CN109234196A - A kind of mixed bacterial and its crude enzyme preparation of the fumonisin B1 that degrades - Google Patents
A kind of mixed bacterial and its crude enzyme preparation of the fumonisin B1 that degrades Download PDFInfo
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- CN109234196A CN109234196A CN201811142897.6A CN201811142897A CN109234196A CN 109234196 A CN109234196 A CN 109234196A CN 201811142897 A CN201811142897 A CN 201811142897A CN 109234196 A CN109234196 A CN 109234196A
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Abstract
The invention discloses the mixed bacterials and its crude enzyme preparation of a kind of fumonisin B1 that degrades.The mixed bacterial of degradation fumonisin B1 of the invention, including following ingredient: pseudomonas (Pseudomonas), Comamonas (Comamonas) and Sphingobacterium (sphingobacterium), total living bacteria count account for 90% or more of mixed bacterial total quantity.The mixed bacterial of degradation fumonisin B1 of the invention amplification cultivation in the MM culture medium containing sucrose, after being centrifuged the thallus ultrasonication of acquisition, supernatant freeze-drying obtains crude enzyme preparation.The mixed bacterial and its crude enzyme preparation for the degradation fumonisin B1 that the present invention obtains are in for 24 hours to fumonisin B1 degradation rate up to 90% or more.The mixed bacterial and its crude enzyme preparation have the characteristics that fermentation is simple, production cost is low, have broad application prospects to the detoxification of fumonisins in fodder B1.
Description
Technical field
The invention belongs to field of biotechnology, mixed bacterial more particularly to a kind of fumonisin B1 that degrades and its thick
Enzyme preparation.
Background technique
Fumonisin (FB) is by the toxic secondary metabolite of one kind of the generations such as fusarium moniliforme and wheel branch sickle-like bacteria.Mesh
Before, it is known that FB have 11 kinds, wherein it is most wide with fumonisin B1 (FB1) pollution range, toxicity is most strong, the health caused by people and animals
Harm is maximum.FB1 can pollute feed and its raw material etc. extensively.Investigation shows that the pollution rate of FB1 in China's feed is universal in recent years
More than 90%, animal husbandry is caused and cannot be neglected harm.FB1 not only interferes plant normal physiological function, but also to livestock and poultry
With various specific toxicities effects, white matter of brain malacosis, the pig pulmonary edema of horse and rabbit can be caused, there is kidney to rodent
Toxicity and hepatotoxicity wind agitation etc..In addition, research shows that FB1 and mankind's cancer of the esophagus have potential relevance, international cancer research aircraft
Structure (IARC) has been classified as 2B class carcinogenic substance.The global pollution of fumonisin and high harmfulness cause people and close extensively
Note, therefore how to remove or reduce FB1 health hazard caused by humans and animals and be of great significance.
Currently, fumonisin often includes chemical method, physical method, absorption method and microbial detoxification method with poison-removing method.Tradition
Chemical method (ammonia, ozone, isothiocyanate and cinnamon oil facture) FB content in feed or cereal can be reduced, but the method is easy
New harmful substance is introduced, nutritional ingredient can be destroyed, increases the security risk of feed and cereal；Physical method (sorts and rinsing, heat
Processing, shelling and grinding) also can remove a certain amount of FB, but the law article part requires high, changeable food quality, application by
Limitation.Absorption detoxicity method is the method when the most effective removal mycotoxin of former.Research find in feed add cholestyramine,
The adsorbents such as AdiDetoxTM can significantly reduce FB to the toxic effect of rat.However, absorption method has specificity not high, easily inhale
In attached feed the disadvantages of nutritional ingredient.Microbial detoxification method can convert low toxicity or nontoxic production for toxin by enzymatic reaction
Object, or achieve the purpose that remove toxin by Adsorption on cell walls effect.Compared with chemically and physically method, microbial method has special
One property is strong, treatment conditions are mild, nutrient component damages are few, and palatability aesthetic on raw material influences that small, to be easy to industrialization etc. excellent
Point, it has also become mycotoxin detoxification main direction of studying is considered having good development and application prospect.Most of microbe pair
The degradation of fumonisin is realized by the key enzyme of its generation after all.Currently, reported to volt horse both at home and abroad
Toxin B1 has the bacterial strain of degradation effect to only have 3 plants, is belonging respectively to sphingol box Pseudomonas (Sphingopyxis), Si Pingnifu outer blank
Handle mould (Exophialaspinifera) and Rhinocladiella (Rhinocladiella atrovirens).But it is limited to produce
The reasons such as technique are not applied in actual production.
Summary of the invention
The purpose of the present invention first consist in provides it is a kind of degrade fumonisin B1 mixed bacterial, the flora include as follows at
Point:
Pseudomonas (Pseudomonas), Comamonas (Comamonas) and Sphingobacterium
(sphingobacterium)；These three Pseudomonas account for 90% or more of mixed bacterial total quantity；
Further, pseudomonas (Pseudomonas) accounts for 85% or more of mixed bacterial total quantity.
The mixed bacterial of above-mentioned degradation fumonisin B1 of the invention, is prepared via a method which to obtain:
(1) bacteria residue of Jiangxi Province straw mushroom production base is acquired, is saved backup under the conditions of 4 DEG C；
(2) it weighs bacteria residue sample and is dissolved in distilled water, vibrate 12h under the conditions of 37 DEG C；Collect oscillation liquid；
(3) it screens the flora of degradable fumonisin B1: B1 containing fumonisin being used to train as the inorganic salts of sole carbon source
It supports base (MM culture medium) and carries out screening domestication culture, adjust PH to 7.0；The flora of acclimation and screening is transferred in MM culture medium,
Domestication culture again, 10 times repeatedly；Resulting bacterium solution will be tamed to cross in MM solid medium separation, trained under the conditions of 28 DEG C
After supporting, picking single bacterium drops down onto MM culture medium and cultivates 48h, obtains the mixed bacterial of fumonisin B1 that degrades a kind of；
The wherein minimal medium formula (MM culture medium) are as follows: K2HPO4(2.5g/L)、KH2PO4(2.5g/L)、
(NH4)2HPO4(1.0g/L)、MgSO4·7H2O(0.2g/L)、FeSO4(0.01g/L)、MnSO4.7H2O (0.004g),
The fumonisin B1 of various concentration can be added as needed in MM culture medium when in use.
When the PH of MM culture medium is 7, degradation efficiency highest of the mixed bacterial to fumonisin B1.
The macro genome analysis of 16SrDNA is carried out for the mixed bacterial of acquisition, the results showed that mixed bacterial specifically includes that vacation
Zygosaccharomyces (Pseudomonas), Comamonas (Comamonas) and Sphingobacterium
(sphingobacterium), total living bacteria count accounts for 90% or more of mixed bacterial total quantity.Wherein, pseudomonas bacterium
Strain accounts for 85% or more of mixed bacterial total quantity.
The present invention also provides the crude enzyme preparations of the mixed bacterial of above-mentioned degradation fumonisin B1, are by the following method
It is prepared:
The mixed bacterial of acquisition is seeded in the MM culture medium containing 1% sucrose (10g sucrose is dissolved in 1L MM culture medium,
Adjust PH to 7, high pressure sterilization 30min under the conditions of 121 DEG C), the shaken cultivation 48h at 28 DEG C is collected after centrifugation thallus and passes through
It crosses and cleans repeatedly, PBS buffer solution is then added and carries out ultrasonication extraction；It collects supernatant and is placed in freeze drier and freeze
It is dry, obtain the crude enzyme preparation of mixed bacterial.
By mixed bacterial or crude enzyme preparation be seeded to the B1 of fumonisin containing 50mg/L MM culture medium (pH value 6-8, preferably
It is 7) 200rpm shaken cultivation 48h under the conditions of 28 DEG C for PH；Using LC-MS/MS measurement culture solution in fumonisin B1 it is dense
Degree, according to the content of fumonisin B1 in culture solution, judges mixed bacterial or crude enzyme preparation to the degradation efficiency of fumonisin B1.
The result shows that: in 48h, mixed bacterial or crude enzyme preparation are to the degradation rate of 50mg/L fumonisin B1 up to 90% or more.
The mixed bacterial and its crude enzyme preparation of a kind of degradation fumonisin B1 of the invention has the following beneficial effects:
The mixed bacterial of degradation fumonisin B1 disclosed by the invention, with pseudomonas strain
(Pseudomonadaceae) it is main component, 90% or more is up to fumonisin B1 degradation rate interior for 24 hours.Meanwhile passing through
The mixed bacterial crude enzyme preparation that ultrasonic fragmentation obtains, to the toxin also degradation efficiency (90% or more) with higher.This
Invention has high application value to the detoxification of mycotoxin in feed.
The mixed bacterial and its crude enzyme preparation of degradation fumonisin B1 disclosed by the invention can for what is reported for the first time both at home and abroad
Flora/enzyme preparation of degradation fumonisin B1.Meanwhile it can be obtained by simple culture medium and fermentation process a large amount of effective
Flora is used to prepare the crude enzyme preparation of degradable fumonisin B1, and fermentation process is simple and production cost is low.In addition, with chemistry and
Physical method is compared, and microbial method is with specificity is strong, treatment conditions are mild, nutrient component damages are few, aesthetic to raw material and suitable
Mouth property influences small advantage, and prevention and control fumonisins in fodder B1 is endangered caused by animal husbandry with before wide application
Scape.
Detailed description of the invention
The present invention is described in more detail with reference to the accompanying drawings and examples
Fig. 1 is flora structural analysis in mixed bacterial
Fig. 2 is for mixed bacterial to the degradation efficiency of fumonisin B1 under the conditions of different PH
Fig. 3 is for crude enzyme preparation to the degradation efficiency of fumonisin B1 under different pH conditions
Specific embodiment
Detailed description is made to implementation process of the present invention below with reference to embodiment.In particular, it should be pointed out that all similar replaces
It changes or changes, be regarded as being included in the present invention.
Raw materials used or preparation source in the following example:
This experiment bacteria residue of the sample from Guangchang County of Jiangxi Province Yanlin planting edible mushroom Specialty Co-operative Organization used, will acquire
Bacteria residue be stored in sterilized transparent sealed bag, saved backup in 4 DEG C of refrigerators.
In following embodiments, unless otherwise specified, used experimental method is conventional method, and reagent used etc. is equal
Can chemically or biological reagent company purchase.
Embodiment 1: the separation screening of the mixed bacterial of degradation fumonisin B1
Prepare MM culture medium:
Weigh 2.5g K2HPO4、2.5g KH2PO4、1.0g(NH4)2HPO4、0.2g MgSO4·7H2O、0.01g FeSO4,
0.004g MnSO4·7H2O is placed in volumetric flask, and water is added to be settled to 1L, adjusts PH to 7.High pressure sterilization under the conditions of 121 DEG C
30min。
The distilled water that 10g bacteria residue sample is dissolved in 100mL sterilizing is weighed, vibrates 12h under the conditions of 37 DEG C.1mL is taken to vibrate
Liquid is diluted to 10mL, takes 100 μ L dilutions to be seeded to the MM culture medium (sterilizing) of 2mL fumonisin containing 50mg/L B1, at 28 DEG C
Under the conditions of 200rpm shaken cultivation 7d.Utilize the content of fumonisin B1 in LC-MS/MS measurement culture solution.The result shows that: in 7d
In culture period, the culture solution is to the degradability of fumonisin B1 up to 99% or more.Further, take the 100 μ L culture solutions be added to
In the MM culture medium (sterilizing) of 2mL fumonisin containing 100mg/L B1, same to above-mentioned steps, enrichment culture 9 times, are enriched with repeatedly
Bacterium solution.
The enrichment bacterium solution of above-mentioned acquisition is subjected to gradient dilution (10 with aqua sterilisa-1With 10-2), coating or scribing line are inorganic
(fumonisin B1 is added after dissolving by heating in minimal medium, is configured to 20 μ g/mL of final concentration, puts down on salt agar medium
Plate), culture is inverted under the conditions of 28 DEG C, after having bacterium colony to grow, picking upgrowth situation in solid medium tablets is good
Single colonie is seeded to the MM culture medium containing 20 μ g/mL fumonisin B1.200rpm shaken cultivation 7d under the conditions of 28 DEG C is utilized
LC-MS/MS measures the concentration of fumonisin B1 in culture medium, evaluates the different bacterium colonies of picking to fumonisin B1 degradation capability.
The result shows that: 1 single colonie of picking is to fumonisin B1 degradation rate up to 96%.The Mixed Microbes that the bacterium colony is obtained as separation
Group, is dissolved in 20% glycerite and is stored at -80 DEG C.
Embodiment 2: the COMMUNITY STRUCTURE of mixed bacterial
Mixed bacterial described in 200 μ L examples 1 is taken to be seeded in the MM culture medium of the B1 of fumonisin containing 50mg/L, 28
200rpm shaken cultivation for 24 hours, is centrifuged 10min under the conditions of 15000rpm under the conditions of DEG C, and thallus is stored under the conditions of -20 DEG C.
It is extracted according to QIAamp Fast DNA Stool Mini Ki kit (German Qiagen company) total in thallus
DNA, and carry out electroresis appraisal and successfully obtain DNA of bacteria.Using the method for high-flux sequence, using primer 926F (5 '-
AAACTYAAAKGAATTGACGG-3 ') and 518R (5 '-ATTACCGCGGCTGCTGG-3 ') amplification bacterial 16 S rDNA V4-
The area V5 segment (primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd).After PCR product is detected with agarose gel electrophoresis, send
The bis- end sequencing analyses of Illumina are carried out to Sangon Biotech (Shanghai) Co., Ltd.., the resulting sequence of high-flux sequence
Data carry out quality control using FLASH and Fastq, then are corrected by Qiime software flow, remove chimera and targeting
Sequence outside region, then carry out OUT clustering, using RPD classifier software bayesian algorithm to 97% it is similar
The horizontal OUT representative series of degree carry out Taxonomic analysis, count group's composition between each sample, and classification confidence level uses
The estimation of Bootstrap method.
Found by carrying out 16SrDNA analysis to the mixed bacterial of acquisition: mixed bacterial is with pseudomonas
(Pseudomonas), based on Comamonas (Comamonas) and Sphingobacterium (sphingobacterium), point
86.43%, 5.57% and the 1.8% of total amount is not accounted for, accounts for 93.8% (Fig. 1 is shown in the accounting distribution of each Pseudomonas) of total bacteria count in total.
Meanwhile this test clearly plays the flora of principal degradation using antibiotic-screening method to fumonisin B1.It investigates
Cefotaxime (French A2S company) (0,1,10,50 and of various concentration level are added in the MM culture medium of inoculation mixed bacterial
100ppm) the variation of Bacterial community afterwards.It is found by the macro gene order-checking analysis of 16S rDNA: not plus in cefotaxime processing group
The quantity of pseudomonas (Pseudomonas) accounts for about the 85% of total bacterium；Pseudomonas in 1ppm cefotaxime processing group
(Pseudomonas) quantity accounts for total amount about 48%；Greater than pseudomonas in 1ppm cefotaxime processing group
(Pseudomonas) quantity accounts for about 20%-35%.In 48h, 0 processing group is 92% to fumonisin B1 degradation rate；It is greater than
1ppm cephalo processing group is lower than 30% to fumonisin B1 degradation rate, shows the Pseudomonas to play a major role of degrading to fumonisin B1
For pseudomonas.
Embodiment 3: degradation efficiency of the mixed bacterial to fumonisin B1
Mixed bacterial is investigated under different culture medium and condition of culture to the degradation capability of fumonisin B1.
(1) mixed bacterial is under the conditions of different culture medium to the degradation efficiency of FB1
Different culture mediums is configured, it is specific as follows: 1. minimal medium (MM): to weigh 2.5g K2HPO4、2.5g
KH2PO4、1.0g(NH4)2HPO4、0.2g MgSO4·7H2O、0.01g FeSO4, 0.004g MnSO4·7H2O is placed in volumetric flask
In, add water to be settled to 1L, adjusts PH to 7.High pressure sterilization 30min under the conditions of 121 DEG C；2. LB culture medium: weighing 5g yeast and mention
It takes object, 10g peptone, 10g NaCl to be placed in volumetric flask, water is added to be settled to 1L, adjust PH to 7, high pressure under the conditions of 121 DEG C
Sterilize 30min；3. nutrient broth medium (NB): weighing 10.0g peptone, 3.0g powdered beef, 5.0g NaCl, be dissolved in 1L
In distilled water, high pressure sterilization 30min under the conditions of 121 DEG C；4. containing the MM culture medium (MM+PT) of 1% glucose: weighing the Portugal 10g
Grape sugar is dissolved in 1L MM culture medium, adjusts PH to 7, high pressure sterilization 30min under the conditions of 121 DEG C；5. the MM containing 1% sucrose is trained
It supports base (MM+GT): weighing 10g sucrose and be dissolved in 1L MM culture medium, adjust PH to 7, high pressure sterilization under the conditions of 121 DEG C
30min；6. containing the MM culture medium of 0.5% peptone: weighing 5g peptone and be dissolved in 1L MM culture medium, adjust PH to 7.121
High pressure sterilization 30min under the conditions of DEG C.
The mixed bacterial in 100 μ L embodiments 1 is taken to be seeded in the different culture medium of 2mL fumonisin containing 50mg/L B1,
200rpm shaken cultivation 48h under the conditions of 28 DEG C.Then, it takes 100 μ L culture solutions to be added isometric acetonitrile, after shake well, surpasses
Sound extracts 15min, and the content of fumonisin B1 in different culture solutions is measured using LC-MS/MS, evaluates mixed bacterial in different trainings
Support the degradation capability in base to fumonisin B1.The result shows that: mixed bacterial is in MM culture medium to fumonisin B1 degradation rate
It is 93%, degradation rate is between 60-80% in the MM culture medium containing sucrose or glucose, the degradation rate in other culture mediums
Respectively less than 50%.Therefore, follow-up test is all made of the research that minimal medium carries out degradation efficiency.
(2) mixed bacterial is under condition of different pH to fumonisin B1 degradation efficiency
The pH value of MM culture medium, respectively 2,4,5,7,8,9 and 10 are adjusted using 0.1M NaOH solution or HCl solution.It will
Mixed bacterial in example 1 is seeded in the MM culture medium containing 10ppm fumonisin B1, shaken cultivation under the conditions of 28 DEG C,
Culture solution is collected with 48h respectively at for 24 hours.100 μ L culture solutions are taken to be added isometric acetonitrile, after shake well, ultrasonic extraction
15min, the content of fumonisin B1 in different culture solutions is measured using LC-MS/MS, to evaluate the pH value of culture medium to volt horse
The influence of toxin B1 degradation efficiency.As shown in Fig. 2, under the conditions of PH=7, degradation of the mixed bacterial to fumonisin B1 in 12h
Rate is up to 95%；To fumonisin B1 degradation rate up to 100% in for 24 hours.Meanwhile under acid or alkaline pH conditions, mixed bacterial
Degradation rate to fumonisin B1 is only 30% or less.It is obtained by this test: mixed bacterial degradation efficiency under the conditions of PH=7
Highest.
Embodiment 4: the preparation of crude enzyme preparation and its degradation efficiency to fumonisin B1
Taking the mixed bacterial in appropriate example 1 to be seeded in MM culture medium of the 100mL containing 1% sucrose, (10g sucrose is dissolved in
1L MM culture medium adjusts PH to 7, high pressure sterilization 30min under the conditions of 121 DEG C) in, 200rpm oscillation training under the conditions of 28 DEG C
Support 48h.After being centrifuged 10min at 4500rpm, bacterial sediment uses 50mM PBS buffer solution repeated flushing 2 times after sterilizing.
After being centrifuged 10min at 4500rpm, thallus is dissolved in 2mL PBS buffer solution, is placed in Ultrasonic Cell Disruptor and is extracted 30min.
It is the crude enzyme preparation of mixed bacterial under the conditions of 4 DEG C after 4500rpm centrifugation, after supernatant freeze-drying.Crude enzyme preparation is dissolved in
In 1mL PBS buffer solution, protein quantification is carried out by Coomassie Brilliant Blue.
The pH value of MM culture medium, respectively 2,4,6,7,8,9 and 10 are adjusted using 0.1M NaOH solution or HCl solution.It takes
Appropriate crude enzyme preparation is into the MM culture medium containing 10ppm fumonisin B1, shaken cultivation under the conditions of 28 DEG C, respectively at for 24 hours
Culture solution is collected with 48h.100 μ L culture solutions are taken to be added isometric acetonitrile, after shake well, ultrasonic extraction 15min utilizes LC-
MS/MS measures the content of fumonisin B1 in different culture solutions, so that the pH value for evaluating culture medium is degraded to fumonisin B1 and imitated
The influence of rate.As shown in figure 3, when PH is in 6-8 range, crude enzyme preparation to fumonisin B1 degradation rate 85%-100% it
Between；When PH is 7, crude enzyme preparation is more than 95% to the degradation rate of fumonisin B1；When PH is between 2-4, to fumonisin B1
Degradation rate is below 65%；When PH is between 9-10, to fumonisin B1 degradation rate within the scope of 68-78%.
Claims (4)
1. a kind of mixed bacterial for the fumonisin B1 that degrades, it is characterised in that the flora includes following ingredient:
Pseudomonas Pseudomonas, Comamonas Comamonas and Sphingobacterium
sphingobacterium；These three Pseudomonas account for 90% or more of mixed bacterial total quantity.
2. the mixed bacterial of degradation fumonisin B1 according to claim 1, it is characterised in that wherein pseudomonas
Pseudomonas accounts for 85% or more of mixed bacterial total quantity.
3. a kind of method for the mixed bacterial for preparing degradation fumonisin B1 as claimed in claim 1 or 2, it is characterised in that this method
Include the following steps:
(1) bacteria residue of straw mushroom production base is acquired, is saved backup under the conditions of 4 DEG C；
(2) it weighs bacteria residue sample and is dissolved in distilled water, vibrate 12h under the conditions of 37 DEG C；Collect oscillation liquid；
(3) it screens the flora of degradable fumonisin B1: carrying out screening domestication culture using the MM culture medium of the B1 containing fumonisin,
Adjust PH to 7.0；The flora of acclimation and screening is transferred in MM culture medium, again domestication culture, 10 times repeatedly；It will domestication gained
Bacterium solution cross in MM solid medium separation, after cultivating under the conditions of 28 DEG C, picking single bacterium drops down onto cultivates in MM culture medium
48h obtains the mixed bacterial of fumonisin B1 that degrades a kind of；
The wherein MM culture medium are as follows: K2HPO4 2.5g/L、KH2PO4 2.5g/L、(NH4)2HPO4 1.0g/L、MgSO4·
7H2O 0.2g/L、FeSO4 0.01g/L、MnSO4.7H2O 0.004g。
4. a kind of crude enzyme preparation of the mixed bacterial of the degradation fumonisin B1 of claims 1 or 2, it is characterised in that it is to pass through
What following method was prepared:
The mixed bacterial of claims 1 or 2 is seeded in the MM culture medium containing 1% sucrose, PH 6-8, training is vibrated at 28 DEG C
48h is supported, thallus is collected after centrifugation and through over cleaning, then addition PBS buffer solution progress ultrasonication extraction；Collect supernatant
It is placed in freeze drier and is lyophilized, obtain the crude enzyme preparation of mixed bacterial.
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Application publication date: 20190118 |