CN107058171A - 具有降低胆固醇降低甘油三酯作用的戊糖乳杆菌及应用 - Google Patents
具有降低胆固醇降低甘油三酯作用的戊糖乳杆菌及应用 Download PDFInfo
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- CN107058171A CN107058171A CN201710091198.2A CN201710091198A CN107058171A CN 107058171 A CN107058171 A CN 107058171A CN 201710091198 A CN201710091198 A CN 201710091198A CN 107058171 A CN107058171 A CN 107058171A
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- bacterial strain
- cholesterol
- lactobacillus pentosus
- norcholesterol
- pentosus
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Abstract
本发明公开了一株从甘肃玛曲县藏獒肠道中分离纯化出的具有降胆固醇、降甘油三酯作用的戊糖乳杆菌(La.Pentosus),保藏号为CGMCC 7426,试验证明,菌株具有很强的耐酸、耐胆盐能力,可显著降低胆固醇(TC),并可降低甘油三酯(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】
本发明涉及一种戊糖乳杆菌,特别是一种具有降胆固醇降甘油三酯作用的戊糖乳杆菌(La.Pentosus)及其应用。
【背景技术】
胆固醇是人体必需的营养成分,在体内承担着重要的生理功能,然而人体血清中胆固醇含量往往过高,血清胆固醇过高所造成的一系列心血管疾病普遍存在。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。WTO曾预测,到2030年心血管疾病将是导致死亡的主要原因,影响着全世界大约2.36亿人们的健康。曾有报道,西欧国家和东欧国家的心脏病发生率是由于血胆脂醇过多造成的。与正常血脂水平人群相比,高血脂人群具有高出3倍的心脏病诱发风险。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。但如果甘油三酯过量,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。目前我国有近1/3的成年人的血脂偏高,高血脂症现在已成为人类健康的第一隐形杀手。随着社会的进步,人们健康意识的增强,减肥降脂迫在眉睫。
目前,主要通过他汀类药物控制高血脂症,其能竞争性抑制HMG-CoA还原酶(胆固醇合成的限速酶)的活性减少胆固醇的自体合成,特异性降低血清胆固醇(总胆固醇TC、低密度脂蛋白胆固醇LDL和极低密度脂蛋白胆固醇VLDL),中度降低甘油三酯水平和升高高密度脂蛋白水平,由此对动脉粥样硬化和冠心病的防治产生作用。但该种药物具有严重的毒副作用,容易造成肌痛、头痛、胃肠不适、血脂紊乱、肝肾功能损害等副作用。他汀类药物的一种较严重的不良反应是横纹肌溶解。西立伐他汀(拜斯亭)由于严重的致横纹肌溶解及肾衰竭,于2001年8月在世界范围内收回,它已导致美国及欧洲部分地区52例病人死亡的事故。因此,开发和利用具有减肥降脂作用,且无毒无害的保健药品,对保护人体健康具有重要的现实意义。
益生菌作为“有生命的绿色药品”已被研究证明具有优效、安全、符合生态等诸多优点,已被越来越多的应用于人类疾病的各个领域。
有关益生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。CN 104818230A植物乳杆菌(L01)、CN 102899276A嗜热链球菌(BLST)、CN102604851A乳酸乳球菌、CN101559082A乳杆菌(MA2)、CN1186855A尿链球菌(DM891128)及CN 104789488A鼠李糖乳杆菌(SCF 20120718)在降胆固醇的作用中都有一定的效果,但降解率都不高,而戊糖乳杆菌在降胆固醇作用中的专利未见报道,且乳酸菌在降甘油三酯方面的菌株报道鲜见。
【发明内容】
本发明的目的是提供一种具有降胆固醇、降甘油三酯作用的乳酸菌及其应用。
为实现上述目的,本发明的技术方案是:
一种分离自藏獒肠道的乳酸菌,为戊糖乳杆菌(La.Pentosus),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CGMCC7426,保藏地址为:中国北京市朝阳区北辰西路1号院3号。
本发明通过将藏獒肠道内容物涂布于MRS培养基上进行分离培养,分纯菌株菌落为乳白色,直径约2mm,表面光滑、边缘整齐、中央***,菌落周围有明显的溶钙圈,镜检形态为短杆状,将分纯菌株进行菌株革兰氏染色、平板菌落形态、菌株对蛋黄中胆固醇及胆固醇标准品的降解率、对甘油三酯的降解率、耐酸试验、耐胆盐试验、毒性试验(小鼠)等实验。采用16S rDNA法对所分纯的菌株进行分子鉴定。其16S rDNA序列在NCBI(National Centerfor Biotechnology Information,美国国立生物技术信息中心)数据库中进行比对,结果发现该菌株的基因序列,在所有相似的序列中,与戊糖乳杆菌(La.Pentosus)的同源性为99%,因此结合生理生化特征,鉴定菌株是戊糖乳杆菌,将其命名为EAM-ZL016T。
本发明提供了一种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食品和保健药品的原料菌株。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:本发明提供的从藏獒肠道中分离纯化出的戊糖乳杆菌(La.Pentosus),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,具有抑菌效果,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为本发明的菌株革兰氏染色结果。
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率。
图4为本发明的菌株对甘油三酯的降解率。
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戊糖乳杆菌EAM-ZL016T的筛选和鉴定。
1菌种的分离、纯化
称取0.5g甘肃玛曲县藏獒肠道内容物,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MRS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。
MRS固体培养基的配方及条件为:蛋白胨10g、牛肉膏10g、酵母粉5g、葡萄糖20g、无水乙酸钠5g、吐温-801mL、柠檬酸二胺2g、磷酸氢二钾2g、硫酸镁0.58g、硫酸锰0.25g、碳酸钙20g、琼脂18g、蒸馏水1000mL,pH6.6~6.8,115℃,灭菌20min。
分纯菌株菌落为乳白色,直径约2mm,表面光滑、边缘整齐、中央***,菌落周围有明显的溶钙圈,镜检形态为短杆状,(1-1.2)μm×(1-1.5)μm,单个、成对、短链排列,将分纯菌株命名为EAM-ZL016T。
2菌株生理生化鉴定
将分纯菌株进行革兰氏染色、运动性检查、氧化酶、过氧化氢酶、明胶液化等实验。
菌株革兰氏染色阳性,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,兼性厌氧。菌株不还原硝酸盐,不液化明胶,接触酶和氧化酶反应均为阴性。
表1 EAM-ZL016T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 |
甘露醇 | + | 乳糖 | + | 硝酸盐(还原) | - | ***醇 | + |
葡萄糖 | + | 七叶苷 | + | 侧金盏花醇 | - | 山梨醇 | - |
棉籽糖 | + | 苯丙氨酸 | + | 胆汁七叶苷 | - | 木糖醇 | + |
麦芽糖 | + | 赤藓醇 | + | 赖氨酸脱羧酶 | - | 苦杏仁苷 | + |
果糖 | + | 松三糖 | + | 鸟氨酸脱羧酶 | - | 酒石酸盐 | - |
淀粉 | - | 蔗糖 | + | 精氨酸脱羧酶 | - | 甘露糖 | + |
硫化氢 | - | 纤维二糖 | + | 精氨酸双水解酶 | - | 密二糖 | + |
醋酸盐 | - | 卫茅醇 | - | β-半乳糖苷 | + | 水杨素 | + |
D-核糖 | + | 肌醇 | - | 甲基红 | + | 鼠李糖 | - |
乙酰胺 | - | ***糖 | - | V.P. | - | 半乳糖 | + |
尿素 | - | 枸橼酸盐 | - | 明胶 | - | 蕈糖 | + |
丙二酸盐 | - | 木糖 | - | 山梨糖 | + | 糊精 | + |
40℃ | - | 15℃ | + | 6.5%NaCl | - | 10%NaCl | - |
316S rDNA分子鉴定
采用16S rDNA法对所分纯的菌株进行分子鉴定。16S 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-ZL016T的16S 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-ZL016T的基因序列,在所有相似的序列中,与戊糖乳杆菌(La.Pentosus)的同源性为99%,因此结合生理生化特征,鉴定菌株EAM-ZL016T是戊糖乳杆菌。该分离自藏獒肠道的戊糖乳杆菌(La.Pentosus),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 7426。
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戊糖乳杆菌EAM-ZL016T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MRS-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-ZL016T的降蛋黄中胆固醇能力为64.84%。
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MRSO-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MRSO-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MRSO-CHOL培养基:以1000mL的MRS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MRS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-ZL016T的降胆固醇能力为22%。
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戊糖乳杆菌EAM-ZL016T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-ZL016T的降甘油三酯能力为2.8%。
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戊糖乳杆菌EAM-ZL016T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MRS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106cfu/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-ZL016T的胆盐耐受能力为15%。人体小肠中胆盐含量在0.03%-0.3%范围内波动,试验所选取胆盐含量为最大浓度。
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戊糖乳杆菌EAM-ZL016T的抑菌试验。
分别取活化好的枯草芽孢杆菌、大肠杆菌和金黄色葡萄球菌接种于营养肉汤中,37℃恒温培养24h,用无菌水稀释至108cfu/ml,备用。
将50mL营养琼脂固体培养基灭菌后冷却到45℃,与指示菌(枯草芽孢杆菌、大肠杆菌和金黄色葡萄球菌)新鲜培养物1000μL混匀后倒平板,待平板凝固后放上灭菌的牛津杯。调整菌悬液浓度为108cfu/ml,用移液枪分别量取100μL的菌培养液及无菌水加入牛津杯中,盖上皿盖,将平板置于4℃冰箱扩散,然后放置37℃恒温培养24h,观察小孔周围抑菌圈直径。每个指示菌做两个皿子,测抑菌圈直径,求平均值。结果见表2。
表2戊糖乳杆菌抑菌结果
实施例6
本发明所提供的具有降胆固醇降甘油三酯能力的戊糖乳杆菌EAM-ZL016T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组灌胃戊糖乳杆菌悬液。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个戊糖乳杆菌的悬浮液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3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,小鼠体重前14天增长较快,后28天趋于稳定,维持在27g左右,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。
结果表明饲喂戊糖乳杆菌EAM-ZL016T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,菌株具有抑菌效果,在降脂降胆固醇同时,可起到整肠及调节肠道菌群的作用,且菌株没有任何毒副作用。
本发明提供的戊糖乳杆菌EAM-ZL016T的上述特性使其能够应用于制备保健食品和药品中。
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 (3)
1.一种分离自藏獒肠道的戊糖乳杆菌(La.Pentosus),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 7426。
2.一种权利要求1所述的戊糖乳杆菌,应用于降脂降胆固醇保健药品。
3.一种权利要求1所述的戊糖乳杆菌,应用于降脂降胆固醇功能的食品。
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