CN109136129B - 一株嗜酸乳杆菌ncu426 - Google Patents
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Abstract
一株嗜酸乳杆菌NCU426：所述嗜酸乳杆菌Lactobacillus acidophilus NCU426，已于2018年4月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心，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，其简称为CGMCC，保藏编号为CGMCC NO15572，嗜酸乳杆菌NCU426具有优良的肠道菌群调节作用；嗜酸乳杆菌NCU426的耐酸性能具有稳定的遗传性且对生物胺有明显的抑制作用，可以有效降低由发酵带来的生物胺过高的问题。
Description
技术领域
本发明属于于微生物领域，具体涉及一株嗜酸乳杆菌NCU426。
背景技术
对果蔬汁进行微生物发酵的过程能够促进果蔬原料中碳水化合物、蛋白质、脂类等物质的代谢反应，大量产生和积累初级及次级代谢产物，生成有机酸、低聚糖、糖醇、酶类、低聚肽、多酚等多种有益成分；同时，经发酵得到的酵素饮料中还包含果蔬原料及微生物自身所含有的营养及功能成分。这些有益成分可以促进肠道有益菌的增殖，抑制有害菌的繁殖及腐败物质形成，起到调节肠道菌群平衡、增强免疫、促进睡眠、延缓衰老等作用，对人体有多种有益功效。因而，利用天然果蔬制备酵素饮料因其营养保健功能和独特的风味深受广大消费者的青睐；由于人体胃里面的环境长期处于酸性状态，在发酵过程中发酵菌往往会由于耐酸性不强而被杀死，大量的发酵有益菌耐胆盐性能也很差，有益菌不能进入肠道，所以寻找一种耐酸能力、耐胆盐性能强的有益发酵菌尤为重要。
生物胺是食品中普遍存在的有害物质，尤其是发酵食品中，食品中的生物胺主要是由微生物产生，同时也受游离氨基酸含量、温度、 pH值、含盐量等影响。各类食品中生物胺的含量不尽相同，对于肉制品和水产品而言，屠宰后的新鲜肉和新鲜的水产品生物胺含量非常低，但在贮藏过程中会有所上升，而经过发酵之后得到的香肠或腌制品中生物胺普遍存在，且部分含量很高；此外，发酵乳制品中奶酪中生物胺浓度很高，调味品(如鱼露、豆豉、醋等)中有些胺的含量很高，橙汁等果汁类、番茄酱等蔬菜罐头类和咖啡豆等中也发现了生物胺。因为不同生物胺的毒性差别较大，且不同人群对生物胺的解毒能力也相差较大，建立统一的生物胺限量标准非常困难。美国FDA规定水产品中的组胺含量不得超过50mg/kg，并建议酪胺含量不要超过 100mg/kg，生物胺总量不要超过1000mg/kg；本发明采用在制备饮料的过程中采用的两种乳杆菌混合发酵，对降低饮料、食品中生物胺有明显的效果，尤其是在发酵过程中产生的。CN105132308 A提供了一种能降低食品中生物胺含量的植物乳杆菌，但是采用的是一种乳杆菌，对总胺的降解率较低为67.3％，效果不够理想。
发明内容
本发明的目的是提供一株嗜酸乳杆菌NCU426及其应用。
一株嗜酸乳杆菌NCU426及其应用，其特征在于：所述嗜酸乳杆菌NCU426Lactobacillus acidophilus NCU426，已于2018年4月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心，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，其简称为 CGMCC，保藏编号为CGMCC NO15572；其16S rRNA序列SEQ ID:1 所示。
所述嗜酸乳杆菌菌株的获得方式由以下步骤组成：
A：对常规的酸性水果清洗去杂，打浆后装瓶密封，所使用的工具需提前灭菌，在37℃恒温培养箱中培养，待果浆自然发酵36h后梯度稀释，选择浓度为10-30％果浆接种于MRS选择性培养基中，此为初筛，在初筛固体培养基上挑取溴甲酚紫变黄的菌落，以该类菌株进行钙溶解圈实验选择出产酸能力及生长繁殖强的目的菌株，选择溴甲酚紫变色最快、透明圈最大的菌落进行平板划线获得单菌落此为复筛；
B：根据菌株的形态和生理生化特征，并参照伯杰氏细菌鉴定手册中有关乳酸菌的鉴定标准对其进行分析，并结合16S rRNA序列测序结果，将其鉴定为嗜酸乳杆菌，命名为嗜酸乳杆菌NCU426。
所述乳杆菌可用于日常发酵果蔬饮料、发酵果蔬原浆、发酵果蔬泥的生产领域中。
本发明的有益效果:
(1)嗜酸乳杆菌NCU426在果蔬汁中优良的产酸性能；
(2)嗜酸乳杆菌NCU426具有优良的耐酸性能及胆盐耐受性能；
(3)嗜酸乳杆菌NCU426具有优良的肠道菌群调节作用；嗜酸乳杆菌 NCU426的耐酸性能具有稳定的遗传性。
(4)嗜酸乳杆菌NCU426对生物胺有明显的抑制作用，可以有效降低由发酵带来的生物胺过高的问题。
附图说明
图1菌落形态及镜检结果；
图2菌在桑葚果浆中的酸度变化；
图3 NCU426耐酸性能的遗传稳定性。
具体实施方式
实施例1
菌株的筛选及获得
本发明的嗜酸乳杆菌NCU426的获得：将常规的酸性水果清洗、去杂、打浆(提前开水烫漂的打浆杯及培养瓶)密封于培养瓶，在 37℃恒温培养箱培养。待果浆自然发酵36h后梯度稀释，选择合适的浓度接种于MRS选择性培养基中，此为初筛。挑取溴甲酚紫变黄且菌落大小不同的菌落单独划线纯化，对于溴甲酚紫变黄且菌落大小相近的菌落混合稀释，涂布重复初筛步骤。对初筛纯化的单菌株进行钙溶解圈实验，通过游标卡尺测量透明圈直径选择出产酸能力、生长繁殖强的目的菌株。目的菌株在pH2.5的条件下优化3月，并进行生理生化实验观察。最终经过提取DNA、PCR，进行16SrRNA测序筛选得到的最优的菌株NCU426。NCU426革兰氏染色呈阳性，专性厌氧，无运动属性，生长周期不产生芽孢，无荚膜和鞭毛。
该菌株已于2018年4月9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，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，菌种保藏编号为CGMCCNO15572，建议分类命名为嗜酸乳杆菌(Lactobacillus acidophilus)。
实施例2
菌株菌落形态及镜检
在平板上观察菌落形态，并无菌条件下接种环挑取嗜酸乳杆菌 NCU426，涂于载玻片上，制片并革兰氏染色，在显微镜下观察菌体形态具体见图1。
实施例3
菌株优良的产酸性能
菌株优良的产酸性能不但通过其应用价值体现，而且要明显优于其它菌株。本实例以桑葚为发酵原料接种NCU426，并以CICC6092为发酵对照菌株对比菌株产酸性能。结果见图2。
实施例4
菌株优良的耐酸性能
用液体培养基活化冻干管保存的NCU426，培养24h。取0.2mL 接种到100mL三角瓶中培养18h即得新鲜种子液。调节PBS缓冲溶液pH至2.5，接种新鲜种子液0.5mL于99.5mLPBS缓冲溶液中，分别在0、0.5、1、1.5、2、2.5h测其菌落数，CICC6092为对照菌株。
表1 NCU426在pH 2.5的PBS缓冲溶液中的耐酸性
实施例5
菌株调节肠道菌群的作用
表2嗜酸乳杆菌NCU426对小鼠肠道菌群的影响(log10 CFU/g)
实施例6
菌株的耐酸性能具有稳定遗传性
嗜酸乳杆菌NCU426连续传代20代，每代菌株用甘油法留样。用 pH2.5的PBS缓冲溶液对留样进行耐酸性测定。将待测定的菌株用MRS 液体培养基复苏并取2mL对数期的新鲜菌液于5mL离心管中，在4500rpm下离心20min获得菌体。将菌体转移至100mL pH2.5的 PBS缓冲溶液中培养3h，在处理时刻0、3h时，用平板计数法涂布测其菌落浓度，结果见图3。
实施例7
菌株优良的胆盐耐受性能
在MRS液体培养基中加入浓度为0、0.5、1.5、2.5、3.5、4.5 g/100mL的牛胆盐，121℃高压蒸汽灭菌20min，冷却至37℃以下备用。将活化后的新鲜菌液以1％的接种量接种到上述处理后的培养基中，在0、1、2、3h涂布测其菌落变化。
表3 NCU426胆盐耐受性
实施例8
本菌株对生物胺的的降解率测定：
采用高效液相色谱法分别测定采用嗜酸乳杆菌NCU426发酵的饮料，和采用普通嗜酸乳杆菌ATCC 4356(从中科院微生物所购买的普通嗜酸乳杆菌)发酵的饮料，测定加入乳杆菌前后饮料中生物胺的降解率，通过积分计算含量；得出对生物胺的的降解率，测定条件为：色谱柱为C18型号；紫外检测波长为254nm，进样量10ul，柱温30℃，流速1.5ml/min，结果具体见表4。
表4
从本表中可以明确的得出采用本发明的两种混合菌株所制备的饮料中生物胺的降解率很高，综合生物胺的降解率在90％以上，未采用本发明指定的乳杆菌所制备饮料对生物胺的降解率很低。
序列表
<110> 南昌大学
<120> 一株嗜酸乳杆菌NCU426
<130> 20180720
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1143
<212> DNA
<213> 嗜酸乳杆菌(Lactobacillus acidophilus)
<400> 2
gcccgtaccg gcttgcgtgt actgcagtcg agcgagctga accaacagat tcacttcggt 60
gatgacgttg ggaacgcgag cggcggatgg gtgagtaaca cgtggggaac ctgccccata 120
gtctgggata ccacttggaa acaggtgcta ataccggata agaaagcaga tcgcatgatc 180
agcttataaa aggcggcgta agctgtcgct atgggatggc cccgcggtgc attagctagt 240
tggtagggta acggcctacc aaggcaatga tgcatagccg agttgagaga ctgatcggcc 300
acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360
aatggacgaa agtctgatgg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa 420
gctctgttgt tggtgaagaa ggatagaggt agtaactggc ctttatttga cggtaatcaa 480
ccagaaagtc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540
gtccggattt attgggcgta aagcgagcgc aggcggaaga ataagtctga tgtgaaagcc 600
ctcggcttaa ccgaggaact gcatcggaaa ctgtttttct tgagtgcaga agaggagagt 660
ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc 720
ggctctctgg tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga 780
taccctggta gtccatgccg taaacgatga gtgctaagtg ttgggaggtt tccgcctctc 840
agtgctgcag ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact 900
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960
cgaagaacct taccaggtct tgacatctag tgcaatccgt agagatacgg agttcccttc 1020
ggggacacta agacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag atgttggggt 1080
taagtcccgc aacgagcgca acccttgtca ttagttgcca gcattaagtt ggggcacttc 1140
taa 1143
Claims (1)
1.一株嗜酸乳杆菌NCU426，其特征在于：所述嗜酸乳杆菌(Lactobacillusacidophilus)NCU426，已于2018年4月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心，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，其简称为CGMCC，保藏编号为CGMCC NO15572；其16S rRNA序列如SEQ ID NO:1所示。
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