CN114075522B - Actinomycetes F8 with broad-spectrum antibacterial activity and application thereof - Google Patents
Actinomycetes F8 with broad-spectrum antibacterial activity and application thereof Download PDFInfo
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- CN114075522B CN114075522B CN202111438934.XA CN202111438934A CN114075522B CN 114075522 B CN114075522 B CN 114075522B CN 202111438934 A CN202111438934 A CN 202111438934A CN 114075522 B CN114075522 B CN 114075522B
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/28—Streptomyces
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
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- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to actinomycetes F8 with broad-spectrum antibacterial activity and application thereof, which can effectively solve the utilization problem of wetland microorganisms, and the technical scheme is that the actinomycetes F8 is classified and named as Streptomyces armeliiStreptomyces amritsarensis) The strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC NO.22041, the preservation date is 2021, 3 months and 19 days, the preservation address is North Silu No. 1, 3 of the Korean area of Beijing, and the 16S rDNA gene sequence of the strain is shown as SEQ ID NO. 1.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to actinomycetes F8 with broad-spectrum antibacterial activity and application thereof.
Background
Actinomycetes are a functional microbial resource with important application value, are named because mycelium at the edge of a colony is often in an actinomycete shape, have the capability of synthesizing abundant bioactive substances, and can produce various antibiotics, bacteriostats, antitumor substances and the like. Of the natural active products found in the world, more than 40% are found in actinomycetes, and about two-thirds of the various antibiotics currently in common use clinically and agriculturally are produced by actinomycetes, of which more than 70% are produced by streptomyces.
The wetland has the dual characteristics of water and land, is an ecological system with the most abundant biodiversity, highest productivity and greatest ecological service value on the earth, has the functions of stabilizing the environment, protecting species genes and the like, and enjoys the reputation of 'the kidney of the earth' and 'biological gene library'. Because of the land and river junction, the wetland is obviously affected by both the river and the land environment, and the wetland soil may have unique actinomycete resources which are different from the land and the water environment, and the uniqueness of the habitat determines the diversity of actinomycetes and the rarity of the resources. The diversity of actinomycetes necessarily results in the diversity of metabolites produced thereby, which provides the ability to synthesize novel active compounds. The discovery of secondary metabolites of the actinomycetes of the wetland with novel structure, multiple functions and special physiological activities and the potential of the application value of the secondary metabolites of the actinomycetes of the wetland lay a foundation for the development of natural products from wetland microorganisms and provide scientific basis for the efficient utilization of the wetland microorganism resources.
Disclosure of Invention
Aiming at the situation, the invention aims to provide actinomycetes F8 with broad-spectrum antibacterial activity and application thereof, which can effectively solve the problem of utilizing wetland microorganisms.
The technical scheme is that the actinomycetes F8 are classified and named as Streptomyces amjensis (Streptomyces amritsarensis), and are preserved in the China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC NO.22041, the preservation date is 2021, 3 months and 19 days, the preservation address is North Silu No. 1, 3 of the Korean area of Beijing, and the 16S rDNA gene sequence of the strain is shown as SEQ ID NO. 1.
The preparation method of the actinomycete F8 sterile fermentation broth comprises the following steps: inoculating the seed solution into 100ml basic fermentation medium or optimized fermentation medium according to 1% inoculum size, shaking and culturing at 35 deg.C and 180rpm for 7d to obtain fermentation liquor, centrifuging at 12000 Xg and 25 deg.C to remove thallus when the supernatant of the fermentation liquor is needed, removing residual thallus from the supernatant by using a filter membrane with pore diameter of 0.22 μm to obtain the sterile fermentation liquor supernatant containing active substances secreted by thallus.
The basic fermentation culture medium is as follows: soluble starch 20g, KNO 3 1g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 O0.01 g, pH7.0, distilled water to 1000mL.
The optimized fermentation medium is as follows: soluble starch 20g, peanut cake powder 50g and NaNO 3 0.5g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 1g，FeSO 4 ·7H 2 O0.01 g, pH7.0, distilled water to 1000mL.
The application of actinomycetes F8 with broad-spectrum antibacterial activity in preparing medicaments for infecting staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), escherichia coli (escherichia coli) and Botrytis cinerea (Botrytis cinerea) serving as a filamentous fungus.
The application of the actinomycetes F8 sterile fermentation broth with broad-spectrum antibacterial activity in preparing medicines for infecting gram-positive bacteria staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), gram-negative bacteria escherichia coli (escherichia coli) and filamentous fungus Botrytis cinerea (Botrytis cinerea) diseases.
The actinomycete F8 can be used for treating diseases infected by gram-positive bacteria staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), gram-negative bacteria escherichia coli (escherichia coli) and filamentous fungus Botrytis cinerea (Botrytis cinerea), and is an innovation on streptomyces amjensis.
Drawings
FIG. 1 is a colony morphology of the F8 strain of the present invention on solid medium of Gao's No. 1.
FIG. 2 is a phylogenetic tree of F8 strains constructed based on the 16S rDNA sequences of the present invention.
FIG. 3 is a graph showing the growth of the F8 strain of the present invention at various temperatures.
FIG. 4 is a graph showing the growth of the F8 strain of the present invention at different pH values.
FIG. 5 is a graph showing the growth of the F8 strain of the present invention at different salinity.
FIG. 6 is a graph showing the inhibitory effect of the fermentation broth of the F8 strain on indicator bacteria; the indicator bacteria shown in the figure are respectively: a, bacillus subtilis; b, bacillus amyloliquefaciens; c, staphylococcus aureus; d, escherichia coli; e, botrytis cinerea.
FIG. 7 is a graph showing the effect of the heat stability test of the fermentation broth of the F8 strain of the present invention.
FIG. 8 shows the bacteriostatic effect of F8 strain fermentation broth after lysozyme and proteinase K treatment.
FIG. 9 F8 is a graph showing the relationship between the bacteriostatic effect of the fermentation broth and the growth of the cells
FIG. 10 is a graph showing the effect of different concentrations of glucose on the bacteriostasis of F8 strain fermentation broth.
FIG. 11 is a graph showing the effect of different concentrations of starch on the bacteriostatic effect of F8 strain fermentation broth.
FIG. 12 NaNO concentration 3 And (5) a graph showing the influence on the bacteriostasis effect of the fermentation liquor of the F8 strain.
FIG. 13 is a graph showing the effect of peanut cake flour of different concentrations on bacteriostasis of F8 strain fermentation broth.
FIG. 14 is a graph showing the extraction effect of different extractants on the active product of fermentation broth of F8 strain.
FIG. 15 is a graph showing the extraction effect of ethyl acetate as an extractant for various extraction times.
FIG. 16 is a graph showing the extraction effect of different amounts of extractant.
Detailed Description
The following detailed description of specific embodiments of the invention is provided in connection with the accompanying drawings and detailed description.
The actinomycetes F8 is obtained by separating and purifying a soil sample of a wetland park in Zhengzhou yellow river country. The relevant experimental data are as follows:
1. isolation and screening of strains
1) Sample collection and pretreatment
The wetland soil sample is collected in a wetland park of Zhengzhou yellow river country, and the sampling time, place and surrounding environment are recorded in time. Drying the sample in shade for 21 days at a ventilation and light-shielding place, grinding the sample into powder, and carrying out dry heat treatment at 110 ℃ for 1-2h;
2) Separation of wetland soil actinomycetes
Weighing 1.0g of a soil sample subjected to dry heat treatment, adding the soil sample into 9.0mL of sterile physiological saline containing 1.5% phenol, vibrating for 1h at 37 ℃ and 150r/min, standing for 20min to obtain supernatant, carrying out gradient dilution, and then taking 100 mu L of the supernatant to be coated on a solid culture medium of Gao's first order containing nalidixic acid (25 mg/L), cycloheximide (50 mg/L) and potassium dichromate (25 mg/L), wherein each gradient is repeated for 3 times; placing the mixture into an incubator at 30 ℃ for inversion culture for 14-21 d; selecting colonies with different forms, culturing on a solid culture medium plate of Gao's first, and repeatedly streaking and purifying the separated colonies on the plate until a culture with single colony form is obtained;
3) Screening of broad-spectrum antibacterial active actinomycetes
Actinomycetes with broad-spectrum antibacterial activity are screened by adopting an oxford cup method, and the specific steps are as follows: the surface of a solid culture medium (the bacteria are LB culture medium and the fungi are PDA culture medium) is uniformly coated with indicator bacteria staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), escherichia coli (escherichia coli) and filamentous fungus Botrytis cinerea (Botrytis cinerea) by aseptic operation, then 3 oxford cups are vertically placed, lightly pressurized, the oxford cups are contacted with the culture medium without gaps, and 200 mu L of aseptic fermentation liquid is added into the oxford cups, so that the aseptic fermentation liquid does not overflow. After the culture is completed, the culture is carried out for 2 to 3 days at 37 ℃ (bacteria) or 28 ℃ (fungi), and the diameter of the inhibition zone is measured by a vernier caliper according to the observation result.
2. Strain culture
Solid medium No. 1 gao: soluble starch 20g, KNO 3 1g，K2HPO4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 O0.01 g, agar 15g, pH 7.2-7.4, distilled water to 1000mL. Sterilizing at 121deg.C with high pressure steam.
Seed culture medium: soluble starch 20g, KNO 3 1g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 O0.01 g, pH 7.2-7.4, distilled water to 1000mL.
Basic fermentation medium: soluble starch 20g, KNO 3 1g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 0.5g，FeSO 4 ·7H 2 O0.01 g, pH7.0, distilled water to 1000mL.
Optimizing a fermentation medium: soluble starch 20g, peanut cake powder 50g and NaNO 3 0.5g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O0.5g，NaCl 1g，FeSO 4 ·7H 2 O0.01 g, pH7.0, distilled water to 1000mL.
LB medium: naC 1g, peptone 10g, yeast extract 5g, agar powder 15g, pH7.0, distilled water to 1000ml.
Potato dextrose agar medium (PDA medium): cutting potato (peeled) into 200g, adding water, boiling for 20-30 min, filtering with eight layers of gauze, glucose 20g, agar 20g, and naturally pH, and fixing volume to 1000ml with tap water.
Sterilizing the culture medium at 121deg.C with high pressure steam for 20 min.
Solid plate culture: the strain was streaked onto solid medium of Gao's No. 1 and cultured at 30℃for 7 days until spores were grown.
And (3) culturing the seed liquid, namely re-suspending strain spores growing on a solid culture medium of Gao's No. 1, inoculating the strain spores into 100ml of seed culture medium, and culturing for 36h at 30 ℃ with shaking at 180rpm to obtain the seed liquid.
And (3) strain fermentation culture: inoculating the seed solution into 100ml basic fermentation medium or optimized fermentation medium according to 1% inoculum size, shaking and culturing at 35 deg.C and 180rpm for 7d to obtain fermentation liquor, centrifuging at 12000 Xg and 25 deg.C to remove thallus when the supernatant of the fermentation liquor is needed, removing residual thallus from the supernatant by using a filter membrane with pore diameter of 0.22 μm to obtain the sterile fermentation liquor supernatant containing active substances secreted by thallus.
3. Identification of strains
1) Morphological feature observations
The strain F8 grows white colony on a solid culture medium of Gaoshan No. 1 for 3-5 days, white aerial hypha and grey pink spore silk are produced, after the culture time is 7d, the colony is aged to be yellow, and the spores are observed to be in a straight chain link shape by a microscope.
2) 16S rDNA sequence determination and phylogenetic tree construction
Extracting genome DNA of the strain F8, amplifying 16SrDNA by using a bacterial universal primer 27F and 1492R, purifying and sequencing, comparing the homology of the sequencing result by using a Blastn program of NCBI, and establishing a phylogenetic tree by using a NJ method by using MEGA7.0 software. By sequence analysis, F8 homology was found to be as high as 99% with Streptomyces amritsarensis 2A. The morphological characteristics and physiological and biochemical characteristics are combined, and the preliminary judgment is Streptomyces amritsarensis, which is shown in fig. 2.
4. Optimization of strain growth conditions
1) Optimization of optimal growth temperature of F8 Strain
100. Mu.L of seed solution was applied to Gaoshi No. one solid medium, and after culturing at 4℃at 16℃at 25℃at 30℃at 37℃at 40℃for 48, the colony growth was as shown in FIG. 3.
As can be seen from FIG. 3, the F8 strain can grow at 25-40 ℃, the growth is slow below 25 ℃, the bacterial colony is less, the bacterial colony growth is slowed down at 40 ℃, the bacterial colony is reduced, and the optimal growth temperature is 37 ℃.
2) Optimization of optimal growth pH of F8 Strain
1mL of the seed solution is inoculated into a basic fermentation medium, and after the culture is carried out for 3d, the growth condition of the strain growing under the condition of pH3-12 is studied by adopting a method for measuring the dry weight of the strain (figure 4)
As can be seen from FIG. 4, the F8 strain can grow at a pH value of 3.0-12.0, the growth state of 6.0-8.0 is better, and the optimal growth pH value is 7.0.
3) Optimization of optimal growth salinity of F8 strain
1mL of seed solution is inoculated into a basic fermentation medium, and after 3d of culture, the growth condition of the strain in a series of NaCl solutions with concentration is studied by adopting a method for measuring the dry weight of the strain, as shown in figure 5.
The result shows that the F8 strain can grow at the NaCl concentration of 0-2.5%, but the growth is obviously inhibited when the concentration is more than 2.0%, and the optimal salinity is 1% of the NaCl concentration.
5. F8 strain fermentation liquor antibacterial effect
The F8 fermentation broth antibacterial effect is detected by adopting an oxford cup method, and the specific steps are as follows: the surface of a solid culture medium (the bacteria are LB culture medium and the fungi are PDA culture medium) is uniformly coated with indicator bacteria staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), escherichia coli (escherichia coli) and filamentous fungus Botrytis cinerea (Botrytis cinerea) by aseptic operation, then 3 oxford cups are vertically placed, lightly pressurized, the oxford cups are contacted with the culture medium without gaps, and 200 mu L of F8 bacterial strain aseptic fermentation liquid is added into the oxford cups, so that the F8 bacterial strain aseptic fermentation liquid does not overflow. After the culture is completed, the culture is carried out for 2 to 3 days at 37 ℃ (bacteria) or 28 ℃ (fungi), and the result is observed, and the diameter of the inhibition zone is measured by a vernier caliper. It can be seen that a distinct zone of inhibition occurred around oxford cup and that the amount of F8 growth was not large (fig. 6), and that F8 synthesized the active substance against the indicator bacteria during fermentation.
6. Stability of active substances in fermentation broths of F8 Strain
1) Thermal stability test: and (3) respectively placing the fermentation broth after 7d culture at 40 ℃,50 ℃,60 ℃,70 ℃,80 ℃ and 90 ℃ for 30min, then using bacillus subtilis (Bacillus subtilis) as indicator bacteria, measuring the antibacterial effect of the F8 strain fermentation broth by using a method of measuring a bacteriostasis zone by using oxford cups, and performing analysis of variance by using SPSS 19.0. The difference of the bacteriostasis effect of the fermentation broth at each temperature was measured to be insignificant. The activity of the F8 strain fermentation broth has good temperature stability and can resist the high temperature of 90 ℃, as shown in figure 7.
2) Stability experiment of F8 Strain fermentation liquor on lysozyme and proteinase K
Taking 2ml of the fermentation broth after 7d culture, respectively adding 200ug of lysozyme and 200ug of proteinase K, preserving heat for 30min in a constant-temperature water bath at 37 ℃, then taking bacillus subtilis (Bacillus subtilis) as indicator bacteria, measuring the antibacterial effect of the F8 strain fermentation broth by using a method of measuring a bacteriostasis zone by using an oxford cup, and performing analysis of variance by using SPSS 19.0. After the F8 strain fermentation broth is treated by lysozyme and proteinase K, the change of the antibacterial effect is not obvious, which indicates that the activity of the fermentation broth is stable to lysozyme and proteinase K, as shown in figure 8.
7. F8 strain fermentation condition optimization
1) F8 strain fermentation temperature optimization
Seed liquid is inoculated to a basic fermentation culture medium according to the inoculation amount of 1%, the antibacterial effect of fermentation liquid at 30 ℃,35 ℃ and 37 ℃ is compared, and the fermentation liquid shows better inhibition effect on bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), staphylococcus aureus (Staphylococcus aureus), escherichia coli (escherichia coli) and Botrytis cinerea (Botrytis cinerea) after being cultured at 35 ℃, and the optimal fermentation temperature is 35 ℃ (table 1).
TABLE 1 influence of different fermentation temperatures on the bacteriostatic effect of F8 strains
2) F8 strain fermentation time optimization
Inoculating seed liquid to a basic fermentation culture medium according to an inoculum size of 1%, carrying out shaking culture at 35 ℃ and 180r/min, taking samples at intervals of 0h,3h,6h and 9h … …, measuring the dry weight of the bacterial strain by adopting a weighing method, taking bacillus subtilis (Bacillus subtilis) as an indicator bacterium, and measuring the antibacterial effect of the F8 strain in different fermentation periods by using a method of measuring a bacteriostasis zone by using oxford cups, wherein the relation between the antibacterial effect of the F8 strain fermentation liquid and the bacterial growth is shown in figure 9: the growth of the antibacterial effect in the slow period and the exponential period (before 60 h) of the thallus growth is slower, the effect of the fermentation liquor for inhibiting the indicator bacteria is gradually increased after the thallus enters the stable period (after 70 h), the bacterial body enters the declining period after 160h, and the increasing liquid of the antibacterial activity of the fermentation liquor is gradually slowed down, so that the collection time of the fermentation liquor is longer than 160h in order to obtain a better antibacterial effect, and the collection time of the fermentation liquor in the test is the seventh day, namely the fermentation time is 168h.
3) F8 strain fermentation medium carbon optimization
The inhibition effect of sucrose, glucose, maltose, fructose, lactose, galactose, trehalose and starch as the only carbon source F8 strain fermentation broth on bacillus subtilis (Bacillus subtilis) of the indicator bacteria was tested. The experimental results show that the F8 strain can not grow well in the culture medium of sucrose, maltose, fructose, lactose, galactose and trehalose and can not produce antibacterial effect (shown in figure 10). Glucose and starch have certain antibacterial effect as the sole carbon source, and 6% of starch has the best effect as the sole carbon source (shown in figure 11).
4) Fermentation medium inorganic nitrogen optimization
Study of NH 4 NO 3 、NaNO 3 、KNO 3 、(NH 4 ) 2 SO 4 Inhibition of Bacillus subtilis (Bacillus subtilis) as indicator by fermentation broth of F8 strain as sole nitrogen sourceAnd (5) fruits. Experimental results show that F8 strain is in NH 4 NO 3 、(NH 4 ) 2 SO 4 Neither good growth nor bacteriostatic effect can be produced in the culture medium. NaNO 3 With KNO 3 Has a certain antibacterial effect as the sole nitrogen source, and 1.5g/L NaNO 3 The effect as the sole nitrogen source is best (as shown in fig. 12).
5) F8 strain fermentation medium composite nitrogen optimization
The inhibition effect of peanut cake powder, corn flour, bran and soybean powder serving as organic nitrogen source F8 strain fermentation liquor on bacillus subtilis (Bacillus subtilis) of indicator bacteria is studied. Experimental results show that the F8 strain can not grow well in the wheat bran and soybean meal culture medium and can not generate antibacterial effect. The peanut cake powder and the corn powder have certain antibacterial effect as nitrogen sources, and 6% of the peanut cake powder has the best effect as nitrogen sources (shown in figure 13).
6) Soluble starch, naNO 3 Optimization of composite fermentation medium of peanut cake powder
Using Bacillus subtilis (Bacillus subtilis) as indicator bacteria, respectively using soluble starch and NaNO 3 And taking peanut cake powder as a carbon source, an inorganic nitrogen source and a composite nitrogen source, and optimizing a culture medium by using an orthogonal test. After culturing for 7d at 35 ℃ and 180rpm, the antibacterial effect of the fermentation broth is measured by adopting an oxford cup method.
TABLE 2 optimization results of composite fermentation Medium
As can be seen from Table 2, the optimized culture medium contains 5% of soluble starch, 5% of NaNO3 and 5% of peanut cake powder, and the fermentation broth with the concentration of 4% and 0.5g/L has the best antibacterial effect, the diameter of the antibacterial zone reaches 22.01cm, and the antibacterial zone is 24% higher than that of the basic culture medium of 17.74 cm.
7) Optimizing inhibition effect of fermentation medium fermentation liquor on indicator bacteria
Seed solutions were inoculated in 100ml of basal fermentation medium and optimized fermentation medium, respectively, at 1% inoculum size, and cultured for 7d at 35℃with shaking at 180 rpm. The antibacterial effect of the fermentation broth was measured by oxford cup method using a sterile operation to uniformly coat the surface of a solid medium (the bacteria are LB, the fungi are PDA) with indicator bacteria such as bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), staphylococcus aureus (Staphylococcus aureus), escherichia coli (escherichia coli) and Botrytis cinerea (Botrytis cinerea), and the results are shown in table 3, and compared with the basic fermentation medium, the antibacterial activities of the fermentation broth on bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), staphylococcus aureus (Staphylococcus aureus), escherichia coli (escherichia coli) and Botrytis cinerea (Botrytis cinerea) are improved by 28%, 24%, 18%, 30% and 19% respectively.
TABLE 3 bacteriostatic Effect of optimized Medium fermentation broths on indicator strains
8. F8 strain fermentation liquor extraction condition optimization
1) F8 strain fermentation liquor extractant selection
The extraction effect of ethyl acetate, carbon tetrachloride, petroleum ether, ethanol, n-butanol and acetone on the fermentation broth is tested by using bacillus subtilis as indicator bacteria, and the test result is shown in fig. 14. The result shows that the extraction effect of the ethyl acetate and the carbon tetrachloride on the active substances is good, the loss of the extraction activity of petroleum ether, ethanol, n-butyl alcohol and acetone is large, and the optimal extraction reagent is determined to be the ethyl acetate.
2) Selection of extraction time of ethyl acetate as extractant
The results of the experiment using Bacillus subtilis (Bacillus subtilis) as indicator bacteria and ethyl acetate as extractant for 10min, 20min, 30min, 40min, 50min and 60min are shown in FIG. 15. The extraction effect at different extraction times is shown in fig. 16, and the result shows that the difference of the extraction effect is not significant when the extraction is performed for more than 30 minutes, so that the extraction time is determined to be the optimal extraction time for 30 minutes.
3) Determination of the amount of extraction agent ethyl acetate
The extraction effect of different ratios (1:1, 1:2, 1:3, 1:4 and 1:5) of fermentation broth and extractant ethyl acetate was studied by using bacillus subtilis (Bacillus subtilis) as indicator bacteria. The results show that the ratio of the fermentation liquor to the extractant ethyl acetate is 1:3, the best effect is achieved, and the difference of the extractant continuous increase is not obvious.
The actinomycetes F8 are separated from soil on the surface layer of a wetland park in Zhengzhou yellow river country, and are pretreated in a combined mode of air drying, dry heat and phenol, and nalidixic acid, cycloheximide and potassium dichromate are used as separation inhibitors. The colony is white, white aerial hypha, grey pink spore silk, the optimum growth temperature is 37 ℃, the optimum pH7.0 and the optimum salinity is 1 percent NaCl. The actinomycetes F8 is primarily identified as Streptomyces amritsarensis by morphological characteristic observation, 16S rDNA sequence determination, systematic evolution analysis and other multiphase taxonomic identification of actinomycetes F8. Preserving in China general microbiological culture Collection center, CGMCC No. 22041. The antibacterial activity of actinomycetes F8 is detected by adopting an oxford cup method, and the results show that the actinomycetes F8 has good antibacterial effects on gram-positive bacteria staphylococcus aureus (Staphylococcus aureus), bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), gram-negative bacteria escherichia coli (escherichia coli) and filamentous fungus Botrytis cinerea. The antibacterial effect of the fermentation liquor is obviously increased from 70h of fermentation, and the fermentation liquor has better stability to temperature, lysozyme and protease. The optimal fermentation temperature is 35 ℃ and the optimal pH is 7.0 through optimizing the fermentation conditions. The most suitable fermentation medium comprises soluble starch 20g/L, peanut cake powder 50g/L, and NaNO 3 0.5g/L，K 2 HPO 4 0.5g/L，MgSO 4 ·7H 2 O 0.5g/L，NaCl1g/L，FeSO 4 ·7H 2 O0.01 g/L. The most suitable extractant of the fermentation broth is ethyl acetate, the extraction time is 30min, the ratio of the most suitable fermentation broth to the extractant is 1:3, and the discovery of the strain has practical production significance and is suitable for large-scale popularization and application.
Sequence listing
<110> biological research all of the Limited liability company of the academy of sciences of Henan province
<120> actinomycetes F8 with broad-spectrum antibacterial activity and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1430
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gggcctggcg gggtcttacc atgcaagtcg aacgatgaag cccttcgggg tggattagtg 60
gcgaacgggt gagtaacacg tgggcaatct gcccttcact ctgggacaag ccctggaaac 120
ggggtctaat accggatacg actgcggaag gcatcttctg cggtggaaag ctccggcggt 180
gaaggatgag cccgcggcct atcagcttgt tggtggggta atggcctacc aaggcgacga 240
cgggtagccg gcctgagagg gcgaccggcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtgggg aatattgcac aatgggcgaa agcctgatgc agcgacgccg 360
cgtgagggat gacggccttc gggttgtaaa cctctttcag cagggaagaa gcgaaagtga 420
cggtacctgc agaagaagcg ccggctaact acgtgccagc agccgcggta atacgtaggg 480
cgcaagcgtt gtccggaatt attgggcgta aagagctcgt aggcggcttg tcacgtcgga 540
tgtgaaagcc cgaggcttaa cctcgggtct gcattcgata cgggctagct agagtgtggt 600
aggggagatc ggaattcctg gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg 660
tggcgaaggc ggatctctgg gccattactg acgctgagga gcgaaagcgt ggggagcgaa 720
caggattaga taccctggta gtccacgccg taaacgttgg gaactaggtg ttggcgacat 780
tccacgtcgt cggtgccgca gctaacgcat taagttcccc gcctggggag tacggccgca 840
aggctaaaac tcaaaggaat tgacgggggc ccgcacaagc ggcggagcat gtggcttaat 900
tcgacgcaac gcgaagaacc ttaccaaggc ttgacatata ccggaaagca ttagagatag 960
tgcccccctt gtggtcggta tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcaac ccttgtcctg tgttgccagc atgcccttcg 1080
gggtgatggg gactcacagg agaccgccgg ggtcaactcg gaggaaggtg gggacgacgt 1140
caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc ggtacaatga 1200
gctgcgatac cgtgaggtgg agcgaatctc aaaaagccgg tctcagttcg gattggggtc 1260
tgcaactcga ccccatgaag tcggagtcgc tagtaatcgc agatcagcat tgctgcggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacgtca cgaaagtcgg taacacccga 1380
agccggtggc ccaacccttg tggagggagc ttcgaagggg ggattcggct 1430
Claims (4)
1. An actinomycete F8 with broad-spectrum antibacterial activity is characterized by being classified and named as Streptomyces amjensisStreptomyces amritsarensis) The strain is preserved in China general microbiological culture Collection center (China Committee) with the preservation number: CGMCC No.22041, the 16S rDNA gene sequence of the strain is shown as SEQ ID No. 1.
2. The process for preparing aseptic fermentation broth of actinomycetes F8 with broad-spectrum antibacterial activity as claimed in claim 1, wherein seed liquid is inoculated into 100ml basic fermentation medium or optimized fermentation medium according to 1% inoculum size, fermentation broth is obtained by shaking culture at 35 ℃ and 180rpm for 7d, thalli is removed by centrifugation at 12000 Xg and 25 ℃, residual thalli is removed from supernatant by a filter membrane with aperture of 0.22 μm, and aseptic fermentation broth supernatant containing active substances secreted by thalli is obtained; the basic fermentation culture medium is as follows: soluble starch 20g, KNO 3 1g，K 2 HPO 4 0.5 g，MgSO 4 ·7H 2 O 0.5 g，NaCl 0.5 g，FeSO 4 · 7H 2 O0.01 g, pH7.0, distilled water to 1000mL；
The optimized fermentation medium is as follows: soluble starch 20g, peanut cake powder 50g and NaNO 3 0.5g，K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 1g，FeSO 4 · 7H 2 O0.01 g, pH7.0, distilled water to 1000mL.
3. The preparation of actinomycetes F8 with broad-spectrum antibacterial activity as claimed in claim 1 for inhibiting gram-positive bacteria staphylococcus aureusStaphylococcus aureus) Bacillus subtilisBacillus subtilis) Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) Gram negative bacteria E.coli @Escherich coli) Botrytis cinereaBotrytis cinerea) Is used in the medicine.
4. The process for preparing aseptic fermentation liquor of actinomycetes F8 with broad-spectrum antibacterial activity as claimed in claim 2 for inhibiting gram-positive bacteria staphylococcus aureusStaphylococcus aureus) Bacillus subtilisBacillus subtilis) Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) Gram negative bacteria E.coli @Escherich coli) Botrytis cinereaBotrytis cinerea) Is used in the medicine.
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| CN108587981A (en) * | 2018-05-30 | 2018-09-28 | 西南大学 | Multi-functional molten algae streptomycete Streptomyces amritsarensis and its application |
| CN110331103A (en) * | 2019-06-04 | 2019-10-15 | 湖南师范大学 | One plant of molten algae streptomycete N1-32, its probiotics and preparation method thereof |
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| WO2017151059A1 (en) * | 2016-02-29 | 2017-09-08 | Agency For Science, Technology And Research | Multiplexable activation of silent biosynthetic clusters in native actinomycete hosts for natural product discovery |
| CN108587981A (en) * | 2018-05-30 | 2018-09-28 | 西南大学 | Multi-functional molten algae streptomycete Streptomyces amritsarensis and its application |
| CN110331103A (en) * | 2019-06-04 | 2019-10-15 | 湖南师范大学 | One plant of molten algae streptomycete N1-32, its probiotics and preparation method thereof |
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