CN113564067B - 一株促肠蠕动动物双歧杆菌亚种f1-7及应用 - Google Patents
一株促肠蠕动动物双歧杆菌亚种f1-7及应用 Download PDFInfo
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Abstract
本发明属于食品微生物技术、生物医药领域，公开了一株促肠蠕动动物双岐杆菌亚种F1‑7及应用，本发明所述动物双歧杆菌亚种F1‑7具有良好的促进肠蠕动功能。本发明还涉及动物双歧杆菌亚种F1‑7在制备乳制品、固体饮料、微生态制剂中的应用及含有该菌株的益生菌产品。
Description
技术领域
本发明属于食品微生物技术、生物医药领域，尤其涉及一株促肠蠕动动物双歧杆菌亚种F1-7及应用。
背景技术
肠蠕动，是指蠕动类似蠕虫的运动，尤指肠的波状收缩。近年来，随着生活节奏的不断加快，越来越多的人群发生肠道蠕动功能障碍。肠道蠕动减缓，不仅无法保证体内毒素尽快排出体内，还会导致有害物质在体内的不断堆积，长期蠕动减缓，不仅会导致肠梗阻、便秘等胃肠道疾病的发生，还会引发心脑血管疾病。由于受到多种因素影响，蠕动功能障碍确切发病机制尚未明确，目前多依靠泻剂改善这一症状，虽见效快但易复发，且无法从根源解决。因此寻找一种能够替代传统治疗方式的新途径具有重要现实意义，微生态疗法从全新的角度解决了传统治疗方式的多种弊端。研究发现肠功能障碍患者的肠道菌群不同于健康人，双歧杆菌属、乳杆菌属丰度下降，而一些潜在致病菌如产甲烷古细菌、梭菌的丰度增加，这些改变或许在一定程度上意味着肠道微生态系统的紊乱，肠道菌群失调同时又加重肠道动力障碍。肠道菌群可通过发酵底物产生短链脂肪酸等多种代谢产物；或通过特定内分泌因子与胃肠激素，调节消化器官的运动。双歧杆菌等作为早期定植于肠道的主要益生菌，对肠道粘膜免疫系统和全身免疫系统的发育成熟以及功能的建立具有重要的作用。O’Mahony等研究发现含有双歧杆菌的VSL#3混合菌制剂可以促进肠蠕动，同时可改变肠道菌群的组成，主要是 VSL#3 促进了肠道内益生菌的定植和抑制致病菌的繁殖，从而改变肠道微环境，促进肠蠕动。尽管针对双歧杆菌的生理功能有大量的研究报道，但是对于有效的促进肠蠕动双歧杆菌产品开发和详细机制报道很少。因此筛选并开发优良的促肠蠕动双歧杆菌产品具有极强的研究意义和应用价值。
在目前已经公开的文献与专利或专利申请中，例如CN111466440A公开了一种可以用于改善肠蠕动的复合菌株发酵乳；CN111493261A公开了一种可以用于改善肠蠕动的复合菌固体饮料；CN111000109A公开了一种防治肠蠕动障碍的合生元固体饮料。针对上述背景技术中已有的实现方案，这些专利或专利申请的共同点都是应用多株混合菌种或菌与多种成分混合使用来预防或促进肠蠕动，而有效促进肠蠕动的单菌株的益生特性，效果和详尽作用机制仍缺乏报道，同时相关产品的制备形式与制品应用仍不够广泛。通过上述分析，现有技术存在的问题及缺陷为：具有优良益生特性和促肠蠕动功能的菌株未被有效开发，同时单菌株的详尽促蠕动机制仍缺乏报道。解决以上问题及缺陷的难度为：益生菌筛选范围广，任务量大，现有益生菌的潜在益生特性良莠不齐；同时肠蠕动功能障碍的形成原因复杂，能够有效促进肠蠕动的单株动物双歧杆菌未被有效开发，同时机制未被有效验证与探究；最后，相关产品的制备形式与应用仍然较少。解决以上问题及缺陷的意义为：获得益生菌性能突出，同时能够有效促进肠蠕动的单菌株，揭示单菌株动物双歧杆菌促进肠蠕动的机制，开发更为高效的产品制备形式，拓宽应用范围，为临床肠道功能障碍的缓解提供可靠的理论依据和有效的干预方式。
发明内容
针对现有技术存在的问题，本发明提供了一株动物双歧杆菌亚种F1-7及应用，尤其涉及一种能够显著促进肠蠕动的动物双歧杆菌亚种F1-7及其应用。本发明的动物双歧杆菌亚种F1-7的基因序列为：
GGGTGGGGGGCGTTCTTACACATGCAGTCGAACGGGATCCCTGGCAGCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCTCCATCGCATGGTGGGGTGGGAAATGCTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGGAGCCGTCTAAGGGTAAGAACTCG
本发明是这样实现的，一株具备促进肠蠕动功能的动物双歧杆菌亚种F1-7（Bifidobacterium animalis subsp F1-7），其在中国典型培养物保藏中心保藏，地址为中国湖北省武汉市武昌珞珈山武汉大学，保藏日期2020年12月2日，保藏号为CCTCC NO：M2020833，状态为存活。
本发明的另一目的在于提供一种益生性能潜力突出的动物双歧杆菌亚种F1-7，其特征在于具有良好的黏附能力，具有突出的耐受模拟胃肠道生存环境能力。
本发明的另一目的在于提供一种利用上述的动物双歧杆菌亚种F1-7在促进肠蠕动中的应用。
本发明的另一目的在于提供一种利用上述的促进肠蠕动动物双歧杆菌亚种F1-7的丸剂制备。
本发明的另一目的在于提供一种利用上述促进肠蠕动动物双歧杆菌亚种F1-7在乳制品中的应用，包括奶片乳制品，但不限于此，还包括豆乳等。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例中所需要使用的附图做简单的介绍，显而易见地，下面所描述的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的改善便秘菌株益生产品的制备方法流程图；图2(a)是本发明实施例提供的益生菌F1-7表面疏水和自聚集能力示意图；图2(b)是本发明实施例提供的益生菌F1-7耐酸和耐胆盐能力的示意图；图2(c)是本发明实施例提供的益生菌F1-7模拟胃液转运和模拟肠液转运示意图；图3是本发明实施例提供的益生菌F1-7在斑马鱼体内的定植效果示意图；图4是本发明实施例提供的F1-7促进斑马鱼肠蠕动的效果示意图；图5是本发明实施例提供的F1-7对促进小鼠肠道蠕动的影响示意图；图6是本发明实施例提供的F1-7在小鼠肠道的定植变化示意图。
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。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
针对现有技术存在的问题，本发明提供了一株动物双歧杆菌亚种F1-7及应用，尤其涉及一种能够显著促进肠蠕动的动物双歧杆菌亚种F1-7及应用，下面结合附图对本发明作详细的描述。
如图1所示，本发明实施例提供的促进肠蠕动F1-7的制备方法包括以下步骤：S101， F1-7培养：将动物双歧杆菌亚种F1-7接种到发酵罐的增菌TPY培养基中培养，得发酵液；S102，F1-7干燥：将F1-7依次进行冷冻前预冷处理、预冻处理、主干燥处理及解析干燥处理，完成动物双歧杆菌冻干产品的制备；S103，F1-7的包装：将粉状F1-7进行丸剂的制备；S104，F1-7的应用：将F1-7合理配比乳、豆浆等，得奶片或豆奶制品。
具体实施例
F1-7的益生特性：（1）具有优良的黏附能力。与模式菌株Bifidobacterium animalis 12(BB12)相比，F1-7的表面疏水能力达到60.74±0.62%，接近BB12的67.77±1.39%；自聚集能力达到13.67±2.57%，低于BB12的93.98±0.06%，因此具有优良的黏附性（图2a）。（2）具有优良的耐酸耐胆盐能力。F1-7在PH为3.00的酸性环境中消化3小时后，不仅能够良好地存活，还能实现一定程度的增长，高达101.61±0.04%,优于BB12的101.53±0.03%；F1-7在0.3%的胆盐性环境中消化4小时后，存活率高达56.61±1.07%，接近BB12的69.03±0.91%（图2b）。（3）具有优良的耐受模拟胃肠液能力。F1-7在PH为3.00的模拟胃液环境中消化3小时后，存活率高达100.58±0.03%，优于BB12的97.52±0.18%；之后转为PH为8.00的模拟肠液环境中消化8小时后，能够良好存活，高达95.01±0.22%，与BB12的95.19±0.19%接近（图2c）。
F1-7功能性：（1）F1-7对斑马鱼具有良好的促蠕动效果。随机选取5dpf的斑马鱼幼苗并分Model+LGG组，Model +F1-7组，每组48尾。将各浓度为108CFU/mL的菌液，浸浴斑马鱼24h。于0,8,16,24h四个时间点，每个时间点随机挑选12尾幼苗，用E3培养液洗涤两次，置于琼脂平板上用5μL 浓度100μg/mL庆大霉素滴洗5分钟以除去表面细菌。将洗过的幼虫破碎并涂布MRS平板上于37 ℃温育过夜，定量菌落并计算平均每尾幼虫的菌体数。每组实验进行三次重复。以活体斑马鱼为模型，进一步评价F1-7的体内滞留能力。F1-7浸浴斑马鱼之后，8h之后能够达到2.70±0.00 Log/CFU/Larvae，接近模式菌株LGG；16h之后能够达到3.69±0.01 Log/CFU/Larvae，显著高于模式菌株LGG；24h之后能够达到3.09±0.02 Log/CFU/Larvae，接近模式菌株LGG。因此， F1-7可以有效定植于斑马鱼体内（见图3）。选取5dpf的斑马鱼幼苗为实验对象。采用盐酸洛哌丁胺法（盐酸洛哌丁胺浓度为10μg/mL）构建蠕动斑马鱼模型（Model），浸浴斑马幼鱼24h后对其进行生理状态镜检。选取构建成功的斑马鱼模型并进行随机分组，每组12尾：分为1个Model组、1个Model+阳性药物对照组（Phenolphthalein Tablets,50μg/mL），1个Model+LGG组，1个Model +F1-7组。同时设置1个正常对照组（Normal）。将各浓度为108 CFU/mL的菌液，浸浴便秘斑马鱼24h后，于倒置荧光显微镜下观察单位时间（1min）内各组斑马鱼肠蠕动次数。在1min的肠蠕动观察中，正常组斑马鱼肠蠕动的首次蠕动波峰出现在28s左右，而模型组在1min内未出现蠕动现象，说明蠕动模型良好。施加阳性药物和菌株干预后，阳性药物组22s左右出现第一次蠕动，42s左右出现了第二次蠕动；而LGG在1min内未见到蠕动现象出现，F1-7在22s左右出现第一次蠕动，42s左右出现了第二次蠕动，与阳性药物效果接近(见图4)。因此， F1-7具有良好的促斑马鱼肠蠕动功能。（2）F1-7通过对小鼠具有良好的促肠蠕动功能。采用盐酸洛哌丁胺法于Day7成功诱导小鼠肠蠕动障碍后，进行为期20天的益生菌干预。干预结束后，与模型组相比，F1-7显著促进了小鼠肠道转运率，由1.04±1.81%提升为88.19±4.10%（p<0.01），接近阳性药物水平，优于模式菌株LGG (图5)。以CK为对照，1h时荧光染料大量分布于从胃至结肠部位；F1-7虽然不如荧光染料强度高，但同样均匀分布于胃至结肠部位。3h时，F1-7回盲肠部位荧光强度增加，说明F1-7由胃、十二指肠逐步向空肠、回盲肠移动。6h时，F1-7十二指肠大量荧光消失，可见空肠荧光残留，回盲肠荧光强度明显增强，结直肠部位可见荧光增加。12h时，F1-7小肠部位仍有少量荧光，说明仍有少量菌株定植，而盲肠与结肠荧光强度与3h，6h较为一致，仍然有很强荧光，说明F1-7可以有效定植于大肠部位。总体而言，益生菌F1-7在小肠包括十二指肠、和空肠部位的定植较弱，可以较长时间地定植于大肠部位，包括回盲肠与结直肠(见图6)。
含有本发明动物双歧杆菌亚种F1-7的益生产品：（1）动物双歧杆菌亚种F1-7丸剂的制备。本发明所述的动物双歧杆菌亚种F1-7丸剂的制备方法，包括以下步骤：选取5%茉莉花蜜作湿润剂，3-5%低聚糖为保护剂，并分散于熔化的食用南极磷虾油中，并形成油脂混合物，添加维生素E作为抗氧化剂；然后，在油脂混合物中加入所述动物双歧杆菌亚种F1-7冻干菌粉，冻干菌粉与油脂混合物比例为1:1-6(m/V)，搅拌混合均匀。加入适量比例的明胶作为基质，在冷却的油相中进行搅拌，同时通过一同心双层喷嘴，滴制温度为50-55℃，滴入流动的冷却玉米油中，形成活菌明胶滴丸。过滤油相，加入CaCl2(0.1-1mol/L)溶液以回收经明胶包衣后的活菌明胶滴丸，用0.9％NaCl溶液清洗并过滤回收得到活菌明胶滴丸。最后于30℃及空气相对湿度为10％的条件下鼓风干燥3个小时，降低产品水分含量至8-10%，得到成品，湿滴丸中活菌数≥109CFU/g。（2）动物双歧杆菌亚种F1-7在乳制品中的应用。
本发明的动物双歧杆菌亚种F1-7可以在制备乳制品中使用，乳制品包括奶片，豆奶等。但本发明的乳制品不限于此，还包括其他形式的乳制品。本发明的各种乳制品可以通过将本发明的动物双歧杆菌亚种F1-7菌粉作为原料，按照本领域常规的方法，以常规的比例添加制成。
实施案例1：动物双歧杆菌亚种F1-7奶片。一种促进肠蠕动的益生菌奶片，由以下重量份数的原料制成：50份全脂牛乳粉、35份脱脂牛乳粉、4份木糖醇粉、3份羧甲基纤维素钠、2份硬脂酸镁和5份益生菌菌粉，上述促进肠蠕动益生菌奶片的制备方法，包括以下步骤：将益生菌接种到含有3％蛋白胨、2.5％酵母浸粉、1％碳酸钙的番茄-卷心菜汁(番茄汁80％，卷心菜汁20％)培养基中，37℃条件下培养15h，然后将增殖培养的菌液在4℃下6000r/min离心5min，除去上清液，并用等体积PBS缓冲液洗涤至菌体无色；用含有10％脱脂乳粉、1.5％木糖醇、2％乙酸钠和6％谷氨酸钠的冻干保护剂重悬收集的菌体，在-25℃预冻1h后放入-80℃冷冻12h，菌体置于冻干机中-50℃，5Pa真空冷冻干燥12h；收集冻干粉置于-80℃冰箱备用；将50份全脂牛乳粉、35份脱脂牛乳粉、5份益生菌菌粉和4份木糖醇粉碎后过60目筛，得到奶片粗混合物；将3份羧甲基纤维素钠和2份硬脂酸镁与奶片粗混合物混匀，然后压片，采用塑料和铝箔板包装得到益生菌奶片。
实施案例2：动物双歧杆菌亚种F1-7豆奶饮品。一种促进肠蠕动的动物双歧杆菌亚种F1-7豆乳饮品，包括以下步骤：将鲜驼乳和豆浆按2：1比例混合并加热至50℃以上，加入7%-9%的白砂糖搅拌至完全溶解，预热温度为55-65℃，20Mpa的压力进行均质化，95℃进行杀菌处理，时间3-5分钟，冷却温度至45℃，接种发酵剂0.005%-0.008%和本发明的动物双歧杆菌F1-7为0.004%-0.006%。之后39-44℃进行发酵处理，pH为4.5-4.8，经冷却和冷藏处理，制成动物双歧杆菌亚种F1-7豆奶饮品。
最后应说明的是：以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。
序列表
<110> 中国海洋大学
<120> 一株促肠蠕动动物双岐杆菌亚种F1-7及应用
<130> 2021
<150> 2020115856034
<151> 2020-12-29
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1444
<212> DNA
<213> Bifidobacterium animalis subsp. lactis strain
<400> 1
gggtgggggg cgttcttaca catgcagtcg aacgggatcc ctggcagctt gctgtcgggg 60
tgagagtggc gaacgggtga gtaatgcgtg accaacctgc cctgtgcacc ggaatagctc 120
ctggaaacgg gtggtaatac cggatgctcc gctccatcgc atggtggggt gggaaatgct 180
tttgcggcat gggatggggt cgcgtcctat cagcttgttg gcggggtgat ggcccaccaa 240
ggcgttgacg ggtagccggc ctgagagggt gaccggccac attgggactg agatacggcc 300
cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag 360
cgacgccgcg tgcgggatgg aggccttcgg gttgtaaacc gcttttgttc aagggcaagg 420
cacggtttcg gccgtgttga gtggattgtt cgaataagca ccggctaact acgtgccagc 480
agccgcggta atacgtaggg tgcgagcgtt atccggattt attgggcgta aagggctcgt 540
aggcggttcg tcgcgtccgg tgtgaaagtc catcgcctaa cggtggatct gcgccgggta 600
cgggcgggct ggagtgcggt aggggagact ggaattcccg gtgtaacggt ggaatgtgta 660
gatatcggga agaacaccaa tggcgaaggc aggtctctgg gccgtcactg acgctgagga 720
gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacggtgg 780
atgctggatg tggggccctt tccacgggtc ccgtgtcgga gccaacgcgt taagcatccc 840
gcctggggag tacggccgca aggctaaaac tcaaagaaat tgacgggggc ccgcacaagc 900
ggcggagcat gcggattaat tcgatgcaac gcgaagaacc ttacctgggc ttgacatgtg 960
ccggatcgcc gtggagacac ggtttccctt cggggccggt tcacaggtgg tgcatggtcg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa ccctcgccgc 1080
atgttgccag cgggtgatgc cgggaactca tgtgggaccg ccggggtcaa ctcggaggaa 1140
ggtggggatg acgtcagatc atcatgcccc ttacgtccag ggcttcacgc atgctacaat 1200
ggccggtaca acgcggtgcg acacggtgac gtggggcgga tcgctgaaaa ccggtctcag 1260
ttcggatcgc agtctgcaac tcgactgcgt gaaggcggag tcgctagtaa tcgcggatca 1320
gcaacgccgc ggtgaatgcg ttcccgggcc ttgtacacac cgcccgtcaa gtcatgaaag 1380
tgggtagcac ccgaagccgg tggcccgacc cttgtggggg gagccgtcta agggtaagaa 1440
ctcg 1444
Claims (2)
1.一株动物双歧杆菌亚种F1-7(Bifidobacterium animalis subsp F1-7)，其特征在于，所述的动物双歧杆菌菌株在中国典型培养物保藏中心保藏，地址为湖北省武汉市武汉大学保藏中心，保藏日期2020年12月2日，保藏号为CCTCC NO：M 2020833。
2.权利要求1所述的动物双歧杆菌亚种F1-7在制备促进肠蠕动乳制品中的应用。
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| CN110923166A (zh) * | 2019-12-11 | 2020-03-27 | 石家庄君乐宝乳业有限公司 | 动物双歧杆菌乳亚种jmcc0025、其分离纯化方法及应用 |
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| CN110577919A (zh) * | 2019-10-14 | 2019-12-17 | 汉臣氏（沈阳）儿童制品有限公司 | 一种分离自婴儿粪便的耐热型动物双歧杆菌乳亚种 |
| CN110923166A (zh) * | 2019-12-11 | 2020-03-27 | 石家庄君乐宝乳业有限公司 | 动物双歧杆菌乳亚种jmcc0025、其分离纯化方法及应用 |
| CN111484957A (zh) * | 2019-12-11 | 2020-08-04 | 石家庄君乐宝乳业有限公司 | 动物双歧杆菌乳亚种i797、其分离纯化方法及应用 |
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