CN109652350B

CN109652350B - Tylosin degrading bacterium and application thereof

Info

Publication number: CN109652350B
Application number: CN201910153969.5A
Authority: CN
Inventors: 李兆君; 冯长青; 冯瑶
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2022-01-28
Anticipated expiration: 2039-03-01
Also published as: CN109652350A

Abstract

The invention discloses a tylosin degrading strain and application thereof, belonging to the technical field of microbial degradation of antibiotic residues. Tylosin degrading bacteria TYL2 is Brevibacillus borstelensis TYL2, is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 29 months and 12 years in 2018, the preservation number is CGMCC No.17051, the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The invention utilizes the medicine dregs as the initial raw material for screening, and the obtained bacterial strain and the prepared microbial inoculum can be directly used for treating the wastes of tylosin enterprises and can also be possibly applied to the remediation of the soil and water body environment polluted by the tylosin at the periphery.

Description

Tylosin degrading bacterium and application thereof

Technical Field

The invention belongs to the field of microbial degradation of antibiotics, and particularly relates to a tylosin degrading bacterium and application thereof.

Background

Tylosin (Tylosin), also known as Tylosin or Tylosin, is a macrolide antibiotic obtained by american scientists in 1959 in a study of a strain of streptomyces fradiae. Tylosin is a white plate-like crystal, slightly soluble in water and alkaline. The molecular formula of tylosin is: c₄₆H₇₇NO₁₇Relative molecular mass: 916.1, the chemical formula is:

tylosin is a broad-spectrum antibiotic with specific effect on mycoplasma and various G⁺The bacteria have strong antibacterial effect, and can also be used for treating part G^－The bacteria, curvularia, spirochete and coccidian have inhibiting effect.

At present, the problem of tylosin environmental residue has become one of hot environmental problems of general concern at home and abroad. To date, many research reports on tylosin biodegradation and degradation strain screening at home and abroad are not available.

Therefore, finding a strain capable of rapidly and efficiently degrading tylosin becomes an urgent problem.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a strain for degrading tylosin quickly and efficiently so as to solve the problem of environmental residue of tylosin.

The technical scheme of the invention is as follows: a strain of tylosin degrading bacteria is Brevibacillus borstelensis TYL2, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation date of 2018, 12 and 29 months, has the preservation number of CGMCC No.17051, and has the preservation address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

The Brevibacillus borstelensis CGMCC No.17051 or the metabolite thereof is applied to the degradation of tylosin.

The Brevibacillus borstelensis CGMCC No.17051 or the metabolite thereof is applied to the remediation of the soil or water body polluted by tylosin.

Compared with the prior art, the invention has the following beneficial effects:

the method utilizes the medicine dregs as the initial raw material for screening to obtain the Brevibacillus borstelensis CGMCC No.17051 capable of efficiently degrading tylosin, and the strain and the prepared microbial inoculum can be directly applied to treatment of wastes of tylosin enterprises and can also be applied to remediation of soil and water environment polluted by the tylosin at the periphery.

Preservation description:

brevibacillus borstelensis TYL2, which is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2018, 12 months and 29 days, the preservation number is CGMCC No.17051, the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

Drawings

FIG. 1 is a growth curve of Brevibacillus borstelensis CGMCC No. 17051;

FIG. 2 shows the tylosin degradation by Brevibacillus borstelensis CGMCC No.17051 under optimized conditions;

FIG. 3 shows the degradation of tylosin by Brevibacillus borstelensis CGMCC No.17051 in sewage.

Detailed Description

Example 1

Separation and screening of tylosin degrading bacteria

Obtaining tylosin medicine residue sample from Turketo county of Tokton city of Huohaite city of autonomous region of inner Mongolia 20/11/2017, weighing 10g of the sample, placing in a triangular flask, adding 90m of the samplel phosphate buffer, placed in a shaker at 30 deg.C, 150rpm oscillation for 2h, and then taken out and left to stand. Diluting the supernatant to 10^-5、10^-6、10^-7And sucking the supernatant with the particle size of 100 mu m, coating the supernatant into a beef extract peptone solid medium (5 g of peptone, 5g of NaCl, 3g of beef extract, 18g of agar and distilled water to a constant volume of 1000mL, adjusting the pH to 7.0-7.2 by using 1mol/L sodium hydroxide, and then sterilizing the mixture), and culturing the mixture for 3 days at the constant temperature of 30 ℃. And selecting bacterial colonies according to the characteristics of the growth speed, the size, the shape, the color and the like of the bacterial colonies, streaking the bacterial colonies on a flat plate of a beef extract peptone solid medium, and repeating the steps for multiple times until a single bacterium is separated. Stored in a 4 ℃ refrigerator.

Bacterial strain degradation function verification

And performing activated culture on the single colony obtained by purification, performing activated culture on the single colony for 2 days by using a beef extract peptone culture medium (5 g of peptone, 5g of NaCl and 3g of beef extract, diluting the volume to 1000mL of distilled water, adjusting the pH to 7.0-7.2 by using 1mol/L sodium hydroxide, and then sterilizing the mixture to obtain a strain culture solution. Taking 100 μ L of culture solution, respectively spreading on tylosin inorganic salt solid culture medium (inorganic salt solid culture medium: NH) with tylosin as sole carbon source and tylosin concentration of 25mg/L₄Cl 1g，NaCl 1g，K₂HPO₄1.5g，KH₂PO₄ 0.5g，MgSO₄0.2g of agar, 18g of agar and 1L of distilled water, wherein the pH value is adjusted to 7.0), culturing for 3 days at 30 ℃ in a shade, and gradually increasing the concentration of tylosin in the culture medium to 100mg/L according to a gradient of 25mg/L, and culturing as above. And finally obtaining a strain which can resist high-concentration tylosin through domestication, and preliminarily identifying the strain as the dominant strain with the degradation capability on the tylosin. Streaking, and storing in refrigerator at 4 deg.C.

Activating the separated and purified high-efficiency bacteria, inoculating the bacteria into a tylosin medium containing 50mg/L in an inoculation amount of 1% (2 multiplied by 108CFU), culturing at 30 ℃ and 150r/min for 7 days, sampling once every 2 days, measuring the degradation rate of each strain to the tylosin by using a high-efficiency liquid chromatography, and selecting the strains with good degradation effect. The blank group was prepared without adding any bacteria, and 3 replicates were set for each of the above treatments.

Tylosin content determination

Tylosin stock solution and standard solution: weighing 0.05g of tylosin standard substance, adding a small amount of methanol to dissolve, transferring into a 10mL brown quantitative bottle, and fixing the volume with methanol to prepare a standard stock solution of 5 mg/mL. The stock solution is prepared by diluting standard solutions (3.125, 6.25, 12.5, 25, 50, 100 and 200mg/L) with methanol. The prepared solution was placed in a 4 ℃ refrigerator.

Sample treatment: adding 8mL of acetonitrile and 8mL of n-hexane into 5mL of sample, vortexing for 1min, performing ultrasonic treatment for 3min, centrifuging for 10min at 5000r/min, and filtering with a 0.2-micron filter membrane.

Detection conditions are as follows: the tylosin content was measured by means of an Alliance 2695 model hplc 2998 model PDA detector (Waters corporation, usa). Chromatographic conditions are as follows: column SunfireC18(150 mm. times.4.6 mm, 3.5 μm, Waters, USA); mobile phase 0.1% formic acid (a): acetonitrile (B) 70: 30; the flow rate is 1.0 mL/min; the column temperature is 40 ℃; an ultraviolet detector with the detection wavelength of 285 nm; the injection volume was 10. mu.L.

Through the screening, a strain with outstanding tylosin degradation effect is obtained, and the strain is numbered as TYL 2.

Identification of Strain TYL2

Morphological observation and identification of physiological and biochemical characteristics

The growth form of the screened degradation strain TYL2 on a beef extract peptone culture medium is circular and milky white, the edge is regular and opaque, and the bacterium is gram-positive, short-rod and smooth by microscopic observation by adopting a conventional bacteria physiological and biochemical identification method and electron microscope observation. And the optimal growth temperature of the bacteria is 35 ℃, the optimal pH value is 7, partial physiological and biochemical experiment operation is carried out on the degrading bacteria, the physiological and biochemical characteristics are identified according to the 'common bacteria system identification manual', and the identification results are shown in a table 1:

TABLE 1 physiological and biochemical characteristics of Strain TYL2

Indicating positive reaction;

indicates that the reaction is negative

Strain growth curve determination

Inoculating the strain TYL2 into a culture medium, culturing at 30 ℃ and 150r/min, taking 1 sample every 2h, detecting the OD600 value of the sample by spectrophotometry to determine the growth amount of the degrading bacteria, and drawing a growth curve of the strain (the result is shown in figure 1).

The strain TYL2 was sent to a professional identification institution (Shanghai Meiji biological medicine science and technology Co., Ltd.) for sequencing identification, and the result was shown in SEQ ID No.1 below.

The 16SrDNA gene sequence is compared with the registered gene sequence in Genbank, the strain is found to have the highest homology of 99 percent with Brevibacillus borstelensis, and the strain is named as Brevibacillus borstelensis (Brevibacillus borstelensis) according to the results, and the strain is named as Brevibacillus borstelensis (Brevibacillus borstelensis) TYL2 and is preserved in the China general microbiological culture Collection center, the preservation date is 12 months and 29 days in 2018, the preservation number is CGMCC No.17051, the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

Example 2

Optimized determination of TYL2 degradation conditions

And determining the optimal culture condition for removing tylosin by using tylosin degrading bacteria by adopting a single-factor test. The content of the measurement comprises the temperature (25-45 ℃), the rotating speed (90-200 r/min), the pH (5-11), the tylosin concentration (25-150 mg/L), the inoculation amount (1-10%), and the culture time is 7 days. Each of the above processes was set to 3 parallels. The results are shown in FIG. 2. As seen in FIG. 2, TYL2 was inoculated at 35 deg.C, 150r/min, pH 7, at an inoculum size of 1.4X 10⁹CFU/mL, when the concentration of tylosin is 25mg/L, the degradation rate reaches about 75% at most after 7 days of treatment under the optimized condition.

Example 3

Determination of degradation efficiency of tylosin degrading strain TYL2 in sewage

Adding the screened efficient degrading bacteria TYL2 into a sewage sample, culturing for 7 days under the optimal optimized condition of the degrading bacteria TYL2, and detecting the residual amount of tylosin. The results are shown in FIG. 3. From fig. 3, it can be seen that TYL2 can degrade tylosin in sewage by more than 60% under the optimized conditions. The bacterium can be used for degrading tylosin. The strain TYL2 and the prepared microbial inoculum can be directly applied to the treatment of tylosin enterprise waste, and can also be applied to the remediation of the soil and water body environment polluted by the tylosin at the periphery.

Sequence listing

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> a strain of tylosin degrading bacterium and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1405

<212> DNA

<213> Brevibacillus borstelensis (Brevibacillus borstelensis)

<400> 1

atacatgcag tcgagcgagt cccttcgggg gctagcggcg gacgggtgag taacacgtag 60

gcaacctgcc cgtaagctcg ggataacatg gggaaactca tgctaatacc ggatagggtc 120

ttctctcgca tgagaggaga cggaaaggtg gcgcaagcta ccacttacgg atgggcctgc 180

ggcgcattag ctagttggtg gggtaacggc ctaccaaggc gacgatgcgt agccgacctg 240

agagggtgac cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300

tagggaattt tccacaatgg acgaaagtct gatggagcaa cgccgcgtga acgatgaagg 360

tcttcggatt gtaaagttct gttgtcagag acgaacaagt accgttcgaa cagggcggta 420

ccttgacggt acctgacgag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 480

gtaggtggca agcgttgtcc ggaattattg ggcgtaaagc gcgcgcaggc ggctatgtaa 540

gtctggtgtt aaagcccggg gctcaacccc ggttcgcatc ggaaactgtg tagcttgagt 600

gcagaagagg aaagcggtat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 660

accagtggcg aaggcggctt tctggtctgt aactgacgct gaggcgcgaa agcgtgggga 720

gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aggtgttggg 780

ggtttcaata ccctcagtgc cgcagctaac gcaataagca ctccgcctgg ggagtacgct 840

cgcaagagtg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900

taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccgctgac cgtcctagag 960

atagggcttc ccttcggggc agcggtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1020

gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt atctttagtt gccagcattc 1080

agttgggcac tctagagaga ctgccgtcga caagacggag gaaggcgggg atgacgtcaa 1140

atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggctggt acaacgggaa 1200

gctagctcgc gagagtatgc caatctctta aaaccagtct cagttcggat tgcaggctgc 1260

aactcgcctg catgaagtcg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1320

cgttcccggg ccttgtacac accgcccgtc acaccacggg agtttgcaac acccgaagtc 1380

ggtgaggaac cgcaaggagc cagcc 1405

Claims

1. A strain of tylosin degrading bacterium is Brevibacillus borstelensis (A)Brevibacillusborstelensis) TYL2, preserved in China general microbiological culture Collection center, with the preservation date of 2018, 12 months and 29 days, the preservation number of CGMCC No.17051, the preservation address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

2. A microbial preparation comprising the tylosin degrading bacterium of claim 1.

3. Use of the tylosin degrading bacterium according to claim 1 or the microbial inoculum according to claim 2 for the degradation of tylosin.

4. Use of the tylosin degradation bacterium according to claim 1 or the microbial inoculum according to claim 2 for remediating a soil or water body contaminated with tylosin.