Ethyl carbamate-degrading bacteria and application thereof

Abstract

The application relates to the field of microbial technology, in particular to an ethyl carbamate degrading bacterium and application thereof, and the ethyl carbamate degrading bacterium is Pichia kudriavzevii (Pichia kudriavzevii ) K44, which is preserved in the China Center for Type Culture Collection, and the preservation date is April 25, 2025, and the biological preservation number is CCTCC NO: M 2025887. The ethyl carbamate degrading bacterium can significantly degrade ethyl carbamate in a yellow rice fermentation system, and provides a scientific basis for safe production and quality improvement of traditional fermented foods.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to an ethyl carbamate degrading bacterium screened from yellow rice wine and application thereof in degrading ethyl carbamate. BACKGROUND

[0002] Ethyl carbamate (EC) is a natural by-product of fermentation, which is widely present in various fermented foods and beverages, including distilled spirits, wine, yellow rice wine, yogurt, cheese and soy sauce. It is mainly metabolized by cytochrome P450 enzyme system to generate intermediate products such as vinyl-ethyl carbamate and N-hydroxyethyl carbamate, which have carcinogenic activity. In 2007, the International Agency for Research on Cancer (IARC) upgraded it to class 2A, which is very likely to be carcinogenic. Each country has implemented strict control on the content of EC in food, such as the Canadian standard of no more than 100 μg / L of EC in alcoholic beverages, and the Japanese import limit of 200 μg / L of EC in yellow rice wine.

[0003] The formation of EC in alcoholic beverages involves a variety of precursor substances, mainly including urea, citrulline, cyanide, carbamoyl phosphate and diethyl pyrocarbonate. In the yellow rice wine brewing system, the mechanism of EC generation has particularity, which is mainly derived from the non-enzymatic chemical reaction of urea and citrulline with ethanol. Among them, the urea pathway is the most important: the yeast produces urea by decomposing arginine through arginase, and accumulates under nitrogen metabolic repression and reacts with ethanol to generate EC; the citrulline pathway is produced by lactic acid bacteria metabolism, which intensifies the formation of EC during aging. The accumulation of these precursor substances is closely related to microbial metabolism. The open fermentation and long-term storage characteristics of yellow rice wine make the problem of EC exceeding the standard particularly prominent, which seriously restricts its international market competitiveness and consumer safety.

[0004] In the prior art, the control strategies of EC mainly include physical methods (such as high temperature treatment), chemical methods (such as adding inhibitors) and biological methods (such as strain improvement). For example, breeding low-urea-producing yeast (such as defective Saccharomyces cerevisiae) can block urea synthesis, but these strains often have poor fermentation performance, resulting in impaired flavor of yellow rice wine; adding acid urease can degrade urea precursors, but the enzyme activity is usually less than 1.0 U / mL, and the stability is poor in the environment of yellow rice wine acidity (pH 3.5-4.5) and high ethanol (15%), which cannot directly degrade the formed EC, and the actual degradation rate is less than 20%; other methods such as metabolic engineering modification of gene expression have made progress, but there are problems of gene stability and industrialization difficulty. These limitations result in that the existing strategies have unsatisfactory application effect in the complex fermentation environment of yellow rice wine, and cannot effectively control the content of EC below the international standard.

[0005] Therefore, the field urgently needs an efficient and highly resistant EC-degrading strain that can directly degrade EC and achieve green control through optimized enzyme production conditions, especially for yellow rice wine production, to improve product safety and quality. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a urethane-degrading strain and its application. The strain is obtained by screening from yellow rice wine fermentation grains and has excellent EC degradation capacity and environmental tolerance (tolerance to 15% ethanol, pH 3.0 and 2.5 g / L EC). After optimization, the enzyme activity reaches 1.93 U / mL, and the EC degradation rate in yellow rice wine reaches 37.25%.

[0007] The technical solution of the present application is as follows:

[0008] The present application discloses a urethane-degrading strain, which is Pichia kudriavzevii (P. Pichia kudriavzevii ) K44, preserved in the China Center for Type Culture Collection on April 25, 2025, with the preservation number CCTCC NO: M 2025887.

[0009] The fermentation culture method of the degrading strain includes using glucose as the carbon source with a concentration of 25 g / L, using peptone as the nitrogen source with a concentration of 10 g / L, setting the initial pH to 5.0, setting the inoculation amount to 5%, setting the culture temperature to 30℃, setting the shaking speed to 200 r / min, and setting the culture time to 24 h.

[0010] The enzyme extraction method of the degrading strain adopts ultrasonic crushing method, and the intermittent treatment is performed for 25 min under the condition of a power of 400 W, wherein the intermittent mode is working for 2 s and intervaling for 4 s.

[0011] In addition, the present application also includes the application of the degrading strain in degrading urethane, especially in degrading urethane in yellow rice wine fermentation system, wherein the EC degradation rate is not less than 35%.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] The strain K44 is uniquely obtained by screening from yellow rice wine fermentation grains, and is identified as P. Pichia Pichia kudriavzevii, with 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 better than existing strains (<20%). The unexpected results were achieved through gradient enrichment and orthogonal experiment to solve the problem of tolerance in yellow rice environment. The application in yellow rice can reduce urea by 14.2%, and EC content to 67.39 μg / L (lower than the international standard of 200 μg / L), without affecting the flavor, thereby solving the long-term problem of poor enzyme stability in existing technology, and having potential commercial value, such as improving the competitiveness of yellow rice exports.

[0014] Biological preservation

[0015] Kodama Pichia pastoris (Pichia pastoris) Pichia kudriavzevii ) K44, preserved in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, Hubei, China, 299, Bayi Road, Wuchang District, Wuhan, Hubei, China, preservation date: April 25, 2025, preservation number: CCTCC NO: M 2025887. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the standard curve of ammonium ion concentration.

[0017] Figure 2 is the colony photo of strain K44.

[0018] Figure 3 is the morphological characteristics of K44 single colony under microscope.

[0019] Figure 4 is the phylogenetic tree of strain K44 based on ITS sequence.

[0020] Figure 5 is the growth curve of strain K44.

[0021] Figure 6 is the effect of different types of carbon sources on the growth and enzyme production of K44.

[0022] Figure 7 is the effect of different glucose concentrations on the growth and enzyme production of K44.

[0023] Figure 8 is the effect of different nitrogen source types on the growth and enzyme production of K44.

[0024] Figure 9 is the effect of peptone concentration on the growth and enzyme production of K44.

[0025] Figure 10 is the effect of initial pH value on the growth and enzyme production of K44.

[0026] Figure 11 Effect of inoculum on K44 growth and enzyme production.

[0027] Figure 12 Effect of incubation temperature on K44 growth and enzyme production.

[0028] Figure 13 Effect of shaker speed on K44 growth and enzyme production.

[0029] Figure 14 Effect of ultrasonic power on enzyme activity.

[0030] Figure 15 Effect of ultrasonic time on enzyme activity.

[0031] Figure 16 Changes of urea content in experimental and control groups during fermentation.

[0032] Figure 17 Changes of EC content in experimental and control groups during fermentation. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0034] Example 1: Screening of ethyl carbamate-degrading bacteria

[0035] I. Experimental materials

[0036] 1. Sample source

[0037] The yellow rice wine fermentation dregs were stored in a refrigerator at -20°C.

[0038] 2. Culture medium

[0039] (1) Primary 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, ddH2O 2 constant volume 250 mL, pH=5.0.

[0040] (2) Flat 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 250 mL, pH=5.0.

[0041] (3) YEPD solid medium: 1% yeast extract powder, 2% peptone, 2% glucose, 2% agar powder.

[0042] (4) EC-degrading yeast screening medium N (EC as the only 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 500 mL, pH=5.0.

[0043] (5) EC-degrading yeast screening medium C (EC as the only 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 500 mL, pH=5.0.

[0044] (6) Degradation medium: peptone 0.5 g, glucose 1 g, potassium dihydrogen phosphate (KH2PO4) 1.25 g, sodium chloride (NaCl) 1 g, EC 1.25 g, bromocresol purple 1 mg, ddH2O2 500 mL, pH=5.0.

[0045] II. Experimental content

[0046] 1. Method for determining the activity of EC-degrading enzyme

[0047] (1) Enzyme activity: the amount of enzyme that decomposes EC to produce 1 μmol of ammonia per minute under the conditions of 35°C and pH=4.5 is one enzyme activity unit (U).

[0048] (2) Substrate: use the mother liquor 0.05 mol / L citric acid buffer to prepare a 3% EC solution with pH=4.5.

[0049] Color reagent I: dissolve 30 g of phenol and 1.25 g of nitrosoferricyanide sodium (SNP) in distilled water, stir until uniform, and then make up to 500 mL.

[0050] Color reagent II: 26.25 g NaOH was added into 15 mL sodium hypochlorite, mixed and diluted with ddH2O2 to 500 mL.

[0051] Termination reagent: 10% trichloroacetic acid solution (w / v).

[0052] (3) 0.2 mL enzyme solution was added into two 25 mL cuvettes, one of which was boiled in water bath for 15 min as control; 0.8 mL substrate solution was added into each cuvette and mixed, and the reaction was carried out at 37°C for 15 min; 1 mL termination reagent was added to terminate the reaction, followed by 1 mL color reagent I and II, mixed and reacted at 37°C for 20 min; the reaction was terminated by adding ddH2O to 25 mL, and the absorbance value at 625 nm was measured, and the enzyme activity was calculated according to the standard curve of ammonium ion.

[0053] (4) Gradient concentrations of NH4 + were prepared using NH4Cl, and the absorbance value at 625 nm was measured, and the standard curve was drawn with NH4 + concentration and OD 625nm absorbance value as the horizontal and vertical coordinates, respectively, as shown in Figure 1 , and the absorbance value and concentration had good linear correlation (R 2 = 0.999), and the enzyme activity calculation formula was: enzyme activity = OD 625nm × n (enzyme solution dilution multiple) × 1 / k (linear standard curve slope) × 1 / 20 (enzyme reaction time, min).

[0054] 2. Screening of EC-degrading enzyme-producing bacteria

[0055] (1) Preliminary screening

[0056] The EC-degrading bacteria were screened by gradient enrichment culture combined with selective plate separation, and the specific operation was as follows: under sterile conditions, 10 g sample was inoculated into selective liquid medium (EC as the only nitrogen source) and cultured at 30°C on a shaking table for 24 h. 1 mL of the culture was transferred to fresh medium for secondary enrichment culture (conditions were the same as above). Then, the enriched bacterial solution was inoculated into the reinforced medium (EC as the only carbon source) at 1% inoculation amount, and cultured at the same conditions for 24 h, and then gradient diluted (10 -3 ~10 -8 ) with sterile saline, and plated on EC screening plates, and incubated at 30°C for 36 h.

[0057] The WL medium morphological identification was carried out on the isolated strains: the yeast formed cream to light green colonies with a diameter of 2-3 mm on the medium, and the surface was smooth and semispherical. The yeast strains with EC degradation potential were screened by the characteristics, and inoculated into YPD slant medium for preservation.

[0058] (2) Rescreening

[0059] The isolated strains were inoculated into the medium for activation, and the crude enzyme solution was prepared, and the enzyme activity of EC degrading enzyme was determined.

[0060] The EC degrading yeast strains screened were inoculated into YPD liquid medium, and cultured at 30°C, 200 rpm to 1×10 7 CFU / mL, and then the tolerance and fermentation performance were detected, and the excellent strain K44 with high EC degradation capacity, strong tolerance and excellent fermentation characteristics was screened.

[0061] 3. Cell culture and enzyme solution preparation

[0062] (1) Preparation of whole cells

[0063] The strain was inoculated into the seed culture medium and placed in a 30°C shaker at a speed of 200 rpm / min for 1d, and then inoculated into 50 mL fermentation medium for further growth. The fermentation broth was centrifuged at low speed and low temperature for 10 min, the bacterial liquid precipitate was washed with 0.05 mol / L PBS buffer for 2 times, and finally the whole cells were obtained.

[0064] (2) Preparation of crude enzyme solution

[0065] The cells were diluted with PBS buffer to 10 mL, and then broken by ultrasonic cell disrupter for 20 min (300 W, 2s on, 4s off), and then centrifuged at 8000 rpm / min for 10 min at 4°C, and the supernatant was transferred to a new EP tube, which was the crude enzyme solution.

[0066] 4. Strain identification

[0067] (1) Morphological observation

[0068] The selected strain K44 was streaked on YEPD solid medium, and the color, transparency, smoothness, moisture and edge of single colony were observed, and the morphological characteristics were observed under a microscope.

[0069] The results are as follows: Figures 2-3As 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.

[0070] (2) DNA extraction

[0071] 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.

[0072] (3) PCR amplification

[0073] 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.

[0074] (4) Sequencing identification

[0075] 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 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.

[0076] Example 2: Optimization of fermentation and enzyme extraction conditions for strain K44

[0077] I. Optimization of fermentation culture conditions for the strain

[0078] 1. Determination of bacterial growth curves

[0079] 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. 600nmand enzyme activity as the ordinate to draw the curve, determine the best inoculation time, and draw the growth curve, as shown in Figure 5

[0080] The results show that the growth kinetics of the strain presents typical S-shaped curve characteristics, which can be clearly divided into three typical growth stages: lag phase, logarithmic phase and stationary phase. Specifically, in the lag growth period of 0-4 h, strain K44 showed typical physiological adaptation characteristics. In this stage, the growth rate remained at a low level because the cells needed to resynthesize ribosomes, enzymes and other key biological macromolecules to adapt to the new environment. When entering the logarithmic growth phase of 4-14 h, the specific growth rate significantly increased to the maximum value, and the cells showed exponential proliferation, accompanied by rapid accumulation of extracellular enzyme activity. Based on this physiological characteristic, it is recommended to select 12 h (mid-log phase) of seed liquid for fermentation inoculation, at which time the cells are in the most vigorous metabolic physiological state. In the stationary phase of 14-26 h, the cell density reached the maximum value, and the enzyme activity remained at a plateau. In this stage, due to nutrient limitation and accumulation of metabolic byproducts, cells entered the active period of secondary metabolism. Therefore, the optimal growth time of the seed medium is determined to be 12 h, and the optimal growth time of the fermentation medium is determined to be 24 h.

[0081] 2. Effect of carbon source on enzyme production capacity

[0082] In the basic fermentation medium, glucose, sucrose, maltose, fructose and lactose were used as single carbon sources (same concentration), and the other components remained the same. After inoculation, the strain was cultured at 30°C, 200 r / min for 24 h, the fermentation broth was collected and the crude enzyme solution was prepared, and the enzyme activity was determined to select the optimal carbon source. Next, further investigate the effect of different carbon source concentration gradient (10-40 g / L, gradient 5 g / L) on enzyme production of the strain. Keep other culture conditions unchanged, measure the change of enzyme activity under different concentrations, to determine the optimal carbon source addition amount.

[0083] The experimental results show that the type of carbon source significantly affects the growth and enzyme production characteristics of K44 strain Figure 6 ​). Among the five carbon sources, glucose, maltose, sucrose, D-fructose and α-lactose, the strain showed the best growth state and the highest EC-degrading enzyme activity under glucose culture conditions, and its biomass was significantly better than that of other carbon sources. Maltose was second, while D-fructose and α-lactose had the worst culture effect. From the metabolic mechanism, glucose as the preferred carbon source for microorganisms can be quickly absorbed and involved in energy metabolism and material synthesis, which may be the main reason for promoting the growth of bacterial cells and enzyme synthesis. In addition, considering the advantages of low price (about 35% cheaper than maltose) and wide sources, the best carbon source for K44 strain fermentation culture is ultimately selected as glucose, considering the biological performance and economic factors.

[0084] Based on the determination of glucose as the best carbon source, the effects of different glucose concentrations (10-40 g / L) on the growth and metabolism of strain K44 were investigated. Experimental data showed (Fig. 2) Figure 7 ), glucose concentration had a significant regulatory effect on the growth of bacterial cells and enzyme synthesis. Within the concentration range of 10-25 g / L, the biomass (OD 600 ) and EC-degrading enzyme activity of the strain increased significantly with the increase of carbon source concentration, and reached the maximum at 25 g / L glucose, at which time the OD 600 was 1.857 and the enzyme activity was 1.678 U / mL. When the glucose concentration was higher than 25 g / L, the bacterial concentration showed a slight downward trend with the increase of glucose concentration, while the enzyme production capacity decreased significantly with the increase of glucose concentration, which may be due to the carbon metabolic repression effect caused by high sugar environment. Based on the above indicators, the optimal glucose concentration was determined to be 25 g / L.

[0085] 3. Effect of nitrogen source on enzyme production capacity

[0086] Proteose peptone, ammonium chloride, ammonium nitrate, potassium nitrate and ammonium sulfate were selected as single nitrogen source (same concentration), and the other medium components and culture conditions (30℃, 200 r / min, 24 h) were kept unchanged. After fermentation, the crude enzyme solution was prepared and the EC-degrading enzyme activity was determined by spectrophotometry. After determining the optimal nitrogen source, the effect of nitrogen source concentration gradient (5, 10, 15, 20, 25, 30, 35 g / L) on enzyme production of the strain was further explored. The enzyme activity under different nitrogen source concentrations was determined to determine the optimal nitrogen source addition amount.

[0087] Using 25 g / L glucose as the basic carbon source, 10 g / L of ammonium citrate, potassium nitrate, ammonium chloride and ammonium sulfate were used to replace proteose peptone in the fermentation medium, and the OD 600 of the cultured bacterial sample was measured and the enzyme activity was calculated. The experimental results showed (Fig. 3) Figure 8 ) that when proteose peptone was used as the nitrogen source, the bacterial cells showed the best biomass accumulation (OD600 The cell growth and enzyme activity were significantly lower than those in the other inorganic nitrogen sources. This phenomenon might be related to the single nutritional composition of inorganic nitrogen sources, which could not provide sufficient nutrients and energy for the rapid proliferation of cells and enzyme synthesis. Thus, peptone was determined to be the optimal nitrogen source for the strain to achieve efficient growth and enzyme production.

[0088] To explore the optimal concentration of peptone, different concentrations of peptone were added to the preliminary optimized medium to study the effects of different concentrations of peptone on cell growth and enzyme production. The experimental concentration gradient of peptone was set to 5-30 g / L, and the results are shown in Figure 9 Within the range of 5-15 g / L, the cell biomass (OD 600 ) and EC-degrading enzyme activity both showed an upward trend with the increase of peptone concentration; however, when the concentration of peptone exceeded 15 g / L, both showed a downward trend, and the decrease in enzyme activity was particularly significant. This might be because the accumulation of metabolic by-products caused by excessive nitrogen sources inhibited the late growth of cells and interfered with the normal synthesis and secretion of exogenous proteins. Based on the growth curve and enzyme activity data, 15 g / L was determined as the optimal concentration of peptone.

[0089] 4. Effect of initial pH on enzyme production

[0090] Under the optimized conditions of carbon and nitrogen sources, the effect of initial pH (4.0-6.5, gradient 0.5) of the medium on the enzyme production of the strain was explored. After adjusting the pH of the fermentation broth to the set value, the fermentation was carried out at 30°C and 200 r / min for 24 h. After the fermentation was completed, the crude enzyme solution was prepared and the enzyme activity was measured to analyze the effect of initial pH on enzyme activity and determine the optimal initial pH condition for enzyme production of the strain.

[0091] The experimental data are shown in Figure 10 When the initial pH was lower than 4.5, the cell biomass accumulation and enzyme activity expression were inhibited to some extent. When the pH value was equal to 4.5, the enzyme production was the strongest, and then it gradually decreased with the increase of pH value. The biomass reached the maximum at pH 5 and then gradually decreased with the increase of pH value.

[0092] 5. Effect of inoculum size on enzyme production

[0093] The effects of different inoculum sizes (1.0-8.0%, v / v, gradient 1.0%) on enzyme production of the strain were investigated. After incubation at 30°C and 200 r / min for 24 h, the crude enzyme solution was prepared and the enzyme activity was measured to determine the optimal inoculum size for enzyme production of the strain by comparing the changes in enzyme activity under different inoculum sizes.

[0094] As shown in Figure 11 , when the inoculum size was in the range of 1%-4%, both the biomass and the enzyme activity showed a significant increasing trend with the increase of inoculum size, which was mainly due to the fact that the appropriate increase of inoculum size was beneficial to shorten the adaptation period of the bacteria and promote the rapid entry into the logarithmic growth phase. However, when the inoculum size exceeded 4%, although the growth of the bacteria was not significantly inhibited, the enzyme activity showed a rapid decline. This phenomenon may be due to the fact that high inoculum size led to high cell density in the fermentation system, resulting in rapid consumption of nutrients and insufficient dissolved oxygen, which in turn triggered the bacteria to enter the decline phase prematurely, affecting the synthesis of secondary metabolites. Therefore, by comprehensively analyzing the growth curve and enzyme activity data, 4% was determined as the optimal inoculum size, at which the growth state of the bacteria and the enzyme production capacity reached the best balance.

[0095] 6. Effect of culture temperature on enzyme production capacity

[0096] The effect of different culture temperatures (26-38℃, gradient 2℃) on enzyme production was investigated. After constant temperature shaking culture at 200 r / min for 24 h, the crude enzyme solution was prepared and the enzyme activity was measured to analyze the effect of temperature on enzyme yield and determine the optimal culture temperature.

[0097] According to the experimental results of Figure 12 , when the culture temperature was 30℃, the biomass and enzyme activity of the bacteria reached the maximum value, indicating that this temperature was most conducive to the growth and metabolism of the strain. Therefore, 30℃ was determined as the optimal temperature for fermentation of the strain.

[0098] 7. Effect of shaking bed speed on enzyme production capacity

[0099] 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 rotation speed.

[0100] As shown in Figure 13 , when the fermentation rotation speed was set to 200 r / min, the enzyme production capacity of the bacteria reached the peak value, indicating that the dissolved oxygen content under this rotation speed condition was most suitable for the enzyme production needs of the strain. Therefore, 200 r / min was determined as the optimal rotation speed for fermentation culture of the strain.

[0101] Based on the results of single factor experiment, four key factors were selected as the investigation variables, including carbon source concentration (A), nitrogen source concentration (B), initial pH (C) and inoculum size (D). Three optimization levels were set for each factor (as shown in Table 1), and L9(34) orthogonal experimental design method was used to optimize the four factors in coordination, with EC-degrading enzyme activity as the evaluation index. The significance level of each factor was statistically evaluated by ANOVA, and the optimal culture condition combination was finally determined.

[0102] Table 1 Factor level table of orthogonal optimization experiment

[0103]

[0104] Table 2 Analysis table of orthogonal experiment results

[0105]

[0106] Table 3 Variance analysis table of orthogonal experiment

[0107]

[0108] Note, F 0.05 (2,18)=3.555;F 0.01 (2,18)=6.013, “*” and “**” represent the difference reaching the significant level of 0.05 and 0.01, respectively.

[0109] The orthogonal experiment results and variance analysis results are shown in Tables 2-3, respectively. The results of range analysis and variance analysis are consistent, and the influence degree of the four investigation factors on acid urease activity presents the same primary and secondary order: carbon source concentration (A) > initial pH (C) > nitrogen source concentration (B) > inoculum size (D). Variance analysis further shows that the influence of the four factors on EC-degrading enzyme activity reaches a very significant level (p<0.01). From the intuitive analysis of the results of the 9 groups of orthogonal experiments, it is found that the optimal culture condition combination is A2B1C2D3, with specific parameters as follows: glucose 25 g / L, protein peptone 10 g / L, initial pH 5.0, and inoculum size 5%. Verification experiment was carried out under this culture condition, and the EC-degrading enzyme activity produced by the strain was measured as 1.93±0.28 U / mL.

[0110] II. Optimization of enzyme extraction conditions

[0111] 1. Effect of ultrasonic power on extraction efficiency

[0112] The bacterial cell pellet was collected and washed three times with 0.1 M phosphate buffer (pH 7.4) to remove the residual culture medium. Then the bacterial cells were resuspended in the same buffer and subjected to cell disruption under ice-water bath condition using gradient ultrasonic power (200, 250, 300, 350, 400, 450 W) with a cycle mode of 3 s working / 2 s interval for 5 min. The disrupted sample was immediately centrifuged at 8000 x g for 10 min at 4 °C to separate the supernatant from the cell debris. The enzyme activity of the supernatant was then determined and the cell disruption rate was calculated to determine the optimal ultrasonic power.

[0113] The experimental results show that Figure 14 ), the ultrasonic power in the range of 200-450 W has a significant regulatory effect on cell disruption. When the power increases from 200 W to 400 W, the cell disruption rate and enzyme activity show a linear growth trend. This phenomenon can be explained from the physical mechanism of ultrasonic cavitation effect: as the power increases, the generation density of cavitation bubbles in unit volume of liquid increases, and the medium energy density also increases, thereby producing stronger mechanical shear effect, promoting effective destruction of cell wall structure and full release of intracellular enzymes. However, when the power exceeds 400 W, the cell disruption rate tends to be stable, while the enzyme activity shows a downward trend. This is because too high power will cause a sharp rise in local temperature, a dramatic increase in pressure and the formation of high-energy shock waves, which may cause conformational changes or structural damage to enzyme protein molecules, thereby affecting their catalytic activity. Based on the above analysis, 400 W is determined as the optimal ultrasonic power condition.

[0114] 2. Effect of ultrasonic time on extraction efficiency

[0115] The effect of different ultrasonic time (5-30 min, gradient 5 min) on disruption effect was investigated. After centrifugation at 4 °C for 10 min, the enzyme activity of the supernatant was determined and the disruption rate was calculated to determine the optimal ultrasonic time.

[0116] Note: The cell disruption rate C was calculated by colony counting method.

[0117] C = (1 - number of colonies after disruption / number of colonies before disruption) x 100%

[0118] As Figure 15As shown, with the extension of ultrasonic treatment time, the cell disruption rate showed a trend of rapid rise first and then gradually flat, while the enzyme activity showed a change rule of first increase and then decrease. Within the treatment time of 10-25 min, the enzyme activity increased significantly with the extension of ultrasonic time, which was mainly because the continuous cavitation effect effectively improved the cell disruption rate, and prompted more urease to be released from the bacterial cells. When the ultrasonic time reached 25 min, the enzyme activity reached the peak, and the cell disruption effect was best at this time. However, beyond this time point, although the cell disruption rate was slightly improved, the enzyme activity began to decrease, which might be due to the long ultrasonic treatment which caused the mechanical shear force and local high temperature produced by cavitation effect to continuously act on the enzyme molecules, so that the spatial structure of the enzyme molecules changed and became inactivated. Therefore, the experiment determined that 25 min was the best ultrasonic treatment time.

[0119] Example 3 Ethyl carbamate degradation by strain K44 during the fermentation process of yellow rice wine

[0120] 1. Preparation of yellow rice wine

[0121] The yellow rice wine samples were prepared according to the following process, and 100 g of japonica rice was soaked in 150 mL of water, soaked at 37°C for 48 h, then filtered, washed, cooked (108°C, 15 min), cooled to room temperature, and then fermented with 500 mL of water, 0.2% of koji, 0.05% of 10 8 cfu / L of active dry yeast for the control group (CK group), and 500 mL of water, 0.2% of koji, 0.05% of 10 8 cfu / L of active dry yeast, and 500 mL of water, 0.2% of koji, 0.05% of 10 8 cfu / L of K44 for the experimental group. The pre-fermentation period was 5-8 days at 25-30°C, and the post-fermentation period was 15-25 days at 18-20°C, with stirring once a day. The fermentation liquid was collected in a clean bench on the 1st, 3rd, 5th, 10th, 20th, and 30th day for determination of physicochemical properties. After collection, the samples were quickly stored at -80°C until analysis.

[0122] 2. Detection of physicochemical indicators

[0123] The total sugar, ethanol, total acid, pH, and amino nitrogen were determined according to the national standard for yellow rice wine (GB / T 13662, 2018).

[0124] Table 4 Changes in physicochemical properties of yellow rice wine in the control group during fermentation

[0125]

[0126] Note: The values are represented by the mean + standard error.

[0127] Table 5 Changes in physicochemical properties of the experimental group during the fermentation process of yellow rice wine

[0128]

[0129] Note: Values are expressed as mean + standard error.

[0130] As shown in Tables 4-5, the total sugar of both the control group and the experimental group decreased over time, indicating that the yeast bacteria during the fermentation process began to proliferate and grow by utilizing fermentable sugars, reflecting the gradual conversion of sugar into ethanol during the fermentation process; the total sugar of the experimental group decreased more rapidly in the early fermentation stage, possibly because the fermentation efficiency of the experimental group was higher. The total acid content increased as the fermentation proceeded, reaching 5.49 g / L and 6.36 g / L at the end of fermentation for the control group and the experimental group, respectively, and the total acid content of the experimental group was always higher than that of the control group, indicating that the accumulation of acidity was more significant in the experimental group, which may be related to the differences in microbial metabolism. The ethanol content of both groups increased over time, but the control group was slightly higher at the end of fermentation, possibly because the control group had a stronger ethanol-producing capacity after fermentation. The pH of both groups increased and then stabilized, with the pH of the control group (3.81 g / L) being slightly higher than that of the experimental group (3.74 g / L), which may be related to the difference in acidity. After 10 days of fermentation, there was no significant fluctuation in the total acid, ethanol concentration, and pH of the control group and the experimental group, and they maintained a relatively stable state. It is worth noting that the amino acid nitrogen content of both groups showed an upward trend, which may be due to the release of protease A and the peptides and amino acids catalyzed by it during the autolysis of yeast cells. The amino acid nitrogen content of the experimental group was generally higher than that of the control group, suggesting that protein decomposition was more complete in the experimental group. 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, and amino acid nitrogen ≥0.3 g / L) all met the limited range of the national standard for yellow rice wine (GB / T 2-1366, 2018), proving that the fermentation process was well controlled, and the resulting samples could be used for further in-depth research.

[0131] 3. Determination of urea content and ethyl carbamate content

[0132] (1) The urea content was determined by diacetyl monoxime colorimetry, and the specific method was as follows: two reagents were prepared in advance, reagent I was composed of 50 mL phosphoric acid (85%, v / v), 120 mL sulfuric acid (98%, v / v), 0.05 g FeCl3, and 330 mL deionized water, and reagent II was an aqueous solution containing 0.5 mg / mL diacetyl monoxime and 0.1 mg / mL aminothiourea.

[0133] The reagent I and reagent II were mixed in the ratio of 2:1 (v / v) before determination, 5 mL of the mixture was added to the test tube containing 1 mL of the sample to be tested, and then the test tube was immediately cooled to room temperature in ice water after heating in boiling water bath for 15 min. The urea and diacetyl monoxime formed a red complex under acidic conditions and in the presence of aminothiourea, and the absorbance was measured at 526 nm.

[0134] (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°C for future use.

[0135] Preparation of working standard solution: Dilute the standard stock solution with 30% methanol solution to four concentration gradients of 4.5 μg / mL, 1.8 μg / mL, 0.9 μg / mL, and 0.18 μg / mL, respectively.

[0136] Sample preparation and derivatization: All samples for ethyl carbamate (EC) detection were set up in five gradients for each type during the validation experiment. Due to the low EC concentration in the original sample, the crude sample needed to be concentrated. The specific operation was as follows: use a rotary evaporator to concentrate the sample to an appropriate volume (10-fold concentration) at 35°C. After concentration, perform derivatization reaction: add 0.02 M 9-anthracenol (200 μL) and 100 μL 1.5 mol / L hydrochloric acid to 1 mL of concentrated sample or standard EC solution, vortex for a few seconds, and then stand for 30 minutes to ensure complete reaction. Finally, inject into the chromatographic system for analysis.

[0137] Chromatographic conditions: The high performance liquid chromatography-fluorescence detection system (HPLC-FLD) used in this study consisted of the following components: two Waters 510 pumps (Waters Corporation, Milford, USA), a Rheodyne 108 injector (Rheodyne Corporation, Cotati, USA), and a Waters 2475 fluorescence detector. The reverse phase Symmetry C18 column (250 mm × 4.6 mm, 4 μm, Waters Corporation) was selected, the column temperature was maintained at 35°C, and the injection volume was 20 μL. The excitation wavelength and emission wavelength of the detector were set to 233 nm and 600 nm, respectively. The mobile phase was a methanol-water system, and the specific gradient elution conditions are shown in Table 6. Quantitative analysis was performed using the external standard method.

[0138] Table 6 Chromatographic conditions for EC determination in yellow rice wine

[0139]

[0140] Changes in urea and EC content and comparison: The changes in urea content and EC content in the experimental group and the control group during fermentation are as followsFigures 16-17 The results are shown in the table 1.

[0141] The control group CK: urea content gradually increased over time, which may be related to the accumulation of microbial metabolites in the late fermentation. The EC content showed an overall upward trend followed by a downward trend. The experimental group T: the urea content was significantly lower than the control group, indicating that the experimental group reduced the generation of urea by inoculating K-44. The EC content of the experimental group was significantly lower than that of the control group, indicating that the inoculation of K-44 in the experimental group effectively inhibited the generation of EC.

[0142] Table 7 Effect of K44 inoculation on urea and EC content in yellow rice wine

[0143]

[0144] By measuring the urea and EC content of the control group and the experimental group at the end of fermentation, the experimental group compared with the control group, the urea content decreased by 14.2%, and the EC content decreased by 37.25%. The experiment showed that the inoculation of strain K44 had a significant effect on the inhibition of EC formation.

[0145] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A urethane-degrading bacterium, characterized by: The degrading bacteria is Pichia kudriavzevii (Pichia kudriavzevii ) K44, deposited with the China Center for Type Culture Collection on April 25, 2025, and assigned the biological preservation number CCTCC NO: M 2025887.

2. The fermentation culture method of the urethane-degrading bacteria according to claim 1, characterized by: The carbon source is glucose with a concentration of 25 g / L; the nitrogen source is peptone with a concentration of 10 g / L; the initial pH is 5.0, and the inoculation amount is 5%.

3. The enzyme extraction method for ethyl carbamate-degrading bacteria according to claim 1, characterized in that: Ultrasonic treatment is adopted, and intermittent treatment is performed for 25 min at a power of 400 W, with an operation of 2 s and an interval of 4 s.

4. The application of the urethane-degrading bacteria in claim 1 in degrading urethane.

5. Use of the urethane-degrading bacteria according to claim 4 for degrading urethane, characterized in that: The application is used for degrading urethane in a yellow rice wine fermentation system, and the EC degradation rate is not less than 35%.

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