CN102199565B - 一种屎肠球菌及其产生雌马酚的方法与应用 - Google Patents
一种屎肠球菌及其产生雌马酚的方法与应用 Download PDFInfo
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Abstract
本发明公开了属于生物化工领域的一种屎肠球菌Enterococcus faeciumBY1及其产生雌马酚的方法与应用,该菌已于2011年3月28日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCCNO.4707。本发明屎肠球菌具有将异黄酮中的大豆苷部分转化为雌马酚的能力。
Description
技术领域
本发明属于生物化工领域,具体涉及一种屎肠球菌及其产生雌马酚的方法与应用。
背景技术
异黄酮类物质属于多酚化合物,故较容易结合葡萄糖,形成β-葡萄糖苷键偶联的形式,这也是异黄酮类物质在豆制品中的主要存在形式。但是,偶联的异黄酮是不具备生物活性的,同时不可被人体吸收,正常情况下必须首先由胃中的β-葡萄糖酶代谢大豆苷至大豆苷元的形式,这一步转化对大豆苷的吸收和后续利用至关重要。
雌马酚(equol)是异黄酮的衍生物,为人体肠道细菌降解大豆苷元的产物。目前人们所认可的雌马酚代谢途径之一可描述为(Juliana,2003):人体摄入大豆苷等以后,偶联的异黄酮在肠内经β-葡萄糖苷酶水解,释放出具主要生物活性的苷元形式的大豆苷元、染料木苷元和黄豆苷元等。糖苷基在肠道末端经微生物群落的脱甲基作用,形成二氢大豆苷元(Dihydrodaidzein, DHD),进一步降解作用后,部分形成雌马酚,部分随细菌代谢物从肠道吸收,转运至肝脏,然后这些物质重新偶联,主要形成葡萄糖醛酸和邻脱甲基安哥拉紫檀素。另一代谢途径是澳大利亚科学家Adlercreutz在对墨尔本的120位更年期女性进行异黄酮类物质代谢调查时所发现的,他通过核磁共振发现在雌马酚生产者的尿样中存在一种新的异黄酮类物质四氢大豆苷元(Thrhydrodaidzein, THD),他认为这是二氢大豆苷元在横结肠和降结肠段微生物的作用下形成的产物,是雌马酚的直接代谢前体。
雌马酚比其它异黄酮具备更强大的功能性,主要表现在其抗氧化作用,雌马酚可以清除多种自由基,抑制铜离子诱发的低密度脂蛋白氧化,减轻由于氧化引起的DNA链应激损伤,减轻紫外线诱发的炎性反应,并解除紫外线诱发的免疫抑制,防止由于机体氧化作用导致的肿瘤,这些作用是其代谢前体大豆苷元所不具备的。雌马酚还能够治疗和预防体内雌激素缺乏引起的疾病,如女性更年期综合症,骨质疏松等。
但是,在人群中只有30%-50%的人是雌马酚生产者,即能在体内将大豆苷元降解为雌马酚者。台湾台安医院的Jou Hei-Jen通过卵泡刺激素的水平筛选适合的女性受试者,即处于后更年期的女性,试验者分为能产雌马酚组,不产雌马酚组,对照组,实验组摄入3克大豆胚芽提取粉末,2次/天,持续6个月。他们的研究表明摄入大豆异黄酮对没有雌马酚生产能力的后更年期女性是无作用的,对雌马酚生产者的作用却是显著的,即大豆异黄酮转变为雌马酚的能力可以衡量大豆异黄酮的利用效率。
陈新采用化学还原法,以市场上有售的大豆异黄酮(从豆粕中提取或化学法合成)为原料,选择性地将4-位上的羟基还原成亚甲基,并使2, 3位双键加氢成异黄烷结构的雌马酚。此发明的特点是无需采用较复杂的从人、动物尿中提取或以技术要求较高的微生物法制备,就可以实现较大规模的雌马酚工业化生产,同时合成工艺比较简单。
内山成人等分离出了含有产生雌马酚的乳酸菌的组合物,鉴定出乳球菌属的一种细菌,具有代谢大豆苷元或二氢大豆苷元产生雌马酚的能力。这株菌是试验者由人体粪便中分离鉴定出来的乳球菌20-92(FERM BP-10036号)。其菌落形状呈正圆,凸圆状突起,表面及边缘平滑。
Yokoyama从健康人的粪便中分离出来一株能产雌马酚的菌株,形态学和生物化学特征进行了鉴定,他们无孢子,不能运动,革兰氏阳性杆菌,多型。菌株有过氧化氢酶活性,氧化酶等反应阴性,能被菌株利用的唯一的糖是甘油,菌株也降解明胶,但不降解七叶苷。基于以上特征,菌株被命名为yy7918,它能转化异黄酮,脱氢大豆异黄酮到S-雌马酚,但是不能转化大豆苷元、染料木素、黄豆黄素、或芒柄花黄素。抗菌敏感性试验表明,其对氨基糖苷等敏感。
Decroos等以四位志愿者的粪样为接种物,体外研究人粪源微生物降解大豆黄酮苷元的情况,结果表明仅两位志愿者的粪源微生物具有降解大豆黄酮苷元的能力,终产物分别为双氢大豆黄酮苷元和O-DMA,双氢大豆黄酮苷元和雌马酚。分子指纹分析技术(DGGE)研究表明其中只有3株细菌可以被纯培养。但这些菌在纯培养中不能产生雌马酚。结果同时表明氢气以及丁酸、丙酸等结肠中的碳水化合物的微生物发酵终产物可以提高混合培养中雌马酚的含量;而添加果寡糖后,雌马酚的生产被抑制;当把该混合培养物接种到原来不能产生雌马酚个体的粪样中时,可以在培养物中检测到雌马酚。
发明内容
本发明的目的是提供一种能将异黄酮部分转化为雌马酚的屎肠球菌(Enterococcus faecium BY1)。
本发明的目的还在于提供该屎肠球菌(Enterococcus faecium BY1)产生雌马酚的方法。
一种屎肠球菌(Enterococcus faecium BY1),其特征在于,其保藏号为CGMCC NO.4707。
该屎肠球菌(Enterococcus faecium BY1)具有以下微生物学特征:
a、形态特征:
屎肠球菌(Enterococcus faecium BY1)在37℃,pH 5.3-7.4条件下,在BHI培养基中培养48小时,菌落呈无色透明,菌落呈圆形,边缘整齐或呈波状,菌落中央稍微凸起;在MRS培养基中培养48小时,菌落呈浅黄色,菌落呈圆形,边缘整齐或呈波状,菌落中央凸起;屎肠球菌(Enterococcus faecium BY1)细胞呈卵圆形,呈链状排列,无芽孢,在显微镜下不运动,有荚膜,在太阳光照下可反光,革兰氏染色为阳性;
b、生理生化特征:
屎肠球菌(Enterococcus faecium BY1)为兼性厌氧菌,能将异黄酮中的大豆苷转化为雌马酚;优选生长温度为37℃,pH值为6.6;不能水解明胶和淀粉;不能利用乙酸钠;可利用葡萄糖、甘露糖、蔗糖、乳糖;硫化氢和靛基质实验阳性。
屎肠球菌(Enterococcus faecium BY1)产生雌马酚的方法,包括如下步骤:
(1)屎肠球菌(Enterococcus faecium BY1)菌株的扩培
将屎肠球菌(Enterococcus faecium BY1)菌株放置在37℃培养箱中活化,按1/20接种量接种于亨盖特厌氧管中已灭菌的BHI培养基中,氮吹仪充入高纯氮气,37℃培养48h,得到扩培发酵液;
(2)细菌发酵代谢产生含雌马酚的异黄酮组合物
配制异黄酮-BHI培养基,调节pH为6.6,氮吹仪充入高纯氮气,灭菌,灭菌注射器将扩培发酵液按1/20接种量接种于亨盖特厌氧管中异黄酮-BHI培养基中,37℃培养12-14d,即可将底物异黄酮中的大豆苷部分转化为雌马酚,得到发酵液;
(3)发酵液中异黄酮成分的萃取
取一定量的发酵液,加2.5倍体积的乙酸乙酯,在培养液pH 6.6,离心转速6000r/min的条件下离心,取上清液,水浴温度60℃条件下蒸干上清液,加入所取发酵液等体积的甲醇。
所述异黄酮-BHI培养基成分和组成分别为:凝胶蛋白胨11.0-14.5 g/L,动物组织4.0-6.0 g/L,脑心浸出粉3.0-6.0 g/L,葡萄糖1.0-3.0 g/L,磷酸氢二钠0.5-2.5 g/L,氯化钠0.1-5.0 g/L,异黄酮0.14-0.56 g/L;其中异黄酮中含大豆苷14%,染料木苷57%。
本发明的有益效果:
本发明的屎肠球菌Enterococcus faecium BY1已于2011年03月28日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号为CGMCC NO.4707,保藏单位地址:北京市朝阳区北辰西路1号院3号。
具体实施方式
下面结合附图和具体实施例对本发明做进一步说明,实施例未说明的实验步骤参照《分子克隆实验指南》第三版,或参照相应试剂盒的说明书。
实施例1屎肠球菌(Enterococcus faecium BY1)的分离
屎肠球菌(Enterococcus faecium BY1)的制备方法如下:
1、样品的采集:
取婴儿新鲜粪便1g,盛放于9g质量分数为0.85%的生理盐水中,4℃冰箱中保存,12h内接种培养。
2、样品的梯度稀释与产雌马酚菌株的筛选:
(1)单一菌落式分离培养
用无菌生理盐水将事先准备好的样品进行梯度稀释,分别稀释到10-1、10-2、10-3、10-4、10-5、10-6、10-7倍,选取10-5、10-6、10-7三个梯度接种到预先备好的含有BHI固体培养基的亨盖特厌氧管中,用氮吹仪冲入高纯氮气,37℃厌氧培养48h即可。
(2)具有产雌马酚功能的单菌落的筛选
将培养获得的单菌落接种到盛有5毫升异黄酮-BHI液体培养基的试管内,该培养基的成分为(g/L):凝胶蛋白胨14.5,动物组织6.0,脑心浸出粉6.0,葡萄糖3.0,磷酸氢二钠2.5,氯化钠5.0,异黄酮0.14(含大豆苷14%,染料木苷57%)。培养48h后,取200μl培养液加入500μl乙酸乙酯进行提取,用高效液相色谱仪检测,检测出有雌马酚生成的试管内接种的单菌落即为有产生雌马酚功能的菌落。
3、单一功能微生物菌种的纯化培养、菌种初步鉴定与保藏
(1)单菌落的纯化培养
将筛选出的单一菌落在BHI固体培养基上进行反复纯化培养3次以上,确保菌落形态一致,将微生物纯菌种包藏在-70℃的超低温冰箱中并定期进行复壮培养和转化活性测定。
(2)对筛选出的单一功能微生物菌株进行菌种鉴定
根据菌落及菌体形态、革兰氏染色、常规生化鉴定以及16S rDNA基因序列等多项指标,将分离到的功能细菌菌株初步鉴定为乳酸链球菌属。
本发明乳酸链球菌具有如下性质:
a、形态特征:
屎肠球菌(Enterococcus faecium BY1)在37℃,pH 5.3-7.4条件下,在BHI培养基中培养48小时,菌落呈无色透明,菌落呈圆形,边缘整齐或呈波状,菌落中央稍微凸起;在MRS培养基中培养48小时,菌落呈浅黄色,菌落呈圆形,边缘整齐或呈波状,菌落中央凸起;屎肠球菌(Enterococcus faecium BY1)细胞呈卵圆形,呈链状排列,无芽孢,在显微镜下不运动,有荚膜,在太阳光照下可反光,革兰氏染色为阳性;
b、生理生化特征:
屎肠球菌(Enterococcus faecium BY1)为兼性厌氧菌,能将异黄酮中的大豆苷转化为雌马酚;优选生长温度为37℃,pH值为6.6;不能水解明胶和淀粉;不能利用乙酸钠;可利用葡萄糖、甘露糖、蔗糖、乳糖;硫化氢和靛基质实验阳性。
实施例2屎肠球菌(Enterococcus faecium BY1)产生雌马酚的方法
该方法按照如下步骤进行:
(1)屎肠球菌(Enterococcus faecium BY1)菌株的扩培
将屎肠球菌(Enterococcus faecium BY1)菌株放置在37℃培养箱中活化,按1/20接种量接种于亨盖特厌氧管中已灭菌的BHI培养基中,氮吹仪充入高纯氮气,37℃培养48h,得到扩培发酵液;
(2)细菌发酵代谢产生含雌马酚的异黄酮组合物
配制异黄酮-BHI培养基,其中大豆苷含量为异黄酮总质量的14%,染料木苷含量为异黄酮总质量的57%,调节pH为6.6,氮吹仪充入高纯氮气,灭菌,灭菌注射器将扩培发酵液按1/20接种量接种于亨盖特厌氧管中异黄酮-BHI培养基中,37℃培养12-14d,即可将底物异黄酮中的大豆苷部分转化为雌马酚,得到发酵液;
(3)发酵液中异黄酮成分的萃取
取一定量的发酵液,加2.5倍体积的乙酸乙酯,在培养液pH 6.6,离心转速6000r/min的条件下离心,取上清液,水浴温度60℃条件下蒸干上清液,加入所取发酵液等体积的甲醇。
Claims (1)
1.一种屎肠球菌(Enterococcus faecium)BY1,其特征在于,其保藏号为CGMCC NO.4707。
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