CN111793594A - 一种曲霉菌分生孢子悬液的制备方法 - Google Patents
一种曲霉菌分生孢子悬液的制备方法 Download PDFInfo
- Publication number
- CN111793594A CN111793594A CN202010776591.7A CN202010776591A CN111793594A CN 111793594 A CN111793594 A CN 111793594A CN 202010776591 A CN202010776591 A CN 202010776591A CN 111793594 A CN111793594 A CN 111793594A
- Authority
- CN
- China
- Prior art keywords
- conidia
- aspergillus
- conidium
- suspension
- test tube
- 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.)
- Granted
Links
- 239000000725 suspension Substances 0.000 title claims abstract description 55
- 241000228212 Aspergillus Species 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000012360 testing method Methods 0.000 claims abstract description 41
- 238000001914 filtration Methods 0.000 claims abstract description 29
- 102000004190 Enzymes Human genes 0.000 claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 claims abstract description 27
- 239000000706 filtrate Substances 0.000 claims abstract description 25
- 239000003480 eluent Substances 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 230000010355 oscillation Effects 0.000 claims abstract description 14
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- 239000013049 sediment Substances 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 8
- 230000004913 activation Effects 0.000 claims abstract description 7
- 108091005804 Peptidases Proteins 0.000 claims abstract description 4
- 239000004365 Protease Substances 0.000 claims abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 16
- 229920000053 polysorbate 80 Polymers 0.000 claims description 16
- 238000007865 diluting Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 8
- 101710091977 Hydrophobin Proteins 0.000 claims description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000007790 scraping Methods 0.000 claims description 7
- 108010067770 Endopeptidase K Proteins 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 230000003287 optical effect Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 238000002703 mutagenesis Methods 0.000 abstract description 11
- 231100000350 mutagenesis Toxicity 0.000 abstract description 11
- 238000002474 experimental method Methods 0.000 abstract description 7
- 239000006185 dispersion Substances 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 4
- 239000013543 active substance Substances 0.000 abstract description 3
- 238000004220 aggregation Methods 0.000 abstract description 3
- 230000002776 aggregation Effects 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 description 20
- 241000228197 Aspergillus flavus Species 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000228245 Aspergillus niger Species 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 241000138954 Aspergillus oryzae AS 3.863 Species 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000004763 spore germination Effects 0.000 description 2
- 229930195730 Aflatoxin Natural products 0.000 description 1
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010059892 Cellulase Proteins 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
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 229940091771 aspergillus fumigatus Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000003008 fumonisin Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- -1 haematochrome Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical compound OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 description 1
- 229960004705 kojic acid Drugs 0.000 description 1
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012795 verification Methods 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
- C12N3/00—Spore forming or isolating processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种曲霉菌分生孢子悬液的制备方法,该方法包括以下步骤:将低温保藏的曲霉菌菌种接种到PDA试管斜面活化后,转接至大号PDA试管斜面上,培养至斜面布满分生孢子;将洗脱液加入布满分生孢子的试管中,振荡使分生孢子脱离菌丝;将振荡后富含分生孢子的洗脱液进行过滤,滤液中加入蛋白酶进行处理,酶处理完毕后进行洗涤,获得分生孢子沉淀;在分生孢子沉淀中加入重悬液重悬,重悬后经过超声振荡混匀,即得分生孢子分散均匀的悬液。用本发明提供的方法制备的曲霉菌分生孢子悬液,分生孢子分散均匀,无聚集,完全适合作为菌种诱变实验和活性物质抑菌实验的材料。
Description
技术领域
本发明涉及一种曲霉菌分生孢子悬液的制备方法,尤其适于用作菌种诱变或用于抑菌物质活性验证实验的材料。
背景技术
曲霉菌是一类在自然界中广泛分布的霉菌,其菌丝体十分发达,有隔膜,多分枝,主要通过分生孢子进行繁殖。常见的曲霉菌主要有黑曲霉、黄曲霉、米曲霉、烟曲霉等种类,这些曲霉菌在酿造及食品加工等产业中具有重要的作用。在食品工业中,曲霉菌具有举足轻重的作用,部分菌种已用于多种食品原辅料的生产,如柠檬酸、葡萄糖酸、曲酸、红色素及淀粉酶、纤维素酶等多种酶类。在工业生产中,为了充分发挥曲霉菌的作用,常常需要通过诱变的方式改造菌种,以提高有机酸、酶等有益产物的产量;但曲霉菌为多细胞的丝状微生物,不可将菌丝作为诱变出发材料,只能以分散均匀的孢子悬液作为诱变材料,以提高诱变的成功率。
曲霉菌也是造成工农业产品腐败、人及动物患病的重要原因。曲霉菌除了能造成工农业产品腐败变质外,其产生的黄曲霉毒素、烟曲霉毒素等毒素严重威胁着人类健康,并造成动物养殖业的巨大损失。为了开发新的抑霉物质,越来越多的天然或合成物质将进行抑霉活性测定,以评价其在工农业生产中的应用潜力。在测定天然或合成物质抑制霉菌活性时,也需要将霉菌孢子制成均匀分散的悬液后,才可用于测定该种物质抑制孢子萌发的抑制率、最小抑菌浓度和最小杀菌浓度等指标。
霉菌孢子具有较高的疏水性,容易聚集,不易在水中充分分散。到目前为止,曲霉菌分生孢子悬液的制备方法存在较大缺陷:在布满孢子的培养皿中加蒸馏水或吐温80水溶液,然后用接种环刮取孢子,因培养皿开口较大,很容易染菌,且会将较多的菌丝刮到孢子悬液中;用脱脂棉或3-6层脱脂纱布过滤孢子悬液除菌丝,易造成大量孢子吸附在脱脂棉或纱布上,降低了孢子收率;用移液器枪头吹吸或在孢子液中加玻璃珠振荡的方法进行分散孢子,孢子分散不充分,往往还有大量的双孢子、三孢子甚至孢子团存在。采用分散不充分的孢子进行诱变获得假阳性正向突变的概率较高,严重影响诱变的效率,同样也不适于活性物质对孢子萌发抑制率的测定实验。
发明内容
本发明的目的是:提供一种曲霉菌分生孢子悬液的制备方法,用该方法制备的孢子悬液孢子分散均匀、无聚集,适用于菌种诱变实验和活性物质的抑菌实验。
本发明的技术解决方案是:一种曲霉菌分生孢子悬液的制备方法,该制备方法包括以下步骤:
(1)曲霉菌菌种活化:将低温保藏的曲霉菌菌种接种到PDA试管斜面活化;
(2)培养分生孢子:活化后转接至大号PDA试管斜面上,培养至斜面布满分生孢子;
(3)洗脱分生孢子:将洗脱液加入布满分生孢子的试管中,振荡使分生孢子脱离菌丝;
(4)洗脱液过滤:将振荡后富含分生孢子的洗脱液进行过滤;
(5)滤液酶处理:滤液中加入蛋白酶进行处理;
(6)洗涤分生孢子:酶处理完毕后进行洗涤,获得分生孢子沉淀;
(7)分生孢子重悬:在分生孢子沉淀中加入重悬液重悬,并超声振荡混匀;
(8)计数、稀释:超声振荡混匀后,经计数、稀释,即得分生孢子分散均匀的悬液。
步骤(1)中,所述的曲霉菌菌种活化是:取4℃保藏的曲霉菌菌种斜面,用接种铲挖取带菌丝菌块,将菌块接种至普通PDA试管斜面上,带菌丝面紧贴培养基,塞好塞子后置于30℃培养箱避光培养3-5天,直至长出分生孢子。
步骤(2)中,所述的培养分生孢子是:采用接种环刮取步骤(1)所培养的分生孢子,划线接种于32×200mm的PDA试管斜面上,塞好塞子后30℃避光培养3-5天,直至试管斜面布满分生孢子。
步骤(3)中,所述的洗脱分生孢子是:步骤(2)所培养的布满分生孢子的曲霉菌试管斜面,加入10-15mL预灭菌洗脱液,倾斜试管使布满分生孢子的固体斜面向上,置于旋涡混合器上,300~500rpm振荡20-30s,将曲霉菌菌丝上的分生孢子充分洗脱下来。
其中,所述的预灭菌洗脱液是:质量浓度0.03-0.05%吐温80的50mmol/L Tris-HCl、pH7.5。
步骤(4)中,所述的洗脱液过滤是:将步骤(3)获得的分生孢子洗脱液采用灭菌的10cm×15cm四层叠加的显微镜擦镜纸进行过滤,滤除洗脱液中的菌丝片段,含分生孢子的滤液收集入50mL的无菌三角瓶中。
步骤(5)中,所述的滤液酶处理是:步骤(4)过滤后分生孢子滤液中加入过滤除菌的20mg/mL蛋白酶K溶液,至酶终浓度为0.2-0.5mg/mL,37℃恒温水浴振荡器60-100rpm酶处理20-40min,去除分生孢子表面疏水蛋白。
步骤(6)中,所述的洗涤分生孢子是:步骤(5)酶处理后的分生孢子滤液置于冷冻离心机中4℃、6000-8000 rpm条件下离心6-10min,沉淀中加入灭菌的20mL的质量浓度0.03-0.05%吐温80水溶液洗涤分生孢子,再用上述冷冻离心条件离心6-10min,得到无杂质的分生孢子沉淀。
步骤(7)中,所述的分生孢子重悬是:步骤(6)所得的分生孢子沉淀中加入已灭菌的20mL无菌的质量浓度0.03-0.05%吐温80和20-30mmol/L尿素的重悬液重悬孢子,将孢子悬液转移至无菌的50mL三角瓶后,放入超声波清洗器中,室温30KHz-50KHz超声振荡20-30s,使孢子充分分散。
步骤(8)中,所述的计数、稀释是:用光学显微镜观察并计数步骤(7)所得的分生孢子重悬液,用无菌水将悬液的分生孢子浓度稀释至106-107CFU/mL,放入4℃冰箱中保存备用。
本发明与现有技术相比的优点在于:
一、用32×200mm试管斜面培养霉菌孢子,因培养基斜面面积较大,可培养足够量的孢子;又因试管口较培养皿开口小,在洗脱孢子时不易染菌;
二、在布满孢子的试管中加入洗脱液,通过旋涡混合器振荡的方式洗脱孢子,既能较彻底的将分生孢子洗脱至洗脱液中,又可避免将霉菌菌丝带入到洗脱液中;
三、用四层显微镜擦镜纸过滤分生孢子悬液,既可除去带入到孢子悬液中的少量菌丝,又可避免用脱脂棉或脱脂纱布过滤造成大量孢子损失、孢子收率低的问题;
四、用蛋白酶K处理分生孢子悬液,可除去分生孢子表面的部分疏水蛋白,避免因孢子表面疏水蛋白的存在引起的孢子间的聚集;
五、应用超声波清洗仪在低频率下振荡分生孢子悬液,可使悬液中聚集的双孢子、三孢子或多孢子团分散开;
六、悬液中的吐温80能使分生孢子在悬液中较好的分散,同时悬液中的尿素能够避免孢子间因孢壁多糖分子形成氢键而引起聚集,在吐温80和尿素的双重作用下,分生孢子悬液更加稳定。
附图说明
图1为用传统方法制备的黄曲霉分生孢子悬液中分生孢子的分散状态;
图2为用本发明方法制备的黄曲霉分生孢子悬液中分生孢子的分散状态。
具体实施方式
下面结合具体实施例进一步说明本发明的技术解决方案,这些实施例不能理解为是对技术方案的限制。
实例1:黑曲霉分生孢子悬液制备
(1)曲霉菌菌种活化:取4℃保藏的黑曲霉AS3.939菌种斜面,用接种铲挖取带菌丝菌块,将菌块接种至普通PDA试管斜面上,带菌丝面紧贴培养基,塞好塞子后置于30℃培养箱避光培养3天,直至长出分生孢子;
(2)培养分生孢子:采用接种环刮取步骤(1)所培养的黑曲霉AS3.939分生孢子,划线接种于32×200mm的PDA试管斜面上,塞好塞子后30℃避光培养3天,直至试管斜面布满分生孢子;
(3)洗脱分生孢子:步骤(2)所培养的布满分生孢子的黑曲霉AS3.939试管斜面,加入10mL预灭菌洗脱液,倾斜试管使布满分生孢子的固体斜面向上,置于旋涡混合器上,300rpm振荡30s,将菌丝上的分生孢子充分洗脱下来;其中,所述的预灭菌洗脱液是:质量浓度0.03%吐温80的50mmol/L Tris-HCl、pH7.5;
(4)洗脱液过滤:将步骤(3)获得的分生孢子洗脱液采用灭菌的10cm×15cm四层叠加的显微镜擦镜纸进行过滤,滤除洗脱液中的菌丝片段,含分生孢子的滤液收集入50mL的无菌三角瓶中;
(5)滤液酶处理:步骤(4)过滤后分生孢子滤液中加入过滤除菌的20mg/mL蛋白酶K溶液,至酶终浓度为0.2mg/mL,37℃恒温水浴振荡器60rpm酶处理40min,去除分生孢子表面疏水蛋白;
(6)洗涤分生孢子:步骤(5)酶处理后的分生孢子滤液置于冷冻离心机中4℃、6000rpm条件下离心10min,沉淀中加入灭菌的20mL的质量浓度0.03%吐温80水溶液洗涤分生孢子,再用上述冷冻离心条件离心10min,得到无杂质的分生孢子沉淀;
(7)分生孢子重悬:步骤(6)所得的分生孢子沉淀中加入已灭菌的20mL无菌的质量浓度0.03%吐温80和20mmol/L尿素的重悬液重悬孢子,将孢子悬液转移至无菌的50mL三角瓶后,放入超声波清洗器中,室温30KHz超声振荡30s,使孢子充分分散;
(8)计数、稀释:用光学显微镜观察并计数步骤(7)所得的分生孢子重悬液,用无菌水将悬液的分生孢子浓度稀释至106-107CFU/mL,放入4℃冰箱中保存,用于黑曲霉AS3.939菌种诱变以提高柠檬酸产量。
实例2:米曲霉分生孢子悬液制备
(1)曲霉菌菌种活化:取4℃保藏的米曲霉AS3.863菌种斜面,用接种铲挖取带菌丝菌块,将菌块接种至普通PDA试管斜面上,带菌丝面紧贴培养基,塞好塞子后置于30℃培养箱避光培养4天,直至长出分生孢子;
(2)培养分生孢子:采用接种环刮取步骤(1)所培养的米曲霉AS3.863分生孢子,划线接种于32×200mm的PDA试管斜面上,塞好塞子后30℃避光培养4天,直至试管斜面布满分生孢子;
(3)洗脱分生孢子:步骤(2)所培养的布满分生孢子的试管斜面,加入12.5mL预灭菌洗脱液,倾斜试管使布满分生孢子的固体斜面向上,置于旋涡混合器上,400rpm振荡25s,将菌丝上的分生孢子充分洗脱下来;其中,所述的预灭菌洗脱液是:质量浓度0.04%吐温80的50mmol/L Tris-HCl、pH7.5;
(4)洗脱液过滤:将步骤(3)获得的分生孢子洗脱液采用灭菌的10cm×15cm四层叠加的显微镜擦镜纸进行过滤,滤除洗脱液中的菌丝片段,含分生孢子的滤液收集入50mL的无菌三角瓶中;
(5)滤液酶处理:步骤(4)过滤后分生孢子滤液中加入过滤除菌的20mg/mL蛋白酶K溶液,至酶终浓度为0.35mg/mL,37℃恒温水浴振荡器80rpm酶处理30min,去除分生孢子表面疏水蛋白;
(6)洗涤分生孢子:步骤(5)酶处理后的分生孢子滤液置于冷冻离心机中4℃、7000rpm条件下离心8min,沉淀中加入灭菌的20mL的质量浓度0.04%吐温80水溶液洗涤分生孢子,再用上述冷冻离心条件离心8min,得到无杂质的分生孢子沉淀;
(7)分生孢子重悬:步骤(6)所得的分生孢子沉淀中加入已灭菌的20mL无菌的质量浓度0.04%吐温80和25mmol/L尿素的重悬液重悬孢子,将孢子悬液转移至无菌的50mL三角瓶后,放入超声波清洗器中,室温40KHz超声振荡25s,使孢子充分分散;
(8)计数、稀释:用光学显微镜观察并计数步骤(7)所得的分生孢子重悬液,用无菌水将悬液的分生孢子浓度稀释至106-107CFU/mL,放入4℃冰箱中保存,用于米曲霉AS3.863菌种诱变以提高蛋白酶产量。
实例3:黄曲霉分生孢子悬液制备
(1)曲霉菌菌种活化:取4℃保藏的黄曲霉AS3.3950菌种斜面,用接种铲挖取带菌丝菌块,将菌块接种至普通PDA试管斜面上,带菌丝面紧贴培养基,塞好塞子后置于30℃培养箱避光培养5天,直至长出分生孢子;
(2)培养分生孢子:采用接种环刮取步骤(1)所培养的黄曲霉AS3.3950分生孢子,划线接种于32×200mm的PDA试管斜面上,塞好塞子后30℃避光培养5天,直至试管斜面布满分生孢子;
(3)洗脱分生孢子:步骤(2)所培养的布满分生孢子的试管斜面,加入15mL预灭菌洗脱液,倾斜试管使布满分生孢子的固体斜面向上,置于旋涡混合器上,500rpm振荡20s,将菌丝上的分生孢子充分洗脱下来;其中,所述的预灭菌洗脱液是:质量浓度0.05%吐温80的50mmol/L Tris-HCl、pH7.5;
(4)洗脱液过滤:将步骤(3)获得的分生孢子洗脱液采用灭菌的10cm×15cm四层叠加的显微镜擦镜纸进行过滤,滤除洗脱液中的菌丝片段,含分生孢子的滤液收集入50mL的无菌三角瓶中;
(5)滤液酶处理:步骤(4)过滤后分生孢子滤液中加入过滤除菌的20mg/mL蛋白酶K溶液,至酶终浓度为0.5mg/mL,37℃恒温水浴振荡器100rpm酶处理20min,去除分生孢子表面疏水蛋白;
(6)洗涤分生孢子:步骤(5)酶处理后的分生孢子滤液置于冷冻离心机中4℃、8000rpm条件下离心6min,沉淀中加入灭菌的20mL的质量浓度0.05%吐温80水溶液洗涤分生孢子,再用上述冷冻离心条件离心6min,得到无杂质的分生孢子沉淀;
(7)分生孢子重悬:步骤(6)所得的分生孢子沉淀中加入已灭菌的20mL无菌的质量浓度0.05%吐温80和30mmol/L尿素的重悬液重悬孢子,将孢子悬液转移至无菌的50mL三角瓶后,放入超声波清洗器中,室温50KHz超声振荡20s,使孢子充分分散;
(8)计数、稀释:用光学显微镜观察并计数步骤(7)所得的分生孢子重悬液,用无菌水将悬液的分生孢子浓度稀释至106-107CFU/mL,放入4℃冰箱中保存,用于抑制黄曲霉AS3.3950活性实验。
Claims (10)
1.一种曲霉菌分生孢子悬液的制备方法,其特征是:该制备方法包括以下步骤:
(1)曲霉菌菌种活化:将低温保藏的曲霉菌菌种接种到PDA试管斜面活化;
(2)培养分生孢子:活化后转接至大号PDA试管斜面上,培养至斜面布满分生孢子;
(3)洗脱分生孢子:将洗脱液加入布满分生孢子的试管中,振荡使分生孢子脱离菌丝;
(4)洗脱液过滤:将振荡后富含分生孢子的洗脱液进行过滤;
(5)滤液酶处理:滤液中加入蛋白酶进行处理;
(6)洗涤分生孢子:酶处理完毕后进行洗涤,获得分生孢子沉淀;
(7)分生孢子重悬:在分生孢子沉淀中加入重悬液重悬,并超声振荡混匀;
(8)计数、稀释:超声振荡混匀后,经计数、稀释,即得分生孢子分散均匀的悬液。
2.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(1)中,所述的曲霉菌菌种活化是:取4℃保藏的曲霉菌菌种斜面,用接种铲挖取带菌丝菌块,将菌块接种至普通PDA试管斜面上,带菌丝面紧贴培养基,塞好塞子后置于30℃培养箱避光培养3-5天,直至长出分生孢子。
3.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(2)中,所述的培养分生孢子是:采用接种环刮取步骤(1)所培养的分生孢子,划线接种于32×200mm的PDA试管斜面上,塞好塞子后30℃避光培养3-5天,直至试管斜面布满分生孢子。
4.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(3)中,所述的洗脱分生孢子是:步骤(2)所培养的布满分生孢子的曲霉菌试管斜面,加入10-15mL预灭菌洗脱液,倾斜试管使布满分生孢子的固体斜面向上,置于旋涡混合器上,300~500rpm振荡20-30s,将曲霉菌菌丝上的分生孢子充分洗脱下来。
5.根据权利要求4所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:所述的预灭菌洗脱液是:质量浓度0.03-0.05%吐温80的50mmol/L Tris-HCl、pH7.5。
6.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(4)中,所述的洗脱液过滤是:将步骤(3)获得的分生孢子洗脱液采用灭菌的10cm×15cm四层叠加的显微镜擦镜纸进行过滤,滤除洗脱液中的菌丝片段,含分生孢子的滤液收集入50mL的无菌三角瓶中。
7.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(5)中,所述的滤液酶处理是:步骤(4)过滤后的分生孢子滤液中加入过滤除菌的20mg/mL蛋白酶K溶液,至酶终浓度为0.2-0.5mg/mL,37℃恒温水浴振荡器60-100rpm酶处理20-40min,去除分生孢子表面的疏水蛋白。
8.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(6)中,所述的洗涤分生孢子是:步骤(5)酶处理后的分生孢子滤液置于冷冻离心机中4℃、6000-8000rpm条件下离心6-10min,沉淀中加入灭菌的20mL的质量浓度0.03-0.05%吐温80水溶液洗涤分生孢子,再用上述冷冻离心条件离心6-10min,得到无杂质的分生孢子沉淀。
9.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(7)中,所述的分生孢子重悬是:步骤(6)所得的分生孢子沉淀中加入已灭菌的20mL无菌的质量浓度0.03-0.05%吐温80和20-30mmol/L尿素的重悬液重悬孢子,将孢子悬液转移至无菌的50mL三角瓶后,放入超声波清洗器中,室温30KHz-50KHz超声振荡20-30s,使孢子充分分散。
10.根据权利要求1所述的一种曲霉菌分生孢子悬液的制备方法,其特征是:步骤(8)中,所述的计数、稀释是:用光学显微镜观察并计数步骤(7)中所得的分生孢子重悬液,用无菌水将悬液的分生孢子浓度稀释至106-107 CFU/mL,放入4℃冰箱中保存备用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010776591.7A CN111793594B (zh) | 2020-08-05 | 2020-08-05 | 一种曲霉菌分生孢子悬液的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010776591.7A CN111793594B (zh) | 2020-08-05 | 2020-08-05 | 一种曲霉菌分生孢子悬液的制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111793594A true CN111793594A (zh) | 2020-10-20 |
CN111793594B CN111793594B (zh) | 2023-05-26 |
Family
ID=72827672
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010776591.7A Active CN111793594B (zh) | 2020-08-05 | 2020-08-05 | 一种曲霉菌分生孢子悬液的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111793594B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103966106A (zh) * | 2014-05-09 | 2014-08-06 | 江苏食品药品职业技术学院 | 用于摇瓶发酵对比实验的霉菌液体菌种的制备方法 |
CN104774905A (zh) * | 2015-04-23 | 2015-07-15 | 河南省农业科学院园艺研究所 | 用于镰刀菌产孢细胞及其分生孢子产生方式的显微分析方法 |
CN107365735A (zh) * | 2017-08-30 | 2017-11-21 | 武汉工控工业技术研究院有限公司 | 一种霉菌孢子制品的制备方法 |
-
2020
- 2020-08-05 CN CN202010776591.7A patent/CN111793594B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103966106A (zh) * | 2014-05-09 | 2014-08-06 | 江苏食品药品职业技术学院 | 用于摇瓶发酵对比实验的霉菌液体菌种的制备方法 |
CN104774905A (zh) * | 2015-04-23 | 2015-07-15 | 河南省农业科学院园艺研究所 | 用于镰刀菌产孢细胞及其分生孢子产生方式的显微分析方法 |
CN107365735A (zh) * | 2017-08-30 | 2017-11-21 | 武汉工控工业技术研究院有限公司 | 一种霉菌孢子制品的制备方法 |
Non-Patent Citations (1)
Title |
---|
储韬 等: "黄曲霉菌原生质体的制备和再生技术优化", 《激光生物学报》 * |
Also Published As
Publication number | Publication date |
---|---|
CN111793594B (zh) | 2023-05-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103789227B (zh) | 一株高产碱性蛋白酶的枯草芽孢杆菌菌株 | |
Kumar et al. | Extracellular xylanase production from T. lanuginosus VAPS24 at pilot scale and thermostability enhancement by immobilization | |
Taskin | Co-production of tannase and pectinase by free and immobilized cells of the yeast Rhodotorula glutinis MP-10 isolated from tannin-rich persimmon (Diospyros kaki L.) fruits | |
CN107488640B (zh) | 一种耐氧化低温葡萄糖氧化酶及其生产方法与应用 | |
CN107488600B (zh) | 一株高产耐氧化低温葡萄糖氧化酶的黑曲霉 | |
CN104975039B (zh) | 一种重组质粒及其在降解纤维素原料中的应用 | |
Ire et al. | Production, purification and characterization of polygalacturonase from Aspergillus niger in solid state and submerged fermentation using banana peels | |
CN105087446B (zh) | 一株高产中性蛋白酶的解淀粉芽孢杆菌 | |
CN111793594B (zh) | 一种曲霉菌分生孢子悬液的制备方法 | |
CN103555597A (zh) | 一种β-半乳糖苷酶的制备及其固定化方法 | |
CN116286565B (zh) | 一株高产碱性蛋白酶的枯草芽孢杆菌菌株及其应用 | |
CN103031289A (zh) | 一种乳糖酶及其重组表达工程菌 | |
CN101717733A (zh) | 一种利用微生物酶系对啤酒酵母细胞进行破壁裂解的方法 | |
Wajeeha et al. | Production, purification, and characterization of alkaline protease from Aspergillus flavus and its compatibility with commercial detergents | |
CN106434476A (zh) | 一种碱性果胶酶高产菌株及其应用 | |
CN103013963A (zh) | 一种蜡蚧菌发酵生产蛋白酶的培养基及方法 | |
ABBASI et al. | Continuous production of polygalacturonases (PGases) by Aspergillus awamori using wheat flour in surface culture fermentation | |
Unger et al. | Continuous aerobic process for distiller's yeast | |
Fiedurek et al. | Glucoamylase Biosynthesis by Cells of Aspergillus niger C58‐III Immobilized in Sintered Glass and Pumice Stones | |
CN114958623B (zh) | 一株高产纤维素酶的盖姆斯木霉及其应用 | |
Bakri et al. | Enzymatic activity of the endophytic Fusarium species strains isolated from wheat | |
Dwi et al. | Extracellular enzymes of Bacillus licheniformis from milkfish gut as degradation agent of Chlorella vulgaris cell wall | |
JPS5918987B2 (ja) | セルラ−ゼの生産方法 | |
CN114032249B (zh) | 一种碱性蛋白酶表达的工程菌、菌剂及在碱性蛋白酶生产中的应用 | |
Sagar et al. | Production of Pectinase by Aspergillus niger Isolated from Different Sites of Kathmandu Valley and Compare the Activity of the Best Strain |
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 | ||
EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20201020 Assignee: The Jiangsu bio tech Ltd. Fu Duomei Assignor: JIANGSU FOOD & PHARMACEUTICAL SCIENCE COLLEGE Contract record no.: X2024980007696 Denomination of invention: Preparation method of Aspergillus spore suspension Granted publication date: 20230526 License type: Common License Record date: 20240624 |