CN106987543B - A kind of serratia marcescens BSFL-6 in stratiomyiid enteron aisle source and its application - Google Patents
A kind of serratia marcescens BSFL-6 in stratiomyiid enteron aisle source and its application Download PDFInfo
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Abstract
The invention belongs to technical field of agricultural microbiology, specifically disclose serratia marcescens BSFL-6 and its application in a kind of insect stratiomyiid enteron aisle source, the serratia marcescens be serratia marcescens (Serratia marcescens) BSFL-6, deposit number: CCTCC NO:M 2017073.Serratia marcescens provided by the invention (Serratia marcescens) BSFL-6 can effectively degrade antibiotic tetracycline, enhancing stratiomyiid is resisted during converting material and degrades the ability of tetracycline, and to harmful bacteria staphylococcus aureus (S.Aureus) and Escherichia coli (E.coli) there is bacteriostatic activity, it is one plant of stratiomyiid beneficial bacteria of intestinal tract, the conversion that can be used for promoting stratiomyiid to the waste polluted containing tetracycline.
Description
Technical field
The invention belongs to technical field of agricultural microbiology, and in particular to a kind of cement sand thunder in insect stratiomyiid enteron aisle source
Salmonella BSFL-6 and its application, serratia marcescens BSFL-6 separate the stratiomyiid enteron aisle of the inverting contaminated material containing tetracycline,
The application for further relating to the serratia marcescens Yu stratiomyiid Synergistic degradation tetracycline further relates to the serratia marcescens and inhibits harmful bacteria
The application of staphylococcus aureus and Escherichia coli Growth.
Background technique
With the rapid development of intensive modern livestock and poultry breeding industry, usage amount is increasingly in its production process for antibiotic
Greatly, the antibiotic of high dose can be used for treating various diseases, and low dosage then can be used for promoting growth of animals or poultry and volume increase, In
The purpose devoted exclusive attention to output, increased economic efficiency all is realized in nearly all area using antibiotic in global range.Wherein, Fourth Ring
Element occupies an important position in drug market, is one of the antibiotic being most widely used in the world.ASSOCIATE STATISTICS shows, 2014
Year, China's tetracycline yield reached more than 4000 tons, in addition, national, there are also the productions of 40,000 tons or so of tetracycline derivant and its salt
Ability.Although at home and abroad on medical market, tetracycline has dropped out anti-infectious mainstream medicine kind, because its is cheap,
Has a broad antifungal spectrum is largely used to livestock and poultry medication for many years and is added in feed.They enter in animal or human body after about 70%
It cannot be absorbed, but be directly excreted in the form of parent compound.
There is certain eco-toxicities to microorganism, plant and animal for tetracycline in environment, and lure microorganism production into
Raw resistant gene increases ecological risk.It can inhibit the generation of microalgae protein synthesis and chloroplaset；By inhibiting chloroplaset to close
The activity of enzyme, to generate inhibiting effect to plant growth；The activity for inhibiting a variety of enzymes of aquatic animal, such as ethoxyresorufin-
O- removes ethyl enzyme, beta galactosidase etc., and may cause the more serious DNA damage of fish；Cause mouse liver cell gene table
The change reached is related to including the inhibition of energetic supersession, the synthesis of protein and inhibition of drug metabolism related enzyme systems gene etc.
Multiple molecular mechanisms.Meanwhile the accumulation of the resistant gene as caused by tetracycline etc. is also resistant bacteria --- " superbacteria " produces
One of raw reason, is extremely serious ecological threat.
In every processing technique for feces of livestock and poultry, stratiomyiid processing technique is a new and effective, inexpensive high yield
Value technology.Stratiomyiid is a kind of resource insect, and larva, which is similar to fly maggot, to be food with feces of livestock and poultry, but bigger than fly maggot head,
Growth cycle is longer, and prepupal period can climb out of automatically, is easier to realize manipulation and control, corresponding treatment effeciency and biomass output are more
It is high；Its adult is cleaner with respect to fly, does not approach the mankind；Stratiomyiid is directly inoculated into the feces of livestock and poultry of accumulation by many researchers
In, it finds it while having the function of walking quickly and keeping away fly maggot, deodorization, inhibition and killing pathogenic microorganism, until stratiomyiid prepupa period (2-3
Week) the clean larva peeled climbed out of from feces of livestock and poultry can also be directly collected, form the byproduct (packet of more high added value
Include polypide albumen, insect oils, residual fertilizer etc.), entire ecologic breeding circulation is formed for feed, therefore receive the world and grind
The concern for the person of studying carefully.
In view of the above-mentioned problems, applicant in the stratiomyiid Study on Transformation to chicken manure and pig manure containing tetracycline, has found stratiomyiid
The antibiotic such as tetracycline can effectively be decomposed.Fourth Ring is being decomposed by constructing sterile stratiomyiid system, comparing stratiomyiid enteric microorganism
Contribution during element, the study found that stratiomyiid enteric microorganism is the key that stratiomyiid decomposes tetracycline participant.The present invention is from water
Isolated one plant of stratiomyiid commensal gut bacterium serratia marcescens BSFL-6 in horsefly enteron aisle has enhancing stratiomyiid in conversion material
The ability of resistance and tetracycline of degrading in the process, and to harmful bacteria staphylococcus aureus (S.Aureus) and Escherichia coli
(E.coli) there is bacteriostatic activity, be one plant of stratiomyiid beneficial bacteria of intestinal tract, currently, there are no have drop from insect gut separation in the world
Solve the report of the insect gut bacterium of antibiotic and the antibiotic that can degrade with insect stratiomyiid symbiosis.
Summary of the invention
The object of the present invention is to provide a kind of serratia marcescens BSFL-6, the bacterial strain can with the good symbiosis of stratiomyiid,
Itself there is the ability of certain degradation tetracycline in aerobic environment, while can effectively inhibit harmful bacteria staphylococcus aureus
(S. Aureus) and Escherichia coli (E.coli), the bacterial strain were sent on 2 28th, 2017 into China typical culture collection
Heart preservation, classification naming: serratia marcescens (Serratia marcescens) BSFL-6, deposit number: CCTCC NO:M
2017073, address: Wuhan, China Wuhan University.
It is another object of the present invention to provide the applications of serratia marcescens BSFL-6, including the use of the bacterial strain system
Allowance resists and/or the stratiomyiid of degradation tetracycline, or the degradation agent of tetracycline is prepared into using the bacterium, or prepare while degrading
Tetracycline and the preparation for inhibiting Escherichia coli and staphylococcus aureus.
In order to reach the goals above, the present invention takes following measures:
By constructing sterile stratiomyiid system, comparing contribution of stratiomyiid enteric microorganism during decomposing tetracycline, apply
People's discovery, stratiomyiid enteric microorganism is the key that stratiomyiid decomposes tetracycline participant, and by separation, applicant is inverting to contain Fourth Ring
Isolated one plant of serratia marcescens BSFL-6 in the stratiomyiid enteron aisle of plain contaminated material, the bacterial strain is on 2 28th, 2017
It send to China typical culture collection center preservation, classification naming: serratia marcescens (Serratia marcescens)
BSFL-6, deposit number: CCTCC NO:M 2017073, address: Wuhan, China Wuhan University.
Serratia marcescens BSFL-6 is cultivated at 28 DEG C generates red pigments, which has cellulose hydrolysis, albumen water
Solution, Starch Hydrolysis, fat splitting, phosphorus decomposing, potassium decomposing and fixed nitrogen isoreactivity, and can antagonism harmful bacteria staphylococcus aureus
(S.Aureus) and Escherichia coli (E.coli).
The culture of serratia marcescens BSFL-6: it in LB culture medium, is cultivated at 28 DEG C.
The application of serratia marcescens BSFL-6 prepares the stratiomyiid resisted and degrade tetracycline including the use of the bacterial strain, or
Be the degradation agent of tetracycline is prepared into using the bacterium, or preparation simultaneously degrade tetracycline and inhibit Escherichia coli and golden yellow Portugal
The preparation of grape coccus.
Compared with prior art, the invention has the following advantages that
1. serratia marcescens BSFL-6 has cellulose hydrolysis, proteolysis, Starch Hydrolysis, fat splitting, phosphorus decomposing, solution
Potassium and fixed nitrogen isoreactivity can promote stratiomyiid growth with stratiomyiid symbiosis；
2. serratia marcescens BSFL-6 can antagonism harmful bacteria staphylococcus aureus (S.Aureus) and Escherichia coli
It (E.coli), is one plant of stratiomyiid beneficial bacteria of intestinal tract；
The tetracycline 3. serratia marcescens BSFL-6 can degrade in vitro, and can with stratiomyiid combined degradation tetracycline,
It is one plant of stratiomyiid beneficial bacteria of intestinal tract with Degradation of Antibiotics application potential.
Detailed description of the invention
Fig. 1 is growth curve signal of the serratia marcescens BSFL-6 in the LB culture medium of the tetracycline containing various concentration
Figure.
Fig. 2 is serratia marcescens BSFL-6 tieback tetracycline degradation rate ratio when sterile stratiomyiid handles material containing tetracycline
Compared with figure.
Specific embodiment
Experimental method in following embodiments is unless otherwise instructed the microbiology conventional practices of report.Institute
Commercial channel is derived from if not otherwise specified with reagent or material.
Embodiment 1:
Serratia marcescens BSFL-6 is separated and identified from stratiomyiid enteron aisle
Applicant uses the LB culture medium of the tetracycline containing 40mg/L, grinds from the stratiomyiid enteron aisle of conversion contaminated material containing tetracycline
Isolated one plant of serratia marcescens BSFL-6 in grinding fluid, produces red pigments, which sent on 2 28th, 2017 into
State's Type Tissue Collection preservation, classification naming: serratia marcescens (Serratia marcescens) BSFL-6 is protected
Hiding number: CCTCC NO:M 2017073, address: Wuhan, China Wuhan University.The Physiology and biochemistry of serratia marcescens BSFL-6
Characteristic and cultural character are as shown in table 1.
The physio-biochemical characteristics and cultural character of 1 serratia marcescens BSFL-6 of table
"+" is the positive, and "-" is feminine gender.
Embodiment 2:
The in vitro culture of serratia marcescens BSFL-6:
Serratia marcescens BSFL-6 is activated in flat lining out, single colonie, which is inoculated into dress 100mL, on plate of making even LB
In the triangular flask of culture medium, cultivation temperature is 28 DEG C, shaking speed 180rpm, incubation time 12h, obtains serratia marcescens
BSFL-6 zymophyte suspension (present invention or abbreviation BSFL-6 bacteria suspension), effective bacteria concentration of the bacteria suspension are 108cfu/mL
(OD600 value 1.5).
Embodiment 3:
The external degradation tetracycline of serratia marcescens BSFL-6:
By 500 μ L BSFL-6 bacteria suspensions, (effective bacteria concentration is 108Cfu/mL) be seeded to respectively equipped with 200mL containing 20,
40, in the triangular flask of the LB culture medium of 80 and 160mg/L tetracycline, cultivation temperature is 28 DEG C, shaking speed 180rpm, cultivates
Time 96h, detection content and result are as follows:
1) its growth curve is detected, as a result as shown in Figure 1, increasing with tetracycline concentration, serratia marcescens BSFL-
6 laundering period gradually extends, but enters logarithmic growth phase after the laundering period, and 24 after entering logarithmic growth phase are small
When interior reach 108Cfu/mL (OD600 value 1.5)；Even if under the concentration conditions of 160mg/L tetracycline, serratia marcescens
BSFL-6 is resistant to grow, and illustrates that it is known as good tolerance to the Fourth Ring of high concentration and acts on.
2) the tetracycline concentration variation in its culture medium is detected, until when 72h, the tetracycline containing 20,40,80 and 160mg/L
Tetracycline degradation rate is respectively as follows: 29.9 ± 0.8%, 42.1 ± 0.5%, 57.3 ± 1.2%, 61.7 ± 0.9% in culture medium.
Embodiment 4:
Serratia marcescens BSFL-6 promotes the application of stratiomyiid conversion tetracycline in stratiomyiid body:
Stratiomyiid used in the present embodiment is the flat angle Minamata Wuhan strain of speck.
1) sterile artificial's material of the tetracycline containing 40mg/L (solid content 50g, moisture content 65%) is packed into sterile triangular flask
In, become material triangular flask；
2) 3 groups of experimental setup, every group 3 are parallel:
First, being inoculated with 1mL BSFL-6 bacteria suspension in material triangular flask, (effective bacteria concentration is 108Cfu/mL) and
100 sterile stratiomyiid larvas (being free of enteric microorganism), are labeled as BSFL-6 tieback processing group；
Second, accessing 100 sterile stratiomyiid larvas (being free of enteric microorganism) in material triangular flask, it is labeled as sterile water
Horsefly processing group；
Third, being inoculated with 1mL BSFL-6 bacteria suspension in material triangular flask, (effective bacteria concentration is 108Cfu/mL), mark
For BSFL-6 ex vivo treatment group；
3) above 3 experimental groups are placed in 28 DEG C of incubators, keep ventilation and gnotobasis, cultivated 12d, take starting material
And each group material of 12d, its Fourth Ring cellulose content is tested, the degradation rate of each group tetracycline is calculated, while measuring stratiomyiid weight；
4) experimental result as shown in Fig. 2, BSFL-6 tieback processing group tetracycline degradation rate be 59.6 ± 7.9%, to show
It writes and is higher than sterile stratiomyiid processing group (its degradation rate is 38.2 ± 7.3%) and BSFL-6 ex vivo treatment group (its degradation rate is 30.8
± 3.2%) nearly twice, illustrate the degradation effect that serratia marcescens BSFL-6 can effectively help stratiomyiid to improve its tetracycline,
And its effect for being better than ex vivo treatment with the effect that stratiomyiid is jointly processed by；The stratiomyiid of BSFL-6 tieback processing group is fresh when 12d
Weight is 185 ± 6 mg/ items, is compared to the data of fresh weight 170 ± 6mg/ item of normal stratiomyiid equal conditions culture 12d, as a result says
Bright BSFL-6 tieback processing is conducive to the growth-weight gain of stratiomyiid itself, and therefore, BSFL-6 is stratiomyiid growth and degradation tetracycline
Beneficial bacteria of intestinal tract.
Claims (1)
1. a kind of isolated serratia marcescens (Serratia marcescens) inhibits big in preparation degradation tetracycline, simultaneously
Answering in the preparation of enterobacteria (Escherichia coli) and staphylococcus aureus (Staphyloccocus aureus)
With；The deposit number of the serratia marcescens is CCTCC NO:M2017073, classification naming are as follows: serratia marcescens
(Serratia marcescens)BSFL-6。
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| CN105483036A (en) * | 2015-10-30 | 2016-04-13 | 西安交通大学第一附属医院 | Serratia marcescens and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| "Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens";Stuart A. Thompson et al.,;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20070430;第73卷(第7期);第2200页，左栏第2-3段 * |
| "亮斑扁角水虻卵携带细菌的分离鉴定及其对成虫产卵行为影响";杨森等;《昆虫学报》;20170228;第60卷(第2期);第163页摘要部分 * |
| "粘质沙雷氏菌 （Serratia marcescens）的研究1-Serratia marcescens 9-2 菌株分离、分类鉴定和形态特征";黄文芳等;《华南师范大学学报》;20030831(第3期);第108-111页 * |
| Stuart A. Thompson et al.,."Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens".《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.2007,第73卷(第7期),第2200页，左栏第2-3段. * |
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