CN104232503B - A strain of Bacillus shachei and its application - Google Patents

Abstract

The invention discloses a Bacillus shacheensis HNA‑14, whose preservation number in the Korean Type Culture Collection is KCTC 33145. Through isolation, screening, and physiological and biochemical identification from the saline-alkali soil in Shache County, Xinjiang, it was determined that the strain was a short rod-shaped (Figure 1) Gram-positive bacterium, strictly aerobic, spore-forming, motile, and the cell size was 0.5 ‑0.8×1.0‑1.5 μm, the colony is yellow. The NaCl tolerance range is 3%‑20% (the optimal salt concentration is 7%‑15%), the pH growth range is 6.5‑10.0 (the optimal pH range is 7.5‑8.0), and the temperature growth range is 5‑40°C ( The optimum growth temperature is 25-30°C). The Xinjiang Shache halophilic bacteria Bacillusshacheensis HNA‑14 of the present invention can effectively decompose gelatin and starch, has Esterase (C4), Esterase lipase (C8), Valinearylamidase, Acid phosphatase and Naphthol‑AS‑B1‑phosphohydrolase enzyme activities, and the strain It has high adaptability to salt concentration and can survive under high salt concentration. The moderately halophilic bacteria can be widely used in the production of secondary metabolites under high salt concentration. The accompanying drawing is the morphology of strain HNA‑14 observed under a scanning electron microscope at 10,000 times.

Description

A strain of Bacillus shachei and its application

technical field

The invention relates to the field of microbial strains and applications thereof, in particular to the technical field of a bacillus and its applications.

Background technique

Moderate halophilic bacteria is not a taxonomic term, but a general term for a class of extremophiles whose best salt-tolerant concentration is between 3% and 15%. It is widely distributed in various high-salt environments, such as oceans, salt lakes, saline-alkali lands, salt ponds, and many fermented foods.

Due to their unique growth environment and salt-tolerant mechanism, moderately halophilic bacteria have many notable features: (1) they can grow at higher salt concentrations, reducing the risk of bacterial contamination during fermentation; (2) they do not require comparison Complex nutritional conditions, a wide range of carbon and nitrogen sources; (3) diverse physiological and biochemical characteristics, and great potential application value.

In recent years, with the continuous deepening of the research on moderate halophilic bacteria, more and more new species have been discovered, and people are excited about their potential application value. Nowadays, the applied research of moderate halophilic bacteria mainly focuses on the following aspects:

(1) Application in food industry. In the traditional food fermentation industry, the application of halophilic microorganisms has a long history. For example, food seasoning sauce is made by sealing and soaking crops such as wheat, millet and soybeans in about 19% NaCl solution and fermenting them for 9-10 months. . The scientists found that during this process, a moderately halophilic bacterium called Tetragenococcus halophilus played a major role in promoting and undertaking the fermentation. Its concentration of 108 CFU/mL of medium cells not only inhibited the growth of other bacteria, but also inhibited the growth of other bacteria. It can ensure that the flavor of the sauce is strong and delicious under the environment of lack of mixed amino acids. In Korea, people often like to pickle seafood, and the strain that plays a major role in this process is also a type of moderate halophilic bacteria called Halomonas alimentaria . Colonies of many moderately halophilic bacteria are also very attractive, often orange or pink, and beneficial species of them are also used to produce natural food additives.

(2) Application in the production of enzyme preparations. Industrial production usually needs to meet many special physical and chemical conditions, but it is often difficult to match the optimal conditions required by the enzyme reaction, which provides higher requirements for the adaptation conditions of the enzyme. The enzymes produced by moderate halophilic bacteria have a wide range of salinity and temperature tolerance, and the advantages of easy purification, so they have attracted widespread attention and have good commercial application prospects.

(3) Applications in the fields of genetic engineering and cosmetics. In a high-salt environment, moderately halophilic bacteria can often accumulate many compatible substances, such as trehalose, ectoine, betaine, proline, hydroxyectoine, etc. Because of its good osmotic protection function, it has attracted extensive attention in the fields of molecular genetic engineering and cosmetics. Among them, ectoine and hydroxyectoine are stabilizers called molecular chaperones, which have the function of stabilizing and protecting biological macromolecular enzymes and DNA in extreme environments such as high salt, high temperature, dryness or low temperature. At the same time, they also have the effect of preventing skin dryness and slowing down skin aging, and are a good cosmetic moisturizer.

(4) Applications in polymer production. After petroleum deposits, high salt concentration is often formed, and general surfactants and bio-emulsifiers are often difficult to adapt to, which increases the difficulty of petroleum recovery and utilization. Extracellular polymers formed by moderately halophilic bacteria undoubtedly have significant advantages.

(5) Applications in environmental protection. Modern industrial wastewater contains many difficult-to-treat chemical substances such as high-salt ions and toxic organic substances, and traditional biological methods often have little effect. Due to their strong adaptability and unique physiological and biochemical characteristics, some moderately halophilic bacteria can degrade toxic substances such as formaldehyde, organic phosphorus, and aromatic hydrocarbons such as phenol in very high salt concentrations, and are environmentally friendly. Pollution is a growing concern.

(6) Other applications. In addition to the above applications, moderately halophilic bacteria still show good application prospects in many other aspects. Such as the production of active substances such as antibiotics, the extraction of new enzymes such as restriction endonucleases, and the synthesis of emulsifiers are widely used.

Contents of the invention

Aiming at the current research status of halophilic bacillus at home and abroad, the present invention provides a new halophilic bacillus and its application. The halophilic bacillus is negative for alkaline phosphatase and positive for valine arylamidase Glucuronidase is negative, and the highest 16S rDNA similarity with all other strains of this genus is distributed between 95.655% and 96.348% (NCBI). These characteristics are obviously different from the effective species of Bacillus halophilus that have been reported publicly.

The present invention specifically provides a Bacillus shacheensis .

The present invention uses different temperature, pH value, salt concentration and culture medium as enrichment conditions, takes samples from Shache Salt Lake in Xinjiang, separates, purifies and cultures multiple strains of bacteria, and determines a strain of halophilic Bacillus ( Bacillus shacheensis HNA-14), named Bacillus shacheensis. The strain has been deposited in Budapest Treaty of Microorganisms International Depository Unit: Korean Collection of Type Cultures (KCTC) before the application date. The preservation time is March 26, 2013, and the preservation number is KCTC 33145, the address of KCTC is the Korea Institute of Bioscience and Technology, Yucheng District, Daejeon City, Korea, the postal code is 305-806, and it was identified as Bacillus shacheensis by multiphase classification of microorganisms. The bacteria is aerobic, Gram-positive, motile, short rod-shaped, with a size range of 0.5–0.8×1.0–1.5 μm, terminal spores, slightly swollen sporangia, yellow colonies, and regular round shapes , with smooth edges. The NaCl tolerance range is 3%-20%, the pH growth range is 6.5-10.0, and the temperature growth range is 5-40°C. Positive for oxidase and catalase, no nitrate reducing ability, can hydrolyze starch and gelatin, but cannot hydrolyze Tween-80 and cellulose. H2S and indole were not produced during its growth, and MR and VP tests were negative. Carbon sources available: D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, tetrazolium violet, D-raffinose, α-D - Lactose, β-methyl-D-glucoside, D-salicin, D-sorbitol, D-arabinol, inositol, pectin, D-gluconic acid, D-glucuronic acid, glucuronamide, L-lactic acid, D-malic acid, L-malic acid, lithium oxide, Tween-40, alpha-hydroxybutyric acid, alpha-hydroxybutyric acid, beta-hydroxybutyric acid, acetoacetic acid, propionic acid, acetic acid, butyric acid Sodium and sodium bromate; unavailable carbon sources: dextrin, vancomycin, tetrazolium blue, D-melibiose, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N -Acetyl-D-galactosamine, N-acetyl-neuraminic acid, α-D-glucose, D-mannose, D-fructose, D-galactose, 3-methyl-glucose, D-fucose, L-fucose, L-rhamnosine, 1% sodium lactate, fusidic acid, D-serine, D-mannitol, glycerin, D-glucose-6-phosphate, D-fructose-6-phosphate, D-Aspartic Acid, D-Serine, Troleandomycin, Rifamycin, Minocycline, Gelatin, Aminoacetyl-L-Proline, L-Alanine, L-Arginine, L-Aspartic Acid, L-L-Glutamic Acid, L-Histidine, L-Pyroglutamic Acid, L-Serine, Lincomycin, Guanidine Hydrochloride, Tetradecyl Sodium Sulfate, D-Galacturonic Acid L-galactonolactone, mucic acid, quinic acid, D-glucaric acid, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid, methyl ester, L-lactic citric acid, alpha-oxo Glutaric acid, bromosuccinic acid, nalidixic acid, potassium tellurite, gamma-aminobutyric acid, alpha-oxobutyric acid, formic acid, and aztreonam. The main fatty acids in cells are anteiso-C15:0 and iso-C15:0, the main quinone type is MK-7, the main type of cell wall peptidoglycan is meso-diaminopimelic acid (meso-DAP), and the main polar lipids are Diphosphatidylglycerol (DPG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI). The DNA (G+C) content was 48.6 mol%.

The enzymatic properties of the Bacillus shacheensis HNA-14 were detected by the API ZYM system, and the carbon source utilization was detected by the Biolog automatic bacterial identification system. For analysis, polar lipid type analysis refers to Actinomycetes Systematics, respiratory quinone components and G+Cmol% are analyzed by high performance liquid phase (HPLC).

In the present invention, by extracting the total DNA, amplification and sequencing of 16S rDNA genes, according to the sequencing results, Blast is used for comparison and analysis on NCBI, and the 16S rDNA sequences of related strains with high similarity are sorted out, and Clustal X1.83 is used for multiple After the sequences were matched and arranged, a phylogenetic tree was constructed by the Neighbor-Joining and Maximum-Likelihood methods in the program MEGA5.0.

It has been determined that the 16S rRNA sequence of Bacillus shacheensis HNA-14 is 1570bp long, as follows:

From the phylogenetic tree constructed based on the 16S rDNA gene, it can be clearly seen that Bacillus shacheensis HNA-14 and the three type strains of the genus Bacillus are clustered together, and the three closest type strains Bacillus clausii KSM- The similarities of K16 ^T , Bacillus clausii DSM 8716 ^T , and Bacillus rhizosphaerae SC-N012 ^T were 96.348%, 95.951% and 95.883%, respectively, all of which were lower than the similarity threshold of 97%, which is commonly used to delineate new species. Based on the analysis of phenotype, physiology, biochemistry and genotype of Bacillus shacheensis HNA-14, Bacillus shacheensis HNA-14 was preliminarily identified as a new species of Bacillus.

Based on the fact that Bacillus shacheensis can effectively decompose starch, it has enzyme activities of Esterase (C4), Esterase lipase (C8), Valinearylamidase, Acid phosphatase and Naphthol-AS-B1-phosphohydrolase, and has high adaptability to salt concentration , can make gelatin liquefaction and milk peptonization, etc., the Bacillus shacheensis HNA-14 provided by the invention can be widely used in the fields of food industry, enzyme preparation and genetic engineering.

The seed liquid of the strain was inoculated into 15% salt concentration TSB medium with 5% inoculum amount, fermented and cultured at 37°C and 200rpm for 3 days, centrifuged at 12000rpm to remove impurities and bacteria, and 65% ammonium sulfate at 1°C Salt out, collect the precipitate after centrifugation at 12,000rpm, redissolve in 25mM Tris-HCl at 1°C, dialyze in 5mM Ca ²⁺ and mM Tris-HCl at 1°C for 10 hours, centrifuge at 12,000rpm to remove impurities, and the supernatant is ready for amylase.

By implementing the specific technical indicators of the present invention, the following effects can be achieved.

The Bacillus shacheensis of the present invention can be used to ferment and produce amylase and gelatinase under high salt concentration conditions, and can also be used in Esterase (C ₄ ), Esterase lipase (C ₈ ), Valinearylamidase, Acid phosphatase and Naphthol-AS -B1-phosphohydrolase-related fields.

Description of the drawings: Figure 1 is the morphology of bacterial strain HNA-14 observed under a scanning electron microscope at 10,000 times. Figure 2 is the 16S rRNA gene sequence phylogenetic tree of strain HNA-14 and related classifications constructed by the neighbor-joining method; Bar indicates that the number of nucleotide substitutions per unit is 0.01.

detailed description

Example 1: Screening and isolation of Bacillus shachei

Sample collection and strain screening and isolation

The samples were collected in Shache County, Xinjiang Uygur Autonomous Region. The samples come from multiple areas, distributed between 76°1'57" to 77°46'30" east longitude and 37°27'30" to 39°15" north latitude. The soil 5-10cm from the surface was collected in a sterile glass bottle, air-dried, ground, sieved, and stored at 4°C for subsequent separation. The screening medium was phenol-enriched medium (PT), humic acid-enriched medium (HVA), sodium molybdate-enriched medium (HNA) and high-salt-enriched medium (YCSS), and the purification medium was pancreatic Peptone Soy Broth (TSB+10% NaCl), strains were screened by inoculating different enrichment media. Add 1 g of the soil sample into a shaker flask filled with 25 ml of liquid medium, and culture it with shaking at 28°C for 7 days. Then use a pipette gun to draw the enriched solution, make a 10-fold serial dilution, take 200 μl of 10 ^-1 ~ 10 ^-4 multiple dilutions and spread them on the corresponding medium plates respectively, and place them at 28°C for a further week of cultivation. Observe the growth status and morphological characteristics, transfer the grown colony to TSB solid medium, and then pick a single colony and transfer it to TSB slant medium after multiple streaks, and store it at 4°C for short-term storage. At the same time, take fresh bacterial liquid mixed with 25% glycerol and store at -80°C for long-term preservation. (All media formulations are listed in the table below)

Phenol enriched medium (PT): 7.5g casein peptone, 100g sodium chloride, 10g yeast extract, 690μl phenol, 1 unit of MS mixed solution, 1000ml sterile water, pH8.2-8.5.

Humic acid-enriched medium (HVA): 1g of humic acid, 150g of sodium chloride, 1 unit of MS mixed solution, 1000ml of sterile water, pH8.2-8.5.

Sodium molybdate enriched medium (HNA): 7.5g of casein peptone, 150g of sodium chloride, 10g of yeast extract, 1 unit of MS mixed solution, 1000ml of sterile water, pH8.2-8.5.

High-salt enrichment medium (YCSS): 7.5g casein peptone, 250g sodium chloride, 10g yeast extract, 1unit MS mixture, 1000ml sterile water, pH8.2-8.5.

MS mixed solution: the concentration is 0.05g/L compatible substance (betaine, proline, glycine, D-sorbitol, glutamate) mixed solution 1.5ml; the concentration is 0.05g/L amino acid (aspartic acid) acid, histidine, lysine, methionine, valine, leucine, tyrosine, glutamic acid) mixture 15ml; the concentration is 0.025mg/L vitamins (VB1, VB2, VB12, VB6, VH, VM, VPP) mixed solution 0.25ml.

Tryptone soybean broth medium (TSB+10% NaCl): tryptone 15g, soybean peptone 5g, NaCl 100g, agar powder 18g, distilled water 1000ml, pH8.0-8.2.

Description of strains:

The Bacillus shachei described in the present invention is an aerobic bacterium, Gram-positive, motile, short-rod-shaped, with a size range of 0.5-0.8×1.0-1.5 μm, terminal spores, slightly swollen sporangia, and colonies in the form of Yellow in color, round and regular in shape with smooth edges. The NaCl tolerance range is 3%-20% (the optimal salt concentration is 7%-15%), the pH growth range is 6.5-10.0 (the optimal pH range is 7.5-8.0), and the temperature growth range is 5-40°C ( The optimum growth temperature is 25-30°C). Positive for oxidase and catalase, no nitrate reducing ability, can hydrolyze starch and gelatin, but cannot hydrolyze Tween-80 and cellulose. It does not produce H2S and indole during its growth, and M.R. and V.P. tests are negative. The main fatty acids in cells are anteiso-C15:0 and iso-C15:0, the main quinone type is MK-7, the main type of cell wall peptidoglycan is meso-diaminopimelic acid (meso-DAP), and the main polar lipids are Diphosphatidylglycerol (DPG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI).

Example 2: Phenotypic characteristics of Bacillus shachei HNA-14

by BIOLOG GEN The MicroStation automatic microbial identification system obtained the carbon source utilization of HNA-14 as shown in the table below. The reference strain of HNA-14 is Bacillus clausii DSM 8716 ^T

Carbon sources available: D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, tetrazolium violet, D-raffinose, α-D - Lactose, β-methyl-D-glucoside, D-salicin, D-sorbitol, D-arabinol, inositol, pectin, D-gluconic acid, D-glucuronic acid, glucuronamide, L-lactic acid, D-malic acid, L-malic acid, lithium oxide, Tween-40, alpha-hydroxybutyric acid, alpha-hydroxybutyric acid, beta-hydroxybutyric acid, acetoacetic acid, propionic acid, acetic acid, butyric acid Sodium and sodium bromate; unavailable carbon sources: dextrin, vancomycin, tetrazolium blue, D-melibiose, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N -Acetyl-D-galactosamine, N-acetyl-neuraminic acid, α-D-glucose, D-mannose, D-fructose, D-galactose, 3-methyl-glucose, D-fucose, L-fucose, L-rhamnosine, 1% sodium lactate, fusidic acid, D-serine, D-mannitol, glycerin, D-glucose-6-phosphate, D-fructose-6-phosphate, D-Aspartic Acid, D-Serine, Troleandomycin, Rifamycin, Minocycline, Gelatin, Aminoacetyl-L-Proline, L-Alanine, L-Arginine, L-Aspartic Acid, L-L-Glutamic Acid, L-Histidine, L-Pyroglutamic Acid, L-Serine, Lincomycin, Guanidine Hydrochloride, Tetradecyl Sodium Sulfate, D-Galacturonic Acid L-galactonolactone, mucic acid, quinic acid, D-glucaric acid, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid, methyl ester, L-lactic citric acid, alpha-oxo Glutaric acid, bromosuccinic acid, nalidixic acid, potassium tellurite, gamma-aminobutyric acid, alpha-oxobutyric acid, formic acid, and aztreonam.

Example 3: Extraction of Bacillus shachei HNA-14 genome and 16S rDNA sequencing

The total bacterial DNA was extracted by SDS method, the 16S rRNA gene sequence of the strain was amplified by PCR method, and the DNA sequencing was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Go to the GenBank (http://www.ncbi.nlm.nih.gov/) database to submit the partial sequence of the 16S rRNA gene measured by each strain for registration to obtain the sequence number. Then perform BLAST comparison on NCBI (National Center for Biotechnology Information) to find the strain with the closest relationship. The corresponding strains with the closest relatives were compared by BioEdit, and then the Neighbor-Joinin (NJ) method and the maximum likelihood method (ML method) were used to construct a phylogenetic tree on the software MEGA 5.0, and the bootstrap of the phylogenetic tree was 1000. The corresponding PCR primers, reaction system, and conditions are as follows: PCR primer: PA ₈ : 5'-CCGTCGACGAGCTCAGAGTTTGATCCTGGCTCAG-3', PB ₁ : 5'-CCCGGGTACCAAGCTTAAGGAGGTGATCCAGCCGCA-3'; reaction system (25μl system): Mg ²⁺ 2.5μl, PA ₈ 2.5 μl, PB ₁ 2.5 μl, dNTP 0.75 μl, H ₂ O 14 μl, Taq enzyme 0.25 μl, reaction conditions 95°C pre-denaturation for 5 minutes, 94°C denaturation for 45 seconds, 50°C annealing for 45 seconds, extension for 1 minute, repeated 35 cycles, 72°C Keep warm for 10 minutes. The constructed phylogenetic tree is shown in Figure 2.

Embodiment 4: the application of Bacillus shachei HNA-14

Prepare gelatin liquefaction medium in a test tube, sterilize at 121°C for 20 minutes, inoculate the strain by puncture, and culture it in a 30°C incubator for 30 days, observe and record the degree of gelatin liquefaction at 5, 10, 20, and 30 days respectively. Cool the test tube for 20-30 minutes or rinse it with tap water for 30 minutes before observation, so that the degree of liquefaction can be observed more clearly. Results Analysis Bacillus sacchari HNA-14 can liquefy gelatin.

Embodiment five: the application of Bacillus shachei HNA-14

Prepare milk coagulation and peptonization medium in a test tube, boil for 30 minutes, then place in a high-speed centrifuge and centrifuge at 3000r/min for 30 minutes, remove the supernatant, and sterilize intermittently for 30 minutes at 112°C 2-3 times. Inoculate the strain to be tested, and culture it statically in a 30°C incubator for 30 days, observe and record the degree of milk coagulation and peptonization at 5, 10, 20, and 30 days respectively. According to the analysis of the results, Bacillus shachei HNA-14 coagulates the milk when it coagulates. After coagulation, the coagulation is hydrolyzed into liquid, which proves that it can peptonize milk.

Embodiment 6: the application of Bacillus shachei HNA-14

Prepare the starch hydrolysis plate medium, sterilize at 121°C for 20 minutes, inoculate the strain to be tested by the spot method, and the inoculation diameter should not exceed 5mm; keep it in a constant temperature box at 30°C for 48 hours, observe the growth situation, and when the growth is good, Iodine solution was added dropwise around the colony for detection, and the results showed that a transparent halo appeared around Bacillus shachei HNA-14 and did not change color, which proved that it had the ability to hydrolyze starch.

TCAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAGCGAACCAAAGGGAGCTTGCTCCCGGAGGTTAGCGGCGGACGGGTGAGTACCACGTGGGCAACCTGCCCCATGGACAGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAAGACCGACCTTCGCCTGGAGGTTGGTTGAAAGATGGCCTTTAAGGCTATCACCAAGGGATGGGCCCGCGGCGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAATGCCGCGTGAGTGAGGAAGGCCTTCGGGTCGTAAAGCTCTGTTGCGAGGGAAGAAGCAAGTACCGGTGGATAACGCCGGTACCCTGACGGTACCTCGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGGGGCTCAACCCCGAGCGGCCATTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGTTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATGCCCGTAGTGCCGAAGTCAACACATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAGGTCTTG ACATCCTTTGACCACCCTGGAGACAGGGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAATCTTAGTTGCCAGCATTGAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGGGTAGCGAAGCCGTGAGGTGAAGCCAATCCCAGAAAGCCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTAAGCTTGGTACCCGGGAATCTCTA

Claims (2)

1. A strain of Bacillus shacheensis HNA-14, the number of which is KCTC 33145 in the Korean Type Culture Collection. 2. Bacillus shacheensis HNA-14 according to claim 1, characterized in that the cells are aerobic, Gram-positive, motile, short rod-shaped, and the size range is 0.5-0.8×1.0- 1.5 μm, terminal spores, slightly swollen sporangia, yellow colony, regular round shape, smooth edges, NaCl tolerance range is 3%-20%, pH growth range is 6.5-10.0, temperature growth range is 5- 40°C, positive for oxidase and catalase, no nitric acid reduction ability, can hydrolyze starch and gelatin, but cannot hydrolyze Tween-80 and cellulose, does not produce H ₂ S and indole, MR and VP during its growth Negative test, able to utilize carbon sources: D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turranose, stachyose, tetrazolium violet, D- raffinose, α-D-lactose, β-methyl-D-glucoside, D-salicin, D-sorbitol, D-arabinol, inositol, pectin, D-gluconic acid, D-glucuronic acid, glucosinolate Romamide, L-Lactic Acid, D-Malic Acid, L-Malic Acid, Lithium Oxide, Tween-40, Alpha-Hydroxybutyrate, Alpha-Hydroxybutyrate, Beta-Hydroxybutyrate, Acetoacetate, Propionic Acid, Acetic Acid , sodium butyrate and sodium bromate; carbon sources that cannot be utilized: dextrin, vancomycin, tetrazolium blue, D-melibiose, N-acetyl-D-glucosamine, N-acetyl-β-D-mannose Amine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, α-D-glucose, D-mannose, D-fructose, D-galactose, 3-methyl-glucose, D-rock Alcohol, L-fucose, L-rhamnosine, 1% sodium lactate, fusidic acid, D-serine, D-mannitol, glycerin, D-glucose-6-phosphate, D-fructose-6 -Phosphate, D-Aspartic Acid, D-Serine, Troleandomycin, Rifamycin, Minocycline, Gelatin, Aminoacetyl-L-Proline, L-Alanine, L-Arginine Amino acid, L-aspartic acid, L-L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, lincomycin, guanidine hydrochloride, tetradecyl sodium sulfate, D-semi Lacturonate L-galactonolactone, mucic acid, quinic acid, D-glucaric acid, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid, methyl ester, L-lactic citric acid, α-oxoglutaric acid, bromosuccinic acid, nalidixic acid, potassium tellurite, γ-aminobutyric acid, α-oxobutyric acid, formic acid and aztreonam, the main fatty acid in cells is anteiso-C _15:0 and iso-C _15:0 , the main quinone type is MK-7, the cell wall peptidoglycan type is mainly meso-diaminopimelic acid (meso-DAP), and the main polar lipid is diphosphatidylglycerol ( DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), the G+C content of DNA is 48.6 mol%.