CN115404169A - Cladosporium sp - Google Patents
Cladosporium sp Download PDFInfo
- Publication number
- CN115404169A CN115404169A CN202110592534.8A CN202110592534A CN115404169A CN 115404169 A CN115404169 A CN 115404169A CN 202110592534 A CN202110592534 A CN 202110592534A CN 115404169 A CN115404169 A CN 115404169A
- Authority
- CN
- China
- Prior art keywords
- cladosporium
- polysaccharide
- strain
- enzyme preparation
- degrading
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 241001207508 Cladosporium sp. Species 0.000 claims description 27
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Abstract
The invention provides Cladosporium and application thereof, and particularly provides a Cladosporium sp, which is found for the first time to be capable of effectively degrading polysaccharides, and also found for the first time to be capable of simultaneously degrading multiple polysaccharides (such as algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate E, carrageenan, kappa-type carrageenan, lambda-type carrageenan and iota-type carrageenan).
Description
Technical Field
The invention relates to the field of microorganisms, in particular to cladosporium and application thereof.
Background
Polysaccharides are polymeric carbohydrate macromolecules having at least 10 monosaccharides linked by glycosidic linkages, and can be represented by the general formula (C) 6 H 10 O 5 ) n represents. The polysaccharide of different species has different pharmacological effects due to different structures, molecular weights and solubilities, and has various biological activities. Most polysaccharides are important components of organisms, some are energy source substances, and part of the polysaccharides are information molecules involved in cell recognition. The glycosaminoglycan or oligosaccharide including hyaluronic acid, chondroitin sulfate/dermatan sulfate, heparin/heparan sulfate, keratan sulfate and the like is widely distributed on the cell surface or extracellular matrix of marine animal and terrestrial animal tissues, and participates in various physiological and pathological behaviors such as morphogenesis, signal conduction, cell proliferation and differentiation, wound repair, virus infection and the like of the developing tissues of the central nervous system, so that the research on the glycosaminoglycan has important significance. In addition, degradation products of polysaccharides have been shown to possess a variety of important physiological activities, such as: the new agaro-oligosaccharide has antioxidation, the pectin oligosaccharide chelated zinc is used as a feed additive, the chitosan oligosaccharide has anti-tumor proliferation activity, and the like. In conclusion, polysaccharide and oligosaccharide products thereof have important application values, so the development of polysaccharide degrading bacteria and polysaccharide degrading enzyme resources thereof is one of the research hotspots for preparing oligosaccharides by an enzyme method, and the discovery of novel polysaccharide degrading bacteria is the source guarantee for developing novel tool enzymes.
The pluripotent polysaccharide-degrading bacteria refer to strains that can grow by using a plurality of polysaccharides as a sole carbon source. The multi-energy polysaccharide degrading bacteria can generate various different types of polysaccharide degrading enzymes, catalyze the breakage of glycosidic bonds between sugar units in substrate molecules and generate a series of oligosaccharides and even monosaccharides, thereby realizing the degradation and utilization of different polysaccharide carbon sources by the bacterial strains. Therefore, the method has important value and significance for the development of novel polysaccharide degrading bacteria and enzyme resources thereof and the research of metabolites. Microorganisms from special niches, such as marine endophytic symbionts, form unique biological metabolic pathways in the long-term evolution process in order to adapt to the special living environment (high salt, high pressure, low oxygen, low light, nutrient limitation and the like) of the marine ecosystem, have abundant enzyme resources, and can utilize various polysaccharide substrates and produce a plurality of natural products with novel structures and good biochemistry. Many bacteria, actinomycetes, fungi, and marine invertebrates and algae, among which bacteria are mainly concentrated in pseudomonas, vibrio, micrococcus, bacillus, enterobacter, and allomonas bacteria; actinomycetes mainly include Streptomyces and Micromonospora; the marine epiphyte mainly comprises cladosporium, alternaria, aspergillus, pediococcus, penicillium, and phoma. And certain substances in the fungal metabolite are novel therapeutic drugs with biological activity, so that the method can provide a choice for continuously exploring low-toxicity and high-efficiency drugs, and has a wide application prospect. However, the only reports on the marine origin of the polysaccharide-degrading strains are Flammeovirga sp.MY04, persicobacter sp.JZB09, pseudoalteromonas sp.A601, vibrio sp.FC509, pseudoalteromonas sp.Q02 and a few foreign strains such as Saccharophugus degrada-ns, zobellia galactitanvorans, etc.
The cladosporium cladosporioides has high concentration in the environment, generally does not threaten the organism, but can be infected by secondary cladosporium cladosporioides such as some primary diseases (leukemia, cancer, AIDS and the like), hypoimmunity (immunosuppressant, chemotherapeutic drugs), organ transplantation and the like, and the chances of infecting the organism by the cladosporium cladosporioides and other fungi are increased while the lives of patients are prolonged by advanced medical technology and various drugs of people. Reports of Cladosporium ramosum infecting human and animals are frequent, the first case of pulmonary mycosis caused by Cladosporium ramosum is reported from 1975Kwon-Chung, and students in the world report diseases such as skin and subcutaneous mycosis, corneal pigmentary mycosis, fungal keratitis and nasosinusitis caused by the bacteria in sequence, the bacteria are separated from a plurality of places such as gingival granulation tissues, cerebrospinal fluid, spinal cords of leukemia patients and bodies of diabetes patients, and skin diseases, encephalitis, nephritis and other diseases caused by the bacteria are also found on animals. Currently, research on cladosporium is mainly focused on the research on cladosporium strains, in contrast, research on other species of cladosporium is rare, and reports on pluripotent polysaccharide-degrading bacteria from which no cladosporium is derived are available.
Studies on polysaccharide-degrading enzymes are relatively rare and focused on studies on single polysaccharide-degrading enzymes produced by strains, such as cellulose-degrading enzymes, pectinases, polygalacturonases, and the like. However, there is no known polysaccharide-degrading bacterium belonging to the genus Cladosporium, which has a capability of degrading various polysaccharides.
Therefore, there is an urgent need in the art to develop cladosporium moulds that facilitate the development of cognitive and functional oligosaccharide drugs for the relevant molecular pathogenic mechanisms.
Disclosure of Invention
The invention aims to provide cladosporium moulds which are beneficial to the development of cognitive and functional oligosaccharide medicines of related molecular pathogenic mechanisms.
In a first aspect, the present invention provides a Cladosporium sp.
In another preferred embodiment, the ITS gene of Cladosporium has the sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the Cladosporium is Cladosporium sp.m02, deposited under the following accession number: CGMCC No 13568.
In another preferred embodiment, the cladosporium is derived from seaweed.
In another preferred embodiment, the cladosporium moulds are from seaweed on the coast of Qingdao.
In a second aspect, the invention provides an enzyme preparation comprising:
cladosporium sp.and/or a metabolite thereof according to the first aspect of the invention.
In another preferred embodiment, the enzyme preparation comprises a polysaccharide degrading enzyme preparation.
In another preferred embodiment, the enzyme preparation is an extracellular enzyme preparation.
In another preferred embodiment, the polysaccharide is derived from microorganisms, seaweeds, plants and animals.
In another preferred example, the polysaccharide comprises microbial polysaccharide, algal polysaccharide, higher plant polysaccharide, and animal polysaccharide. In another preferred embodiment, the microbial polysaccharide comprises xanthan gum.
In another preferred example, the algal polysaccharides include algin (Alginate), agarose, carrageenan, kappa carrageenan, iota carrageenan, lambda carrageenan.
In another preferred example, the higher plant polysaccharide comprises cellulose, microcrystalline cellulose, carboxymethyl cellulose, starch, xylan, mannan, pectin.
In another preferred example, the animal polysaccharide comprises Hyaluronic Acid (HA), chondroitin Sulfate A (CSA), chondroitin Sulfate C (CSC), chondroitin sulfate E, heparan sulfate, dermatan sulfate, chitin, chitosan.
In another preferred embodiment, the enzyme preparation comprises Cladosporium and/or its metabolites in an amount of 1X 10 to 1X 10 15 cfu/mL or cfu/g Cladosporium sp., preferably 1X 10 4 -1×10 9 cfu/mL or cfu/g Cladosporium sp., based on the total volume or total weight of the enzyme preparation.
In another preferred embodiment, the enzyme preparation comprises 0.0001 to 99 wt.%, preferably 0.1 to 90 wt.%, of component (a), based on the total weight of the enzyme preparation.
In another preferred embodiment, the enzyme preparation further comprises a substance (e.g. a protective agent) which helps to maintain the viability of cladosporium.
In another preferred embodiment, the substance (e.g. protective agent) that helps to maintain the viability of cladosporium is selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidant, D-erythorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
In another preferred embodiment, the enzyme preparation is in a dosage form selected from the group consisting of: solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.
In a third aspect, the present invention provides a use of a cladosporium according to the first aspect of the invention and/or a metabolite thereof, or an enzyme preparation according to the second aspect of the invention, for the preparation of a composition or a preparation for degrading a polysaccharide.
In another preferred embodiment, the composition or formulation can degrade multiple polysaccharides (e.g., algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate a, chondroitin sulfate E, carrageenan, kappa carrageenan, lambda carrageenan, iota carrageenan) simultaneously.
In another preferred embodiment, the polysaccharide is selected from the group consisting of: algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate E, carrageenan, kappa carrageenan, lambda carrageenan, iota carrageenan, or a combination thereof.
In a fourth aspect, the present invention provides a use of the cladosporium and/or a metabolite thereof according to the first aspect of the invention for the preparation of an enzyme preparation for degrading a polysaccharide.
In another preferred embodiment, the enzyme preparation comprises a polysaccharide degrading enzyme preparation.
In another preferred embodiment, the enzyme preparation is an extracellular enzyme preparation.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows photographs of the basic morphological characteristics of the M02 strain of Cladosporium sp;
a, culturing the bacterial strain for 4 days, wherein the bacterial colony morphology of the bacterial strain on a primary screening culture medium is positive; B. culturing for 4 days, and then, putting the strain on a primary screening culture medium to obtain the reverse side of the colony morphology; C. lugol iodine staining pattern;
FIG. 2 shows a microscopic photograph of M02 strain of Cladosporium sp;
wherein, A, the hyphal form of a microscope (insert method, 10 multiplied by 40); B. microscopic hypha morphology (slide printing, 10 × 40); C. microscopic hyphal morphology (slide printing, 10 × 100);
FIG. 3 shows a cladosporium M02 strain plotted on the basis of a partial ITS gene sequence;
FIG. 4 shows a polyacrylamide gel electrophoresis (SDS-PAGE) of a complex polysaccharide-degrading enzyme preparation of M02 strain of Cladosporium sp;
in the figure: lanes M1, 10 μ L protein molecular weight standards, bands are from top to bottom: 116kD,66.2kD,45kD,35kD,25kD,18.4kD,14.4kD; lane 1, 20 μ LM02 strain ammonium sulfate 80% saturation ectoenzyme preparation; lane 2, 20 μ L of M02 strain ammonium sulfate 30% saturation extracellular enzyme preparation; lanes M2, 10. Mu.L protein molecular weight standards, bands are from top to bottom: 170kD,130kD,100kD,70kD,55kD,40kD,35kD,25kD,15kD,10kD; lane 3, 20. Mu.L of supernatant enzyme preparation after breaking cell wall of M02 strain; lane 4, 20. Mu.L of precipitated enzyme preparation after breaking cell wall of M02 strain;
FIG. 5 shows a histogram of analysis of polysaccharide-degrading ability of a complex-type polysaccharide-degrading enzyme preparation of M02 strain of Cladosporium sp.sp.as measured by the DNS-reducing sugar method;
wherein 30% is ammonium sulfate 30% saturation level extracellular enzyme preparation; 80% is ammonium sulfate 80% saturation level extracellular enzyme preparation; super is supernatant enzyme preparation after breaking the cell wall of M02 strain;
FIG. 6 shows a TLC analysis chart of the product obtained by degrading a polysaccharide with the complex-type polysaccharide-degrading enzyme preparation;
wherein, M, mannooligosaccharide: m, mannose; m2, mannite pond; m3, mannotriose; m4, mannotetrasaccharide; m5, mannopentaose; m6, mannose hexaose;
A. mannan (1, 2,3, 4), pectin (5, 6, 7, 8); B. xanthan gum (1, 2,3, 4), lambda-carrageenan (5, 6, 7, 8);
1.5 groups are negative control groups without enzyme preparation; 2. 6 groups are added with 30 percent saturation extracellular enzyme preparation of ammonium sulfate; 3. adding ammonium sulfate 80% saturation extracellular enzyme preparation into 7 groups; 4. adding 8 groups of supernatant enzyme preparation after breaking cell wall;
FIG. 7 shows a HPLC analysis of the products of the degradation of pectin and mannan polysaccharides by an extracellular enzyme preparation with 80% saturation of ammonium sulfate;
wherein, A is pectin; B. mannan; 1. a monosaccharide; 2. a disaccharide; 3. a trisaccharide; 4. tetrasaccharide; 5. a pentasaccharide; 6. a hexasaccharide;
FIG. 8 shows HPLC analysis of products from degradation of HA, alginate, CSA, CSC by complex polysaccharide-degrading enzymes;
wherein, E (-) is not added with compound polysaccharide degrading enzyme preparation, and is a negative control group; e (30%) ammonium sulfate 30% saturation exoenzyme preparation; e (80%) ammonium sulfate 80% saturation exoenzyme preparation; e (sup) adding the supernatant enzyme preparation after the M02 strain is subjected to wall breaking.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies and experiments, and unexpectedly found that the Cladosporium sp of the present invention can effectively degrade polysaccharides, and that the Cladosporium sp of the present invention can simultaneously degrade various polysaccharides (such as algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate a, chondroitin sulfate E, carrageenan, kappa carrageenan, lambda carrageenan, and iota carrageenan) for the first time, and that the inventors have unexpectedly isolated a new Cladosporium strain, cladosporium sp.m. 02. The present invention has been completed based on this finding.
Term(s) for
As used herein, the term "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.
As used herein, the terms "strain of the invention", "Cladosporium sp.M02" of the invention "," Cladosporium sp.M02"," Cladosporium sp. ", and" Cladosporium sp.M02 "of the invention are used interchangeably and refer to the deposit number: CGMCC No 13568 and its subculture strain or derivative strain.
Cladosporium sp
In the present invention, a novel strain screened for effective degradation of polysaccharides is provided.
The strain belongs to the family of davidiella aceraceae, and is called Cladosporium sp.M02. The strain is a novel strain with polysaccharide degradation property which is separated and identified from seaweed by the inventor.
Specifically, the Cladosporium strain of the present invention belongs to Cladosporium (Cladosporium sp.) as identified by ITS sequence.
Specifically, the physiological properties of Cladosporium sp.M02 of Cladosporium of the invention are as follows: the bacterial strain is a circular bacterial colony on a flat plate, the surface of the bacterial colony is velvet, the middle of the bacterial colony is raised, white hypha which is not transversely separated and has branches grows around the bacterial colony, the bacterial colony grows fast and is milky white, the color of the bacterial colony is gradually deepened along with the time, and the color of the bacterial colony is changed into navy blue after the bacterial colony grows for 40 hours. The conidiophores are observed by a microscope to form a cross chain spherical spore branch from the top. The solid culture medium for observing the thallus morphology is a primary screening solid culture medium and comprises the following components: 0.5M NaCl,0.1M KCl,0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl，1％Yeast Extract(w/v)，2％Tryptone(w/v)，2％Glucose(w/v)，2％Agar(w/v)，pH 7.0。
M02 strain of Cladosporium (Cladosporium sp.) has ITS gene sequence length of 551bp as shown in SEQ ID NO. 1.
DNA of ITS(SEQ ID NO.:1)
The gene sequence of the ITS of the strain of the present invention was proved to have high homology with the gene sequence of the ITS of the standard strain Cladosporium species (Cladosporium) registered for NCBI, in which the sequence identity was 100.00% most recently with the standard strain Cladosporium silenes CBS 109082, by searching for an alignment using the BLASTN program of the National Center for Biotechnology Information (NCBI).
M02 strain of Cladosporium sp.was used to construct phylogenetic trees using ITS genes, and the results are shown in FIG. 2. The results show that the M02 strain clustered with the model strain of Cladosporium, and was located inside this branch. Therefore, the M02 strain was identified to Cladosporium.
In the present invention, the method for culturing M02 strain of Cladosporium sp comprises the steps of:
(1) Streaking a frozen and preserved M02 strain sample of Cladosporium sp.to a primary screening solid culture medium, and performing inverted culture at 25-30 ℃ for 18-48h to obtain an activated strain;
(2) Selecting the activated strain prepared in the step (1), inoculating the activated strain into a primary screening liquid culture medium, and performing shake culture for 86-96 hours at the temperature of 25-30 ℃ and the rotating speed of 180-220 r/min to prepare a seed solution;
(3) Inoculating the seed solution prepared in the step (2) into an amplification culture medium according to the volume percentage of 1-5%, and performing amplification culture for 4-6 days at the temperature of 25-30 ℃ and the rotating speed of 180-220 r/min to obtain M02 bacterial liquid of Cladosporium sp.
Preferably, the primary screening solid culture medium in the step (1) comprises the following components per liter:
1% Yeast Extract (w/v), 2% Tryptone (w/v), 2% glucose (w/v), 2% agar (w/v), balance artificial seawater, pH 7.0;
the artificial seawater comprises the following components:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl。
preferably, the primary screening medium in step (2) comprises the following components per liter:
1% Yeast Extract (w/v), 2% tryptone (w/v), 2% glucose (w/v), balance artificial seawater, pH 7.0;
the artificial seawater comprises the following components:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl。
preferably, the expanding medium in step (3) comprises the following components per liter:
1% Yeast Extract (w/v), 2% tryptone (w/v), 2% glucose (w/v), balance artificial seawater, pH 7.0;
the artificial seawater comprises the following components:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl。
according to the invention, the application comprises the following steps:
(i) Inoculating the M02 bacterial liquid of the Cladosporium sp to a fermentation culture medium according to the volume percentage of 1-5%, and carrying out amplification culture for 3-7 days at the temperature of 25-30 ℃ and the rotating speed of 180-220 r/min to obtain a Cladosporium M02 strain fermentation liquid;
(ii) And (ii) taking the fermentation liquor of the cladosporium M02 strain prepared in the step (i), carrying out solid-liquid separation, taking the liquid, adding ammonium sulfate to ensure that the concentration reaches 30 percent and 80 percent of saturation degree respectively, centrifuging, collecting precipitate, carrying out heavy suspension on the precipitate by using 20-50 mL of TGE buffer solution, and dialyzing to remove the ammonium sulfate to prepare the compound polysaccharide degrading enzyme preparation.
Preferably, the fermentation medium in step (i) comprises the following composition per liter:
1% Yeast Extract (w/v), 2% tryptone (w/v), 2% glucose (w/v), balance artificial seawater, pH 7.0;
the artificial seawater comprises the following components:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl。
preferably, in step (ii), the solid-liquid separation conditions are as follows: centrifuging at 12,000 Xg for 5-20 min at 4 ℃.
Preferably according to the invention, in step (ii), the centrifugation is: 15,000 Xg, and centrifuging for 15-30 min at 4 ℃.
Preferably, in step (ii), the TGE buffer has the following composition:
50mMtris,50mM NaCl,0.5mM EDTA,5mM dithiothreitol, 5.0% (v/v) glycerol, ddH 2 O is metered to 1000mL, and the pH is 7.9.
According to the present invention, in the step (ii), the dialysis is performed by stirring dialysis using a dialysis bag having a molecular cut-off of 12 to 14 kDa.
The application of the enzyme preparation for degrading algal polysaccharide in degrading microbial polysaccharide, algal polysaccharide, higher plant polysaccharide and animal polysaccharide. Preferably according to the invention, the microbial polysaccharide is xanthan gum; the algal polysaccharide is algin (Alginate), agarose, carrageenan, kappa-carrageenan, iota-carrageenan and lambda-carrageenan; the higher plant polysaccharide is cellulose, microcrystalline cellulose, carboxymethyl cellulose, starch, xylan, mannan, and pectin; the animal polysaccharide is Hyaluronic Acid (HA), chondroitin Sulfate A (CSA), chondroitin Sulfate C (CSC), chondroitin sulfate E, heparan sulfate, dermatan sulfate, chitin, and chitosan.
The Cladosporium sp.M02 strain is used for degrading microbial polysaccharide, higher plant polysaccharide and/or animal polysaccharide.
Preferably according to the present invention, the microbial polysaccharide is xanthan gum; the algal polysaccharides are algin, agarose, carrageenan, kappa carrageenan, iota carrageenan and lambda carrageenan; the higher plant polysaccharide is cellulose, microcrystalline cellulose, carboxymethyl cellulose, starch, xylan, mannan, and pectin; the animal polysaccharide is hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate E, heparan sulfate, dermatan sulfate, chitin, and chitosan.
Enzyme preparation
The active substances according to the invention (Cladosporium sp.) can be prepared in a conventional manner into enzyme preparations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substance, microcapsules in polymers.
These preparations can be produced by known methods, for example, microbial fermentation.
In the present invention, the "enzyme preparation" contains an active ingredient of Cladosporium sp.
The enzyme preparations of the invention can be mixed with other substances, such as cysteine, glutathione, butylhydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-erythorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E in a commercial preparation thereof or in a dosage form prepared from these preparations.
Furthermore, the enzyme preparations of the invention can also be present in their commercial preparations in a mixture with synergists, which are compounds which increase the action of the active bacteria, or in the use forms prepared from these preparations, it being possible for synergists not to be added, since the active bacteria themselves are active.
The dosage form of the enzyme preparation of the present invention may be various, and the preferred enzyme preparation includes spray, solution preparation from the standpoint of easy preparation and application.
The enzyme preparations according to the invention generally contain 1X 10 to 1X 10 15 cfu/mL or cfu/g Cladosporium sp., preferably 1X 10 4 -1×10 9 cfu/mL or cfu/g Cladosporium sp., based on the total volume or total weight of the enzyme preparation. The concentration of the strains according to the invention in commercial preparations or in dosage forms used can vary within wide limits.
Strain preservation
The Cladosporium sp.M02 (same as the preservation name) of the strain of Cladosporium sp.M02 is preserved in China general microbiological culture Collection center (CGMCC) in 2017 in 3, 8 and 8 days, and the No. 3 Hospital No.1 Xilu Beijing of the Beijing market in the morning area of Chaoyang has the preservation number: CGMCC No.13568.
The main advantages of the present invention include:
(1) The Cladosporium sp can effectively degrade polysaccharides, and the Cladosporium sp can also degrade various polysaccharides (such as algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate E, carrageenan, kappa-type carrageenan, lambda-type carrageenan and iota-type carrageenan) for the first time.
(2) The Cladosporium sp is a new strain obtained by the first separation and identification of the inventor, and the inventor discovers that the Cladosporium sp can effectively degrade polysaccharides for the first time and can degrade a plurality of polysaccharides (such as algin, xanthan gum, pectin, mannan, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate E, carrageenan, kappa carrageenan, lambda carrageenan and iota carrageenan) simultaneously.
(3) The invention separates and obtains a Cladosporium sp M02 strain from seaweed, the strain can grow by taking various types of polysaccharides from microorganisms, seaweed, land plants and animals as unique carbon sources, the variety of the produced polysaccharide degrading enzymes is rich, the strain is a multifunctional polysaccharide degrading strain, and an extracellular enzyme preparation prepared by the strain can degrade the microorganisms, the seaweed, the plants and the animal polysaccharides, and particularly has obvious degrading activity on pectin, mannan, carrageenan and lambda carrageenan of the seaweed and xanthan gum from the microorganisms, and has potential application value, and the function is completely different from the function of the existing known Cladosporium sp.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the protocol of microorganisms: the conditions described in the handbook of experiments (James Cappuccino and Natalie Sherman eds., pearson Edurion Press) or as recommended by the manufacturer.
The materials and reagents used in the examples were all commercially available products unless otherwise specified.
Example 1
Separation and purification of seaweed microorganisms
Taking seaweed near Qingdao tress, taking 1g of sample, placing the sample in agarose, algin, kappa-carrageenan and iota-carrageenan with the volume of 100mLCulturing in a unique carbon source culture medium of gum, lambda-type carrageenan or carrageenan at the temperature of 28 ℃ and 210rmp until the solution is turbid; when the turbidity degree of the solution is higher and the viscosity is obviously reduced, transferring the culture solution into a corresponding unique carbon source culture medium according to 2 percent (v/v), and culturing at the temperature of 28 ℃ and 210rmp until the solution is turbid; adding 1mL of the solution into 9mL of sterile water, and respectively diluting to a concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 5 concentration gradients. The diluted bacterial suspension was spread on a primary screening medium, each concentration was made in duplicate, and cultured at 28 ℃ for 7 days. Selecting single colony with large colony size, color and morphology difference, performing plate streaking separation and purification for 3 times, selecting single colony in primary screening liquid culture medium, culturing at 28 deg.C and 210rmp for 24 hr, adding 400 μ L glycerol into 1.6mL culture, mixing, and storing in refrigerator at-80 deg.C for a long time.
The primary screening culture medium comprises the following components per liter:
1% Yeast Extract (w/v), 2% Tryptone (w/v), 2% glucose (w/v), 2% agar (w/v), balance artificial seawater, pH 7.0.
Agar was added to the solid medium at a mass concentration of 1.5% (w/v).
Artificial seawater, each liter of which comprises the following components:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl。
example 2
Screening of polysaccharide-degrading bacteria
The single clone of the strain obtained in example 1 was inoculated into a liquid medium containing a single carbon source, cultured at 210rmp and 28 ℃ for 72 hours, and the turbidity of the culture broth and the change in the viscosity of the culture broth were observed. Selecting enzyme-producing strains according to the two indexes. Selecting strains with large changes of thallus turbidity and viscosity in each unique carbon source culture medium, streaking and culturing on a primary screening solid culture medium, respectively preserving and numbering and recording M01, M02 and M03, \ 8230, and the like; after culturing the single colony for 24h, the single colony is stained by modified Lugol iodine solution, and the strain is observed by a microscope.
The preparation method of the unique carbon source culture medium comprises the following steps:
polysaccharide substrates were added separately to artificial seawater to a final concentration of 0.10% (w/v).
The polysaccharide substrate is selected from: cellulose, microcrystalline cellulose, carboxymethyl cellulose, xylan, mannan, starch, pectin, chitin, chitosan, agarose, sodium alginate, hyaluronic acid, chondroitin sulfate a, chondroitin sulfate C, chondroitin sulfate E, heparin, carrageenan, xanthan gum, kappa carrageenan, iota carrageenan, lambda carrageenan; autoclaving at 115 deg.C for 20min.
The artificial seawater comprises the following components per liter:
0.5M NaCl，0.1M KCl，0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl，pH7.0。
the improved Lugol iodine solution comprises the following components per liter:
KI 2.0g，I 2 1.0g, and the volume of water is up to 1L.
A Cladosporium sp.M02 strain was isolated as described above and shown in FIG. 1A. The strain is on a solid culture medium, the front surface of the strain is in a white colony round shape, the surface is in a velvet shape, the middle part is raised, and white hyphae grow around the strain, which indicates that the M02 strain is a fungus; but the center of the colony is observed to be dark green from the reverse side of the colony, and a circle of white hyphae are arranged at the edge of the colony, as shown in figure 1B; after Lugol's iodine staining, a clear ring appeared around the strain as shown in FIG. 1C. This indicates that the M02 strain is able to secrete extracellular agarase and produce water-soluble reducing sugars after degrading agarose. Microscopic observation of the strain revealed that the strain had hyphae with no cross-hatching and branching, as shown in FIG. 2A; and conidiophores of the sporophytes are formed from the top ends and form cross chain spherical spores, as shown in figures 2B and 2C, the sporophytes have the characteristics of Cladosporium spp.
As shown in table 1, the Cladosporium sp M02 strain can grow by using a plurality of polysaccharides as a unique carbon source, wherein the Cladosporium sp M grows more vigorously in xanthan gum, mannan, carrageenan and lambda-carrageenan, grows less strongly in glycosaminoglycan (hyaluronic acid, chondroitin sulfate) and the like, and does not grow significantly in plant polysaccharides such as cellulose, carboxymethyl cellulose, microcrystalline cellulose, starch and the like. The analysis shows that the M02 strain can degrade at least 18 polysaccharides, is a multi-energy polysaccharide degrading strain and has the potential of efficiently converting the polysaccharides into biological energy sources.
TABLE 1 statistics of growth of M02 strains of Cladosporium in a Medium with sole carbon source
Wherein "+": represents the growth of the M02 strain in the culture medium; "-": representing that the M02 strain did not grow in the medium. ++: represents that the M02 strain grows weakly in the culture medium; +++: represents that the M02 strain grows normally in the culture medium; ++++: the M02 strain grows better in the culture medium; +++++: represents that the M02 strain grows vigorously in the culture medium; ++++++: the M02 strain is represented to grow vigorously in the culture medium.
Example 3
Extraction of Cladosporium sp.M02 strain genome DNA (deoxyribonucleic acid)
Inoculating M02 strain of Cladosporium sp.into primary screening culture medium, and performing shaking culture at 28 deg.C and 200rpm until the solution is turbid; the culture broth was centrifuged at 20mL,28 ℃ and 12,000X (g, constant of global gravity) for 20min, and the pellet was collected.
The primary screening culture medium comprises the following components: 1% Yeast Extract (w/v), 2% tryptone (w/v), 2% glucose (w/v), 2% agar (w/v), 0.5M NaCl,0.1M KCl,0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl，pH7.0。
Adding 12.0mL of lysozyme buffer solution into the thallus sediment to obtain about 14.0mL of bacterial liquid, and respectively adding 560 mu L of lysozyme with the concentration of 20mg/mL to obtain the final concentration of about 800 mu g/mL; placing in ice water bath for 1.0h, transferring to water bath at 37 deg.C, and warm-bathing for 2h until the reaction system is viscous; adding 0.82 mL of hexadecyl sodium sulfonate solution with the concentration of 100mg/mL and 60 mu L of proteinase K solution with the concentration of 100mg/mL, and bathing for 1.0h at the temperature of 52 ℃; adding 15mL of Tris-equilibrated phenol/chloroform/isoprene (volume ratio of 25: 24); centrifuging at 10,000 Xg and 4 deg.C for 10min, collecting supernatant, adding 2.0mL NaAc-HAc (pH 5.2, 3.0M) buffer and 17.0mL absolute ethanol (stored at-20 deg.C), and mixing; picking out filamentous DNA by a gun head, transferring the filamentous DNA into a centrifugal tube with the volume of 1.5 ml, washing for 2 times by using 70% ethanol, and discarding a supernatant after microcentrifugation; centrifuging at 10,000 Xg and 4 deg.C for 2min, and completely discarding supernatant; the DNA precipitate was air-dried in a sterile bench and then the DNA sample was dissolved overnight at 4 ℃ with sterile deionized water to prepare genomic DNA.
Example 4
Molecular identification of Cladosporium sp.M02 strain based on ITS gene cloning
The genomic DNA of the M02 strain obtained in example 3 was used as a template, amplified with universal primers for fungal ITS genes (ITS 1 and ITS 4), subjected to agarose gel electrophoresis, and then the PCR amplification product was purified using a gel recovery kit (Tiangen), and after electrophoresis verification, ligated to pEASY-Blunt Simple Cloning Vector and transformed into E.coli Trans 1T 1 competent cells. Positive clones were obtained by ampicillin resistance screening. Sequencing of the ITS gene was carried out by Biotechnology engineering (Shanghai) Inc., and the sequence was compared with the ITS gene sequence of a standard strain included in the National Center for Biotechnology information (NCBI, national Center for Biotechnology information, ttp:// www.ncbi.nlm.nih.gov /), and phylogenetic trees were constructed using MEGA 7.0.
The universal primers for strain ITS gene amplification are as follows:
the forward primer is ITS 1:5 'TCCGTAGGTGAA CCTGCGG-3';
the reverse primer is ITS 4:5 'TCCTCCGCTTATTGATATGC-3'.
The reaction system for the ITS gene amplification of the strain is as follows, and the total volume is 20 mu L:
the gene amplification reagent Prime STAR HS DNA polymerase, dNTP mixture and buffer are purchased from Dalibao biotechnology limited.
The above procedure for the amplification of the ITS gene of the strain was:
pre-denaturation at 95 ℃ for 4min; denaturation at 94 ℃ for 40s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s for 35 cycles; extending for 15min at 72 ℃; cooling to 4 deg.C, and standing for 10min.
The construction method of the phylogenetic tree based on the ITS genes comprises the following steps:
the measured ITS gene sequence and a similar sequence of a standard strain obtained from an NCBI gene database are subjected to multi-sequence comparison by using Clustal W in a MEGA7.0 software package, a phylogenetic tree is constructed by using a Neighbor-Joining method, and 1000 Bootstraps tests are carried out to obtain a statistical tree.
As shown in FIG. 3, the M02 strain selected by the present invention has the closest relationship with Cladosporium silene CSB 109082 and Cladosporium aff.Cladosporioides CSB 673.69 in the database, and the similarity between ITS genes is 100% and 99.79%, respectively. The statistical tree constructed on the basis of the ITS genes shows that the M02 strain is clustered with a plurality of standard strains of Cladosporium sp. Thus, the M02 strain was identified to the genus Cladosporium.
Example 5
Preparation of Cladosporium sp.M02 strain compound polysaccharide degrading enzyme preparation
(1) Culturing Cladosporium M02 strain at-80 deg.C in a primary solid culture medium under inversion at 28 deg.C for 18-48 hr;
(2) Picking M02, dropping into a primary screening liquid culture medium, and performing shake cultivation for 4d at the temperature of 28 ℃ and 210rmp to obtain 100mL of seed liquid;
(3) Inoculating the seed solution prepared in the step (2) into a fermentation medium 100 mL/bottle (only liquid is sucked) according to the volume percentage of 1%, and performing amplification culture at the temperature of 28 ℃ and the rpm of 210 for 6.5 days to obtain 1000mL of cladosporium M02 strain fermentation liquor;
(4) And (4) filtering the M02 strain fermentation liquor of the cladosporium M02 strain prepared in the step (3) by using sterile filter paper, centrifuging for 20min at 4 ℃ at 12,000 Xg, taking the supernatant, adding ammonium sulfate, centrifuging for 30min at 4 ℃ until the concentrations respectively reach 30% and 80% of saturation, adding 15,000 Xg and collecting precipitates obtained under the saturation of 30% and 80% of ammonium sulfate, resuspending the precipitates by using 20 times of volume of TGE buffer solution, and dialyzing by using a dialysis bag with the molecular interception amount of 12-14 KDa to remove the ammonium sulfate to obtain the extracellular enzyme preparation.
(5) And (5) centrifuging the M02 strain obtained by filtering in the step (4) for 15min at the temperature of 4 ℃ at the speed of 8,000 Xg, collecting thalli, resuspending the thalli by using a buffer solution A, and ultrasonically crushing in an ice-water bath environment. Further centrifugation was carried out at 15,000 Xg at 4 ℃ for 30min, and the water-soluble supernatant fraction and the precipitate fraction were collected, respectively. The compound polysaccharide degrading enzyme preparation which comprises 30 percent of saturation degree extracellular enzyme of ammonium sulfate, 80 percent of saturation degree extracellular enzyme of ammonium sulfate, intracellular supernatant and intracellular sediment is prepared.
The primary screening and enzyme production culture medium in the steps (1) and (2) comprises the following components per liter:
1% Yeast Extract (w/v), 2% Tryptone (w/v), 2% glucose (w/v), balance Artificial seawater (0.5M NaCl,0.1M KCl,0.001M CaCl 2 ，0.001M MgSO 4 ，0.02M NH 4 Cl), pH 7.0, agar was added to the solid medium at a mass concentration of 1.5% (w/v).
The components of the above TGE buffer were as follows:
50mM Tris,50mM NaCl,0.5mM EDTA,5mM DTT (dithiothreitol), 5% (v/v) Glycerol (Glycerol), ddH 2 O 1000mL，pH 7.9。
The dialysis is to use a dialysis bag with molecular cut-off of 12-14 KDa, stir and dialyze TGE buffer solution with 20-50 times volume in low temperature environment, and then detect the extracellular enzyme preparation compound polysaccharide degrading enzyme preparation of the M02 strain of the cladosporium by polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that 80% of the precipitated complex polysaccharide-degrading enzyme preparation of M02 strain of Cladosporium sp.contained multiple protein bands, as shown in FIG. 4 (A, B).
Example 6
Analysis of degradation capability of M02 compound type polysaccharide degrading enzyme preparation to different polysaccharides
Preparing a polysaccharide substrate with the concentration of 0.1% (w/v) by using artificial seawater, a compound polysaccharide degrading enzyme preparation, sterile water and a PBS buffer solution according to the weight ratio of 10:3:7:10 Mixing the components according to the volume ratio, reacting for 24h at 30 ℃ and pH 7.4, incubating in boiling water bath for 10min to inactivate enzyme, centrifuging at 15,000 Xg at 4 ℃ for 10min, transferring supernatant, centrifuging for two times, collecting supernatant as enzymolysis product of the compound polysaccharide-degrading enzyme preparation, and detecting the generated reducing sugar by DNS method. Control experiments were performed with enzymes previously inactivated in a boiling water bath.
The PBS buffer comprises the following components per liter:
NaCl 8g，KCl 0.2g，Na 2 HPO 4 1.44g，KH 2 PO 4 0.24g，ddH 2 O 1000mL，pH 7.4。
the results are shown in fig. 5, and the analysis of the reducing sugar products shows that the compound polysaccharide degrading enzyme preparation prepared by the cladosporium M02 strain can not only degrade the polysaccharides from terrestrial higher plants, such as: carboxymethyl cellulose, mannan, and pectin; it also degrades polysaccharides of seaweed origin, such as: algin, lambda-carrageenan; can also degrade animal-derived polysaccharides, and especially has high degradation activity on hyaluronic acid, chondroitin sulfate A and chondroitin sulfate C derived from connective tissues of higher animals; in addition, the xanthan gum derived from microorganisms also has certain degradation capability. Wherein 80% of the precipitated protein fraction has a higher degradation capacity in the same volume of the enzyme preparation. Therefore, the compound polysaccharide degrading enzyme preparation prepared by using the cladosporium M02 strain can degrade various polysaccharides, has obvious degrading activity on hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C and algal polysaccharide from connective tissues of higher animals, and has potential application value.
Example 7
TLC analysis of product obtained by degrading polysaccharide with compound polysaccharide-degrading enzyme preparation
mu.L of the enzymatic hydrolysate of mannan, pectin, xanthan gum and lambda-carrageenan of example 6 was subjected to Thin Layer Chromatography (TLC) analysis using TLC Silica gel 60F 254 The developing solvent is respectively n-butyl alcohol: formic acid: water (v: v) 4:6:1 and n-butanol: acetic acid: water (v: v) 2:1:1, color development by heating at 110 ℃ for 10min after dyeing with a developer (diphenylamine: aniline: phosphoric acid: acetone (v: v) 1g.
As shown in FIG. 6, when compared with the negative control group, the complex polysaccharide-degrading enzyme preparation reacted with mannan, pectin (FIG. 6-A), alginate, and lambda-carrageenan (FIG. 6-B), and a series of oligosaccharide products were observed, and the fraction of the extracellular enzyme preparation was 80% saturated with ammonium sulfate in the main group.
Example 8
HPLC analysis of product obtained by degrading polysaccharide with compound polysaccharide-degrading enzyme preparation
Taking 100 mu L of enzymolysis product of pectin and mannan obtained in example 6 for High Performance Liquid Chromatography (HPLC) analysis, wherein the gel column is Superdex TM 30Increatase 10/300GL, the mobile phase is 0.2M ammonium bicarbonate, and the flow rate is 0.4mL/min; the detector is a differential refractometer detector (RID). Another 100. Mu.L of the enzymatic hydrolysate of hyaluronic acid, algin, chondroitin sulfate A and chondroitin sulfate C of example 6 was subjected to High Performance Liquid Chromatography (HPLC) analysis using Superdex as the gel column TM 30Increatase 10/300GL, with a mobile phase of 0.2M ammonium bicarbonate and a flow rate of 0.4mL/min; the detection conditions were UV232nm or UV235nm.
As shown in FIG. 7, the peak time of oligosaccharide fragment having the highest absorption value among the main products after the reaction of the M02 ammonium sulfate 80% saturation exoenzyme preparation with pectin (FIG. 7-A) and mannan (FIG. 7-B) was about 33.6min, indicating pentasaccharide. In contrast, no oligosaccharide component with characteristic absorption was detected in the control product. Therefore, the extracellular enzyme preparation prepared from M02 strain of Sclerotinia (Cladosporum sp.) contains pectin hydrolase and mannan hydrolase, and can be used for producing pectin oligosaccharide and mannan oligosaccharide.
As shown in FIG. 8, in the reaction products of the complex polysaccharide-degrading enzyme preparation with hyaluronic acid (FIG. 8-A), algin (FIG. 8-B), CSA (FIG. 8-C) and CSC (FIG. 8-D), a characteristic absorption oligosaccharide signal peak was observed within 20-45 min, indicating that a series of unsaturated oligosaccharide products were produced. In contrast, no oligosaccharide component with characteristic absorption was detected in the control product. Therefore, the compound polysaccharide degrading enzyme preparation prepared from M02 strain of Cladosporium sp.has glycosaminoglycan degrading enzyme and algin lyase, and provides a potential tool for the research of structural functions of glycosaminoglycan and algin in the later period.
Strain preservation
The Cladosporium sp.M02 strain (same as the preservation name) is preserved in China general microbiological culture Collection center (CGMCC) at 3, 8 days in 2017 and has the preservation number of No. 3 Hospital No.1 of Xilu-Chen-Yang district, beijing city, and the preservation number is: CGMCC No.13568.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Green grain pharmaceutical Co., ltd
<120> Cladosporium sp
<130> P2021-1308
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 551
<212> DNA
<213> Cladosporium sp
<400> 1
tccgtaggtg aacctgcgga gggatcatta caagtgaccc cggtctaacc accgggatgt 60
tcataaccct ttgttgtccg actctgttgc ctccggggcg accctgcctt cgggcggggg 120
ctccgggtgg acacttcaaa ctcttgcgta actttgcagt ctgagtaaac ttaattaata 180
aattaaaact tttaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcgaaat 240
gcgataagta atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc 300
gccccctggt attccggggg gcatgcctgt tcgagcgtca tttcaccact caagcctcgc 360
ttggtattgg gcaacgcggt ccgccgcgtg cctcaaatcg accggctggg tcttctgtcc 420
cctaagcgtt gtggaaacta ttcgctaaag ggtgttcggg aggctacgcc gtaaaacaac 480
cccatttcta aggttgacct cggatcaggt agggataccc gctgaactta agcatatcaa 540
taagcggagg a 551
Claims (10)
1. A Cladosporium sp.sp.sp.is a Cladosporium sp.sp.sp.sp.sp.sp.sp..
2. The Cladosporium sp.sp.sp.as claimed in claim 1, wherein the ITS gene of Cladosporium sp.sp.has the sequence as shown in SEQ ID No. 1.
3. Cladosporium sp.M02 as claimed in claim 1, deposited under the number Cladosporium sp.M02: CGMCC No 13568.
4. An enzyme preparation, comprising:
cladosporium sp.sp.and/or a metabolite thereof as claimed in any of claims 1 to 3.
5. The enzyme preparation of claim 4, wherein the enzyme preparation comprises a polysaccharide degrading enzyme preparation.
6. The enzyme preparation of claim 4, wherein the enzyme preparation is an extracellular enzyme preparation.
7. The enzyme formulation of claim 5, wherein the polysaccharide comprises a microbial polysaccharide, algal polysaccharide, higher plant polysaccharide, animal polysaccharide.
8. As claimed in claim4, wherein the content of Cladosporium species and/or metabolites thereof in the enzyme preparation is 1X 10 to 1X 10 15 cfu/mL or cfu/g Cladosporium sp., preferably 1X 10 4 -1×10 9 cfu/mL or cfu/g Cladosporium sp, based on the total volume or total weight of the enzyme preparation.
9. Use of a cladosporium and/or a metabolite thereof according to claim 1, or of an enzyme preparation according to claim 4, for the preparation of a composition or preparation for degrading a polysaccharide.
10. Use of a cladosporium and/or a metabolite thereof according to claim 1 for the preparation of an enzyme preparation for degrading polysaccharides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110592534.8A CN115404169A (en) | 2021-05-28 | 2021-05-28 | Cladosporium sp |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110592534.8A CN115404169A (en) | 2021-05-28 | 2021-05-28 | Cladosporium sp |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116376708A (en) * | 2022-12-05 | 2023-07-04 | 青岛农业大学 | Cladosporium fungus and application thereof |
| CN117736944A (en) * | 2024-02-20 | 2024-03-22 | 云南省农业科学院农业环境资源研究所 | Streptomyces griseus as well as microbial inoculum and application thereof |
-
2021
- 2021-05-28 CN CN202110592534.8A patent/CN115404169A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116376708A (en) * | 2022-12-05 | 2023-07-04 | 青岛农业大学 | Cladosporium fungus and application thereof |
| CN116376708B (en) * | 2022-12-05 | 2024-06-07 | 青岛农业大学 | Cladosporium fungus and application thereof |
| CN117736944A (en) * | 2024-02-20 | 2024-03-22 | 云南省农业科学院农业环境资源研究所 | Streptomyces griseus as well as microbial inoculum and application thereof |
| CN117736944B (en) * | 2024-02-20 | 2024-04-26 | 云南省农业科学院农业环境资源研究所 | Streptomyces griseus as well as microbial inoculum and application thereof |
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