CN114145337B - Method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate and application - Google Patents
Method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate and application Download PDFInfo
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- CN114145337B CN114145337B CN202111261995.3A CN202111261995A CN114145337B CN 114145337 B CN114145337 B CN 114145337B CN 202111261995 A CN202111261995 A CN 202111261995A CN 114145337 B CN114145337 B CN 114145337B
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- mushrooms
- epsilon
- tolaasii
- methyl jasmonate
- polylysine
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- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 58
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 title claims abstract description 55
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
- A23B7/144—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of gases, e.g. fumigation; Compositions or apparatus therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate, which comprises the following steps: preprocessing, namely selecting and grading mushrooms, selecting fresh mushrooms, and orderly placing the selected mushrooms; preparing 100 mug/mL epsilon-polylysine solution, and spraying the surface of the mushrooms; fumigating with 100 mu mol/L methyl jasmonate for 12h; after fumigation, the mushrooms are scattered to breathe heat, then the mushrooms are sealed with preservative films, and a plurality of holes are punched for storage. The method starts from three layers of bacteriostasis, brown reduction and resistance induction, effectively inhibits the bacterial brown spot of mushrooms by utilizing the bacteriostasis effect of epsilon-polylysine and the resistance induction effect of methyl jasmonate, maintains the quality of mushrooms, inhibits the incidence rate of mushrooms, is a novel method for inhibiting the bacterial diseases of mushrooms, and can also provide a reference for the control mode of the diseases of edible mushrooms.
Description
Technical Field
The invention belongs to the technical field of edible fungus disease resistance, and in particular relates to a method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate and application thereof.
Background
Bacterial brown spot is an important factor that seriously affects the storage quality and edible value of the edible fungi after harvest, and pseudomonas tolaasii (Pseudomonas tolaasii, p.tolaasii) is a main pathogenic bacterium causing bacterial brown spot of the edible fungi.
Among bacterial diseases of edible fungi, bacterial brown spot of edible fungi caused by p.tolaasii infection is common in various edible fungi, such as agaricus bisporus, oyster mushroom, agaricus blazei, pleurotus ostreatus, lentinus edodes, flammulina velutipes, phoenix mushroom and the like. Taking agaricus bisporus as an example, the surface of the agaricus bisporus is small (diameter is 1-4 mm) and is dispersed as light brown spots, the color and the sinking degree of the spots are gradually deepened along with the aggravation of the disease, and the spots are gradually enlarged to enable a plurality of spots to be combined and even cover the whole surface of the agaricus bisporus and to be gradually rotten, and a strong and unpleasant smell is formed.
At present, the prevention and treatment of bacterial diseases of edible fungi are chemical prevention and treatment. Although the chemical control is simple, convenient and effective, the ecological environment is polluted, and the potential food safety risk exists, and even the drug resistance is generated, so that the chemical control is more difficult to control. The edible fungi are used as food, and the prevention and control means for diseases of the edible fungi are high-efficiency, safe, nontoxic, green and environment-friendly, and have no harm to human bodies or the edible fungi, so that green, safe and efficient measures are needed to be searched for controlling bacterial diseases of the edible fungi after harvest. Epsilon-polylysine (epsilon-polylysine, epsilon-PL) is a natural and safe cationic polypeptide and has the advantages of wide antibacterial spectrum, good water solubility, good thermal stability and the like. epsilon-PL has an inhibiting effect on common bacteria and fungus post-harvest diseases of fruits and vegetables, and in recent years, epsilon-PL has been used in post-harvest preservative and fresh-keeping researches on various fruits and vegetables, however, no application of epsilon-PL for inhibiting bacterial brown spot of post-harvest edible fungi has been known so far. Methyl jasmonate (MeJA) is a novel plant hormone, can improve the resistance of fruits and vegetables, prolong the storage time of the fruits and vegetables, and has no toxicity to human bodies and no pollution to the environment.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
A method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate comprises the following steps:
firstly, preprocessing, namely selecting and grading mushrooms, namely selecting fresh mushrooms which have no mechanical injury, no disease spots, white fruiting bodies, complete mushroom bodies and diameters of 3+/-0.5 cm and have no umbrella opening, cutting off petioles, and orderly placing the selected mushrooms;
Step two, preparing 100 mug/mL epsilon-PL solution, and spraying the mushroom surface, wherein the specific method is as follows:
Placing the selected mushrooms in order, preparing 100 mug/mL epsilon-PL solution, spraying the mushrooms at room temperature by using a sprayer, spraying 2mL of each mushroom on average, and then absorbing for 10-20min;
step three, 100 mu mol/L MeJA fumigation is carried out for 12 hours, and the specific method is as follows:
placing mushrooms sprayed with 100 mu g/mL epsilon-PL solution in order, and fumigating with 100 mu mol/L MeJA in a light-proof closed environment at room temperature for 12h;
and fourthly, after fumigation is finished, ventilation is carried out on the mushrooms, the respiratory heat is dispersed, then the mushrooms are sealed with preservative films, a plurality of holes are punched, the mushrooms are guaranteed to breathe, and the mushrooms are stored at the relative humidity of 85% -95% and the temperature of 2-4 ℃.
Further, the stipe after the stipe is excised in the first step remains 0.5.+ -. 0.2cm.
Further, in the third step, 100 mu M MeJA fumigation is carried out for 12 hours, and then the mushrooms are ventilated, and the respiratory heat is dispersed for 10 minutes.
The application of the method in the aspect of preventing and treating bacterial brown spot of the edible fungi after harvest.
The beneficial effects obtained by the invention are as follows:
1. The method starts from three layers of bacteriostasis, brown reduction and resistance induction, effectively inhibits the bacterial brown spot of mushrooms by utilizing the bacteriostasis effect of epsilon-PL and the resistance induction effect of MeJA, maintains the quality of mushrooms, inhibits the incidence rate of mushrooms, is a novel method for inhibiting the bacterial diseases of mushrooms, and can also provide a reference for the control mode of the diseases of edible mushrooms.
2. The method utilizes the combination of natural epsilon-polylysine (epsilon-PL) antibacterial peptide and natural micromolecular signal substance methyl jasmonate (MeJA), and can cooperatively inhibit the incidence of mushrooms, improve the relative enzyme activities of disease resistance and oxidation resistance, have good inhibition effect on bacterial brown spot of the edible mushrooms after picking, and provide a reference for safe, efficient and durable disease control mode of the edible mushroom industry from three layers of directly inhibiting the growth of pathogenic bacteria (bacteriostasis), regulating and controlling the browning process of the pathogenic bacteria (browning) and inducing the disease resistance of fruiting bodies.
3. Bacterial pathogen infection is an important cause of brown spot of fruiting bodies of the edible fungi after harvest and causes the quality of the edible fungi to be reduced after harvest. The method of the invention is based on the important requirements of domestic edible fungus disease control, and the natural micromolecular signal substances MeJA and epsilon-PL are used for the first time, so that a safe, efficient and durable disease control mode is provided for the edible fungus industry from three aspects of directly inhibiting the growth (bacteriostasis) of pathogenic bacteria, regulating and controlling the browning process (browning) of the pathogenic bacteria and inducing the disease resistance (resistance induction) of fruiting bodies, and cooperatively inhibiting the bacterial brown spot of the picked mushrooms.
4. Earlier studies by the present inventors showed that MeJA inhibited the occurrence of bacterial brown spot in mushrooms mainly by inducing endogenous disease resistance of fruiting bodies. The combination of two or more kinds of antistaling agents with different disease-resistant mechanisms can obtain better disease-resistant effect than a single antistaling agent, and the possibility of drug resistance of microorganisms can be greatly reduced. Especially, after epsilon-PL is combined with other antibacterial agents, antistaling agents and physical antistaling methods, the antistaling effect can be effectively and synergistically enhanced. Therefore, the inventor utilizes epsilon-PL to have good antibacterial performance, and combines the MeJA induced fruit body endogenous disease defense capability, so that a safe, efficient and durable disease control mode can be provided for the fresh-keeping of the edible fungi after the picking.
Drawings
FIG. 1 is a graph showing the inhibition of P.tolaasii by ε -PL in the present invention; wherein A is a minimum inhibitory concentration diagram of epsilon-PL to P.tolaasii; b is a minimum sterilization concentration graph of epsilon-PL to P.tolaasii; c is a sterilization kinetic diagram of epsilon-PL to P.tolaasii; wherein, PC: a positive control; the concentration unit of epsilon-PL is mug/mL;
FIG. 2 is a graph showing the effect of ε -PL on P.tolaasii cell membrane integrity and ultrastructure in the present invention; wherein A is an influence diagram of epsilon-PL on the integrity of a P.tolaasii cell membrane, and B is an influence diagram of epsilon-PL on a P.tolaasii ultrastructure; wherein, control: blank control; 100PL:100 μg/mL ε -PL;200PL:200 μg/mL ε -PL;
FIG. 3 is a graph showing the effect of ε -PL on P.tolaasii protein synthesis and DNA in the present invention; wherein A is an influence diagram of epsilon-PL on P.tolaasii protein synthesis; b is the influence diagram of epsilon-PL on P.tolaasii DNA; wherein, lane 1: a Marker;2: control;3:100PL;4:200PL;
FIG. 4 is a graph showing the inhibition of P.tolaasii by ε -PL and MeJA in accordance with the present invention; wherein A is a graph of inhibition of P.tolaasii by 100 μg/mL ε -PL and MeJA at different concentrations; b is a graph of inhibition of P.tolaasii by 150 μg/mL ε -PL and MeJA at various concentrations; graph of P.tolaasii inhibition by 180 μg/mL ε -PL with different concentrations of MeJA; wherein the method comprises the steps of ,100m MJ：100mmol/L MeJA;100+0.5：100μg/mLε-PL+0.5mmol/LMeJA;100+2：100μg/mLε-PL+2mmol/LMeJA;100+100：100μg/mLε-PL+100mmol/LMeJA;150PL：150μg/mLε-PL;180PL：180μg/mLε-PL;
FIG. 5 is a graph of appearance, incidence and index of incidence of mushrooms treated by the method of the present invention; wherein A is an apparent graph of mushrooms treated by the method, B is a graph of the incidence rate of mushrooms treated by the method, and C is a graph of the incidence index of mushrooms treated by the method; wherein, 100 mu MJ: 100. Mu. Mol/L MeJA; 100PL+100. Mu.MJ: 100 μg/mL ε -PL+100 μmol/LMeJA;
FIG. 6 is a graph showing the effect of disease-resistant related substances in mushrooms treated by the method of the present invention; wherein A is an influence diagram of the total phenol content in the mushrooms treated by the method of the invention, and B is an influence diagram of the flavonoid content in the mushrooms treated by the method of the invention;
FIG. 7 is a graph showing the effect of disease-resistant related enzyme activity in mushrooms treated by the method of the present invention; wherein A is an influence diagram of enzyme activity of Chitinase (CHI) in the mushrooms treated by the method, B is an influence diagram of enzyme activity of beta-1, 3-glucanase (Glu) in the mushrooms treated by the method, and C is an influence diagram of enzyme activity of Phenylalanine Ammonia Lyase (PAL) in the mushrooms treated by the method;
FIG. 8 is a graph showing the effect of browning related enzyme activity and antioxidant related enzyme activity in mushrooms treated by the method of the present invention; wherein A is an influence diagram of polyphenol oxidase (PPO) enzyme activity in mushrooms treated by the method, B is an influence diagram of Peroxidase (POD) enzyme activity in mushrooms treated by the method, C is an influence diagram of Catalase (CAT) enzyme activity in mushrooms treated by the method, and D is an influence diagram of superoxide dismutase (SOD) enzyme activity in mushrooms treated by the method;
fig. 9 is a flow chart of the method of the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.
A method for inhibiting bacterial diseases of edible fungi by combining epsilon-PL and MeJA comprises the following specific steps (taking agaricus bisporus as an example):
Step one, preprocessing, namely selecting and grading mushrooms, selecting the mushrooms without mechanical injury, avoiding disease spots, enabling fruiting bodies to be white, enabling the fruiting bodies not to be opened completely, cutting fresh agaricus bisporus with the diameter of 3+/-0.5 cm, cutting off stipes (keeping about 0.5 cm), orderly placing the selected mushrooms in a frame, and taking and putting the mushrooms lightly in the picking process to avoid mechanical damage on the surface of the mushrooms as much as possible.
Step two, preparing 100 mug/mL epsilon-PL solution, and spraying the mushroom surface, wherein the specific method is as follows:
placing the selected mushrooms in a frame in order, preparing a certain amount of 100 mug/mL epsilon-PL solution, spraying the mushrooms at room temperature by using a sprayer, spraying 2mL of each mushroom on average, and absorbing for 10-20min.
Step three, 100 mu mol/L MeJA fumigation is carried out for 12 hours, and the specific method is as follows:
the mushrooms sprayed with 100. Mu.g/mL of epsilon. -PL solution were placed in a frame in order, and 100. Mu. Mol/L MeJA was fumigated for 12 hours in a light-tight closed environment at room temperature (about 200 mushrooms were placed in a 40L closed dark environment, with a MeJA content of about 880. Mu.L).
And fourthly, after fumigation is finished, ventilation is carried out on the mushrooms, the respiratory heat is dispersed, then the mushrooms are sealed with preservative films, a plurality of holes are punched, the mushrooms are guaranteed to breathe, and the mushrooms are stored at the relative humidity of 85% -95% and the temperature of 2-4 ℃. (P.tolaasii inoculation of mushrooms: P.tolaasii bacterial suspension is uniformly spread on the surface of mushrooms, 2mL per mushroom on average, for better observation of the use of epsilon-PL in combination with MeJA to inhibit bacterial diseases of mushrooms). As shown in fig. 9.
Preferably, in the third step, 100 mu M MeJA fumigation is performed for 12 hours, and then the mushrooms are ventilated, and the respiratory heat is dissipated for 10 minutes.
The application of the method in the aspect of preventing and treating bacterial brown spot of the edible fungi after harvest.
Specifically, the preparation and detection of the correlation are as follows:
1. the bacteriostasis and sterilization effect of epsilon-PL on P.tolaasii is as follows:
Step one: determining Minimum Inhibitory Concentration (MIC) of epsilon-PL on P.tolaasii, picking single colony of P.tolaasii into 1mL beef extract peptone culture medium, shake culturing at 28 ℃ until OD 600 is about 1.0, diluting 1000 times, adding 100 mu L of test bacteria into each well of a 96-well plate, adding 100 mu L of epsilon-PL solution with different concentrations, setting negative and positive control, culturing at 28 ℃ for 12-16h, and measuring absorbance at 595nm on an enzyme-labeled instrument. ( 1L beef extract peptone culture medium formula: 10g of peptone, 3g of beef extract and 5g of sodium chloride, and distilled water is dissolved to be constant volume to 1L )
Step two: determining Minimum Bactericidal Concentration (MBC) of epsilon-PL on P.tolaasii, and preparing test bacteria by the same method as the step one. 50 mu L of tested bacteria are added into a 1.5mL EP tube, 50 mu L of epsilon-PL with different concentrations are added into each tube, negative and positive controls are set, the tube is subjected to stationary culture for 2 hours at 28 ℃,10 mu L of bacteria liquid after the action is taken, the tube is subjected to gradient dilution, 100 mu L of bacteria liquid is respectively coated on a beef extract peptone flat plate, after stationary culture for 12-16 hours at 28 ℃, single colonies on the flat plate are counted, and the minimum protein concentration during aseptic colony growth on the flat plate is MBC.
Step three: the sterilization kinetics of epsilon-PL on Pseudomonas tolaciensis is studied, and the preparation method of the test bacteria is the same as that of the step one. 50 mu L of bacterial liquid is added into a 1.5mL EP tube, 50 mu L of epsilon-PL with different concentrations is added into each tube, negative and positive controls are set, 10 mu L of samples are respectively taken for dilution by 10 times at 20min, 40min, 60min and 90min, plating is carried out on beef extract peptone flat plates, and the beef extract peptone flat plates are placed in a 28 ℃ incubator for overnight culture. The colony count was calculated.
As shown in FIG. 1A, the higher the sterilization ability against P.tolaasii, the higher the inhibition rate of P.tolaasii was 90% when the concentration of ε -PL reached 200. Mu.g/mL, and the inhibition rate of P.tolaasii was 99% or more when the concentration of ε -PL was 250. Mu.g/mL, so that the MIC of ε -PL against P.tolaasii was 200. Mu.g/mL. As shown in FIG. 1B, the number of P.tolaasii colonies decreased in sequence with increasing concentration of ε -PL over 2 hours. Until the concentration of ε -PL was 300. Mu.g/mL, the colony count of P.tolaasii was zero, i.e., 300. Mu.g/mL of ε -PL was able to completely kill P.tolaasii (MBC=300. Mu.g/mL) within 2 hours. As can be seen from FIG. 1C, as the concentration of epsilon-PL increases, the epsilon-PL sterilization time of P.tolaasi decreases in sequence, the sterilization capacity increases in sequence, and 800 mug/mL of epsilon-PL sterilization capacity is superior to gentamicin (PC) with 2MIC, which indicates that epsilon-PL has good antibacterial sterilization capacity on P.tolaasi.
2. The effect of ε -PL on P.tolaasii on cell membrane integrity and ultrastructural activity was tested as follows:
Step one: effect on cell membrane integrity log phase p.tolaasii was washed and resuspended in phosphate buffer (PBS, 0.02mol/L pH 7.2). The bacterial suspension is mixed with different concentrations of epsilon-PL in equal volumes. After incubation at 28℃for 2h, washing with PBS and resuspension were performed. Propidium Iodide (PI) was added to give final concentrations of 2. Mu. Mol/L, incubated at 28℃for 30min in the dark, washed with PBS, resuspended, and the stained cells were photographed by observation using a fluorescence microscope.
Step two: effect on ultrastructural function of P.tolaasii cells, P.tolaasii and ε -PL treatment methods are as in step one. After incubation for 2h at 28℃the cells were washed with PBS, resuspended, then fixed overnight at 4℃with 4% glutaraldehyde, washed with ethanol of different concentrations, air-dried and photographed using transmission electron microscopy.
As shown in FIG. 2B, the epsilon-PL treatment had a significant effect on P.tolaasii morphology. The control group has full thallus with smooth surface, complete structure and regular shape and is in normal growth state. Whereas P.tolaasii treated with 100. Mu.g/mL ε -PL had a rough surface and had shrinkage distortion. In contrast, the cell morphology of the 200. Mu.g/mL epsilon. -PL treated group was more severely changed, the cell surface was severely wrinkled, and the leakage of cell content material was severe. This suggests that epsilon-PL can disrupt the P.tolaasii cell membrane and that high concentrations of epsilon-PL have a significant effect on P.tolaasii morphology.
3. Effect of ε -PL on P.tolaasii cell protein Synthesis and DNA
Step one: effect on protein synthesis by p.tolaasii cells, treatment of p.tolaasii with epsilon-PL was as in step one. After incubation for 4h at 28℃the samples were washed and resuspended in PBS and visualized by SDS-PAGE.
Step two: effect on p.tolaasii DNA p.tolaasii genomic DNA was extracted using a DNA kit, DNA (final concentration 25 μg/mL) was mixed with bacteriostatic agents at various concentrations, incubated at 28 ℃ for 2 hours, subjected to nucleic acid electrophoresis and photographed using a gel imager.
As a result, as shown in FIG. 3A, the whole of the protein band became shallow after 200. Mu.g/mL. Epsilon. -PL treatment, indicating a decrease in protein concentration, and it was found that the protein band was successively deepened after 100. Mu.g/mL. Epsilon. -PL and 200. Mu.g/mL. Epsilon. -PL treatments at a molecular weight of less than 10 kDa. The results show that epsilon-PL has an inhibitory effect on the synthesis of macromolecular proteins or degrades macromolecular proteins into small molecular proteins. As shown in FIG. 3B, 100. Mu.g/mL. Epsilon. -PL and 200. Mu.g/mL. Epsilon. -PL treated DNA were retained in the wells in this order, probably because of the change in the electrical properties of the charges or the change in the double helix structure of the DNA, and thus the mobility of the DNA was affected.
4. Sterilization of P.tolaasii by treatment of epsilon-PL and MeJA
Shaking culture P.tolaasii to OD 600 at 28deg.C of about 1.0, dilution 1000 times, adding 50 μl of bacterial liquid into 1.5mL EP tube, mixing 50 μl of epsilon-PL and MeJA mixed liquid at different concentrations, setting negative and positive control, culturing at 28deg.C for 2h, plating, and culturing at 28deg.C overnight to count colony.
As shown in FIG. 4, with the increase of the concentration of epsilon-PL and MeJA, the effect of completely sterilizing P.tolaasii is not achieved, and as can be seen from the graph, the maximum inhibition rate of epsilon-PL+MeJA can reach 93.9%, and bacteria are not killed, namely, the epsilon-PL+MeJA does not have the effect of sterilizing P.tolaasii in vitro, and the respective effects of epsilon-PL and MeJA cannot be achieved due to incompatibility of epsilon-PL and epsilon-PL are utilized to spray and MeJA fumigate mushrooms, so that the epsilon-PL bacteriostasis and MeJA induction can be well achieved, and bacterial brown spot of mushrooms can be effectively inhibited.
5. The appearance, incidence and incidence index of mushrooms treated by the method
A tolaasii infestation causes browning of the mushrooms, dishing of the tissues, and a rotting taste, as shown in fig. 5A. At 48h post-infection, the whole caps of the fruiting bodies of the mushrooms of the control group were dark brown spots, and the meat portions of the fruiting bodies were consumed with unpleasant taste. Compared with the method, the epsilon-PL and MeJA entity has no soft rot phenomenon, and the fruiting body is fuller than that of the epsilon-PL and MeJA independently treated, the fungus cover is white and has no dent, and the mushroom fragrance and the putrefactive flavor are emitted. To better verify the severity of brown spot produced by the method of the present invention on mushrooms, we quantified this as disease index and incidence. As shown in fig. 5b and c, the morbidity and the disease index are gradually increased with the increase of the storage time, the morbidity and the disease index of the control group, epsilon-PL and MeJA are up to 50% and 30% or more at 24 hours, the morbidity of the control group reaches 100% and the disease index of the control group reaches 60% or more even at 72 hours, and the morbidity and the disease index of the epsilon-pl+meja group are far lower than those of the control group at 24-72 hours, so that the epsilon-PL and MeJA can effectively inhibit the occurrence of mushroom bacterial diseases.
6. Determination of disease-resistant related substances (Total phenols, flavonoids) in mushrooms treated by the method of the invention
1.0G of the sample was weighed, added with 4.0mL of 1% hydrochloric acid-methanol solution, ground in an ice bath, and transferred into a 20mL graduated tube. Light was prevented from being removed at 4℃for 2 hours, and the supernatant was collected by centrifugation at 10,000rpm/min at 4℃for 15 minutes. Taking 1% hydrochloric acid-methanol solution as blank, taking supernatant, and measuring absorbance at 280nm and 325nm respectively. And a standard curve of gallic acid and catechol is prepared, and the contents of total phenols and flavonoids are calculated.
Total phenols and flavonoids are important secondary metabolites in plants and are closely related to disease-resistant defenses. As shown in FIG. 6, the total phenolic flavonoids content of the epsilon-PL+MeJA groups are significantly higher than that of the control group, the epsilon-PL group and the MeJA group, wherein the total phenols and flavonoids of the epsilon-PL+MeJA group reach the maximum value at 48 hours and are 1.8 times and 1.5 times that of the control group respectively. The results prove that the epsilon-PL and MeJA cooperative treatment can accumulate the resistant substances of total phenols and flavonoids in the fruiting body, thus indicating that the epsilon-PL and MeJA cooperative treatment can promote the increase of the upstream enzyme activity of the metabolic pathway of the phenylpropane substances and the accumulation of the downstream secondary metabolites, and effectively induce the start of the disease-resistant defense reaction.
7. Determination of disease-resistant related enzyme Activity (CHI, GLU, PAL) in mushrooms treated by the method of the invention
Preparing an enzyme extracting solution: 1.0g of the sample was taken, 5mL of 100mmol/L acetic acid buffer (pH 5.2, containing 8% (w/v) PVP,1mmol/L Na 2EDTA·2H2 O,5mmol/L beta-mercaptoethanol) was added, the slurry was ground in an ice bath, centrifuged at 10000rpm at 4℃for 5min, and the supernatant was collected.
CHI activity assay: taking 0.5mL of crude enzyme solution, adding 0.5mL of 10mg/mL colloidal chitin, preserving heat at 37 ℃ for 1h, adding 0.1mL of 3% desalination snailase, preserving heat at 37 ℃ for 1h, adding 0.4mL of 0.6mol/L potassium tetraborate, boiling in boiling water for 5min, and cooling. 2mL of p-dimethylaminobenzaldehyde diluted 5-fold with glacial acetic acid was added. Incubate at 37℃for 10min and measure at 585 nm. 1X 10 -9 mol Glc-NAc per hour of the chitin is produced as one unit of enzyme activity (U).
GLU Activity assay: taking 250 mu L of enzyme solution, adding 250 mu L of 0.5% laminarin, reacting for 1h at 37 ℃, adding 400 mu L of DNS, and boiling in boiling water for 5min. The absorbance at 500nm was measured. 1X 10 -9 mol of glucose is produced as one enzyme activity unit (U) per hour of the split laminarin.
PAL Activity assay: 2.0g of the sample was taken, 6.0mL of 200mmol/L boric acid buffer pH 8.8 (containing 1% PVP,1mmol/L Na 2EDTA·2H2 O,5mmol/L beta-mercaptoethanol) was added, the mixture was ground, centrifuged at 10000rpm at 4℃for 15min, and the supernatant was collected. The enzymatic reaction system consisted of 2.0mL 200mmol/L pH 8.8 boric acid buffer, 1.0mL20mmol/L L-phenylalanine and 0.5mL crude enzyme solution. The reaction system was incubated at 37℃for 5min before the addition of the enzyme solution, and immediately after the addition of the enzyme solution, the absorbance was measured at 290nm and incubated at 37℃for 1h. The absorbance of the mixture at 290nm was again measured as the termination value of the reaction. PAL enzyme activity was varied by 1 activity unit (u=Δa290 h -1) with absorbance change per hour.
Chitinase (CHI) and beta-1, 3-glucanase (Glu) play a key role in plant disease resistance, and are important disease course related proteins in plants. Phenylalanine Ammonia Lyase (PAL) is an important resistance-related enzyme involved in secondary metabolism in plants. As shown in FIG. 7, four groups CHI, glu, PAL all show a tendency of rising and then falling, CHI and Glu reach the highest values at 72h, 24h and 48h respectively, and the enzyme activities of the epsilon-PL+MeJA group CHI, glu, PAL are obviously higher than those of the control group, the epsilon-PL group and the MeJA group, wherein the enzyme activities of the epsilon-PL+MeJA group CHI, glu, PAL are 1.7 times, 3.2 times and 1.8 times of those of the control group respectively. It is shown that the synergistic treatment of epsilon-PL and MeJA can induce the increase of the enzymatic activity of the fruiting body CHI, glu, PAL, thereby resisting bacterial infection and improving the disease resistance of the fruiting body.
8. The browning related enzyme activity (PPO) and the antioxidation related enzyme activity (POD, CAT, SOD) in the mushrooms treated by the method are measured, and the measuring method is as follows:
1g of the sample is weighed, 9mL of 50mmol/L phosphate buffer (pH 7.0, 1% PVP,1 mmol/LDTT) is added, ground in an ice bath and centrifuged at 8500rpm/min at4℃for 5min. The supernatant is taken for standby, and four enzyme assays are all carried out by adopting corresponding enzyme kits developed by Nanjing built biological engineering research institute.
As shown in FIG. 8, the result shows that the PPO enzyme activity of epsilon-PL and MeJA is remarkably reduced compared with that of a control group, epsilon-PL group and MeJA group by utilizing the method for synergistically inhibiting mushroom bacterial diseases, and is stable in the whole storage process, the difference is maximum at 72 hours, and the PPO activity of the control group is 1.6 times that of epsilon-PL and MeJA. The activities of antioxidant enzymes POD, CAT and SOD are in a trend of ascending and descending, and the activities of the antioxidant enzymes of epsilon-PL and MeJA are obviously higher than those of a control group, epsilon-PL group and MeJA group, and the activities of the antioxidant enzymes of epsilon-PL and MeJA are respectively 1.6 times, 1.5 times and 1.7 times of those of the control group in the next day, so that the synergistic treatment of epsilon-PL and MeJA can effectively inhibit PPO activity, improve the antioxidant enzyme activities of POD, CAT, SOD and the like, and effectively alleviate browning caused by pathogenic bacteria infection.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (2)
1. A method for inhibiting bacterial diseases of edible fungi by combining epsilon-polylysine and methyl jasmonate is characterized by comprising the following steps of: the method comprises the following steps:
firstly, preprocessing, namely selecting and grading mushrooms, namely selecting fresh mushrooms which have no mechanical injury, no disease spots, white fruiting bodies, complete mushroom bodies and diameters of 3+/-0.5 cm and have no umbrella opening, cutting off petioles, and orderly placing the selected mushrooms;
step two, preparing 100 mug/mL epsilon-polylysine solution, and spraying the mushroom surface, wherein the specific method is as follows:
placing the selected mushrooms in order, preparing 100 mug/mL epsilon-polylysine solution, spraying the mushrooms at room temperature by using a sprayer, spraying 2mL of each mushroom on average, and then absorbing for 10-20min;
step three, fumigating 100 mu mol/L methyl jasmonate for 12 hours, wherein the specific method is as follows:
the mushrooms sprayed with 100 mu g/mL epsilon-polylysine solution are orderly placed, and 100 mu mol/L methyl jasmonate fumigation is carried out for 12 hours in a light-proof closed environment at room temperature;
fourthly, after fumigation is finished, ventilation is carried out on the mushrooms, the respiratory heat is dispersed, then the mushrooms are sealed with preservative films, holes are punched, the mushrooms are guaranteed to breathe, and the mushrooms are stored at the relative humidity of 85% -95% and the temperature of 2-4 ℃;
The stipe after the stipe is cut off in the first step is reserved for 0.5+/-0.2 cm;
In the third step, 100 mu M methyl jasmonate fumigation is carried out for 12 hours, and then mushrooms are ventilated and breathed heat is dispersed for 10 minutes;
the bacteria are pseudomonas tolaciensis (Pseudomonas tolaasii, p.tolaasii).
2. Use of the method according to claim 1 for the control of bacterial brown spot in post harvest edible mushrooms, said bacterium being pseudomonas tolaciensis (Pseudomonas tolaasii, p.
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| Arginase participates in the methyl jasmonate-regulated quality maintenance of postharvest Agaricus bisporus fruit bodies;Meng D，et al;Postharvest Biology and Technology;7-14 * |
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| ε-聚赖氨酸在果蔬采后贮藏保鲜中的应用研究进展;袁帅，等;食品研究与开发(第17期);196-203 * |
| 壳聚糖、聚赖氨酸对滑菇保鲜的影响;陈晓宁;轻纺工业与技术(第2期);164-166 * |
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