CN112143668B - Tetracycline antibiotic degrading strain, preparation method and application thereof - Google Patents

Abstract

The minocycline degrading strain is Kluyvera sp.Kluyvera, which has been preserved in China general microbiological culture Collection center (CGMCC) at 6-22.2020, and the preservation number is CGMCC No. 20117. The strain can be applied to the preparation of minocycline degrading microbial inoculum, is beneficial to enriching a strain resource library of tetracycline antibiotic degrading bacteria, and provides an effective biodegradation method for the treatment of tetracycline antibiotic pollution. The strain used in the invention has high minocycline degradation efficiency, and the 72h degradation rate of the strain reaches over 90 percent under the condition of laboratory conditions and the initial minocycline content of 50 mg/L. The invention provides an effective biological way for removing minocycline in the environmental water body.

Description

Tetracycline antibiotic degrading strain, preparation method and application thereof

Technical Field

The invention relates to the technical field of antibiotic pollution treatment by a biological method, relates to a degrading microorganism, and particularly relates to a tetracycline antibiotic degrading strain, a preparation method and application thereof.

Background

Tetracycline antibiotics (TCs) are a broad spectrum antibiotic used to treat infections caused by rickettsia, chlamydia, mycoplasma, spirochetes, and bacteria. Because of low price and wide antibacterial spectrum, the compound is widely used all over the world and is mainly used as a therapeutic drug for animal breeding and human beings. Minocycline (Minocycline), also known as Minocycline or mecycline, is a semi-synthetic tetracycline antibiotic with broad-spectrum antibacterial activity. Can be combined with RNA to achieve the bacteriostatic effect. Compared with the similar medicines, minocycline has wider antibacterial spectrum, strongest antibacterial activity and small side effect on human body. Due to these properties, minocycline is widely used clinically and is one of the highest antibiotics in cosmetics.

In 2013, the usage amount of the tetracycline antibiotics in China reaches 1.2 ten thousand tons. However, since the tetracycline antibiotics are not completely metabolized by humans or animals, 70% of the antibiotics are excreted into the environment through urine and feces, and their high hydrophilicity and low volatility make them significantly persistent in aquatic environments. Tetracycline antibiotic residues have been currently detected in a variety of environments, such as rivers, sewage treatment plants, landfill leachate. Higher concentrations of minocycline have been detected in hospital wastewater and wastewater treatment plants, with the highest concentrations of minocycline detected in the wastewater from a particular hospital of grapevine and the influent from a wastewater treatment plant being 531.7 μ g/L and 915.3 μ g/L, respectively. The presence of minocycline has also been detected in surface waters somewhere in Anhui province. In the wastewater treatment process, tetracycline antibiotics cannot be effectively removed, and the average removal rate is only 30%.

The residual antibiotics in the environmental water body can directly or indirectly enter the human body through various exposure ways such as drinking water or food chain enrichment and the like, and can be accumulated and enriched in the human body through the biological amplification effect of the food chain. TCs have an ecotoxic effect, easily cause enrichment of drug-resistant flora and generation of resistance genes, reduce autoimmune functions and treatment effects of various drugs, inhibit development of teeth and bone growth of infants and the like, so that potential threats of TCs widely accumulated in an environmental medium to the environment and human health cannot be ignored.

Currently, methods for pollution control of TCs include a high-temperature composting method, an advanced oxidation method, a photoelectrocatalysis method, and a microbial degradation method, and compared with other methods, the microbial degradation method has the advantages of low cost, strong specificity, no secondary pollution and the like. However, microbial degradation methods are mainly focused on natural tetracycline, and no literature report is available for microbial degradation of minocycline.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a tetracycline antibiotic degrading strain, a preparation method and application thereof.

A tetracycline antibiotic degradation strain is Kluyvera sp, which is preserved in China general microbiological culture Collection center on 6-22.2020, with the preservation number of CGMCC No.20117 and the address: beijing, Chaoyang district, Beichen Xilu No. 1 institute, institute of microbiology, China academy of sciences.

Specifically, the Kluyvera sp is obtained from sewage of municipal sewage through enrichment, separation and purification.

Specifically, the method comprises the steps of enriching, separating and purifying the sewage from municipal sewage pipelines.

Specifically, the method comprises the following steps:

the method comprises the following steps: enrichment of strains

Adding a supernatant in the sewage into an enrichment culture medium, wherein the enrichment culture medium is a common culture medium containing 8-12 mg/L of minocycline and used for enriching degradation strains, placing the common culture medium into a shaking table at the temperature of 28-32 ℃ for dark culture, sucking a culture solution after 6-8 days, inoculating the culture solution into a common culture medium containing 18-22 mg/L of minocycline again, culturing for 6-8 days without changing other conditions, and repeating the above operation steps until the final concentration of minocycline in the enrichment culture medium is 48-52 mg/L;

step two: isolation and purification of the strains

After enrichment is finished, taking the culture solution for gradient dilution, taking the diluted bacterial solution, separating by adopting a plate coating method, selecting single bacteria with different colony morphologies and appearances, placing the single bacteria on a solid culture medium, streaking, further separating and purifying, selecting the single bacteria colony, inoculating the single bacteria colony to an inclined plane, numbering and storing;

step three: screening of degrading strains

And (3) respectively carrying out a minocycline degradation experiment on each separated single strain, quantitatively detecting the minocycline residual concentration by using a high performance liquid chromatography to obtain the strain with the minocycline degradation capability, and naming the strain with the strongest degradation capability as DM-13.

Further, the preparation method of the enrichment medium comprises the following steps: adding peptone, beef extract and sodium chloride into distilled water, adjusting the pH value to 7.0, and sterilizing at 120-122 ℃.

The invention also provides a tetracycline-degrading antibiotic agent, which contains the Kluyvera sp.

Specifically, the preparation method of the tetracycline antibiotic degrading agent comprises the steps of performing conventional activation culture on the Kluyvera sp.

The tetracycline antibiotic degrading strain is applied to degrading minocycline.

The application of the tetracycline antibiotic degrading agent in degrading minocycline is provided.

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

the minocycline high-efficiency degrading bacterium obtained by screening is identified as Kluyvera sp (Kluyverella) through physiological and biochemical characteristics and 16S rRNA sequence analysis, and is named as DM-13. The preservation number is-CGMCC No 20117. The strain used in the invention has high minocycline degradation efficiency, and the 72h degradation rate of the strain reaches over 90 percent under the condition of laboratory conditions and the initial minocycline content of 50 mg/L. The invention provides an effective biological approach for removing minocycline in the environmental water body.

The present invention will be described in further detail with reference to specific embodiments.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a colony morphology of strain DM-13;

FIG. 2 is a scanning electron micrograph of strain DM-13;

FIG. 3 is a phylogenetic tree of strain DM-13;

fig. 4 shows the result of HPLC quantitative detection of minocycline degradation by strain DM 13.

Detailed Description

The structures, proportions, sizes, and other dimensions shown in the drawings and described in the specification are for understanding and reading the present disclosure, and are not intended to limit the scope of the present disclosure, which is defined in the claims, and are not essential to the art, and any structural modifications, changes in proportions, or adjustments in size, which do not affect the efficacy and attainment of the same are intended to fall within the scope of the present disclosure.

The high performance liquid chromatography determination method comprises the following steps: high Performance Liquid Chromatography (HPLC) was used to quantitatively analyze minocycline concentration. A C18 chromatographic column (4.6X 250mm, 5 μ M, Agilent technique) was run at 25 ℃ with a mobile phase consisting of 88% (v/v)0.2M phosphoric acid, 12% (v/v) acetonitrile, and a sample introduction rate of 1 mL/min. The amount of sample was 10. mu.L. The column isocratic elution was monitored with a UV detector at 252 nm.

Minocycline standard (C23H27N3O 7. HCl) in the present invention was obtained from shanghai folk biotechnology limited, and Acetonitrile (ACN) (HPLC grade) was obtained from tianjinke mior chemical reagent. Other reagents are analytically pure. The water was Millipore ultrapure water.

The apparatus employed in the present invention is conventional in the art unless otherwise specified.

The preparation method of the culture medium used by the invention comprises the following steps:

1. enrichment culture medium: 5g of peptone, 3g of beef extract, 5g of sodium chloride and 1000ml of distilled water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20 min.

2. Ordinary solid medium: 5g of peptone, 3g of beef extract, 5g of sodium chloride, 25g of agar and 1000mL of distilled water, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 20min, and pouring the mixture into a flat plate.

3. Ordinary liquid medium: 5g of peptone, 3g of beef extract, 5g of sodium chloride and distilled water are supplemented to 1000ml, the pH is adjusted to 7.0, and the sterilization is carried out for 20min at 121 ℃.

4. Inorganic salt culture medium: MgSO (MgSO)₄·7H₂O 0.15g,FeSO₄·7H₂O 0.01g,CaCl₂ 0.02g,KH₂PO₄1g,Na₂HPO₄1g, 1000ml of distilled water

Carbon source: 0.5g of glucose is added into the mixture,

nitrogen source: NH (NH)₄NO₃ 1g

Adjusting pH to 7.0, sterilizing at 115 deg.C for 30min

Example 1:

the embodiment provides a tetracycline antibiotic degrading strain, which is Kluyvera sp.Kluyvera, and is stored in China general microbiological culture Collection center (CGMCC) at 22/6/2020 with the storage number of CGMCC No.20117 and the address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The Kluyvera sp is obtained from sewage of a certain municipal sewer pipeline in Xian city through enrichment, separation and purification.

Specifically, the method comprises the following steps:

the method comprises the following steps: enrichment of strains

Adding a supernatant in the sewage into an enrichment medium, wherein the enrichment medium is a common medium containing 8-12 mg/L of minocycline and used for enriching degradation strains, placing the common medium into a shaking table at 28-32 ℃ for dark culture, absorbing a culture solution after 6-8 days, inoculating the culture solution into the common medium containing 18-22 mg/L of minocycline again, culturing for 6-8 days without changing other conditions, and repeating the above operation steps until the final concentration of minocycline in the enrichment medium is 48-52 mg/L;

as a preferred embodiment, 10mL of sewage can be taken out from a certain sewage pipeline, the sewage is mixed evenly and then stands, supernatant in 1mL of sewage is taken out and added into an enrichment medium, the enrichment medium is a common medium containing minocycline with the concentration of 10mg/L and used for enriching degradation strains, the common medium is placed into a shaking table with the temperature of 30 ℃ for dark culture, 1mL of culture solution is sucked after 7 days and inoculated into the common medium containing minocycline with the concentration of 20mg/L again, other conditions are unchanged, the operation steps are cultured for 7 days, and the operation steps are repeated until the final concentration of the minocycline in the enrichment medium is 50 mg/L;

Step two: isolation and purification of the strains

After enrichment is finished, 1mL of culture solution is subjected to gradient dilution, 2-3 bacteria solutions with proper dilution are taken and separated by adopting a plate coating method, single bacteria with different colony morphologies and appearances are selected to fall on a solid culture medium and are streaked for further separation and purification, and then the single bacteria colony is selected and inoculated to an inclined plane, and the number is stored;

step three: screening of degrading strains

And (3) respectively carrying out a minocycline degradation experiment on each separated single strain, quantitatively detecting the residual concentration of minocycline by using High Performance Liquid Chromatography (HPLC), obtaining the strain with the minocycline degradation capability, and naming the strain with the strongest degradation capability as DM-13.

High performance liquid chromatography determination method

High Performance Liquid Chromatography (HPLC) was used to quantitatively analyze minocycline concentration. A C18 chromatographic column (4.6X 250mm, 5 μ M, Agilent technique) was run at 25 ℃ with a mobile phase consisting of 88% (v/v)0.2M phosphoric acid, 12% (v/v) acetonitrile, and a sample introduction rate of 1 mL/min. The amount of sample was 10. mu.L. The column isocratic elution was monitored with a UV detector at 252 nm.

Minocycline standard (C23H27N3O 7. HCl) from shanghai-derived lobe biotechnology limited, and Acetonitrile (ACN) (HPLC grade) from tianjin komi chemical reagent. Other reagents were analytically pure. The water was Millipore ultrapure water.

The strain has strong degradation effect on minocycline, and has the following characteristics:

(1) the purified DM-13 strain is inoculated on a common solid culture medium and cultured for 48 hours at the temperature of 30 ℃, the colony shape is round, the edge is semitransparent, the middle is raised and milk white, and the surface is glossy, as shown in figure 1.

(2) The colony after 48h of culture was pretreated, and observed by scanning electron microscopy (10000 times) to be rod-shaped, with a size (0.5-0.7) μm x (2.5-4) μm, and without spores, as shown in FIG. 2.

(3) The purified DM-13 strain is taken to carry out physiological and biochemical determination such as gram staining, the determination of physiological and biochemical characteristics refers to the handbook of identifying common bacterial systems, and the physiological and biochemical characteristics are shown in the following table.

Physiological and biochemical identification of DM-13 strain

Example 2 identification of 16S rRNA of Strain

(1) Genome extraction

The genomic DNA of strain DM-13 was extracted using a bacterial genome extraction kit (SK8255) from Shanghai Biotech Ltd, according to the instructions.

(2) PCR amplification

16S rDNA Universal primer amplification:

and (3) PCR reaction system:

PCR cycling conditions:

(3) gel electrophoresis

Finally, the PCR product was electrophoresed on 1% agarose gel at 150V for 20min at 100mA (see DNA Ladder Mix make). According to the result of agarose gel electrophoresis, the band of the desired DNA is cut by the electrophoresis band of the PCR product, and the PCR product is directly sequenced by using the PCR primer. The nucleotide sequence of the 16S rDNA gene of the strain DM-13 was obtained, and the nucleotide sequence of the 16S rDNA gene of the strain DM-13 was shown in the following figure.

Strain DM-13 gene sequence table

CAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTTGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCCGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCACACCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCATTGTGGTTAATAACCGCAGTGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAA

The sequence consists of 1467 bases (bp).

The measured 16S rDNA sequence was subjected to BLAST search at the National Center for Biotechnology Information (NCBI) website to find a strain sequence having a high similarity, and a phylogenetic tree was constructed using MEGA6.0 software, as shown in FIG. 3. The comparative analysis result shows that the strain DM-13 is identified as the Kluyveromyces.

Example 3 demonstration of the degradation Effect of DM-13 on minocycline

A loop of the DM-13 strain was picked from the plate and inoculated into 150ml of a common liquid medium, and cultured with shaking at 120rpm at 30 ℃ for 36 hours to prepare a seed solution. 5mL of the seed solution was taken and 200mL of an inorganic salt medium (0.15g of MgSO4 and 0.2g of CaC 1) was added to the seed solution₂) Adding 1g/L glucose into the culture medium, adding 50mg/L minocycline (reagents are purchased from Shanghai-source leafy organisms), uniformly mixing, oscillating for 72h, centrifuging to obtain a supernatant, filtering the supernatant by using a filter head with a pore diameter of 0.22mm, and carrying out HPLC quantitative detection on the filtered supernatant. The experiment was repeated three times and a blank control without added bacteria was set. The HPLC quantitative detection result is shown in figure 4, and the result shows that after 72 hours, the degradation rate of minocycline is 25% in the culture medium without the DM-13 strain, the degradation rate of minocycline is more than 90% in the culture medium with the DM-13 strain, and the contribution rate of bacteria to minocycline degradation is more than 65%.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, it should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of the various embodiments of the present disclosure can be made, and the same should be considered as the inventive content of the present disclosure, as long as the combination does not depart from the spirit of the present disclosure.

Claims (6)

1. The minocycline degrading strain is Kluyvera sp.Kluyvera, which is preserved in China general microbiological culture Collection center (CGMCC) at 6-22.2020, and the preservation number is CGMCC No. 20117.

2. The minocycline degrading strain of claim 1, wherein said Kluyvera sp.

3. A minocycline degrading agent comprising Kluyvera sp.

4. The minocycline degrading microbial inoculum of claim 3, wherein the preparation method comprises the steps of performing conventional activation culture on Kluyvera sp.

5. Use of a minocycline degrading strain according to any one of claims 1 or 2 to degrade minocycline.

6. Use of the minocycline degrading microbial inoculum of claim 3 for degrading minocycline.

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