CN106190869B - Humicola and application thereof - Google Patents

Humicola and application thereof Download PDF

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Publication number
CN106190869B
CN106190869B CN201610812401.6A CN201610812401A CN106190869B CN 106190869 B CN106190869 B CN 106190869B CN 201610812401 A CN201610812401 A CN 201610812401A CN 106190869 B CN106190869 B CN 106190869B
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humicola
extract
strain
fermentation
filter paper
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CN106190869A (en
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李浩华
章卫民
李赛妮
谭国慧
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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Guangdong Institute of Microbiology
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/645Fungi ; Processes using fungi
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
    • C12N1/145Fungal isolates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts

Abstract

The invention discloses a Humicola strain and application thereof. Humicola sp A695, deposit No.: GDMCC No: 60025. the Humicola sp A695 extract has obvious antibacterial activity, and the extract has good application and development prospects, so that the Humicola sp A695 extract provides a basis for researching and developing new antibacterial drugs and provides a scientific basis for developing and utilizing natural active substances from endophytic fungi.

Description

Humicola and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a medicinal plant fructus amomi endophytic fungus Humicola sp.A695, an extract prepared from a fermentation liquid of the strain and application of the extract in preparation of antibacterial drugs.
Background
Endophytic fungi (endophytic fungi) refer to fungi that live in healthy plant tissues at some or all stages of their life history, but do not cause significant disease symptoms to plant tissues (Tan RX and Zou WX, 2001). Endophytic fungi have abundant species diversity and genetic diversity, so that a plurality of new or rare fungal species are discovered from plant endophytic fungi so far, and the endophytic fungi become research hotspots at home and abroad. The endophytic fungi grow in the special environment inside plants, produce active compounds far beyond the range of plant metabolites, can produce various active substances with unique structure types and potential application prospects, and become important resources for discovering new natural active substances.
Disclosure of Invention
A first object of the present invention is to provide an endophytic fungus Humicola (Humicola sp.) a695 deposited at the guangdong province collection of microorganisms (GDMCC) 3, 21 days 2016, addresses: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC No: 60025.
the second purpose of the invention is to provide a Humicola sp A695 extract with antibacterial effect, which is characterized in that the Humicola sp A695 extract is used as a fermentation strain, liquid fermentation is carried out to obtain a fermentation culture, mycelium and fermentation liquor are separated, the fermentation liquor is extracted by ethyl acetate, and the Humicola sp A695 fermentation extract is obtained after the extraction liquid is concentrated.
The liquid fermentation is to inoculate Humicola (Humicola sp.) A695 into a fermentation culture medium, and perform liquid fermentation culture at 28 ℃ and 120r/min to obtain a fermentation culture, wherein each liter of the fermentation culture medium is prepared by the following method: decocting 200g potato in 500mL distilled water for 20min, filtering to obtain juice, adding glucose 20g and KH2PO43g、MgSO40.75g, vitamin B110mg, adding water to 1L, and naturally adjusting pH to obtain fermentation medium.
Antibacterial experiments were carried out using the Humicola sp A695 extract of the present invention, and it was found through experiments that the inhibition zones for Staphylococcus aureus and Bacillus subtilis were 19.5 mm and 16.25mm, respectively, when each filter paper sheet (diameter: 6mm) contained 250. mu.g of the extract. The Minimum Inhibitory Concentration (MIC) was determined at 7 concentrations of 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078mg/mL to determine the MIC values of Humicola sp A695 extract for Staphylococcus aureus and Bacillus subtilis at 0.321mg/mL and 0.625mg/mL, respectively.
Therefore, a third object of the present invention is the use of a Humicola sp A695 extract for the preparation of an antibacterial medicament.
An antibacterial agent comprising the above Humicola sp A695 extract as an active ingredient.
The antibacterial drug is a drug for resisting Staphylococcus aureus (Staphylococcus aureus) or Bacillus subtilis (Bacillus subtilis).
The Humicola sp A695 extract has obvious antibacterial activity, and the extract has good application and development prospects, so that the Humicola sp A695 extract provides a basis for researching and developing new antibacterial drugs and provides a scientific basis for developing and utilizing natural active substances from endophytic fungi.
Humicola (Humicola sp.) a695 was deposited at 2016 at 21/3 with the collection of microbial cultures of guangdong province (GDMCC), address: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC No: 60025.
description of the drawings:
FIG. 1 is a phylogenetic tree of Humicola sp A695 strain and related strains according to the invention, wherein A695 represents Humicola sp A695.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: isolation, purification and characterization of Humicola sp A695
(1) Separating and purifying strains:
washing the root of fructus Amomi collected from Shuizhou of Yangchun city of Guangdong province with tap water, and naturally air drying on filter paper. Soaking in 0.1% by mass of mercuric chloride solution for 5-7 min, rinsing with sterile water for 4 times, soaking in 75% by volume of ethanol aqueous solution for 3-5 min, and rinsing with sterile water for 3 times. The head and tail parts are cut off by scissors, and the middle part is cut into small sections with the length of about 1 cm. Placing the strain in a separation culture medium (containing 20 mug/mL kanamycin sulfate and 20 mug/mL ampicillin), culturing for 3-7 days at a constant temperature box at 28 ℃, selecting marginal hyphae, transferring the marginal hyphae to a fresh separation culture medium, and continuously purifying until a pure strain is obtained, namely the strain A695.
The separation culture medium is obtained by the following method: decocting 200g potato in 500mL distilled water for 20min, filtering to obtain juice, adding glucose 20g and KH2PO43g、MgSO40.75g, vitamin B110mg of agar and 20g of water, the solution is made up to 1L with natural pH and sterilized for later use.
(2) Identification of the strains:
and extracting the genome DNA of the strain A695 by adopting a fungus genome DNA extraction kit to serve as a template for PCR amplification. PCR amplification the rDNA ITS region of the isolated strain was amplified using the fungal rDNA internal transcribed spacer (rDNA ITS) universal primers ITS1(5'-TCCGTAGGTGAACCTGCGG-3', forward) and ITS4(5'-TCCTCCGCTTATTGATATGC-3', reverse). PCR was carried out using Ex Taq (TaKa-Ra) using a 20. mu.L reaction system. Reaction conditions are as follows: pre-denaturation at 93 deg.C for 3 min; denaturation at 93 deg.C for 45s, renaturation at 55 deg.C for 45s, and extension at 72 deg.C for 1.5min for 30 cycles; finally, extension is carried out for 10min at 72 ℃. The PCR product is sequenced by Shenzhen Hua Dagen science and technology service Limited company, and the sequence is shown as SEQ ID NO. 1. The obtained sequences were subjected to a similarity sequence search on GenBank by BLAST program, the related sequences were downloaded, and phylogenetic trees were constructed by using MEGA 5 software by the Neighbor-Joining method (Neighbor-Joining) with Bootstrap set to 1000 repeats (fig. 1). The base sequence of the ITS region of the strain A695 is shown in SEQ ID NO. 1. Sequence information obtained from sequencing was BLAST-performed, and BLAST results showed that strain a695 was 99.3% similar to Humicola sp. (KF 472159). From the constructed phylogenetic tree, strain a695 was clustered with 5 Humicola sp. sequences into a highly self-expanding branch with minimal genetic distance from each other (fig. 1). Therefore, the strain was identified as Humicola insolens (Humicola sp.) by molecular phylogenetic analysis, and named Humicola sp A695.
The Humicola sp 695 was deposited at 2016, 3/21 days, in the Guangdong province collection of microorganisms (GDMCC), address: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC No: 60025.
example 2: humicola sp A695 activation, fermentation culture and extract preparation
(1) The pure Humicola strain (Humicola sp.) A695 obtained in example 1 was inoculated into a slant culture medium and cultured at 28 ℃ for 5 days to obtain an activated strain. The slant culture medium is obtained by the following method: decocting 200g potato in 500mL distilled water for 20min, filtering to obtain juice, adding glucose 20g and KH2PO43g、MgSO40.75g, vitamin B110mg of agar and 20g of water, the solution is made up to 1L with natural pH and sterilized for later use.
(2) Inoculating the strain subjected to the activation culture in the step (1) into a seed culture medium, and culturing for 7d under the conditions of 28 ℃ and 120r/min oscillation to obtain the strainTo a seed solution, the seed culture medium is obtained by the following method: decocting 200g potato in 500mL distilled water for 20min, filtering to obtain juice, adding glucose 20g and KH2PO43g、MgSO40.75g, vitamin B110mg, adding water to 1L, naturally adjusting pH, and sterilizing.
(3) Inoculating the seed liquid obtained in the step (2) into a fermentation culture medium in an inoculation amount of 10%, and culturing at 28 ℃ and 120r/min for 7d to obtain a fermentation culture. The fermentation medium is the same as the seed medium.
(4) Preparing an extract: and (3) extracting the filtrate obtained after the fermentation culture obtained in the step (3) is subjected to degerming with ethyl acetate for 3 times, and concentrating the extract under reduced pressure to obtain the Humicola sp A695 extract.
Example 3: measurement of antibacterial Activity of Humicola sp A695 extract
The antibacterial activity of the extract of Humicola sp A695 against Staphylococcus aureus (Staphylococcus aureus) or Bacillus subtilis (Bacillus subtilis) was measured by a filter paper method.
The Humicola insolens (Humicola sp.) A695 extract obtained in example 2 was dissolved in DMSO and diluted to a concentration of 500. mu.g/mL.
Respectively diluting the staphylococcus aureus and bacillus subtilis liquid obtained by liquid fermentation with normal saline to the concentration of 106CFU/mL, suction concentration 1061mL of CFU/mL bacterial solution is put into a plate, and an LB culture medium cooled to a proper temperature is poured into the plate and mixed uniformly. Clamping a filter paper sheet with the thickness of 1.5mm and the diameter of 6mm by using a sterile forceps, placing the filter paper sheet on a bacteria-containing plate, simultaneously dropwise adding 5 mu L of a Humicola sp A695 extract diluent on the filter paper sheet respectively to obtain a sample group, using DMSO instead of the Humicola sp A695 extract diluent to obtain a blank control group, using ampicillin with the concentration of 200 mu g/mL as a positive control group, placing the plate in a 37 ℃ incubator for culturing for 24h, measuring the diameter of a bacteriostatic ring by using a cross measurement method, repeating the steps for three times, and calculating the average value of the diameter of the bacteriostatic ring.
The results of the experiment are shown in table 1:
TABLE 1 inhibition of bacteria by Humicola sp A695 extract ((n=3)
Example 4: determination of the minimum inhibitory concentration of Humicola sp A695 extract on Staphylococcus aureus and Bacillus subtilis
The paper filter disc method is adopted to determine the minimum inhibitory concentration of the Humicola sp A695 extract on staphylococcus aureus and bacillus subtilis.
The Humicola insolens (Humicola sp.) A695 extract of example 2 was diluted with DMSO to 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078mg/mL, respectively. Respectively absorb the concentration of 1061mL of CFU/mL of staphylococcus aureus and bacillus subtilis liquid is put into a culture dish, and an LB culture medium cooled to a proper temperature is poured into the culture dish and is uniformly mixed. A filter paper piece with the thickness of 1.5mm and the diameter of 6mm is clamped by a sterile forceps and placed on a bacteria-containing flat plate, 5 mu L of Humicola sp A695 extract diluent with the concentration is respectively dripped on the filter paper piece, the filter paper piece is placed in a 37 ℃ incubator for culturing for 24 hours, the bacteriostatic circle around the filter paper piece is observed, and the lowest concentration of a sample contained in the filter paper piece with the bacteriostatic circle around the filter paper piece is taken as the minimum bacteriostatic concentration (MIC value).
The results of the experiment are shown in table 2:
table 2: minimum inhibitory concentration (MIC value) of Humicola sp A695 extract on bacterian=3)
Note: "+" indicates the presence of zone of inhibition, and "-" indicates the absence of zone of inhibition
The experimental result shows that the Humicola sp A695 extract has obvious antibacterial effect on staphylococcus aureus or bacillus subtilis, and can be used for preparing antibacterial drugs. Therefore, the invention provides a material basis for researching and developing new antibacterial drugs and provides a scientific basis for developing and utilizing natural active substances from endophytic fungi of plants.

Claims (1)

1. Humicola insolens (A) and (B)Humicola sp.) A695, accession no: GDMCC No: 60025.
CN201610812401.6A 2016-09-08 2016-09-08 Humicola and application thereof Active CN106190869B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102492629A (en) * 2011-12-09 2012-06-13 广东省微生物研究所 Marine fungi penicillium thomii, extract and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102492629A (en) * 2011-12-09 2012-06-13 广东省微生物研究所 Marine fungi penicillium thomii, extract and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
培养条件对细脚拟青霉抑制金黄色葡萄球菌的影响;桂琳等;《食品工业科技》;20101231(第10期);216-218 *
药用植物马蔺的内生真菌分离及其抑菌活性的研究;毕江涛等;《安徽农业科学》;20121231(第32期);15651-15654 *

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