CN108315266A - Paecilomyces cicadae and application - Google Patents
Paecilomyces cicadae and application Download PDFInfo
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Abstract
The invention discloses a kind of Paecilomyces cicadae and the applications in preparing antiseptic, probiotics preparation and antimicrobial preservative film, isolated one plant of new strains-Paecilomyces cicadae CH 113.1 from wild entomogenous fungi cicada fungus for the first time, the strain fermentation product is inhibited to encountered pathogenic microorganism, to the inhibiting rates of Escherichia coli up to 70% or more, which can apply in food fresh keeping.The present invention shows that the bacterial strain has a good application prospect in fields such as food.
Description
(1) technical field
The present invention relates to a kind of Paecilomyces cicadae and its as the application of bacteriostatic agent.
(2) background technology
Pathogenic bacteria refer to the microorganism that can cause disease, include mainly bacterium, virus, conveyor screw, rickettsia, clothing original
Body, mycoplasma, fungi and actinomyces etc..General described pathogenic bacteria refer to the bacterium in pathogenic microorganism.Bacterium is caused a disease
Property with its virulence, intrusion quantity and portal of entry it is related.Although most bacteriums are harmless or even beneficial, quite big
A part can cause a disease, to generate harm to human body.Bacteriostatic agent is exactly the substance that bacterium or fungi can be inhibited to grow.Bacteriostatic agent
Possibly thalline can not be killed, but it can inhibit growth, harmful bacterium or fungi is prevented to grow excessive, harm health.Often
Bacteriostatic agent (Bacteriostat) is some antibiotic agents, includes mainly sulfamido, tetracycline, chloramphenicol, red mould
Element, lincomycin etc..In recent years, with antibiotic overuse, the drug resistance of bacterium and fungi is continuously increased, and causes some anti-
Raw element curative effect reduces and the appearance of multidrug resistant pathogenic bacteria, clinically causes great threat, therefore there is an urgent need to screen
Novel antibacterial active material is to meet the needs of clinical.
Probiotics can inhibit harmful bacteria, balance host's microecological balance.Beneficial bacterium or fungi mainly have in animal body：
Clostridium butyricum, lactic acid bacteria, Bifidobacterium, actinomyces, saccharomycete etc..So far, the probiotics that scientist has found is generally
It can be divided into three categories：Lactobacillus class, Bifidobacterium class and gram-positive cocci class.With the appearance of probiotics, bacteriostatic agent one
Word has obtained new annotation, and this biology based bacteriostat has thoroughly broken the treatment of mankind's traditional medicine and cleaning-nursing mode, special
The infection of pathogenic bacteria (harmful bacteria) is inhibited using active probiotic (beneficial bacterium), with the biological theory of " beneficial bacteria is antibacterial " for the mankind
A brand-new period is brought in sound development into.The study found that antibacterial substance mainly passes through cell membrane, the cell wall of destruction microorganism
And DNA structure, it influences the approach such as gene expression, cellular respiration and energetic supersession and plays bacteriostasis.
Currently, bacteriostatic agent used in the market is mostly chemicals, human body and environment can be caused centainly to injure, and given birth to
Object bacteriostatic agent is increasingly becoming the best substitute of chemical bacteriostatic agent with low toxicity, feature efficiently, environmentally friendly.Based on background above, grind
One of the research hotspot in the field will be become by studying carefully new bio based bacteriostat.Cicada fungus (Cordyceps cicadae) also known as cicada
Pupa grass, refers to the fungi Paecilomyces cicadae of Clavicipitaceae (Clavicipttaceus) Cordyceps (Cordyceps)
(Paecilomyces cicadae) is colonized on cicada nymph and is formed by compound.Paecilomyces cicadae was once considered as " Chinese medicine eight delicacies "
One of.Cicada fungus and cordyceps sinensis, Cordceps militaris are eponymous, are traditional one of the three big medicinal cordyceps sinensis in China, and cicada fungus and cordyceps sinensis
Active constituent is similar, containing a variety of active ingredients such as fungi polysaccharide, cordycepic acid, cordycepin, adenosines, has and adjusts immune, anti-swell
Tumor, anti-aging, improve renal function, it is hypoglycemic and antibacterial the effects that.For this purpose, this seminar, from wild cicada fungus, screening is provided
The Paecilomyces cicadae for having good bacteriostatic activity studies its bioactivity, and inquires into its application in food fresh keeping, is given birth to for cicada fungus source
The antibacterial product depth exploitation of object provides theoretical foundation.
(3) invention content
It is an object of the present invention to provide one plant of new strains-Paecilomyces cicadae (Paecilomyces cicadae) CH 113.1, with
And its application of the tunning as bacteriostatic agent, which, which has, inhibits staphylococcus aureus, bacillus subtilis
The activity of the pathogenic microorganisms such as bacterium and Escherichia coli, can destroy the eucaryotic cell structure of Escherichia coli, and the wall film for changing Escherichia coli is logical
Permeability destroys thalline memebrane protein.In addition, the present invention has determined the formula of Paecilomyces cicadae bacteriostatic agent, and it is inquired into antibacterial
The application of preservative film.
The technical solution adopted by the present invention is：
The present invention provides one plant of new strains-Paecilomyces cicadae (Paecilomyces cicadae) CH 113.1, in being preserved in
State's Type Tissue Collection, the deposit date is on 01 04th, 2017, deposit number was CCTCC NO：M2017004 is protected
Tibetan address is Wuhan, China, Wuhan University, postcode 430072.
The present invention also provides a kind of applications of Paecilomyces cicadae CH113.1 in preparing antiseptic, the antiseptic is
Bacillus subtilis antiseptic, staphylococcus glucose coccus antiseptic or Escherichia coli antiseptic.Paecilomyces cicadae of the present invention
Antiseptic in the supernatant that the fermented cultures of CH 113.1 obtain, can destroy the eucaryotic cell structure of Escherichia coli, change large intestine
The wall membrane permeability of bacillus destroys thalline memebrane protein.Antiseptic pair in 113.1 fermented supernatant fluids of Paecilomyces cicadae CH
The minimal inhibitory concentration of Escherichia coli is 0.050mg/mL.
Antiseptic of the present invention is zymotic fluid centrifugations of the Paecilomyces cicadae CH113.1 after 28 DEG C, 200rpm fermented and cultureds,
Supernatant a is taken to isolate and purify acquisition.
Antiseptic of the present invention is prepared as follows：
(1) inclined-plane culture：Paecilomyces cicadae CH 113.1 is seeded to slant medium, 28 DEG C are cultivated 5 days, and inclined-plane bacterium is obtained
Body；The slant medium composition：Potato 200g/L, glucose 20g/L, agar powder 10g/L, solvent are tap water, pH value
It is natural；
(2) fermented and cultured：It is seeded to fermentation medium, 28 DEG C, 200rpm trainings from one oese thalline of inclined-plane thalline picking
5d is supported, supernatant a is collected in zymotic fluid centrifugation (4 DEG C, 12000g centrifuges 30min)；The fermentation medium composition：Potato
200g/L, glucose 20g/L, solvent are tap water, and pH value is natural.
(3) antiseptic：50mL supernatant a are taken, the absolute ethyl alcohol of 3 times of volumes are added, 4 DEG C stand overnight precipitate polysaccharides；4
DEG C, 12000g centrifuge 30min, collect supernatant b, Rotary Evaporators are evaporated, and sulfuric acid is slowly added in 4 DEG C with after sterile water dissolution
Ammonium makes ammonium sulfate quality final concentration of 60%, stands overnight；4 DEG C, 12000g centrifugation 30min, collect supernatant b, rotary evaporation
Instrument is evaporated, and is slowly added to ammonium sulfate in 4 DEG C with after sterile water dissolution, is made ammonium sulfate quality final concentration of 60%, stand overnight；4
DEG C, 12000g centrifuge 30min, collect supernatant c and precipitation c respectively, 0.22 μm of membrane filtration degerming of supernatant c filtered
Slag a；After the sterile water dissolutions of filter residue a, 0.45 μm of filter membrane is crossed, obtains filter residue b；0.050mol/L NaAc- are used after filter residue b freeze-dryings
HAc buffer solutions and upper CM- fibre columns 52, it is flat with pH 4.0~5.0,0.010~0.10mol/L NaAc-HAc buffer solutions
Weigh pillar, carries out gradient elution with pH 5.0~5.5,0.10~0.50mol/L NaAc-HAc buffer solutions, collects target components
(be preferably a pipe per 10mL, measure antibacterial activity, merge collection vigor peak) is super for 1000Da ultrafiltration membranes through molecular weight cut-off value
Filter concentration, obtains after trapped fluid a be lyophilized at -50 DEG C and is with distilling water dissolution and upper Sephadex G-25/G-50 columns, eluent
PH 6.0~7.0,0.010~0.10mol/L phosphate buffer collect target components and (are preferably a pipe per 10mL, measure antibacterial
Activity merges collection vigor peak) it is concentrated by ultrafiltration for 1000Da ultrafiltration membranes through molecular weight cut-off value, the trapped fluid b of acquisition is at -50 DEG C
With distillation water dissolution and upper preparative HPLC, 10~80% acetonitrile water of volumetric concentration of the 0.010%TFA containing volumetric concentration after freeze-drying
Solution elutes, and collects -50 DEG C of target components (be preferably a pipe per 5.0mL, measure antibacterial activity, merge collection vigor peak) and freezes
It is dry, in -20 DEG C of preservations, obtain antiseptic.
The present invention also provides a kind of applications of Paecilomyces cicadae CH113.1 in preparing probiotics preparation.
It is described antibacterial the invention further relates to a kind of applications of Paecilomyces cicadae CH113.1 in preparing bacteriostasis, preservation film
For preservative film by Paecilomyces cicadae CH113.1 through 28 DEG C, the zymotic fluid centrifugation after 200rpm fermented and cultureds takes supernatant a to isolate and purify
The bacteriostatic agent of acquisition is made.
Further, the bacteriostasis, preservation film is prepared as follows：By dry pectolysis in distilled water, and it is added anti-
Microbial inoculum and glycerine are uniformly mixed and film liquid are made, and are sufficiently stirred and pave with after vacuum outgas, pouring into film tool to be paved with, are subsequently placed at true
40 DEG C of dryings in empty drying box obtain bacteriostasis, preservation film；Pectin and qualities of glycerin content distinguish 2.5% He in the film liquid
3.0%, bacteriostatic agent addition is 0.050mg/mL.It is preferred that pour mask amount 50.0g, takes off film and is put into closed container for use, film thickness
0.15mm。
Compared with prior art, the beneficial effects are mainly as follows：Divide from wild entomogenous fungi cicada fungus for the first time
From one plant of new strains-Paecilomyces cicadae CH 113.1 is obtained, which, which has encountered pathogenic microorganism, inhibits to make
With to the inhibiting rates of Escherichia coli up to 70% or more, which can apply in food fresh keeping.The present invention is shown
The bacterial strain has a good application prospect in fields such as food.
(4) it illustrates
Fig. 1 cicada fungus molecular biology of fungi is identified；A：The gel electrophoresis figure of rDNA-ITS amplified productions, M are standard protein,
ITS is bacterial strain CH113.1 genome amplification PCR products；B：Target fragment sequencing result；C：Blast sequence alignment results D：Bacterium
The systematic evolution tree of strain CH113.1, X indicate the cicada fungus fungi that separation screening arrives.
The morphological observation of Fig. 2 Paecilomyces cicadaes；A is growthform of the Paecilomyces cicadae in PDA culture medium；B-D is quasi- for cicada
The scanning electron microscope (SEM) photograph of mould, respectively 5K, 7K, 8K times.
The antibacterial collection of illustrative plates of Fig. 3 Paecilomyces cicadaes, A:Fermentation medium, B:Paecilomyces cicadae zymotic fluid.
Influence of Fig. 4 Paecilomyces cicadaes bacteriostatic agent to Escherichia coli structure；A is control (sterile water process Escherichia coli)；B-D
For the Escherichia coli electron microscope of Paecilomyces cicadae bacteriostatic agent processing, amplify 5K, 7K respectively, 8K times.
Fig. 5 Paecilomyces cicadaes bacteriostatic agent is on the active influences of Escherichia coli bacteria liquid AKP.
Influence of Fig. 6 Paecilomyces cicadaes bacteriostatic agent to coli somatic total protein；M:Standard molecular weight albumen, A:At water
Reason, B:16 times of MIC processing, C:8 times of MIC processing, D:4 times of MIC processing.
Influence of Fig. 7 Paecilomyces cicadaes bacteriostatic agent to the extracellular albumen concentration of Escherichia coli.
Influence of Fig. 8 Paecilomyces cicadaes bacteriostatic agent to Escherichia coli bacteria liquid conductivity.
Influence of Fig. 9 Paecilomyces cicadaes bacteriostatic agent to Escherichia coli bacteria liquid betagalactosidase activity.
Influence of Figure 10 Paecilomyces cicadaes bacteriostatic agent to Escherichia coli breakage memebrane protein；M:Standard molecular weight albumen, A:At water
Reason, B:16 times of MIC processing, C:8 times of MIC processing, D:4 times of MIC processing.
Influence of Figure 11 Paecilomyces cicadaes bacteriostatic agent to Escherichia coli functional gene expression quantity.
(5) specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
1 strain isolation of embodiment purifies and identification
(1) it operates
1, strain isolation and screening
The fresh cicada fungus of acquisition is cleaned into surface, impregnates 1min, volume with the mercuric chloride solution of volumetric concentration 0.10%
75% ethanol water of concentration impregnates 1min and disinfects, and aseptic filter paper blots, and after carrying out tissue separation with sterile scalpel, connects
Kind is placed in 28 DEG C of culture 5d in (containing 10 μ g/mL chloramphenicol) on potato dextrose agar (PDA) tablet.With method of scoring and painting
Cicada fungus sample after culture is carried out strain separating by cloth method, and the inoculation of picking different shape is on new PDA plate respectively,
28 DEG C of culture 3d are placed in, it is purebred continuously to detach 3 acquisitions.Strain is inoculated in PDA liquid medium after expanding culture, 28 DEG C,
160rpm fermented and cultured 5d, are collected by centrifugation fermentating metabolism product, with Escherichia coli, staphylococcus aureus and bacillus subtilis
For indicator bacteria, there is the cicada fungus bacterial strain of bacteriostasis with Odontothrips loti screening.Sterile Oxford cup is gently put into containing 1.0% instruction
It in the LB solid medium tablets of bacterium, draws in 200 μ L to Oxford cup of fermentating metabolism product, equivalent PDA cultures are added in control group
Base, 4 DEG C stand after sample fully penetrates into culture medium, for 24 hours in 37 DEG C of cultures, observe the size of inhibition zone around Oxford cup,
Screening has the bacterial strain CH113.1 of bacteriostatic activity.
In the method for above-mentioned screening, it is as follows to be related to culture medium：
(1) meat soup (Luria-Bertani, LB) liquid (solid) culture medium
Tryptone 5.0g, yeast extract 10g, sodium chloride 10g, (agar powder 10g) add water to be settled to 1.0L, and 121
DEG C, 0.10MPa sterilizings 20min, 4 DEG C of preservations.
(2) potato glucose (PDA) liquid (solid) culture medium
Potato 200g, glucose 20g, (agar powder 10g), by peeling potatoes, chopping after adding boiling to boil 20min, is used
Double gauze filters to take supernatant, and 20g glucose is added, and water is added to be settled to 1.0L, 121 DEG C, and 0.10MPa sterilizes 20min, 4 DEG C
It preserves.
2, thalline is identified
2.1 extracting genome DNA
With fungal gene group extracts kit extraction bacterial strain CH113.1DNA：Take the 20 fresh mycelia of μ g, be transferred to 1.5mL from
In heart pipe, 400 μ L Buffer Digestion (cell pyrolysis liquid) and 4.0 μ L beta -mercaptoethanols are added, the two are shaken mixed
It is even, 1h is kept the temperature at 65 DEG C and is cracked completely to cell, is added 200 μ L Buffer PF (split-phase buffer solution) immediately, mixing, -20 DEG C
Refrigerator places 5min；Room temperature 10000g centrifuges 5min, and supernatant (500~550 μ L) is transferred in new 1.5mL centrifuge tubes, is added
Enter isometric isopropanol, mix well, is placed at room temperature for 2-3min；Room temperature 10000g centrifuges 5min, abandons supernatant；It is added
1.0mL75% ethyl alcohol, rinses 1-3min, and 10000g centrifuges 2min, abandons supernatant；1.0mL75% ethyl alcohol is added, 1- is rinsed
3min, 10000g centrifuge 2min, abandon supernatant, are repeated once；After remaining ethyl alcohol completely volatilization, dissolved with 50 μ L nucleic acid slow
Fliud flushing (Tris-EDTA buffer, TE buffer) dissolving DNA, -20 DEG C of preservations.
2.2ITS fragment amplification
RDNA-ITS sequences use universal primer ITS 1 and ITS 4, and primer sequence is shown in Table 1.
1 fungi ITS of table identifies primer
PCR amplification system (20 μ L)：Taq PCR StarMix：10μL；Primer(10μmol/L)：Each 1.0 μ L；DNA：
1.0μL；ddH2O：7.0μL.
PCR amplification program：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30s, anneal 30s, and 72 DEG C of extension 1min, totally 35 are followed
Ring；72 DEG C of final extension 5min.Amplified production is detected with 1.0% agarose gel electrophoresis containing ethidium bromide.
2.3PCR product purification
(1) electrophoresis：1.0% agargel electrophoresis on pcr amplification product, 20 μ L of point sample.
(2) glue is cut：After the completion of electrophoresis, target fragment is cut out rapidly in the UV lamp, removal agarose gel block edge is free of
The part of DNA, and blob of viscose is shredded.
(3) it weighs：Blob of viscose is fitted into the 1.5mL centrifuge tubes that one weighs in advance, weighs weight, calculates glue weight.Often manage
700mg is not to be exceeded in interior blob of viscose.
(4) colloidal sol：The ratio (1 of 1.0 μ L film combination liquid is added in every 1.0mg gels:1) film combination liquid is added, after mixing
55 DEG C are placed in, it is primary at interval of 1-2min mixings, until blob of viscose is completely dissolved (about 5min).
(5) DNA is combined：It after gel solution is cooled to room temperature, is transferred in the centrifugal adsorbing column for being inserted into collecting pipe, stands
1min, at room temperature 12000g centrifuge 1min, the waste liquid in reject collecting pipe turns back to centrifugal adsorbing column in collecting pipe again.
(6) it cleans：600 μ L film rinsing liquids are added in centrifugal adsorbing column, 12000g centrifuges 30s at room temperature, and reject is collected
Waste liquid in pipe turns back to centrifugal adsorbing column in collecting pipe again.
(7) it cleans again：600 μ L film rinsing liquids are added in centrifugal adsorbing column, room temperature 12000g centrifuges 30s, and reject is received
Waste liquid in collector turns back to centrifugal adsorbing column in collecting pipe again.Centrifugal adsorbing column uncapping is centrifuged into 2min again, is thoroughly removed
Remaining rinsing liquid.
(8) it elutes：It is careful to take out centrifugal adsorbing column, it is inserted in a 1.5mL centrifuge tube, into silica gel absorption film
30 μ L elution buffers are added in centre, and after being stored at room temperature 1min, 12000g centrifuges the DNA fragmentation that 1min collects purifying.
(9) it stores：Reject centrifugal adsorbing column, the DNA fragmentation of acquisition is for subsequent experimental or -20 DEG C of preservations.
2.4 sequencings and comparison
Target DNA after purification is served extra large Sani company and is sequenced, and nucleotides sequence is classified as shown in SEQ ID NO.1.It surveys
Sequence result is in GenBank (http://blast.ncbi.nlm.nih.gov/) in carry out Blast gene comparisons, it is similar to obtain
Spend higher sequence；Multiple sequence alignments are carried out by CLUSTALX, with 6.0 software building systematic evolution trees of MEGA, identify bacterium
Kind.
2.5 strain morphological observations
The bacterial strain CH113.1 that screening obtains is inoculated on new PDA plate, 28 DEG C of 5~7d of constant temperature incubation is placed in, waits growing
Morphological observation is carried out after going out spore.In addition, fungus colony is cut from culture medium with blade, with the penta 2 of volumetric concentration 2.5%
Aldehyde aqueous solution fixes 20min, and distilled water washs 2 times, each 15min, Gradient elution using ethanol (successively with 50%, 70%, 80%,
90% ethanol dehydration, each 10-15min, then with 100% ethanol dehydration 3 times, each 10-15min), isoamyl acetate is critical
It is dry, with ion film plating instrument metal spraying, scanning electron microscopic observation.
(2) conclusion
The molecular biology identification of 1 Paecilomyces cicadae
Bacterial strain CH113.1DNA is extracted with fungal DNA Rapid extraction kit, is expanded using universal primer ITS1 and ITS4
Fungi conserved sequence rDNA-ITS.Amplified production is detected with 1.0% agarose gel electrophoresis containing EB, as a result, it has been found that PCR
Primer size is 600bp or so (A in Fig. 1).Purifying recycling target DNA after, be sent to Shanghai Sani bio tech ltd into
Row sequencing.Sequencing result (B in Fig. 1) carries out Blast gene comparisons in GenBank, after obtaining the higher sequence of similarity, leads to
It crosses CLUSTALX and carries out Multiple sequence alignments, comparison result passes through 6.0 software building systematic evolution trees of MEGA (D in Fig. 1) again.
The result shows that bacterial strain CH113.1 and cicada fungus fungi (Paecilomyces cicadae) BA-001 matching rates that separation screening obtains
99% (C in Fig. 1) is reached.
The morphological observation of 2 Paecilomyces cicadaes
Shown in A in Fig. 2, bacterial strain CH113.1 bacterium colonies are white, and mycelia is calm, felted, combines closely with culture medium, no
Easy picking, quality is close, the radial rill of bacterium colony, ellipse, and spore is in ecru.In the bacterium of electric microscopic observation Paecilomyces cicadae
Silk and spore shape, as shown in B in Fig. 2, mycelia is without every, multi-branched, base portion amacrine.Base portion is generated in mycelia upper end to expand
Deviation main shaft bottle stalk, bottle stalk is different in size, and operative tip germination is in herringbone, and conidiophore branch generates broom shape
Phialide (C in Fig. 2).Conidium is round, is in tufted or catenation, is distributed on sporophore or vegetative hyphae, has no
Germ tube and appresorium, spore surface have no slime layer (D in Fig. 2).
To sum up, bacterial strain CH113.1 is accredited as Paecilomyces cicadae, is named as Paecilomyces cicadae (Paecilomyces cicadae)
CH113.1 is preserved in China typical culture collection center, the deposit date is on 01 04th, 2017, deposit number CCTCC
NO：M 2017004, preservation address are Wuhan, China Wuhan University.
The preparation of 2 Paecilomyces cicadae CH113.1 bacteriostatic agents of embodiment
(1) inclined-plane culture：Paecilomyces cicadae CH 113.1 is seeded to slant medium, 28 DEG C are cultivated 5 days, and inclined-plane bacterium is obtained
Body.Slant medium is PDA solid mediums, is formed with embodiment 1.
(2) fermented and cultured：It is seeded to seed culture medium, 28 DEG C, 200rpm trainings from one oese thalline of inclined-plane thalline picking
5d is supported, seed liquor is obtained；Seed culture medium is PDA liquid medium, is formed with embodiment 1.
(3) by seed liquor with 5.0% inoculum concentration of volumetric concentration switching fermentation medium, 28 DEG C, 200rpm fermentation 5d acquisitions
Zymotic fluid.Fermentation medium is PDA liquid medium, is formed with embodiment 1.
(4) bacteriostatic agent：113.1 zymotic fluids of Paecilomyces cicadae CH prepared by step (3) filter mycelia, filtrate 4 with double gauze
DEG C, 12000g centrifuge 30min, collect supernatant a, that is, obtain 113.1 fermentating metabolism product of Paecilomyces cicadae CH；Take 50mL supernatants
The absolute ethyl alcohol of 3 times of volumes, 4 DEG C of overnight precipitation polysaccharide are added in liquid a；4 DEG C, 12000g centrifugation 30min, collect supernatant b, rotation
Turn evaporimeter to be evaporated, with sterile water dissolution；Sample after weight is molten is slowly added to ammonium sulfate in 4 DEG C, makes ammonium sulfate quality final concentration
It is 60%, stands overnight；4 DEG C, 12000g centrifuges 30min, collects supernatant c respectively and precipitation c, supernatant c are filtered with 0.22 μm
Filter residue a is collected in membrane filtration degerming, after the filter residue a sterile water dissolutions of 5.0mL, is crossed 0.45 μm of filter membrane, is obtained filter residue b.Filter residue b-
It is pH 4.0~5.0 that 0.50mol/L NaAc-HAc buffer solutions and upper CM- fibre columns 52, equilibrium liquid are used after 50 DEG C of freeze-dryings,
0.010~0.10mol/L NaAc-HAc buffer solutions, eluent are pH 5.0~5.5,0.10~0.50mol/L NaAc-HAc
Buffer solution for gradient elution (gradient 0.10,0.20,0.30,0.40,0.50mol/L) is a pipe per 10mL, measures antibacterial and lives
Property, merge collection vigor peak, vigor peak is concentrated by ultrafiltration, and ultrafiltration membrane molecular weight cut-off value is 1000Da, the trapped fluid a of acquisition-
With distillation water dissolution and upper Sephadex G-25/G-50 columns after 50 DEG C of freeze-dryings, elution requirement be pH 6.0~7.0,0.010~
0.10mol/L phosphate buffers (gradient 0.010,0.020,0.030,0.040,0.050,0.060,0.070,0.080,
0.090,0.10mol/L), it is a pipe per 10mL, measures antibacterial activity, merges collection vigor peak, vigor peak is retained through molecular weight
Value is that 1000Da ultrafiltration membranes are concentrated by ultrafiltration, the trapped fluid b of acquisition be lyophilized at -50 DEG C after with distilling water dissolution and upper preparative
HPLC, with the elution of 10~80% acetonitrile solution of volumetric concentration of the 0.010%TFA containing volumetric concentration (gradient 10,20,30,
40,50,60,70,80%), it is a pipe per 5.0mL, measures antibacterial activity, merges collection vigor peak, the freeze-drying of -50 DEG C of vigor peak,
In -20 DEG C of preservations, 113.1 bacteriostatic agents of Paecilomyces cicadae CH prepared from 113.1 zymotic fluids of Paecilomyces cicadae CH are obtained.
The bacteriostasis property of 3 Paecilomyces cicadae CH113.1 bacteriostatic agents of embodiment
With Escherichia coli, Bacillus cereus, staphylococcus aureus, staphylococcus epidermis, streptococcus pyogenes, excrement intestines ball
Bacterium, pseudomonas aeruginosa, Friedlander's bacillus, moscow' paratyphi B, Candida albicans, alternaric bacteria, extension are green
Mould and aspergillus niger is indicator bacteria, and the bacteriostatic activity of 113.1 bacteriostatic agents of Paecilomyces cicadae CH is measured with Odontothrips loti.Take OD600For
0.80 indicator bacteria bacterium solution prepares indicator bacteria LB tablets by the inoculum concentration of volumetric concentration 2.0%, places to Oxford cup horizontal homogeneous
In on tablet, the aseptic aqueous solution of 200 μ L Paecilomyces cicadaes CH, 113.1 bacteriostatic agents prepared by embodiment 2 is added in experimental group, and 4 DEG C quiet
After setting 2h, 37 DEG C are incubated overnight, and measure antibacterial circle diameter, establish antibacterial collection of illustrative plates, the results are shown in Table 2.
The antibacterial collection of illustrative plates of 2 Paecilomyces cicadae bacteriostatic agent of table
aNo inhibition zone be-, antibacterial circle diameter<15mm is+, antibacterial circle diameter 15-20mm is ++, antibacterial circle diameter>20mm
For +++
The present invention relates to pathogen relevant informations for table 3
Influence of the 4 Paecilomyces cicadae bacteriostatic agent of embodiment to Escherichia coli structure
113.1 bacteriostatic agents of Paecilomyces cicadae CH (sterile water dissolution) prepared by embodiment 3 handle the Escherichia coli of 9h
(CMCC 44115) bacterium solution centrifuges, and collects thalline, is washed 3 times with the PBS of 0.10mol/L, pH 7.2, washs 30min every time, from
The heart abandons supernatant, and the glutaraldehyde water solution that 1.0mL volumetric concentrations 4.0% are added is fixed, and shakes up thalline, 4 DEG C of fixed 72h will consolidate
The thalline set takes out, and is placed at room temperature for 2h, is washed 3 times with the PBS of 0.10mol/L, pH 7.2, washs 30min every time.Use PBS
Thalline suspension is prepared, by a small amount of bacteria suspension on glass slide, uniform spreadable, -20 DEG C of vacuum freeze drying 5h, after dehydration and drying
Sample sputters metal spraying, scanning electron microscopic observation somatic cells form, operating voltage 25kV under vacuum.
As shown in figure 4, A is untreated control group Escherichia coli in Fig. 4, somatic cells are complete, in typical rod-shaped or
Rod-short, after birth surface is smoother, and thalline more disperses, and is in stock through antibacterial substance treated Escherichia coli part cell
Shape, thalline are more assembled, and part thalline intermediate recess (B, C in Fig. 4), film surface is imperfect, and part thalline swelling, form occurs
Distortion, after birth become fold, are seriously damaged (D in Fig. 4).
Influence of the 5 Paecilomyces cicadae bacteriostatic agent of embodiment to Bacillus coli cells wall stability
Cicada prepared by the embodiment 3 of 4 × MIC, 8 × MIC and 16 × MIC concentration is obtained with sterile water dissolution and dilution respectively
113.1 antiseptics of Paecilomyces varioti CH (sterile water dissolution) handle Escherichia coli (CMCC 44115) 9h, compare sterile water process,
With the extracellular AKP activity of kit (green skies bio tech ltd, product identification P0321) detection Escherichia coli.Use reagent
It is y=0.2165x+ that box Plays product (10mM p-nitrophenols), which do light absorption value and the standard curve of AKP concentration correlativities,
0.0054, calculate the content of extracellular AKP.The changes of contents of the outer AKP of Bacillus coli cells can reflect the impaired feelings of bacteria cell wall
Condition.The Escherichia coli that Paecilomyces cicadae bacteriostatic agent through 4 × MIC, 8 × MIC and 16 × MIC handles 9h are calculated by AKP standard curves
The content of extracellular AKP is respectively 0.134,0.389 and 1.206mM, and a concentration of 0.067mM of control group A KP, by life per minute
It is defined as an enzyme activity unit at the amount of the AKP needed for 1 μM of p-nitrophenol, AKP enzyme activity is as shown in Figure 5 after conversion.As a result
Show that Paecilomyces cicadae bacteriostatic agent can cause the damage of Bacillus coli cells wall, and the concentration of degree of injury and antibacterial substance is in just
It is related.
Influence of the 6 Paecilomyces cicadae bacteriostatic agent of embodiment to Bacillus coli cells membrane permeability
1 bacterial protein
Respectively with 4 × MIC (D in Fig. 6), the preparation of embodiment 3 of 8 × MIC (C in Fig. 6) and 16 × MIC (B in Fig. 6) concentration
113.1 bacteriostatic agents of Paecilomyces cicadae CH (sterile water dissolution) handle Escherichia coli (CMCC 44115) 9h, control group sterile water
It handles (A in Fig. 6), culture solution 5000g centrifuges 10min, abandons supernatant, by thalline 0.10mol/L, the PBS washings 2 of pH 7.2
After secondary, it is resuspended in cell pyrolysis liquid (RIPA lysates, green skies bio tech ltd, product identification P0013C), makes
OD600=1, add 400 μ L protease inhibitors (PMSF) and 80 μ L lysozymes, after 37 DEG C are incubated 40min, 20 μ L DNase/ are added
RNase, 37 DEG C of culture 10min, 4 DEG C, 3000g centrifuges 30min, collects supernatant and is placed in new pipe, the thalline as extracted is total
Albumen.Mycoprotein carries out 10%SDS-Page detection (10% separation gels：4671μL ddH2O；2625 μ L 40%Acryl/
Bis；2500 μ L 1.5M Tris-HCl, pH8.8；100 μ L 10%SDS；100 μ L 10%APS；4.0μL TEMED.4.0%
Spacer gel：3791μL ddH2O；582 μ L 40%Acryl/Bis；1500 μ L 0.5M Tris-HCl, pH6.8；60 μ L 10%
SDS；60 μ L 10%APS；6.0μL TEMED).
As shown in fig. 6, after the processing of the Paecilomyces cicadae antibiotic preparation of various concentration, coli somatic total protein is slightly poor
It is different, relative to untreated control group, there are the bands of a spectrum that part obviously shoals, and antibacterial substance concentration is higher, this phenomenon is brighter
It is aobvious.After the processing of high concentration antibacterial substance, the band of the e. coli total protein 97.4kDa or more extracted obviously shoals, this
Show that antibacterial substance may influence the normal expression of mycoprotein or influence its function, change the certain keys of thalline structure or
The synthesis of functional protein.
2 cellular protein concentrations
113.1 bacteriostatic agents of Paecilomyces cicadae CH prepared respectively with the embodiment 3 of 4 × MIC, 8 × MIC and 16 × MIC concentration
(sterile water dissolution) handles Escherichia coli (CMCC 44115) 9h, compares sterile water process, measures thalline cellular protein concentration.
According to OD562(protein standard curve is made with the standard curve y=1822.6x-7.2015 of albumen concentration：Take 8 1.5mL from
The BSA protein standard solutions and PBS of corresponding volume are added into every centrifuge tube for heart pipe, are obtained after mixing well different dense
The protein standard solution of degree is finally separately added into 1.0mL BCA working solutions, keeps the temperature 30min after mixing well at 60 DEG C, cooling
OD is surveyed after to room temperature562Value.With OD562For abscissa, protein concentration is ordinate, draws protein content calibration curve equation and is
Y=1822.6x-7.2015, R2=0.9929) bacteriostatic peptide treated the extracellular albumen concentration of Escherichia coli, is calculated.
As shown in Figure 7, the cellular protein concentration of Escherichia coli is got higher compared with control group after processing, and antibacterial agent concentration is got over
Height, cellular protein concentration are bigger.The above result shows that the Substance in Paecilomyces cicadae zymotic fluid can hinder Escherichia coli
The expression of Partial Protein, it is also possible to which antibacterial substance destroys the cell envelope structure of Escherichia coli, makes its permeability increase, draws
It plays intracellular protein to dissociate to extracellular, to play bacteriostasis.
3 conductivity
Logarithmic phase Escherichia coli (CMCC 44115) 0.10M, pH7.4PBS are washed 3 times, bacterial concentration is adjusted to
108CFU/mL is inoculated in by the inoculum concentration of volumetric concentration 2.0% in LB liquid medium, is separately added into 4 × MIC of 5.0mL, and 8
113.1 bacteriostatic agents of Paecilomyces cicadae CH (sterile water dissolution) prepared by the embodiment 3 of × MIC and 16 × MIC, control group addition etc.
Sterile water is measured, 37 DEG C, 200rpm fermented and cultured 9h, each bacterium solution conductivity is measured with conductivity gauge.When cell encounters biocidal property substance
And when making cell membrane by destroying, the protective barrier of thalline is broken, and the electrolyte inside thalline can leak, to make culture solution
Conductivity rise.As shown in Figure 8, there were significant differences between the bacterium solution conductivity and control group of experimental group, antibacterial substance concentration
It is higher, cause Escherichia coli Electrolyte Leakage degree bigger, bacteriostasis is stronger.
4 betagalactosidase activities
113.1 bacteriostatic agents of Paecilomyces cicadae CH prepared respectively with the embodiment 3 of 4 × MIC, 8 × MIC and 16 × MIC concentration
(sterile water dissolution) handles Escherichia coli (CMCC 44115) 9h, compares sterile water process, the extracellular β-of detection Escherichia coli half
Gal activity (takes the indicator bacteria bacterium solution 10mL being incubated overnight, 4 DEG C, 3000g centrifuges 10min, must precipitate, as large intestine bar
Bacterium thalline；The thalline is fully transferred to 100mL M9 lactose inducing cultures, 37 DEG C, 200rpm cultivates 9h, in 4 DEG C, 3000g
10min is centrifuged, thalline is washed 2 times with sterile saline, and is resuspended in 100mL beta galactosidase buffer solutions, makes its OD630
About 0.20.Take beta galactosidase buffer solutions of the 4.0mL containing Escherichia coli, sequentially add 0.50mL cicada fungus polysaccharide solution and
0.50mL ortho-nitrophenyl-β-D- synthesis (ONPG, 1.0mg/mL), after mixing well, in 37 DEG C, 160rpm is cultivated,
An OD is measured every 30min415), often change 0.05 with OD values and be defined as an enzyme activity unit, calculates enzyme activity.Work as cell membrane
When by destroying, a large amount of beta galactosidases are released to extracellular, and are reacted immediately with ONPG, generate the ONP of yellow, therefore,
The stability of cell membrane can be assessed using the change of the extracellular betagalactosidase activity of indicator bacteria.
As shown in Figure 9, control group enzyme activity has almost no change, and maintains 30U or so.But through antibacterial substance treated refer to
Show bacterium, the enhancing of enzyme activity conspicuousness.The result illustrates that antibacterial substance can destroy Bacillus coli cells film, makes Escherichia coli intracellular substance
It is discharged into culture medium.In addition, antibacterial substance concentration is bigger, then the extracellular beta galactosidase enzyme activity of thalline is higher, illustrates antibacterial
It acts on and being proportionate with the concentration of antibacterial substance.
Influence of the 7 Paecilomyces cicadae bacteriostatic agent of embodiment to Escherichia coli breakage memebrane protein
Respectively with 4 × MIC (D in Figure 10), the embodiment 3 of 8 × MIC (C in Figure 10) and 16 × MIC (B in Figure 10) concentration
113.1 bacteriostatic agents of Paecilomyces cicadae CH (sterile water dissolution) of preparation handle Escherichia coli (CMCC44115) 9h, compare with sterile
Water process (A in Figure 10), 4500g centrifuge 15min and obtain thalline, washed 2 times with 1 × PBS, and be resuspended in 4.0mL PBS and (contain
8.0%Triton X-114) in, 4 DEG C of preservation 3h.Bacteria suspension centrifuging and taking supernatant, after being placed in 37 DEG C of culture 2h, room temperature, 3000g
15min is centrifuged, bottom organic phase is taken, 36mL absolute ethyl alcohols are added, 10h is preserved in 4 DEG C.4 DEG C, 10000g centrifuges 30min, and it is heavy to take
It forms sediment, as epicyte protein.After the 20 sterile water dissolutions of μ L of the memebrane protein extracted, 5.0 μ 5 × sample-loading buffers of L are added, mix
10min, SDS-Page electrophoresis are boiled in heating after even.
Such as Figure 10, Paecilomyces cicadae antimicrobial peptide preparation changes its permeability of cell membrane by destroying epicyte protein.Control
Histone group is divided into blank, and experimental group protein component is significantly different with treated, this shows Paecilomyces cicadae antimicrobial peptide preparation energy
It enough destroys cell membrane and the extraction of epicyte protein, i.e. antibacterial substance is promoted to destroy the structure of Bacillus coli cells film, in turn
Play bacteriostasis.Meanwhile antibacterial peptide concentration is higher, it is bigger to the extent of the destruction of cell membrane.
The influence that 8 Paecilomyces cicadae bacteriostatic agent of embodiment expresses bacillus coli gene
Sample treatment：The quasi- blueness of cicada prepared by the embodiment 3 of sterile water, 4 × MIC, 8 × MIC and 16 × MIC concentration is used respectively
Mould bacteriostatic agent handles Escherichia coli (CMCC 44115) 9h, and thalline were collected by centrifugation, respectively label A, B, C, D.Sample RNA extractions：
Every 5 × 1070.50mL Trizol are added in a bacterial cell, and the sample after cracking is placed at room temperature for 5-10min, makes nucleoprotein and core
Acid is kept completely separate, and 0.20mL chloroforms are added, acutely shake 30s, are placed at room temperature for 3min, 4 DEG C, and 12000g centrifuges 10min, in absorption
Layer water phase is transferred in centrifuge tube, and the absolute ethyl alcohol of 0.5 times of volume is added, and mixing obtains sample to be tested.It is used according to adsorption column
Adsorption column (UNIQ-10, raw work Sangon Biotech) is put into collecting pipe, stands 2min, 12000g centrifugations by specification
3min abandons waste liquid in collecting pipe.Adsorption column is put back in collecting pipe, 500 μ L RNA combinations liquid (RPE Solution) are added, it is quiet
2min is set, 10000g centrifuges 30s, abandons waste liquid in collecting pipe, repeats this step 2 time.Adsorption column is put back in collecting pipe, 12000g
2min is centrifuged, waste liquid in collecting pipe is abandoned, adsorption column is put into centrifuge tube, 30 μ L DEPC- are added in adsorbed film center
treated ddH2O stands 5min, and 12000g centrifuges 2min, and -70 DEG C of the RNA solution of gained, which preserves, is used for subsequent experimental.It reverses
Record：The first chains of cDNA synthesize：Total RNA are added according to 800ng reverse transcriptions in RNA in the nuclease-free PCR pipes of ice bath
(being determined according to extraction RNA concentration), 1.0 μ L Random Primer p (dN)6(100pmoL), 1.0 μ L dNTP Mix
(0.50mM final concentration), with RNase-free ddH2O is settled to 14.5 μ L.Gently after mixing centrifugation 3~
5s, reaction mixture is after 65 DEG C of warm bath 5min, ice bath 2min, is then centrifuged for 3~5s, and ice bath adds 4.0 5 × RT of μ L
Buffer, 0.50 μ L Thermo Scientific RiboLock RNase Inhibitor (20U), 1.0 μ L RevertAid
Premium Reverse Transcriptase (200U) gently centrifuge 3~5s after mixing, it is anti-that reverse transcription is carried out in PCR instrument
It answers：25 DEG C of incubation 10min；50 DEG C, 30min synthesizes cDNA；85 DEG C, 5min terminates reaction, -20 DEG C of preservations of solution.Fluorescent quantitation
PCR：It is loaded in 96 orifice plates by table 4, cDNA, which is diluted 10 times as machine in target, to react.Using 16S-rDNA as reference gene,
Each gene does 3 technologies and repeats, and measures the Relative gene of Escherichia coli argC, zraP, fliP and dkgA after bacteriostatic agent processing
Expression quantity.Setting PCR amplification program is 95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 7s, 57 DEG C of annealing 10s, 72 DEG C of extension 15s, altogether
45 cycles, primer 5.0 Software for Design of Primer, the synthesis of commission Shanghai Sani company, sequence are shown in Table 5.
As a result as shown in figure 11, fliP is flagellin related gene in Escherichia coli, encodes the synthesis of flagellin.Whip
Hair is the locomotive organ of bacterium, and thalline can be made to generate tropism, encode flagellin gene be suppressed can interfere it is thin
The normal movement of going after profits and advoiding disadvantages of bacterium.ArgC regulates and controls the synthesis of bacterium nucleoprotein, and expression, which is suppressed, can influence thalline heredity object
The normal transmission of matter, to inhibit the breeding of cell.DkgA is a kind of and thalline ATP is metabolized relevant gene, the conjunction with ATP enzyme
At correlation, ATP enzyme can provide energy for many metabolism of cell.At the Substance in Paecilomyces cicadae zymotic fluid
After reason, the expression of fliP, argC and dkgA genes are suppressed, and show as gene expression amount downward.Wherein, fliP and argC bases
Concentration of the downward degree of cause independent of antibacterial substance.Under the action of antibacterial substance, Escherichia coli dkgA synthesis is pressed down
System, and then inhibit the activity of ATP enzyme, it is suppressed that somatic cells eubolism.In addition, Substance significantly raises large intestine bar
It can lead to the non-of somatic cells endoplasm protein content with cytoplasmic protein synthesis related gene zraP, such gene overexpression in bacterium
It is normal to increase, the export-oriented secretion of thalline intracellular protein may be promoted.
4 quantitative fluorescent PCR reaction system of table
5 quantitative fluorescent PCR of table reacts primer sequence
Application of the 9 Paecilomyces cicadae bacteriostatic agent of embodiment in bacteriostasis, preservation
Will dry pectolysis in distilled water, and the bacteriostatic agent of 2 method of embodiment preparation is added and glycerine is uniformly mixed
It is made film liquid, wherein glycerine input is 1.5g, is sufficiently stirred and paves with after vacuum outgas, pouring into be paved in mold, pour mask amount
50.0g is subsequently placed at 40 DEG C of dryings in vacuum drying chamber, obtains bacteriostasis, preservation film, takes off film and be put into closed container for use, film thickness
Spend 0.15mm；Pectin and qualities of glycerin content difference 2.5% and 3.0%, antiseptic addition are 0.050mg/ in the film liquid
mL。
The fungistatic effect of Paecilomyces cicadae CH113.1 preservative films is measured by filter paper inhibition zone method.It is drawn with liquid-transfering gun
0.10mL bacterium number concentration is all 106The Escherichia coli of CFU/mL and staphylococcus aureus suspension, are inoculated in PDA solid cultures
In base, even spread.Then it spreads and is stated on Paecilomyces cicadae CH113.1 preservative films (a diameter of 6mm) respectively, tablet is placed in 37 DEG C of trainings
It supports case culture for 24 hours, using aseptic filter paper piece as blank control, measures antibacterial region, replication 3 times.
Antibacterial region/mm2=inhibition zone area/mm2Filter paper area/mm2
The result shows that Paecilomyces cicadae CH113.1 preservative films can preferably inhibit Escherichia coli (32.13mm2) and it is golden yellow
Grape ball (28.64mm2) growth.The film shows good application potential in field of food preservation.
Sequence table
<110>Zhejiang University
<120>Paecilomyces cicadae and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 581
<212> DNA
<213>Unknown (Unknown)
<400> 1
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 (10)
1. Paecilomyces cicadae (Paecilomyces cicadae) CH113.1, is preserved in China typical culture collection center, preservation
Date is on 01 04th, 2017, and deposit number is CCTCC NO：M 2017004, preservation address is Wuhan, China, Wuhan is big
It learns, postcode 430072.
2. applications of the Paecilomyces cicadae CH113.1 in preparing antiseptic described in a kind of claim 1.
3. application as claimed in claim 2, it is characterised in that the antiseptic is bacillus subtilis antiseptic, golden yellow Portugal
Grape sugar ball bacterium antiseptic or Escherichia coli antiseptic.
4. application as claimed in claim 2, it is characterised in that the antiseptic is Paecilomyces cicadae CH113.1 through 28 DEG C,
Zymotic fluid centrifugation after 200rpm fermented and cultureds, takes supernatant a to isolate and purify acquisition.
5. application as claimed in claim 3, it is characterised in that the supernatant a is prepared as follows：By Paecilomyces cicadae CH
113.1 are seeded to slant medium, and 28 DEG C are cultivated 5 days, and inclined-plane thalline is obtained；The slant medium composition：Potato 200g/
L, glucose 20g/L, agar powder 10g/L, solvent are tap water, and pH value is natural；It is connect from one oese thalline of inclined-plane thalline picking
Kind is to fermentation medium, and 28 DEG C, 200rpm cultivates 5d, and supernatant a is collected in zymotic fluid centrifugation；The fermentation medium composition：Horse
Bell potato 200g/L, glucose 20g/L, solvent is tap water, and pH value is natural.
6. application as claimed in claim 4, it is characterised in that the supernatant a isolation and purification methods are：Supernatant a is taken, is added
Enter the absolute ethyl alcohol of 3 times of volumes, 4 DEG C stand overnight after in 4 DEG C, 12000g centrifuge 30min, collect supernatant b, Rotary Evaporators
It is evaporated, is slowly added to ammonium sulfate in 4 DEG C with after sterile water dissolution, makes ammonium sulfate quality final concentration of 60%, stand overnight；4℃、
12000g centrifuges 30min, collects supernatant c and precipitation c respectively, and 0.22 μm of membrane filtration degerming of supernatant c obtains filter residue a；
After the sterile water dissolutions of filter residue a, 0.45 μm of filter membrane is crossed, obtains filter residue b；It is slow with 0.050mol/L NaAc-HAc after filter residue b freeze-dryings
Fliud flushing dissolves and upper CM- fibre columns 52, with pH 4.0~5.0,0.010~0.10mol/L NaAc-HAc buffer solution balance columns
Son carries out gradient elution with pH 5.0~5.5,0.10~0.50mol/L NaAc-HAc buffer solutions, collects target components through dividing
Son amount cutoff value is concentrated by ultrafiltration for 1000Da ultrafiltration membranes, in distillation water dissolution after obtaining trapped fluid a and being lyophilized at -50 DEG C
Sephadex G-25/G-50 columns, eluent are pH 6.0~7.0,0.010~0.10mol/L phosphate buffers, collect target
Group lease making molecular weight cut-off value is that 1000Da ultrafiltration membranes are concentrated by ultrafiltration, and the trapped fluid b of acquisition uses distillation water-soluble after be lyophilized at -50 DEG C
It solves and mesh is collected in upper preparative HPLC, 10~80% acetonitrile solution of the volumetric concentration elution of the 0.010%TFA containing volumetric concentration
Mark component is lyophilized at -50 DEG C, in -20 DEG C of preservations, obtains antiseptic.
7. applications of the Paecilomyces cicadae CH113.1 in preparing probiotics preparation described in a kind of claim 1.
8. applications of the Paecilomyces cicadae CH113.1 in preparing bacteriostasis, preservation film described in a kind of claim 1.
9. application as claimed in claim 8, it is characterised in that the bacteriostasis, preservation film by Paecilomyces cicadae CH113.1 through 28 DEG C,
Zymotic fluid centrifugation after 200rpm fermented and cultureds, takes the antiseptic that supernatant a isolates and purifies acquisition to be made.
10. application as claimed in claim 9, it is characterised in that the bacteriostasis, preservation film is prepared as follows：By dried fruit
Glue is dissolved in distilled water, and antiseptic and glycerine is added is uniformly mixed and film liquid is made, and is sufficiently stirred with after vacuum outgas, is poured into
It is paved with and paves in mold, be subsequently placed in vacuum drying chamber dry, acquisition bacteriostasis, preservation film；Pectin and glycerine matter in the film liquid
Content difference 2.5% and 3.0% is measured, antiseptic addition is 0.050mg/mL.
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| CN111434342A (en) * | 2018-12-25 | 2020-07-21 | 葡萄王生技股份有限公司 | Cordyceps cicadae mycelium active substance for preventing and/or improving acute lung injury |
| WO2021093425A1 (en) * | 2019-11-15 | 2021-05-20 | 滨州医学院 | Root of membranous milkvetch-paecilomyces cicadae fermentative bacterium fermentation substance and application thereof |
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| WO2021093425A1 (en) * | 2019-11-15 | 2021-05-20 | 滨州医学院 | Root of membranous milkvetch-paecilomyces cicadae fermentative bacterium fermentation substance and application thereof |
| US11866692B2 (en) | 2019-11-15 | 2024-01-09 | Binzhou Medical University | Fermentation fungal substance of astragalus membranaceus paecilomyces cicadae and its use |
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