CN109536403B - TF-06 bacterium for degrading patulin and application thereof - Google Patents

TF-06 bacterium for degrading patulin and application thereof Download PDF

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CN109536403B
CN109536403B CN201811391247.5A CN201811391247A CN109536403B CN 109536403 B CN109536403 B CN 109536403B CN 201811391247 A CN201811391247 A CN 201811391247A CN 109536403 B CN109536403 B CN 109536403B
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patulin
bacteria
pseudomonas
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CN109536403A (en
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李博强
田世平
张占全
徐勇
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Institute of Botany of CAS
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Abstract

The invention discloses TF-06 bacteria for degrading patulin and application thereof. The invention provides Pseudomonas (Pseudomonas sp.) TF-06 with the preservation number of CGMCC NO. 16575. The invention also provides application of the Pseudomonas (Pseudomonas sp.) TF-06 or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof in degrading patulin. Experiments prove that the bacteria TF-06 has the effect of degrading patulin, is high in degradation efficiency and specificity, is easy to culture, high in propagation speed and easy to prepare, and lays a foundation for researching the degradation of patulin.

Description

TF-06 bacterium for degrading patulin and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to TF-06 bacteria for degrading patulin and application thereof.
Background
Patulin (patulin), also called patulin, is a toxic fungal secondary metabolite. Fruits such as apples and the like are infected by pathogenic fungi such as penicillium expansum and the like to generate serious patulin pollution, and the patulin pollution can enter food circulation along with the fruits and processed products thereof, thereby threatening the health of human bodies. Therefore, there is a need to develop effective toxin contamination control techniques. At present, the existing patulin removal technology mostly relies on the principles of physics (such as resin adsorption, ultrasonic crushing, ray irradiation and the like) or chemistry (such as oxidant, sulfydryl chemical agent and the like). Physical treatment, although simple to operate, can affect the nutritional value of food; the chemical reagent has good removal effect, but is easy to generate secondary pollution.
Disclosure of Invention
An object of the present invention is to provide TF-06 belonging to the genus Pseudomonas sp.
The preservation number of the Pseudomonas (Pseudomonas sp.) TF-06 is CGMCC NO. 16575.
The application of the above-mentioned Pseudomonas TF-06 or its culture solution or its bacterial suspension or its fermentation product in degrading patulin is also within the protection scope of the present invention.
Another object of the present invention is to provide a method for degrading patulin.
The method provided by the invention is to use the Pseudomonas (Pseudomonas sp.) TF-06 or the culture solution thereof or the bacterial suspension thereof or the fermentation product thereof to act on the patulin so as to realize the degradation of the patulin.
In the above process, the step of treating patulin with TF-06 of the genus Pseudomonas (Pseudomonas sp.) or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof is carried out by co-culturing TF-06 of the genus Pseudomonas sp or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof with patulin.
In the method, the co-culture system is a buffer solution with a pH value of 4 or a buffer solution with a pH value of 7 or apple juice.
In the above method, the concentration of the patulin in the co-culture system is more than 0.
In the above method, the concentration of the patulin in the co-culture system is specifically 0 to 500ppm and is not 0.
In the above method, the Pseudomonas TF-06 or the culture solution or the bacterial suspension thereof or the fermentation product thereof is a viable bacterium or a viable bacterium culture solution or a viable bacterium suspension or a viable bacterium fermentation product.
Experiments prove that the bacteria TF-06 has the effect of degrading patulin, is high in degradation efficiency and specificity, is easy to culture, high in propagation speed and easy to prepare, and lays a foundation for researching the patulin degradation technology.
Deposit description
The strain name is as follows: TF-06
Latin name: pseudomonas sp.
And (3) classification and naming: pseudomonas sp
The strain number is as follows: 16575
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 10 and 12 months in 2018
Registration number of the preservation center: CGMCC No.16575
Drawings
FIG. 1 shows the result of microscopic examination of TF-06 bacteria under a 100-fold microscope.
FIG. 2 shows the results of detection of TF-06 bacteria degrading patulin in different reaction systems.
FIG. 3 shows the growth of TF-06 bacteria at different patulin levels.
FIG. 4 shows the results of the detection of active bacteria of Aspergillus clavatus.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Patulin (Macklin, P816488).
Example 1 isolation and identification of bacteria TF-06 degrading patulin
1. Isolation of bacteria TF-06 degrading patulin
Taking pulp tissues at the focus of a rotten apple, mashing the pulp tissues in a sterile mortar, centrifuging the leached fruit juice, removing residual pulp tissues, performing gradient dilution on the obtained centrifuged fruit juice, coating the fruit juice on a plate culture medium containing 200ppm patulin for screening, and performing patulin degradation capability detection on the screened strain to find that one strain of bacteria (TF-06) has better degradation capability on the patulin.
TF-06 bacteria were serially streaked until a single bacterial colony was cultured.
2. Identification of bacteria TF-06 degrading patulin
1) Form of the composition
The TF-06 bacteria isolated in 1 above were examined microscopically under a 100-fold microscope.
The result is shown in figure 1, the left figure is the colony morphology of TF-06 bacteria on a solid LB plate, the colony is milky white, and the surface is smooth; the right image shows the microscopic morphology of the TF-06 bacterium under a common light microscope of 100 times, and it can be seen that the bacterium is in a ball-bar shape under a microscope of 100 times.
2) Molecular identification
And (3) carrying out 16s rDNA identification on the TF-06 bacteria, wherein the 16s rDNA sequence of the strain is sequence 1 in the sequence table. Comparing the strain with genbank sequences, finding that the strain has extremely high similarity with most bacteria of the Pseudomonas, and judging the strain to be Pseudomonas sp.
TF-06 bacteria are preserved in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC) No.16575 in 2018, 10 months and 12 days, and are classified and named as pseudomonas.
Example 2 use of TF-06 bacteria for degrading patulin
One, TF-06 bacteria degradation patulin in different reaction systems
Obtaining TF-06 bacteria: after overnight activation, TF-06 bacteria were transferred to 20ml LB (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L) medium in a volume ratio of 1:100, and cultured continuously in a shaker at 25 ℃ and 200rpm until the OD of the bacteria600Reaches 0.6(3 × 10)8cfu/ml), centrifuging and removing supernatant, and collecting precipitate to obtain TF-06 thallus.
Preparation of MES buffer System containing TF-06 bacteria at pH 4 the TF-06 cells were resuspended in 6ml of 50mM MES buffer (pH 4; 9.76g MES in 1L distilled water, pH 4 adjusted with NaOH) to give a MES buffer system containing TF-06 bacteria at pH 4, the TF-06 bacteria concentration being 1 × 109cfu/ml。
pH 7MES buffer System containing TF-06 bacteria was prepared by suspending the TF-06 cells in 50mM MES buffer (9.76g MES in 1L distilled water, pH 7 adjusted with NaOH) at pH 7 to give a pH 7MES buffer system containing TF-06 bacteria at a concentration of 1 × 109cfu/ml。
Preparation of a concentrated source apple juice system containing TF-06 bacteria comprises resuspending the above TF-06 bacteria in commercially available concentrated source apple juice (soft box packaged in 200 ml/box) to obtain concentrated source apple juice system containing TF-06 bacteria, wherein the concentration of TF-06 bacteria is 1 × 109cfu/ml。
pH 4MES buffer set: mixing the above 0.5ml withTF-06 bacteria pH 450 mM MES buffer system and 25 μ g patulin were mixed to form 500 μ l reaction system (TF-06 bacteria and patulin ratio is 2 × 10)7cfu is 1 mug), and is co-cultured for 72h at 25 ℃;
pH 7MES buffer group the 0.5ml of the TF-06 bacteria-containing pH 750mM MES buffer system was mixed with 25. mu.g of patulin to prepare a 500. mu.l reaction system (the ratio of TF-06 bacteria to patulin was 2 × 10)7cfu is 1 mug), and is co-cultured for 72h at 25 ℃;
the Huiyuan apple juice group is prepared by mixing the above 0.5ml Huiyuan apple juice system containing TF-06 bacteria with 25 μ g patulin to form 500 μ l reaction system (the ratio of TF-06 bacteria to patulin is 2 × 107cfu is 1 mug), and is co-cultured for 72h at 25 ℃;
CK groups corresponding to the respective experimental groups: mu.g of patulin was added to 0.5ml of MES buffer (pH 450 mM), 0.5ml of MES buffer (pH 750 mM) and 0.5ml of convergent apple juice, respectively, to form a 500. mu.l reaction system, which was mixed with each other and cultured together with the experimental group as a control group.
The culture products of the above groups were collected, and the patulin content in each group was determined by High Performance Liquid Chromatography (HPLC). The chromatograph used was a high performance liquid chromatograph from Waters corporation, USA, equipped with an autosampler (Waters2498), a two-way HPLC pump (Waters 1525), an ultraviolet detector (Waters 2487); the chromatographic column used was Shimadzu ODS reversed-phase column (C18 column, 5 μm, 250X 4.6 mm); in the detection, the mobile phase was acetonitrile/water at a flow rate of 1.0mL/min and the column temperature, detection wavelength and sample injection amount were 25 ℃, 276nm and 10. mu.L, respectively.
After the reaction of the sample, the reaction mixture was filtered through a 0.22 μm filter, and 200. mu.l of the reaction mixture was placed in a liquid phase vial and subjected to HPLC analysis.
Efficiency of degradation [ [ (C)0-C1)/C0]100% of C0Content of patulin in control group, C1The content of patulin is shown in the experimental group.
As shown in FIG. 2, it can be seen that the strain of bacteria has a better patulin degradation ability in MES buffer solutions under different pH conditions and in commercially available fruit juice; the residual amount of patulin in the solution after the bacterial strain is treated in the MES buffer solution with the pH value of 7 is lower than the detection limit, the degradation efficiency of the bacterial strain to the patulin in the MES buffer solution with the pH value of 4 is 46 percent, and the degradation efficiency of the bacterial strain to the patulin in the commercial fruit juice is 75 percent.
Second, the growth of TF-06 bacteria at different patulin contents
TF-06 bacteria are activated overnight and transferred into 20ml of LB culture medium according to the volume ratio of 1:100 to obtain TF-06 bacteria transfer culture solution, and then the following operations are carried out:
0ppm group: 20ml of TF-06 bacteria transfer culture solution is not added with patulin;
25ppm group: 20ml of TF-06 bacteria transfer culture solution is added with patulin with the final concentration of 25 ppm;
50ppm group: 20ml of TF-06 bacteria transfer culture solution is added with patulin with the final concentration of 50 ppm;
100ppm group: 20ml of TF-06 bacteria transfer culture solution is added with 100ppm patulin;
culturing the above culture solutions in a shaker at 25 deg.C and 200rpm, and respectively detecting OD of the strain of bacteria at different culture time (0h, 24h, 48h, 72h, and 96h)600(model 756 uv-vis spectrophotometer);
as a result, as shown in FIG. 3, it can be seen that the bacterial strain was able to grow normally at a patulin concentration of 100 ppm.
Third, degradation mechanism research of bacteria
Activating TF-06 bacteria overnight, transferring into 20ml LB medium at a ratio of 1:100, and culturing in a shaker at 25 deg.C and 200rpm until bacteria OD6000.6(3×108cfu/ml) to obtain a culture solution containing TF-06 live bacteria.
The culture solution containing the inactivated TF-06 bacteria is obtained by inactivating the culture solution containing the TF-06 live bacteria at 121 ℃ for 20 min.
And (3) 50mM MES buffer solution with pH 6 of active TF-06 bacteria, namely, 5000g of culture solution containing live TF-06 bacteria is centrifuged for 10min, supernatant is removed, and precipitates are suspended in 50mM MES buffer solution with pH 6 to obtain 50mM MES buffer solution with pH 6 of active TF-06 bacteria.
And (3) 50mM MES buffer solution with the pH value of 6 of the inactivated TF-06 bacteria, namely, the culture solution containing the inactivated TF-06 bacteria is centrifuged, the supernatant is removed, and the precipitate is resuspended in the 50mM MES buffer solution with the pH value of 6 to obtain the 50mM MES buffer solution with the pH value of 6 of the inactivated TF-06 bacteria.
Viable cell group (viable-cell group) 500. mu.l of 50mM MES buffer solution having pH 6 containing viable TF-06 bacteria and 25. mu.g patulin were mixed to form a 500. mu.l reaction system (ratio of TF-06 bacteria to patulin was 2 × 10)7cfu is 1 mug), and the mixture is cultured for 72 hours;
inactivated cell group (inactive cell group) 500. mu.l of 50mM MES buffer solution having pH 6 containing inactivated TF-06 bacteria and 25. mu.g patulin were mixed to form a 500. mu.l reaction system (ratio of TF-06 bacteria to patulin was 2 × 10)7cfu is 1 mug), and the mixture is cultured for 72 hours;
cell culture broth group (active-broth group): centrifuging 5000g of culture solution containing TF-06 live bacteria for 10min, collecting supernatant, mixing 500ul of supernatant with 25 mu g of patulin uniformly to form a 500 mu l reaction system, and culturing for 72 h;
an inactivated cell culture solution (inactive culture solution) is prepared by centrifuging culture solution containing inactivated TF-06 bacteria for 10min to collect supernatant, mixing 500ul supernatant with 25 μ g patulin, and forming 500 μ l reaction system (the ratio of TF-06 bacteria to patulin is 2 × 10)7cfu is 1 mug), and the mixture is cultured for 72 hours;
and (3) cell group CK: 50mM MES buffer (pH 6) was mixed with 25. mu.g patulin to prepare a 500. mu.l reaction system;
culture medium group CK: LB medium was mixed with 25. mu.g patulin to form a 500. mu.l reaction system.
The patulin content in different groups was determined (same as above);
as shown in FIG. 4, it can be seen that both the viable cells and the viable cell culture fluid of TF-06 bacteria can degrade patulin, and the degradation effect disappears after the cells and the culture fluid of TF-06 bacteria are inactivated at high temperature, indicating that the degradation effect of TF-06 bacteria on patulin is completed by the active substances in the cells or the fermentation fluid.
Sequence listing
<110> institute of plant of Chinese academy of sciences
<120> TF-06 bacterium for degrading patulin and application thereof
<160>1
<170>PatentIn version 3.5
<210>1
<211>1417
<212>DNA
<213> Pseudomonas sp
<400>1
ctacacatgc aagtcgagcg gatgaaggga gcttgctcct ggattcagcg gcggacgggt 60
gagtaatgcc taggaatctg cctggtagtg ggggataacg tccggaaacg ggcgctaata 120
ccgcatacgt cctgagggag aaagtggggg atcttcggac ctcacgctat cagatgagcc 180
taggtcggat tagctagttg gtggggtaaa ggcctaccaa ggcgacgatc cgtaactggt 240
ctgagaggat gatcagtcac actggaactg agacacggtc cagactccta cgggaggcag 300
cagtggggaa tattggacaa tgggcgaaag cctgatccag ccatgccgcg tgtgtgaaga 360
aggtcttcgg attgtaaagc actttaagtt gggaggaagg gcagtaagtt aataccttnc 420
tgttttgacg ttaccaacag aataagcacc ggctaacttc gtgccagcag ccgcggtaat 480
acgaagggtg caagcgttaa tcggaattac tgggcgtaaa gcgcgcgtag gtggttcagc 540
aagttggatg tgaaatcccc gggctcaacc tgggaactgc atccaaaact actgagctag 600
agtacggtag agggtggtgg aatttcctgt gtagcggtga aatgcgtaga tataggaagg 660
aacaccagtg gcgaaggcga ccacctggac tgatactgac actgaggtgc gaaagcgtgg 720
ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgtcg actagccgtt 780
gggatccttg agatcttagt ggcgcagcta acgcgataag tcgaccgcct ggggagtacg 840
gccgcaaggt taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg 900
tttaattcga agcaacgcga agaaccttac ctggccttga catgctgaga actttccaga 960
gatggattgg tgccttcggg aactcagaca caggtgctgc atggctgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgtaacg agcgcaaccc ttgtccttag ttaccagcac 1080
ctcgggtggg cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140
caagtcatca tggcccttac ggccagggct acacacgtgc tacaatggtc ggtacaaagg 1200
gttgccaagc cgcgaggtgg agctaatccc ataaaaccga tcgtagtccg gatcgcagtc 1260
tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgt gaatcagaat gtcacggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgctccagaa 1380
gtagctagtc taaccgcaag ggggacggta ccacgga 1417

Claims (8)

1. Pseudomonas sp TF-06 with a preservation number of CGMCC NO. 16575.
2. Use of Pseudomonas TF-06 or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof according to claim 1 for degrading patulin.
3. A method for degrading patulin, which comprises the step of acting the Pseudomonas TF-06 or a culture solution or a bacterial suspension thereof or a fermentation product thereof of claim 1 on the patulin to degrade the patulin.
4. The method of claim 3, wherein: the use of Pseudomonas TF-06 or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof according to claim 1 for the action of patulin with Pseudomonas sp is characterized in that TF-06 or a culture solution thereof or a bacterial suspension thereof or a fermentation product thereof according to claim 1 is co-cultured with patulin.
5. The method of claim 4, wherein:
the co-culture system is a buffer solution with the pH value of 4 or a buffer solution with the pH value of 7 or apple juice.
6. The method according to claim 4 or 5, characterized in that: the concentration of the patulin in the co-culture system is more than 0.
7. The method of claim 6, wherein:
the concentration of patulin in the co-culture system is specifically 0-500ppm and is not 0.
8. The method of claim 4, wherein:
the Pseudomonas (Pseudomonas sp.) TF-06 or the culture solution or the bacterial suspension or the fermentation product thereof are all live bacteria or live bacteria culture solution or live bacteria suspension or live bacteria fermentation product.
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