Ethyl carbamate degrading bacterium and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an ethyl carbamate degrading bacterium and application thereof.The ethyl carbamate degrading bacterium is pichia kudriavzevii K44 and is preserved in the China Center for Type Culture Collection on April 25, 2025, the preservation date is April 25, 2025, and the biological preservation number is CCTCC NO: M 2025887. The ethyl carbamate degrading bacterium can obviously degrade ethyl carbamate in a yellow rice wine fermentation system, and a scientific basis is provided for safe production and quality improvement of traditional fermented food.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an ethyl carbamate-degrading bacterium screened from rice wine mash and its application in the degradation of ethyl carbamate. Background Technology

[0002] Ethyl carbamate (EC), a natural byproduct of fermentation, is widely found in various fermented foods and beverages, including distilled spirits, wine, rice wine, yogurt, cheese, and soy sauce. It is primarily activated by cytochrome P450 enzymes, producing carcinogenic intermediates such as vinyl-ethyl carbamate and N-hydroxyethyl carbamate. In 2007, the International Agency for Research on Cancer (IARC) upgraded it to a Group 2A likely carcinogen. Various countries have implemented strict controls on EC content in food; for example, Canada's standard is no more than 100 μg / L for EC in alcoholic beverages, and Japan's limit for imported rice wine is 200 μg / L.

[0003] The formation of eclectic acid (EC) in alcoholic beverages involves various precursors, primarily including urea, citrulline, cyanide, carbamoyl phosphate, and diethyl pyrocarbonate. In the rice wine brewing system, the EC formation mechanism is unique, mainly originating from the non-enzymatic chemical reactions of urea and citrulline with ethanol. The urea pathway is the most significant: yeast produces urea by breaking down arginine with arginase, which accumulates under nitrogen metabolism repression and reacts with ethanol to generate EC. The citrulline pathway, on the other hand, is produced by lactic acid bacteria metabolism, exacerbating EC formation during the aging stage. The accumulation of these precursors is closely related to microbial metabolism. The open fermentation and long-term storage characteristics of rice wine make the problem of excessive EC particularly prominent, severely restricting its international market competitiveness and consumer safety.

[0004] Existing technologies primarily employ physical methods (such as high-temperature treatment), chemical methods (such as adding inhibitors), and biological methods (such as strain improvement). For instance, selecting low-urea-producing yeasts (such as defective Saccharomyces cerevisiae) can block urea synthesis, but these strains often exhibit poor fermentation performance, leading to impaired flavor in rice wine. Adding acidic urease can degrade urea precursors, but the enzyme activity is typically below 1.0 U / mL, and its stability is poor in the acidic (pH 3.5-4.5), high-ethanol (15%) environment of rice wine, making it unable to directly degrade already formed EC, with an actual degradation rate of less than 20%. Other methods, such as metabolic engineering to modify gene expression, have made progress, but face challenges in gene stability and industrialization. These limitations result in unsatisfactory application of existing strategies in the complex fermentation environment of rice wine, failing to effectively control EC content below international standards.

[0005] Therefore, there is an urgent need in this field for a highly efficient and tolerant EC-degrading strain that can directly degrade EC and achieve green control through optimized enzyme production conditions, especially suitable for rice wine production, to improve product safety and quality. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an ethyl carbamate (EC)-degrading bacterium and its application. This strain, screened from rice wine mash, exhibits excellent EC degradation ability and environmental tolerance (tolerant to 15% ethanol, pH 3.0, and 2.5 g / L EC). After optimization, its enzyme activity reaches 1.93 U / mL, and its EC degradation rate in rice wine reaches 37.25%.

[0007] The technical solution of the present invention is as follows: This invention discloses a carbamate-degrading bacterium, namely, *Pichia kudrica*. Pichia kudriavzevii K44, deposited at the China Center for Type Culture Collection, date of deposit: April 25, 2025, accession number: CCTCC NO: M 2025887.

[0008] The fermentation culture method of this degrading bacteria includes: using glucose as a carbon source at a concentration of 25 g / L; using peptone as a nitrogen source at a concentration of 10 g / L; initial pH of 5.0; inoculum size of 5%; culture temperature of 30℃; shaking speed of 200 r / min; and culture time of 24 h.

[0009] The enzyme extraction method for this degrading bacteria was ultrasonic disruption, with intermittent treatment at a power of 400 W for 25 min, wherein the intermittent mode was 2 s working and 4 s interval.

[0010] Furthermore, the present invention also includes the application of the degrading bacteria in the degradation of ethyl carbamate, particularly in the degradation of ethyl carbamate in rice wine fermentation systems, wherein the EC degradation rate is not less than 35%.

[0011] Compared with the prior art, the beneficial effects of the present invention are: This strain K44 was uniquely screened from rice wine mash and identified as [specific strain name] by ITS sequence analysis. Pichia kudriavzeviiThis enzyme exhibits specific tolerance and degradation characteristics. By optimizing fermentation and extraction conditions, enzyme activity was increased by 72.3% (from 1.12 U / mL to 1.93 U / mL), and EC degradation rate reached 37.25%, significantly superior to existing strains (<20%). This was an unexpected achievement in addressing the environmental tolerance challenges of rice wine through gradient enrichment and orthogonal experiments. Its application in rice wine reduced urea by 14.2% and EC content to 67.39 μg / L (below the international standard of 200 μg / L) without affecting flavor. This solves the long-standing problem of poor enzyme stability in existing technologies and has potential commercial value, such as enhancing the export competitiveness of rice wine.

[0012] Biological Preservation

[0013] Kudria zwibichi yeast ( Pichia kudriavzevii K44, deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on April 25, 2025, accession number: CCTCC NO: M 2025887. Attached Figure Description

[0014] Figure 1 This is the standard curve for ammonium ion concentration.

[0015] Figure 2 This is a colony photograph of strain K44.

[0016] Figure 3 The morphological characteristics of a single K44 colony under a microscope.

[0017] Figure 4 A phylogenetic tree for strain K44 constructed based on its ITS sequence.

[0018] Figure 5 The growth curve of strain K44 is shown.

[0019] Figure 6 The effects of different types of carbon sources on the growth and enzyme production of K44.

[0020] Figure 7 The effect of different glucose concentrations on the growth and enzyme production of K44.

[0021] Figure 8 The effects of different nitrogen sources on the growth and enzyme production of K44.

[0022] Figure 9 The effect of peptone concentration on K44 growth and enzyme production.

[0023] Figure 10 The effect of initial pH on K44 growth and enzyme production.

[0024] Figure 11 The effect of inoculum size on K44 growth and enzyme production.

[0025] Figure 12 To investigate the effect of culture temperature on the growth and enzyme production of K44.

[0026] Figure 13 The effect of shaking speed on the growth and enzyme production of K44.

[0027] Figure 14 The effect of ultrasonic power on enzyme activity.

[0028] Figure 15 The effect of ultrasound time on enzyme activity.

[0029] Figure 16 The changes in urea content in the experimental and control groups during fermentation are shown.

[0030] Figure 17 The changes in EC content in the experimental and control groups during fermentation are shown. Detailed Implementation

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1 Screening of ethyl carbamate-degrading bacteria I. Experimental Materials 1. Sample Source The fermented rice wine mash is stored in a refrigerator at -20℃.

[0033] 2. Culture medium (1) Initial screening culture medium: glucose 0.5 g, EC 1.25 g, potassium dihydrogen phosphate (KH2PO4) 0.5 g, sodium acetate (NaAc) 0.5 g, sodium chloride (NaCl) 1.25 g, ddH2O2 to a final volume of 250 mL, pH adjusted to 5.0.

[0034] (2) Plate screening medium: glucose 0.5 g, EC 1.25 g, potassium dihydrogen phosphate (KH2PO4) 0.5 g, sodium acetate (NaAc) 0.5 g, sodium chloride (NaCl) 1.25 g, agar powder 0.25 g, bromocresol purple 0.005 g, ddH2O2 to a final volume of 250 mL, pH adjusted to 5.0.

[0035] (3) YEPD solid medium: 1% yeast extract, 2% peptone, 2% glucose, 2% agar powder.

[0036] (4) Screening medium for EC-degrading yeast (EC as the sole nitrogen source): EC 1.25 g, glucose 1 g, sodium acetate (NaAc) 1 g, sodium chloride (NaCl) 1 g, bromocresol purple 0.001 g, potassium dihydrogen phosphate (KH2PO4) 1 g, agar powder 10 g, ddH2O2 to a final volume of 500 mL, pH adjusted to 5.0.

[0037] (5) Screening medium C for EC-degrading yeast (EC as the sole carbon source): EC 1.25 g, ammonium sulfate 2 g, sodium chloride (NaCl) 1 g, diammonium hydrogen phosphate 1.25 g, magnesium sulfate 0.25 g, potassium dihydrogen phosphate (KH2PO4) 1.25 g, agar powder 10 g, ddH2O2 to a final volume of 500 mL, pH adjusted to 5.0.

[0038] (6) Degradation capacity medium: 0.5 g peptone, 1 g glucose, 1.25 g potassium dihydrogen phosphate (KH2PO4), 1 g sodium chloride (NaCl), 1.25 g EC, 1 mg bromocresol purple, ddH2O2 to a final volume of 500 mL, pH adjusted to 5.0.

[0039] II. Experiment Content 1. Method for determining the activity of EC degrading enzymes (1) Enzyme activity: The amount of enzyme that produces 1 µmol of ammonia per minute by decomposing EC at 35℃ and pH=4.5 is one enzyme activity unit (U).

[0040] (2) Substrate: Prepare a 3% EC solution with a mother solution of 0.05 mol / L citrate buffer and adjust the pH to 4.5.

[0041] Colorimetric reagent I: Dissolve 30 g of phenol and 1.25 g of sodium nitrosoferricyanide (SNP) in distilled water, stir well, and then bring the volume to 500 mL.

[0042] Colorimetric reagent II: Add 26.25 g NaOH to 15 mL of sodium hypochlorite, mix well, and then dilute to 500 mL with ddH2O2.

[0043] Terminating agent: 10% trichloroacetic acid solution (w / v).

[0044] (3) Add 0.2 mL of enzyme solution to two 25 mL colorimetric tubes respectively, and boil one of the enzyme solutions in a water bath for 15 minutes as a control; add 0.8 mL of substrate solution to each tube and mix well, and react at 37℃ for 15 min; quickly add 1 mL of terminator to terminate the reaction, and add 1 mL of colorimetric reagent I and II in sequence, mix well and continue to react at 37℃ for 20 min; make up to 25 mL with distilled water, measure the absorbance value at 625 nm, and calculate the enzyme activity according to the ammonium ion standard curve.

[0045] (4) Prepare gradient concentrations of NH4 using NH4Cl + The standard solution was used to measure the absorbance at 625 nm, and the absorbance was measured using NH4+. + Concentration and OD 625nm A standard curve was plotted using absorbance values ​​as the x and y axes, and the results are as follows: Figure 1 As shown, there is a good linear correlation between absorbance and concentration (R0). 2 =0.999), enzyme activity calculation formula: enzyme activity = OD 625nm ×n (dilute factor of enzyme solution) ×1 / k (slope of fitted linear standard curve) ×1 / 20 (time of enzyme-catalyzed reaction, min).

[0046] 2. Screening of EC-degrading enzyme-producing bacteria (1) Initial screening The experiment used a gradient enrichment culture combined with selective plate isolation to screen EC-degrading strains. The specific procedures were as follows: Under aseptic conditions, 10 g of sample was inoculated into selective liquid medium (with EC as the sole nitrogen source) and cultured at 30°C with shaking for 24 h. 1 mL of the culture was then transferred to fresh medium for a second enrichment culture (under the same conditions). Subsequently, the enriched bacterial solution was transferred to enhanced medium (with EC as the sole carbon source) at a 1% inoculum size and cultured under the same conditions for 24 h. Then, it was serially diluted (10 g / mL) with sterile physiological saline. -3 ~10 -8 The sample was spread on EC screening plates and incubated at 30°C for 36 h.

[0047] The isolated strains were morphologically identified on WL medium: the yeast formed cream-colored to light green colonies with a diameter of 2-3 mm on this medium, with a smooth, hemispherical surface. Yeast strains with EC degradation potential were screened using this characteristic and then inoculated onto YPD slant medium for preservation.

[0048] (2) Secondary screening The isolated strain was inoculated into a culture medium for activation, crude enzyme solution was prepared, and the enzyme activity of EC degrading enzyme was measured.

[0049] The selected EC-degrading yeast strains were inoculated into YPD liquid medium and cultured at 30℃ and 200 rpm until the bacterial concentration reached 1×10⁻⁶. 7 CFU / mL was then tested for tolerance and fermentation performance, and the superior strain K44, which combines high EC degradation ability, strong tolerance and excellent fermentation characteristics, was selected.

[0050] 3. Cell culture and enzyme solution preparation (1) Preparation of whole cells The bacterial strain was inoculated into seed culture medium and cultured for 1 day at 30°C on a shaker at 200 rpm / min. It was then inoculated into 50 mL of fermentation medium for further proliferation. The fermentation broth was centrifuged at low temperature and medium speed for 10 min, and the bacterial precipitate was retained. The precipitate was washed twice with 0.05 mol / L PBS buffer to obtain whole cells.

[0051] (2) Preparation of crude enzyme solution Dilute the cells to 10 mL with PBS buffer, then disrupt them with an ultrasonic cell disruptor for 20 min (300 W, 2 s on, 4 s off). After that, centrifuge at 8000 rpm / min for 10 min at 4 °C, and transfer the supernatant to a new EP tube to obtain the crude enzyme solution.

[0052] 4. Strain identification (1) Morphological observation The selected strain K44 was streaked onto YEPD solid medium, and the characteristics of single colonies, such as color, transparency, smoothness, moisture, and whether the edges were neat, were observed. Single colonies were also selected for morphological observation under a microscope.

[0053] The results are as follows Figure 2-3 As shown, the colonies are relatively regular and round, with a uniform milky white color and are opaque. The surface is rough with raised morphological features, and typical filamentous structures are visible in the edge area, while the surrounding growth area is relatively flat. Under a microscope, the cells are mainly long oval single-celled structures with typical budding reproduction characteristics. These morphological features are consistent with the biological characteristics of yeast.

[0054] (2) DNA extraction The selected strains were inoculated into YEPD liquid medium and cultured at 30°C for 1 day. 1 mL of the bacterial culture was centrifuged at room temperature for 1 min, the supernatant was discarded, and the bacterial cells were collected. Total yeast DNA was extracted using a yeast genomic DNA rapid extraction kit according to the manufacturer's instructions.

[0055] (3) PCR amplification The 26S rDNA gene of the strain was amplified by PCR using universal PCR primers (ITS1 and ITS4R). The PCR products were detected by 2% agarose gel electrophoresis.

[0056] (4) Sequencing identification Sequencing was commissioned to General Biotechnology (Anhui) Co., Ltd. Homology alignment analysis was performed using the Basic Local Alignment Search (BLAST) tool, and sequences with over 99% homology were selected for phylogenetic tree construction. Figure 4 As shown, strain K44 and LC289008 Pichia kudriavzevii IFM strain 51980 is from the same branch as KP674595. Pichia kudriavzevii strain d63a shares the same root, and this strain is related to... Pichia kudriavzevii The strain shared the highest homology with the genus, therefore it was named... Pichia kudriavzevii K44.

[0057] Example 2: Optimization of fermentation and enzyme extraction conditions for strain K44 I. Optimization of fermentation culture conditions for the strain 1. Determination of bacterial growth curves The activated seed culture was inoculated into the fermentation medium at a 1% (v / v) inoculation rate and cultured in a constant temperature shaker (30±0.5℃, 200±5 rpm). Samples were taken every 2 h after inoculation, and the absorbance (OD600) and EC degrading enzyme activity of the samples were immediately measured at 600 nm. OD600 was plotted on the x-axis. 600nm Plot a curve with enzyme activity on the ordinate to determine the optimal inoculation time, and then plot the growth curve, as shown below. Figure 5 As shown.

[0058] The results showed that the growth kinetics of this strain exhibited a typical S-shaped curve, which could be clearly divided into three typical growth phases: lag phase, logarithmic phase, and stationary phase. Specifically, during the lag phase (0-4 h), strain K44 exhibited typical physiological adaptation characteristics. During this phase, the growth rate remained low because cells needed to resynthesize key biomolecules such as ribosomes and enzymes to adapt to the new environment. Upon entering the logarithmic phase (4-14 h), the specific growth rate significantly increased to its maximum value. During this phase, the cells exhibited exponential proliferation, accompanied by a rapid accumulation of extracellular enzyme activity. Based on these physiological characteristics, it is recommended to select seed culture at 12 h (mid-logarithmic phase) for fermentation inoculation, as the cells are in their most metabolically active state at this time. During the stationary phase (14-26 h), the cell density reached its maximum, while enzyme activity remained at a plateau. During this phase, due to nutrient limitation and the accumulation of metabolic byproducts, the cells entered a period of secondary metabolic activity. Therefore, the optimal growth time for the seed culture medium was determined to be 12 h, and the optimal growth time for the fermentation medium was 24 h.

[0059] 2. The effect of carbon source on fermentation enzyme production capacity In the basal fermentation medium, glucose, sucrose, maltose, fructose, and lactose were used as single carbon sources (at the same concentration), while other components remained consistent. After inoculation, the strain was cultured at 30℃ and 200 r / min with shaking for 24 h. The fermentation broth was collected, and crude enzyme solution was prepared. Enzyme activity was measured to screen for the optimal carbon source. Next, the effect of different carbon source concentration gradients (10–40 g / L, gradient 5 g / L) on enzyme production by the strain was further investigated. With other culture conditions kept constant, changes in enzyme activity at different concentrations were measured to determine the optimal carbon source addition.

[0060] Experimental results showed that the type of carbon source significantly affected the growth and enzyme production characteristics of strain K44. Figure 6 Among the five carbon sources—glucose, maltose, sucrose, D-fructose, and α-lactose—the strain exhibited the best growth and highest EC-degrading enzyme activity under glucose culture conditions, with significantly higher biomass than other carbon sources. Maltose was the second best, while D-fructose and α-lactose showed the worst culture results. From a metabolic perspective, glucose, as a preferential carbon source for microorganisms, can be rapidly absorbed and participate in energy metabolism and substance synthesis, which is likely the main reason for its promotion of cell growth and enzyme synthesis. Furthermore, considering the advantages of glucose's low price (approximately 35% cheaper than maltose) and wide availability, and taking into account both biological performance and economic factors, glucose was ultimately selected as the optimal carbon source for the fermentation culture of strain K44.

[0061] Based on the determination that glucose is the optimal carbon source, the effects of different glucose concentrations (10-40 g / L) on the growth and metabolism of strain K44 were systematically investigated. Experimental data showed ( Figure 7 Glucose concentration has a significant regulatory effect on bacterial growth and enzyme synthesis. Within the concentration range of 10-25 g / L, bacterial biomass (OD)... 600 Both the activity of α and EC degradative enzymes showed a significant increasing trend with increasing carbon source concentration, reaching a maximum at 25 g / L glucose, at which point OD 600 The optimal glucose concentration was 1.857 g / L, and the enzyme activity was 1.678 U / mL. When the glucose concentration exceeded 25 g / L, the cell concentration showed a slight decreasing trend with increasing glucose concentration, while the enzyme production capacity decreased significantly with increasing glucose concentration. This may be due to the carbon metabolism repression effect induced by the high glucose environment. Based on the above indicators, the optimal glucose concentration was determined to be 25 g / L.

[0062] 3. The effect of nitrogen source on fermentation enzyme production capacity Peptone, ammonium chloride, ammonium nitrate, potassium nitrate, and ammonium sulfate were selected as single nitrogen sources (at the same concentration), while other culture medium components and conditions (30℃, 200 r / min, 24 h) remained consistent. After fermentation was terminated, crude enzyme solution was prepared, and the EC-degrading enzyme activity was determined spectrophotometrically. After determining the optimal nitrogen source, the effect of nitrogen source concentration gradients (5, 10, 15, 20, 25, 30, 35 g / L) on enzyme production by the strain was further investigated. Keeping other culture conditions constant, changes in enzyme activity at different nitrogen source concentrations were measured to determine the optimal nitrogen source addition amount.

[0063] Using 25 g / L glucose as the basic carbon source, and replacing peptone in the fermentation medium with 10 g / L ammonium citrate, potassium nitrate, ammonium chloride, and ammonium sulfate, the OD was measured after culturing the bacterial samples. 600 And the enzyme activity was calculated. The experimental results showed that ( Figure 8 When peptone is used as the nitrogen source, the cells exhibit optimal biomass accumulation (OD). 600 The strain exhibited the highest enzyme activity (1.75 U / mL). In contrast, cell growth and enzyme production levels were significantly reduced under other inorganic nitrogen source culture conditions. This phenomenon may be related to the fact that inorganic nitrogen sources have limited nutritional components and cannot provide sufficient nutrients and energy support for rapid cell proliferation and enzyme synthesis. Therefore, peptone was determined to be the optimal nitrogen source for this strain to achieve efficient growth and enzyme production.

[0064] To investigate the optimal concentration of peptone, different concentrations of peptone were added to a preliminarily optimized culture medium to study the effects of different peptone concentrations on bacterial growth and enzyme production capacity. An experimental peptone concentration gradient of 5–30 g / L was established, and the results are as follows: Figure 9As shown. Within the range of 5–15 g / L, with increasing peptone concentration, the bacterial biomass (OD) increased. 600 Both the growth curve and the activity of EC degradative enzymes showed an increasing trend; however, when the peptone concentration exceeded 15 g / L, both showed a decreasing trend, with the decrease in enzyme activity being particularly significant. This may be because excessive nitrogen source led to the accumulation of metabolic byproducts, inhibiting late-stage cell growth and interfering with the normal synthesis and secretion of exogenous proteins. Based on the combined growth curves and enzyme activity data, 15 g / L was finally determined to be the optimal concentration of peptone for addition.

[0065] 4. Effect of initial pH on fermentation enzyme production capacity Under optimized carbon and nitrogen source conditions, the effect of initial culture medium pH (4.0–6.5, gradient 0.5) on the enzyme production capacity of the strain was investigated. After adjusting the pH of the fermentation broth to the set values, the cultures were incubated at 30℃ and 200 r / min with shaking for 24 h. After fermentation, crude enzyme solutions were prepared and enzyme activity was measured. The effect of initial pH on enzyme activity was analyzed to determine the optimal initial pH conditions for enzyme production by the strain.

[0066] Experimental data such as Figure 10 As shown, when the initial pH is below 4.5, both bacterial biomass accumulation and enzyme activity expression are inhibited to some extent. The enzyme production capacity is strongest when the pH is equal to 4.5. After that, the enzyme production capacity gradually decreases as the pH increases. The biomass reaches its maximum at pH 5 and then gradually decreases as the pH increases.

[0067] 5. Effect of inoculum size on fermentation enzyme production capacity The effects of different inoculum amounts (1.0–8.0%, v / v, gradient 1.0%) on enzyme production by the strain were investigated. After culturing at 30℃ and 200 r / min for 24 h, crude enzyme solutions were prepared and their enzyme activities were measured. By comparing the changes in enzyme activity under different inoculum amounts, the optimal inoculum amount for enzyme production by the strain was determined.

[0068] Depend on Figure 11 As shown, when the inoculum size is in the range of 1%-4%, both cell biomass and EC degradative enzyme activity show a significant increasing trend with increasing inoculum size. This is mainly because appropriately increasing the inoculum size helps shorten the cell adaptation period and promotes rapid entry into the logarithmic growth phase. However, when the inoculum size exceeds 4%, although cell growth is not significantly inhibited, enzyme activity decreases rapidly. This phenomenon may be due to the excessively high cell density in the fermentation system caused by a high inoculum size, resulting in rapid consumption of nutrients and insufficient dissolved oxygen, which in turn causes the cells to enter the death phase prematurely, affecting the synthesis of secondary metabolites. Therefore, through comprehensive analysis of growth curves and enzyme activity data, 4% was determined to be the optimal inoculum size, at which point the cell growth state and enzyme production capacity reach the best balance.

[0069] 6. Effect of culture temperature on fermentation enzyme production capacity The effects of different culture temperatures (26-38℃, gradient 2℃) on enzyme production by the strain were investigated. After isothermal shaking culture at 200 r / min for 24 h, crude enzyme solution was prepared and enzyme activity was measured. The effect of temperature on enzyme yield was analyzed to determine the optimal culture temperature.

[0070] according to Figure 12 The experimental results showed that when the culture temperature was 30℃, both the bacterial biomass and enzyme activity reached their maximum values, indicating that this temperature was most favorable for the growth and metabolism of the strain. Therefore, 30℃ was determined to be the optimal fermentation temperature for this strain.

[0071] 7. Effect of shaking speed on fermentation enzyme production capacity The effect of different rotation speeds (140-240 r / min, gradient 20 r / min) on enzyme production was investigated. After shaking culture at the optimal temperature for 24 h, the enzyme activity of the crude enzyme solution was measured to determine the optimal culture speed.

[0072] like Figure 13 As shown, the enzyme production capacity of the bacteria reached its peak when the fermentation speed was set to 200 r / min, indicating that the dissolved oxygen level at this speed best met the enzyme production requirements of the strain. Therefore, 200 r / min was determined as the optimal fermentation speed for this strain.

[0073] Based on the results of previous single-factor experiments, this study selected four key factors as variables: carbon source concentration (A), nitrogen source concentration (B), initial pH value (C), and inoculum size (D). Three optimization levels were set for each factor (as shown in Table 1). An L9(34) orthogonal experimental design was used, with EC degrading enzyme activity as the evaluation index for multi-factor synergistic optimization. The significance level of each factor was statistically evaluated using analysis of variance (ANOVA), and the optimal combination of culture conditions was finally determined.

[0074] Table 1. Factor Level Table for Orthogonal Optimization Experiment

[0075] Table 2 Analysis of Orthogonal Experiment Results

[0076] Table 3. Analysis of Variance for Orthogonal Experiments

[0077] Note, F 0.05 (2,18)=3.555;F 0.01 (2,18)=6.013, where “*” and “**” indicate that the difference reached a significance level of 0.05 and 0.01, respectively.

[0078] The results of the orthogonal experiment and the analysis of variance are shown in Tables 2-3. The range analysis and analysis of variance results are consistent: the four factors affecting acid urease activity show the same order of importance: carbon source concentration (A) > initial pH (C) > nitrogen source concentration (B) > inoculum size (D). Further analysis of variance shows that all four factors have a highly significant effect on EC degrading enzyme activity (p<0.01). A direct analysis of the nine experimental results from the orthogonal experiment reveals that the optimal culture conditions are A2B1C2D3, with the following parameters: glucose 25 g / L, peptone 10 g / L, initial pH 5.0, and inoculum size 5%. Under these culture conditions, the EC degrading enzyme activity produced by the strain was measured to be 1.93 ± 0.28 U / mL.

[0079] II. Optimization of Enzyme Extraction Conditions 1. Effect of ultrasonic power on extraction efficiency The bacterial pellet was collected and washed three times with 0.1 M phosphate buffer (pH 7.4) to remove residual culture medium. The bacterial cells were then resuspended in the same buffer and subjected to cell disruption using gradient sonication (200, 250, 300, 350, 400, 450 W) under ice-water bath conditions. Each sonication session was performed in a 3-second cycle with a 2-second interval, for a total treatment time of 5 min. The disrupted sample was immediately centrifuged at 8000 × g for 10 min at 4°C to separate the supernatant from the cell debris. Enzyme activity in the supernatant was then measured, and the cell disruption rate was calculated to determine the optimal sonication power.

[0080] Experimental results show that ( Figure 14 The ultrasonic power range of 200-450 W significantly modulates the cell disruption effect. When the power increases from 200 W to 400 W, both cell disruption rate and enzyme activity show a linear increasing trend. This phenomenon can be explained by the physical mechanism of ultrasonic cavitation: as the power increases, the density of cavitation bubbles per unit volume of liquid increases, and the energy density of the medium correspondingly increases, thereby generating a stronger mechanical shearing effect, promoting the effective destruction of cell wall structure and the full release of intracellular enzymes. However, when the power exceeds 400 W, the cell disruption rate tends to stabilize, while enzyme activity shows a decreasing trend. This is because excessively high power can lead to a sudden increase in local temperature, a surge in pressure, and the formation of high-energy shock waves. These extreme conditions may cause conformational changes or structural damage to enzyme protein molecules, thus affecting their catalytic activity. Based on the above analysis, 400 W was determined to be the optimal ultrasonic power condition.

[0081] 2. Effect of ultrasonic time on extraction efficiency The effect of different sonication times (5-30 min, gradient 5 min) on the disruption effect was investigated. After high-speed centrifugation at 4℃ for 10 min, the enzyme activity in the supernatant was measured and the disruption rate was calculated to determine the optimal sonication time.

[0082] Note: Cell disruption rate C was calculated using colony counting.

[0083] C = (1 - number of colonies after disruption / number of colonies before disruption) × 100% like Figure 15 As shown, with the extension of ultrasonic treatment time, the cell disruption rate initially increased rapidly and then gradually leveled off, while enzyme activity showed a pattern of first increasing and then decreasing. Within the treatment time of 10-25 min, enzyme activity significantly increased with prolonged ultrasonic time. This was mainly because the continuous cavitation effectively improved the cell disruption rate, prompting the release of more urease from the bacterial cells. Enzyme activity reached its peak at 25 min, indicating the optimal cell disruption effect. However, beyond this point, although the cell disruption rate slightly increased, enzyme activity began to decline. This may be because the prolonged ultrasonic treatment caused the mechanical shear force and localized high temperature generated by the cavitation effect to continuously act on the enzyme molecules, altering their spatial structure and leading to inactivation. Therefore, the experiment determined 25 min to be the optimal ultrasonic treatment time.

[0084] Example 3: Strain K44 degrades ethyl carbamate during the fermentation of Shaoxing rice wine. 1. Preparation of Shaoxing wine The rice wine samples were prepared according to the following process, in triplicate (independent brewing process): 100 g of japonica rice was soaked in 150 mL of water at 37℃ for 48 h. The rice was then filtered, washed, steamed (108℃, 15 min), and cooled to room temperature. The control group (CK group) was prepared by adding 500 mL of water, 0.2% yeast, and 0.05% 10... 8 Fermentation was carried out using CFU / L active dry yeast. The experimental group was set as K44 group with 500 mL water, 0.2% yeast starter, and 0.05% 10 8 CFU / L active dry yeast, 0.05% of 10 8 Fermentation was carried out using CFU / L K44. The pre-fermentation period (5-8 days) was conducted at 25-30°C with daily stirring, followed by the post-fermentation period (15-25 days) at 18-20°C. Fermentation broth was collected in a clean bench on days 1, 3, 5, 10, 20, and 30 for physicochemical property determination. After collection, all samples were immediately stored at -80°C until analysis.

[0085] 2. Testing of physicochemical indicators The physicochemical indicators such as total sugar, ethanol, total acid, pH, and amino nitrogen were determined in accordance with the national standard for rice wine (GB / T 13662, 2018).

[0086] Table 4. Changes in physicochemical properties of the control group's rice wine during fermentation.

[0087] Note: Values ​​are expressed as mean plus standard error.

[0088] Table 5. Changes in physicochemical properties of rice wine during fermentation in the experimental group.

[0089] Note: Values ​​are expressed as mean plus standard error.

[0090] As shown in Table 4-5, the total sugar content in both the control and experimental groups decreased over time, indicating that the yeast began to utilize fermentable sugars for proliferation and growth during fermentation, reflecting the gradual conversion of sugars into ethanol. The experimental group showed a faster decrease in total sugar content in the early stages of fermentation, possibly due to its higher fermentation efficiency. Total acid content increased with fermentation, reaching 5.49 g / L in the control group and 6.36 g / L in the experimental group at the end of fermentation. The experimental group consistently had higher total acid content than the control group, indicating more significant acid accumulation, possibly related to differences in microbial metabolism. Ethanol content increased in both groups over time, but was slightly higher in the control group at the end of fermentation, possibly due to the control group's stronger ethanol production capacity after fermentation. The pH in both groups increased with fermentation time and then stabilized. The pH in the control group (3.81 g / L) was slightly higher than that in the experimental group (3.74 g / L), possibly related to differences in acidity. After 10 days of fermentation, the total acid, ethanol concentration, and pH value in both the control and experimental groups showed no significant fluctuations, maintaining a relatively stable state. It is noteworthy that the amino acid nitrogen content of both groups of samples showed an increasing trend, which may be due to the release of protease A and the peptides and amino acids catalyzed by it during yeast cell autolysis. The amino acid nitrogen content of the experimental group was generally higher than that of the control group, suggesting that the protein decomposition in the experimental group was more complete. According to the test results, the five key physicochemical indicators of the samples (total sugar 15.1~40.0 g / L, acidity 3.0~7.5 g / L, alcohol content ≥8.0% vol, pH 3.5~4.6, amino acid nitrogen ≥0.3 g / L) all met the limits of the national standard for rice wine (GB / T 2-1366, 2018), proving that the fermentation process was well controlled and the obtained samples can be used for further in-depth research.

[0091] 3. Determination of urea content and ethyl carbamate content (1) The urea content was determined by the diacetyl monooxime colorimetric method. The specific method is as follows: two reagents were prepared in advance. Reagent I consisted of 50 mL of phosphoric acid (85%, v / v), 120 mL of sulfuric acid (98%, v / v), 0.05 g of FeCl3 and 330 mL of deionized water. Reagent II was an aqueous solution containing 0.5 mg / mL diacetyl monooxime and 0.1 mg / mL aminothiourea.

[0092] During the determination, reagent I and reagent II were mixed at a ratio of 2:1 (v / v). 5 mL of the mixture was added to a test tube containing 1 mL of the sample to be tested. The mixture was heated in a boiling water bath for 15 min and then immediately cooled to room temperature with ice water. Urea and diacetyl monooxime formed a red complex under acidic conditions and in the presence of aminothiourea. The absorbance was measured at a wavelength of 526 nm.

[0093] (2) Preparation of ethyl carbamate standard stock solution: Dissolve the standard in 30% methanol solution (v / v) to prepare a standard stock solution with a concentration of 18 μg / mL, and store at 4℃ for later use.

[0094] Preparation of working standard solutions: Dilute the standard stock solution sequentially with 30% methanol solution to four concentration gradients: 4.5 μg / mL, 1.8 μg / mL, 0.9 μg / mL and 0.18 μg / mL.

[0095] Sample preparation and derivatization: All samples used for ethyl carbamate (EC) detection were prepared using the methods described above (five gradients were set up for each type during the rice wine fermentation process in the validation experiment). Due to the low EC concentration in the original samples, the crude extract needed to be concentrated. The specific procedure was as follows: The sample was vacuum concentrated to a suitable volume (concentration factor 10-fold) at 35℃ using a rotary evaporator. After concentration, a derivatization reaction was performed: 0.02 M 9-anthraphenol (200 μL) and 100 μL 1.5 mol / L hydrochloric acid were added to 1 mL of concentrated sample or standard EC solution. The mixture was vortexed for several seconds and then allowed to stand for 30 minutes to ensure complete reaction. Finally, the sample was injected into the chromatographic system for analysis.

[0096] Chromatographic conditions: The high-performance liquid chromatography-fluorescence detection (HPLC-FLD) system used in this study consisted of the following components: two Waters 510 pumps (Waters Corporation, Milford, USA), a Rheodyne 108 injector (Rheodyne Corporation, Cortati, USA), and a Waters 2475 fluorescence detector. A reversed-phase Symmetry C18 column (250 mm × 4.6 mm, 4 μm, Waters Corporation) was used, maintained at 35℃, with an injection volume of 20 μL. The excitation and emission wavelengths of the detector were set to 233 nm and 600 nm, respectively. The mobile phase was a methanol-water system; specific gradient elution conditions are shown in Table 6. Quantitative analysis was performed using the external standard method.

[0097] Table 6 Chromatographic conditions for EC determination in Shaoxing wine

[0098] Changes and comparisons of urea and EC content: Changes in urea and EC content in the experimental and control groups during fermentation are as follows: Figure 16-17 As shown.

[0099] In the control group (CK), urea content gradually increased over time, which may be related to the accumulation of microbial metabolites in the later stages of fermentation. EC content generally showed a trend of first increasing and then decreasing. In the experimental group (T), urea content was significantly lower than that in the control group, indicating that K-44 inoculation reduced urea production in the experimental group. EC content in the experimental group was significantly lower than that in the control group, indicating that K-44 inoculation effectively inhibited EC production in the experimental group.

[0100] Table 7. Effects of K44 inoculation on urea and EC content in rice wine

[0101] By measuring the urea and EC content in the control and experimental groups at the end of fermentation, the experimental group showed a 14.2% decrease in urea content and a 37.25% decrease in EC content compared to the control group. The experiment showed that inoculation with strain K44 had a significant effect on inhibiting EC formation.

[0102] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A carbamate-degrading bacterium, characterized in that: The degrading bacteria is *Pichia kudrica*. (Pichia kudriavzevii K44, deposited at the China Center for Type Culture Collection on April 25, 2025, with accession number CCTCC NO: M 2025887.

2. The fermentation culture method for ethyl carbamate-degrading bacteria according to claim 1, characterized in that: The carbon source was glucose at a concentration of 25 g / L; the nitrogen source was peptone at a concentration of 10 g / L; the initial pH was 5.0; and the inoculum size was 5%.

3. The enzyme extraction method for ethyl carbamate-degrading bacteria according to claim 1, characterized in that: Ultrasonic treatment was performed intermittently at 400 W power for 25 minutes, with the intermittent treatment consisting of 2 seconds of operation followed by a 4-second interval.

4. The application of the ethyl carbamate-degrading bacteria of claim 1 in the degradation of ethyl carbamate.

5. The application of the ethyl carbamate-degrading bacteria according to claim 4 in the degradation of ethyl carbamate, characterized in that: It is used to degrade ethyl carbamate in the rice wine fermentation system, wherein the EC degradation rate is not less than 35%.

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