CN1116932A

CN1116932A - Biological bacteriostat and its preparing method

Info

Publication number: CN1116932A
Application number: CN94111370A
Authority: CN
Inventors: 秦生巨
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-09-22
Filing date: 1994-06-28
Publication date: 1996-02-21
Anticipated expiration: 2014-06-28
Also published as: CN1049823C

Abstract

The present invention discloses a biological prepn and the said biological bacteriostat is used for preventing and curing animals' bacterial diseases. The present invention adopts heating or chloroform process to obtain deactivated host bacterium and culture phage bdellovibrio in the environment at 37-43 deg.C. The phage bdellovibrio inoculum and the deactivated host bacterium in a certain proportion are cultured in dilute liquid which contains the trace elements Ca, Mg, Zn and Fe at 37-43 deg.C for a certain period, then it is regulated in the pH value of 3.0-9.8 to obtain the said biological bacteriostat.

Description

Bactericide prepared from biology and production method thereof

The present invention relates to a kind of control animal bacteria sick and promotion metabolic biological preparation of animal and production method thereof, belong to biological product and manufacturing field thereof.

The present both at home and abroad control medicine of animal bacteria disease mainly contains the chemical synthetic drug of antibiotics.The use of this type of medicine can make constantly to produce drug resistance strain in the animal body, and effectively dose increases gradually, suppresses the normal growth of animal, and in animal body residual of medicine, can bring disaster to health.Therefore, method and the goods of developing the Biological control animal bacteria disease of high effect nontoxic cause very big interest of people and concern, and the research of all kinds of ecological agents is extremely paid attention to the biological control method of bacterium system bacterium just at turn up.

Bacteriophagic Bdellovibrio finds that from the sixties it has just caused people's great attention to the unique biological characteristic of many kinds of animals and plants pathogenic bacterium " parasitism " and " cracking ".But for a long time, people think that always the growth temperature of bacteriophagic Bdellovibrio is 25-30 ℃, just can not growth and breeding more than 40 ℃, and can only grow on viable bacteria.And the normal temperature in the animal body (remove human body) is generally about 40 ℃, and bacteriophagic Bdellovibrio is responsive especially to temperature, and the bacteriophagic Bdellovibrio of cultivating in 25-30 ℃ of environment is easy inactivation in animal body; On viable bacteria, cultivate again in the production process of bacteriophagic Bdellovibrio, the easy contaminated environment of viable bacteria, and cultivation cycle long (48-72 hours), harvest yield is low by (＜10 ⁶Pfu/ml), the residual viable bacteria of culture is harmful to animal body.Therefore, people utilize the development and application of bacteriophagic Bdellovibrio control animal bacteria disease to be subjected to very big restriction.

The object of the present invention is to provide a kind of is main component with the bacteriophagic Bdellovibrio culture, is used to prevent and treat animal bacteria sick and the metabolic biological preparation of promotion animal---bactericide prepared from biology and production method thereof.

The technical solution that realizes the object of the invention is: produce host bacteria suspension with conventional method, and with the sterile purified water centrifuge washing more than three times; Then, add sterile purified water and be mixed with the bacteria suspension of bacteria containing amount more than 2,000,000,000/ml; Under 70-150 ℃, heating perhaps used 1/5-1/10 amount (volume) chloroform give with more than 1 hour more than 15 minutes, made inactivation host bacterium again; Be the host with this inactivation host bacterium then, produce its culture with the bacteriophagic Bdellovibrio general culture method, select in 25-30 ℃ of environment the typical bacteriophagic Bdellovibrio of plaque on the double-deck agar plate of tap water again, picking list plaque be soaked in the sterilization tap water in 4-6 hours, then, get supernatant and make double-deck agar plate again, heighten 3-5 ℃ of cultivations of temperature, and repeatedly for several times, until selecting the bacteriophagic Bdellovibrio that in 37-43 ℃ of environment, can grow; At last with the inactivation host bacterium that makes in the said process and the bacteriophagic Bdellovibrio that selects 1: 10-1 in proportion: 100,000 be suspended in contain trace elements of Ca (1.0-10.0mg/l), Mg (2.0-11.0mg/l), Zn (2.0-15.0mg/l), Fe is (in 0.1-1.5mg/l) the diluent, under 37-43 ℃ of temperature conditions, cultivated 10-24 hours, proofread and correct its pH value in 3.0-9.8 scopes, it is sick and promote the metabolic bactericide prepared from biology of animal that being used to of obtaining promptly that the present invention will provide prevented and treated animal bacteria.

Bactericide prepared from biology provided by the present invention to the clearance rate of salmonella typhi, escherichia coli, dysentery bacterium, vibrio cholera, Aeromonas hydrophila all more than 92.74%.Through Agricultural University Of Nanjing's test, it is 88.3% that bactericide prepared from biology is used to prevent the protective rate of the white scour of chicken, is 8.23-15.66% to the chick rate of body weight gain; Through the TanJin Agricultural College test, its effect and antibiotic that is used for the treatment of the intestine of young pigs bacterial disease is basic identical.

The method of production bactericide prepared from biology disclosed by the invention avoided cultivating the environmental pollution that bacteriophagic Bdellovibrio caused on viable bacteria, and cultivation cycle is short, output is high; Particularly make bacteriophagic Bdellovibrio that the control that the unique biological characteristic of animals and plants pathogenic bacterium " parasitism " and " cracking " is applied to the animal bacteria disease is become a reality.

Below in conjunction with embodiment the present invention is further elaborated:

At first, obtaining the bacteriophagic Bdellovibrio sensitivity, not toxogenic, non-pathogenic bacteria with conventional method is the host bacterium, and reuse heating or the lethal method of chloroform make host bacterium inactivation.Specific practice is: ask for or separate from environment to culture presevation mechanism and obtain escherichia coli, with its separation of on the Nutrient agar plate, ruling, cultivated 18 hours at 37 ℃, with corresponding serum coagulation, get the male single bacterium colony of agglutination, be inoculated in the nutrient broth, cultivated 4-6 hours at 37 ℃, then, the transferred species agar slant is in 37 ℃ of cultivations 18-20 hours, with aseptic distillation water elution lawn, and the centrifugation washing is more than three times, remove the nutrient substance in the culture medium, add people's sterile purified water again and be mixed with the bacteria suspension of bacteria containing amount more than 2,000,000,000/ml, heating sterilization at least 15 minutes under 70-150 ℃ of temperature conditions then, perhaps use 1/5-1/10 amount (volume) chloroform give with more than 1 hour, promptly obtain the escherichia coli of inactivation.

Secondly, be the host with the escherichia coli of above-mentioned inactivation, produce its culture with the bacteriophagic Bdellovibrio general culture method after, again it is carried out anti-37-43 ℃ instructionization.Specific practice is: ask for or separate from environment from culture presevation mechanism and obtain bacteriophagic Bdellovibrio, and the escherichia coli of itself and inactivation are added in 4ml, 46 ℃ 0.7% top-layer agar mix, be poured on then on the double-deck agar plate of tap water, make it solidify one deck; Then, in 25-30 ℃ of environment, cultivated 48-72 hours, choose the typical bacteriophagic Bdellovibrio of plaque, picking list plaque be soaked in the sterilization tap water in 4-6 hours; Then, get supernatant and make double-deck agar plate again, heighten 3-5 ℃ of cultivations of temperature, and repeatedly for several times, until selecting the bacteriophagic Bdellovibrio that in 37-43 ℃ of environment, can grow.

At last, with the escherichia coli of inactivation and bacteriophagic Bdellovibrio 1: 10 in proportion ³---1: 10 ⁴Be suspended in and contain trace elements of Ca, Mg, Zn, Fe and be respectively in the diluent of 3.0mg/l, 3.5mg/l, 3.2mg/l and 0.25mg/l, cultivated 10-24 hours for 37-43 ℃, proofreading and correct its pH value is 7.4, promptly obtains bactericide prepared from biology.

Claims

It is 1, a kind of that to be used to prevent and treat animal bacteria sick and promote the metabolic biological preparation of animal---bactericide prepared from biology, its Main Ingredients and Appearance is: bacteriophagic Bdellovibrio, host bacterium and trace elements of Ca, Mg, Zn, Fe, and wherein the ratio of bacteriophagic Bdellovibrio and host bacterium is 1; 10---1; 100,000; Micronutrient levels is respectively Ca=1.0-10.0mg/l, Mg=2.0-11.0mg/l, Zn=2.0-15mg/l, Fe=0.1-1.5mg/l; PH value is 3.0-9.8.
2, bactericide prepared from biology according to claim 1 is characterized in that: the ratio of bacteriophagic Bdellovibrio and host bacterium is 1: 10 ³-1: 10 ⁴Ca content is 3.0mg/l, and Mg content is 3.5mg/l, and Zn content is 3.2mg/l, and Fe content is 0.25mg/l; PH value is 7.4.
3, bactericide prepared from biology according to claim 1 and 2 is characterized in that the host bacterium is to the inactivation thalline bacteriophagic Bdellovibrio sensitivity, not toxogenic, nonpathogenic.
4, bactericide prepared from biology according to claim 3 is characterized in that the host bacterium is escherichia coli.
5, bactericide prepared from biology according to claim 3 is characterized in that bacteriophagic Bdellovibrio can be on the host bacterium of inactivation, growing multiplication in 37-43 ℃ of environment.
6, a kind of method of bactericide prepared from biology of production claim 1 comprises following processing step:

(1) produces host bacteria suspension and use the sterile purified water centrifuge washing more than three times with conventional method; Then,

(2) add sterile purified water and be mixed with the bacteria suspension of bacteria containing amount more than 2,000,000,000/ml; Then,

(3) under 70-150 ℃, heated at least 15 minutes, perhaps use 1/5-1/10 amount (volume) chloroform give with more than 1 hour, make inactivation host bacterium;

(4) the inactivation host bacterium that makes with step (1)-(3) is the host, produces its culture with the bacteriophagic Bdellovibrio general culture method; Then,

(5) select in 25-30 ℃ of environment on the double-deck agar plate of tap water the typical bacteriophagic Bdellovibrio picking of plaque list plaque to be soaked in the sterilization tap water 4-6 hours; Then,

(6) get supernatant and make double-deck agar plate again, heighten 3-5 ℃ of cultivations of temperature, repeatedly for several times, until selecting the bacteriophagic Bdellovibrio that in 37-43 ℃ of environment, can grow;

(7) the inactivation host bacterium that will make by step (1)-(3) and by bacteriophagic Bdellovibrio that step (4)-(6) make 1: 10-1 in proportion: 100,000 be suspended in contain trace elements of Ca (1.0-10.0mg/l), Mg (2.0-11.0mg/l), Zn (2.0-15mg/l), Fe is (in 0.1-1.5mg/l) the diluent, 37-43C cultivated 10-24 hours, proofread and correct its pH value in 3.0-9.8 scopes, promptly get bactericide prepared from biology.