Schizosaccharomyces japonicus with high urease yield and application of schizosaccharomyces japonicus

Abstract

The invention belongs to the technical field of microorganisms, and relates to schizosaccharomyces japonicus with high urease yield and application of the schizosaccharomyces japonicus. The schizosaccharomyces japonicus FBKL2.9792 is preserved in the China Center for Type Culture Collection on March 18, 2024, the preservation number is CCTCC M 2024466, the strain shows the high-yield urease characteristic during urease qualitative and quantitative screening, and the urease activity is 1.26 + / -0.10 U / mL. Compared with a saccharomyces cerevisiae strain without urease and a schizosaccharomyces japonicus strain with low yield of urease, the strain shows higher urea degradation capacity in simulated fermentation, the urea degradation capacity reaches 24.74 mmol / L (1486.2 mg / L) after the strain is subjected to static culture for 10 days in a simulated fermentation culture medium with the urea addition amount of 1.5 g / L at 28 DEG C, and 99.08% of urea is degraded; after standing fermentation is performed for 10 days in a simulated fermentation culture medium with the addition amount of arginine being 1.5 g / L at 28 DEG C, the urea generation amount is (2.2 mg / L), which is about 12% of the urea generation amount of the saccharomyces cerevisiae strain under the same condition, so that the content of ethyl carbamate in the stored wine liquid is effectively reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and relates to a high-urease-producing Schizochytrium japonicum and application thereof. BACKGROUND

[0002] Ethyl carbamate (EC) is a carcinogenic by-product formed in the brewing of alcoholic beverages. Research reports have found that the detection rate of EC content in Chinese liquor and rice wine is relatively high. In the "Research Status and Prospect of Quality and Safety of Traditional Beverage Liquor and Rice Wine in China in the Past 10 Years", Fan Wenlai et al. explored the EC level in different flavor types of liquor and found that the average mass concentration was between 44.48 and 214 μg / L. Among them, the clear, sauce, medicinal and special flavor types of liquor showed lower EC content, while the strong, sesame and phoenix flavor types contained more EC, and some of them were higher than the international limit standard (150 μg / L) of EC in distilled wine. In the brewing of wine, the content of EC in rice wine is relatively high. Based on the research reports of 890 rice wine samples (including finished products and base wine) in various regions of China, the National Center for Food Safety Risk Assessment found that the EC content of 468 finished rice wine ranged from 6.3 to 776 μg / L (average 233 μg / L), which limited the import and export trade of Chinese wine.

[0003] Urea is the main precursor of EC in brewing wine such as rice wine, sake and grape wine. Part of the urea in wine comes from raw materials and processes, and most of it comes from the metabolism of microorganisms during the fermentation process. Saccharomyces cerevisiae produces urea from arginine through the urea cycle under the action of arginase, and is regulated by nitrogen metabolic repression. Saccharomyces cerevisiae cannot metabolize urea itself, and urea accumulates in the fermentation broth, spontaneously reacts with ethanol to form EC. The content of urea can reflect the potential content of EC in fermented wine. By limiting the formation of urea, the content of EC can be effectively reduced.

[0004] The main EC control strategies currently used in the food industry include process control, genetically engineered bacteria construction, physical adsorption, and enzymatic degradation. Process optimization reduces the amount of precursor urea by precisely controlling fermentation parameters such as temperature, pH, and nitrogen source addition. This method can only remove a small amount of urea. Genetic engineering techniques control by targeting key metabolic genes for knockout or overexpression, but the biosafety evaluation system for engineered strains has not been fully developed and validated. Physical adsorption is direct and efficient, but it may adsorb trace flavor substances in wine, which can significantly affect the quality of the wine. Enzymatic degradation utilizes the high catalytic properties of urease or EC hydrolase, which is green and efficient, but the industrial production and cost of enzyme preparations limit its large-scale application. Current research should focus on reducing EC production from the fermentation source and process control. Therefore, it is necessary to screen yeast strains with good fermentation performance and high urease activity and to study urea degradation.

[0005] In recent years, the interest in specific applications of Schizosaccharomyces in modern wine research has continued to increase. Urease can specifically catalyze the decomposition of urea. One important feature of Schizosaccharomyces is its urease activity, which can reduce urea during fermentation. Benito S. et al. (2016) combined the use of heat-resistant Lachancea fermentati and Schizosaccharomyces pombe as substitutes for traditional malolactic fermentation. Compared with traditional wine produced by traditional fermentation technology, the final concentration of urea after alcohol fermentation was lower, less than 0.2 mg / L, and this study first proposed that the urease activity of Schizosaccharomyces could have great potential in reducing ethyl carbamate in wine. Patent No. CN 106635848 B discloses a Schizosaccharomyces pombe that can degrade arginine and urea. After 54 h of static culture at 30℃ in a highland juice medium with an urea addition amount of 200 mg / L, the urea degradation capacity was 2.03 mmol / L, and the degradation rate was about 60.9%. The arginine degradation capacity was 1.01 mmol / L, but the urease activity was not tracked. The present inventors found that Japanese Schizosaccharomyces generally produces urease, but there is no report on Japanese Schizosaccharomyces for urea degradation in wine brewing in the domestic public. The application potential of high-urease-producing Japanese Schizosaccharomyces strains is great. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application provides a high-urease-producing Japanese Schizosaccharomyces and its application, as follows: A high-urease-producing Japanese Schizosaccharomyces, which is classified and named as Schizosaccharomyces japonicus, has a laboratory strain preservation bank number record of FBKL2.9792, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC M 2024466 and a preservation time of March 18, 2024.

[0007] Further, the Schizosaccharomyces japonicus CCTCC M 2024466 is screened from the natural fermentation process of Guizhou Vitis quinquangularis. The high-urease-producing Schizosaccharomyces strain is isolated and screened from the natural fermentation of Guizhou Vitis quinquangularis, and coexists with Saccharomyces cerevisiae, proving its excellent competitiveness and tolerance.

[0008] Further, the high-urease-producing is high in urease activity.

[0009] Further, the high-urease-producing is high in urease activity, specifically effectively reducing the urea content in the fermentation liquor during fermentation, and effectively reducing the ethyl carbamate content in the fermentation liquor after storage.

[0010] The application of the high-urease-producing Schizosaccharomyces japonicus CCTCC M 2024466 in wine brewing.

[0011] Further, the wine brewing includes fermented wine and distilled wine.

[0012] The application of the high-urease-producing Schizosaccharomyces japonicus CCTCC M 2024466 in urea degradation.

[0013] A wine brewing method, which uses the above-mentioned high-urease-producing Schizosaccharomyces japonicus as a fermentation strain.

[0014] A screening and identification method of high-urease-producing Schizosaccharomyces japonicus, the specific steps are as follows: 2 μL of cultured bacteria liquid is inoculated on a natural pH urease qualitative culture medium, and after the droplet water of the inoculum is volatilized, it is transferred to a 28℃ constant temperature incubator for culture for 48h, and the ratio (D / d) of the discoloration circle diameter (D) to the colony diameter (d) is used to judge the urease production capacity of the strain; D / d≥10 is high-yield, 4≤D / d<10 is medium-yield, 1<D / d<4 is low-yield, and no discoloration circle or D / d≤1 is defined as not producing.

[0015] Further, the urease qualitative culture medium is prepared by 20 g / L glucose, 20 g / L agar, 2 g / L yeast extract powder, 0.12 g / L phenol red, 0.005 g / L nickel sulfate hexahydrate and 20 g / L urea, and the reagents except urea are sterilized at 115℃ for 20 min, and the urea is sterilized by filtering with a 0.45 μm sterile filter membrane.

[0016] A quantitative screening method of high-urease-producing Schizosaccharomyces japonicus, which uses improved Berthelot colorimetric method for urease quantitative analysis on the basis of the above-mentioned method, and uses inactivated enzyme liquid as blank control, and the specific way is to add 10 % TCA for inactivation for 10 min.

[0017] Furthermore, the quantitative detection method for urease activity of the high-urease-producing *Schizosaccharomyces cerevisiae* strain is (referencing the Berthelot colorimetric method with appropriate modifications): the strain is used in a 10-1... 6 Cells / mL were inoculated into 5 mL of urea liquid medium and cultured at 28 °C for 48 h. 1000 μL of the bacterial culture was centrifuged for 5 min (10000 rpm), and the supernatant was collected and placed at 4 °C (as the extracellular crude enzyme solution). The cell pellet was washed twice with citrate buffer (pH 3.5) (0.05 mol / L citric acid monohydrate and 0.05 mol / L trisodium citrate C6H5Na3O7, volume ratio 3:1, adjusted to pH 3.5), resuspended in 250 μL of citrate buffer, and prepared using a cell disruptor and 0.2 g sterile glass beads (0.8–1.0 mm in diameter). After disruption, the sample was centrifuged for 5 min (10000 rpm), and the supernatant was collected and placed at 4 °C (as the intracellular crude enzyme solution). Add 100 μL of enzyme solution (appropriately diluted) to 350 μL of urea substrate solution (prepared as a 20 g / L urea substrate solution using pH 3.5 citrate buffer), and react at 300 rpm and 37℃ for 15 min. Immediately add 100 μL of stop agent (10% TCA), followed by 200 μL of colorimetric reagent A (3 g phenol and 0.125 g sodium nitrosoferricyanide, adjusted to 50 mL) and colorimetric reagent B (2.625 g NaOH, 2 mL 5% sodium hypochlorite solution, adjusted to 50 mL), and react at 300 rpm and 37℃ for 20 min. Measure the absorbance at 625 nm. Use inactivated enzyme solution as a blank control (inactivated by adding 10% TCA for 10 min beforehand). Establish a standard curve for 0–1 μmol / mL ammonium ions according to the enzyme activity assay method, where the enzyme solution is replaced by NH4Cl standard solution and the urea substrate solution is replaced by citrate buffer.

[0018] Furthermore, the urea liquid culture medium is prepared as follows: 20 g / L glucose, 20 g / L urea, 20 g / L yeast extract powder, and 0.005 g / L nickel sulfate hexahydrate are mixed and the pH is adjusted to 3.5. The mixture is then sterilized at 115°C for 20 min, and the urea is filtered through a 0.45 μm sterile filter membrane for sterilization.

[0019] Furthermore, enzyme activity is defined as the amount of enzyme that produces 1 μmol of ammonium ions per minute by decomposing urea under normal pressure, 37°C, and pH 3.5 conditions. One unit of enzyme activity is defined as such.

[0020] Further, the high-urease-producing Schizochytrium japonicum strain has high urease production on urease qualitative medium, and the Berthelot colorimetric urease activity quantitative result shows that the enzyme activity is greater than 1.00 U / mL. Specifically, the qualitative and quantitative results of the high-urease-activity Schizochytrium japonicum CCTCC M 2024466 are D / d=14, high production, and the total urease activity is 1.26±0.10 U / mL.

[0021] Advantages of the present application: 1. The Schizochytrium japonicum provided by the present application is a newly developed wild strain, which is isolated and screened from the late stage (5th day) of natural fermentation of Vitis vinifera (variety: purple autumn), and has a total urease activity of 1.26±0.10 U / mL, and has the characteristics of high urease production. In the process of simulated medium alcohol fermentation, the urease activity is intermittently detected, and the concentrated formation period is 6-9 d. When the urea concentration is low (0.15 g / L), the urease activity is low, and the enzyme activity is intermittently detected (concentrated in 5-7 d), and with the increase of urea content, the urease activity increases significantly, and when the urea addition amount is 6.0, 20.0 g / L, the urease activity reaches the highest level, and stable urease activity is detected throughout the fermentation process, and the change of enzyme activity shows a parabolic trend.

[0022] 2. The strain of the present application can significantly reduce the urea content in the fermentation liquor, thereby reducing the risk of formation of ethyl carbamate in the wine, and using the strain of the present application for simulated medium alcohol fermentation, the urea content of the obtained fermentation liquor is reduced from 13.5 mg / L to 8.8 mg / L. The urea degradation rate increases with the increase of urea level, and the urea degradation rate is as high as 90% or more when the urea level is 1.5 g / L and 6.0 g / L.

[0023] 3. The strain of the present application has excellent comprehensive fermentation ability, and under appropriate urea content (1.50 g / L-6.00 g / L), the strain has the same fermentation ethanol production capacity as Saccharomyces cerevisiae, and the alcohol content of the obtained fermentation liquor is 10.3 %vol-9 %vol. The strain has good alcohol tolerance and competition ability with Saccharomyces cerevisiae, has high urease activity and good urea degradation ability, and is expected to play an important role in the safe production of wine brewing. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : Colony morphology and cell morphology of Schizochytrium japonicum FBKL2.9792; Figure 2 : Change of urease activity of Schizochytrium japonicum FBKL2.9792 in fermentation with different initial urea concentrations; Figure 3: Urease activity change of Schizosaccharomyces japonicus FBKL2.9792 in fermentation with different amounts of arginine addition. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with specific examples, which are intended to explain but not limit the application.

[0026] Example 1 A method for obtaining a Schizosaccharomyces japonicus strain with high urease activity comprises the following steps: (1) Collecting Vitis davidii grape grains in Ziyun County, Guizhou Province, crushing them into juice, and placing them in a sterile fermentation bottle for sealed fermentation at 28°C without adding exogenous microorganisms. The addition amount of Vitis davidii grape mash in the fermentation bottle is 800 mL / L. The fermentation process is tracked by the amount of carbon dioxide generated. The Vitis davidii grape is naturally fermented, and the Schizosaccharomyces japonicus is obtained by dilution, coating, classification of yeasts, and identification using culturable separation technology.

[0027] (2) Qualitative screening of urease activity of the Schizosaccharomyces japonicus strain by urease qualitative medium: 2 μL of bacterial liquid is inoculated on the natural pH urease qualitative medium, and after the water droplets in the inoculum are evaporated, it is transferred to a 28°C constant temperature incubator for 48 h of culture. The ratio (D / d) of the discoloration circle diameter (D) to the colony diameter (d) is used to judge the urease production capacity of the strain. D / d≥10 is high yield, 4≤D / d<10 is medium yield, 1<D / d<4 is low yield, and no discoloration circle or D / d≤1 is defined as no production.

[0028] (3) The urease activity is quantitatively detected by the improved Berthelot colorimetric method. The quantitative result of enzyme activity greater than 1 U / mL is defined as high urease activity. The morphological characteristics of the Schizosaccharomyces japonicus strain with high urease activity FBKL2.9792 are as follows: the colony on the YPD medium is spherical with a raised middle and a tray-shaped bottom. Under a microscope, the cells are elliptical or round rod-shaped, with 6-8 ascospores inside. Figure 1 On the urease qualitative medium, D / d=14 is defined as high urease production. The quantitative result of enzyme activity is 1.26±0.10 U / mL, which is defined as high urease activity.

[0029] Example 2 Alcohol fermentation characteristics, urease activity change, and urea degradation of a Schizosaccharomyces japonicus strain with high urease production in alcohol fermentation.

[0030] Referring to the urea and arginine content in grape juice and wine, urea addition levels were set at 0 g / L, 0.01 g / L, 0.15 g / L, 1.5 g / L, 6 g / L, and 20 g / L, and arginine addition levels were set at 0.5 g / L, 1.0 g / L, and 1.5 g / L. A simulated culture medium was used to conduct urea alcoholic fermentation metabolism experiments on the experimental strains. One strain of *Schizosaccharomyces nigra* with high urease activity (FBKL2.9792), one strain of *Schizosaccharomyces nigra* with low urease activity (FBKL2.9886), and one strain of *Saccharomyces cerevisiae* without urease activity (FBKL2.9126) were selected for urea and arginine alcoholic fermentation metabolism experiments.

[0031] Changes in urease activity during alcoholic fermentation under different amounts of urea and arginine addition are shown in the figure. Figure 2 , 3 Compared with *Saccharomyces cerevisiae* and low-urease-producing strains, FBKL2.9792 exhibited higher urease activity during alcohol fermentation. At urea concentrations of 0.01 g / L and 0.15 g / L, enzyme activity was generally below 0.20 U / mL, with intermittent detection (concentrated over 5–7 days). Urease activity significantly increased at urea concentrations of 1.50 g / L, 6.00 g / L, and 20.00 g / L, maintaining a range of 0.20–0.79 U / mL. Enzyme activity was continuously detected throughout the fermentation period, exhibiting a parabolic trend. Arginine addition (0.5, 1.0, and 1.5 g / L) did not significantly increase urease activity; enzyme activity was intermittently detected during fermentation without a clear pattern, but the urease activity was higher than that of FBKL2.9886.

[0032] Table 1. Urea and arginine metabolism of three yeast strains during simulated fermentation with different amounts of urea and arginine.

[0033] Note: Lowercase superscript letters indicate significant differences in the same indicator under different yeast fermentation conditions. The three strains of yeast can degrade urea and arginine, but their abilities are different, as shown in Table 1. The urea degradation rate of FBKL2.9792 with high urease activity is the highest, and the urea degradation rate in the simulated fermentation medium with urea addition is more than 50%. After 10 days of static culture at 28°C in the simulated fermentation medium with 1.5 g / L urea addition, the urea degradation capacity is 24.74 mmol / L, and 99.08% of the urea is degraded. In the simulated culture medium with 20 g / L urea, the urea degradation rate decreases to 31-55%, but the actual metabolic urea content of the strain is 10.81 g / L, which is higher than the above-mentioned urea addition, indicating that there is an upper limit to the metabolic capacity of the strain to urea. Saccharomyces cerevisiae FBKL2.9126 has a certain decomposition ability for urea, but it is far inferior to Schizosaccharomyces japonicus. The addition of arginine promotes the generation of urea, and the high-urease strain further degrades the generated urea. Under different arginine addition amounts, the urea concentration at the end of fermentation is increased to different degrees compared with the initial fermentation. The urea increase amplitude of FBKL2.9972 is the smallest under different arginine addition amounts, and even when the arginine addition amount is 1.5 g / L, the urea increase amplitude is still less than 20%. The urea increase amplitude of Saccharomyces cerevisiae FBKL2.9126 is the largest. Arginine promotes the generation of urea, and the high-urease strain FBKL2.9792 can degrade more new urea.

[0034] After the fermentation of the simulated culture medium with 20 g / L urea addition was completed, the fermentation supernatant was centrifuged and stored at 28°C for 2 months, and ethyl carbamate detection was performed, as shown in Table 2. No ethyl carbamate was detected in the fermentation broth just after fermentation and 1 month later, and it was detected after 2 months. The ethyl carbamate content of FBKL2.9792 is the lowest. When the urea addition amount is 20 g / L, the urease activity of FBKL2.9792 is the highest, and the urea degradation rate is also the largest among the three strains (55.48%), which shows the correlation trend between the ability to reduce urea and the generation of ethyl carbamate.

[0035] Table 2 Ethyl carbamate content of three strains of yeast fermented in simulated culture medium with 20 g / L urea addition and stored for 2 months

[0036] ND indicates not detected Schizosaccharomyces japonicus FBKL2.9792 was deposited at the China Center for Type Culture Collection on March 18, 2024, and the deposit number is CCTCC M 2024466.

[0037] The fermentation ability levels of three strains of yeast were compared, and it was found that different strains had different urea utilization ability and fermentation characteristics. The results are shown in Table 3. Urea concentration had an effect on the alcohol fermentation of Schizochytrium sp. (FBKL2.9SZJ3, FBKL2.9792, FBKL2.9886), but had no significant effect on the alcohol fermentation of S. cerevisiae. The addition of low-concentration urea (0.01, 0.15 g / L) inhibited the alcohol fermentation. Appropriate amount of urea could promote the alcohol fermentation. The alcohol content was significantly higher than that of other urea concentrations (≥9%vol) when the addition amount of urea was 1.50 g / L and 6.00 g / L. The alcohol fermentation was severely inhibited (4.7%vol) when the addition amount of urea was 20.00 g / L. S. cerevisiae FBKL2.9126 had strong tolerance to urea concentration, and the alcohol fermentation was less affected by urea concentration. The reducing sugar was almost completely utilized. The alcohol content of each strain showed obvious differences when the addition amount of urea was 20 g / L, which reflected the different tolerance degrees of the strains to high-concentration urea. The addition of arginine promoted the alcohol fermentation of Schizochytrium sp.

[0038] Table 3 Alcohol fermentation of three strains of yeast under different urea and amino acid addition levels

[0039] Note: The superscript lowercase letters represent the results of significant difference analysis of the same index under different fermentation conditions of the same yeast Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, and there can be many variations. All variations that can be directly derived or inferred by those of ordinary skill in the art from the disclosed content should be considered as falling within the scope of protection of the present application.

Claims

1. A high urease-producing Schizochytrium japonicum, characterized in that, It is preserved in China Center for Type Culture Collection, the preservation number is CCTCC M 2024466, and the preservation time is March 18, 2024.

2. The high-urease-producing Schizochytrium sp. of claim 1, wherein, The high-urease-producing strain has high urease activity.

3. The high-urease-producing Schizochytrium sp. of claim 2, wherein, The high urease activity specifically refers to effectively reducing the urea content in the fermentation liquor during fermentation and effectively reducing the ethyl carbamate content in the fermentation liquor after storage.

4. Application of the high-urease-producing Schizochytrium japonicum in wine brewing according to claim 1.

5. Use according to claim 4, characterized in that, The wine brewing aspect includes fermented wine and distilled wine.

6. Application of the high-urease-producing Schizochytrium japonicum in urea degradation according to claim 1.

7. A method of brewing an alcoholic beverage, characterized by, The high-urease-producing Schizochytrium japonicum according to claim 1 is used as a fermentation strain.

8. A method for the primary screening and identification of high urease-producing Schizochytrium japonicum, characterized by, The specific steps are as follows: 2 μL of cultured bacteria liquid is inoculated on urease qualitative culture medium at natural pH value, the inoculum is dried after water evaporation, and then it is transferred into a 28℃ constant temperature incubator for 48 h of culture. The ratio (D / d) of the discoloration circle diameter (D) to the colony diameter (d) is used to judge the urease production capacity of the strain; D / d≥10 is high production, 4≤D / d<10 is medium production, 1<D / d<4 is low production, and no discoloration circle or D / d≤1 is defined as no production.

9. The method for the primary screening and identification of high-urease-producing P. japonica according to claim 8, characterized in that, The urease qualitative culture medium is prepared by using 20 g / L glucose, 20 g / L agar, 2 g / L yeast extract powder, 0.12 g / L phenol red, 0.005 g / L nickel sulfate hexahydrate, and 20 g / L urea. The reagents except urea are sterilized at 115℃ for 20 min, and the urea is sterilized by filtering through a 0.45 μm sterile filter.

10. A method for quantitative screening of a high urease-producing Schizosaccharomyces japonicus, characterized by, On the basis of claim 8, the urease quantitative analysis is performed by using the improved Berthelot colorimetric method.

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