CN109619183B - 植物乳杆菌cqpc03在制备预防肝脏氧化损伤的药品中的应用 - Google Patents
植物乳杆菌cqpc03在制备预防肝脏氧化损伤的药品中的应用 Download PDFInfo
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Abstract
本发明公开了保藏编号为CGMCC NO.14492的植物乳杆菌(Lactobacillus plantarum)CQPC03在制备预防肝脏氧化损伤的药品中的应用，不仅丰富了乳酸菌菌种资源，而且扩大了植物乳杆菌CQPC03的应用范围，有助于功能保健制品的开发，也给肝脏氧化损伤的预防带来了新的希望。
Description
技术领域
本发明属于微生物技术领域，涉及一种乳酸菌，还涉及该乳酸菌在制备食品或药品中的应用。
背景技术
乳酸菌广泛存在于发酵食品、动物和人体体内。研究表明，乳酸菌与机体健康密切相关，具有提高免疫力、预防龋齿、缓解乳糖不耐受症和促进机体消化吸收等功能。此外其在提高食品营养价值、改善食品风味、延长保存时间和改善食品功能特性方面也发挥重要作用。为了更好的利用这些微生物资源，应开展更广泛的分离和鉴定工作，积累丰富的菌种资源，开发出丰富的工业用益生菌种类。
四川泡菜的生产过程是将新鲜的泡菜洗净，密封于坛内，在盐水中厌氧发酵浸泡的泡菜。泡菜水中含有丰富的天然乳酸菌，对泡菜风味和品质的形成起着关键作用。其使用可溶性成分(主要是糖和含氮物质)进行增殖，产生酸性物质并代谢出风味成分，使泡菜产生独特的酸脆口味。
肝脏在维持机体健康方面具有重要作用，包括代谢、解毒、造血、免疫、胆道和肝再生等。同时，肝脏也容易受到病毒、毒素、药物、酒精和创伤等一系列的刺激，最终都导致肝脏的急性或慢性损伤。临床研究发现，长期肝损伤可能导致肝纤维化、肝硬化和肝细胞癌。
发明内容
本发明的目的在于对泡菜水中的乳酸菌进行分离鉴定，并考察其对肝脏氧化损伤的作用效果，以丰富乳酸菌菌种资源、开发功能保健制品。
经研究，本发明提供如下技术方案：
1.植物乳杆菌(Lactobacillus plantarum)CQPC03，保藏编号为CGMCC NO.14492。
2.植物乳杆菌CQPC03在制备食品或预防肝脏氧化损伤的药品中的应用。
优选的，所述食品为发酵食品。
优选的，所述发酵食品为乳酸菌奶饮料、发酵乳、乳粉或乳粉胶囊。
本发明对泡菜水中的乳酸菌进行了分离鉴定，将其中一株植物乳杆菌(Lactobacillus fermentum)命名为CQPC03，于2017年8月4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(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC NO.14492。
本发明的植物乳杆菌CQPC03具有很好的抗胃酸能力，经pH3.0人工胃液处理3h后的存活率达到91.99％；在0.3％胆盐中该菌也可缓慢生长，生长效率达到无胆盐培养的19.60％。
四氯化碳诱导肝脏氧化损伤小鼠模型实验结果显示，植物乳杆菌CQPC03能明显减轻小鼠肝脏的氧化损伤，显著降低血清中ALT、AST和MDA的水平并提高SOD和GSH的水平，还能显著降低肝脏中IL-1β、TNF-α和Bax基因的表达水平并增加Bcl-2基因的表达水平。由此可见，植物乳杆菌CQPC03对肝脏氧化损伤具有较好的预防作用。
本发明的有益效果在于：本发明提供了一种植物乳杆菌CQPC03，消化道抗性强，而且可以有效地预防肝脏氧化损伤，不仅丰富了乳酸菌菌种资源，而且扩大了植物乳杆菌CQPC03 的应用范围，有助于功能保健制品的开发，也给肝脏氧化损伤的预防带来了新的希望。
附图说明
图1为植物乳杆菌CQPC03的菌落形态。
图2为植物乳杆菌CQPC03的细胞形态。
图3为植物乳杆菌CQPC03的16S rDNA PCR扩增产物的琼脂糖凝胶电泳图，其中M为DNA分子量标准，0为阴性对照，1为植物乳杆菌CQPC03。
图4为实验各组小鼠肝脏病理学变化。
图5为实验各组小鼠血清中相关因子水平。
图6为实验各组小鼠肝脏中相关基因表达水平。
图5和图6中，##表示与正常组比较存在显著差异(p<0.05)；**表示与模型组比较存在显著差异(p<0.05)。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明的优选实施例进行详细的描述。
实施例1、植物乳杆菌CQPC03的分离与鉴定
1、实验材料
采自重庆市南岸区农家自然发酵泡菜水6份，分别吸取40mL泡菜水放入无菌离心管中，置于食品采样箱内，放于实验室4℃冰箱保存备用。
2、乳酸菌的分离纯化
分别取1mL泡菜水样品，用无菌生理盐水进行10倍梯度稀释至10-6，然后分别取10-4、 10-5、10-6 3个梯度的稀释液100μL进行平板涂布，37℃培养24-48h，观察并记录菌落形态。挑取平板上不同形态的菌落进行划线分离，经37℃培养48h后，再次挑取平板上不同形态的单菌落进行划线分离，如此重复2至3次，直至得到形态一致的纯的单菌落。
编号为CQPC03的菌株菌落形态如图1所示，菌落颜色多为白色或乳白色，形状为圆形，边缘整齐，表面湿润光滑。
3、乳酸菌的初步鉴定
挑取平板上的纯菌落接种于5mL MRS液体培养基中，37℃培养24h。取上述含菌培养基1mL于无菌离心管中，4000r/min离心10min后弃去上层培养基，菌体沉淀重悬于无菌生理盐水并进行革兰氏染色镜检，革兰氏染色镜检为阳性的初步鉴定为乳酸菌。
编号为CQPC03的菌株革兰氏染色呈现阳性，在100倍油镜下，菌株细胞形态如图2所示，细胞杆状，成单、成对或者成链，不形成芽孢，两端圆形。
4、乳酸菌DNA提取
将已纯化的疑似目标菌株接种于MRS肉汤中，37℃培养18-24h后，采用细菌基因组DNA提取试剂盒进行DNA提取。提取的DNA放于-20℃冰柜保藏备用。
5、基因组DNA PCR扩增及琼脂糖凝胶电泳检测
取提取的DNA，PCR扩增16S rDNA，其中上游引物27F(5'－AGAGTTTGATCCTGGCTCAG－3'，SEQ ID No.1)1μL、下游引物1495R(5'－CTACGGCTACCTTGTTACGA－ 3'，SEQ IDNo.2)1μL，2×Taq plus Buffer 12.5μL、模板DNA 1μL，用无菌dd H2O将体系补足至25μL。并以无菌超纯水替代模板DNA作为阴性对照。扩增条件为：94℃5min；94℃ 30s，55℃30s，72℃1min，共29个循环；最后72℃延伸5min。然后取5μL扩增产物进行琼脂糖凝胶电泳检测，琼脂糖浓度为1.5％，电泳条件为110V，45min。
编号为CQPC03的菌株16S rDNA扩增产物的琼脂糖凝胶电泳检测结果如图3所示，阴性对照组的泳道无条带，表明PCR扩增过程中未受到污染；编号为CQPC03的菌株的泳道有一条长度约为1500bp的条带，符合预期扩增片段的长度。
将编号为CQPC03的菌株的16S rDNA扩增产物委托北京擎科生物技术有限公司进行测序，测得序列如SEQ ID No.3所示。使用NCBI中的BLAST(Basic Local AlignmentSearch Tool) 程序对测得序列进行同源性比对分析，结果显示，编号为CQPC03的菌株为乳酸菌中的植物乳杆菌(Lactobacillus plantarum)，其与Gene Bank数据库中已知乳酸菌的同源性均达99％。
6、乳酸菌体外抗性筛选
(1)耐受0.3％胆盐的能力
在MRS-THIO培养基(含0.2％巯基乙酸钠的MRS肉汤)中添加猪胆盐使其浓度为0.3％， 121℃灭菌15min；将活化好的5mL菌种以2％(v/v)的接种量分别接入不含胆盐(0.0％)的 MRS-THIO培养基和含0.3％胆盐的MRS-THIO培养基中，以空白培养基(未接菌的MRS-THIO培养基)为对照，37℃培养24h后，分别测定上述不同浓度培养基的OD600nm值，按公式(1)计算菌株对胆盐的耐受力：
结果显示，编号为CQPC03的菌株在0.3％胆盐中能够缓慢生长，生长效率达到无胆盐培养的19.60±5.06％。
(2)人工胃液耐受性试验
人工胃液的配制：由0.2％NaCl和0.35％胃蛋白酶组成，用1mol/L HCl调节pH为3.0，再用孔径为0.22μm的滤膜过滤除菌备用。
在超净工作台中吸取5mL培养好的含菌培养基于10mL无菌离心管中，经3000r/min离心10min，弃去上层培养基并收集菌体，加入等体积(5mL)无菌生理盐水混匀制成菌悬液，然后取1mL菌悬液与9mL pH 3.0的人工胃液混匀，此时取1mL上述混合液作为人工胃液处理0h的样品，剩余9mL混合液置于恒温水浴摇床(37℃，150r/min)中培养3h。0 h和3h的样品分别经10倍梯度稀释，选择合适梯度采用平板涂布的方法测定活菌数，在MRS 固体培养基上37℃培养48h，按公式(2)计算存活率(％)。
结果显示，编号为CQPC03的菌株具有很好的抗胃酸能力，经pH3.0人工胃液处理3h后的存活率达到91.99±6.91％。
实施例2、植物乳杆菌CQPC03对肝脏氧化损伤的预防作用
1、实验动物
健康6周龄雄性昆明小鼠，30只，购于重庆医科大学实验动物中心。饲养于室温25±2℃、相对湿度50±10％、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。
2、实验方法
30只小鼠根据体重随机分为3组，每组10只，分别为正常组、模型组和L.plantarum组。实验共持续14天，正常组和模型组小鼠每天自由摄食基础饲料和饮水，L.plantarum组小鼠每天除自由摄食基础饲料和饮水外，还按1.0×109CFU/kg·BW灌胃植物乳杆菌CQPC03；实验最后一天，对模型组和L.plantarum组小鼠按10mL/kg·BW腹腔注射1％四氯化碳的大豆油溶液，正常组腹腔注射等量大豆油。注射完毕后，所有小鼠禁食不禁水，16h后脱脊椎处死小鼠，取适量肝脏固定于10％福尔马林溶液中，按照HE染色流程制作其HE染色切片，另取适量肝脏匀浆；取小鼠血液，于4℃、3000r/min离心15min，收集血清。
3、小鼠肝脏病理学变化观察
实验各组小鼠肝脏病理学变化如图4所示，相对于正常组，模型组小鼠肝脏出现严重损伤，伴有肝细胞坏死、炎性细胞浸润和肝细胞在中央静脉周围无规则排列，而L.plantarum 组小鼠肝脏的损伤程度明显减弱，说明植物乳杆菌CQPC03对四氯化碳引起的肝脏氧化损伤具有一定的预防作用。
4、小鼠血清中相关因子水平测定
按照ALT、AST、SOD、GSH和MDA试剂盒说明书测定小鼠血清中上述因子的水平。
结果如图5所示，相对于模型组，L.plantarum组小鼠血清中ALT、AST、SOD、GSH 和MDA的水平均得到明显改善，血清中ALT、AST和MDA的水平均显著低于模型组，而 SOD和GSH的水平均显著高于模型组，说明植物乳杆菌CQPC03对四氯化碳引起的肝脏氧化损伤具有一定的预防作用。
5、小鼠肝脏中相关基因表达水平的测定
取小鼠肝脏匀浆，按照Trizol试剂盒说明书提取RNA，然后将RNA反转录为cDNA，以得到的cDNA为模板，按照
Green实时荧光定量PCR方法测定基因IL-1β、TNF-α、 Bcl-2和Bax相对于β-actin基因的表达水平。
结果如图6所示，与模型组相比，L.plantarum组小鼠肝脏中IL-1β、TNF-α和Bax基因的表达水平显著降低，而Bcl-2基因的表达水平显著增加，说明植物乳杆菌CQPC03对四氯化碳引起的肝脏氧化损伤及炎症相关基因的表达具有一定的调节作用。
实施例3、利用植物乳杆菌CQPC03制备发酵食品
植物乳杆菌CQPC03原始菌种的保存：在温度-75℃下以30wt％甘油悬液形式保存，或在温度4℃下以冷冻干燥菌粉的形式保存。
植物乳杆菌CQPC03工作发酵剂的制备，采用下述两种方法中的任意一种：
第一种方法：将植物乳杆菌CQPC03的原始菌种接种于经110℃灭菌10min的12wt％脱脂乳中，37℃培养14-16h至凝乳，连续培养活化两代，用作母发酵剂；然后将母发酵剂按3-5vol％接种于灭菌乳中，培养14-16h至凝乳，凝乳中的活菌数约109cfu/mL，用作工作发酵剂。
第二种方法：将植物乳杆菌CQPC03的原始菌种接种于MRS液体培养基中，37℃培养12-16h进行活化，连续活化两代，然后将活化培养物按2-4vol％接种于MRS培养基中，培养16-18h，4℃、4000r/min离心15min，去除上清夜，细胞沉淀用无菌脱脂乳制成悬浮液，用作工作发酵剂。
1、制备乳酸菌奶饮料
将原料乳(选自脱脂奶、鲜奶和复原奶中的一种或多种)在95℃下加热杀菌20min或在 140℃下高温热杀菌2s，冷却到4℃，加入植物乳杆菌CQPC03工作发酵剂使其浓度达到106cfu/ml以上，即得到含植物乳杆菌CQPC03的乳酸菌奶饮料，4℃冷藏保存。
2、制备发酵乳
将原料乳(选自脱脂奶、鲜奶和复原奶中的一种或多种)在95℃下加热杀菌20min或在 140℃下高温热杀菌2s，冷却到37℃，按照原料乳体积的4％加入植物乳杆菌CQPC03工作发酵剂，37℃发酵16h，即得到植物乳杆菌CQPC03发酵乳，4℃冷藏保存。
或者，将原料乳(选自脱脂奶、鲜奶和复原奶中的一种或多种)在95℃下加热杀菌20min 或在140℃下高温热杀菌2s，冷却到37℃，按照原料乳体积的4％加入植物乳杆菌CQPC03 工作发酵剂，再按照原料乳体积的4％加入其它可共生的制备发酵乳的商业发酵剂(如保加利亚乳杆菌和嗜热链球菌)，混匀，37℃混菌发酵至滴定酸度以乳酸计为0.6-0.7％，即得到混菌发酵乳，4℃冷藏保存。
3、制备乳粉
将原料乳(选自脱脂奶、鲜奶和复原奶中的一种或多种)在95℃下加热杀菌20min或在 140℃下高温热杀菌2s，冷却到37℃，按照体积比3：1加入植物乳杆菌CQPC03发酵乳，均质，真空浓缩，喷雾干燥，即得到含植物乳杆菌CQPC03的乳粉。
4、制备乳粉胶囊
将含植物乳杆菌CQPC03的乳粉装入胶囊壳中，即得到乳粉胶囊。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管通过参照本发明的优选实施例已经对本发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。
序列表
<110> 重庆第二师范学院
<120> 植物乳杆菌CQPC03及其在制备预防肝脏氧化损伤的食品或药品中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctacggctac cttgttacga 20
<210> 3
<211> 1411
<212> DNA
<213> 植物乳杆菌CQPC03(Lactobacillus plantarum CQPC03)
<400> 3
ggctctaaag gttaccccac cgactttggg tgttaaaact ctcatggtgt gacgggcggt 60
gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta ctagcgattc 120
cgacttcgtg caggcgagtt gcagcctgca gtccgaactg agaacggttt taagagattt 180
gcttgccctc gcgagttcgc gactcgttgt accgtccatt gtagcacgtg tgtagcccag 240
gtcataaggg gcatgatgat ctgacgtcgt ccccaccttc ctccggtttg tcaccggcag 300
tctcactaga gtgcccaact taatgctggc aactagtaac aagggttgcg ctcgttgcgg 360
gacttaaccc aacatctcac gacacgagct gacgacgacc atgcaccacc tgtcattgcg 420
ttcccgaagg aaacgcccta tctctagggt tggcgcaaga tgtcaagacc tggtaaggtt 480
cttcgcgtag cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc 540
ctttgagttt caaccttgcg gtcgtactcc ccaggcggag tgcttaatgc gttagctccg 600
gcactgaagg gcggaaaccc tccaacacct agcactcatc gtttacggca tggactacca 660
gggtatctaa tcctgttcgc tacccatgct ttcgagtctc agcgtcagtt gcagaccagg 720
tagccgcctt cgccactggt gttcttccat atatctacgc attccaccgc tacacatgga 780
gttccactac cctcttctgc actcaagtta tccagtttcc gatgcacttc tccggttaag 840
ccgaaggctt tcacatcaga cttagaaaac cgcctgcact ctctttacgc ccaataaatc 900
cggataacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtgactt 960
tctggttaaa taccgtcaac gtatgaacag ttactctcat acgtgttctt ctttaacaac 1020
agagctttac gagccgaaac ccttcttcac tcacgcggtg ttgctccatc aggcttgcgc 1080
ccattgtgga agattcccta ctgctgcctc ccgtaggagt atgggccgtg tctcagtccc 1140
attgtggccg atcagtctct caactcggct atgcatcatc gccttggtag gccgttaccc 1200
caccaacaag ctaatgcacc gcaggtccat ccagaagtga tagcgagaag ccatctttta 1260
agcgttgttc atgcgaacaa cgctgttatg cggtattagc atctgtttcc aaatgttgtc 1320
ccccgcttct gggcaggtta cctacgtgtt actcacccgt ccgccactcg ttggcgacca 1380
aaatcaatca ggtgcaagca ccatcaatca a 1411
Claims (1)
1.保藏编号为CGMCC NO.14492的植物乳杆菌(Lactobacillus plantarum)CQPC03在制备预防肝脏氧化损伤的药品中的应用。
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