CN110408561B - 一种丁酸梭菌及其应用与含有该丁酸梭菌的组合物 - Google Patents
一种丁酸梭菌及其应用与含有该丁酸梭菌的组合物 Download PDFInfo
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Abstract
本发明公开了一种丁酸梭菌(Clostridium butyricum)KMBGI 02，该丁酸梭菌可有效抑制α‑葡萄糖苷酶的活性，可应用于调节肠道功能和血糖血脂。本发明还提供了包含该丁酸梭菌或其代谢产物的组合物，可以用作降血糖、降血脂和减肥类的食品或药品。
Description
技术领域
本发明涉及微生物工程技术领域，具体涉及一种丁酸梭菌及其应用与含有该丁酸梭菌的组合物。
背景技术
丁酸梭菌，又名酪酸菌、丁酸梭状芽孢杆菌或丁酸菌，是一种革兰氏阳性厌氧杆菌，最适生长温度为35-40℃。丁酸梭菌厌氧培养的过滤物中含有较多的短链脂肪酸，具有极强的整肠作用，它可抑制肠道中的致病菌，促进肠道中有益菌如双歧杆菌和乳杆菌的生长。因此，被广泛应用于改善肠道不适等症状的药品或动物饲料中。同时，由于丁酸梭菌能激活免疫系统，可以将其作为抗肿瘤免疫激活剂使用。
丁酸梭菌是厌氧芽孢杆菌，产生内生芽孢，稳定性好。能耐热、耐酸、耐胆汁，通过消化道而不失活，同时对部分抗生素有较强的抗性，可与之合用，提高效率。大量实验表明他没有毒副作用，被广泛应用于医药、功能性保健品和动物饲料添加剂等。丁酸梭菌具有强大的整肠作用，能调节肠道微生态，促进有益物质吸收并且具有较强的稳定性，且其发酵工艺设备简单，所以成本低廉，市场应用前景广泛。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种有效抵制α-葡萄糖苷酶活性丁酸梭菌(Clostridium butyricum)KMBGI 02。该丁酸梭菌(Clostridiumbutyricum)KMBGI 02具有广谱的抑菌性、较强的耐药性，耐酸、耐碱盐能力、耐胃肠液能力强；能有效调节肠道微生态，降血糖血脂。
本发明的目的之二在于提供该丁酸梭菌(Clostridium butyricum)KMBGI 02的应用。
本发明的目的之三在于提供包含该丁酸梭菌(Clostridium butyricum)KMBGI02的组合物。
本发明的目的之一采用如下技术方案实现：
一种丁酸梭菌(Clostridium butyricum)KMBGI 02，保藏号为：CGMCC17176。保藏于中国普通微生物菌种保藏管理中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101，电话：010-64807355；传真：010-64807288；保藏日期为2019年01月14日；分类命名为Clostridiumbutyricum。
本发明的目的之二采用如下技术方案实现：
该丁酸梭菌在抑制α-葡萄糖苷酶活性上的应用或在调节肠道功能上的应用。
本发明的目的之三采用如下技术方案实现：
一种组合物，含有上述的丁酸梭菌。
进一步地，所述丁酸梭菌的菌含量大于106CFU/mL，即106CFU/mL的菌含量为起效浓度。
进一步地，所述丁酸梭菌的菌含量为108-1010CFU/mL。
进一步地，还含有丁酸梭菌的代谢产物。
进一步地，所述代谢产物为蛋白、多糖、多肽和生物素中的一种或两种以上。
进一步地，该组合物为降血糖食品或药品。
进一步地，该组合物为降血脂食品或药品。
进一步地，该组合物为减肥食品或药品。
相比现有技术，本发明的有益效果在于：
本发明提供了一种能有效抵制α-葡萄糖苷酶活性的丁酸梭菌，该丁酸梭菌具有广谱的抑菌活性和耐药性，能有效调节肠道微生态、降低血糖；
该丁酸梭菌耐酸、耐碱盐能力、耐胃肠液能力强，可在胃部环境中保持菌活力、发挥作用；
该丁酸梭菌经培养后培养上清液的pH值为3.8，产生了大量的有机酸，其知合于胃肠道的pH值环境，且能在胃肠道具有较佳的活性；经体外模拟胃肠液转运发现，其能在低pH环境和胆汁中存能，通过胃和十二指肠，保持足够高的活力到达小肠，即包含该丁酸梭菌组合物的食品或药品均有较佳的小肠缓释活性；
该丁酸梭菌能有效调节胃肠道微生态，抵制肠道致病菌、降低血糖；同时喂食高脂食物和丁酸梭菌的小鼠，8周后其体增重明显低于仅喂食高脂食物的模型组、甚至低于喂食普通食物组的对照组，说明该丁酸梭菌能有效抑制体增重。
附图说明
图1为实施例2的α-葡萄糖苷酶活性测试结果；
图2为实施例4的体增重结果；
图3为实施例4的荧光定量PCR测定菌群组成变化的结果；
图4为实施例4的空腹血糖值测试结果；
图5为实施例4的空腹葡萄糖含量测试结果。
具体实施方式
下面，结合附图和具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
以下是本发明具体的实施例，在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过购买方式获得。
实施例1：丁酸梭菌KMBGI 02的分离与鉴定
采集：采集广东省深圳市新生儿粪便，封样留存，低温保存送回实验室；
分离：于实验室在厌氧工作站中进行肠道微生物的分离，将粪便悬液以1-10倍的梯度稀释，涂布于哥伦比亚血琼脂平板上，厌氧培养48h，挑取黄褐色的单菌落，在平板上划线分离，反复进行纯化培养，用革兰氏染色法观察菌落形态选出革兰氏阳性菌；
分离菌株：将分离得到的菌株液体培养后，用pH计检测培养24h时pH值，筛选出产酸能力强的菌株，用接种环挑取单一的菌落作为模板；
测序比对：进行菌落PCR；将阳性PCR产物送至公司测序；将16s rRNA碱基序列如SEQ ID No.1所示，与数据库进行比对分析，所分离菌菌株的碱基序列与丁酸梭菌菌株TK520菌株的16s rRNA基因序列具有高的同源性(99％)，因此被确认为丁酸梭菌，命名为丁酸梭菌KMBGI 02；
保存：于2019年01月14日保存于中国科学院微生物研究所菌种保藏中心，保藏号为CGMCC 17176。
经纯培养48小时后丁酸梭菌KMBGI 02的培养上清液pH值为3.8，能产生大量有机酸。革兰氏染色结果呈阳性，可观察得出KMBGI 02呈直杆状、两端钝圆的棒状杆菌，具有良好的生长性能。
实施例2：抑制α-葡萄糖苷酶活性测试
本实施例筛选具有抑制α-葡萄糖苷酶活性的菌株，通过评价已分离菌株对α-葡萄糖苷酶活性的抑制率来筛选具有潜在降血糖功效的菌株。
本实施例采用PNPG法检测α-葡萄糖苷酶活性，其原理为4-硝基酚-2-D吡喃葡萄糖苷(PNPG)在α-葡萄糖苷酶的作用下能水解产生葡萄糖和对硝基苯酚(PNP)，PNP在405nm处有最大吸收。通过加入样品，检测其对α-葡萄糖苷酶活性的抑制性。因此，通过检测候选株的不同组分对α-葡萄糖苷酶的抑制率来评价其体外降血糖活性。如图1所示，丁酸梭菌KMBGI02对α-葡萄糖苷酶活性的抑制率达到13％。与其他从肠道中分离得到的细菌相比，丁酸梭菌KMBGI02对α-葡萄糖苷酶活性的抑制率最高。
实施例3：体外模拟胃肠液转运
将108CFU/mL的丁酸梭菌接种于模拟胃液，pH调为3.0，孵育90min，计数活菌数和存活率，将培养液离心用生理盐水洗一次后加入模拟十二指肠液，孵育10min，计数活菌数和存活率，将培养液离心用生理盐水洗一次，离心加入模拟肠液，孵育120min，计数活菌数和存活率。
结果表明，丁酸梭菌在经胃液、十二指肠液和肠液连续孵育后存活率达到25.78％，活菌数高于益生菌在胃肠道中定植和发挥益生功能的最低要求溶度(106CFU/mL)。经模拟胃肠转运实例，可得出丁酸梭菌KMBGI 02具有更好的耐受性，在低pH和胆汁中能存活，顺利通过胃和十二指肠，保持足够高的活力到达小肠。同时也说明丁酸梭菌KMBGI02具有长时间在胃肠道中存活的潜能，且当其应用在生产加工中时，对加工条件的酸碱性要求更低。
实施例4：小鼠8周喂养试验
为了验证菌株对糖尿病小鼠模型的影响，取正常雄性C57小鼠30只饲养于鼠笼中，鼠笼置于可控环境中：每日光照12h，保持温度22℃。小鼠可自由进食及饮水，适应性饲养2周改喂高脂食物，持续8周。在第4周时，连续两天腹膜注释链脲佐菌素(60mg/kg，STZ)，在此接下来的4周之后，选取禁食血糖含量高于10.0mmol/L的小鼠，随机分组，分别为对照组(CK)，实验组，模型组(DM)。实验组每天灌胃0.2mL菌悬液(1×109CFU/mL)，对照组和模型组灌胃等剂量的生理盐水，持续8周。所有小鼠喂食相同的高脂食物，并处于相同的生活环境下。分别体增重、菌群组成和血液参数进行分析。数据结果表示为平均值±标准差。经过方差分析后进行显著性分析，p<0.05即认为在统计学上具有显著性差异(*)。
1)体增重
经过8周的喂养后，体增重结果如图2所示，结果显示，在小鼠的生长曲线中，经过8周的喂养，仅喂食高脂食物小鼠的体重显著高于普通饲料小鼠的体重。同时喂食丁酸梭菌的小鼠体重显著低于仅喂食高脂食物的小鼠。从以上结果可知，灌胃丁酸梭菌可以有效控制小鼠肥胖的发生。
2)菌群组成
用粪便DNA提取试剂盒对粪便中的DNA进行提取。通过实时荧光定量PCR技术分析肠道菌群的变化。
荧光定量PCR结果显示如图3所示，实验组小鼠在接受完丁酸梭菌连续灌胃处理后，与模型组相比，肠道和粪便中的微生态发生了明显的改变，主要趋势为益生菌数量上升，致病菌数量下降，肠道健康得到明显的改善。灌胃了丁酸梭菌的小鼠，粪便中的菌群组成发生了明显的变化。这种变化与其潜在的益生性有关，如产短链脂肪酸、抑制致病菌等。有益菌数量的增加，具有维持肠道内菌群平衡，提高机体免疫力，促进营养物质吸收等多种功能。
3)血液参数
小鼠空腹16小时后，分别从眼球后及眼眶内的毛细血管中抽取血液，并立即放入4℃冰箱保存。分离血浆后保藏于-20℃备用。用葡萄糖仪测定血清葡萄糖水平。
结果如图4和图5所示。在实验进行之前，模型组和实验组的血清葡萄糖水平没有差异。结果显示灌胃8周后，保持普通维持饲料喂食的CK组小鼠的空腹血糖值和血清葡萄糖含量没有明显变化。而喂食高脂食物的小鼠血清葡萄糖含量大幅增加。实验组的血清葡萄糖水平虽然没有对照组低，但是显著低于模型组。结果表明，丁酸梭菌可显著降低高脂喂食小鼠的血清葡萄糖水平。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
序列表
<110> 康美华大基因技术有限公司
<120> 一种丁酸梭菌及其应用与含有该丁酸梭菌的组合物
<130> 1
<141> 2019-07-08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1383
<212> DNA
<213> KMBGI 02 16s rRNA
<400> 1
gctgcctcct tgcggttagc tcacgaactt tgggtattgc caactctcat ggtgtgacgg 60
gcggtgtgta caaggcccgg gaacgtattc accgcgacat tctgattcgc gattactagc 120
aactccagct tcatgtaggc gagtttcagc ctacaatccg aactgagatc ggttttatag 180
ttttgctcac tctcgcgagg ttgcatctca ttgtaccgac cattgtagca cgtgtgtagc 240
cctagacata aggggcatga tgatttgacg tcatccccac cttcctcccg gttaacccgg 300
gcagtctcgc tagagtgctc aactaaatgg tagcaactaa caataagggt tgcgctcgtt 360
gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtctt 420
cctgccaccg aagtggcttc ctccattaca gagtaattca ggagatgtca agtctaggta 480
aggttcttcg cgttgcttcg aattaaacca catgctccgc tgcttgtgcg ggcccccgtc 540
aattcctttg agttttaatc ttgcgaccgt actccccagg cggaatactt aatgcgttag 600
cggcggcaca gaggtcatga caacccctac acctagtatt catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgag cctcagtgtc agttacagtc 720
cagaaaggcg ccttcgccac tggtattctt cctaatctct acgcatttca ccgctacact 780
aggaattctc ctttcctctc ctgcactcta gatatccagt ttggaatgca gcacccaggt 840
taagcccggg tatttcacat cccacttaaa tatccaccta cgctcccttt acgcccagta 900
aatccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960
gcttcctcct taggtaccgt cattatcgtc cctaaagaca gagctttaca atccgaagac 1020
cgtcatcact cacgcggcgt tgctgcatca gggtttcccc cattgtgcaa tattccccac 1080
tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca atgtggccga tcaccctctc 1140
aggtcggcta cgcatcgtcg ccttggtgag ccgttacctc accaactagc taatgcgacg 1200
cgggtccatc tcatagcgga ttactccttt aattgctgta ccatgcggta ctacaatctt 1260
atgcggtatt aatcttcctt tcgaaaggct attcccctct atgaggcagg ttacccacgt 1320
gttactcacc cgtccgccgc taatccactc ccgaaggagc ttcatcgctc gacttgcatg 1380
tgt 1383
Claims (7)
1.一种丁酸梭菌(Clostridium butyricum)KMBGI 02，保藏号为：CGMCC 17176。
2.一种组合物，其特征在于，含有如权利要求1所述的丁酸梭菌。
3.如权利要求2所述的组合物，其特征在于，所述丁酸梭菌的菌含量为108-1010CFU/mL。
4.如权利要求2所述的组合物，其特征在于，还包括丁酸梭菌代谢产物。
5.如权利要求4所述的组合物，其特征在于，所述代谢产物为蛋白、多糖、多肽和生物素中的一种或两种以上。
6.如权利要求2所述的组合物，其特征在于，该组合物为辅助降血糖食品或药品。
7.如权利要求2所述的组合物，其特征在于，该组合物为减肥食品或药品。
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