CN113930466B - 一种类芽孢杆菌源金雀异黄素及其制备方法 - Google Patents
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- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229940045109 genistein Drugs 0.000 title claims abstract description 35
- 235000006539 genistein Nutrition 0.000 title claims abstract description 35
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 title claims abstract description 35
- 241000179039 Paenibacillus Species 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 78
- 238000010828 elution Methods 0.000 claims description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 238000002390 rotary evaporation Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000012264 purified product Substances 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000000287 crude extract Substances 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 239000012074 organic phase Substances 0.000 claims description 4
- 239000012071 phase Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 2
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims description 2
- 239000012137 tryptone Substances 0.000 claims description 2
- 239000000047 product Substances 0.000 claims 1
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- 238000000926 separation method Methods 0.000 abstract description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002953 preparative HPLC Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 4
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Abstract
本发明涉及一种类芽孢杆菌源金雀异黄素及其制备方法,基于类芽孢杆菌代谢产物中分离得到金雀异黄素,为金雀异黄素开创了新的来源,建立通过类芽孢杆菌发酵制备金雀异黄素的新途径。填补金雀异黄素的微生物制备途径可以降低生产成本,缩短生产周期,扩大生产率。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种类芽孢杆菌源金雀异黄素,同时还提供了其制备方法。
背景技术
金雀异黄素(Genistein,GEN)又称为染料木素或染料木黄酮,分子式为C15H10O5,化学名为4', 5, 7-三羟基异黄酮,广泛存在于大豆等豆科植物和齿状植物中,作为天然有机化合物,凭借其独特的化学结构已作为靶向抗癌制剂在临床应用,并在其他一些疾病治疗中展示出极大的应用潜力。国内外对金雀异黄素的需求量巨大,然而当前主要从豆类植物中提取,也可化工合成。微生物合成因低成本、易于大规模生产、便于遗传操作等优势,在制药中占重要地位。开发微生物源金雀异黄素,利用发酵生产克服当前大规模生产金雀异黄素的固有缺陷,在医药以及食品领域具有良好的应用前景。
发明内容
本发明针对金雀异黄素大规模生产的不足,提供了类芽孢杆菌源金雀异黄素及其制备方法。
基于上述目的,类芽孢杆菌源金雀异黄素及其制备方法包括以下步骤,并且以下步骤顺次进行:
S1.将细菌发酵液冷冻离心去除菌体和发酵残渣,收集上清液;
S2.将上清液进行酸化沉淀,冷冻离心收集沉淀部分,冷冻干燥,对干燥粉末进行甲醇萃取,收集有机相进行减压旋转蒸发至浸膏,获得粗提取物;
S3.以200-300目硅胶柱对粗提取物进行低压层析,用不同梯度的二氯甲烷甲醇溶液进行梯度洗脱,收集洗脱液,依次对洗脱液进行减压旋蒸发,获得初步纯化的提取物FrⅠ、FrⅡ、FrⅢ、FrⅣ;
S4.以半制备高效液相色谱法对初步纯化物FrⅡ进一步纯化,获得纯化物F2,其结构式如下:
优选的,所述细菌发酵液为类芽孢杆菌YPG26细菌发酵液,培养基为胰蛋白胨大豆肉汤液体培养基。
优选的,所述酸化沉淀是利用6M盐酸将上清液pH调至2.0,静置沉淀(4℃,12h~16h)。
优选的,所述半制备高效液相色谱法包含如下工艺参数:
色谱柱: COSMOSIL 5C18-MS-II柱,规格为: 10×250mm I.D.,粒径为:5μm ;流动相:甲醇(A)和水(B),柱温:RT,检测波长为280nm。
优选的,所述半制备高效液相色谱法包含如下操作:
梯度洗脱:0~15min采用20% ~50%甲醇溶液进行洗脱,15~16min采用50%~80%甲醇溶液进行洗脱,16~40min采用80% ~100%甲醇溶液进行洗脱,40~50min采用100%甲醇溶液进行洗脱。
检测并收集各流出物洗脱峰,得到保留时间Rt为22.035min的洗脱峰,然后减压旋转蒸发冻干,得到纯化物F2(金雀异黄素)。
本发明的积极效果在于:基于类芽孢杆菌代谢产物中分离得到金雀异黄素,为金雀异黄素开创了新的来源,建立通过类芽孢杆菌发酵制备金雀异黄素的新途径。填补金雀异黄素的微生物制备途径可以降低生产成本,缩短生产周期,扩大生产率。
附图说明
图1为本发明类芽孢杆菌源金雀异黄素的半制备液相洗脱峰图;
图2为本发明类芽孢杆菌源金雀异黄素的分析型高效液相色谱图;
图3为本发明类芽孢杆菌源金雀异黄素的UPLC/MS-MS二级阳离子质谱图;
图4为本发明类芽孢杆菌源金雀异黄素的1H NMR谱图;
图5为本发明类芽孢杆菌源金雀异黄素的13C NMR谱图。
具体实施方式
下面由特定的具体实施例进一步说明本发明,所描述的实施例是本发明一部分实施例,而不是全部实施例,但也并不因此将本发明限制在所述的实施例范围之中。下述实施例中未注明具体条件的实验方法,按照常规方法进行,或按照商品说明书进行。
实施例1
1、发酵培养:取对数生长期末期类芽孢杆菌YPG26菌液为种子液,按1%(v/v)转接扩大培养,获得细菌发酵液。10000×g,20min,4℃离心去除菌体和发酵残渣,收集上清液;
2、粗提取:利用6M盐酸将上清液pH调至2.0,边加盐酸边搅拌、实时测定pH,静置沉淀(4℃,12h)。8000×g,15min,4℃离心收集沉淀部分,4M NaOH调节pH=6.8,冷冻干燥,干燥粉末进行甲醇萃取,收集有机相进行减压旋转蒸发至浸膏,获得粗提取物;
3、初步纯化:以200-300目硅胶柱对粗提取物进行低压层析,分别以二氯甲烷:甲醇比例为10:0;9:1;8:2;7:3;6:4;5:5;0:10每个洗脱比例洗涤一个柱体积来进行梯度洗脱,收集洗脱液,依次对洗脱液进行减压旋蒸发,获得初步纯化的提取物FrⅠ(10:0洗脱组分)、FrⅡ(9:1、8:2洗脱组分合并)、FrⅢ(7:3、6:4洗脱组分合并)、FrⅣ(5:5、0:10洗脱组分合并);
4、制备纯化:以半制备高效液相色谱法对初步纯化物FrⅡ进一步纯化,获得纯化物F2。色谱柱: COSMOSIL 5C18-MS-II柱,规格为: 10×250mm I.D.,粒径为:5μm;流动相:甲醇(A)和水(B),柱温:RT,检测波长为280nm。
梯度洗脱:0~15min采用20% ~50%甲醇溶液进行洗脱,15~16min采用50%~80%甲醇溶液进行洗脱,16~40min采用80% ~100%甲醇溶液进行洗脱,40~50min采用100%甲醇溶液进行洗脱。
检测并收集各流出物洗脱峰,如图1所示得到保留时间Rt为22.035min的洗脱峰,然后减压旋转蒸发冻干,得到纯化物F2。
实施例2
1、发酵培养:取对数生长期末期类芽孢杆菌YPG26菌液为种子液,按1%(v/v)转接扩大培养,获得细菌发酵液。10000×g,20min,4℃离心去除菌体和发酵残渣,收集上清液;
2、粗提取:利用6M盐酸将上清液pH调至2.0,边加盐酸边搅拌、实时测定pH,静置沉淀(4℃,16h)。8000×g,15min,4℃离心收集沉淀部分,4M NaOH调节pH=6.8,冷冻干燥,干燥粉末进行甲醇萃取,收集有机相进行减压旋转蒸发至浸膏,获得粗提取物;
3、初步纯化:以200-300目硅胶柱对粗提取物进行低压层析,分别以二氯甲烷:甲醇比例为10:0;9:1;8:2;7:3;6:4;5:5;0:10每个洗脱比例洗涤一个柱体积来进行梯度洗脱,收集洗脱液,依次对洗脱液进行减压旋蒸发,获得初步纯化的提取物FrⅠ(10:0洗脱组分)、FrⅡ(9:1、8:2洗脱组分合并)、FrⅢ(7:3、6:4洗脱组分合并)、FrⅣ(5:5、0:10洗脱组分合并);
4、制备纯化:以半制备高效液相色谱法对初步纯化物FrⅡ进一步纯化,获得纯化物F2。
色谱柱: COSMOSIL 5C18-MS-II柱,规格为: 10×250mm I.D.,粒径为:5μm ;流动相:甲醇(A)和水(B),柱温:RT,检测通道波长为280nm.
梯度洗脱:0~15min采用20% ~50%甲醇溶液进行洗脱,15~16min采用50%~80%甲醇溶液进行洗脱,16~40min采用80% ~100%甲醇溶液进行洗脱,40~50min采用100%甲醇溶液进行洗脱。
检测并收集各流出物洗脱峰,半制备高效液相色谱图如图1所示,手动收集保留时间Rt为22.035min的洗脱峰,然后减压旋转蒸发冻干,得到纯化物F2。
纯化物F2由分析型高效液相色谱法检测纯度如图2所示,经高效液相色谱峰面积归一化法分析,F2纯度高于95%。
通过UPLC/MS-MS和1H NMR ,13C NMR,对F2进行组分鉴定,确定为金雀异黄素,具体的数据如下:
如图3,UPLC/MS-MS二级阳离子质谱图结果显示F2分子量为270.05908。
如图4,1H NMR (400 MHz, DMSO) δ 8.02 (s, 1H), 7.31 (d, J = 8.6 Hz,2H), 6.82 (d, J = 8.6 Hz, 2H), 5.83 (d, J = 2.3 Hz, 1H), 5.77 (d, J = 2.3 Hz,1H);
如图5,13C NMR (101 MHz, DMSO) δ 157.09 (1C,s), 138.91 (1C,s), 130.43(2C,s), 122.02 (2C,s), 114.88 (2C,s), 103.36 (1C,s), 87.38 1C,s) , 69.77 (1C,s)。
根据上述信息,F2分子式确定为C15H10O5,结构式推演为如下所示:
确定其为金雀异黄素,证实了类芽孢杆菌(Paenibacillus jilinensis sp. nov.YPG26)次级代谢产物中金雀异黄素的产生,且此制备方法切实可行。
由上述实施例可以看出,本发明提供了金雀异黄素新来源,并对类芽孢杆菌源金雀异黄素制备方法进行了说明,但也可以看出,在本发明基础上,可以对之做出一些修改,因此,在不偏离本发明精神和原则的基础上所做的修改或改进,均属于本发明要求保护的范围。
Claims (1)
1.一种类芽孢杆菌源金雀异黄素的制备方法,其特征在于包括以下步骤:
S1.将类芽孢杆菌Paenibacillus jilinensis sp. nov. YPG26细菌发酵液冷冻离心去除菌体和发酵残渣,收集上清液;培养基为胰蛋白胨大豆肉汤液体培养基;
S2.将上清液进行酸化沉淀,冷冻离心收集沉淀部分,冷冻干燥,对干燥粉末进行甲醇萃取,收集有机相进行减压旋转蒸发至浸膏,获得粗提取物;所述酸化沉淀是利用6M盐酸将上清液pH调至2.0,4℃静置沉淀12h~16h;
S3.以200-300目硅胶柱对粗提取物进行低压层析,用不同梯度的二氯甲烷甲醇溶液进行梯度洗脱,收集洗脱液,依次对洗脱液进行减压旋蒸发,获得初步纯化的提取物 FrⅠ、FrⅡ、FrⅢ、FrⅣ,10:0 洗脱组分为 FrⅠ,9:1、8:2 洗脱组分合并得到 FrⅡ,7:3、6:4 洗脱组分合并得到 FrⅢ,5:5、0:10 洗脱组分合并得到 FrⅣ;
S4.以半制备高效液相色谱法对初步纯化物FrⅡ进一步纯化,具体制备方法包含如下工艺参数:色谱柱: COSMOSIL 5C18-MS-II柱,规格为:10×250mm I.D.,径为:5μm ;流动相:甲醇(A)和水(B),柱温:RT,检测波长:280nm,梯度洗脱:0~15min采用20% ~50%甲醇溶液进行洗脱,15~16min采用50%~80%甲醇溶液进行洗脱,16~40min采用80% ~100%甲醇溶液进行洗脱,40~50min采用100%甲醇溶液进行洗脱;检测并收集各流出物洗脱峰,得到保留时间Rt为22.035min的洗脱峰,然后减压旋转蒸发冻干,即得。
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