CN107227281B - Salt lake streptomycete TRM20170601 and application thereof in disinfectant - Google Patents

Abstract

The invention discloses streptomyces salina TRM20170601 and application thereof in disinfectants, wherein a strain of streptomyces salina (Streptomyces salina) with higher capability of producing biological enzyme and remarkable biomembrane inhibition effect of fermentation liquor is obtained by separating, screening and culturing in soil samples collected in the region of Nitroculter lake, XinjiangStreptomyces salilacus) TRM20170601CGMCC No.14260, the bacterium has the strongest capability of inhibiting biofilm formation under the conditions of pH value of 11, temperature of 60 ℃ and ether treatment, can reduce the relative biofilm formation capability of staphylococcus epidermidis to 10.98 percent, and has average killing logarithm value of more than 5.00 on staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa in bacterial suspension after acting for 5min in a sterilization test.

Description

Salt lake streptomycete TRM20170601 and application thereof in disinfectant

Technical Field

The invention belongs to the technical field of microorganisms, and relates to the technical field of a novel streptomycete strain and application thereof in disinfectants.

Technical Field

The disinfection refers to removing or killing pathogenic microorganisms and other harmful microorganisms on the body surfaces of the livestock and poultry and in the living environment thereof by using a proper method according to different production links and objects. Under the current trend of higher and higher intensification degree, diseases of the breeding farm are more and more complicated, and the problem of animal epidemic disease prevention and treatment is very prominent. The occurrence of epidemic diseases in large-scale farms is often the result of a multi-factor combination effect, but the most important of the epidemic diseases is caused by invasion and diffusion of pathogenic microorganisms in the external environment or pollution and diffusion of pathogenic microorganisms of animals in the farms. Effective disinfection is one of important measures for stopping and reducing pathogens in the environment of a farm, cutting off epidemic disease transmission ways and preventing and controlling infectious diseases of the farm. However, the existing disinfectant often has residual drug-resistant pathogenic bacteria after disinfection, is not ideal in use effect, cannot effectively prevent and treat certain scale farm epidemic diseases such as recurrent attacks of cow recessive mastitis and the like, is difficult to cure, and becomes an aeipathia disease of the livestock breeding industry.

In recent years, researches show that biofilm is the main reason causing the pathogenic bacteria to be difficult to eradicate, bacteria in the form of biofilm is different from planktonic bacteria, the bacteria have strong resistance to bactericides such as antibiotics and the like, severe environment and host immune defense mechanism, the bacteria in the biofilm have unique properties in the aspects of physiology, metabolism, degradation or utilization of substrates and resistance to environment, the ideal disinfection effect is difficult to achieve by using the existing disinfectants to eliminate the pathogenic bacteria, the disinfectant containing biofilm inhibitors can well solve the problem and greatly improve the disinfection effect, the biofilm inhibitors produced by microorganisms are one of the types, some actinomycetes can generate active substances such as indole, protease and the like for inhibiting the biofilm formation of bacteria, and the action mechanism is completely different from the traditional antibiotics, the active substances realize the inhibition effect by interfering the expression of genes related to the biofilm formation of pathogenic bacteria on the premise of not influencing the growth of the pathogenic bacteria. Compared with the traditional antibacterial disinfectant, the novel biological membrane inhibiting active substance can relieve the problem of bacterial drug resistance to a certain extent. Therefore, the bacterial biofilm formation inhibitor as a novel disinfectant component has a wide application prospect in the field of prevention of microorganisms, particularly biofilm infection, but at present, reports about the application of the microbial biofilm inhibitor to the disinfectant are few, so that the biofilm inhibition effect of the microorganisms needs to be further researched, a resistant strain with the biofilm formation inhibition effect and stable performance is provided for the inhibition of the microbial biofilm, and the bacterial biofilm formation inhibitor has a potential application development prospect in the field of the disinfectant.

Disclosure of Invention

Aiming at the current situation that the related reports about the new streptomycete strain and the application of the streptomycete strain in the disinfectant are not found in the prior art, the existing disinfectant has residual drug-resistant pathogenic bacteria after disinfection, the using effect is not ideal, and the scale farm epidemic disease cannot be effectively prevented and treated, the invention aims to provide a new resistant strain with biofilm formation inhibiting effect and stable performance for the disinfectant. The invention separates and screens out a new streptomycete strain in a soil sample, and has higher capability of producing biological enzyme, so that the fermentation liquor has obvious biological membrane inhibition effect. Provides a Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 and an application technical scheme of fermentation liquor of the Streptomyces salina in disinfectant.

The invention adopts the main technical scheme that:

the soil sample adopted by the invention is collected from Nitrocurone lake, Xinjiang by the national institute of Life sciences of Tarim university, a large amount of well-grown streptomyces strains are selected and optimized from the collected soil sample, the 16S rRNA gene sequence is determined, and a new streptomyces strain (streptomyces salinalacus) with strong capability of inhibiting biofilm formation is selected from the streptomyces strain.

The invention specifically provides a salt lake Streptomyces (Streptomyces salilaceus) TRM20170601CGMCC No.14260, which is characterized in that a certain screening method is provided, a batch of Streptomyces microbial strains are obtained by separating, screening and culturing collected soil samples, a Streptomyces strain TRM20170601 with strong capability of inhibiting biofilm formation is screened out, and the Streptomyces strain TRM20170601 belongs to a new strain in Streptomyces (Streptomyces salilaceus) through microbiological classification and identification and is tentatively named as Streptomyces salilaceus TRM 20170601.

The strain is deposited in the Budapest treaty microorganism international preservation unit before application, namely the China general microbiological culture Collection center (CGMCC) of culture management committee, the address: Beijing Korean area, No. 3 of West Lu 1, institute of microbiology, the code: 100101, 6.21 days of preservation in 2017, the strain preservation number is CCNo.14260, the strain is identified as Streptomyces (Streptomyces salilaccus) by microbiological identification, the strain specification is identified as Streptomyces salina (Streptomyces salilaccus) by systematic classification, the strain specification is named as Streptomyces salina (Streptomyces salilaccus) by adopting a high-temperature culture medium (mannitol 10g, magnesium sulfate 10g, white phosphate medium, calcium sulfate, calcium phosphate, calcium sulfate, calcium phosphate, calcium sulfate, calcium phosphate, calcium sulfate, calcium phosphate, calcium sulfate, calcium phosphate, calcium sulfate, calcium phosphate, calcium sulfate, magnesium sulfate, magnesium sulfate, magnesium chloride, magnesium sulfate, magnesium sulfate, magnesium chloride, magnesium sulfate, magnesium chloride, magnesium chloride, magnesium sulfate, magnesium.

Meanwhile, the total DNA of the strain TRM20170601 is extracted, a universal primer for bacterial 16SrDNA PCR amplification is adopted for PCR amplification, and a PCR product is subjected to gel cutting and purification and then is sequenced. The tested 16SrRNA gene sequence is compared with the sequence in the GenBank database, and the result shows that: the maximum homology of the strain TRM20170601 with a model strain Streptomyces macrosporirus NBRC 14748T (AB184616) is 98.0 percent, the homology with other strains in the same genus is less than 97.0 percent, the strain can not be determined to be exactly classified, the strain is determined to be a new strain, and the strain is tentatively named Streptomyces salilaceus from the taxonomy point of view.

Further, the invention provides application of the Streptomyces salina TRM20170601CGMCC No.14260 fermentation liquid in disinfectants. By researching the influence of Streptomyces salinilacus fermentation liquor on the inhibition of the biofilm formation under different pH values, temperatures and organic reagent treatment conditions, the optimal inhibition condition of the strain TRM20170601 on the biofilm formation is obtained by treating the strain with diethyl ether at the temperature of 60 ℃ and the pH value of 11.

By implementing the specific technical indexes of the invention, the content of the invention is realized, and the following beneficial effects can be achieved:

(1) the Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 provided by the invention has the advantages of simple culture conditions, rapid propagation, stable genetic characteristics and strong capability of inhibiting biofilm formation.

(2) When the Streptomyces salinus (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation liquor provided by the invention is applied to a disinfectant, the capability of inhibiting biofilm formation is strongest under the conditions of pH value of 11, temperature of 60 ℃ and ether treatment, the capability of forming staphylococcus epidermidis relative to the biofilm is reduced to 10.94%, and the average killing logarithm value of the staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa in a bacterial suspension is more than 5.00 after the fermentation liquor acts for 5min in a sterilization test.

Drawings

FIG. 1 shows the colony and bacteria photographs of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No. 14260.

FIG. 2 shows a phylogenetic dendrogram of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No. 14260.

FIG. 3 shows the effect of temperature on the ability of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation broth to inhibit the formation of Staphylococcus epidermidis biofilm.

FIG. 4 shows the effect of pH value on the ability of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation broth to inhibit the formation of Staphylococcus epidermidis biofilm.

FIG. 5 shows the effect of organic reagent treatment on the ability of Streptomyces salina TRM20170601CGMCC No.14260 fermentation broth to inhibit the formation of Staphylococcus epidermidis biofilm.

Detailed Description

The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All the raw and auxiliary materials (except Staphylococcus epidermidis ATCC35984 (biofilm formation positive strain), which is given by the college of medicine of Shanghai Compound denier university) and the culture methods of the selected strains are well known and selected in the art, and the percentages referred to in the present invention are percentages by weight unless otherwise indicated.

The first embodiment is as follows: separation, screening and identification of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260

1. Isolation and selection of bacterial species

The soil sample is collected from Nierku lake Xinjiang by the national institute of Life sciences of Tarim university, and a large number of Streptomyces strains with good growth are selected from the collected soil sample, and the 16S rRNA gene sequence is determined, so that a strain with the number of TRM20170601 is selected from the collected soil sample.

A separation step:

a series of gradient diluents are obtained by a gradient dilution method of microbial separation, mixed bacteria diluents 0.lm L with I0-1-10-3 dilution degrees are respectively sucked by a sterilization suction pipe, transferred to a plate poured with a Hirschhorn-number-I culture medium, evenly coated by a sterile glass rod, then placed in a constant temperature incubator at 28 ℃ upside down to be cultured until single bacterial colonies grow out, the single bacterial colonies growing on the plate are picked up by a sterile inoculating loop in a sterile operating platform and inoculated on the plate filled with the Hirschhorn-number-I culture medium to be streaked and separated after the single bacterial colonies grow out, the single bacterial colonies are transferred to a sterilization test tube inclined plane again, each bacterial strain is repeatedly cultured for 3-5 days at 28 ℃, the consistency of the bacterial colonies is observed by utilizing light rays of transmitted light, reflected light and dark background, the consistency of the bacterial colonies is observed by an optical microscope for smear dyeing, and the consistency is repeatedly separated and purified for multiple times until the bacterial colonies and individuals are even and consistent for later use.

2. Culture conditions of the strains

(1) The growth medium of the strain with the number of TRM20170601 is Gao's first medium which comprises 10g of mannitol, 1g of magnesium sulfate heptahydrate, 0.2g of calcium carbonate, 1g of alanine, 1g of dipotassium phosphate, 0.01g of ferrous sulfate heptahydrate, 15g of sodium chloride, 50mg of potassium dichromate, 18g of agar powder, 1L of water, 1m L (0.001 g/L) of trace salt, 1m L (0.5 mg/L) of vitamin complex and has the pH value of 7.2-7.4.

(2) The strain with the number of TRM20170601 can grow under the condition of 25-50 ℃, the optimal growth temperature is 28 ℃, and the culture time is 3-5 days.

(3) The strain numbered TRM20170601 had an optimal growth pH of 7.

Specifically, the strain with the number of TRM20170601 is screened out by separating, screening and culturing collected soil samples, and is classified and identified microbiologically, the strain belongs to a Streptomyces salilacus strain, the strain is stored in Budapest treaty international preservation unit of microorganisms before application date, the China general microbiological culture Collection center (CGMCC) has the address of No. 3 North Chenxi Luo No.1 of Beijing Korean area, the institute of microbiology, the zip code of 100101, the preservation date of 2017 and 6-21 days, the strain preservation number is CGMCC No.14260, the new strain identified as Streptomyces by microbiology is identified, the strain is classified into Streptomyces salilacus TRM20170601 from the classification temporary point, the optimal growth conditions of the strain are that the temperature is 28 ℃, the culture medium adopts Gao's primary culture medium (mannitol 10g, heptahydrate 1g, magnesium sulfate, calcium carbonate 0.2g, calcium carbonate 1.2 g, potassium phosphate 1.7 g, sodium chloride 0.7 g, sodium phosphate 0.7 g, sodium chloride 5 g/35 g, 364 g, sodium phosphate, sodium chloride 5.7 g and 364 mg/15 mg of sodium chromate 0.3 and 364 mg.

3. Physiological and biochemical identification of strain TRM20170601

Morphological characteristics: after the TRM20170601 is cultured for 4 days, on the surface of a Gao's I culture medium, bacterial colonies are round, neat in edge, convex, smooth, white, opaque, sticky and not easy to pick up; the bacteria are gram-positive bacteria, aerobic bacteria, thallus rod, flagellum-free bacteria, and endospore, and their colony and thallus morphology are shown in figure 1.

Physiological and biochemical characteristics of the strain are that a Biolog GN2 plate detects that carbon sources available for the strain are L-fucose, glucose and D-fructose.

The strain TRM20170601 is a typical new strain, and is assigned to a member of Streptomyces salilacus from the taxonomy perspective and is named as the TRM20170601 temporarily as the Streptomyces salilacus TRM20170601 through the strain morphology, culture characteristic observation and physiological and biochemical index measurement of the strain TRM20170601CGMCC No.14260, namely through tests such as the strain morphology observation, the strain culture characteristic observation and growth temperature measurement by referring to methods of Bergey's systematic bacteriology identification handbook, eighth edition and common bacterial system identification handbook, wherein the strain with the number of TRM20170601 has obvious physiological and biochemical characteristic difference compared with common Streptomyces strains and has stronger capability of inhibiting biofilm formation.

Example two: molecular level identification of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260

Extracting total DNA of a strain TRM20170601, adopting a bacterial 16S rDNA PCR amplification universal primer to carry out PCR amplification, carrying out sequencing after PCR products are subjected to gel cutting purification, referring to a whole gene SEQUENCE of the strain TRM20170601 provided after the attachment, a SEQUENCE obtained by an experimental strain is compared with a known SEQUENCE in a GenBank database by B L AST, determining the species relationship closest to the genetic relationship of the experimental strain, combining SEQUENCE alignment in ezTaxon (http:// www.ezbiocloud.net/ezTaxon) and adjusting a related pattern strain SEQUENCE to carry out phylogenetic tree analysis of a pattern strain SEQUENCE, carrying out cluster analysis and phylogenetic tree construction by using MEGA5.0 software package by using an adjacency method (Neighbor-Joingmethod), showing that the strain TRM20170601 and the pattern strain are named as Streptomyces maltospos 14748T (1840.616), the maximum homologous evolution is 98.616%, and the strain is classified into a new strain, and the strain has obvious difference from the related molecular evolution of Streptomyces strain NBlaclossariosporus, namely the new strain NBlacosase accession number, and the strain is shown in the attached drawing, and the new strain NBlacosase of the Streptomyces strain.

Example three: preparation of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation liquor

(1) Activation of the strain: the strain preserved in the glycerin tube is inoculated on a Gao's first culture medium plate and cultured for 5 days at 28 ℃.

(2) The preparation of seed liquid, which is to select a single colony from the solid plate of Gao's I after activation and transfer the single colony to a 500m L triangular flask filled with 150m L liquid Gao's medium, and culture the single colony at 185r/min for 5-7 days at 28 ℃ of constant temperature shaking table.

(3) Initial fermentation culture conditions: inoculating the seed liquid into a liquid Gao's No. I culture medium according to the inoculation amount of 4%, and culturing for 15d at the constant temperature of a shaking table at 28 ℃ and 185 r/min.

Example four: influence of streptomyces salina (streptomyces salilacus) fermentation liquor on inhibiting biofilm formation under different pH values, temperatures and organic reagent treatment conditions

1. Test method

Diluting the bacterial liquid according to a ratio of 1:100, filtering the fermentation liquid by using an aseptic filter membrane, mixing the treated fermentation liquid with the bacterial liquid according to different concentrations (10% -50%), sucking 20 mu L to a 96-pore plate after mixing, keeping the plate stand and culturing for 24h at 37 ℃ in a constant-temperature incubator with 4 pores per gradient, measuring OD590, slowly washing the plate for 3 times by flowing water to wash off the non-adhered bacteria, putting the plate into an oven, drying for 1h at 56 ℃, dyeing for 5min by using 0.5% crystal violet, washing by flowing water to wash off redundant crystal violet dye, naturally drying, and measuring OD 490.

Relative biofilm formation ability (treatment group OD 490/blank OD490) × 100.

2. Influence of different temperatures on capability of Streptomyces salina TRM20170601CGMCC No.14260 fermentation broth in inhibiting staphylococcus epidermidis biofilm formation

The temperature of-20 ℃ is set as an untreated group, and the temperatures of the treated groups are respectively as follows: after the fermentation broth was treated with water baths of different temperatures at 30 deg.C, 40 deg.C, 60 deg.C, 80 deg.C, 100 deg.C for 30min, the results are shown in FIG. 3, where the film forming ability to the biological film was 51.36% when the protein temperature was-20 deg.C; the film forming ability to the biological film at the temperature of 30 ℃ is 52.02%; the film forming capability to the biological film is 49.88% at the temperature of 40 ℃; the film forming capability to the biological film is 37.89% when the temperature is 60 ℃; the film forming capability to the biological film is 54.68% when the temperature is 80 ℃; the film forming ability to the biological film at 100 ℃ is 59.04%; the fermentation liquor has the strongest capability of inhibiting the formation of staphylococcus epidermidis biofilm at the temperature of 60 ℃.

3. Influence of different pH values on capability of Streptomyces salinensis (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation broth in inhibiting staphylococcus epidermidis biofilm formation

The pH value of the untreated group was 7, the fermentation solutions were adjusted to different pH values, and the influence of the fermentation solutions on the biofilm formation ability of Staphylococcus epidermidis ATCC35984 was measured, and the results are shown in FIG. 4, in which the biofilm formation ability was 27.47% when the pH value of the protein was 3; the film forming capability to the biological film is 30.24% when the pH value is 5; the film forming ability to the organism at pH7 was 38.54%; the film forming capability to the biological film is 25.74% when the pH value is 9; the film forming capability to the biological film is 10.94% when the pH value is 11; when the pH value is 11, the biofilm forming capability is weakest, and the biofilm inhibition rate is highest.

4. Influence of different organic reagents on capability of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation broth in inhibiting staphylococcus epidermidis biofilm formation

The untreated group was prepared by adding only the fermentation broth without adding the organic reagent, and the treated group was prepared by adding acetone, chloroform, ethyl acetate, and diethyl ether, as shown in FIG. 4, the acetone-treated group had a relative biofilm formation ability of 14.47%; the relative biofilm forming capacity of chloroform treated was 38.4%; its relative biofilm formation ability with ethyl acetate treatment was 11.21%; the relative biofilm formation capacity of the ether treated product was 10.98%; its relative biofilm formation ability without organic agent treatment was 45.27%; the experiment shows that the biofilm forming ability of the fermentation liquor after the treatment of the ether is the weakest, and the biofilm inhibiting activity of the fermentation liquor on the staphylococcus epidermidis is the strongest.

Example five: disinfectant prepared based on Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 fermentation broth and having killing effect on different bacteria

Diluting a disinfectant prepared from a fermentation broth of Streptomyces salina (Streptomyces salilacus) TRM20170601CGMCC No.14260 into the disinfectant with the test concentration of 2 times by using sterile standard hard water, and keeping the temperature of the disinfectant in a water bath at 60 +/-1 ℃ for 10 min. 1.0ml of the bacterial suspension and 4.0ml of a disinfectant (the positive control group is a diluent) are added into a sterile test tube and mixed for a predetermined time. Adding 1ml of the mixed solution into a test tube containing 10ml of neutralizer, mixing, and neutralizing for 15 min. Through mixing, sucking 1.0ml sample liquid as pouring inoculation culture, counting live bacteria and calculating killing log value. The test was repeated 2 times, and the results are shown in Table 1.

Table 1: disinfectant prepared based on new strain fermentation liquor has effect of killing different bacteria

The disinfectant diluent prepared based on Streptomyces salinilacus TRM20170601CGMCC No.14260 fermentation liquor acts for 5min, and average killing logarithm value of staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa in the bacterial suspension is more than 5.00; it acts on Candida albicans for 5min, and has average killing log value of 3.57.

The experiments show that the Streptomyces (Streptomyces salinilacus) TRM20170601CGMCC No.14260 fermentation liquid provided by the invention is developed into the disinfectant, the capability of inhibiting the formation of the biofilm is strongest under the conditions of pH value of 11 and temperature of 60 ℃ and ether treatment, the relative biofilm formation capability of staphylococcus epidermidis can be reduced to 10.94%, and the average killing logarithm value of the staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa in a bacterial suspension is more than 5.00 after the Streptomyces salinilacus is acted for 5min in a sterilization test.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.

SEQUENCE LISTING

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Claims (2)

1. The Streptomyces salinosus (Streptomyces salilacus) TRM20170601 is characterized in that the Streptomyces salinosus (Streptomyces salilacus) TRM20170601 has the CGMCC (CGMCC) preservation number of No. 14260.

2. Use of Streptomyces salina (Streptomyces salilacus) TRM20170601 as defined in claim 1 in the preparation of disinfectants.

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