorgeous fruity aroma, and an elegant, mellow taste.

[0292] Compared to the sake in Comparative Examples 1-8, Example 3 showed a lower total acid content, particularly a lower acetic acid content, thus achieving a better balance in taste and aroma. Acids are important flavor compounds in sake; appropriate amounts can regulate flavor. Most acids are produced by microbial metabolism during fermentation. Among these, volatile acids, primarily acetic acid, are the main contributors to the pungent taste of sake. The total acid content in sake should be controlled below 2.4 g / L. Representative acids produced by yeast include malic acid and succinic acid. Malic acid is a refreshing acid; in sweeter sake, its presence can provide balance, resulting in a more balanced fruity flavor. Succinic acid is a umami component found in shellfish, offering a savory taste within its acidity. Sake with a high succinic acid content may exhibit bitterness and astringency.

[0293] The results of Comparative Example 4 indicate that low-temperature fermentation is beneficial to the formation of esters in rice wine and, to some extent, inhibits the growth of acid-producing bacteria. In the early stages of fermentation, sufficient carbon and nitrogen sources are first provided for yeast reproduction through microbial cultivation. Then, *Issa mesosoma orientalis* and *Saccharomyces cerevisiae* are sequentially inoculated for low-temperature fermentation. Low-temperature fermentation increases the activity of ester synthases, resulting in higher ester content in the sake, a cleaner taste, less bitterness, and a richer fruity aroma. The proliferation of *Rhizopus oryzae* and *Rhizopus chinensis* forms highly active ester synthases and abundant nutrients, creating conditions for low-temperature controlled fermentation, resulting in a fuller taste and aroma in the sake.

[0294] In Comparative Example 6, the sake fermented with *Saccharomyces cerevisiae* had the highest content of higher alcohols, with isobutanol and isoamyl alcohol content significantly higher than other sakes. Higher alcohols constitute the unique aroma framework of sake, but if the content is too high, it will affect the quality of the sake and cause harm to the human body, leading to headaches after drinking. Therefore, it is necessary to strictly control the content of higher alcohols.

[0295] The analysis results of the content of ethyl hexanoate and isoamyl acetate in Examples 2, 3 and 4 show that the addition of Rhizopus huananensis rice koji and Isaac's yeast plays a key role in the content of ethyl hexanoate and isoamyl acetate, important flavor compounds of sake ginjo aroma.

[0296] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for producing sake based on multi-strain mixed fermentation, characterized in that, The multi-species includes Rhizopus huassiformis ( Rhizopus chinensis ), Rhizopus oryzae ( Rhizopus oryzae ), Oriental Isaac yeast ( Issatchenkia orientalisi ) and brewer's yeast ( Saccharomyces cerevisiae The production method includes the following steps: (1) Steamed rice; (2) Cultivation of bacteria: After steaming the rice, add rice koji for fermentation to obtain fermented rice; the rice koji is made by mixing pure Rhizopus oryzae rice koji and pure Rhizopus oryzae rice koji in a ratio of 1:1.0 to 1:1.

5. The preparation methods of the pure Rhizopus oryzae rice koji and pure Rhizopus huassifolia rice koji are as follows: Rhizopus oryzae and Rhizopus huassifolia are respectively inoculated into steamed rice and fermented. The Rhizopus oryzae rice koji has a saccharification power ≥1900 U / g koji, an acidic protease activity ≥28 U / g koji, and a liquefaction power ≥190 U / g koji. The Rhizopus huassifolia rice koji has an ester synthesis lipase activity ≥25 U / g koji, an acidic protease activity ≥8 U / g koji, a liquefaction power ≥45 U / g koji, and a saccharifying enzyme activity ≥200 U / g koji. (3) Primary fermentation: a. Inoculating with yeast: Add Oriental Isaac yeast liquid and brewing water to the rice after fermentation with cultured bacteria, control the temperature at 18~25℃, and ferment for 24~26 hours; b. Inoculate with brewer's yeast: After step a is completed, pump brewer's yeast liquid into the fermentation tank, control the temperature at 18~25℃, and ferment for 3~5 days; (4) Post-fermentation: After the main fermentation is completed, the product temperature is controlled at 8~10℃, and the post-fermentation lasts for 30~40 days; (5) After the post-fermentation is completed, the product sake is obtained by pressing, clarification, sterilization, aging, fine filtration, bottling, and sterilization.

2. The production method according to claim 1, characterized in that, In step (2), the Rhizopus oryzae includes: Rhizopus oryzae CICC 41203, Rhizopus oryzae CICC 40865, Rhizopus oryzae CICC 41440, Rhizopus oryzae CICC 3083, and Rhizopus oryzae CICC 40469. All of the Rhizopus oryzae have strong abilities to hydrolyze starch and protein. The Rhizopus oryzae is CICC 41505. This strain has high ester synthesis lipase activity, which is beneficial to increasing the content of esters in sake fermentation.

3. The production method according to claim 2, characterized in that, In step (2), the preparation methods of Rhizopus oryzae koji and Rhizopus huassiformis koji are as follows: Rhizopus oryzae spores and Rhizopus huassiformis spores are respectively inoculated into rice, mixed, cultured at 30-35℃ for 48-72 h, and dried at 40-45℃ for 8-12 h to obtain koji seeds; after steaming the japonica rice, it is cooled to 33-38℃, and 0.5-1.0% of the above koji seeds are inoculated into it. After being cultured at 30-35℃ and 80-90% humidity for 48-72 h, it is cultured at 35-42℃ and 70-80% humidity for 18-24 h, and finally dried at 40-45℃ for 8-12 h to obtain Rhizopus oryzae koji and Rhizopus huassiformis koji.

4. The production method according to claim 3, characterized in that, In step (2), the amount of rice koji added is 8~10 kg / 100 kg of raw rice; before adding the rice koji, add water at 3~5 times the weight of the rice koji to mix the rice koji.

5. The production method according to any one of claims 1 to 4, characterized in that, In step (2), the fermentation conditions for adding rice koji after steaming rice are: 28~30℃ for 36~48 hours, and the sugar content of the mash is 40~44%w / w.

6. The production method according to claim 5, characterized in that, In the main fermentation, step a is: pumping into the fermenter 8-10 kg / 100 kg of raw rice, 1.8-2.2 times the weight of the raw rice of brewing water, mixing uniformly with sterile air, controlling the product temperature at 18-25℃, the initial density of the Isayama orientalis being 0.5-1.5 x 10 7 CFU / mL, the sugar content being controlled at 20-24% w / w, stirring once every 12 hours with sterile air, and controlling the temperature for fermentation.

7. The production method according to claim 6, characterized in that, In step (3), 8-10 kg of brewer's yeast seed liquid per 100 kg of raw rice is pumped into the fermenter, and the brewer's yeast density after inoculation is 0.5-1.5 × 10⁻⁶. 7 CFU / mL, with sterile air rake applied every 12 hours for temperature-controlled fermentation.

8. The production method according to claim 7, characterized in that, In step (3), the Oriental Isaac yeast includes: Oriental Isaac yeast CICC 31693, Oriental Isaac yeast CICC 1273, Oriental Isaac yeast CICC 31129, Oriental Isaac yeast CICC 32163, and Oriental Isaac yeast CICC 31431; the brewing yeast includes: brewing yeast CICC 1299, brewing yeast CICC 1009, brewing yeast CICC 1206, brewing yeast CICC 1442, brewing yeast CICC 31324, brewing yeast CICC 32937, and brewing yeast CICC 33487.

9. The production method according to claim 8, characterized in that, The preparation methods of the *Isabella orientalis* and *Saccharomyces cerevisiae* bacterial cultures include the following steps: (1) Inoculate *Issa mesasura* or *Saccharomyces cerevisiae* into rice koji juice medium and culture at 25-28℃ for 36-48 h to obtain seed culture; (2) After steaming the rice, add water, the pure Rhizopus oryzae koji, and saccharifying enzyme, and keep warm at 60-65℃ for 3-5 hours for saccharification. Add brewing water to adjust the sugar content of the system to 10-12% w / w, and then sterilize and cool. (3) After cooling, the seed liquid obtained in step (1) is inoculated into the system obtained in step (2), and fermented at 28~30℃ to prepare the Oriental Isaac yeast liquid and the brewing yeast liquid respectively.

10. The production method according to claim 9, characterized in that, The seed solution mentioned in step (1) is obtained by gradually expanding the seed solution to meet the inoculation requirements of the yeast tank.

11. The application of the production method according to any one of claims 1 to 10 in increasing the content of flavor substances in sake, characterized in that, The flavoring substance is an ester or β-phenylethanol, and the ester is ethyl acetate, isoamyl acetate, or ethyl hexanoate.

12. A method for increasing the content of flavor compounds in sake, characterized in that, The method involves inoculating Rhizopus chinensis and Isaac's orientalis separately during the sake fermentation process; the method specifically includes the following steps: (1) Steamed rice; (2) Cultivation of bacteria: After steaming the rice, add rice koji for fermentation to obtain fermented rice; the rice koji is made by mixing pure Rhizopus oryzae rice koji and pure Rhizopus oryzae rice koji in a ratio of 1:1.0 to 1:1.

5. The preparation methods of the pure Rhizopus oryzae rice koji and pure Rhizopus huassifolia rice koji are as follows: Rhizopus oryzae and Rhizopus huassifolia are respectively inoculated into steamed rice and fermented. The Rhizopus oryzae rice koji has a saccharification power ≥1900 U / g koji, an acidic protease activity ≥28 U / g koji, and a liquefaction power ≥190 U / g koji. The Rhizopus huassifolia rice koji has an ester synthesis lipase activity ≥25 U / g koji, an acidic protease activity ≥8 U / g koji, a liquefaction power ≥45 U / g koji, and a saccharifying enzyme activity ≥200 U / g koji. (3) Primary fermentation: a. Inoculating with yeast: Add Oriental Isaac yeast liquid and brewing water to the rice after fermentation with cultured bacteria, control the temperature at 18~25℃, and ferment for 24~26 hours; b. Inoculate with brewer's yeast: After step a is completed, pump brewer's yeast liquid into the fermentation tank, control the temperature at 18~25℃, and ferment for 3~5 days; (4) Post-fermentation: After the main fermentation is completed, the product temperature is controlled at 8~10℃, and the post-fermentation lasts for 30~40 days; (5) After the post-fermentation is completed, the product sake is obtained by pressing, clarification, sterilization, aging, fine filtration, bottling, and sterilization.

13. The method according to claim 12, characterized in that, The flavor compounds are esters.

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