CN111849836B - 一种具有抗氧化的鼠李糖乳杆菌及其应用 - Google Patents
一种具有抗氧化的鼠李糖乳杆菌及其应用 Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
本发明属于微生物技术领域，具体涉及一种具有抗氧化的鼠李糖乳杆菌及其应用。所述鼠李糖乳杆菌（Lactobacillus rhamnosus）菌株是从新疆天然发酵老酸奶中分离获得的具有耐酸、耐胆盐的益生菌菌株；该菌株已保藏于中国普通微生物菌种保藏中心，保藏号：CGMCC No.20012。本发明鼠李糖乳杆菌除具备一般乳酸菌的益生作用，还具有抗氧化活性，能提高线虫寿命。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一株分离自新疆自然发酵老酸奶的具有抗氧化、延长线虫寿命的鼠李糖乳杆菌及其在抗氧化食品、保健品应用。
背景技术
衰老是指在生命过程中,随着时间的推移,机体出现的组织形态发生退行性改变，生理功能的降低以及心理功能的衰退，且常伴随着心脏病、高血压、高血脂等多种疾病。目前，全球总人口60多亿，预测到2025年全球人口将达到80亿，届时全球将步入老龄化社会，人们将面对两大健康难题——衰老和疾病。“人之情,莫不恶死而乐生”(《灵枢·师传》),因此长寿是每个人的愿望。
随着世界范围内,特别是我国人口老龄化趋势的不断发展,抗衰老这一世界性医学课题在我国也日益受到关注。近年来关于衰老的研究以及根据衰老的机制研究抗衰老的药物和食品已成为当前生命科学及医学研究领域的热点。氧化是肌肤衰老的最大威胁,饮食不健康、日晒、压力、环境污染等都能让肌肤自由基泛滥,从而产生面色黯淡、缺水等氧化现象,都是身体产生氧化的“罪魁祸首”。在正常情况下，人体内的自由基是处于不断产生与清除的动态平衡之中。自由基产生过多或清除过慢,它通过攻击生命大分子物质及各种细胞,会造成机体在分子水平、细胞水平及组织器官水平的各种损伤,加速机体的衰老进程并诱发各种疾病。人体在不可避免地产生自由基的同时，也在自然产生着抵抗自由基的抗氧化物质,以抵消自由基对人体细胞的氧化攻击。研究证明，人体的抗氧化***是-一个可与免疫***相比拟的、具有完善和复杂的功能的***,机体抗氧化的能力越强，就越健康,生命也越长。机体的抗氧化作用，可保持自由基的产生与消除的平衡,对预防疾病与抗衰老等有重要意义。
最被认可的衰老的自由基学说认为生物体衰老过程是机体组织细胞不断产生自由基积累结果,自由基是正常代谢的中间产物,反应能力强,使细胞中多种物质发生氧化，损害生物膜。还能使蛋白质、核酸等大分子交联,影响正常生物功能,增加衰变,导致体内抗氧化酶活性降低,抗氧化能力下降,最终引起机体衰老。抗氧化作用是抗衰老的重要指标和途径。
随着对乳酸菌研究的深入，发现一些乳酸菌具有调节肠道菌群稳态，降低血清中胆固醇含量、抗菌、增强免疫外，越来越多的研究表明乳酸菌更是天然的抗氧化剂，一些乳酸菌具有抗氧化的能力，能够清除肠道内的自由基，帮助机体维持自由基处于相对稳定的状态。乳酸菌通过产生抗氧化活性物质，清除活性氧自由基，抑制脂质过氧化及螯合金属离子，调控宿主细胞氧化应激相关细胞通路，以及氧化损伤修复***发挥抗氧化能力。
理想的可以作为益生菌的菌株应具有以下特性：对宿主无害，不易与病原菌发生杂交且不易产生突变，能耐酸、耐胆盐，不具有耐药基因，能够在肠道中定植以及能在产品中存货等。
本发明针对上述问题，从传统发酵乳制品老酸奶中分离出对酸和胆盐的耐受，且能在肠道中生存，并具有抗氧化、延长线虫寿命的菌株，将其应用抗氧化延长线虫寿命的食品、保健品。
发明内容
本发明的目的在于提供一种鼠李糖乳杆菌菌株，所述鼠李糖乳杆菌菌株是从新疆自然发酵老酸奶中分离的能耐酸、耐胆盐的具有益生菌特性的菌株，并具有抗氧化、延长线虫寿命的特性。
鼠李糖乳杆菌(Lactobacillus rhamnosus)在中国微生物菌种保藏管理委员会普通微生物中心保藏，北京市朝阳区北辰西路1号院3号，保藏时间2020.6.3，保藏号：CGMCCNo.20012。
本发明的的另一目的在于所述鼠李糖乳杆菌在抗氧化、延长线虫寿命的作用。
所述的鼠李糖乳杆菌在制备抗氧化或延长线虫寿命的食品、保健品中的应用。
附图说明
图1本发明鼠李糖乳杆菌在MRS平板上的菌落形态；
图2本发明鼠李糖乳杆菌质谱鉴定图；
图3本发明鼠李糖乳杆菌延长线虫寿命。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明，本发明不受这些实力的限制。
实施例1：鼠李糖乳杆菌(Lactobacillus rhamnosus)在中国微生物菌种保藏管理委员会普通微生物中心保藏，北京市朝阳区北辰西路1号院3号，保藏时间2020.6.3，保藏号：CGMCC No.20012。
1.样品采集和乳酸菌的分离鉴定
样品为新疆牧民采用传统方法自然发酵的老酸奶，采集于中国新疆伊犁州，样品置于装有冰袋的泡沫盒中运输至实验室用于乳酸菌分离。
将样品进行倍比稀释后涂布于含有碳酸钙的MRS平板上。置于厌氧袋中，37℃培养48-72小时，待长出菌落，用接种环挑取明显溶碳酸钙的菌落转接到新的MRS平板上，获得纯培养。
将获得的纯培养物接种到MRS液体培养基制成供试菌液，吸取菌悬液10μL接种于5mLpH值为3.5的MRS液体培养基，置于37℃条件下培养24h；吸取培养液液1.0mL与9.0mL pH值为3.0的人工胃液[制备法：NaCl0.2％、胃蛋白酶(pepsin，sigma)0.35％,用1mol/L HCl调整pH值为3.0后,0.45um滤膜过滤除菌备用]混合后，置于37℃条件下进行培养，分别在开始(0h)和3h后取样用MRS琼脂培养基测定活菌数，并计算菌株存活率。通过以上方法筛选出具有高耐酸性生长和耐胃酸的乳杆菌菌株。
将获得的纯培养物经乳杆菌生化鉴定盒(广东环凯微生物科技有限公司)进行生化鉴定，并通过质谱鉴定，获得一株鼠李糖乳杆菌。
乳杆菌的鉴定：分离纯化的乳杆菌，经生化特性分析和质谱分析的方法鉴定，其属于鼠李糖乳杆菌，其在MRS平板上的菌落形态如图1所示，乳杆菌生化鉴定见表1，质谱检测生成的蛋白指纹图谱如图2所示，其结果与鼠李糖乳杆菌图谱对比的Score Value为1.85，可确定分离得到的菌株为鼠李糖乳杆菌。
本发明的鼠李糖乳杆菌(Lactobacillus rhamnosus)除了具有一般乳杆菌所具备的对人体健康有益的作用外，该菌株还具有抗氧化、延长线虫寿命的作用，可用于抗氧化食品、保健品的生产。
表1分离的乳杆菌生化鉴定结果
实施例2：鼠李糖乳杆菌抗氧化作用和延长线虫寿命作用
将待测乳酸菌(实施例1获得)的新鲜培养物接种到液体培养基中，37℃过夜培养。将含菌液体培养基在12000r/min下离心10min，上清液经0.22μm滤膜过滤，获得无细胞上清液。
1.DPPH自由基清除活性
取0.4mL无细胞上清液与1.6mL新鲜配制的0.2mM DPPH无水乙醇溶液混合，避光反应25分钟。对照组用等量MRS培养基代替无细胞上清液。反应结束后，测OD517的值。对照组记为A0，实验组记为A1。则DPPH自由基清除率为：
Scavenging rate＝(A0-A1)/A0×100％
2.羟基自由基清除活性
反应体系包括水杨酸乙醇溶液(5mM)，硫酸亚铁溶液(5mM)，过氧化氢(3mM)。各取1.4mL上述溶液，与100μL无细胞上清液混合。对照组用等量MRS培养基代替无细胞上清液。室温反应50min，8000r/min下离心1min后，用紫外分光光度计测OD510的值。对照组记为A0，实验组记为A1。则羟基清除率为:
Scavenging rate＝(A0-A1)/A0×100％
3.超氧阴离子清除活性
取1mL无细胞上清液和2.8mL Tris-HCl缓冲液(0.1M，pH8.0)混合，再加入0.1mL邻苯三酚(0.05M)，室温条件下避光反应4min。对照组用等量MRS培养基代替无细胞上清液。加入80μL盐酸溶液(0.1M)终止反应，测定OD325的值。对照组记为A0，实验组记为A1。则超氧阴离子清除率为：
Scavenging rate＝(A0-A1)/A0×100％
结果如表2所示：
表2鼠李糖乳杆菌自由基清除能力
鼠李糖乳杆菌延长线虫寿命能力测定：以秀丽隐杆线虫为动物模型进行寿命延长测定。将同步化的线虫在NGM平板上20℃培养2天至L4期。各取50μL过夜培养的OP50和乳酸菌菌液(浓度均为5×108CFU/mL)涂布至补充5-氟脱氧尿苷(终浓度为70mM)的NGM平板(4cm)上，37℃下过夜培养。每个平板上转移10～20条线虫，每24小时在显微镜下统计线虫的存活数，直至线虫全部死亡。每组实验平行三次。结果显示鼠李糖乳杆菌能显著提高线虫寿命，结果如表3和图3所示。
表3.鼠李糖乳杆菌对线虫寿命的影响
结论：鼠李糖乳杆菌(Lactobacillus rhamnosus)CGMCC No.20012具有抗氧化活性，并能提高线虫寿命。
实施例3：含有鼠李糖乳杆菌CGMCC No.20012食品、保健品的制备将实施例1分离获得的鼠李糖乳杆菌CGMCC No.20012接种于MRS培养基中进行发酵，离心获得菌泥，经真空冷冻干燥获得冻干菌粉，将菌粉按照如下配方配置成食品添加剂(w/w)：鼠李糖乳杆菌CGMCC No.20012冻干菌粉：50％，麦芽糊精：20％，菊粉30％，将含鼠李糖乳杆菌CGMCCNo.20012的食品添加剂按5-10％的添加量添加到各种食品、保健品中，即得用于抗衰老、延长寿命的食品及保健品。
Claims (2)
1.一种鼠李糖乳杆菌，其特征在于：所述的鼠李糖乳杆菌(Lactobacillus rhamnosus)于2020年6月3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，保藏地址为北京市朝阳区北辰西路1号院3号，保藏号：CGMCC No. 20012。
2.根据权利要求1所述的一种鼠李糖乳杆菌在制备抗氧化或延长线虫寿命的食品、保健品中的应用。
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