CN111334455B - Filamentous fungus inhibitor and application thereof - Google Patents
Filamentous fungus inhibitor and application thereof Download PDFInfo
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Abstract
The invention discloses a filamentous fungus inhibitor and application thereof, belonging to the technical field of biology. The invention provides a filamentous fungus inhibitor, which comprises the components of lactobacillus plantarum CGMCC No.5494 and/or lactobacillus plantarum CGMCC No.5494 fermentation supernatant, wherein the germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti or penicillium digitatum spores can be remarkably inhibited within 7 days after the lactobacillus plantarum CGMCC No.5494 is co-cultured with the penicillium expansum spores, the aspergillus niger spores, the penicillium roqueforti spores or the penicillium digitatum spores respectively; after the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 is co-cultured with penicillium expansum spore, aspergillus niger spore, penicillium roqueforti spore or penicillium digitatum spore, the germination of the penicillium expansum spore, the aspergillus niger spore, the penicillium roqueforti spore or the penicillium digitatum spore can be obviously inhibited.
Description
Technical Field
The invention relates to a filamentous fungus inhibitor and application thereof, belonging to the technical field of biology.
Background
The spoilage of fruits and vegetables can cause appearance defects and nutrient loss of fruits and vegetables. In developing countries with imperfect refrigeration and transportation facilities, the problem of fruit and vegetable spoilage is particularly prominent, and the loss of the fruit and vegetable industry is also particularly serious.
Filamentous fungi such as Penicillium expansum, Aspergillus niger, Penicillium roqueforti, Penicillium digitatum, and the like, are among the potential causes of fruit and vegetable spoilage, primarily by erosion of fruit and vegetable leaves, fruits, and the like, and further cause storage diseases in fruits and vegetables, and they produce toxins such as patulin and citrinin (see in particular: Hammmami W, Al Thani R, et Al. Patulin and patulin reduction Pen. and apple-based products including basic food, J. Journal of Infection in development and cosmetics, 2017,11(4):343-, S. Banndoh, M. takeuchi, et Al. Patulin reduction and apple products, Biofertilizer, et Al. repair and apple, 2010,143(3):109-117), these toxins remain in the fruits and vegetables and enter human body through food chain, and are potentially harmful. Therefore, it is also important to inhibit the filamentous fungi such as Penicillium expansum, Aspergillus niger, Penicillium roqueforti, Penicillium digitatum, etc. to prevent the spoilage of fruits and vegetables, and further to reduce the loss of the fruit and vegetable industry.
At present, fruit and vegetable spoilage caused by filamentous fungi is mainly controlled by chemical bactericides, but in view of the emergence of drug resistance of filamentous fungi and the potential health threat to human bodies caused by residual amounts of chemical bactericides, people tend to develop eco-friendly methods for controlling postharvest decay of fruits.
Compared with chemical bactericides, both physical control methods and biological control methods have certain advantages in the aspect of ecological friendliness. The physical prevention and control method is mainly used for inhibiting filamentous fungus infection by microwave heating and refrigeration of fruits and vegetables, but the physical prevention and control method has no continuity because the physical means such as microwave heating and refrigeration are short in stay on the surfaces of the fruits and vegetables.
The biological control method is mainly to inhibit filamentous fungi infection by antagonistic microorganisms, and the method is better in persistence compared with the physical control method, but the biological control method has the defect of poor effect, mainly because many antagonistic microorganisms belong to fungi and can cause fungal spoilage on the surface of fruits. Therefore, it is urgently needed to find a non-fungal antagonistic microorganism having a strong ability to inhibit filamentous fungi such as Penicillium expansum and the like so as to solve the defects of the existing biological control methods.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a filamentous fungus inhibitor.
[ solution ]
In order to solve the technical problem, the invention provides a filamentous fungus inhibitor, and the components of the inhibitor comprise lactobacillus plantarum CGMCC No.5494 and/or fermentation supernatant of lactobacillus plantarum CGMCC No. 5494. The fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is obtained by inoculating the lactobacillus plantarum CGMCC No.5494 to a culture medium for fermentation.
In one embodiment of the invention, the components of the inhibitor further comprise a carrier.
In one embodiment of the invention, the carrier is a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier is a filler, binder, wetting agent, disintegrant, lubricant and/or flavoring agent.
In one embodiment of the present invention, the inhibitor is in the form of powder, emulsion, granule or fine granule.
In one embodiment of the invention, the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 is heated; or treating the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 by protease.
The invention also provides a method for inhibiting filamentous fungi, which is to co-culture the filamentous fungi inhibitor and the filamentous fungi.
In one embodiment of the invention, inhibiting the filamentous fungus comprises reducing the germination rate of spores of the filamentous fungus, disrupting the cell membrane permeability of the filamentous fungus, and/or inhibiting the growth of mycelia of the filamentous fungus.
The invention also provides the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 or the filamentous fungus inhibitor or the application of the method in inhibiting filamentous fungi without the aim of diagnosing and treating diseases.
The invention also provides fermentation supernatant of lactobacillus plantarum CGMCC No.5494 or the filamentous fungus inhibitor or the application of the filamentous fungus inhibitor in preventing fruit and vegetable putrefaction.
[ advantageous effects ]
(1) The invention provides a filamentous fungus inhibitor, which comprises the components of lactobacillus plantarum CGMCC No.5494 and/or lactobacillus plantarum CGMCC No.5494 fermentation supernatant, wherein the germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti or penicillium digitatum spores can be remarkably inhibited within 7 days after the lactobacillus plantarum CGMCC No.5494 is co-cultured with the penicillium expansum spores, the aspergillus niger spores, the penicillium roqueforti spores or the penicillium digitatum spores respectively; after the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 is co-cultured with penicillium expansum spore, aspergillus niger spore, penicillium roqueforti spore or penicillium digitatum spore respectively, the germination of the penicillium expansum spore, the aspergillus niger spore, the penicillium roqueforti spore or the penicillium digitatum spore can be obviously inhibited, and the spore germination inhibition rates can be respectively up to 99.68 +/-0.97%, 98.45 +/-0.13%, 96.35 +/-0.35% and 96.26 +/-0.54%; after the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 and penicillium expansum spores are co-cultured, the cell membrane permeability of penicillium expansum mycelia can be remarkably damaged; after the penicillium expansum spores are inoculated to a culture medium containing the lactobacillus plantarum CGMCC No.5494 fermentation supernatant for culture, the growth of penicillium expansum mycelia is obviously inhibited, and the inhibition rate can reach 35.8 percent, so the lactobacillus plantarum CGMCC No.5494 has extremely high application prospect in inhibiting penicillium expansum and preventing fruit and vegetable spoilage.
(2) The invention provides a penicillium expansum inhibitor, which comprises the components of lactobacillus plantarum CGMCC No.5494 and/or fermentation supernatant of lactobacillus plantarum CGMCC No.5494, wherein the inhibition capability of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 on the growth of penicillium expansum mycelium after heating treatment is improved compared with the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 without heating treatment, so that the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 can inhibit penicillium expansum and has good thermal stability.
(3) The invention provides a penicillium expansum inhibitor, which comprises the components of lactobacillus plantarum CGMCC No.5494 and/or fermentation supernatant of lactobacillus plantarum CGMCC No.5494, wherein the pH of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is 4, and the inhibition capability of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 on the growth of penicillium expansum mycelium is stronger, so that the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 can inhibit penicillium expansum and has good acid stability.
(4) The invention provides a penicillium expansum inhibitor, which comprises the components of lactobacillus plantarum CGMCC No.5494 and/or fermentation supernatant of lactobacillus plantarum CGMCC No.5494, wherein the inhibition capability of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 on the growth of penicillium expansum mycelium after protease treatment is not obviously changed compared with the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 without protease treatment, so that the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 can inhibit penicillium expansum and has good protease stability.
Drawings
FIG. 1: the effect of Lactobacillus plantarum CGMCC No.5494 on the growth of Penicillium expansum mycelium.
FIG. 2: the influence of the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 with different concentrations on the permeability of mycelium cell membranes.
FIG. 3: the lactobacillus plantarum CGMCC No.5494 treated by different modes has the influence of fermentation supernatant on the growth of penicillium expansum mycelium.
Detailed Description
The invention is further elucidated with reference to a specific embodiment and a drawing.
Penicillium expansum referred to in the following examples was purchased from China center for Industrial culture Collection of microorganisms with product number CICC 40658; the aspergillus niger related in the following examples is purchased from China center for culture Collection of industrial microorganisms, and the product number is CICC 2089; the Penicillium roqueforti related in the following examples is purchased from China center for culture Collection of industrial microorganisms with product number CICC 40663; penicillium digitatum referred to in the examples below was purchased from North Nay, product number BNCC 336887; lactobacillus plantarum CGMCC No.5494 (published in patent application No. CN 102586148A) in the following examples was deposited in the China general microbiological culture Collection center (CGMCC), and no patent procedure was repeated.
The media involved in the following examples are as follows:
MRS solid medium: adding 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5g of magnesium sulfate heptahydrate, 2g of diammonium citrate and 18g of agar powder into 1L of distilled water, wherein the pH value is 6.2-6.5.
MRS liquid medium: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5g of magnesium sulfate heptahydrate and 2g of diammonium citrate are added into 1L of distilled water, and the pH value is 6.2-6.5.
PDA culture medium: 20g of glucose and 18g of agar were added to 1L of potato juice, and the pH was adjusted to the natural pH.
Example 1: lactobacillus plantarum CGMCC No.5494 and influence of fermentation supernatant thereof on germination rate of filamentous fungus spores
1. Influence of Lactobacillus plantarum CGMCC No.5494 on the germination rate of filamentous fungi spores (double-layer plate-growth inhibition method)
Dipping lactobacillus plantarum CGMCC No.5494 in a glycerin tube by using an inoculating loop, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a single colony; and selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 3 times to obtain a bacterial liquid cultured to the third generation.
Dipping the penicillium expansum solution in the ampoule tube by using an inoculating ring, inoculating the penicillium expansum solution on a PDA culture medium, and culturing at 28 ℃ and 200rpm for 7d to obtain mycelia and spores; selecting spores to inoculate on a PDA inclined plane, culturing at 28 ℃ and 200rpm for 7d, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; adding 5mL of sterile water into a PDA culture medium in which penicillium expansum grows and is cultured to the third generation, scraping spores by using an inoculating loop, and filtering by using 4 layers of sterile gauze to obtain penicillium expansum spore suspension; diluting Penicillium expansum spore suspension with sterile water to concentration of 1 × 104cfu/mL。
Dipping two parallel lines of two centimeters on MRS solid culture medium of bacterial suspension by using inoculating loop, culturing for 48h at 37 ℃, and adding 8mL of MRS solid culture medium with the concentration of 1 multiplied by 104After cfu/mL penicillium expansum spore suspension is cultured for 2d and 7d respectively at 28 ℃, an inhibition area (namely a streak area on an MRS solid culture medium) is observed, the inhibition capacity of the lactobacillus plantarum CGMCC No.5494 on the germination rate of the penicillium expansum spores is detected by taking the percentage of the lactobacillus plantarum CGMCC No.5494 colony in the inhibition area and the area without spore germination around the colony to the total area of the MRS solid culture medium as an index, and the detection result is shown in Table 1 and figure 1.
The inhibition capability of lactobacillus plantarum CGMCC No.5494 on the germination rates of spores of Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is respectively detected by referring to the same method, and the detection results are shown in Table 1.
As can be seen from Table 1 and FIG. 1, when the inhibition ability of Lactobacillus plantarum CGMCC No.5494 on the germination rate of spores of Penicillium expansum, Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is detected, 2d of cultivation is carried out, no spores are germinated in a colony of Lactobacillus plantarum CGMCC No.5494 in the inhibition area and in an area which is not less than 70% of the area of MRS solid medium around the colony, 7d of cultivation is carried out, and no spores are germinated in a colony of Lactobacillus plantarum CGMCC No.5494 in the inhibition area and in an area which is not less than 30% of the area of MRS solid medium around the colony. Therefore, the lactobacillus plantarum CGMCC No.5494 has stronger inhibition capability on the germination rate of spores of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum.
TABLE 1 inhibition ability of Lactobacillus plantarum CGMCC No.5494 on spore germination rate of different filamentous fungi
Note: no spore germination exists in the bifidobacterium colonies and the area which is not less than 30% of the area of the plate around the bifidobacterium colonies ++; no spore germination in the bifidobacterium colony area and the area around the colony which is less than 30 percent of the plate area ++; no spore germination in the colony area of bifidobacterium only +; no zone of inhibition of spore germination-.
2. Influence of Lactobacillus plantarum CGMCC No.5494 fermentation supernatant on filamentous fungus spore germination rate (96-well plate-spore germination inhibition method)
Dipping lactobacillus plantarum CGMCC No.5494 in a glycerin tube by using an inoculating loop, scribing on an MRS solid culture medium, scribing on the MRS solid culture medium, and culturing for 48h at 37 ℃ in an anaerobic environment to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS liquid culture medium in an inoculation amount of 2% (v/v), culturing at 37 ℃ for 48h in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; the fermentation broth was centrifuged at 8000rpm for 10min and then filtered through a 0.2 μm filter to obtain a fermentation supernatant.
Dipping the penicillium expansum solution in the ampoule tube by using an inoculating ring, inoculating the penicillium expansum solution on a PDA culture medium, and culturing at 28 ℃ and 200rpm for 7d to obtain mycelia and spores; selecting spores to inoculate on a PDA inclined plane, culturing at 28 ℃ and 200rpm for 7d, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; adding 5mL of sterile water into PDA culture medium containing Penicillium expansum cultured to the third generation, scraping spores with inoculating loop, and culturingFiltering with 4 layers of sterile gauze to obtain penicillium expansum spore suspension; diluting Penicillium expansum spore suspension with sterile water to concentration of 1 × 104cfu/mL。
mu.L of fermentation supernatant was added to sterile 96-well plates and 10. mu.L of 1X 104culturing cfu/mL penicillium expansum spore suspension at 28 ℃ for 48h to obtain a culture solution; using MRS liquid culture medium as control, and measuring OD of culture solution and MRS liquid culture medium580Calculating the spore germination inhibition rate of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant to penicillium expansum, wherein the calculation result is shown in table 2; wherein the spore germination inhibition rate (%) is (1- (. DELTA.OD fermentation supernatant-. DELTA.ODMRS)/. DELTA.ODMRS) × 100%.
The spore germination inhibition rates of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on Aspergillus niger, Penicillium roqueforti and Penicillium digitatum are respectively detected by referring to the same method, and the detection results are shown in Table 2.
As can be seen from Table 2, the spore germination inhibition rates of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum can be respectively as high as 99.68 +/-0.97%, 98.45 +/-0.13%, 96.35 +/-0.35% and 96.26 +/-0.54%, and thus the lactobacillus plantarum CGMCC No.5494 fermentation supernatant has stronger inhibition capability on the spore germination inhibition rates of the penicillium expansum, the aspergillus niger, the penicillium roquefortii and the penicillium digitatum.
TABLE 2 inhibitory potency of Lactobacillus plantarum CGMCC No.5494 on spore germination of different filamentous fungi
| Bacterial strains | Spore germination inhibition (%) |
| Penicillium expansum | 99.68±0.97 |
| Aspergillus niger | 98.45±0.13 |
| Blue mould of Mongolian blue | 96.35±0.35 |
| Penicillium digitatum | 96.26±0.54 |
Example 2: influence of lactobacillus plantarum CGMCC No.5494 fermentation supernatant on cell membrane permeability of expanded penicillium mycelium
Mixing MRS liquid culture medium with PDA culture medium at volume ratio of 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5, 4:6(MRS liquid culture medium: PDA culture medium) to obtain control group mixed solution with MRS liquid culture medium concentration of 10, 15, 20, 25, 30, 35, 45% (v/v); mixing fermentation supernatants of lactobacillus plantarum CGMCC No.5494 obtained in example 1 with PDA culture medium at volume ratio of 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5 and 4:6 respectively to obtain experimental group mixed liquid with fermentation supernatant concentration of 10, 15, 20, 25, 30, 35 and 45% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; dripping 10 μ L of Penicillium expansum spore suspension obtained in example 2 into the center of the plate, culturing at 28 deg.C and 200rpm for 1d and 1d, scraping Penicillium expansum mycelium 1 on the plate, washing with PBS buffer solution twice, centrifuging at 8000rpm for 10min, and collecting mycelium 2; adding PI dye (purchased from Shanghai-constructed biological Co., Ltd.) with concentration of 1 μ M into the mycelium 2, incubating at room temperature (25 deg.C) for 30min, centrifuging at 8000rpm for 10min, and collecting mycelium 3; washing the mycelium 3 with PBS buffer solution twice, centrifuging at 8000rpm for 10min, and collecting mycelium 4; the staining of the mycelium 4 was observed with a fluorescence microscope and a picture was taken, which is shown in FIG. 2.
As shown in FIG. 2, the mycelium 4 of the control group had no red fluorescence signal, indicating that the integrity of the cell membrane of Penicillium expansum mycelium which was not treated with the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 was not destroyed; in the mycelium 4 of the experimental group, red fluorescence can be observed from the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 being 10%, but the fluorescence signal under the treatment is weaker, while a stronger fluorescence signal can be observed when the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is 25%, which shows that the cell membrane of the penicillium expansum can be damaged and the permeability of the penicillium expansum can be increased by the treatment of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494, and in addition, the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is in positive correlation with the change condition of the cell membrane permeability and the intensity of the fluorescence signal.
Example 3: influence of Lactobacillus plantarum CGMCC No.5494 fermentation supernatant on growth of penicillium expansum mycelium
Mixing the MRS liquid culture medium with a PDA culture medium according to the volume ratio of 1.5:8.5(MRS liquid culture medium: PDA culture medium) to obtain a control group mixed solution with the concentration of the MRS liquid culture medium of 15 (v/v); mixing the fermentation supernatant obtained in example 1 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain experimental group mixtures with respective fermentation supernatant concentrations of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; dripping 10 μ L of Penicillium expansum spore suspension obtained in example 2 into the center of the plate, culturing at 28 deg.C for 6d, measuring the diameter of mycelium on each plate every 2d during the culturing period, and detecting the influence of fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of Penicillium expansum mycelium, wherein the detection result is shown in FIG. 3; wherein the mycelium growth inhibition ratio (%) - (1- (D)Control group-DTreatment group)/DControl group) X 100%, wherein D: diameter of mycelium.
As shown in FIG. 3, it can be seen from the measurement of the diameter of the mycelia on the plate at the 6 th day of culture that the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 can significantly inhibit the growth of the mycelia of Penicillium expansum with an inhibition rate as high as 35.8% (diameter of mycelia of 9.8 mm).
Example 4: influence of temperature on ability of lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit growth of penicillium expansum mycelium
Mixing the fermentation supernatant obtained in example 1 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); heating the fermentation supernatant obtained in example 1 at 121 ℃ for 20min, and mixing the fermentation supernatant with PDA culture medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA culture medium) to obtain an experimental group mixture with a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was added dropwise to the center of the plate and cultured at 28 ℃ for 6 days, during which the effect of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of the Penicillium expansum mycelia was examined by measuring the diameter of the mycelia on each plate every 2 days, and the results are shown in FIG. 3.
As shown in FIG. 3, it can be seen from the measurement of the diameter of the mycelia on the plate at the 6 th day of culture, the inhibitory activity of the fermentation supernatant (the diameter of the mycelia is 9.4mm) of Lactobacillus plantarum CGMCC No.5494 on the growth of the mycelia of Penicillium expansum after the heat treatment was slightly improved compared with the fermentation supernatant (the diameter of the mycelia is 9.8mm) of Lactobacillus plantarum CGMCC No.5494 without the heat treatment.
Example 5: influence of pH on ability of lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit growth of penicillium expansum mycelium
Mixing the fermentation supernatant (pH 4) obtained in example 1 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); after adjusting the pH of the fermentation supernatant obtained in example 1 to 7 (adjusted with 1mol/L NaOH and 1mol/L HCl), the fermentation supernatant was mixed with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a mixture of experimental groups at a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 mu.L of the Penicillium expansum spore suspension obtained in example 2 was added dropwise to the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of pH on the ability of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of mycelia on each plate every 2 days, and the results are shown in FIG. 3.
As shown in FIG. 3, the pH of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 was 4, as shown by measuring the diameter of mycelium on the plate at 6d, and at this time, it showed strong inhibition of growth of extended penicillium mycelium (diameter of mycelium was 9.4 mm); when the pH value is 7, the inhibition capability (the diameter of the mycelium is 15.3mm) of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on the growth of the expanded penicillium mycelium is obviously reduced compared with the lactobacillus plantarum CGMCC No.5494 fermentation supernatant without pH adjustment.
Example 6: effect of protease treatment on the ability of Lactobacillus plantarum (Lactobacillus plantarum) fermentation supernatant to inhibit the growth of Penicillium expansum mycelium
Adjusting the pH of the fermentation supernatant obtained in example 1 to 7 (by using 1mol/L NaOH and 1mol/L HCl), adding 1mg/mL protease (purchased from SIGMA company, product number is P3910) into the fermentation supernatant, carrying out water bath at 37 ℃ for 2h, boiling the fermentation supernatant at 100 ℃ to inactivate the enzyme for 3min, and obtaining the fermentation supernatant treated by the protease; mixing the fermentation supernatant obtained in example 1 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); mixing the fermentation supernatant with PDA culture medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA culture medium) to obtain an experimental group mixed solution with a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was added dropwise to the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of protease treatment on the ability of Lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of mycelia on each plate every 2 days, and the results are shown in FIG. 3.
As shown in FIG. 3, by measuring the diameter of the mycelia on the plate at the 6d, the inhibitory activity of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of extended penicillium (diameter of mycelia: 9.7mm) was reduced after the treatment with protease compared with the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 without the treatment with protease (diameter of mycelia: 9.4 mm).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. Application of fermentation supernatant of lactobacillus plantarum CGMCC number 5494 and/or lactobacillus plantarum CGMCC number 5494 in preparation of filamentous fungus inhibitor; the filamentous fungi are Penicillium expansum, Aspergillus niger, Penicillium roqueforti or Penicillium digitatum; the preparation method of the fermentation supernatant comprises the steps of inoculating lactobacillus plantarum CGMCC number 5494 into an MRS liquid culture medium, and culturing at 37 ℃ for 48 hours in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS liquid culture medium in an inoculation amount of 2% by volume, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; and centrifuging the fermentation liquor at 8000rpm for 10min, and filtering the fermentation liquor through a 0.2 mu m filter to obtain fermentation supernatant.
2. The use of claim 1, wherein the inhibitor component further comprises a carrier.
3. The use of claim 2, wherein the carrier is a pharmaceutically acceptable carrier.
4. Use according to claim 2 or 3, wherein the carrier is a filler, binder, wetting agent, disintegrant, lubricant and/or flavouring agent.
5. The use of claim 1, wherein the inhibitor is in the form of a powder, emulsion, granule or microgranule.
6. The use as claimed in claim 1, wherein the fermentation supernatant of lactobacillus plantarum CGMCC number 5494 is heated; the heating is carried out at 121 ℃ for 20 min.
7. A method for inhibiting filamentous fungi is characterized in that the method comprises the step of carrying out in-vitro co-culture on fermentation supernatant of lactobacillus plantarum CGMCC number 5494 and/or lactobacillus plantarum CGMCC number 5494 and the filamentous fungi; the filamentous fungi are Penicillium expansum, Aspergillus niger, Penicillium roqueforti or Penicillium digitatum; the preparation method of the fermentation supernatant comprises the steps of inoculating lactobacillus plantarum CGMCC number 5494 into an MRS liquid culture medium, and culturing at 37 ℃ for 48 hours in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS liquid culture medium in an inoculation amount of 2% by volume, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; and centrifuging the fermentation liquor at 8000rpm for 10min, and filtering the fermentation liquor through a 0.2 mu m filter to obtain fermentation supernatant.
8. The application of the fermentation supernatant of lactobacillus plantarum CGMCC number 5494 or CGMCC number 5494 in the aspect of inhibiting filamentous fungi is not aimed at the diagnosis and treatment of diseases; the filamentous fungi are Penicillium expansum, Aspergillus niger, Penicillium roqueforti or Penicillium digitatum; the preparation method of the fermentation supernatant comprises the steps of inoculating lactobacillus plantarum CGMCC number 5494 into an MRS liquid culture medium, and culturing at 37 ℃ for 48 hours in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS liquid culture medium in an inoculation amount of 2% by volume, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; and centrifuging the fermentation liquor at 8000rpm for 10min, and filtering the fermentation liquor through a 0.2 mu m filter to obtain fermentation supernatant.
9. The fermentation supernatant of lactobacillus plantarum CGMCC number 5494 or the application of the method of claim 7 in preventing fruit and vegetable spoilage; the preparation method of the fermentation supernatant comprises the steps of inoculating lactobacillus plantarum CGMCC number 5494 into an MRS liquid culture medium, and culturing at 37 ℃ for 48 hours in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS liquid culture medium in an inoculation amount of 2% by volume, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; and centrifuging the fermentation liquor at 8000rpm for 10min, and filtering the fermentation liquor through a 0.2 mu m filter to obtain fermentation supernatant.
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