CN112795559A - 一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 - Google Patents
一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 Download PDFInfo
- Publication number
- CN112795559A CN112795559A CN202110324796.6A CN202110324796A CN112795559A CN 112795559 A CN112795559 A CN 112795559A CN 202110324796 A CN202110324796 A CN 202110324796A CN 112795559 A CN112795559 A CN 112795559A
- Authority
- CN
- China
- Prior art keywords
- nscs
- nanospheres
- god
- glucose oxidase
- colletotrichum gloeosporioides
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002077 nanosphere Substances 0.000 title claims abstract description 54
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 15
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 15
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 14
- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 14
- 229920001661 Chitosan Polymers 0.000 claims abstract description 19
- 241001529387 Colletotrichum gloeosporioides Species 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000005764 inhibitory process Effects 0.000 claims abstract description 8
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 6
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000001965 potato dextrose agar Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000007987 MES buffer Substances 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 235000012055 fruits and vegetables Nutrition 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 8
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 230000004580 weight loss Effects 0.000 abstract description 5
- 239000003899 bactericide agent Substances 0.000 abstract description 4
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
- 230000005540 biological transmission Effects 0.000 abstract description 2
- 238000004587 chromatography analysis Methods 0.000 abstract 2
- 230000001939 inductive effect Effects 0.000 abstract 1
- 229940079593 drug Drugs 0.000 description 7
- 238000002329 infrared spectrum Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000427940 Fusarium solani Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000002070 germicidal effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000035322 succinylation Effects 0.000 description 2
- 238000010613 succinylation reaction Methods 0.000 description 2
- KJPRLNWUNMBNBZ-QPJJXVBHSA-N (E)-cinnamaldehyde Chemical compound O=C\C=C\C1=CC=CC=C1 KJPRLNWUNMBNBZ-QPJJXVBHSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 241001133184 Colletotrichum agaves Species 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 241000881860 Paenibacillus mucilaginosus Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 244000294611 Punica granatum Species 0.000 description 1
- 235000014360 Punica granatum Nutrition 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 239000007633 bacillus mucilaginosus Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000004112 carboxyamino group Chemical group [H]OC(=O)N([H])[*] 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940117916 cinnamic aldehyde Drugs 0.000 description 1
- KJPRLNWUNMBNBZ-UHFFFAOYSA-N cinnamic aldehyde Natural products O=CC=CC1=CC=CC=C1 KJPRLNWUNMBNBZ-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 235000021022 fresh fruits Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Images
Classifications
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用。首先，向壳聚糖中加入琥珀酸酐，制备N‑琥珀酰壳聚糖纳米球。通过透射电镜、红外色谱、XRD、热失重等结果显示NSCS纳米球是大小均一、外形规则的纳米球，并且结构中酰胺键特征峰的出现，说明了NSCS纳米球的水溶性和稳定性增加。其次，通过EDC和NHS活化NSCS的‑COOH基团,与葡萄糖氧化酶分子结构上的‑NH2发生共价结合制备GOD‑NSCS纳米球，红外色谱、热失重等结果均表明GOD成功固载到NSCS的表面。最后，GOD‑NSCS纳米球抑菌试验结果显示，GOD‑NSCS能够有效抑制诱发炭疽病主要病菌之一胶孢炭疽菌菌丝的生长，当药物浓度为256ug/mL时，菌丝抑制率超过50％。为开发新型天然杀菌剂在抑制果蔬炭疽病方面的实际应用提供参考。
Description
技术领域
本发明设计一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用，特别设计一种改性壳聚糖纳米球固载葡萄糖氧化酶在抑制胶孢炭疽菌方面的应用
背景技术
炭疽病菌是引起真菌性炭疽病的最常见的植物病原体之一。这种真菌能够通过多种方式侵染新鲜果蔬，极大地限制了果蔬的贮藏期和上市时间。其中，胶孢炭疽菌是引起许多热带水果炭疽病的主要菌种之一，其中芒果，香蕉、石榴、木瓜等每年都因炭疽病的发生造成重大的经济损失。市面上，控制炭疽病最常见的杀菌剂是化学杀菌剂。随着消费者对减少水果中化学杀菌剂残留的迫切需求和炭疽病耐药性的日益增强，亟需研发新型、绿色安全的天然杀菌剂。
近年来，纳米技术在采后果蔬保鲜上受到关注。一方面，在果蔬包装材料上使用纳米颗粒(如纳米TiO2、纳米ZnO、纳米CuO等)可以间接避免果蔬在贮运过程中受到病菌污染；另一方面，在果蔬表面直接喷涂纳米化的抗菌剂(如纳米化肉桂醛、纳米化葡聚糖)可以直接抵抗果蔬的腐败。纳米球的直径通常在50-500nm之间，呈现固态或胶态，将溶解的药物包裹于纳米球的内部或吸附在纳米球的表面，可制成纳米载体药物。用纳米球作载体，具有药物包封率高、稳定性强、释药时间长、体内特异性分布等特点。
壳聚糖因其具有无毒、良好的生物相容性、生物可降解性及生物黏附性的特点适宜做纳米球载体，但其水溶性差，在果蔬保鲜上受到了一定的限制。为了提高壳聚糖的溶解性，可在其骨架上引入亲水基团，如羧甲基，烷基，琥珀酰基等。其中，N-琥珀酰壳聚糖(NSCS)是通过琥珀酸酐与壳聚糖反应制备的，其结构稳定并带有活性基团，具有良好的溶解性和生物相容性，已被广泛研究用于药物输送、伤口敷料、生物医学材料和食品领域。依据以上特点，将NSCS作为载体应用到果蔬保鲜当中，具有重要意义。
葡萄糖氧化酶(GOD；EC 1.1.3.4)是一种非水解酶；主要提取自真菌曲霉属和青霉属。GOD利用分子氧作为电子受体，催化β-D-葡萄糖氧化为葡萄糖酸和H2O2。GOD非常便宜，它已经广泛应用于工业领域，包括非食品和食品领域。根据美国食品和药物管理局的分类，GOD通常被认为是安全的(GRAS)。据报道，GOD能够抑制茄病镰刀菌的生长和孢子的产生，并且它可以作为针对番茄植株的茄病镰刀菌病害的生物控制措施。GOD-葡萄糖系统能够用到鸡蛋的保存，以抑制腐败菌群的生长，如肠炎沙门氏菌、蜡样芽孢杆菌和荧光假单胞菌。也有报道称，GOD-葡萄糖系统能够有效抑制草莓灰霉病致病菌灰葡萄孢菌的菌丝生长。
NSCS的-COOH基团通过EDC和NHS活化，可与GOD分子结构上的-NH2发生共价结合，可制备一种新型的NSCS固载GOD(GOD-NSCS)的杀菌剂。通过菌丝抑制试验，发现其对胶孢炭疽菌表现出良好的抑菌性。
发明内容
本发明是为提供一种新型NSCS固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用。将葡萄糖氧化酶与溶解范围广，稳定性强的NSCS共价连接，制备GOD-NSCS纳米球。将GOD-NSCS纳米球用于控制胶孢炭疽菌，发现具有良好的抑菌效果。
本发明的技术方案由以下部分组成：
(1)NSCS纳米球的制备：壳聚糖粉末溶于冰乙酸溶液中，35℃条件下搅拌均匀。在室温条件下慢速滴加丁二酸酐的丙酮溶液。然后，在50℃水浴锅中匀速搅拌5h后放置至室温，用0.1mM/L NaOH滴加至PH＝10后，加入过量丙酮沉淀20min后，抽滤条件下，分别用丙酮和乙醇清洗沉淀，50℃烘箱干燥12h。
(2)GOD-NSCS纳米球的制备：NSCS粉末溶于2-(N-吗啉代)乙磺酸缓冲液(MES，PH＝6)，待搅拌完全溶解之后，分别加入5、10、20mg EDC和NHS(EDC:NHS＝1；1)，匀速搅拌1h，充分活化羧基。再向上述溶液中分别添加5、10、20mg GOD，冰水浴匀速搅拌5h后，将固载溶液于冷冻干燥机内冷冻干燥12h后得到粉末。
(3)GOD-NSCS纳米球对胶孢炭疽菌的抑制：将GOD-NSCS纳米球溶于水中，用65℃以下的马铃薯葡萄糖琼脂培养基(PDA)将纳米球分别稀释系列浓度，待PDA培养基凝固之后，取培养皿边缘培养好的胶孢炭疽菌菌盘(6mm)，于28℃恒温培养箱培养至空白对照组长满培养皿，对菌盘进行拍照。
本发明制得的NSCS纳米球水溶性好，并能与GOD共价结合。所得的GOD-NSCS纳米球固载酶的含量高，对胶孢炭疽菌也具有良好抑制效果。
附图说明
图1为本发明制得的NSCS纳米球电镜透射照片；
图2为本发明制得的NSCS纳米球的傅里叶红外光谱图；
图3为本发明制得的NSCS纳米球的X-衍射图；
图4为本发明制得的GOD-NSCS纳米球的的傅里叶红外光谱图；
图5为本发明制得的NSCS纳米球和GOD-NSCS纳米球固载酶的热失重图；
图6为本发明制得的GOD-NSCS纳米球对胶孢炭疽菌的抑制效果照片；
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
称取1g壳聚糖粉末溶于200mL(1％w/t)的冰乙酸溶液中，35℃条件下搅拌均匀。慢速滴加含有0.2g丁二酸酐的丙酮溶液20mL。然后，在50℃水浴锅中匀速搅拌5h后放置至室温，用0.1mM/L NaOH滴加至PH＝10后，加入过量丙酮沉淀20min后，抽滤条件下，分别用丙酮和乙醇清洗沉淀，50℃烘箱干燥12h。根据图1可以看出，壳聚糖经过酰化之后形成的NSCS纳米球，大小均一，表面没有明显粗糙，纳米球平均直径为102.73nm；根据图2可以看出，和壳聚糖傅里叶红外光谱图比较，在1656cm-1处，由于C＝O的伸缩振动产生酰胺I带振动峰；并且，NSCS光谱图显示在1567cm-1处N-H弯曲导致的峰偏移出现酰胺II振动峰。根据图3可以看出2θ＝11°和2θ＝20°是壳聚糖特征衍射峰，而经过琥珀酰化得到的NSCS，2θ＝11°处的峰消失，2θ＝20°处的峰变弱。这是因为壳聚糖中的氨基经过琥珀酰化转变为-NH-CO-,破坏了壳聚糖分子中的氢键，结晶性降低，水溶性增加。
实施例2
称取0.1g NSCS粉末溶于50mLMES缓冲液(PH＝6)，待完全溶解之后，添加分别加入5、10、20mg EDC和NHS(EDC:NHS＝1；1)，匀速搅拌1h，充分活化羧基。向上述溶液中分别添加5mg GOD，冰水浴匀速搅拌5h后，将固载溶液置于冷冻干燥机内冷冻干燥12h后得到粉末。根据图4可以看出和GOD的红外光谱图比较，GOD在1500-1530cm-1之间的氨基振动峰，在GOD-NSCS的红外图谱中位置发生偏移，出现在1546cm-1处，表明酰胺键的形成，说明NSCS经EDC和NHS活化之后的羧基和GOD的氨基发生了羧氨反应。并且，GOD-NSCS形成了大量的-CH3。以上均表明GOD成功固载到NSCS上。根据图5我们可以看出壳聚糖发生失重的最大速率在303.24℃，NSCS在温度为262.3℃，而GOD-NSCS则是在312.24℃时失重速率最大，说明GOD-NSCS的稳定性更高。
实施例3
GOD-NSCS纳米球对胶孢炭疽菌的抑制：将纳米球溶于水中，用65℃以下的马铃薯葡萄糖琼脂培养基(PDA)将其分别稀释系列浓度(4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL、128ug/mL、256ug/mL)，待PDA培养基凝固之后，取培养皿边缘培养好的胶孢炭疽菌菌盘(6mm)，于28℃恒温培养箱培养至空白对照组长满培养皿，对菌盘进行拍照。从图6我们可以看出，在去除游离酶之后的纳米球依旧能够很好的抑制胶孢炭疽菌的生长，当EDC和NHS添加量为5mg，GOD添加量为5和10mg，固载酶药物浓度为128ug/mL时，菌丝抑制率将近50％。当GOD-NSCS纳米球浓度为256ug/mL时，菌丝抑制率超过50％。由此可见，GOD-NSCS纳米球能够较好地抑制胶孢炭疽菌的菌丝生长。
Claims (4)
1.一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用，其特征是，包括步骤如下：
(1)N-琥珀酰壳聚糖(NSCS)纳米球的制备：壳聚糖粉末溶于冰乙酸溶液中，35℃条件下搅拌均匀；在室温条件下慢速滴加丁二酸酐的丙酮溶液；然后，在50℃水浴锅中匀速搅拌5h后放置至室温，用0.1mM/L NaOH滴加至PH＝10后，加入过量丙酮沉淀20min后，抽滤条件下，分别用丙酮和乙醇清洗沉淀，50℃烘箱干燥12h；
(2)N-琥珀酰壳聚糖固载葡萄糖氧化酶(GOD-NSCS)纳米球的制备：NSCS粉末溶于MES缓冲液(PH＝6)，待搅拌完全溶解之后，分别加入5、10、20mg EDC和NHS，匀速搅拌1h，充分活化羧基；向上述溶液中分别添加5、10、20mg葡萄糖氧化酶(GOD)，冰水浴匀速搅拌5h后，将固载溶液于冷冻干燥机内冷冻干燥12h后得到粉末；
(3)GOD-NSCS纳米球对胶孢炭疽菌的抑制：将纳米球溶于水中，用65℃以下的马铃薯葡萄糖琼脂培养基(PDA)将纳米球分别稀释系列浓度，待PDA培养基凝固之后，取培养皿边缘培养好的胶孢炭疽菌菌盘(6mm)，于28℃恒温培养箱培养至空白对照组长满培养皿，对菌盘进行拍照。
2.根据权利要求1所述的一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用，其特征是：所述的(1)中所制备的纳米球为NSCS，壳聚糖的称取量为1g，丁二酸酐的添加量为0.2g，丙酮溶液为20mL。
3.根据权利要求1所述的一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用，其特征是：所述的(2)中所制备的纳米球为GOD-NSCS，NSCS的添加量为0.1g，EDC:NHS为1；1。
4.根据权利要求1所述的一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用，其特征是：所述的(3)中所述得纳米球的浓度分别为4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL、128ug/mL、256ug/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110324796.6A CN112795559A (zh) | 2021-03-26 | 2021-03-26 | 一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110324796.6A CN112795559A (zh) | 2021-03-26 | 2021-03-26 | 一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112795559A true CN112795559A (zh) | 2021-05-14 |
Family
ID=75815740
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110324796.6A Pending CN112795559A (zh) | 2021-03-26 | 2021-03-26 | 一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112795559A (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1599557A (zh) * | 2001-12-04 | 2005-03-23 | 诺和酶股份有限公司 | 杀死孢子的方法 |
| CN105451573A (zh) * | 2013-08-06 | 2016-03-30 | 比恩卡有限公司 | 抗微生物组合物以及其在食品保存中的用途 |
| CN106519271A (zh) * | 2016-11-29 | 2017-03-22 | 吉林大学 | 一种n‑琥珀酰壳聚糖固载溶菌酶制备抑菌膜的方法 |
-
2021
- 2021-03-26 CN CN202110324796.6A patent/CN112795559A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1599557A (zh) * | 2001-12-04 | 2005-03-23 | 诺和酶股份有限公司 | 杀死孢子的方法 |
| CN105451573A (zh) * | 2013-08-06 | 2016-03-30 | 比恩卡有限公司 | 抗微生物组合物以及其在食品保存中的用途 |
| RU2665094C2 (ru) * | 2013-08-06 | 2018-08-28 | Биенка Н.В. | Противомикробная композиция и ее применение для консервирования пищевых продуктов |
| CN106519271A (zh) * | 2016-11-29 | 2017-03-22 | 吉林大学 | 一种n‑琥珀酰壳聚糖固载溶菌酶制备抑菌膜的方法 |
Non-Patent Citations (6)
| Title |
|---|
| MOUNA KRIAA 等: "Biocontrol of tomato plant diseases caused by Fusarium solani using a new isolated Aspergillus tubingensis CTM 507 glucose oxidase" * |
| XIAODI NIU 等: "An antimicrobial agent prepared by N-succinyl chitosan immobilized lysozyme and its application in strawberry preservation" * |
| XIAOJING LI 等: "Glucose oxidase as a control agent against the fungal pathogen Botrytis cinerea in postharvest strawberry" * |
| YGOR R G ELOY 等: "H2O2 plays an important role in the lifestyle of Colletotrichum gloeosporioides during interaction with cowpea [Vigna unguiculata (L.) Walp]" * |
| ZHU AIPING 等: "Synthesis and characterization of N-succinyl-chitosan and its self-assembly of nanospheres" * |
| 周钦威: "改性壳聚糖微球固定化脂肪酶及其应用研究" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jeon et al. | Antimicrobial effect of chitooligosaccharides produced by bioreactor | |
| Chen et al. | Application of functionalized chitosan in food: A review | |
| Ziani et al. | Antifungal activity of films and solutions based on chitosan against typical seed fungi | |
| Elsabee et al. | Surface modification of polypropylene films by chitosan and chitosan/pectin multilayer | |
| Younes et al. | Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities | |
| CN106962498B (zh) | 乳酸链球菌素/壳聚糖纳米粒子抗菌膜及制备方法和用途 | |
| CN111286057B (zh) | 一种草莓保鲜壳聚糖基抑菌膜的制备方法 | |
| Mesgari et al. | A comprehensive review of the development of carbohydrate macromolecules and copper oxide nanocomposite films in food nanopackaging | |
| US20120108540A1 (en) | Preservatives from Chitin Derivatives | |
| Sharma et al. | Chitosan: Properties and roles in postharvest quality preservation of horticultural crops | |
| CN112314838A (zh) | 一种抑菌食品保鲜Pickering乳液及其制备方法 | |
| Iseppi et al. | Anti-listerial activity of coatings entrapping living bacteria | |
| De Azeredo et al. | Chitosan edible films and coatings-a review. | |
| CN110558376B (zh) | 一种壳聚糖-Zn复合物纳米粒子保鲜膜的制备方法和用途 | |
| Hashem et al. | Bio-based antimicrobial food packaging films based on hydroxypropyl starch/polyvinyl alcohol loaded with the biosynthesized zinc oxide nanoparticles | |
| Liu et al. | Preparation and properties of chitosan‐based bacteriostatic agents and their application in strawberry bacteriostatic preservation | |
| CN112795559A (zh) | 一种固载葡萄糖氧化酶纳米球在抗胶孢炭疽菌方面的应用 | |
| Yu et al. | Antimicrobial activity of gamma-poly (glutamic acid), a preservative coating for cherries | |
| EP1484982B1 (en) | Hydrolysed chitosan as antimicrobial coumpound and uses thereof | |
| Bosquez-Molina et al. | New bioactive biomaterials based on chitosan | |
| Bayazidi et al. | Immobilized lysozyme onto bacterial cellulose nanofibers as active and reinforcing agent of sodium caseinate based films: physical characteristics and antimicrobial activity | |
| EP3932202A2 (en) | Antimicrobial material, especially packaging material, method of obtaining the antimicrobial material and application thereof | |
| Aktuganov et al. | Chitosan resistance of Bacteria and Micromycetes differing in ability to produce extracellular Chitinases and Chitosanases | |
| Al-Soufi | Extending the storage life of some fruits by using pullulan produced from locally isolate Aureobasidium pullulans | |
| Badawy et al. | Bactericidal and fungicidal activities of different molecular weight chitosan samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210514 |
|
| WD01 | Invention patent application deemed withdrawn after publication |