CN102719365B - Paecilomyces lilacinus with bacteriostatic activity - Google Patents
Paecilomyces lilacinus with bacteriostatic activity Download PDFInfo
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- CN102719365B CN102719365B CN 201210229012 CN201210229012A CN102719365B CN 102719365 B CN102719365 B CN 102719365B CN 201210229012 CN201210229012 CN 201210229012 CN 201210229012 A CN201210229012 A CN 201210229012A CN 102719365 B CN102719365 B CN 102719365B
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Abstract
The invention provides a paecilomyces lilacinus with a bacteriostatic activity and relates to the paecilomyces lilacinus. The invention provides the paecilomyces lilacinus which has a better inhibition effect on gram-positive bacteria and provides evidences for developing a new antibacterial medicament. The paecilomyces lilacinus with the bacteriostatic activity is the paecilomyces lilacinus WG20and is preserved in China Center for Type Culture Collection (CCTCC); and the preservation date is April 24th, 2012 and the preservation number is CCTCC NO: M2012140. The paecilomyces lilacinus WG20 has a better inhibition effect on the gram-positive bacteria.
Description
Technical field
The present invention relates to a strain Paecilomyces lilacinus.
Background technology
In recent years, along with SARS and H
1N
1Etc. the continuous appearance of novel disease, and cancer, major diseases such as acquired immune deficiency syndrome (AIDS) still do not have specifics and can treat, and people press for and seek new medicine and deal with the significant damage that these diseases are brought human health.Along with deepening continuously of endogenetic fungus research, and the report of isolated pharmaceutically active substance is more and more from the secondary metabolite of endogenetic fungus, and endogenetic fungus has become the important source of seeking newtype drug.
Endogenetic fungus has fast growth as a kind of special microorganism, breeding cycle is short, breeding coefficient is big, be easy to characteristics such as suitability for industrialized production, and because the living environment of endogenetic fungus uniqueness and and host plant between long-term interaction, make it more be easy to generate special secondary metabolite, in these meta-bolitess, often can find the activeconstituents of novel structure, therefore, endogenetic fungus is a kind of resource-type microorganism with broad prospect of application.
Summary of the invention
The invention provides a strain Paecilomyces lilacinus, gram-positive microorganism is had good inhibitory effect, provide foundation for developing new antibacterials.
The Paecilomyces lilacinus that the present invention has bacteriostatic activity is Paecilomyces lilacinus (Paecilomyces lilacinus) WG20, be deposited in Chinese typical culture collection center (CCTCC), the preservation address is Wuhan City Wuhan University, preservation date is on April 24th, 2012, and deposit number is CCTCC No:M 2012140.
Paecilomyces lilacinus WG20 of the present invention belongs to hyphomycete section (Hyohomycetaceae) paecilomyces (Paecilomyces) Paecilomyces lilacinus (Paecilomyces lilacinus)
Paecilomyces lilacinus WG20 of the present invention cultivates at the PDA solid medium, and bacterium colony begins to be white in color, and the growth with incubation time gradually becomes pale pink, mycelia is fine and close, is panniform, and colony edge is neat, be close to substratum, produce conidium, the spherical in shape or elliposoidal of spore.It is light brown that substrate mycelium is, and do not secrete soluble pigment.
Paecilomyces lilacinus WG20 of the present invention can grow at the PDA substratum, 28 ℃ of optimum growth temperatures.
The gram-positive microorganism of Paecilomyces lilacinus WG20 of the present invention has good inhibitory effect.The active substance that extracts from Paecilomyces lilacinus WG20 fermented liquid is respectively 19 μ g/mL to the minimal inhibitory concentration of streptococcus aureus, Listeria monocytogenes, bacillus pumilus and subtilis, 48 μ g/mL, 50 μ g/mL and 35 μ g/mL.
Paecilomyces lilacinus WG20 of the present invention belongs to paecilomyces (Paecilomyces), be deposited in Chinese typical culture collection center (CCTCC), the preservation address is Wuhan City Wuhan University, and preservation date is on April 24th, 2012, and deposit number is CCTCC No:M2012140.
Description of drawings
Fig. 1 is the colonial morphology photo of Paecilomyces lilacinus WG20 initial growth of the present invention; Fig. 2 is the colonial morphology photo in Paecilomyces lilacinus WG20 growth later stage of the present invention; Fig. 3 is Paecilomyces lilacinus WG20 mycelia form photo of the present invention; Fig. 4 carries out the systematic evolution tree that the homology comparison makes up for the ITS sequence of Paecilomyces lilacinus WG20 of the present invention and close bacterial strain; Fig. 5 is active substance to the antibacterial change curve of four strains for the examination bacterium, among the figure-◆-expression streptococcus aureus ,-■-expression Listeria monocytogenes ,-●-expression bacillus pumilus ,-▲-subtilis represented.
Embodiment
Embodiment one: the Paecilomyces lilacinus that present embodiment has bacteriostatic activity is Paecilomyces lilacinus (Paecilomyces lilacinus) WG20, be deposited in Chinese typical culture collection center (CCTCC), the preservation address is Wuhan City Wuhan University, preservation date is on April 24th, 2012, and deposit number is CCTCC No:M 2012140.
Present embodiment Paecilomyces lilacinus WG20 cultivates at the PDA solid medium, bacterium colony begin to be white in color (as Fig. 1), growth with incubation time, gradually become pale pink (as Fig. 2), mycelia is fine and close, is panniform, colony edge is neat, be close to substratum, produce conidium, the spherical in shape or elliposoidal of spore.Substrate mycelium is light brown (as Fig. 3), does not secrete soluble pigment.
Present embodiment Paecilomyces lilacinus WG20 can grow at the PDA substratum, 28 ℃ of optimum growth temperatures.
Embodiment two: present embodiment Paecilomyces lilacinus (Paecilomyces lilacinus) WG20 obtains by screening in the regional healthy wild Schisandra chinensis fruit in Heilongjiang Province of China cap mountain.Screening is carried out according to the following steps: chooses healthy wild Schisandra chinensis fruit water and rinses well, and with the aseptic filter paper suck dry moisture, 75% alcohol-pickled 3-5min, aseptic water washing 4 times; Shell into 2 parts with aseptic operation hilt fruit, place the PDA substratum, cultivate 3～5d in 28 ℃ of water bath with thermostatic control shaking tables, treat to grow thalline around the experiment material, the picking thalline changes flat board (PDA solid medium) separation and purification of repeatedly ruling over to, bacterial strain behind the purifying is placed slant medium (PDA solid medium), and 4 ℃ of preservations are Paecilomyces lilacinus WG20.
PDA substratum (1000mL) is made up of the distilled water of 200g potato, 20g sucrose and surplus.PDA solid medium (1000mL) is made up of the distilled water of 200g potato, 20g sucrose, 16g agar powder and surplus.
Negative control: whether thorough in order to check surface sterilization, the sterilized water of last flushing Schisandra chinensis fruit is coated on the PDA solid culture primary surface with aseptic spreading rod, Schisandra chinensis fruit after will sterilizing in addition rolls a week at the PDA solid medium,, cultivate under identical condition as negative control with this.
Through repeatedly repetition, the negative control flat board does not all have any bacterium colony and grows, and proves the sterilization of Schisandra chinensis fruit surface thoroughly, and the bacterium that is separated to is Schisandra chinensis endogenetic fungus-Paecilomyces lilacinus WG20, rather than the epiphyte on plant surface.
The Paecilomyces lilacinus WG20 that separation and purification is obtained carries out molecular biology identification, carry out according to the following steps: the total DNA that extracts bacterial strain, adopt fungi universal primer ITS1, ITS4 amplification Paecilomyces lilacinus WG20ITS sequence, check order behind the PCR product purification, its sequence is shown in SEQ ID NO:1, with sequencing result through the ncbi database sequence alignment, then by DNAMAN software building systematic evolution tree (as shown in Figure 4), all reached more than 99% with the similarity of Paecilomyces_lilacinus_strain_MCCF_58.06 and Paecilomyces_lilacinus_genes bacterial strain, by in conjunction with morphological features, growth conditions determines that Paecilomyces lilacinus WG20 belongs to paecilomyces (Paecilomyces).
(1) fermented liquid to present embodiment Paecilomyces lilacinus WG20 carries out bacteriostatic activity mensuration, and concrete steps are as follows:
1, the activation of Paecilomyces lilacinus WG20: aseptic technique in Bechtop with transfering loop picking Paecilomyces lilacinus WG20, is seeded in the plate culture medium (PDA solid medium), at 28 ℃ of constant temperature culture 5d.
2, the activation of strains tested:,, under gnotobasis, rule at slant medium (PDA solid medium) for examination fungi--Candida albicans bacterium with the transfering loop picking, in 28 ℃ of constant incubators, cultivate 3d.
Supply examination bacterium (subtilis, bacillus pumilus, streptococcus aureus, enterococcus faecalis, faecium, Listeria monocytogenes, streptococcus pyogenes, intestinal bacteria, pseudomonas aeruginosa, Acinetobacter bauamnnii, Klebsiella pneumonia) with 11 strains of transfering loop difference picking, under gnotobasis, rule at slant medium (beef extract-peptone nutrient agar), in 37 ℃ of constant incubators, cultivate 2d.
3, the preparation of Paecilomyces lilacinus WG20 fermented sample: the Paecilomyces lilacinus WG20 that separation is obtained is inoculated in the 50mLPDA substratum, does 3 repetitions, places 28 ℃ of airbath shaking tables of 140r/min, cultivates 24h and takes out, as seed liquor; The 20mL seed liquor is inoculated in the Erlenmeyer flask that 200mL PDA substratum is housed, places 28 ℃ of airbath shaking tables of 140r/min to cultivate 7 days, get fermented liquid.Fermented liquid with the centrifugal 10min of 4500r/min, is got supernatant liquid as Paecilomyces lilacinus WG20 fermented sample.
4, contain the preparation of bacterium flat board: will activate 2-3 time 11 strains confession examination bacterium according to above-mentioned steps 2, be inoculated on the slant medium (beef extract-peptone nutrient agar), in 37 ℃ of incubators, cultivate 24h, use the blood cell counting plate counting process, in the slant medium test tube of 11 strains for the examination bacterium, add the 5mL sterilized water respectively, fully vibration shakes up, get in the one after another drop of nucleonics between blood cell counting plate slide glass and cover glass, count at microscopically, being diluted to cell concentration then is 1 * 10
7CFU/mL obtains 11 strains for the bacteria suspension of examination bacterium; The bacteria suspension of each bacterial strain is joined in the beef extract-peptone nutrient agar about 50 ℃ in the ratio that adds the 1mL bacteria suspension in every 100mL substratum, behind the mixing, divide rapidly to install to and contain (the symmetry placement of two Oxford cups, apart from plate edge 20cm) aseptic empty flat board in, cooling is made 11 strains and is contained the bacterium flat board for the examination bacterium.
Same method, get activation and supply examination fungi--Candida albicans bacterium for 2-3 time, be inoculated on the slant medium (PDA solid medium), in 28 ℃ of incubators, cultivate 1-2d, use the blood cell counting plate counting process, in the slant medium test tube for the examination fungi, add 5mL physiological saline respectively, fully vibration shakes up, get in the one after another drop of nucleonics between blood cell counting plate slide glass and cover glass, count at microscopically, being diluted to cell concentration then is 1 * 10
7CFU/mL obtains the bacteria suspension for the examination fungi; Bacteria suspension is joined in the PDA solid medium about 50 ℃ in the ratio that adds the 1mL bacteria suspension in every 100mL substratum, behind the mixing, divide rapidly in the empty flat board that installs to after the sterilization that contains two Oxford cups, cooling is made and is contained the bacterium flat board for the examination fungi.
5, adopt cylinder plate method to detect bacteriostatic activity: measure Paecilomyces lilacinus WG20 fermented sample 150 μ L with liquid-transfering gun respectively and join and contain in the cup of the dull and stereotyped Oxford of bacterium, place 4 ℃ of refrigerator 6h, treat the fermented sample basic absorption after, flat board is put in 37 ℃ cultivates 24h.Observe antibacterial circle diameter, and do respective record.The bacterium flat board that contains of every strain strains tested is made 3 parallel samples, and negative control is the PDA substratum, and positive control adopts 0.1mg/mL Streptomycin sulphate and 0.2mg/mL nysfungin solution respectively.Detected result is as shown in table 1.
As can be seen from Table 1, Paecilomyces lilacinus WG20 fermented sample has bacteriostatic action preferably to gram-positive microorganisms such as streptococcus aureus, bacillus pumilus, subtilis, Listeria monocytogenes and streptococcus pyogeness, and to the almost not influence of growth of Gram-negative bacteria such as intestinal bacteria, Klebsiella pneumonia, Acinetobacter bauamnnii, pseudomonas aeruginosa and fungi (Candida albicans).
A is streptococcus aureus in the table 1; B is bacillus pumilus; C is subtilis; D is Listeria monocytogenes; E is streptococcus pyogenes; F is enterococcus faecalis; G is faecium; H is intestinal bacteria; I is Klebsiella pneumonia; J is Acinetobacter bauamnnii; K is pseudomonas aeruginosa; L is Candida albicans; Str is Streptomycin sulphate; Nys is nysfungin.--the expression non-activity.
Described streptococcus aureus (Staphylococcus aureus), bacillus pumilus (Bacillus pumilus), subtilis (Bacillus subtilis), Listeria monocytogenes (Listeria monocytogenes), streptococcus pyogenes (Streptococcus pyogenes), enterococcus faecalis (Enterococcus facalis), faecium (Enterococcus faecium), intestinal bacteria (Escherichia coli), Klebsiella pneumonia (Klebsiella pneumoniae), Acinetobacter bauamnnii (Acinetobacter baummanii), pseudomonas aeruginosa (Pseudomonas aeruginosa) and Candida albicans (Candida albicans) are all bought from Institute of Microbiology, Heilongjiang Academy of Sciences.
(2) activity of active substance detects
Utilize the TLC method to separate active substance in the Paecilomyces lilacinus WG20 fermented sample of above-mentioned steps 3 preparations, the WG20 fermented sample is adopted hexyl acetate extraction 2 times, behind the combining extraction liquid, obtain WG20 fermented liquid medicinal extract after reclaiming hexyl acetate, adopt silicagel column to separate WG20 fermented liquid medicinal extract, get active substance with methyl alcohol-chloroform gradient elution.
The active substance that separation is obtained is made into the sample solution of 0.1mg/mL with methyl alcohol, adopts filter paper method detection of active material to the restraining effect of 12 strains for the examination bacterium, and choose restraining effect stronger do the check of minimal inhibitory concentration for the examination bacterium.
Active substance is dissolved in 10%DMSO to final concentration 1mg/mL, precision is measured 100 μ L solution to 1mL centrifuge tube 1, precision pipettes 50 μ L solution to centrifuge tube 2 and adds 50 μ L10%DMSO and supplies from centrifuge tube 1, dilution successively according to said method, strength of solution is respectively 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL to the 1-6 centrifuge tube, and 50 μ L are not contained bacterium PDA substratum adds the 10%DMSO of 50 μ L as positive control in addition; The bacteria suspension of 50 μ L strains testeds adds the 10%DMSO of 50 μ L as negative control.Place 37 ℃ of constant incubators to cultivate 18h 96 orifice plates, take out 96 orifice plates, add the resazurin of the 0.4mg/mL of 40 μ L in each hole, cultivate 30min in 37 ℃ of constant incubators, microplate reader is measured its OD value, and analytical data is observed antibacterial result.
Active substance is as shown in table 2 for the inhibition result of examination bacterium to 12 strains, can see that from table 2 active substance has bacteriostatic activity preferably to streptococcus aureus, bacillus pumilus, subtilis and Listeria monocytogenes, the inhibition zone size all more than 10cm, is therefore chosen this 4 strain bacterium as checking active substance to its minimal inhibitory concentration for the examination bacterium.
A is streptococcus aureus in the table 1; B is bacillus pumilus; C is subtilis; D is Listeria monocytogenes; E is streptococcus pyogenes; F is enterococcus faecalis; G is faecium; H is intestinal bacteria; I is Klebsiella pneumonia; J is Acinetobacter bauamnnii; K is pseudomonas aeruginosa; L is Candida albicans; Str is Streptomycin sulphate; Nys is nysfungin.--the expression non-activity.
Active substance to four strains for the antibacterial change curve that tries bacterium as shown in Figure 5.Among the figure-◆-expression streptococcus aureus ,-■-expression Listeria monocytogenes ,-●-expression bacillus pumilus ,-▲-the expression subtilis.Active substance all has stronger restraining effect to four strains for the examination bacterium, and its restraining effect strengthens with the rising of concentration.Minimal inhibitory concentration (MIC) to streptococcus aureus, Listeria monocytogenes, bacillus pumilus and subtilis is respectively 19 μ g/mL, 48 μ g/mL, 50 μ g/mL and 35 μ g/mL.
Claims (1)
1. a strain has the Paecilomyces lilacinus (Paecilomyces lilacinus) of bacteriostatic activity, it is characterized in that this bacterial strain is Paecilomyces lilacinus WG20, be deposited in Chinese typical culture collection center (CCTCC), the preservation address is Wuhan City Wuhan University, preservation date is on April 24th, 2012, and deposit number is CCTCC No:M2012140.
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