CN109897788B - 一种芸薹链格孢及在制备抑菌剂中的应用 - Google Patents
一种芸薹链格孢及在制备抑菌剂中的应用 Download PDFInfo
- Publication number
- CN109897788B CN109897788B CN201910174849.3A CN201910174849A CN109897788B CN 109897788 B CN109897788 B CN 109897788B CN 201910174849 A CN201910174849 A CN 201910174849A CN 109897788 B CN109897788 B CN 109897788B
- Authority
- CN
- China
- Prior art keywords
- alternaria brassicae
- bacteriostatic agent
- fermentation
- concentrating
- brassicae
- 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.)
- Active
Links
- 241001149961 Alternaria brassicae Species 0.000 title claims abstract description 39
- 239000000022 bacteriostatic agent Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 239000002207 metabolite Substances 0.000 claims abstract description 17
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 14
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 239000000706 filtrate Substances 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 6
- 238000000967 suction filtration Methods 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 4
- 239000002024 ethyl acetate extract Substances 0.000 claims description 4
- 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 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 235000010344 sodium nitrate Nutrition 0.000 claims description 3
- 239000004317 sodium nitrate Substances 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 238000002525 ultrasonication Methods 0.000 claims description 2
- 229960004793 sucrose Drugs 0.000 claims 1
- 241000233866 Fungi Species 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 5
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 2
- 235000013399 edible fruits Nutrition 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 244000194101 Ginkgo biloba Species 0.000 description 18
- 235000008100 Ginkgo biloba Nutrition 0.000 description 17
- 235000011201 Ginkgo Nutrition 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 239000000203 mixture Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000003385 bacteriostatic effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229910021642 ultra pure water Inorganic materials 0.000 description 5
- 239000012498 ultrapure water Substances 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241000269913 Pseudopleuronectes americanus Species 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 235000011869 dried fruits Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003810 ethyl acetate extraction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- YXHVCZZLWZYHSA-UHFFFAOYSA-N (Z)-6-[8-pentadecenyl]salicylic acid Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-UHFFFAOYSA-N 0.000 description 1
- HGUFODBRKLSHSI-UHFFFAOYSA-N 2,3,7,8-tetrachloro-dibenzo-p-dioxin Chemical compound O1C2=CC(Cl)=C(Cl)C=C2OC2=C1C=C(Cl)C(Cl)=C2 HGUFODBRKLSHSI-UHFFFAOYSA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000218791 Ginkgoaceae Species 0.000 description 1
- YXHVCZZLWZYHSA-FPLPWBNLSA-N Ginkgoic acid Chemical compound CCCCCC\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-FPLPWBNLSA-N 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241001089723 Metaphycus omega Species 0.000 description 1
- 206010039424 Salivary hypersecretion Diseases 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000003749 cleanliness Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- QCCDLTOVEPVEJK-UHFFFAOYSA-N phenylacetone Chemical compound CC(=O)CC1=CC=CC=C1 QCCDLTOVEPVEJK-UHFFFAOYSA-N 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 208000026451 salivation Diseases 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- -1 terpene lactones Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种芸薹链格孢Gb.PY‑F2及在制备抑菌剂中的应用,所述抑菌剂是芸薹链格孢Gb.PY‑F2经发酵培养获得的发酵液超声破碎后抽滤,取滤液经微孔滤膜过滤后浓缩,再用乙酸乙酯萃取,浓缩至恒重,获得芸薹链格孢Gb.PY‑F2代谢产物,即为抑菌剂。本发明提供一株新菌株‑‑银杏果内生真菌Gb.PY‑F2,芸薹链格孢Gb.PY‑F2代谢产物对金黄色葡萄球菌(CMCC(B)26003)的MIC=1.5625mg/mL,通过微生物发酵得到具有抑菌活性的成分,产量大,工艺简单,更加安全,环保。
Description
(一)技术领域
本发明涉及一种金黄色葡萄球菌抑菌剂,具体涉及一种内生真菌芸薹链格孢Gb.PY-F2及在制备抗金黄色葡萄球菌药物的应用。
(二)背景技术
银杏是晚古生代的孑遗植物,具有“活化石”的美称,集食用、药用、绿化、观赏为一身。中国银杏种植面积位居世界首位,银杏叶产量约占全球总产量的70%。银杏种子甜而苦,与医药、食品同源,含有多种活性底物,如黄酮类、萜内酯、银杏酸、苯丙酮类、酚类等。因此,它具有促进唾液分泌、止渴祛痘、改善脑功能、增强记忆、治疗阿尔茨海默病、扩张微血管、促进血液循环、平滑血管、治疗脑供血等多种保健功能。银杏果一直被誉为高级滋补品,应用范围从食品领域扩展到医药、保健品、化妆品等领域。内生植物在各种植物中普遍存在。许多内生植物可以产生与宿主植物相同或相似的代谢产物,而且许多代谢产物是尚未发现的新物质。
(三)发明内容
本发明目的是提供一种芸薹链格孢Gb.PY-F2及在制备抑菌剂中的应用。
本发明采用的技术方案是:
本发明提供一种芸薹链格孢(Altemaria brassicae)Gb.PY-F2,保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M 219126,保藏日期为2019年3月6日,保藏地址为中国武汉武汉大学,邮编:430072。
本发明所述芸薹链格孢Gb.PY-F2,菌落灰黑色,铺散状,菌落背面为黑色。
本发明还提供一种所述芸薹链格孢Gb.PY-F2在制备抑菌剂中的应用,所述抑菌剂为金黄色葡萄球菌(Staphylococcus aureus)抑菌剂,优选金黄色葡萄球菌 CMCC(B)26003。
进一步,所述抑菌剂是芸薹链格孢Gb.PY-F2经发酵培养获得的发酵液超声破碎后抽滤,取滤液经微孔滤膜过滤后浓缩(优选浓缩至1/4体积),再用乙酸乙酯萃取 (优选萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相),有机相浓缩至恒重,获得芸薹链格孢Gb.PY-F2代谢产物,即为抑菌剂。
更进一步,所述抑菌剂按如下方法制备:将芸薹链格孢Gb.PY-F2接种于发酵培养基中,28℃,180r/min培养7d,获得发酵液;将发酵液超声破碎后抽滤,取滤液经微孔滤膜过滤后浓缩至原体积的1/4,用1倍体积乙酸乙酯萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,浓缩至恒重,获得芸薹链格孢Gb.PY-F2 粗提物,即为抑菌剂;所述发酵培养基组成:硝酸钠3g/L,磷酸氢二钾1g/L,硫酸镁(MgSO4·7H2O)0.5g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,蔗糖30g/L,溶剂为蒸馏水,pH7.0~7.2;利用高压蒸汽灭菌锅对其进行灭菌,条件为121℃,20分钟。
进一步,所述超声破碎条件为:在405W条件下,每工作3s,间歇4s,进行300 次循环。
进一步,所述芸薹链格孢Gb.PY-F2发酵前先活化培养,然后再接入发酵培养基,所述活化培养为:将芸薹链格孢Gb.PY-F2接种于PDA培养基中,置于28℃恒温培养箱中培养7d;所述PDA培养基组成:马铃薯200g/L,葡萄糖20g/L,琼脂15~20g/L,溶剂为蒸馏水,自然pH。
与现有技术相比,本发明有益效果主要体现在:本发明提供一株新菌株--芸薹链格孢Gb.PY-F2,通过微生物发酵得到具有抑菌活性的成分,芸薹链格孢Gb.PY-F2代谢产物对金黄色葡萄球菌(CMCC(B)26003)的MIC=1.5625mg/mL。传统抑菌剂为抗生素,国内抗生素滥用情况普遍,现已加大管控,天然的抑菌剂将是未来市场的趋势,该抑菌剂产量大,工艺简单,更加安全,环保。
(四)附图说明
图1为Gb.PY-F2菌株形态。
图2为Gb.PY-F2菌株***发育树。
图3为芸薹链格孢Gb.PY-F2发酵物乙酸乙酯萃取相的抗真菌活性抑菌圈照片; A为卡那霉素阳性对照;B为空白对照(DMSO);C样品浓度为0.750mg/mL;D 样品浓度为1.500mg/mL;E样品浓度为12.500mg/mL;F样品浓度为6.250mg/mL; G样品浓度为3.125mg/mL;H为空白对照(无菌超纯水)。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明所述银杏(学名:Ginkgo biloba L.),为银杏科、银杏属落叶乔木。银杏树的种子俗称白果,因此银杏又名白果树。
本发明所述超纯水是指电阻率达到18MΩ*cm(25℃)的水。水中除了水分子外,几乎没有什么杂质,更没有细菌、病毒、含氯二噁英等有机物,也没有人体所需的矿物质微量元素。
实施例1:芸薹链格孢Gb.PY-F2的典型分离
1.植物样本采集:新鲜健康的银杏果采于江苏省无锡市惠山大道,银杏果用自来水清洗10分钟,然后用体积浓度75%乙醇水溶液漂洗一次。用质量浓度2%次氯酸钠水溶液浸泡10分钟后,用无菌水反复洗涤种子,再用体积浓度75%乙醇水溶液浸泡10分钟,然后用无菌水漂洗三次,合并漂洗液,用干燥的无菌吸收纸吸去表面水分,获得表面消毒后的银杏果。在超净工作台中,放置PDA培养基的无菌平板作为空白对照1,该对照为检查超净工作台的洁净程度;将漂洗液接种于PDA培养基的无菌平板作为空白对照2,该对照为漂洗液的检查;将表面消毒后的银杏果,置于PDA 培养基的无菌平板中滚动后取出,作为空白对照3,该对照为植物组织印迹法筛选无菌的组织块。
2.内生真菌的筛选以及分离纯化:于无菌超净工作台,将步骤1中表面消毒后的银杏果从中间切成薄片,作为无菌组织,然后接种在PDA培养基中,在28℃倒置培养,待出现菌丝沿着组织切口向外生长时,与空白对照1、2以及3均进行比较,采用尖端菌丝挑选的方法,将不同形态的菌落划线接种于PDA培养基中,待长出单菌落后,将单菌落再次划线接种于无菌PDA培养基中,反复多次接种,直到菌落形态一致且只有一种真菌生长时说明纯化完成,得到1株菌株为灰黑色,铺散状,菌落背面为黑色的真菌,见于图1,将该菌株记为菌株Gb.PY-F2。
PDA培养基组成:马铃薯200g,葡萄糖20g,琼脂15~20g,蒸馏水1000mL,自然pH。
3.总DNA的提取:将菌株Gb.PY-F2接种于PDA培养基中,于28℃恒温培养箱中倒置培养7d。采用真菌基因组DNA快速抽提试剂盒(购自生工生物工程(上海) 股份有限公司,产品编号:B518229)以及相关操作说明提取基因组DNA:①取50-100 mg新鲜真菌或20mg干燥子实体或菌丝,液氮中充分研磨成粉末后放入到1.5mL离心管中,依次加入400μL BufferDigestion和4μlβ-巯基乙醇,震荡混匀。65℃水浴1 h至细胞完全裂解。②加入200μlBuffer PF,充分颠倒混匀,-20℃冰箱放置5min。③室温10000rpm离心5min,将上清液(500~550μl)转移到新的1.5ml离心管中。④加入等体积的异丙醇,颠倒5~8次使之充分混匀,室温放置2~3min。室温10000 rpm离心5min,弃上清。⑤加入1ml 75%乙醇,颠倒漂洗1~3min,10,000rpm离心2min,弃上清。⑥重复步骤⑤一次。⑦开盖室温倒置5~10min至残留的乙醇完全挥发。⑧得到的DNA用50-100μl TE Buffer溶解。提取的DNA可立即进行下一步实验或-20℃保存。
4.菌株Gb.PY-F2的ITS序列扩增:采用真菌扩增通用引物ITS1 (5’-TCCGTAGGTGAACCTGCGC-3’)和ITS4(5’-TCCTCCGCTTATTGATATGC-3’) 扩增内部转录区间序列,反应体系如下:
DNA模板1μL、上游引物1μL、下游引物1μL、PCRMix 12.5μL、ddH2O 9.5μL。
PCR扩增程序:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸1min, 35个循环,72℃延伸10min,4℃低温保存。
5.PCR反应产物确认:取5μLPCR产物与1μLDAN Green染料混合后点样于 1.2%琼脂糖凝胶,110V条件下电泳15分钟,于凝胶成像***中观察条带,如出现 500bp左右条带,则初步判断扩增成功。
6.PCR反应产物测序:将PCR产物送样生工生物工程(上海)股份有限公司进行测序,菌株Gb.PY-F2的ITS序列见SEQ ID NO.1所示。
7.数据分析:用Blast比对,将菌株Gb.PY-F2的序列与GenBank中的序列进行同源性比对,BLAST检索表明,菌株Gb.PY-F2的ITS序列与芸薹链格孢(Altemaria brassicae)(GenBank登录号KU204772)的序列相似度为99%,绘制***发育树,见图2所示。由图2可知支持率为100%,根据基因亲缘性对比,确定菌株Gb.PY-F2 为交链孢酶(Altemaria)属,命名为芸薹链格孢(Altemaria brassicae)Gb.PY-F2,该菌株保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M 219126,保藏日期为 2019年3月6日。
实施例2:芸薹链格孢Gb.PY-F2发酵液抑菌能力筛选
1、菌株复苏活化:将保存在4℃冰箱中的芸薹链格孢Gb.PY-F2接种于PDA培养基中,置于28℃恒温培养箱中培养7d;
2、芸薹链格孢Gb.PY-F2代谢产物的制备:在超净工作台中,用无菌打孔器将步骤1的Gb.PY-F2菌株沿着菌落边缘打直径为5mm的菌饼,接种于含200mL发酵培养基的500mL锥形瓶中,28℃,180r/min发酵培养7d,以未接种菌饼的发酵培养基为空白对照。取发酵完成的发酵液,采用Y92-IIDN型超声波细胞粉碎机在405W条件下,每工作3s,间歇4s,进行300次循环对发酵液进超声破壁处理,然后抽滤得滤液,经孔径为0.45μm的微孔滤膜过滤后,微滤液利用旋转蒸发仪将其浓缩至原体积的1/4,获得浓缩液。用1倍体积乙酸乙酯对浓缩液进行多次萃取,直至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,再次利用旋转蒸发仪将乙酸乙酯萃取相浓缩干燥至恒重,获得芸薹链格孢Gb.PY-F2代谢产物31.582g,于-20℃保存。
发酵培养基组成:硝酸钠3g,磷酸氢二钾1g,硫酸镁(MgSO4·7H2O)0.5g,氯化钾0.5g,硫酸亚铁0.01g,蔗糖30g,蒸馏水1000mL,pH7.0~7.2。
3、抑菌实验:在超净工作台中将金黄色葡萄球菌(CMCC(B)26003,南京茂捷微生物科技有限公司)接种于液体LB培养基中,180r/min,37℃培养1d,作为供试菌;称取步骤2获得的25mg芸薹链格孢Gb.PY-F2代谢产物,溶于1mL DMSO中,配置 25mg/mL的样品储备液。采用双层平板牛津杯法对抑菌活性进行测定:在超净工作台中,样品储备液经微孔滤膜(0.22μm)过滤后,用无菌超纯水稀释得到浓度为0.750 mg/mL、1.500mg/mL、12.500mg/mL、6.250mg/mL、3.125mg/mL的样品。在无菌条件下,将牛津杯放置在无菌平皿中后倒入无菌LB培养基;取另一瓶无菌LB培养基,待其温度冷却至45℃左右,以体积浓度1%(例:100mL培养基,接种量为1mL)的接种量将供试菌接种于其中,摇匀后,倒入已放置牛津杯且底层无菌LB培养基已经凝固的培养皿中,待其冷却凝固用镊子将牛津杯取出后,在孔中加入150μL不同浓度的样品,分别以DMSO和无菌超纯水为空白对照,以1.500mg/mL卡那霉素水溶液为阳性对照,将平皿置于37℃恒温培养箱中培养1d,观察芸薹链格孢Gb.PY-F2代谢产物对供试菌金黄色葡萄球菌CMCC(B)26003的抑制效果,各浓度样品及对照的透明抑菌圈的直径见表1和图3所示,当透明圈直径大于8mm则说明该菌株发酵液中含有抑菌活性成分。
LB培养基组成:胰蛋白胨10g,酵母提取物5g,NaCl 10g,琼脂15g,1000mL 去离子水,pH7.0。
LB液体培养基组成:胰蛋白胨10g,酵母提取物5g,NaCl 10g,1000mL离子水,pH7.0。
4、实验结果:芸薹链格孢Gb.PY-F2的抗真菌活性见图3和表1所示。
表1
实施例3:芸薹链格孢Gb.PY-F2发酵液对金黄色葡萄球菌的最低抑制浓度测定供试菌株的活化:将金黄色葡萄球菌CMCC(B)26003接种于无菌的牛肉膏蛋白胨培养基(牛肉膏3.0g/L、蛋白胨10g/L、氯化钠5.0g/L、琼脂20g/L,溶剂为蒸馏水, pH 7.4~7.6)中,置于37℃倒置培养1d;将活化后的供试菌接种于LB液体培养基中,置于37℃,180r/min的恒温摇床中发酵培养1d,取发酵液用无菌水稀释使得菌悬液浓度为106cfu/mL,作为供试菌液。
称取实施例2方法制备的芸薹链格孢Gb.PY-F2代谢产物50mg,溶于2mL DMSO 中,配置25mg/mL的储备液,在超净工作台中,储备液经微孔滤膜(0.22μm)过滤后,用无菌水半倍稀释得到不同浓度的样品(12.500,6.250,3.125,1.562,0.781mg/mL)。在无菌条件下,将牛津杯放置在无菌平皿中后将无菌LB培养基倒入平皿;取另一瓶无菌LB培养基,待其温度冷却至45℃左右,以体积浓度1%的接种量将供试菌液接种于其中,摇匀后,倒入已放置牛津杯且底层无菌LB培养基已经凝固的培养皿中,待其冷却凝固用镊子将牛津杯取出后,在孔中加入150μL不同浓度的样品,分别以 DMSO和无菌超纯水为空白对照,以1.500mg/mL卡那霉素水溶液为阳性对照,将平皿置于37℃恒温培养箱中培养1d,观察各浓度代谢产物对供试菌的抑制效果,记录各浓度样品的透明抑菌圈直径,见表2,当透明圈直径大于8mm则说明该浓度样品有抑菌效果,小于等于8mm则说明该浓度样品中没有抑菌效果,每组实验重复操作 3次。
表2
实验结果:
芸薹链格孢Gb.PY-F2代谢产物对金黄色葡萄球菌(CMCC(B)26003)的 MIC=1.5625mg/mL。
序列表
<110> 浙江工业大学
<120> 一种芸薹链格孢及在制备抑菌剂中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 552
<212> DNA
<213> 芸薹链格孢(Altemaria brassicae)
<400> 1
gtaacctgcg gagggatcat tacacaaata tgaaggcggg ctggaacctc tcggggttac 60
agccttgctg aattattcac ccttgtcttt tgcgtacttc ttgtttcctt ggtgggttcg 120
cccaccacta ggacaaacat aaaccttttg taattgcaat cagcgtcagt aacaaattaa 180
taattacaac tttcaacaac ggatctcttg gttctggcat cgatgaagaa cgcagcgaaa 240
tgcgataagt agtgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg 300
cgccctttgg tattccaaag ggcatgcctg ttcgagcgtc atttgtaccc tcaagctttg 360
cttggtgttg ggcgtcttgt ctctagcttt gctggagact cgccttaaag taattggcag 420
ccggcctact ggtttcggag cgcagcacaa gtcgcactct ctatcagcaa aggtctagca 480
tccattaagc ctttttttca acttttgacc tcggatcagg tagggatacc cgctgaactt 540
aagcatatca at 552
Claims (5)
1.一种芸薹链格孢Gb.PY-F2在制备抑菌剂中的应用,其特征在于所述抑菌剂为金黄色葡萄球菌(Staphylococcus aureus)抑菌剂;所述芸薹链格孢(Altemaria brassicae)Gb.PY-F2,保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO: M 2019126,保藏日期为2019年3月6日,保藏地址:中国武汉,武汉大学,邮编:430072。
2.如权利要求1所述的应用,其特征在于所述抑菌剂是芸薹链格孢Gb.PY-F2经发酵培养获得的发酵液超声破碎后抽滤,取滤液经微孔滤膜过滤后浓缩,再用乙酸乙酯萃取,有机相浓缩至恒重,获得芸薹链格孢Gb.PY-F2代谢产物,即为抑菌剂。
3.如权利要求2所述的应用,其特征在于所述抑菌剂按如下方法制备:将芸薹链格孢Gb.PY-F2接种于发酵培养基中,28℃,180r/min培养7d,获得发酵液;将发酵液超声破碎后抽滤,取滤液经微孔滤膜过滤后浓缩至原体积的1/4,用1倍体积乙酸乙酯萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,浓缩至恒重,获得芸薹链格孢Gb.PY-F2代谢产物,即为抑菌剂;所述发酵培养基组成:硝酸钠3g/L,磷酸氢二钾1g/L,硫酸镁0.5g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,蔗糖30g/L,溶剂为蒸馏水,pH7.0~7.2。
4.如权利要求3所述的应用,其特征在于所述超声破碎条件为:在405W条件下,每工作3s,间歇4s,进行300次循环。
5.如权利要求3所述的应用,其特征在于所述芸薹链格孢Gb.PY-F2发酵前先活化培养,然后再接入发酵培养基,所述活化培养为:将芸薹链格孢Gb.PY-F2接种于PDA培养基中,置于28℃恒温培养箱中培养7d;所述PDA培养基组成:马铃薯200g/L,葡萄糖20g/L,琼脂15~20g/L,溶剂为蒸馏水,自然pH。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910174849.3A CN109897788B (zh) | 2019-03-08 | 2019-03-08 | 一种芸薹链格孢及在制备抑菌剂中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910174849.3A CN109897788B (zh) | 2019-03-08 | 2019-03-08 | 一种芸薹链格孢及在制备抑菌剂中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109897788A CN109897788A (zh) | 2019-06-18 |
CN109897788B true CN109897788B (zh) | 2021-04-06 |
Family
ID=66946943
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910174849.3A Active CN109897788B (zh) | 2019-03-08 | 2019-03-08 | 一种芸薹链格孢及在制备抑菌剂中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109897788B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111471721B (zh) * | 2020-03-16 | 2022-10-04 | 浙江工业大学 | 一种芸薹链格孢菌代谢物在制备黄曲霉菌抑菌剂中的应用 |
CN111676143A (zh) * | 2020-06-29 | 2020-09-18 | 新疆农业科学院植物保护研究所 | 一种交链格孢(Alternaria alternata)在瓜列当生防中的应用 |
CN112029775B (zh) * | 2020-08-18 | 2022-07-19 | 浙江大学 | 芥蓝BoWRKY33基因及其应用 |
CN113980816B (zh) * | 2021-08-19 | 2023-03-07 | 杨凌未来中科环保科技有限公司 | 一种芸苔链格孢菌及其应用 |
-
2019
- 2019-03-08 CN CN201910174849.3A patent/CN109897788B/zh active Active
Non-Patent Citations (3)
Title |
---|
Sinalexin, a phytoalexin from white mustard elicited by destruxin B and Alternaria brassicae;Soledade, M.et al;《Phytochemistry》;19971231;第46卷(第5期);833-837 * |
戴氏绿僵菌变种拮抗耐甲氧西林金黄色葡萄球菌的作用;余 瑛等;《重庆理工大学学报(自然科学)》;20130430;第27卷(第4期);22-26 * |
茄链格孢培养滤液抗菌活性物质的研究;李树正等;《植物病理学报》;19971231;第27卷(第2期);161-165 * |
Also Published As
Publication number | Publication date |
---|---|
CN109897788A (zh) | 2019-06-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109897788B (zh) | 一种芸薹链格孢及在制备抑菌剂中的应用 | |
CN110066830B (zh) | 银杏内生真菌代谢产物及在制备抑菌剂中的应用 | |
CN110024696B (zh) | 黄盖小脆柄菇及在制备天然抑菌剂中的应用 | |
CN108504594A (zh) | 一株拟无枝菌酸菌及其在制备抗三七根腐病剂中的应用 | |
CN110343625B (zh) | 一种酿酒酵母菌株及其应用 | |
CN109294961B (zh) | 一株防治荔枝霜疫病的生防菌株pnc25及其应用 | |
CN107236820B (zh) | 一种诺丽内生真菌的分离鉴定方法 | |
CN110129376B (zh) | 银杏内生真菌代谢产物及在制备抗氧化剂中的应用 | |
CN111117900B (zh) | 一种高效降解黄曲霉毒素b1的菌株及其应用 | |
CN113151086A (zh) | 一种暹罗芽孢杆菌、菌剂、萃取物及其应用 | |
CN116218694B (zh) | 扣囊覆膜孢酵母、具有紧致嫩肤、提亮光泽和修护功效的半乳糖酵母样菌发酵产物滤液 | |
CN116869870A (zh) | 一种具有保湿、紧致和舒缓功效的半乳糖酵母样菌发酵产物滤液及其制备方法和应用 | |
CN115806913A (zh) | 野尻链霉菌(Streptomyces nojiriensis)菌株9-13及应用 | |
CN111334441B (zh) | 一种降酸酵母菌株及其应用 | |
CN111979157B (zh) | 一种马铃薯干腐病拮抗菌jz3-1-15及其应用 | |
CN108315266A (zh) | 蝉拟青霉及应用 | |
CN115418326A (zh) | 一种复合菌剂及其应用 | |
CN111004727B (zh) | 一株在高盐环境下增加木麻黄生物量的内生真菌z1 | |
CN113684158A (zh) | 一种暹罗芽孢杆菌jy-1及其制剂和应用 | |
CN114467978B (zh) | 刺梨内生真菌的代谢产物在作为广谱抗菌剂中的应用 | |
CN102352326B (zh) | 一种利用气单胞菌去除水华蓝藻的方法 | |
CN113355247B (zh) | 地耳草内生真菌菌株hubu0121及其应用 | |
CN114621981B (zh) | 香榧内生真菌代谢产物的制备方法及其作为抑菌剂的应用 | |
CN114214205B (zh) | 牛樟芝共生真菌AcEF007及其分离方法 | |
CN110272828B (zh) | 一株产石杉碱甲的蛇足石杉内生宁博炭疽菌 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |