CN110408567B - Saline-alkali-tolerant methylotrophic bacillus and viable bacteria preparation and application thereof - Google Patents
Saline-alkali-tolerant methylotrophic bacillus and viable bacteria preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a saline-alkali-tolerant methylotrophic bacillus and a live bacterial preparation and application thereof. The invention separates a strain of Bacillus methylotrophicus YJJJK-5 from the rhizosphere soil of the capsicum in the saline-alkali soil, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC NO. 17952. The strain has strong saline-alkali resistance, high planting rate in saline-alkali soil, good degradation effect on benzoic acid, p-hydroxybenzoic acid, cinnamic acid, ferulic acid and the like, and can relieve continuous cropping obstacles caused by phenolic acid autotoxic substances. After the bacterial strain is fermented, propyl gallate, beta-cyclodextrin and biochemical fulvic acid powder are added into fermentation liquor, and spray drying is carried out to obtain the viable bacteria preparation. The preparation method is scientific, the concentration of the fermentation broth is high, the loss of viable bacteria in the spray drying process is less, and the stability of the viable bacteria preparation in the storage process is high.
Description
Technical Field
The invention relates to a saline-alkali-tolerant methylotrophic bacillus, a live bacterial preparation and application thereof, and belongs to the technical field of agricultural biology.
Background
Hot pepper, another name: the cayenne pepper, the long pepper, the vegetable pepper and the lantern pepper are herbaceous plants growing in one year or limited years in the capsicum genus of the magnoliaceae class and the solanaceae class, and are one of the most widely planted vegetables in China. Continuous cropping obstacles are one of the main limiting factors influencing the yield and quality of pepper, and the problem of continuous cropping obstacles is increasingly serious with the increase of continuous planting years. The causes of continuous cropping obstacles are complex and mainly include: soil hardening and secondary salinization, unbalanced distribution of soil nutrients, deterioration of soil micro-ecosystem, aggravation of soil-borne diseases and insect pests, self-toxicity of plants and the like. Among them, phytoautotoxicity has been receiving increasing attention as one of the major factors causing continuous cropping obstacles.
The so-called autotoxicity is a chemical substance produced by a plant that may have a toxic effect on the same species or family of plants. The chemical substances can be released into the environment through the leaching of the overground part of the plant body, root secretion, plant stubble and other ways, and further the growth of the next crop is influenced. The phenolic acid substances are main autotoxic substances generated by the hot pepper, and comprise benzoic acid, p-hydroxybenzoic acid, cinnamic acid, ferulic acid and the like, have a remarkable inhibiting effect on the growth of the hot pepper, and can cause continuous cropping obstacles.
The main pathway of phenolic acid decomposition in soil is biodegradation, wherein microorganisms are the main biological group initiating biodegradation. The active microbial preparation is prepared by screening microbes with strong degradation effect on phenolic acid substances from soil, is used for degrading the phenolic acid autotoxic substances in the soil to reduce continuous cropping obstacles, and is a promising biological control measure. Many microorganisms have been reported to have the ability to degrade phenolic acids, including more than 20 genera, such as Phanerochaete chrysosporium, Serratia marcescens, Azospirillum lipolyticum, Bacillus amyloliquefaciens, Paenibacillus polymyxa and Streptomyces ochrolens, but there are rarely reports of phenolic acid degradation by saline-alkali tolerant methylotrophic bacilli.
Disclosure of Invention
The invention aims to provide a Bacillus methylotrophicus (Bacillus methylotrophicus) YJJJK-5 and a live bacterial preparation and application thereof. The bacillus methylotrophicus YJJJK-5 is separated from pepper rhizosphere soil in saline-alkali soil, has multiple excellent characteristics of salt and alkali resistance, degradation of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid, promotion of plant growth and the like, and can be used for producing microbial fertilizers.
The purpose of the invention is realized by adopting the following technical scheme:
bacillus methylotrophicus YJJK-5 separated from rhizosphere soil of saline-alkali soil pepper, wherein the preservation number of the strain in China general microbiological culture Collection center (CGMCC) is CGMCC NO. 17952; the preservation address is microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the preservation date is 2019, 6 months and 18 days.
The colony and thallus characteristics of the bacillus methylotrophicus YJJJK-5 strain are as follows: after 3 days of culture in NA medium at 37 ℃, the colony is round, convex, opaque, smooth and milky white. The thallus is rod-shaped, produces spores, has round spores, does not expand, has no parasporal crystal and is gram-positive.
The physiological and biochemical characteristics of the bacillus methylotrophicus YJJJK-5 strain are as follows: gram stain test positive, motility test positive, catalase test positive, oxidase test negative, nitrate reduction test negative, casein hydrolysis test positive, gelatin hydrolysis test positive, starch hydrolysis test negative, mannitol test positive, fructose test positive, galactose test positive, maltose test positive, and trehalose test positive.
The invention also discloses a live bacterial preparation taking the bacillus methylotrophicus YJJJK-5 as a main effective component.
The preparation method of the viable bacteria preparation of the bacillus methylotrophicus YJJJK-5 strain is characterized in that the bacillus methylotrophicus YJJK-5 is inoculated in a fermentation medium (the inoculum size is 2-5%) after amplification culture (an NB culture medium is adopted), and the bacillus methylotrophicus YJJJK-5 fermentation liquor is obtained after culture at 35-37 ℃ for 24-28 h. Adding 0.01-0.02% (w/w) of propyl gallate, 1-2% (w/w) of beta-cyclodextrin and 5-7% (w/w) of biochemical fulvic acid powder into the fermentation liquor of bacillus methylotrophicus YJJJK-5, stirring uniformly, and spray drying to obtain raw powder of viable bacteria preparation; and uniformly mixing the raw powder of the viable bacteria preparation and the soluble starch according to the mass ratio of 1 (5-10) to obtain the viable bacteria preparation of the bacillus methylotrophicus YJJJK-5.
The formula of the fermentation medium is as follows: 20-25g of soybean meal, 20-25g of corn steep liquor dry powder, 15-20g of corn meal, 1.0g of monopotassium phosphate, 1.5g of magnesium sulfate, 0.1g of manganese sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 8.5. The complex enzyme preparation comprises the following components: by weight, 50% of alkaline protease, 20% of neutral protease, 20% of cellulase and 10% of xylanase.
The use method of the live bacillus methylotrophicus YJJJK-5 bacterial preparation comprises the following steps: the bacillus methylotrophicus YJJJK-5 viable bacteria preparation is diluted by 100-200 times by adding water, and is applied by dipping roots during seedling transplanting or watering along with water.
The invention has the beneficial effects that:
the bacillus methylotrophicus YJJJK-5 can simultaneously degrade plant autotoxic substances such as benzoic acid, p-hydroxybenzoic acid, cinnamic acid, ferulic acid and the like, and after the bacillus methylotrophicus is cultured for 7 days, the degradation rates of the four phenolic acid substances are 81.9%, 77.2%, 68.6% and 91.4% respectively. The strain has strong saline-alkali tolerance, can survive in saline-alkali soil, and the live bacterial preparation is suitable for degrading the self-toxic substances generated by the hot pepper and reducing the continuous cropping obstacle of the hot pepper.
The production method of the viable bacteria preparation of the bacillus methylotrophicus YJJJK-5 is scientific, the viable bacteria number of the fermentation liquor is high, and the production cost is low. The complex enzyme preparation is added into the fermentation medium, and raw materials such as soybean meal, corn steep liquor dry powder, corn flour and the like are hydrolyzed in the temperature rise process during sterilization, so that the fermentation delay period can be shortened, the utilization rate of the raw materials is improved, and the viable bacteria concentration and the spore generation rate in the fermentation liquid are improved. During spray drying, antioxidant propyl gallate, embedding agent cyclodextrin and biochemical fulvic acid powder with protection effect on viable bacteria are added, so that loss of viable bacteria in spray drying process can be reduced, and stability of viable bacteria preparation in preservation process can be improved.
Drawings
FIG. 1 shows the colony morphology of Bacillus methylotrophicus strain YJJJK-5;
FIG. 2 shows the form of Bacillus methylotrophicus YJJJK-5;
FIG. 3 is a phylogenetic tree constructed based on a 16S rDNA partial sequence.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: screening of Bacillus methylotrophicus YJJJK-5
(1) Screening of saline-alkali tolerant strains
Bacillus methylotrophicus YJJJK-5 was isolated from the pepper rhizosphere soil. The soil is sampled in reclamation areas of Dongying cities in Shandong province and is saline-alkali soil. The specific separation method comprises the following steps: a10 g sample of the soil was weighed, added to a triangular flask containing 90mL of 0.9% physiological saline and a small amount of glass beads, and shaken at 180rpm at 37 ℃ for 30 min. Taking 1mL of soil suspension for 10-1-10-7Serial concentration gradient dilutions were made and then 10 taken-5、10-6、10-7The three dilutions were spread onto plates containing selection medium and cultured in an inverted format at 35-37 ℃ for 2 d. Single colonies were picked individually and streaked onto plates containing selection medium and cultured in an inverted format at 37 ℃ for 2 d. Picking single colony, transferring to the slant of the preservation culture medium test tube, culturing at 37 deg.C for 2d, growing with thallus Porphyrae, and storing in 4 deg.C refrigerator. And co-screening 194 saline-alkali tolerant strains.
The formula of the selective culture medium is as follows: 10g of peptone, 5g of yeast extract and composite inorganic salt (NaCl: KCl: MgCl)210: 1), 20g of agar, 1000mL of distilled water, pH9.0, and sterilizing at 121 ℃ for 20 min.
The preservation culture medium adopts an NA culture medium, namely a nutrient agar culture medium, and the formula of the culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000mL of water, and the pH value is 7.0-8.0.
(2) Screening of bacterial strains for degrading plant autotoxic substances such as benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid
And (3) respectively selecting single colonies of the halotolerant alkalium bacteria screened in the step (1), streaking the single colonies onto a flat plate containing a selective culture medium, and carrying out inverted culture at 35 ℃ for 3 d. Selecting single colony, transferring to the slant of storage medium test tube, culturing at 35 deg.C for 3d, growing with thallus Porphyrae, and storing in 4 deg.C refrigerator. 7 strains with different colony characteristics and bacterial morphology are separated out.
The formula of the selective culture medium is as follows: phthalic acid 1000mg, ammonium sulfate 0.5g, composite inorganic salt (NaCl: KCl: MgCl)210: 1), 20g, 1.5g dipotassium phosphate, 1000mL distilled water, 20g agar, and pH 7.0. The formula of the preservation culture medium is as follows: 1000mg of p-hydroxybenzoic acid, 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000ml of water, and pH 7.0.
Transferring 7 isolates into a basic culture medium added with 1000mg/L p-hydroxybenzoic acid, 1000mg/L benzoic acid, 1000mg/L cinnamic acid or 1000mg/L ferulic acid, carrying out shake culture at 35 ℃ and 180rpm for 3 days, measuring the degradation rate of the benzoic acid, the p-hydroxybenzoic acid, the cinnamic acid or the ferulic acid by adopting a high performance liquid chromatography, and screening 1 strain with better degradation effect on four self-toxic substances, namely YJJK-5.
The basic culture medium comprises the following components in percentage by weight: 5g of peptone, 0.5g of ammonium sulfate, 0.1g of magnesium sulfate, 0.2g of potassium chloride, 0.5g of sodium chloride, 1000ml of water and pH 7.0.
The conditions adopted when the degradation rates of the benzoic acid, the p-hydroxybenzoic acid, the cinnamic acid and the ferulic acid are measured by the high performance liquid chromatography are as follows: acetonitrile and water (pH 2.8 adjusted by acetic acid) are used as mobile phases, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 280 nm. Gradient elution is adopted, wherein acetonitrile is increased from 5% to 40% for 30-40min, acetonitrile is maintained for 40% for 40-45min, and acetonitrile is decreased from 40% to 5% for 0-30 min.
Example 2 identification of Bacillus methylotrophicus YJJJK-5
(1) Morphological and physiological biochemical characteristics
The colony and thallus characteristics of the bacillus methylotrophicus YJJJK-5 strain are as follows: after 3 days of incubation in NA medium at 37 deg.C, the colonies were round, prominent, opaque, smooth, milky white as shown in FIG. 1. The thallus is rod-shaped, produces spores, has round spores, does not expand, has no parasporal crystal, and is gram-positive, as shown in figure 2. The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and 7.0-8.0 of pH.
The physiological and biochemical characteristics of the bacillus methylotrophicus YJJJK-5 strain are as follows: gram stain test positive, motility test positive, catalase test positive, oxidase test negative, nitrate reduction test negative, casein hydrolysis test positive, gelatin hydrolysis test positive, starch hydrolysis test negative, mannitol test positive, fructose test positive, galactose test positive, maltose test positive, and trehalose test positive.
(2)16S rDNA sequence analysis
The YJJK-5 strain is inoculated into NB medium and cultured for 24h at 37 ℃ and 180r/min in a shaking way. The NB culture medium comprises the following components in percentage by weight: 3g of beef extract, 10g of peptone, 5g of NaCl, 1000ml of water and pH 7.0. Collecting thallus, extracting total DNA, and performing PCR amplification of 16S rDNA gene under the guide of universal primers F27: 5'-AGA GTT TGA TCA TGG CTC AG-3' and F27: 5'-AGA GTT TGA TCA TGG CTC AG-3' of prokaryotic 16S rRNA gene by using the total DNA as a template. After the amplified product is separated by 1% agarose gel electrophoresis, the amplified product is recovered by a gel recovery kit and is handed over to Shanghai Biotechnology Limited company for sequencing, and the obtained sequence is shown as a sequence table SEQ No. 1. The 16S rDNA sequences tested were aligned to sequences in the GenBank database and multiple sequence homology analysis was performed with MEGA 7.0 software and a phylogenetic tree was constructed as shown in figure 3.
Through morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain is Bacillus methylotrophicus and named as Bacillus methylotrophicus YJJJK-5. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 6 months and 18 days, and the preservation number is CGMCC NO. 17952.
Example 3 saline tolerance of Bacillus methylotrophicus strain YJJJK-5
And (3) verifying the saline-alkali tolerance of the bacillus methylotrophicus YJJJK-5 strain by adopting selective culture media with different saline-alkali degrees. The selective cultureThe formula of the base is as follows: 10g of peptone, 5g of yeast extract and composite inorganic salt (NaCl: KCl: MgCl)210: 1), 20-100 g of agar, 20g of distilled water, 1000mL of pH 8-10, and sterilizing at 121 ℃ for 20 min. And streaking the bacillus methylotrophicus YJJJK-5 strain from a slant culture medium onto selective culture medium plates with different salinity and alkalinity, culturing for 2-3 d at 35-37 ℃, and judging whether the strain is resistant to the corresponding salinity and alkalinity according to whether the bacillus methylotrophicus YJJJK-5 grows out. The growth conditions of Bacillus methylotrophicus YJJJK-5 on a high-salt and high-alkali culture medium are shown in the following table 1, and the Bacillus methylotrophicus YJJK-5 has strong saline-alkali resistance and can grow on a high-salt and alkali culture medium with the salt content of 10% and the pH value of 10.
TABLE 1 growth of Bacillus methylotrophicus YJJK-5 on high salt, high alkaline medium
Example 4 preparation of Bacillus methylotrophicus YJJJK-5 microbial inoculum
The preparation method of the viable bacteria preparation of the bacillus methylotrophicus YJJJK-5 strain comprises the following steps:
1) activating strains: inoculating Bacillus methylotrophicus YJJJK-5 to a test tube slant of NA culture medium, and culturing at 35 deg.C for 24h for activation. The formula of the NA culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000ml of water, and the pH value is 7.0.
2) Preparing seeds in a triangular flask: scraping the activated bacillus methylotrophicus YJJJK-5 lawn by using an inoculating ring, inoculating the lawn in an NB culture medium, and culturing for 24h at 35 ℃. The NB culture medium is a nutrient broth culture medium, and the formula of the NB culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl and 1000ml of water, and the pH value is 7.0.
3) Preparing strains in a seeding tank: inoculating 2% of seed in a triangular flask into a 10L seed tank containing 6L NB culture medium, culturing at 35 deg.C for 20h, stirring at 200rpm, ventilation rate of 3L/min for 0-6h, and ventilation ratio of 6L/min for 6-20 h.
4) Fermentation culture: inoculating the strain in seed tankThe seed was transferred to a 500L fermentor at 2% inoculum size. 300L of fermentation medium is filled in the fermentation tank, and the fermentation is carried out for 28h at 35 ℃ to obtain the fermentation liquor of the bacillus methylotrophicus YJJJK-5. The whole stirring speed is 180rpm, the ventilation rate is 200L/min for 0-6h, and the ventilation rate is 300L/min for 6-28 h. After the fermentation is finished, the viable count of the bacillus methylotrophicus YJJJK-5 in the fermentation liquor is (1-2) multiplied by 1010cfu/ml, the spore generation rate is 90-95%.
The formula of the fermentation medium in the step 4) is as follows: 20g of soybean meal, 20g of corn steep liquor dry powder, 15g of corn meal, 1.0g of monopotassium phosphate, 1.5g of magnesium sulfate, 0.1g of manganese sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 8.5. The complex enzyme preparation comprises the following components: by weight, 50% of alkaline protease, 20% of neutral protease, 20% of cellulase and 10% of xylanase. The neutral protease and the alkaline protease are produced by Tai' an Xin Deli biological technology limited company, the product specifications are 5 ten thousand U/g and 10 ten thousand U/g respectively, and the enzyme activity unit definition and the detection method thereof execute the national standard GB/T23527-2009 protease preparation. The cellulase and the xylanase are produced by Shandong Long Dabiol technology Co., Ltd, the product specifications are respectively 10 ten thousand U/g and 5 ten thousand U/g, the enzyme activity unit definition and the detection method of the cellulase execute the light industry standard QB 2583-.
The preparation method of the fermentation medium in the step 4) comprises the following steps: weighing raw materials according to the formula, dissolving in water, heating to 52 deg.C, maintaining the temperature for 1.5h, then heating to 121 deg.C, maintaining the temperature for 25min, and sterilizing.
5) Preparation of the viable bacteria preparation: adding 0.01% (w/w) of Propyl Gallate (PG), 1% (w/w) of beta-cyclodextrin and 5% (w/w) of biochemical fulvic acid powder into the fermentation liquor of the bacillus methylotrophicus YJJJK-5 in the step 4), uniformly stirring, and then carrying out spray drying at the inlet temperature of 200 ℃ and the outlet temperature of 80 ℃ to obtain the raw powder of the viable bacteria preparation. The biochemical fulvic acid powder is produced by fertilizer Limited liability company of Shandong quanlima, and the fulvic acid content of the biochemical fulvic acid powder is 40%.
Uniformly mixing the raw powder of the live bacillus methylotrophicus YJJJK-5 preparation and soluble starch according to the mass ratio of 1:7.5 to obtain the live bacillus methylotrophicus YJJJK-5 preparation, wherein the content of live bacteria is preferably 1.5 multiplied by 1010cfu/g。
Example 5 degradation test of Bacillus methylotrophicus YJJJK-5 for benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid
The preparation of the culture medium containing benzoic acid comprises the following components: benzoic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, complex inorganic salts (NaCl: KCl: MgCl)210: 1)20g, 1000ml of water, pH 7.0.
Preparing a culture medium containing p-hydroxybenzoic acid, wherein the formula is as follows: 1000mg of p-hydroxybenzoic acid, 5g of peptone, 0.5g of ammonium sulfate, and complex inorganic salts (NaCl: KCl: MgCl)210: 1)20g, 1000ml of water, pH 7.0.
Preparing a culture medium containing cinnamic acid, wherein the formula is as follows: cinnamic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, composite inorganic salt (NaCl: KCl: MgCl)210: 1)20g, 1000ml of water, pH 7.0.
Preparing a culture medium containing ferulic acid, wherein the formula is as follows: ferulic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, composite inorganic salt (NaCl: KCl: MgCl)210: 1)20g, 1000ml of water, pH 7.0.
Bacillus methylotrophicus YJJJK-5 was transferred into the above four media and cultured at 35 ℃ for 7 days with shaking at 180 rpm. 10mL of culture solution is taken, 3500r/min is centrifuged for 15min, the precipitate is discarded, and the supernatant is extracted by 10mL of dichloromethane. The extract phase was evaporated to dryness using a vacuum rotary evaporator and the residue was dissolved in 1mL of methanol for further use.
Detecting the residual amounts of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid in the sample by adopting a high performance liquid chromatography, and calculating the degradation rate. The parameter conditions are as follows: acetonitrile and water (pH 2.8 adjusted by acetic acid) are used as mobile phases, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 280 nm. Gradient elution is adopted, wherein acetonitrile is increased from 5% to 40% for 30-40min, acetonitrile is maintained for 40% for 40-45min, and acetonitrile is decreased from 40% to 5% for 0-30 min.
The detection result of the high performance liquid chromatography shows that the degradation rates of the autotoxic substances benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid are 81.9%, 77.2%, 68.6% and 91.4% respectively when the bacillus methylotrophicus YJJJJK-5 is cultured for 7 days.
SEQUENCE LISTING
<110> Shandong Zongtian Biotech Co., Ltd
<120> saline-alkali-tolerant methylotrophic bacillus, live bacterial preparation and application thereof
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1414
<212> DNA
<213> 16S rDNA gene of Bacillus methylotrophicus YJJJK-5
<400> 1
tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420
ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200
aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260
cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380
ccgaagtcgg tgaggtaacc ttttaggagc cagc 1414
Claims (7)
1. Saline-alkali-tolerant methylotrophic bacillus（Bacillus methylotrophicus）YJJK-5, and the preservation number of the strain is CGMCC NO. 17952.
2. The use of Bacillus methylotrophicus YJJJK-5 according to claim 1 for degrading benzoic acid, p-hydroxybenzoic acid, cinnamic acid or ferulic acid.
3. A live bacterial preparation comprising the Bacillus methylotrophicus YJJJK-5 according to claim 1 as a main active ingredient.
4. The method for preparing the viable bacteria preparation of claim 3, wherein the viable bacteria preparation is further prepared by culturing Bacillus methylotrophicus YJJJK-5 in an expansion way, inoculating the culture medium to the fermentation medium, culturing the culture medium at 35-37 ℃ for 24-28h to obtain a fermentation liquid of the Bacillus methylotrophicus YJJJK-5.
5. The method according to claim 4, wherein the fermentation medium is: 20-25g of soybean meal, 20-25g of corn steep liquor dry powder, 15-20g of corn flour, 1.0g of monopotassium phosphate, 1.5g of magnesium sulfate, 0.1g of manganese sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 8.5; the compound enzyme preparation comprises the following components in parts by weight: 50% of alkaline protease, 20% of neutral protease, 20% of cellulase and 10% of xylanase.
6. The method for preparing the viable bacteria preparation according to claim 4 or 5, characterized in that 0.01-0.02% of propyl gallate, 1-2% of beta-cyclodextrin and 5-7% of biochemical fulvic acid powder are added into the fermentation broth of bacillus methylotrophicus YJJJK-5, and the raw powder of the viable bacteria preparation is obtained after even stirring and spray drying; and uniformly mixing the raw powder of the viable bacteria preparation and the soluble starch according to the mass ratio of 1 (5-10) to obtain the viable bacteria preparation of the bacillus methylotrophicus YJJJK-5.
7. Use of the live bacterial preparation according to claim 3 for degrading benzoic acid, p-hydroxybenzoic acid, cinnamic acid or ferulic acid.
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