Method for preparing yellow rice wine, yellow rice wine and application

Abstract

The application discloses a preparation method of yellow rice wine, the yellow rice wine and application. The preparation method of the yellow rice wine comprises the following steps: gelatinizing starch raw materials to prepare gelatinized starch; uniformly mixing the gelatinized starch, yellow rice wine starter, acidic protease, saccharomyces cerevisiae, yellow rice wine and water to prepare fermentation material, and performing fermentation; adding lactic acid bacteria at 72h-74h of fermentation, and continuing fermentation; and performing solid-liquid separation after the fermentation is completed, collecting clear liquid, decocting wine, aging, and preparing the yellow rice wine. The preparation method of the yellow rice wine breaks through the technical bottleneck that the metabolic time sequence of strains is uncontrollable in traditional mixed fermentation, optimizes the inoculation time of lactic acid bacteria, guarantees the acid production capacity of lactic acid bacteria under the condition of controlling alcohol metabolism, and obtains the yellow rice wine with the characteristics of high sugar, high acid and high ester, which can be used for seasoning.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation technology, and relates to the preparation method of rice wine, rice wine, and its application. Background Technology

[0002] Yellow rice wine is generally divided into basic yellow rice wine and specialty yellow rice wine. Basic yellow rice wine, such as Yuanhong wine, Jiafan wine, and dry wines of different vintages, has a large production volume, low production cost, and high production efficiency. Specialty yellow rice wine includes high-acid yellow rice wine, sweet yellow rice wine, and high-ester aroma yellow rice wine, which have complex production processes. In the preparation of drinking yellow rice wine, specialty yellow rice wine is often used to adjust the flavor. However, current specialty yellow rice wines have limited flavoring functions; for example, sweet yellow rice wine can only adjust sweetness, high-acid yellow rice wine can only adjust acidity, and high-ester aroma yellow rice wine can only enhance ester aroma, and cannot simultaneously possess the characteristics of high sugar, high acid, and high ester. Summary of the Invention

[0003] Therefore, it is necessary to provide a method for preparing rice wine that simultaneously possesses high sugar, high acid, and high ester properties, as well as the rice wine itself and its applications.

[0004] In some embodiments, a method for preparing rice wine is provided, comprising the following steps:

[0005] Gelatinized starch is prepared by gelatinizing starch raw materials.

[0006] The gelatinized starch, rice wine yeast, acidic protease, brewing yeast, rice wine, and water are mixed to prepare a fermentation material, which is then fermented. Lactic acid bacteria are added after 72-74 hours of fermentation to continue fermentation.

[0007] After fermentation, solid-liquid separation is performed, the clear liquid is collected, heated, aged, and the rice wine is prepared.

[0008] In some embodiments, the lactic acid bacteria in the provided method for preparing rice wine include one or more of the following: fruit and vegetable fermentation strain PE331, lactic acid bacteria strain BE-01, *Lactobacillus plantarum* Lp90, and *Lactobacillus acidophilus* LA18.

[0009] In some embodiments, the lactic acid bacteria in the provided method for preparing rice wine are selected from Lactobacillus plantarum Lp90.

[0010] In some embodiments, the brewing yeast in the provided method for preparing rice wine is selected from rice wine yeast.

[0011] In some embodiments, the provided method for preparing rice wine satisfies one or more of the following conditions:

[0012] (1) The mass ratio of the starch raw material to the rice wine yeast is 1000:(3~6);

[0013] (2) The mass ratio of the starch raw material to the acidic protease is 1000:(0.01~0.03);

[0014] (3) The mass-to-volume ratio of the starch raw material to the rice wine is 1 kg: (0.5~1.5) L;

[0015] (4) The ratio of the starch raw material to the lactic acid bacteria is 1 kg: (1 × 10⁻⁶) 6 ~1×10 8 CFU;

[0016] (5) The ratio of the starch raw material to the brewing yeast is 1 kg: (1 × 10⁻⁶) 7 ~1×10 10 CFU;

[0017] (6) Based on 1 kg of the starch raw material, the amount of water used is such that the mass of the fermentation material reaches 2.5 kg to 3 kg;

[0018] (7) The starch raw material includes rice;

[0019] (8) The alcohol content of the rice wine is 16% to 18% by volume percentage.

[0020] In some embodiments, the fermentation conditions in the provided method for preparing rice wine include a temperature of 25°C to 30°C and a time of 15 to 20 days.

[0021] In some embodiments, the aging conditions in the provided method for preparing rice wine include: a temperature of 5°C to 30°C and a aging time of 12 to 15 months.

[0022] In some embodiments, a rice wine is provided, prepared by the aforementioned preparation method.

[0023] In some embodiments, the rice wine is provided for use in the blending of drinking rice wine.

[0024] In some embodiments, a drinking rice wine is provided, comprising the aforementioned rice wine;

[0025] Optionally, the drinking rice wine further comprises base rice wine, wherein the volume ratio of the base rice wine to the rice wine is (5~10):1.

[0026] In some embodiments, the use of Lactobacillus plantarum Lp90 in the fermentation of rice wine is provided.

[0027] The provided method for preparing rice wine overcomes the technical bottleneck of uncontrollable bacterial metabolism in traditional mixed fermentation by optimizing the inoculation time of lactic acid bacteria. Under the condition of controlling alcohol metabolism, it ensures the acid production capacity of lactic acid bacteria, resulting in a rice wine that is high in sugar, high in acid, and high in esters and can be used for flavoring. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0029] Figure 1 This is a flowchart illustrating the preparation process of rice wine in some implementation methods. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0035] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0037] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0038] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0039] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0040] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0041] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0043] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0044] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0045] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0046] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0047] Modern mechanized processes widely use enzyme preparations and pure yeast strains. The microbial community structure and function during the fermentation of rice wine are less complex, resulting in severe homogenization of aroma and taste, which greatly hinders the transformation and upgrading of the rice wine industry.

[0048] Mixed-culture fermentation is the core driving force behind substrate consumption and flavor formation, playing a crucial role in improving flavor quality, enhancing process stability, and reducing safety risks. Among the microorganisms, *Saccharomyces cerevisiae*, as the dominant microorganism in the fermentation process of rice wine, possesses strong fermentation power and good tolerance, while also producing metabolites such as ethanol to inhibit the growth of other microorganisms. Lactic acid bacteria, in addition to producing lactic acid during rice wine brewing, also use lactic acid as a substrate to produce other organic acids such as acetic acid and succinic acid. The ethyl ester compounds generated by the esterification reaction of these organic acids are important components of the flavor of rice wine. Therefore, a mixed-culture fermentation system using *Saccharomyces cerevisiae* and lactic acid bacteria can be constructed to enhance the acidity and ester aroma of mechanized sweet rice wine, thereby improving the flavor of the product when used in combination with base rice wine and meeting the development needs of high-quality drinking rice wine.

[0049] To address the issue of weak flavor in existing Shaoxing wine, a mixed fermentation system is constructed using a mechanized Shaoxing wine fermentation process. This system utilizes selected high-acid-producing lactic acid bacteria strains and brewing yeast to enhance the acidity and ester aroma of sweet wine. This system is then applied to the flavoring of basic Shaoxing wine, thereby improving the product's flavor.

[0050] In some embodiments, a method for preparing rice wine is provided, comprising the following steps:

[0051] Gelatinized starch is prepared by gelatinizing starch raw materials.

[0052] Gelatinized starch, rice wine yeast, acidic protease, brewing yeast, rice wine, and water are mixed to prepare a fermentation material. Fermentation is then carried out, and lactic acid bacteria are added after 72-74 hours of fermentation to continue fermentation.

[0053] After fermentation, solid-liquid separation is carried out, the clear liquid is collected, heated, aged, and then rice wine is prepared.

[0054] During the brewing process of Shaoxing wine, microorganisms participate in the transformation of substances by secreting a large number of enzymes, ultimately forming flavor compounds. At different stages of fermentation, various microorganisms play different roles. In the early stages of fermentation, molds secrete various enzymes (such as amylase and protease) to hydrolyze starch and protein, yeast metabolizes sugar to produce ethanol, and lactic acid bacteria produce organic acids to lower the pH value. In the later stages of fermentation, fungi, represented by yeast, and bacteria, represented by lactic acid bacteria, cooperate metabolically to produce flavor substances such as esters.

[0055] It is understandable that the added rice wine is used to increase the initial alcohol content of fermentation, which to some extent inhibits yeast fermentation, resulting in incomplete fermentation and a high sugar content in the wine at the end of fermentation.

[0056] Inoculating lactic acid bacteria too early can lead to dual inhibition of yeast by alcohol and acid, resulting in failure of ethanol fermentation. Adding lactic acid bacteria at 72-74 hours of fermentation avoids both excessive inhibition of yeast due to inoculation too early and excessive inhibition of lactic acid bacteria due to inoculation too late, ensuring that the metabolic activity of both lactic acid bacteria and yeast remains at a controllable and suitable level.

[0057] In some embodiments, the provided method for preparing rice wine includes one or more of the following lactic acid bacteria: fruit and vegetable fermentation strain PE331, lactic acid bacteria strain BE-01, *Lactobacillus plantarum* Lp90, and *Lactobacillus acidophilus* LA18. Fruit and vegetable fermentation strain PE331 and lactic acid bacteria strain BE-01 are available from Microcare Probiotics (Suzhou) Co., Ltd.

[0058] In some embodiments, the lactic acid bacteria are selected from *Lactobacillus plantarum* Lp90. By screening existing lactic acid bacteria, *Lactobacillus plantarum* Lp90, which has high acid-producing properties, is used to construct a mixed-culture fermentation system with *Saccharomyces cerevisiae*.

[0059] In some embodiments, the brewing yeast in the provided method for preparing rice wine is selected from rice wine yeast.

[0060] In some embodiments, in the provided method for preparing rice wine, the mass ratio of starch raw material to rice wine yeast is 1000:(3~6). For example, the mass ratio of starch raw material to rice wine yeast can be 1000:3, 1000:4, 1000:5, 1000:6, etc., or it can be any range of the two aforementioned ratios.

[0061] In some embodiments, in the provided method for preparing rice wine, the mass ratio of starch raw material to acidic protease is 1000:(0.01~0.03). For example, the mass ratio of starch raw material to acidic protease can be 1000:0.01, 1000:0.02, 1000:0.03, etc., or it can be a range of any two of the aforementioned ratios.

[0062] In some embodiments, the mass-volume ratio of starch raw material to rice wine in the provided method for preparing rice wine is 1 kg: (0.5~1.5) L. For example, the mass-volume ratio of starch raw material to rice wine can be 1 kg: 0.5 L, 1 kg: 1 L, 1 kg: 1.5 L, etc., or it can be a range of any two of the aforementioned ratios.

[0063] In some embodiments, the ratio of starch raw material to lactic acid bacteria in the provided method for preparing rice wine is 1 kg: (1 × 10⁻⁶) 6 ~1×10 8For example, the ratio of starch raw material to lactic acid bacteria can be 1 kg: 1 × 10⁻⁶ CFU. 6 CFU, 1kg: 1×10 7 CFU, 1kg: 1×10 8 CFU, etc., can also be a range consisting of any two of the aforementioned ratios.

[0064] In some embodiments, the ratio of starch raw material to brewing yeast in the provided method for preparing rice wine is 1 kg: (1 × 10⁻⁶) / kg. 7 ~1×10 10 For example, the ratio of starch raw material to brewing yeast can be 1 kg: 1 × 10 CFU. 7 CFU, 1kg: 1×10 8 CFU, 1kg: 1×10 9 CFU, 1kg: 1×10 10 CFU, etc., can also be a range consisting of any two of the aforementioned ratios.

[0065] In some embodiments, in the provided method for preparing rice wine, the amount of water used is such that the mass of the fermentation material reaches 2.5kg to 3kg, for example, 2.5kg, 2.6kg, 2.7kg, 2.8kg, 2.9kg, 3kg, etc., based on 1kg of starch raw material, or it can be a range composed of any two of the aforementioned values.

[0066] In some embodiments, the starch raw material in the provided method for preparing rice wine includes rice.

[0067] In some embodiments, the alcohol content of the provided rice wine, by volume percentage, is 16% to 18%, for example, 16%, 16.5%, 17%, 17.5%, 18%, etc., or it can be a range composed of any two of the aforementioned values.

[0068] In some embodiments, the fermentation conditions in the provided method for preparing rice wine include: a temperature of 25°C to 30°C and a time of 15 to 20 days. For example, the fermentation temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc., or any range of two of the aforementioned values; the fermentation time can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, etc., or any range of two of the aforementioned values.

[0069] In some embodiments, the aging conditions in the provided method for preparing rice wine include: a temperature of 5°C to 30°C and a time of 12 to 15 months. For example, the aging temperature can be 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, etc., or it can be any range of two of the aforementioned values; the aging time can be 12 months, 13 months, 14 months, 15 months, etc., or it can be any range of two of the aforementioned values.

[0070] In some embodiments, a rice wine is provided, prepared by the aforementioned preparation method.

[0071] In some implementations, the use of rice wine in the blending of drinking rice wine is provided.

[0072] In some embodiments, a drinking rice wine is provided, comprising the aforementioned rice wine.

[0073] In some embodiments, the drinking rice wine also includes base rice wine, and the volume ratio of base rice wine to rice wine is (5~10):1. For example, the volume ratio of base rice wine to rice wine can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc., or it can be any range of the two ratios mentioned above.

[0074] Understandably, basic Shaoxing wine refers to dry Shaoxing wine prepared by fermenting base materials with brewing yeast and Shaoxing wine koji, with a total sugar (calculated as glucose) content of less than 15g / L, and which has not been blended after fermentation. Examples include Yuanhong wine, Jiafan wine, and dry Shaoxing wines from different years.

[0075] In some embodiments, the use of Lactobacillus plantarum Lp90 in the fermentation preparation of rice wine is provided.

[0076] In some embodiments, the application of *Lactobacillus plantarum* Lp90 in the fermentation preparation of rice wine includes the following steps:

[0077] Gelatinized starch is prepared by gelatinizing starch raw materials.

[0078] Gelatinized starch, rice wine yeast, acidic protease, brewing yeast, rice wine, and water were mixed to prepare a fermentation mixture. Fermentation was then carried out, and *Lactobacillus plantarum* Lp90 was added after 72-74 hours of fermentation to continue fermentation.

[0079] After fermentation, solid-liquid separation is carried out, the clear liquid is collected, heated, aged, and then rice wine is prepared.

[0080] The prepared rice wine met the required standards for alcohol content, amino acid nitrogen, and other indicators, with total sugar exceeding 150 g / L and total acid exceeding 10 g / L. Compared to rice wine obtained through single-strain fermentation with brewing yeast, the total acid content increased by over 180%, and the ethyl ester content increased by over 100%. Specifically, the contents of ethyl lactate, ethyl propionate, ethyl butyrate, ethyl octanoate, ethyl nonanoate, and ethyl decanoate showed significant increases. The overall flavor was harmonious and pleasant, with a rich floral and fruity aroma.

[0081] The drinking rice wine prepared by blending the provided rice wine with the base rice wine has improved total sugar, total acid and ester aroma, with a rich overall flavor, harmonious aroma, mellow taste and mild acidity.

[0082] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0083] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0084] The fruit and vegetable fermentation strains PE331, lactic acid bacteria strain BE-01, Lactobacillus plantarum Lp90, and Lactobacillus acidophilus LA18 were all provided by Microcare Probiotics (Suzhou) Co., Ltd.; the brewing yeast was commercially available rice wine yeast; and the rice wine koji was brewing koji provided by Angel Yeast Co., Ltd.

[0085] Example 1: Screening of high-acid-producing lactic acid bacteria

[0086] Fruit and vegetable fermentation strain PE331, lactic acid bacteria strain BE-01, *Lactobacillus plantarum* Lp90, and *Lactobacillus acidophilus* LA18 were inoculated into MRS liquid medium and cultured anaerobically at 37°C. When the OD600 of the culture medium reached 600... nm When the concentration of the seed culture is 1.0, it is considered the seed culture. 1 μL of the seed culture was spread onto rice saccharification mash solid medium containing 2% CaCO3 and anaerobically cultured at 37°C for 24 h. The diameter of the calcium dissolution zone of the strain was then measured.

[0087] The results showed that *Lactobacillus lactis* Lp90 had the largest calcification zone diameter, and the colonies were round and raised with smooth, dense, and milky white edges.

[0088] Example 2: Tolerance analysis of Lactobacillus plantarum Lp90

[0089] ① Strain activation: Take out Lp90 of *Lactobacillus plantarum* that has been frozen at -80℃, thaw it at room temperature, and shake it well. Use an inoculation loop to streak the bacterial solution on MRS solid medium, incubate at 37℃ for 48h, pick a single colony and inoculate it into MRS liquid medium, mix well by pipetting, and incubate anaerobically at 37℃ for 24h.

[0090] ②Tolerance test: At an inoculation ratio of 3%, the bacterial culture was aspirated into MRS liquid culture medium containing different concentrations of ethanol and glucose, and anaerobic cultured at 37℃ for 72h. The turbidity of the culture medium under each condition was observed, and the OD600 of the culture medium was measured at the same time.

[0091] The results showed that Lactobacillus plantarum Lp90 could withstand an alcohol content of 14% vol and a glucose concentration of 400 g / L, and could maintain its activity during the fermentation of sweet rice wine.

[0092] Example 3: Fermentation preparation of high-concentration flavored rice wine

[0093] ① Strain activation: Take out the *Saccharomyces cerevisiae* that was frozen at -80℃, thaw it at room temperature, and shake it well. Use an inoculation loop to dip the bacterial solution into YPD solid medium and streak it onto the medium. Incubate at 28℃ for 48 hours. Pick a single colony and transfer it to YPD liquid medium, mix it thoroughly, and incubate at 28℃ and 150 rpm for 24 hours. Activate *Lactobacillus plantarum* Lp90 according to the method in Example 2.

[0094] Seed culture preparation: Saccharomyces cerevisiae and Lactobacillus plantarum Lp90 bacterial suspensions were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile water to remove residual culture medium. A small amount of sterile water was added to the bacterial cells, and the mixture was thoroughly shaken and diluted serially. The original bacterial concentration was obtained by counting cells using a hemocytometer, yielding 1×10⁻⁶ cells respectively. 8 Seed culture of CFU / mL.

[0095] ② The base rice wine is made from locally produced dry rice wine. The preparation method is as follows: Take 1 kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and firm outside, with no white center." Spread it out in a ventilated place to cool to 45℃. Add 8±0.8g of rice wine yeast, 0.02±0.002g of acidic protease, and 5±0.5mL of brewing yeast seed liquid. Add water to a final volume of 3000g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. After 15 days of fermentation, filter the fermented mash and collect the clear liquid. Heat the liquid to obtain the base rice wine.

[0096] ③ Fermentation: Take 1 kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and hard outside, with no white center". Spread it out in a ventilated place to cool to 45℃. Add 5±0.5g of rice wine yeast, 0.02±0.002g of acidic protease, 10±1mL of brewing yeast seed liquid, and 1±0.1L of self-produced dry rice wine. Add water to make up to 2800g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. On the 3rd day of fermentation (72-74h), inoculate the fermentation mash with 0.1±0.01mL of Lactobacillus plantarum Lp90 seed liquid. After 15 days of fermentation, filter the fermentation mash, collect the clear liquid, and boil it to make wine.

[0097] ④Aging: After aging naturally at room temperature for 1 year, rice wine is obtained.

[0098] Physicochemical indicators were determined in accordance with GB / T 13662-2018 Yellow Rice Wine.

[0099] The determination of organic acids was performed using high performance liquid chromatography (HPLC). The sample was centrifuged, and the supernatant was collected. 10% trichloroacetic acid was added at a volume ratio of 1:1. After protein removal and membrane filtration, the detection conditions were as follows: Waters e2695 HPLC system, SB-C18 Analytical column (4.6 mm × 250 mm, 5 μm), mobile phase Na2HPO4 (concentration 0.02 mol / L, pH 2.70), injection volume 10 μL, flow rate 0.8 mL / min, column temperature 30℃, and UV detector (210 nm).

[0100] The determination of major esters was performed using gas chromatography-mass spectrometry (GC-MS). 5 mL of sample was placed in a 20 mL headspace vial, incubated at 40 °C and 300 RPM for 10 min with shaking, followed by extraction with a DVB / CAR / PDMS three-phase extraction head for 30 min. The sample was then thermally desorbed at 250 °C for 10 min and injected. Detection conditions were as follows: HP-INNOWAX column (60 m × 0.25 mm, 0.25 μm), injection port temperature 250 °C; single-stage temperature program: 40 °C for 5 min, then ramped to 250 °C at 5 °C / min and held for 10 min; constant flow mode, column flow rate 1.2 mL / min; mass spectrometry interface temperature 250 °C; ion source 250 °C, quadrupole 150 °C; full scan mode for signal acquisition (29-550 amu).

[0101] As shown in Tables 1-3, after aging, the total acid of the high-concentration flavored rice wine reached 12.28 g / L, the total sugar remained above 150 g / L, and the alcohol content and ammonia nitrogen were within the normal range. Regarding flavor compounds, the total organic acid content of the high-concentration flavored rice wine reached 12.19 g / L, of which lactic acid content was 7.86 g / L, which was the main component of organic acids; the total ester content reached 43.84 mg / L, exhibiting a rich ester aroma.

[0102] Table 1 Physicochemical Indicators of High-Concentration Flavored Shaoxing Wine

[0103]

[0104] Table 2 Organic acid composition (mg / L) of high-concentration flavored Shaoxing wine

[0105]

[0106] Table 3. Ester composition (mg / L) of high-concentration flavored Shaoxing wine

[0107]

[0108] Example 4: Fermentation Preparation of High-Concentration Flavored Rice Wine

[0109] ① Strain activation: Take out the *Saccharomyces cerevisiae* that was frozen at -80℃, thaw it at room temperature, and shake it well. Use an inoculation loop to dip the bacterial solution into YPD solid medium and streak it onto the medium. Incubate at 28℃ for 48 hours. Pick a single colony and transfer it to YPD liquid medium, mix it thoroughly, and incubate at 28℃ and 150 rpm for 24 hours. Activate *Lactobacillus plantarum* Lp90 according to the method in Example 2.

[0110] Seed culture preparation: Saccharomyces cerevisiae and Lactobacillus plantarum Lp90 bacterial suspensions were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile water to remove residual culture medium. A small amount of sterile water was added to the bacterial cells, and the mixture was thoroughly shaken and diluted serially. The original bacterial concentration was obtained by counting cells using a hemocytometer, yielding 1×10⁻⁶ cells respectively. 8 Seed culture of CFU / mL.

[0111] ② The base rice wine is made from locally produced dry rice wine. The preparation method is as follows: Take 1 kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and firm outside, with no white center." Spread it out in a ventilated place to cool to 45℃. Add 8±0.8g of rice wine yeast, 0.02±0.002g of acidic protease, and 5±0.5mL of brewing yeast seed liquid. Add water to a final volume of 3000g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. After 15 days of fermentation, filter the fermented mash and collect the clear liquid. Heat the liquid to obtain the base rice wine.

[0112] ③ Fermentation: Take 1kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and hard outside, with no white center". Spread it out in a ventilated place to cool to 45℃. Add 3g of rice wine yeast, 0.01g of acidic protease, 100mL of brewing yeast seed liquid, and 1.5L of self-produced dry rice wine. Add water to make up to 2800g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. On the 3rd day of fermentation (72-74h), inoculate the fermentation mash with 1mL of Lactobacillus plantarum Lp90 seed liquid. After 15 days of fermentation, filter the fermentation mash, collect the clear liquid, and boil it to make wine.

[0113] ④Aging: After aging naturally at room temperature for 1 year, rice wine is obtained.

[0114] The physicochemical indicators, organic acids, and major esters were determined according to the method in Example 3. The results are shown in Tables 4-6. After aging, the total acid of the high-concentration flavored rice wine reached 12.80 g / L, the total sugar remained above 150 g / L, and the alcohol content and ammonia nitrogen were within the normal range. Regarding flavor compounds, the total organic acid content of the high-concentration flavored rice wine reached 12.41 g / L, of which the lactic acid content was 7.91 g / L, resulting in a mellow acidity; the total ester content reached 42.55 mg / L, exhibiting a rich ester aroma.

[0115] Table 4 Physicochemical Indicators of High-Concentration Flavored Yellow Wine

[0116]

[0117] Table 5 Organic acid composition (mg / L) of high-concentration flavored Shaoxing wine

[0118]

[0119] Table 6. Ester composition (mg / L) of high-concentration flavored Shaoxing wine

[0120]

[0121] Example 5: Fermentation Preparation of High-Concentration Flavored Rice Wine

[0122] ① Strain activation: Take out the *Saccharomyces cerevisiae* that was frozen at -80℃, thaw it at room temperature, and shake it well. Use an inoculation loop to dip the bacterial solution into YPD solid medium and streak it onto the medium. Incubate at 28℃ for 48 hours. Pick a single colony and transfer it to YPD liquid medium, mix it thoroughly, and incubate at 28℃ and 150 rpm for 24 hours. Activate *Lactobacillus plantarum* Lp90 according to the method in Example 2.

[0123] Seed culture preparation: Saccharomyces cerevisiae and Lactobacillus plantarum Lp90 bacterial suspensions were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile water to remove residual culture medium. A small amount of sterile water was added to the bacterial cells, and the mixture was thoroughly shaken and diluted serially. The original bacterial concentration was obtained by counting cells using a hemocytometer, yielding 1×10⁻⁶ cells respectively. 7 Seed culture of CFU / mL.

[0124] ② The base rice wine is made from locally produced dry rice wine. The preparation method is as follows: Take 1 kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and firm outside, with no white center." Spread it out in a ventilated place to cool to 45℃. Add 8±0.8g of rice wine yeast, 0.02±0.002g of acidic protease, and 5±0.5mL of brewing yeast seed liquid. Add water to a final volume of 3000g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. After 15 days of fermentation, filter the fermented mash and collect the clear liquid. Heat the liquid to obtain the base rice wine.

[0125] ③ Fermentation: Take 1kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and hard outside, with no white center". Spread it out in a ventilated place to cool to 45℃. Add 6g of rice wine yeast, 0.03g of acidic protease, 1mL of brewing yeast seed liquid, and 0.5L of self-produced dry rice wine. Add water to make up to 2800g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. On the 3rd day of fermentation (72-74h), inoculate the fermentation mash with 0.1mL of Lactobacillus plantarum Lp90 seed liquid. After 15 days of fermentation, filter the fermentation mash, collect the clear liquid, and boil it to make wine.

[0126] ④Aging: After aging naturally at room temperature for 1 year, rice wine is obtained.

[0127] The physicochemical indicators, organic acids, and major esters were determined according to the method in Example 3. The results are shown in Tables 7-9. After aging, the total acid of the high-concentration flavored rice wine reached 12.06 g / L, the total sugar remained above 150 g / L, and the alcohol content and ammonia nitrogen were within the normal range. Regarding flavor compounds, the total organic acid content of the high-concentration flavored rice wine reached 11.98 g / L, of which the lactic acid content was 7.63 g / L, resulting in a mellow acidity; the total ester content reached 43.09 mg / L, exhibiting a rich ester aroma.

[0128] Table 7 Physicochemical Indicators of High-Concentration Flavored Yellow Wine

[0129]

[0130] Table 8 Organic acid composition (mg / L) of high-concentration flavored Shaoxing wine

[0131]

[0132] Table 9. Ester composition (mg / L) of high-concentration flavored Shaoxing wine

[0133]

[0134] Comparative Example 1: Fermentation Preparation of High-Concentration Flavored Shaoxing Wine

[0135] The fermentation preparation method of the high-concentration flavored rice wine provided in this comparative example is similar to that in Example 3. The difference is that in this comparative example, during the fermentation step, 0.1±0.01mL of Lactobacillus plantarum Lp90 seed liquid is inoculated into the fermentation mash on the 6th and 9th days of fermentation, respectively.

[0136] The physicochemical properties and organic acid components were determined according to the method described in Example 3. The test results are shown in Tables 10 and 11.

[0137] Compared with Tables 1 and 2, it can be found that the total acid of the high-concentration flavored rice wine inoculated with Lactobacillus plantarum Lp90 in the middle and late stages of fermentation, as shown in Tables 10 and 11, is lower than that inoculated in the early stages of fermentation. The difference in lactic acid content is the most significant. This may be because in the middle and late stages of fermentation, ethanol accumulates to a higher level, which inhibits the metabolism of lactic acid bacteria.

[0138] Table 10 Physicochemical properties of high-concentration flavored Shaoxing wine inoculated with Lactobacillus plantarum Lp90 at different time points

[0139]

[0140] Table 11 Organic acid composition (mg / L) of high-concentration flavored Shaoxing wine inoculated with Lp90 of *Lactobacillus plantarum* at different time points.

[0141]

[0142] Comparative Example 2: Fermentation Preparation of High-Concentration Flavored Shaoxing Wine

[0143] The fermentation preparation method of the high-concentration flavored rice wine provided in this comparative example is similar to that in Example 3. The difference is that in this comparative example, during the fermentation step, when adding the brewing yeast seed liquid, 0.1±0.01mL of Lactobacillus plantarum Lp90 seed liquid is inoculated into the fermentation mash.

[0144] The physicochemical properties were determined according to the method described in Example 3. The test results are shown in Table 12.

[0145] Table 12 shows that the alcohol content of the high-concentration flavored sweet wine inoculated with both *Saccharomyces cerevisiae* and *Lactobacillus plantarum* Lp90 was only 8.90% vol, while the total acid content exceeded 20 g / L and the total sugar content reached as high as 264.34 g / L. Comparison with Tables 1 and 2 shows that under these conditions, the lactic acid bacteria were excessively vigorous, leading to uncontrolled total acidity and inhibiting yeast metabolism. Only a very small amount of sugar was converted into ethanol, resulting in low alcohol content and ammonia nitrogen.

[0146] Table 12 Physicochemical properties of high-concentration flavored rice wine under simultaneous inoculation with *Saccharomyces cerevisiae* and *Lactobacillus plantarum* Lp90.

[0147]

[0148] Comparative Example 3: Single-strain fermentation of ordinary sweet wine using brewer's yeast

[0149] ① Strain activation: Activate the Saccharomyces cerevisiae according to the method in Example 3.

[0150] ② Seed culture preparation: Prepare the seed culture of Saccharomyces cerevisiae according to the method in Implementation 3, and dilute it to 1×10⁻⁶. 8 CFU / mL.

[0151] ③ Fermentation: Take 1kg of glutinous rice, soak it for 1 hour, and then steam it for 30 minutes. The rice grains should be "soft inside and hard outside, with no white center". Spread it out in a ventilated place to cool to 45℃. Add 5±0.5g of rice wine yeast, 0.02±0.002g of acidic protease, 10±1mL of brewing yeast seed liquid, and 1±0.1L of self-produced dry rice wine. Add water to make up to 2800g, stir well, and place it in a 28℃ incubator for fermentation. Stir twice a day for the first 3 days of fermentation. After 15 days of fermentation, filter the fermented mash, collect the clear liquid, and boil it to make wine.

[0152] ④ Aging: After one year of natural aging, the physicochemical properties, organic acids, and major esters were determined according to the method described in Example 3. The test results are shown in Tables 13 to 15.

[0153] As shown in Tables 13-15, after aging, the total acid of ordinary sweet wine is 4.35 g / L, which is only 35.42% of that of high-concentration flavored rice wine, while the total sugar is slightly higher. In terms of flavor compounds, the total amount of organic acids and esters in ordinary sweet wine is only 34.46% and 49.62% of that in high-concentration flavored rice wine, respectively. In particular, the content of lactic acid, ethyl lactate (cream aroma), ethyl propionate (pineapple aroma), ethyl butyrate (pineapple aroma), ethyl caprylate (pineapple aroma), ethyl nonanoate (grape aroma), and ethyl decanoate (coconut aroma) is significantly lower than that in high-concentration flavored rice wine.

[0154] Table 13 Physicochemical Indicators of Ordinary Sweet Wine

[0155]

[0156] Table 14 Organic acid composition of ordinary sweet wine (mg / L)

[0157]

[0158] Table 15 Ester composition (mg / L) of ordinary sweet wines

[0159]

[0160] Example 6: Application of high-concentration flavored rice wine in the blending of drinking rice wine products

[0161] ① Strain activation: Activate the Saccharomyces cerevisiae according to the method in Example 3.

[0162] ② Seed culture preparation: Prepare the Saccharomyces cerevisiae seed culture according to the method in Example 3, with a concentration of 1×10⁻⁶. 8 CFU / mL.

[0163] ③ Basic Shaoxing Rice Wine Fermentation and Aging: Take 1 kg of glutinous rice, soak it for 1 hour, then steam it for 30 minutes, ensuring the rice grains are "soft inside and firm outside, with no white center." Let it cool to 45℃ in a ventilated area. Add 8±0.8 g of Shaoxing rice wine starter, 0.02±0.002 g of acidic protease, and 5±0.5 mL of brewing yeast seed liquid. Add water to a final volume of 3000 g, stir well, and place in a 28℃ incubator for fermentation. Stir twice daily for the first 3 days of fermentation. After 15 days of fermentation, filter the fermented mash and collect the clear liquid. After heating, naturally age for 1 year.

[0164] ④ Fermentation and aging of high-concentration flavored rice wine: Same as in Example 3.

[0165] ⑤ Product blending: High-concentration flavored rice wine is added to base rice wine to prepare drinking rice wine products. The proportion of high-concentration flavored rice wine in drinking rice wine products is 10% (v / v).

[0166] The physicochemical properties, organic acids, and major esters were determined according to the method described in Example 3, and a preference assessment was performed. The test results are shown in Tables 16-18.

[0167] The results shown in Tables 16-18 indicate that the physicochemical properties of the drinking rice wine products blended with high-concentration flavored rice wine meet the requirements of GB13662 for refreshing semi-dry rice wine. Compared with the base rice wine before blending, the total acid and total ester content of the blended drinking rice wine products are increased by more than 20%, especially the lactic acid content, which is increased by more than 130%.

[0168] A total of 12 people participated in the sensory evaluation. 11 people believed that the overall flavor of the drinking rice wine product after being blended with high-concentration flavored rice wine was better than that of the base rice wine before blending. Specifically, it was characterized by a rich floral and fruity aroma, a complex and harmonious fragrance, a mellow taste, and a mild acidity.

[0169] Table 16 Physicochemical properties of drinking rice wine after blending with high-concentration flavored rice wine

[0170]

[0171] Table 17 Organic acid composition (mg / L) of drinking rice wine after blending with high-concentration flavored rice wine

[0172]

[0173] Table 18. Ester composition (mg / L) of drinking rice wine after blending with high-concentration flavored rice wine.

[0174]

[0175] Comparative Example 4: Application of Ordinary Sweet Wine in Drinking-Type Yellow Wine Products

[0176] ① Basic rice wine fermentation and aging: Same as ③ Basic rice wine fermentation and aging in Example 6.

[0177] ② Fermentation and aging of ordinary sweet wine: Same as comparative ratio 3.

[0178] ③ Product blending: 10% (v / v) of ordinary sweet wine was added to the base rice wine. The physicochemical indicators, organic acids, and main esters were determined according to the method in Example 3, and a preference assessment was conducted. The test results are shown in Tables 19 to 21.

[0179] The results shown in Tables 19-21 indicate that the physicochemical properties of the blended rice wine products meet the requirements of GB13662 for refreshing semi-dry rice wine. Compared with the base rice wine before blending, the total acidity of the blended rice wine products is basically not increased, while the total esters are increased by 10.59%. The sensory evaluation results show that the flavor improvement is not significant.

[0180] Table 19 Physicochemical Indicators of Drinking Yellow Wine Blended with Ordinary Sweet Wine

[0181]

[0182] Table 20 Organic acid composition (mg / L) of drinking-grade Shaoxing wine after blending with regular sweet wine

[0183]

[0184] Table 21. Ester composition (mg / L) of drinking-grade Shaoxing wine after blending with regular sweet wine.

[0185]

[0186] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0187] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing rice wine, characterized in that, Includes the following steps: Gelatinized starch is prepared by gelatinizing starch raw materials. The gelatinized starch, rice wine yeast, acidic protease, brewing yeast, rice wine and water are mixed to prepare a fermentation material, which is then fermented. Lactic acid bacteria are added at 72-74 hours of fermentation to continue fermentation. as well as, After fermentation, solid-liquid separation is performed, the clear liquid is collected, heated, aged, and the rice wine is prepared.

2. The method for preparing rice wine according to claim 1, characterized in that, The lactic acid bacteria include one or more of the following: fruit and vegetable fermentation strain PE331, lactic acid bacteria strain BE-01, *Lactobacillus plantarum* Lp90, and *Lactobacillus acidophilus* LA18.

3. The method for preparing rice wine according to claim 1, characterized in that, The lactic acid bacteria are selected from Lactobacillus plantarum Lp90, and / or the brewing yeast is selected from rice wine yeast.

4. The method for preparing rice wine according to claim 1, characterized in that, One or more of the following conditions must be met: (1) The mass ratio of the starch raw material to the rice wine yeast is 1000:(3~6); (2) The mass ratio of the starch raw material to the acidic protease is 1000:(0.01~0.03); (3) The mass-to-volume ratio of the starch raw material to the rice wine is 1 kg : (0.5~1.5) L; (4) The ratio of the starch raw material to the lactic acid bacteria is 1 kg: (1 × 10⁻⁶) 6 ~1×10 8 CFU; (5) The ratio of the starch raw material to the brewing yeast is 1 kg: (1 × 10⁻⁶) 7 ~1×10 10 CFU; (6) Based on 1 kg of the starch raw material, the amount of water used is such that the mass of the fermentation material reaches 2.5 kg to 3 kg; (7) The starch raw material includes rice; (8) The alcohol content of the rice wine is 16% to 18% by volume percentage.

5. The method for preparing rice wine according to any one of claims 1 to 4, characterized in that, Fermentation conditions include a temperature of 25℃~30℃ and a time of 15~20 days.

6. The method for preparing rice wine according to any one of claims 1 to 4, characterized in that, The aging conditions include a temperature of 5°C to 30°C and a time of 12 to 15 months.

7. A type of rice wine, characterized in that, Prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the rice wine according to claim 7 in the blending of drinking rice wine.

9. A type of rice wine for drinking, characterized in that, Includes the rice wine as described in claim 7; Optionally, the drinking rice wine further comprises base rice wine, wherein the volume ratio of the base rice wine to the rice wine is (5~10):

1.

10. Application of Lactobacillus plantarum Lp90 in the fermentation preparation of rice wine.

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