CN105925513B - Composite bacteria agent and preparation method thereof for degrading aflatoxin B 1 - Google Patents
Composite bacteria agent and preparation method thereof for degrading aflatoxin B 1 Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of composite bacteria agent for degrading aflatoxin B 1 and the prepared composite bacteria agents of this method, the preparation method includes: first to cultivate pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus respectively, obtains pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution.Pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution are mixed again, obtain compound bacteria liquid.Then compound bacteria liquid is put into compound bacterium culture medium and carries out mixed fermentation, to obtain the composite bacteria agent for degrading aflatoxin B 1, which can efficiently degrade to aflatoxin B1, and inexpensive, without secondary pollution.
Description
Technical field
The present invention relates to aflatoxin degradation technique fields, are used for aflatoxin degradation in particular to one kind
Composite bacteria agent of B1 and preparation method thereof.
Background technique
Aflatoxin is the toxic metabolic products as caused by aspergillus flavus and aspergillus parasiticus.In nature, aspergillus flavus
Grow of less demanding, under aerobic conditions, peanut and corn are best breeding places.Feedstuff (such as corn, wheat, rice
Paddy) etc. (such as 25 DEG C~30 DEG C of temperature, moisture content 17%~20%) easily during storage and transport, when condition is suitble to
It breeds aspergillus flavus group fungi and generates aflatoxin contamination.
Aflatoxin is to be difficult to avoid in Feed Manufacturing and extremely harmful pollution, and be difficult to remove.And aspergillus flavus
Toxin (especially aflatoxin B1) has strong toxic effect to most animals.Aflatoxin can lead to feed
The nutritive value of palatability and feed reduces, and influences Reproduction, leads to Reproduction disorder, also has immune poison
Property, hepatotoxicity wind agitation, livestock and poultry can be caused large quantities of dead and strong carcinogenesises, or even influence human health.
It is existing to generally require higher cost using the method for physics and chemistry removal aflatoxin B1, and be easy to make
The destruction and secondary pollution of pairs of raw material.
Summary of the invention
The purpose of the present invention is to provide a kind of composite bacteria agents for degrading aflatoxin B 1, to realize to aspergillus flavus
Toxin B1 low cost, free of contamination degradation.
Another object of the present invention is to provide a kind of preparation method of composite bacteria agent for degrading aflatoxin B 1,
To obtain the composite bacteria agent for capableing of effectively degrading aflatoxin B 1, and preparation method is simple, and cost is relatively low.
The present invention solves its technical problem and adopts the following technical solutions to realize.
A kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising: by pseudomonad, Flavobacterium,
Rhodococcus sp, Stenotrophomonas and bacillus are cultivated respectively, obtain pseudomonad 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, oligotrophy
Monad bacterium solution and the mixing of bacillus bacterium solution, obtain compound bacteria liquid.Compound bacteria liquid is accessed in compound bacterium culture medium
Carry out mixed fermentation.
Further, pseudomonad bacterium solution, Flavobacterium bacterium in presently preferred embodiments of the present invention, in above-mentioned compound bacteria liquid
Liquid, Rhodococcus sp bacterium solution, the cell number of Stenotrophomonas bacterium solution and bacillus bacterium solution are followed successively by compound bacteria liquid carefully respectively
10~20%, 5~15%, 10~35%, 5~20%, the 25~35% of born of the same parents' sum.
Further, in presently preferred embodiments of the present invention, it is above-mentioned by pseudomonad, Flavobacterium carry out culture be by false unit cell
Bacterium, Flavobacterium are respectively connected in pseudomonad culture medium and Flavobacterium culture medium, are 27~32 DEG C and aerobic condition in temperature
Lower culture 45~50 hours.
Further, in presently preferred embodiments of the present invention, above-mentioned pseudomonad culture medium and Flavobacterium culture medium are by weight
Part meter include 4~6 parts of peptone, beef leaching 2~4 parts of object, 4~6 parts of sodium chloride, 0.004~0.006 part of manganese sulfate and
950~1050 parts of distilled water.
Further, in presently preferred embodiments of the present invention, it is above-mentioned by Rhodococcus sp carry out culture be that Rhodococcus sp is accessed to red ball
In bacterium culture medium, 26~30 DEG C at a temperature of cultivate 45~50 hours.
Further, in presently preferred embodiments of the present invention, above-mentioned Rhodococcus sp culture medium include by weight glucose 3~
5 parts, 3~5 parts of yeast powder, 9~11 parts of malt extract, 1.5~2.5 parts of calcium carbonate and 950~1050 parts of distilled water.
Further, in presently preferred embodiments of the present invention, it is above-mentioned by Stenotrophomonas and bacillus carry out culture be by
Stenotrophomonas and bacillus are respectively connected in Stenotrophomonas culture medium and bacillus culture medium, in 27~32 DEG C of temperature
Degree lower culture 45~50 hours.
Further, in presently preferred embodiments of the present invention, above-mentioned Stenotrophomonas culture medium and bacillus culture medium are pressed
Poidometer includes 4~6 parts of peptone, 2~4 parts of beef extract and 950~1050 parts of distilled water.
Further, in presently preferred embodiments of the present invention, above-mentioned carry out mixed fermentation be compound bacteria liquid is accessed it is compound
It is 26~34 DEG C in temperature, pH value is cultivated 45~50 hours under conditions of being 7.0, and compound bacterium culture medium is by weight in bacterium culture medium
Meter include 3~5 parts of glucose, 4~6 parts of peptone, beef leaching 2~4 parts of object, 4~6 parts of sodium chloride, 3~5 parts of yeast powder,
1.5~2.5 parts of calcium carbonate, 0.004~0.006 part of manganese sulfate and 950~1050 parts of distilled water.
A kind of composite bacteria agent for degrading aflatoxin B 1 passes through above-mentioned answering for degrading aflatoxin B 1
The preparation method of combined bacteria agent is made.
The beneficial effect of the composite bacteria agent for degrading aflatoxin B 1 of the embodiment of the present invention and preparation method thereof is:
By the way that pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are cultivated respectively, so that pseudomonad,
Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are largely bred, and activity and growth conditions reach fine
Afterwards, pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution are obtained.Then
By pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution according to certain ratio
Example is mixed, and compound bacteria liquid is obtained.Compound bacteria liquid is put into compound bacterium culture medium again and carries out mixed fermentation, thus
To the composite bacteria agent that can be used for degrading aflatoxin B 1.By pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas with
And the mixing coordinated of bacillus, which efficiently and rapidly degrades to aflatoxin B1,
And make that its is at low cost, not will cause secondary pollution by biodegradable mode.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Below to a kind of composite bacteria agent for degrading aflatoxin B 1 of the embodiment of the present invention and preparation method thereof into
Row illustrates.
A kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising:
S1, pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are cultivated respectively, obtains vacation
Monad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution.
Wherein, pseudomonad (Pseudomonas sp.) comes from bacterial strain deposit number CICC 23439, Flavobacterium
(Flavobacterium sp.) comes from bacterial strain deposit number CICC 20907, and Rhodococcus sp (Rhodococcus sp.) comes from bacterial strain
Deposit number DSM 763, Stenotrophomonas (Stenotrophomonas sp.) come from bacterial strain deposit number DSM 21257, gemma bar
Bacterium (Bacillus sp.) comes from bacterial strain deposit number ATCC 21228.
Specifically, pseudomonad, Flavobacterium are respectively connected in pseudomonad culture medium and Flavobacterium culture medium, in temperature
To be cultivated 45~50 hours under 27~32 DEG C and aerobic condition.Wherein, aerobic condition refers to ventilation or shake culture.
Wherein, pseudomonad culture medium and Flavobacterium culture medium include 4~6 parts of peptone, beef leaching by weight
Take 2~4 parts of object, 4~6 parts of sodium chloride, 0.004~0.006 part of manganese sulfate and 950~1050 parts of distilled water.Pseudomonad training
The pH value for supporting base and Flavobacterium culture medium is 6.8~7.2.
By Rhodococcus sp access Rhodococcus sp culture medium in, 26~30 DEG C at a temperature of cultivate 45~50 hours.
Wherein, Rhodococcus sp culture medium includes 3~5 parts of glucose, 3~5 parts of yeast powder by weight；Malt extract 9~11
Part, 1.5~2.5 parts of calcium carbonate and 950~1050 parts of distilled water.
Stenotrophomonas and bacillus are respectively connected in Stenotrophomonas culture medium and bacillus culture medium, 27
It is cultivated 45~50 hours at a temperature of~32 DEG C.
Wherein, Stenotrophomonas culture medium and bacillus culture medium include 4~6 parts of peptone, beef by weight
2~4 parts of cream and 950~1050 parts of distilled water.
It, can be right before cultivating pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus
Corresponding culture medium carries out disinfecting action, i.e., 118~123 DEG C at a temperature of, sterilize 3~5min.
By by pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus respectively in corresponding culture medium
In cultivated so that above-mentioned strain carry out mass propagation, while make bacterium activity and vitality reach good state,
Be conducive to subsequent mixed mixed fermentation.
S2, by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium
Liquid mixing, obtains compound bacteria liquid.
Specifically, by pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and gemma bar
Bacterium bacterium solution is mixed according to the ratio that cell number content is 10~20%, 5~15%, 10~35%, 5~20%, 25~35%
It closes, obtains compound bacteria liquid.Wherein, cell number content refers to pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, widow
The cell number of feeding monad bacterium solution and every kind of bacterium solution in bacillus bacterium solution accounts for the percentage of the total cell number of compound bacteria liquid
Than.
S3, it compound bacteria liquid is put into compound bacterium culture medium carries out mixed fermentation.
Specifically, compound bacteria liquid is accessed in compound bacterium culture medium, is 26~34 DEG C in temperature, the item that pH value is 7.0
After cultivating 45~50 hours under part, the composite bacteria agent for degrading aflatoxin B 1 is obtained.Wherein, compound bacterium culture medium is by weight
Meter include 3~5 parts of glucose, 4~6 parts of peptone, beef leaching 2~4 parts of object, 4~6 parts of sodium chloride, 3~5 parts of yeast powder,
1.5~2.5 parts of calcium carbonate, 0.004~0.006 part of manganese sulfate and 950~1050 parts of distilled water.
A kind of composite bacteria agent for degrading aflatoxin B 1 passes through above-mentioned answering for degrading aflatoxin B 1
The preparation method of combined bacteria agent is made.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
In the present embodiment, a kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising:
Firstly, will from 23439 pseudomonad of bacterial strain deposit number CICC be linked by peptone 4g, beef leaching object 3g,
It is 7.0 in pH value in the pseudomonad culture medium of sodium chloride 5g, manganese sulfate 5mg and distilled water 1000mL composition, temperature 28
DEG C and ventilation under conditions of cultivate 48 hours.
Flavobacterium from bacterial strain deposit number CICC 20907 is linked by peptone 5g, beef leaching object 3g, chlorination
It is 7.1 in pH value in the Flavobacterium culture medium of sodium 5g, manganese sulfate 5mg and distilled water 1000mL composition, the item that temperature is 30 DEG C
Shake culture 48 hours under part.
Rhodococcus sp from bacterial strain deposit number DSM 763 is linked by glucose 3g, yeast powder 3g, malt extract 9g, carbon
Sour calcium 1.5g and distilled water 1000mL composition Rhodococcus sp culture medium in, 28 DEG C at a temperature of cultivate 48 hours.
Stenotrophomonas from bacterial strain deposit number DSM 21257 is linked by peptone 4g, beef extract 2g and steaming
Distilled water 1000mL composition Stenotrophomonas culture medium in, 28 DEG C at a temperature of cultivate 48 hours.
Bacillus from bacterial strain deposit number ATCC 21228 is linked by peptone 5g, beef extract 3g and distillation
Water 1000mL composition bacillus culture medium in, 30 DEG C at a temperature of cultivate 48 hours.
Before cultivating pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus, it will correspond to
Pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, Stenotrophomonas culture medium and bacillus culture medium
It is placed at a temperature of 121 DEG C, sterilize 3min.
Secondly, in obtained pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bud
A certain amount is taken to be mixed in spore bacillus bacterium solution according to the ratio that cell number content is 15%, 10%, 28%, 15%, 32%,
Obtain compound bacteria liquid.
Then, compound bacteria liquid is accessed in compound bacterium culture medium, is 26 DEG C in temperature, pH value is trained under conditions of being 7.0
After supporting 48 hours, the composite bacteria agent for degrading aflatoxin B 1 is obtained.Wherein, compound bacterium culture medium include glucose 3g,
Peptone 4g, beef leaching object 2g, sodium chloride 4g, yeast powder 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
Liquid and the finally obtained composite bacteria agent for degrading aflatoxin B 1 individually or are combined to aflatoxin
B1 carries out Degrading experiment.Wherein, A is set as pseudomonad bacterium solution, and B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is oligotrophy list
Born of the same parents' bacterium bacterium solution, E is bacillus bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method are as follows: A, B, C, D, E of equivalent or the bacterium solution being combined are linked into containing aflatoxin B1
By glucose 4.0g, peptone 5.0g, beef leach object 3.0g, sodium chloride 5.0g, yeast powder 4.0g, calcium carbonate 2.0g, sulphur
It is 7.0 in pH value in the culture medium of sour manganese 5mg and distilled water 1000mL composition, under conditions of temperature is 30 DEG C, shake culture
24 hours.The initial concentration of aflatoxin B1 is 1mg/L.
Test result such as table 1:
Table 1
| Bacterium solution and combinations thereof | Aflatoxin B1 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, Stenotrophomonas bacterium
The degradation that wherein several in liquid, bacillus bacterium solution are conducive to when being mixed to aflatoxin B1, and by pseudomonad
Bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution, bacillus bacterium solution are mixed according to the ratio in the present embodiment
Being combined the composite bacteria agent for degrading aflatoxin B 1 for fermenting and being formed in the present embodiment can be to greatest extent to drop
Solution aflatoxin B1 is degraded, and degradation rate reaches 92.4%.To greatly increase the degradation to aflatoxin B1
Ability, while cost also can be preferably reduced, since biodegrade will not generate secondary pollution.
Embodiment 2
In the present embodiment, a kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising:
Firstly, will from 23439 pseudomonad of bacterial strain deposit number CICC be linked by peptone 6g, beef leaching object 4g,
It is 7.2 in pH value in the pseudomonad culture medium of sodium chloride 6g, manganese sulfate 6mg and distilled water 1050mL composition, temperature 30
DEG C and ventilation under conditions of cultivate 50 hours.
Flavobacterium from bacterial strain deposit number CICC 20907 is linked by peptone 5g, beef leaching object 4g, chlorination
It is 6.9 in pH value in the Flavobacterium culture medium of sodium 4g, manganese sulfate 5mg and distilled water 1000mL composition, the item that temperature is 29 DEG C
Shake culture 48 hours under part.
Rhodococcus sp from bacterial strain deposit number DSM 763 is linked by glucose 5g, yeast powder 5g, malt extract 11g, carbon
Sour calcium 2.5g and distilled water 1050mL composition Rhodococcus sp culture medium in, 30 DEG C at a temperature of cultivate 50 hours.
Stenotrophomonas from bacterial strain deposit number DSM 21257 is linked by peptone 6g, beef extract 4g and steaming
Distilled water 1000mL composition Stenotrophomonas culture medium in, 29 DEG C at a temperature of cultivate 48 hours.
Bacillus from bacterial strain deposit number ATCC 21228 is linked by peptone 4g, beef extract 4g and distillation
Water 1000mL composition bacillus culture medium in, 29 DEG C at a temperature of cultivate 50 hours.
Before cultivating pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus, it will correspond to
Pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, Stenotrophomonas culture medium and bacillus culture medium
It is placed at a temperature of 120 DEG C, sterilize 4min.
Secondly, in obtained pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bud
A certain amount is taken to be mixed in spore bacillus bacterium solution according to the ratio that cell number content is 20%, 12%, 25%, 16%, 27%,
Obtain compound bacteria liquid.
Then, compound bacteria liquid is accessed in compound bacterium culture medium, is 30 DEG C in temperature, pH value is trained under conditions of being 7.0
After supporting 49 hours, the composite bacteria agent for degrading aflatoxin B 1 is obtained.Wherein, compound bacterium culture medium include glucose 5g,
Peptone 6g, beef leaching object 4g, sodium chloride 6g, yeast powder 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
Liquid and the finally obtained composite bacteria agent for degrading aflatoxin B 1 individually or are combined to aflatoxin
B1 carries out Degrading experiment.Wherein, A is set as pseudomonad bacterium solution, and B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is oligotrophy list
Born of the same parents' bacterium bacterium solution, E is bacillus bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method are as follows: A, B, C, D, E of equivalent or the bacterium solution being combined are linked into containing aflatoxin B1
By glucose 4.0g, peptone 5.0g, beef leach object 3.0g, sodium chloride 5.0g, yeast powder 4.0g, calcium carbonate 2.0g, sulphur
It is 7.0 in pH value in the culture medium of sour manganese 5mg and distilled water 1000mL composition, under conditions of temperature is 30 DEG C, shake culture
24 hours.The initial concentration of aflatoxin B1 is 1mg/L.
Test result such as table 2:
Table 2
| Bacterium solution and combinations thereof | Aflatoxin B1 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 analyzing table 2, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, oligotrophy list
The degradation that wherein several in born of the same parents bacterium bacterium solution, bacillus bacterium solution are conducive to when mixing to aflatoxin B1, and will be false
Monad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution, bacillus bacterium solution are according in the present embodiment
Ratio mixes the composite bacteria agent for degrading aflatoxin B 1 that fermentation is formed in the present embodiment can be to greatest extent
Degrade to degrading aflatoxin B 1, degradation rate reaches 96.2%.To greatly increase to aflatoxin B1
Degradation capability, while cost also can be preferably reduced, since biodegrade will not generate secondary pollution.
Embodiment 3
In the present embodiment, a kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising:
Firstly, will from 23439 pseudomonad of bacterial strain deposit number CICC be linked by peptone 5g, beef leaching object 3g,
It is 7.0 in pH value in the pseudomonad culture medium of sodium chloride 5g, manganese sulfate 5mg and distilled water 1000mL composition, temperature 27
DEG C and ventilation under conditions of cultivate 47 hours.
Flavobacterium from bacterial strain deposit number CICC 20907 is linked by peptone 4g, beef leaching object 2g, chlorination
It is 6.8 in pH value in the Flavobacterium culture medium of sodium 5g, manganese sulfate 4mg and distilled water 950mL composition, the item that temperature is 28 DEG C
Shake culture 48 hours under part.
Rhodococcus sp from bacterial strain deposit number DSM 763 is linked by glucose 4g, yeast powder 4g, malt extract 10g, carbon
Sour calcium 2g and distilled water 1000mL composition Rhodococcus sp culture medium in, 30 DEG C at a temperature of cultivate 48 hours.
Stenotrophomonas from bacterial strain deposit number DSM 21257 is linked by peptone 5g, beef extract 3g and steaming
Distilled water 1000mL composition Stenotrophomonas culture medium in, 28 DEG C at a temperature of cultivate 48 hours.
Bacillus from bacterial strain deposit number ATCC 21228 is linked by peptone 5g, beef extract 4g and distillation
Water 1000mL composition bacillus culture medium in, 30 DEG C at a temperature of cultivate 48 hours.
Before cultivating pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus, it will correspond to
Pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, Stenotrophomonas culture medium and bacillus culture medium
It is placed at a temperature of 122 DEG C, sterilize 5min.
Secondly, in obtained pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bud
A certain amount is taken to be mixed in spore bacillus bacterium solution according to the ratio that cell number content is 18%, 15%, 30%, 12%, 25%,
Obtain compound bacteria liquid.
Then, compound bacteria liquid is accessed in compound bacterium culture medium, is 34 DEG C in temperature, pH value is trained under conditions of being 7.0
After supporting 48 hours, the composite bacteria agent for degrading aflatoxin B 1 is obtained.Wherein, compound bacterium culture medium include glucose 4g,
Peptone 5g, beef leaching object 3g, sodium chloride 5g, yeast powder 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
Liquid and the finally obtained composite bacteria agent for degrading aflatoxin B 1 individually or are combined to aflatoxin
B1 carries out Degrading experiment.
Wherein, A is set as pseudomonad bacterium solution, and B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is Stenotrophomonas bacterium
Liquid, E is bacillus bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method are as follows: A, B, C, D, E of equivalent or the bacterium solution being combined are linked into containing aflatoxin B1
By glucose 4.0g, peptone 5.0g, beef leach object 3.0g, sodium chloride 5.0g, yeast powder 4.0g, calcium carbonate 2.0g, sulphur
It is 7.0 in pH value in the culture medium of sour manganese 5mg and distilled water 1000mL composition, under conditions of temperature is 30 DEG C, shake culture
24 hours.The initial concentration of aflatoxin B1 is 1mg/L.
Test result such as table 3:
Table 3
| Bacterium solution and combinations thereof | Aflatoxin B1 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% |
Pass through table 3, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
The degradation that wherein several in bacillus bacterium solution are conducive to when being mixed to aflatoxin B1, and by pseudomonad bacterium
Liquid, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution, bacillus bacterium solution are mixed according to the ratio in the present embodiment
The composite bacteria agent for degrading aflatoxin B 1 that fermentation is formed in the present embodiment together can be to greatest extent to degradation
Aflatoxin B1 is degraded, and degradation rate reaches 93.5%.To greatly increase the degradation energy to aflatoxin B1
Power, while cost also can be preferably reduced, since biodegrade will not generate secondary pollution.
Embodiment 4
In the present embodiment, a kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1, comprising:
Firstly, will from 23439 pseudomonad of bacterial strain deposit number CICC be linked by peptone 5g, beef leaching object 3g,
It is 7.0 in pH value in the pseudomonad culture medium of sodium chloride 5g, manganese sulfate 5mg and distilled water 1000mL composition, temperature 28
DEG C and ventilation under conditions of cultivate 48 hours.
Flavobacterium from bacterial strain deposit number CICC 20907 is linked by peptone 5g, beef leaching object 3g, chlorination
It is 7.0 in pH value in the Flavobacterium culture medium of sodium 5g, manganese sulfate 5mg and distilled water 1000mL composition, the item that temperature is 30 DEG C
Aerlbic culture 48 hours under part.
Rhodococcus sp from bacterial strain deposit number DSM 763 is linked by glucose 4g, yeast powder 4g, malt extract 10g, carbon
Sour calcium 2g and distilled water 1000mL composition Rhodococcus sp culture medium in, 28 DEG C at a temperature of cultivate 48 hours.
Stenotrophomonas from bacterial strain deposit number DSM 21257 is linked by peptone 5g, beef extract 3g and steaming
Distilled water 1000mL composition Stenotrophomonas culture medium in, 28 DEG C at a temperature of cultivate 48 hours.
Bacillus from bacterial strain deposit number ATCC 21228 is linked by peptone 5g, beef extract 3g and distillation
Water 1000mL composition bacillus culture medium in, 30 DEG C at a temperature of cultivate 48 hours.
Before cultivating pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus, it will correspond to
Pseudomonad culture medium, Flavobacterium culture medium, Rhodococcus sp culture medium, Stenotrophomonas culture medium and bacillus culture medium
It is placed at a temperature of 118 DEG C, sterilize 5min.
Secondly, in obtained pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bud
A certain amount is taken to be mixed in spore bacillus bacterium solution according to the ratio that cell number content is 15%, 15%, 35%, 15%, 20%,
Obtain compound bacteria liquid.
Then, compound bacteria liquid is accessed in compound bacterium culture medium, is 28 DEG C in temperature, pH value is trained under conditions of being 7.0
After supporting 48 hours, the composite bacteria agent for degrading aflatoxin B 1 is obtained.Wherein, compound bacterium culture medium include glucose 4g,
Peptone 5g, beef leaching object 3g, sodium chloride 5g, yeast powder 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
Liquid and the finally obtained composite bacteria agent for degrading aflatoxin B 1 individually or are combined to aflatoxin
B1 carries out Degrading experiment.Wherein, A is set as pseudomonad bacterium solution, and B is Flavobacterium bacterium solution, and C is Rhodococcus sp bacterium solution, and D is oligotrophy list
Born of the same parents' bacterium bacterium solution, E is bacillus bacterium solution, and A+B+C+D+E is the composite bacteria agent for degrading aflatoxin B 1.
Test method are as follows: A, B, C, D, E of equivalent or the bacterium solution being combined are linked into containing aflatoxin B1
By glucose 4.0g, peptone 5.0g, beef leach object 3.0g, sodium chloride 5.0g, yeast powder 4.0g, calcium carbonate 2.0g, sulphur
It is 7.0 in pH value in the culture medium of sour manganese 5mg and distilled water 1000mL composition, under conditions of temperature is 30 DEG C, shake culture
24 hours.The initial concentration of aflatoxin B1 is 1mg/L.
Test result such as table 4:
Table 4
| Bacterium solution and combinations thereof | Aflatoxin B1 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% |
Pass through table 4, it can be seen that when pseudomonad bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution,
The degradation that wherein several in bacillus bacterium solution are conducive to when being mixed to aflatoxin B1, and by pseudomonad bacterium
Liquid, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution, bacillus bacterium solution are mixed according to the ratio in the present embodiment
The composite bacteria agent for degrading aflatoxin B 1 that fermentation is formed in the present embodiment together can be to greatest extent to degradation
Aflatoxin B1 is degraded, and degradation rate reaches 97.8%.To greatly increase the degradation energy to aflatoxin B1
Power, while cost also can be preferably reduced, since biodegrade will not generate secondary pollution.
In conclusion by the way that pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are carried out respectively
Culture, so that pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are largely bred, and in work
Property and growth conditions reach very well after, obtain pseudomonad 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
Bacillus bacterium solution is mixed according to a certain percentage, obtains compound bacteria liquid.Compound bacteria liquid is put into compound bacteria culture again
Mixed fermentation is carried out in base, to obtain the composite bacteria agent that can be used for degrading aflatoxin B 1.Pass through pseudomonad, yellow bar
Bacterium, Rhodococcus sp, Stenotrophomonas and bacillus mixing coordinated, enable the composite bacteria agent efficiently and rapidly right
Aflatoxin B1 is degraded, and degradation effect is very good, at low cost, pollution-free.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (8)
1. a kind of preparation method of the composite bacteria agent for degrading aflatoxin B 1 characterized by comprising
Pseudomonad, Flavobacterium, Rhodococcus sp, Stenotrophomonas and bacillus are cultivated respectively, obtain pseudomonad
Bacterium solution, Flavobacterium bacterium solution, Rhodococcus sp bacterium solution, Stenotrophomonas bacterium solution and bacillus bacterium solution；
By the pseudomonad bacterium solution, the Flavobacterium bacterium solution, the Rhodococcus sp bacterium solution, the Stenotrophomonas bacterium solution and institute
The mixing of bacillus bacterium solution is stated, compound bacteria liquid is obtained；
The compound bacteria liquid is accessed in compound bacterium culture medium and carries out mixed fermentation；
Wherein, the pseudomonad bacterium solution in the compound bacteria liquid, the Flavobacterium bacterium solution, the Rhodococcus sp bacterium solution, institute
Stating the cell number of Stenotrophomonas bacterium solution and the bacillus bacterium solution, to be followed successively by cell in the compound bacteria liquid respectively total
Several 10~20%, 5~15%, 10~35%, 5~20%, 25~35%, the Rhodococcus sp come from bacterial strain deposit number DSM
763, pseudomonad comes from bacterial strain deposit number CICC 23439, and Flavobacterium comes from bacterial strain deposit number CICC 20907, oligotrophy unit cell
Bacterium comes from bacterial strain deposit number DSM 21257, and bacillus comes from bacterial strain deposit number ATCC 21228；
Carrying out the mixed fermentation is to access the compound bacteria liquid in the compound bacterium culture medium, is 26~34 in temperature
DEG C, pH value be 7.0 under conditions of cultivate 45~50 hours, the compound bacterium culture medium include by weight 3~5 parts of glucose,
4~6 parts of peptone, beef leach 2~4 parts of object, 4~6 parts of sodium chloride, 3~5 parts of yeast powder, 1.5~2.5 parts of calcium carbonate, sulfuric acid
0.004~0.006 part of manganese and 950~1050 parts of distilled water.
2. the preparation method of the composite bacteria agent according to claim 1 for degrading aflatoxin B 1, which is characterized in that
It is that the pseudomonad, the Flavobacterium are respectively connected to pseudomonad that the pseudomonad, the Flavobacterium, which are carried out culture,
In culture medium and Flavobacterium culture medium, cultivated 45~50 hours in the case where temperature is 27~32 DEG C and aerobic condition.
3. the preparation method of the composite bacteria agent according to claim 2 for degrading aflatoxin B 1, which is characterized in that
The pseudomonad culture medium and the Flavobacterium culture medium include 4~6 parts of peptone, beef leaching object 2 by weight
~4 parts, 4~6 parts of sodium chloride, 0.004~0.006 part of manganese sulfate and 950~1050 parts of distilled water.
4. the preparation method of the composite bacteria agent according to claim 1 for degrading aflatoxin B 1, which is characterized in that
By the Rhodococcus sp carry out culture be by the Rhodococcus sp access Rhodococcus sp culture medium in, 26~30 DEG C at a temperature of cultivate 45
~50 hours.
5. the preparation method of the composite bacteria agent according to claim 4 for degrading aflatoxin B 1, which is characterized in that
The Rhodococcus sp culture medium includes 3~5 parts of glucose, 3~5 parts of yeast powder, 9~11 parts of malt extract, calcium carbonate by weight
1.5~2.5 parts and 950~1050 parts of distilled water.
6. the preparation method of the composite bacteria agent according to claim 1 for degrading aflatoxin B 1, which is characterized in that
It is that the Stenotrophomonas and the bacillus are respectively connected to widow that the Stenotrophomonas and bacillus, which are carried out culture,
Support unit cell bacterium culture medium and bacillus culture medium in, 27~32 DEG C at a temperature of cultivate 45~50 hours.
7. the preparation method of the composite bacteria agent according to claim 6 for degrading aflatoxin B 1, which is characterized in that
The Stenotrophomonas culture medium and the bacillus culture medium include 4~6 parts of peptone, beef extract 2~4 by weight
Part and 950~1050 parts of distilled water.
8. a kind of composite bacteria agent for degrading aflatoxin B 1, which is characterized in that it is by claim 1~7 any one
The preparation method of the composite bacteria agent for degrading aflatoxin B 1 is made.
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| CN106636017A (en) * | 2017-02-27 | 2017-05-10 | 河南工业大学 | Preparation method of compound enzyme preparation for efficiently degrading aflatoxin B1 |
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| CN109182205B (en) * | 2018-10-09 | 2021-02-02 | 北京林业大学 | Rhodococcus with carbon fixing capacity and application thereof |
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| CN113462589A (en) * | 2021-04-17 | 2021-10-01 | 苏州优利莱生物科技有限公司 | Leaven for degrading aflatoxin in feed and preparation method thereof |
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