CN115322912A - 麦角硫因高产菌株及其筛选方法和应用 - Google Patents
麦角硫因高产菌株及其筛选方法和应用 Download PDFInfo
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- CN115322912A CN115322912A CN202210982607.9A CN202210982607A CN115322912A CN 115322912 A CN115322912 A CN 115322912A CN 202210982607 A CN202210982607 A CN 202210982607A CN 115322912 A CN115322912 A CN 115322912A
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Abstract
本发明公开了麦角硫因高产菌株及其筛选方法和应用,属于微生物和食品生物技术领域。本发明筛选到一株麦角硫因高产菌株圆红冬孢酵母DL‑XSY01,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,该菌株麦角硫因产量高,将该菌株接种至发酵培养基中,摇瓶发酵水平即可达到26mg/L(胞内产量)。当进行5‑L罐发酵时,发酵培养基中补充前体组氨酸,麦角硫因产量可达到145mg/L(胞内产量)。与同样天然来源的食用菌深层发酵提取法的产量相当,但发酵时间短,工艺相对简单。本发明还涉及一种高产麦角硫因微生物菌株的筛选方法,该方法简单有效,筛选成本低。
Description
技术领域
本发明涉及麦角硫因高产菌株及其筛选方法和应用,属于微生物和食品生物技术领域。
背景技术
麦角硫因(Ergothioneine,EGT)又叫2-硫基-L-组氨酸三甲基内盐,是一种稀有天然手性组氨酸衍生类硫醇化合物,最早由Tamcet于1909年从麦角菌(Claviceps purpurea)中分离。麦角硫因作为机体内的一种重要活性分子,抗氧化、抗炎症、延长细胞生存周期或抗细胞衰老活性,改善神经细胞生成等多种生理功效;同时在多种疾病模型中包括阿兹海默、糖尿病等的并发症中,具有较好的保护细胞和抗击损伤的功效,因而也被认为是一种特有的、多功能的细胞生理保护剂,在食品、化妆品、医药等领域应用前景广阔。
动物和植物本身不能产生麦角硫因,动物必须从膳食来源,以及植物需要从其周围环境中获得麦角硫因。目前,麦角硫因可以通过化学合成、食用真菌提取和微生物发酵生产而获得。
现有技术中生产麦角硫因的方法,主要还是化学合成,但是,化学合成法由于左旋麦角硫因合成困难,原料昂贵,难以达到预期的产量。
提取法主要通过培养和提取担子菌纲菌菇类等子实体细胞以获取麦角硫因,但有着生产率低、原料来源不足、培养周期长、提取方法复杂等问题;提取法制备麦角硫因的产率较低。比如,专利CN102978121B公开了利用白香蘑菌作为全菌催化剂,催化组氨酸底物生成麦角硫因,底物转化率为70%,;CN103184246A公开了利用食用菌花脸香蘑摇瓶发酵10d,麦角硫因产量为51mg/L。
众多细菌物种枯草芽孢杆菌、大肠杆菌、乳杆菌、葡萄球菌、普通变形杆菌和链球菌,以及酿酒酵母、毕赤酵母、马克斯克鲁维酵母、粘菌等属于子囊菌纲和半知菌纲的真菌等,均不能产生麦角硫因;只有少数微生物被证实具有麦角硫因的合成能力,诸如耻垢分枝杆菌(Mycobacterium smegmatis)、链霉菌和粗糙脉孢菌(Neurospora crassa)等,因筛选方法限制,麦角硫因天然生产菌株目前报道有限。所以研究人员通过代谢工程手段对部分遗传操作体系相对完善的微生物进行改造,以获得高效的麦角硫因产率。例如:文献(Bioscience,Biotechnology,and Biochemistry,2018:1-4.)公开了代谢工程改造米曲霉的麦角硫因产量为231mg/L;公开号为CN107250347B的中国发明专利公开了通过代谢工程改造的基因工程曲霉的麦角硫因产量为438mg/L,而改造的大肠杆菌工程菌株麦角硫因产量可达640mg/L;公开号为CN110358719B的中国发明专利公开了代谢工程改造的枯草芽孢杆菌麦角硫因产量为568.4mg/L。文献(BioRxiv,2019:667592.)公开了代谢工程酿酒酵母麦角硫因的产量为630mg/L;公开号为CN106661585B的中国发明专利公开了代谢工程改造的大肠杆菌的麦角硫因产量为12mg/L;文献(Scientific reports 2019,9(1):1895-1895.)报道了其代谢工程改造大肠杆菌通过持续性补加前体物质组氨酸,进行长达216h即9d的发酵,最终可获得1.3g/L麦角硫因产量。
但作为转基因菌株,菌株本身不能应用于传统发酵食品,同时,其麦角硫因产品应用前景也受影响。因此,建立有效筛选方法,寻找天然来源且具有食源安全属性的麦角硫因高产微生物,并开发合适的生产工艺,对解决麦角硫因高效生产尤为重要。
发明内容
本发明建立了麦角硫因高产的天然菌株有效筛选方法,获得了高产麦角硫因的天然菌株。本发明提供的菌株以及应用本发明筛选方法获得的麦角硫因高产菌株,可以扩充麦角硫因生产菌株资源库,为发酵食品提质减害提供有效手段,为保障发酵食品安全提供技术支持。
针对现有麦角硫因天然生产菌株较少且多数非食品来源、麦角硫因产量低等问题,本发明目的之一在于提供一株分离自发酵食品的天然麦角硫因生产酵母菌株DL-XSY01。
所述圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏日期为2021年10月08日。
所述圆红冬孢酵母DL-XSY01分离采集自山东小米醋醋厂的醋醅中,将测序结果进行Blastn分析,发现该菌与Rhodosporidium toruloides和Rhodotorula mucilaginosa的同源性最高,均达到99%。经形态学和26s rRNA鉴定,该菌株DL-XSY01为Rhodosporidiumtoruloides,命名为圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01。
所述圆红冬孢酵母DL-XSY01在YPD固体平板中生长良好,28℃培养48h,形成湿润、圆形、突起、光滑、浅粉色菌落;镜检发现菌体呈椭圆型长棒状,在4℃静置2d后呈现橘红色。最适生长温度28℃,最适pH5~6。
本发明还提供了一种微生物菌剂,所述微生物菌剂中含有上述圆红冬孢酵母DL-XSY01或其发酵液,或所述圆红冬孢酵母DL-XSY01裂解液。
在本发明的一种实施方式中,所述微生物菌剂中,圆红冬孢酵母DL-XSY01的含量至少为:1010CFU/mL或1010CFU/g。
本发明还提供了上述圆红冬孢酵母DL-XSY01在制备麦角硫因或含有麦角硫因的产品中的应用。
在本发明的一种实施方式中,所述产品包括但不限于:药品、化妆品、饲料、饲料添加剂。
在本发明的一种实施方式中,所述药品、化妆品、饲料包括圆红冬孢酵母DL-XSY01的发酵液、相应的载体和/或辅料。
本发明还提供了一种化学品,所述化学品中含有上述圆红冬孢酵母DL-XSY01,或含有上述微生物制剂。
在本发明的一种实施方式中,所述化学品中,圆红冬孢酵母DL-XSY01的含量至少为:1010CFU/mL或1010CFU/g。
在本发明的一种实施方式中,所述化学品包括但不限于化妆品、药品、饲料、饲料添加剂。
针对于缺乏天然高产麦角硫因菌株筛选方法的问题,本发明的另一个目的是提供一种高产麦角硫因天然菌株的有效筛选方法。
本发明提供了一种快速筛选高产麦角硫因的菌株的方法,所述方法包括以下步骤:
S1.将待筛选的担子门酵母菌接种于YPD液体培养基中进行活化,在25-32℃、150-250rpm恒温振荡培养24~48h,获得活化后的种子液。
S2.将所述种子液按1%接种量接种于含有4mM强氧化剂的YPD液体培养基中,在25~32℃、150~250rpm恒温振荡培养48~96h,得到培养液。
S3.将S2的培养液浑浊组进行适当稀释,涂布在含有5mM强氧化剂的YPD平板,在25~32℃条件下,培养48~96h。
S4.挑取S3平板上的单菌落,进行YPD液体培养,在25~32℃,150~250rpm恒温振荡培养72~120h,得到培养物。
S5.S4培养物10000rpm离心5min,弃上清,记录湿菌体质量,每100mg湿菌体加入1mL无菌水重悬,90℃温浴30min,温浴结束后12000rpm离心10min,取上清,0.22μm水系滤膜过滤,样品进行HPLC检测,即得到高产麦角硫因的菌株。
在本发明的一种实施方式中,所述强氧化剂为过氧化氢、次氯酸钠、次氯酸钙中的任一种。
在本发明的一种实施方式中,所述高产麦角硫因菌株的筛选方法中,S2所述接种于含有强氧化剂的YPD液体培养基,使用的是96孔细胞培养板或Bioscreen全自动生长曲线分析仪的培养板,可批量测定培养物OD。
在本发明的一种实施方式中,所述红酵母DL-XSY01、采用上述筛选方法筛选得到的菌株在发酵食品中的应用,是以发酵菌剂形式直接应用于食醋、酱油、大酱、豆瓣酱等发酵食品,以增加相应产品的抗氧化性能。
本发明还提供了一种麦角硫因的制备方法,采用上述圆红冬孢酵母DL-XSY01发酵制备得到。
在本发明的一种实施方式中,所述圆红冬孢酵母DL-XSY01的添加量至少为:104CFU/mL或104CFU/g。
有益效果
(1)本发明筛选到一株源自小米醋醋醅的麦角硫因高产菌株圆红冬孢酵母DL-XSY01,该菌株麦角硫因产量高,将该菌株接种至发酵培养基中,摇瓶发酵水平即可达到26mg/L(胞内产量)。当进行5-L罐发酵时,发酵培养基中补充前体组氨酸,麦角硫因产量可达到145mg/L(胞内产量)。与同样天然来源的食用菌深层发酵提取法的产量相当,但发酵时间短,工艺相对简单,并有更进一步的发酵优化空间。
(2)由于本发明所获的菌株DL-XSY01分离自发酵食品,该菌株及其制成的发酵剂应用于发酵食品,可以有效提发酵食品的抗氧化性性能。
(3)安全性评价表明:菌株DL-XSY01是安全的。抗生素敏感性中,菌株对抗生素头孢氨苄中度敏感,对10种抗生素头孢唑林、红霉素、庆大霉素、四环素、阿米卡星氨苄西林、链霉素、万古霉素、米诺环素和青霉素G敏感;溶血性实验中,DL-XSY01在羊血平板上28℃培养时未表现出溶血(γ-溶血)。然而,金黄色葡萄球菌ATCC 25923显示出β-溶血作用,阳性对照成立。此外大肠杆菌Nissle 1917显示α-溶血。因此,DL-XSY01被认为是对人体健康无危害的安全的生物体。
(4)益生性评价表明:菌株DL-XSY01在人工模拟胃液中和人工模拟肠液中有良好的存活率;对DPPH和ABTS自由基具有较好清除能力,清除率分别可达90.02%和93.67%。
生物材料保藏
一株圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,分类命名为圆红冬孢酵母Rhodosporidium toruloides,已于2021年10月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
附图说明
图1:本发明圆红冬孢酵母DL-XSY01的YPD培养基菌落照片。
图2:本发明圆红冬孢酵母DL-XSY01的显微照片。
具体实施方式
下述实施例仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,本发明主要阐述菌株以及基于所述菌株的应用思想,实施方式中简单参数的替换不能一一在实施例中赘述,其它的任何改变未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,应被视为等效的置换方式,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,都应涵盖在本发明的保护范围之内
以下结合说明书附图和具体实施例进一步说明本发明。除非特殊说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备;除非特别说明,以下实施例所用试剂和材料均为市购。
下述实施例中所涉及的Rhodotorula mucilaginosa CGMCC 2.0022、Rhodosporidium sphaerocarpum CGMCC 2.2423、Rhodotorula graminis CGMCC 2.4202、Rhodotorula marina CGMCC 2.4203、Rhodotorula mucilaginosa CGMCC 2.2506、Rhodosporidium toruloides CGMCC2.1389购自中国普通微生物菌种保藏管理中心,下述实施例中所涉及的Rhodotorula glutinis GIM 2.28、Saccharomyces cerevisiae BY4741分别购自:广东省微生物研究所微生物菌种保藏中心和美国典型菌种保藏中心。
本发明中使用的各种培养基均采用常规方法配制,实施例中涉及的分子生物学操作如未注明具体试验条件和方法,均参照SambrookJ等主编,科学出版社,2002,分子克隆实验指南(第三版);或参照产品说明书。
下述实施例中所涉及的培养基的配制如下:
YPD液体培养基:蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,蒸馏水补至1L,调pH至7.0,高压灭菌20min。
YPD固体培养基:蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,15g琼脂,蒸馏水补至1L,调pH至7.0,高压灭菌20min,然后倒平板。
His-YPD液体培养基:组氨酸3.0g,蛋白胨20.0g,酵母粉10.0g,葡萄糖20.0g,蒸馏水补至1L,调pH至7.0,高压灭菌20min。
下述实施例中所涉及的麦角硫因检测方法:
麦角硫因标准品,购自阿拉丁aladdin。
HPLC检测条件为:安捷伦高效液相色谱仪1260infinity II,依利特ODS-BP色谱柱,柱温40℃,流动相:A,磷酸二氢铵(配置方法,称取1.1503g磷酸二氢铵加400mL纯化水,氨水调pH至5.0,再加100mL纯化水);B:乙腈。A:B=99:1,流速1mg/min,进样量10μL,检测波长258nm。
胞外麦角硫因检测:发酵菌液进行10000rpm,4min离心,取上清,用2μm过滤器进行过滤处理后,样品进行HPLC检测。
胞内麦角硫因检测:发酵菌液进行10000rpm,4min离心,收集菌体,称取100mg湿菌体,转移至干净的1.5mL离心管中,加1mL无菌水,涡旋,重悬菌体,然后于90℃进行温浴30min,温浴结束,12000rpm,10min离心,取上清,用2μm过滤器进行过滤处理后,样品进行HPLC检测。
下述实施例中所涉及的抗氧化物质含量和抗氧化能力测定
DPPH测定:分别取5mL发酵后的产品(醋、黄酒、酱油、豆瓣酱),在10000rpmrpm条件下离心10min。取1mL离心上清液,按照1(上清):9(水)的比例加入超纯水稀释,再加入1mL0.2mM DPPH自由基乙醇溶液,室温下放人黑暗的环境下反应30min;4000rpm离心10min取上清,于517nm测上清液吸光度。按公式计算DPPH自由基清除率:
DPPH自由基清除率%=[1-(Ai-Aj)Ac]×100%;
式中:Ai为1mL DPPH+1mL样品的吸光度;
Aj为1mL蒸馏水+1mL样品的吸光度;
Ac为1mLDPPH+1mL蒸馏水的吸光度。
ABTS测定:分别取2mL豆浆,在10000rpm条件下离心10min。取1mL离心上清液,按照1(上清):3(水)的比例加入超纯水稀释,作为样品;将14mM的ABTS和5mM的过硫酸钾溶解于去离子水中,以1:1的比例混合,并在室温下反应12-16h,反应结束后将其OD调至1,作为ABTS溶液。将样品与ABTS溶液按1:9混合,在25℃下黑暗中培养15min,在734nm处测量上清液的吸光度。
自按公式计算ABTS自由基清除率:
AS和AC:样品和对照品的吸光度。
实施例1:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01的分离制备
1、菌株的分离纯化
(1)样品:采集自山东小米醋醋厂的醋醅。
(2)分离筛选方法采用富集培养法,具体如下:
取醋醅1g,加入到10mL含有2mM过氧化氢的YPD液体培养基中,并将上述样品移入均质袋,拍打30min,无菌条件下,移取均质袋中的部分液体至50mL离心管中,在30℃,200rpm条件下培养48h。由于在强氧化环境中菌体的生长会受到抑制甚至不生长,而麦角硫因具有强抗氧化性,因此能够产生较高水平麦角硫因的菌株会在培养基中正常生长,而不能产生麦角硫因或产量较低的菌株则会被筛选压力淘汰。待培养基浑浊后,稀释涂布于YPD固体培养基上,液体吸收后置于30℃倒置培养48h,挑单菌落进行YPD固体培养基上平板划线,连续纯化三代,挑取单菌落进行镜检,确定未污染后,进行菌株冻存保藏,菌株命名为DL-XSY01。
2、菌株DL-XSY01的鉴定
(1)提取菌株DL-XSY01基因组进行26s rRNA PCR鉴定,基因组提取方法按照《精编分子生物学实验指南》中的玻璃珠法进行提取。
PCR条件如下:
扩增体系为:2×Taq Master Mix 25μL、引物D1 2μL、引物D2 2μL、灭菌水19μL、模板2μL。PCR反应条件为:95℃预变性3min;95℃变性15S;50℃退火15S;72℃延伸1min;72℃15min;30个循环;4℃无穷。
PCR结束后进行琼脂糖凝胶(1.0%)电泳检测酵母菌样品PCR产物,跑出条带且条带明亮的为PCR扩增成功的基因组,成功的基因组即可送测序。引物序列D1:GCATATCAATAAGCGGAGGAAAAG,D2:GGTCCGTGTTTCAAGACGG。
所述菌株DL-XSY01的26S rRNA序列如下:
TTTACGGCATTCCCTAGTAGCGGCGAGCGAAGCGGGAAGAGCTCAAATTTATAATCTGGCACCTTCGGTGTCCGAGTTGTAATCTCTAGAAATGTTTTCCGCGCTGGACCGCACACAAGTCTGTTGGAATACAGCGGCATAGTGGTGAGACCCCCGTATATGGTGCGGACGCCCAGCGCTTTGTGATACATTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAATTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAACAGTACGTGAAATTGTTGGAAGGGAAACGCTTGAAGTCAGACTTGCTTGCCGAGCAATCGGTTTGCAGGCCAGCATCAGTTTTCCGGGATGGATAATGGTAGAGAGAAGGTAGCAGTTTCGGCTGTGTTATAGCTCTCTGCTGGATACATCTTGGGGGACTGAGGAACGCAGTGTGCCTTTGGCGGGGGTTTCGACCTCTTCACACTTAGGATGCTGGTGGAATGGCTTTAAACGACCCGTCTTGAAACACGGACCCAAA。
将上述测序结果进行Blastn分析,发现该菌与Rhodosporidium toruloides和Rhodotorula mucilaginosa的同源性最高,均达到99%。经形态学和26s rRNA鉴定,该菌株DL-XSY01为Rhodosporidium toruloides,命名为圆红冬孢酵母(Rhodosporidiumtoruloides)DL-XSY01。并已于2021年10月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC No.23534。
(2)菌株DL-XSY01在YPD固体培养基中生长良好,28℃培养48h,如图2所示,形成湿润、圆形、突起、光滑、浅粉色菌落;镜检发现菌体呈椭圆型长棒状,在4℃静置2d后呈现橘红色。最适生长温度28℃,最适pH5~6。
实施例2:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01菌株的性质
具体步骤如下:
1、DL-XSY01菌株安全性评价
(1)抗生素敏感性实验
使用纸片扩散法对圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01的抗生素敏感性谱进行表征。
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取200μL DL-XSY01菌液涂布于YPD-琼脂固体平板中,分别缓慢放置庆大霉素(10μg)、链霉素(10μg)、红霉素(15μg)、四环素(30μg)、头孢氨苄(30μg)、万古霉素(30μg)、头孢唑林(30μg)、氨苄西林(10μg)、青霉素(10μg)、米诺环素(30μg)及阿米卡星(30μg)11种抗生素药敏纸片,每个纸片的间距不小于24mm;
将上述放有纸片的培养基,在28℃静置培养24h后测量并统计抑菌圈直径,分析其耐药性抗性R(≤14毫米),中期I(14-20毫米)或敏感S(≥20毫米),每组设置三个重复,DL-XSY01的抗生素敏感性如表1所示。
表1:DL-XSY01的抗生素敏感性
结果显示,菌株对抗生素头孢氨苄中度敏感,对10种抗生素头孢唑林、红霉素、庆大霉素、四环素、阿米卡星氨苄西林、链霉素、万古霉素、米诺环素和青霉素G敏感。因此,根据CLSI指南,这些结果证实DL-XSY01是安全的。
(2)溶血性实验
溶血性可分为三种,其中α溶血:又称草绿色溶血,菌落周围培养基出现1-2mm的草绿色环,为高铁血红蛋白所致,α溶血环中的红细胞未完全溶解,可形成α溶血环的细菌如甲型溶血性链球菌、肺炎链球菌;β溶血是在固体平板上培养时,菌落周围形成的宽大(2-4mm)、界限分明、完全透明的溶血环,β溶血环中的红细胞完全溶解,是菌体产生的溶血素使红细胞完全溶解所致,又称完全溶血,可形成β溶血环的包括乙型溶血性链球菌、金黄色葡萄球菌等;γ溶血:又称不溶血,在菌落周围无溶血环。
具体步骤如下:
培养基配置:哥伦比亚琼脂+5%脱纤维羊血。
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取菌液划线于含有5%脱纤维羊血的哥伦比亚平板中,28℃培养24h,之后观察是否有透明圈。金黄色葡萄球菌ATCC 25923作为阳性对照,每组设置三个重复。
结果显示,在此项溶血试验中,金黄色葡萄球菌ATCC 25923被用作阳性对照。DL-XSY01在羊血平板上28℃培养时未表现出溶血(γ-溶血)。然而,金黄色葡萄球菌ATCC25923显示出β-溶血作用,阳性对照成立。此外大肠杆菌Nissle 1917显示α-溶血。因此,DL-XSY01被认为是对人体健康无危害的安全的生物体。
2、DL-XSY01菌株益生性评价
(1)人工模拟胃液实验
益生菌要定植于胃肠道发挥益生作用,首先要对消化道环境具有一定的耐受性,对胃液的耐受性是其中重要筛选标准。
配制模拟胃液:稀盐酸16.4mL,加水约900mL,胃蛋白酶10g,均匀混合后加水定容至1000mL。分别调节pH为2.5,用0.22μm滤膜过滤除菌,4℃保存。
模拟胃液耐受性实验:
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL),取5mL菌液于10000rpm,4℃离心5min收集菌体,用pH 7.4的无菌磷酸盐缓冲液洗涤2次,重复如上操作。
将菌体重新悬浮于5mL的pH 2.5的人工胃液中。采用平板涂布法计算在pH 2.5的人工模拟胃液中孵育0h和3h的活菌数量,每组设置三个重复。
存活率(%)=logCFU(N1)/logCFU(N0)*100%。
其中,N0表示在人工模拟胃液中孵育0h的活菌数,N1表示在人工模拟胃液中孵育3h后的活菌数。
同时,以大肠杆菌Nissle 1917作为阳性对照。
结果显示,在人工模拟胃液条件(含有0.3%胃蛋白酶,pH值为2.5)培养3h以后,DL-XSY01的存活率为86.73%,而阳性对照组大肠杆菌Nissle 1917的存活率为86.14%。结果表明,与大肠杆菌Nissle 1917相比,DL-XSY01在人工模拟胃液中有良好的存活率。
(2)人工模拟肠液实验
益生菌要定植于胃肠道发挥益生作用,首先要对消化道环境具有一定的耐受性,对肠液的耐受性是其中重要标准。
配置模拟肠液:用灭菌后的磷酸盐缓冲液(pH 7.4)将胰蛋白酶配制成浓度为1mg/mL溶液,在加入0.3%的牛胆盐,并用1mol/L氢氧化钠调节pH值至7.4,而后经0.22μm微孔滤膜过滤除菌后备用。
模拟肠液耐受性实验:
在YPD-琼脂固体平板上挑取DL-XSY01单菌落,转移至YPD液体培养基培养,获得菌液(约1×108cfu/mL);
取5mL菌液于10000rpm,4℃离心5min收集菌体,用pH 7.4的无菌磷酸盐缓冲液洗涤2次,重复如上操作。将菌体重新悬浮于5mL的pH 7.4的人工模拟肠液中。采用平板涂布法计算在pH 7.4人工模拟肠液中0h和4h的活菌数量,每组设置三个重复。
存活率(%)=logCFU(N1)/logCFU(N0)*100%
其中,N0代表在人工模拟肠液中孵育0h的活菌数,N1代表在人工模拟肠液中孵育3h后的活菌数。
同时,以大肠杆菌Nissle 1917作为阳性对照。
结果显示,在人工模拟肠液条件(含有0.3%胰蛋白酶和0.3%牛胆盐)培养4h以后,DL-XSY01的存活率为85.92%,而阳性对照组大肠杆菌Nissle 1917的存活率为84.64%。结果表明,与大肠杆菌Nissle 1917相比,DL-XSY01在人工模拟肠液中有良好的存活率。
(3)抗氧化实验
DPPH自由基清除率试验:
采用0.0078g DPPH,用无水乙醇溶解,定容至100ml,配制得到0.2mmol/L DPPH,避光放置,现配现用。
按照上述步骤(1)制备得到菌浓为108CFU/mL的DL-XSY01菌液;
将上述108CFU/mL的DL-XSY01菌液按照体积比为1:1的比例与100%乙醇DPPH溶液(0.2mM)混合,在25℃的黑暗中培养30min。
单独使用DL-XSY01菌液和100%乙醇作为空白,而DPPH乙醇溶液作为对照。在2330×g(4120rpm)下离心10分钟后收集上清液。在517nm处测量吸光度一式三份。
ABTS自由基清除率试验:
将ABTS(14mM)和过硫酸钾(5mM)溶解在0.1M磷酸钾缓冲液(pH 7.4)中,以1:1的比例混合,并在25℃下反应12~16h。
将100μL菌株DL-BJ01(108CFU/mL)添加到900μL ABTS溶液中,并在25℃下黑暗中培养15min。离心(14000g,1分钟)后,在734nm处测量上清液的吸光度。
结果显示,DL-XSY01的DPPH清除活性为90.02%,ABTS清除活性为93.67%,说明菌株DL-XSY01具有很好的抗氧化活性。
实施例3:圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01菌株的发酵和麦角硫因检测
具体步骤如下:
1、摇瓶发酵制备麦角硫因
(1)使用接种环取圆红冬孢酵母DL-XSY01单菌落接种至10mL YPD液体培养基(50mL离心管)中,在28℃,200rpm条件下培养24h,制备得到一级种子液;
将上述一级种子液按10%(v/v)的接种比例,转接至50mL YPD液体培养基(250mL锥形瓶)中,在28℃,200rpm条件下培养24h,制备得到二级种子液;
(2)将上述二级种子液按10%(v/v)的接种比例,分别转接至50mL YPD液体培养基(250mL锥形瓶)和50mL His-YPD液体培养基(250mL锥形瓶)中,分别将上述体系置于28℃,200rpm培养96h。每组设置3个平行,培养结束后,分别检测进行胞内麦角硫因检测和胞外麦角硫因检测,结果如表2所示。
2、5L罐发酵制备麦角硫因。
(1)使用接种环取圆红冬孢酵母DL-XSY01单菌落接种至10mL YPD液体培养基(50mL离心管)中,在28℃,200rpm条件下培养24h,制备得到一级种子液;
将上述一级种子液按10%(v/v)的接种比例,转接至100mL YPD液体培养基(500mL锥形瓶)中,在28℃,200rpm条件下培养24h,制备得到二级种子液;
将上述二级种子液按10%(v/v)的接种比例,转接至500mL YPD液体培养基(2500mL锥形瓶),在28℃,200rpm条件下培养24h,制备得到三级种子液;
(2)将上述三级种子液按10%(v/v)的接种比例,转接3L His-YPD液体培养基(5L发酵罐),发酵罐搅拌转速为600rpm,溶氧偶联,溶氧控制40%,温度为28℃,空气流量3L/min,控制pH 6.5,发酵96h。
其间,在24~72h,以5mL/h的速率流加500g/L的葡萄糖和3g/L组氨酸溶液。
培养结束后,分别检测进行胞内麦角硫因检测和胞外麦角硫因检测,结果如表2所示。
表2:圆红冬孢酵母DL-XSY01胞内、胞外麦角硫因含量
结果显示:本发明的圆红冬孢酵母DL-XSY01无需经过改造,即可发酵制备得到麦角硫因,并且在摇瓶阶段,麦角硫因的产量可达26mg/L,并且,在5L发酵罐阶段,麦角硫因的产量可达145mg/L。
实施例4:圆红冬孢酵母(Rhodosporidium toruloides)的应用
具体步骤如下:
(1)圆红冬孢酵母DL-XSY01菌株在小米醋发酵中的应用
以山西小米为原料,参考SB/T 10306-1999香醋酿制工艺规程和T/QGCML 288-2022制作小米醋,区别在于,在糖化结束后和酒精发酵结束后分别按104CFU/ml的比例接种入DL-XSY01菌株,以正常发酵组为对照。
陈酿发酵结束,分别进行DPPH测定和ABTS测定,分析其抗氧化值。
结果显示:添加DL-XSY01菌株的实验组DPPH清除活性为91.11%,ABTS清除活性为92.34%;而不添加的对照组则分别为89.23%和88.79%。
(2)圆红冬孢酵母DL-XSY01菌株在黄酒发酵中的应用
根据参考文献(酿酒科技,2013,3:65-66;中国酿造,2021,40(3):54-63),按如下工艺:小米、糯米→除杂→浸渍→沥水→蒸煮→冷却→添加酒曲糖化、酵母发酵→压榨→澄清→过滤→成品,进行小米黄酒的酿造,并在“添加酒曲糖化、酵母发酵”这一步,按105CFU/ml的比例接种入DL-XSY01菌株,以正常发酵组为对照。过滤后的成品,分别进行DPPH测定和ABTS测定,分析其抗氧化值。
结果显示:添加DL-XSY01菌株的实验组DPPH清除活性为90.07%,ABTS清除活性为93.13%;而不添加的对照组则分别为85.66%和89.0%。
(3)DL-XSY01菌株在酱油发酵中的应用
按照SB/T 10312-1999高盐稀态发酵酱油酿造工艺规程,进行酱油发酵,原料豆粕∶麸皮=7:3进行混合,经润水处理、蒸煮处理、冷却处理和接种处理,接种沪酿3.042米曲霉,然后在制曲24h按106CFU/g干料的比例接种入DL-XSY01菌株,30℃恒温制曲42h,松散、秤重,加入盐和水(最终14%盐度),30℃恒温进行发酵6个月,压榨、灭菌、过滤、分装等工序获得成品酱油,对照组和实验组分别进行DPPH测定和ABTS测定,分析其抗氧化值。
结果显示:添加DL-XSY01菌株的实验组DPPH清除活性为91.18%,ABTS清除活性为92.67%;而不添加的对照组则分别为84.56%和87.43%。
(4)DL-XSY01菌株在郫县豆瓣酱发酵中的应用
按照GB/T 20560-2006地理标志产品郫县豆瓣标准,进行郫县豆瓣酱发酵,在甜瓣子阶段,豆瓣曲装入发酵容器中时,控制3-5%的食盐和15-25%的水,按107CFU/ml的比例接种入DL-XSY01菌株,继续进行后续发酵,氨基酸态氮(以氮计)/(g/100g)大于0.18后结束发酵。成品分别进行DPPH测定和ABTS测定,分析其抗氧化值。
结果显示:添加DL-XSY01菌株的实验组DPPH清除活性为91.69%,ABTS清除活性为93.62%;而不添加的对照组则分别为84.56%和87.43%。
综上,和不添加DL-XSY01菌株的对照组相比,所有添加了DL-XSY01菌株的实验组,其DPPH自由基清除率均≥90%(对照组为80%-90%之间大小不等),其ABTS自由基清除率均≥92(对照组为86%-89%之间大小不等)。使得传统发酵食品的抗氧化能力得到提升。
实施例5:麦角硫因高产菌株的筛选方法
具体步骤如下:
(1)将待筛选的担子门酵母菌株、本发明菌株圆红冬孢酵母DL-XSY01和Saccharomyces cerevisiae BY4741阴性对照菌株接种于YPD液体培养基中进行活化,在25-32℃、150-250rpm恒温振荡培养24-48h,获得活化后的种子液。
(2)使用96孔细胞培养板或Bioscreen全自动生长曲线分析仪的培养板,将所述将待筛选的担子门酵母菌株和阴性对照菌株的种子液按超始OD=0.2的接种量接种于分别含有3-5mM强氧化剂的YPD液体培养基中,在25-32℃、150-250rpm恒温振荡培养48-96h。设置每12h进行OD检测。强氧化剂分别是过氧化氢、次氯酸钠、次氯酸钙、高锰酸钾。
结果显示,次氯酸钠和次氯酸钙的筛选结果和过氧化氢相似,但弱于过氧化氢的筛选效果;而高锰酸钾的筛选下,只有本发明菌株DL-XSY01实现轻微生长,其它菌株OD值均在0.1-0.2间,说明3-5mM高锰酸钾的筛选作用不强。
强氧化剂过氢化氢具有较好的筛选作用,凡是摇瓶水平His-YPD液体培养120h麦角硫因产量在5mg/L以上的菌株(如表3所示)。
表3:不同菌株在强氧化剂过氢化氢存在时的发酵终点OD值
结果显示,在3-4mM过氧化氢浓度下都可达到OD值倍增,5mM过氧化氢浓度下虽然OD值虽然没有倍增,但有增加。
(3)选择步骤(2)的培养液OD明显增加的组,将其对应的菌株,挑取单菌落,接种至His-YPD液体培养基中进行培养,在25-32℃、150-250rpm恒温振荡培养120h,得到培养物。
(4)将步骤(3)得到的培养物10000rpm离心5min,弃上清,收集5mL含菌发酵液,离心收集菌体,记录湿菌体质量,每100mg湿菌体加入1mL无菌水重悬,90℃温浴30min,温浴结束后12000rpm离心10min,取上清,0.22μm水系滤膜过滤,将得到的样品沉淀进行HPLC检测不同菌株的胞内麦角硫因含量,结果如表4所示。
表4:不同菌株的胞内麦角硫因含量
结果显示,采用本发明的方法能够快速的筛选出麦角硫因高产菌株。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株圆红冬孢酵母(Rhodosporidium toruloides)DL-XSY01,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23534,保藏日期为2021年10月08日。
2.一种微生物菌剂,其特征在于,所述微生物菌剂中含有权利要求1所述的圆红冬孢酵母DL-XSY01或其发酵液。
3.权利要求1所述的圆红冬孢酵母DL-XSY01在制备麦角硫因或含有麦角硫因的产品中的应用。
4.一种化学品,其特征在于,所述化学品中含有权利要求1所述的圆红冬孢酵母DL-XSY01,或含有权利要求2所述的微生物制剂。
5.如权利要求4所述的化学品,其特征在于,所述化学品中,圆红冬孢酵母DL-XSY01的含量至少为:104CFU/mL或104CFU/g。
6.如权利要求4或5所述的化学品,其特征在于,所述化学品包括但不限于化妆品、药品、饲料、饲料添加剂。
7.一种快速筛选高产麦角硫因的菌株的方法,其特征在于,所述方法包括以下步骤:
(1)将待筛选的担子门酵母菌接种于YPD液体培养基中进行活化,在25-32℃、150-250rpm恒温振荡培养24~48h,获得活化后的种子液;
(2)将步骤(1)得到的种子液按1%的接种量接种于含有4mM强氧化剂的YPD液体培养基中,在25~32℃、150~250rpm恒温振荡培养48~96h,得到培养液;
(3)将步骤(2)得到的培养液稀释后,涂布在含有5mM强氧化剂的YPD平板,在25~32℃条件下,培养48~96h;
(4)挑取步骤(3)培养结束后的平板上的单菌落,接种至YPD液体培养基中,在25~32℃,150~250rpm恒温振荡培养72~120h,得到培养物;
(5)将步骤(4)得到的培养物在10000rpm条件下离心5min,弃上清,记录湿菌体质量,每100mg湿菌体加入1mL无菌水重悬,90℃温浴30min,温浴结束后,在12000rpm条件下离心10min,取上清,0.22μm水系滤膜过滤,样品进行HPLC检测,即得到高产麦角硫因的菌株。
8.如权利要求7所述的方法,其特征在于,所述强氧化剂为过氧化氢、次氯酸盐中的任一种。
9.一种麦角硫因的制备方法,其特征在于,采用权利要求1所述的圆红冬孢酵母DL-XSY01发酵制备得到。
10.如权利要求9所述的制备方法,其特征在于,所述圆红冬孢酵母DL-XSY01的添加量至少为:104CFU/mL或104CFU/g。
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