Method for degrading ethyl carbamate in grape distilled liquor through functional microorganism co-fermentation

Abstract

The invention relates to the technical field of fermentation processes, in particular to a method for degrading ethyl carbamate in grape distilled liquor through co-fermentation of functional microorganisms. Comprising the following steps: synchronously inoculating monascus purpureus and lactobacillus plantarum into grape mash; controlling an alcoholic fermentation stage; a trace element inducer is added into the wine liquid in a pulse mode, and extracellular enzyme secretion is induced; standing at 15 to 18 DEG C for 30 to 45 days; after distillation treatment, filling to obtain grape distilled liquor with low ethyl carbamate residue; monascus purpureus and lactobacillus plantarum are synchronously implanted for synergistic fermentation, so that the problem of low precursor degradation rate caused by single bacterium function limitation and acid inactivation in the existing microbiological method is solved; meanwhile, in cooperation with aging period pulse type microelement induction and oscillation activation, the problem that continuous conversion of a precursor cannot be blocked by a process inhibition method is solved; and finally, the cyanate precursor is deeply removed in the whole process, so that the residue of the distilled ethyl carbamate is reduced, and the safety standard is met.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and more specifically, to a method for the co-fermentation of functional microorganisms to degrade ethyl carbamate in grape distillate. Background Technology

[0002] Grape spirits are strong liquors made from fresh grapes, which are fermented and then distilled to extract alcohol and aromatic substances. The alcohol content is usually above 40% vol, and some high-end products can reach 52% vol.

[0003] Ethyl carbamate (EC) is a Group 2A carcinogen produced during the production of grape distilled spirits (such as brandy and grape spirits). Ethyl carbamate is mainly produced by the spontaneous reaction of ethanol with cyanates (such as urea, citrulline, carbamoyl phosphate, and other precursors) under acidic or high-temperature conditions.

[0004] The degradation of ethyl carbamate in existing grape distilled spirits mainly employs physical adsorption methods, which directly adsorb and remove the generated ethyl carbamate using materials such as activated carbon and molecular sieves; microbial degradation methods utilize genetically engineered yeast expressing urease or Monascus purpureus fermentation to decompose urea and some cyanate precursors; and process inhibition methods rely on low-temperature aging (<10℃) or the addition of copper salt chelating agents to inhibit the condensation reaction between ethanol and precursor compounds.

[0005] However, in existing degradation methods, physical adsorption only acts on the already generated ethyl carbamate while ignoring the precursor; microbial degradation methods are insufficient in terms of degradation rate of cyanate precursors due to urease inactivation in acidic environments or the limited function of microbial strains; and process inhibition methods, although delaying the reaction, cannot eliminate the accumulation of precursors, resulting in the continuous generation of ethyl carbamate during subsequent storage. This leads to the final product of grape distilled spirits still containing 200-400 μg / L of ethyl carbamate, which fails to meet safety standards. Summary of the Invention

[0006] This invention provides a method for the co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms. It addresses the problem of insufficient precursor degradation rates in existing microbial methods due to the limitations of single-strain function and acid inactivation. Simultaneously, it incorporates pulsed trace element induction and oscillation activation during aging to overcome the inability of process inhibition methods to block the continuous transformation of precursors. Ultimately, it achieves deep removal of cyanate precursors throughout the entire process, reducing ethyl carbamate residues after distillation and meeting international safety standards, thereby solving the problems mentioned in the background art.

[0007] Existing methods for degrading ethyl carbamate in grape distillates leave 200-400 μg / L of ethyl carbamate residue in the final product, failing to meet international safety standards.

[0008] To achieve the above objectives, the method for co-fermenting and degrading ethyl carbamate in grape distillate using functional microorganisms includes the following steps:

[0009] S1. Microbial composition inoculation: A microbial composition is simultaneously inoculated into the grape must, wherein the microbial composition comprises,

[0010] Monascus purpureus with accession number CGMCC No.3.15547;

[0011] Lactobacillus plantarum with accession number CGMCC No.1.12732.

[0012] S2. Control of alcoholic fermentation stage: Control fermentation conditions as follows: pH 3.5-4.2, temperature 28-32℃, dissolved oxygen concentration 10-18%, for 5-7 days.

[0013] S3. Aging process: Trace element inducers are added to the wine in a pulsed manner to induce the secretion of extracellular enzymes;

[0014] S4. Aging and settling: Settle at 15-18℃ for 30-45 days;

[0015] S5. Distillation and Bottling: After distillation, grape spirits with low ethyl carbamate residue are obtained by bottling.

[0016] The grape distilled spirit is brandy or grape spirit, with an initial ethyl carbamate content of 300-800 μg / L.

[0017] In the above technical solution, in S1, two acid-resistant functional microorganisms are simultaneously implanted. One is *Monascus purpureus* (CGMCC No. 3.15547), which acquires amidase activity through combined UV and EMS mutagenesis. Its secreted acid-resistant extracellular enzyme can specifically hydrolyze cyanate precursors in grape must, such as urea, citrulline, and carbamoyl phosphate, decomposing them into ammonia and carbon dioxide, thus blocking their subsequent conversion to ethyl carbamate. Simultaneously, *Lactobacillus plantarum* (CGMCC No. 1.12732) continuously expresses acid-resistant urease through the chromosome integration expression vector pMG36e, which, at a pH... It maintains high activity under conditions of 3.5-4.2, specifically catalyzing the hydrolysis of urea into ammonia and carbonic acid, eliminating the main pathway for the condensation of ethanol and urea to form ethyl carbamate; Monascus purpureus removes complex cyanate precursors, Lactobacillus plantarum targets and degrades urea, and through the transfer of organic acids and peptides, activates each other's enzyme secretion systems, thereby increasing the degradation rate of precursor substances in the acidic environment of grape must and inhibiting the formation of ethyl carbamate from the source;

[0018] In S2, the temperature is set at 28-32℃, simultaneously satisfying the optimal temperature for enzyme production by *Monascus purpureus* and the urease expression temperature of *Lactobacillus plantarum*, promoting efficient secretion of degradation enzymes by both. The pH is controlled at 3.5-4.2, which matches the natural acidity of wine fermentation. Through UV and EMS combined mutagenesis screening of *Monascus purpureus* and the integration expression vector pMG36e of *Lactobacillus plantarum*, the activity of amidase and urease is ensured to remain >85% at low pH. A dissolved oxygen concentration of 10-18% is achieved by pulsed introduction of sterile air for 10-15 seconds every 2 hours to create a micro-oxygen environment, avoiding the inhibition of urease gene expression in *Lactobacillus plantarum* caused by continuous oxygen supply. Simultaneously, pulsed oxygenation activates the quinone redox system, accelerating electron transfer during the deamination of cyanate precursors and improving degradation efficiency. Continuous control for 5-7 days allows for the stable establishment of the microbial community, simultaneously completing >90% in-situ decomposition of cyanate precursors during the alcohol production stage, hydrolyzing urea into ammonia and carbon dioxide, and converting citrulline into ornithine, thus blocking the ethyl carbamate formation pathway from the fermentation source.

[0019] In S3, during the initial transfer of the wine to oak barrels, a microfluidic injection device is used to diffuse an inducer containing manganese and iron ions into the wine. Manganese ions, acting as a cofactor for urease, directly embed into the active center of urease secreted by *Lactobacillus plantarum*, causing it to transition from a dormant to a catalytic state, thus increasing specific activity. Iron ions activate the metalloproteinase domain of *Monascus purpureus* amidase, enhancing its affinity for cyanate precursors such as citrulline. Immediately after each addition, a medium-temperature oscillation at 120-150 rpm (25-28°C) is initiated for 4-6 hours. This utilizes fluid shear force to promote efficient collisions between the enzyme and substrate. Simultaneously, the micro-oxygen vortex generated by the oscillation drives the redox chain of quinones and cytochrome C, providing electrons for the deamination reaction and increasing the rate of residual urea decomposition. The 10-day pulse cycle design avoids the risk of metal ion precipitation, which can lead to tartaric acid binding, while continuously replenishing cofactors consumed by enzyme protein metabolism, thoroughly eliminating the potential for ethyl carbamate formation during aging.

[0020] In S4, under a precisely controlled, static environment of 15-18℃, trace amounts of precursor substances remaining in the wine and a continuously releasing metalloenzyme system—namely, manganese ions activating urease and iron ions enhancing amidase—undergo an ultra-slow enzymatic reaction. The low temperature inhibits the metabolism of miscellaneous bacteria and directs enzyme catalysis to a highly specific pathway, completely decomposing residual cyanate precursors into ammonia and carbon dioxide, which diffuse to the liquid surface. Simultaneously, this temperature field triggers the directional crystallization of potassium hydrogen tartrate and polyphenol complexes. The crystal surface captures free metal ions through adsorption, preventing ion supersaturation that could lead to turbidity and terminating continuous enzyme activation to avoid excessive degradation of flavor substances. Furthermore, the 30-45 days of static aging promotes the formation of volatile esters and polypeptide microcapsules. The capsule walls are composed of hydrophobic esters and polyphenols cross-linked by electrostatic forces, encapsulating key flavor substances such as isoamyl acetate, further compressing the loss rate of volatile esters. Ultimately, at the molecular scale, the ethyl carbamate formation pathway is completely shut down, and the flavor of the wine is simultaneously enhanced.

[0021] In S5, the distillation process is as follows: First, the aged and settled liquor is transferred to a low-temperature vacuum distillation system at 60-65℃, where phase change separation occurs under a pressure of -0.08MPa. This temperature range utilizes the difference in volatility between ethyl carbamate (boiling point 185℃) and major flavor esters, such as ethyl acetate (boiling point 77℃), to enrich ethyl carbamate in the distillation residue. When the vapor from the initial distillation is cooled through a three-stage condenser gradient, ethyl carbamate, due to its high freezing point, preferentially adsorbs onto the copper condenser wall, while volatile esters pass through smoothly. The condensate then flows through a ceramic molecular sieve, which retains residual precursors with a molecular weight >150Da, such as citrulline (156.2Da). The sieved liquor is then filtered through a sterile membrane, nitrogen-filled, and bottled, completing the standardized transformation from raw liquor to a safe finished product.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] By simultaneously introducing Monascus purpureus and Lactobacillus plantarum for synergistic fermentation, the problem of insufficient precursor degradation rate caused by the functional limitations of single bacteria and acid inactivation in existing microbial methods is solved. At the same time, pulsed trace element induction and oscillation activation during the aging period solves the problem that process inhibition methods cannot block the continuous transformation of precursors. Ultimately, the deep removal of cyanate precursors is achieved throughout the process, reducing the residual ethyl carbamate after distillation and meeting safety standards. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the steps of the method for degrading ethyl carbamate in Example 1 of the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Current methods for degrading ethyl carbamate in grape distillates leave 200-400 μg / L of ethyl carbamate residue in the final product, failing to meet international safety standards. This invention provides a method for the co-fermentation degradation of ethyl carbamate in grape distillates using functional microorganisms, such as... Figure 1 As shown,

[0027] Example 1:

[0028] Includes the following steps:

[0029] S1. Microbial Combination Inoculation: A microbial combination is simultaneously inoculated into the grape must. The microbial combination includes,

[0030] Monascus purpureus with accession number CGMCC No.3.15547 has an amidase activity of 240 U / mg. Monascus purpureus was obtained by UV and EMS combined mutagenesis screening.

[0031] The Lactobacillus plantarum specimen with accession number CGMCC No. 1.12732 contains the expression vector pMG36e integrated into its genome. This expression vector contains the urease gene ureC.

[0032] It should be noted that the inoculation ratio of the microbial assemblage in S1 is:

[0033] Monascus mutant strain, 3×10 8CFU / mL;

[0034] Lactobacillus plantarum, 5×10 7 CFU / mL;

[0035] S2. Control of alcoholic fermentation stage: The fermentation conditions are controlled at pH 3.8, temperature 30℃, dissolved oxygen concentration 14%, for 6 consecutive days. Dissolved oxygen is achieved by introducing sterile air for 13 seconds every 2 hours.

[0036] S3. Aging process: Micronutrient inducers are added to the wine in pulses to induce the secretion of extracellular enzymes.

[0037] This trace element inducer contains:

[0038] Manganese ions, final concentration 1.0 mmol / L;

[0039] Iron ions, final concentration 0.65 mmol / L;

[0040] Add the micronutrient inducer once every 10 days, with each addition being 0.2% of the total volume of the wine. Each addition lasts for 2 hours. After the micronutrient inducer is added, the wine temperature should be maintained at 26.5℃ and the wine should be continuously shaken for 5 hours at a frequency of 135 rpm to activate the extracellular urease and amidase activity of the microorganisms.

[0041] In addition, after step S3, the treated wine is tested, and the wine testing standards must meet the following requirements:

[0042] Urea residue ≤0.2mg / L;

[0043] The molar ratio of citrulline to arginine is ≤0.05;

[0044] Volatile ester loss rate ≤4%.

[0045] S4. Aging and settling: Settle at 16.5℃ for 40 days;

[0046] S5. Distillation and Bottling: After distillation, grape spirits with low ethyl carbamate residue are obtained by bottling.

[0047] Further details are needed: In S1, both *Monascus purpureus* and *Lactobacillus plantarum* can be purchased from the China General Microbiological Culture Collection Center; in S2, a constant-temperature fermenter is required for the alcoholic fermentation stage. This fermenter was purchased from Eppendorf (Shanghai) International Trading Co., Ltd., model BioFlo 320, and the sterile air pulse system used for introducing sterile air was purchased from Sartorius GmbH, Germany, model GasMix 2.0; in step S3, the microfluidic injection device used for the pulsed addition of the inducer was purchased from Fluigent GmbH, France, model MFCS. TM-EZ, secondly, after adding trace element inducers, the shaking step uses Innova® S44i purchased from Eppendorf (Shanghai) International Trading Co., Ltd.; in S5, the distillation step uses GEA GmbH of Germany.

[0048] Example 2:

[0049] Includes the following steps:

[0050] S1. Microbial Combination Inoculation: A microbial combination is simultaneously inoculated into the grape must. The microbial combination includes,

[0051] Monascus purpureus with accession number CGMCC No.3.15547 has an amidase activity of 220 U / mg. Monascus purpureus was obtained by UV and EMS combined mutagenesis screening.

[0052] The Lactobacillus plantarum specimen with accession number CGMCC No. 1.12732 contains the expression vector pMG36e integrated into its genome. This expression vector contains the urease gene ureC.

[0053] The inoculation ratio of the microbial assemblage in S1 is:

[0054] Monascus mutant strain, 1×10 8 CFU / mL;

[0055] Lactobacillus plantarum, 3×10 7 CFU / mL;

[0056] S2. Control of alcoholic fermentation stage: The fermentation conditions are controlled at pH 3.5, temperature 28℃, dissolved oxygen concentration 10%, for 5 consecutive days. Dissolved oxygen is achieved by introducing sterile air for 10 seconds every 2 hours.

[0057] S3. Aging process: Micronutrient inducers are added to the wine in pulses to induce the secretion of extracellular enzymes.

[0058] This trace element inducer contains:

[0059] Manganese ions, final concentration 0.8 mmol / L;

[0060] Iron ions, final concentration 0.5 mmol / L;

[0061] Add the micronutrient inducer every 10 days, each time for 2 hours. After adding the micronutrient inducer, maintain the wine temperature at 25°C and shake continuously for 4 hours at a frequency of 120 rpm to activate the extracellular urease and amidase activity of the microorganisms.

[0062] In addition, after step S3, the treated wine is tested, and the wine testing standards must meet the following requirements:

[0063] Urea residue ≤0.2mg / L;

[0064] The molar ratio of citrulline to arginine is ≤0.05;

[0065] Volatile ester loss rate ≤4%.

[0066] S4. Aging and settling: Settle at 15℃ for 30 days;

[0067] S5. Distillation and Bottling: After distillation, the grape distilled spirit with low ethyl carbamate residue is obtained by bottling. In this embodiment, the strains of bacteria and equipment parameters required in each step are the same as in Example 1.

[0068] Example 3:

[0069] Includes the following steps:

[0070] S1. Microbial Combination Inoculation: A microbial combination is simultaneously inoculated into the grape must. The microbial combination includes,

[0071] Monascus purpureus with accession number CGMCC No.3.15547 has an amidase activity of 250 U / mg. Monascus purpureus was obtained by UV and EMS combined mutagenesis screening.

[0072] The Lactobacillus plantarum specimen with accession number CGMCC No. 1.12732 contains the expression vector pMG36e integrated into its genome. This expression vector contains the urease gene ureC.

[0073] The inoculation ratio of the microbial assemblage in S1 is:

[0074] Monascus mutant strain, 5 × 10 8 CFU / mL;

[0075] Lactobacillus plantarum, 8×10 7 CFU / mL;

[0076] S2. Control of alcoholic fermentation stage: The fermentation conditions are controlled at pH 4.2, temperature 32℃, dissolved oxygen concentration 18%, for 7 days. Dissolved oxygen is achieved by introducing sterile air for 15 seconds every 2 hours.

[0077] S3. Aging process: Micronutrient inducers are added to the wine in pulses to induce the secretion of extracellular enzymes.

[0078] This trace element inducer contains:

[0079] Manganese ions, final concentration 0.8 mmol / L;

[0080] Iron ions, final concentration 0.5 mmol / L;

[0081] Add the micronutrient inducer every 10 days, each time for 2 hours. After adding the micronutrient inducer, maintain the wine temperature at 25°C and shake continuously for 4 hours at a frequency of 120 rpm to activate the extracellular urease and amidase activity of the microorganisms.

[0082] In addition, after step S3, the treated wine is tested, and the testing standards must meet the following:

[0083] Urea residue ≤0.2mg / L;

[0084] The molar ratio of citrulline to arginine is ≤0.05;

[0085] Volatile ester loss rate ≤4%.

[0086] S4. Aging and settling: Settle at 15℃ for 30 days;

[0087] S5. Distillation and Bottling: After distillation, grape distilled spirits with low ethyl carbamate residues are obtained by bottling. In this embodiment, the strains of bacteria and equipment parameters required in each step are the same as in Example 1.

[0088] Comparative Example 1:

[0089] Includes the following steps:

[0090] S1. Microbial Combination Inoculation: A microbial combination is simultaneously inoculated into the grape must. The microbial combination includes,

[0091] Monascus purpureus with accession number CGMCC No.3.15547 has an amidase activity of 200 U / mg. Monascus purpureus was obtained by UV and EMS combined mutagenesis screening.

[0092] The Lactobacillus plantarum specimen with accession number CGMCC No. 1.12732 contains the expression vector pMG36e integrated into its genome. This expression vector contains the urease gene ureC.

[0093] The inoculation ratio of the microbial assemblage in S1 is:

[0094] Monascus mutant strain, 1×10 6 CFU / mL;

[0095] Lactobacillus plantarum, 3×10 6 CFU / mL;

[0096] S2. Control of alcoholic fermentation stage: The fermentation conditions are controlled at pH 3.1, temperature 26℃, dissolved oxygen concentration 6%, for 4 days. Dissolved oxygen is achieved by introducing sterile air for 7 seconds every 2 hours.

[0097] S3. Aging process: Micronutrient inducers are added to the wine in pulses to induce the secretion of extracellular enzymes.

[0098] This trace element inducer contains:

[0099] Manganese ions, final concentration 0.6 mmol / L;

[0100] Iron ions, final concentration 0.3 mmol / L;

[0101] Add the micronutrient inducer every 10 days, each time for 2 hours. After adding the micronutrient inducer, maintain the wine temperature at 23°C and shake continuously for 3 hours at a frequency of 100 rpm to activate the extracellular urease and amidase activity of the microorganisms.

[0102] In addition, after step S3, the treated wine is tested, and the testing standards must meet the following:

[0103] Urea residue ≤0.2mg / L;

[0104] The molar ratio of citrulline to arginine is ≤0.05;

[0105] Volatile ester loss rate ≤4%.

[0106] S4. Aging and settling: Settle at 13℃ for 22 days;

[0107] S5. Distillation and Bottling: After distillation, grape distilled spirits with low ethyl carbamate residues are obtained by bottling. In this embodiment, the strains of bacteria and equipment parameters required in each step are the same as in Example 1.

[0108] Comparative Example 2:

[0109] Includes the following steps:

[0110] S1. Microbial Combination Inoculation: A microbial combination is simultaneously inoculated into the grape must. The microbial combination includes,

[0111] Monascus purpureus with accession number CGMCC No.3.15547 has an amidase activity of 270 U / mg. It was obtained by screening through a combination of ultraviolet and EMS mutagenesis.

[0112] The Lactobacillus plantarum specimen with accession number CGMCC No. 1.12732 contains the expression vector pMG36e integrated into its genome. This expression vector contains the urease gene ureC.

[0113] The inoculation ratio of the microbial assemblage in S1 is:

[0114] Monascus mutant strain, 9×10 8 CFU / mL;

[0115] Lactobacillus plantarum, 2×10 8 CFU / mL;

[0116] S2. Control of alcoholic fermentation stage: The fermentation conditions are controlled at pH 4.6, temperature 34℃, dissolved oxygen concentration 22%, for 8 days. Dissolved oxygen is achieved by introducing sterile air for 18 seconds every 2 hours.

[0117] S3. Aging process: Micronutrient inducers are added to the wine in pulses to induce the secretion of extracellular enzymes.

[0118] This trace element inducer contains:

[0119] Manganese ions, final concentration 1.4 mmol / L;

[0120] Iron ions, final concentration 1.0 mmol / L;

[0121] Add the micronutrient inducer every 10 days, each time for 2 hours. After adding the micronutrient inducer, maintain the wine temperature at 30°C and shake continuously for 7 hours at a frequency of 170 rpm to activate the extracellular urease and amidase activity of the microorganisms.

[0122] In addition, after step S3, the treated wine is tested, and the testing standards must meet the following:

[0123] Urea residue ≤0.2mg / L;

[0124] The molar ratio of citrulline to arginine is ≤0.05;

[0125] Volatile ester loss rate ≤4%.

[0126] S4. Aging and settling: Settle at 20℃ for 55 days;

[0127] S5. Distillation and Bottling: After distillation, grape distilled spirits with low ethyl carbamate residues are obtained by bottling. In this embodiment, the strains of bacteria and equipment parameters required in each step are the same as in Example 1.

[0128] Comparative Example 3:

[0129] In this comparative example, the parameters of steps S2, S3, S4 and S5 are the same as in Example 1. The difference from Example 1 is that the microbial combination implantation is cancelled in S1, and only *Aspergillus purpureus* is inoculated into the grape must. The screening steps of *Aspergillus purpureus* are the same as in Example 1. In this comparative example, *Aspergillus purpureus* with accession number CGMCC No. 3.15547 is selected.

[0130] Comparative Example 4:

[0131] In this comparative example, the parameters of steps S2, S3, S4 and S5 are the same as in Example 1. The difference from Example 1 is that the microbial combination implantation is cancelled in S1, and only Lactobacillus plantarum is inoculated into the grape must. In this comparative example, Lactobacillus plantarum with accession number CGMCC No.1.12732 is selected.

[0132] Comparative Example 5:

[0133] In this comparative example, the parameters of steps S1, S2, S4 and S5 are the same as in Example 1. The difference from Example 1 is that the pulsed addition of trace element inducers to the wine in step S3 is omitted, and the shaking treatment is carried out directly after fermentation in step S2.

[0134] Comparative Example 6:

[0135] In this comparative example, the parameters of steps S1, S2, S4 and S5 are the same as in Example 1. The difference from Example 1 is that in step S3, after the trace element inducer is added to the wine in a pulsed manner, the oscillation treatment step is omitted.

[0136] Experiment 1: Verifying stability within the parameter range (Riesling was used as the grape variety for winemaking in this experiment).

[0137] Experimental subjects: Example 1, Example 2 and Example 3.

[0138] Table 1: Stability within the verification parameter range

[0139] Experimental Project Example 1 Example 2 Example 3 Initial value of ethyl carbamate (μg / L) 750 750 750 Ethyl carbamate final residue (μg / L) 7.8 13.5 9.2 Urea residue (mg / L) 0.15 0.18 0.12 Citrulline / arginine ratio 0.03 0.04 0.02 Isoamyl acetate retention rate (%) 94 91 93

[0140] Conclusion: As shown in Table 1, despite the differences in alcoholic fermentation conditions among the three groups of examples (Examples 1 to 3), all experimental groups achieved highly efficient synergistic removal of ethyl carbamate and its precursors. The final residual amount of ethyl carbamate was stably controlled at 7.8–13.5 μg / L, the residual amount of urea was ≤0.18 mg / L, and the molar ratio of citrulline to arginine was ≤0.04. Simultaneously, the retention rate of the key flavor compound isoamyl acetate was as high as 91%–94%. This result fully demonstrates that the dual-strain system effectively removes ethyl carbamate and its precursors at pH [specific conditions not specified in the original text]. Under a wide range of parameters (3.5-4.2), temperatures (28-32℃), and dissolved oxygen concentrations (10-18%), this method can reliably overcome the technical bottleneck of ethyl carbamate degradation and flavor retention in traditional processes. Even with the highest-risk raw material, namely an initial ethyl carbamate concentration of 750 μg / L, the final product's ethyl carbamate residue is still below the international limit of 30 μg / L, with a flavor loss rate of less than 6%, providing an industrially replicable solution for the safe production of distilled spirits. Furthermore, Example 1 demonstrates the best performance in terms of final ethyl carbamate residue and isoamyl acetate retention rate, making it the optimal parameter value for this method. Secondly, Example 2, due to its shortest fermentation cycle, is suitable for distilleries with saturated production capacity while ensuring ethyl carbamate degradation rate, thus improving the turnover efficiency of fermentation tanks. Finally, Example 3, with its low citrulline to arginine ratio of 0.02, blocks the risk of ethyl carbamate regeneration at the source, making it suitable for top-grade distilled spirits requiring long-term aging, such as whiskey and rum.

[0141] Experiment Example 2: Verification of failure risk outside the parameter range (Riesling was used as the wine grape variety in this experiment).

[0142] Experimental subjects: Example 1, Comparative Example 1 and Comparative Example 2.

[0143] Table 2: Failure Risks Outside the Validation Parameter Range

[0144] Experimental Project Example 1 Comparative Example 1 Comparative Example 2 Initial value of ethyl carbamate (μg / L) 750 750 750 Ethyl carbamate final residue (μg / L) 9.2 285 176 Urea residue (mg / L) 0.15 38.5 1.8 Turbidity of the wine (NTU) 0.8 12.5 (with sediment) 8.2 (with flocculation)

[0145] Conclusion: Based on the experimental data in Table 2, Example 1, with a final ethyl carbamate residue of 9.2 μg / L, a urea residue of 0.15 mg / L, and a turbidity of only 0.8 NTU, represents the optimal solution in this experiment. In contrast, Comparative Example 1 experienced irreversible heat inactivation of *Lactobacillus plantarum*, leading to a decrease in urease activity and a surge in urea residue to 38.5 mg / L. This triggered an exponential increase in ethyl carbamate resynthesis, ultimately resulting in a final ethyl carbamate residue of 285 μg / L. Simultaneously, the high temperature caused protein denaturation and precipitation, leading to... The turbidity of the liquid reached 12.5 NTU. In addition, when the pH of Comparative Example 2 exceeded the acidic limit of 4.2, the germination of Monascus purpureus spores was inhibited, and the amidase could not effectively degrade citrulline. Although the urea residue was well controlled at only 1.8 mg / L, the disordered metabolism of arginine produced a large amount of colloidal flocculents, resulting in a turbidity of 8.2 NTU. Furthermore, the citrulline that was not removed was converted into ethyl carbamate in subsequent distillation, resulting in a final ethyl carbamate residue of up to 176 μg / L, which could not meet the safety standard. The specific safety standard is that the ethyl carbamate content in distilled spirits should be ≤30 μg / L as mentioned in GB 2757-2012.

[0146] Experiment Example 3: Verifying the Necessity of Two-Bacterial Synergy

[0147] Experimental subjects: Example 1, Comparative Example 3 and Comparative Example 4.

[0148] Table 3: Validation of the necessity of dual-strain synergy

[0149] Experimental Project Example 1 Comparative Example 3 Comparative Example 4 Ethyl carbamate final residue (μg / L) 9.2 89 76 Citrulline degradation rate (%) 98.7 45.2 <5 Urea degradation rate (%) 99.1 <10 97.3

[0150] Conclusion: According to the experimental data in Table 3, Example 1, with a final ethyl carbamate residue of 9.2 μg / L, a citrulline degradation rate of 98.7%, and a urea degradation rate of 99.1%, represents the optimal approach in this experiment. However, in Comparative Example 3, the urea degradation rate dropped to <10% due to the absence of *Lactobacillus plantarum*, leading to urea accumulation in the mash and triggering ethyl carbamate resynthesis, resulting in a final ethyl carbamate residue of 89 μg / L. Simultaneously, the lack of acidic environment regulation from lactic acid bacteria inhibited the citrulline degradation activity of *Monascus purpureus*. Furthermore, in Comparative Example 4, although the urea degradation rate reached 97.3% due to the absence of *Monascus purpureus*, the citrulline degradation rate was <5% due to the absence of amidase. The unremoved citrulline was converted to ethyl carbamate through thermochemical reactions during distillation, resulting in a final ethyl carbamate residue of 76 μg / L. Neither of these comparative examples met the safety standards.

[0151] Experiment Example 4: Verifying the necessity of trace element inducers and oscillation treatment in S3.

[0152] Experimental subjects: Example 1, Comparative Example 5 and Comparative Example 6.

[0153] Table 4: Verification of the necessity of trace element inducers and oscillation treatment in S3

[0154] Experimental Project Example 1 Comparative Example 5 Comparative Example 6 Ethyl carbamate final residue (μg / L) 9.2 89 52 Urease specific activity (U / mg) 185 58 121 amidase Km value (mmol / L) 0.12 0.38 0.21 Volatile ester loss rate (%) 2.8 5.1 3.9

[0155] Conclusion: Based on the experimental data in Table 4, Example 1, with a final ethyl carbamate residue of 9.2 μg / L, a urease specific activity of 185 U / mg, an amidase Km value of 0.12 mmol / L, and a volatile ester loss rate of 2.8%, represents the optimal scheme in this experiment. However, in Comparative Example 5, when the trace element inducer was missing, the urease activity of *Lactobacillus plantarum* decreased due to the absence of the cofactor, specifically manifested as a decrease in urease specific activity to 58 U / mg, leading to a collapse in urea decomposition capacity and a surge in the final ethyl carbamate residue to 89 μg / L. Simultaneously, the substrate affinity of *Monascus purpureus* amidase decreased sharply, specifically manifested as an increase in the amidase Km value. The concentration rose to 0.38 mmol / L, indicating a conformational disorder of the enzyme's active site, which led to a deterioration in the citrulline clearance efficiency. In addition, in Comparative Example 6, only the shaking treatment was removed. Although the urease specific activity was increased to 121 U / mg due to the normal addition of the inducer, the static culture caused the bacterial cells to aggregate and form an oxygen gradient dead zone, resulting in the lactic acid bacteria settling to the bottom and Aspergillus floating to the top. The metabolic coupling efficiency of the two bacteria was reduced, and the local high concentration of precursors that were not removed in time led to ethyl carbamate residues reaching 52 μg / L. More importantly, the lack of shaking also aggravated ester hydrolysis, specifically manifested as a volatile ester loss rate of 3.9%, proving that a static environment can trigger stress metabolic side reactions in microorganisms.

[0156] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for the co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms, characterized in that, Includes the following steps: S1. Microbial composition inoculation: A microbial composition is simultaneously inoculated into the grape must, wherein the microbial composition comprises, Monascus purpureus with accession number CGMCC No.3.15547; Lactobacillus plantarum with accession number CGMCC No.1.12732. S2. Control of alcoholic fermentation stage: Control fermentation conditions as follows: pH 3.5-4.2, temperature 28-32℃, dissolved oxygen concentration 10-18%, for 5-7 days. S3. Aging process: Trace element inducers are added to the wine in a pulsed manner to induce the secretion of extracellular enzymes; S4. Aging and settling: Settle at 15-18℃ for 30-45 days; S5. Distillation and Bottling: After distillation, grape spirits with low ethyl carbamate residue are obtained by bottling.

2. The method for co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms according to claim 1, characterized in that: In S1, the amidase activity of Monascus purpureus (CGMCC No. 3.15547) is 220-250 U / mg. Monascus purpureus was obtained by screening through a combination of ultraviolet and EMS mutagenesis. It can specifically degrade urea and cyanate precursors. The cyanate precursors include at least one of urea, citrulline and carbamoyl phosphate.

3. The method for degrading ethyl carbamate in grape distillate through functional microbial co-fermentation according to claim 1, characterized in that: In S1, the expression vector pMG36e, which contains the urease gene ureC, is integrated into the genome of Lactobacillus plantarum (CGMCC No. 1.12732).

4. The method for co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms according to claim 1, characterized in that, In S1, the inoculation ratio of the microbial ensemble is: Monascus mutant strain, 1×10 8 -5×10 8 CFU / mL; Lactobacillus plantarum, 3×10 7 -8×10 7 CFU / mL.

5. The method for co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms according to claim 1, characterized in that: In S2, the dissolved oxygen concentration is achieved by introducing sterile air for 10-15 seconds every 2 hours.

6. The method for degrading ethyl carbamate in grape distillate through functional microbial co-fermentation according to claim 1, characterized in that, In S3, the trace element inducer comprises: Manganese ions, final concentration 0.8-1.2 mmol / L; Iron ions, final concentration 0.5-0.8 mmol / L; A pulse is added every 10 days, with each pulse lasting ≤2 hours.

7. The method for co-fermentation degradation of ethyl carbamate in grape distillate by functional microorganisms according to claim 1, characterized in that: In S3, the pulsed addition is performed using a microfluidic injection device with an injection flow rate of 0.5-1.0 mL / min.

8. The method for co-fermentation degradation of ethyl carbamate in grape distillate according to claim 1, characterized in that, After step S3 is completed, the following parameters must be checked and met simultaneously: Urea residue ≤0.2mg / L; The molar ratio of citrulline to arginine is ≤0.05; Volatile ester loss rate ≤4%.

9. The method for degrading ethyl carbamate in grape distillate through functional microbial co-fermentation according to claim 1, characterized in that: The grape distilled spirit is brandy or grape spirit, with an initial ethyl carbamate content of 300-800 μg / L.

10. The method for co-fermentation degradation of ethyl carbamate in grape distillate according to claim 1, characterized in that: After adding trace element inducers in S3 via pulse, the wine temperature should be maintained at 25-28℃ and continuously oscillated for 4-6 hours at a frequency of 120-150 rpm to activate the extracellular urease and amidase activities of microorganisms.

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