CN105925513A - Composite microbial inoculant for degrading aflatoxin B1 and preparation method thereof - Google Patents
Composite microbial inoculant for degrading aflatoxin B1 and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a composite microbial inoculant for degrading aflatoxin B1 and a composite microbial inoculant prepared by the method. The method comprises the following steps: respectively culturing pseudomonad, Flavobacterium, Rhodococcus, Stenotrophomonas and Bacillus to obtain a pseudomonad solution, a Flavobacterium solution, a Rhodococcus solution, a Stenotrophomonas solution and a Bacillus solution; mixing the pseudomonad solution, Flavobacterium solution, Rhodococcus solution, Stenotrophomonas solution and Bacillus solution to obtain a composite bacterium solution; and putting the composite bacterium solution into a composite bacterium culture medium, and carrying out mixed culture fermentation to obtain the composite microbial inoculant for degrading aflatoxin B1. The composite microbial inoculant can efficiently degrade aflatoxin B1, and has the advantages of low cost and no secondary pollution.
Description
Technical field
The present invention relates to aflatoxin degradation technique field, be used for degrading in particular to one
Composite bacteria agent of AFB1 and preparation method thereof.
Background technology
Aflatoxin is by toxic metabolic products produced by aspergillus flavus and aspergillus parasiticus.At nature,
The growth of aspergillus flavus is less demanding, and under aerobic conditions, peanut and corn are best breeding places.
Feedstuffs (such as corn, wheat class, paddy) etc. are storing and during transport, when condition is suitable for
(such as temperature 25 DEG C～30 DEG C, moisture 17%～20%) is easily grown aspergillus flavus group fungi and is produced Huang
Aspertoxin pollutes.
Aflatoxin is to be difficult in Feed Manufacturing avoid and extremely harmful pollution, and is very difficult to remove.
And aflatoxin (particularly AFB1) has strong toxic action to most animals.
The nutritive value of palatability and feed that aflatoxin may result in feed reduces, and affects animal reproduction machine
Can, cause Reproduction disorderly, also there is immunotoxicity, hepatotoxicity wind agitation, livestock and poultry can be caused large quantities of
Dead and strong carcinogenesis, even affects human health.
Existing utilize physics with chemistry method remove AFB1 generally require higher becoming
This, and easily cause the destruction to raw material and secondary pollution.
Summary of the invention
It is an object of the invention to provide a kind of composite bacteria agent for degrading aflatoxin B 1, with reality
Now to AFB1 low cost, free of contamination degraded.
Another object of the present invention is to provide a kind of composite bacteria agent for degrading aflatoxin B 1
Preparation method, with obtain can the composite bacteria agent of degrading aflatoxin B 1 effectively, and prepare
Method is simple, and cost is relatively low.
The present invention solves it and technical problem is that and realize by the following technical solutions.
A kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, including: by vacation unit cell
Bacterium, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are cultivated respectively, obtain false single
Born of the same parents' bacterium bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution.
By pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and gemma bar
Bacterium bacterium solution mixes, and obtains compound bacteria bacterium solution.Compound bacteria bacterium solution is accessed in compound bacteria culture medium and mixes
Close fermentation.
Pseudomonad bacterium further, in presently preferred embodiments of the present invention, in above-mentioned compound bacteria bacterium solution
Liquid, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and the cell of bacillus bacterium solution
Number be followed successively by respectively TCS in compound bacteria bacterium solution 10～20%, 5～15%, 10～35%, 5～20%,
25～35%.
Further, in presently preferred embodiments of the present invention, above-mentioned pseudomonad, Flavobacterium are trained
Supporting is pseudomonad, Flavobacterium to be respectively connected in pseudomonad culture medium and Flavobacterium culture medium,
Temperature is to cultivate 45～50 hours under 27～32 DEG C and aerobic condition.
Further, in presently preferred embodiments of the present invention, above-mentioned pseudomonad culture medium and Flavobacterium training
Foster base the most all include peptone 4～6 parts, beef leaching thing 2～4 parts, sodium chloride 4～6 parts,
Manganese sulfate 0.004～0.006 part and distilled water 950～1050 parts.
Further, in presently preferred embodiments of the present invention, above-mentioned Rhodococcus sp carries out cultivation is by red ball
Bacterium is accessed in Rhodococcus sp culture medium, cultivates 45～50 hours at a temperature of 26～30 DEG C.
Further, in presently preferred embodiments of the present invention, above-mentioned Rhodococcus sp culture medium wraps by weight
Include glucose 3～5 parts, dusty yeast 3～5 parts, malt extract 9～11 parts, calcium carbonate 1.5～2.5 parts and
Distilled water 950～1050 parts.
Further, in presently preferred embodiments of the present invention, above-mentioned Stenotrophomonas and bacillus are entered
Row cultivation is that Stenotrophomonas and bacillus are respectively connected to Stenotrophomonas culture medium and bacillus
In culture medium, cultivate 45～50 hours at a temperature of 27～32 DEG C.
Further, in presently preferred embodiments of the present invention, above-mentioned Stenotrophomonas culture medium and gemma bar
Bacterium culture medium the most all includes peptone 4～6 parts, beef extract 2～4 parts and distilled water
950～1050 parts.
Further, in presently preferred embodiments of the present invention, above-mentioned to carry out mixed fermentation be by compound bacteria bacterium
Liquid accesses in compound bacteria culture medium, is 26～34 DEG C in temperature, and pH value is cultivation 45～50 under conditions of 7.0
Hour, compound bacteria culture medium includes glucose 3～5 parts, peptone 4～6 parts, beef leaching by weight
Take thing 2～4 parts, sodium chloride 4～6 parts, dusty yeast 3～5 parts, calcium carbonate 1.5～2.5 parts, manganese sulfate
0.004～0.006 part and distilled water 950～1050 parts.
A kind of composite bacteria agent for degrading aflatoxin B 1, it is by above-mentioned for degrading aspergillus flavus
The preparation method of the composite bacteria agent of toxin B1 is made.
The composite bacteria agent for degrading aflatoxin B 1 of the embodiment of the present invention and preparation method thereof
Provide the benefit that: by by pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and gemma bar
Bacterium is cultivated respectively so that pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and gemma
After bacillus carries out substantial amounts of breeding, and activity and growth conditions reach very well, obtain pseudomonad bacterium
Liquid, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution.Then
By pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and gemma bar
Bacterium bacterium solution mixes according to a certain percentage, obtains compound bacteria bacterium solution.Again compound bacteria bacterium solution is put into multiple
Close and bacterium culture medium carries out mixed fermentation, thus obtain may be used for the compound of degrading aflatoxin B 1
Microbial inoculum.Mixing by pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus
Coordinated so that AFB1 can efficiently and rapidly be degraded by this composite bacteria agent, and
Make its low cost by biodegradable mode, do not result in secondary pollution.
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below will be to this
Technical scheme in bright embodiment is clearly and completely described.Unreceipted actual conditions in embodiment
Person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production of instrument
Manufacturer person, being can be by the commercially available conventional products bought and obtain.
Below to a kind of composite bacteria agent for degrading aflatoxin B 1 of the embodiment of the present invention and
Preparation method is specifically described.
A kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, including:
S1, pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are entered respectively
Row is cultivated, and obtains pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution
And bacillus bacterium solution.
Wherein, pseudomonad (Pseudomonas sp.) is from bacterial strain preserving number CICC 23439, Huang bar
Bacterium (Flavobacterium sp.) is from bacterial strain preserving number CICC 20907, Rhodococcus sp (Rhodococcus
Sp.) from bacterial strain preserving number DSM 763, Stenotrophomonas (Stenotrophomonas sp.) from
Bacterial strain preserving number DSM 21257, bacillus (Bacillus sp.) is from bacterial strain preserving number ATCC
21228。
Specifically, by pseudomonad, Flavobacterium is respectively connected to pseudomonad culture medium and Flavobacterium is cultivated
In base, cultivate 45～50 hours under temperature is 27～32 DEG C and aerobic condition.Wherein, aerobic condition
Refer to ventilation or concussion is cultivated.
Wherein, pseudomonad culture medium and Flavobacterium culture medium the most all include peptone 4～6
Part, beef leaching thing 2～4 parts, sodium chloride 4～6 parts, manganese sulfate 0.004～0.006 part and distilled water
950～1050 parts.The pH value of pseudomonad culture medium and Flavobacterium culture medium is 6.8～7.2.
Rhodococcus sp is accessed in Rhodococcus sp culture medium, cultivate 45～50 hours at a temperature of 26～30 DEG C.
Wherein, Rhodococcus sp culture medium includes glucose 3～5 parts, dusty yeast 3～5 parts by weight；
Malt extract 9～11 parts, calcium carbonate 1.5～2.5 parts and distilled water 950～1050 parts.
Stenotrophomonas and bacillus are respectively connected to Stenotrophomonas culture medium and bacillus is cultivated
In base, cultivate 45～50 hours at a temperature of 27～32 DEG C.
Wherein, Stenotrophomonas culture medium and bacillus culture medium the most all include peptone
4～6 parts, beef extract 2～4 parts and distilled water 950～1050 parts.
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are being cultivated
Before, corresponding culture medium can be carried out disinfecting action, i.e. at a temperature of 118～123 DEG C, sterilizing
3～5min.
By pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are existed respectively
Corresponding culture medium is cultivated so that above-mentioned bacterial classification carries out amount reproduction, makes bacterium simultaneously
Activity and vitality reach good state, follow-up mixed mixed fermentation.
S2, by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution with
And the mixing of bacillus bacterium solution, obtain compound bacteria bacterium solution.
Specifically, by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium
Liquid and bacillus bacterium solution are 10 according to cell number content～20%, 5～15%, 10～35%, 5～20%,
25～35% ratio mix, obtain compound bacteria bacterium solution.Wherein, cell number content refers to false single
Born of the same parents' bacterium bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution
In the cell number of every kind of bacterium solution account for the percentage of total cell number of compound bacteria bacterium solution.
S3, compound bacteria bacterium solution is put into compound bacteria culture medium carries out mixed fermentation.
Specifically, compound bacteria bacterium solution is accessed in compound bacteria culture medium, is 26～34 DEG C in temperature, PH
Value be cultivate 45～50 hours under conditions of 7.0 after, obtain for degrading aflatoxin B 1 is compound
Microbial inoculum.Wherein, compound bacteria culture medium include by weight glucose 3～5 parts, peptone 4～6 parts,
Beef leaching thing 2～4 parts, sodium chloride 4～6 parts, dusty yeast 3～5 parts, calcium carbonate 1.5～2.5 parts,
Manganese sulfate 0.004～0.006 part and distilled water 950～1050 parts.
A kind of composite bacteria agent for degrading aflatoxin B 1, it is by above-mentioned for degrading aspergillus flavus
The preparation method of the composite bacteria agent of toxin B1 is made.
Below in conjunction with embodiment, inventive feature and performance are described in further detail.
Embodiment 1
In the present embodiment, the preparation method of a kind of composite bacteria agent for degrading aflatoxin B 1, bag
Include:
First, will be linked into by peptone 4g, ox from bacterial strain preserving number CICC 23439 pseudomonad
Meat leaching thing 3g, sodium chloride 5g, manganese sulfate 5mg and the pseudomonad of distilled water 1000mL composition
In culture medium, be 7.0 at pH value, temperature be 28 DEG C and ventilation under conditions of cultivate 48 hours.
Flavobacterium from bacterial strain preserving number CICC 20907 is linked into by peptone 5g, beef leaching
In the Flavobacterium culture medium of thing 3g, sodium chloride 5g, manganese sulfate 5mg and distilled water 1000mL composition,
Being 7.1 at pH value, under conditions of temperature is 30 DEG C, concussion is cultivated 48 hours.
Rhodococcus sp from bacterial strain preserving number DSM 763 is linked into by glucose 3g, dusty yeast 3g,
In the Rhodococcus sp culture medium of malt extract 9g, calcium carbonate 1.5g and distilled water 1000mL composition, at 28 DEG C
At a temperature of cultivate 48 hours.
To be linked into by peptone 4g, beef from the Stenotrophomonas of bacterial strain preserving number DSM 21257
In the Stenotrophomonas culture medium of cream 2g and distilled water 1000mL composition, train at a temperature of 28 DEG C
Support 48 hours.
To be linked into by peptone 5g, beef from the bacillus of bacterial strain preserving number ATCC 21228
In the bacillus culture medium of cream 3g and distilled water 1000mL composition, cultivate at a temperature of 30 DEG C
48 hours.
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are being cultivated
Before, by corresponding pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, oligotrophy list
Born of the same parents' bacterium culture medium and bacillus culture medium are positioned at a temperature of 121 DEG C, sterilizing 3min.
Secondly, at the pseudomonad bacterium solution obtained, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy unit cell
Bacterium bacterium solution and bacillus bacterium solution take a certain amount according to cell number content be 15%, 10%, 28%,
15%, the ratio of 32% mixes, and obtains compound bacteria bacterium solution.
Then, compound bacteria bacterium solution being accessed in compound bacteria culture medium, be 26 DEG C in temperature, pH value is
After cultivating 48 hours under conditions of 7.0, obtain the composite bacteria agent for degrading aflatoxin B 1.Its
In, compound bacteria culture medium include glucose 3g, peptone 4g, beef leaching thing 2g, sodium chloride 4g,
Dusty yeast 3g, calcium carbonate 1.5g, manganese sulfate 4mg and distilled water 950mL.
By the pseudomonad bacterium solution produced, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
Bacillus bacterium solution and the composite bacteria agent for degrading aflatoxin B 1 finally given are the most single
Solely or be combined AFB1 is carried out Degrading experiment.Wherein, A is set as pseudomonad bacterium
Liquid, B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is Stenotrophomonas bacterium solution, and E is gemma bar
Bacterium bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method is: A, B, C, D, E of equivalent or the bacterium solution that is combined is linked into and contains
Have AFB1 is leached thing 3.0g, chlorination by glucose 4.0g, peptone 5.0g, beef
Sodium 5.0g, dusty yeast 4.0g, calcium carbonate 2.0g, manganese sulfate 5mg and distilled water 1000mL group
In the culture medium become, being 7.0 at pH value, under conditions of temperature is 30 DEG C, concussion is cultivated 24 hours.
The initial concentration of AFB1 is 1mg/L.
Result of the test such as table 1:
Table 1
| Bacterium solution and combinations thereof | AFB1 degradation rate |
| A | 67.2% |
| B | 45.7% |
| C | 43.0% |
| D | 53.3% |
| E | 47.6% |
| A+B | 70.1% |
| A+C | 51.6% |
| A+D | 46.2% |
| A+E | 75.4% |
| A+B+C | 66.3% |
| A+B+D | 53.7% |
| A+B+E | 86.5% |
| A+B+C+D | 69.3% |
| A+B+C+E | 72.6% |
| A+C+D+E | 62.3% |
| A+B+C+D+E | 92.4% |
As shown in table 1, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution,
Be conducive to aspergillus flavus when the most several in Stenotrophomonas bacterium solution, bacillus bacterium solution mix
The degraded of toxin B1, and by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy unit cell
Bacterium bacterium solution, bacillus bacterium solution mix fermentation according to the ratio in the present embodiment and form this enforcement
The composite bacteria agent for degrading aflatoxin B 1 in example can be to greatest extent to degrading aspergillus flavus
Toxin B1 degrades, and degradation rate reaches 92.4%.Thus greatly increase aflatoxin
The degradation capability of B1, also is able to preferably reduce cost simultaneously, will not produce secondary due to biodegradable
Pollute.
Embodiment 2
In the present embodiment, the preparation method of a kind of composite bacteria agent for degrading aflatoxin B 1, bag
Include:
First, will be linked into by peptone 6g, ox from bacterial strain preserving number CICC 23439 pseudomonad
Meat leaching thing 4g, sodium chloride 6g, manganese sulfate 6mg and the pseudomonad of distilled water 1050mL composition
In culture medium, be 7.2 at pH value, temperature be 30 DEG C and ventilation under conditions of cultivate 50 hours.
Flavobacterium from bacterial strain preserving number CICC 20907 is linked into by peptone 5g, beef leaching
In the Flavobacterium culture medium of thing 4g, sodium chloride 4g, manganese sulfate 5mg and distilled water 1000mL composition,
Being 6.9 at pH value, under conditions of temperature is 29 DEG C, concussion is cultivated 48 hours.
Rhodococcus sp from bacterial strain preserving number DSM 763 is linked into by glucose 5g, dusty yeast 5g,
In the Rhodococcus sp culture medium of malt extract 11g, calcium carbonate 2.5g and distilled water 1050mL composition,
Cultivate 50 hours at a temperature of 30 DEG C.
To be linked into by peptone 6g, beef from the Stenotrophomonas of bacterial strain preserving number DSM 21257
In the Stenotrophomonas culture medium of cream 4g and distilled water 1000mL composition, train at a temperature of 29 DEG C
Support 48 hours.
To be linked into by peptone 4g, beef from the bacillus of bacterial strain preserving number ATCC 21228
In the bacillus culture medium of cream 4g and distilled water 1000mL composition, cultivate at a temperature of 29 DEG C
50 hours.
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are being cultivated
Before, by corresponding pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, oligotrophy list
Born of the same parents' bacterium culture medium and bacillus culture medium are positioned at a temperature of 120 DEG C, sterilizing 4min.
Secondly, at the pseudomonad bacterium solution obtained, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy unit cell
Bacterium bacterium solution and bacillus bacterium solution take a certain amount according to cell number content be 20%, 12%, 25%,
16%, the ratio of 27% mixes, and obtains compound bacteria bacterium solution.
Then, compound bacteria bacterium solution being accessed in compound bacteria culture medium, be 30 DEG C in temperature, pH value is
After cultivating 49 hours under conditions of 7.0, obtain the composite bacteria agent for degrading aflatoxin B 1.Its
In, compound bacteria culture medium include glucose 5g, peptone 6g, beef leaching thing 4g, sodium chloride 6g,
Dusty yeast 5g, calcium carbonate 2.5g, manganese sulfate 6mg and distilled water 1050mL.
By the pseudomonad bacterium solution produced, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
Bacillus bacterium solution and the composite bacteria agent for degrading aflatoxin B 1 finally given are the most single
Solely or be combined AFB1 is carried out Degrading experiment.Wherein, A is set as pseudomonad bacterium
Liquid, B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is Stenotrophomonas bacterium solution, and E is gemma bar
Bacterium bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method is: A, B, C, D, E of equivalent or the bacterium solution that is combined is linked into and contains
Have AFB1 is leached thing 3.0g, chlorination by glucose 4.0g, peptone 5.0g, beef
Sodium 5.0g, dusty yeast 4.0g, calcium carbonate 2.0g, manganese sulfate 5mg and distilled water 1000mL group
In the culture medium become, being 7.0 at pH value, under conditions of temperature is 30 DEG C, concussion is cultivated 24 hours.
The initial concentration of AFB1 is 1mg/L.
Result of the test such as table 2:
Table 2
| Bacterium solution and combinations thereof | AFB1 degradation rate |
| A | 66.5% |
| B | 46.7% |
| C | 43.2% |
| D | 52.3% |
| E | 49.1% |
| A+B | 70.3% |
| A+C | 51.8% |
| A+D | 47.1% |
| A+E | 74.9% |
| A+B+C | 67.1% |
| A+B+D | 54.2% |
| A+B+E | 85.9% |
| A+B+C+D | 70.2% |
| A+B+C+E | 71.8% |
| A+C+D+E | 62.3% |
| A+B+C+D+E | 96.2% |
By table 2 is analyzed, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, red ball
Be conducive to when the most several in bacterium bacterium solution, Stenotrophomonas bacterium solution, bacillus bacterium solution mix
Degraded to AFB1, and by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution,
Stenotrophomonas bacterium solution, bacillus bacterium solution mix fermentation shape according to the ratio in the present embodiment
The composite bacteria agent for degrading aflatoxin B 1 in cost implementation can be to greatest extent to fall
Solving AFB1 to degrade, degradation rate reaches 96.2%.Thus greatly increase yellow bent
The degradation capability of mould toxin B1, also is able to preferably reduce cost simultaneously, will not produce due to biodegradable
Raw secondary pollution.
Embodiment 3
In the present embodiment, the preparation method of a kind of composite bacteria agent for degrading aflatoxin B 1, bag
Include:
First, will be linked into by peptone 5g, ox from bacterial strain preserving number CICC 23439 pseudomonad
Meat leaching thing 3g, sodium chloride 5g, manganese sulfate 5mg and the pseudomonad of distilled water 1000mL composition
In culture medium, be 7.0 at pH value, temperature be 27 DEG C and ventilation under conditions of cultivate 47 hours.
Flavobacterium from bacterial strain preserving number CICC 20907 is linked into by peptone 4g, beef leaching
Take thing 2g, sodium chloride 5g, manganese sulfate 4mg and the Flavobacterium culture medium of distilled water 950mL composition
In, it is 6.8 at pH value, under conditions of temperature is 28 DEG C, concussion is cultivated 48 hours.
Rhodococcus sp from bacterial strain preserving number DSM 763 is linked into by glucose 4g, dusty yeast 4g,
In the Rhodococcus sp culture medium of malt extract 10g, calcium carbonate 2g and distilled water 1000mL composition, at 30 DEG C
At a temperature of cultivate 48 hours.
To be linked into by peptone 5g, beef from the Stenotrophomonas of bacterial strain preserving number DSM 21257
In the Stenotrophomonas culture medium of cream 3g and distilled water 1000mL composition, train at a temperature of 28 DEG C
Support 48 hours.
To be linked into by peptone 5g, beef from the bacillus of bacterial strain preserving number ATCC 21228
In the bacillus culture medium of cream 4g and distilled water 1000mL composition, cultivate at a temperature of 30 DEG C
48 hours.
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are being cultivated
Before, by corresponding pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, oligotrophy list
Born of the same parents' bacterium culture medium and bacillus culture medium are positioned at a temperature of 122 DEG C, sterilizing 5min.
Secondly, at the pseudomonad bacterium solution obtained, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy unit cell
Bacterium bacterium solution and bacillus bacterium solution take a certain amount according to cell number content be 18%, 15%, 30%,
12%, the ratio of 25% mixes, and obtains compound bacteria bacterium solution.
Then, compound bacteria bacterium solution being accessed in compound bacteria culture medium, be 34 DEG C in temperature, pH value is
After cultivating 48 hours under conditions of 7.0, obtain the composite bacteria agent for degrading aflatoxin B 1.Its
In, compound bacteria culture medium include glucose 4g, peptone 5g, beef leaching thing 3g, sodium chloride 5g,
Dusty yeast 4g, calcium carbonate 2g, manganese sulfate 5mg and distilled water 1000mL.
By the pseudomonad bacterium solution produced, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
Bacillus bacterium solution and the composite bacteria agent for degrading aflatoxin B 1 finally given are the most single
Solely or be combined AFB1 is carried out Degrading experiment.
Wherein, setting A as pseudomonad bacterium solution, B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, D
For Stenotrophomonas bacterium solution, E is bacillus bacterium solution, and A+B+C+D+E is for being used for yellow song of degrading
The composite bacteria agent of mould toxin B1.
Test method is: A, B, C, D, E of equivalent or the bacterium solution that is combined is linked into and contains
Have AFB1 is leached thing 3.0g, chlorination by glucose 4.0g, peptone 5.0g, beef
Sodium 5.0g, dusty yeast 4.0g, calcium carbonate 2.0g, manganese sulfate 5mg and distilled water 1000mL group
In the culture medium become, being 7.0 at pH value, under conditions of temperature is 30 DEG C, concussion is cultivated 24 hours.
The initial concentration of AFB1 is 1mg/L.
Result of the test such as table 3:
Table 3
| Bacterium solution and combinations thereof | AFB1 degradation rate |
| A | 65.1% |
| B | 45.8% |
| C | 42.9% |
| D | 53.1% |
| E | 48.6% |
| A+B | 70.8% |
| A+C | 51.2% |
| A+D | 46.3% |
| A+E | 74.0% |
| A+B+C | 67.7% |
| A+B+D | 55.2% |
| A+B+E | 86.3% |
| A+B+C+D | 71.1% |
| A+B+C+E | 75.9% |
| A+C+D+E | 64.1% |
| A+B+C+D+E | 93.5% |
By table 3, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy
Be conducive to aflatoxin when the most several in monad bacterium solution, bacillus bacterium solution mix
The degraded of B1, and by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium
Liquid, bacillus bacterium solution mix fermentation according to the ratio in the present embodiment and are formed in the present embodiment
The composite bacteria agent for degrading aflatoxin B 1 can be to greatest extent to aflatoxin degradation
B1 degrades, and degradation rate reaches 93.5%.Thus greatly increase AFB1
Degradation capability, also is able to preferably reduce cost simultaneously, will not produce secondary pollution due to biodegradable.
Embodiment 4
In the present embodiment, the preparation method of a kind of composite bacteria agent for degrading aflatoxin B 1, bag
Include:
First, will be linked into by peptone 5g, ox from bacterial strain preserving number CICC 23439 pseudomonad
Meat leaching thing 3g, sodium chloride 5g, manganese sulfate 5mg and the pseudomonad of distilled water 1000mL composition
In culture medium, be 7.0 at pH value, temperature be 28 DEG C and ventilation under conditions of cultivate 48 hours.
Flavobacterium from bacterial strain preserving number CICC 20907 is linked into by peptone 5g, beef leaching
In the Flavobacterium culture medium of thing 3g, sodium chloride 5g, manganese sulfate 5mg and distilled water 1000mL composition,
Be 7.0 at pH value, temperature be 30 DEG C under conditions of aerlbic culture 48 hours.
Rhodococcus sp from bacterial strain preserving number DSM 763 is linked into by glucose 4g, dusty yeast 4g,
In the Rhodococcus sp culture medium of malt extract 10g, calcium carbonate 2g and distilled water 1000mL composition, at 28 DEG C
At a temperature of cultivate 48 hours.
To be linked into by peptone 5g, beef from the Stenotrophomonas of bacterial strain preserving number DSM 21257
In the Stenotrophomonas culture medium of cream 3g and distilled water 1000mL composition, train at a temperature of 28 DEG C
Support 48 hours.
To be linked into by peptone 5g, beef from the bacillus of bacterial strain preserving number ATCC 21228
In the bacillus culture medium of cream 3g and distilled water 1000mL composition, cultivate at a temperature of 30 DEG C
48 hours.
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are being cultivated
Before, by corresponding pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, oligotrophy list
Born of the same parents' bacterium culture medium and bacillus culture medium are positioned at a temperature of 118 DEG C, sterilizing 5min.
Secondly, at the pseudomonad bacterium solution obtained, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy unit cell
Bacterium bacterium solution and bacillus bacterium solution take a certain amount according to cell number content be 15%, 15%, 35%,
15%, the ratio of 20% mixes, and obtains compound bacteria bacterium solution.
Then, compound bacteria bacterium solution being accessed in compound bacteria culture medium, be 28 DEG C in temperature, pH value is
After cultivating 48 hours under conditions of 7.0, obtain the composite bacteria agent for degrading aflatoxin B 1.Its
In, compound bacteria culture medium include glucose 4g, peptone 5g, beef leaching thing 3g, sodium chloride 5g,
Dusty yeast 4g, calcium carbonate 2g, manganese sulfate 5mg and distilled water 1000mL.
By the pseudomonad bacterium solution produced, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
Bacillus bacterium solution and the composite bacteria agent for degrading aflatoxin B 1 finally given are the most single
Solely or be combined AFB1 is carried out Degrading experiment.Wherein, A is set as pseudomonad bacterium
Liquid, B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is Stenotrophomonas bacterium solution, and E is gemma bar
Bacterium bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method is: A, B, C, D, E of equivalent or the bacterium solution that is combined is linked into and contains
Have AFB1 is leached thing 3.0g, chlorination by glucose 4.0g, peptone 5.0g, beef
Sodium 5.0g, dusty yeast 4.0g, calcium carbonate 2.0g, manganese sulfate 5mg and distilled water 1000mL group
In the culture medium become, being 7.0 at pH value, under conditions of temperature is 30 DEG C, concussion is cultivated 24 hours.
The initial concentration of AFB1 is 1mg/L.
Result of the test such as table 4:
Table 4
| Bacterium solution and combinations thereof | AFB1 degradation rate |
| A | 65.9% |
| B | 47.1% |
| C | 42.9% |
| D | 52.8% |
| E | 49.7% |
| A+B | 69.9% |
| A+C | 52.5% |
| A+D | 46.8% |
| A+E | 74.2% |
| A+B+C | 68.3% |
| A+B+D | 55.2% |
| A+B+E | 84.8% |
| A+B+C+D | 70.7% |
| A+B+C+E | 78.6% |
| A+C+D+E | 60.1% |
| A+B+C+D+E | 97.8% |
By table 4, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy
Be conducive to aflatoxin when the most several in monad bacterium solution, bacillus bacterium solution mix
The degraded of B1, and by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium
Liquid, bacillus bacterium solution mix fermentation according to the ratio in the present embodiment and are formed in the present embodiment
The composite bacteria agent for degrading aflatoxin B 1 can be to greatest extent to aflatoxin degradation
B1 degrades, and degradation rate reaches 97.8%.Thus greatly increase AFB1
Degradation capability, also is able to preferably reduce cost simultaneously, will not produce secondary pollution due to biodegradable.
In sum, by by pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and gemma
Bacillus is cultivated respectively so that pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bud
Spore bacillus carries out substantial amounts of breeding, and after activity and growth conditions reach very well, obtains false unit cell
Bacterium bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution.
Then by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bud
Spore bacillus bacterium solution mixes according to a certain percentage, obtains compound bacteria bacterium solution.Again compound bacteria bacterium solution is put
Enter and compound bacteria culture medium carries out mixed fermentation, thus obtain may be used for degrading aflatoxin B 1
Composite bacteria agent.By pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus
Mixing coordinated so that AFB1 can efficiently and rapidly be dropped by this composite bacteria agent
Solve, and degradation effect is the best, low cost, pollution-free.
Embodiments described above is a part of embodiment of the present invention rather than whole embodiments.
The detailed description of embodiments of the invention is not intended to limit the scope of claimed invention, but
It is merely representative of the selected embodiment of the present invention.Based on the embodiment in the present invention, ordinary skill
The every other embodiment that personnel are obtained under not making creative work premise, broadly falls into this
The scope of bright protection.
Claims (10)
1. the preparation method for the composite bacteria agent of degrading aflatoxin B 1, it is characterised in that
Including:
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are trained respectively
Support, obtain pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and
Bacillus bacterium solution；
By described pseudomonad bacterium solution, described Flavobacterium bacterium solution, described Rhodococcus sp bacterium solution, described oligotrophy
Monad bacterium solution and the mixing of described bacillus bacterium solution, obtain compound bacteria bacterium solution；
Described compound bacteria bacterium solution is accessed in compound bacteria culture medium and carries out mixed fermentation.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 1
Method, it is characterised in that the described pseudomonad bacterium solution in described compound bacteria bacterium solution, described Flavobacterium
Bacterium solution, described Rhodococcus sp bacterium solution, described Stenotrophomonas bacterium solution and described bacillus bacterium solution thin
Born of the same parents' number be followed successively by respectively TCS in described compound bacteria bacterium solution 10～20%, 5～15%, 10～35%,
5～20%, 25～35%.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 1
Method, it is characterised in that it is by described false single that described pseudomonad, described Flavobacterium carry out cultivation
Born of the same parents bacterium, described Flavobacterium are respectively connected in pseudomonad culture medium and Flavobacterium culture medium, in temperature are
Cultivate 45～50 hours under 27～32 DEG C and aerobic condition.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 3
Method, it is characterised in that described pseudomonad culture medium and described Flavobacterium culture medium are by weight
All include peptone 4～6 parts, beef leaching thing 2～4 parts, sodium chloride 4～6 parts, manganese sulfate
0.004～0.006 part and distilled water 950～1050 parts.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 1
Method, it is characterised in that it is that described Rhodococcus sp accesses Rhodococcus sp training that described Rhodococcus sp carries out cultivation
Support in base, cultivate 45～50 hours at a temperature of 26～30 DEG C.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 5
Method, it is characterised in that described Rhodococcus sp culture medium includes glucose 3～5 parts, ferment by weight
Female powder 3～5 parts, malt extract 9～11 parts, calcium carbonate 1.5～2.5 parts and distilled water 950～1050 parts.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 1
Method, it is characterised in that described Stenotrophomonas and bacillus are carried out cultivation is by described oligotrophy
Monad and described bacillus are respectively connected in Stenotrophomonas culture medium and bacillus culture medium,
Cultivate 45～50 hours at a temperature of 27～32 DEG C.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 7
Method, it is characterised in that described Stenotrophomonas culture medium and described bacillus culture medium are by weight
Meter all includes peptone 4～6 parts, beef extract 2～4 parts and distilled water 950～1050 parts.
The preparation of the composite bacteria agent for degrading aflatoxin B 1 the most according to claim 1
Method, it is characterised in that carrying out described mixed fermentation is that described compound bacteria bacterium solution accesses described being combined
In bacterium culture medium, being 26～34 DEG C in temperature, pH value is to cultivate 45～50 hours under conditions of 7.0,
Described compound bacteria culture medium includes glucose 3～5 parts, peptone 4～6 parts, beef leaching by weight
Thing 2～4 parts, sodium chloride 4～6 parts, dusty yeast 3～5 parts, calcium carbonate 1.5～2.5 parts, manganese sulfate
0.004～0.006 part and distilled water 950～1050 parts.
10. the composite bacteria agent for degrading aflatoxin B 1, it is characterised in that it is by right
The preparation method of the requirement composite bacteria agent for degrading aflatoxin B 1 described in 1～9 any one
It is made.
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| CN108102971A (en) * | 2018-01-26 | 2018-06-01 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | One plant can heat-resisting, efficient degradation aflatoxin Meng Shi pseudomonads |
| CN109182205A (en) * | 2018-10-09 | 2019-01-11 | 北京林业大学 | Rhodococcus sp and its application with carbon sequestration capacity |
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