CN117025710A - 一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法 - Google Patents
一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法 Download PDFInfo
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Abstract
本发明提供了一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,其是将该香紫苏内生真菌接种至含有三七皂苷的PDB液体培养基中,发酵生产人参皂苷。其中所述的香紫苏内生真菌为散囊菌纲曲霉属,分类命名为Aspergillus tubingensis XY17,于2022年11月07日在中国典型培养物保藏中心登记保藏,保藏号为CCTCC No:M20221729。本发明方法克服了过去使用不属于食品药品安全生产的细菌及霉菌作为发酵菌种而不能用于食品及药品生产的缺点,同时克服了传统物理方法及化学方法反应步骤繁多、副产物较难去除及环境污染等缺点,可实现规模化生产,具有较高应用价值。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法。
背景技术
人参皂苷是人参、三七、西洋参中的主要活性成分,具有独特的生理和药理活性,特别是在抗癌、抗氧化及抗衰老方面的疗效使其成为最有开发潜力的化合物之一。人参皂苷因其骨架上连接的糖基数量越多其极性越大,导致其细胞膜透过性较差,很难被胃肠道吸收,生物利用度较低。通常来说,人参皂苷药理学活性随着糖基数量的减少而增强。人参皂苷Rd具有保护心血管系统、抗肿瘤、免疫调节等作用,是原人参二醇型皂苷经代谢后被人体肠道吸收利用的重要次级皂苷之一。
三七是我国特有品种,以广西、云南、四川等地为主要种植地区。目前,我国对三七产品的开发及加工,主要集中于传统入药部位三七根、根茎及三七花,而三七茎叶通常作为废物被丢弃。据初步调查,目前三七总产量达到3万吨左右,每年三七茎叶产量达到2000万公斤以上,有效利用率不到5%,大量资源被浪费。三七茎叶含总皂苷约4~6%,以原人参二醇型皂苷为主,其中人参皂苷Rb1等含量较高。由于人参皂苷Rd在人参属植物中的含量较低,采用植物提取方法成本高昂且造成植物资源的极大浪费。
次级人参皂苷通常由主要人参皂苷经物理法、化学法和微生物转化法获得。物理法和化学法在转化过程中存在转化率低、反应剧烈、副产物多、环境不友好等缺陷。微生物转化法相对于物理法和化学法具备以下主要优势:(1)反应定向,目的产物明确,副产物少,发酵产物相对单一利于分离纯化,(2)生产周期短、底物转化效率高;(3)条件温和,可利用廉价碳源,环境友好及成本低廉等优势,易实现大规模绿色工业化生产。
多年来,研究者们对人参皂苷的微生物转化途径及机理进行了大量研究,为各类型人参皂苷的制备奠定了基础。但是目前报道的多数微生物(包括细菌及真菌)不属于美国食品药品监督管理局(FDA)认定为一般认可安全(GRAS, Generally Recognized as Safe)微生物,因此不能用于食品及药品的生产。综上所述,采用安全的微生物转化通常作为废弃物的三七茎叶中的二醇型人参皂苷生成药理活性更好的Rd,对大规模工业化生产次级人参皂苷具有较为广阔的应用前景和一定的现实意义。
发明内容
本发明的目的旨在提供一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,该方法克服了过去使用不属于食品药品安全生产的细菌及霉菌作为发酵菌种而不能用于食品及药品生产的缺点,同时克服了传统物理方法及化学方法反应步骤繁多、副产物较难去除及环境污染等缺点,可实现规模化生产,具有较高应用价值。
具体的,一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,是将该香紫苏内生真菌接种至含有三七皂苷的PDB液体培养基(即马铃薯葡萄糖水培养基)中,发酵生产人参皂苷;其中所述的香紫苏内生真菌为散囊菌纲曲霉属,分类命名为Aspergillus tubingensis XY17,于2022年11月07日在中国典型培养物保藏中心登记保藏,保藏号为CCTCC No:M20221729。
作为上述方法的进一步说明,所述香紫苏内生真菌的rDNA-ITS序列如SEQ IDNo.1所示。
香紫苏内生真菌Aspergillus tubingensis XY17是从香紫苏植株的根、茎、叶、花序筛选分离的真菌,其能够以香紫苏醇为原料制备香紫苏醇及其衍生物。同时该菌株还能够以三七皂苷为原料发酵生产人参皂苷。
作为上述方法的进一步说明,所述三七皂苷为三七茎叶提取得到的总皂苷,是至少包括人参皂苷Rb1的混合物;其发酵主要产物为人参皂苷Rd。
与现有技术相比,本发明具有以下有益效果:
1、本发明涉及的菌株Aspergillus tubingensis XY17是首次分离自香紫苏植物的散囊菌纲曲霉属的内生真菌,属于安全用于食品和药品生产的微生物(GRAS),克服了过去使用不属于食品药品安全生产的细菌及霉菌作为发酵菌种的缺点,为转化主要人参皂苷生产稀有人参皂苷提供了新的菌种。
2、本发明涉及对三七茎叶总皂苷进行微生物转化,转化时间短,可实现定向、专一的生产人参皂苷Rd,且成本低廉。同时克服了传统物理方法及化学方法反应步骤繁多、副产物较难去除及环境污染等缺点,为生产制备人参皂苷提供了绿色、高效的途径,具有较高应用价值。
附图说明
图1为基于ITS基因序列构建的香紫苏内生真菌的系统发育树。
图2为所述内生真菌XY17转化三七茎叶总皂苷的时间过程曲线。
图3为所述内生真菌XY17转化三七茎叶总皂苷的高效液相色谱分析结果。
图4为人参皂苷Rb1在内生真菌XY17作用下的转化途径。
实施方式
下面结合附图对本发明作进一步说明。
实施例1:香紫苏内生真菌的筛选分离及分子生物学鉴定
将从新疆昭苏县采集回来的香紫苏植株用自来水冲洗去掉表面的泥土杂质后室温晾干,然后将香紫苏植株用无菌刀片分割为根、茎、叶、花序,依次用75%乙醇漂洗1 min、无菌水漂洗3次、3%次氯酸钠溶液漂洗2 min、无菌水漂洗3次进行表面消毒;将消毒后的根和茎用无菌刀片切成直径约1 cm的小片组织并放置于固体分离培养基表面,和将消毒后的叶和花序用无菌手术刀切成小块并在无菌研钵中碾碎,加入适量无菌水稀释后取100 µL涂布于固体分离培养基表面,于24-28℃恒温培养箱中培养5-7天。固体分离培养基组成成分为:1.851 g/L香紫苏醇,2.44 g/L KH2PO4,14.04 g/L Na2HPO4•12H2O,2 g/L NH4Cl,0.2 g/L MgCl2•6H2O,1 g/L CaCl2•2H2O,0.01 g/L酵母提取物,5 g/L混合金属离子溶液(包含0.5g/L FeCl2•4H2O,0.1 g/L NaMoO4•2H2O,0.5 g/L MnCl2,0.05 g/L CuCl2,120 mM HCl),0.2mL/L 维生素混合溶液(包含2 g/L泛酸钙,1 g/L肌酸,2 g/L烟酸,2 g/L维生素B6,2.5 mg/L钴胺素,1 g/L对氨基苯甲酸),9.255 ml/L吐温80,1.5% (w/v)琼脂,培养基pH为6.2。重复操作3次以上对菌株进行纯化。
将上述筛选获得的形态差别较大的菌株分批次接种于液体分离培养基(该培养基成分与固体分离培养基一致,但不含琼脂)中,进行香紫苏醇转化实验。在28℃恒温摇床中180 rpm培养48-144 h,每12 h取发酵液0.5-1 mL,用等体积乙酸乙酯进行萃取,于12000rpm条件下离心15 min,取上清液经直径0.22 µm有机滤膜过滤后,利用HPLC及GC-MS进行定性检测。在所有分离获得的香紫苏内生菌中,有1株可以转化香紫苏醇产生其结构类似物。将分离到的菌株命名为XY17并进行甘油管保存,置于-80℃冰箱中保存。
提取上述菌株XY17基因组DNA,通过PCR扩增ITS基因序列。对PCR产物进行测序,将测序结果在NCBI数据库中进行BLAST同源性比对,经分析发现该菌与塔宾曲霉(Aspergillus tubingensis)的ITS序列(如SEQ ID No.1所示)的相似度为100%。上传该菌株的ITS基因序列至GenBank数据库,其登录号为ON973989。利用MEGA11软件及邻接法构建基于ITS序列的系统发育树,如图1所示,确定目标菌株的分类地位。最后将该菌株分类命名为Aspergillus tubingensis XY17,并于2022年11月07日在中国典型培养物保藏中心登记保藏,保藏号为CCTCC No:M20221729。
按1-10%的接种量将内生真菌XY17接种到50 mL PDB液体培养基(组分包括马铃薯浸出粉6 g/L、葡萄糖20 g/L,初始pH为6-7)中,28℃恒温摇床(180 rpm)培养2-3天;然后加入0.1-5 g/L三七茎叶总皂苷,继续发酵7天,期间每24 h取样0.5-1 mL,用等体积正丁醇萃取后12000 rpm离心15 min,收集上层有机相,挥干有机相后用等体积甲醇复溶,经直径0.22 µm有机滤膜过滤后,利用HPLC进行定性、定量检测。高效液相色谱检测转化产物的色谱条件为:岛津高效液相色谱仪LC-20A,色谱柱InfinityLab Poroshell 120 EC-C18(4.6mm×250 mm×4 μm,Agilent),流动相:水(A)-乙腈(B),柱温30℃,检测波长203 nm,流速1mL/min,进样量10 μL,梯度洗脱条件:0 min(22.5% B),0-55 min(22.5%-62.5% B),55-60min(62.5% B),60-65 min(62.5%-22.5% B)。
结果如图2-4所示。其中图2显示所述内生真菌XY17转化三七茎叶总皂苷的时间过程曲线,图3显示所述内生真菌XY17转化三七茎叶总皂苷的高效液相色谱分析结果,图4为人参皂苷Rb1在内生真菌XY17作用下的转化途径。实验表明,该散囊菌纲曲霉属的真菌能够转化三七茎叶总皂苷中的主要人参皂苷Rb1为人参皂苷Rd,在28℃、180 rpm恒温摇床中发酵3天后,Rd平均收率可达25.53%。
以上实施例仅为本发明的示例性实施例,不用于限制本发明,本发明的保护范围由权利要求书限定。本领域人员可以在本发明的实质和保护范围内,对本发明做出各种修改或等同替换,这种修改或等同替换也应视为落在本发明的保护范围内。
Claims (3)
1.一种香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,其特征在于,将该香紫苏内生真菌接种至含有三七皂苷的PDB液体培养基中,发酵生产人参皂苷;所述的香紫苏内生真菌为散囊菌纲曲霉属,分类命名为Aspergillus tubingensis XY17,于2022年11月07日在中国典型培养物保藏中心登记保藏,保藏号为CCTCC No:M20221729。
2.根据权利要求1所述香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,其特征在于,所述香紫苏内生真菌的rDNA-ITS序列如SEQ ID No.1所示。
3.根据权利要求1所述香紫苏内生真菌转化三七皂苷生成人参皂苷的方法,其特征在于,所述三七皂苷为三七茎叶提取得到的总皂苷,是至少包括人参皂苷Rb1的混合物;发酵产物为人参皂苷Rd。
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