CN106754441B - 具有降低胆固醇、甘油三酯作用的解脂亚罗酵母菌及应用 - Google Patents
具有降低胆固醇、甘油三酯作用的解脂亚罗酵母菌及应用 Download PDFInfo
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Abstract
本发明公开了一株从啤酒渣中分离纯化出的具有降胆固醇、降甘油三酯作用的解脂亚罗酵母菌株(Yarrowia lipolytica),保藏号为CGMCC 7427,试验证明,菌株具有很强的耐酸、耐胆盐能力,可明显降低胆固醇(TC)及甘油三酯(TG),小鼠体内试验发现,该菌株无任何毒副作用,因此,本发明菌株可用于降脂降胆固醇保健药品及功能食品的制备。
Description
【技术领域】
本发明涉及一种解脂亚罗酵母菌,特别是一种具有降胆固醇降甘油三酯作用的解脂亚罗酵母菌(Yarrowia lipolytica)及其应用。
【背景技术】
甘油三酯(Triglyceride,缩写TG)是长链脂肪酸和甘油形成的脂肪分子,是人体内含量最多的脂类,大部分组织均可以利用甘油三酯分解产物供给能量。甘油三酯来自食物中脂肪的分解,肝脏也能将血液中的某些糖类转化为甘油三酯。如果甘油三酯过量,囤积于皮下就会使身体肥胖,囤积于血管壁则造成动脉硬化,囤积于心脏就会导致心脏肥大,囤积于肝脏则会造成脂肪肝。甘油三酯高的后果无论发生在哪个部位,对人体损伤都很严重。如果在心脏,可引起冠心病、心梗;在大脑,可发生脑卒中、中风;发生在眼底,会导致视力下降、失明;如在肾脏,可引起肾衰;发生在下肢,则出现肢体血流不畅导致坏死。此外,甘油三酯高的危害还包括引发高血压、胆结石、胰腺炎;还能够加重肝炎、致使男性性功能障碍、导致老年痴呆等。目前我国有近1/3的成年人血脂偏高,高脂血症现在已成为人类健康的第一隐形杀手。胆固醇在体内有着广泛的生理作用,但当其过量时便会导致高胆固醇血症,对机体产生不利的影响。现代研究已发现,动脉粥样硬化、静脉血栓形成与高胆固醇血症有密切的相关性。
有关乳酸菌在降胆固醇方面的研究,国内外有大量的论文报道和专利成果。CN101864375A植物乳杆菌(C88)、CN102604851A乳酸乳球菌、CN101559082A乳杆菌(MA2)、CN101724589A乳酸菌、CN1186855A尿链球菌(DM891128)及鼠李糖乳杆菌(grx10)在降胆固醇的作用中都有一定的效果,但酵母菌在降胆固醇方面的研究报道鲜见、在降甘油三酯方面更是未见报道。
【发明内容】
本发明的目的是提供一种具有降低胆固醇、甘油三酯作用的酵母菌及其应用。
为实现上述目的,本发明的技术方案是:
一种分离自啤酒渣的酵母菌,为解脂亚罗酵母菌株(Yarrowia lipolytica),菌株已于2013年 04月07日保藏于中国北京的中国微生物菌种保藏管理委员会普通微生物中心,其保藏编 号为CGMCC 7427。
本发明通过将啤酒渣涂布于麦氏培养基上进行分离,培养分纯菌株菌落为乳白色,镜检形态为卵圆形,将分纯菌株进行简单染色、运动性检查,进行糖发酵试验、硝酸盐还原试验、明胶液化等实验。采用26S rDNA法对所分纯的菌株进行分子鉴定。其26S rDNA序列在NCBI(National Center for Biotechnology Information,美国国立生物技术信息中心)数据库中进行比对,结果发现菌株EAM-ZT003T的基因序列,在所有相似的序列中,与解脂亚罗酵母菌株(Yarrowia lipolytica)的同源性为100%,因此结合生理生化特征,鉴定该菌株为解脂亚罗酵母菌株,将其命名为EAM-ZT003T。
本发明提供能用于进一步研究开发预防和治疗由于胆固醇甘油三酯过高所引起的心脑血管疾病的功能性保健食品和药品的原料菌株。通过磷硫铁比色法等方法测定菌株EAM-ZT003T的降胆固醇、甘油三酯以及耐酸、耐胆盐效果,并且测定了菌株在小鼠体内是否具有毒副作用。
结果发现:本发明提供的来源于啤酒渣的解脂亚罗酵母菌株,具有降胆固醇、降甘油三酯作用,并具有很好的安全性,且小鼠体内试验发现无毒无害,同时具有良好的胆盐、强酸耐受性,其降胆固醇降甘油三酯能力强,能够应用在功能性食品和保健药品的制备中。所谓功能性食品是指具有特定功能的食品,适宜于特定人群食用,可调节机体的功能,又不以治疗为目的。
【附图说明】
图1为本发明的菌株简单染色结果。
图2为本发明的菌株平板菌落形态。
图3为本发明的菌株对蛋黄中胆固醇及胆固醇标准品的降解效果。
图4为本发明的菌株对甘油三酯的降解效果。
图5为本发明的菌株耐酸试验结果。
图6为本发明的菌株耐胆盐试验结果。
图7为本发明的菌株对毒性试验小鼠的体重变化。
图8为本发明的菌株毒性试验结果(小鼠肝脏切片)。
图9为本发明的菌株毒性试验结果(小鼠肾脏切片)。
【具体实施方式】
以下结合具体实施例,对本发明进行详细说明。
实施例1
本发明所提供的具有降胆固醇降甘油三酯能力的解脂亚罗酵母菌EAM-ZT003T的筛选和鉴定。
1菌种的分离、纯化
称取0.5g啤酒渣样品,加入到装有4.5ml无菌水的试管中,逐次稀释至10-6,取10-4、10-5、10-6三管稀释液各0.1ml,采用涂布法,涂布于麦氏培养基上进行分离,37℃恒温培养48h。挑取菌落,连续划线分离纯化,最后选择单菌落,经镜检确认为纯种后,接种到斜面培养基培养24h后,置4℃冰箱保存备用。
麦氏培养基的配方及条件为:葡萄糖1.0g、氯化钾1.8g、酵母粉2.5g、无水乙酸钠8.2g、蒸馏水1000mL,113℃,灭菌20min。
分纯菌株菌落为乳白色,直径约2mm,边缘整齐,容易挑起,镜检形态为卵圆形,出芽生殖,大小为(3-1.5)μm×(4-5.5)μm。将分纯菌株命名为EAM-ZT003T。
2菌株生理生化鉴定
将分纯菌株进行简单染色、运动性检查,进行糖发酵试验、硝酸盐还原试验、明胶液化等实验。
菌株经半固体培养基穿刺培养发现,菌株沿穿刺线生长,边缘清晰,菌株无运动性。菌株不能液化明胶,不还原硝酸盐。
表1 EAM-ZT003T生理生化鉴定结果
检测项目 | 结果 | 检测项目 | 结果 | 检测项目 | 结果 | 检测项目 | 结果 |
葡萄糖 | + | 山梨醇 | + | 鼠李糖 | - | 山梨糖 | - |
棉籽糖 | - | 核糖 | - | 半乳糖 | - | D-木糖 | + |
果糖 | - | 松三糖 | - | 半乳糖 | - | 硝酸盐(还原) | - |
淀粉 | - | 蔗糖 | - | 明胶 | - | 尿素 | + |
硫化氢 | - | 纤维二糖 | - | 阿拉伯糖 | - | 密二糖 | - |
醋酸盐 | - | 卫茅醇 | - | β-半乳糖苷 | - | 蕈糖 | - |
45℃ | - | 15℃ | + | 6.5%NaCl | - | 10%NaCl | - |
3 26S rDNA分子鉴定
采用26S rDNA法对所分纯的菌株进行分子鉴定。26S rDNA扩增及序列测定由宝生物工程(大连)有限公司完成。
测序完成提交菌株EAM-ZT003T的26S rDNA序列在NCBI(National Center forBiotechnology Information,美国国立生物技术信息中心)数据库中进行比对,结果发现菌株EAM-ZT003T的基因序列,在所有相似的序列中,与解脂亚罗酵母菌株(Yarrowialipolytica)的同源性为100%,因此结合生理生化特征,鉴定菌株EAM-ZT003T为解脂亚罗酵母菌株。分离自啤酒渣的解脂亚罗酵母菌株(Yarrowia lipolytica),菌株已于2013年04月07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,其保藏编号为CGMCC7427。
实施例2
本发明所提供的具有降胆固醇降甘油三酯能力的解脂亚罗酵母菌EAM-ZT003T的降胆固醇效果。
1降蛋黄中胆固醇菌的效果
无菌条件下,于灭菌的培养基中加入一定量鸡蛋黄作为胆固醇源,无菌均匀分装到试管中。以2%(菌体浓度为108cfu/ml)的接种量加入菌液,37℃恒温培养24h,在8000r/min下离心10min,取上清液按照磷硫铁比色法,测定上清液中胆固醇含量。每组实验设定2个重复。
硫磷铁显色剂的配置——三氯化铁溶液:称取5.0g FeCl3·6H2O溶于200ml浓磷酸中;磷硫铁试剂(P-S-Fe试剂):取40ml的三氯化铁溶液用浓硫酸定容到500mL。
磷硫铁比色法:取容量为10ml的离心管,加入4.8ml无水乙醇及0.2ml发酵上清液,置室温内10min后,8000r/min离心5min。另取干净试管,用移液枪准确移取上清液2ml加入试管中,再沿管壁缓慢加入2ml配置好的硫磷铁试剂,立即摇匀后,室温冷却30min。待所生颜色稳定后,用分光光度计在560nm下进行比色,用无水乙醇作空白调零,以未接菌的麦氏-蛋黄培养基作对照。
胆固醇降低率按照以下公式计算:
胆固醇降低率=(C-A)/C×100%
A:菌株发酵后培养液在560nm处的OD值
C:对照在560nm处的OD值
经试验测得菌株EAM-ZT003T的降蛋黄中胆固醇能力为32.8%。
2降高纯度胆固醇菌的效果
将菌株以2%(菌体浓度为108cfu/ml)的接种量接种于高胆固醇MeclaryO-CHOL培养基中,37℃恒温培养24h,在8000r/min下离心10min,取上清液按照磷硫铁比色法,测定上清液中胆固醇含量。用无水乙醇作空白调零,以未接种的高胆固醇MeclaryO-CHOL培养基作对照。
胆固醇降低率按照以下公式计算:
胆固醇降低率=(C-A)/C×100%
A:菌株发酵后培养液在560nm处的OD值
C:对照在560nm处的OD值
高胆固醇MeclaryO-CHOL培养基:以1000mL的麦氏液体培养基计,含1.0mg/mL胆固醇胶束溶液制备:准确称量胆固醇0.1g放入小烧杯中,加入0.2g牛胆盐、0.1g蔗糖酯、1mL吐温80搅拌均匀,再移取5mL的冰乙酸加热溶解,把溶解液用超声波处理15min后,快速加入到配制好的麦氏液体培养基中,边加入边搅拌,使其形成均匀稳定的胶体溶液。
经试验测得菌株EAM-ZT003T的降胆固醇能力为12%。
实施例3
本发明所提供的具有降胆固醇降甘油三酯能力的解脂亚罗酵母菌EAM-ZT003T的降甘油三酯效果。
无菌条件下,于灭菌的培养基中加入一定量的甘油三酯标准品,无菌分装到试管中。以2%(菌体浓度为108cfu/ml)的接种量加入菌液(未接菌的试管作为空白对照),37℃恒温培养24h,于420nm处测定甘油三酯降解率。
甘油三酯降低率=(C-A)/C×100%
式中的A为菌株发酵后培养液在420nm处的OD值,C为空白对照在420nm处的OD值。
经试验测得菌株EAM-ZT003T的降甘油三酯能力为80%。
实施例4
本发明所提供的具有降胆固醇降甘油三酯能力的解脂亚罗酵母菌EAM-ZT003T的耐酸耐胆盐试验。
1菌株的耐酸试验
将菌株活化后制成菌悬液,采用稀释涂板法测定菌株的浓度。将活化菌株分别接种于pH 6.8、pH 3.0、pH 2.0的麦氏液体培养基中,37℃恒温培养10h,利用梯度稀释平板法测定活菌数,做5个平行求平均值。实验结果表明,菌株具有较强的耐酸性,在pH3.0的条件下存活率比较高,pH2.0时,培养10h,菌株的存活率有所下降,但仍能达到105cfu/ml。
2菌株对0.3%胆盐环境的耐受力测定
将菌株活化后制成菌悬液(菌体浓度为108cfu/ml),按2%的接种量分别接种于含0.0%牛胆盐(即空白)、0.3%牛胆盐的复合液体培养基中。37℃培养24h后,分别测定其OD值,做5个平行求平均值,按下述公式计算菌株对胆盐的耐受力。
胆盐耐受力(%)=含胆盐培养基的OD/空白培养基的OD×100%
经试验测得菌株EAM-ZT003T的胆盐耐受能力为91.7%。
实施例5
本发明所提供的具有降胆固醇降甘油三酯能力的解脂亚罗酵母菌EAM-ZT003T的小鼠毒性试验。
将20只小鼠随机分成2组,A组灌胃生理盐水,B组灌胃解脂亚罗酵母菌悬液。A组小鼠每只每天灌胃生理盐水0.5ml,B组小鼠每只每天灌胃含1×108个酵母菌的悬浮液0.5ml。为了保证菌体溶液中一定的活菌数,每次灌胃都采用新鲜处理的菌株。自由进水,每周记录体重。在给药42天后,处死解剖,将心脏、肝脏、脾脏、肾脏、肺取出用生理盐水冲洗并擦拭干净后称重,计算小鼠脏器指数,此外,内脏分离做石蜡切片,观察脏器是否发生病变。实验结果见图7、8、9。
表1毒性试验小鼠脏器指数比较
为期42天的毒性试验发现,小鼠毛色光洁,食欲正常,灌胃42天未出现异常体征,未发现异常死亡,小鼠体重增长前14天较快,14天后趋于缓慢,但试验过程中未出现消瘦、死亡现象。小鼠解剖后内脏正常,颜色红润、光滑,切片结果显示,小鼠脏器无气质性改变。
结果表明饲喂解脂亚罗酵母菌EAM-ZT003T对小鼠没有毒副作用。
结论:本发明提供的菌株具有极好的降胆固醇降甘油三酯的功效,并具有非常好的耐酸、耐胆盐性,进入人体胃肠道后,有较多的活菌存在,确保菌株的降胆固醇降甘油三酯活性,且菌株没有任何毒副作用。
本发明提供的解脂亚罗酵母菌EAM-ZT003T的上述特性使其能够应用于制备保健药品和具有降脂降胆固醇功能的食品中。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.一种分离自啤酒渣的解脂亚罗酵母菌株(Yarrowia lipolytica),其特征在于,所述菌株已于2013年04月07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,其保藏编号为CGMCC 7427。
2.权利要求1所述的解脂亚罗酵母菌株(Yarrowia lipolytica)在制备具有降脂降胆固醇功能的食品方面的应用。
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