CN115010737A - Method for collecting volatile components in microbial fermentation process and application - Google Patents
Method for collecting volatile components in microbial fermentation process and application Download PDFInfo
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- CN115010737A CN115010737A CN202210690665.4A CN202210690665A CN115010737A CN 115010737 A CN115010737 A CN 115010737A CN 202210690665 A CN202210690665 A CN 202210690665A CN 115010737 A CN115010737 A CN 115010737A
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- volatile components
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- volatile
- bacillus
- components
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Abstract
The invention provides a method for collecting volatile components in a microbial fermentation process and application thereof, belonging to the technical field of biology. The invention puts the liquid culture medium inoculated with the microorganism into a culture container for culture; and (3) after the culture is finished, moving the culture container into a heater, heating and balancing at 29-35 ℃, and then collecting volatile components by adopting a solid phase microextraction technology. According to the invention, the heating temperature is adjusted, so that the obtained volatile components do not contain volatile components generated by the culture medium, and the sensitivity and accuracy of microbial metabolic component detection are ensured. According to the invention, the volatile components of the Bacillus beiLeisi LT1 collected by the method are found for the first time, and the unique components of the Bacillus beiLeisi LT1 and the unique components of the Bacillus beiLeisi LT1 have the inhibiting effect on various plant pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for collecting volatile components in a microbial fermentation process and application of the volatile components.
Background
MVOCs (volatile organic compounds) are generated by metabolism in the biological growth and development process, are closely related to the growth activity of the compounds, have volatile properties at normal temperature and normal pressure, are most lipophilic substances, have low water solubility, and can be rapidly volatilized at 20 ℃ and 0.01Pa to enter a gas phase state. Research shows that MVOCs mainly have the following functions: (1) as intracolony and interpenomic signaling species; (2) cell-to-cell signaling substances; (3) possible carbon release channels; (4) growth promoting or inhibiting factors. Microorganisms can produce a large number of volatile substances of various kinds and functions. Statistically, it has been found that the bacteria produce more than 346 kinds of volatile organic compounds, mainly including olefin, alcohol, ketone, terpene, benzene, pyrazine, acid, ester, etc. While the 250 identified volatile organic species produced by fungi were primarily alcohols, benzenes, aldehydes, olefins, acids, esters, ketones, and the like.
Research on MVOCs has been conducted in a closed environment. MVOCs are relatively complex in composition and often contain multiple substances of different chemical structures and different polarities. The first step of the research on MVOCs which is of crucial importance is the acquisition of as much and as complete as possible of the volatile substance components during the metabolism of the microorganisms. Therefore, the desired sampling technique for MVOCs should be efficient, suitable for collecting MVOCs components of different chemical structures and different polarities, and avoid sample contamination. The techniques used to date for sampling MVOCs are mainly liquid-liquid extraction (LLE), Steam Distillation (SD), Simultaneous Distillation Extraction (SDE), purge and trap (P & T), Supercritical Fluid Extraction (SFE) and Solid Phase Microextraction (SPME). Of these, LLE, SD and SDE are classical volatile organic compound collection techniques, but these techniques not only require multiple steps, use large amounts of organic solvents, take long time, but also some unstable VOC components (such as olefinic, ester and unsaturated VOC components) decompose or degrade during heat extraction or distillation. Nowadays, P & T, SFE and SPME are considered to be an advanced and environment-friendly technology for collecting volatile organic compounds, especially SPME, which is characterized by high sensitivity, short preparation time, fast collection speed, etc. However, at present, the above-mentioned techniques still have the problems of long time consumption, complex instrument and equipment requirements, high cost and large operation difficulty, and are very unfavorable for the rapid detection of volatile components (MVOCs) of microorganisms, and the sample extraction pretreatment also destroys the original volatile component composition of the sample, and part of the volatile components are subjected to physicochemical property change, and part of the volatile components are released in a culture medium, so that errors occur in the result, and the identification of the volatile components is very unfavorable. Therefore, a collection and treatment method capable of ensuring the stability of gas components in the microbial metabolism process is needed to realize the detection accuracy.
Disclosure of Invention
In view of this, the present invention provides a method for collecting volatile components in a microbial fermentation process, which can ensure the stability of gas components in the microbial metabolism process, thereby realizing the accurate detection of microbial metabolism components.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for collecting volatile components in a microbial fermentation process, which comprises the following steps:
placing the liquid culture medium inoculated with the microorganisms into a culture container for culture; and (3) after the culture is finished, heating the culture container at 29-35 ℃ for balancing, and then collecting volatile components by adopting a solid phase microextraction technology.
Preferably, the temperature of the heater is 30-32 ℃.
Preferably, after the culture is finished, the culture container is kept still at room temperature for 1.5-2.5 h and then heated.
Preferably, the heating balance time is 8-12 min.
Preferably, the microorganism comprises bacillus belgii LT 1.
The invention also provides the application of the method in the separation or analysis of volatile components generated in the microbial metabolism process.
The invention also provides the volatile components of the Bacillus belgii LT1 collected by the method.
Preferably, the components include ethylboronic acid, methylketene, vinyl acetate, 2-butanone, isobutyric acid, 2-decanone, 2-dodecadione, 2-tetradecanone, 2-decanol, methylbutanoic acid, isovaleric acid, 5-methyl-2-hexanone, 2-terfenadone, 2-undecanone, 2-heptanone, 6-methyl-2-heptanone, 2-nonanol, and 1-iodododecane.
The invention also provides application of the Bacillus belgii LT1 volatile component in inhibiting phytopathogens.
Preferably, the plant pathogenic bacteria comprise largehead atractylodes rhizome leaf spot, coptis southern blight, tobacco root rot, panax japonicus circular spot, dwarf lilyturf root rot, pinellia ternate sclerotinia, tobacco brown spot, tobacco anthracnose and paris polyphylla gray mold.
Compared with the prior art, the invention has the following beneficial effects:
the invention has high precision and small experimental error, avoids the problem that impurity gas is possibly generated in the treatment process and volatile components in the culture medium are released, can ensure the stability of gas components in the microbial metabolism process, thereby realizing the precise detection of the microbial metabolism components, improving the detection sensitivity, effectively knowing the change condition of the volatile components of the microbes in the metabolism process and providing technical support for the comprehensive utilization and research of the microbes.
The gas collection device is simple in gas collection operation, low in cost and short in operation time, only a simple headspace bottle is needed for collecting gas, and the gas collection device is suitable for large-scale high-flux detection of volatile components of microorganisms and convenient for rapid determination and analysis.
Bacillus belgii LT1 (Latin name Bacillus velezensis LT1), depository: china Center for Type Culture Collection (CCTCC) for short, the address of a preservation unit: wuhan, Wuhan university, China, accession number: CCTCC NO: m2020023, preservation date is 2020, 1, 7.
Drawings
FIG. 1: bacterial inhibition spectrum of volatile product VOCs of strain LT 1;
FIG. 2: the VOCs generated by the strain LT1 has the effect of inhibiting the growth of fungal filaments of different pathogenic bacteria;
FIG. 3: the volatile components generated by the strain LT1 have the effect of preventing and treating the southern blight of coptis;
FIG. 4: the single volatile component produced by the strain LT1 has the effect of inhibiting hypha of sclerotium rolfsii;
FIG. 5: the influence of the volatile components of the LT1 strain on the hypha morphology of sclerotium rolfsii;
FIG. 6: the mycelium ultrastructure of the sclerotium rolfsii is treated by the volatile components of LT1 strain.
Detailed Description
The invention provides a method for collecting volatile components in a microbial fermentation process, which comprises the following steps: placing the liquid culture medium inoculated with the microorganisms into a culture container for culture; and (3) after the culture is finished, moving the culture container into a heater, heating and balancing at 29-35 ℃, and then collecting volatile components by adopting a solid phase microextraction technology.
The invention prepares the volatile components generated in the microbial metabolic process: firstly, inoculating microbial strains into a liquid culture medium, then placing the liquid culture medium into a culture container, sealing and then shaking for culture. The invention can promote the microorganisms to generate a large amount of volatile components through sealed shake culture, and ensures that the variety of the collected volatile components is complete.
The culture vessel of the present invention is preferably a chromatography headspace bottle, more preferably a screw-top chromatography headspace bottle.
The addition amount of the liquid culture medium is preferably 35-50% of the volume of the culture container.
The sealing mode of the invention is preferably sealing by adopting a paraffin sealing film.
The invention collects the volatile components generated in the microbial metabolism process: and taking out the headspace bottle after the culture is finished from the incubator, moving the headspace bottle into a heater, heating and balancing at 29-35 ℃, and then collecting volatile components by adopting a solid phase microextraction technology.
After the sealed shake culture is finished, the taken-out headspace bottle is preferably kept stand at room temperature for 1.5-2.5 h, and then is transferred to a heater for heating balance. The standing time is more preferably 2 hours.
The temperature of the heater is preferably 30-32 ℃. According to the invention, the temperature of the heater is limited, so that the generation of impurity gas in the treatment process can be avoided, the gas components in the microbial metabolism process are stable, the volatile components of the culture medium are not contained, and the accurate collection of the microbial metabolism components is realized.
The heating balance time is 8-12 min, preferably 9-10 min.
According to the invention, before solid-phase microextraction, the fiber head is aged for 3-8 min at 245-255 ℃, and more preferably aged for 5min at 250 ℃ for subsequent extraction.
The invention adopts solid phase microextraction technology to collect volatile components generated in the microbial metabolism process after heating balance treatment: and inserting the fiber head into a headspace bottle, absorbing for 15-25 min while swirling without contacting a liquid sample, then inserting the fiber head into a chromatographic sample inlet for analyzing for 5-6 min, and performing separation detection by adopting gas chromatography. The solid phase micro-extraction time is preferably 20-22 min.
The method for collecting volatile components in the microbial fermentation process is suitable for various microorganisms, and most preferably bacillus beiLeisi LT 1.
The invention also provides the application of the method in the separation or analysis of volatile components generated in the microbial metabolism process.
The invention analyzes the volatile components generated in the microbial metabolic process: and performing mass spectrum qualitative and quantitative analysis on the metabolites of the sample to be detected based on the NIST database. Opening a sample lower machine profile file by MassHunter quantitative software, integrating and correcting chromatographic peaks, wherein the peak Area (Area) of each chromatographic peak represents the relative content of a corresponding substance, and finally exporting integral data of all chromatographic peak areas for storage.
The invention adopts GC-MS to analyze the acquired original data file. First, peak extraction is carried out by MassHunter software (Agilent), information such as mass-to-charge ratio, retention time and peak area of a characteristic peak is obtained, and then statistical analysis is carried out on data. The preparation and arrangement of the original data mainly comprises the following steps: (1) calculating a retention index; (2) filtering the single Peak, and only retaining Peak area data with a single null value of no more than 50% or with null values of no more than 50% in all groups; (3) and (6) carrying out data standardization processing.
According to the invention, a liquid culture medium without microorganisms is set as a blank control, and volatile components measured by a control group are removed from volatile components of microorganisms measured by a sample to be measured in an experimental group, namely the volatile components unique to the microorganisms.
The volatile components of the Bacillus beijerinckii LT1 are collected by the method, and the volatile components of the Bacillus beijerinckii LT1 are measured and analyzed.
The invention detects 18 belonged exclusively to Bacillus belgii LT1, including 10 ketones, 4 organic acids, 2 alcohols, 1 ester and 1 alkane. Specifically, ethyl boric acid, methyl ketene, vinyl acetate, 2-butanone, isobutyric acid, 2-decanone, 2-decadione, 2-tetradecone, 2-decanol, methyl butyric acid, isovaleric acid, 5-methyl-2-hexanone, 2-terfenazone, 2-undecanone, 2-heptanone, 6-methyl-2-heptanone, 2-nonanol and 1-iodododecane. And it was found for the first time that Bacillus beleisi can produce vinyl acetate, 5-methyl-2-hexanone, 2-butanone and 1-iodododecane.
The invention provides application of the Bacillus belgii LT1 volatile component in inhibiting phytopathogens. The plant pathogenic bacteria include Atractylodes macrocephala leaf spot, southern blight of Coptis, tobacco root rot, Panax japonicus circular spot, radix Ophiopogonis root rot, pinellia ternate sclerotinia, tobacco brown spot, tobacco anthracnose and Paris polyphylla gray mold.
The invention discovers that the suppression efficiencies of volatile components generated by the Bacillus belgii LT1 strain on sclerotium rolfsii and botrytis cinerea are respectively 90.42% and 91.86% to the maximum, and the suppression efficiencies on leaf spot of white atractylodes, sclerotinia sclerotiorum of pinellia tuber, root rot of dwarf lilyturf tuber, colletotrichum gloeosporioides, root rot of tobacco, root rot of panax japonicus, and alternaria alternata are all 70-80%.
Bacillus belgii LT1 (Latin name Bacillus velezensis LT1), depository: china Center for Type Culture Collection (CCTCC) for short, the address of a preservation unit: wuhan, Wuhan university, China, accession number: CCTCC NO: m2020023, preservation date is 2020, 1, 7.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In a specific example, the strain used is bacillus belgii LT1, accession number: CCTCC NO: m2020023, the preservation date is 1 month and 7 days 2020; the culture medium is LB liquid culture medium; the instrument used was GC-MS/MS7890B-7000 DAgilent.
Example 1
The embodiment provides a method for collecting volatile components in a microbial fermentation process, which comprises the following steps:
1. 20 mu L of pure cultured Bacillus belgii LT1 fermentation liquid is inoculated into 10mL of liquid culture medium, the inoculated liquid culture medium is placed into a sterile screw-top chromatographic headspace bottle with the capacity of 20mL, the bottle mouth is screwed down, the bottle is sealed by paraffin for 5 circles, and the mixture is placed at 28 ℃ and 180 r/min for culture for 1 week.
2. The chromatographic headspace bottle of step 1 was removed and allowed to stand at room temperature for 2 hours. Before sample extraction, a fiber head is aged for 5min at 250 ℃, the sample is moved to a heater, the sample is heated and balanced for 10min at 30 ℃, the fiber head is inserted into a bottle and does not contact with a liquid sample, the liquid sample is sucked for 20min while being swirled, the fiber head is inserted into a sample inlet and is analyzed for 5min, and gas chromatography and mass spectrometry are carried out for separation.
Example 2
This example differs from example 1 in that: the amount of the liquid medium used was 8 mL.
Example 3
This example differs from example 1 in that: standing at room temperature for 1.5 h.
Example 4
This example differs from example 1 in that: heating and balancing for 9min at 31 ℃.
Example 5
This example differs from example 1 in that: the heater is heated and balanced at 32 ℃ for 11 min.
Example 6
This example differs from example 1 in that: the sample was sucked up for 18min while vortexing.
Comparative example 1
This comparative example differs from example 1 in that: heating and balancing for 10min at 60 ℃ by using a heater.
Comparative example 2
The comparative example differs from example 1 in that: the liquid medium was not inoculated with the strain.
Comparative example 3
The present comparative example differs from comparative example 1 in that: the liquid medium was not inoculated with the strain.
Example 7
This example was conducted to analyze the volatile components obtained in example 1 and comparative examples 1 to 3 in comparison.
The production of volatile components (VOCs) by Bacillus belgii LT1 was determined by gas chromatography-mass spectrometry (GC-MS). Working conditions of the GC-MS gas chromatograph: the method adopts a non-shunting sample introduction mode, helium is taken as carrier gas, a chromatographic column DB-5MS (30m multiplied by 0.25mm multiplied by 0.25 mu m), the flow rate is 1.0mL/min, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, the transmission line temperature is 280 ℃, the electron energy is 70eV, and the detection is carried out by adopting a full scanning mode, wherein the detection range is 50-600 amu. The original data file obtained by GC-MS analysis is subjected to peak extraction by MassHunter software (Agilent) to obtain information such as mass-to-charge ratio, retention time and peak area of characteristic peaks, and the differences of volatile components in example 1 and comparative examples 1-2 are statistically analyzed, and the results are shown in tables 1-2.
1. The samples taken from example 1 and comparative example 2 were subjected directly to GC-MS analysis with 3 replicates in the test set-up and the results are shown in table 1.
TABLE 1 identification of total volatile Components
As can be seen from Table 1, 18 species uniquely belonging to Bacillus belgii LT1 were detected in example 1, including 10 species of ketones, 4 species of organic acids, 2 species of alcohols, 1 species of esters and 1 species of alkanes. Specifically, ethyl boric acid, methyl ketene, vinyl acetate, 2-butanone, isobutyric acid, 2-decanone, 2-dodecadione, 2-tetradecanone, 2-decanol, methyl butyric acid, isovaleric acid, 5-methyl-2-hexanone, 2-terfenadone, 2-undecanone, 2-heptanone, 6-methyl-2-heptanone, 2-nonanol, and 1-iodododecane.
2. The samples taken from comparative example 1 and comparative example 3 were subjected directly to GC-MS analysis and the test set up was 3 replicates and the results are shown in table 2.
TABLE 2 identification of total volatile Components
As can be seen from Table 2, there is no difference between comparative example 1 and comparative example 3 in the volatile components in 62, which indicates that the volatile components in the fermentation process of the strain cannot be accurately detected after heating treatment at 60 ℃, and interference of the components of the culture medium occurs.
Example 8
This example demonstrates the inhibitory effect of volatile components produced by bacillus belgii LT1 on various phytopathogens.
1. Adopting an inverted dish method: taking fresh activated LT1 thallus, uniformly coating on a solid LB plate, inoculating the cake (phi is 5mm) of the plant pathogenic fungi to be detected to the center of a PDA plate, buckling the plate in a 2-dish manner, sealing the plate with a sealing film, culturing at 28 ℃, and setting a control. Wherein largehead atractylodes rhizome leaf spot (P.exigua), southern blight of coptis (S.rolfsii), tobacco root-rot pathogen (F.oxysporum), panax japonicus circular spot (M.acetina) and radix ophiopogonis root-rot pathogen (F.acettina) are cultured at 28 ℃, sclerotinia pinea (S.sclerotiorum), alternaria alternata (A.alternata), anthracnose pathogen (C.micola) and botrytis cinerea (B.cinerea) are cultured at 22 ℃, the colony diameter is measured when the control strains are full of dishes, 3 repeats are set for each strain, and the hypha inhibition rate is calculated. Hypha inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%. The results are shown in FIGS. 1-2.
As can be seen from FIGS. 1-2, the maximum inhibition efficiency of volatile components generated by the Bacillus belgii LT1 strain on sclerotium rolfsii and botrytis cinerea is 90.42% and 91.86%, respectively, and the inhibition efficiency on leaf spot of white atractylodes, sclerotinia sclerotiorum, rhizoctonia rot of dwarf lilyturf root, colletotrichum nicotianae, rhizoctonia solani, physalospora japonica and alternaria alternata is 70-80%.
2. Preparing a solid LB culture medium plate by using a culture dish with the diameter of 7cm, coating 20 mu L of Bacillus belgii LT1 fermentation liquor, sealing, and culturing for 24h in a constant-temperature incubator at 28 ℃. Respectively placing 10, 15 and 20 Bacillus LT1 LB plates cultured in a dish cover on the bottom layer of a dryer with the diameter of 300mm, potting coptis chinensis on the upper layer, selecting young leaves, inoculating a sclerotium rolfsii LC1 cake (phi is 3mm), covering the dryer with a cover, and sealing with a sealing film. The lesion diameter was measured after 72h incubation at 28 ℃ and the lesion inhibition was calculated and the experiment repeated 3 times. LB plates of 10, 15 and 20 non-inoculated B.belief LT1 with the lid removed were placed on the bottom of the desiccator as controls. Control effect (%) - (control lesion diameter-treatment lesion diameter)/control lesion diameter × 100%. The results are shown in FIG. 3(A: lesion diameter; B: inhibition rate).
As can be seen from FIG. 3, the volatile components produced by the strain LT1 were effective in inhibiting southern blight of Coptis, and the inhibitory effect increased with the increase in the concentration of the volatile components.
Example 9
For the 18 volatile components identified in example 7, which are unique to bacillus belgii LT1, 12 volatile organic compounds were purchased as individual products: ethyl boric acid, methyl ketene, vinyl acetate, 2-butanone, 2-decanone, 2-dodecadione, methyl butyric acid, isovaleric acid, 5-methyl-2-hexanone, 2-undecanone, 2-heptanone, 6-methyl-2-heptanone.
The inhibition effect of 12 single products on sclerotium rolfsii determined in volatile organic compounds generated by the Bacillus belgii LT1 strain was determined by a two-cell method. Two separate dishes of culture medium were prepared, the same PDA medium was added to each side, the activated fresh cake of southern blight strain LC1 (phi 5mm) was inoculated into the center of one side of the dish, a sterilized small filter paper (phi 5mm) was placed in the center of the other side of the dish, and 20. mu.L of each volatile organic substance was dropped onto the filter paper. After sealing, the colony diameter was measured after incubation in a constant temperature incubator at 28 ℃ for 72h, and each individual experiment was repeated 3 times. Two dishes with 20 mu L of sterile water dropped on the filter paper sheets are used as a control group, the colony diameter of each southern sclerotium treatment bacterium is measured, and the inhibition effect of each volatile substance single product on the growth of the mycelium of the southern sclerotium rolfsii LC1 is calculated. Hypha inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%. The results are shown in FIG. 4.
As can be seen from FIG. 4, most of the 12 types of volatile organic compounds tested exhibited a good inhibitory effect against southern blight fungus LC 1.8 of the ketone volatile components except 6-methyl-2-heptanone have better inhibition effect on hypha growth of sclerotium rolfsii, the most effective ketone volatile component in the ketone volatile components is 2-decadione, the inhibition rate reaches 81.67%, and the inhibition rate is 2-undecanone, 80.08%, 75.69%, 68.13%, 58.96% and less than 40% of other 3 ketone volatile components, wherein the lowest ketone volatile component is 6-methyl-2-heptanone, and the inhibition rate is 9.56%. The organic acid and organic acid ester volatile components have poor inhibition effect on the growth of hyphae of the southern sclerotium rolfsii, 2-methylbutyric acid is the best inhibition effect on the growth of the hyphae of the southern sclerotium rolfsii in the organic acid volatile components, the inhibition effect is only 32.27%, and the inhibition rate of other volatile components is lower than 20%. The volatile component which belongs to the Bacillus belgii LT1 and can be measured by the invention is the component which plays the inhibition effect of the Bacillus belgii LT 1.
Selecting normal sclerotium rolfsii LC1 hyphae and sclerotium rolfsii hyphae stressed by volatile components for 5 days, and observing the hypha ultrastructure by adopting a scanning electron microscope and a transmission electron microscope.
And (3) observing the mycelium state of the southern blight fungus by a scanning electron microscope: selecting mycelium, rapidly adding glutaraldehyde electron microscope stationary liquid, fixing for 2 hr, and rinsing the mycelium with 0.1M phosphate buffer PB (pH 7.4) for 3 times. Gradient dehydrating the mycelium with alcohol, drying by critical point method, and spraying gold for 30 s. The morphology of the treated and control hyphae was observed by scanning electron microscopy (HITACHI Regulus8100) and photographed, and the results are shown in FIG. 5 (A: hyphal morphology of the strain LC1 of southern sclerotium and B: hyphal morphology of the strain LC1 under stress of volatile components).
As can be seen from FIG. 5, hyphae of the southern blight strain LC1 were seriously wrinkled under the stress of the volatile components of the Bacillus LT1 strain, and were closely attached to the surface of the medium, some of the hyphae were broken on the surface, cytoplasm was leaked, and the hyphae were fragmented, and were degraded.
The hypha form of the sclerotium rolfsii is observed by a transmission electron microscope: selecting above sclerotium rolfsii and control mycelium, adding glutaraldehyde solution and 1% osmium tetroxide electron microscope fixing solution, fixing at 4 deg.C for 2 hr, adding 0.1M phosphate buffer PB (pH 7.4) into the mycelium, and rinsing. Alcohol gradient dehydration and acetone rinsing. The sample is placed in 812 embedding medium for embedding, sliced into an 80nm ultrathin section by a slicer, dyed in a dark place by 2% uranium acetate saturated alcohol solution, dyed in a carbon dioxide-free manner by 2.6% lead citrate solution, cleaned, dried overnight at room temperature, observed in a transmission electron microscope (HITACHI HT7800) for hypha morphology and photographed, and the result is shown in FIG. 6 (A: transverse section of healthy southern blight hypha, B: longitudinal section of healthy southern blight hypha, C: transverse section of southern blight hypha treated by volatile components of Bacillus LT1 strain, F: longitudinal section of southern blight hypha treated by volatile components of Bacillus LT1 strain, CM: cell membrane, CW: cell wall, M: mitochondria, N: cell nucleus, N: nucleolus and V: vacuole).
As can be seen from FIG. 6, the hyphal cell wall of the Bacillus LT1 is thickened obviously under the stress of volatile components, the cytoplasm is concentrated and is dyed deeply, a large amount of white granular substances appear, the nuclear structure is unclear, no obvious nuclear membrane boundary is seen, the vacuole is highly fragmented, the mitochondria are reduced obviously, and the dead cells are increased obviously.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for collecting volatile components in a microbial fermentation process is characterized by comprising the following steps:
placing the liquid culture medium inoculated with the microorganisms into a culture container for culture; and (3) after the culture is finished, heating the culture container at 29-35 ℃ for balancing, and then collecting volatile components by adopting a solid phase microextraction technology.
2. The method of claim 1, wherein the heater temperature is 30-32 ℃.
3. The method according to claim 1, wherein the culture vessel is left at room temperature for 1.5 to 2.5 hours and then heated after the completion of the culture.
4. The method of claim 1, wherein the heating equilibration time is 8-12 min.
5. The method according to any one of claims 1 to 4, wherein the microorganism comprises Bacillus belgii LT 1.
6. Use of the method of any one of claims 1 to 4 for the separation or analysis of volatile components produced by microbial metabolic processes.
7. The volatile fraction of Bacillus belgii LT1 collected by the method of claim 5.
8. The volatile component of claim 7, wherein the component comprises ethylboronic acid, methylketene, vinyl acetate, 2-butanone, isobutyric acid, 2-decanone, 2-dodecadione, 2-tetradecanone, 2-decanol, methylbutanoic acid, isovaleric acid, 5-methyl-2-hexanone, 2-terfenadone, 2-undecanone, 2-heptanone, 6-methyl-2-heptanone, 2-nonanol, and 1-iodododecane.
9. Use of a volatile component of bacillus beijerinckii LT1 according to any one of claims 7 to 8 for the inhibition of phytopathogens.
10. The use according to claim 9, wherein the phytopathogens comprise largehead atractylodes rhizome leaf spot, southern blight of coptis, tobacco root rot, panax japonicus teres, dwarf lilyturf root rot, sclerotinia sclerotiorum, alternaria alternata, tobacco anthracnose and botrytis cinerea.
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| CN110988184A (en) * | 2019-12-20 | 2020-04-10 | 海南科大林业有限公司 | Method for detecting bacillus subtilis Y13 volatile antibacterial substance based on HS-SPME and GC-MS |
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