CN108315266B - Paecilomyces cicadae and application thereof - Google Patents
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- CN108315266B CN108315266B CN201810184052.7A CN201810184052A CN108315266B CN 108315266 B CN108315266 B CN 108315266B CN 201810184052 A CN201810184052 A CN 201810184052A CN 108315266 B CN108315266 B CN 108315266B
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Abstract
The invention discloses a Paecilomyces cicadae and application thereof in preparing antibacterial agents, probiotic preparations and antibacterial preservative films, wherein a new strain Paecilomyces cicadae CH113.1 is separated from wild entomogenous fungi cicada fungus for the first time, a fermentation product of the strain has an inhibition effect on common pathogenic microorganisms, the inhibition rate on escherichia coli can reach more than 70%, and the biological bacteriostatic agent can be applied to food preservation. The invention shows that the strain has good application prospect in the fields of food and the like.
Description
(I) technical field
The invention relates to paecilomyces cicadae and application thereof as a bacteriostatic agent.
(II) background of the invention
Pathogenic bacteria refer to microorganisms which can cause diseases, and mainly comprise bacteria, viruses, spirochetes, rickettsiae, chlamydia, mycoplasma, fungi, actinomycetes and the like. By pathogenic bacteria is generally meant bacteria of the group of pathogenic microorganisms. The pathogenicity of bacteria is related to their virulence, number of invasion and portal of invasion. Although most bacteria are harmless and even beneficial, a significant proportion can be pathogenic and thus harmful to humans. Bacteriostatic agents are substances that inhibit the growth of bacteria or fungi. The bacteriostatic agent may not kill the bacteria, but it can inhibit the growth of the bacteria, prevent the harmful bacteria or fungi from breeding too much and endanger health. Commonly used bacteriostatic agents (Bacteriostat) are some antibiotic substances, mainly including sulfonamides, tetracycline, chloramphenicol, erythromycin, lincomycin and the like. In recent years, with the overuse of antibiotics, the drug resistance of bacteria and fungi is continuously increased, so that the curative effect of some antibiotics is reduced and multiple drug-resistant pathogenic bacteria appear, which poses great threat in clinic, and therefore, a novel antibacterial active substance is urgently needed to be screened to meet the clinical requirement.
The probiotics can inhibit harmful bacteria and balance the microecological balance of the host. The beneficial bacteria or fungi in animals are mainly: clostridium butyricum, lactobacillus, bifidobacterium, actinomycete, yeast, etc. To date, probiotics found by scientists can be broadly divided into three broad categories: lactobacillus, bifidobacterium and gram-positive coccus. With the emergence of probiotics, the bacteriostatic agent is newly explained, the biogenic bacteriostatic agent thoroughly breaks through the traditional medical treatment and clean care modes of human beings, particularly adopts active probiotics (beneficial bacteria) to inhibit the infection of pathogenic bacteria (harmful bacteria), and brings a brand-new period for the healthy development of human beings by the biological concept of beneficial bacteria bacteriostasis. Researches show that the antibacterial substance plays a role in inhibiting bacteria mainly by destroying cell membranes, cell walls and DNA structures of microorganisms and influencing the ways of gene expression, cell respiration, energy metabolism and the like.
Currently, most of bacteriostats used in the market are chemical preparations which can cause certain damage to human bodies and the environment, and biological bacteriostats gradually become the best substitute of chemical bacteriostats due to the characteristics of low toxicity, high efficiency and environmental protection. Based on the background, the research on novel biogenic bacteriostats is bound to become one of the research hotspots in the field. Cordyceps sobolifera (Cordyceps cicadae), also known as Cordyceps cicadae, refers to a complex formed by the fungus Paecilomyces cicadae (Paecilomyces cicadae) of Cordyceps (Cordyceps) of Clavicitaceae parasitizing on the nymph cicadae. Paecilomyces cicadae was regarded as one of the "eight delicacies of Chinese medicine". The cordyceps sobolifera is one of three traditional medicinal cordyceps sinensis in China, is similar to active ingredients of cordyceps sinensis, contains various active ingredients such as fungal polysaccharide, cordycepic acid, cordycepin, adenosine and the like, and has the effects of regulating immunity, resisting tumors, resisting aging, improving kidney functions, reducing blood sugar, inhibiting bacteria and the like. Therefore, the subject group screens the paecilomyces cicadae with good antibacterial activity from wild cordyceps sobolifera, researches the biological activity of the paecilomyces cicadae, discusses the application of the paecilomyces cicadae in food preservation and provides a theoretical basis for deep development of cordyceps sobolifera-derived biological antibacterial products.
Disclosure of the invention
The invention aims to provide a new strain, namely Paecilomyces cicadae CH113.1 and application of a fermentation product thereof as a bacteriostatic agent, wherein the fermentation product of the strain has the activity of inhibiting pathogenic microorganisms such as staphylococcus aureus, bacillus subtilis, escherichia coli and the like, can destroy the cell structure of the escherichia coli, change the wall membrane permeability of the escherichia coli and destroy the mycoplasmal membrane protein. In addition, the invention researches and determines the formula of the paecilomyces cicadae bacteriostatic agent and discusses the application of the bacteriostatic agent in the bacteriostatic preservative film.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-Paecilomyces cicadae (Paecilomyces cicadae) CH113.1, which is preserved in China center for type culture collection with the preservation date of 2017, 04 months and the preservation number of CCTCC NO: m2017004, the preservation address is Wuhan, Wuhan university, postcode 430072.
The invention also provides an application of the paecilomyces cicadae CH113.1 in preparing an antibacterial agent, wherein the antibacterial agent is a bacillus subtilis antibacterial agent, a staphylococcus aureus antibacterial agent or an escherichia coli antibacterial agent. The antibacterial agent in the supernatant obtained by fermentation culture of the paecilomyces cicadae CH113.1 can destroy the cell structure of escherichia coli, change the wall membrane permeability of the escherichia coli and destroy mycoplasmal membrane proteins. The minimum inhibitory concentration of the antibacterial agent in the Paecilomyces cicadae CH113.1 fermentation supernatant to escherichia coli is 0.050 mg/mL.
The antibacterial agent is obtained by centrifuging fermentation liquor obtained by fermenting and culturing paecilomyces cicadae CH113.1 at 28 ℃ and 200rpm, and separating and purifying supernatant a.
The antibacterial agent is prepared by the following method:
(1) slant culture: inoculating Paecilomyces cicadae CH113.1 to a slant culture medium, and culturing at 28 ℃ for 5 days to obtain slant thallus; the slant culture medium comprises the following components: 200g/L of potato, 20g/L of glucose, 10g/L of agar powder and a natural pH value, wherein the solvent is tap water;
(2) fermentation culture: selecting one strain of thallus from the inclined plane thallus, inoculating the thallus to a fermentation culture medium, culturing at 28 ℃ and 200rpm for 5d, centrifuging the fermentation liquor (at 4 ℃ and 12000g for 30min), and collecting supernatant a; the fermentation medium comprises the following components: 200g/L of potato, 20g/L of glucose and a natural pH value, wherein the solvent is tap water.
(3) Antibacterial agents: taking 50mL of supernatant a, adding 3 times of volume of absolute ethyl alcohol, and standing overnight at 4 ℃ to precipitate polysaccharide; centrifuging at 4 deg.C and 12000g for 30min, collecting supernatant b, evaporating to dryness with rotary evaporator, dissolving with sterile water, slowly adding ammonium sulfate at 4 deg.C to make the final concentration of ammonium sulfate be 60%, standing overnight; centrifuging at 4 deg.C and 12000g for 30min, collecting supernatant b, evaporating to dryness with rotary evaporator, dissolving with sterile water, slowly adding ammonium sulfate at 4 deg.C to make the final concentration of ammonium sulfate be 60%, standing overnight; centrifuging at 4 deg.C and 12000g for 30min, respectively collecting supernatant c and precipitate c, and filtering the supernatant c with 0.22 μm filter membrane for sterilization to obtain residue a; dissolving the filter residue a with sterile water, and filtering with a 0.45-micrometer filter membrane to obtain a filter residue b; freeze-drying the filter residue b, dissolving the filter residue b by using 0.050mol/L NaAc-HAc buffer solution, loading the filter residue b on a CM-fiber column 52, balancing the column by using pH 4.0-5.0 and 0.010-0.10 mol/L NaAc-HAc buffer solution, carrying out gradient elution by using pH 5.0-5.5 and 0.10-0.50 mol/L NaAc-HAc buffer solution, collecting target components (preferably one tube per 10mL for determining antibacterial activity, combining and collecting activity peaks), carrying out ultrafiltration concentration by using an ultrafiltration membrane with the molecular weight cut-off value of 1000Da to obtain a cut-off solution a, dissolving the cut-off solution a by using distilled water after freeze-drying at the temperature of-50 ℃, loading the cut-off solution a Sephadex G-25/G-50 column, determining the eluent by using pH 6.0-7.0.010-0.10 mol/L phosphate buffer solution, collecting target components (preferably one tube per 10mL for determining the antibacterial activity, combining and collecting activity peaks) and carrying out ultrafiltration concentration by using the molecular weight cut, freeze-drying the obtained trapped fluid b at-50 ℃, dissolving the trapped fluid b with distilled water, performing preparative HPLC, eluting with acetonitrile water solution with volume concentration of 10-80% and volume concentration of 0.010% TFA, collecting target components (preferably, each 5.0mL of the target components is a tube, measuring antibacterial activity, combining and collecting activity peaks), freeze-drying at-50 ℃, and storing at-20 ℃ to obtain the antibacterial agent.
The invention also provides application of the paecilomyces cicadae CH113.1 in preparation of probiotic preparations.
The invention also relates to application of the paecilomyces cicadae CH113.1 in preparation of the bacteriostatic preservative film, wherein the bacteriostatic preservative film is prepared from a bacteriostatic agent obtained by centrifuging fermentation liquor obtained by fermenting and culturing the paecilomyces cicadae CH113.1 at 28 ℃, 200rpm, and separating and purifying supernatant a.
Further, the antibacterial preservative film is prepared by the following steps: dissolving dry pectin in distilled water, adding an antibacterial agent and glycerol, uniformly mixing to prepare a membrane liquid, fully stirring, vacuum degassing, pouring into the membrane liquid, paving, and then drying in a vacuum drying oven at 40 ℃ to obtain the antibacterial preservative membrane; the mass contents of pectin and glycerol in the membrane liquid are respectively 2.5% and 3.0%, and the addition amount of the bacteriostatic agent is 0.050 mg/mL. Preferably, the amount of the film is 50.0g, the film is peeled and put into a closed container for standby, and the thickness of the film is 0.15 mm.
Compared with the prior art, the invention has the following beneficial effects: the new strain Paecilomyces cicadae CH113.1 is separated from wild entomogenous fungus cicada fungus for the first time, the fermentation product of the strain has the inhibition effect on common pathogenic microorganisms, the inhibition rate on escherichia coli can reach more than 70%, and the biological bacteriostatic agent can be applied to food preservation. The invention shows that the strain has good application prospect in the fields of food and the like.
(IV) description of the drawings
FIG. 1 molecular biological identification of Cordyceps sobolifera fungi; a: gel electrophoresis picture of rDNA-ITS amplification product, M is standard protein, ITS is PCR product of strain CH113.1 genome amplification; b: sequencing the target fragment; c: blast sequence alignment result D: phylogenetic tree of strain CH113.1, X represents isolated and screened cicada fungus.
FIG. 2 morphological observation of Paecilomyces cicadae; a is the growth form of paecilomyces cicadae on a PDA culture medium; B-D are scanning electron micrographs of paecilomyces cicadae, which are respectively 5K, 7K and 8K times.
FIG. 3 shows the bacteriostasis spectrum of Paecilomyces cicadae, wherein A is fermentation medium and B is Paecilomyces cicadae fermentation liquid.
FIG. 4 effect of Paecilomyces cicadae bacteriostats on E.coli structure; a is control (sterile water-treated E.coli); B-D are electron micrographs of Escherichia coli treated by the paecilomyces cicadae bacteriostat and magnified by 5K, 7K and 8K times respectively.
FIG. 5 influence of the bacteriostatic agent of Paecilomyces cicadae on AKP activity of Escherichia coli liquid.
FIG. 6 the effect of a Paecilomyces cicadae bacteriostatic agent on the total protein of E.coli bacteria; m is standard molecular weight protein, A is water treatment, B is 16 times of MIC treatment, C is 8 times of MIC treatment, and D is 4 times of MIC treatment.
FIG. 7 Effect of Paecilomyces cicadae bacteriostats on extracellular protein concentration of E.coli.
FIG. 8 shows the effect of the bacteriostatic agent of Paecilomyces cicadae on the conductivity of the Escherichia coli liquid.
FIG. 9 influence of the bacteriostatic agent against Paecilomyces cicadae on the activity of Escherichia coli bacterial liquid β -galactosidase.
FIG. 10 Effect of Paecilomyces cicadae bacteriostats on E.coli disrupted membrane proteins; m is standard molecular weight protein, A is water treatment, B is 16 times of MIC treatment, C is 8 times of MIC treatment, and D is 4 times of MIC treatment.
FIG. 11 shows the effect of a bacteriostatic agent against Paecilomyces cicadae on the expression level of a functional gene of Escherichia coli.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
EXAMPLE 1 isolation, purification and characterization of the Strain
Operation (a)
1. Strain isolation and selection
Cleaning the surface of the collected fresh cordyceps sobolifera, soaking the cordyceps sobolifera in 0.10% mercuric chloride aqueous solution for 1min, soaking the cordyceps sobolifera in 75% ethanol aqueous solution for 1min for disinfection, sucking the cordyceps sobolifera dry by using sterile filter paper, performing tissue separation by using a sterile scalpel, inoculating the cordyceps sobolifera on a potato glucose agar (PDA) plate (containing 10 mu g/mL chloramphenicol), and culturing the cordyceps sobolifera at 28 ℃ for 5 d. Separating strain from cultured Cordyceps sobolifera sample by scribing and coating, respectively selecting strains with different forms, inoculating on new PDA plate, culturing at 28 deg.C for 3d, and continuously separating for 3 times to obtain pure strain. After the strain is subjected to amplification culture, inoculating the strain to a PDA liquid culture medium, fermenting and culturing at 28 ℃ and 160rpm for 5d, centrifugally collecting fermentation metabolites, taking escherichia coli, staphylococcus aureus and bacillus subtilis as indicator bacteria, and screening a cordyceps sobolifera strain with an antibacterial effect by an Oxford cup method. Gently placing a sterile oxford cup into an LB solid culture medium flat plate containing 1.0% of indicator bacteria, sucking 200 mu L of fermentation metabolite into the oxford cup, adding an equivalent amount of PDA culture medium into a control group, standing at 4 ℃ until a sample completely permeates into the culture medium, culturing at 37 ℃ for 24h, observing the size of a bacteriostatic zone around the oxford cup, and screening a strain CH113.1 with bacteriostatic activity.
In the above screening method, the following media are involved:
(1) broth (Luria-Bertani, LB) liquid (solid) medium
5.0g of tryptone, 10g of yeast extract, 10g of sodium chloride and 10g of agar powder, adding water to a constant volume of 1.0L, sterilizing at 121 ℃ and 0.10MPa for 20min, and storing at 4 ℃.
(2) Potato Dextrose (PDA) liquid (solid) culture medium
200g of potato, 20g of glucose and 10g of agar powder, peeling the potato, cutting, adding water, boiling for 20min, filtering by using double-layer gauze, taking supernate, adding 20g of glucose, adding water to a constant volume of 1.0L, sterilizing at 121 ℃, 0.10MPa for 20min, and storing at 4 ℃.
2. Identification of bacteria
2.1 genomic DNA extraction
Extracting strain CH113.1DNA with fungus genome extraction kit, taking 20 μ g fresh hypha, transferring into 1.5mL centrifuge tube, adding 400 μ L Buffer Digestion and 4.0 μ L β -mercaptoethanol, shaking and mixing the two, keeping the temperature at 65 ℃ for 1h until the cells are completely cracked, immediately adding 200 μ L Buffer PF (split-phase Buffer), mixing, placing in 20 ℃ refrigerator for 5min, centrifuging at room temperature 10000g for 5min, transferring the supernatant (500-550 μ L) into a new 1.5mL centrifuge tube, adding equal volume of isopropanol, mixing thoroughly, placing at room temperature for 2-3min, centrifuging at room temperature 10000g for 5min, discarding the supernatant, adding 1.0mL 75% ethanol, rinsing for 1-3min, centrifuging at 10000g for 2min, discarding the supernatant, adding 1.0mL 75% ethanol, rinsing for 1-3min, 10000g for 2min, discarding the supernatant, repeating once, after the residual ethanol is completely volatilized, using 50 μ L EDTA (EDTA-Buffer) to dissolve DNA, and centrifuging at-20 ℃ to dissolve DNA.
2.2ITS fragment amplification
rDNA-ITS sequences the universal primers ITS1 and ITS4 were used, and the primer sequences are shown in Table 1.
TABLE 1 fungal ITS identification primers
PCR amplification System (20. mu.L): taq PCR StarMix: 10 mu L of the solution; primer (10. mu. mol/L): 1.0. mu.L each; DNA: 1.0 μ L; ddH2O：7.0μL。
PCR amplification procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing for 30s, and extension at 72 ℃ for 1min for 35 cycles; final extension at 72 ℃ for 5 min. The amplification products were detected by electrophoresis on a 1.0% agarose gel containing ethidium bromide.
2.3PCR product purification
(1) Electrophoresis: the PCR amplification product was electrophoresed on 1.0% agar gel and spotted at 20. mu.L.
(2) Cutting the glue: after completion of electrophoresis, the desired fragment was cut out rapidly under an ultraviolet lamp, the DNA-free portion of the agarose gel edge was removed, and the gel was cut up.
(3) Weighing: the gel block was placed in a pre-weighed 1.5mL centrifuge tube, and the weight was weighed to calculate the gel weight. The mass of glue in each tube should not exceed 700 mg.
(4) Sol: adding 1.0 μ L of membrane binding solution into 1.0mg gel at a ratio of 1:1, mixing, standing at 55 deg.C, and mixing once every 1-2min until the gel block is completely dissolved (about 5 min).
(5) DNA binding: and after the gel solution is cooled to room temperature, transferring the gel solution into a centrifugal adsorption column inserted into the collecting pipe, standing for 1min, centrifuging at 12000g for 1min at room temperature, discarding waste liquid in the collecting pipe, and reinserting the centrifugal adsorption column into the collecting pipe.
(6) Cleaning: adding 600 μ L of membrane rinsing liquid into centrifugal adsorption column, centrifuging at room temperature 12000g for 30s, discarding waste liquid in collection tube, and reinserting centrifugal adsorption column into collection tube.
(7) And (3) cleaning again: adding 600 μ L of membrane rinsing liquid into centrifugal adsorption column, centrifuging at room temperature 12000g for 30s, discarding waste liquid in collection tube, and reinserting centrifugal adsorption column into collection tube. The centrifugal adsorption column was uncapped and centrifuged again for 2min to completely remove the residual rinse.
(8) And (3) elution: the centrifugation and adsorption column was carefully removed and put into a 1.5mL centrifuge tube, 30. mu.L of elution buffer was added to the center of the silica gel adsorption membrane, and after standing at room temperature for 1min, 12000g was centrifuged for 1min to collect the purified DNA fragment.
(9) And (3) storage: the centrifugal adsorption column was discarded and the obtained DNA fragments were used for subsequent experiments or stored at-20 ℃.
2.4 sequencing and alignment
The purified target DNA is sent to Shanghai Sangni company for sequencing, and the nucleotide sequence is shown as SEQ ID NO. 1. Performing Blast gene comparison in GenBank (http:// blast.ncbi.nlm.nih.gov /) to obtain a sequence with higher similarity; performing multiple sequence alignment by CLUSTALX, constructing phylogenetic tree by MEGA 6.0 software, and identifying strains.
2.5 morphological Observation of strains
Inoculating the screened strain CH113.1 on a new PDA plate, culturing at the constant temperature of 28 ℃ for 5-7 days, and carrying out morphological observation after spores grow out. In addition, a fungal colony is cut from the culture medium by a blade, is fixed for 20min by glutaraldehyde aqueous solution with the volume concentration of 2.5%, is washed for 2 times and 15min each time by double distilled water, is subjected to ethanol gradient dehydration (sequentially dehydrated by 50%, 70%, 80% and 90% of ethanol for 10-15min each time and then dehydrated by 100% of ethanol for 3 times and 10-15min each time), is subjected to critical drying of isoamyl acetate, is sprayed with gold by an ion plating instrument, and is observed by a scanning electron microscope.
(II) conclusion
1 molecular biological identification of Paecilomyces cicadae
A fungus DNA rapid extraction kit is used for extracting a strain CH113.1DNA, and universal primers ITS1 and ITS4 are used for amplifying a fungus conserved sequence rDNA-ITS. The amplified product was detected by 1.0% agarose gel electrophoresis containing EB, and the size of the PCR product was found to be about 600bp (A in FIG. 1). After the target DNA is purified and recovered, it is sent to Shanghai Sangni Biotech Co., Ltd for sequencing. And performing Blast gene comparison in GenBank on the sequencing result (B in figure 1), obtaining a sequence with higher similarity, performing multiple sequence comparison through CLUSTALX, and constructing a phylogenetic tree (D in figure 1) through MEGA 6.0 software on the comparison result. The result shows that the matching rate of the strain CH113.1 obtained by separation and screening and cicada fungus (Paecilomyces cicadae) BA-001 reaches 99 percent (C in figure 1).
Morphological observation of Paecilomyces cicadae
As shown in A in figure 2, bacterial colony of the strain CH113.1 is white, has flat and felty hypha, is tightly combined with the culture medium, is not easy to pick, has tight texture, is in radial furrow and oval shape, and has beige spore. The shapes of the hyphae and spores of the paecilomyces cicadae were observed under an electron microscope, and as shown in B in FIG. 2, the hyphae had no septa, had many branches, and had no podocytes at the base. The upper end of the hyphae generates bottle peduncles with expanded bases deviating from the main shaft, the bottle peduncles are different in length, partial tops of the bottle peduncles germinate in a herringbone shape, and conidiophore branches generate broom-shaped bottle peduncles (C in figure 2). Conidia are round, arranged in cluster or chain shape, distributed on spore peduncles or vegetative hyphae, and have no germ tube and attached cell, and no mucus layer on spore surface (D in FIG. 2).
In conclusion, the strain CH113.1 is identified as Paecilomyces cicadae (Paecilomyces cicadae) named as Paecilomyces cicadae CH113.1, is preserved in China center for type culture Collection with the preservation date of 2017, day 01 and day 04, and the preservation number is CCTCCNO: m2017004, the preservation address is Wuhan university in Wuhan, China.
EXAMPLE 2 preparation of Paecilomyces cicadae CH113.1 bacteriostatic agent
(1) Slant culture: the paecilomyces cicadae CH113.1 is inoculated to a slant culture medium and cultured for 5 days at the temperature of 28 ℃ to obtain slant thalli. The slant medium was PDA solid medium, and the composition was the same as in example 1.
(2) Fermentation culture: selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 28 ℃ and 200rpm for 5d to obtain a seed solution; the seed medium was PDA liquid medium and the composition was the same as in example 1.
(3) Inoculating the seed solution into a fermentation culture medium at a volume concentration of 5.0%, and fermenting at 28 deg.C and 200rpm for 5d to obtain a fermentation solution. The fermentation medium was PDA broth and the composition was the same as in example 1.
(4) Bacteriostatic agent: filtering hypha of the fermentation liquor of the paecilomyces cicadae CH113.1 prepared in the step (3) by using double-layer gauze, centrifuging the filtrate at 4 ℃ for 30min at 12000g, and collecting supernatant a to obtain a fermentation metabolite of the paecilomyces cicadae CH 113.1; taking 50mL of supernatant a, adding 3 times of volume of absolute ethyl alcohol, and precipitating polysaccharide at 4 ℃ overnight; centrifuging at 4 deg.C and 12000g for 30min, collecting supernatant b, evaporating to dryness with rotary evaporator, and dissolving with sterile water; slowly adding ammonium sulfate into the re-dissolved sample at 4 ℃ until the final mass concentration of the ammonium sulfate is 60%, and standing overnight; centrifuging at 4 ℃ and 12000g for 30min, respectively collecting supernatant c and precipitate c, filtering and sterilizing the supernatant c with a 0.22 mu m filter membrane, collecting filter residue a, dissolving the filter residue a with 5.0mL of sterile water, and then filtering with a 0.45 mu m filter membrane to obtain filter residue b. Freeze-drying the filter residue b at-50 ℃, dissolving the filter residue b by using 0.50mol/L NaAc-HAc buffer solution, applying the filter residue b to a CM-fiber column 52, performing gradient elution by using a balance solution of pH 4.0-5.0, 0.010-0.10 mol/L NaAc-HAc buffer solution and an eluent of pH 5.0-5.5 and 0.10-0.50 mol/L NaAc-HAc buffer solution (the gradient is 0.10, 0.20, 0.30, 0.40 and 0.50mol/L), determining antibacterial activity by using one tube per 10mL, combining and collecting activity peaks, performing ultrafiltration concentration on the activity peaks, dissolving the obtained retention solution a by using distilled water after freeze-drying at-50 ℃, applying the retention solution a Sephadex G-25/G-50 column, wherein the elution condition is pH 6.0-7.0, 0.010-0.10 mol/L phosphate buffer solution (the gradient is 0.010, 0.030, 0.020, 0.080, 0.040, 0.090, 0.10mol/L), measuring antibacterial activity by taking one tube per 10mL, combining and collecting activity peaks, ultrafiltering and concentrating the activity peaks by an ultrafiltration membrane with the molecular weight cut-off value of 1000Da, freeze-drying the obtained cut-off solution b at the temperature of minus 50 ℃, dissolving the freeze-dried cut-off solution b by using distilled water, applying preparative HPLC, eluting by using acetonitrile water solution with the volume concentration of 10-80% and the volume concentration of 0.010% TFA (the gradient is 10, 20, 30, 40, 50, 60, 70 and 80%), taking one tube per 5.0mL, measuring the antibacterial activity, combining and collecting the activity peaks, freeze-drying the activity peaks at the temperature of minus 50 ℃, and storing at the temperature of minus 20 ℃ to obtain the Paecilomyces cicadae CH113.1 bacteriostatic agent prepared from Paecilomyces cicadae CH113.1 fermentation.
EXAMPLE 3 bacteriostatic Properties of Paecilomyces cicadae CH113.1 bacteriostatic Agents
Escherichia coli, bacillus cereus, staphylococcus aureus, staphylococcus epidermidis, streptococcus pyogenes, enterococcus faecalis, pseudomonas aeruginosa, klebsiella pneumoniae, salmonella paratyphi B, candida albicans, alternaria alternate, penicillium expansum and aspergillus niger are used as indicator bacteria, and the antibacterial activity of the paecilomyces cicadae CH113.1 antibacterial agent is measured by an Oxford cup method. Taking OD600An indicator LB plate is prepared for 0.80 indicator bacterium liquid according to the inoculation amount of 2.0% of volume concentration, Oxford cups are horizontally and uniformly placed on the plate, 200 mu L of the sterile aqueous solution of the Paecilomyces cicadae CH113.1 bacteriostatic agent prepared in example 2 is added into an experimental group, after standing for 2h at 4 ℃, the mixture is cultured overnight at 37 ℃, the diameter of a bacteriostatic ring is measured, and a bacteriostatic map is established, wherein the result is shown in Table 2.
TABLE 2 antibacterial spectrum of Paecilomyces cicadae bacteriostat
aNo zone of inhibition-diameter<15mm is + and the diameter of the bacteriostatic circle is 15-20mm ++, and the diameter of the bacteriostatic circle is>20mm is ++
TABLE 3 information relating to pathogenic bacteria
Example 4 Effect of Paecilomyces cicadae bacteriostats on Escherichia coli Structure
The bacterial liquid of escherichia coli (CMCC44115) treated by the bacteriostatic agent CH113.1 prepared in example 3 (dissolved in sterile water) for 9h is centrifuged, the thalli is collected and washed 3 times by PBS with 0.10mol/L and pH 7.2 for 30min each time, the supernatant is discarded by centrifugation, 1.0mL of glutaraldehyde aqueous solution with the volume concentration of 4.0% is added for fixation, the thalli is shaken evenly and fixed at 4 ℃ for 72h, the fixed thalli is taken out, placed for 2h at room temperature and washed 3 times by PBS with 0.10mol/L and pH 7.2 for 30min each time. Preparing thallus suspension with PBS, uniformly coating a small amount of thallus suspension on a glass slide, carrying out vacuum freeze drying for 5h at-20 ℃, sputtering gold spraying on a dehydrated and dried specimen under vacuum, and observing thallus cell morphology by a scanning electron microscope with the working voltage of 25 kV.
As shown in FIG. 4, A in FIG. 4 is untreated control group Escherichia coli, the thallus cells are complete and in a typical rod shape or a short rod shape, the cell surface is smooth, the thallus is relatively dispersed, while part of the Escherichia coli cells treated by the bacteriostatic substance are in a long rod shape, the thallus is relatively aggregated, the middle of part of the thallus is sunken (B and C in FIG. 4), the membrane surface is incomplete, part of the thallus is swelled, the shape is distorted, the cell membrane becomes wrinkled, and the thallus is seriously damaged (D in FIG. 4).
Example 5 Effect of Paecilomyces cicadae bacteriostats on the stability of E.coli cell walls
Escherichia coli (CMCC44115) was treated with a Paecilomyces cicadae CH113.1 antibacterial agent (sterile water-soluble) prepared in example 3 at concentrations of 4 × MIC, 8 × MIC and 16 × MIC, respectively, and treated with sterile water for 9h, and a kit (Byunshi Biotechnology Co., Ltd., product No. P0321) was used to detect extracellular AKP activity.Eschericia extracellular AKP content was calculated by using a standard curve obtained by correlating the absorbance with the AKP concentration using a standard substance (10mM paranitrophenol) in the kit.y 0.2165x + 0.0054. the change in extracellular AKP content in Escherichia coli cell is reflected in the damage of the bacterial cell wall.A change in the extracellular AKP content of Escherichia coli was calculated by using a standard curve of AKP for 9h which the antibacterial agent was treated with 4 × MIC, 8 × MIC and 16 × MIC and the concentration of the control group AKP was 0.134 mM and 1 mM respectively, and the enzyme activity was calculated as a change in the enzyme activity of the corresponding to 1. mu. the enzyme activity, and the enzyme activity was defined as a graph.
Example 6 Effect of Paecilomyces cicadae bacteriostats on the permeability of E.coli cell membranes
1 Total protein of the thallus
Escherichia coli (CMCC44115) was treated with 4 × MIC (D in FIG. 6), 8 × MIC (C in FIG. 6) and 16 × MIC (B in FIG. 6) each with the bacteriostatic CH113.1 (dissolved in sterile water) prepared in example 3 for 9h, the control group was treated with sterile water (A in FIG. 6), the culture was centrifuged at 5000g for 10min, the supernatant was discarded, the cells were washed 2 times with 0.10mol/L of pH 7.2 PBS, and then resuspended in cell lysate (RIPA lysate, Biyuntian Biotech Ltd., product No. P001 0013C) to make OD into OD600Adding 400 mu L protease inhibitor (PMSF) and 80 mu L lysozyme, incubating at 37 ℃ for 40min, adding 20 mu L DNase/RNase, culturing at 37 ℃ for 10min, centrifuging at 4 ℃ and 3000g for 30min, collecting supernatant, and placing in a new tube to obtain the total protein of the thallus. The mycoprotein was detected by 10% SDS-Page (10% separation gel: 4671. mu.L ddH)2O; 2625 μ L of 40% Acryl/Bis; 2500 μ L of 1.5M Tris-HCl, pH 8.8; 100 μ L10% SDS; 100 μ L of 10% APS; 4.0 μ L TEMED. 4.0% lamination glue: 3791 μ L ddH2O；582μL 40％Acryl/Bis；1500μL 0.5M Tris-HCl，pH6.8；60μL 10％SDS；60μL 10％APS；6.0μL TEMED)。
As shown in FIG. 6, after treatment with different concentrations of the Paecilomyces cicadae antibacterial agent, the total protein of the Escherichia coli thallus is slightly different, and compared with the untreated control group, the total protein of the Escherichia coli thallus has a partially obvious light band, and the phenomenon is more obvious when the concentration of the antibacterial substance is higher. After being treated by the high-concentration antibacterial substance, the band above 97.4kDa of the total protein of the extracted escherichia coli becomes obviously shallow, which shows that the antibacterial substance can influence the normal expression of mycoprotein or influence the function of the mycoprotein, and change the synthesis of certain key structures or functional proteins of the mycoprotein.
Extracellular protein concentration
Escherichia coli (CMCC44115) was treated with the bacterial inhibitors CH113.1 (in sterile water) prepared in example 3 at concentrations of 4 × MIC, 8 × MIC and 16 × MIC, respectively, for 9h, and the control was treated with sterile water to determine extracellular protein concentration, according to OD562And protein concentration standard curve y is 1822.6x-7.2015 (protein standard curve is prepared by taking 8 centrifuge tubes of 1.5mL, adding BSA protein standard solution and PBS with corresponding volume into each centrifuge tube, and mixing well to obtain protein standard with different concentrationsAdding 1.0mL BCA working solution into the solution, mixing, keeping the temperature at 60 deg.C for 30min, cooling to room temperature, and measuring OD562The value is obtained. By OD562The abscissa and the ordinate represent protein concentration, and a standard curve equation of protein content is plotted, wherein y is 1822.6x-7.2015, R20.9929), calculating the concentration of the escherichia coli extracellular protein after the antibacterial peptide treatment.
As is clear from FIG. 7, the extracellular protein concentration of E.coli after the treatment was higher than that of the control group, and the extracellular protein concentration was higher as the bacteriostatic agent concentration was higher. The results show that the bacteriostatic active substances in the paecilomyces cicadae fermentation liquor can block the expression of partial proteins of the escherichia coli, and the bacteriostatic substances can also damage the cell wall membrane structure of the escherichia coli, so that the permeability of the escherichia coli is increased, intracellular proteins are caused to be dissociated to the outside of cells, and the bacteriostatic action is exerted.
3 conductivity of
Logarithmic phase E.coli (CMCC44115) was washed 3 times with 0.10M, pH7.4PBS and the concentration of the bacterial solution was adjusted to 108CFU/mL is inoculated into LB liquid culture medium according to the inoculation amount with the volume concentration of 2.0%, 5.0mL of the bacterial inhibitor of Paecilomyces Cicadae CH113.1 (dissolved in sterile water) prepared in example 3 with 4 × MIC, 8 × MIC and 16 × MIC is respectively added, the same amount of sterile water is added into a control group, fermentation and culture are carried out for 9h at 37 ℃ and 200rpm, and the conductivity of each bacterial liquid is measured by a conductivity meter.
4 β -galactosidase Activity
Respectively treating Escherichia coli (CMCC44115) with bacteriostatic agents of Cicada penicillium CH113.1 (dissolved in sterile water) with concentrations of 4 × MIC, 8 × MIC and 16 × MIC prepared in example 3 for 9h, treating with sterile water as a control, detecting extracellular β -galactosidase activity of Escherichia coli (10 mL of indicator bacterium liquid cultured overnight, centrifuging at 4 deg.C and 3000g for 10min to obtain precipitate as Escherichia coli thallus, transferring the thallus to 100mL of M9 lactose-induced medium, culturing at 37 deg.C and 200rpm for 9h, centrifuging at 4 deg.C and 3000g for 10min, washing the thallus with sterile physiological saline for 2 times, and resuspending in 100mL of β -galactosidase buffer solution to make OD630About 0.20. taking 4.0mL β -galactosidase buffer solution containing Escherichia coli, adding 0.50mL Cordyceps sobolifera polysaccharide solution and 0.50mL o-nitrobenzene- β -D-galactopyranoside (ONPG, 1.0mg/mL) in sequence, mixing well, culturing at 37 deg.C and 160rpm, measuring OD once every 30min415) When the cell membrane is disrupted, a large amount of β -galactosidase is released extracellularly and reacts immediately with ONPG to produce yellow ONP, and thus, the stability of the cell membrane can be assessed by indicating a change in extracellular β -galactosidase activity.
As can be seen from FIG. 9, the enzyme activity of the control group is almost unchanged and maintained at about 30U, but the enzyme activity of the indicator bacteria treated by the antibacterial substance is remarkably enhanced, and the result shows that the antibacterial substance can destroy the cell membrane of the Escherichia coli and release the substances in the Escherichia coli into the culture medium.
Example 7 Effect of Paecilomyces cicadae bacteriostats on E.coli disrupted membrane proteins
Escherichia coli (CMCC44115) was treated with 4 × MIC (D in FIG. 10), 8 × MIC (C in FIG. 10) and 16 × MIC (B in FIG. 10) each with the bacteriostatic agent of Paecilomyces cicadae CH113.1 (dissolved in sterile water) prepared in example 3 (FIG. 10) for 9h, and the control was treated with sterile water (A in FIG. 10), and 4500g was centrifuged for 15min to obtain the cells, washed with 1 × PBS for 2 times, and resuspended in 4.0mL of PBS containing 8.0% Triton X-114, and the cell suspension was stored at 4 ℃ for 3h, and the supernatant was centrifuged, after culturing at 37 ℃ for 2h, at room temperature, 3000g was centrifuged for 15min, and the bottom organic phase was taken, and 36mL of anhydrous ethanol was added, and stored at 4 ℃ for 10 h.4 ℃, 10000g was centrifuged for 30min, and the precipitate was taken, i.e., the cell membrane protein, and the extracted membrane protein was dissolved in 20. mu.l of sterile water, and then dissolved in 5.0. mu.l of 5 × loading buffer, and mixed.
As shown in FIG. 10, the antibacterial peptide preparation of Paecilomyces cicadae changes the permeability of cell membrane by destroying the protein of the cell membrane. The contrast histone component is blank and is obviously different from the treated experimental histone component, which shows that the paecilomyces cicadae antibacterial peptide preparation can destroy cell membranes and promote the extraction of cell membrane protein, namely, bacteriostatic substances destroy the structure of escherichia coli cell membranes, thereby playing a role in bacteriostasis. Meanwhile, the higher the concentration of the antibacterial peptide is, the greater the damage degree to the cell membrane is.
Example 8 Effect of Paecilomyces cicadae bacteriostats on E.coli Gene expression
Sample treatment, namely, treating Escherichia coli (CMCC44115) with sterile water and the bacterial inhibitors of Paecilomyces cicadae prepared in example 3 at concentrations of 4 × MIC, 8 × MIC and 16 × MIC for 9h, centrifuging to collect thalli, and respectively carrying out sample RNA extraction at the indexes of A, B, C and D of every 5 × 107Adding 0.50mL of Trizol into each bacterial cell, standing the cracked sample at room temperature for 5-10min to completely separate the nucleoprotein and the nucleic acid, adding 0.20mL of chloroform, violently shaking for 30s, standing at room temperature for 3min, centrifuging at 4 ℃, 12000g for 10min, absorbing the upper aqueous phase, transferring the upper aqueous phase into a centrifuge tube, adding 0.5-time volume of absolute ethyl alcohol, and uniformly mixing to obtain a sample to be detected. According to the using instruction of the adsorption column, the adsorption column (UNIQ-10, unprocessed Sangon Biotech) is placed in a collecting pipe, is kept stand for 2min, is centrifuged at 12000g for 3min, and is discarded with waste liquid in the collecting pipe. The adsorption column was returned to the collection tube, 500. mu.L of RNA binding Solution (RPE Solution) was added, left to stand for 2min, centrifuged at 10000g for 30s, the waste Solution in the collection tube was discarded, and the procedure was repeated 2 times. Placing the adsorption column into the collection tube, centrifuging at 12000g for 2min, discarding the waste liquid in the collection tube, placing the adsorption column into the centrifuge tube, and adding 30 μ L DEPC-treated ddH into the center of the adsorption membrane2And O, standing for 5min, centrifuging 12000g for 2min, and storing the obtained RNA solution at-70 ℃ for subsequent experiments. Reverse transcription: first strand cDNA Synthesis: RNA Total RNA (determined by the concentration of extracted RNA), 1.0. mu.L Random Primer p (dN) was added to a 800ng reverse-transcribed, ice-cooled, nuclear-free PCR tube6(100pmoL), 1.0. mu.L dNTP Mix (0.50mM final concentration), with RNase-free ddH2O constant volume is 14.5 mu L, the mixture is gently mixed and then centrifuged for 3-5 s, the reaction mixture is subjected to warm bath at 65 ℃ for 5min, ice bath is carried out for 2min, then centrifugation is carried out for 3-5 s, ice bath is carried out, and then 4.0 mu L of 5 × RTBuff is addeder, 0.50. mu.L Thermo Scientific riboLock RNase Inhibitor (20U), 1.0. mu.L RevertaIdpremium Reverse Transcriptase (200U), gently mixed and centrifuged for 3-5 s, and Reverse transcription reaction was performed on a PCR instrument: incubating at 25 deg.C for 10 min; synthesizing cDNA at 50 ℃ for 30 min; the reaction was terminated at 85 ℃ for 5min and the solution was stored at-20 ℃. Fluorescent quantitative PCR: the sample was applied to a 96-well plate as shown in Table 4, and the cDNA was diluted 10-fold to prepare a target computer reaction. And (3) taking 16S-rDNA as an internal reference gene, repeating each gene by 3 techniques, and determining the relative gene expression amounts of the escherichia coli argC, zraP, fliP and dkgA after treatment by the bacteriostatic agent. Setting PCR amplification program as 95 deg.c pre-denaturation for 3 min; denaturation at 95 ℃ for 7s, annealing at 57 ℃ for 10s, and extension at 72 ℃ for 15s for 45 cycles, primers were designed using Primer 5.0 software, and the sequences were synthesized by Shanghai Sangni, Inc., and are shown in Table 5.
As a result, as shown in FIG. 11, fiiP is a flagellin-related gene in E.coli, encoding synthesis of flagellin. Flagella are moving organs of bacteria, can make thalli generate tendency movement, and inhibition of genes encoding flagellin can prevent normal tendency and harmful movement of bacteria. argC regulates the synthesis of bacterial nucleoprotein, and inhibition of its expression can affect the normal transmission of bacterial genetic material, thereby inhibiting cell proliferation. dkgA is a gene associated with cellular ATP metabolism, and is involved in the synthesis of atpase, which provides energy for many cellular metabolism. After being treated by antibacterial active substances in the paecilomyces cicadae fermentation liquor, the expression of the genes fiiP, argC and dkgA is inhibited, and the expression is expressed as the down-regulation of the gene expression quantity. Wherein, the down regulation degree of the fliP and argC genes does not depend on the concentration of the bacteriostatic substance. Under the action of the antibacterial substance, the synthesis of the escherichia coli dkgA is inhibited, so that the activity of ATP enzyme is inhibited, and the normal metabolism of somatic cells is inhibited. In addition, the bacteriostatic active substance remarkably up-regulates a gene zraP related to the synthesis of cytoplasmic protein in escherichia coli, and the over-expression of the gene can cause the abnormal increase of the content of the cytoplasmic protein in a thallus cell, possibly promoting the outward secretion of the protein in the thallus cell.
TABLE 4 fluorescent quantitative PCR reaction System
TABLE 5 fluorescent quantitative PCR reaction primer sequences
Example 9 application of Paecilomyces cicadae bacteriostat in bacteriostasis and fresh keeping
Dissolving dry pectin in distilled water, adding the bacteriostatic agent prepared in the method of example 2 and glycerol, uniformly mixing to prepare a membrane liquid, wherein the addition amount of the glycerol is 1.5g, fully stirring and vacuum degassing, pouring the mixture into a mold, fully spreading the mixture, pouring the mixture into a vacuum drying oven, drying the mixture at 40 ℃ to obtain a bacteriostatic preservative film, and uncovering the film and placing the bacteriostatic preservative film into a closed container for later use, wherein the thickness of the film is 0.15 mm; the mass contents of pectin and glycerol in the membrane liquid are respectively 2.5% and 3.0%, and the addition amount of the antibacterial agent is 0.050 mg/mL.
The antibacterial effect of the paecilomyces cicadae CH113.1 preservative film is measured by a filter paper sheet antibacterial ring method. 0.10mL of bacteria were aspirated by pipette with the concentration of 106CFU/mL suspension of Escherichia coli and Staphylococcus aureus, inoculated into PDA solid medium, and uniformly coated. Then spreading the Paecilomyces cicadae CH113.1 preservative film (diameter is 6mm) respectively, placing the flat plate in an incubator at 37 ℃ for culturing for 24h, taking a sterile filter paper sheet as a blank control, determining a bacteriostatic area, and repeating the determination for 3 times.
Zone of inhibition/mm2Area of bacteriostatic circle/mm2Area of filter paper/mm2
The result shows that the Paecilomyces cicadae CH113.1 preservative film can better inhibit escherichia coli (32.13 mm)2) And golden yellow grape ball (28.64 mm)2) The growth of (2). The film has good application potential in the field of food preservation.
Sequence listing
<110> Zhejiang university
<120> paecilomyces cicadae and application thereof
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cttccgtagg ggtgaacctg cggaaggatc attacaacaa ggccacgggc ccccaagctt 60
cggcggaagg ggtcccagcc tgactttata cccacccttt gcctatgtgt acctctatcg 120
cttcctcggc gggaaccccc gccgatagca acttatcaaa ccctttgcat tatacattaa 180
cacttctgat acaaaaacaa attattacaa ctttcaacaa tggatctctt ggttctggca 240
tcgatgaaga acgcagcgaa atgcgataag tagtgtgaat tgcagaattc agtgaatcat 300
cgaatctttg aacgcacatt gcgcccctcg gtattccgtg gggcatgcct gttcgagcgt 360
catttacacc ctcaagctct gcttggtgtt gggcgtctgt cccgccttcg tgcgcggact 420
cgcctcaaag tcattggcag cggtctcgcc ggcttctcgc gcagcacatt tgcgcttctc 480
gaagccccgg cggatctgcg tccagcaagc catttcacga cttgacctcg gatcaggtag 540
ggatacccgc tgaacttaag catatcaata agccggagga a 581
Claims (9)
1. Paecilomyces cicadae (Paecilomyces cicadae) CH113.1, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2017, 01-04 and the preservation number of CCTCC NO: m2017004, the preservation address is Wuhan, Wuhan university, China, zip code 430072.
2. Use of the paecilomyces cicadae CH113.1 according to claim 1 in the preparation of an antibacterial agent.
3. The use of claim 2, wherein the antimicrobial agent is a bacillus subtilis antimicrobial agent, a staphylococcus aureus antimicrobial agent, or an escherichia coli antimicrobial agent.
4. The use according to claim 2, wherein the antibacterial agent is obtained by centrifuging fermentation liquor obtained by fermenting and culturing paecilomyces cicadae CH113.1 at 28 ℃ and 200rpm, and separating and purifying supernatant a.
5. The use according to claim 4, characterized in that the supernatant a is prepared by the following steps: inoculating Paecilomyces cicadae CH113.1 to a slant culture medium, and culturing at 28 ℃ for 5 days to obtain slant thallus; the slant culture medium comprises the following components: 200g/L of potato, 20g/L of glucose, 10g/L of agar powder and a natural pH value, wherein the solvent is tap water; selecting one strain of the thallus on the inclined plane, inoculating the strain to a fermentation culture medium, culturing at 28 ℃ and 200rpm for 5d, centrifuging the fermentation liquor, and collecting a supernatant a; the fermentation medium comprises the following components: 200g/L of potato, 20g/L of glucose and a natural pH value, wherein the solvent is tap water.
6. The use according to claim 4, wherein the separation and purification method of the supernatant a comprises the following steps: taking the supernatant a, adding 3 times of volume of absolute ethyl alcohol, standing at 4 ℃ overnight, centrifuging at 4 ℃ and 12000g for 30min, collecting the supernatant b, evaporating to dryness by a rotary evaporator, dissolving with sterile water, slowly adding ammonium sulfate at 4 ℃ to ensure that the final mass concentration of the ammonium sulfate is 60%, and standing overnight; centrifuging at 4 deg.C and 12000g for 30min, respectively collecting supernatant c and precipitate c, and filtering the supernatant c with 0.22 μm filter membrane for sterilization to obtain residue a; dissolving the filter residue a with sterile water, and filtering with a 0.45-micrometer filter membrane to obtain a filter residue b; freeze-drying the filter residue b, dissolving the filter residue b by using 0.050mol/L NaAc-HAc buffer solution, loading the filter residue b on a CM-fiber column 52, balancing the column by using pH 4.0-5.0 and 0.010-0.10 mol/L NaAc-HAc buffer solution, carrying out gradient elution by using pH 5.0-5.5 and 0.10-0.50 mol/L NaAc-HAc buffer solution, collecting a target component, carrying out ultrafiltration concentration by using an ultrafiltration membrane with the molecular weight cut-off value of 1000Da to obtain a cut-off solution a, dissolving the cut-off solution a on a Sephadex G-25/G-50 column by using distilled water after freeze-drying at the temperature of-50 ℃, dissolving the obtained cut-off solution b by using distilled water after freeze-drying at the temperature of-50 ℃ and carrying out preparative HPLC, eluting by using 10-80% acetonitrile water solution containing TFA with the volume concentration of 0.010-percent, collecting target components, lyophilizing at-50 deg.C, and storing at-20 deg.C to obtain antibacterial agent.
7. Use of paecilomyces cicadae CH113.1 according to claim 1 in the preparation of a probiotic formulation.
8. Application of the paecilomyces cicadae CH113.1 in preparation of antibacterial preservative films according to claim 1.
9. The use according to claim 8, wherein the bacteriostatic preservative film is prepared by the following steps: dissolving dry pectin in distilled water, adding an antibacterial agent and glycerol, uniformly mixing to prepare a membrane liquid, fully stirring, vacuum degassing, pouring into a mold, fully paving, and then placing in a vacuum drying oven for drying to obtain the antibacterial preservative film; the mass contents of pectin and glycerol in the membrane liquid are respectively 2.5% and 3.0%, and the addition amount of the antibacterial agent is 0.050 mg/mL; the antibacterial agent is obtained by centrifuging fermentation liquor obtained by fermenting and culturing paecilomyces cicadae CH113.1 at 28 ℃ and 200rpm, and separating and purifying supernatant a.
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