CN102349548A - Environment biological disinfectant with phages as main components and application thereof - Google Patents
Environment biological disinfectant with phages as main components and application thereof Download PDFInfo
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- CN102349548A CN102349548A CN2011102340767A CN201110234076A CN102349548A CN 102349548 A CN102349548 A CN 102349548A CN 2011102340767 A CN2011102340767 A CN 2011102340767A CN 201110234076 A CN201110234076 A CN 201110234076A CN 102349548 A CN102349548 A CN 102349548A
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Abstract
The invention provides an environment biological disinfectant with phages as main components and application thereof. The biological disinfectant is composed of Escherichia coli phage CCTCC (China Center for Type Culture Collection) M209208, Bacterium enteritidis phage CCTCC M2010226, Salmonella pullorum phage CCTCC M2011042, Shigella flexneri phage CCTCC M209029, cinnamon extract, Chinese pulsatilla extract and auxiliary agents, wherein the purified phages are prepared into liquid with the concentration of more than or equal to 10<4> pfu (plaque forming unit)/mL, and are used for sterilizing and cleansing environment places and devices. The biological disinfectant has the characteristics of strong sterilization capability, rapid sterilization, no toxicity or harm, no irritation, no corrosion, no colorizing performance and the like, is especially suitable for disinfection and cleansing of breeding farms, slaughter houses, livestock product processing factories, food warehouses and other environments and devices, and has better popularization and use values.
Description
Technical field
The environment disinfected agent that it is key component that the present invention provides a kind of lytic phage that separates with many strains and on the environment in places such as plant, slaughter house, food processing factory, hospital and relevant vessel thereof kill pathogenic bacteria and purification applications such as Escherichia coli, salmonella, Shigella.Belong to bioengineering field.
Background technology
At present; Multiple, occurred frequently and mixed infection trend that plant's bacteriosis spreads, is; Plant's eqpidemic disease control and livestock breeding culture benefit and the pollution of animal product pathogenic bacteria etc. are produced very big harm; Eqpidemic disease in plant (pig farm, chicken house, cattle farm and duck field etc.) takes place and epidemic characteristic investigation discovery in recent years: the bacteriosis of plant is very severe, is the key factor that directly influences animal production, animal production safety.
Mostly the environment disinfected agent is the chemical classes disinfectant, and the most toxicity of these preparations are bigger, and like glutaraldehyde, formalin etc., or smell is more unpleasant, like Peracetic acid, formalin etc.Though the chemosterilant Disinfection Effect is good, exists poor stability, need dilute and dilute the back during use and use up in one week, there are certain weak points such as stimulation peculiar smell that its scope of application is restricted.Chemosterilant is sterilization through the toxic action of pair cell own.Some disinfectant is to the effect of toxic elements in human body property; Some disinfectant biodegradation rate is extremely slow, causes environmental pollution easily; Some disinfectant is corrosive to article and other detrimental effects.Simultaneously, these chemosterilant prolonged application also can cause residual in the sterilization of plant's food animal, and the sterilization that is used for vessel can produce corrosiveness.In addition, along with the long-term use of a large amount of disinfectants, the part bacterium of plant offsets toxic agent and has produced certain drug resistance, and Disinfection Effect is died down gradually.Therefore, people hope to obtain can reach sterilization effect in a kind of short time, free from extraneous odour, and long action time, easy to use, nontoxic or low toxicity, excitant is little and the good disinfectant of sterilization virus killing effect.Along with people's health care and the reinforcement gradually of sterilizing prevent disease consciousness in recent years, the agent of new bio environment disinfected becomes a kind of development trend.
Phage is the dependent virus of a bacterioid, claims bacterial virus again.Target bacteria being had very strong specificity splitting action, the environment other biological is not had harm, is the jinx of target bacteria in the ideal environment.Various target bacterias to existing in the environment have very strong bactericidal action, play the purpose that purifies sterilization.
The applicant once separated in plant's sewage and ight soil and obtained colibacteriophage; Salmonella enteritidis bacteriophage; White diarrhea salmonella phage; Many strains such as shigella flexneri phage lytic phage; Wherein, The preserving number of colibacteriophage is CCTCC M 209208; Open by No. 200910263295.0 patent applications; The preserving number of Salmonella enteritidis bacteriophage is CTCC M 2010226; Open by No. 201010508259.9 patent applications; The preserving number of white diarrhea salmonella phage is CCTCC M 2011042; Open by No. 201110053518.8 patent applications; The preserving number of shigella flexneri phage is CCTCC M 209029, and is open by No. 200910029306.9 patent applications.
At present; In pathogens such as Escherichia coli, salmonella, Shigella being had the preparation of killing with catharsis; Adopt described colibacteriophage, Salmonella enteritidis bacteriophage, white diarrhea salmonella phage, shigella flexneri phage to carry out compound composition, do not report.
Summary of the invention
The objective of the invention is to: provide a kind of with many strains lytic phage be the environmental organism disinfectant of key component and in the environment in places such as plant, slaughter house, animal product processing factory and vessel the killing and purification applications of bacterium.
Another object of the present invention is: control and the purification method and the measure of germ contamination in a kind of new environment are provided.
A kind of is the environmental organism disinfectant of key component with the phage; It is characterized in that: percentage meter by volume; Its prescription is colibacteriophage CCTCC M 209208 10-30%; Salmonella enteritidis bacteriophage CTCC M 2010226 10-30%; White diarrhea salmonella phage CCTCC M 2011042 10-30%, shigella flexneri phage CCTCC M 209029 10-30%, Chinese cassia tree extract 5-8%; Chinese pulsatilla extract 5-8%, surplus is an assistant agent; Wherein the phage of various purifying is prepared into concentration>=10
4Pfu/ml.
Described assistant agent is the conventional assistant agent of biological disinfectant.
Described biological disinfectant is meant water or glycerine with conventional assistant agent
Described Chinese cassia tree extract is cleaned chopping for selecting Chinese cassia tree, adds 95% ethanol in 1: 10 by volume, 70 ℃ of lixiviates 5 hours, and vacuum filters, and Rotary Evaporators steams near to be done, and filters, and with aqueous solution dissolving, obtains the Chinese cassia tree extract.
Described Chinese pulsatilla extract is the plant extracts that obtains by in the Pulsatilla plant, and described Pulsatilla plant is to select a Chinese pulsatilla of selecting, the golden county Chinese pulsatilla, the Xingan Chinese pulsatilla, the Mongolian Chinese pulsatilla, the southwestern Chinese pulsatilla, the palm leaf Chinese pulsatilla, the chrysanthemum Chinese pulsatilla, the clock calyx Chinese pulsatilla, the purple stamen Chinese pulsatilla, the spire Chinese pulsatilla, pasque flower and the renal lobe Chinese pulsatilla.
A kind of is the environmental organism Application of disinfectants of key component with the phage, it is characterized in that: this environment disinfected agent is used for suppressing or the pollution of the corresponding Escherichia coli of environment, salmonella and Shigella extremely that disappears.
Advantage of the present invention is: 1) because lytic phage involved in the present invention is in the sewage of plant and ight soil, to separate acquisition; And fragmentation pattern is wider; Special pathogenic bacteria to plant have very strong specific aim; Disinfection effect is stronger, also the pathogenic bacteria of carrying in the animal product is had very strong killing action equally; 2) because this invention is the compound of several lytic phages; Compare with single phage; Widened its phagocytosis spectrum greatly; Can kill the Escherichia coli in the environment; Also can kill the salmonella and the Shigella pathogenic bacteria that exist in the environment, improve killing effect greatly plant's common pathogen.3) this invention has overcome the corrosivity and the penetrating odor of environment disinfected agent in the past, and is effective to environment and vessel sterilizing and purifying, odorless, tasteless, also has fragrance, the safety non-toxic of some Chinese medicine, can drive the use that carries out disinfection of thing and band utensil.4) the present invention simultaneously has good killing action to the drug resistance Escherichia coli on place such as plant and slaughter house and the utensil and salmonella and Shigella.
Embodiment
The composition of embodiment 1, environmental organism disinfectant
With 4 kinds of purifying phages of this laboratory preservation, dilute after the activation respectively, make its concentration difference>=10
4Pfu/ml gets final product, and mixes according to following volume ratio then, mixes, and adds Chinese pulsatilla extract and Chinese cassia tree extract according to following volume ratio then, fully mixes, and is adding an amount of assistant agent then.Prescription is formed as follows:
Salmonella enteritidis bacteriophage (CTCC M 2010226): 10-30%;
White diarrhea salmonella phage (CCTCC M 2011042): 10-30%;
Colibacteriophage (CCTCC M 209208): 10-30%;
Shigella flexneri phage (CCTCC M 209029): 10-30%;
Chinese cassia tree extract (outsourcing) 5-8%;
Chinese pulsatilla extract (self-control) 5-8%;
Assistant agent is supplemented to 100%.Be prepared into liquid, subsequent use;
Wherein: described Chinese pulsatilla extract is to obtain like this.
Described assistant agent is a water.
During practical implementation, described Pulsatilla plant can be selected a Chinese pulsatilla of selecting, the golden county Chinese pulsatilla, the Xingan Chinese pulsatilla, the Mongolian Chinese pulsatilla, the southwestern Chinese pulsatilla, the palm leaf Chinese pulsatilla, the chrysanthemum Chinese pulsatilla, the clock calyx Chinese pulsatilla, the purple stamen Chinese pulsatilla, the spire Chinese pulsatilla, pasque flower and the renal lobe Chinese pulsatilla.Described assistant agent can also adopt glycerine, or is employed in other the conventional assistant agents in the biological disinfectant.
Embodiment 2: the environment disinfected agent is to the bactericidal purifying effect of space air natural bacteria
A kind of commercial disinfectant of this experimental selection: peroxyacetic acid disinfectant is studied the evolution disinfecting power of disinfectant of the present invention to the space air natural bacteria as a comparison.
Environmental organism disinfectant of the present invention is become solution, cause aerosol, airtight door and window 1h before and after the spraying through sprayer.Also airtight 1h before and after the Peracetic acid spraying.Above-mentioned 2 kinds of different disinfectants all adopt the aerosol spray method, carry out bacterial culture respectively at 30min, the 30min sampling of sterilization back before the sterilization.
Air sterillization monitoring method: according to " disinfection technology standard " requirement that the Ministry of Public Health issues, all adopt the plate exposure method, press clean room area size and place number of plates; At sustained height, same position is layouted at every turn, before and after sterilization, carries out air sampling; Culture dish exposes 5min, closes lid and detects.
Disinfection Effect: testing result shows; Two kinds of sterile products all demonstrate good disinfection efficacy; Environmental organism disinfectant sterilizing rate provided by the invention is a little less than Peracetic acid; But difference is not remarkable; Environmental organism disinfectant provided by the invention has no irritating odor, does not have corrosiveness, also has certain Chinese medicine fragrance.The phage disinfectant solution causes aerosol through sprayer, and the tiny diffusion of its particle not only soon but also strong.This method not only has disinfective action, can also make the dust sedimentation, and humidifying, fresh air.
The result shows: environment disinfected agent provided by the invention and Peracetic acid have the equal fungicidal effectiveness to natural bacteria; And have no irritating odor; The use of can spraying has in public places solved sterilization problems and the problem of drug-fast bacteria under the unsolved for a long time dynamic environment.
The agent of table 1 environment disinfected is to room air natural bacteria killing effect
Embodiment 3: disinfectant is in the Disinfection Effect experiment of hen house
This experiment selects 3 laying hen houses to experimentize.
Sterilization method: hen house 1: adopt the sprayer thorough disinfection, comprise ground and wall, soup is spread fully come.Sampled in environmental air, wall and ground, carry out the bacterium separation determination.Hen house interior 2: cage, egg basket and feed go-cart wiping are sterilized with the gauze that soaks into disinfectant.Hen house 3: adopt the Peracetic acid spraying disinfection, gather the environmental air sample and carry out the bacterium separation determination.
Disinfection Effect detects: respectively before spraying disinfection or wiping sterilization after 30min and the sterilization behind 30min, the 24h to the air sampling in hen house 1 and the hen house 3; Analyze the bacterium situation of change; Simultaneously wall in the hen house 1 and ground are sampled; Carry out the bacterium separation detection; To the cage in the hen house 2, egg basket and feed go-cart sampling, carry out bacterial culture and detection.
The detection of bacterium in the air: bacterial sampling adopts natural sedimentation in the air; Layout at same position and sustained height; 8 points of each hen house cloth are positioned over 8 sampled points with aseptic culture medium is uncovered, before sterilization with behind sterilization back 30min or the 24h, take a sample respectively; Medium exposes 5 minutes; Lid is taken back the laboratory, puts the incubator constant temperature culture; Calculate the bacterium number that falls of each bacterioid, fall the mean of bacterium in the counting chamber.
The sampling of ground, wall, egg basket and feed go-cart: before and after sterilization; Use aseptic cotton swab respectively; Respectively get 3 points on wall in hen house and the ground; Every some obliterating area is 5 * 5cm; The sterilization post-sampling is adjacent with the front position of sterilizing and do not overlap; Cotton formula is dipped in the bottle of stroke-physiological saline solution of 5.0ml (in add 0.1% casein, to improve the utilization percent of sample).After the sealing, take back the laboratory and handle.Cage in the hen house is with the sampling of disinfecting silk or cotton swab, and aseptic inoculation is cultivated mensuration to culture dish.
The agent of table 2 environment disinfected purifies Disinfection Effect to chicken coop air
Experimental result shows: find out from table 2: environmental organism disinfectant provided by the invention has the good sterilization effect to pathogenic bacteria such as total plate count the chicken coop air and Escherichia coli, salmonella and Shigellas, and sterilizing rate all reaches more than 88%.And peroxyacetic acid disinfectant is better to natural bacterium colony Disinfection Effect, but undesirable to the sterilization effect of the Escherichia coli in the hen house, salmonella and Shigella, far below environment disinfected agent of the present invention.This explanation: environment disinfected agent provided by the invention has the good sterilization effect to the specific pathogenic bacteria of plant, and very strong specific aim is arranged.And the Peracetic acid specific aim is not strong, or because long-term use bacterium has produced chemical sproof cause to it, causes Disinfection Effect undesirable.
Also find simultaneously; In the process of hen house band chicken spraying disinfection; In the peracetic acid disinfectant process, the chicken crowd has significantly discomfort reactions such as closing one's eyes, shake the head, acutely shake health and head, and the incompatible reaction of tangible chicken crowd is not found in environment disinfected agent of the present invention in spraying.
Table 3 environment disinfected agent spraying is to the purification Disinfection Effect on hen house wall and ground
The wiping of table 4 environmental organism disinfectant is to surface disinfection effects such as cage, egg basket and feed go-carts
Find out that from table 3 and table 4 environmental organism disinfectant provided by the invention article such as air in the hen house and wall, ground, cage and egg basket and feed go-cart are carried out bactericidal purifying, and sterilizing rate is all above more than 87% through spraying and wiping sterilization.
Embodiment 4: the safety evaluatio of disinfectant
Experimental Animal Center Nanjing Medical University buys 30 of kunming mices in Jiangsu Province, male and female each half, body weight 20 ± 2g.Carry out mouse and suck and irritate stomach environmental organism disinfectant provided by the invention, observe the toxicity of disinfectant mouse.
Result of the test shows: the chmice acute oral toxicity of disinfectant: LD50＞5000mg/kg, acute toxicity on inhalation: LD50＞2.5mg/L; Acute dermal toxicity: LD50＞3000mg/kg shows that disinfectant is the avirulence medicine.
Above embodiment only supplies to illustrate the present invention's usefulness; But not to qualification of the present invention; The technical staff in relevant technologies field; In not breaking away from spirit of the present invention and technical scope; Can prepare the product of various proportionings and various modification; Therefore, the technical scheme that all technical fields are equal to all should belong to interest field of the present invention.
Claims (5)
1. one kind is the environmental organism disinfectant of key component with the phage; It is characterized in that: percentage meter by volume; Its prescription is colibacteriophage CCTCC M 209208 10-30%; Salmonella enteritidis bacteriophage CTCC M 2010226 10-30%; White diarrhea salmonella phage CCTCC M 2011042 10-30%, shigella flexneri phage CCTCC M 209029 10-30%, Chinese cassia tree extract 5-8%; Chinese pulsatilla extract 5-8%, surplus is an assistant agent; Wherein the phage of various purifying is prepared into concentration>=10
4Pfu/ml.
According to claim 1 said a kind of be the environmental organism disinfectant of key component with the phage, it is characterized in that: described assistant agent is the conventional assistant agent of biological disinfectant.
According to claim 2 said a kind of be the environmental organism disinfectant of key component with the phage, it is characterized in that: described biological disinfectant is meant water or glycerine with conventional assistant agent.
According to claim 1 said a kind of be the environmental organism disinfectant of key component with the phage; It is characterized in that: described Chinese pulsatilla extract is the plant extracts that obtains by in the Pulsatilla plant, and described Pulsatilla plant is to select a Chinese pulsatilla of selecting, the golden county Chinese pulsatilla, the Xingan Chinese pulsatilla, the Mongolian Chinese pulsatilla, the southwestern Chinese pulsatilla, the palm leaf Chinese pulsatilla, the chrysanthemum Chinese pulsatilla, the clock calyx Chinese pulsatilla, the purple stamen Chinese pulsatilla, the spire Chinese pulsatilla, pasque flower and the renal lobe Chinese pulsatilla.
5. environmental organism Application of disinfectants according to claim 1; It is characterized in that: this environment disinfected agent is used for suppressing or the pollution of the corresponding Escherichia coli of environment, salmonella and Shigella extremely that disappears, in particular for the purification sterilization of plant, animal house, slaughter house, food warehouse and animal product process plant environments and utensil.
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| CN2011102340767A CN102349548A (en) | 2011-08-16 | 2011-08-16 | Environment biological disinfectant with phages as main components and application thereof |
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| CN2011102340767A CN102349548A (en) | 2011-08-16 | 2011-08-16 | Environment biological disinfectant with phages as main components and application thereof |
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Cited By (5)
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| CN104472550A (en) * | 2014-12-02 | 2015-04-01 | 江苏省农业科学院 | Broad-spectrum salmonella bacteriophage biological bactericide and application thereof |
| CN105002144A (en) * | 2015-09-03 | 2015-10-28 | 青岛农业大学 | Bacteriophage capable of entering blood through oral administration and application of bacteriophage |
| CN109730235A (en) * | 2019-02-01 | 2019-05-10 | 珠海横琴普罗恩能医用食品有限公司 | A kind of preservative for medical food |
| CN111296493A (en) * | 2020-03-09 | 2020-06-19 | 苏州十一方生物科技有限公司 | Phage disinfectant and preparation method thereof |
| CN111316999A (en) * | 2020-03-04 | 2020-06-23 | 苏州十一方生物科技有限公司 | Spray type environmental disinfectant containing bacteriophage and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104472550A (en) * | 2014-12-02 | 2015-04-01 | 江苏省农业科学院 | Broad-spectrum salmonella bacteriophage biological bactericide and application thereof |
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| CN109730235A (en) * | 2019-02-01 | 2019-05-10 | 珠海横琴普罗恩能医用食品有限公司 | A kind of preservative for medical food |
| CN111316999A (en) * | 2020-03-04 | 2020-06-23 | 苏州十一方生物科技有限公司 | Spray type environmental disinfectant containing bacteriophage and preparation method and application thereof |
| CN111316999B (en) * | 2020-03-04 | 2022-02-08 | 苏州十一方生物科技有限公司 | Spray type environmental disinfectant containing bacteriophage and preparation method and application thereof |
| CN111296493A (en) * | 2020-03-09 | 2020-06-19 | 苏州十一方生物科技有限公司 | Phage disinfectant and preparation method thereof |
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Application publication date: 20120215 |