CN113969298B - Preparation method of 6-carboxyl chitosan oligosaccharide-copper complex and application of prepared product - Google Patents
Preparation method of 6-carboxyl chitosan oligosaccharide-copper complex and application of prepared product Download PDFInfo
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- CN113969298B CN113969298B CN202111416689.2A CN202111416689A CN113969298B CN 113969298 B CN113969298 B CN 113969298B CN 202111416689 A CN202111416689 A CN 202111416689A CN 113969298 B CN113969298 B CN 113969298B
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 claims abstract description 61
- 229910052802 copper Inorganic materials 0.000 claims abstract description 48
- 239000010949 copper Substances 0.000 claims abstract description 48
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 28
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 13
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 108010029541 Laccase Proteins 0.000 claims abstract description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000001301 oxygen Substances 0.000 claims abstract description 10
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 10
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 230000000844 anti-bacterial effect Effects 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000003899 bactericide agent Substances 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- 230000008961 swelling Effects 0.000 claims description 9
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 claims description 7
- 150000007524 organic acids Chemical class 0.000 claims description 7
- 241000589615 Pseudomonas syringae Species 0.000 claims description 6
- 241000207199 Citrus Species 0.000 claims description 5
- 235000020971 citrus fruits Nutrition 0.000 claims description 5
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 5
- 230000006196 deacetylation Effects 0.000 claims description 5
- 238000003381 deacetylation reaction Methods 0.000 claims description 5
- 241000196324 Embryophyta Species 0.000 claims description 4
- 241001123668 Verticillium dahliae Species 0.000 claims description 4
- 230000001276 controlling effect Effects 0.000 claims description 4
- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 claims description 4
- 230000003301 hydrolyzing effect Effects 0.000 claims description 4
- 241000223218 Fusarium Species 0.000 claims description 3
- 241000209140 Triticum Species 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 208000025865 Ulcer Diseases 0.000 claims description 2
- 231100000397 ulcer Toxicity 0.000 claims description 2
- 241000123650 Botrytis cinerea Species 0.000 claims 1
- 230000000855 fungicidal effect Effects 0.000 claims 1
- 239000000417 fungicide Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 59
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- 230000000694 effects Effects 0.000 description 23
- 239000000523 sample Substances 0.000 description 20
- 239000012065 filter cake Substances 0.000 description 16
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 15
- 229910001431 copper ion Inorganic materials 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 239000004563 wettable powder Substances 0.000 description 8
- 240000008067 Cucumis sativus Species 0.000 description 7
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 230000021523 carboxylation Effects 0.000 description 6
- 238000006473 carboxylation reaction Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 238000000967 suction filtration Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- MGIWDIMSTXWOCO-UHFFFAOYSA-N butanedioic acid;copper Chemical compound [Cu].OC(=O)CCC(O)=O MGIWDIMSTXWOCO-UHFFFAOYSA-N 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 3
- 108010089807 chitosanase Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- GDOPTJXRTPNYNR-UHFFFAOYSA-N methyl-cyclopentane Natural products CC1CCCC1 GDOPTJXRTPNYNR-UHFFFAOYSA-N 0.000 description 3
- 238000011056 performance test Methods 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000536 complexating effect Effects 0.000 description 2
- 238000010668 complexation reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 244000053095 fungal pathogen Species 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000000954 titration curve Methods 0.000 description 2
- 238000003828 vacuum filtration Methods 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 239000005739 Bordeaux mixture Substances 0.000 description 1
- 239000005740 Boscalid Substances 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- JJLJMEJHUUYSSY-UHFFFAOYSA-L Copper hydroxide Chemical compound [OH-].[OH-].[Cu+2] JJLJMEJHUUYSSY-UHFFFAOYSA-L 0.000 description 1
- 239000005750 Copper hydroxide Substances 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 241000097097 Laurella Species 0.000 description 1
- 241001469654 Lawsonia <weevil> Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 244000273928 Zingiber officinale Species 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- WYEMLYFITZORAB-UHFFFAOYSA-N boscalid Chemical compound C1=CC(Cl)=CC=C1C1=CC=CC=C1NC(=O)C1=CC=CN=C1Cl WYEMLYFITZORAB-UHFFFAOYSA-N 0.000 description 1
- 229940118790 boscalid Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- -1 chitosan oligosaccharide copper complex Chemical class 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 150000004699 copper complex Chemical class 0.000 description 1
- 229910001956 copper hydroxide Inorganic materials 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 235000008397 ginger Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 238000005142 microbroth dilution method Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
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- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- 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
- A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
- A01N55/02—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur containing metal atoms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- 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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- Chemical & Material Sciences (AREA)
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of antibacterial agents, and particularly provides a preparation method of a 6-carboxyl chitosan oligosaccharide-copper complex, which comprises the following steps: adjusting the pH value of the chitosan oligosaccharide solution to 4.0-6.0 to obtain the chitosan oligosaccharide solution with the pH value adjusted; mixing the chitosan oligosaccharide solution with the pH value regulated, 2, 6-tetramethyl piperidine-1-oxyl and laccase, and introducing oxygen at 20-40 ℃ for reacting for 2-10 h to obtain a 6-carboxyl chitosan oligosaccharide solution; mixing the 6-carboxyl chitosan oligosaccharide solution with a copper-containing compound, and reacting for 2-4 hours at 44-55 ℃ to obtain the 6-carboxyl chitosan oligosaccharide solution-copper complex. The 6-carboxyl chitosan oligosaccharide-copper complex prepared by the invention can obviously improve the complex capacity of sugar chains, and has stronger inhibitory activity on various pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of antibacterial agents, and particularly relates to a preparation method of a 6-carboxyl chitosan oligosaccharide-copper complex and application of a prepared product thereof.
Background
The chitosan oligosaccharide, also called as amino-oligosaccharin, is a green biological pesticide with the functions of activating the immune system and inhibiting bacteria of crops. It is mainly obtained by deacetylation and hydrolysis of chitin in shrimp and crab offal, the polymerization degree is generally 2-20 (molecular weight 340-3238 Da). Chitosan oligosaccharide is the only natural alkaline oligosaccharide discovered so far, and has been widely applied in the aspects of preventing and controlling crop virus, bacteria, fungal diseases and the like. However, the chitosan oligosaccharide has weak direct bacteriostasis and sterilization capability, the minimum bacteriostasis concentration (minimum inhibitory concentration, MIC) of the chitosan oligosaccharide on pseudomonas syringae is 1000-3000ppm, the MIC value on verticillium dahliae is 20000-40000ppm, and the chitosan oligosaccharide is difficult to be directly used as a bactericide.
The chitosan oligosaccharide molecule contains hydroxyl, amino and amide groups, can form a complex with divalent metal ions, and can also retain the immune induction activity of the chitosan oligosaccharide. Copper ions have stronger bactericidal capability, copper preparations such as copper hydroxide, copper succinate and bordeaux mixture are applied at home and abroad for a long time, but the safety of the copper preparations is low, so that phytotoxicity is often caused, and the copper preparations become main barriers for limiting the further expansion application of the products. In order to improve the bacteriostatic ability of chitosan oligosaccharide and the safety of copper preparations, patent CN201410174256.4 discloses a preparation method of a chitosan oligosaccharide copper complex, wherein the mass ratio of chitosan oligosaccharide to copper ions is 5:1-1:1.2, and in fact, the highest copper content in the complex is not more than 15% due to more coordination number of copper, which is far lower than the copper content in the copper preparation products on the market.
Disclosure of Invention
The invention provides a preparation method of a 6-carboxyl chitosan oligosaccharide-copper complex and application of a prepared product thereof, wherein the prepared 6-carboxyl chitosan oligosaccharide-copper complex has higher copper content and has stronger inhibitory activity on various pathogenic bacteria.
The invention provides a preparation method of a 6-carboxyl chitosan oligosaccharide-copper complex, which comprises the following steps:
adjusting the pH value of the chitosan oligosaccharide solution to 4.0-6.0 to obtain the chitosan oligosaccharide solution with the pH value adjusted;
mixing the chitosan oligosaccharide solution with the pH value regulated, 2, 6-tetramethyl piperidine-1-oxyl and laccase, and introducing oxygen at 20-40 ℃ for reacting for 2-10 h to obtain a 6-carboxyl chitosan oligosaccharide solution;
mixing the 6-carboxyl chitosan oligosaccharide solution with a copper-containing compound, and reacting for 2-4 hours at 44-55 ℃ to obtain a 6-carboxyl chitosan oligosaccharide solution-copper complex;
the copper-containing compound is copper sulfate pentahydrate, copper acetate monohydrate or copper chloride dihydrate.
Preferably, the chitosan oligosaccharide solution is prepared by the following steps:
mixing chitosan with deacetylation degree more than or equal to 85%, water and organic acid/hydrochloric acid, and swelling to obtain swelled chitosan;
and hydrolyzing the swelled chitosan by adopting chitosan enzyme to obtain a chitosan oligosaccharide solution.
Preferably, the mass ratio of the chitosan to the water is 1:5-20; the dosage of the organic acid/hydrochloric acid is 5-50% of the mass of the chitosan.
Preferably, the temperature during swelling is 30-65 ℃; the time is 4-24 hours.
Preferably, the dosage of the chitosanase is 2-16U/g chitosan.
Preferably, the addition amount of the 2, 6-tetramethyl piperidine-1-oxyl is 1-8% of the chitosan oligosaccharide according to the weight percentage.
Preferably, the mass ratio of the 6-carboxyl chitosan oligosaccharide in the 6-carboxyl chitosan oligosaccharide solution to the copper-containing compound is 1:1-5.
The invention provides application of the 6-carboxyl chitosan oligosaccharide-copper complex prepared by any one of the methods as a bactericide.
Preferably, the bactericide is used for preventing and controlling plant diseases caused by pseudomonas syringae, bacterial wilt, verticillium dahliae, gray mold, citrus canker pathogenic bacteria, apple rot pathogenic bacteria or gibberella wheat.
Compared with the prior art, the invention has the advantages and positive effects that:
according to the invention, the hydroxy group at the C6 position of the chitosan oligosaccharide is converted into the carboxyl group by a biocatalysis method, so that the complex capacity of a sugar chain to copper ions and the stability of a complex are improved, the maximum copper ion content in a 6-carboxyl chitosan oligosaccharide-copper sample can reach 27.1%, and the copper ion content in a conventional copper preparation is at the same level. The complex has good inhibition effect on various pathogenic bacteria and fungi, and can maintain the immune induced resistance activity of chitosan oligosaccharide.
Drawings
FIG. 1 shows the mass spectrometry analysis of chitosan oligosaccharide of example 1,
wherein 1 is chitosan, 2 is chitosan, 3 is chitosan, 4 is chitosan, 5 is chitosan, 6 is chitosan, 7 is chitosan, 8 is chitosan;
FIG. 2 shows the NMR of chitosan oligosaccharide obtained in example 1 1 H spectrum analysis;
FIG. 3 IR spectra of Chitosan Oligosaccharide (COS) and 6-carboxychitosan oligosaccharide (C-COS).
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a preparation method of a 6-carboxyl chitosan oligosaccharide-copper complex, which comprises the following steps:
adjusting the pH value of the chitosan oligosaccharide solution to 4.0-6.0 to obtain the chitosan oligosaccharide solution with the pH value adjusted;
mixing the chitosan oligosaccharide solution with the pH value regulated, 2, 6-tetramethyl piperidine-1-oxyl and laccase, and introducing oxygen at 20-40 ℃ for reacting for 2-10 h to obtain a 6-carboxyl chitosan oligosaccharide solution;
mixing the 6-carboxyl chitosan oligosaccharide solution with a copper-containing compound, and reacting for 2-4 hours at 44-55 ℃ to obtain a 6-carboxyl chitosan oligosaccharide solution-copper complex;
the copper-containing compound is copper sulfate pentahydrate, copper acetate monohydrate or copper chloride dihydrate.
The pH value of the chitosan oligosaccharide solution is adjusted to 4.0-6.0, so that the chitosan oligosaccharide solution with the adjusted pH value is obtained. In the present invention, the pH is preferably adjusted to 5.0.
In the invention, the chitosan oligosaccharide solution is preferably prepared by the following steps:
mixing chitosan with deacetylation degree more than or equal to 85%, water and organic acid/hydrochloric acid, and swelling to obtain swelled chitosan;
and hydrolyzing the swelled chitosan by adopting chitosan enzyme to obtain a chitosan oligosaccharide solution.
In the invention, the mass ratio of the chitosan to the water is preferably 1:5-20; more preferably 1:10. In the present invention, the amount of the organic acid/hydrochloric acid is preferably 5% to 50% by mass of chitosan, more preferably 20% to 30%. In the present invention, the organic acid is preferably one or two of glacial acetic acid, lactic acid, formic acid, citric acid, butyric acid and succinic acid.
In the present invention, the temperature at the time of swelling is preferably 30 to 65 ℃, more preferably 40 to 50 ℃; the swelling time is preferably 4 to 24 hours, more preferably 10 to 16 hours.
In the present invention, the amount of the chitosanase is preferably 2 to 16U/g chitosan, more preferably 6 to 10U/g chitosan. In the present invention, the temperature at the time of the hydrolysis is preferably 30 to 65 ℃, more preferably 40 to 60 ℃; the hydrolysis time is preferably 3 to 6 hours. In the present invention, filtration is preferably performed after the hydrolysis is completed to obtain a clarified chitosan oligosaccharide solution. In the present invention, the filtration is preferably vacuum filtration, preferably using diatomaceous earth as a filter aid.
In the invention, the chitosan enzyme is purchased from the chemical industry institute of Beijing and Bo, inc. of yellow river delta, the product is in the form of liquid, and the enzyme activity is more than or equal to 1000U/mL.
In the present invention, the enzyme activity of the chitosan enzyme is preferably determined by the following method: (1) chitosan swelling. 2.0g (to the nearest 0.01 g) of the chitosan sample (noted m) was weighed into a beaker, 100mL of water was added and stirred to thoroughly wet the chitosan. Then 0.75mL glacial acetic acid was added and the mixture was swollen for 30min with stirring at 35℃until the mass became a viscous, homogeneous liquid. And (2) enzymolysis. A volume (designated V) of enzyme solution was added at the expected enzyme activity level, and the time was counted from the addition of enzyme solution. And (3) judging the end point. Sampling is carried out regularly, enzymolysis liquid is dripped into 0.1mol/L NaOH solution, and the reaction end point is regarded when no white precipitate is generated. The time taken (noted t) was recorded. The reaction time is controlled to be 10-20 min. (4) calculating the result. Enzyme activity = m 1000/(V t), unit is U/mL.
After the chitosan oligosaccharide solution with the pH value adjusted is obtained, the chitosan oligosaccharide solution with the pH value adjusted, 2, 6-tetramethyl piperidine-1-oxyl and laccase are mixed and subjected to oxygen introduction reaction for 2-10 hours at 20-40 ℃ to obtain the 6-carboxyl chitosan oligosaccharide solution. In the present invention, the amount of the 2, 6-tetramethylpiperidine-1-oxyl added is preferably 1 to 8% by weight, more preferably 3 to 6% by weight of the chitosan oligosaccharide.
In the invention, the addition amount of laccase is preferably 2.5-5.0U/g chitosan oligosaccharide. In the present invention, the laccase is purchased from the company limited of the chemical industry institute of yellow river delta, genia-jingbo. The enzyme activity of the laccase is more than or equal to 100U/mL.
In the present invention, after the completion of the oxygen supplying reaction, vacuum filtration is preferably performed. To obtain 6-carboxyl chitosan oligosaccharide solution, if not urgent to prepare 6-carboxyl chitosan oligosaccharide solution-copper complex, for convenient storage, the 6-carboxyl chitosan oligosaccharide solution can also be subjected to spray drying or vacuum drying to obtain a solid sample.
After obtaining a 6-carboxyl chitosan oligosaccharide solution, mixing the 6-carboxyl chitosan oligosaccharide solution with a copper-containing compound, and reacting for 2-4 hours at 44-55 ℃ to obtain a 6-carboxyl chitosan oligosaccharide solution-copper complex; the copper-containing compound is copper sulfate pentahydrate or copper acetate monohydrate or copper chloride dihydrate.
In the invention, the mass ratio of the 6-carboxyl chitosan oligosaccharide to the copper-containing compound in the 6-carboxyl chitosan oligosaccharide solution is preferably 1:1-5.
In the invention, after the 6-carboxyl chitosan oligosaccharide solution-copper complex or the 6-carboxyl chitosan oligosaccharide solution is obtained, the invention can be further processed into products such as suspending agents, wettable powder, soluble solutions and the like.
According to the invention, the biocatalytic method of the reaction by adding laccase is used for converting the hydroxyl at the C6 position of the chitosan oligosaccharide into carboxyl, and the complex capacity of the sugar chain on copper ions and the stability of the complex can be improved after the hydroxyl at the C6 position is converted into carboxyl, so that the copper ion content in a 6-carboxyl chitosan oligosaccharide-copper sample can reach 27.1% to the maximum, and the copper ion content in the chitosan oligosaccharide-copper sample is at the same level as that in a conventional copper preparation. The complex has good inhibition effect on various pathogenic bacteria and fungi, and can maintain the immune induced resistance activity of chitosan oligosaccharide.
The invention provides application of the 6-carboxyl chitosan oligosaccharide-copper complex prepared by any one of the methods as a bactericide.
In the present invention, the bactericide is used for controlling plant diseases caused by pseudomonas syringae, lauteromyces subtilis, verticillium dahliae, gray mold, citrus canker pathogenic bacteria, apple rot pathogenic bacteria or gibberella wheat.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
55g of chitosan powder (deacetylation degree 91%, ash content 1.5%, moisture content 8.5%) is dispersed into 450g of deionized water, and stirred to be fully wetted; adding 20g of glacial acetic acid, and swelling chitosan at 40 ℃ for 6h under stirring; then adding 0.2mL of chitosanase with the enzyme activity of 1000U/mL, and hydrolyzing for 3h at 40 ℃; adding 4g of diatomite into the hydrolysate and dispersing the diatomite uniformly, and carrying out vacuum suction filtration to obtain a chitosan oligosaccharide solution with the content of 10% (according to mass percent), or further carrying out freeze drying to obtain chitosan oligosaccharide powder, wherein the chitosan oligosaccharide content in the powder is 90%. As shown in FIG. 1, the polymerization degree of the obtained chitosan oligosaccharide is 2-10 through mass spectrometry. FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the obtained chitosan oligosaccharide, which shows that the obtained chitosan oligosaccharide sample has a part of acetyl groups, and the acetyl content is 9.2%.
Example 2
(1) 200g of the 10% chitosan oligosaccharide solution prepared in example 1 (pH value is adjusted to 5.0) was taken, 0.8g of TEMPO (2, 6-tetramethylpiperidine-1-oxyl) was added, and the mixture was placed in a water bath kettle at 35 ℃ to be stirred and reacted for 15min; adding 0.8mL laccase (enzyme activity 105U/mL) to perform oxygen ventilation reaction for 8 hours; and after oxygen introduction, regulating the reaction solution to be neutral by using liquid alkali, adding 4g of diatomite, uniformly dispersing, and carrying out vacuum suction filtration to obtain a 10% 6-carboxyl chitosan oligosaccharide solution. Concentrating the reaction solution to 100mL by rotary evaporation, transferring to a beaker, adding 400mL of absolute ethyl alcohol, and standing for 30min; after the reaction product is separated out, carrying out vacuum suction filtration, repeatedly washing the filtered product with alcohol until the alcohol washing liquid is clear; and (5) placing the filter cake in a vacuum drying oven to be dried for 24 hours at 50 ℃, and grinding the dried filter cake to obtain sample powder. The sample was determined to have a carboxylation rate of 80% -85%, indicating that most of the hydroxyl groups at the C6 position are oxidized to carboxyl groups. FIG. 3 is an infrared spectrum of the chitosan oligosaccharide obtained in example 1 and the 6-carboxychitosan oligosaccharide obtained in this example.
Carboxylation rate determination in 6-carboxychitosan oligosaccharide powder: 0.2g of the sample was weighed and dissolved in 10mL of 0.1mol/L HCl solution for acidification, and after dissolution, 90mL of water was added for dilution. The automatic potentiometric titrator was turned on and titration was performed using 0.1mol/L NaOH standard solution. The consumption and conductivity of NaOH was recorded and the carboxylation rate of the sample was calculated by formula (1).
Wherein: v (V) 1 、V 2 、V 3 And V 4 The metering points of the titration curves of the chitosan oligosaccharide solution and the sample solution represent the consumption volume of NaOH and mL; c is the concentration of NaOH solution and mol/L; m is the mass of the sample, g;45 is the relative molecular mass of the carboxyl group; 0.257 is the theoretical ratio of carboxyl groups after all the hydroxymethyl groups at the C6 position are converted into carboxyl groups.
FIG. 3 is an infrared spectrum of the obtained 6-carboxychitosan oligosaccharide (C-COS) and Chitosan Oligosaccharide (COS). The comparison shows that: the peak positions of the two are approximately the same and are 2 880cm -1 And 2930cm -1 The stretching vibration peak of C-H mainly represents methylene at C6 position and methyl on acetamido connected with C2 position; 1600cm of C-COS -1 And 1410cm -1 A new and obvious asymmetric C=O stretching vibration absorption peak appears, which indicates that carboxyl is newly generated after the chitosan oligosaccharide is oxidized.
(2) 200g of 10% 6-carboxyl chitosan oligosaccharide solution is taken, 28g of copper acetate hydrate is added, and the mixture is reacted for 2 hours at 40 ℃ under stirring, so as to obtain the 6-carboxyl chitosan oligosaccharide-copper complex solution.
Taking 50mL of the prepared 6-carboxyl chitosan oligosaccharide-copper complex solution, adding 200mL of absolute ethyl alcohol to precipitate 6-carboxyl chitosan oligosaccharide-copper, filtering, flushing a filter cake 2 times with 50mL of 80% ethyl alcohol (V/V), and respectively collecting filtrate and filter cake; detecting the content and total amount of copper ions in the filtrate, and calculating the complexation rate to be 87.9%; drying the filter cake at 50 ℃ and crushing the filter cake (the water content is less than or equal to 5%) to obtain 6-carboxyl chitosan oligosaccharide-copper powder, wherein the copper ion content in the sample is 27.1%.
Example 3
The preparation method is the same as in example 2, except that parameters such as the addition amount of TEMPO, the copper-containing compound and the reaction conditions are different, and the specific steps are as follows:
(1) 200g of the 10% chitosan oligosaccharide solution prepared in example 1 (pH value is adjusted to 5.0) was taken, 1.6g of TEMPO (2, 6-tetramethylpiperidine-1-oxyl) was added, and the mixture was placed in a water bath kettle at 35 ℃ to be stirred and reacted for 15min; adding 0.8mL laccase (enzyme activity 105U/mL) to perform oxygen introducing reaction for 6 hours; and after oxygen introduction, regulating the reaction solution to be neutral by using liquid alkali, adding 4g of diatomite, uniformly dispersing, and carrying out vacuum suction filtration to obtain a 10% 6-carboxyl chitosan oligosaccharide solution. Concentrating the reaction solution to 100mL by rotary evaporation, transferring to a beaker, adding 400mL of absolute ethyl alcohol, and standing for 30min; after the reaction product is separated out, carrying out vacuum suction filtration, repeatedly washing the filtered product with alcohol until the alcohol washing liquid is clear; and (5) placing the filter cake in a vacuum drying oven to be dried for 24 hours at 50 ℃, and grinding the dried filter cake to obtain sample powder. The carboxylation rate in this sample was determined to be 86.8% -92.2%, indicating that most of the hydroxyl group at the C6 position was oxidized to carboxyl group.
Carboxylation rate determination in 6-carboxychitosan oligosaccharide powder: 0.2g of the sample was weighed and dissolved in 10mL of 0.1mol/L HCl solution for acidification, and after dissolution, 90mL of water was added for dilution. The automatic potentiometric titrator was turned on and titration was performed using 0.1mol/L NaOH standard solution. The consumption and conductivity of NaOH was recorded and the carboxylation rate of the sample was calculated by formula (1).
Wherein: v (V) 1 、V 2 、V 3 And V 4 The metering points of the titration curves of the chitosan oligosaccharide solution and the sample solution represent the consumption volume of NaOH and mL; c is the concentration of NaOH solution and mol/L; m is the mass of the sample, g;45 is the relative molecular mass of the carboxyl group; 0.257 is the theoretical ratio of carboxyl groups after all the hydroxymethyl groups at the C6 position are converted into carboxyl groups.
(2) 200g of 10% 6-carboxyl chitosan oligosaccharide solution is taken, 35g of copper sulfate pentahydrate is added, and the mixture is reacted for 4 hours at 50 ℃ under stirring, so as to obtain the 6-carboxyl chitosan oligosaccharide-copper complex solution.
Taking 50mL of the prepared 6-carboxyl chitosan oligosaccharide-copper complex solution, adding 200mL of absolute ethyl alcohol to precipitate 6-carboxyl chitosan oligosaccharide-copper, filtering, flushing a filter cake 2 times with 50mL of 80% ethyl alcohol (V/V), and respectively collecting filtrate and filter cake; detecting the content and total amount of copper ions in the filtrate, and calculating the complexation rate to be 87.2%; drying the filter cake at 50 ℃ and crushing the filter cake (the water content is less than or equal to 5%) to obtain 6-carboxyl chitosan oligosaccharide-copper powder, wherein the copper ion content in the sample is 26.1%.
Example 4
Preparation of 6-carboxyl chitosan oligosaccharide-copper wettable powder
(1) 50 parts of 6-carboxyl chitosan oligosaccharide-copper powder prepared in example 2,6 parts of glucose, 5 parts of starch, 0.03 part of carboxymethyl cellulose, 1004 parts of wetting agent, 1 part of dispersant NNO and 30 parts of kaolin are taken and uniformly mixed.
(2) And (3) crushing the uniformly mixed material in the step (1) to more than 150 meshes in an airflow crusher to obtain the 6-carboxyl chitosan oligosaccharide-copper wettable powder (the moisture of the wettable powder is below 5wt percent, and the pH value is between 6.5 and 7.5).
Comparative example 1
200g of the 10% chitosan oligosaccharide solution prepared in example 1 was taken, and copper acetate monohydrate was gradually added with stirring until the addition was stopped after precipitation, and the total addition amount of copper acetate monohydrate was 13.3g. Reacting for 2 hours at 40 ℃ under stirring to obtain the chitosan oligosaccharide-copper complex solution. Taking 50mL of the reaction solution, adding 200mL of absolute ethyl alcohol to precipitate chitosan oligosaccharide-copper, carrying out vacuum suction filtration, flushing a filter cake with 50mL of 80% ethyl alcohol (V/V) for 2 times, and respectively collecting filtrate and filter cake; detecting the content and total amount of copper ions in the filtrate, and calculating the complexing rate of the copper ions to be 82.3%; drying the filter cake at 50 ℃ and crushing the filter cake (the water content is less than or equal to 5%) to obtain the chitosan oligosaccharide-copper powder, wherein the copper ion content of the powder is 12.9%.
As is clear from comparison with example 2, the method provided by the invention can improve the complexing capacity of the sugar chain to copper.
Performance test 1
MIC value determination for crop pathogenic bacteria
The chitosan oligosaccharide powder prepared in example 1, the 6-carboxyl chitosan oligosaccharide prepared in example 2, the 6-carboxyl chitosan oligosaccharide-copper powder, the 6-carboxyl chitosan oligosaccharide-copper wettable powder prepared in example 4, the chitosan oligosaccharide-copper powder prepared in comparative example 1 and the commercially available wettable powder were respectively used for measurement, and specific types and basic compositions are shown in table 1:
TABLE 1 types and basic compositions of agents to be tested
The MIC of the agents of Table 1 above for Pseudomonas syringae (isolated from cucumber) and Lawsonia solanacearum (isolated from ginger), citrus canker pathogens were tested using a micro broth dilution method.
The method comprises the following specific steps:
A. preparation of antibacterial agent: each of the reagents was prepared according to Table 1, and the reagents were diluted appropriately to a concentration of 8192. Mu.g/mL, and the diluted solution was used as a stock solution. The prepared stock solution should be stored in-20deg.C environment with shelf life of no more than 6 months.
B. Preparation of bacteria to be tested: (1) bacterial activation: 50mL of LB seed medium was added to a 250mL Erlenmeyer flask, inoculated with a seed of bacterial slant at 4℃under environmental conditions, 160r/min, and cultured overnight at 37 ℃. (2)And (3) determining the bacterial load: 1mL of the activated bacterial liquid is added into 9mL of sterile water, 6 gradients are diluted in sequence, 100 mu L of the diluted bacterial liquid is evenly coated on a flat plate of a solid LB culture medium, and the flat plate is cultivated overnight at 37 ℃ for counting. If the number of bacteria in the culture solution is 1×10 8 CFU/mL, diluting with LB medium 100 times to obtain about 1×10 bacteria 6 CFU/mL bacterial liquid is reserved.
C. 200. Mu.L of bactericide is added to the first well, 100. Mu.L of broth medium is added to the second to tenth wells, 100. Mu.L of broth medium is added to the second well from the first well, the mixture is homogenized, 100. Mu.L to third well is again aspirated, and the tenth well is aspirated for 100. Mu.L discarding. 200. Mu.L of the bacterial liquid was added to the eleventh well, and 200. Mu.L of the broth medium was added to the twelfth well.
D. Then 100 mu L of prepared bacterial liquid is added to each of 1 to 10 holes, and the final bacterial liquid concentration of each tube is about 5 multiplied by 10 5 CFU/mL. And (3) placing the inoculated 96-well plate in a 37 ℃ incubator for culturing, and observing the growth condition of the bacterial liquid for 24 hours.
The concentration of the bactericide contained in the bacteria-free growth hole (the total amount of the copper complex is calculated as the basis) is the MIC value of the medicament for the bacteria. MIC values of each drug against the bacteria are shown in table 2.
TABLE 2 MIC values (μg/mL) of each agent for three pathogenic bacteria
| Numbering device | Pseudomonas syringae | Laurella solanacearum | Pathogenic bacteria for citrus ulcer |
| 1 | 1024 | 2048 | 4096 |
| 2 | 512 | 512 | 2048 |
| 3 | 1024 | 2048 | 4096 |
| 4 | 256 | 256 | 2048 |
| 5 | 128 | 256 | 1024 |
| 6 | 128 | 256 | 1024 |
As can be seen from table 2:
(1) The chitosan oligosaccharide and the 6-carboxyl chitosan oligosaccharide have weaker inhibition capability to three bacteria, and the MIC value is 4-8 times of that of copper succinate.
(2) The inhibition capability of the 6-carboxyl chitosan-copper on three bacteria is superior to that of the chitosan-copper, which is related to the higher copper content in the former aspect; on the other hand, it may be related to the microstructure of the complex.
(3) The inhibition capability of the 6-carboxyl chitosan oligosaccharide-copper on three bacteria is equivalent to that of the commercial 30% copper succinate.
(4) The copper content in the 6-carboxyl chitosan oligosaccharide-copper wettable powder prepared in example 4 is lower than that in the 6-carboxyl chitosan oligosaccharide-copper obtained in example 3, but the antibacterial effect is better than that of example 3, probably because the 6-carboxyl chitosan oligosaccharide-copper wettable powder is prepared into a preparation, and the auxiliary agent has a synergistic effect.
Performance test 2
Inhibition effect on crop pathogenic fungi
The specific test agents and test results are shown in table 3.
(1) Activation of fungi: fungal seeds stored on a 4℃slope were picked, transferred to a cooled and solidified PDA plate, and cultured in a 28℃incubator for 6 days.
(2) The solid samples listed in Table 1 were sterilized by irradiation with an ultraviolet lamp for 30min.
(3) PDA solid culture mediums with different concentration samples are respectively prepared, and the PDA solid culture mediums are respectively prepared into plates with different concentration gradients.
(4) Bacterial cakes with the diameter of 6mm are taken from the colony edges of each bacterial strain cultured on the PDA for 6 days, the mycelium faces downwards, the bacterial cakes are inoculated in the center of a solidified PDA culture medium, the bacterial cakes are placed in a constant temperature incubator with the temperature of 28 ℃ for 5 days, the colony diameters are measured by a crisscross method, and each treatment is repeated for 3 times. PDA medium without any sample was used as a blank control and boscalid as a positive control.
(5) Calculation of inhibition rate: inhibition ratio = (control medium colony diameter-treatment medium colony diameter)/control medium colony diameter x 100%
TABLE 3 required concentration of each agent for 100% inhibition of pathogenic fungi (%)
From the results in Table 3, it can be seen that the inhibition effect of 6-carboxychitosan-copper on four fungi is significantly better than that of chitosan-copper.
Performance test 3
Test of control effect on cucumber angular leaf spot
The test medications and the amounts of each are shown in table 4.
Table 4 test agents
The control effect of the agents in table 4 on greenhouse cucumber angular leaf spot was tested, and the planted cucumber was managed according to conventional production.
The method comprises the following specific steps: the backpack spray was used, the bactericide sample liquid of table 4 was used to spray the crops in each block, and the blank group was sprayed with the same amount of clear water. The disease conditions are investigated 1 day before and 7 days after the pesticide is applied, and the specific test method is carried out according to pesticide field efficacy test criterion. The control effect was calculated according to the formula, and the specific results are shown in table 5.
Cucumber angular leaf spot disease is recorded in 6 grades according to the leaf area ratio of the disease spots:
level 0: no disease spots;
stage 1: the area of the disease spots accounts for less than 1% -20% of the whole leaf area;
2 stages: the area of the lesion accounts for 21% -40% of the whole leaf area;
3 stages: the area of the lesion accounts for 41% -60% of the whole leaf area;
4 stages: the area of the lesion accounts for 61% -80% of the whole leaf area;
5 stages: the area of the lesion accounts for 81% -100% of the whole leaf area.
And calculating the disease index and the prevention and treatment effect of each treatment according to the investigation data.
TABLE 5 control of cucumber angular leaf spot by treatments
As can be seen from Table 5, the control effect of chitosan oligosaccharide-copper on cucumber angular leaf spot is low and is not more than 60%; the 6-carboxyl chitosan oligosaccharide-copper and 30% copper succinate fertilizer have equivalent control effects when the dosage is respectively 10 g/mu and 200 g/mu, and the control effects are all over 70%; the 6-carboxyl chitosan oligosaccharide-copper has higher control effect under the condition of lower dosage, and can be related to the synergistic effect of the 6-carboxyl chitosan oligosaccharide on the immune activation of plant cells besides higher copper content.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. The preparation method of the 6-carboxyl chitosan oligosaccharide-copper complex is characterized by comprising the following steps:
adjusting the pH value of the chitosan oligosaccharide solution to 4.0-6.0 to obtain the chitosan oligosaccharide solution with the pH value adjusted;
mixing the chitosan oligosaccharide solution with the pH value regulated, 2, 6-tetramethylpiperidine-1-oxyl and laccase, and introducing oxygen at 20-40 ℃ for reacting for 2-10 hours to obtain a 6-carboxyl chitosan oligosaccharide solution;
the addition amount of the 2, 6-tetramethyl piperidine-1-oxyl is 3% -6% of that of chitosan oligosaccharide;
mixing the 6-carboxyl chitosan oligosaccharide solution with a copper-containing compound, and reacting at 44-55 ℃ for 2-4 hours to obtain a 6-carboxyl chitosan oligosaccharide solution-copper complex;
the copper-containing compound is copper sulfate pentahydrate or copper acetate monohydrate or copper chloride dihydrate;
the mass ratio of the 6-carboxyl chitosan oligosaccharide to the copper-containing compound in the 6-carboxyl chitosan oligosaccharide solution is 1: 1-5.
2. The preparation method according to claim 1, wherein the chitosan oligosaccharide solution is prepared by the following steps:
mixing chitosan with deacetylation degree more than or equal to 85%, water and organic acid/hydrochloric acid, and swelling to obtain swelled chitosan;
and hydrolyzing the swelled chitosan by adopting chitosan enzyme to obtain a chitosan oligosaccharide solution.
3. The preparation method according to claim 2, wherein the mass ratio of chitosan to water is 1: 5-20 parts; the dosage of the organic acid/hydrochloric acid is 5% -50% of the mass of the chitosan.
4. The preparation method according to claim 2, wherein the temperature at the time of swelling is 30-65 ℃; the time is 4-24 hours.
5. The preparation method of claim 2, wherein the chitosan enzyme is used in an amount of 2-16U/g chitosan.
6. The use of the 6-carboxyl chitosan oligosaccharide-copper complex prepared by the method according to any one of claims 1-5 as a bactericide.
7. The use according to claim 6, wherein the fungicide is for controlling plant diseases caused by pseudomonas syringae, lauteromyces subtilis, verticillium dahliae, botrytis cinerea, citrus ulcer pathogenic bacteria, apple rot pathogenic bacteria or gibberella wheat.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103947671A (en) * | 2014-04-28 | 2014-07-30 | 牛赡光 | Chitosan oligosaccharide copper complex fungicide and application of chitosan oligosaccharide copper complex fungicide |
| CN105418799A (en) * | 2015-12-18 | 2016-03-23 | 黄河三角洲京博化工研究院有限公司 | Chitosan oligosaccharide-iodine complex as well as preparation method and application thereof |
| CN108623706A (en) * | 2017-03-21 | 2018-10-09 | 广西大学 | Microwave and complexing synergistic oxidation prepare the copper complex formazan method of chitosan oligosaccharide- |
| CN109354633A (en) * | 2018-11-14 | 2019-02-19 | 黄河三角洲京博化工研究院有限公司 | A kind of chitosan oligosaccharide chelates of zinc and its preparation method and application |
-
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- 2021-11-25 CN CN202111416689.2A patent/CN113969298B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103947671A (en) * | 2014-04-28 | 2014-07-30 | 牛赡光 | Chitosan oligosaccharide copper complex fungicide and application of chitosan oligosaccharide copper complex fungicide |
| CN105418799A (en) * | 2015-12-18 | 2016-03-23 | 黄河三角洲京博化工研究院有限公司 | Chitosan oligosaccharide-iodine complex as well as preparation method and application thereof |
| CN108623706A (en) * | 2017-03-21 | 2018-10-09 | 广西大学 | Microwave and complexing synergistic oxidation prepare the copper complex formazan method of chitosan oligosaccharide- |
| CN109354633A (en) * | 2018-11-14 | 2019-02-19 | 黄河三角洲京博化工研究院有限公司 | A kind of chitosan oligosaccharide chelates of zinc and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 6-羧基壳寡糖清除活性氧的能力及作用机理;杨晓莉等;天津科技大学学报;第36卷(第4期);73-80 * |
| 羧基化壳寡糖络合碘(CCOS-I)对水稻纹枯病菌的抑制作用及机理初讨;胡雪芳等;热带作物学报;第39卷(第8期);1590-1595 * |
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