CN116179402A - 一株类胡萝卜素合成菌株及其应用 - Google Patents
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Abstract
本发明公开了一株类胡萝卜素合成菌株及其应用,所述菌株分类命名为食醚红球菌(Rhodococcus aetherivorans),菌株命名为N1,已保藏于中国典型培养物保藏中心,保藏日期为2022年8月11日,保藏号为:CCTCC NO:M 20221270。菌株N1在摇瓶发酵中利用葡萄糖,红球菌细胞干重达到5.0 g/L,合成类胡萝卜素含量达到6.1 mg/g。菌株N1还能够直接利用未经脱毒的玉米芯水解液作为碳源,合成类胡萝卜素含量达到5.5 mg/g。食醚红球菌N1做为一株具有类胡萝卜素合成能力的细菌菌株,能够利用广泛的糖类碳源及未经脱毒的玉米芯水解液,可为类胡萝卜素的工业化生产提供优良的菌株资源。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株类胡萝卜素合成菌株及其应用。
背景技术
类胡萝卜素是由植物、藻类、酵母、真菌和一些细菌产生的一系列天然生物分子。因其不饱和度和两端结构不同它们的颜色呈现从红色、黄色到橙色。目前已发现超过1100种来自不同物种的不同的类胡萝卜素,不同的类胡萝卜素具有不同的颜色和不同的生物功能特性。
此前,类胡萝卜素一直被作为维生素A合成前体被研究,直到最近,科学界才意识到它们具有很高的抗氧化潜力,这使它们能够对抗威胁生命的疾病,如癌症、黄斑变性等,人类牙齿或骨骼发育迟缓,粗糙鳞状皮肤受损等。有效的抗氧化活性提高了对感染的免疫反应,并在生态功能中发挥着独特的作用。这些生物活性化合物还能够避免与光氧化损伤相关的问题。因此类胡萝卜素不仅作为营养食品,还作为生物活性化合物而广受欢迎。2017年全球类胡萝卜素市场为15亿美元,2020年达到约20亿美元。全球市场调查(2018-2024年)估计,类胡萝卜素在食品和饮料、药品、化妆品、动物饲料和膳食补充剂中的市场份额分别为26.1%、9.2%、6.5%、34.8%和23.5%。目前,约80–90%的类胡萝卜素供应由化学合成完成。因此,与化学合成类胡萝卜素相比,天然类胡萝卜素的市场份额要低得多,因为其成本较高。化学合成类胡萝卜素的市场价值相对较低位250-2000美元酶公斤,而天然植物类胡萝卜素的市场价值为350-7500美元每公斤。由于植物来源的类胡萝卜素价格昂贵,近年来,由于其经济可持续性和成本效益,市场对微生物类胡萝卜素生产的兴趣不断上升。
目前已有一些关于微生物产类胡萝卜素的报道,包括藻类、霉菌、酵母、细菌。但是不同来源的类胡萝卜素种类有明显的差异,且菌株生成类胡萝卜素的能力也明显不同。目前关于细菌产类胡萝卜素工业化应用的研究还是较少的。红球菌作为一种能够产生类胡萝卜素的革兰氏阳性菌株,被科研人员进行相关研究。但是目前报道最多的类胡萝卜素合成菌株Rhodococcus opacusPD630通过分批培养类胡萝卜素含量仅为0.99 mg/L,产量低,无法实现大规模的制备类胡萝卜素。
发明内容
本发明旨在提供高产类胡萝卜素的菌株及其应用。
本发明中的食醚红球菌N1在摇瓶发酵中利用葡萄糖,红球菌细胞干重达到5.0 g/L,合成类胡萝卜素含量达到6.1 mg/g。食醚红球菌还能够直接利用未经脱毒的玉米芯水解液作为碳源,合成类胡萝卜素含量达到5.5 mg/g。菌株N1不仅能够利用多种碳源,生产高浓度的类胡萝卜素,还能够直接利用低劣生物质玉米芯水解液合成较高浓度的类胡萝卜素,降低了发酵过程中的成本,为菌株工业化合成类胡萝卜素提供有利条件。
为了实现上述技术目的,本发明采用的技术方案如下:
一株产类胡萝卜素的细菌菌株,其分类命名为食醚红球菌(Rhodococcus aetherivorans),菌株命名为N1,已保藏于中国典型培养物保藏中心,保藏日期为2022年8月11日,保藏号为:CCTCC NO:M 20221270。保藏地址为:中国武汉。
本发明所述的菌株N1,其16S rDNA的核苷酸序列如序列表中SEQ ID NO:1所示。
本发明所述的Rhodococcus aetherivoransN1筛选方法为:在LB培养基中,对从江苏南京采集的土样进行筛选。具体地,在LB培养基中,30℃培养3-5天,进行稀释涂布,挑取橙黄色单菌落在LB培养中进行培养48 h,5000 rpm离心3min收集菌体,进行菌株合成类胡萝卜素验证,筛选得到一株类胡萝卜素产量最高的细菌。
高产类胡萝卜素的菌株Rhodococcus aetherivoransN1的鉴定:利用引物27F :5’-AGAGTTTGATCCTGGCTCAG-3’和1492R:5’-TACCTTGTTACGACTT-3’扩增菌株N1的16S rDNA,通过T/A克隆的方式连接至克隆载体pMD19T,构建重组克隆载体pMD19T-16S,将其转化到克隆宿主菌Escherich coliDH5α获得重组微生物Escherich coliDH5α ( pMD19T-16S ),将所获得的重组微生物外源片段进行测序,NCBI数据库比对该16SrDNA序列,在分子水平上将菌株N1鉴定至Rhodococcus aetherivoransN1菌属。
所述食醚红球菌N1的生理学特征:N1菌株在LB培养基中呈橙色,表面光滑;生长温度为22-30℃,生长pH为5-10,生长NaCl浓度为0-40g/L。
本发明所述食醚红球菌发酵培养的方式如下:
1)平板培养:将食醚红球菌划线至LB固体培养基培养,培养温度28-32℃,培养时间45-50 h;
2)种子培养:将固体培养基上的菌落接种到LB种子培养基中培养,培养温度28-32℃,培养时间28-32 h;
3)发酵培养:将种子培养液接种到发酵培养基中,接种量3% -5%v/v,发酵温度28-32℃,发酵培养时间为120-125 h。
进一步的,所述食醚红球菌在好氧条件下发酵培养。
进一步的,所述食醚红球菌以葡萄糖为碳源发酵培养。
进一步的,所述食醚红球菌的发酵培养基配方为碳源10-80 g/L、尿素0.5-1 g/L、NaCl 1-3 g/L、K2HPO4·3H2O 1.5-2.0 g/L、KH2PO40.5-1.0 g/L、MgSO4·7H2O 0.2-0.3 g/L、微量元素 1 mL/L(FeCl2·4H2O 1.5 g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl20.07 g/L、NiCl2·6H2O 0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L),调节pH至7.0。
其中,所述的碳源为葡萄糖、木糖、果糖、乳糖或蔗糖。最优选的是,所述的碳源为葡萄糖。
其中,所述葡萄糖浓度优选为60 g/L。
本发明中菌株Rhodococcus aetherivoransN1直接利用未脱毒的玉米芯水解液为碳源发酵方法如下:
发酵培养基配方为:玉米芯水解液100 mL、尿素0.5 g/L、NaCl 1 g/L、K2HPO4·3H2O 1.5 g/L、KH2PO40.5 g/L、MgSO4·7H2O 0.2 g/L、微量元素 1 mL/L(FeCl2·4H2O 1.5g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl20.07 g/L、NiCl2·6H2O 0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L),调节pH至7.0。
本发明中玉米芯水解液的制备方法为:玉米芯与质量分数2%的H2SO4混合,固液比(m/V)为 1:7.5,130℃下水解1 h,对得到的固型物使用0.22 μm的滤膜抽滤,抽滤同时加入一倍体积去离子水。用NaOH将pH调节至7.0,即得玉米芯稀酸水解液。
本发明中使用LB培养基配方如下:
LB固体培养基配方:胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,琼脂粉15-20g/L。
LB液体培养基配方:胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L。
本发明具体发酵过程及类胡萝卜素检测方法如下:
将食醚红球菌划线至LB固体培养基培养,培养温度28-32℃,培养时间45-50 h;然后将固体培养基上的菌落接种到LB种子培养基中培养,培养温度28-32℃,培养时间28-32h;最后将种子培养液接种到发酵培养基中,接种量3% -5%v/v,发酵温度28-32℃,发酵培养时间为120-125 h。取2 mL发酵结束后的菌液一式两份,10000 rpm、3 min;一份菌泥至于105℃烘干过夜至恒重,用于检测其生物量;一份菌泥加入400 uL,3moL/L HCL,沸水浴3min 后再冰水浴3 min,13000 rpm,5 min;弃去上清液,向菌体中加入400 uL 丙酮,浸提1h,取带有类胡萝卜素的上清保存好,重复此操作3-4 次,直至菌体无色结束,最后合并丙酮提取液,用分光光度计测检测455 nm处的吸光度。
有益效果
1. 本发明以南京森林土壤为分离材料,通过一系列的筛选分离纯化得到一株可以利用高产类胡萝卜素的菌株Rhodococcus aetherivoransN1,该菌株能够利用多种碳源生长及合成类胡萝卜素。
2. 发明中菌株N1在摇瓶发酵中利用葡萄糖,细胞干重达到5.0 g/L,合成类胡萝卜素含量达到6.1 mg/g。 菌株N1还能够直接利用未经脱毒的玉米芯水解液作为碳源,合成类胡萝卜素含量达到5.5mg/g,是目前报道的直接利用玉米芯水解液合成类胡萝卜素的最高水平。Rhodococcus aetherivoransN1做为一株具有高产类胡萝卜素能力的细菌菌株,可为类胡萝卜素的工业化生产提供优良的菌株资源。
附图说明
图1菌株N1在LB固体培养基中生长的菌落形态。
图2菌株N1利用不同碳源的生长及类胡萝卜素合成。
图3菌株N1利用不同浓度葡萄糖的生长及类胡萝卜素合成。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
高产类胡萝卜素的Rhodococcus aetherivoransN1的分离筛选:
称取5 g南京森林采取的土样,用生理盐水稀释,吸取200 μL至以LB固体培养基上,于30℃培养5天。选取生长出的具有橙黄色的菌落划线纯化5代,并将其在LB培养基中发酵,考察其发酵产物和性能,发现该菌株可以利用多种碳源进行生长,并且可以产生类胡萝卜素,最终筛选得到一株高产类胡萝卜素的菌株N1,菌株N1在LB固体培养基中形态如图1所示。
上述LB培养基配方为胰蛋白胨10 g/L,酵母粉5 g/L,NaCl 5 g/L,LB固体培养基加入琼脂粉15 g/L,121℃灭菌15 min。
上述类胡萝卜素的检测方法为:
称取β-胡萝卜素标样15.5 mg,用丙酮定容至25 mL,得到620g/L浓度的母液,将β-胡萝卜素母液分别稀释10倍,20倍,50倍,100倍,200倍,分别用分光光度计测455 nm处的吸光度,制作β-胡萝卜素的标准曲线,用于检测样品中β-胡萝卜素的含量。
取2 mL发酵结束后的菌液一式两份,10000 rpm、3 min;一份菌泥至于105 ℃烘干过夜至恒重,用于检测其生物量;一份菌泥加入400 uL,3 moL/L HCL,沸水浴3 min 后再冰水浴3 min,13000 rpm,5 min;弃去上清液,向菌体中加入400 uL 丙酮,浸提1 h,取带有类胡萝卜素的上清保存好,重复此操作3-4 次,直至菌体无色结束,最后合并丙酮提取液,用分光光度计检测455nm处的吸光度。
实施例2
高产类胡萝卜素的菌株Rhodococcus aetherivoransN1的鉴定:
对菌株N1进行16S rDNA鉴定:利用引物27F 5 ’-AGAGTTTGATCCTGGCTCAG-3’和1492R:5’-TACCTTGTTACGACTT-3’扩增菌株N1的16S rDNA,通过T/A克隆的方式连接至克隆载体pMD19T,构建重组克隆载体pMD19T-16S,将其转化到克隆宿主菌Escherich coliDH5α获得重组微生物Escherich coliDH5α ( pMD19T-16S ),将所获得的重组微生物外源片段进行测序,NCBI数据库比对该16S rDNA序列,在分子水平上将菌株N1鉴定至Rhodococcus aetherivorans菌属,其16S rDNA的核苷酸序列如序列表中SEQ ID NO:1所示。
实施例3
菌株Rhodococcus aetherivoransN1利用不同碳源生长及发酵特性。
菌株Rhodococcus aetherivoransN1可以利用葡萄糖、木糖、果糖、乳糖或蔗糖为碳源生长及产类胡萝卜素(图2)。从固体培养基上挑取菌株N1单菌落接种到100 mL LB液体培养基中,30℃,180 r·min-1培养48 h,然后以接种量4% v/v接种到发酵培养基中,30℃,180 r·min-1震荡培养,发酵120 h。
上述发酵培养基配方为:碳源30 g/L、尿素0.5 g/L、NaCl 1 g/L、K2HPO4·3H2O1.5 g/L、KH2PO40.5 g/L、MgSO4·7H2O 0.2 g/L、微量元素 1 mL/L(FeCl2·4H2O 1.5 g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl20.07 g/L、NiCl2·6H2O 0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L),调节pH至7.0。由图2所示,分别以葡萄糖、木糖、果糖、乳糖或蔗糖为碳源培养Rhodococcus aetherivoransN1,以葡萄糖为碳源生产类胡萝卜素的量最高,达到4.03 mg/g。
实施例4
菌株Rhodococcus aetherivoransN1利用不同葡萄糖浓度的生长及发酵特性。
从固体培养基上挑取菌株N1单菌落接种到100 mL LB液体培养基中,30℃,180r·min-1培养48 h,然后以接种量4% v/v接种到不同葡萄糖浓度的发酵培养基中,30 ℃,180 r·min-1震荡培养,发酵120 h。
上述发酵培养基配方为:葡萄糖10-80 g/L、尿素0.5 g/L、NaCl 1 g/L、K2HPO4·3H2O 1.5 g/L、KH2PO40.5 g/L、MgSO4·7H2O 0.2 g/L、微量元素 1 mL/L(FeCl2·4H2O 1.5g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl20.07 g/L、NiCl2·6H2O 0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L),调节pH至7.0。由图3所示,分别以不同浓度的葡萄糖为碳源培养Rhodococcus aetherivoransN1,以60 g/L葡萄糖为碳源生产类胡萝卜素的量最高,达到6.1mg/g。
实施例5
菌株Rhodococcus aetherivoransN1直接利用未脱毒的玉米芯水解液为碳源的生长及发酵特性。
从固体培养基上挑取菌株N1单菌落接种到100 mL LB液体培养基中,30℃,180r·min-1培养48 h,然后以接种量4% v/v接种到不同葡萄糖浓度的发酵培养基中,30 ℃,180 r·min-1震荡培养,发酵120 h。
上述发酵培养基配方为:玉米芯水解液100 mL、尿素0.5 g/L、NaCl 1 g/L、K2HPO4·3H2O 1.5 g/L、KH2PO40.5 g/L、MgSO4·7H2O 0.2 g/L、微量元素 1 mL/L(FeCl2·4H2O 1.5 g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl20.07 g/L、NiCl2·6H2O0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L),调节pH至7.0。菌株N1能够以未脱毒的玉米芯水解液为碳源,在100 mL摇瓶发酵合成类胡萝卜素,产量达到5.5 mg/g,菌体干重达2.5 g/L。
上述玉米芯水解液的制备方法为:玉米芯与质量分数2%的H2SO4混合,固液比(m/V)为 1:7.5,130℃下水解1 h,对得到的固型物使用0.22 μm的滤膜抽滤,抽滤同时加入一倍体积去离子水。用NaOH将pH调节至7.0,即得玉米芯稀酸水解液。
Claims (9)
1.一株类胡萝卜素合成菌株,其分类命名为食醚红球菌(Rhodococcus aetherivorans),菌株命名为N1,已保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221270。
2.权利要求1所述菌株在生产类胡萝卜素中的应用。
3.根据权利要求2所述的应用,其特征在于,在营养培养基上,好氧条件下发酵培养食醚红球菌生产类胡萝卜素。
4.根据权利要求3所述的应用,其特征在于,包括如下步骤:
1)平板培养:将食醚红球菌N1划线至LB固体培养基培养,培养温度28-32℃,培养时间45-50 h;
2)种子培养:将固体培养基上的菌落接种到LB种子培养基中培养,培养温度28-32℃,培养时间28-32 h;
3)发酵培养:将种子培养液接种到发酵培养基中,接种量3-5% v/v,发酵温度28-32℃,发酵培养时间为120-125 h。
5.根据权利要求4所述的应用,其特征在于,所述的发酵培养基配方为:碳源10-80 g/L、尿素0.5-1 g/L、NaCl 1-3 g/L、K2HPO4·3H2O 1.5-2.0 g/L、KH2PO4 0.5-1.0 g/L、MgSO4·7H2O 0.2-0.3 g/L、微量元素 1 mL/L,调节pH至7.0。
6.根据权利要求5所述的应用,其特征在于,所述的碳源为葡萄糖、木糖、果糖、乳糖、蔗糖。
7.根据权利要求4所述的应用,其特征在于,所述的发酵培养基配方为玉米芯水解液100 mL、尿素4 g/L、NaCl 1 g/L、K2HPO4·3H2O 1.5 g/L、KH2PO4 0.5 g/L、MgSO4·7H2O 0.2g/L、微量元素 1 mL/L,调节pH至7.0。
8.根据权利要求5或者7所述的应用,其特征在于,所述微量元素为FeCl2·4H2O 1.5 g/L、CoCl2·6H2O 0.19 g/L、MnCl2·4H2O 0.1 g/L、ZnCl2 0.07 g/L、NiCl2·6H2O 0.024 g/L、Na2MO4·2H2O 0.036 g/L、CuCl2·2H2O 0.002 g/L。
9.根据权利要求7所述的应用,其特征在于,所述玉米芯水解液是将玉米芯与H2SO4混合,130℃下水解1 h,对得到的固型物进行抽滤,抽滤同时加入一倍体积去离子水,通过NaOH条件pH至7.0,即得到未经脱毒的玉米芯稀酸水解液。
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