NL2034102B1 - Bacillus velezensis ye-1 antagonistic to aspergillus flavus and application thereof - Google Patents
Bacillus velezensis ye-1 antagonistic to aspergillus flavus and application thereof Download PDFInfo
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Abstract
Disclosed is a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and an application thereof, and relates to the technical field of microbial control. In the invention, an Aspergillus flavus antagonistic bacterium, namely Bacillus velezensis YE-1, is screened from lichens, and the preservation number is CCTCC NO: M 20221143. The experimental verification shows that the Bacillus velezensis strain antagonizes Aspergillus flavus, and has good resistance to high temperature, osmotic pressure, artificial gastric juice and artificial intestinal juice. Kunming mice are gavaged with Bacillus velezensis YE-1 solution, and the results show that the Bacillus velezensis YE-1 strain is safe and reliable. Therefore, the screened Bacillus velezensis YE-1 is used to prevent and control Aspergillus flavus pollution in food, feed and grain storage.
Description
BACILLUS VELEZENSIS YE-1 ANTAGONISTIC TO ASPERGILLUS FLAVUS AND
APPLICATION THEREOF
The invention relates to the technical field of microbial control, and in particular to a
Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and an application thereof.
Aspergillus flavus is a common saprophyte fungi, and pollutes food and feed at all stages of the food/feed chain. The Food and Agriculture Organization of the United States estimates that 25% of the global food supply is contaminated by Aspergillus flavus. Aflatoxin is produced in the late growth stage of Aspergillus flavus. Aflatoxin is a group of compounds with similar chemical structures. The basic structure of aflatoxin is coumarin and difuran ring. Aflatoxin that has been isolated and identified includes b1, b2, g1, g2, m1, m2, p1, gq, h1, gm, b2a and toxol.
Aflatoxin is a highly toxic substance that has been proven to have carcinogenic, teratogenic and mutagenic effects on human body. As early as 1993, it was classified as a class of carcinogens by the Cancer Research Institute of the World Health Organization (WTO). Not only because of their toxicity, but also because of their stability under many conditions, they have brought great health risks and international trade losses. Therefore, Aspergillus flavus pollution has always been an urgent problem to be solved in the agricultural market and food industry.
The control measures of Aspergillus flavus mainly include physical control, chemical control and biological control. Physical methods include dehumidification, temperature control, oxygen removal and ultraviolet irradiation, etc., but physical methods cause adverse effects or even damage to the nutritional components of grain and feed, and consume a lot of energy.
Chemical methods mainly use chemical mildew-proof agents to treat products; they are easy to produce by-products and residues of treatment agents, and bring potential safety hazards to health. A large number of studies have shown that many microorganisms inhibit the growth of
Aspergillus flavus, including bacteria, actinomycetes, yeast, algae and so on. Biological control has attracted more and more attention because of its advantages of good specificity, environmental friendliness, mild treatment conditions and easy industrialization.
Bacillus has entered the public’s vision because of its high stress resistance and good environmental adaptability, and has been widely used as a biological control agent. Bacillus velezensis is a new type of biocontrol bacteria discovered by Spanish scholars in 2005. It is very important to screen new microbial strains and obtain reliable results in vitro and in vivo for expanding probiotic germplasm resources. Although many scholars at home and abroad have studied antagonistic bacteria to prevent and control Aspergillus flavus and published papers, there are few researches on Bacillus velezensis to prevent and control Aspergillus flavus at present and there are some shortcomings.
The objective of the present invention is to provide a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus and an application thereof, to solve the problems existing in the prior art.
The strain effectively antagonizes Aspergillus flavus, has safety, and may be applied to food, feed and grain storage.
To achieve the above objective, the present invention provides the following solutions.
The invention provides a Bacillus velezensis YE-1 antagonistic to Aspergillus flavus; the preservation number of this bacterium is CCTCC NO: M 20221143, and the preservation time is
July 21, 2022; the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, China.
The invention also provides a microbial agent for antagonizing Aspergillus flavus, and the microbial agent includes the Bacillus velezensis YE-1 or its metabolites.
Preferably, active ingredients of the microbial agent include the living bacteria, solid culture or liquid culture solution of the Bacillus velezensis.
The invention also provides an application of the Bacillus velezensis YE-1 or the microbial agent, and the application includes any of the following: (1) inhibiting Aspergillus flavus growth; and (2) preventing or controlling aflatoxin pollution in food, grains or feeds.
Preferably, inhibiting Aspergillus flavus growth includes an inhibition of Aspergillus flavus spore germination, an inhibition of Aspergillus flavus bud tube elongation and an inhibition of
Aspergillus flavus mycelium expansion.
Preferably, the grains include corn and peanuts.
The invention also provides an application of the Baciilus velezensis YE-1 or the microbial agent in a preparation of feed additives.
The invention also provides a feed additive for antagonizing Aspergillus flavus, and the feed additive includes the Bacillus velezensis YE-1 or its metabolite.
Preferably, active ingredients of the feed additive are living bacteria, solid culture or liquid culture solution of Bacillus velezensis YE-1.
The invention discloses the following technical effects.
Firstly, in the invention, Bacillus velezensis YE-1 is screened, may antagonize Aspergillus flavus, and has a rod-shaped cell shape.
Secondly, Bacillus velezensis YE-1 has a better ability to resist adversity. The survival rate is 33.60% after 20 min treatment at 80°C. The survival rate is 19.75% after 2 h treatment with 7.5% sodium chloride. The survival rate is 33.57% after 3 h treatment in artificial gastric juice.
The survival rate is 64.24% after 6 h treatment in artificial intestinal juice. It is sensitive to most antibiotics.
Thirdly, Bacillus velezensis YE-1 significantly inhibits the spore germination, bud tube elongation and mycelium expansion of Aspergillus flavus.
Fourthly, Bacillus velezensis YE-1 obviously eliminates or inhibits the pollution of
Aspergillus flavus in peanuts and corn.
Fifthly, Bacillus velezensis YE-1 obviously eliminates or inhibits the pollution of Aspergillus flavus in complete feed.
Sixthly, Bacillus velezensis YE-1 has no potential safety hazard to Kunming mice.
Biochemical indexes, haematological indexes and anatomical observation of the mice are normal.
In order to explain the embodiment of the invention or the technical scheme in the prior art more clearly, the drawings used in the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the invention. For ordinary technicians in the field, other drawings may be obtained according to these drawings without making creative efforts.
FIG. 1 shows a morphology of Bacillus velezensis YE-1; (A) Bacillus velezensis YE-1 grows on LB medium at 37°C for 1 day, and forms milky white, oval, wavy, low concave, dull, opaque and sticky colony; (B) it is a morphology of Bacillus velezensis YE-1 under the thermal field scanning electron microscope at 3.00KX, and the bacterium is rod-shaped; the diagram is with a scale of 3 um.
FIG. 2 shows bacteriostatic effects of Bacillus velezensis YE-1 on Aspergillus flavus; (A) control group, (B) experimental group.
FIG. 3 shows inhibitory effect of Bacillus velezensis YE-1 on spore germination, bud tube elongation and mycelium expansion of Aspergillus flavus; (A) inhibition results of spore germination, (B) inhibition results of bud tube elongation, and (C) inhibition results of mycelium expansion; in the figure, a, b, ¢, d indicate significant differences, and p<0.05.
FIG. 4 shows inhibition effects of Bacillus velezensis YE-1 on Aspergillus flavus in corns and peanuts; (A) peanut control group; (B) peanut treatment group; (C) corn control group; and (D) corn treatment group.
FIG. 5 shows inhibition effects of Bacillus velezensis YE-1 on complete feed of pigs; (A) control group; and (B) experimental group.
FIG. 6 shows tolerance test results of Bacillus velezensis YE-1 to temperature, osmotic pressure, artificial gastric juice and artificial intestinal juice; a is a tolerance graph at different temperatures, b is a tolerance graph at different osmotic pressures, c is a tolerance graph at different treatment times in artificial gastric juice, and d is a tolerance graph at different treatment times in artificial intestinal juice. * means there is significant difference, and p < 0.05; ** indicates a highly significant difference, and p < 0.01.
FIG. 7 is a graph showing the changes in body weight and food intake of mice in each group by Bacillus velezensis YE-1 during 14 days of intragastric administration; a is a result chart of female mice's weight change, b is a result chart of male mice’s weight change, c is a result chart of female mice’s food intake change, and d is a result chart of male mice’s weight intake change.
FIG. 8 shows pathological effects of Bacillus velezensis YE-1 on liver and kidney tissues of
Kunming mice.
Various exemplary embodiments of the present invention will now be described in detail, which should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terms described in the present invention are only for describing specific embodiments, and are not intended to limit the present invention. In addition, as for the numerical range in the present invention, it should be understood that every intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range and every smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention relates. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.
Without departing from the scope or spirit of the invention, it is obvious to those skilled in the art that many modifications and changes may be made to the specific embodiments of the specification of the invention. Other embodiments derived from the description of the present invention will be apparent to the skilled person. The specification and examples of this application are only exemplary.
As used herein, the terms “including”, “comprising”, “having”, “containing”, etc. are all open terms, which mean including but not limited to.
Embodiment 1 Isolation, screening and identification of a Bacillus velezensis YE-1 antagonizing Aspergillus flavus (1) Isolation of antagonistic bacteria
Taking lichens from Baiyin City, Gansu Province, and putting in a sterilized 500 ml conical flask containing 300 ml of sterile water; shaking at 160 r/min for 15 min to obtain suspended bacteria solution, subpackaging the suspended bacteria solution into a 1.5 ml EP tube and treating in a water bath at 85°C for 20 min; coating 100 pl of suspended bacteria solution on LB 5 agar medium, and culturing in an incubator at 37°C upside down overnight; selecting single colony into LB broth medium and culturing overnight at 37°C and 160 r/min; mixing the suspension liquid with 50% glycerol in equal ratio and storing in glycerol storage tube, and storing in -80°C refrigerator for later use. (2) Screening of antagonistic bacteria
Preparation of Aspergillus flavus spore suspension: activating and culturing Aspergifius flavus strain in potato glucose agar medium (PDA), and then culturing in an incubator at 25°C for 10 days; taking Aspergillus flavus mycelium cake and putting it in a conical flask containing 30 ml of sterile water, shaking it evenly with a vortex meter, and filtering it twice with sterile gauze to remove mycelium and impurities; counting the spores with a blood cell counting plate under an optical microscope, and adjusting the spore concentration to 10%/ml; coating 100 HI of Aspergillus flavus spore solution on PDA culture medium, culturing in 25°C incubator for 24 h, and punching Aspergillus flavus mycelium cake on Petri dish with a 7 mm hole punch; transferring Aspergillus flavus mycelium cake to a centre of a new PDA plate, culturing in an incubator at 25°C for 24 h, drawing 20 pl of activated bacteria liquid on two sides about 2 cm away from the Aspergillus flavus mycelium cake, and culturing in an incubator at 25°C for 48 h. (3) Identification of antagonistic bacteria
Morphological identification: the single colony state of the strains in logarithmic growth period with stable colony size is described, mainly including colony size, colour, transparency, colony surface state and colony edge state. Then, the colony is observed by scanning electron microscope. As shown in FIG. 1, after Bacillus velezensis YE-1 grows on LB medium at 37°C for 1 day, it forms milky white, oval, wavy, low concave, dull, opaque and sticky colony. The morphological scale of Bacillus velezensis YE-1 at 3.00KX of thermal field scanning electron microscope is 3 um, and the bacterium is rod-shaped.
As shown in FIG. 2, in the growth direction of Bacillus velezensis YE-1, the mycelium growth of Aspergillus flavus is obviously inhibited. The strains that inhibit the growth of
Aspergillus flavus are selected, and the 16s rDNA, translation initiation factor IF-2 (infB) and topoisomerase (topA) genes are amplified by extracting the DNA of the strains, and then sent to
Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.
The primers, PCR reaction conditions and amplified fragment sequences used in the above identification are as follows:
PCR amplification system:
Table 1 Amplification system
Table 1 lists the PCR systems used to amplify the 716s rDNA, infB and topA gene sequences of biocontrol bacteria Bacillus velezensis YE-1 strain.
Gene primers and PCR conditions:
Table 2 Amplification primers and PCR conditions
Annealing time (s) (°C)
F: AGAGTTTGATCCTGGCTCAG rDNA R: TACGGCTACCTTGTTACGACTT
FATGGCTAAAATGAGAGTTTACGAATATGS
R: TCACTTTCTTTCGATTTCCTGCATGACAT
F: AACGCGGTCAACOGAATGATTGAA (OPA IR TGTAGGAATGTCGAGOGTATCATEE > 0
The primers used to amplify 765 rDNA, infB and topA gene sequences of Bacillus velezensis YE-1 strain, and the annealing temperature and extension time during PCR amplification are listed in Table 2. The specific conditions of PCR are: pre-denaturating 94°Cx5 min, [94°Cx30 s, annealing temperaturex30 s, 72°Cx1 extension time] x30 cycles, 72°Cx5 min.
After the PCR, gel electrophoresis verifies that the fragment size is correct, and the PCR products are sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.
The amplified nucleotide sequence is shown in SEQ ID NO: 1-3, and the sequencing results are compared in NCBI (National Center for Biological Information) database by Blast.
The comparison result shows that the strain belongs to Bacillus velezensis, and the strain number is YE-1. The Bacillus velezensis YE-1 antagonistic to Aspergillus flavus has been deposited in China Center for Type Culture Collection on July 21, 2022, with the preservation address of Wuhan University, China, and the preservation number is CCTCC NO: M 20221143.
SEQ ID NO: 1 is as follows: gaaagtggcgggcgtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtg agtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtctgaaccgcatggtt cagacataaaaggtggctteggctaccacttacagatggacccgcggcgcattagctagttagtgaggtaacggctcaccaaggcg acgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagg gaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaa gaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggta atacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctca accggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagaga tgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattag ataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactc cgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcga agcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtg gtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgg gcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacaca cgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtct gcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgc ccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgacaagaagg.
SEQ ID NO: 2 is as follows: aacgcccttcaatgtttcagcaggaattttgactgcgctgaaaaacatggatatagtagtaaataaccatatggcaatgcttgaa gaaaagaccattaagcagcttgatgcgaaatttaaaaaaggcggcgccggcgttacatctcagaagcctgcggaaacgaacaaa aacaagccgcaagggattaatcagcagcctgctgggaatcaaccaaacaaaattcgagacggaaagaagaatgacgtgcagaa taatcaatttaacaaaaacaagaagaacaacaacaacaaaaacaaaaacaaacgcaatcataacaacaaaaatcagtatcagc aaaaaccgctgaagccgaaaaaagagcttcctgagaaaattacgttttccggctctttaacagtcggggccttagccgaagaacttg gcaaagaaccgtctgaactcattaaaaaactgatgcttctgggcgtaatggctacgattaaccaggagcttgacaaagacacaatcg aactgatcgcatctgaatacggtgtggaaacagaagaagtgattgtgctcgaagaaactgagcttgaaaaatatgaagaggctgac aaagaagaagatcttcaaatccgtccgcctgtcgtgacgatcatgggccacgttgaccacgggaaaacgaccctccttgacagcatc agaaaaacaaaagttgttgaaggtgaagccggcggaatcacccagcatatcggggcgtaccagatcgaagaaaacggcaagaa aatcacgttcttagatactccgggacacgcggcgttcacaacaatgcgtgcgcgcggtgcggaagtaactgatattaccattttagtcgt agcggctgatgacggcgttatgccgcaaacagtcgaagccatcaaccatgcgaaagcggctgaggttccgattatcgttgccgtgaa taaagtggataaagaaagcgcgaaccctgaccgtgtcatgcaggagctgactgaatacggactcgttcctgaagcatggggagga gaaacgattttcgttcctctgtccgctcttacaggcaagggaattgacgagcttgtcgaaatgattctgcttgtcagtgaagtggaagagc tgaaagccaatccgaaccgtcaggcaaaaggaacggttattgaagctgagctcgataaaggaagaggatctgtcgcgacacttctc gttcaaacgggaacattgcatgtcggagatccgatcgttgtcggaaatacattcggccgcgtgcgggccatggtcaatgatctcggcc gccgcgtgaaaacagccggaccgtccacacctgtagaaatcacgggcttaaacgacgtaccgcaggctggcgatcaattcctcgtc tttaaagatgaaaaaacagcccgttctgtcggtgaagcacgcgcttccaagcaactggaagaacagcgcagcgacaaagcgaag ctgagcctcgatgatctgtttgaacaaattaaacaaggcgatgttaaagacatcaacttaatcgtaaaagctgacgttcaaggttcagc cgaagcgttaacagccgctcttcaaaaaattgaagtagaaggcgtaaaagtgaaaatcattcacacaggcgtcggtgcgattacgg aatcagacatcattctcgcatctgcttcaaatgcaatcgttatcgggtttaatgtgcgcccagacggaaatgctaagagcacggctgaa gctgaaaatgtagatatccgtcttcaccgtatcatttacaaagtcatcgaagaaattgaagcggcgatgaaaggaatgcttgatcctga gtacgaagaaaaagtcatcggtcaggttgaagtgcgccaaacattcaaagtgtcgaaaatcggcacgattgccggcggatatgtaa cagacggccacattacacgtgacagcggactccgcctgatccgtgacggcgtggtcatcttcgaaggggaagtagacgttctgaaa cgcttcaaagatgatgtgaaagaagtttcacaaggatatgaatgtggtataacgattaagaaatacaatgacattcgcgaaggcgact cacttactgcgc.
SEQ ID NO: 3 is as follows: ccggggcacgcgaaacgattgacgttatttagggaagaaatataaagtcaaagcttcaatgggacatgtccgggatcttccga aaagtcaaatgggagttgacatcgaacagaactttgaaccgaaatatattacgatccgcggaaaaggccctgtittaaaagaattaa aaacagcggcaaagaaagcaaaaaaagtctatcttgcggccgaccccgacagagaaggggaagcgattgcatggcatctcgca cacagccttgatctggatctcagctctgactgccgggtcgtctttaatgaaataacgaaagacgccattaaagaatcatttaaacatccg cgcatgattaacatggatcttgtcgatgcgcagcaggcgcgccgcatactagacagactcgtcggatacaaaatcagcccgattcttt ggaaaaaagtaaaaaaaggcctcagtgcgggccgtgttcaatccgttgcgctccggctgatcatcgaccgggaaaaggaaatcaa tgactttaaacctgaagagtactggacgattaccggctcatttttaaagggaaaagagacgtttgaagccggatttttcgggaaaaacg gcaaaaagcttcctttaaaaagcgaagaagacgtaaaagccgtccttgctcagctgaaaggcaataagtatacagttgacaaggtg acgaaaaaagagcgcaaacgaaatcccgctctgccgtttacaacgtccactctgcagcaggaggctgccaggaagctgaatttca gagcgaagaaaacgatgatgatcgctcagcaattatatgaaggaattgatcttggcaaagaagggacagtcggactcatcacgtat atgagaacggactcaacgcggatttccaatacagccgttgaagaggcgtcagctittattgatcaggcatacggaaaagagtacttag cgggaaaacggaaaccggcaaagaaaaatgaaaacgctcaagatgcccacgaagcgatccgcccgacttcggtgctcagaaa acctgccgacttaaaagcggtgcttggaagagaccagctcagactatacaagctgatttgggagcgttttgttgcaagccagatggcg cctgccgtccttgatacgatgagcgtcgatctcagcaataacggattgacattccgggcaaacggaagtaaagtcaaattcgccggct tcatgaaagtctatgtagagggtaaagacgatcagctggaagaaaaagaccgcatgcttccggaccttaaagagggagacacggt tttatcaaaagatattgagcctgagcagcactttactcagccgcctccgcgttacacagaggcgcgcctggttaaaacgcttgaagaa ctggggatcggccgtccttcgacatatgcgccgactcttgacaccatccagcgccgcggttacgtgggattagacaataaacggtttgt gccgactgagcttgggcagatcgtgctggatcttatcatggagtttttcccggaaatcatcaacgttgaattcacggctaaaatggaaag ggaccttgaccatgttgaagatggcgagacagaatgggttcaaatcattgacagcttctataccgatttcgaaaaacgcgtgaaaaaa gcggaagctgaaatgaaagaagtggaaattgaacctgaatacgcagatgaagattgtgaattatgcggctcccggatggtttataaa atgggccgatacggtaaatttatggcgtgttccaacttccctgactgccggaatacaaaaccgattgttaaacaaatcggcgtaaaatg tcctaagtgccatgaaggaaatattgtcgaacgaaaatcaaaaaagaaacgcgtcttttacggctgtgaccgttatcctgaatgcgact tcgtatcttgggacaaaccgattgaacgaaaatgcccgaaatgtgaaaatatgctcgtagagaaaaagctaaaaaaggcatacaa gtccaagctcgaacc.
Embodiment 2 Detection of stress resistance of Bacillus velezensis YE-1 (1) Detection of temperature and osmotic pressure tolerance
Taking 1 ml of bacterial solution in 1.5 ml EP tube, putting it in 40, 50, 60, 70 and 80°C water baths for 20 min, diluting it with physiological saline at a gradient of 10 times to an appropriate concentration, taking 100 pL of bacterial solution, coating it on the solidified PCA medium, and culturing it upside down in an incubator at 37°C for 12 h; inoculating the activated bacterial solution into 5 ml of LB broth culture medium with different concentrations of sodium chloride (1, 2, 3, 4, 5, 6, 7.5%) for 2 h, and then placing it on the plate for anaerobic culture at 37°C for 12 h; repeating each group of experiments for three times, and counting and calculating the survival rate of bacteria by plate counting method:
Survival rate = rT x 100% where, Nb is the number of viable bacteria (CFU/ml) in the control group; N: is the number of viable bacteria (CFU/mI) in the experimental group.
As shown in FIG. 8, the results show that the survival rate of Bacillus velezensis YE-1 is 33.60% after it is treated in the water bath at 80°C for 20 min, and the survival rate is 19.75% after it is treated in the 7.5% sodium chloride for 2 h. (2) Detection of tolerance of artificial gastric juice and artificial intestinal juice
To prepare artificial gastric juice, potassium chloride (7 mmol/l), sodium bicarbonate (45 mmol/l), sodium chloride (125 mmol/l) and pepsin (3 g/l) (Shanghai yuanye Bio-Technology Co.,
Ltd.) are prepared. Finally, the pH is adjusted to 3.0 with hydrochloric acid, and the bacteria are removed by filtration with 0.22 um water system microporous membrane. The secondary activated bacterial solution is centrifuged at 6000 r/min for 8 min. After the supernatant is removed, the same volume of artificial gastric juice is added and cultured at 37°C. After 1, 2 and 3 h of culture, the bacterial solution is taken out and counted on the plate. Similarly, to prepare artificial intestinal juice, trypsin (1 g/l) (Shanghai yuanye Bio-Technology Co., Ltd.) and pig bile salt (3 g/l) are added, the pH is adjusted to 7.5 with sodium hydroxide, and 0.22 um water system microporous membrane is used for filtration and sterilization. The artificial intestinal juice is added to the bacterial precipitate prepared above and cultured at 37°C. After 2, 4 and 6 h of culture, the survival rate is calculated by taking out the inverted plate (the calculation formula is the same as above). Each group of experiments is repeated three times to evaluate the tolerance of the strains to artificial gastric juice and artificial intestinal juice.
The results shown in FIG. 6 show that the survival rate of artificial gastric juice is 33.57% after 3 h of treatment, and the survival rate of artificial intestinal juice is 64.24% after 6 h of treatment. (3) Detection of antibiotic sensitivity
Taking 100 pL of Bacillus velezensis YE-1 with a cell concentration of 107 CFU/ml and coating it on the solidified LB agar medium, then placing the drug sensitive paper (Thermo
Fisher Scientific Co., Ltd.) evenly on the LB agar medium, placing it in a 37°C incubator for 24 h, and accurately measuring the diameter of the bacteriostatic ring of the drug sensitive paper with a vernier calliper; if there is no bacteriostatic ring, recording the diameter of the antibiotic paper; by comparing with the area diameter provided in the antimicrobial disc sensitivity performance standard, the results are expressed as resistance (R), intermediate sensitivity (1) and sensitivity (S).
Table 3 Sensitivity results of Bacillus velezensis YE-1 to different antibiotics
Test index (Hg) mm) ring/mm
RIT [S [Coven |W | || 7 | Reem |R
Towaen | 0 |S [| | ios |v
Ke EN 1
Naphthalic 30 <13 14-18 219 S nn] 7
As shown in Table 3, the drug sensitivity test results show that Bacillus velezensis YE-1 is sensitive to most antibiotics, so it is safe to use as a probiotic.
Embodiment 3 Antagonism of Bacillus velezensis YE-1 against Aspergillus flavus and its application (1) Effects of antagonistic bacteria on spore germination and bud tube elongation of
Aspergillus flavus
Inoculating Bacillus velezensis YE-1 in LB broth medium and culturing at 37°C overnight; adding the bacterial solution into PDA culture medium with proper temperature and mixing it evenly, so that the final bacterial solution concentration is 107, 108 and 105 CFU/ml, and pouring it on the plate; coating 100 ul of Aspergillus flavus spore solution on the plate after the plate is solidified, and then culturing in an incubator at 25°C for 11 h, and detecting the spore germination rate and bud tube length; each repetition containing 9 parallels, and each parallel containing 900 spores; at the same time, setting PDA without any treatment and PDA control with LB broth.
It can be seen from A-B in FIG. 3 that the fermentation broth of Bacillus velezensis YE-1 has a highly significant inhibition effect on spore germination and bud tube elongation of
Aspergillus flavus. There is no significant difference in spore germination rate and bud tube length between CK group and LB group (p>0.05), except that LB broth, the solvent of bacterial solution, has influences on spore germination rate and bud tube elongation. There is a significant difference between CK group and 105 CFU/ml group (p<0.01), the spore germination rate between 103 CFU/ml group and 10° CFU/ml group (p<0.01), and the spore germination rate and bud tube length between 108 CFU/ml group and 107 CFU/ml group (p<0.01). To sum up, it is concluded that Bacillus velezensis YE-1 has a highly significant inhibition effect on spore germination and bud tube elongation of Aspergillus flavus, and this inhibition effect is significantly enhanced with the increase of Bacillus velezensis YE-1 content. (2) Inhibition effects of antagonistic bacteria on mycelium expansion of Aspergillus flavus
Inoculating Bacillus velezensis YE-1 in LB broth medium and culturing at 37°C overnight; adding the bacterial solution into PDA culture medium with proper temperature and mixing it evenly, so that the final bacterial solution concentration is 107, 10° and 10° CFU/ml, and pouring it on the plate; coating 100 ul of Aspergillus flavus spore solution on PDA culture medium, culturing in 25°C incubator for 24 h, and punching Aspergillus flavus mycelium cake on Petri dish with a 7 mm hole punch; transferring Aspergillus flavus mycelium cake to PDA plate containing different concentrations of biocontrol bacteria solution, and placing it in an incubator at 25°C for cultivation; at the same time, setting PDA without any treatment and PDA control with LB broth; every 24 h, measuring the diameter of Aspergillus flavus mycelium cake perpendicular to each other with vernier calliper until 96 h.
It can be seen from C in FIG. 3 that there is no significant difference in the diameter of
Aspergillus flavus mycelium between CK group and LB group at 24, 48, 72 and 96 h (p>0.05), so that LB broth, the solvent of antagonistic bacteria, is excluded from inhibiting the growth of
Aspergillus flavus mycelium. There are significant differences between CK group and 107, 10°, 105 CFU/ml groups after 24 h treatment (p<0.05), but there is no significant difference among the bacterial liquid treatment groups (p>0.05). At 48, 72 and 96 h, there are significant differences between 107, 10° and 10° CFU/ml groups and CK group (p<0.05), and there are significant differences between 107, 10° CFU/ml groups and 105 CFU/ml groups (p<0.05), but, there is no significant difference between 107 CFU/ml group and 10° CFU/ml group (p>0.05). To sum up, it may be seen that antagonistic bacteria significantly inhibit the growth of Aspergillus flavus mycelium, but this inhibition does not increase with the increase of antagonistic bacteria concentration. (3) Inhibition effect of antagonistic bacteria on Aspergillus flavus of corn and peanut
Selecting corns and peanuts without damage and disease, washing them with clean water, soaking them in 1% sodium hypochlorite for 3 min, then rinsing them with sterile water and drying them; soaking the treated corns and peanuts in Aspergillus flavus spore solution (10%/ml)
for 1 min, then drying, placing a wet filter paper on a Petri dish, evenly placing corns or peanuts on the filter paper, and spraying antagonistic bacteria on the experimental group; placing the corns and peanuts in an incubator at 30°C for 6 days; each repetition containing 6 repetitions, each repetition containing 3 parallels, and each parallel containing 7 corns or peanuts.
It may be seen from FIG. 4 that compared with the control group (inoculated with
Aspergillus flavus spore solution only}, the treatment group (inoculated with Aspergifius flavus spore solution and Bacillus velezensis YE-1 strain solution) has a lower incidence of corns and peanuts.
As corns and peanuts are the most susceptible to Aspergillus flavus contamination, the experimental results of corns and peanuts reflect the antagonistic effect of Bacillus velezensis
YE-1 antagonistic bacteria in grain storage. (4) Application of antagonistic bacteria in complete feed for pigs
Weighing 10 g of pig complete feed, inoculating 1 ml of bacterial fermentation broth in the experimental group, 1 ml of normal saline in the control group, and treating at 60°C for 5 h, then spraying 1 ml of 10%/ml| Aspergillus flavus spore solution into the control group and the experimental group respectively, cultivating in an incubator at 25°C for 6 days, and taking photos every 2 days.
It can be seen from FIG. 5 that compared with the control group (inoculated with
Aspergillus flavus spore solution only), the treatment group (inoculated with Aspergillus flavus spore solution and Bacillus velezensis YE-1 bacterial solution) has a relatively mild incidence of pig complete feed.
Embodiment 4 Safety evaluation of Bacillus velezensis YE-1 bacterial solution
In this embodiment, Kunming mice are used as experimental animals, and the safety of
Bacillus velezensis YE-1 is evaluated by oral gavage. The specific methods are as follows. 1. Centrifuging the activated bacterial solution at 6000 r/min for 8 min after the activated culture of Bacillus velezensis YE-1, and resuspending the lower bacterial precipitate by adding normal saline. 2. Kunming mice (license number SCXK (Yu) 2020-0005), weighing 20 + 2 g, being purchased from Henan Skbex Biotechnology Co., Ltd. 3. Purchasing 64 healthy mice, half male and half female; after 5 days’ adaptation to the experimental environment, randomly dividing them into 8 groups, with 8 mice in each group; there is a blank control group for each female/male, and three groups for each experimental group, namely, low dose group (107 CFU/mouse), middle dose group (102 CFU/mouse) and high dose group (10° CFU/mouse). 4. The administration route is oral gavage with a volume of 20 ml/Kg; the mice have to fast for 6 h before gavage, and continue to fast for 2 h after gavage, the gavage lasts for 14 days, and the mice drink freely during feeding.
5. Detection index (1) Clinical observation: all mice show no signs of abnormal gait, lethargy, hair, eyes, skin, salivation and local injury and poisoning. (2) Weight and feed intake of mice: weighing and recording the weight of each mouse before gavage of bacteria solution every day; calculating and recording the food intake of mice from the time of feeding this time to the next time of fasting. There is no significant difference in body weight and feed intake of each group (p>0.05), and the results are shown in FIG. 7. (3) Biochemical indicators and haematological indicators: mice fasting overnight after 14 days of gavage of bacteria solution, and the next day, taking blood by removing the eyeballs; keeping the whole blood of mice in a refrigerator at 4°C for 4 h, and centrifuging at 3000 r/min at 4°C for 15 min. The biochemical indexes of the upper serum are determined by a commercial kit (Nanjing Jiancheng Institute of Bioengineering). Blood urea nitrogen (BUN) and creatinine (CRE) are used to evaluate renal function. Direct bilirubin (DBIL) and total bilirubin (TBIL) are used to evaluate liver function. Total cholesterol (T-CHO), glucose (GLU) and triglyceride (TG) are used to evaluate cholesterol, blood sugar and blood lipid, respectively. The whole blood of each group of mice is stored in EDTA (Kz) micro blood collection tube and sent to Wuhan Service Biological
Company as soon as possible for haematological parameters detection.
Table 4 Biochemical results of mice in each group veen group
Low dose Middle dose High dose group
TCHO 3.18+0.29 | 3.37+0.80 | 3.51 +1.45 | 3.30 0.61
DBIL | ymol/l | 6.01 £ 0.31 7.06 +£0.16 | 6.06 £2.08 5.23 +£2.53
TBIL | ymol/l | 118.86+6.17 | 102.50+3.86 | 101.67+6.86 | 116.30+12.76 pmol/l | 72.79 + 2.63 | 67.08 + 4.41 | 68.82+12.17 | 73.33 + 7.87
As can be seen from Table 4, there is no significant difference in all indexes of mice with different concentrations of bacterial liquid (9>0.05), indicating that there are no adverse effects on liver function, kidney function, cholesterol, blood sugar and blood lipid levels.
Table 5
WBC / LYM/ MON / Gran / LYM% / Gran% /
Gender | Group 10%/1 10%/1 10%/1 109 % %
Control 7.8011.47 | 5.851+1.87 | 0.38+10.20 | 1.08+£0.84 | 74.4313.60 | 21.651£2.91 group
Low q 6.7510.25 | 4.8212.08 | 0.23+0.19 | 0.97+£0.37 | 77.33+4.52 | 26.2512.48 ose
Female
Middle q 5.4011.62 | 3.95+1.52 | 0.23+0.04 | 1.231+0.19 | 75.00+4.17 | 21.10£3.22 ose
High q 6.7310.71 | 4.931+0.47 | 0.30+0.08 | 1.50+0.22 | 73.0712.45 | 22.331£2.16 ose
Control 7.7612.28 | 5.53+3.12 | 0.37+40.19 | 2.05+£0.87 | 67.5514.63 | 25.5313.38 group
Low q 8.671+0.42 | 4.95+2.27 | 0.30+40.12 | 1.63+£0.79 | 71.10+3.64 | 23.00+£3.40 ose
Middle q 6.881£2.27 | 4.8011.73 | 0.23+0.08 | 1.65+0.50 | 69.40+3.47 | 25.031£3.18 ose
High q 6.9511.15 | 4.0411.45 | 0.2740.17 | 2.2710.57 | 72.40+2.04 | 23.0311.43 ose
Table 6
Gen- Gc RBC / HGB / HCT / MCV / MCH / MCHC / rou der p 10124 g/l % FL Pg g/l
Control 10.99 162.00 £ 55.93 + 51.13 14.73 289.75 group 0.92 11.36 4.25 3.80 0.44 15.69
Low 10.82 184.751 52.80 + 48.88 + 15.181 295.751
Fe- dose 0.85 23.92 4.46 1.37 1.66 31.36 male Middle 10.33 148.251 52.38 50.78 + 14.281 282.751 dose 0.86 12.75 4.49 1.90 0.25 6.42
High 10.391 143.671 50.50 + 49.10 13.93 283.67 + dose 0.99 12.04 3.83 2.97 0.82 2.63
Control 9.661 141.751 51.43 53.38 14.63 + 275.75 1 group 0.66 8.69 4.34 3.98 0.59 11.28
Low 10.80 + 157.251 53.951 50.00 + 14.48 + 290.75 dose 1.65 25.75 8.49 2.03 0.33 8.35
Middle 11.15 + 155.00 £ 56.43 + 50.75 13.88 + 274.00 dose 0.79 15.44 2.83 1.70 1.22 17.98
High 9.23 149.00 t 56.90 + 51.351 13.45 + 262.00 + dose 1.28 6.00 2.40 1.13 1.84 31.80
Table 7
RDW/ PLT/ MPV/
Gender Group PDW PCT (%) (10%) (fL) 1498+ | 1368.75 5.781 16.45 + 0.50
Control group 1.14 210.85 0.69 0.48 0.01 1543 + 842.50 + 5.581 16.30 + 0.451
Low dose « 0.49 418.27 0.54 0.31 0.22
Female 1583+ | 1196.75 5.801 16.15 0.51%
Middle dose w 0.27 317.16 0.35 G.11 0.03 17.13 + | 1202.00 + 5.331 15.70 + 0.531
High dose = 1.89 262.27 0.31 0.30 0.01 16.05+ | 1096.50 + 6.10 £ 16.95 + 0.451
Control group 1.02 409.17 0.16 0.49 0.06 15.03+ | 1173.67 + 5.851 16.48 + 0.421
Low dose ml 0.48 282.03 0.38 0.33 0.28 15.40 + | 1034.67+ 6.401 16.65 + 0.50 +
Middle dose Ww 0.46 116.44 0.76 0.46 0.19 15.73 + | 1235.00 + 6.081 16.95 + 0.371
High dose . 0.65 79.09 0.29 1.02 0.26
Note: “**” in the table indicates significant difference, p<0.01.
Table 5-Table 7 show the haematological results of each group of mice. From the results, it can be seen that there is a significant difference in platelet (PLT) between the control group and the treatment group (p<0.01), but this index has no dose-dependent relationship. Combined with the results of other 16 haematological indexes that are not significantly different between the treatment group and the control group (p>0.05), comprehensive analysis shows that the highly significant change of PLT has no biological and toxicological significance. To sum up, different concentrations of bacteria have no adverse effects on haematological indexes of mice in each group. (4) Anatomical and pathological examination of mice: the mice are killed by cervical dislocation and dissected, and no abnormal oedema or congestion is found in heart, lung,
intestine, stomach, liver, thymus and other organs. The heart, liver, spleen, lung and kidney of each mouse are weighed, and the organ index is calculated; histopathological observation shows that liver and kidney cells are clear in structure and regular in shape. The results are shown in
FIG. 8.
Table 8 Results of organ index of mice in each group
Control 26.141 0.61% 4181 0.36 0.761 1.11 +
Low 27.821 0.60 450+ 0.31% 0.791 1.02 +
Middle 27.82% 0.731 4401 0.35% 0.771 1.22 +
High 271671 0.661 4.261 0.361 0.85% 1.43 +
Control 30.75 + 0.60% 4881 0.381 0.811 1.56 +
Low 3222 + 0.661 445+ 0.331 0.861 1.45 +
Middle 3232+ 0.581 4571 0.331 0.861 1.53 +
High 31.86 0.641 4.351 0.271 0.691 1.45 +
It can be seen from Table 8 that there is no significant difference in all organ indexes between the treatment group and the control group {(p>0.05), indicating that Bacillus velezensis
YE-1 has no adverse effects on mouse organs.
The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art make various contributions to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.
i Alum. overslon=T1.0" encoding=TUTF-Qn Tx 2 <!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing 1.3//EN" "ST265equenceListing V1 3.dtd"> 3 <ST26Sequencelisting drdvVersicn="NWL 37 filsName="Bacverelensis NL Seghist, ami” soïtwaceNems=*WIPO Segvencen zscoïtwareVension="2 8,05 vrodoolicnDeie=P2edS-dndD7N>
A <Applicatioconidentiiflcatlon> <IPCfficelode>CN</IPOfficaelode> & <ApplicerionNumberText>202211304472.7</ApplicationNumberText> 7 <FilingDate>2022-10-24</FilingDatex> 8 </Ppplicationidentification> 3 <Applicant¥FileReference>»SHX-Bacvezelensis NL</ApplicantFileReference> <BarliiestPricritvipplicationidentification> il <IPOfEiceloderCN</TIPOfflceloderx 12 <ApplicationNumberText>202211304472.7</ApplicaticnlunberText> 13 <FilinglDate>2022-10-24</Filingiates> 14 </FarliestPriorityApplicationidentification> in <ApplicaniNeme languagelode="an">Hefeli University of Technology /ApplicantNems> is <InpvaentionTitle lsnguagecode="en"2BACILLUS VELEZENSIS YE-1 ANTAGONISTIC TO
ASPERGILLUS FLAVUS AND APPLICATION THEREOF/InventionTitle>
Lj <Seguenceïotaluantityr9</SeguenceTotalQuantitys» is <SeguenceData semuenceIDNumber=Nln> 1% <“INSDSeq>
Ze <INSDSeq lengch>1460</INSDSeg length» “1 <INSDSeq molitype>DNA</IN3DSeq moltype> dd <INSDSeq division>PAT</INSDSeq divisicn> 23 <INSDSeq [eatureriabie> 24 <IMNSDFeature> <IN3DFeature key>source</IiN3DFeature key» 28 <IN3DFeature lowation»l..1460</INSDFeature location 27 <INSDFeature guals> 25 <INSDOualifier> 2% <INSDOQualifier name>mol type</INSDQualifier name>
Si <INSDQualifler valverother DNA</INSDQualifier value» 3h </INSDOualifier> zl <INSDOualifier id="gi"> 32 <IN3DQualifier namevorganism“/INSDQualifier name> 34 <INSDQualifier valuersynthetic construct</INSDQuallifier value» <{INSDQualifier»
Ki </INSDFeaturs quals> 37 </IN3DFeature> 38 “/INSDSeqg fesature-table> <iN3D3eq sequence>gaaagtggcgggcgtgcctaatacatgcaagtcgagcggacagatgggagcttgct ccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaac cggggctaataccggatggttgtctgaaccgcatggttcagacataaaaggtggettcggctaccacttacaga tggacccgecggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgecgtagccgacctgagagg gtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatg gacgaaagtctgacggagcaacgccgecgtgagtgatgaaggtttteggategtaaagctctgttgttagggaag aacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagca gccgcggtaatacgtaggtggcaagecgttgtceggaattattgggcgtaaagggctcgcaggcggtttcttaag tctgatgtgaaagccccecggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagag tggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtet gtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacga tgagtgctaagtgttagggggtttcecgccccttagtgctgcagctaacgcattaagcactccgcctggggagta cggtegcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaag caacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcaga gtgacaggtggtgcatggttgtegtcagectegtgtecgtgagatgttgggttaagtcccgcaacgagcgcaacce ttgatcttagttgccagcattcagttgggcactctaaggtgactgceggtgacaaaccggaggaaggtggggat gacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaa ccgcgaggttaagccaatcccacaaatctgttctcagttecggatcgcagtctgcaactcgactgcgtgaagctg gaatcgctagtaatcgcggatcagcatgccgeggtgaatacgttccegggeccttgtacacaccgccecgtcacac cacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagcegcegaaggtgacaagaagg-/ iNSDSeg sequencer </INSDSegr 41 </SaquenceData> 42 “SequenceData seguencellNMumber="27> a2 <INSDSeq> 44 ZINSDSegq length»2087</INSDSeq length» 4% <IN3DSeq moltype>DNA</INSDSeq moltyper 48 <INSDSeq division>PAT</INSDSeg division 47 <INSDSeq feature-table>
43 <IN3DFeature> 4% <INSDFeaturs keyrsource</INSDFeaturs key» 54 <INSDFeature location>l..2087</INSDFeature location bi <INSDFeature guels> 52 <INSDOQualifier> 53 <IN3DQualifier name>mol type“/INSDQuali fier name>
Sá <INSDQualifier valuerother DNA</INSDQualifier value»
EE </INSDOualifier>
LE <INSDQualifler id="gd™> 57 <INSDQualifier name>organism</INSDQualifier name> 58 <IN3DQualifier value>synthetic construct</INSDQualifier value» 38 </INSDOQuali fier a0 </INSDFsature guals> al </INSDFeature> eZ </INSDSeqg feature-tabhlex <INSDSeq sequencevaacgcccttcaatgtttcagcaggaattttgactgcgctgaaaaacatggatatag tagtaaataaccatatggcaatgcttgaagaaaagaccattaagcagcttgatgcgaaatttaaaaaaggcggc gccggegttacatctcagaagcctgcggaaacgaacaaaaacaagccgcaagggattaatcagcagcctgctgg gaatcaaccaaacaaaattcgagacggaaagaagaatgacgtgcagaataatcaatttaacaaaaacaagaaga acaacaacaacaaaaacaaaaacaaacgcaatcataacaacaaaaatcagtatcagcaaaaaccgctgaagccg aaaaaagagcttcctgagaaaattacgttttceggctctttaacagtcggggccttagccgaagaacttggcaa agaaccgtctgaactcattaaaaaactgatgcttctgggecgtaatggctacgattaaccaggagcttgacaaag acacaatcgaactgatcgcatctgaatacggtgtggaaacagaagaagtgattgtgctcgaagaaactgagctt gaaaaatatgaagaggctgacaaagaagaagatcttcaaatccgtecegectgtecgtgacgatcatgggccacgt tgaccacgggaaaacgaccctccttgacagcatcagaaaaacaaaagttgttgaaggtgaagccggcggaatca cccagcatatcggggcgtaccagatcgaagaaaacggcaagaaaatcacgttecttagatactccgggacacgcg gegttcacaacaatgcgtgcgcgecggtgecggaagtaactgatattaccattttagtcgtagcggctgatgacgg cgttatgccgcaaacagtcgaagccatcaaccatgcgaaagcggctgaggtteccgattatecgttgccgtgaata aagtggataaagaaagcgcgaaccctgacecgtgtcatgcaggagctgactgaatacggactcgttectgaagca tggggaggagaaacgattttegttecctetgtecgetcttacaggcaagggaattgacgagcttgtcgaaatgat tctgcttgtcagtgaagtggaagagctgaaagccaatccgaaccgtcaggcaaaaggaacggttattgaagctg agctecgataaaggaagaggatctgtcgecgacacttctecgttcaaacgggaacattgcatgtcggagatccgatc gttgtcggaaatacattcggcegegtgcgggccatggtcaatgatcteggcecgccgecgtgaaaacagccggacc gtccacacctgtagaaatcacgggcttaaacgacgtaccgcaggctggecgatcaattcctegtectttaaagatg aaaaaacagccecgttctgtcggtgaagcacgecgcttccaagcaactggaagaacagcgcagcgacaaagcgaag ctgagcctcgatgatctgtttgaacaaattaaacaaggcgatgttaaagacatcaacttaatcgtaaaagctga cgttcaaggttcagccgaagcgttaacagcecgctcttcaaaaaattgaagtagaaggcgtaaaagtgaaaatca ttcacacaggcgtcggtgcgattacggaatcagacatcattctegcatctgcttcaaatgcaatcgttatcggg tttaatgtgecgcccagacggaaatgctaagagcacggctgaagctgaaaatgtagatatcegtcttcacegtat catttacaaagtcatcgaagaaattgaagcggcgatgaaaggaatgcttgatcctgagtacgaagaaaaagtca teggtcaggttgaagtgcgccaaacattcaaagtgtcgaaaatcggcacgattgccggcggatatgtaacagac ggccacattacacgtgacagcggacteccgcctgatccgtgacggecgtggtcatcttcgaaggggaagtagacgt tctgaaacgcttcaaagatgatgtgaaagaagtttcacaaggatatgaatgtggtataacgattaagaaataca atgacattcgcgaaggcgactcacttactgege</INEDS ag sequence 4 </INSDSeg>
En </Zequencebata>
G8 <SequernceData seguancaiDNunbhao="3%> 67 <INSDSeqg> af <INSDSeq Iength>»2013</INSDSey length> 53 <INSDSeq moltype>DNA-/INSDSeg moltype> io ZINSDSeq division>PAT</INSDSeq division»
EL <INSDSeq feabure-table>
Fa <INSDreature> js <INSDFeature key>source</INSDFeature keys 74 <INSDFeature location>l..2013</INSDFzature location 75 <INSDFealurse guals>
Ta <INSDOQualifier»
Gi <IN3DQualifier namedmol type</INSDQualifisr name> ia <INSDQualifier value>other DNA</IN3DGualifier value>
To </INSDQuali fier» <INSDQuaiifler id="g3">
Sl <INSDQualifier namerorganism</INSDQualifier name>
SE <INSDgQualifier value>synthetic construct“/INSDGualifier value» 82 </INSDOualifier> £4 </INBDFeature gvals> £5 </INSDFealure>
Se </INSDSegy featurs-table> <INSDSeg sequencerceggggcacgcgaaacgattgacgttatttagggaagaaatataaagtcaaagctt caatgggacatgtcecgggatcttccgaaaagtcaaatgggagttgacatcgaacagaactttgaaccgaaatat attacgatccgcggaaaaggccctgttttaaaagaattaaaaacagcggcaaagaaagcaaaaaaagtctatct cgcggccgaccccgacagagaaggggaagcgattgcatggcatctcgcacacagccttgatctggatctcagct ctgactgccgggtcgtctttaatgaaataacgaaagacgccattaaagaatcatttaaacatccgcgcatgatt aacatggatcttgtecgatgcgcagcaggcgegccgcatactagacagactcgtcggatacaaaatcagcccgat tctttggaaaaaagtaaaaaaaggcctcagtgcgggcegtgttcaatecgttgegctceggctgatcategacc gggaaaaggaaatcaatgactttaaacctgaagagtactggacgattaccggctcatttttaaagggaaaagag acgtttgaagccggatttttcgggaaaaacggcaaaaagcttcctttaaaaagcgaagaagacgtaaaagccgt ccttgctcagctgaaaggcaataagtatacagttgacaaggtgacgaaaaaagagcgcaaacgaaatcccgcte tgccgtttacaacgtccactctgcagcaggaggctgccaggaagctgaatttcagagcgaagaaaacgatgatg atcgctcagcaattatatgaaggaattgatctcggcaaagaagggacagtcggactcatcacgtatatgagaac ggactcaacgcggatttccaatacagccgttgaagaggcgtcagcttttattgatcaggcatacggaaaagagt acttagcgggaaaacggaaaccggcaaagaaaaatgaaaacgctcaagatgcccacgaagcgatcegcccgact teggtgctcagaaaacctgccgacttaaaagcggtgcttggaagagaccagctcagactatacaagctgatttg ggagecgttttgttgcaagccagatggegectgeecgtccttgatacgatgagegtcgatctcagcaataacggat tgacattcecgggcaaacggaagtaaagtcaaattegcecggcttcatgaaagtctatgtagagggtaaagacgat cagctggaagaaaaagaccgcatgctteccggaccttaaagagggagacacggttttatcaaaagatattgagcc tgagcagcactttactcagccgcctcecgegttacacagaggcgcgcectggttaaaacgcttgaagaactgggga teggcecgtecttcgacatatgegcegactcttgacaccatccagcgccgcggttacgtgggattagacaataaa cggtttgtgcecgactgagcttgggcagategtgctggatcttatcatggagtttttcceggaaatcatcaacgt tgaattcacggctaaaatggaaagggaccttgaccatgttgaagatggcgagacagaatgggttcaaatcattg acagcttctataccgatttcgaaaaacgcgtgaaaaaagcggaagctgaaatgaaagaagtggaaattgaacct gaatacgcagatgaagattgtgaattatgecggectcccggatggtttataaaatgggccgatacggtaaatttat ggcgtgttccaacttccctgactgcecggaatacaaaaccgattgttaaacaaatcggcgtaaaatgtcctaagt gccatgaaggaaatattgtcgaacgaaaatcaaaaaagaaacgcgtecttttacggctgtgaccgttatcctgaa tgcgacttegtatcttgggacaaaccgattgaacgaaaatgcccgaaatgtgaaaatatgctcgtagagaaaaa gctaaaaaaggcatacaagtccaagctcgaacc“/INSDSeg sequenced ge </INSDSeg> </SeguencaData>
Go <SequenceData zaquencaldDNumben=n4gn>
Si <INSDSeq>
Ge <INSDSeq length>20</INSD5eq length» 25 <INSDSeq moltype>DNA</INSDSeg moltype> 34 <INSDSeq divislion»PAT</INSDSeqg division» <IN3D3eq feature-table> 98 <INEZDFeature>
Si <IN3DFeature key>source</IN3DFeature key> 85 <IN3DFeature location»l..20</INSDFeaturs locations
EN <INSDFsature qualsg>
LO <INSDQusliifier> [Re <INSDoualifier name>mol type“ /INSDQualifier name>
Ll <INSDQualifisr value>other DNA</INSDQualifier valuer 102 </INSDOualifier> ijd <INSDOualifier id="gàr> 10h <IN3DQualifier name>organism</INSDQualifisr name> 10a <INSDQualifier valuersynthetic construct</INSDQualifier valued
TOT </INSDOualifier> ies </INSDFeaturs auals>
LOS </INSDF=aturex 11h “/INSDSeg feature-table>
Lil <IN3D3eq sequence>agagtttgatcctggctcag</INSD5eq sequence
Lie </INSDSeg> 143 </SeguenceDala>
Liá <SequenceData sequencelDNumer="B">
LiL <INSDSeq>
LLG <“INSDSeqg length>22</INSDSeq lengths 17 <INSDSeq moltype>DNA</INSDSea moliype> iis <INSDSeq divislon»PAT</INSDSey division iis <IN3D3eq feature-table> 1240 <INSDFeatura>
Lal <IN3DFeature key>source</IN3DFeature key» 122 <INSDFeature location>l..22</INSDFeature location»
Las <INSDFeature quals>
Lod <IN3DQualifiers 125 <INSDQualifier name>mol type</INSDQualifier name> 128 <INSDQualifier value>other DNA</INSDQualifier valued 127 </INSDQualifier> i28 <INSDOQualifier 1d=Vghu» 12% <IN3DQualifier namerorganism“/INSDQuali fier name> 130 <INSDQualifier valuersynthetic construct</INSDQualifier valued
13% </INSDOualifiers se </INSDFeaturs duals? 132 </TNSDFeaturer 124 </INBDSeq feature-table> 125 ZINSDSeq sequenas>tacggetaccttgttacgactt</INSDSeg sequence i38 </INSDSeg> 137 </Zequencebatar
RYE <Sequencebata seguenasibNumbar=ngys 13% <INSDSegr 140 <INSDSeq length>29</IN3DSeq length» 141 <INSDSeq moltype>DNA</INSDSeq moltype> 142 <IN3DSeq division»PAT</INSD3eq division» 143 <INSDSeq feature-table> 144 <INSDFeabture> ids <INSDFeature key>source“/INSDFeature key> 146 <INSDFeature location>l..29</INSDFeature location> 147 <INSDFeature qguals> u 148 <INSDQualifier»> 143 <INSDQualifier name>mol type</iNSDQualifier name> 154 <INS3DQualifier value>other DNA</INSDQualifier valued
LSL </INSDOQuali fier 152 <INSDQualifler id='qgs"> 153 <INSDQualifier namerorganism</INSDQualiifier name>
Lhd <INSDQualifier valuersynthetic construct“/INSDQualifien value» 155 </INSDOualifier> 1548 </IN3DFeature gualsd 157 </INSDFeature> u 158 </INSDSeg features table» 15% <IN3DSeqy sequence>atggetaaaatgagagtttacgaatatge</IN3LSeq sequenca>
Led </INSDSeg>
TEL </SequenceData>
Led <SequernceData sequenceliNuDec="N?> 163 <INSDSeqg> 164d <INSDSeq length>29</INSDSeq length> eh <INSDSeq moltype>DNA-/INSDSeg moltype> 188 ZINSDSeq division>PAT</INSDSeq division»
Le <INSDSeq feabure-table>
Los <iNSDPFeacure»>
Low <INSDFeature key>source</INSDFeature key>
LEO <INSDFeature location>l..29</INSDFeature locations jl <INSDFealurse guals> iz <INSDOQualifier» 172 <IN3DQualifier namedmol type</INSDQualifisr name> 174 <INSDUvaelifier valuerother DNA</INSDGualifier value> iS </INSDQualifier» u ie <INSDOQualifier id="g7*x>
LET <INSDOQualifier namerorganism</INSDQualifier name>
LEE <INSDQualiflsr value>synthetic construct /INSDQualifier value> 173 </INSDOualifier> 18a </INBDFeature gvals> iel </THSDFeatura> 182 </INSDSegy featurs-table> 183 <INSDSeq sequence>tcactttcetttegatttectgeatgacat</INSDSeq sequence>
Tid </INSDSeg> ies </SequenceData> 188 “<SequenceData segusnceliNumec=NS"> 187 <INSDSeq> ies <IN3DSeq length»24</INSDSeq length» igs ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 180 <IN3DSeq divisior>PAT</INSDIeqg division»
Lei <INSDSeq feature-table>
Led <INS3DFesature>
Les <INSDFeaturs keyrsource</INSDFeaturs key> 194 <INSDFeature location>l..24</IN3DFeature location» 125 <INSDhFeature quels» 156 <INSDQualifier> 187 <IN3DQualifier name>mol type</INSDQualifisr name>
LSB <INSDQualifler valuerother DNA</INSTGualifier value» 10% </INSDQualifier> 200 <INSDQualifier id="g8"> gol <INSDoualifier namerorganism</INsSDQualifier name> 207 <INSDQualifier valus>synthetic construct /INSDGualifier value 202 </INSDQualifier> u 203 </INSDFeature guals> 20% </INSDFeaturs> 208 </INSDSey feature-tabled 207 <INSDSeg sequenceraacgcggtcaaccgaatgattgaa-/IN3D5eq sequencer
Us </INSDSear> 200 </SaquenceData> zin “SequenceData seguencelóNumbec=NB%> 21 <INSDSeg> 212 <INSDSeg length>»25</INSDSeq lengths» 213 <IN3DSeq moltype>DNA</INSDSeq moltyper 244 <INSDSeq division>PAT</INSDSeg division 215 <INSDSeq feature-table> ais <INSDFeature> 217 <INSDFeaturs keyrsource</INSDFeaturs key 218 <INSDFeature location>l..2B</INSlFeature location» 219 <INSDFeature guals> 220 <INSDQualifiar> 224 <IN3DQualifier name>mol type“/INSDQuali fier name>
ZZE <INSDQualifier valuerother DNA</INSDQualifier value» zes «</INSDOQualifier»>
AA <INSDQualifler ia=’gS">
HES <INSDQualifier name>organism</INSDQualifier name>
Zin <IN3DQualifier value>synthetic construct</INSDQualifier value» 22 </INSDOQuali fier 228 </INSDFesature duals» 228 </INSDFeature> 230 </INSDSeg feature-tabhle> «St “INSDSeq sequsncertgtaggaatgtecgagggcatcatce/INSDSeq sequences 252 </INSDSeg> 232 </Seguencedata> 234 </3T268eguencelisting> 225
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|---|---|---|---|---|
| CN112358995A (en) * | 2020-11-17 | 2021-02-12 | 山东省花生研究所 | Biocontrol bacillus beleisi ZHX-12 and application thereof |
| CN114058549A (en) * | 2021-11-29 | 2022-02-18 | 中国农业科学院农产品加工研究所 | Bacillus belgii Shui-6, production method and application |
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| CN112358995A (en) * | 2020-11-17 | 2021-02-12 | 山东省花生研究所 | Biocontrol bacillus beleisi ZHX-12 and application thereof |
| CN114058549A (en) * | 2021-11-29 | 2022-02-18 | 中国农业科学院农产品加工研究所 | Bacillus belgii Shui-6, production method and application |
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| LI SHENGJIE ET AL: "Screening of Bacillus velezensis E2 and the Inhibitory Effect of Its Antifungal Substances on Aspergillus flavus", FOODS, vol. 11, no. 2, 6 January 2022 (2022-01-06), pages 140, XP093053875, DOI: 10.3390/foods11020140 * |
| LIU Y. ET AL: "Antimicrobial Bacillus velezensis HC6: production of three kinds of lipopeptides and biocontrol potential in maize", JOURNAL OF APPLIED MICROBIOLOGY, vol. 128, no. 1, 1 January 2020 (2020-01-01), GB, pages 242 - 254, XP093053952, ISSN: 1364-5072, Retrieved from the Internet <URL:https://watermark.silverchair.com/jambio0242.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAtwwggLYBgkqhkiG9w0BBwagggLJMIICxQIBADCCAr4GCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMzYr3EnK4hzYPNm37AgEQgIICjxJd_syZ3eMQo_Ki1DSUIM9W6m4x0SJZ6SYkYsrrH3_XNY5MMhzoBzBTkIDHO0rhn_Whb3A2K_tiUMWyBN2STbDhr> DOI: 10.1111/jam.14459 * |
| VAHIDINASAB MALIHEH ET AL: "Bacillus velezensis UTB96 Is an Antifungal Soil Isolate with a Reduced Genome Size Compared to That of Bacillus velezensis FZB42", MICROBIOLOGY RESOURCE ANNOUNCEMENTS, vol. 8, no. 38, 19 September 2019 (2019-09-19), XP093053941, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753265/pdf/MRA.00667-19.pdf> DOI: 10.1128/MRA.00667-19 * |
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