CN117100772A - 植物乳杆菌shy21-2在增加耐力缓解疲劳中的应用 - Google Patents
植物乳杆菌shy21-2在增加耐力缓解疲劳中的应用 Download PDFInfo
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Abstract
本发明公开了植物乳杆菌SHY21‑2在增加耐力缓解疲劳中的应用,涉及微生物技术领域,包括植物乳杆菌SHY21‑2用于制备增加耐力缓解疲劳的口服剂;植物乳杆菌SHY21‑2的保藏编号为CCTCC NO:M2020043。口服剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。本发明提供了植物乳杆菌SHY21‑2可以降低肌酸激酶CK、乳酸LA和血尿素氮BUN的含量,提高乳酸脱氢酶LDH的活性;并且,能够增加腓肠肌组织中肌糖原含量,显著上调AMPK、PGC‑1α、NRF1、TFAM的mRNA表达,上调pAMPK/AMPK、PGC‑1α蛋白表达,从而实现能够增强运动耐力缓解疲劳。并且其对胃液有着较好的耐受力,以及具有较好的小肠内胆盐耐受力,能够直接口服使用。
Description
技术领域
本发明涉及微生物技术领域,具体涉及植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用。
背景技术
运动疲劳是机体超负荷运动刺激后无法维持特定水平的一种生理现象,常常伴随着运动能力的降低,分为外周疲劳和中枢神经疲劳。适度的运动能够增强耐力并可通过一定手段恢复,但极端高强度或长时间运动会诱发多种不良身心健康问题,如肠道菌群紊乱、肠粘膜通透性增加、上呼吸道感染、氧化应激等,并最终影响竞技运动表现。因此,急需寻找一种安全的营养补充剂缓解运动疲劳,延长运动耐力。
运动性疲劳的发病机制涉及到外周肌肉疲劳和中枢神经疲劳两个方面,包括能源物质的消耗、代谢产物的累积、中枢神经的抑制等。随着“微生物肠脑轴”的提出和运动性疲劳机制研究的深入,益生菌与运动性疲劳的关系不容忽视,益生菌通过“微生物肠脑轴”影响中枢神经疲劳来缓解运动性疲劳成为可能。在2022年的保健食品功能检验于评价方法中,运动性疲劳归类于体力疲劳,开发具有缓解体力疲劳的功能保健益生菌食品刻不容缓。
发明内容
针对现有技术的上述不足,本发明提供了一种植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用。
本发明所采用的技术方案为:
提供植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的口服剂;植物乳杆菌SHY21-2的保藏编号为CCTCC NO:M2020043。
进一步的,口服剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。
进一步的,植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的食品。
进一步的,植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的保健品。
进一步的,植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的药品。
进一步的,植物乳杆菌SHY21-2由发酵牦牛酸乳中提取得到。
本发明的有益效果为:
本发明提供了植物乳杆菌SHY21-2可以降低肌酸激酶CK、乳酸LA和血尿素氮BUN的含量,提高乳酸脱氢酶LDH的活性;并且,能够增加腓肠肌组织中肌糖原含量,显著上调AMPK、PGC-1α、NRF1、TFAM的mRNA表达,上调pAMPK/AMPK、PGC-1α蛋白表达,从而实现能够增强运动耐力缓解疲劳。并且其对胃液有着较好的耐受力,以及具有较好的小肠内胆盐耐受力,能够直接口服使用。
附图说明
图1为小鼠力竭跑步时间对比图;
图2为小鼠负重游泳时间对比图;
图3为实施例6的活性测定对比图;
图4为小鼠肌糖原测定对比图;
图5为实施例8的表达量测定对比图;
图6为实施例9的占比测定对比图。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保护之列。
提供植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的口服剂;植物乳杆菌SHY21-2的保藏编号为CCTCC NO:M2020043。
口服剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。
植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的食品。
植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的保健品。
植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的药品。
植物乳杆菌SHY21-2由发酵牦牛酸乳中提取得到。
实施例1植物乳杆菌SHY21-2的分离
取四川红原的牦牛酸乳,以1%的接种量接种到脱脂乳培养基中于37℃培养,待其凝乳后进行10倍梯度稀释,依次稀释至10-7。选择4个适宜稀释度分别取100μL涂布在MRS固体平板上,37℃培养48h后,选取形态各异的单菌落采用平板划线法分离得到纯化菌株。
实施例2菌株在pH=3.0的人工胃液中存活率的测定
消化道是机体抵御外来物质侵扰的天然屏障,胃是人体重要消化器官,胃蛋白酶和胃液中强酸环境成为大多数微生物进入肠道的阻碍。通常人体中胃液pH值维持在3.0左右,食物在胃中消化1-3h,且乳杆菌只有在人体胃肠道中生存并大量繁殖才能够发挥有益作用。
将实施例1中得到的纯化菌株分离适量,在37℃培养18h,于3000r/min、15min的条件下离心收集菌体,用无菌生理盐水洗涤2次后,重悬于等体积无菌生理盐水中制成菌悬液。将所得菌悬液与人工胃液(0.2%NaCl、0.35%胃蛋白酶1︰10000,用1mol/L的HCl调节pH至3.00)按体积比1︰9混合,于37℃培养,取培养0h和培养3h的菌液采用平板涂布法测定活菌数。
并计算得到菌株存活率:其中,H3h为培养3h后菌液中的活菌数,H3h为培养0h后菌液中的活菌数,两者的单位为CFU/mL。
植物乳杆菌SHY21-2在人工胃液中存活率为91.56±3.48%,明显大于60.00%,则植物乳杆菌SHY21-2在胃液中有较好的存活率,具有较佳的胃液耐受力。
实施例3菌株对胆盐耐受力的测定
当胆盐到达一定浓度后可以通过疏水作用将细菌的细胞膜脂膜溶解,具有抗菌能力。在人体小肠中胆盐的质量浓度约为0.03-0.3%,故采用0.3%胆盐来模拟肠液筛选不同乳杆菌对胆盐耐受能力。
将实施例1中得到的纯化菌株分离适量,在37℃培养18h,以2%的接种量分别接种于含0.00%、0.30%牛胆盐的MRS-THIO液体培养基(含0.2%巯基乙酸钠的MRS肉汤)中,37℃培养18h后测定其生长率,以不接种菌液的液体培养基作为空白对照。
计算得到菌株在胆盐中的生长率:其中,为纯化菌株在0.30%牛胆盐的MRS-THIO液体培养基中培养后的菌密度,/>为纯化菌株在0.00%牛胆盐的MRS-THIO液体培养基中培养后的菌密度,/>为未接种菌株的0.30%牛胆盐的MRS-THIO液体培养基的菌密度,/>为未接种菌株的0.00%牛胆盐的MRS-THIO液体培养基的菌密度。
植物乳杆菌SHY 21-2在0.30%牛胆盐中存活率为27.60±1.09%,明显大于10.00%,则植物乳杆菌SHY21-2具有较好胆盐耐受力。
实施例4建立小鼠模型
购置湖南斯莱克景达实验动物有限公司7周龄雄性ICR小鼠,饲养于室温25±2℃、相对湿度50±5%、12h光照/12h黑暗的标准化实验室中,适应性喂养一周后开始实验。
挑选若干只健康的小鼠随机分成静坐组(SED)、静坐+乳杆菌组(SED+21-2)、跑步训练组(Running)、跑步训练+乳杆菌组(Running+21-2)4组,每组数量相等,进行7周的对照训练,跑步训练组(Running)和跑步训练+乳杆菌组(Running+21-2)采用相同的跑步训练方案,跑步训练方案如下表1所示。
表1
四组小鼠每天除饲喂基础饲料外,四组小鼠每天在相同时间点均会进行一次灌胃,其中,非乳杆菌的两组(SED、Running)使用10%脱脂乳粉进行灌胃;乳杆菌的两组(SED+21-2、Running+21-2)使用10%脱脂乳粉加上1×109CFU/kg·BW的植物乳杆菌SHY21-2进行灌胃;且进行跑步训练的两组(Running、Running+21-2)在灌胃1小时后再进行训练。
实施例5菌株提高耐力的测定
分别在实施例4的四组实验中每组随机抽取10只小鼠,对共40只小鼠分别执行一次对应的灌胃后,等待1h后进行运动耐力测试。运动耐力测试包括跑步测试和负重游泳试验。
跑步测试为:将小鼠置于动物平板跑步机(ZH-PT/5S,安徽正华生物仪器设备有限公司)中,让小鼠以14m/min的速度开始跑步,以2m/min的速度递增,直到30m/min时停止增加,并保持这个速度直到力竭,即不能持续跑10s,记录力竭时间。如图1所示,运动训练后即Running组和Running+21-2组小鼠力竭跑步时间比SED组和SED+21-2组小鼠跑步时间显著增加,且相比于SED组和Running组小鼠,摄入植物乳杆菌SHY21-2后力竭跑步时间显著增加。
负重游泳试验为:在小鼠尾部负自身重量的5%,将小鼠放入恒温水池中开始计时,小鼠使始终保持游动,直至小鼠沉于水面下8s后不能浮出水面的时间作为力竭游泳时间。如图2所示,相较于SED组,摄入植物乳杆菌SHY21-2后负重游泳时间显著增加,Running组和Running+SHY21-2组负重游泳时间显著增长。且与Running组相比,Running+21-2游泳时间显著增加。
综上可知,摄入植物乳杆菌SHY21-2后,小鼠在跑步测试和负重游泳的测试中均明显比未摄入植物乳杆菌SHY21-2的小鼠坚持时间长,证明了植物乳杆菌SHY21-2能够增加运动耐力。
实施例6对小鼠血清中CK、LA、BUN和LDH活性进行测定
肌酸激酶(CK)是肌肉损伤的重要临床生物标志。大部分CK存在于肌肉中,当损伤发生时,细胞膜通透性增加,肌肉细胞中分解CK进入到血液中,导致血液中CK升高。乳酸(LA)是厌氧条件下的糖酵解产物,LA解离产生的H+会降低肌肉和血液中的pH导致酸中毒,从而抑制肌肉收缩、干扰肌肉神经信号。血尿素氮(BUN)是蛋白质和氨基酸的代谢产物。当机体大负荷运动,糖和脂肪分解代谢产生的能量不足以供身体使用时,蛋白质和氨基酸分解代谢将会增加来补偿能量消耗。乳酸脱氢酶(LDH)能够催化丙酮酸和LA相互转化,其活性代表乳酸代谢程度。
分别在实施例4的四组实验中随机抽取相同数量的小鼠,抽取小鼠的血清,分别使用北京索莱宝科技有限公司货号BC1145试剂盒、货号BC2235试剂盒、货号BC1535试剂盒和货号BC0685试剂盒测定小鼠血清中CK、LA、BUN和LDH的浓度。如图3所示,小鼠摄入植物乳杆菌SHY21-2后,CK、LA、BUN的浓度降低,而LDH的浓度增加。即摄入植物乳杆菌SHY21-2后,能够清除血清中CK、LA和BUN含量,并提高LDH活性,增强运动耐力缓解疲劳。
实施例7对小鼠肌糖原进行测定
在长时间剧烈运动中,糖原被快速分解为身体提供能量。高水平的糖原含量储存可以增强剧烈运动至疲劳的时间。因此,糖原水平直接决定了运动耐力。
分别在实施例4的四组实验中随机抽取相同数量的小鼠,使用南京建成生物工程研究所序号A043-1-1的肝/肌糖原测定试剂盒测定小鼠腓肠肌中肌糖原含量。如图4所示,小鼠摄入植物乳杆菌SHY21-2后,腓肠肌中肌糖原含量明显增加,从而增加了小鼠的运动耐力。
实施例8对小鼠腓肠肌中的AMPK、PGC-1α蛋白、NRF1蛋白和TFAM蛋白进行测定
AMP活化蛋白激酶(AMPK)是一种异源的丝氨酸/苏氨酸蛋白激酶三聚体复合物,能够促进脂肪酸氧化并加速糖原生成。AMPK磷酸化能够直接调节过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC-1α),促进肌纤维从Ⅱ型(快肌)转化为更耐疲劳Ⅰ型(慢肌),从而为肌肉收缩提供能量。PGC-1α蛋白能够激活跨膜转录因子,即核呼吸因子1(NRF1蛋白)。NRF1激活后进一步上调线粒体转录因子A(TFAM蛋白)表达,从而启动线粒体DNA复制和转录。
使用实时荧光定量PCR的定量方法对小鼠进行测量,具体操作为:采用TRIZOL试剂(Invitrogen,美国)分别提取实施例4中四组实验小鼠腓肠肌总RNA,用超微量分光光度计测定总RNA的纯度和浓度,将每个样本的RNA浓度调整到同一水平(1μg/μL);然后取1μg/μL的RNA样品1μL,加入1μL的通用引物oligo(primer dT)和10μL无菌超纯水,混合后于65℃反应5min,反应完成后,在反应体系中加入1μL的Ribolock RNase抑制剂、2μL的100mM dNTPmix、4μL的5×Reaction buffer和1μL的Revert Aid M-mu/v RT,混合均匀后,在42℃、60min和70℃、5min条件下合成cDNA;接着以下表2所述引物序列对目标基因进行反转录和扩增,在八联管中将1μL的cDNA、10μL的Master Mix、1μL的上游引物、1μL的下游引物和7μL的ddH2O混匀后,盖上八联管盖,简短离心心后放于StepOnePlus Real-Time PCR实时荧光定量仪上,95℃预变性15min后,分别在95℃、15s,60℃、1min,72℃、30s条件下进行40次循环;最后以DAPDH作为持家基因,通过2-ΔΔCT计算目的基因的相对表达量。
表2
测定结果如图5所示,由图可知,与未运动组小鼠相比,运动训练后小鼠腓肠肌AMPK、PGC-1α、NRF1、TFAM均显著提升;并且,摄入植物乳杆菌SHY21-2的小鼠的AMPK、PGC-1α、NRF1、TFAM表达相较于未摄入的小鼠,表达也有提升。从而证明摄入植物乳杆菌SHY21-2能够提高运动耐力。
实施例9对小鼠腓肠肌pAMPK和PGC-1α蛋白进行测定
采用Western Blot法研究植物乳杆菌SHY21-2对小鼠腓肠肌线粒体生物合成表达的作用。图6示出了植物乳杆菌SHY21-2对腓肠肌中AMPK、pAMPK和PGC-1α的蛋白表达的影响,由图可知,小鼠摄入植物乳杆菌SHY21-2后,SED+21-2组和Running+21-2组比SED组和Running组小鼠pAMPK/AMPK表达量有上升趋势,且PGC-1α显著提升。说明植物乳杆菌SHY21-2能够显著提高pAMPK/AMPK、PGC-1α蛋白表达。
Claims (6)
1.植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的口服剂;植物乳杆菌SHY21-2的保藏编号为CCTCCNO:M2020043。
2.根据权利要求1所述的植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述口服剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。
3.根据权利要求1所述的植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的食品。
4.根据权利要求1所述的植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的保健品。
5.根据权利要求1所述的植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述植物乳杆菌SHY21-2用于制备增加耐力缓解疲劳的药品。
6.根据权利要求1所述的植物乳杆菌SHY21-2在增加耐力缓解疲劳中的应用,其特征在于,所述植物乳杆菌SHY21-2由发酵牦牛酸乳中提取得到。
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