CN113215035A - 长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 - Google Patents
长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用 Download PDFInfo
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Abstract
本发明提供长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,涉及双歧杆菌技术领域,由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris‑HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。本发明,可以缓解D-半乳糖衰老小鼠的记忆障碍,增加了小鼠血清中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH‑PX)活性,降低了血清中脂质过氧化物丙二醛的浓度,减少细胞的氧化速度,有效提高单个细胞的寿命。
Description
技术领域
本发明涉及双歧杆菌技术领域,尤其涉及长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用。
背景技术
双歧杆菌是一个细菌属名。双歧杆菌属(Bifidobacterium)是一种革兰氏阳性、不运动、细胞呈杆状、一端有时呈分叉状、严格厌氧的细菌属,广泛存在于人和动物的消化道、阴道和口腔等生境中。双歧杆菌属的细菌是人和动物肠道菌群的重要组成成员之一。一些双歧杆菌的菌株可以作为益生菌而用在食品、医药和饲料方面。
衰老几乎是所有生物都会经历的过程,衰老的特征在于随着时间推移,机体细胞、组织、器官功能逐渐衰退,同时伴随着认知和记忆功能减退。随着人口的老龄化,包括神经退行性疾病和神经精神疾病在内的脑功能障碍的发生率预计会迅速增加。寻找可行性的抗衰老药物成为对老龄化问题的研究热点。目前关于衰老的机制主要有氧化应激、细胞凋亡、DNA损伤、免疫、炎性衰老等。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用。
为了实现上述目的,本发明采用了如下技术方案:长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L 过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
优选的,所述MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,所述长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
优选的,所述长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以 1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
优选的,所述粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
优选的,所述长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
优选的,所述长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
优选的,所述双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D 的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
与现有技术相比,本发明的优点和积极效果在于,
1、本发明中,涉及的长双歧杆菌和动物双歧杆菌来源于百岁老人粪便样本中分离得到的,该菌株经菌种测序分析,其序列如SEQIDNO.1所示,将得到的序列经NCBI核酸序列对比,结果显示菌株为双歧杆菌,命名为长双歧杆菌 BAMA-B05/BAu-B1024和动物双歧杆菌LPL-RH。所述长双歧杆菌和动物双歧杆菌,它可以缓解D-半乳糖衰老小鼠的记忆障碍,增加了小鼠血清中超氧化物歧化酶 (SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,降低了血清中脂质过氧化物丙二醛的浓度,减少细胞的氧化速度,有效提高单个细胞的寿命。
附图说明
图1为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌耐酸结果;
图2为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌耐胆盐结果;
图3为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌抗氧化结果;
图4为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌抑菌结果;
图5为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中长双歧杆菌和动物双歧杆菌HT-29细胞黏附结果;
图6为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠旷场实验测试结果;
图7为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠爬杆实验测试结果;
图8为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠巴恩斯迷宫实验测试结果;
图9为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠海马中SOD、GSH-Px、MDA测定实验结果;
图10为本发明提出长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用中实验小鼠β-半乳糖苷酶染色实验结果。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和实施例对本发明做进一步说明。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开说明书的具体实施例的限制。
实施例1:本发明提供了长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,由以下重量份的原料制备而成:MRS液体培养基,去离子水; 0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液, 6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以 1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
长双歧杆菌和动物双歧杆菌的获得及其鉴定
一、长双歧杆菌和动物双歧杆菌的分离纯化
百岁老人的粪便采用50%的甘油保存,使用无菌PBS(1%,w/v)稀释,涂布于MRS琼脂平板上,37℃静置培养24h后,根据菌落形态,在MRS琼脂平板上反复划线纯化,直到固体培养基上长出纯单一菌落,将分离的乳酸菌连续传代3次,然后以50%甘油与菌液1:1混合保存于-80℃。
二、长双歧杆菌和动物双歧杆菌的鉴定
将挑选的菌落连续培养后,取部分菌液离心,按照天根细菌DNA提取试剂盒说明书提取DNA,然后送于公司,由公司进行16SrDNA序列扩增、纯化,然后测序。
双歧杆菌形态学上主要定义为两种形态,分叉形态定义为Ⅰ型,命名为乳杆菌;杆状则定义为Ⅱ型,命名为副分叉乳杆菌。在肠道内,双歧杆菌多呈直杆状,极少以分叉状、弯杆状的形态呈现。初次分离时,由于相应的培养条件不足,通常呈现为Ⅰ型,菌株形态主要包括不分叉、分叉两种形态,其中,分叉状呈现为V字形、Y字形,但于后续的培养中,分叉状通常呈现为弯曲、杆状。显微镜下菌体呈Y字形、勺状、V字形、弯曲状、棍棒状等,经由一定程度培养后,将转变为Ⅱ型,且长时间、稳定性的遗传。因此,临床上认为Ⅱ型向Ⅰ型的转变是由于对环境、温度等不适应造成的退化,而Ⅰ型向Ⅱ型的转变则是由于可更好地适应环境、温度等而出现的进化。
实施例2:长双歧杆菌和动物双歧杆菌的耐酸实验
从-80℃取出长双歧杆菌和动物双歧杆菌接种于MRS培养基中,37℃培养过夜,再按2%接种至新鲜MRS培养基中,37℃培养24h,用PBS缓冲液梯度稀释101, 103,105倍(取100ul加入到900ul的PBS缓冲液,再从中取10ul加入到990ul 的PBS中,再从中取10ul加入到990ul的PBS中),4-5000rpm离心10min,弃掉上清。加入pH为3、5、7、9的PBS缓冲液,放置4小时,混匀各取10uL涂板, 37℃培养箱培养过夜,活菌计数。结果如图1所示。
实施例2:长双歧杆菌和动物双歧杆菌的耐胆盐实验
从-80℃取出长双歧杆菌和动物双歧杆菌接种于MRS培养基中,37℃培养过夜,再按2%接种分别至0%胆盐、0.1%胆盐、0.3%胆盐和0.5%胆盐的MRS培养基中,37℃培养24h,用PBS缓冲液梯度稀释102,104,106,108倍,混匀各取 10uL涂板,37℃培养箱培养过夜。活菌计数。结果如图2所示。
双歧杆菌是一种重要的肠道有益微生物。双歧杆菌作为一种生理性有益菌,对人体健康具有生物屏障、营养作用、抗肿瘤作用、免疫增强作用、改善胃肠道功能、抗衰老等多种重要的生理功能。
人体肠道中定植着大量的微生物。肠道微生物与人体健康与疾病之间存在着十分密切的关系。根据所知道的肠道微生物对人体健康的影响,肠道微生物可分为有益、无害和有害3大类。
实施例3:长双歧杆菌和动物双歧杆菌的抗氧化实验
溶液配制:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L 硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0 的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC, 0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
灭菌:以上的溶液
活化:各取100uL的双歧杆菌液加到装有5mL的MRS培养基的玻璃试剂管内, 37℃培养箱培养过夜。第二天,离心,取上清液。
①DPPH自由基清除能力的测定
各取2mL的上清液加到玻璃试管内,再加2mL配制好的DPPH甲醇溶液,黑暗下室温反应30分钟,离心取上清。另一根玻璃试管内加入2mL的去离子水和2mL 的DPPH甲醇溶液作为对照。517nm测OD值。记录对照组和实验组数据。
DPPH自由基清除率=[1-A517(样品)/A517(空白)]×100%
②羟基自由基清除能力的测定
各取1mL的细菌上清液加到玻璃试管内,加入1mL的硫酸亚铁溶液,1mL的过氧化氢溶液,1mL的水杨酸溶液,静置30分钟。另一根玻璃管内加入上述溶液,并加入1mL的去离子水。30分钟后510nm处测吸光度。记录对照组和实验组数据。
羟基自由基清除率=[1-A510(样品)/A510(空白)]×100%
③超氧自由基清除能力
取0.5m将培养的不同菌分别作涂片(注意涂片切不可过于浓厚)、干燥、固定。固定时用甲醇固定。
(2)初染:加草酸铵结晶紫1滴,约1min,使用纯水对样本进行清洗。
(3)媒染:加碘液覆盖约1min,使用纯水对样本进行清洗。
(4)脱色:将载玻片上面的水甩净,并衬以白背景,用95%酒精滴洗至流出酒精刚刚不出现紫色时为止,约20-30s,立即用水冲洗。
(5)复染:用番红染1-2min,使用纯水对样本进行清洗。
(6)镜检:待样本干燥后,置油镜下观察,革兰氏阴性菌呈红色,革兰氏阳性菌呈紫色;以分散开的细菌的革兰氏染色反应为准。
在正常情况下,人体内的肠道微生物形成了一个相对平衡的状态。一旦平衡受到破坏,如服用抗生素、放疗、化疗、情绪压抑、身体衰弱、缺乏免疫力等,就会导致肠道菌群失去平衡,某些肠道微生物如产气荚膜梭菌等在肠道中过度增殖并产生氨、胺类、硫化氢、粪臭素、吲哚、亚硝酸盐、细菌毒素等有害物质,从而进一步影响机体的健康。
实施例6:长双歧杆菌和动物双歧杆菌菌株制剂的制备
A.将-80℃保存的长双歧杆菌和动物双歧杆菌菌株接种于10mLMRS培养基, 37℃静置培养16小时;
B.然后按1:100接种于新鲜培养基37℃震荡或静置培养24小时左右;
C.采用大体积离心机离心,离心前将离心筒清洗干净并用75%酒精或紫外消毒,4700rpm/20min;
D.弃上清,PBS重悬后采用50mL离心管,6000rpm/5-10min;
E.弃上清,称重菌泥重量,按1:1加入1%灭菌的海藻酸钠重悬菌泥,再按 1:1体积加入16%灭菌的脱脂牛奶重悬,-20℃冻存后再置于-80℃冻存24小时左右,至样品完全冷冻;
F.置于真空冷冻干燥机,24-48小时,至样本完全冻干成粉末;
G.研钵使用前清洗干净并消毒,将冻干粉与少许红薯粉一起研磨;
H.称取0.01g混合物溶解于1mL无菌PBS梯度稀释后,点板于相应固体培养基中,待菌液晾干后,37℃倒置培养过夜;
I.计数,根据数量进行混合包装,干酪乳杆菌菌株制剂总数不少于5× 109cfu/g。
双歧杆菌和乳酸菌等有益细菌则能抑制人体有害细菌的生长,抵抗病原菌的感染,合成人体需要的维生素,促进人体对矿物质的吸收,产生醋酸、丙酸、丁酸和乳酸等有机酸刺激肠道蠕动,促进排便,防止便秘以及抑制肠道腐败作用、净化肠道环境、分解致癌物质、刺激人体免疫系统,从而提高抗病能力等方面有着重要作用。具体地,双歧杆菌的有益作用包括以下几个方面。
治疗慢性腹泻与抗生素相关性腹泻
双歧杆菌具有调节肠道菌群的作用。通过用双歧杆菌对慢性腹泻患者临床观察研究表明,在服药两周以后,患者大便次数、形状异常,临床症状消失,对总有效率为90.3%,复发率低。许多国内医院已将双歧杆菌制剂作为治疗慢性腹泻的首选药物。此外,双歧杆菌还可以治疗因过量使用抗生素而导致的抗生素相关性腹泻疾病。有人采用双歧杆菌制剂治疗伪膜性肠炎380例,临床总治愈率无明显差异,但临床副作用和复发率均明显降低。双歧杆菌对儿童急慢性腹泻具有很好的治疗作用。
营养作用
双歧杆菌缺少醛酶、葡萄糖,因此其分解代谢途径不同于乳酸菌。双歧杆菌最主要产物主要包括乳酸、乙酸等,可改善机体pH值,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌可以通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。临床上,机体在缺乏乳糖酶的情况下,摄入的乳糖或纯牛奶不能被消化吸收进血液,仍然留在肠道内,肠道细菌就会在发酵分解乳糖的过程中产生大量气体,造成腹泻、胃胀和气胀等症状。动物生产过程中,豆粕为最主要的蛋白来源,但由于寡糖不易消化,其使用受到一定的限制,主要为棉籽糖、水苏糖。而双歧杆菌含有活性较多数细菌高的乳糖酶,这种酶能发酵乳糖产生半乳糖。因此,乳糖酶缺乏者,饮用经双杆菌发酵的乳制品,既可获得乳制品中丰富的营养,又免受胃肠道病痛之苦。
拮抗作用
COLLADO等研究表明,当两歧双歧杆菌(S17)与致病性大肠埃希菌、福氏志贺菌、沙门菌等肠道致病菌共同竞争培养时,对HeLa细胞的黏附能力均明显下降。双歧杆菌除与病原菌争夺营养物质和空间位置外,还可通过其代谢产物,以及产生抗生素、细菌素等阻止病原菌的生长。双歧杆菌可以阻断致病菌和毒素的特异性结合位点,可能是由胞外酶能够降解肠上皮细胞表面多糖来实现的。
通便功能
便秘是指粪便干燥难解或排便次数减少。临床上根据病因可分为功能性便秘和器质性便秘,双歧杆菌对功能性的便秘有明显作用。有研究报道,双歧杆菌在人体肠道定植数量随人年龄和健康状态的变化而改变,母乳喂养儿童肠道中的双歧杆菌可占肠道总菌数的91%,而老年人的肠道菌群中双歧杆菌数量下降较明显,很多老年人口服双歧杆菌微生态制剂可明显改善因缺乏双歧杆菌而引发的便秘。
增强免疫力
双歧杆菌具有调节免疫功能的作用,主要通过对肠道黏膜的刺激,激活肠道黏膜的免疫系统,使其产生抗体、细胞因子,进而更好地提高肠道黏膜的免疫、抗感染能力。
抗肿瘤的作用
双歧杆菌具有抗结肠癌作用,可能是通过影响肠道菌群代谢、提高宿主免疫应答;黏附及降解潜在致癌物,预防肠道癌症;改变肠道菌群;产生抗癌抗诱变物质;提高宿主的免疫应答;影响宿主的生理活动来实现。
抗衰老功能
人体的衰老往往从肠道老化先表现出来。双歧杆菌在维持机体肠道健康和减缓肠道老化方面具有重要作用。VERENA等将含有乳酸双歧杆菌牛乳添加至人体肠道细胞DNA的培养基中,可使肠道细胞避免氧化损伤,具有延缓肠道衰老作用。研究证明,双歧杆菌能明显增加血液中过氧化物歧化酶的含量及其生物活性,有效促进机体内自由基的清除,将体内有害物质毒性降低90%,抑制血浆脂质过氧化反应,延缓机体衰老。
实施例7:长双歧杆菌和动物双歧杆菌对小鼠的抗衰应用
A.实验菌株的制备:分别将长双歧杆菌和动物双歧杆菌按1%(v/v)接种于 MRS培养基,37℃培养过夜,至OD600=0.6,8000rpm离心3min收集菌体,用无菌生理盐水清洗2次,再用含0.1%的明胶生理盐水重悬,调整菌数为1× 109cfu/mL;
B.实验动物及分组治疗:将6周龄的Balb/c雄性小鼠55只(购买自湖南思湖王实验动物有限公司),适应性饲养一周,标记称重分组后开始实验,其中M 组、A组、L组、LA组小鼠腹腔注射D-半乳糖(150mg/kg/d),连续注射12周,同时C组(连续灌胃含0.1%明胶生理盐水治疗,n=11),M组(连续灌胃含0.1%明胶生理盐水治疗,n=11),A组(连续灌胃长双歧杆菌1×109cfu/只/天,n=11), L组(连续灌胃动物双歧杆菌1×109cfu/只/天,n=11),LA组(连续灌胃长双歧杆菌和动物双歧杆菌1×109cfu/只/天,n=11)。12周以后,在进行旷场实验、爬杆实验以及巴恩斯迷宫实验测试后处死小鼠,并收集小鼠脑组织。
C.旷场实验:将每只小鼠置于(90cm×90cm×30cm)暗箱活动室10分钟,使用activitymonitor视频跟踪软件测量小鼠运动的总距离及去到中央区域的时间。为了最大程度地减少气味干扰,每次运行后都要同75%乙醇清洁暗室。结果如图6所示。
D.爬杆实验:老鼠被放置在直径为10毫米高度为55厘米的粗糙木杆顶部。每只老鼠接受下杆动作训练3天(3-5分钟/次/天),只有那些在预试验(通常在试验前2.5h进行)中运动时间<18s的老鼠才能继续进行后续实验。结果如图 7所示。
E.巴恩斯迷宫实验:实验开始前一天,将动物单个从目标洞置于目标箱内适应4min。将动物置于迷宫中央的塑料圆桶(直径20cm,高27cm)内限制活动 5s。移开圆桶,启动计时器,实验者在挡帘后进行观察。动物四肢均进入目标箱,则计为一次逃避(escape),并让动物在箱内停留30s。每一动物一次最多观察4min。在此期间如果动物仍然找不到目标箱,则将动物从迷宫移开,放入目标箱内并停留30s。利用这一间隙清洁迷宫。动物每天训练两次,连续5~6d。从第二次训练开始,每次训练之前将迷宫随机转动一至数个洞的位置,但目标箱始终固定在同一方位。这样做的目的是防止动物依靠气味、而非凭借记忆来确定目标洞的位置。实验记录以下参数:探究任何一个洞的潜伏期、到达目标箱的潜伏期和每只动物的错误次数(一次错误定义为动物把头伸向或探究任何一个非目标洞,包括专注于探究同一个非目标洞)。结果如图8所示。
F.小鼠海马组织SOD活力的测定:
(1)分光光度计预热30min以上,调节波长至560nm,蒸馏水调零;
(2)测定前将试剂一、三和五37℃水浴5min以上;
(3)把试管编号,分别编为测定管、对照管、空白管1和空白管2;试管编号后按下表加入试剂:
充分混匀,37℃水浴30min后,置于1mL玻璃比色皿测定560nm下的吸光度,分别记为A测定、A对照、A空1、A空2,计算△A测定=A测定-A对照,△A 空白=A空1-A空2。
(4)SOD活性计算:
①抑制百分率的计算:抑制百分率=(ΔA空白-ΔA测定)÷ΔA空白×100%
②SOD酶活性单位:在上述黄嘌呤氧化酶藕联反应体系中抑制百分率为50%时,反应体系中的SOD酶活力定义为一个酶活力单位。
③SOD酶活性计算:血清(浆)SOD活性(U/mL)=[抑制百分率÷(1-抑制百分率)×V反总]÷V样×F=11.4×抑制百分率÷(1-抑制百分率)×F。结果如图 9所示。
G.小鼠海马组织MDA的测定:采用硫代巴比妥法检测血浆中MDA的浓度,操作步骤如下:
(1)把试管编号,分别编为标准管、标准空白管、测定管和测定空白管,试管编号后按下表加入试剂:
(2)漩涡混匀器混匀,试管口用保鲜薄膜扎紧,用针头刺一小孔,95℃水浴 40min;
(3)取出后流水冷却,然后4000r/min,离心10min,使沉淀完全;
(4)取上清,吸取上清比色时用移液器吸取上清加入比色皿中,尽量避免倾倒,以免沉淀进入比色皿,影响吸光度;
(5)把比色皿置于532nm处,1cm光径,蒸馏水调零,测各管吸光度值;
(6)计算血浆中MDA的含量:血浆中MDA含量(ng/ml)=(测定管吸光度-测定空白管吸光度)/(标准管吸光度-标准空白管吸光度)×标准品浓度 (10ng/ml)×样品测试前稀释倍数。结果如图9所示。
H.小鼠海马组织GSH-Px的测定:
(1)酶促反应:
(2)血清中GSH—PX酶活力的计算:规定每O.1ml血清在37℃反应5分钟,扣除非酶促反应作用,使反应体系中GSH浓度降低19mol/L为一个酶活力单位;血清GSH-PX活力=(非酶管OD值.酶管OD值)÷(标准管OD值-空白管OD 值)×标准品浓度(20umol/L)×稀释倍数(6)×样本测试前稀释倍数。结果如图9所示。
I.小鼠海马组织β-半乳糖苷酶染色阳性水平的测定行为学实验后处死小鼠,各组解剖并保藏三只小鼠的海马组织。制备海马组织冷冻切片(6μm;-25℃),切片用PBS洗涤两次,在室温下用固定液固定15min,在避光和隔绝CO2情况下,37℃用X-Gal染色液染色24小时,切片用PBS洗涤,孵育后在明视野显微镜下观察。使用ImageJImageProPlus软件进行定量图像分析。结果如图10所示。
序列表
应用方法:促进体内双歧杆菌增殖的方法有两种。第一种办法是喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂。此外,还可以摄入含有双歧因子的产品来促进人体自身固有双歧杆菌的生长繁殖。所谓双歧因子,主要是指些功能性的非消化性的低聚糖,这些低聚糖可以选择性地促进双歧杆菌的增殖。具有使双歧杆菌增殖作用的低聚糖主要有异麦芽低聚糖、低聚果糖、低聚半乳糖、大豆低聚糖、棉子糖混合乳蔗糖、低聚木糖等。
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域,但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
Claims (7)
1.长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:由以下重量份的原料制备而成:MRS液体培养基,去离子水;0.2mmol/LDPPH甲醇溶液;2mmol/L硫酸亚铁溶液,6mmol/L过氧化氢溶液,6mmol/L水杨酸溶液;150mmol/LpH=8.0的Tris-HCL溶液,1.2mmol/L邻苯三酚溶液;0.4%的硫酸亚铁溶液,1%的VC,0.2mol/L的氢氧化钠溶液,10%的三氯乙酸溶液,0.1%邻二氮菲溶液;pH=6.6的磷酸缓冲液,1%铁氰化钾溶液,0.1%的三氯化铁溶液。
2.根据权利要求1所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述MRS液体培养基内部接种有长双歧杆菌和动物双歧杆菌,所述长双歧杆菌和动物双歧杆菌均提取与百岁老人的粪便中分离的耐酸、耐胆盐和抑菌的益生菌株。
3.根据权利要求1所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌的制备方法包括:
步骤一:取定量的百岁老人的粪便加入量筒中,并采用50%比例的甘油进行封存保存;
步骤二:取出一定量的粪便样本至托盘上并通过无菌PBS(1%,w/v)进行稀释,将稀释后的样本涂抹在MRS琼脂平板上;
步骤三:将处理后的MRS琼脂平板放置于无菌室中,并设置室内温度至37℃,培养24小时,并逐步观察MRS琼脂平板上的菌落情况;
步骤四:观察MRS琼脂平板上的菌落状态,使用消毒后的划针对菌落进行划线纯化操作;
步骤五:取上述操作中菌落中的样本并再次进行接种,并对接种后的样本再次进行接种操作,重复接种三次,并提取操作样本中的菌液使其与50%甘油以1:1的比例搅拌混合,将样本存放在-80℃;
步骤六:即可对样本进行后续操作。
4.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述粪便取固体状,并采用玻璃棒捣碎搅拌,将内部的大颗粒状杂物剔除。
5.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌每日补充量各为109CFU/mL,可通过喝含双歧杆菌的酸奶或饮料来补充双歧杆菌,也可直接摄入含有双歧杆菌的微生态制剂进行补充。
6.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述长双歧杆菌和动物双歧杆菌可进一步制成流体乳品、饮料或益生菌菌片。
7.根据权利要求2所述的长双歧杆菌和动物双歧杆菌的制备及其在抗衰老方面的应用,其特征在于:所述双歧杆菌最主要产物包括乳酸、乙酸等,促进铁和维生素D的吸收并提高磷、铁、钙的利用率;双歧杆菌通过磷蛋白磷酸酶分解α-酪蛋白,促进蛋白吸收。
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