CN115247141B - Bacillus amyloliquefaciens strain A9 and application thereof - Google Patents

Bacillus amyloliquefaciens strain A9 and application thereof Download PDF

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CN115247141B
CN115247141B CN202210835029.6A CN202210835029A CN115247141B CN 115247141 B CN115247141 B CN 115247141B CN 202210835029 A CN202210835029 A CN 202210835029A CN 115247141 B CN115247141 B CN 115247141B
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bacillus amyloliquefaciens
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周礼芹
王小虎
朱绮霞
叶柳健
何双
韦圣博
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Guangxi Academy of Sciences
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Abstract

The invention relates to the technical field of microorganisms, in particular to a bacillus amyloliquefaciens strain A9 and application thereof, wherein the bacillus amyloliquefaciens strain has good inhibition effect on bacteria, protein separation is carried out on a fermentation product by utilizing a protein separation column Superdextm7510/300GL, gamma-glutamyltransferase with bacteriostasis effect on penicillium is obtained at the No. 1 peak, and diglyceride phosphate phosphodiesterase with good bacteriostasis effect on escherichia coli is obtained at the No. 5 peak; further application research is carried out on the metabolite, and experimental results show that the gamma-glutamyl transferase obtained by the No. 1 peak has good bacteriostatic effect in the aspect of food preservation.

Description

Bacillus amyloliquefaciens strain A9 and application thereof
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microorganisms, in particular to a bacillus amyloliquefaciens strain A9 and application thereof.
[ background of the invention ]
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a Bacillus, has high affinity with Bacillus subtilis, can produce a series of metabolites capable of inhibiting bacteria or fungi in the growth process, but the research on the metabolites is wide at present, and the specific bacteriostatic component is not deeply researched.
Although there are reports that bacillus amyloliquefaciens can produce bacteriostatic active substances at present, researches show that the components of the bacteriostatic substances in fermentation products of different bacillus amyloliquefaciens are very complex, and different components have different bacteriostatic effects on different bacteria; it is difficult to distinguish which substance has a corresponding inhibitory effect on the corresponding bacterium; moreover, not all Bacillus amyloliquefaciens bacteria have a bacteriostatic effect on all bacteria.
Because the content of the fermentation product of the bacillus amyloliquefaciens is low, the crude fermentation product is difficult to exert the bacteriostatic effect, so that the fermentation product needs to be further deeply researched to obtain specific bacteriostatic substances, the bacteriostatic substances are enriched to obtain more efficient bacteriostatic action, and more efficient products are provided for the fields of medical treatment, health care, food processing and the like in the later period and are efficiently utilized.
[ summary of the invention ]
In view of the above, it is necessary to deeply research the fermentation product of bacillus amyloliquefaciens, study the bacteriostatic mechanism, and efficiently utilize, ferment and enrich the bacteriostatic substance of bacillus amyloliquefaciens to obtain the bacteriostatic substance with high bacteriostatic effect.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the strain Bacillus amyloliquefaciens A9 has a preservation number of GDMCC NO.62173.
The invention also comprises a secondary metabolite of the strain Bacillus amyloliquefaciens A9.
The invention also comprises protease with bacteriostatic activity, which is obtained by secreting the strain Bacillus amyloliquefaciens A9, wherein the preservation number of the strain A9 is GDMCC NO.62173.
Further, the protease is gamma-glutamyl transferase; the gamma-glutamyltransferase inhibits the growth of penicillium.
Further, the protease is a glycerol phosphodiesterase; the glycerophosphodiesterase inhibits escherichia coli growth.
The invention also comprises the application of the protease with bacteriostatic activity in fruit preservation.
Further, the fruit is orange.
The invention also comprises a method for preparing the protease with bacteriostatic activity, which comprises the following steps: culturing the strain Bacillus amyloliquefaciens A9 on an LB culture medium, centrifuging, taking supernatant, freeze-drying, extracting by methanol, separating and purifying by a protein separation column Superdextm7510/300GL, and recovering a peak 1 and a peak 5 to obtain the protease with the antibacterial activity.
The invention has the following beneficial effects:
in the research process, the Bacillus amyloliquefaciens strain has an inhibiting effect on bacteria, but the fermentation product is complex, and the specific protein with the inhibiting effect is difficult to distinguish and obtain, and when the antibacterial effect of the strain Bacillus amyloliquefaciens A9 is researched, a subject group discovers that after protein separation is carried out by using a protein separation column Superdextm7510/300GL, gamma-glutamyltransferase with the inhibiting effect on penicillium is obtained at the No. 1 peak, and diglyceride phosphodiesterase with good antibacterial effect on escherichia coli is obtained at the No. 5 peak; further application research is carried out on the metabolite, and experimental results show that the gamma-glutamyl transferase obtained from the No. 1 peak has good bacteriostatic effect on food preservation (inhibiting penicillium aurantium).
[ description of the drawings ]
FIG. 1 is a diagram showing the morphology of the A9 strain of the present invention on a plate;
FIG. 2 is a phylogenetic tree diagram of the A9 strain of the present invention;
FIG. 3 is a graph showing the results of the separation of metabolites of A9 strain by Superdextm7510/300GL protein separation column;
FIG. 4 is a graph of the plate antagonism of Penicillium strain A9;
FIG. 5 is a photomicrograph of the results of the strain A9 confronting Penicillium;
FIG. 6 is a diagram showing the results of a plate inhibition experiment of a strain A9 secondary metabolite on Penicillium;
FIG. 7 is a diagram showing the results of bacteriostatic plate experiments on E.coli with the purified product of peak 5 of strain A9;
FIG. 8 is a graph showing the results of a bacterial inhibition plate experiment of the peak 1 purified product of the strain A9 against Penicillium;
FIG. 9 is a graph showing the results of inhibition of Penicillium aurantiacum by the strain A9;
FIG. 10 is a comparison plate experiment of the inhibition effect of Bacillus amyloliquefaciens JY-863 and the strain A9 in the experiment on Penicillium.
[ detailed description ] A
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.
Example 1:
screening of the strain Bacillus amyloliquefaciens A9:
bacillus amyloliquefaciens strain Bacillus amyloliquefaciens A9 with the preservation number of GDMCC NO.62173 is preserved in: guangdong province microbial strain preservation center, address: the preservation date of the No. 59 building 5 of the No. 100 college of the Pieli Zhou city is 2021, 12 months and 30 days.
The above strains were isolated in 2021 from rotten woolly oranges in the south China Guangxi market.
The separation method comprises the following steps: after suspension of the Or epidermis in sterile water, 100. Mu.l of the suspension were spread on a petri dish containing a selective medium, the dish was incubated at 28 ℃ for up to 1 day and colony forming units were then calculated. Each bacterial colony was picked from all tissue samples and transferred to growth medium. After acute observation and subculture, pure bacteria were isolated according to phenotype, morphology, colony color, size texture, opacity, height, edges and surface.
The isolated strains were morphologically classified and molecularly biologically identified as follows:
1. morphological Classification of strains
Morphological classification of strains:
as shown in FIG. 1, the morphological characteristics of the colonies observed for strain A9 in LB medium are: the bacterial colony is light yellow and opaque, has smooth surface and regular edges and is provided with bulges.
2. Molecular biological identification
Sequencing and verifying the strains: and (3) amplifying the gene sequence of the strain by adopting a universal primer, and sequencing the purified PCR product. Using Mega 6.0 to generate a multiple sequence alignment of A9 and other strain sequences, a phylogenetic tree of strains was constructed: as shown in FIG. 2, the strain A9 has 99.7% similarity with Bacillus sp, belongs to the genus Bacillus sp, and is considered to be more similar to Bacillus amyloliquefaciens species through morphological identification, so that the strain is named as Bacillus amyloliquefaciens.
Example 2:
isolation of the secondary metabolite of the strain A9:
culturing Bacillus amyloliquefaciens A9 in LB culture medium for 24h, centrifuging supernatant, freeze-drying for 48h, extracting with 50% methanol, separating and purifying with protein separation column Superdextm7510/300GL, and obtaining the experimental result shown in FIG. 3;
sequentially recovering products of 1-8 wave crests from left to right, recovering 500ul of each wave crest, freeze-drying and concentrating to 100ul, taking 5ul to perform bacteriostatic experiments on penicillium and escherichia coli:
1. bacteriostatic experiment of metabolite on penicillium:
the results of the plate confrontation experiments show that: the product with 8 peaks only has the inhibiting effect of peak 1 on penicillium, and the specific results are shown in figures 4-5:
FIG. 4 shows the plate confrontation experiment of strain A9 against Penicillium, in which the A is a control group; the figure B shows that the control group contains the whole plate, and the plate inoculated with the strain A9 has obviously reduced penicillium hyphae, thereby indicating that the strain A9 has inhibition effect on penicillium.
The test result of the purified product of peak 1 on the bacteriostatic flat plate of penicillium is shown in fig. 8, wherein the left side of the figure is a control group, and the right side is an experimental group, so that the experimental group has an obvious bacteriostatic zone, while the control group has no bacteriostatic zone; the average size of the inhibition zone of the experimental group is 12.5mm.
The result of microscopic photography of the opposite penicillium is shown in fig. 5, the left side of the figure is a control group, the right side of the figure is an experimental group, the penicillium hyphae of the control group in the figure grows strongly, and the A9 experimental group shows that the penicillium hyphae grows well, thereby the strain A9 has good inhibition effect on the penicillium.
Adding the secondary metabolite of the No. 1 peak into a PDA soft agar culture medium according to the adding amount of 0 (Control group), 20%, 40%, 60%, 80% and 100% of the volume of the PDA culture medium, then subpackaging the PDA culture medium containing the No. 1 peak secondary metabolite into 24-well plates, arranging 4-well plates for each concentration gradient, then inoculating penicillium into a flat dish containing cell extracts with different concentrations, incubating and culturing for 2d and 2d, and observing the growth condition of hyphae on different well plates.
As shown in fig. 6, it can be seen that when the metabolite addition amount of A9 reaches 20%, the effect of inhibiting penicillium is started: when 40% concentration was reached, the metabolite of A9 completely inhibited the growth of mycelium.
2. Bacteriostatic experiment of metabolite on escherichia coli:
the results of the plate bacteriostasis zone experiments show that: the product with 8 peaks only has a No. 5 peak which has an inhibiting effect on Escherichia coli, and the specific method is as follows:
(1) preparation of E.coli indicator plates:
symmetrically placing 2 Oxford cups on an LB solid culture medium plate, uniformly mixing Escherichia coli and an LB agar culture medium which is melted and cooled to 60 ℃, pouring 1ml of the mixture into the upper surface of the LB solid culture medium according to a ratio of 1;
(2) loading:
experimental groups: the recovered product from peak 5 was removed and added to the Oxford cup wells of the indicated plate of step (1).
Control group: equal amount of LB medium was added to the Oxford cup wells of the indicator plate of step (1).
(3) Culturing:
after the loading was complete, the E.coli indicator plates were incubated at 37 ℃ for 24h.
(4) And (3) processing an experimental result:
after the culture is finished, the flat plate is photographed and observed, the indication result of the flat plate is shown in figure 7, the left side in the figure is a control group, the right side in the figure is an experimental group, and therefore the experimental group has an obvious transparent inhibition zone, and the control group does not have an inhibition zone; the average size of the inhibition zone of the experimental group is 13.17mm.
Example 3:
as can be seen from example 2, peak 1 had an inhibitory effect on Penicillium and peak 5 had an inhibitory effect on Escherichia coli; thus, the recovered products from peak 1 and peak 5 were further analyzed as follows:
mass spectrometry was performed for peak 1 and peak 5 using Q active Plus liquid chromatography-mass spectrometry system from Thermo. The samples were separated by a nanoliter flow rate liquid phase UltiMate 3000RSLCnano system. The peptide fragment sample was dissolved in loading buffer, aspirated by an autosampler, bound to a C18 capture column (3 μm,
Figure BDA0003747456020000051
100 μm.times.20 mm) and then eluted onto an analytical column (2 μm, in each case>
Figure BDA0003747456020000052
75 μm × 150 mm). The% by using two mobile phases (mobile phase A:3% DMSO,0.1% 2 O and mobile phase B:3% DMSO,0.1% formic acid,97% ACN) establishing an analytical gradient. The flow rate of the liquid phase was set at 300nL/min. Mass spectrometry DDA mode analysis included one MS full scan per scan cycle (R =70k, agc =3e6, max it =20ms, scan range =350-1800 m/z), followed by 15 MS/MS scans (R =17.5k, agc =2e5, max it = 100ms). The HCD collision energy was set at 28. The screening window of the quadrupole was set to 1.6Da. The dynamic exclusion time for repeated ion acquisitions was set at 35s.
Sequencing after the peptide fragments of the No. 1 peak and the No. 5 peak are broken to find that: peak 1 has high homology with gamma-glutamyltransferase; peak 5 has high homology with glycerophosphodiesterases. The method comprises the following specific steps:
table 1 sequencing results for Peak protein
N terminal Description Conf Sequence
1 Gamma-glutamyltransferase 99.00000095 DGMVATAHPLASQIGADVLK
1 Gamma-glutamyltransferase 99.00000095 ENGTFTGVADSSR
1 Gamma-glutamyltransferase 99.00000095 GFPIDSVLADAISDYKDK
1 Gamma-glutamyltransferase 99.00000095 GLLNPDYINAR
1 Gamma-glutamyltransferase 99.00000095 NGAAIGVNLK
1 Gamma-glutamyltransferase 99.00000095 TAPPPSSGGVFLLQMLNLLDDFK
1 Gamma-glutamyltransferase 99.00000095 TIIDSRERA
1 Gamma-glutamyltransferase 99.00000095 AFAGDPEFVNIPLKGLLNPDYINAR
Sequencing results for Peak protein No. 5 in Table 2
N terminal Description Conf Sequence
2 Glycerophosphodiesterphosphodiesterase 99.00000095 ALNAQNVR
2 Glycerophosphodiesterphosphodiesterase 99.00000095 APETYPGMEEK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 DHTLAEIQK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 HANYYIETK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 LLNWGVTGVFTNYADIFQK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 LPAVQLLEAEQMTSMTDADLTDIK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 MKADYIELDLQMTK
2 Glycerophosphodiesterphosphodiesterase 98.8499999 VPTLDDVLK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 ADYIELDLQMTK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 LPAVQLLEAEQMTSMTDADLTDIK
2 Glycerophosphodiesterphosphodiesterase 99.00000095 LPAVQLLEAEQMTSMTDADLTDIK
Example 4:
the inhibition effect of the strain A9 on oranges is as follows:
inhibition experiments on penicillium aurantium:
inoculating penicillium hyphae to the epidermis of the orange by using an inoculating needle, and inoculating 6 oranges in total, wherein 3 of the penicillium hyphae are experimental groups, 3 of the penicillium hyphae are control groups, and the control groups and the experimental groups are respectively treated as follows:
experimental groups: spraying suspension containing No. 1 peak of A9 strain on the surface of the orange, and repeating the dropwise application once every 72h, wherein the dropwise application amount is 10ul, and the content of the effective component in the suspension is 0.3% by mass.
Control group: distilled water is dripped to the surface of the orange, and the dripping is repeated every 72 hours, wherein the dripping amount is 10ul.
Culturing the orange at room temperature, and observing the growth of mould plaque on the surface of the orange after 15 days.
The experimental results are shown in fig. 9, and it can be seen from the figure that the left side is the orange of the control group, and the right side is the orange of the experimental group, and it is obvious that the plaque size of the control group is significantly larger than that of the experimental group, thereby demonstrating that the peak 1 recovered product (gamma-glutamyltransferase) of the strain A9 of the present application has a significant inhibitory effect on penicilliosis of the orange.
Example 5:
the inhibition effect of the bacillus on the penicillium is as follows:
inhibition assay for penicillium:
experimental groups: will 10 6 Filtering the bacillus culture solution with the concentration of 0.22um, adding the bacillus secondary metabolite into a penicillium PDA soft agar culture medium containing dug holes according to 100ul, incubating and culturing 2d and 2d, and observing the growth condition of hyphae.
Negative control group: 100ul of distilled water was added to the medium containing the excavated Penicillium PDA soft agar to incubate and culture 2d and 2d, and then the growth of hyphae was observed.
Positive control group: adding 100ul A9 secondary metabolite or 100ul106 bacteria liquid into Penicillium PDA soft agar culture medium containing dug holes, incubating and culturing 2d and 2d, and observing hypha growth.
The experimental results are shown in fig. 10, and it can be seen from the figure that the right side is a bacillus amyloliquefaciens TY-863 filtrate control group, the middle is an A9 bacterial solution experimental group, the left side is an A9 filtrate experimental group, the transparent circle of the experimental group can be obviously seen, and the control group has no transparent circle, so that not all bacillus amyloliquefaciens have inhibition effect on penicillium, but the strain A9 of the present application has obvious inhibition effect on orange penicillium.
In conclusion, the Bacillus amyloliquefaciens A9 strain and the metabolite thereof have obvious inhibition effects on penicillium and escherichia coli, and simultaneously have a protection effect on orange penicilliosis, and analysis on the metabolite shows that the metabolite of the strain A9: the gamma-glutamyltransferase has good inhibition effect on penicillium; the diacylglycerol phosphodiesterase has an inhibiting effect on escherichia coli, so that the strain can be widely applied to: the strain is widely applied in various fields of plant protection, food preservation and the like, and is an excellent strain suitable for plant protection and food preservation.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (3)

1. Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens ) Strain A9 having the accession number GDMCC No.62173.
2. The Bacillus amyloliquefaciens of claim 1 (C: (C.))Bacillus amyloliquefaciens ) The application of the strain A9 in inhibiting escherichia coli and penicillium in vitro.
3. The Bacillus amyloliquefaciens of claim 1 (C: (C.))Bacillus amyloliquefaciens ) The application of the strain A9 in orange preservation.
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