Bacillus subtilis producing acid urease and application thereof

Abstract

The application discloses a kind of acid urease-producing bacillus subtilis and application thereof, belong to microbial technical field.In order to provide a kind of high safety and high enzyme activity acid urease, the application provides a kind of bacillus subtilis, under the condition of 37 DEG C, at least fermentation 40h hour acid urease is produced, and its acid urease enzyme activity reaches 6224U / L, the application finds that Bacillus subtilis has the ability to produce acid urease, on the other hand, after the enzyme treatment, the EC content of yellow rice is 22.23 μg / L, only 17.9% of the control group, so the strain shows excellent EC control ability in yellow rice, and provides an effective method for the control of EC content in yellow rice.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an acid urease-producing Bacillus subtilis and its applications. Background Technology

[0002] Ethyl carbamate (EC) is a carcinogenic and mutagenic substance commonly found in fermented foods (GOWD V, SU H, KARLOVSKY P, et al. Ethyl carbamate: An emerging food and environmental toxicant [J]. Food Chem, 2018, 248: 312-321.). In 1943, EC was confirmed to have carcinogenic effects (NETTLESHIP A, HENSHAW PS, MEYER HL. Induction of Pulmonary Tumors in Mice With Ethyl Carbamate (Urethane) [J]. JNCI: Journal of the National Cancer Institute, 1943, 4(3): 309-319.). EC is produced by the reaction of carbamate compounds and ethanol. Carbamate compounds are produced in fermented wines such as rice wine, wine, and sake. Urea is one of the main carbamate compounds and an important precursor of EC (EBER JV, SHARYPOV V I. Ethyl carbamate in foods and beverages: a review [J]. Environ Chem Lett, 2009, 7(3): 233-247.).

[0003] Based on the formation mechanism of ethyl carbamate (EC), current research on solving the EC problem in alcoholic beverages, both domestically and internationally, mainly focuses on reducing its sources (raw materials, low-urea-producing yeast, acidic urease, etc.), controlling the formation process (process conditions), and subsequent removal (acidic EC-degrading enzymes, etc.) (Yang Jia, Li Xinsheng, Geng Jingzhang, et al. Research progress on ethyl carbamate in rice wine [J]. China Brewing, 2020, 39(3): 7-11.). However, the acidic urease substrate removal method is still the most widely accepted internationally. Urease can degrade urea and release NH3 and CO2. Urease is divided into acidic urease and neutral urease. Since the fermentation environment of alcoholic beverages is acidic, neutral urease has no particular value in alcoholic beverage production. Therefore, acidic urease is used to control urea in alcoholic beverages. Adding urea-degrading enzymes to alcoholic beverages has several significant advantages: ① It does not require changing the yeast strain; ② It does not require changing the production process; ③ It is convenient to use and increases the safety of alcoholic beverages during later storage; ④ As an FDA (U.S. Food and Drug Administration) recommended method for controlling ethyl carbamate (ZHOU W, FANG R, CHEN Q. Effect of gallic and protocatechuic acids on the metabolism of ethyl carbamate in Chinese yellow rice wine brewing [J]. Food Chem, 2017, 233:174-181.), finding a method to prepare acidic uricase is a very important technology. Summary of the Invention

[0004] The purpose of this invention is to provide an acid uricase with high enzyme activity and high safety.

[0005] This invention provides a Bacillus subtilis ( Bacillus subtilis The Bacillus subtilis was deposited at the China Center for Type Culture Collection on November 1, 2021, with accession number CCTCC NO: M 20211344, and named Bacillus subtilis Au-14.

[0006] The present invention provides a microbial preparation of the above-mentioned Bacillus subtilis.

[0007] This invention provides the application of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in the preparation of acid urease.

[0008] This invention provides a method for preparing acidic urease, wherein the above-mentioned Bacillus subtilis is fermented at 37 °C for at least 40 h.

[0009] Further specified, the culture medium used for fermentation uses glucose as the carbon source; the culture medium used for fermentation uses urea, peptone and yeast extract as nitrogen sources.

[0010] Further specifying, the culture medium used for fermentation includes: glucose, urea, peptone, yeast extract, sodium chloride, potassium dihydrogen phosphate, sodium acetate, manganese sulfate tetrahydrate, and nickel sulfate hexahydrate.

[0011] The present invention provides urease obtained by the above preparation method.

[0012] This invention provides the application of the above-mentioned Bacillus subtilis in the degradation of urea.

[0013] This invention provides a method for degrading urea, which utilizes Bacillus subtilis to produce acidic urease, and then adds the acidic urease to the urea system at a rate of 0.2 U / mL, and reacts at 37 °C for 24 h.

[0014] This invention provides the application of the above-mentioned Bacillus subtilis in reducing ethyl carbamate in the brewing industry.

[0015] Beneficial effects: This invention provides an acid urease-producing strain. This strain, fermented using a liquid shaking method, achieves an acid urease activity of 6224 U / L. This strain belongs to... Bacillus subtilis There is currently no information regarding Bacillus subtilis Based on reports of acid urease production and the fact that this genus is non-pathogenic, this invention has discovered... Bacillus subtilis It has the ability to produce acid urease. On the other hand, after being treated with this enzyme, the EC content of rice wine was 22.23 μg / L, which was only 17.9% of that of the control group. Therefore, this strain showed excellent EC control ability in rice wine, providing an effective method for controlling the EC content in rice wine.

[0016] [Preservation of Biological Materials] A type of Bacillus subtilis ( Bacillus subtilis The Bacillus subtilis strain was deposited at the China Center for Type Culture Collection on November 1, 2021, with accession number CCTCC NO: M 20211344, named Bacillus subtilis Au-14, and deposited at Wuhan University, China. Attached Figure Description

[0017] Figure 1 The phylogenetic tree of the isolated Bacillus subtilis; Figure 2 Plate image for initial screening of Bacillus subtilis; Figure 3 The graph shows the results of acid urease activity produced by Bacillus subtilis, where the horizontal axis represents the group and the vertical axis represents the enzyme activity. Figure 4 The effect of initial sugar concentration on enzyme production is shown, where the horizontal axis represents time and the vertical axis represents enzyme activity. Figure 5 The effect of peptone concentration on enzyme production is shown, where the horizontal axis represents time and the vertical axis represents enzyme activity. Figure 6 The effect of fermentation temperature on enzyme production is shown, where the horizontal axis represents time and the vertical axis represents enzyme activity. Figure 7 To compare the enzyme production capacity of strains before and after fermentation condition optimization, the horizontal axis represents the group and the vertical axis represents enzyme activity. Figure 8 The urea-degrading capacity of Au-14 acid urease is represented by the x-axis, which is the group, and the y-axis is the urea concentration. Figure 9 The effect of Au-14 acid urease on EC control in rice wine is shown, where the horizontal axis represents the group and the vertical axis represents the EC concentration. Detailed Implementation

[0018] Enrichment medium pH=4.0 (components g / L): glucose 20, urea 20, sodium chloride 5, potassium dihydrogen phosphate 2, sodium acetate 2.

[0019] Acidic primary screening plate pH=5.0 (components g / L): glucose 20, urea 20, peptone 10, beef extract 5, sodium chloride 5, yeast extract 3, potassium dihydrogen phosphate 2, sodium acetate 2, bromocresol purple 0.02, agar 25.

[0020] Neutral rescreening plate pH=6.8 (components g / L): glucose 20, urea 20, peptone 2, beef extract 10, sodium chloride 5, yeast extract 3, potassium dihydrogen phosphate 2, sodium acetate 2, phenol red 0.005, agar 20.

[0021] Fermentation medium pH=5.5 (components g / L): glucose 20, urea 5, peptone 10, yeast extract 5, sodium chloride 5, potassium dihydrogen phosphate 2, sodium acetate 2, manganese sulfate tetrahydrate 0.05, nickel sulfate hexahydrate 0.05.

[0022] LB medium pH=7.0 (components g / L): peptone 10, sodium chloride 10, yeast extract 5.

[0023] Example 1. Obtaining Bacillus subtilis that produces acid urease 1. Weigh 2 g of soil sample, dissolve it in sterile physiological saline, filter, and add the filtrate to enrichment medium. Incubate at 37 ℃ with shaking at 120 rpm / min for 48 h. Spread an appropriate amount of bacterial solution onto an acidic primary screening plate and incubate at 37 ℃ for 48 h. Pick colonies with a blue-purple transparent zone and inoculate them onto a neutral secondary screening plate. Incubate at 37 ℃ for 24 h. If the area around the colony on the neutral secondary screening plate does not change color, it confirms that the urease produced by this strain is acidic urease. Streak the strain onto LB slant medium and incubate at 37 ℃, then store at 4 ℃.

[0024] The target strain was inoculated into LB liquid medium and cultured at 37 °C with shaking at 200 rpm / min for 16 h. The bacterial genome was extracted from the cultured culture according to the instructions of the rapid genome extraction kit from Sangon Biotech (Shanghai) Co., Ltd. The extracted genomic DNA was dissolved in 100 μL of TE buffer. Primers 27-F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO.1) and 1492-R (5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO.2) were synthesized by Sangon Biotech (Shanghai).

[0025] Using genomic DNA as a template, PCR amplification was performed under the following conditions: 95 ℃ for 10 min; 95 ℃ for 30 s; 51 ℃ for 30 s; 72 ℃ for 90 s; for a total of 30 cycles; 72 ℃ for 10 min; and storage at 4 ℃. The amplified products were examined by 1% agarose gel electrophoresis. The target band was excised and recovered according to the instructions of the DNA gel recovery kit. The recovered DNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST alignment in the GenBank database to identify the target strain.

[0026] The 16S rDNA sequence is shown in SEQ D NO.3. The target bacterium obtained after sequencing belongs to the genus Bacillus. The phylogenetic tree is constructed as follows: Figure 1 As shown, it was found to be similar to Bacillus subtilis It is most closely related, with a maximum sequence similarity of 100%, therefore it is... Bacillus subtilis . Some colonies growing on the acidic screening plate have a purple transparent ring, as shown in the results. Figure 2 As shown, the portion indicated by the arrow entered the secondary screening colony. After secondary screening, 9 strains producing acid urease were obtained and then proceeded to the subsequent enzyme activity test.

[0027] 2. Bacterial culture and enzyme activity test Culture and collection of bacterial cells: The slant culture was inoculated into the fermentation medium and cultured with shaking at 37 ℃ for 24 h. After centrifugation, the bacterial cells were washed with citrate buffer (0.05 mol / L, pH 4.5), and the whole cells were obtained by centrifugation. The whole cells were resuspended in citrate buffer to the original volume to obtain the crude enzyme solution.

[0028] Acid urease activity was calculated based on the amount of ammonia produced, as follows: The enzyme solution and urea (3%) solution were preheated at 37 °C for 10 min. 0.2 mL of crude enzyme solution was mixed with 0.8 mL of urea solution and precisely incubated at 37 °C for 30 min. Immediately after incubation, 1 mL of trichloroacetic acid (10%) solution was added to terminate the reaction. 1 mL of colorimetric reagent A (concentration per 100 mL component: 6 g phenol, 0.25 g sodium nitrosoferricyanide) and 1 mL of colorimetric reagent B (concentration per 100 mL component: 3 mL sodium hypochlorite, 5.25 g sodium hydroxide) were added. The mixture was incubated in the dark for 20 min and then measured at 625 nm. The control group enzyme solution was pre-boiled for 30 min, and the rest of the operation was performed as usual. The enzyme activity unit (U) was defined as: 1 μmol of ammonia produced per minute at 37 °C and pH 4.5.

[0029] After 24 hours of fermentation, the nine strains obtained from the screening were further analyzed to determine their urease activity and identify the target enzyme-producing strains. Figure 3 The acid urease activity of nine strains was shown, with strain Au-14 exhibiting the highest activity of 840 U / L. This strain was identified as an enzyme-producing strain and was deposited in a cultural heritage institution.

[0030] Example 2. Method for producing acid urease using Bacillus subtilis Au-14 To facilitate the industrial production of acid urease, fermentation experiments were conducted on Au-14 to produce enzymes. The experiments optimized the initial sugar concentration, peptone concentration, and fermentation temperature. Enzyme activity was measured every 4 hours during Au-14 fermentation. It was found that Au-14 exhibited the highest enzyme activity (4565 U / L) at an initial sugar concentration of 30 g / L. Optimization of peptone concentration also yielded the following results: Figures 4-7 As shown, Au-14 reached its peak enzyme activity at a peptone concentration of 10 g / L. The effect of fermentation temperature on the enzyme production capacity of Au-14 was investigated; enzyme production was inhibited at 30 ℃ and 40 ℃, with the optimal enzyme production temperature being 37 ℃. The enzyme production capacity of Au-14 at 40 h in optimized and unoptimized media showed that the enzyme activity in the optimized medium reached 6224 U / L, a 160.2% increase compared to 3884 U / L in the unoptimized medium, significantly enhancing its commercial application value.

[0031] Example 3. A method for decomposing urea using acidic urease produced by Bacillus subtilis Au-14. Determination of urea content: Take 5 mL of solution A (concentration per 100 mL: 2.5 g diacetyl oxime); 2 mL of solution B (concentration per 100 mL: 0.25 g thiourea); and 100 mL of solution C (concentration per 100 mL: 60 mL 85% concentrated phosphoric acid, 2 mL concentrated sulfuric acid) and mix them to form a colorimetric reagent. Measure 1 mL of the sample to be tested into a 25 mL stoppered colorimetric tube, add 15 mL of the colorimetric reagent to the tube, and dilute to 25 mL with distilled water. Mix well, boil in a water bath for 30 min, cool, and then measure at 527 nm.

[0032] The acidic urease obtained in Example 2 was added to urea (100 mg / L) at pH 4.5 at a final concentration of 0.2 U / mL, and the reaction was carried out at 37 °C for 24 h. After the reaction, the urea content was determined using the diacetylthiourea method. Figure 8 The results showed that the urea content of the urea solution decreased by 84.2% after treatment with Au-14 acid urease, demonstrating its good degradation ability for urea.

[0033] Example 4. Application of acidic urease produced by Bacillus subtilis Au-14 in the brewing of rice wine, wine, sake, and other wines. The acid urease obtained in Example 2 was added to the pressed rice wine at a final concentration of 0.2 U / mL and treated at 37°C for 24 h. The treated rice wine was then sterilized by boiling. Subsequently, the EC content of the Au-14 treated and untreated rice wine was determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry.

[0034] The specific method for determining EC content is as follows: 8 mL of wine sample is pipetted into a 20 mL headspace vial, 8 μL of PC internal standard solution (concentration per 100 mL component: 0.015 g propyl carbamate, diluted to volume with ethanol) is added, followed by 3.1 g sodium chloride. The vial cap is tightened, the extraction head is inserted, and extraction is performed at 70 ℃ with stirring at 250 rpm for 45 min. After extraction, the extraction head is removed, and the sample is subjected to gas chromatography at 250 ℃ for thermal desorption for 5 min. High-purity helium is used as the carrier gas at a flow rate of 2 mL / min. The chromatographic column is a DB-FFAP capillary column (60 m × 0.25 mm id × 0.25 μm, Agilent, CA, USA). The separation temperature program is as follows: initial temperature 50 ℃ held for 2 min, then increased to 230 ℃ at a rate of 5 ℃ / min and held for 10 min. MS conditions were: EI ionization source, electron energy 70 eV, ion source temperature 230 ℃, and characteristic ion m / z 62 in SIM mode.

[0035] Figure 9 The effect of Au-14 acid urease on EC control in rice wine was investigated. The results showed that the EC content of rice wine after Au-14 treatment was 22.23 μg / L, which was only 17.9% of that of the control group. Therefore, Au-14 showed excellent EC control ability in rice wine, providing an effective method for controlling the EC content in rice wine. SEQUENCE LISTING <110> Shaoxing University <120> A Bacillus subtilis species producing acid urease and its applications <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> artificial synthesis <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> artificial synthesis <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1435 <212> DNA <213> Bacillus subtilis <400> 3 tgctatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac 60 gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg aaccggggc 120 tataccgga tgcttgtttg aaccgcatgg ttcaaacata aaaggtggct tcggctacca 180 cttacagatg gacccgcggc gcattagcta gttggtgagg taatggctca ccaaggcgac 240 gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300 cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360 cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtacc 420 gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta actacgtgcc 480 agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct 540 cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg 600 aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc 660 gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga 720 ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga 780 tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact 840 ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa 900 gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 960 ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg 1020 ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga 1080 tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga 1140 aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa 1200 tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca 1260 gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc 1320 agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag 1380 tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga aggtg 1435

Claims

1. A type of Bacillus subtilis (Bacillus subtilis) Its features are, The Bacillus subtilis was deposited at the China Center for Type Culture Collection on November 1, 2021, with accession number CCTCC NO: M 20211344, and named Bacillus subtilis Au-14.

2. A microbial preparation containing the Bacillus subtilis of claim 1.

3. The use of Bacillus subtilis according to claim 1 or the microbial preparation according to claim 2 in the preparation of acid urease.

4. A method for preparing acid urease, characterized in that, The Bacillus subtilis of claim 1 is fermented at 37 °C for at least 40 h.

5. The method according to claim 4, characterized in that, The culture medium used for fermentation uses glucose as the carbon source; the culture medium used for fermentation uses peptone as the nitrogen source.

6. The method according to claim 4, characterized in that, The culture media used for fermentation include: glucose, urea, peptone, yeast extract, sodium chloride, potassium dihydrogen phosphate, sodium acetate, manganese sulfate tetrahydrate, and nickel sulfate hexahydrate.

7. The application of Bacillus subtilis as described in claim 1 in the degradation of urea.

8. A method for degrading urea, characterized in that, Acid urease was produced using Bacillus subtilis as described in claim 1, and then the acid urease was added to a urea system at a rate of 0.2 U / mL, and the reaction was carried out at 37 °C for 24 h.

9. The use of Bacillus subtilis as described in claim 1 in reducing ethyl carbamate in the brewing industry.

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