CN113813292A - Marine protozoan pillworm extract with antibacterial activity - Google Patents
Marine protozoan pillworm extract with antibacterial activity Download PDFInfo
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- CN113813292A CN113813292A CN202111262517.4A CN202111262517A CN113813292A CN 113813292 A CN113813292 A CN 113813292A CN 202111262517 A CN202111262517 A CN 202111262517A CN 113813292 A CN113813292 A CN 113813292A
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- 239000000284 extract Substances 0.000 title claims abstract description 58
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 9
- 230000003385 bacteriostatic effect Effects 0.000 claims abstract description 22
- 241000237858 Gastropoda Species 0.000 claims abstract description 15
- 241000607598 Vibrio Species 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 241000589876 Campylobacter Species 0.000 claims abstract description 6
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims abstract description 6
- 241000700141 Rotifera Species 0.000 claims abstract description 6
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 6
- 241000607265 Vibrio vulnificus Species 0.000 claims abstract description 6
- 229960003085 meticillin Drugs 0.000 claims abstract description 6
- 238000003113 dilution method Methods 0.000 claims abstract description 3
- 244000005700 microbiome Species 0.000 claims description 6
- 244000000010 microbial pathogen Species 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 12
- 239000000022 bacteriostatic agent Substances 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- 229910052651 microcline Inorganic materials 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 229940088710 antibiotic agent Drugs 0.000 description 8
- 244000052616 bacterial pathogen Species 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 240000004507 Abelmoschus esculentus Species 0.000 description 3
- 235000003934 Abelmoschus esculentus Nutrition 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000101515 Staphylotrichum Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 241001075517 Abelmoschus Species 0.000 description 1
- 241000251557 Ascidiacea Species 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000422450 Gastrostyla Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241000222722 Leishmania <genus> Species 0.000 description 1
- 241000248487 Moneuplotes crassus Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000842 anti-protozoal effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003904 antiprotozoal agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229930183674 euplotin Natural products 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
Classifications
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- 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
- A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
-
- 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
-
- 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
- C12N1/10—Protozoa; Culture media therefor
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- 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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Abstract
The invention discloses a marine protozoan gastrodiae extract with antibacterial activity, and relates to the technical field of protozoan extracts. The invention comprises a preparation method of the ocean pillworm extract and a microdilution method for measuring the bacteriostatic activity of the ocean pillworm extract; the preparation method of the ocean pillworm extract is used for preparing the extract of protozoan pillworm; the application of the microcline dilution method for determining the bacteriostatic activity of the extract of the marine gastropod worm in preparing the bacteriostatic agent of the extract with the bacteriostatic action of the marine gastropod worm. The ocean pillworm extract has high antibacterial activity, can be used for preparing bacteriostatic agents, is derived from ocean protozoa, can continuously obtain raw materials by expanding culture, and has the advantages of environmental friendliness and low application cost; the antibacterial agent is used for developing antibacterial agents of methicillin-resistant staphylococcus aureus, rotifer vibrio, campylobacter vibrio and vibrio vulnificus, and has a very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of protozoan extracts, and particularly relates to a marine protozoan gastrodiae extract with antibacterial activity.
Background
Pathogenic microorganisms have great harm to the production and life of human beings, and antibiotics, as a type of secondary metabolites generated by microorganisms or higher animals and plants in the life process, can kill bacteria and also have good inhibition and killing effects on other pathogenic microorganisms such as mould, mycoplasma, chlamydia and the like, but in the past decades, the common use of antibiotics enables pathogenic bacteria to generate drug resistance on the antibiotics, and the development of novel antibiotics becomes more urgent due to the appearance of antibiotic-resistant bacteria;
over the past decades, the acquisition of antibiotics has been mainly focused on terrestrial resources, however the possibility of finding new bioactive compounds from terrestrial organisms is constantly decreasing; marine environmental organisms become important sources of novel active secondary metabolites, and due to ecological pressure, including competition of space and predation, and physical characteristics such as salinity, pressure and temperature change, the marine environment develops a unique metabolic system and a body defense mechanism in the high-demand ecosystem to adapt to the environment, so that the characteristics of the marine microorganisms are certainly greatly different from those of land microorganisms, and therefore, the marine organisms can generate various new bioactive metabolites different from the land, and the marine environment becomes a main production place for searching and discovering new product resources;
although protists are eukaryotic unicellular animals without cell walls, and have all functions necessary for life maintenance and progeny continuation, research in related fields focuses on several aspects such as morphological taxonomy, systematics, cytogenesis, zoology and ecology, and in recent years researchers have extracted some pathogenic antagonistic active substances from some marine protozoa, for example, the thick posterior servant of the order Abelmoschus (Euplotes crassus) can produce a unique terpenoid named Euplotin, which can resist promastigotes with antagonistic effect on Leishmania, and can be used as a synergist of antiprotozoal chemotherapeutic drugs. The study of antibacterial active ingredients of protozoa is of great significance.
Disclosure of Invention
Aiming at the problem that pathogenic bacteria generate drug resistance to the existing antibiotics due to the common use of the antibiotics, the invention provides the extract of the ocean protozoan gastrodiae extract with bacteriostatic activity, and provides resources for the research and development of novel antibiotics.
The invention is realized by the following technical scheme: a marine protozoan Staphylotrichum extract with antibacterial activity comprises preparing extract of Staphylotrichum marinum and measuring antibacterial activity of the extract by microdilution;
the preparation method of the ocean pillworm extract is used for preparing the extract of protozoan pillworm;
the micro-dilution method is used for determining the bacteriostatic activity of the ocean pillworm extract and is used for detecting the bacteriostatic effect of the ocean pillworm extract.
Further, the preparation method of the ocean pillworm extract comprises the following steps:
the first step is as follows: separating and purifying the pillworm population from the environment to obtain a single population of the pillworms;
the second step is that: performing expanded culture on the pillworm, namely inoculating a single population of the pillworm into a liquid culture medium for expanded culture;
the third step: and (4) preparing an extract.
Further, the method for determining the bacteriostatic activity of the extract of the marine gastropod worms by the microdilution method is characterized in that the bacteriostatic activity of the extract of the marine gastropod worms is determined by detecting the inhibition percentage of thalli added into the extract of the marine gastropod worms in the cultured pathogenic microorganisms.
Further, the microorganism is methicillin-resistant staphylococcus aureus, vibrio rotifer, vibrio campylobacter and vibrio vulnificus.
The invention has the following beneficial effects:
the ocean pillworm extract has high antibacterial activity, can be used for preparing bacteriostatic agents, is derived from ocean protozoa, can continuously obtain raw materials by expanding culture, and has the advantages of environmental friendliness and low application cost; the antibacterial agent is used for developing antibacterial agents of methicillin-resistant staphylococcus aureus, rotifer vibrio, campylobacter vibrio and vibrio vulnificus, and has a very wide application prospect.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a marine protozoan pillworm extract with bacteriostatic activity, which comprises a preparation method of a marine pillworm extract and a microdilution method for measuring the bacteriostatic activity of the marine pillworm extract;
the preparation method of the ocean pillworm extract is used for preparing the extract of protozoan pillworm;
the preparation method of the ocean pillworm extract comprises the following steps:
the first step is as follows: separating and purifying the pillworm population from the environment;
protozoan communities are collected from seawater, and the communities are expanded by wheat grain culture solution in a laboratory, and the method comprises the following operation steps: sucking a gastrocylic insect (Gastrostyla sp.) under a microscope, and carrying out amplification culture by using a wheat grain culture solution to obtain a single population of the gastrocylic insect;
the second step is that: carrying out expanded culture on the Abelmoschus esculentus;
inoculating the single population of the Abelmoschus esculentus into a liquid culture medium, carrying out shake culture at 25 ℃ for 2 months at a low speed, carrying out scale enlargement culture to 5L, centrifuging the obtained sample (3,000rpm for 10min), collecting the polypide, and inoculating the single population of the Abelmoschus esculentus into the liquid culture medium for enlargement culture;
the third step: preparing an extract; extracting with methanol for 3 times, wherein the extraction method comprises adding methanol into the centrifuged sediment, homogenizing the sediment with high speed disperser, centrifuging to 12000r/min, collecting supernatant methanol sample, concentrating with rotary evaporator to obtain extract, dissolving with methanol to 5mL, and storing at 4 deg.C;
the high-speed dispersion machine is purchased from product number of Guangzhou laboratory technology GmbH of IKA Ka Germany: t25DS 25;
the rotary evaporator was purchased from Heidolph, germany, product number: Hei-VAP;
the application of the microclutition method for determining the bacteriostatic activity of the marine gastropod worm extract in preparing the bacteriostatic agent of the extract with the marine gastropod worm bacteriostatic action.
The method for determining the bacteriostatic activity of the extract of the marine gastropod worms adopts a method for determining the growth inhibition condition of thalli added with the extract of the marine gastropod worms in the cultured pathogenic microorganisms to determine the bacteriostatic activity of the extract of the marine gastropod worms.
The microorganisms are methicillin-resistant staphylococcus aureus, vibrio rotifer, vibrio campylobacter and vibrio vulnificus;
two strains of methicillin-resistant Staphylococcus aureus, Vibrio rotifer, Vibrio campylobacter and Vibrio vulnificus. By OD600Determining the minimum inhibitory concentration MIC when the inhibition rate of each bacterium is 90%;
the above strains can be purchased from ATCC, CGMCC and MCCC;
the minimum inhibitory concentration MIC of the ocean pillworm extract to the pathogenic bacteria is shown in Table 1, and as can be seen from Table 1, the ocean pillworm extract effectively inhibits the proliferation of the pathogenic bacteria, has higher activity of inhibiting the bacterial proliferation, can be used for preparing bacteriostatic agents, and has very wide development and application prospects;
TABLE 1 MIC result table of minimum inhibitory concentration of ocean ascidian extract
The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (4)
1. An extract of ocean protozoa gastrodiae with bacteriostatic activity is characterized in that: comprises a preparation method of the extract of the marine gastropod worm and a microdilution method for measuring the bacteriostatic activity of the extract of the marine gastropod worm;
the preparation method of the ocean pillworm extract is used for preparing the extract of protozoan pillworm;
the micro-dilution method is used for measuring the bacteriostatic activity of the ocean pillworm extract.
2. The extract of the marine protozoan gastrodiae having bacteriostatic activity according to claim 1, characterized in that: the preparation method of the ocean pillworm extract comprises the following steps:
the first step is as follows: separating and purifying the pillworm population from the environment to obtain a single population of the pillworms;
the second step is that: performing expanded culture on the pillworm, namely inoculating a single population of the pillworm into a liquid culture medium for expanded culture;
the third step: and (4) preparing an extract.
3. The extract of the marine protozoan gastrodiae having bacteriostatic activity according to claim 1, characterized in that: the method for determining the bacteriostatic activity of the extract of the marine gastropod worms by the microdilution method is characterized in that the bacteriostatic activity of the extract of the marine gastropod worms is determined by detecting the thallus inhibition percentage of the extract of the marine gastropod worms added into cultured pathogenic microorganisms.
4. The extract of the marine protozoan gastrodiae having bacteriostatic activity according to claim 3, characterized in that: the microorganism is methicillin-resistant staphylococcus aureus, vibrio rotifer, vibrio campylobacter and vibrio vulnificus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111262517.4A CN113813292B (en) | 2021-10-28 | 2021-10-28 | Marine protozoan armillar extract with antibacterial activity |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111262517.4A CN113813292B (en) | 2021-10-28 | 2021-10-28 | Marine protozoan armillar extract with antibacterial activity |
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| CN113813292B CN113813292B (en) | 2024-01-16 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034489A1 (en) * | 1996-03-20 | 1997-09-25 | Aquaculture Technology, Inc. | Antibacterially active marine algae extracts from the class diatomatae and methods of use |
| US20070098745A1 (en) * | 2004-04-30 | 2007-05-03 | Bruno Cynthia K | Antagonistic properties of reef fish microflora |
| JP2010059100A (en) * | 2008-09-04 | 2010-03-18 | Yokohama City Univ | Antibacterial agent for gram-positive bacteria and antimicrobial activity potentiating agent |
| KR20120063245A (en) * | 2010-12-07 | 2012-06-15 | 전남대학교산학협력단 | Anti-bacterial and anti-protozoal composition containing bromophenol derivatives |
| CN108283653A (en) * | 2018-04-05 | 2018-07-17 | 吉林大学 | It is a kind of using Taxus extracts prepare plant-based bacteriostat method and its in the application of antibiosis |
| CN109576341A (en) * | 2018-12-05 | 2019-04-05 | 海南省海洋与渔业科学院（海南省海洋开发规划设计研究院） | A kind of lead compound screening technique inhibiting grouper pathogenic vibrio |
| US20200347345A1 (en) * | 2019-05-02 | 2020-11-05 | University Of Rhode Island Board Of Trustees | Marine Bacteria Formulation Useful in Aquaculture |
-
2021
- 2021-10-28 CN CN202111262517.4A patent/CN113813292B/en active Active
Patent Citations (7)
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|---|---|---|---|---|
| WO1997034489A1 (en) * | 1996-03-20 | 1997-09-25 | Aquaculture Technology, Inc. | Antibacterially active marine algae extracts from the class diatomatae and methods of use |
| US20070098745A1 (en) * | 2004-04-30 | 2007-05-03 | Bruno Cynthia K | Antagonistic properties of reef fish microflora |
| JP2010059100A (en) * | 2008-09-04 | 2010-03-18 | Yokohama City Univ | Antibacterial agent for gram-positive bacteria and antimicrobial activity potentiating agent |
| KR20120063245A (en) * | 2010-12-07 | 2012-06-15 | 전남대학교산학협력단 | Anti-bacterial and anti-protozoal composition containing bromophenol derivatives |
| CN108283653A (en) * | 2018-04-05 | 2018-07-17 | 吉林大学 | It is a kind of using Taxus extracts prepare plant-based bacteriostat method and its in the application of antibiosis |
| CN109576341A (en) * | 2018-12-05 | 2019-04-05 | 海南省海洋与渔业科学院（海南省海洋开发规划设计研究院） | A kind of lead compound screening technique inhibiting grouper pathogenic vibrio |
| US20200347345A1 (en) * | 2019-05-02 | 2020-11-05 | University Of Rhode Island Board Of Trustees | Marine Bacteria Formulation Useful in Aquaculture |
Non-Patent Citations (3)
| Title |
|---|
| 季宇彬，等: "海洋真菌Aspergillus violaceus WZXY-m64-17中的抗菌活性成分", vol. 37, no. 4 * |
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