A brewing method for reducing the content of ethyl carbamate precursors in rice wine and its application

Abstract

A brewing method and its application for reducing the content of ethyl carbamate precursors in rice wine includes: soaking, steaming, and cooling rice to obtain cooked rice; inoculating the cooked rice with a synthetic microbial community consisting of Rhizopus oryzae M5, Saccharomyces cerevisiae G2, Lactobacillus fermentum d6, and Wickham's yeast 1D6, and mixing with water; and sequentially performing pre-fermentation and post-fermentation. A continuous inoculation method is used, with Rhizopus oryzae M5 inoculated first, followed by the other three strains 24 hours later. Through the synergistic metabolism of the four strains, the simultaneous and efficient control of the two key ethyl carbamate precursors, urea and citrulline, during rice wine fermentation is achieved. This invention intervenes at the source of brewing, without the need for adding exogenous enzymes or chemical reagents, avoiding the biosafety risks of genetic engineering modification. Furthermore, the strain combination is clearly defined, the proportions are controllable, and the fermentation batches exhibit good stability, making it suitable for safe rice wine production.

Description

Technical Field

[0001] This invention relates to a brewing method and its application for reducing the content of ethyl carbamate precursors in rice wine. Background Technology

[0002] Yellow wine is a traditional alcoholic beverage made from rice, using wheat koji and yeast as saccharifying and fermenting agents, and fermented with various microorganisms. It has a unique flavor and rich nutritional value. However, during the fermentation and storage process, yellow wine produces ethyl carbamate (EC), which has been classified as a Group 2A carcinogen by the International Agency for Research on Cancer.

[0003] Studies have shown that ethyl carbamate (EC) is mainly formed by the spontaneous reaction of carbamoyl compounds with ethanol, and its precursors include urea, citrulline, and carbamoyl phosphate. Urea is mainly produced by the metabolism of arginine by Saccharomyces cerevisiae, while citrulline is mainly produced by lactic acid bacteria through the arginine deiminase (ADI) pathway. During the brewing process of Shaoxing wine, EC is primarily produced by the spontaneous reaction of ethanol with carbamoyl compounds such as urea, carbamoyl phosphate, and citrulline. Long-term intake may pose potential health risks.

[0004] Currently, methods to reduce EC content in Shaoxing rice wine mainly include production process optimization, enzymatic methods, and metabolic engineering. Production process optimization reduces EC precursor formation by refining raw materials or optimizing fermentation conditions, but this easily leads to raw material waste and increased costs. Enzymatic methods mainly decompose urea by adding acidic urease, but the active site of urease contains nickel ions, which are harmful to humans, limiting its widespread application. Metabolic engineering mainly modifies brewing yeast through genetic engineering, such as knocking out the CAR1 gene or overexpressing the DUR3 gene to reduce urea accumulation, but this method may affect the taste of Shaoxing rice wine, and the metabolically engineered strains face issues of biosafety and strain stability. However, most existing strain improvement or process optimization methods focus on single strains, i.e., controlling only urea or only citrulline. For example, the acidic urease method can only decompose already generated urea and is ineffective against citrulline; while knocking out the ADI pathway of lactic acid bacteria may reduce citrulline, it disrupts the original metabolic network of the microbial community. Therefore, designing a microbial strategy from the brewing source that can efficiently metabolize urea while simultaneously blocking the citrulline synthesis pathway and reducing these two major EC precursors is a technical challenge to be solved. Synthetic microbial community technology offers a possibility for this. (Chinese Invention Patent No.: ZL202111438894.9, Authorization Announcement Date: July 18, 2023). A method for reducing ethyl carbamate in fermented foods using synthetic starter cultures is disclosed. The synthetic starter cultures used include *Rhizopus oryzae* M5 (CCTCC NO: M20211357), *Saccharomyces cerevisiae* G2 (CCTCC NO: M20211359), *Lactobacillus fermentum* d6 (CCTCC NO: M20211358), and *Wickerhamomyces anomalus* 1D6 (CCTCC NO: M2020333). This patent achieves an effective reduction in urea and ethyl carbamate content in rice wine through simultaneous or continuous inoculation of these four strains. However, this patent primarily focuses on controlling the total amount of ethyl carbamate in the final product, without systematically studying the dynamic changes of the two main precursors, urea and citrulline, during fermentation. It also fails to design specific microbial community combinations and fermentation processes to reduce the accumulation of urea and citrulline. Therefore, how to effectively control urea and citrulline, the two main EC precursors, by optimizing the combination of microbial communities, inoculation timing, and fermentation parameters based on the known strains mentioned above remains an unsolved technical problem.

[0005] In the fermentation of Shaoxing rice wine, reducing the content of ethyl carbamate (EC) precursors is difficult. Therefore, developing a brewing method that can effectively reduce the content of urea and citrulline in Shaoxing rice wine is of significant industrial importance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a brewing method for reducing the content of ethyl carbamate precursors in rice wine. The method constructs a synthetic microbial community composed of Rhizopus oryzae M5, Saccharomyces cerevisiae G2, Lactobacillus fermentum d6 and Wickham's yeast 1D6, and combines it with a continuous inoculation process to achieve simultaneous and efficient control of the two key precursors of ethyl carbamate in rice wine: urea and citrulline.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned brewing method for reducing the content of ethyl carbamate precursor substances in rice wine in the production of rice wine. The method intervenes from the source of brewing, without the need to add exogenous enzymes or chemical reagents, avoiding the biosafety risks brought about by genetic engineering modification. At the same time, the strain combination is clear and the ratio is controllable, ensuring the stability of fermentation batches.

[0008] To solve the first technical problem mentioned above, the present invention provides a brewing method for reducing the content of ethyl carbamate precursors in rice wine, comprising the following steps:

[0009] (1) Soak, steam, and cool the rice to obtain cooked rice;

[0010] (2) The synthetic microbial colony is inoculated into cooked rice, and water is added and mixed evenly to obtain a mixture. The synthetic microbial community consists of Rhizopus oryzae M5, Saccharomyces cerevisiae G2, Lactobacillus fermentum d6 and Wickerhamomyces anomalus 1D6.

[0011] (3) The mixture obtained in step (2) is subjected to pre-fermentation and post-fermentation in sequence to finally obtain fermented mash.

[0012] Preferably, step (2) uses a continuous inoculation method: first inoculate with Rhizopus oryzae M5, and then inoculate with Saccharomyces cerevisiae G2, Lactobacillus fermentum d6 and Vickham's yeast 1D6 24 hours later.

[0013] Preferably, the inoculum size of Rhizopus oryzae M5 is 2.0 × 10⁻⁶. 10 ~2.0×10 12 Spores / mL, the inoculum size of Saccharomyces cerevisiae G2 was 1.0 × 10⁻⁶.10 ~1.0×10 12 CFU / mL, Lactobacillus fermentum d6 inoculum size 1.0 × 10⁻⁶ 9 ~1.0×10 10 CFU / mL, abnormal Vicham yeast 1D6 inoculum size 1.0 × 10⁻⁶ 9 ~1.0×10 10 CFU / mL.

[0014] Preferably, in step (1), the amount of rice used is 100g, the amount of water used is 160~180mL, the soaking time is 2~3 days, and the cooking is carried out under normal pressure.

[0015] Preferably, in step (3), both the pre-fermentation and post-fermentation are static fermentations. The temperature of the pre-fermentation is 28~30℃, and the time is 5 days. The temperature of the post-fermentation is 15℃, and the time is 15 days.

[0016] To address the aforementioned technical problem, this invention provides a brewing method for reducing the content of ethyl carbamate precursors in rice wine, which is applied in rice wine production.

[0017] The bacterial strains used in this invention are all previously deposited publicly available strains, and the deposit information is as follows:

[0018] The yeast strain G2 provided by this invention, classified as Saccharomyces cerevisiae G2, was deposited at the China Center for Type Culture Collection on November 2, 2021, with accession number CCTCCNO: M 20211359, and the deposit address is Wuhan University, Wuhan, China.

[0019] The mold M5 provided by this invention, classified and named Rhizopus oryzae M5, was deposited at the China Center for Type Culture Collection on November 2, 2021, with accession number CCTCCNO: M 20211357, and the deposit address is Wuhan University, Wuhan, China.

[0020] The yeast strain d6 provided by this invention, classified and named Lactobacillus fermentum d6, was deposited at the China Center for Type Culture Collection on November 2, 2021, with accession number CCTCCNO: M 20211358, and the deposit address is Wuhan University, Wuhan, China.

[0021] The yeast strain 1D6 provided by this invention, classified and named Wickerhamomyces anomalus 1D6, was deposited at the China Center for Type Culture Collection on November 2, 2021, with accession number CCTCCNO: M 2020333, and the deposit address is Wuhan University, Wuhan, China.

[0022] The principle of strain screening and functional design in this invention: The construction of the synthetic microbial community described in this invention is not an arbitrary combination of strains, but a rational design based on in-depth analysis of the EC precursor metabolic pathway in the rice wine fermentation system. Traditional fermented rice wine flavor formation depends on the saccharification activity of Rhizopus. The selected Rhizopus oryzae M5 not only possesses highly efficient saccharification capabilities, providing a carbon source for subsequent yeast and lactic acid bacteria fermentation, but its metabolic activities can also regulate the fermentation microenvironment, creating suitable growth conditions for subsequent inoculated strains. Urea is mainly produced by Saccharomyces cerevisiae via the arginase pathway. Compared to ordinary Saccharomyces cerevisiae, the Saccharomyces cerevisiae G2 selected in this invention has a stronger urea utilization capacity, efficiently absorbing and decomposing urea produced by other microbial metabolism, thereby reducing the total amount of urea. Citrulline is mainly produced by lactic acid bacteria via the ADI pathway and is another core precursor of EC. The selected Lactobacillus fermentum d6 in this invention has highly efficient EC hydrolase activity and citrulline metabolism capacity. The strain selected in this invention is Wickham yeast 1D6, which has excellent ester production capacity in the middle and late stages of fermentation. Its introduction can compensate for the loss of typical floral and fruity aromas that may be caused by the enhancement of functional strains, ensuring food safety without sacrificing the traditional flavor of rice wine.

[0023] The combination of four strains was designed based on the natural fermentation sequence of saccharification, alcoholic fermentation, lactic acid fermentation, and flavor modification. By continuously inoculating (first M5, then G2, d6, and 1D6 after 24 hours), the traditional rice wine fermentation process was simulated to avoid early nutrient competition and achieve functional synergy.

[0024] Advantages of this invention: This invention constructs a synthetic microbial community composed of Rhizopus oryzae M5, Saccharomyces cerevisiae G2, Lactobacillus fermentum d6, and V. aberrantis 1D6, and combines this with a continuous inoculation process to achieve simultaneous and efficient control of urea and citrulline, two key precursors of ethyl carbamate in rice wine. At the end of fermentation, the urea content is as low as 0.70 ± 0.11 mg / L, and the citrulline content is as low as 4.22 ± 1.04 mg / L. Compared with single-strain fermentation, urea is reduced by 91.7%~92.7%, and citrulline by 66.1%~73.8%. This invention utilizes the urea-decomposing ability of Saccharomyces cerevisiae G2 and the citrulline-metabolizing ability of Lactobacillus fermentum d6 to form a functional complementarity, combined with the saccharification effect of Rhizopus oryzae M5 and the flavor-modifying effect of V. aberrantis 1D6, to achieve synergistic effects on metabolic pathways and significantly reduce the residues of byproducts ornithine, high-arginine, and high-citrulline. This method intervenes at the source of brewing, without the need to add exogenous enzymes or chemical reagents, thus avoiding the biosafety risks brought about by genetic engineering. At the same time, the strain combination is clear and the proportion is controllable, ensuring the stability of fermentation batches.

[0025] This invention provides a safe, efficient, and low-cost technical solution for reducing the ethyl carbamate content in fermented foods (yellow wine production), and has broad prospects for industrial application. Attached Figure Description

[0026] Figure 1 This is a graph showing the change in urea content during the simulated fermentation process of Shaoxing wine.

[0027] Figure 2 This is a graph showing the change in citrulline content during the simulated fermentation process of Shaoxing wine in an example.

[0028] Figure 3 This is a graph illustrating the changes in arginine content during the simulated fermentation process of Shaoxing wine, as shown in the example.

[0029] Figure 4 This is a graph showing the change in ornithine content during the simulated fermentation process of Shaoxing wine in an example.

[0030] Figure 5 This is a graph showing the change in arginine content during the simulated fermentation process of Shaoxing wine in an example embodiment.

[0031] Figure 6 This is a graph showing the change in citrulline content during the simulated fermentation process of Shaoxing wine in an example. Detailed Implementation

[0032] Preparatory work

[0033] 1. Culture medium

[0034] PDA medium: 200 g / L potato, 20 g / L glucose, solid medium with 20 g / L agar.

[0035] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, with an additional 2% agar added to the solid medium.

[0036] MRS medium: peptone 10 g / L, beef meal 8 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, solid medium with 15 g / L agar.

[0037] The sterilization conditions for all the above culture media were 115℃ for 15 min. Each culture medium was prepared using a 250 mL Erlenmeyer flask containing 50 mL of liquid.

[0038] 2. Inoculation

[0039] M5 was streaked onto PDA solid medium and incubated at 28°C for 2-3 days. G2 and 1D6 were streaked onto YPD solid medium and incubated at 30°C for 2-3 days. d6 was streaked onto MRS solid medium and incubated at 37°C for 1 day. Single colonies of *S. cerevisiae* G2 and *W. anomalus* 1D6 were picked and inoculated into YPD liquid medium, and cultured at 30°C and 200 rpm for 16 h to obtain seed culture. Single colonies of *L. fermentum* d6 were picked and inoculated into MRS liquid medium, and incubated statically at 37°C for 24 h to obtain seed culture. The spores of M5 were washed off with sterile water for later use.

[0040] 3. The detection methods involved in the embodiments are as follows:

[0041] a. Urea detection method

[0042] Take a sample of fermented rice wine and shake well. Transfer an appropriate amount of sample to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 10 min at 4℃. Collect the supernatant for testing. Follow the instructions for the blood urea nitrogen (BUN) kit, adding reagents sequentially according to the ratio and mixing well. Heat the mixture in a 92℃ water bath for 10 min, then cool to room temperature. Transfer 200 μL of the reaction solution to a 96-well plate and measure the absorbance (A) of each well at 540 nm using a microplate reader. Perform three replicates for each sample and calculate the average value. Calculate ΔA = Ameasured - Ablank. The kit instructions provide the standard curve formula: y = 4.3204x + 0.0225, R² = 0.9976, where x is the standard concentration (mg / mL) and y is ΔA. According to the kit instructions, the urea nitrogen content (mg / mL) for liquid samples is calculated by volume as follows: (ΔA - 0.0225) ÷ 4.3204 = 0.2315 × (ΔA - 0.0225).

[0043] b. Amino acid detection methods

[0044] Accurately weigh 136 amino acid standards to prepare a 5 mg / mL linear standard stock solution. Dilute the linear stock solution with methanol to obtain a series of concentrations. Prepare L-Alanine-d4 and Phenylanine-d2 solutions of specific concentrations and mix well to obtain the internal standard solution (IS). The linear, internal standard, and quality control stock solutions and working solutions were all stored at -20℃. Add 100 μL of sample to the mass spectrometer water and vortex to mix, which is the diluted sample. Take 50 μL of the diluted sample and add 200 μL of precipitant containing the mixed internal standard (acetonitrile:methanol = 1:1), vortex to mix, incubate on ice for 30 min, centrifuge at 12000 rpm at 4℃ for 10 min, and collect all supernatant for LC-MS analysis. Mix equal volumes of all sample metabolites to prepare a quality control sample (QC). During the instrument analysis, insert one QC sample for every 10 samples to examine the repeatability of the entire analysis process. The experimental samples were replaced with a 53% methanol aqueous solution, and the pretreatment process was the same as that for the experimental samples.

[0045] LC-MS analysis instrument: QTRAP 6500+ mass spectrometer

[0046] Chromatographic conditions

[0047] Column: ACQUITY UPLC BEH Amide (2.1×100mm, 1.7μm)

[0048] Mobile phase: Phase A: 0.1% formic acid water, 5mM ammonium acetate; Phase B: 0.1% formic acid, acetonitrile

[0049] Column temperature: 50℃

[0050] Injection volume: 1 μL

[0051] Flow rate: 0.3 mL / min

[0052] Mass spectrometry conditions: Electrospray ionization (ESI) source, negative ionization mode. Ion source temperature 550°C, ion source voltage -4500V, sheath gas 35 psi, auxiliary gas 50 psi, collision gas 55 psi. Multiple reaction monitoring (MRM) was used for scanning.

[0053] Example:

[0054] Step 1: Preparation of fermentation cells

[0055] Seed cultures of *Saccharomyces cerevisiae* G2 and *V. aberrantis* 1D6 were transferred to YPD liquid medium and cultured for 2-3 days at 30°C and 200 rpm in a shaker. Cells were collected by centrifugation at 4°C and 10,000 rpm for 5 minutes, washed twice with sterile water, and counted. The *Saccharomyces cerevisiae* G2 culture was then adjusted to a concentration of 10⁻⁶ cells / mL. 10 CFU / mL, adjust abnormal Vicham yeast 1D6 to 10. 9 CFU / mL.

[0056] The seed culture of *Lactobacillus fermentum* d6 was transferred to MRS liquid medium and incubated statically at 37°C for 1 day. The cells were collected by centrifugation at 10,000 rpm for 5 min at 4°C, washed twice with sterile water, and the bacterial count was adjusted to 10-1. 9 CFU / mL.

[0057] The collected Rhizopus oryzae M5 was transferred to PDA solid medium and cultured at 28°C for 2-3 days. Sterile water was poured onto the solid plates, spores were scraped off, and the spores were counted. The concentration of Rhizopus oryzae M5 was then adjusted to 10-1. 10 CFU / mL.

[0058] Step 2: Sample preparation and collection

[0059] Take 100 g of rice, soak it at room temperature for 2-3 days, steam it frequently under pressure and then cool it. Divide the rice into 500 mL Erlenmeyer flasks, add 180 mL of water and mix with the steamed rice. Add the activated Rhizopus oryzae M5 at a rate of 2.0 × 10⁻⁶. 10 Spores / mL, Saccharomyces cerevisiae G2 at 1.0 × 10⁻⁶ 10 CFU / mL, Lactobacillus fermentum d6 at 1.0×10 9 CFU / mL and abnormal Vicham yeast 1D6 at 1.0×10 9 The inoculation dose of CFU / mL was applied to cooked rice, and the inoculation combination was as follows:

[0060] Synthetic microbial community group (I): Rhizopus oryzae M5+ Saccharomyces cerevisiae G2+ Lactobacillus fermentum d6+ Wickerhamomyces anomalus 1D6 (continuous inoculation fermentation: i.e., after inoculating Rhizopus oryzae M5 for 24 hours, the other three strains were inoculated).

[0061] Single-strain fermentation group (II): Only Lactobacillus fermentum d6 was inoculated, with the addition of wheat koji and yeast, of which wheat koji accounted for 10% of the rice weight and yeast accounted for 7% of the rice weight.

[0062] Single-strain fermentation group (III): Only Saccharomyces cerevisiae G2 was inoculated, with the addition of wheat koji and yeast, of which wheat koji accounted for 10% of the rice weight and yeast accounted for 7% of the rice weight.

[0063] The above-mentioned malt and yeast were purchased from:

[0064] Wine yeast: Hubei Chuzhai Fermentation Products Co., Ltd.;

[0065] Maiqu: Liangshan County Hualiang Bioengineering Co., Ltd.

[0066] The initial fermentation was carried out at 28-30℃ for 5 days, followed by continuous static fermentation in Erlenmeyer flasks at 15℃ for another 15 days, after which the secondary fermentation ended. Samples were taken at five different time points during fermentation (1 day, 3 days, 5 days, 10 days, and 20 days) and immediately stored at -80℃. After fermentation, the fermentation mash was filtered. The filtrate (yellow wine) was stored at 4℃ for further analysis. All these procedures were performed in triplicate.

[0067] Detection and comparison experiment

[0068] 1. Detection of urea content during the fermentation of rice wine;

[0069] The specific method is as follows:

[0070] Shake the rice wine fermentation liquid sample obtained in the above embodiment well, take an appropriate amount of sample into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 10 min, and take the supernatant for testing.

[0071] The urea content of the fermentation liquid of rice wine in different groups was detected separately, and the results are shown in Table 1 and 2. Figure 1 As shown.

[0072] Table 1: Urea content in rice wine at the end of fermentation in different groups

[0073]

[0074] The rice wine fermented using the synthetic microbial community group I in this embodiment had a urea content of only 0.70±0.11 mg / L at the end of fermentation, while the urea contents of the single-strain fermentation groups II and III were 8.44±0.16 mg / L and 9.54±0.04 mg / L, respectively. Compared with the single-strain groups, the synthetic microbial community of this invention reduced the urea content by approximately 91.7%~92.7%. This indicates that the accumulation of urea is relatively high when fermented by a single strain alone. However, during fermentation using the synthetic microbial community, urea is efficiently degraded through metabolic synergy among strains, especially the urea-decomposing ability of Saccharomyces cerevisiae G2 combined with the metabolism of other strains. This demonstrates the advantage of the synthetic microbial community in reducing urea in rice wine fermentation broth.

[0075] 2. Detection of citrulline content during the fermentation of rice wine;

[0076] The specific method is as follows:

[0077] Shake the rice wine fermentation liquid sample obtained in the above embodiment well, take an appropriate amount of sample into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 10 min, and take the supernatant for testing.

[0078] The citrulline content of the fermentation liquid of rice wine in different groups was detected separately, and the results are shown in Table 2 and 3. Figure 2 As shown.

[0079] Table 2: Citrulline content in rice wine fermentation liquid at the end of fermentation in different groups

[0080]

[0081] In this embodiment, the citrulline content at the end of fermentation in the synthetic microbial community group I was 4.22 ± 1.04 mg / L, while the citrulline contents in the single-strain fermentation groups II and III were 16.08 ± 0.22 mg / L and 12.43 ± 1.80 mg / L, respectively. Compared with the single-strain groups, this invention reduced the citrulline content by approximately 66.1% to 73.8%. Citrulline is another key precursor of EC, mainly produced by lactic acid bacteria via the arginine deiminase (ADI) pathway. In group II, due to the absence of lactic acid bacteria, the citrulline content was relatively low. Although lactic acid bacteria were present in group III, the lack of other strains resulted in an incomplete fermentation system, leading to the highest accumulation of citrulline. In group SC, *Lactobacillus fermentatus* d6 converted arginine to citrulline via the complete ADI pathway, which was then further metabolized into ornithine, releasing ammonia and carbon dioxide. Citrulline was consumed, resulting in a significant reduction in its final content. This indicates that the synthetic microbial community can effectively solve the problem of excessive citrulline accumulation during single-strain fermentation.

[0082] 3. Detection of arginine content during the fermentation process of rice wine;

[0083] The specific method is as follows:

[0084] Shake the rice wine fermentation liquid sample obtained in the above embodiment well, take an appropriate amount of sample into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 10 min, and take the supernatant for testing.

[0085] The arginine content of the fermentation liquid of rice wine in different groups was detected separately, and the results are shown in Table 3 and 4. Figure 3 As shown.

[0086] Table 3: Arginine content in rice wine fermentation liquid at the end of fermentation in different groups

[0087]

[0088] In this embodiment, the arginine content at the end of fermentation in the synthetic microbial community group I was 4.61 ± 0.78 mg / L, significantly lower than that in the single-strain group. This indicates that arginine was more fully metabolized and utilized in the synthetic microbial community. In group I, *Saccharomyces cerevisiae* G2 decomposed arginine into urea and ornithine via the arginase pathway, while *Lactobacillus fermentum* d6 converted arginine to ornithine via the ADI pathway through citrulline. This dual pathway resulted in highly efficient arginine decomposition with minimal residue, reducing EC precursor substances at the source.

[0089] 4. Detection of ornithine content during rice wine fermentation;

[0090] The specific method is as follows:

[0091] Shake the rice wine fermentation liquid sample obtained in the above embodiment well, take an appropriate amount of sample into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 10 min, and take the supernatant for testing.

[0092] The ornithine content of the rice wine fermentation liquid was measured at different time points, and the results are shown in Table 4. Figure 4 As shown.

[0093] Table 4: Ornithine content in rice wine fermentation liquid at the end of fermentation in different groups

[0094]

[0095] Ornithine is one of the end products of arginine metabolism via the urea cycle or the ADI pathway. Results showed that the ornithine content in single-strain fermentation groups II and III was as high as 194.92 mg / L and 236.96 mg / L, respectively, while the ornithine content in the synthetic microbial community group I of this invention was only 89.39 mg / L, a reduction of approximately 54.1%–62.3%. This invention significantly reduced the final concentration of ornithine, indicating that the synthetic microbial community effectively converted intermediate products of the arginine metabolic pathway.

[0096] 5. Detection of high arginine and high citrulline content during the fermentation process of rice wine;

[0097] The specific method is as follows:

[0098] Shake the rice wine fermentation liquid sample obtained in the above embodiment well, take an appropriate amount of sample into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm and 4℃ for 10 min, and take the supernatant for testing.

[0099] The contents of high arginine and high citrulline in the fermentation broth of rice wine in different groups were detected separately, and the results are shown in Table 5. Figure 5-6 As shown.

[0100] Table 5: Arginine content in rice wine fermentation liquid at the end of fermentation in different groups

[0101]

[0102] High arginine and high citrulline are derivatives of arginine and citrulline, respectively, and their content changes reflect the overall activity of amino acid metabolism. Table 5 and Figure 5 and Figure 6 The results showed that at the end of fermentation, the content of high-arginine in group I was 8.64 ± 1.32 ng / mL, significantly lower than that in groups II and III; the content of high-citrulline in group I was 85.28 ± 3.33 ng / mL, significantly lower than that in groups II and III. The lowest levels of both derivatives in group I indicate that the synthetic microbial community regulates arginine and citrulline, reducing the accumulation of EC precursor-related substances.

[0103] This invention constructs a synthetic microbial community composed of *Rhizopus oryzae* M5, *Saccharomyces cerevisiae* G2, *Lactobacillus fermentum* d6, and *V. anomala* 1D6, and employs a continuous inoculation process to effectively control the levels of urea (≤0.70 mg / L) and citrulline (≤4.22 mg / L), key precursors of endocrine disruptors (ECs), during the fermentation of rice wine. Compared with single-strain fermentation, the four-strain synergistic system exhibits significant advantages in urea degradation, citrulline elimination, arginine consumption, and accumulation of metabolic byproducts (urea reduced by over 92%, and citrulline reduced by over 66%), fully demonstrating the synergistic effect of this synthetic microbial community. This method solves the problem of EC precursor accumulation in rice wine from the brewing source, providing a reliable technical solution for the safe production of rice wine.

Claims

1. A brewing method for reducing the content of ethyl carbamate precursors in rice wine, characterized in that, Includes the following steps: (1) Soak, steam, and cool the rice to obtain cooked rice; (2) The synthetic microbial colony is inoculated into cooked rice, and water is added and mixed evenly to obtain a mixture. The synthetic microbial community consists of Rhizopus oryzae M5, Saccharomyces cerevisiae G2, Lactobacillus fermentum d6 and Wickerhamomyces anomalus 1D6. (3) The mixture obtained in step (2) is subjected to pre-fermentation and post-fermentation in sequence to finally obtain fermented mash.

2. The brewing method for reducing the content of ethyl carbamate precursors in rice wine according to claim 1, characterized in that: In step (2), a continuous inoculation method is adopted: first inoculate with Rhizopus oryzae M5, and then inoculate with Saccharomyces cerevisiae G2, Lactobacillus fermentum d6 and Vickham's abnormal yeast 1D6 24 hours later.

3. The brewing method for reducing the content of ethyl carbamate precursors in rice wine according to claim 1, characterized in that: The inoculum size of Rhizopus oryzae M5 was 2.0 × 10⁻⁶. 10 ~2.0×10 12 Spores / mL, the inoculum size of Saccharomyces cerevisiae G2 was 1.0 × 10⁻⁶. 10 ~1.0×10 12 CFU / mL, Lactobacillus fermentum d6 inoculum size 1.0 × 10⁻⁶ 9 ~1.0×10 10 CFU / mL, abnormal Vicham yeast 1D6 inoculum size 1.0 × 10⁻⁶ 9 ~1.0×10 10 CFU / mL.

4. The brewing method for reducing the content of ethyl carbamate precursors in rice wine according to claim 1, characterized in that: In step (1), the amount of rice used is 100g, the amount of water used is 160~180mL, the soaking time is 2~3 days, and the steaming is carried out under normal pressure.

5. The brewing method for reducing the content of ethyl carbamate precursors in rice wine according to claim 1, characterized in that: In step (3), both the pre-fermentation and post-fermentation are static fermentations. The temperature of the pre-fermentation is 28~30℃, and the time is 5 days. The temperature of the post-fermentation is 15℃, and the time is 15 days.

6. The application of the brewing method for reducing the content of ethyl carbamate precursors in rice wine according to any one of claims 1-5 in the production of rice wine.

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