CN111466440A - 一种可缓解便秘的益生菌发酵乳 - Google Patents
一种可缓解便秘的益生菌发酵乳 Download PDFInfo
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- CN111466440A CN111466440A CN202010493077.2A CN202010493077A CN111466440A CN 111466440 A CN111466440 A CN 111466440A CN 202010493077 A CN202010493077 A CN 202010493077A CN 111466440 A CN111466440 A CN 111466440A
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- fermented milk
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- bifidobacterium animalis
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Abstract
本申请公开了一种可缓解成人便秘的益生菌发酵乳,所述发酵乳由具有益生功能的发酵剂菌种发酵而成。其中,发酵剂菌株包含保加利亚乳杆菌ND02(Lactobacillus bulgaricus ND02)、嗜热链球菌ND03(Streptococcus thermophilus ND03)、干酪乳杆菌Zhang(Lactobacillus casei Zhang)和动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)。本申请通过临床人群实验证实了该益生菌发酵乳可改善肠道菌群、提高肠内有益菌丰度、调节免疫因子及促进维生素E和肉毒碱穿梭系统代谢途径,达到增强肠道蠕动和改善便秘的效果。
Description
技术领域
本申请涉及生物技术领域,尤其涉及一种可缓解便秘的益生菌发酵乳。
背景技术
便秘,在临床上是指出现排便困难或费力、排便不畅、排便次数减少、粪便干、结量少等症状,发病率呈逐年上升。便秘可导致肠道蠕动减缓,肠道内有害菌增加,有益菌减少,微生态环境失衡,是临床较为常见的功能性胃肠病之一。研究表明,便秘患者肠道菌群产生紊乱,标志性菌群如双歧杆菌和乳杆菌丰度或数量降低,致病菌和条件致病菌丰度或数量增加。短期便秘可影响人们的正常工作和生活,长时间便秘不仅降低生活质量还可能会引起如结肠癌、乳腺癌、阿尔兹海默症等严重疾病,甚至引起猝死。因此,一种能够有效缓解便秘,并且对人体不造成其他有害作用的有效方法显然十分必要。
益生菌是目前消费者广泛接受、认可和促进自身健康的膳食补充剂之一,在现代生活中具有重要地位。益生菌是指当人体摄入足够量后,会对人体健康产生明确益处的一类活的微生物总称。发酵乳可促进人体健康已成为共识,但多数只含有不耐酸性环境的嗜热链球菌和保加利亚乳杆菌。益生菌发酵乳不仅能保持普通发酵乳的健康作用,更能结合益生菌独有的益生功效,进而产生多重健康作用。本发明所述的发酵乳产品由具有益生功能的发酵剂发酵而成,所添加干酪乳杆菌Zhang(Lactobacillus casei Zhang)和动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)具有良好的肠道定植能力,能够改善人体肠道菌群,使得该产品具有明显的缓解和治疗便秘的益生功能。
发明内容
本申请的目的之一在于提供一种益生菌发酵乳;
本申请的目的之二在于提供一种益生菌发酵乳在缓解便秘症状的应用;
本申请的目的之三在于提供所述干酪乳杆菌Zhang(Lactobacillus caseiZhang)和所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)在缓解便秘症状的应用;
进一步的,所述干酪乳杆菌Zhang(Lactobacillus casei Zhang)和所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)在制备食品、保健品或药品中的应用。
为达到上述目的,本申请通过以下技术方案来实现:
本申请中益生菌发酵乳是由基础发酵剂保加利亚乳杆菌ND02(Lactobacillusbulgaricus ND02),嗜热链球菌ND03(Streptococcus thermophilus ND03);同时添加了干酪乳杆菌Zhang(Lactobacillus casei Zhang)和动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)在42℃环境下恒温协同发酵而成。
保加利亚乳杆菌ND02(Lactobacillus bulgaricus ND02)和嗜热链球菌ND03(Streptococcus thermophilus ND03)是从我国自然发酵酸牛奶中分离得到的具有产酸、产香、产粘等优良发酵特性的发酵剂菌株。其中,所述保加利亚乳杆菌ND02保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17757;分类名称为保加利亚乳杆菌(Lactobacillus bulgaricus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。所述嗜热链球菌ND03保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNo.17756。分类名称为嗜热链球菌(Streptococcus thermophilus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。
干酪乳杆菌Zhang(Lactobacillus casei Zhang)是2002年分离自内蒙古大草原自然发酵酸马奶中的1株具有多种益生功能的益生菌,是我国第1株完成全基因组测序的益生菌,所述干酪乳杆菌Zhang(Lactobacillus casei Zhang)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5469。分类名称为干酪乳杆菌(Lactobacilluscasei);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。适应性进化研究表明干酪乳杆菌Zhang(Lactobacillus casei Zhang)基因组稳定,是一株可定植于人体肠道,改善排便频率,使肠道菌群年轻化的优良菌株。
动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)是2005年分离自健康蒙古族儿童肠道的1株益生菌,所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5470。分类名称为动物双歧杆菌乳亚种(Bifidobacterium lactis);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。基因组学研究发现该菌株发生重组、突变几率极小,遗传稳定。试验研究表明:动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)可拮抗肠道致病菌、可使人体肠道菌群结构健康化,对便秘治疗有效率为95.3%。
本申请所得结论是结合血液代谢组、粪便代谢组及调查问卷(通过粪便次数、排便类型和排便不尽感等指标衡量),综合评估益生菌发酵乳产品对便秘人群症状缓解的情况,以部分免疫、炎症因子、血糖、血脂代谢相关指标,链接肠道微生物宏基因组水平探讨益生菌发酵乳产品对便秘患者肠道菌群的影响,以期建立益生菌发酵乳、肠道菌群与便秘三者之间相关性。
本发明的有益效果在于:提供富含益生菌的发酵乳,消化道抗性增强,可有效缓解便秘的不良反应,进而达到改善便秘效果。同时,也有助于功能性保健食品的开发。
附图说明
为了更清楚地说明本申请的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1.益生菌发酵乳缓解便秘的影响实验设计流程图;
图2.食用益生菌发酵乳后排便不尽感随食用益生菌发酵乳时间的变化;
图3.食用益生菌发酵乳后疼痛感随食用益生菌发酵乳时间的变化;
图4.食用益生菌发酵乳后不成功次数随食用益生菌发酵乳时间的变化;
图5.食用益生菌发酵乳后排便时间随食用益生菌发酵乳时间的变化;
图6.食用益生菌发酵乳后排便次数随食用益生菌发酵乳时间的变化。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细说明。
保加利亚乳杆菌ND02(Lactobacillus bulgaricus ND02)和嗜热链球菌ND03(Streptococcus thermophilus ND03)是从我国自然发酵酸牛奶中分离得到的具有产酸、产香、产粘等优良发酵特性的发酵剂菌株。其中,所述保加利亚乳杆菌ND02保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17757;分类名称为保加利亚乳杆菌(Lactobacillus bulgaricus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。所述嗜热链球菌ND03保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNo.17756。分类名称为嗜热链球菌(Streptococcus thermophilus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。
干酪乳杆菌Zhang(Lactobacillus casei Zhang)是2002年分离自内蒙古大草原自然发酵酸马奶中的1株具有多种益生功能的益生菌,是我国第1株完成全基因组测序的益生菌,所述干酪乳杆菌Zhang(Lactobacillus casei Zhang)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5469;分类名称为干酪乳杆菌(Lactobacilluscasei);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。适应性进化研究表明干酪乳杆菌Zhang(Lactobacillus casei Zhang)基因组稳定,是一株可定植于人体肠道,改善排便频率,使肠道菌群年轻化的优良菌株。
动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)是2005年分离自健康蒙古族儿童肠道的1株益生菌,所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5470;分类名称为动物双歧杆菌乳亚种(Bifidobacterium lactis);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。基因组学研究发现该菌株发生重组、突变几率极小,遗传稳定。试验研究表明:动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)可拮抗肠道致病菌、可使人体肠道菌群结构健康化,对便秘治疗有效率为95.3%。
实施例1
益生菌发酵乳产品制作工艺及贮藏稳定性评估实验
1.将牛奶(94%,温度为60℃)和白砂糖(6%)混和均匀后,经63℃、20Mpa均质,95℃、5min灭菌后冷却至42℃接种发酵剂菌种,恒温发酵至pH值低于4.50时终止,急速冷却后转至4℃备用。具体发酵剂菌种接种量如表1所示:
表1.发酵剂菌种接种量明细
2.益生菌发酵乳产品贮藏评估
益生菌发酵乳产品在货架期pH值、滴定酸度和益生菌活菌数的变化稳定性是衡量其质量的重要指标。本申请所述贮藏评估,是将益生菌发酵乳分别在4℃、10℃和25℃贮藏,测定其pH值、滴定酸度和活菌数(每7d进行一次)变化情况,并进行数据统计。具体地,操作如下:
称取10g(精确到0.001g)已混匀的发酵乳试样,置于150mL锥形瓶中,加20mL新煮沸冷却至室温的水,混匀,用浓度为0.1000mol/L的氢氧化钠标准溶液电位滴定至pH 8.3为终点:记录消耗的氢氧化钠标准溶液毫升数,代入公式:计算可得滴定酸度(°T)。公式如下:
Lactobacillus casei Zhang活菌数测定采用固体MRS+万古霉素(1mg/1000mL)培养基对Lactobacillus casei进行选择培养,倾注法厌氧37℃培养(72±2)h。
Bifidobacterium lactis V9活菌数测定采用固体MRS+莫匹罗星锂盐(5mg/100mL)培养基对Bifidobacterium lactis V9进行选择培养,倾注法厌氧37℃培养(72±2)h。
表2.益生菌发酵乳样品4℃下贮藏pH值、滴定酸度及益生菌活菌数检测结果
表3.益生菌发酵乳样品10℃下贮藏pH值、滴定酸度及益生菌活菌数检测结果
表4.益生菌发酵乳样品25℃下贮藏pH值、滴定酸度及益生菌活菌数检测结果
结果如上述表中所示:富含益生菌发酵乳在贮藏期间(0~28d)具有较高的活菌数,且在常温长时间储藏仍能保持较高的活菌数,成品酸度适宜,味道可口。
实施例2
益生菌发酵乳便秘人群临床实验
2.1志愿者纳入标准
A.20-40岁
B.将排便出现下面两项症状纳入研究对象:
a排便费力(至少每4次排便中有一次);
b排便为干块状或硬便(至少每4次排便中有一次);
c有排便不尽感(至少每4次排便中有一次);
d有肛门直肠梗阻或阻塞感(至少每4次排便中有一次);
e需要用手操作(如手指辅助排便、盆底支撑排便)以促进排便(至少每4次排便中有一次);
f排便少于每周3次。
2.2排除标准
A器质性便秘、神经性便秘和顽固性便秘不纳入此次研究;
B试验前一个月内食用发酵乳或抗生素;
C未能按时填写问卷调查和提供粪便;
D未按时服用益生菌发酵乳。
最终招募到满足如上标准的便秘等肠道健康问题的受试者26人,行7天排空期,连续服用4周益生菌发酵乳(200g/日)。采集试验开始(0d)和结束(28d)的血液和粪便样品,志愿者每周填写肠道健康调查问卷和饮食调查问卷并结合免疫组学、代谢组学和宏基因组学等指标,综合评估益生菌发酵乳对便秘患者的影响。
具体实验方案参照图1。
结果如下:
1.益生菌发酵乳改善便秘症状
问卷调查结果具体如下所示,服用益生菌发酵乳显著降低了排便过程中的不尽感(3.08分至1.67分,P<0.0001)、用力程度(3.00分至1.40分,P<0.0001)和疼痛感(2.80分至1.64分,P<0.0001);并且显著降低排便不成功次数(2.12至1.4次/周,P=0.0084),显著缩短排便持续时间(缩短22.53%,P=0.0022),排便次数由每周3.76次增加到5.46次(但是无显著性差异,P=0.059),参见图2-图6。
综上所述,食用益生菌发酵乳可有效缓解便秘症状。
2.益生菌发酵乳对免疫因子的影响
取患者晨起空腹静脉血一部分用于血常规检测,一部分血液静置,待血液凝固后,将其离心(3000rpm,10min),得到的上清液即为血清。检测患者血清免疫指标:免疫球蛋白如IgG1,IgG2,IgM和IgA;慢性炎症因子:C反应蛋白(CRP)、脂多糖结合蛋白(LBP)、脂多糖(LPS)、可溶性CD14;激素类物质:皮质醇(Cortisol)。
构建OPLSDA模型发现,服用益生菌发酵乳28天后,血液中免疫因子含量与0天相比区分明显。
食用益生菌发酵乳显著降低了血液中脂多糖LPS(P<0.01)和C反应蛋白CRP(P<0.05)的含量,它们与便秘症状存在较强的负相关,即血液中LPS和C反应蛋白CRP含量越少,便秘症状缓解和改善程度越大;食用益生菌发酵乳还增加了脂多糖结合蛋白LBP(P<0.05)的含量,与便秘症状呈正相关,即LBP含量越高,便秘症状越少。
3.益生菌发酵乳对便秘人群血液和粪便代谢物整体的影响
将血清和粪便样品进行前处理,通过LC-MS技术非靶向测定血清和粪便样品全谱,以确定不同治疗组间代谢差异物质。
饮用发酵乳前后,存在明显聚类趋势,表明食用发酵乳后便秘人群粪便和血液中的代谢物发生了明显的变化,尤其是血液代谢物。
食用发酵乳后志愿者血液代谢物差异明显,涉及到胆汁酸合成、C21类固醇激素合成及代谢等。结果显示胆汁酸合成水平减少。据报道,胆汁酸次级代谢产物对人体危害众多,是恶性肿瘤的启动子。表明益生菌发酵乳能显著减少胆汁酸含量,对人体健康具有保护作用。
食用发酵乳后,志愿者粪便中代谢物区分较明显;Vitamin E、Glycosphingolipid(糖脂)在服用发酵乳后含量发生了显著变化。这些特征代谢物促进脂肪酸代谢水平增加,调节Treg细胞分化、具有抗氧化和抑制炎症的作用;并其对增加肠血流、维持肠道形态及功能有重要作用,由此推测益生菌发酵乳能改善肠道健康。
综上所述,益生菌发酵乳缓解便秘的机理是通过对肠道菌群进行微调,调控炎症因子,降低血液中次级胆汁酸代谢物,产生对宿主有利的代谢物。
4.益生菌发酵乳对肠道菌群的影响
提取粪便样品DNA,将符合测序要求的DNA依次进行随机片段化、末端修复、连接接头盒构建文库,之后利用Illumina GAIIx和HiSeq 2000对样品进行测序。以分析益生菌对菌群功能基因及代谢通路的影响。
饮用发酵乳后不存在明显的聚类趋势,即食用发酵乳不会影响便秘人群肠道菌群整体结构,饮用发酵乳后,便秘人群肠道菌群多样性有升高,但是差异不显著(P=0.61)。采用LefSe分析比较了在饮用发酵乳0周和4周时肠道中相对含量存在显著差异的菌群,发现食用发酵乳后乳球菌属和噬纤维素菌属的部分有害菌菌种相对含量降低,肠道中乳杆菌属、链球菌属、双歧杆菌属等有益菌相对含量显著升高,而肠道中双歧杆菌、乳杆菌属含量的升高具有缓解便秘症状的作用。
志愿者肠道中主要的双歧杆菌为链状双歧杆菌、青春双歧杆菌和短双歧杆菌;服用益生菌发酵乳后,青春双歧杆菌和动物双歧杆菌增加。由此推测检测到的动物双歧杆菌来源于发酵乳发酵剂动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactisV9),即动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)可在大部分志愿者肠道中定殖。
服用益生菌发酵乳后肠道中嗜热链球菌和嗜酸乳杆菌相对含量增加,乳酸杆菌的丰度和多样性增加(P<0.05)。
综上所述,食用发酵乳后,可以对肠道菌群微调,增加肠道中有益菌、减少有害菌,对于维持肠道菌群稳态有积极作用。
经过人体试验表明,该产品对于便秘有显著的缓解功效。
本领域技术人员在考虑说明书及实践这里公开的发明后,将容易想到本发明的其它实施方案。本申请旨在涵盖本发明的任何变型、用途或者适应性变化,这些变型、用途或者适应性变化遵循本发明的一般性原理并包括本发明未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的,本发明的真正范围和精神由下面的权利要求指出。
Claims (8)
1.一种可缓解便秘的益生菌发酵乳,其特征在于,所述益生菌发酵乳是由基础发酵剂保加利亚乳杆菌ND02(Lactobacillus bulgaricus ND02)和嗜热链球菌ND03(Streptococcus thermophilus ND03);同时添加了干酪乳杆菌Zhang(Lactobacilluscasei Zhang)和动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)协同发酵而成。
2.权利要求1所述的可缓解便秘的益生菌发酵乳,其特征在于,所述保加利亚乳杆菌ND02保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17757;分类名称为保加利亚乳杆菌(Lactobacillus bulgaricus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。
3.权利要求1所述的可缓解便秘的益生菌发酵乳,其特征在于,所述嗜热链球菌ND03保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.17756;分类名称为嗜热链球菌(Streptococcus thermophilus);保藏时间:2019年5月10日;保藏地址:中国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;该菌株的检测结果为存活。
4.权利要求1所述的可缓解便秘的益生菌发酵乳,其特征在于,所述干酪乳杆菌Zhang(Lactobacillus caseiZhang)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5469;分类名称为干酪乳杆菌(Lactobacillus casei);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。
5.权利要求1所述的可缓解便秘的益生菌发酵乳,其特征在于,所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.5470;分类名称为动物双歧杆菌乳亚种(Bifidobacterium lactis);保藏时间:2011年11月18日;保藏地址:中国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;该菌株的检测结果为存活。
6.权利要求1所述的可缓解便秘的益生菌发酵乳在缓解便秘症状上的应用。
7.权利要求4或5所述干酪乳杆菌Zhang(Lactobacillus caseiZhang)和所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactis V9)在缓解便秘症状上的应用。
8.根据权利要求7所述的应用,其特征在于,所述干酪乳杆菌Zhang(Lactobacilluscasei Zhang)和所述动物双歧杆菌乳亚种V9(Bifidobacterium animalis subsp lactisV9)在制备食品、保健品或药品中的应用。
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