CN113337403A - Chaetomium globosum HJF 13 strain and application thereof - Google Patents
Chaetomium globosum HJF 13 strain and application thereof Download PDFInfo
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Abstract
The invention provides chaetomium globosum and application thereof, belonging to the technical field of applied microbiology. The Chaetomium coccolium (Chaetomium cochliodes) HJF 13 bacterial strain is preserved in China center for type culture Collection in 2021, 4 months and 9 days, and the preservation number is CCTCC NO: m2021343. The anti-bacillus subtilis agent prepared by the liquid fermentation culture of the strain has better inhibition effect on bacillus subtilis, and can be used for preventing and treating the bacillus subtilis. The invention has the advantages of high efficiency, low toxicity, low raw material cost and simple production process, and has potential and practical application value in the fields of daily chemical products, foods, industrial products and the like.
Description
Technical Field
The invention relates to Chaetomium cochliodes HJF 13 and application thereof in preparing a bacillus subtilis resistant agent, belonging to the technical field of applied microbiology.
Background
Chaetomium globosum belongs to a species of chaetomium, is used as an effective biocontrol fungus and can generate antagonism to various pathogenic bacteria. The chaetomium globosum not only can reduce the incidence rate of plant diseases such as tomato wilt caused by fusarium and apple spot caused by venturia nigripes, but also has good inhibitory activity on common pathogenic bacteria such as candida albicans, escherichia coli, bacillus subtilis, pseudomonas fluorescens and the like in daily life, and has wide application prospect in research and development of medicines and pesticides.
Although bacillus subtilis has good application value in the aspects of biological control of animal and plant diseases, plant growth promoters, industrial enzymes, fertilizer fermentation and the like, the bacillus subtilis still is a harmful bacterium in the fields of daily chemical products, food, industrial products and the like. At present, more and more daily chemical products are added with high-nutrition components such as collagen, hyaluronic acid, polypeptide and the like, the high-nutrition components create more convenient conditions for the propagation of microorganisms while protecting the skin, and consumers use cosmetics with the excessive microorganisms and can cause adverse reactions such as skin redness and swelling, allergy and the like, so that an efficient and low-toxicity biological control method is urgently needed to be found for reducing the pollution of bacillus subtilis to products in some special fields. In earlier researches, Chaetomium globosum HJF 13(Chaetomium cochliodes HJF 13) with good inhibition effect on bacillus subtilis is screened from endophytic fungi separated from polygonatum plants, can be used for biological control of the bacillus subtilis, and is not reported in related documents through literature search.
Disclosure of Invention
The invention aims to provide Chaetomium cochliodes HJF 13 (HJF 13) and application thereof in preparation of a bacillus subtilis resistant agent.
The purpose of the invention is realized as follows:
the strain Chaetomium globosum HJF 13(Chaetomium cochliodes HJF 13) used in the invention, strain HJF 13, which was deposited at 9.4.2021 in the China center for type culture Collection, addresses: the preservation number of Wuhan university No. 16 Lojia mountain road in Wuchang district, Wuhan City, Hubei province of China is CCTCC NO: m2021343.
The morphological characteristics of the strain are as follows: hyphae are interlaced and transparent, the ascocarp is epibiotic, the medium size is egg-shaped, and the colony is initially white and gradually light brown on the PDA culture medium. Extracting DNA of the strain, amplifying ITS sequences by adopting fungus universal primers ITS1 and ITS4, then sequencing, and carrying out BLAST search comparison on the sequenced DNA sequences in NCBI database, wherein the ITS sequences of the strain and Chaetomium coccolioides effectively release Chaetomium coccoliodes HMAS (244354 similarity is 100 percent), so the strain is named Chaetomium coccoliodes HJF 13(Chaetomium coccoliodes HJF 13).
The chaetomium globosum HJF 13 is a polygonatum plant endophytic fungus.
The Chaetomium coccolii HJF 13(Chaetomium cochliodes HJF 13) is applied to preparing the anti-bacillus subtilis agent.
The steps for preparing the microbial anti-bacillus subtilis agent are as follows:
1. culture of test tube species
The strain HJF 13 was inoculated onto a slant of a test tube solid medium and cultured at 28 ℃ for 6 days to obtain a test tube seed. The solid culture medium is common PDA culture medium, and is prepared by peeling potato 200g, cutting into small pieces, adding 1000ml water, boiling for 20 min, filtering, adding glucose 20g and agar 15g into the filtrate, adding water to desired volume of 1000ml, and sterilizing at 121 deg.C for 30 min.
2. Liquid seed culture
Adopting a 500ml triangular flask to culture in a shake flask, the method is to firstly prepare a liquid seed culture medium which is a common PDB culture medium, and the method comprises the following steps: peeling potato 200g, cutting into small pieces, adding water 1000ml, boiling for 20 min, filtering, adding glucose 20g into the filtrate, adding water to desired volume of 1000ml, placing 100ml liquid seeds into each triangular flask, and culturingThe mixture was sterilized at 121 ℃ for 30 minutes. Cooling and connecting to 1cm2Large test tube seeds were incubated at 28 ℃ for 3 days on a shaker at 220rpm to obtain seed solutions.
3. Preparation of anti-Bacillus subtilis agent
Adopting 1000ml triangular flasks for shake flask culture, wherein the liquid fermentation medium is a common PDB medium, the preparation method is the same as the preparation method of the liquid seed culture medium, 200ml of the liquid fermentation medium is filled into each triangular flask, the triangular flasks are sterilized at 121 ℃ for 30 minutes, 20ml of the seed solution obtained by the method 2 is inoculated after cooling, and the strain fermentation broth is obtained after culture on a shaking table with the rotating speed of 220rpm at 28 ℃ for 7 days. Adding equal volume of ethanol into the fermentation liquor, filtering mycelium after shaking for 6 hours by a shaking table, evaporating the ethanol in the fermentation liquor to obtain a strain fermentation extract, and diluting the fermentation extract to the volume of the original fermentation liquor by water to obtain the usable anti-bacillus subtilis microbial inoculum.
The microbial anti-bacillus subtilis agent prepared by the method is used for testing the inhibition effect of the agent on bacillus subtilis by adopting a plate bacteriostasis test.
The plate bacteriostasis test result shows that: the average diameter of the inhibition zone of the strain HJF 13 anti-bacillus subtilis agent prepared by the method on bacillus subtilis is 18.0 mm.
The plate bacteriostasis test shows that the preparation has stronger inhibition effect on resisting bacillus subtilis.
The invention provides chaetomium globosum HJF 13 and application thereof in preparing a bacillus subtilis resistant agent through fermentation. The invention has the advantages of high efficiency, low toxicity, low raw material cost and simple production process, and has potential and practical application value in the fields of daily chemical products, foods, industrial products and the like.
Detailed Description
The invention will be further illustrated with reference to specific examples:
the first embodiment is as follows: identification of Chaetomium coccolifera HJF 13(Chaetomium cochliodes HJF 13)
DNA of a strain is extracted by a conventional CTAB method, ITS sequences are amplified by fungus universal primers ITS1 and ITS4, a PCR product is sent to Beijing Optimalaceae (Kunming) New science and technology Limited company for sequencing, the DNA sequence obtained by sequencing is subjected to BLAST search comparison in NCBI database, the ITS sequence of the strain and Chaetomium cochliodes HMAS which is an effective published species of Chaetomium cochliodes are 100 percent in similarity, therefore, the strain is named as Chaetomium cochliodes HJF (Chaetomium Cochliodes H13), the strain is preserved in China center for type culture collection at 4 and 9 months 2021, the address: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO: m2021343.
Example two: preparation method of microbial anti-bacillus subtilis agent
1. Culture of test tube species
The strain HJF 13 was inoculated onto a slant of a test tube solid medium and cultured at 28 ℃ for 6 days to obtain a test tube seed. The solid culture medium is common PDA culture medium, and is prepared by peeling potato 200g, cutting into small pieces, adding 1000ml water, boiling for 20 min, filtering, adding glucose 20g and agar 15g into the filtrate, adding water to desired volume of 1000ml, and sterilizing at 121 deg.C for 30 min.
2. Liquid seed culture
Adopting a 500ml triangular flask to culture in a shake flask, the method is to firstly prepare a liquid seed culture medium which is a common PDB culture medium, and the method comprises the following steps: peeling 200g of potato, cutting into small pieces, adding 1000ml of water, boiling for 20 minutes, filtering, adding 20g of glucose into the obtained filtrate, adding water to a constant volume of 1000ml, filling 100ml of liquid seed culture medium into each triangular flask, and sterilizing at 121 ℃ for 30 minutes. Cooling and connecting to 1cm2Large test tube seeds were incubated at 28 ℃ for 3 days on a shaker at 220rpm to obtain seed solutions.
3. Preparation of anti-Bacillus subtilis agent
Adopting 1000ml triangular flasks for shake flask culture, wherein the liquid fermentation medium is a common PDB medium, the preparation method is the same as the preparation method of the liquid seed culture medium, 200ml of the liquid fermentation medium is filled into each triangular flask, the triangular flasks are sterilized at 121 ℃ for 30 minutes, 20ml of the seed solution obtained by the method 2 is inoculated after cooling, and the strain fermentation broth is obtained after culture on a shaking table with the rotating speed of 220rpm at 28 ℃ for 7 days. Adding equal volume of ethanol into the fermentation liquor, filtering mycelium after shaking for 6 hours by a shaking table, evaporating the ethanol in the fermentation liquor to obtain a strain fermentation extract, and diluting the fermentation extract to the volume of the original fermentation liquor by water to obtain the usable anti-bacillus subtilis microbial inoculum.
Example three: plate bacteriostasis test
1. Preparation of microbial anti-Bacillus subtilis agent for experiment
The microbial anti-bacillus subtilis microbial agent is prepared from Chaetomium cochliodes HJF 13 strains of HJF 13(Chaetomium cochliodes HJF 13) by a liquid fermentation method. The concrete preparation process and conditions are the same as the description part of the invention content.
2. Cultivation of Bacillus subtilis
Inoculating Bacillus subtilis to test tube slant culture medium, and culturing at 25 deg.C for 5-6 days until the thallus grows well. The culture medium is a conventional ISP2 culture medium, and the formula of the culture medium is as follows: 4g of yeast powder; 4g of glucose; 10g of malt extract; 1000ml of water, 15g of agar powder was added to the medium, and the medium was sterilized at 121 ℃ for 30 minutes.
3. Flat plate bacteriostasis test method
Taking 0.5cm of cultured Bacillus subtilis2The large test tube seed inclined plane and 20ml ISP2 culture medium are mixed evenly, poured into a sterilized culture dish with the inner diameter of 9cm to be made into a bacterium carrying plate, the conventional cup and dish method is adopted in the plate bacteriostasis experiment, namely an Oxford cup is placed on each bacterium carrying plate, 0.2ml of prepared anti-bacillus subtilis agent is added into the Oxford cup, sterile clean water is used as blank control, the treatment is repeated for 3 times, and the diameter of a bacteriostasis ring is measured by a cross method after the culture is carried out in a thermostat at 25 ℃ for 48 hours.
TABLE 1 bacteriostatic results of the microbial anti-Bacillus subtilis agent on Bacillus subtilis
The experimental results show that: the average diameter of the inhibition zone of the strain HJF 13 anti-bacillus subtilis agent to bacillus subtilis is 18.0 mm.
Claims (4)
1. Chaetomium globosum HJF 13 strain, wherein Chaetomium cochliodes HJF 13 strain has been preserved in China Center for Type Culture Collection (CCTCC) at 9.4.2021 with the preservation number of M2021343.
2. The strain chaetomium globosum HJF 13 of claim 1, wherein the form characteristics of the strain HJF 13 are: hyphae are interlaced and transparent, the ascocarp is epibiotic, the medium size is egg-shaped, and the colony is initially white and gradually light brown on the PDA culture medium.
3. The Chaetomium globosum HJF 13 strain of claim 1, wherein the HJF 13 strain is a polygonatum plant endophytic fungus.
4. Use of the chaetomium globosum HJF 13 strain of claim 1, 2 or 3 in the preparation of an anti-bacillus subtilis agent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322192A (en) * | 2021-07-15 | 2021-08-31 | 云南省农业科学院药用植物研究所 | Polygonatum sibiricum endophytic fungi escherichia coli inhibiting agent and preparation method and application thereof |
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|---|---|---|---|---|
| US3917820A (en) * | 1969-05-29 | 1975-11-04 | Canadian Patents Dev | Antibiotic cochliodinol and production by chaetomium, cochliodes and chaetomium globsum |
| CN107163067A (en) * | 2017-07-05 | 2017-09-15 | 山东省科学院生物研究所 | Wide spectrum antimicrobial agent lead compound and preparation and application |
| CN107164233A (en) * | 2016-12-28 | 2017-09-15 | 贵州省植物保护研究所 | A kind of chaetomium globosum LJ S2L1 bacterial strains and its application |
| CN107721908A (en) * | 2017-09-21 | 2018-02-23 | 黄河科技学院 | A kind of method that chaetomium globosum A precursor compounds are extracted from chaetomium globosum |
-
2021
- 2021-05-20 CN CN202110550481.3A patent/CN113337403A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3917820A (en) * | 1969-05-29 | 1975-11-04 | Canadian Patents Dev | Antibiotic cochliodinol and production by chaetomium, cochliodes and chaetomium globsum |
| CN107164233A (en) * | 2016-12-28 | 2017-09-15 | 贵州省植物保护研究所 | A kind of chaetomium globosum LJ S2L1 bacterial strains and its application |
| CN107163067A (en) * | 2017-07-05 | 2017-09-15 | 山东省科学院生物研究所 | Wide spectrum antimicrobial agent lead compound and preparation and application |
| CN107721908A (en) * | 2017-09-21 | 2018-02-23 | 黄河科技学院 | A kind of method that chaetomium globosum A precursor compounds are extracted from chaetomium globosum |
Non-Patent Citations (2)
| Title |
|---|
| KATARÍNA CHOVANOVÁ 等: "Detection of the antibacterial effect of Chaetomium cochliodes Palliser CCM F-232 based on agar plugs and unprocessed fungal substances from cultivation media", 《BIOLOGIA》 * |
| 杨恩超 等: "螺卷毛壳霉代谢产物的抗菌活性研究(英文)", 《天然产物研究与开发》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322192A (en) * | 2021-07-15 | 2021-08-31 | 云南省农业科学院药用植物研究所 | Polygonatum sibiricum endophytic fungi escherichia coli inhibiting agent and preparation method and application thereof |
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