CN104397036B - Microbial disinfectant used in livestock and poultry field, preparation method and application thereof - Google Patents
Microbial disinfectant used in livestock and poultry field, preparation method and application thereof Download PDFInfo
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- CN104397036B CN104397036B CN201410534150.0A CN201410534150A CN104397036B CN 104397036 B CN104397036 B CN 104397036B CN 201410534150 A CN201410534150 A CN 201410534150A CN 104397036 B CN104397036 B CN 104397036B
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Abstract
The invention relates to a microbial disinfectant used in a livestock and poultry field, a preparation method and an application thereof. The microbial disinfectant comprises following components, by volume, 1-3 parts of a bacillus pumilus fermentation liquid, 1-3 parts of a bacillus polymyxa fermentation liquid and 25-50 parts of water. The invention also relates to a preparation method and an application of the microbial disinfectant used in the livestock and poultry field. In the microbial disinfectant, the bacillus pumilus and the bacillus polymyxa are employed as microorganism strains for inhibiting or killing harmful pathogenic bacteria, wherein both the bacillus pumilus and the bacillus polymyxa have a synergistic effect on inhibiting or killing the harmful pathogenic bacteria in the fields of advantageous growth of the strain and various antimicrobial substances and enzymes in metabolites, thereby greatly enhancing a sterilizing effect.
Description
Technical field
The present invention relates to a kind of livestock and poultry farm microbial disinfectant and preparation method and application, belong to herding disinfectant skill
Art field.
Background technology
In the world, for livestock and poultry breeding industry, the loss that pathogenic microorganism causes is huge, at present, livestock and poultry farm
Environment disinfected work mainly adopt chemical method, disinfectant, such as Operand, chlorinated product, formaldehyde etc., through spray, spilling, fumigate etc. on the way
Footpath, is used for environment disinfected, and this kind of method plays an important role to the Environmental security guaranteeing livestock and poultry farm, but, this kind of side
In method, some disinfectant have certain hazardness in itself, and also have residue problem, with safe to food increasingly heavy of people
Depending on the use of chemosterilant is limited by more and more severeer.
Chinese patent literature cn103190449a (application number 201310133696.0) discloses a kind of bacillus subtilises
Livestock and poultry farm microbial air disinfectant, adopts and prepares with the following method: (1) bacillus subtilises are inoculated in fluid medium
Carry out activation culture to exponential phase, activation thalline is obtained；(2) activation thalline is inoculated in liquid fermentation medium, sends out
Ferment is cultivated, prepared microbial fermentation solution；(3) microbial fermentation solution is spray-dried, prepared microbial disinfectant.
Mentioned microorganism disinfectant has to person poultry safety because of it, low in the pollution of the environment, degradable, and controlling object is with strong points
The features such as, gradually obtained the favor of people, but due in microbial disinfectant strain and its metabolite multiple antibiotic substance and
The many factors such as enzyme are not consistent to the action direction of harmful bacteria, thus, the complex microorganism sterilization of suppression pathogenic microorganism
The bactericidal range of agent is more extensive, is that the extensive application of microbial disinfectant lays the first stone.
Content of the invention
The present invention is directed to deficiency of the prior art, provides a kind of preparation method of livestock and poultry farm microbial disinfectant and answers
With.This microbial disinfectant can effectively suppress the livestock and poultry farm common pathogen such as escherichia coli, staphylococcuses, streptococcus, real
The technique effect now safe and reliable, benefit is good.
The technical solution used in the present invention is as follows:
A kind of livestock and poultry farm microbial disinfectant, component is as follows, is parts by volume:
1～3 part of Bacillus pumilus fermentation liquid, 1～3 part of Paenibacillus polymyxa fermentation liquid, 25～50 parts of water；
Described Bacillus pumilus fermentation liquid cell concentration is not less than 1.0 × 108Individual/milliliter；
Paenibacillus polymyxa fermentation liquid cell concentration is not less than 0.5 × 108Individual/milliliter；
In described livestock and poultry farm microbial disinfectant, the content of antibiotic substance is not less than 0.2 mg/ml.
Antibiotic substance is the general name of many kinds of substance, based on lipopeptid class, can detect activity by inhibition zone size: i.e.
On the flat board of even spread pathogenic bacterium, place the sterilized filter paper of 0.8cm, the active sample of Deca 15 μ l on filter paper
Product, and with sterilized water for comparison, after 37 DEG C of culture 24h, observe inhibition zone size.The content detection of antibiotic substance can adopt ability
The general extraction methods of domain fat peptide matters, such as organic solvent extraction etc..
According to currently preferred, it is general that described Bacillus pumilus derive from China Committee for Culture Collection of Microorganisms
Logical microorganism center (cgmcc), bacterium numbering is 1.937 Bacillus pumilus.
According to currently preferred, described Paenibacillus polymyxa derives from China Committee for Culture Collection of Microorganisms
Common micro-organisms center (cgmcc), bacterium numbering is 1.224 Paenibacillus polymyxa.
The preparation method of above-mentioned livestock and poultry farm microbial disinfectant, step is as follows:
(1) Bacillus pumilus are inoculated in fluid medium i and carry out activation culture to exponential phase, activation is obtained
Thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium ii by percent by volume 3～5%, 25～35
At DEG C, fermentation culture 36～48 hours, prepared Bacillus pumilus fermentation liquid；
(2) Paenibacillus polymyxa is inoculated in fluid medium i and carries out activation culture to exponential phase, be obtained and live
Change thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium iii by percent by volume 3～5%, 25～35
At DEG C, fermentation culture 36～48 hours, prepared Paenibacillus polymyxa fermentation liquid；
(3) the Paenibacillus polymyxa fermentation that the Bacillus pumilus fermentation liquid that step (1) is obtained is obtained with step (2)
Liquid is mixed in proportion, prepared livestock and poultry farm microbial disinfectant.
According to currently preferred, the fluid medium i component in described step (1) is as follows, is all weight percentage:
Yeast extract 0.5～1.5%, peptone 0.7～1%, sodium chloride 0.5～1.2%, glucose 0.5～2%, ph7.0
～7.2.
According to currently preferred, the activation culture condition in described step (1) is: the strain that inclined-plane is preserved is inoculated in
In fluid medium, 25～35 DEG C, 180 revs/min of shaken cultivation 12～18 hours, as activate thalline；
According to currently preferred, in described step (2), liquid fermentation medium ii component is as follows, is weight percent
Than:
Peptone 1%, Semen Maydis powder 2%, bean cake 0.5%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, glucose
0.5%, ph7.0～7.2.
According to currently preferred, the fluid medium iii component in described step (3) is as follows, is all weight percentage:
Glucose 0.5%, peptone 0.7～1%, sodium chloride 0.5～1.2%, bean cake 1%, Semen Maydis pulp 0.5%, phosphoric acid
Potassium dihydrogen 0.1%, magnesium sulfate 0.03%, Calcium Carbonate 0.1%, ph6.5.
Application in livestock and poultry farm sterilization for the above-mentioned livestock and poultry farm microbial disinfectant.
Above-mentioned application, step is as follows:
Livestock and poultry farm microbial disinfectant is sprayed in livestock and poultry farm facility surface to be sterilized, Application is 10～
20ml/m3, act on more than 1 hour, sterilization frequency is 10～15 days/time.
Beneficial effect
1st, the present invention adopts Bacillus pumilus, bacillus polymyxa to suppress or kill to be harmful to the micro- of pathogen as main
Biological bacterial strain, the aspect such as the two multiple antibiotic substance in the dominant growth and metabolite of strain and enzyme all in suppression or is killed
Harmful bacteria aspect of going out has synergism, thus substantially increasing sterilization effect.
2nd, livestock and poultry farm microbial disinfectant of the present invention, has safety no residual in terms of the Disinfection Effect of livestock and poultry farm
Stay, the advantage of effect stability.
Brief description
Fig. 1 is the Bactericidal test result of the livestock and poultry farm microbial disinfectant that embodiment 1 is obtained；
Specific embodiment
With reference to embodiment, technical scheme is described further, but institute of the present invention protection domain is not limited to
This.
In detection livestock and poultry farm microbial disinfectant in embodiment, the method for antibiotic substance activity is as follows: i.e. in even spread
On the flat board of pathogenic bacterium, the sterilized filter paper of placement 0.8cm, the active sample of Deca 15 μ l on filter paper, and with aseptic
Water is comparison, observes inhibition zone size after 37 DEG C of culture 24h.In embodiment, the detection of antibiotic substance (fat peptide matters) is using having
Machine solvent extraction method.
Bacillus pumilus described in embodiment derive from China Committee for Culture Collection of Microorganisms's common micro-organismss
Center (cgmcc), bacterium numbering is 1.937 Bacillus pumilus；Described Paenibacillus polymyxa derives from China Microbiological
Culture presevation administration committee common micro-organisms center (cgmcc), bacterium numbering is 1.224 Paenibacillus polymyxa.
Embodiment 1
The preparation method of above-mentioned livestock and poultry farm microbial disinfectant, step is as follows:
(1) Bacillus pumilus are inoculated in fluid medium i and carry out activation culture to exponential phase, activation is obtained
Thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium ii by percent by volume 3%, at 35 DEG C, sends out
Ferment is cultivated 48 hours, prepared Bacillus pumilus fermentation liquid；After testing, described Bacillus pumilus fermentation liquid cell concentration is
3.2×109Individual/milliliter.
(2) Paenibacillus polymyxa is inoculated in fluid medium i and carries out activation culture to exponential phase, be obtained and live
Change thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium iii by percent by volume 3%, at 35 DEG C, sends out
Ferment is cultivated 48 hours, prepared Paenibacillus polymyxa fermentation liquid；After testing, described Paenibacillus polymyxa fermentation liquid cell concentration
For 1.8 × 109Individual/milliliter.
(3) the Paenibacillus polymyxa fermentation that the Bacillus pumilus fermentation liquid that step (1) is obtained is obtained with step (2)
The ratio mixing of liquid and water 1:1:30 by volume, prepared livestock and poultry farm microbial disinfectant.
Fluid medium i component in described step (1) is as follows, is all weight percentage:
Yeast extract 0.5%, peptone 1%, sodium chloride 1%, glucose 2%, excess water, ph7.0.
Activation culture condition in described step (1) is: the strain that inclined-plane is preserved is inoculated in fluid medium, 25
DEG C, 180 revs/min of shaken cultivation 18 hours, as activate thalline；
In described step (2), liquid fermentation medium ii component is as follows, is all weight percentage:
Peptone 1%, Semen Maydis powder 2%, bean cake 0.5%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, glucose
0.5%, excess water, ph7.0.
Fluid medium iii component in described step (3) is as follows, is all weight percentage:
Glucose 0.5%, peptone 1%, sodium chloride 1.2%, bean cake 1%, Semen Maydis pulp 0.5%, potassium dihydrogen phosphate
0.1%, magnesium sulfate 0.03%, Calcium Carbonate 0.1%, excess water, ph6.5.
After testing, in described livestock and poultry farm microbial disinfectant, the content of antibiotic substance is 0.23 mg/ml.
Above-mentioned prepared livestock and poultry farm microbial disinfectant is taken to carry out Bactericidal test to pathogenic bacterium, result is as shown in Figure 1.
Embodiment 2
The preparation method of above-mentioned livestock and poultry farm microbial disinfectant, step is as follows:
(1) Bacillus pumilus are inoculated in fluid medium i and carry out activation culture to exponential phase, activation is obtained
Thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium ii by percent by volume 5%, at 35 DEG C, sends out
Ferment is cultivated 36 hours, prepared Bacillus pumilus fermentation liquid；After testing, described Bacillus pumilus fermentation liquid cell concentration is
3.5×109Individual/milliliter.
(2) Paenibacillus polymyxa is inoculated in fluid medium i and carries out activation culture to exponential phase, be obtained and live
Change thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium iii by percent by volume 5%, at 35 DEG C, sends out
Ferment is cultivated 36 hours, prepared Paenibacillus polymyxa fermentation liquid；After testing, described Paenibacillus polymyxa fermentation liquid cell concentration
For 2.1 × 109Individual/milliliter.
(3) the Paenibacillus polymyxa fermentation that the Bacillus pumilus fermentation liquid that step (1) is obtained is obtained with step (2)
Liquid is mixed with the ratio of 3:2:50 by volume, prepared livestock and poultry farm microbial disinfectant.
Fluid medium i component in described step (1) is as follows, is all weight percentage:
Yeast extract 1.5%, peptone 0.7%, sodium chloride 0.5%, glucose 0.5%, excess water, ph7.2.
Activation culture condition in described step (1) is: the strain that inclined-plane is preserved is inoculated in fluid medium, 35
DEG C, 180 revs/min of shaken cultivation 12 hours, as activate thalline；
In described step (2), liquid fermentation medium ii component is as follows, is all weight percentage:
Peptone 1%, Semen Maydis powder 2%, bean cake 0.5%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, glucose
0.5%, excess water, ph7.2.
Fluid medium iii component in described step (3) is as follows, is all weight percentage:
Glucose 0.5%, peptone 0.7%, sodium chloride 0.5%, bean cake 1%, Semen Maydis pulp 0.5%, potassium dihydrogen phosphate
0.1%, magnesium sulfate 0.03%, Calcium Carbonate 0.1%, excess water, ph6.5.
After testing, in described livestock and poultry farm microbial disinfectant, the content of antibiotic substance is 0.3 mg/ml.
Embodiment 3
The preparation method of above-mentioned livestock and poultry farm microbial disinfectant, step is as follows:
(1) Bacillus pumilus are inoculated in fluid medium i and carry out activation culture to exponential phase, activation is obtained
Thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium ii by percent by volume 4%, at 30 DEG C, sends out
Ferment is cultivated 40 hours, prepared Bacillus pumilus fermentation liquid；After testing, described Bacillus pumilus fermentation liquid cell concentration is
3.1×109Individual/milliliter.
(2) Paenibacillus polymyxa is inoculated in fluid medium i and carries out activation culture to exponential phase, be obtained and live
Change thalline i；
The activation thalline i being obtained is inoculated in liquid fermentation medium iii by percent by volume 4%, at 30 DEG C, sends out
Ferment is cultivated 40 hours, prepared Paenibacillus polymyxa fermentation liquid；After testing, described Paenibacillus polymyxa fermentation liquid cell concentration
For 1.5 × 109Individual/milliliter.
(3) the Paenibacillus polymyxa fermentation that the Bacillus pumilus fermentation liquid that step (1) is obtained is obtained with step (2)
The ratio mixing of liquid and water 2:3:40 by volume, prepared livestock and poultry farm microbial disinfectant.
Fluid medium i component in described step (1) is as follows, is all weight percentage:
Yeast extract 1.0%, peptone 0.9%, sodium chloride 0.8%, glucose 1.5%, excess water, ph7.1.
Activation culture condition in described step (1) is: the strain that inclined-plane is preserved is inoculated in fluid medium, 30
DEG C, 180 revs/min of shaken cultivation 16 hours, as activate thalline；
In described step (2), liquid fermentation medium ii component is as follows, is all weight percentage:
Peptone 1%, Semen Maydis powder 2%, bean cake 0.5%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, glucose
0.5%, excess water, ph7.2.
Fluid medium iii component in described step (3) is as follows, is all weight percentage:
Glucose 0.5%, peptone 0.8%, sodium chloride 0.9%, bean cake 1%, Semen Maydis pulp 0.5%, potassium dihydrogen phosphate
0.1%, magnesium sulfate 0.03%, Calcium Carbonate 0.1%, excess water, ph6.5.
After testing, in described livestock and poultry farm microbial disinfectant, the content of antibiotic substance is 0.32 mg/ml.
Test example
By pouity dwelling place a, b, c, d, e tetra- of 14 meters wide, long 150 meters long, every only raises table hens more than 10000, and a, b, c is
Test group, d, e is matched group, is 20ml/m according to fountain height3, the poultry that embodiment 1 is obtained were sprayed in every 15 days to a hen house
Field microbial disinfectant carries out disinfection once, sterilizes latter 24 hours, measures the pathogenic microorganism amount in feces and environment；To b
Hen house sprays the livestock and poultry farm microbial disinfectant that embodiment 2 is obtained for every 15 days and carries out disinfection once, sterilizes latter 24 hours, surveys
Determine the pathogenic microorganism amount in feces and environment；Spray within every 15 days the prepared livestock and poultry farm microorganism of embodiment 3 to c hen house to disappear
Toxic agent carries out disinfection once, sterilizes latter 24 hours, measures the pathogenic microorganism amount in feces and environment；To every 15 days of d hen house
Spray aseptic fermentation culture once, sterilize latter 24 hours, measure the pathogenic microorganism amount in feces and environment；To e hen house
Every 15 days spray embodiment 1 be obtained Bacillus pumilus fermentation liquid carry out disinfection once, sterilize latter 24 hours, measure feces with
Pathogenic microorganism amount in environment.
Detection method adopts plate count method.
Testing result is as shown in table 1, with the amount of d middle pathogenic microorganism for 100%.
Table 1
Claims (10)
1. a kind of livestock and poultry farm microbial disinfectant is it is characterised in that component is as follows, is parts by volume:
1～3 part of Bacillus pumilus fermentation liquid, 1～3 part of Paenibacillus polymyxa fermentation liquid, 25～50 parts of water；
Described Bacillus pumilus fermentation liquid cell concentration is not less than 1.0 × 108Individual/milliliter；
Paenibacillus polymyxa fermentation liquid cell concentration is not less than 0.5 × 108Individual/milliliter；
In described livestock and poultry farm microbial disinfectant, the content of antibiotic substance is not less than 0.2 mg/ml.
2. livestock and poultry farm microbial disinfectant as claimed in claim 1 is it is characterised in that described Bacillus pumilus derive from
China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium numbering is 1.937 Bacillus pumilus.
3. livestock and poultry farm microbial disinfectant as claimed in claim 1 is it is characterised in that described Paenibacillus polymyxa is originated
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium numbering is 1.224 Paenibacillus polymyxa.
4. the preparation method of the livestock and poultry farm microbial disinfectant described in claim 1 is it is characterised in that step is as follows:
(1) Bacillus pumilus are inoculated in fluid medium i and carry out activation culture to exponential phase, activation thalline is obtained
i；
The activation thalline i being obtained is inoculated in liquid fermentation medium ii by percent by volume 3～5%, at 25～35 DEG C,
Fermentation culture 36～48 hours, prepared Bacillus pumilus fermentation liquid；
(2) Paenibacillus polymyxa is inoculated in fluid medium i and carries out activation culture to exponential phase, activation bacterium is obtained
Body i；
The activation thalline i being obtained is inoculated in liquid fermentation medium iii by percent by volume 3～5%, at 25～35 DEG C
Under, fermentation culture 36～48 hours, prepared Paenibacillus polymyxa fermentation liquid；
(3) the Paenibacillus polymyxa fermentation liquid that Bacillus pumilus fermentation liquid that step (1) be obtained is obtained with step (2) is pressed
Ratio mixes, prepared livestock and poultry farm microbial disinfectant.
5. preparation method as claimed in claim 4 it is characterised in that the fluid medium i component in described step (1) such as
Under, all it is weight percentage:
Yeast extract 0.5～1.5%, peptone 0.7～1%, sodium chloride 0.5～1.2%, glucose 0.5～2%, excess water, ph7.0
～7.2.
6. preparation method as claimed in claim 4 is it is characterised in that the activation culture condition in described step (1) is: will be tiltedly
The strain that face preserves is inoculated in fluid medium, 25～35 DEG C, 180 revs/min of shaken cultivation 12～18 hours, as activates bacterium
Body.
7. preparation method as claimed in claim 4 is it is characterised in that liquid fermentation medium ii component in described step (1)
As follows, all it is weight percentage:
Peptone 1%, Semen Maydis powder 2%, bean cake 0.5%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.05%, glucose 0.5%, excess water,
Ph7.0～7.2.
8. preparation method as claimed in claim 4 it is characterised in that the fluid medium iii component in described step (2) such as
Under, all it is weight percentage:
Glucose 0.5%, peptone 0.7～1%, sodium chloride 0.5～1.2%, bean cake 1%, Semen Maydis pulp 0.5%, potassium dihydrogen phosphate
0.1%, magnesium sulfate 0.03%, Calcium Carbonate 0.1%, excess water, ph6.5.
9. application in livestock and poultry farm sterilization for the livestock and poultry farm microbial disinfectant described in claim 1.
10. application as claimed in claim 9 is it is characterised in that step is as follows:
Livestock and poultry farm microbial disinfectant is sprayed in livestock and poultry farm facility surface to be sterilized, Application is 10～20ml/m3,
Effect more than 1 hour, sterilization frequency is 10～15 days/time.
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| CN201410534150.0A CN104397036B (en) | 2014-10-11 | 2014-10-11 | Microbial disinfectant used in livestock and poultry field, preparation method and application thereof |
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