Thermophilic micrococcus ylw106 and application thereof

Abstract

This invention provides a thermostable microvesicle YLW106, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 7, 2023, with accession number CGMCC 27855. The thermostable microvesicle YLW106 provided by this invention can efficiently synthesize tetrahydropyrimidine using marine polysaccharides as a carbon source. Furthermore, this thermostable microvesicle YLW106 exhibits high utilization of marine polysaccharides and can synthesize tetrahydropyrimidine in high yields, demonstrating broad prospects for development and application.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a thermostable microbubble bacterium YLW106 and its applications. Background Technology

[0002] Tetrahydropyrimidine is a cyclic amino acid derivative with excellent protective effects on biological macromolecules and cells. It has been developed as a protective agent in cosmetics, skin care products, and enzyme molecules, and is used to treat Alzheimer's disease, inflammation, and other conditions. Tetrahydropyrimidine has wide applications in biotechnology, cosmetics, and pharmaceuticals, and has developed into a high-demand, high-commercial-value product with a market value of billions of dollars. The annual market demand is approximately 15,000 tons, and the retail price is approximately US$1,000 per kilogram.

[0003] Tetrahydropyrimidine's molecular structure contains a single chiral carbon atom, making chemical synthesis extremely difficult; currently, it is primarily obtained through biosynthesis. Existing methods for tetrahydropyrimidine biosynthesis include batch fermentation, fed-batch fermentation, bacterial milking / lactation, and combined growth and resting cell synthesis. However, current methods still suffer from high raw material costs and low carbon source conversion rates. Current research reports both domestically and internationally on carbon sources used in tetrahydropyrimidine production include glucose, glycerol, sucrose, yeast extract, aspartate, glutamate, methane, and molasses. Glucose is the most frequently reported carbon source. While using glucose as a substrate for fermentation to produce tetrahydropyrimidine demonstrates outstanding yield and efficiency, glucose is primarily derived from dietary starch, raising concerns about the economic viability of large-scale tetrahydropyrimidine production using glucose. Therefore, finding low-cost, high-carbon-source-conversion non-grain biomass feedstocks for tetrahydropyrimidine biosynthesis is of significant importance.

[0004] Thermostable microvesicles are a group of rod-shaped, strictly aerobic, Gram-negative bacteria that are positive for oxidases and catalases. Some microvesicles can produce tetrahydropyrimidine, but only a few species have been isolated and screened so far. Therefore, microvesicle microorganisms still need further development. Summary of the Invention

[0005] This invention provides a thermostable microbubble bacterium YLW106 that can effectively synthesize tetrahydropyrimidine using marine polysaccharides as a carbon source, and has broad prospects for development and application.

[0006] According to a first aspect of the present invention, a thermostable microvesicular bacterium, YLW106, is provided and deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 27855. The thermostable microvesicular bacterium YLW106 provided by the present invention can efficiently synthesize tetrahydropyrimidine using marine polysaccharides as a carbon source. Furthermore, this thermostable microvesicular bacterium YLW106 exhibits high utilization of marine polysaccharides and can synthesize tetrahydropyrimidine in high yields, thus possessing broad prospects for development and application.

[0007] Preferably, the 16S rDNA nucleotide sequence of thermostable microbubble bacteria YLW106 is shown in SEQ ID NO: 1.

[0008] According to another aspect of the invention, the use of the above-mentioned thermostable microbubble bacterium YLW106 in the synthesis of tetrahydropyrimidine is provided.

[0009] According to another aspect of the present invention, a method for synthesizing tetrahydropyrimidine using the aforementioned thermostable microbubble bacterium YLW106 is provided, comprising the following steps: activating the thermostable microbubble bacterium YLW106 to obtain a seed culture; then inoculating the seed culture into a fermentation medium for fermentation, thereby synthesizing tetrahydropyrimidine, wherein the fermentation medium uses marine polysaccharides as a carbon source. Marine polysaccharides are mainly derived from marine crustaceans, brown algae, red algae, etc., and are abundant globally, making them an important non-food biomass resource. Compared with carbon sources such as glucose, marine polysaccharides are readily available and have the advantages of sustainability, diversity, and not competing with food crops for land. Therefore, the method for synthesizing tetrahydropyrimidine using the thermostable microbubble bacterium YLW106 provided by the present invention, using marine polysaccharides as a carbon source, can effectively synthesize tetrahydropyrimidine, and the thermostable microbubble bacterium YLW106 has a high utilization rate of marine polysaccharides, enabling the synthesis of tetrahydropyrimidine with a high yield. Furthermore, this method is low-cost, simple to operate, suitable for large-scale production, and has broad development and application prospects.

[0010] Preferably, the inoculation amount of seed liquid is 3-10% (v / v).

[0011] Preferably, the fermentation temperature is 40–50°C, and the fermentation time is not less than 24 hours. The above-mentioned suitable fermentation temperature and fermentation time can promote the synthesis of tetrahydropyrimidine by the thermostable microbubble bacterium YLW106 using alginate.

[0012] Preferably, the marine polysaccharide includes at least one of colloidal chitin, alginate, and agar. Chitin is a linear macromolecule composed of N-acetylglucosamine linked by repeating β-1,4 glycosidic bonds. It is the second largest biomass on Earth after cellulose and is widely found in the shells of crustaceans, insect carapaces, and fungal cell walls. Alginate is a long polymer chain composed of α-L-guluronicacid (G) and β-D-mannuronicacid (M) linked by 1,4 glycosidic bonds. It is mainly derived from marine brown algae, which is an abundant and renewable resource. Agar is a long chain composed of alternating β-D-galactose linked by 1,3 glycosidic bonds and 3,6-endo-L-galactose linked by 1,4 glycosidic bonds. It is mainly derived from red algae, which is an important component of marine algae. Chitin, alginate, and agar are all marine polysaccharides with huge reserves. Fermentation media composed of these polysaccharides as carbon sources can fully ferment the thermostable microbubble bacteria YLW106, resulting in a high yield of synthesized tetrahydropyrimidine, which has broad prospects for development and application.

[0013] Preferably, the marine polysaccharide is colloidal chitin. Fermentation media using chitin as a carbon source can promote thorough fermentation, improve the utilization rate of carbon source by the thermostable microbubble bacteria YLW106, thereby increasing the yield of tetrahydropyrimidine, which is beneficial for industrial production.

[0014] Preferably, the concentration of colloidal chitin in the fermentation medium is 10–130 g / L. On the one hand, the above-mentioned concentration of chitin, as a fermentation substrate, can promote the growth of thermostable microbubble bacteria YLW106; on the other hand, the above-mentioned concentration of chitin can improve the carbon source conversion rate and the carbon source utilization rate of thermostable microbubble bacteria YLW106, that is, promote the synthesis of tetrahydropyrimidine by thermostable microbubble bacteria YLW106, reduce the generation and accumulation of by-products, and improve the yield and purity of tetrahydropyrimidine.

[0015] The concentration of colloidal chitin in the fermentation medium was 110 g / L.

[0016] Preferably, the fermentation temperature is 40–50°C and the fermentation time is 48–96 hours. These fermentation conditions facilitate the full utilization of marine polysaccharides by the thermostable microbubble bacteria YLW106, reduce the production of byproducts, and increase the yield and purity of tetrahydropyrimidine.

[0017] Preferably, the fermentation medium also includes NaCl, with a concentration of 30–60 g / L. These low-salt conditions are beneficial to the growth and reproduction of the thermostable microbubble bacterium YLW106, and also promote the synthesis of tetrahydropyrimidine, thereby increasing the yield of tetrahydropyrimidine.

[0018] Preferably, the specific steps for strain activation are as follows: Thermoresistant microbubble bacteria YLW106 are inoculated into 2216E solid medium for a first-stage culture. Then, single colonies from the 2216E liquid medium are picked and inoculated into 2216E liquid medium for a second-stage culture, thereby obtaining the seed culture. The seed culture obtained by the above method has high strain activity and a large quantity, which can improve the utilization rate of thermoresistant microbubble bacteria YLW106, thereby increasing the yield of subsequent tetrahydropyrimidine synthesis.

[0019] Preferably, the first stage of cultivation is carried out at a temperature of 28–35°C for 36–54 hours; the second stage is carried out at a temperature of 42–48°C for 12–36 hours. These suitable cultivation conditions result in high activity and a large quantity of microorganisms in the activated seed culture, which is beneficial for promoting the synthesis of tetrahydropyrimidine and increasing its yield.

[0020] Preferably, the first stage of cultivation is carried out at a temperature of 30°C for 48 hours; the second stage of cultivation is carried out at a temperature of 45°C for 24 hours.

[0021] According to another aspect of the present invention, a microbial agent is provided, the active ingredient of which comprises the above-mentioned thermostable microbubble bacteria YLW106. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of strain YLW106 from Example 1.

[0023] Figure 2 This is the phylogenetic tree of strain YLW106 from Example 1.

[0024] Figure 3 This is a characteristic peak diagram of the tetrahydropyrimidine standard detected by high performance liquid chromatography (HPLC) in Example 2.

[0025] Figure 4 This is a characteristic peak diagram of tetrahydropyrimidine released by strain YLW106 as detected by high performance liquid chromatography (HPLC) in Example 2.

[0026] Figure 5 This describes the metabolic pathway by which strain YLW106 in Example 4 produces tetrahydropyrimidine using marine polysaccharides. Detailed Implementation

[0027] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Isolation and Identification of Strain YLW106

[0029] In this embodiment, a novel microbubble bacterium was isolated from Sargassum fusiforme collected from the beach of Yilong Bay, Qinglan Peninsula, Wenchang. Based on morphological characteristics, physiological and biochemical characteristics, and genetic characteristics (16S rDNA), the microbubble bacterium was identified as thermoresistant microbubble bacterium YLW106. This thermoresistant microbubble bacterium YLW106 was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC 27855.

[0030] 1. Separation

[0031] In this embodiment, Sargassum fusiforme was collected from the beach of Yilong Bay on Qinglan Peninsula in Wenchang. 2g of Sargassum fusiforme was weighed and placed in a 1.5mL sterile centrifuge tube containing 0.8mL of sterile water. The mixture was then ground into a homogenate using an electric grinder. This homogenate was used as the stock solution. The stock solution was then serially diluted to prepare a 10... -2 10 -3 Diluted solutions were prepared by spreading 100 μL of each solution onto sodium alginate medium and incubating them upside down in a 30°C incubator for 2-5 days. Colonies with good growth, different morphologies, and colors were selected for purification culture until pure cultures were obtained. The sodium alginate medium (1 L) formula is as follows: (NH4)2SO4 5g, K2HPO4 1g, NaCl 15g, MgSO4 1g, H2SO4·7H2O 0.01g, sodium alginate 6g, agar 17g, pH 7.5.

[0032] 2. Identification

[0033] (1) Morphological characteristics of strain YLW106

[0034] The isolated and purified strain YLW106 was inoculated onto 2216E solid medium and cultured in a 30°C incubator for 3 days. The colony morphology is as follows: Figure 1 As shown. By Figure 1 It can be seen that after culturing on 2216E solid medium for 3 days, strain YLW106 showed good growth, with slightly uneven edges, relatively translucent, smooth surface, and brown colonies.

[0035] (2) 16S rDNA identification

[0036] DNA Extraction: In this example, genomic DNA of strain YLW106 was extracted using the Chelex-100 method. 50 μL of 10% Chelex-100 solution was added to a 1.5 mL centrifuge tube. A small amount of bacterial cells was picked up with a sterile toothpick and added to the Chelex-100 solution. Simultaneously, the cells were ground several times with a sterile toothpick, vortexed to mix, heated at 100°C for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 10 min. The supernatant was then used for subsequent DNA amplification.

[0037] 16S rDNA sequencing: DNA extracted using universal 16S rDNA primers (27F 5'GAGTTTGATCCTGGCTCAG 3', 1492R 5'GGTTACCTTGTTACGACTT 3') was amplified by PCR. The PCR products were detected by gel electrophoresis and sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing. The 16S rDNA sequence of strain YLW106 is shown in SEQ ID NO:1.

[0038] Phylogenetic tree construction: The 16S rDNA gene sequence of strain YLW106 was compared and analyzed using the BLAST software tool on the NCBI (National Center for Biotechnology Information) database. Highly homologous 16S rDNA sequences were selected, and a phylogenetic tree was constructed using MEGA 7 (Neighbor-Joining) method. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that strain YLW106 is related to Microbulbifer thermotolerans JAMB A94 T The homology reaches 100%.

[0039] Based on the above morphological and 16S rDNA sequence homology analyses, the isolated and purified strain YLW106 was identified as a thermotolerant microbubble bacterium and named *Microbulbifer thermotolerans* YLW106. *Microbulbifer thermotolerans* YLW106 was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC 27855.

[0040] Example 2: Synthesis of Tetrahydropyrimidine from Strain YLW106 using Colloidal Chitin

[0041] 1. Synthesis of tetrahydropyrimidine

[0042] S1. Activation of microorganisms and preparation of seed culture:

[0043] The thermostable microbubble bacteria strain YLW106, preserved in glycerol tubes, was streaked onto 2216E solid medium and incubated at 30°C for the first stage of culture for 48 hours. Then, a single colony was picked and inoculated into 50 mL of medium. The seed culture was obtained by second-stage culture in 2216E liquid medium (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; the medium formula is as follows: peptone 5 g / L, yeast extract 1 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, pH 7.6 ± 0.2) at 45℃ and 200 r / min on a shaker for 24 h.

[0044] S2. Fermentation and preparation of fermentation broth:

[0045] The seed culture was inoculated into the fermentation medium at a rate of 5% (v / v) and fermented on a shaker at 45°C and 200 rpm / min for 36 h to obtain the fermentation broth. The fermentation medium was formulated as follows: colloidal chitin 10 g / L, NaCl 50 g / L, (NH4)2SO4 5 g / L, K2HPO4 1 g / L, MgSO4·7H2O 9.7 g / L, KCl 2 g / L, and anhydrous CaCl2 0.2 g / L. The preparation method of colloidal chitin is as follows: Chitin (purchased from Shanghai Sangon Biotech, item number: A500659-0500) is pulverized into powder by a pulverizer. The chitin powder is then added to concentrated hydrochloric acid at a ratio of 5g chitin powder to 30mL concentrated hydrochloric acid (35.5%) and soaked overnight at 4℃. Pure water is added while stirring at 4℃, and the chitin precipitate is washed with pure water until the pH is neutral to obtain colloidal chitin.

[0046] 2. Detection of bacterial biomass

[0047] The total protein content of the fermentation broth was determined using a modified Bradford protein assay kit from Shanghai Sangon Biotech Co., Ltd. 1 mL of fermentation broth was centrifuged at 12,000 rpm for 10 min. The precipitate was collected, resuspended in ultrapure water, and the cells were disrupted using an ultrasonic cell disruptor. After centrifugation at 12,000 rpm, the supernatant was collected for total protein determination. The total protein content reflects the growth status of the bacterial strain.

[0048] 3. Detection of tetrahydropyrimidine content

[0049] S1. Collection of intracellular tetrahydropyrimidine: Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, and collect the bacterial cells. Resuspend the bacterial cells in 1 mL of sterile water, and sonicate them using an ultrasonic disruptor (working conditions: sonication for 4 s, interval for 6 s, working for 5 min). After disruption, centrifuge at 12000 rpm for 2 min, and collect the supernatant. The supernatant is the sample to be tested for intracellular tetrahydropyrimidine.

[0050] S2. Collection of Tetrahydropyrimidine released extracellularly: Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, and collect the bacterial cells. Resuspend the bacterial cells in a hypotonic solution (10 g / L NaCl solution), and shake at 200 rpm for 20 min on a shaker at 30℃. Centrifuge at 12000 rpm for 2 min, and collect the supernatant. The supernatant is the test sample for tetrahydropyrimidine released extracellularly.

[0051] S3. Determination of tetrahydropyrimidine content using high performance liquid chromatography (HPLC): A Waters e2695 HPLC system equipped with a 2998 PDA detector was used for detection. The chromatographic conditions are as follows:

[0052] a) Liquid chromatography column: Waters C18 column (column length 250mm, column inner diameter 4.6mm, packing particle size 5um);

[0053] b) Mobile phase: 10% acetonitrile: 90% water;

[0054] c) Flow rate: 0.5 mL / min;

[0055] d) Column temperature: 30℃;

[0056] e) Injection volume: 10 μL;

[0057] f) Detection wavelength: 210nm.

[0058] g) Tetrahydropyrimidine standard was purchased from Aladdin, product number E292674-1g.

[0059] A stock solution of tetrahydropyrimidine standard was prepared at a concentration of 1 mg / mL and serially diluted to various concentrations. The tetrahydropyrimidine standard was first analyzed by HPLC under the chromatographic conditions described above, and a standard curve was plotted. Then, the sample to be tested was analyzed under the same chromatographic conditions, and the content of tetrahydropyrimidine in the sample was calculated based on the peak area and the standard curve.

[0060] 4. Test Results

[0061] The characteristic peak diagrams of the standard and the test sample containing tetrahydropyrimidine released extracellularly are shown below. Figure 3 , 4As shown, the intracellular production of tetrahydropyrimidine was 0.33 g / L, and the amount of tetrahydropyrimidine released extracellularly was 0.25 g / L. This indicates that the thermostable microbubble bacterium YLW106 provided by this invention has the ability to synthesize tetrahydropyrimidine, and the yield of synthesized tetrahydropyrimidine is high.

[0062] Example 3: Effect of NaCl concentration in fermentation medium on tetrahydropyrimidine synthesis by strain YLW106

[0063] 1. Synthesis of tetrahydropyrimidine

[0064] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The difference from Example 2 is that the concentrations of NaCl in the fermentation medium are 0 g / L, 10 g / L, 30 g / L, 40 g / L, 60 g / L, and 70 g / L, respectively. Apart from the above differences, the culture medium formulation and preparation procedures used in this embodiment are strictly consistent with those in Example 2.

[0065] 2. Detection of bacterial biomass

[0066] The test method for detecting bacterial biomass is the same as the test method performed in Example 2.

[0067] 3. Detect the amount of tetrahydropyrimidine released extracellularly.

[0068] The test method for detecting the amount of tetrahydropyrimidine released into the extracellular space is the same as the test method performed in Example 2.

[0069] 4. Test Results

[0070] Table 1. Effects of NaCl concentration on the growth and tetrahydropyrimidine synthesis of strain YLW106

[0071]

[0072] The test results are shown in Table 1. As can be seen from Table 1, under conditions of no NaCl or NaCl concentration higher than 70 g / L, strain YLW106 can hardly utilize colloidal chitin for growth; when the NaCl concentration is between 10 and 60 g / L, strain YLW106 can efficiently utilize colloidal chitin for growth. However, when the NaCl concentration was below 30 g / L, strain YLW106 produced almost no tetrahydropyrimidine. At NaCl concentrations of 40–60 g / L, strain YLW106 could produce tetrahydropyrimidine using colloidal chitin. Specifically, at NaCl concentrations of 50 g / L and 60 g / L in the culture medium, the intracellular tetrahydropyrimidine content was 0.33 g / L and 0.32 g / L, respectively, with little difference in yield. Furthermore, after shock with a hypotonic NaCl (10 g / L) solution, the tetrahydropyrimidine released by the cells was 0.25 g / L and 0.27 g / L, respectively. The calculated release rate of tetrahydropyrimidine was 75–85%. This indicates that the thermostable microbubble bacterium YLW106 can synthesize tetrahydropyrimidine through fermentation using colloidal chitin under low salinity conditions, and the yield of tetrahydropyrimidine synthesized in this way is even better when the NaCl concentration in the fermentation medium is 50 g / L.

[0073] Example 4: Effect of the type of marine polysaccharide in the fermentation medium on the synthesis of tetrahydropyrimidine by strain YLW106

[0074] 1. Synthesis of tetrahydropyrimidine

[0075] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The differences in composition compared to Example 2 are: (1) the carbon sources in the fermentation medium are alginate and agar, respectively; (2) the fermentation time is 24 hours during S2 fermentation and fermentation broth preparation. Apart from the above differences, the fermentation medium formulation and preparation operation used in this embodiment are strictly consistent with those in Example 2.

[0076] 2. Detection of bacterial biomass

[0077] The test method for detecting bacterial biomass is the same as the test method performed in Example 2.

[0078] 3. Detect the amount of tetrahydropyrimidine released extracellularly.

[0079] The test method for detecting the amount of tetrahydropyrimidine released into the extracellular space is the same as the test method performed in Example 2.

[0080] 4. Test Results

[0081] Table 2. Growth of strain YLW106 using marine polysaccharides as a carbon source and the amount of tetrahydropyrimidine released.

[0082] carbon source Total protein content (μg / mL) Tetrahydropyrimidine content (g / L) Brown algin 343.471 0.17 Agar 376.198 0.14 Colloidal chitin 447.080 0.21

[0083] The test results are shown in Table 2. Table 2 shows that strain YLW106 can synthesize tetrahydropyrimidine using various marine polysaccharides. Among them, the best yield was achieved by fermentation using colloidal chitin as the sole carbon source, releasing 0.21 g / L of tetrahydropyrimidine after hypotonic solution shock. The second best yield was achieved using alginate as the sole carbon source, releasing 0.17 g / L of tetrahydropyrimidine. The lowest yield was achieved using agar as the carbon source, at 0.14 g / L. This indicates that strain YLW106 can synthesize tetrahydropyrimidine using alginate, agar, and chitin. In other words, the thermostable microbubble bacterium YLW106 possesses a metabolic pathway for synthesizing tetrahydropyrimidine using alginate, agar, and chitin, as detailed below. Figure 5 As shown. By Figure 5 It is known that the thermostable microbubble bacterium YLW106, to adapt to a high-salt, high-osmotic-pressure environment, can synthesize alginate lyase, agarase, and chitinase to degrade marine polysaccharides such as alginate, agar, and chitin into monosaccharides that can be directly utilized by the cells. Through a series of enzymatic reactions, these monosaccharides are converted into acetyl-CoA, entering the tricarboxylic acid cycle to synthesize oxaloacetic acid, which is then converted into aspartic acid under the action of transaminases. Aspartic acid is a precursor for the biosynthesis of tetrahydropyrimidine. Aspartate kinase catalyzes its conversion into phosphorylated L-aspartic acid, which is then catalyzed by aspartate semialdehyde dehydrogenase to generate L-aspartate-β-semialdehyde (ASA), thus entering the specific synthetic pathway of tetrahydropyrimidine. First, L-diaminobutyric acid transaminase (EctB) catalyzes the production of L-diaminobutyric acid (DABA) from ASA. DABA is then acetylated to Nγ-acetyldiaminobutyric acid (ADABA) by L-diaminobutyric acid acetyltransferase (EctA). Finally, tetrahydropyrimidine synthase (EctC) catalyzes the synthesis of tetrahydropyrimidine from ADABA.

[0084] Example 5: Effect of colloidal chitin concentration on tetrahydropyrimidine production by strain YLW106

[0085] 1. Synthesis of tetrahydropyrimidine

[0086] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The differences in composition compared to Example 2 are: (1) the concentrations of colloidal chitin in the fermentation medium are 10 g / L, 30 g / L, 50 g / L, 70 g / L, 90 g / L, and 130 g / L, respectively; (2) the fermentation time is 72 hours during S2 fermentation and fermentation broth preparation. Apart from the above differences, the fermentation medium formulation and preparation operation used in this embodiment are strictly consistent with those in Example 2.

[0087] 2. Detection of bacterial biomass

[0088] The test method for detecting bacterial biomass is the same as the test method performed in Example 2.

[0089] 3. Detect the amount of tetrahydropyrimidine released extracellularly.

[0090] The test method for detecting the amount of tetrahydropyrimidine released into the extracellular space is the same as the test method performed in Example 2.

[0091] Table 3. Effects of different colloidal chitin concentrations on bacterial growth and the amount of tetrahydropyrimidine released.

[0092]

[0093] The test results are shown in Table 3. Table 3 shows that when using colloidal chitin at different concentrations as the sole carbon source for tetrahydropyrimidine fermentation, the tetrahydropyrimidine content synthesized by strain YLW 106 increased with increasing chitin concentration when the colloidal chitin concentration in the culture medium was between 10 and 110 g / L. The highest tetrahydropyrimidine yield (1.45 g / L) was achieved at a colloidal chitin concentration of 110 g / L. Conversely, when the colloidal chitin concentration in the culture medium was between 110 and 130 g / L, the tetrahydropyrimidine yield decreased with increasing chitin concentration, dropping to 1.38 g / L at a concentration of 130 g / L. Therefore, the tetrahydropyrimidine yield was optimal when the thermostable microbubble bacterium YLW106 fermented with 110 g / L colloidal chitin for 72 h.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A strain of microaerophilic thermophilic bacteria (Deinococcus sp.), Microbulbifer thermotolerans) YLW106 , deposited on July 7, 2023 at the China General Microbiological Culture Collection Center, and assigned accession number CGMCC 27855.

2. The application of the thermostable microbubble bacterium YLW106 as described in claim 1 in the synthesis of tetrahydropyrimidine.

3. A method for synthesizing tetrahydropyrimidine by using the heat-resistant microbubble bacteria YLW106 according to claim 1, characterized in that, The process includes the following steps: activating the thermostable microbubble bacteria YLW106 to obtain a seed culture; then inoculating the seed culture into a fermentation medium for fermentation to synthesize tetrahydropyrimidine, wherein the fermentation medium uses marine polysaccharides as a carbon source.

4. The method for synthesizing tetrahydropyrimidine by using heat-resistant microbubble bacteria YLW106 according to claim 3, characterized in that, The fermentation temperature is 40~50℃, and the time is not less than 24 hours.

5. The method for synthesizing tetrahydropyrimidine using the thermostable microbubble bacterium YLW106 as described in claim 3, characterized in that, The marine polysaccharide includes at least one of colloidal chitin, alginate, and agar.

6. The method for synthesizing tetrahydropyrimidine by using heat-resistant microbubble bacteria YLW106 according to claim 5, characterized in that, The concentration of the colloidal chitin in the fermentation medium is 10~130 g / L.

7. The method for synthesizing tetrahydropyrimidine by using heat-resistant microbubble bacteria YLW106 according to claim 4, characterized in that, The specific operation for activating the strain is as follows: the thermostable microbubble bacteria YLW106 is inoculated into 2216E solid medium for the first stage of culture, and then a single colony in the 2216E solid medium is picked and inoculated into 2216E liquid medium for the second stage of culture, thereby obtaining the seed culture.

8. An inoculant characterized in that, The active ingredient of the bacterial agent includes the thermostable microbubble bacteria YLW106 as described in claim 1.

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