Alkali-resistant budding cocci, bacterial agent and application thereof

Abstract

The present invention discloses an alkali-resistant Blastococcus, a microbial agent, and applications thereof. The present invention provides Blastococcus alkalitolerans H2206, which is registered with the General Microbiology Center of the China Culture Collection Administration under the registration number CGMCC No. 26165. Blastococcus alkalitolerans H2206 can produce xylanase, and a microbial agent prepared using this strain as a main component can be used in food processing, feed additives, soil conditioning, organic waste degradation, organic fertilizer additives, and aquaculture.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to an alkali-resistant budding coccus and a bacterial agent and application thereof. Background Art

[0002] In terms of phylogenetic relationships, Blastococcus is a branch of actinomycetes in the family Geodermatophilaceae (Normand P. Geodermatophilaceaefam.nov., a formal description. Int J Syst Evol Microbiol 2006; 56: 2277–2278.).The genus Blastococcus was originally proposed by Ahrens and Moll (Ahrens R, Moll G. Ein neues knospendes Bakterium aus der Ost see. Archiv Mikrobiologie 1970; 70: 243–265.). It was later revised and supplemented by Urzi, Lee, and Hezbri et al. (Urzi C, Salamone P, Schumann P, Rohde M, Stackebrandt E. Blastococcus saxobsidens sp. nov., and emended descriptions of the genus Blastococcus Ahrens and Moll 1970 and Blastococcus aggregatus Ahrens and Moll 1970. Int J Syst Evol Microbiol 2004; 54: 253–259; Lee SD. Blastococcus jejuensis sp. nov., an actinomycete from beach sediment, and The genus Blastococcus is morphologically characterized by rod-shaped or spherical cells. Rod-shaped cells are often motile, dispersed, and not aggregated. Spherical cells are non-motile and often aggregate in clusters.(Urzi C, Salamone P, Schumann P et al (2004) Blastococcus saxobsidens sp.nov., and emended descriptions of the genusBlastococcus Ahrens and Moll 1970and Blastococcus aggregatus Ahrens and Moll1970. Int J Syst Evol Microbiol54:253–259; Lee SD(2006)Blastococcus jejuensissp.nov.,an actinomycete from beach sediment,and emended description of thegenus Blastococcus Ahrens and Moll 1970.Int J Syst Evol Microbiol 56:2391–2396;Normand P,Daffonchio D,Gtari M(2014)The Family Geodermatophilaceae.Procaryote.Springer,Berlin,pp 361–379.). Blastococcus has an optimal growth temperature of 28°C and an optimal pH of 7.0-8.0, and utilizes a broad spectrum of carbon and nitrogen sources. Cytochemically, the polar lipid composition of Blastococcus strains primarily consists of diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylinositol (PI); the primary respiratory quinone is MK-9 (H4) (Hezbri K, Louati M, Nouioui I, et al (2016) Blastococcus capsensis sp. nov., isolated from an archaeological Roman swimming pool and emended description of the genus Blastococcus, B. aggregatus, B. saxobsidens, B. jejuensis and B. endophyticus. Int J Syst Evol Microbiol. IJSEM-D-16-00597).

[0003] Currently, the genus Blastococcus contains 11 described species (https: / / lpsn.dsmz.de / genus / blastococcus). Some of these species were isolated from marine and coastal habitats (Blastococcus jejuensis sp.nov., an actinomycete from beach sediment, and emended description of the genus Blastococcus Ahrens and Moll 1970. Int J Syst Evol Microbiol 2006;56:2391–2396; Xi L,Ruan J,Huang Y.Diversity and biosynthetic potential of culturable actinomycetes associated with marine sponges in the China Seas. Int J Mol Sci 2012;13:5917–5932; Lee DW,Lee H,Kwon BO,Khim JS,Yim UH,Kim BS,Kim JJ.Blastococcus litoris sp.nov., isolated from sea-tidal flat sediment. Int JSyst Evol Microbiol 2018;68:3435-3440), and also isolated from plant endophytic habitats (Zhu WY, Zhang JL, Qin YL, Xiong ZJ, Zhang DF et al. Blastococcus endophyticus sp.nov., anactinobacterium isolated from Camptotheca acuminata. Int J Syst Evol Microbiol 2013;63:3269–3273.) and desert soil (Yang ZW, Asem MD, Li X, Li LY, Salam N, Alkhalifah DHM, Hozzein WN, Nie GX, Li WJ. Blastococcus deserti sp.nov., isolated from a desert sample. Arch Microbiol 2019;201:193-198.),UrzìC,Salamone P,Schumann P,Rohde M,Stackebrandt E.Blastococcus saxobsidenssp.nov.,and emended descriptions of the genus Int J Syst EvolMicrobiol 2004,54:253–259;Hezbri K,Louati M,Nouioui I,Gtari M,Rohde M etal.Blastococcus capsensis sp.nov.,isolated from an Int J Syst Evol Microbiol 2016:66:4864–4872;Hezbri K, Nouioui I, Rohde M, Schumann P, Gtari M et al.Blastococcus coliseisp.nov,isolated from an archaeological amphitheatre.Antonie van Leeuwenhoek2017:110:339–346;UrzìC,Brusetti L,Salamone P,Sorlini C,Stackebrandt E etal Environ Microbiol 2001:3:471–479;GtariM,Essoussi I,Maaoui R,Sghaier H,Boujmil R et al.Contrasted resistance of stone-dwelling Geodermatophilaceae species to stresses known to give rise toreactive oxygen species. FEMS Microbiol Ecol 2012;80:566–577; Hezbri K, NouiouiI, Rohde M, Sproer C, Schumann P, Gtari M, Klenk HP, Montero-Calasanz MDC, Ghodhbane-Gtari F.Blastococcus xanthinilyticus sp.nov.,isolated frommonument.Int J Syst Evol Microbiol 2018;68:1177-118;Louati M,Hezbri K,Montero-Calasanz MDC,Rohde M,Goker M,Ghodhbane-Gtari F,Klenk HP,Nouioui I,Gtari M.Blastococcus tunisiensis sp.nov.,isolated from limestone collected inTunisia.Int J Syst Evol Microbiol 2022;72:5441.). .

[0004] Most members of the genus Blastococcus are isolated from nutrient-poor, harsh stone surfaces, so this type of bacteria is often considered an excellent material for studying the adaptation mechanism of life to environmental pressure (Sghaier H, Hezbri K, Ghodhbane-Gtari F, Pujic P, Sen A et al. Stone-dwelling actinobacteria Blastococcussaxobsidens, Modestobacter marinus and Geodermatophilus obscurusproteogenomes. Isme J 2016; 10:21–29.). Summary of the Invention

[0005] The purpose of the present invention is to provide an alkali-resistant budding coccus and a bacterial agent and application thereof.

[0006] In a first aspect, the present invention claims a new species of the genus Synecococcus.

[0007] The new species of the genus Blastococcus claimed in the present invention is specifically Blastococcus alkalitolerans H2206, and its registration number in the General Microbiology Center of the China Culture Collection Administration of Microorganisms is CGMCC No. 26165.

[0008] The alkali-resistant Blastococcus alkalitolerans H2206 is an aerobic, mesophilic, alkali-resistant bacterium. It has no aerial hyphae differentiation, and its cells are rod-shaped with a cell size of about 0.4-0.6 μm×1.8-2.1 μm. Figure 1 Middle a; some cells are motile. Forms light yellow colonies on PYG medium with a diameter of 0.5-1.1 mm. The strain's growth temperature, salt tolerance, and acid-base tolerance range are 10-40°C, 0-8% NaCl, and pH 7.0-9.0, with optimal growth conditions at 28°C, 0-5% NaCl, and pH 7.0-8.0. Strain SD203 tests negative for oxidase and nitrate reduction. It utilizes a wide range of carbon sources, including most monosaccharides and polysaccharides.

[0009] In a second aspect, the present invention claims a culture.

[0010] The culture claimed in the present invention is the culture of the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, which is a substance obtained by culturing the alkali-resistant Blastococcus alkalitolerans H2206 in a bacterial culture medium.

[0011] In the above culture, the substances include the alkali-resistant Blastococcus alkalitolerans H2206 (the bacteria itself) and metabolites of the alkali-resistant Blastococcus alkalitolerans H2206.

[0012] In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0013] The term "culture" refers to any liquid or solid culture medium containing a microbial population after artificial inoculation and cultivation. This refers to the product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes subcultures obtained by subculturing microorganisms, which can be cultures of a single generation or a mixture of several generations.

[0014] In a specific embodiment of the present invention, the bacterial culture medium is specifically PYG medium.

[0015] In a third aspect, the present invention claims a metabolite.

[0016] The metabolite claimed in the present invention is the metabolite of Blastococcus alkalitolerans H2206 described in the first aspect above.

[0017] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are primary metabolites, such as monomers such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, during a certain growth period, use primary metabolites as precursors to synthesize substances with no clear function in the microorganism's life activities. The products of secondary metabolism are secondary metabolites, which are mostly compounds with relatively complex molecular structures. Based on their functions, they can be divided into types such as antibiotics, hormones, alkaloids, and toxins.

[0018] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0019] The bacterial agent claimed in the present invention contains the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, the culture described in the second aspect above, and / or the metabolites described in the third aspect above.

[0020] The bacterial agent is a bacterial agent for producing xylanase.

[0021] In the above-mentioned microbial agent, in addition to the active ingredient, the agent also contains a carrier. The carrier can be a biologically inert carrier commonly used in the pesticide field. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material, or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soy flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0022] The above-mentioned microbial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0023] As needed, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. may be added to the bacterial agent.

[0024] In a fifth aspect, the present invention claims protection for the use of the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above, in any of the following:

[0025] (A1) producing xylanase;

[0026] (A2) preparing a product for producing xylanase;

[0027] (A3) preparing a product having xylanase activity.

[0028] Furthermore, the application is application under normal conditions or stress conditions; the stress is salt stress and / or alkali stress.

[0029] The salt stress is a condition of less than 8% (8 g / 100 mL) NaCl, and the alkali stress is a condition of pH 7.0-9.0.

[0030] In a sixth aspect, the present invention claims protection for the use of the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above, in any of the following:

[0031] (B1) Food processing;

[0032] (B2) preparing feed additives;

[0033] (B3) Soil conditioning;

[0034] (B4) organic waste degradation;

[0035] (B5) Organic fertilizer additives;

[0036] (B6) Aquaculture.

[0037] Xylan is a hemicellulose composed of xylopyranose units connected by β-1,4 bonds, and naturally accounts for 30% of biomass. Xylanases are a class of xylan-degrading enzymes belonging to the hydrolase family, including endo-β-1,4-xylanases, exo-β-xylanases, and β-xylobiosidases. Xylanases can efficiently degrade cellulose, hemicellulose, and other substances found in natural environments, such as food, feed, organic fertilizers, agricultural byproducts, and soil, thereby improving biomass conversion efficiency. Therefore, the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect, the culture described in the second aspect, the metabolites described in the third aspect, or the bacterial agents described in the fourth aspect can be used in the above-mentioned (B1)-(B6).

[0038] Furthermore, the application is application under normal conditions or stress conditions; the stress is salt stress and / or alkali stress.

[0039] The salt stress is a condition of less than 8% (8 g / 100 mL) NaCl, and the alkali stress is a condition of pH 7.0-9.0.

[0040] In a seventh aspect, the present invention claims a product for producing xylanase.

[0041] The product for producing xylanase claimed in the present invention has the active ingredient of the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0042] In an eighth aspect, the present invention claims a product having xylanase activity.

[0043] The product having xylanase activity claimed in the present invention has as its active ingredient the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0044] In a ninth aspect, the present invention claims a method for producing xylanase.

[0045] The method for producing xylanase claimed in the present invention may comprise the following steps: fermenting and culturing the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above, and obtaining xylanase from the culture product.

[0046] In the tenth aspect, the present invention claims protection for the use of the alkali-resistant Blastococcus alkalitolerans H2206 described in the first aspect above in preparing the culture described in the second aspect above or the metabolite described in the third aspect above or the bacterial agent described in the fourth aspect above.

[0047] Experiments have shown that strain H2206, a new species of xylanase-producing Blastococcus, has been named Blastococcus alkalitolerans. Microbial agents prepared using this strain as a primary component can be used in food processing, feed additives, soil conditioning, organic waste degradation, and as an organic fertilizer additive in aquaculture.

[0048] Preservation Instructions

[0049] Classification and nomenclature: Blastococcus alkalitolerans;

[0050] Reference biological material: H2206;

[0051] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0052] Abbreviation of depository institution: CGMCC;

[0053] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0054] Deposit date: December 7, 2022;

[0055] The registration number of the CGMCC Collection Center is: CGMCC No.26165. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Figures 1 and 2 show the morphological observation results of strain H2206. a) shows the cell morphology of strain H2206 observed under a transmission electron microscope; b) shows the colony morphology of strain H2206 cultured on PYG medium at 28°C for 72 hours.

[0057] Figure 2 This is the analysis result of polar lipid components in strain H2206 cells.

[0058] Figure 3 A phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain H2206 and related strains, with Acidothermus cellulolyticus DSM 8971T (AJ007290) as the outgroup. DETAILED DESCRIPTION

[0059] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0060] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0061] Example 1. Isolation, screening and identification of strain H2206

[0062] 1. Isolation of strain H2206

[0063] Isolation medium: 2 g / L xylan, 5 g / L yeast extract, 2 g / L CaCO₃, 1 g / L K₂HPO₄, 0.5 g / L MgSO₄·7H₂O, 20 g / L agar powder, pH 7.2. Aztreonam (50 μg / L) and potassium dichromate (50 μg / L) were added as inhibitors.

[0064] Soil samples for bacterial strain isolation were collected from rhizosphere soil of Laji Mountain in Guide County, Hainan Tibetan Autonomous Prefecture, Qinghai Province. Fresh soil samples were air-dried at room temperature for 2 weeks and then dry-heated at 120°C for 15 minutes. Two grams of dry-heated soil were added to 18 mL of sterile saline and placed in a shaker at 28°C for 40 minutes at 200 rpm to fully suspend the soil particles. A gradient dilution was then prepared to obtain 10 -4 Dilution of soil sample suspension.

[0065] Isolation and Purification: 0.2 mL of the culture was spread onto a separation medium plate and incubated upside down at 28°C for 4 weeks. After 4 weeks, individual colonies were selected based on their characteristics (shape, color, size, surface gloss, etc.) and plated onto PYG medium plates (medium composition: 3 g / L peptone, 5 g / L yeast extract powder, 10 g / L glycerol, 1.25 g / L betaine, 1.25 g / L sodium pyruvate, 15 g / L agar, pH 7.5). The culture was purified by the quarter streak method. The resulting pure strain was stored in liquid nitrogen and frozen at -80°C using 20% ​​(v / v) glycerol as a protective agent.

[0066] In this experiment, strain H2206 was isolated and purified.

[0067] 2. Screening of xylanase-producing bacteria and determination of xylanase activity of strain H2206

[0068] Xylanases have been widely used in the paper industry for pulp biobleaching, the brewing industry, and the feed and food processing industries (Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev, 2005, 29: 3-23; Begq K, Kapoor M, Ahajan I, et al. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol, 2001, 56: 326-338; Fang Luoyun, Zou Xiaoting. Molecular biology and genetic engineering of xylanase genes. Chinese Feed, 2002, 7: 11-13.). Currently, the most promising application of xylanases is in fuel production. The synergistic action of cellulase and xylanase can completely hydrolyze plant fibers into monosaccharides, and then use genetic engineering and fermentation engineering to produce alcohol. This is currently a hot topic in fuel alcohol research and development and the most recommended production process.

[0069] Screening of xylanase-producing bacteria and determination of xylanase activity:

[0070] (1) Xylanase-producing bacteria screening medium: Xylan 10 g / L, MgSO4 1 g / L, K2HPO4 3 g / L, (NH4)2SO4 5 g / L, CaCl2 0.1 g / L, FeSO4 0.04 g / L, ZnSO4 0.03 g / L, KCl 0.02 g / L, CuSO4 0.001 g / L, MnSO4 0.0001 g / L. pH 7.0.

[0071] (2) Primary screening: The strain was inoculated into the screening culture medium and cultured at 28°C in a constant temperature shaker at 200 r / min for 72 h. The fermentation broth was centrifuged at 1500 g for 10 min, and the supernatant was retained as the crude enzyme solution. Take 0.5 ml of the above crude enzyme solution, add 0.5 ml of 0.1 mol / LMops-NaOH buffer to adjust the pH to 7.0. After oscillation and mixing, mix evenly with 0.5 ml of Azo-wheat arabinoxylan substrate at 50°C and incubate for 10 minutes. After 10 minutes, 2.5 ml of 95% ethanol was added to the system to terminate the reaction, and the mixture was vigorously shaken with a Vortex. Let it stand at room temperature for 10 minutes. Centrifuge at 1500 g for 10 minutes and measure the OD value of the supernatant. 590 nmThe absorbance value was calculated by referring to the standard curve. Under the above assay conditions, the amount of enzyme required to release 1 μmol of reducing sugar (calculated as xylose) per minute was defined as one enzyme activity unit (IU / ml). Strains with an enzyme activity unit greater than 10 were defined as positive strains in the initial screening.

[0072] (3) Optimization of enzyme activity conditions: The enzyme production conditions and enzyme activity of the positive strains screened initially were optimized according to the methods in the literature (Chen Hongge, Zhu Jing, Liang Gaiqin, Yan Zizheng, Zhang Shuzheng. Screening of acid xylanase-producing bacteria and enzyme production conditions. Acta Microbiologica Sinica, 1999, 39, 350-353; Le Yilin, Xiong Tao, Zeng Zheling, Cheng Chi. Ultraviolet mutagenesis of Trichoderma reesei Rut C-30 to improve xylanase. Food and fermentation industries, 2005, 74-76; Hu Yuanyuan, Zhang Shouwen, Xie Yinggen. Ultraviolet mutagenesis of Bacillus to select high-yielding xylanase strains. China Food Additives, 2006, 113-117), including the optimization of the fermentation culture medium, fermentation temperature and time, and enzyme reaction conditions (temperature, pH value, reaction time).

[0073] (4) Rescreening: The positive strains obtained from the initial screening were rescreened. The screened strains were inoculated into the screening medium and cultured at 28°C, 200 rpm, for 72 h. The fermentation broth was centrifuged at 1500 g for 10 minutes and then the enzyme activity was determined. The average value of the three replicates was used as the enzyme activity of the strain.

[0074] result:

[0075] In initial screening, strain H2206 produced a xylanase activity of 55 IU / ml. Optimized conditions for enzyme production and activity determination revealed that the optimal fermentation conditions for strain H2206 were xylan as the carbon source, ammonium nitrate as the nitrogen source, pH 7.0, and 28°C for 72 hours, with the highest enzyme activity. The optimal reaction temperature for xylanase production by strain H2206 was 55°C and the optimal pH was 7.5. Three independent replicates of these conditions were performed, yielding a xylanase activity of 75 IU / ml.

[0076] 3. Cell morphology observation and physiological and biochemical characteristics of strain H2206

[0077] Strain H2206 was cultured on PYG medium at 28°C for 72 h, and cell morphology was observed using a transmission electron microscope (JEDL, JEM-1400). The growth temperature range for strain H2206 was 4, 10, 28, 30, 32, 37, 40, 42, and 45°C; the growth salt concentration (NaCl) range was 0, 1, 3, 5, 7, 8, 9, and 10% (g / 100 mL); and the growth pH range was 8 steps (4, 5, 6, 7, 8, 9, 10, and 11) between pH 4 and 11. Physiological and biochemical characteristics of the strain were determined using API 50CH, API ZYM, and BiOLOG GEN III carbon source assay kits. Other physiological characteristics of the strains, including Gram staining properties, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined with reference to the Manual of Systematic Identification of Actinomycetes (Xu LH (2007). Actinomycete systematics: principles, methods and practices. Beijing: Science Press, 93-108.).

[0078] The identification results showed that strain H2206 is an aerobic, mesophilic, alkali-resistant bacterium. It has no aerial hyphae differentiation, and the cells are rod-shaped with a cell size of approximately 0.4-0.6μm×1.8-2.1μm. Figure 1 Middle a: Some cells can move. They form light yellow colonies on PYG medium, such as Figure 1 b. Colony diameter ranges from 0.5 to 1.1 mm. The strain's growth temperature, salt tolerance, and acid-base tolerance ranges are 10-40°C, 0-8% NaCl, and pH 7.0-9.0, with optimal growth conditions at 28°C, 0-5% NaCl, and pH 7.0-8.0. Strain H2206 tested negative for oxidase and nitrate reduction. It has a broad carbon source utilization range, capable of utilizing most monosaccharides and polysaccharides. The distinguishing physiological and biochemical characteristics of strain H2206 and its related strains are shown in Table 1.

[0079] Table 1. Comparison of the distinctive characteristics of strain H2206 and the closely related strains Blastococcus endophyticus DSM 45413(T) and Blastococcus aggregatus DSM 4725(T)

[0080]

[0081] Note: +, positive; -, negative; w, weakly positive.

[0082] 4. Detection of cytochemical composition of strain H2206

[0083] The cellular chemical components of strain H2206, such as fatty acids, menaquinone types, and polar lipids, were determined by GC gas chromatography, HPLC liquid chromatography, and TLC thin-layer chromatography (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDIinc; 1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M et al. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984; 2: 233–241.). The fatty acid composition of strain H2206 is shown in Table 2. The main fatty acid of strain H2206 is iso-C 16:0 and iso-C 17:1 ω9c. In strain H22064, the dominant menaquinone is MK-9 (H4). Polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), unknown glycophospholipids (GPL) and unknown phospholipids (PL) ( Figure 2 The cytochemical classification characteristics of the strains all conform to the taxonomic characteristics of the genus Blastococcus, supporting the classification of strain H2206 into the genus Blastococcus (Hezbri K, Louati M, Nouioui I, et al (2016) Blastococcus capsensis sp.nov., isolated from anarchaeological Roman swimming pool and emended description of the genus Blastococcus, B. aggregatus, B. saxobsidens, B. jejuensis and B. endophyticus. Int JSyst Evol Microbiol. IJSEM-D-16-00597).

[0084] Table 2. Fatty acid composition of strain H2206

[0085] fatty acid(%) H2206 <![CDATA[iso-C 14:0 ]]> 0.4 <![CDATA[C 14:0 ω5c]]> 0.3 C14:0 0.6 <![CDATA[iso-C 15:0 ]]> 6.8 <![CDATA[anteiso-C 15:0 ]]> 6.9 <![CDATA[iso-C 16:1 H]]> 7.8 <![CDATA[iso-C 16:0 ]]> 24.8 C16:0 8.3 <![CDATA[iso-C 17:0 ]]> 0.5 <![CDATA[anteiso-C 17:0 ]]> 1.4 <![CDATA[iso-C 17:1 ω9c]]> 21.2 <![CDATA[C 18:1 ω9c]]> 3.8 <![CDATA[C 16:0 2OH]]> 2.1 <![CDATA[10-Methyl C 17:0 ]]> 6.5 <![CDATA[iso-C 18:0 ]]> 1.1 <![CDATA[C 18:1 ω9c]]> 4.3 C18:0 1.2

[0086] 5. Determination of the phylogenetic status of strains

[0087] Genomic DNA from strain H2206 was extracted and sequenced, and the 16S rRNA gene sequence (SEQ ID No. 1) was compared online with the internationally recognized bacterial taxonomy analysis database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that strain H2206 of the present invention had the highest similarity to species of the genus Blastococcus (Table 3). The highest similarity of the 16S rRNA gene sequence of strain H2206 of the present invention to known species was: Blastococcus endophyticus DSM 45413 (T) (98.34%) and Blastococcus aggregatus DSM 4725 (T) (98.06%). These similarity values ​​are all below the 98.65% threshold for distinguishing prokaryotic microbial species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351.). This result suggests that strain H2206 does not belong to any known species within the genus Agrobacterium but rather represents a new species of the genus. A phylogenetic tree was constructed using 16S rRNA gene sequences from all valid species of the genus Agrobacterium and from species of neighboring genera in the family Geodermatophilaceae. The results showed that strain H2206 clustered with Blastococcus endophyticus DSM 45413 (T) (98.34%) and Blastococcus aggregatus DSM 4725 (T) (98.06%) in a stable subclade within the genus Blastococcus. Figure 3To further clarify the taxonomic status of the strain, the average nucleotide identity (ANI) between the whole genome sequence of strain H2206 and its closest relative, Blastococcus endophyticus DSM 45413(T), was compared and calculated on EZbiocloud (Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 2017;110:1281–1286.). Whole genome sequence comparison analysis showed that the average nucleotide identity (ANI) between the whole genome sequence of strain H2206 and its closest relative, Blastococcus endophyticus DSM 45413(T), was 80.1%. This value is well below the 95% ANI threshold for distinguishing prokaryotic microbial genotypes (Kim, M., Oh, HS, Park, SC, and Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64, 346–351.). This result further supports the conclusion that strain H2206 represents a new species within the genus Agrobacterium. The genomic GC content of strain H2206 was calculated to be 72.8% based on the complete genome sequence.

[0088] Table 3. 16S rRNA gene sequence comparison results of strain H2206 and its closely related bacteria

[0089]

[0090]

[0091] Based on the phenotypic and genotypic taxonomic data of strain H2206 and its comparison with closely related strains, we determined that the strain represents a new species of the genus Blastococcus and named it Blastococcus alkalitolerans. Because it can produce xylanase, microbial agents prepared with this strain as a primary component have applications in food processing, feed additives, soil conditioning, organic waste degradation, organic fertilizer additives, and aquaculture.

[0092] Blastococcus alkalitolerans H2206 was deposited in the China General Microorganism Culture Collection Center on December 7, 2022, and its deposit number is CGMCC No.26165.

[0093] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Alkali-resistant budding cocci ( Blastococcus alkalitolerans ) H2206, whose registration number in the General Microbiology Center of China Culture Collection Administration is CGMCC No.26165.

2. The alkali-resistant budding coccus according to claim 1 ( Blastococcus alkalitolerans ) H2206 culture, is the alkali-resistant budding cocci of claim 1 ( Blastococcus alkalitolerans ) The substance obtained by culturing H2206 in bacterial culture medium.

3. A bacterial agent, characterized in that: The bacterial agent contains the alkali-resistant blastococcus according to claim 1 ( Blastococcus alkalitolerans ) H2206 and / or the culture described in claim 2.

4. The microbial agent according to claim 3, characterized in that: The bacterial agent is a bacterial agent for producing xylanase.

5. The alkali-resistant budding coccus according to claim 1 ( Blastococcus alkalitolerans ) Use of H2206 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4 in any of the following: (A1) Production of xylanase; (A2) preparing a product for producing xylanase; (A3) Preparing a product having xylanase activity.

6. The use according to claim 5, characterized in that: The application is under normal conditions or stress conditions; the stress is salt stress and / or alkali stress.

7. The alkali-resistant budding coccus according to claim 1 ( Blastococcus alkalitolerans ) Use of H2206 or the culture according to claim 2 or the bacterial agent according to claim 3 or 4 in any of the following: (B1) Food processing; (B2) Preparation of feed additives; (B3) Soil conditioning; (B4) Organic waste degradation; (B5) Organic fertilizer additives; (B6) Aquaculture.

8. The use according to claim 7, characterized in that: The application is under normal conditions or stress conditions; the stress is salt stress and / or alkali stress.

9. A product for producing xylanase, the active ingredient of which is the alkali-resistant budding coccus ( Blastococcus alkalitolerans ) H2206 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

10. A product having xylanase activity, wherein the active ingredient is the alkali-resistant budding coccus according to claim 1 ( Blastococcus alkalitolerans ) H2206 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

11. A method for producing xylanase, comprising the steps of: treating the alkali-resistant budding cocci according to claim 1 ( Blastococcus alkalitolerans ) H2206 is fermented and cultured to obtain xylanase from the culture product.

12. The alkali-resistant budding coccus according to claim 1 ( Blastococcus alkalitolerans ) Use of H2206 in preparing the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

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